Isochlorogenic acid A and PD-1 inhibitor combination and application of pharmaceutical composition

Abstract

The invention belongs to the technical field of medicine and disease treatment, and particularly relates to combination of isochlorogenic acid A and a PD-1 inhibitor and application of a pharmaceutical composition. The invention provides an application of combination of isochlorogenic acid A and a PD-1 inhibitor in preparation of a medicine for preventing and / or treating breast cancer. The breast cancer is triple negative breast cancer. Research results show that in a mouse model, the combination of the isochlorogenic acid A and the PD-1 inhibitor can significantly enhance the infiltration of macrophages and CD8 < + > T cells to tumors, and reduce the number of failure T cells in a tumor immune microenvironment at the same time. In addition, the isochlorogenic acid A can also inhibit the activity of FAK / PI3K / AKT / mTOR signal channels, so that the growth of tumors is reduced.

Description

Technical Field

[0001] The present invention belongs to the technical field of drugs and disease treatment, and specifically relates to the application of a combination of isochlorogenic acid A and a PD-1 inhibitor and a pharmaceutical composition. Background Art

[0002] Triple-negative breast cancer (TNBC), as a highly aggressive subtype of breast cancer, is characterized by the lack of expression of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2). This lack of targetable receptors increases the complexity of treatment strategy selection, leading to poor prognosis and limited treatment options. Although immune checkpoint blockade (ICB) therapy has shown certain efficacy in the treatment of various cancers, its objective response rate (ORR) is usually less than 10% in invasive cancers such as TNBC, which remains a major challenge in the field of tumor treatment. Existing studies have shown that this low ORR may be partly attributed to the lack of functional T cell infiltration in the tumor microenvironment. Therefore, it is particularly urgent to explore combination therapies to improve clinical outcomes. Recent studies have revealed that natural compounds have shown potential effects in enhancing immune responses and synergizing traditional cancer treatments. In particular, in the field of immunotherapy, immune checkpoint inhibitors, such as PD-1 inhibitors, have become new treatment options for the treatment of various cancers, including TNBC. However, the response rate of single-drug treatment suggests that further research and development of new treatment strategies are needed to improve treatment efficacy and overcome the immune escape mechanism of tumors.

[0003] Dandelion (Taraxacum officinale) has gained attention in cancer research due to its various pharmacological properties, including anti-inflammatory, antioxidant, and anticancer properties. Bioactive compounds in dandelion, such as sesquiterpene lactones, flavonoids, and phenolic acids, have shown immunomodulatory effects, making them potential therapeutic options in cancer treatment.

[0004] Focal adhesion kinase (FAK) is a non-receptor tyrosine protein kinase that plays a key role in various stages of tumorigenesis, development, migration and invasion. FAK was identified in 1992 as a highly phosphorylated protein associated with the oncogene v-src, which localizes to integrin-enriched adhesion sites in normal cells. Overexpression of FAK, as well as enhanced phosphorylation and increased enzymatic activity, provide strong evidence for the tumorigenesis of FAK in a variety of cancers, including colon cancer, breast cancer, and ovarian cancer. Various receptors on the cell surface activate FAK, which then transmits signals inside the cell. Because FAK is overexpressed in most tumor cells, it has been used as a marker for diagnosing tumor invasion and metastasis, and targeting FAK regulation may provide a new strategy for cancer treatment.

[0005] In recent years, the research field of chlorogenic acid has been continuously expanded and deepened, and it has shown extensive application potential in many fields such as medicine, chemical industry and food, indicating the broadness of its application prospects. As a water-soluble phenolic compound, chlorogenic acid is ubiquitous in plants in nature and exhibits significant biological activity and diverse pharmacological effects. Chlorogenic acid is a desuccinic acid formed by the condensation reaction of caffeic acid and quinic acid. Chlorogenic acid has a variety of isomers depending on the binding position and number of caffeoyl groups on the quinic acid molecule. Among them, 3-caffeoylquinic acid (also known as neochlorogenic acid), 4-caffeoylquinic acid (also known as cryptochlorogenic acid) and 5-caffeoylquinic acid are more common isomer forms.

[0006] In a 2017 study, Martínez et al. reported that chlorogenic acid showed antifungal activity against a variety of plant pathogenic fungi and had the potential to be used as a biofungicide. Its mechanism of action may involve inhibiting the early membrane permeabilization process of fungal spores, thereby controlling the growth of different plant pathogenic fungi. Jiang's research team found that chlorogenic acid has a significant inhibitory effect on liver cancer, lung cancer and glioma cells. Its mechanism of action is not to directly kill tumor cells, but to inhibit tumor growth by inducing tumor cell differentiation. They also proposed that chlorogenic acid is expected to become a safe and effective tumor differentiation inducer. In 2017, Chen Xiaoguang's research team first reported that chlorogenic acid inhibits the growth of brain gliomas by regulating the M1 / M2 polarization state of macrophages, that is, promoting the activation of M1 macrophages and inhibiting the phenotype of M2 macrophages.

[0007] Chlorogenic acid and its isomers are widely found in a variety of foods and herbs, such as apples, grapes, tea leaves, coffee beans, Eucommia leaves, honeysuckle and mulberry leaves. Isochlorogenic acid, as a class of isomers of chlorogenic acid, includes isochlorogenic acid A (3,5-dicaffeoylquinic acid), isochlorogenic acid B (3,4-dicaffeoylquinic acid) and isochlorogenic acid C (4,5-dicaffeoylquinic acid). These compounds are widely distributed in plants and exhibit diverse biological activities. Studies have shown that small molecule drugs can synergize with PD-1 inhibitors to enhance the immune response to tumors. PD-1 inhibitors restore the ability of T cells to recognize and kill tumor cells by blocking the PD-1 / PD-L1 signaling pathway. Some small molecule drugs may produce synergistic effects with PD-1 inhibitors and improve the therapeutic effect by other mechanisms such as regulating the immune microenvironment or directly acting on tumor cells. Considering that tumors may develop resistance to monotherapy, the combination of small molecule drugs may help overcome this resistance. Through different mechanisms of action, small molecule drugs may interfere with the drug resistance mechanism of tumor cells, allowing PD-1 inhibitors to work more effectively. Therefore, the combination of these small molecule drugs with PD-1 inhibitors may be based on their anti-tumor activity and immunomodulatory effects in order to achieve better therapeutic effects. Therefore, it is very important to conduct research on the combination of small molecule drugs and PD-1 inhibitors in the anti-tumor field. At present, there is an urgent need to develop new applications of small molecule drugs combined with PD-1 inhibitors in the anti-tumor field. Summary of the invention

[0008] In order to develop a new application of a small molecule drug in combination with a PD-1 inhibitor in anti-tumor aspects, the present invention provides a combination of isochlorogenic acid A and a PD-1 inhibitor, and the use of the pharmaceutical composition in the preparation of a drug for preventing and / or treating breast cancer.

