7,2'-dihydroxy-3',4'-dimethoxyisoflavan in the preparation of anticancer drugs

Abstract

The research provides application of 7,2'-dihydroxy-3',4'-dimethoxyisoflavan in preparation of anticancer drugs, and belongs to the technical field of medicine.The pharmacodynamic experiment result of the application shows that 7,2'-dihydroxy-3',4'-dimethoxyisoflavan can inhibit M2 type (tumor development promotion) polarization of breast cancer cells (4T-1) induced mouse peritoneal macrophages (RAW264.7) in vitro, and then play an anti-tumor role; in vivo experiment, 7,2'-dihydroxy-3',4'-dimethoxyisoflavan has obvious therapeutic effect on breast cancer animal model, and can be used for preparation of anticancer drugs.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically the application of 7,2′-dihydroxy-3′,4′-dimethoxyisoflavanane in the preparation of anticancer drugs. Background Technology

[0002] Breast cancer is one of the most common malignant tumors, ranking first in incidence among female cancers. In 2020, there were 2.3 million new cases and 685,000 deaths from breast cancer globally. Breast cancer accounts for approximately 24.5% of all female cancer cases and 15.5% of cancer deaths, with both incidence and mortality showing an increasing trend year by year. The standard treatment for breast cancer has always been a combination of surgery, radiotherapy, and chemotherapy. However, these treatments have significant limitations, and to date, the treatment outcomes for breast cancer remain unsatisfactory. This has prompted researchers both domestically and internationally to shift their focus to exploring newer treatment methods, such as immunotherapy targeting the tumor microenvironment.

[0003] Studies have shown that the extensive infiltration of macrophages in the tumor microenvironment is closely related to the occurrence, development, and prognosis of tumors such as breast cancer and colorectal cancer. Macrophages play a central role in the development of organisms, engulfing microorganisms and providing antigens to T cells, thus forming the first line of defense against pathogen invasion. They also regulate tissue growth, homeostasis, repair, and remodeling through the expression of a large number of cytokines, chemokines, and growth factors. In the tumor microenvironment, the release of inflammatory mediators from tumor cells, such as interleukin-34 and interleukin-6, can cause macrophages to polarize into the M2 type, which promotes tumor development. M2-polarized macrophages usually exacerbate tumor cell proliferation, invasion, and angiogenesis, promoting tumor growth and metastasis. It is evident that macrophages play an important regulatory role in promoting malignant tumor development. Macrophages differentiate from monocytes and differentiate into the M1 type when stimulated by external factors such as interferon and lipopolysaccharide, and into the M2 type when stimulated by IL-4 and IL-3. The M1 / M2 macrophage pattern plays a crucial role in tumor progression. M1-polarized macrophages have long been considered to have anti-tumor effects, while M2-polarized macrophages, generally considered tumor-associated macrophages, participate in many pro-tumorigenic outcomes in cancer by regulating angiogenesis and lymphangiogenesis, immunosuppression, hypoxia induction, tumor cell proliferation, and metastasis. Research has found that macrophage phenotypes are in a dynamic transformation process; therefore, modulating the polarization of tumor-infiltrating macrophages through drug intervention has become an important breakthrough in cancer treatment.

[0004] In order to develop new drugs for the treatment of breast cancer, this invention systematically studied 7,2′-dihydroxy-3′,4′-dimethoxyisoflavan, and obtained results showing that it has a significant inhibitory effect on breast cancer. Summary of the Invention

[0005] The purpose of this invention is to provide a drug that can be used to treat cancer, particularly a drug for treating breast cancer.

[0006] The present invention relates to the application of 7,2′-dihydroxy-3′,4′-dimethoxyisoflavone in the preparation of drugs for treating breast cancer. The 7,2′-dihydroxy-3′,4′-dimethoxyisoflavone exerts its anti-tumor effect indirectly by inhibiting M2 polarization of macrophages.

[0007] The 7,2′-dihydroxy-3′,4′-dimethoxyisoflavone described herein has the following structural formula:

[0008]

[0009] Compared with the prior art, the beneficial effects of the present invention are as follows: Pharmacodynamic experiments of the present invention show that 7,2′-dihydroxy-3′,4′-dimethoxyisoflavone can inhibit M2 polarization of mouse peritoneal macrophages induced by breast cancer cells in vitro, thereby exerting an anti-tumor effect; in vivo experiments show that 7,2′-dihydroxy-3′,4′-dimethoxyisoflavone has a significant inhibitory effect on breast cancer animal models and can be used to prepare drugs for the treatment of breast cancer. Attached Figure Description

[0010] Figure 1 The results of the CCK8 assay for detecting the cytotoxicity of 7,2′-dihydroxy-3′,4′-dimethoxyisoflavones in Example 1 of this invention are shown.

