阅读说明：本技术 Sms2抑制剂在制备治疗高侵袭性乳腺癌药物中的应用 (Application of SMS2 inhibitor in preparation of medicine for treating highly invasive breast cancer ) 是由 董继斌 余科 邓燕 叶德泳 于 2020-05-06 设计创作，主要内容包括：本发明公开了SMS2抑制剂在制备治疗高侵袭性乳腺癌药物中的应用。利用4T1细胞建立小鼠三阴性乳腺癌模型的整体实验以及采用小鼠原代骨髓巨噬细胞进行的体外实验,证实所述化合物I作为SMS2特异性抑制剂,通过降低体内鞘磷脂合酶2活性和鞘磷脂水平,能够有效抑制巨噬细胞的2型极化,并通过减少三阴性乳腺癌肿瘤微环境中M2型巨噬细胞和其他骨髓源性免疫抑制细胞的浸润程度,重塑肿瘤微环境,从而抑制三阴性乳腺的原位生长以及经血行带来的肺转移灶的形成和生长。抑制SMS2活性可用于预防和治疗包括三阴性乳腺癌在内的高侵袭性恶性乳腺癌。(The invention discloses application of an SMS2 inhibitor in preparation of a medicine for treating highly invasive breast cancer. The overall experiment for establishing a mouse triple negative breast cancer model by using 4T1 cells and the in vitro experiment by using mouse primary bone marrow macrophages prove that the compound I is used as an SMS2 specific inhibitor, can effectively inhibit 2-type polarization of macrophages by reducing in-vivo sphingomyelin synthase 2 activity and sphingomyelin level, and remodels a tumor microenvironment by reducing infiltration degree of M2-type macrophages and other bone marrow-derived immunosuppressive cells in a triple negative breast cancer tumor microenvironment, so that in-situ growth of triple negative mammary glands and formation and growth of lung metastases brought by hematopoiesis are inhibited. Inhibition of SMS2 activity is useful for the prevention and treatment of highly aggressive malignant breast cancers, including triple negative breast cancer.)

The application of an SMS2 inhibitor in the preparation of a medicament for treating highly invasive breast cancer.

2. The use of claim 1, wherein the SMS2 inhibitor has a chemical formula as shown in formula (I),

3. the use of claim 2, wherein the SMS2 inhibitor is effective to inhibit macrophage type 2 polarization by decreasing sphingomyelin synthase 2 activity and sphingomyelin levels in vivo, and to remodel the tumor microenvironment by decreasing the degree of infiltration of M2 macrophages and other myeloid-derived immunosuppressive cells in the triple-negative breast cancer tumor microenvironment, thereby inhibiting in situ growth of the triple-negative breast and the formation and growth of blood-borne lung metastases.

4. The medicine for resisting highly invasive breast cancer is characterized in that the medicine is a preparation prepared by taking an SMS2 inhibitor as an active ingredient and adding pharmaceutically acceptable auxiliary materials or auxiliary ingredients.

5. The medicament of claim 4, wherein the SMS2 inhibitor has a chemical structure according to formula (I),

6. the medicament of claim 5, wherein the medicament is administered orally or intravenously.

7. The medicament of claim 5, wherein the SMS2 inhibitor is effective in inhibiting macrophage type 2 polarization by decreasing sphingomyelin synthase 2 activity and sphingomyelin levels in vivo, and in remodeling the tumor microenvironment by decreasing infiltration of M2 macrophages and other myeloid-derived immunosuppressive cells in the triple-negative breast cancer tumor microenvironment, thereby inhibiting in situ growth of triple-negative breast and the formation and growth of blood-borne lung metastases.

Technical Field

The invention relates to the field of pharmacy, in particular to application of an SMS2 inhibitor in preparation of a medicine for treating high invasive breast cancer.