[0009] To achieve the above object, the present invention adopts the following technical solution:

[0010] The present invention provides the use of isochlorogenic acid A and a PD-1 inhibitor in combination in the preparation of a drug for preventing and / or treating breast cancer. The breast cancer is triple-negative breast cancer.

[0011] The present invention has found that in a mouse model, the combination of isochlorogenic acid A and PD-1 inhibitor can significantly enhance the expression of macrophages and CD8 + T cells infiltrate tumors, while reducing the number of exhausted T cells in the tumor immune microenvironment. That is, the combination of isochlorogenic acid A and PD-1 inhibitors can produce a synergistic effect for the prevention and / or treatment of breast cancer. Therefore, the present invention has developed a new application of a small molecule drug combined with a PD-1 inhibitor in anti-tumor. Among them, isochlorogenic acid A is also called ICGA-A.

[0012] Preferably, the molar ratio of the isochlorogenic acid A to the PD-1 inhibitor is 1.6242 to 1.6244:1. In a study, isochlorogenic acid A was administered at doses of 5 and 10 mg / kg orally once a day for 3 weeks to improve TMT-induced cognitive impairment in ICR male mice. This shows that a dose of 10 mg / kg is safe and effective in animal models. In animal models, the dose of PD-1 inhibitors is generally determined based on pharmacodynamic studies to ensure that the drug can reach sufficient concentrations in the body to exert an anti-tumor effect. 5 mg / kg was found in preliminary studies of the present invention to effectively promote tumor infiltration of CD8 + The dosage of the drug can effectively inhibit tumor growth without causing excessive side effects. The dosage level with no obvious toxic side effects determined in the safety evaluation can ensure the efficacy without causing unacceptable toxicity to animals.

[0013] The structural similarities and differences between isochlorogenic acid A and chlorogenic acid are mainly reflected in the different positions of the caffeoyl groups to which they are connected. Isochlorogenic acid A is 3,5-dicaffeoylquinic acid, that is, two caffeoyl groups are connected to the 3rd and 5th positions of quinic acid respectively. Chlorogenic acid is 3-caffeoylquinic acid, that is, caffeoyl groups are connected to the 3rd position of quinic acid. The differences in biological activity between isochlorogenic acid A and chlorogenic acid are mainly reflected in the following aspects:

[0014] Isochlorogenic acid A has stronger antioxidant activity than chlorogenic acid. Studies have shown that the scavenging rates of isochlorogenic acid A for DPPH and hydroxyl radicals are as high as 97.04% and 60.01%, respectively, and it exhibits good antioxidant activity in the skin fluorescent zebrafish model. In addition, studies on the differences in antioxidant properties between different chlorogenic acid isomers have shown that the antioxidant activity of dicaffeoylquinic acid (such as isochlorogenic acid) is better than that of monocaffeoylquinic acid (such as chlorogenic acid). Isochlorogenic acid A shows good in vitro anti-inflammatory effect and can inhibit leukocyte migration and the production of superoxide anions during inflammation. The anti-inflammatory activity of isochlorogenic acid A may be related to reducing the activation of the NLRP3 inflammasome and the phosphorylation expression of NF-κB. Isochlorogenic acid A has a stronger inhibitory effect on Escherichia coli than chlorogenic acid. Isochlorogenic acid A can destroy the bacterial cell wall and membrane structure, leading to increased cell permeability, thereby causing the leakage of cell contents. Its destructive effect on the bacterial cell wall and membrane structure is stronger than that of chlorogenic acid.

[0015] In summary, the biological activities of isochlorogenic acid A in terms of anti-oxidation, anti-inflammatory, and antibacterial are generally stronger than those of chlorogenic acid, which may be related to the additional caffeoyl groups in its molecular structure, which provide more hydroxyl groups to scavenge free radicals and participate in biological activities. Therefore, the present invention selects the above-mentioned molar ratio of isochlorogenic acid A and the PD-1 inhibitor for use in combination to prepare a drug for preventing and / or treating breast cancer.

[0016] The present invention also provides a pharmaceutical composition for preventing and / or treating breast cancer, wherein the pharmaceutical composition is prepared by using a raw material composition including the isochlorogenic acid A and a PD-1 inhibitor as active ingredients and adding pharmaceutically acceptable excipients.

[0017] Preferably, the molar ratio of the isochlorogenic acid A to the PD-1 inhibitor is 1.6243:1.

[0018] Preferably, the dosage form of the pharmaceutical composition is a solid preparation or a liquid preparation.

[0019] The solid preparation is a powder, and the liquid preparation is an oral solution.

[0020] Preferably, the dosage form of the pharmaceutical composition is a liquid preparation.

[0021] The concentration of isochlorogenic acid A in the pharmaceutical composition is 3.8726 mmol / L to 3.8728 mmol / L, and the concentration of the PD-1 inhibitor in the pharmaceutical composition is 2.3838 mmol / L.

[0022] Preferably, the concentration of isochlorogenic acid A in the pharmaceutical composition is 3.8726 mmol / L, and the concentration of the PD-1 inhibitor in the pharmaceutical composition is 2.3838 mmol / L.

[0023] Preferably, the excipients include any one or more of physiological saline, PEG300, Tween 80 and DMSO. In the present invention, when preparing the DMSO mother solution of isochlorogenic acid A, 2.4 mg of the drug is dissolved in 60 μL DMSO solution (mother solution concentration 40 mg / mL), and then the in vivo formula is prepared, 60 μL DMSO mother solution is taken, 360 μL PEG300 is added, 60 μL Tween 80 is added after mixing and clarifying, 720 μL ddH2O is added after mixing and clarifying, and mixing and clarifying. Therefore, the above reagents are selected as excipients for the preparation of drugs.

[0024] The present invention also provides use of a pharmaceutical composition for preventing and / or treating breast cancer in preparing a drug for preventing and / or treating breast cancer, wherein the breast cancer is triple-negative breast cancer.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] 1. The present invention provides a combination of isochlorogenic acid A and a PD-1 inhibitor, and the use of the pharmaceutical composition in the preparation of a drug for preventing and / or treating breast cancer, and provides a new method for preventing and / or treating breast cancer. The present invention develops a new application of a small molecule drug combined with a PD-1 inhibitor in anti-tumor.