[0011] Figure 2 Example 1 of this invention describes the intervention of 7,2′-dihydroxy-3′,4′-dimethoxyisoflavones in the change of Raw264.7CD206 expression ratio.

[0012] Figure 3 Example 1 of this invention describes the intervention of 7,2′-dihydroxy-3′,4′-dimethoxyisoflavones in the morphological changes of Raw264.7.

[0013] Figure 4 Example 1 of this invention describes the intervention of 7,2′-dihydroxy-3′,4′-dimethoxyisoflavones on the changes in the phagocytic capacity of Raw264.7.

[0014] Figure 5 Example 1 of this invention describes the detection of IL-10 and TGF-β cytokine levels in Raw264.7 cells using ELISA (model group vs. blank group: P < 0.05). * P < 0.01 ** Compared with the model group, the drug-treated group showed a p-value of <0.05. # P < 0.01 ##Compared with different dosage groups: P < 0.05 & P < 0.01 && ).

[0015] Figure 6 The results of tumor weight and volume in animal experiments using 7,2′-dihydroxy-3′,4′-dimethoxyisoflavone in Example 2 of this invention (compared to the model group: P < 0.05) are shown. * P < 0.01 ** Comparison of different dosage groups: P < 0.05 # ).

[0016] Figure 7 The results of the organ index in animal experiments using 7,2′-dihydroxy-3′,4′-dimethoxyisoflavone in Example 2 of this invention are shown.

[0017] Figure 8 This is a pathological section of tumor tissue from an animal experiment involving 7,2′-dihydroxy-3′,4′-dimethoxyisoflavones, as described in Example 2 of this invention.

[0018] Figure 9 Example 2 of this invention describes the detection of IL-10 and TGF-β cytokine levels in animal serum samples using ELISA (model group vs. blank group: P < 0.05). * P < 0.01 ** Compared with the model group, the drug-treated group showed a p-value of <0.05. # P < 0.01 ## Compared with different dosage groups: P < 0.05 & P < 0.01 && ). Detailed Implementation

[0019] Example 1: Effects of 7,2′-dihydroxy-3′,4′-dimethoxyisoflavanone on macrophage polarization model in vitro

[0020] 1. Cells: mouse peritoneal macrophages RAW264.7; mouse breast cancer cells 4T-1; Source: Wuhan Pronosei Biotechnology Co., Ltd.; Culture medium: DMEM high glucose medium, RPMI 1640 medium; Culture conditions: 5% CO2, 37℃ constant temperature incubator.

[0021] 2. Test drug: 7,2′-dihydroxy-3′,4′-dimethoxyisoflavone, purchased from Chengdu Pusi Biotechnology Co., Ltd. 7,2′-dihydroxy-3′,4′-dimethoxyisoflavone was dissolved in DMEM medium with 1‰ DMSO to prepare a 1000 μmol / L solution for later use.

[0022] 3 Experimental Methods

[0023] 3.1 In vitro experimental grouping, dosing regimen and drug cytotoxicity

[0024] Macrophages without drug intervention served as the blank group, M2 polarized macrophages induced by tumor supernatant served as the control group, and macrophages induced by tumor supernatant and treated with 7,2′-dihydroxy-3′,4′-dimethoxyisoflavone served as the drug-treated group. Cytotoxicity was assessed using specific drug doses of 32 μmol / L, 64 μmol / L, 128 μmol / L, 256 μmol / L, and 512 μmol / L. RAW264.7 cells were seeded in 96-well plates and cultured for 12 h, then treated with different concentrations of 7,2′-dihydroxy-3′,4′-dimethoxyisoflavone for 36 h. 10 μL of LCK-8 solution was added to each 96-well plate. After 3 h of incubation, the absorbance was measured at 450 nm using a microplate reader.

[0025] 3.2 Establishment of macrophage M2 polarization model and CD206 expression in macrophages

[0026] RAW264.7 macrophages were co-cultured with the supernatant of 4T-1 breast cancer cells collected after 48 hours to induce an M2 polarization model. The drug treatment groups were treated with 32 μmol / L, 64 μmol / L, 128 μmol / L, and 256 μmol / L 7,2′-dihydroxy-3′,4′-dimethoxyisoflavin. After 48 hours, the cells in each group were collected and incubated with CD206+-M2 type flow cytometry antibody and detected by flow cytometry.