Background

Highly aggressive breast cancer is the major type of breast cancer with high mortality among women. Among them, Triple-Negative Breast Cancer (TNBC) is the most aggressive Breast Cancer subtype, and is characterized by Estrogen Receptor (ER) negativity, Progesterone Receptor (PR) negativity, and human epidermal growth factor receptor (HER2) negativity in clinical pathology, and most of genomics shows basal-like Breast Cancer characteristics (basal-like Breast Cancer). TNBC has high malignancy, common lung and brain metastasis, and currently lacks targeted effective treatment, and the average survival time of patients is maintained at about 15 months, which is the most main subtype lethal to breast cancer (N Engl J Med 2009; 360: 790-. The high invasive breast cancer represented by TNBC has the defects of receptor expression, ideal curative effect cannot be obtained by novel targeted drugs and immune drugs, the first-line clinical treatment means is cytotoxic chemotherapy, but the benefit of treatment does not exceed 20 percent of that of TNBC patients, and the search for a new effective treatment means becomes urgent.

Sphingomyelin (SM) is the most abundant sphingolipid in mammalian cells, and its abnormal biological regulation in vivo can be directly or indirectly involved in the development and progression of disease (Am J Physiol 275: L843-L851; Adv Exp Med biol. 2010; 688: 72-85). Sphingomyelin Synthase 2(Sphingomyelin synthsase 2, SMS2) is a key enzyme in the de novo SM synthesis pathway. Previous studies have shown that SMS2 is abundantly expressed in macrophages and plays an important regulatory role in inflammatory states; downregulation of SMS2 activity relieved LPS-induced inflammation of peritoneal macrophages, whereas overexpression of SMS2 induced expression of aortic inflammatory biomarker molecules (Life Sci.2012Jun 6; 90(21-22): 867-73).

In recent years, the study of macrophages in the field of tumor biology and tumor therapeutics has received much attention. Macrophages can appear as type M2, which suppresses inflammation and promotes cancer, and type M1, which suppresses pro-inflammatory tumors. The tumor microenvironment is mainly enriched with M2 type macrophages (also called tumor infiltrating macrophages and TAMs), and becomes an important mechanism for promoting the development and drug resistance of tumors. Based on SM being one of the most predominant phospholipids on macrophage membranes, it is contemplated that the activity of SM2 is likely linked to the enrichment of M2-type macrophages in the tumor microenvironment and their oncogenic function. The invention researches and researches the hypothesis, finds that the SMS2 has an important regulation and control function in M2 type macrophages (TAMs) in tumor microenvironments, and the SMS2 inhibitor is effective in treating TNBC and has application and development values.

The selected 4- (fluorobenzyloxy) -3- (pyridylamino) benzisoxazole compound is discovered to have an SMS2 inhibition effect in the early stage (an alkoxy benzo five-membered (six-membered) heterocyclic amine compound and a pharmaceutical application thereof, the Chinese patent application No. 201810124854.9, application date: 2018, 2 and 7 days; PCT/CN2019/072468, international application date 2019, 1 and 21 days, international priority date 2018, 2 and 27 days), contains the compound I, and the compound I has good bioavailability (the bioavailability is 56% when the compound I is administered to ICR mice by gastric lavage, J.Med.Chem.2018, 61: 8241-54). The SMS2 inhibitor can be used for preventing and treating atherosclerosis and inflammatory bowel disease (Y9 is applied to anti-atherosclerosis, Chinese invention patent with application number of 201610392529.1, application date of 2016 (6 months and 3 days), and application of 4- (2, 6-dichlorobenzyloxy) -3- (3-aminopyridine) benzo [ d ] isoxazole in preparation of drugs for preventing and treating inflammatory bowel disease, and Chinese invention patent with application number of 2019100859357, application date of 2019 (1 month and 29 days), but the influence of the inhibitor on macrophage and tumor microenvironment and the effect of the inhibitor in prevention and treatment of human and animal triple negative breast cancer are not proved by experiments.

Disclosure of Invention

In order to achieve the purpose, the invention provides an application of an SMS2 inhibitor in preparing a medicine for treating highly invasive breast cancer.