[0027] 2. The present invention studies the effect of isochlorogenic acid A on TNBC tumor growth. The research results of the present invention show that in a mouse model, the combination of isochlorogenic acid A and PD-1 inhibitors can significantly enhance the infiltration of macrophages and CD8+T cells into tumors, while reducing the number of exhausted T cells in the tumor immune microenvironment. In addition, isochlorogenic acid A can also inhibit the activity of the FAK / PI3K / AKT / mTOR signaling pathway, thereby reducing tumor growth. Therefore, isochlorogenic acid A is expected to effectively target TNBC cells by inhibiting the FAK / PI3K / AKT / mTOR pathway and enhancing the efficacy of immunotherapy. The synergistic application of isochlorogenic acid A and PD-1 inhibitors in anti-tumor therapy deserves further study.

[0028] 3. The present invention found that the combined use of dandelion crude extract (TOE) and PD-1 inhibitors effectively inhibited the growth of triple-negative breast cancer. Further analysis showed that in the tumor immune microenvironment, the number of M1 macrophages and CD8+T cells increased, and the number of exhausted T cells decreased, indicating that TOE and PD-1 inhibitors have anti-tumor potential in combination. Dandelion contains a variety of bioactive compounds, including sesquiterpene lactones, flavonoids, and phenolic acids, which have been reported to have immunomodulatory properties. Preliminary studies have shown that these compounds can affect various aspects of the immune response, such as enhancing T cell activation and regulating cytokine production. In the present invention's research, the present invention found that TOE is rich in isochlorogenic acid A and chicoric acid, both of which can inhibit the growth of TNBC in animal models when used in combination with PD-1 inhibitors. The combined administration of isochlorogenic acid A and PD-1 inhibitors in the tumor microenvironment increased the number of M1 macrophages and CD8+T cells, while reducing the number of exhausted T cells, thereby exerting an anti-tumor effect. In this study, the present invention investigated the hypothesis that TOE and its active ingredient, isochlorogenic acid A, may reprogram the immune microenvironment of TNBC through FAK regulation. By targeting FAK, the present invention further evaluated the synergistic effect of combining TOE with PD-1 inhibitors, aiming to enhance the therapeutic efficacy against TNBC. Through comprehensive in vitro and in vivo analyses, the present invention attempts to elucidate the underlying mechanisms and therapeutic potential of this combined strategy, providing a new approach to improve the treatment outcomes of TNBC patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a schematic diagram of the tumor size of each group on the 14th day in the TOE combined with immune checkpoint inhibitor (PD-1) experiment in the present invention; wherein, multiple samples in each row in the figure are parallel test samples.

[0030] Figure 2It is a schematic diagram of the tumor size of each group in the experiment of combining isochlorogenic acid A and chicoric acid with immune checkpoint PD-1 inhibitor in the present invention; wherein, Figure 2 Figure A shows the body weight of mice in the control group and the treatment group on the 14th day of drug treatment; Figure 2 Figure B shows the renal index of mice in the control group and the treatment group on the 14th day of drug treatment; Figure 2 Figure C shows the liver index of mice in the control group and the treatment group on the 14th day of drug treatment; Figure 2 Figure D shows the lung weight index of mice in the control group and the treatment group on the 14th day of drug treatment.

[0031] Figure 3 The figure is a schematic diagram of the in vivo imaging of mice during the treatment cycle and the statistical results; wherein, Figure 3 Figure A is a schematic diagram of live animal imaging of mice on day 0, day 7, and day 14 of treatment; Figure 3 Figure B is the quantitative analysis of fluorescence intensity of in vivo imaging after successful modeling of mice; Figure 3 Figure C shows the quantitative analysis of fluorescence intensity during in vivo imaging of mice in the middle stage of treatment; Figure 3 Figure D is the quantitative analysis of fluorescence intensity of live imaging of mice after the end of treatment.

[0032] Figure 4 The TOE in the present invention inhibits the cell viability of triple-negative breast cancer cell lines; wherein, Figure 4 Figure A shows that TOE inhibits the cell viability of human breast cancer cells MDA-MB-231; Figure 4 Figure B shows that TOE inhibits the viability of mouse breast cancer cell 4T1.

[0033] Figure 5 The TOE in the present invention inhibits the migration and invasion ability of triple-negative breast cancer cells; wherein, Figure 5 Figure A shows the ability of TOE to inhibit the migration and invasion of human breast cancer cells MDA-MB-231; Figure 5 Figure B shows that TOE inhibits the migration and invasion of mouse breast cancer cell line 4T1; Figure 5 Figure C is a statistical graph showing the ability of TOE to inhibit the migration of human breast cancer cells MDA-MB-231; Figure 5 Figure D in the figure is a statistical graph showing the ability of TOE to inhibit the invasion of human breast cancer cells MDA-MB-231; Figure 5 Figure E in the figure is a statistical graph showing the ability of TOE to inhibit the migration of mouse breast cancer cell line 4T1; Figure 5 Figure F is a statistical graph showing the ability of TOE to inhibit the invasion of mouse breast cancer cell line 4T1.

[0034] Figure 6The ratio of tumor infiltrating lymphocytes in the control group and the treatment group of mice was quantitatively analyzed by flow cytometry in the present invention; wherein, Figure 6 Panel A in the figure shows quantification of tumor-associated macrophage levels by flow cytometry analysis; Figure 6 Panel B in the figure quantifies the level of M1 macrophages by flow cytometry analysis; Figure 6 Panel C in the figure quantifies the level of M2 macrophages by flow cytometry analysis; Figure 6 Panel D in the figure quantifies the level of Th cells by flow cytometry analysis; Figure 6 Panel E in the figure quantifies the level of CTLs cells by flow cytometry analysis; Figure 6 Panel F shows the levels of LAG-3+ exhausted T cells in mice in the control and treatment groups; Figure 6 Panel G in the figure shows the levels of TIM-3+ exhausted T cells in mice in the control and treatment groups; Figure 6 Figure H shows the levels of PD-1+ exhausted T cells in mice in the control and treatment groups.

