Application of orientin in preparation of medicine for treating breast cancer

Abstract

The invention belongs to the technical field of medicines, and particularly relates to application of orientin in preparation of a medicine for treating breast cancer. An MMTV-PyMT breast cancer transgenic mouse is adopted, it is found that the tumor weight inhibition rate of 31.2% is achieved when the PD-L1 monoclonal antibody is independently used, the inhibition rate of orientin single drug treatment is 25.6%, after the PD-L1 monoclonal antibody and the orientin single drug are jointly used, tumor growth can be remarkably inhibited, the tumor inhibition rate reaches 70.0%, and the combined treatment effect is remarkably superior to simple superposition of the drug effects of all the single drugs. In addition, the combined treatment can also effectively promote the infiltration of CD4 + and CD8 + T cells in tumor tissues, and improve the expression level of key anti-tumor immune factors such as granzyme B (GZMB) and interferon-gamma (IFN-gamma).

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine, and particularly relates to application of ostruthin in preparation of a drug for treating breast cancer. BACKGROUND

[0002] As the fourth treatment method after surgery, radiotherapy and chemotherapy, tumor immunotherapy is to activate the host immune system to fight against tumors. In the field of breast cancer, the application of PD-L1 inhibitor still faces great challenges: although its combination with chemotherapy has become the first-line treatment for breast cancer, the spatiotemporal heterogeneity and non-uniform detection standard of PD-L1 expression as a biomarker lead to inaccurate efficacy prediction and limited response rate. The use of PD-L1 inhibitor alone generally has little effect, which highlights the great challenges of current immunotherapy in the field of breast cancer, such as patient screening, drug resistance and how to expand the effective population. Therefore, it is of great significance to develop new drug treatment strategies for treating breast cancer. SUMMARY

[0003] In order to solve the above technical problems, the application provides application of ostruthin in preparation of a drug for treating breast cancer.

[0004] The application of ostruthin in preparation of a drug for treating breast cancer.

[0005] The structural formula of ostruthin is as follows: .

[0006] The application finds that ostruthin has an inhibitory effect on the growth of breast cancer, and therefore proposes the application of ostruthin in preparation of a drug for treating breast cancer. In the application, ostruthin enhances the effect of tumor immunotherapy by one or more of the following mechanisms: significantly increasing the infiltration level of immune effector cells in the tumor microenvironment, especially the infiltration level of CD8+ T cells and CD4+ T cells; and significantly improving the expression or secretion level of immune effector factors (such as IFN-γ and granzyme B (GZMB)) in the tumor microenvironment.

[0007] Preferably, the drug takes ostruthin as the only effective component and is used for inhibiting the growth of tumors.

[0008] Preferably, the drug takes ostruthin and a tumor immunotherapy drug as effective components, and the ostruthin is used for enhancing the effect of the tumor immunotherapy drug on breast cancer. The application finds that the inhibitory effect of PD-L1 monoclonal antibody alone on the growth of breast cancer is limited, the tumor weight inhibition rate is 31.2%, the inhibition rate of ostruthin single-drug treatment is 25.6%, and the combination of the two can significantly inhibit the growth of tumors, and the tumor inhibition rate reaches 70.0%. Therefore, it is proposed to use ostruthin to enhance the effect of tumor immunotherapy on breast cancer.

[0009] Preferably, the immunotherapy comprises a PD-1 / PD-L1 signaling pathway inhibitor.

[0010] Preferably, the PD-1 / PD-L1 signaling pathway inhibitor is a PD-L1 monoclonal antibody.

[0011] Preferably, the medicine further comprises a pharmaceutically acceptable carrier.

[0012] Preferably, the medicine is in the form of an oral dosage form or an injection dosage form.

[0013] Preferably, the oral dosage form is a tablet, a capsule, a granule or an oral liquid. In order to improve the delivery efficiency and targeting of the medicine, the dosage form can also be a novel targeted preparation such as a nanoliposome or a polymer micelle.