Furthermore, the chemical structural formula of the SMS2 inhibitor is shown as the formula (I),

furthermore, the SMS2 inhibitor can effectively inhibit 2-type polarization of macrophage by reducing the activity of sphingomyelin synthase 2 and sphingomyelin level in vivo, and can remodel tumor microenvironment by reducing the infiltration degree of M2-type macrophage and other marrow-derived immunosuppressive cells in triple negative breast cancer tumor microenvironment, thereby inhibiting in-situ growth of triple negative breast and formation and growth of lung metastasis caused by menstruation

The invention also provides a medicine for resisting high invasive breast cancer, which is a preparation prepared by taking the SMS2 inhibitor as an active ingredient and adding pharmaceutically acceptable auxiliary materials or auxiliary ingredients.

Furthermore, the chemical structural formula of the SMS2 inhibitor is shown as the formula (I),

further, the administration route of the medicament is oral administration or intravenous injection.

Furthermore, the SMS2 inhibitor can effectively inhibit 2-type polarization of macrophages by reducing the activity of sphingomyelin synthase 2 and the level of sphingomyelin in vivo, and can remodel tumor microenvironment by reducing the infiltration degree of M2-type macrophages and other marrow-derived immunosuppressive cells in the tumor microenvironment of triple negative breast cancer, thereby inhibiting the in-situ growth of triple negative breast and the formation and growth of lung metastases brought by menstruation.

Technical effects

The invention proves that the SMS2 inhibitor has the structure shown in the formula I, has the effect of inhibiting highly invasive breast cancer, and provides a new effective treatment medicament for treating the highly invasive breast cancer.

The conception, the specific structure and the technical effects of the present invention will be further described with reference to the accompanying drawings to fully understand the objects, the features and the effects of the present invention.

Drawings

FIG. 1 is the half-maximal inhibition ratio IC of Compound I in mouse-derived macrophage strain RAW264.7 and BALBC mouse bone marrow primary macrophage BMDM₅₀The measurement is carried out, the numerical value is expressed by mean +/-standard error, and n is 9;

figure 2 is a graph of the cytotoxicity of compound I on mouse-derived macrophage strain RAW264.7 and BALBC mouse bone marrow primary macrophage BMDM, values are expressed as mean ± standard error, n-9;

FIG. 3 is a high expression of SMS2 in invasive breast cancer;

fig. 4 is a sample data analysis of 522 breast cancer tumors in the TCGA database: A. in the tumor of the breast cancer patient, the high expression of SMS2 is related to the high ratio of CD206/CD 68; B. in the tumor of the breast cancer patient, the high expression of SMS2 is related to the high expression of FOXP3, CD11b and CD33, and P is less than 0.001; p < 0.0001;

FIG. 5 is a graph of high expression of SMS2 in relation to patient prognosis: high expression of smss 2 in "basal-like" breast cancer; B. in "basal-like" breast cancers, high expression of SMS2 was associated with low RFS, { t, { P < 0.001;

FIG. 6 is a graph of the effect of lack of SMS2 activity on IL-4 induced macrophage type 2 polarization: A. identifying the expression of F4/80 on the surface of the cell by a flow cytometer; B. flow cytometry detects the percentage of F4/80+ CD206+ cells and the MFI of cell surface CD 206; western Blot to detect the expression of Arg1 protein in cells; D.Q-PCR detection of cell CD206, Arg1, YM1, TGF-beta mRNA expression; p <0.01, P < 0.001;

FIG. 7 is the absence of the effect of SMS2 activity on macrophage type 1 polarization under IL-4 induced conditions;

figure 8 is a graph showing that compound I significantly reduced IL-4-induced expression of M2-type surface protein in BMDM cells: A. detecting the percentage of F4/80+ CD206+ cells and CD206 MFI in BMDM cells by a flow cytometer; B. detecting CD206 MFI of RAW264.7 cells by a flow cytometer, wherein P is less than 0.01; p < 0.001;

figure 9 is that compound I significantly reduced IL-4-induced protein expression of BMDM and RAW264.7 cells CD206 and Arg 1. Western Blot to detect protein expression of Arg1 and CD206 in BMDM cells; western Blot to detect protein expression of Arg1 and CD206 in RAW264.7 cells; p < 0.01; p < 0.001;