[0035] Figure 7 The total ion flow diagram and mass spectra of each component of TOE components identified by UPLC-Q-Orbitrap MS in the present invention; wherein, Figure 7 Figure A in the figure is the total ion chromatogram; Figure 7 Figure B in the figure is the mass spectrum of p-coumaric acid; Figure 7 Figure C in the figure is the mass spectrum of gallic acid; Figure 7 Figure D in the figure is the mass spectrum of syringic acid; Figure 7 Figure E in the figure is the mass spectrum of ferulic acid; Figure 7 Figure F in the figure is the mass spectrum of chlorogenic acid;

[0036] Figure 7 Figure G in the figure is the mass spectrum of caffeic acid; Figure 7 The H in the figure is monocaffeoyltartaric acid; Figure 7 Figure L in the figure is the mass spectrum of chicoric acid; Figure 7 Figure M in the figure is the mass spectrum of isochlorogenic acid A.

[0037] Figure 8 The isochlorogenic acid A and chicoric acid in the present invention are used in combination with a PD-1 immune checkpoint inhibitor to inhibit the growth of breast cancer in mice; wherein, Figure 8 Figure A in the figure is a schematic diagram of the tumor size of each group in the experiment of combining isochlorogenic acid A and chicoric acid with immune checkpoint inhibitor (PD-1); wherein, the multiple samples in each row of the figure are parallel test samples; Figure 8 Figure B shows that the combined treatment of PD-1 inhibitor with isochlorogenic acid A and chicoric acid inhibits the growth of tumor volume in mice.

[0038] Fig. 9The present invention provides a safety assessment of the combined use of isochlorogenic acid A and chicoric acid with PD-1 immune checkpoint inhibitors; wherein, Fig. 9 Figure A shows the body weight of mice in the control group and the treatment group on the 14th day of drug treatment; Fig. 9 Figure B shows the renal index of mice in the control group and the treatment group on the 14th day of drug treatment; Fig. 9 Figure C shows the liver index of mice in the control group and the treatment group on the 14th day of drug treatment; Fig. 9 Figure D shows the lung weight index of mice in the control group and the treatment group on the 14th day of drug treatment.

[0039] Fig.10 The effect of the combined use of isochlorogenic acid A and chicoric acid with PD-1 immune checkpoint inhibitors on tumor-infiltrating lymphocytes in mice in the present invention; wherein, Fig.10 Panel A in the figure shows quantification of tumor-associated macrophage levels by flow cytometry analysis; Fig.10 Panel B in the figure quantifies the level of M1 macrophages by flow cytometry analysis; Fig.10 Panel C in the figure is the quantification of CTLs cell levels by flow cytometry analysis; Fig.10 Panel D in the figure shows the levels of LAG-3+ exhausted T cells in mice in the control and treatment groups; Fig.10 Panel E in the figure shows the levels of TIM-3+ exhausted T cells in mice in the control and treatment groups; Fig.10 Figure F shows the levels of PD-1+ exhausted T cells in the control and treatment group mice.

[0040] Fig.11 The isochlorogenic acid A and chicoric acid in the present invention inhibit the activity of the FAK / PI3K / AKT / mTOR signaling pathway; wherein, Fig.11 Figure A is a diagram showing the molecular mechanism of the inhibitory effect of isochlorogenic acid A on the FAK / PI3K / AKT / mTOR signaling pathway in human breast cancer MDA-MB-231 cells; Fig.11 Figure B is a diagram showing the molecular mechanism of the inhibitory effect of isochlorogenic acid A on the FAK / PI3K / AKT / mTOR signaling pathway in mouse breast cancer 4T1 cells; Fig.11 Figure C shows that isochlorogenic acid A and chicoric acid reduced the phosphorylation level of FAK in MDA-MB-231 cells; Fig.11 Figure D shows that isochlorogenic acid A and chicoric acid reduced the phosphorylation level of PI3K in MDA-MB-231 cells; Fig.11 Figure E shows that isochlorogenic acid A and chicoric acid reduced the phosphorylation level of AKT in MDA-MB-231 cells; Fig.11 Figure F shows that isochlorogenic acid A and chicoric acid reduced the phosphorylation level of mTOR in MDA-MB-231 cells; Fig.11Figure G shows that isochlorogenic acid A and chicoric acid reduced the phosphorylation level of FAK in 4T1 cells; Fig.11 Figure H shows that isochlorogenic acid A and chicoric acid reduced the phosphorylation level of PI3K in 4T1 cells; Fig.11 Figure L shows that isochlorogenic acid A and chicoric acid reduced the phosphorylation level of AKT in 4T1 cells; Fig.11 Figure M shows that isochlorogenic acid A and chicoric acid reduced the phosphorylation level of mTOR in 4T1 cells. DETAILED DESCRIPTION

[0041] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0042] Example 1

[0043] 1. Methods

[0044] 1. TOE can inhibit the activity of TNBC tumors in animal models and the malignant phenotype in vitro

[0045] In order to evaluate the in vivo anti-tumor efficacy of TOE and its effect on TNBC cancer immunotherapy, the present invention purchased 5-6 week old female Balb / c mice from Jiangsu Huachuang Xinnuo Pharmaceutical Technology Co., Ltd. and conducted the following experiments:

[0046] 5×10 5 4T1 mouse mammary adenocarcinoma cells labeled with firefly luciferase were suspended in 0.04 mL of DMEM medium and injected into the fourth mammary fat pad of Balb / c mice to induce primary mammary tumor growth. Tumor volume was measured with a vernier caliper and calculated as follows: volume = [width 2 × length] / 2 to generate an allogeneic mouse model of breast cancer.

[0047] The unit of volume in the above calculation formula is mm 3 ; The units of width and length are both mm.

[0048] When the TNBC tumor size reached about 100 cubic millimeters, the mice were randomly divided into six groups and received different treatments: PD-1 inhibitor group alone, low-dose TOE (100 mg / kg) group, high-dose TOE (200 mg / kg) group, low-dose TOE combined with PD-1 inhibitor group, high-dose TOE combined with PD-1 inhibitor group and saline control group. Among them, the PD-1 inhibitor group, low-dose TOE (100 mg / kg) group, high-dose TOE (200 mg / kg) group, low-dose TOE combined with PD-1 inhibitor group, and high-dose TOE combined with PD-1 inhibitor group were all referred to as treatment groups.

[0049] The preparation method of TOE is as follows:

[0050] 500 g of whole dried dandelion plant was heated to reflux at 60°C for 3 hours, and the whole dried dandelion plant was extracted three times with 75%wt ethanol, each time for 2 hours. After the three extracts were concentrated, they were loaded into a D-101 macroporous resin column and eluted with pure water to remove sugars. Subsequently, 100%wt ethanol was used for elution, and the 100%wt ethanol eluate was collected, evaporated and spray-dried to obtain a dandelion extract (TOE).