[0014] Compared with the prior art, the present application has the following beneficial effects: The present application uses MMTV-PyMT breast cancer transgenic mice, finds that the inhibitory effect of PD-L1 monoclonal antibody alone on breast cancer growth is limited, the tumor weight inhibition rate is 31.2%, the inhibitory rate of astrasieversin A single drug treatment is 25.6%, and after the combination of the two, the tumor growth can be significantly inhibited, the tumor inhibition rate reaches 70.0%, and the combination treatment effect is significantly better than the simple superposition of the effects of each single drug. In addition, the combination treatment can also effectively promote the infiltration of CD4+ and CD8+ T cells in the tumor tissue, and improve the expression level of key immune factors such as granzyme B (GZMB) and interferon-γ (IFN-γ). BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 The effects of astrasieversin A and PD-L1 monoclonal antibody on the tumor volume and weight of breast cancer are shown, wherein A is the change trend of tumor volume, B is the tumor photo, and C is the change trend of relative tumor weight.

[0016] Figure 2 The effects of astrasieversin A and PD-L1 monoclonal antibody on the pathological changes of breast cancer tumor tissue are shown, wherein A is tumor HE staining, B is a necrosis area statistical chart, C is tumor proliferation marker Ki67 staining, and D is a Ki67 positive cell proportion statistical chart.

[0017] Figure 3 The fluorescence immunization of CD31 shows the effects of the drugs on tumor blood vessels and vascular integrity, wherein A is the co-localization of CD31 and VE-Cad, B is the co-localization of CD31 and Col IV, C is the statistical proportion of CD31 positive area, D is the statistical proportion of VE-Cad positive blood vessels, and E is the statistical proportion of Col IV positive blood vessels.

[0018] Figure 4The effects of the drugs on the immune cell infiltration of tumor blood vessels were shown by fluorescence immunization, wherein A is the CD31 and CD8 localization, B is the CD31 and CD4 localization, C is the CD8 positive proportion statistics, and D is the CD4 positive cell proportion statistics.

[0019] Figure 5 The effects of the drugs on the anti-tumor immune cell factor expression were analyzed, wherein A is the expression of granzyme B (GZMB) in tumor tissue, and B is the expression of interferon-γ (IFN-γ). DETAILED DESCRIPTION

[0020] The specific embodiments of the present application are described in detail below, but it should be understood that the scope of protection of the present application is not limited by the specific embodiments. Based on the examples in the present application, all other examples obtained by those of ordinary skill in the art without creative labor are within the scope of protection of the present application. The experimental methods described in the embodiments of the present application are conventional methods unless otherwise specified.

[0021] Example 1 1. Experimental materials and animal models Experimental animals: 24 female 8-week-old spontaneous breast cancer MMTV-PyMT transgenic mice (C57BL / 6 background) were selected. The mice of this strain spontaneously develop breast tumors at 5-6 weeks of age, and the tumor nodules can be palpated at 8 weeks of age.

[0022] Grouping: 24 mice were randomly divided into 4 groups, 6 in each group: (1) Control group: given normal saline.

[0023] (2) Sophoraflavescens Ait single-drug group: given sophoraflavescens Ait.

[0024] (3) PD-L1 inhibitor single-drug group: given PD-L1 monoclonal antibody.

[0025] (4) Combination group: sophoraflavescens Ait and PD-L1 monoclonal antibody were given at the same time.

[0026] Main reagents: sophoraflavescens Ait (purity ≥98%, item number 28608-75-5, purchased from Meilunbio Company); anti-mouse PD-L1 monoclonal antibody (item number A2115, purchased from Selleck Chemicals Company).