figure 10 is a graph of compound I significantly reduced IL-4 induced expression of M2 type marker genes in BMDM and RAW264.7 cells: A.Q-PCR detection of mRNA expression of CD206, Arg1, YM1 and TGF-beta in BMDM; Q-PCR detects mRNA expression of CD206, Arg1, YM1 and TGF-beta in RAW264.7 cells; p < 0.01; p < 0.001;

figure 11 is that compound I significantly reduced TNBC cell conditioned medium-induced expression of M2-type surface protein in BMDM cells, but had no effect on M1-type: A. the compound I remarkably reduces the expression of cell surface CD206 of BMDMs induced by 4T1 cell conditioned medium without obvious inhibition on the expression of CD 86; B. the compound I remarkably reduces the expression of BMDMs cell surface CD206 induced by MDA-MB-231 cell conditioned medium without obvious inhibition on the expression of CD 86; p < 0.01;

figure 12 is a graph of compound I significantly reduced TNBC cell-conditioned medium-induced secretion of IL-10 and TGF- β by BMDM cells: A.4T1-CM induces BMDM cells to secrete IL-10 and TGF-beta; MDA-MB-231-CM induces BMDM cells to secrete IL-10 and TGF-beta; p < 0.01; p < 0.001; p < 0.0001;

figure 13 is that compound I significantly inhibited TNBC cell-conditioned medium-induced activation of the mTOR1 signaling pathway; p < 0.001;

figure 14 is the ability of compound I to reduce migration of 4T1 cells induced by M2-type macrophages: A. macrophage conditioned media collection schematic; B.4T1 cell migration assay; p < 0.001;

figure 15 is that compound I reduced total SM levels in mouse plasma; p < 0.001;

figure 16 is a graph showing that compound I is effective in inhibiting hematogenous metastasis of 4T1 cells in vivo: A. imaging a mouse living body; B. lung tissue HE staining; p < 0.01; p <0.001, n ═ 8;

figure 17 is that compound I was effective in reducing the number of M2-type macrophage infiltrates in TNBC lung metastases, but had no effect on macrophage polarization state at normal physiological states: A. the compound I has no obvious inhibition effect on the macrophage polarization of the abdominal cavity of a mouse in a normal physiological state; B. compound I is effective in reducing the proportion of M2-type macrophages in lung metastases; p <0.01, n ═ 8;

figure 18 is that compound I is effective in reducing infiltration of multiple immunosuppressive cells in TNBC lung metastases: A. compound I was effective in reducing FOXP3+ CD4+ cell infiltration in lung metastases; B. compound I was effective in reducing CD11b + Gr1+ cell infiltration in lung metastases; p < 0.01; p <0.0001, n ═ 8;

figure 19 is that compound I is effective in reducing in situ growth of triple negative breast cancer in a mouse model, and in promoting necrosis and apoptosis of in situ tumor tissue: A. the tumor body weight of the compound I administration group is obviously reduced; B. the tumor necrosis and apoptosis of the compound I administration group are obviously increased; C. the collagen fiber content of the compound I administration group was significantly reduced (200 ×); p < 0.01; p < 0.001; p <0.0001, n ═ 8;

figure 20 is that compound I effectively increased effector T cells in the tumor microenvironment and decreased immunosuppressive M2-type macrophages: A. compound I is effective in increasing infiltrating CD8+ T cells in the tumor stroma; B. the compound I effectively reduces the proportion of M2 type macrophages infiltrated in a tumor stroma; p <0.001, n ═ 8;

figure 21 is that compound I significantly inhibited normal development of blood vessels in tumor tissues: A. compound I effectively reduces the luminal area of blood vessels in tumor tissue; B. the compound I obviously inhibits the normal mature development of blood vessels in tumor tissues. P <0.001, n ═ 8.

Detailed Description

The technical contents of the preferred embodiments of the present invention will be more clearly and easily understood by referring to the drawings attached to the specification. The present invention may be embodied in many different forms of embodiments and the scope of the invention is not limited to the embodiments set forth herein.

SMS2 herein refers to sphingomyelin synthase 2.

As the basis of the experiment, the Oncomine and TCGA online database analysis is utilized successively to discover that SMS2 has abnormal expression in human triple negative breast cancer, and the SMS2 high expression is related to M2 type macrophage high infiltration and to the poor prognosis of tumor patients.