[0051] In the experiment to study the effect of TOE on the phenotype of MDA-MB-231 and 4T1 cells, 1 mg TOE was dissolved in 1 mL DMSO to prepare a stock solution.

[0052] Among them, dandelion (product standard number: GHT1091) was purchased from Liaoning Senkangyuan Ecological Agriculture Co., Ltd.

[0053] The PD-1 inhibitor (BMS202) was purchased from Selleck. When used, the PD-1 inhibitor was dissolved in dimethyl sulfoxide (DMSO) to prepare a solution with a concentration of 2.3838 mmol / L.

[0054] The specific treatment methods for the above groups are as follows:

[0055] PD-1 inhibitor alone group: PD-1 inhibitor was intraperitoneally injected once every two days at a dose of 5 mg / kg.

[0056] Low-dose TOE (100 mg / kg) group: TOE was administered orally once a day at a dose of 100 mg / kg.

[0057] High-dose TOE (300 mg / kg) group: TOE was administered orally once a day at a dose of 200 mg / kg.

[0058] Low-dose TOE combined with PD-1 inhibitor group: TOE was administered orally once a day at a dose of 100 mg / kg; PD-1 inhibitor was injected intraperitoneally once every two days at a dose of 5 mg / kg.

[0059] High-dose TOE combined with PD-1 inhibitor group: TOE was administered orally once a day at a dose of 200 mg / kg; PD-1 inhibitor was injected intraperitoneally once every two days at a dose of 5 mg / kg.

[0060] Normal saline control group: 0.9% wt normal saline was intragastrically administered once a day, with a dose of 45 mg / kg.

[0061] The tumor sizes of the groups in the high- and low-dose TOE combined with immune checkpoint inhibitors were as follows: Figure 1 As shown, compared with TOE alone or PD-1 inhibitor alone, the combined treatment of PD-1 inhibitor and TOE has a significantly stronger inhibitory effect on tumor growth, can significantly reduce tumor volume and weight, and significantly inhibit tumor progression. The inhibitory effect of high-dose treatment is significantly higher than that of low-dose treatment.

[0062] The present invention mainly explores the anti-tumor effect of high-dose TOE. Figure 2 As shown, there was no significant difference in body weight between mice treated with high-dose TOE alone and mice treated with TOE and PD-1 inhibitors. In addition, no significant changes were observed in the visceral indexes of the heart, liver, or kidney in each treatment group compared with the saline control group, indicating that TOE does not induce systemic toxicity.

[0063] The substrate sodium fluorescein was injected intraperitoneally into mice. A 20g mouse needed to be injected with 200μL of 15mg / mL sodium fluorescein solution. Before in vivo imaging, the experimental animals were anesthetized with isoflurane gas. The anesthetized experimental animals were placed in the darkroom of the in vivo imaging instrument for imaging. The signal peaked 1 minute after the injection of fluorescein, and images needed to be collected near this time point.

[0064] Bioluminescence data collected using an in vivo imaging system were analyzed to assess the distribution and activity of luciferase-labeled cells in vivo, such as Figure 3 As shown, quantitative analysis found that the combination treatment of PD-1 inhibitors and high-dose TOE significantly inhibited tumor growth.

[0065] These research results indicate that TOE has an effective inhibitory effect on TNBC in vivo.

[0066] In order to further clarify the anti-tumor mechanism of TOE, the present invention used TNBC cell lines MDA-MB-231 and 4T1 for in vitro experiments. The cells were treated with different concentrations of TOE, and the cell survival rate was evaluated by CCK-8 detection. The specific method is: CCK8 detection uses the CCK8 kit produced by Elabscience. In short, MDA-MB-231 or 4T1 cells were seeded in a 96-well plate at a density of 2.0×10^5 cells / mL and cultured in an incubator at 37°C and 5% CO2 for 24 hours. Then, the cells were pretreated for 24 hours and given TOE at different gradient concentrations. After the stimulation, the culture medium was removed, 20 microliters of CCK8 reagent was added to each well, and the incubation continued for 1 hour. The absorbance of each well was measured at 450nm.

[0067] like Figure 4 As shown, the results of CCK-8 showed that the decrease in cell viability was dose-dependent, which demonstrated the cytotoxicity of TOE. The 24-hour IC50 values ​​of MDA-MB-231 and 4T1 cells were 135.9 μg / mL and 150.2 μg / mL, respectively, indicating that TOE could inhibit cell proliferation.

[0068] In addition, the Transwell assay was used to evaluate cell migration and invasion. 4 Cells were seeded into the Transwell in the upper chamber, the upper chamber was treated with TOE, serum-free medium (Opti-MEM) was used, and 750 μL complete medium was added to the lower chamber; in the invasion experiment, 30 μg Corning Matrigel matrix was added to the upper chamber, incubated at 37°C for 1 hour, and then cells treated with TOE were inoculated. After incubation for 20 hours at 37°C and 5% CO2, the culture medium was removed and the cells were washed twice with PBS. The cells were fixed in paraformaldehyde for 15 minutes and then washed twice with PBS. Stained with 0.1% wt fuchsin for 10 minutes, five fields of view were selected, photographed and counted under a microscope to evaluate migration and invasion ability. Figure 5 The results showed that TOE significantly reduced the migration and invasion abilities of MDA-MB-231 and 4T1 cells.

[0069] 2. TOE can enhance the efficacy of in vivo ICB therapy for TNBC

[0070] Tumor-associated macrophages are one of the most active immune cells and play a pivotal role in the tumor microenvironment. In order to study the effect of TOE on macrophage infiltration, the present invention performed flow cytometry detection on transplanted tumors. The specific operation is as follows:

[0071] After cutting the tumor tissue into a slurry, grind the tumor tissue pieces left on the filter with the back of a 1 mL syringe, then filter with a 70 μm cell strainer, and rinse the tumor tissue pieces on the filter with PBS. The resulting tumor suspension was centrifuged at 4°C, 850×g for 5 minutes, and the supernatant was discarded to collect the cell pellet. Add two volumes of red blood cell lysis buffer to the cell pellet, place the mixed sample on ice for 10 minutes, and gently vortex mix twice to promote the lysis of red blood cells. The resulting tumor suspension was centrifuged at 4°C, 850×g for 5 minutes, and the supernatant was discarded. Wash the cells once with PBS, and then resuspend the cells in 1 mL PBS to obtain a cell suspension.