[0027] 2. Experimental methods Dosing regimen: Mice were dosed starting at 10 weeks of age, with a dosing period of 56 days. Prunusiflorus group: Prunusiflorus was dissolved in normal saline to prepare a solution with a concentration of 5 mg / mL, and was administered at a dose of 50 mg / kg by oral gavage, with a volume of 10 mL / kg body weight, once a day. PD-L1 inhibitor group: PD-L1 mAb was dissolved in PBS to prepare a solution with a concentration of 2 mg / mL, and was administered at a dose of 20 mg / kg by intraperitoneal injection, with a volume of 10 mL / kg body weight, twice a week. Combination group: Prunusiflorus solution and PD-L1 mAb solution with the above concentrations were administered simultaneously, with the same dose, route, volume, and frequency as the single-agent groups. Control group: The same volume of PBS solvent was administered by intraperitoneal injection, with a volume of 10 mL / kg body weight, with the same frequency as the PD-L1 mAb group (twice a week).

[0028] Tumor volume monitoring: The long diameter and short diameter of the tumor were measured once a week using a vernier caliper, and the tumor volume was calculated according to the formula Tumor volume (mm3) = (long diameter x short diameter2) / 2. Tumor growth curves were plotted Figure 1 A).

[0029] Sample collection: After the last administration, the mice were euthanized, and the tumor tissue was completely stripped and weighed. Representative tumor photographs are shown in Figure 1 B.

[0030] Histological and immunological analysis: Hematoxylin-eosin (H&E) staining: The necrotic area of the tumor tissue was observed. Immunohistochemical (IHC) staining: The expression level of the tumor cell proliferation marker Ki67 was detected. Immunofluorescence (IF): The tumor vascular integrity and the tumor infiltration of CD4+ and CD8+ T cells were detected, and the results are shown in Figures 2-4 .

[0031] Enzyme-linked immunosorbent assay (ELISA): Commercial ELISA kits were used to quantitatively detect the contents of granzyme B (GZMB) and interferon-γ (IFN-γ) in tumor tissue homogenate, and the results are shown in Figure 5 .

[0032] Statistical analysis: All data are expressed as mean ± standard error. One-way ANOVA was used for multiple group comparisons, followed by Tukey's post-hoc test. Differences with P <0.05 were considered statistically significant.

[0033] Results Prunusiflorus significantly enhances the anti-tumor effect of PD-L1 inhibitors: As shown in Figure 1As shown, compared with the Control group, the treatment of breviscapine and PD-L1 inhibitor alone for 56 days could inhibit the growth of breast cancer tumor to a certain extent. The tumor volume inhibition rate of the breviscapine group was only 21.7%, and the tumor weight inhibition rate was 25.6%. However, breviscapine could enhance the anti-tumor effect of PD-L1 inhibitor, and the tumor volume inhibition rate was significantly increased from 30.5% to 64.7%, and the tumor weight inhibition rate was significantly increased from 31.2% to 70.0%. Moreover, the anti-tumor effect of the combination therapy was much greater than the simple superposition of the effects of the two drugs. This result proves that breviscapine can enhance the anti-tumor effect of PD-L1 inhibitor and play the role of an immune potentiator for breast cancer.

[0034] Combination therapy effectively reduces tumor necrosis area and inhibits tumor proliferation: pathological analysis of tumor tissue by H&E and Ki67 staining Figure 2 H&E staining showed that the tumor cell necrosis area ratio of the combination therapy group was 6.41 times lower than that of the PD-L1 single drug treatment group, indicating that the tumor blood supply and microenvironment were improved. More importantly, compared with the PD-L1 single drug treatment group, the proliferation activity (Ki67 positive rate) of tumor cells in the combination therapy group was reduced by 2.21 times, indicating that the combination regimen could more effectively inhibit tumor proliferation.