According to the invention, the SMS2 activity deletion is verified by using primary bone marrow macrophages of an SMS2 gene knockout mouse, so that the macrophage 2 type polarization induced by IL4 can be obviously reduced, and an experimental foundation is laid for the invention. Then, human and mouse most representative Triple Negative Breast Cancer (TNBC) cell strains 4T1 and MDA-MB-231 are selected to carry out in vitro tests aiming at primary bone marrow macrophages (BMDM in the subsequent examples, BMDM in the subsequent examples are all defined in the same way) derived from BALBC mice; in vivo experiments were performed using a mouse triple negative breast cancer in situ and a lung hematogenous metastasis model established with 4T 1.

The result shows that the compound I can obviously inhibit the SMS2 activity of macrophages in-vitro experiments, and obviously reduce macrophage M2 type polarization induced by TNBC cells; and this inhibition may be associated with the mTOR signaling pathway, as under the same conditions inhibition of macrophage SMS2 activity by compound I does not alter macrophage M1 type polarization levels.

In vivo experiments, a therapeutic administration scheme is adopted, and the compound I can effectively inhibit the growth of tumors and the normal development of blood vessels in an in situ tumor model; in a blood circulation metastasis model, the compound I can effectively inhibit the formation of lung metastasis; the treatment effect is closely related to that the compound I can effectively reduce the plasma sphingomyelin level in vivo, reduce the infiltration of immunosuppressive cells such as tumor-related macrophages (TAMs), regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) in a tumor microenvironment, and reshape the tumor microenvironment.

SMS2 can be used as a drug target for treating highly invasive breast cancer represented by TNBC. The compound I can be used for preventing and treating triple negative breast cancer of human and animals.

Example 1: the experimental dose of compound I is effective and safe.

Determination of IC of Compound I (shown as ComdI in the figure) on mouse-derived macrophage Strain (RAW264.7) and Primary bone marrow macrophage (BMDM)₅₀Value, determine 3 times half inhibition rate (IC)₅₀) The concentration is used as the experimental dosage of the invention, the dosage has obvious inhibition effect on the SMS2 activity of the same experimental cells (shown in figure 1), and the cytotoxicity effect at the dosage is negligible (shown in figure 2), so the dosage of the compound selected by the invention is safe and effective.

Example 2: SMS2 is highly expressed in basal-like breast cancer patients and is positively correlated with high infiltration of immunosuppressive cells such as M2-type macrophages in cancer tissues and poor prognosis of the patients.

In order to lay the experimental foundation of the invention, the Oncorine database (https:// www.oncomine.org) is used for comparatively analyzing the expression difference of SMS2 in the breast cancer tissue and the normal breast tissue; it was found that SMS2 expression was significantly elevated in invasive breast cancer (invasive breast cancer) compared to normal breast tissue (as shown in fig. 3). Next, the TCGA database (Cancer Genome Atlas N. comprehensive molecular portals of human breast Cancer. Nature 2012; 490:61-70.) was used to analyze the relationship between SMS2 expression and immunosuppressive cell infiltration in tumors in 522 human breast cancers. mRNA expression of SMS2 in the samples was ranked from high to low, and tumor samples were classified as "SMS 2 high" and "SMS 2 low" according to median. The mRNA expression ratio of the M2-type macrophage marker gene mannose receptor 1(CD206) and the macrophage universal gene CD68(CD68) was compared in different SMS 2-expressed tumor samples. Meanwhile, the expression of regulatory T cell (Treg) cell marker gene forkhead transcription factor 3(FOXP3) and the expression of myeloid-derived suppressor cell (MDSC) cell marker genes CD11b (CD11b) and CD33(CD33) were also compared in the analysis. The statistical analysis result is shown in FIG. 20, the ratio of CD206/CD68 is higher in the sample with high expression of SMS2 gene, which indicates that the high expression of SMS2 is related to the high infiltration degree of M2 type macrophages. Similarly, the higher the expression of FOXP3, CD11b and CD33 in the sample with high expression of the SMS2 gene, indicating that the high expression of SMS is related to high infiltration of regulatory T cells and myeloid-derived suppressor cells, as shown in fig. 4A, the high expression of SMS2 (SMS2-H) in the tumor of breast cancer patients is related to high ratio of CD206/CD 68; as shown in fig. 4B, high expression of SMS2 in the tumor of breast cancer patients was associated with high expression of FOXP3, CD11B, and CD 33. In the figure, SMS2-L represents low expression of SMS 2. P < 0.001; p < 0.0001.