[0072] Prepare the antibody mix tube and add the flow cytometry intracellular antibody to the flow cytometry staining buffer. Add the antibody mix to the cell suspension and incubate at room temperature for 30 minutes, protected from light. Wash the sample with flow cytometry staining buffer and centrifuge at 500 × g for 5 minutes at 4°C, discard the supernatant. Then resuspend the cells in flow cytometry staining buffer.

[0073] The surface antigens of tumor-associated macrophages are CD45.2+, CD11b+, F4 / 80+, the surface antigens of M1-like macrophages are CD11b+, F4 / 80+, CD86+; the surface antigens of M2-like macrophages are CD11b+, F4 / 80+, CD206+; the surface antigens of cytotoxic T lymphocytes are CD45+, CD3+, CD8+; the surface antigens of helper T cells are CD45+, CD3+, CD4+; the surface antigens of LAG-3+ exhausted T cells are CD3+, CD8+, LAG-3+, the surface antigens of TIM-3+ exhausted T cells are CD3+, CD8+, TIM-3+; the surface antigens of PD-1+ exhausted T cells are CD3+, CD8+, PD-1+. Flow cytometry was used to analyze tumor-infiltrating lymphocytes and phenotypic characteristics. Antibodies were provided by Thermo Fisher Scientific.

[0074] like Figure 6 As shown, PD-1 inhibitor monotherapy has little effect on macrophage infiltration, while PD-1 inhibitor and TOE combined treatment can significantly enhance the trafficking of macrophages to the tumor microenvironment. Specifically, whether used alone or in combination with PD-1 inhibitors, TOE can significantly increase the number of M1 macrophages in tumor-infiltrating lymphocytes. In addition, TOE can also increase the number of M2 macrophages.

[0075] These findings indicate that crude dandelion extract can promote the polarization of macrophages toward the M1 phenotype, but the underlying mechanism remains to be elucidated.

[0076] Flow cytometry analysis further demonstrated that PD-1 monotherapy and TOE monotherapy had a significant effect on CD8 + However, the combination of PD-1 inhibitors and TOE increased the number of CD8 T cells in the tumor microenvironment. + The proportion of T cells increased significantly, while CD4 + No significant changes were observed in the proportion of T cells. These data suggest that TOE can enhance the effective CD8 + Infiltration of T cells contributes to the partial reprogramming of the TNBC immune microenvironment from a “cold” to a “hot” state.

[0077] Immune checkpoint pathways are negative regulatory mechanisms that maintain immune balance; however, tumor cells use these immune checkpoint mechanisms to evade immune surveillance. In the tumor microenvironment (TME), reversing T cell exhaustion is critical for restoring effective anti-tumor immune responses. The present invention aims to explore the immunoregulatory effects of TOE on T cell-mediated anti-tumor immune responses. Flow cytometry analyzed the expression of immune checkpoint molecules on T cells, with particular attention to the expression and function of PD-1, LAG-3, and TIM-3. The results showed that compared with the saline control group, PD-1 inhibitor monotherapy had a significant effect on LAG-3 + 、TIM-3 + or PD-1 + There was no significant effect on the antitumor activity of T cell subsets. In contrast, both TOE alone and the combination of PD-1 and TOE significantly reduced the exhausted LAG-3 + 、TIM-3 + and PD-1 + T cell numbers. Notably, the combination therapy was more effective in reversing LAG-3 + and TIM-3 + T cell exhaustion, thereby enhancing the anti-tumor activity of T cells.

[0078] These research results show that the combination of TOE and PD-1 inhibitors can not only enhance the anti-tumor efficacy of PD-1 therapy, but also limit tumor immune escape and improve the tumor microenvironment, thereby improving the overall treatment effect of cancer. This combination therapy can reprogram the tumor microenvironment from "cold" to "hot", thereby activating immune cells to target and eliminate tumor cells, greatly improving the efficacy of immunotherapy.

[0079] 3. Identification of ingredients in TOE

[0080] 0.50 g of a TOE sample was mixed with 10 ml of a solvent (a mixed solvent prepared by mixing methanol, acetonitrile and water in a volume ratio of 2:2:1), centrifuged at 13,000 rpm for 5 minutes, and then filtered with a 0.22 μm filter membrane to obtain a TOE solution.

[0081] The components of TOE samples were identified by high performance liquid chromatography-mass spectrometry (HPLC-MS).

[0082] For mass spectrometry, thermal electrospray ionization (HESI) was used in conjunction with a Q-Orbitrap MS (Thermo Fisher Scientific). The main parameters included: scan range 100.0 to 1300.0 m / z; spray voltage 2.80 kV (negative mode); capillary temperature 300 °C; auxiliary gas heater temperature 300 °C; sheath gas flow 40 arbitrary units; auxiliary gas flow 10 arbitrary units; maximum injection time 50 ms; automatic gain control target 1.0e6. Molecular weight [MH] - and [MH] + Used for qualitative analysis.

[0083] Ultra-high performance liquid chromatography separation was performed on an AccucoreaQ C18 column (150 mm × 2.1 mm, 2.6 μm, ThermoFisher Scientific) at a flow rate of 0.3 μL / min. The optimized mobile phase was a mixture of water and 0.9% acetic acid (A) and methanol (B) by volume. For the identification samples, the gradient operation was as follows: 0-9 minutes, 20% B to 100% B by volume; 9 minutes to 10 minutes, 100% B by volume; 10 minutes to 11 minutes, 100% B to 20% B by volume; 11 minutes to 14 minutes, 20% B by volume.

[0084] like Figure 7 As shown in Table 1, UPLC-Q-Orbitrap MS analysis identified nine compounds in TOE. The specific components are as follows: chicoric acid, isochlorogenic acid A, chlorogenic acid, monocaffeoyltartaric acid, caffeic acid, ferulic acid, p-coumaric acid, gallic acid, and syringic acid.

[0085] Table 1 Mass spectrometry data of active ingredients determined from TOE

[0086]

[0087] 4. Speculate the potential regulatory mechanism of TOE active ingredients in TNBC based on network pharmacology

[0088] To evaluate whether these compounds in TOE can directly interact with FAK, the researchers performed molecular docking simulations, and the compound structure files were obtained from the PubChem database. The three-dimensional structure of FAK protein (PDB code: 6dj6) was retrieved from the Protein Data Bank (PDB). Molecular docking and virtual screening were performed using AutoDockVina, and visualization images were generated using PyMOL.

[0089] As shown in Table 2, the molecular docking results show that the binding affinities of the nine compounds to FAK are different, among which isochlorogenic acid A (ICGA-A) and chicoric acid (CRA) have the strongest binding affinities.