[0035] Breviscapine significantly enhances the remodeling of tumor immune microenvironment by PD-L1 inhibitor through inducing breast cancer tumor vascular normalization and promoting T cell infiltration: in-depth analysis of tumor microenvironment Figures 3-4 revealed the potential synergistic mechanism. The combination therapy induced a typical "vascular normalization" phenotype, characterized by a 3.15-fold decrease in CD31-positive microvessel density compared with the PD-L1 single drug treatment group, and a 4.26-fold and 3.52-fold increase in the expression of vascular integrity markers VE-cad and Col IV, respectively. This structural improvement was consistent with a significant increase in T cell infiltration: compared with the PD-L1 single drug group, the infiltration levels of CD8+ T cells and CD4+ T cells in the combination therapy group were increased by 1.51 and 2.10 times, respectively. The above results indicate that breviscapine may promote breast cancer tumor vascular normalization, thereby synergizing with PD-L1 inhibitor to promote T cell infiltration into tumor tissue, and thus remodeling the immune microenvironment.

[0036] Combination therapy increases the level of anti-tumor immune factors: detection of the content of key effector anti-tumor immune factors in tumor tissue by ELISA Figure 5 The results showed that the contents of effector immune factors GZMB and IFN-γ in the tumor tissue of the combination therapy group were 1.49 times and 1.43 times higher than those in the PD-L1 single drug group, respectively. This result indicates that combination therapy not only promotes T cell infiltration, but more importantly, strongly activates its tumor killing function.

[0037] The present embodiment proves that ostruthin not only can inhibit tumor growth, but also can be a synergist of PD-L1 inhibitor, and the combined treatment shows a significant anti-tumor effect. The synergistic mechanism lies in that ostruthin can remodel the tumor immune microenvironment, promote T cell infiltration by inducing vascular normalization, and synergistically enhance the killing function of T cells, thereby producing a strong synergistic anti-tumor effect with the PD-L1 inhibitor.

[0038] Ostruthin is a natural flavonoid compound extracted from Trollius chinensis Bunge. The source plant recorded in the Compendium of Materia Medica and other classics is often used to treat rheumatism, dysentery, diarrhea and other symptoms, which embodies its traditional medicinal value of anti-inflammatory and antibacterial. Modern research has confirmed that the compound has multiple pharmacological activities such as antioxidant, anti-inflammatory and anti-tumor. Its unique C-glycoside structure endows it with excellent chemical and metabolic stability. The prior art discloses that ostruthin has pharmacological activities such as antioxidant and anti-inflammatory, but so far, no literature or patent has disclosed or suggested that ostruthin has the effect of enhancing tumor immunotherapy. The present application first discovers this new medical use of ostruthin.

[0039] It should be noted that when the present application claims involving numerical ranges, both endpoints of each numerical range and any number between the two endpoints can be selected. In order to prevent repetition, the present application describes preferred embodiments.

[0040] Although preferred embodiments of the present application have been described, those skilled in the art, once they know the basic creative concept, can make additional changes and modifications to these embodiments. Therefore, the appended claims are intended to include the preferred embodiments and all changes and modifications falling within the scope of the present application.

[0041] Obviously, those skilled in the art can make various modifications and variations to the present application without departing from the spirit and scope of the present application. Thus, if these modifications and variations of the present application fall within the scope of the claims of the present application and their equivalents, the present application also intends to include these modifications and variations.

Claims

1. Use of ostruthin in the preparation of a medicament for treating breast cancer.

2. Use according to claim 1, characterized in that, The medicament uses ostruthin as the only effective component for inhibiting the growth of tumors.

3. Use according to claim 1, characterized in that, The medicament uses ostruthin and a tumor immunotherapy drug as effective components, and the ostruthin is used for improving the effect of the immunotherapy drug in treating breast cancer.

4. Use according to claim 3, characterized in that, The tumor immunotherapy drug includes a PD-1 / PD-L1 signal pathway inhibitor.

5. Use according to claim 4, characterized in that, The PD-1 / PD-L1 signal pathway inhibitor is a PD-L1 monoclonal antibody.

6. Use according to claim 1, characterized in that, The medicament further includes a pharmaceutically acceptable carrier.

7. Use according to claim 6, characterized in that, The medicament is in the form of an oral dosage form or an injection dosage form.

8. Use according to claim 7, characterized in that, The oral dosage form is a tablet, a capsule, a granule or an oral liquid.

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