Using the on-line bioinformatics tool Kaplan-Meyer ((https:// kmplot. com), ratios of mRNA expression of 522 breast cancer samples in the TCGA database were calculated according to the following formula (KRT5+ KRT14)/(KRT8+ KRT18), dividing the breast cancer into two classes of "Basal-like" and "ductal-like" (Luminal-like) according to the median of the samples, higher than the median defined as "Basal-like" breast cancer and lower than the median defined as "ductal-like" breast cancer, analyzing and comparing the difference in expression of SMS2 in the two breast cancers, finding that SMS2 is significantly highly expressed in the "Basal-like" breast cancer sample, and higher expression in the "Basal-like" breast cancer patient 2 suggests that the patient has a lower SMS recurrence-free survival Rate (RFS), FIG. 5 shows that SMS2 is highly expressed in relation to the patient prognosis, FIG. 5A shows that SMS2 is highly expressed in the "Basal-like" breast cancer ", FIG. 5B shows that in the Basal-like" breast cancer, SMS2 high expression is associated with low RFS. P < 0.001.

Example 3: the deletion of SMS2 activity can effectively inhibit IL-4 induced macrophage M2 type polarization

Primary bone marrow macrophage BMDM from SMS2 knock-out mice (KO group) was isolated and cells were dispersed to a final concentration of 2X106 cells/mL using 1640 complete medium containing 20ng/mL macrophage colony stimulating factor (M-CSF). Cells were plated in 10cm dishes or 12-well plates for three days. On the third day, fresh BMDM growth medium was changed. After 7 days of cell culture, the formation of mature BMDM was assessed using flow cytometry to detect the expression of CD11b and the macrophage universal marker protein molecule F4/80 (as were BMDM-induced differentiation conditions in the subsequent examples). Then, the cells were polarized for 48 hours (48h) by replacement with 1640 complete medium containing 20ng/mL interleukin-4 (IL-4, a classical macrophage polarization-inducing factor of type 2). The expression of M2 type macrophage surface marker protein (F4/80 and CD206) is detected by using a flow cytometer, the expression level of M2 type related marker gene is detected by a real-time fluorescence quantitative PCR method, wild type C57bl/6j mouse (WT group) BMDM is used as a control group, and SMS2 knockout mouse BMDM is expressed by KO group.

The results show that the SMS2 gene knockout did not affect the differentiation and maturation of BMDM cells (as shown in fig. 6A), but significantly inhibited the expression of IL-4-induced type 2 polarization marker protein molecule CD206 (fig. 6B) and marker protein arginase 1(Arg1) (fig. 6C) (with β -actin (β -actin) as an internal reference), while the gene expression levels of the type 2 polarized series marker molecules (chitinase 3-like molecule (Ym1), transforming growth factor- β (TGF- β)) also showed significant decrease (fig. 6D), but the effect of IL-4 did not have a significant effect on the expression of macrophage type I polarization marker protein molecule CD86 (fig. 7). In the figure, control represents a normal control group, DMSO is an inhibitor solvent, and the control group is a sample control group, and the subsequent examples do not particularly indicate the above meanings.

Example 4: compound I is effective in inhibiting macrophage type 2 polarization induced by IL 4.