[0027] 3.3 Changes in macrophage morphology and phagocytic capacity

[0028] RAW264.7 macrophages were co-cultured with the supernatant of 4T-1 breast cancer cells collected at 48 h to induce an M2 polarization model. Simultaneously, the drug-treated groups were treated with 256 μmol / L 7,2′-dihydroxy-3′,4′-dimethoxyisoflavin. Cell morphological changes were observed after 36 h. Macrophages were stained green with Cell Trance Red CMTPX dye, and tumor cells were stained green with FITC-CFDA-SE dye. Macrophages from the control group, model group, and drug-treated group were co-cultured with 4T-1 cells for 4 hours, fixed with tissue fixation solution, and observed under a fluorescence microscope.

[0029] 3.4 IL-10 and TGF-β in macrophage supernatant

[0030] Macrophages RAW264.7 were co-cultured with the supernatant of breast cancer cells 4T-1 collected at 48 h to induce an M2 polarization model. The treatment groups were treated with 32 μmol / L, 64 μmol / L, 128 μmol / L, and 256 μmol / L 7,2′-dihydroxy-3′,4′-dimethoxyisoflavin, respectively. After 24 h, the supernatant was removed, the cell surface was washed with PBS, and fresh culture medium was added. After 36 h, the cell culture medium of each group was collected, and 10 μL of each was taken to test the IL-10 and TGF-β cytokine levels according to the ELISA kit (Andy Gene).

[0031] 4 Experimental Results

[0032] 4.1 Compared with the control group, macrophage cell viability was significantly reduced at a drug concentration of 512 μmol / L. Figure 1 Therefore, it was determined that the drug is safe and non-toxic when the concentration range is 32 μmol / L, 64 μmol / L, 128 μmol / L, and 256 μmol / L.

[0033] 4.2 In the flow cytometry experiment, compared with the blank group, the model group CD206 + (M2 type) Macrophages were significantly increased. There were no significant differences between the 32 μmol / L, 64 μmol / L, and 128 μmol / L drug treatment groups and the model group. The 256 μmol / L drug treatment group showed a significant reduction in CD206. + Cell ratio ( Figure 2 This indicates that an M2 polarization model was successfully constructed using pro-macrophage M2 polarization factors secreted by tumor cells, and that the 256 μmol / L drug administration group could significantly inhibit its M2 polarization, determining that the effective drug concentration was 256 μmol / L. 7,2′-dihydroxy-3′,4′-dimethoxyisoflavanane plays an important role in regulating macrophage polarization.

[0034] 4.3 In the blank control group, macrophages were scattered and mostly round; in the model group, cells were aggregated and most were oval or elongated spindle-shaped. In the drug-treated group, the cells reverted to round shapes and aggregation was inhibited. Figure 3 In the phagocytic function test, macrophages in the blank group could phagocytose most tumor cells, while phagocytosis in the model group was significantly weakened, and phagocytosis in the drug-treated group could be reversed. Figure 4 This indicates that 7,2′-dihydroxy-3′,4′-dimethoxyisoflavone exerts its antitumor therapeutic effect by inhibiting the transformation of macrophages to the M2 type.

[0035] 4.4 Compared with the blank group, the levels of IL-10 and TGF-β in the model group were significantly increased. Among them, the level of the IL-10 factor was significantly decreased when the drug concentration was 64, 128, and 256 μmol / L, and the level of the TGF-β factor was significantly reversed at four doses of 32, 64, 128, and 256 μmol / L of the drug ( Figure 5 ). IL-10 and TGF-β are immunosuppressive cytokines secreted by M2 macrophages and have the functions of promoting tumor growth and tumor immune escape. The decrease in the levels of IL-10 and TGF-β factors secreted by macrophages is beneficial to inhibiting tumor development.

[0036] Example 2: Effects of 7,2'-dihydroxy-3',4'-dimethoxyisoflavane on a mouse breast cancer model

[0037] 1 Animals

[0038] Strain: BALB / c mice; Gender: Female; Weight: 18 - 22; Source: Beijing Vital River Laboratory Animal Technology Co., Ltd., Animal Certificate Number: SCXK (Beijing) 2021 - 0006, Feeding: Regular feeding in a SPF-class animal room.

[0039] 2 Test drugs

[0040] 7,2'-dihydroxy-3',4'-dimethoxyisoflavane, purchased from Chengdu Bioscience Co., Ltd. Take 7,2'-dihydroxy-...