[0090] Table 2 Binding scores of the docking interaction between the compounds in dandelion extract and FAK molecule

[0091] Ranking Ligand Binding fraction (kcal / mol) 1 Chicoric acid -8.2 2 Isochlorogenic acid A -7.8 3 Chlorogenic acid -7.6 4 Caffeic acid -7.1 5 Caffeic acid -6.7 6 Ferulic acid -6.6 7 p-Coumaric acid -6.5 8 gallic acid -6 9 Syringic acid -5.9

[0092] 5. Isochlorogenic acid A and chicoric acid in TOE can inhibit the growth of triple-negative breast cancer (TNBC) tumors in mice and inhibit the malignant phenotype of cells in vitro

[0093] To evaluate the potential of isochlorogenic acid A and chicoric acid to inhibit tumorigenesis and tumor growth in TNBC, 5- to 6-week-old female Balb / c mice were purchased from Jiangsu Huachuang Xinnuo Pharmaceutical Technology Co., Ltd. 5 4T1 mouse mammary adenocarcinoma cells labeled with firefly luciferase were suspended in 0.04 mL of DMEM medium and injected into the fourth mammary fat pad of BALB / C mice to induce primary mammary tumor growth.

[0094] When the tumor volume of female Balb / c mice reached 100 cubic millimeters, the mice were randomly assigned to six groups, nine in each group, and received different treatment regimens: PD-1 inhibitor monotherapy group, isochlorogenic acid A (10 mg / kg / day) group, chicoric acid (20 mg / kg, once every two days) group, isochlorogenic acid A combined with PD-1 inhibitor group, chicoric acid combined with PD-1 inhibitor group and saline control group. Among them, the PD-1 inhibitor monotherapy group, isochlorogenic acid A (10 mg / kg / day) group, chicoric acid (20 mg / kg, once every two days) group, isochlorogenic acid A combined with PD-1 inhibitor group, and chicoric acid combined with PD-1 inhibitor group were all referred to as treatment groups.

[0095] Treatment was given daily for 14 consecutive days, with PD-1 inhibitors injected intraperitoneally every two days for two weeks at a dose of 5 mg / kg.

[0096] Among them, the PD-1 inhibitor (BMS-202) is an effective non-peptide PD-1 / PD-L1 complex inhibitor with an IC50 of 18nM and a KD of 8μM. It directly binds to PD-L1 and blocks the interaction of human PD-1 / PD-L1, and has anti-tumor activity. The formula of PD-1 inhibitor injection is 5%wt DMSO + 30%wt PEG300 + 5%wt Tween80 + 60%ddH2O.

[0097] The specific treatment methods for the above groups are as follows:

[0098] PD-1 inhibitor monotherapy group: PD-1 inhibitor was intraperitoneally injected once every two days at a dose of 5 mg / kg.

[0099] Isochlorogenic acid A group: Isochlorogenic acid A was intraperitoneally injected once a day at a dose of 10 mg / kg.

[0100] Isochlorogenic acid A (ICGA-A) was purchased from TOPSCIENCE. When used, isochlorogenic acid A was dissolved in dimethyl sulfoxide to prepare a solution with a concentration of 3.8726 mmol / L.

[0101] Chicoric acid group: Chicoric acid was intraperitoneally injected once every two days at a dose of 20 mg / kg. Chicoric acid (CRA) was purchased from TOPSCIENCE. When used, chicoric acid was dissolved in dimethyl sulfoxide to prepare a solution with a concentration of 8.4322 mmol / L.

[0102] Isochlorogenic acid A combined with PD-1 inhibitor group: Isochlorogenic acid A was injected intraperitoneally once a day at a dose of 10 mg / kg; PD-1 inhibitor was injected intraperitoneally once every two days at a dose of 5 mg / kg.

[0103] Chicoric acid combined with PD-1 inhibitor group: Chicoric acid was injected intraperitoneally once every two days at a dose of 20 mg / kg; PD-1 inhibitor was injected intraperitoneally once every two days at a dose of 5 mg / kg.

[0104] Normal saline control group: 0.9% normal saline was intragastrically administered once a day at a dose of 45 mg / kg.

[0105] Drug administration began when the tumor volume reached 100 mm3, recorded as day 0, and the tumor volume was measured every two days after treatment.

[0106] The tumor volume of each group after drug treatment was analyzed. Figure 8 As shown, both isochlorogenic acid A and chicoric acid significantly inhibited the growth of 4T1 TNBC tumors compared with the saline control group. In addition, the combination of isochlorogenic acid A and PD-1 had a stronger tumor inhibitory effect compared with PD-1 alone, suggesting that this combination may offer therapeutic potential for TNBC treatment.

[0107] Subsequently, the potential effects of the combination therapy on normal organs were evaluated. Fig. 9 As shown, there were no significant differences in the spleen, kidney, and liver indexes of tumor mice receiving different treatments, and there was no abnormal change in the body weight of mice in the combined treatment group, indicating that this therapy is safe. In vivo imaging of luciferase-labeled 4T1 tumors at different time points showed that on the 7th day after tumor formation in female Balb / c mice, the fluorescence intensity of the isochlorogenic acid A monotherapy group and the combined treatment group was significantly lower than that of the saline group.

[0108] 6. Combination therapy of isochlorogenic acid A, chicoric acid and PD-1 inhibitors enhances anti-tumor immune response

[0109] Fresh mouse tumor tissue was obtained after treatment. After the tumor tissue was cut into a slurry, the tissue pieces left on the filter were ground with the back of a 1 mL syringe, and then filtered with a 70 μm cell strainer. The tissue pieces on the filter were rinsed with PBS. The resulting tumor suspension was centrifuged at 4°C, 850×g for 5 minutes, and the supernatant was discarded. Two volumes of red blood cell lysis buffer were added to the cell pellet, and the mixed sample was placed on ice for 10 minutes. During this period, the mixed sample was gently vortexed twice to promote the lysis of red blood cells. The resulting tumor suspension was centrifuged at 4°C, 850×g for 5 minutes, and the supernatant was discarded. Wash the cells once with PBS and then resuspend the cells in 1 mL PBS. Prepare an antibody mixing tube and add the flow cytometry intracellular antibody to the flow cytometry staining buffer. Add the antibody mixture to the cell suspension and incubate at room temperature for 30 minutes, protected from light. Wash the sample with flow cytometry staining buffer and centrifuge it at 500×g for 5 minutes at 4°C, and discard the supernatant. Then resuspend the cells with flow cytometry staining buffer. The present invention selects appropriate excitation light and detection channel on the flow cytometer for analysis.