Under the safe experimental dose, the compound I can effectively inhibit macrophage type 2 polarization induced by IL 4. Compound I was added to a cell culture medium (1640 complete medium containing 10% fetal bovine serum, the same medium in the subsequent examples unless otherwise specified) which induced mature BALLC mouse bone marrow primary macrophages (BMDM, in the subsequent examples BMDM is derived therefrom) and mouse-derived macrophage strain RAW264.7 to a final concentration of 10. mu.M, and 20ng/mL IL-4 was added after 1 hour of cell incubation to continue culturing the cells for 48 hours, and then the expression of BMDM and RAW264.7 cell surface proteins F4/80 and CD206 was detected by flow cytometry. The results show that after pretreatment of BMDM cells with Compound I, F4/80⁺CD206⁺The percentage of cells was significantly reduced and the Mean Fluorescence Intensity (MFI) of CD206 was significantly reduced. Similarly, CD206 MFI was significantly reduced after pretreatment of RAW264.7 cells with compound I (fig. 8). Western blotting assay also confirmed that the expression of the polarization marker proteins CD206 and Arg1 of type 2 on the surface of BMDM and RAW264.7 cells showed a significant decrease after compound I treatment (FIG. 9). Under the same experimental conditions, the expression of M2 type polarization marker genes CD206, Arg1, YM1 and TGF-beta is detected by a reverse transcription fluorescence real-time quantitative PVR (Q-PCR) method, the same result is obtained, and the mRNA level of the marker genes of BMDM and RAW264.7 cells pretreated by the compound I is obviously reduced (figure 10).

Example 5: the compound I can obviously reduce M2 type polarization of BMDM cells induced by TNBC cell conditioned medium without influencing M1 type polarization.

Respectively culturing the selected representative triple negative breast cancer cell strains (4T1 and MDA-MB-231) in 1640 complete culture medium until the cell fusion degree is 80%, collecting and re-suspending the cells to make the final concentration be 2x10⁶And (2) spreading 10mL of cell suspension in a 10CM culture dish, collecting cell culture supernatant after continuously culturing for 72 hours, and performing centrifugal filtration to obtain conditioned medium (4T1-CM and MDA-MB-231-CM) for culturing BMDM so as to simulate the microenvironment for tumor growth.

After 1 hour of pretreatment of BMDM cells with Compound I (final concentration of 10uM), the medium was changed to 4T1-CM and MDA-MB-231-CM and cells were cultured for an additional 72 hours with Compound I added. BMDM cell surface CD86 and CD206 protein expression was analyzed by flow cytometry. The results show that the cell surface CD206 MFI and CD86 MFI of BMDM cells are remarkably increased after the BMDM cells are induced by 4T1-CM and 231-CM. After further treatment with compound I, CD206 MFI was significantly reduced, while CD86 MFI was not significantly changed (fig. 11).

Example 6: the compound I can inhibit IL-10 and TGF-beta secretion of BMDM cells induced by TNBC cell conditioned medium.

Macrophages in the tumor microenvironment are stimulated by a variety of factors, which on the one hand produce type 2 polarization and on the other hand secrete a variety of anti-inflammatory, tumor-promoting cytokines such as interleukin-10 (IL-10) and TGF- β. The secretion of IL-10 and TGF- β was determined after BMDM cells were induced to culture in conditioned media 4T1-CM and MDA-MB-231-CM using ELISA methods. The results show that upon treatment of BMDM with compound I, secreted IL-10 and TGF- β were significantly reduced. (FIG. 12)

Example 7: the modulatory effects of compound I on BMDM cell differentiation in the tumor microenvironment may be associated with the mTOR1 signaling pathway.

In order to preliminarily explore the mechanism of the participation of SMS2 in the regulation of TNBC cell-induced macrophage M2 type differentiation, the expression changes of mTORC1 signal channel-related marker proteins, namely phosphorylated S6 ribosomal protein (P-S6) and phosphorylated protein kinase B (P-Akt) in BMDM are detected by using a Western Blotting method. As a result, the protein expression of P-S6 and P-Akt was increased after BMDM cells were cultured with 4T1-CM and MDA-MB-231-CM, while the effect of compound I significantly reduced the protein increase trend of P-S6 and P-AKT (FIG. 13).

Example 8: compound I reduced the migratory capacity of M2-type macrophages induced 4T1 cells.