[0041] 3 Experimental methods

[0042] 3.1 Grouping of experimental animals, model establishment and drug administration methods

[0043] Ten out of 60 mice were used as the normal group, and 50 mice were used for model establishment. For the mice in the normal group, 0.1 mL of normal saline was drawn with a 1 mL sterile syringe and inoculated subcutaneously in the chest of the mice. For the remaining 50 mice, 0.1 mL of breast cancer cell suspension (about 1×10 6(Number of mice) were subcutaneously injected into the chest of mice to establish a tumor-bearing mouse model. Successful modeling was defined as the palpable nodule the size of a soybean at the injection site after modeling. Mice were randomly divided into four groups (n=10 per group): model group, 7,2′-dihydroxy-3′,4′-dimethoxyisoflavone 50 mg / kg group, 7,2′-dihydroxy-3′,4′-dimethoxyisoflavone 25 mg / kg group, 7,2′-dihydroxy-3′,4′-dimethoxyisoflavone 12.5 mg / kg group, and 7,2′-dihydroxy-3′,4′-dimethoxyisoflavone 6.25 mg / kg group. The intraperitoneal injection dose of 7,2′-dihydroxy-3′,4′-dimethoxyisoflavone in the 7,2′-dihydroxy-3′,4′-dimethoxyisoflavone group was 6 mL / kg. -1 The model group received the same dose of saline. From the second day after successful model establishment, the mice were administered the drug once daily for 21 consecutive days. They were fasted on the evening of the last administration and sacrificed on day 22.

[0044] 3.2 Tumor tissue volume, weight, and organ index

[0045] Every 3 days starting from the first day of administration, the long diameter (a) and short diameter (b) of the tumor in the tumor-bearing mice were measured using calipers. The tumor volume was calculated, and a tumor growth curve was plotted (tumor volume = (a × b²) / 2). 24 hours after the last administration, the mice were sacrificed, the tumor was dissected and weighed (W), and the tumor inhibition rate of each administration group was calculated (tumor inhibition rate = 1 - W administered / W model).

[0046] 3.3 Tumor tissue

[0047] Tumor tissues from each group of mice were fixed in 10% paraformaldehyde solution, dehydrated, embedded, sectioned, stained with hematoxylin and eosin (HE), mounted, and finally observed under a microscope for morphological changes.

[0048] 3.4 Serum IL-10, TGF-β

[0049] Mice were administered the drug continuously for 21 days. On day 22, blood was collected from the eyeballs. The blood volume was 1-2 mL per mouse. The mice were centrifuged at 3500 r / min for 15 min. The supernatant was stored for later use. 10 μL of the supernatant was taken and the levels of IL-10 and TGF-β cytokines were tested according to the instructions of the ELISA kit (Andy Gene).

[0050] 4 Experimental Results

[0051] 4.1 Compared with the model group, all four doses of 7,2′-dihydroxy-3′,4′-dimethoxyisoflavone reduced tumor volume and weight significantly (p<0.05). However, there was no significant difference in tumor volume among the 50 mg / kg, 25 mg / kg, 12.5 mg / kg, and 6.25 mg / kg groups of 7,2′-dihydroxy-3′,4′-dimethoxyisoflavone. Figure 6 a, b). The tumor inhibition rates of each treatment group (low dose to high dose) were 29.0%, 51.7%, 41.0%, and 48.3%, respectively. Figure 6 (c, d) Organ indices showed no significant differences in cardiac, lung, kidney, and thymus indices between the control group, model group, and the three treatment groups. Compared with the control group, the spleen index changed more significantly in the model group and the treatment groups, with the treatment groups showing a reversal of the changes observed in the model group. Compared with the control group, the liver index was significantly increased in the model group, but none of the four treatment groups had a significant effect on the liver index. Figure 7 ).

[0052] 4.2 Tumor pathological sections, such as Figure 8 As shown, HE staining revealed a large number of tumor cells infiltrating the model group, with clear nuclei, intact morphology, and tight arrangement. Compared with the model group, the number of tumor cells infiltrating in each dose group of 7,2′-dihydroxy-3′,4′-dimethoxyisoflavan decreased, the arrangement was sparse, and some areas showed incomplete tumor cell structure and necrosis.

[0053] 4.3 Compared with the blank group, the levels of IL-10 and TGF-β in the model group were significantly increased. After treatment with 6.25 mg / kg of 7,2′-dihydroxy-3′,4′-dimethoxyisoflavone, the levels of IL-10 and TGF-β were not significantly different from those in the model group. Treatment with three doses of 7,2′-dihydroxy-3′,4′-dimethoxyisoflavone (12.5 mg / kg, 25 mg / kg, and 50 mg / kg) significantly reduced the levels of IL-10 and TGF-β. Figure 9 ).

Claims

1.7,2'-Dihydroxy-3',4'-Dimethoxyisoflavane in the preparation of anticancer drugs, wherein the structural formula of the 7,2'-dihydroxy-3',4'-dimethoxyisoflavane is as follows: The anticancer drug mentioned is an anti-breast cancer drug.

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