[0110] like Fig.10 As shown, the anti-tumor immune response stimulated by the combination therapy of isochlorogenic acid A and PD-1 inhibitor was further verified by flow cytometric analysis of macrophages and T cells. To evaluate the reprogramming effect of the combination therapy on the polarization of tumor-associated macrophages (TAMs) within 4T1 tumors and its potential to modulate the immunosuppressive environment, the researchers quantified the total number of macrophages, M1-like TAMs, and M2-like TAMs using flow cytometry. The combination of isochlorogenic acid A + PD-1 inhibitor significantly increased the number of anticancer M1-like TAMs, while having no substantial effect on immunosuppressive M2-like TAMs. In addition, the total number of tumor-associated macrophages was significantly increased compared with the saline control group.

[0111] In the 4T1 tumor model, quantitative evaluation of effector T cells by flow cytometry revealed that the effector CD8 + T cells increased significantly, performing better than the PD-1 inhibitor monotherapy group. In addition, the number of effector T cells in the chicoric acid group was also higher than that in the PD-1 inhibitor monotherapy group. These findings suggest that the isochlorogenic acid A combination therapy strategy can effectively enhance the immune response to immune checkpoint blockade (ICB) therapy.

[0112] The present invention also further studies the effects of isochlorogenic acid A and chicoric acid alone or in combination with PD-1 inhibitors on T cell exhaustion.

[0113] Flow cytometry results showed that both compounds, alone or in combination with PD-1 inhibitors, were effective in reducing the number of exhausted T cells expressing LAG-3. In addition, the combination therapy showed a synergistic effect in reducing TIM-3 expression levels. Specifically, isochlorogenic acid A, alone or in combination with PD-1 inhibitors, was effective in reducing the number of PD-1-positive exhausted T cells.

[0114] These results not only highlight the potential immunomodulatory effects of isochlorogenic acid A and chicoric acid in regulating T cell exhaustion, but also suggest that their combination with PD-1 inhibitors may enhance T cell-mediated tumor immune responses. These findings provide strong experimental evidence to support the development of novel tumor immunotherapy strategies, especially those that use natural products to modulate the tumor microenvironment and enhance existing immunotherapy.

[0115] 7. Isochlorogenic acid A and chicoric acid relieve immunosuppression by inhibiting the FAK / PI3K / AKT / mTOR signaling pathway

[0116] After treating cells with isochlorogenic acid A and chicoric acid for 48 hours, cells were lysed on ice using RIPA buffer (Solarbio) containing 1% PMSF and 1% phosphatase inhibitor (Proteintech). Protein concentration was determined using the Pierce BCA protein assay kit (Thermo Fisher Scientific). Equal amounts of protein (20 μg each) were loaded on 10% SDS-PAGE (Vazyme) and FuturePAGE TM 4%-12% 12 Wells (Ace Biotechnology), and then transferred to a PVDF membrane (Millipore). The membrane was blocked with 5% skim milk at room temperature and then incubated with the primary antibody at 4°C overnight. The membrane was washed with TBST and then incubated with the secondary antibody at room temperature. The protein signal in BG-gdsAUTO730 was detected using a high-sensitivity ECL chemiluminescent detection kit (Proteintech). The Western blot results are shown in Fig.11 As shown, isochlorogenic acid A and chicoric acid reduced the phosphorylation levels of FAK, PI3K, AKT, and mTOR, suggesting that these compounds may be key bioactive components in TOE

[0117] The above experimental results show that two bioactive compounds extracted from dandelion, isochlorogenic acid A and chicoric acid, significantly improve the efficacy of PD-1 inhibitors by regulating the tumor microenvironment (TME) of triple-negative breast cancer (TNBC). +"Through their potent combination of natural compounds and immune checkpoint inhibitors (ICIs), we are confident that the combination will expand the therapeutic potential of this highly aggressive malignancy," said Dr.

[0118] The present invention provides a combination of isochlorogenic acid A and a PD-1 inhibitor, and the use of the pharmaceutical composition in the preparation of a drug for preventing and / or treating breast cancer, and provides a new method for preventing and / or treating breast cancer. The present invention develops a new application of a small molecule drug combined with a PD-1 inhibitor in anti-tumor.

[0119] It should be noted that when the present invention involves a numerical range, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.

[0120] Although preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they understand the basic inventive concepts, and all such changes and modifications fall within the scope of the present invention.

Claims

1. Use of isochlorogenic acid A and PD-1 inhibitor in combination in the preparation of a drug for preventing and / or treating breast cancer, characterized in that: The breast cancer is triple-negative breast cancer.

2. The use according to claim 1, characterized in that: The molar ratio of the isochlorogenic acid A to the PD-1 inhibitor is 1.6242-1.6244:

1.

3. A pharmaceutical composition for preventing and / or treating breast cancer, characterized in that: The pharmaceutical composition is prepared by using the raw material composition comprising the isochlorogenic acid A and the PD-1 inhibitor as claimed in claim 1 as active ingredients, and adding pharmaceutically acceptable excipients.

4. The pharmaceutical composition according to claim 3, characterized in that The molar ratio of the isochlorogenic acid A to the PD-1 inhibitor is 1.6243:

1.

5. The pharmaceutical composition according to claim 3, characterized in that The dosage form of the pharmaceutical composition is a solid preparation or a liquid preparation; The solid preparation is a powder, and the liquid preparation is an oral solution.

6. The pharmaceutical composition according to claim 5, characterized in that The dosage form of the pharmaceutical composition is a liquid preparation; The concentration of the isochlorogenic acid A in the pharmaceutical composition is 3.8726 mmol / L to 3.8728 mmol / L, and the concentration of the PD-1 inhibitor in the pharmaceutical composition is 2.3837 mmol / L to 2.3839 mmol / L.

7. The pharmaceutical composition according to claim 6, characterized in that The concentration of the isochlorogenic acid A in the pharmaceutical composition is 3.8726 mmol / L, and the concentration of the PD-1 inhibitor in the pharmaceutical composition is 2.3838 mmol / L.

8. The pharmaceutical composition according to claim 3, characterized in that The auxiliary materials include any one or more of physiological saline, PEG300, Tween 80 and DMSO.

9. Use of a pharmaceutical composition for preventing and / or treating breast cancer according to claim 3 in preparing a drug for preventing and / or treating breast cancer, characterized in that: The breast cancer is triple-negative breast cancer.

Images