After isolation and induction of differentiation maturation of BMDM, BMDM was resuspended in media and adjusted to a density of 2.5x10⁵one/mL. Plating in 6-well plates at 2 mL/well, changing the medium after 24h incubation, pretreating the SMS2 inhibitor group with compound I at 10 μ M for 1 h, adding IL-4 at 20ng/mL to induce BMDM polarization, pretreating the control group with DMSO for 1 h, adding isotonic Phosphate Buffer (PBS), and further incubating for 48hThen (c) is performed. After that, the culture medium of each well was replaced with 2mL of fresh 1640 medium, the culture was continued for 24 hours, and the culture supernatant of each well was collected and filtered for use in the transmembrane migration assay (Transwell assay) of 4T1 cells. 4T1 cells were cultured in collected medium conditioned by different macrophages, after 48 hours the cells were starved overnight (about 16 hours) by changing to serum-free 1640 medium, then the collected cells were resuspended in serum-free 1640 medium, 100. mu.L of the collected cells were pipetted and plated in 8 μm-pore transwell cells previously placed in 24-well plates, 600. mu.L of 1640 complete medium was placed in the lower layer of the wells, after 12 hours of further culture, the cells that did not penetrate in the upper layer of the transwell cells were gently wiped off with a cotton swab, 95% ethanol was added to the chamber, and the cells were fixed for 20 minutes. Taking out the small chamber, adding 0.2% crystal violet dye solution, and dyeing for 20 min. And (5) slightly rinsing the redundant dye liquor in the small chamber by using distilled water, and standing and airing.

The chamber was placed on a glass slide and five fields of view in the chamber were photographed using a 200-fold microscope and counted and analyzed by ImageJ software. The results are shown in FIG. 14, which shows that the conditioned medium of BMDM treated with IL-4 is effective in increasing the migration ability of 4T1 cells; in contrast, the conditioned medium of BMDM co-treated with compound I and IL-4 significantly reduced the ability of 4T1 to induce migration compared to the former.

Example 9: oral administration of compound I was effective in reducing plasma SM levels in mice.

BALBC mice 6-8 weeks old were randomly divided into two groups, 8-9 mice per group, mice in the SMS 2-inhibited group were gavaged with 20mg/kg of compound I daily, mice in the control group were gavaged with 0.5% sodium carboxymethylcellulose (control) daily, and the SM content in the plasma of the mice was monitored twice weekly (ELISA kit test), and after the mice were gavaged with compound I for 14 days, a significant decrease in the total SM level in the plasma was shown (fig. 15).

Example 10: the oral administration of the compound I can effectively inhibit the hematogenous metastasis of TNBC cells in a model mouse and improve the infiltration degree of TAMs and MSDCs in a tumor microenvironment, and the effect has no influence on macrophage states in a non-tumor microenvironment.

The experimental mice were randomly divided into two groups, compound I (20mg/kg/day) was administered by intragastric administration for two weeks in advance, and the fluorescein-labeled cells 4T1-luc cultured in vitro were collected, resuspended in precooled PBS, and then injected into the tail vein of the mice at a cell volume of 1 × 106. After 6 hours of cell inoculation, fluorescence signals in the mouse lungs were detected using a biopsy instrument. Two weeks after the experiment, the fluorescence signal in the mouse lung was again detected. After inoculation, the SMS2 inhibitor group was administered compound I at 20mg/kg/day by continuous gavage to the end of the experiment, and the control group was administered an equivalent amount of solvent (DMSO).

After the experiment is finished, taking lung tissues of mice to prepare tissue sections, and passing through H&E staining observation and counting the number of lung metastases. IHC staining analyzed the expression of macrophage marker protein F4/80 and macrophage marker protein CD206 type M2 in lung metastases. Immunohistochemistry (IHC) and Immunofluorescence (IF) double staining analysis the expression of marker proteins FOXP3 and CD4 of regulatory T cells, and the expression of myeloid-derived suppressor cell marker proteins CD11b and Gr1 (myeloid differentiation antigen 1). The results show that the lung metastasis tumor focus of the mice of the compound I administration group is obviously reduced (figure 16), and the CD206 expression amount and the ratio of CD206 to F4/80 in the lung metastasis focus are both obviously reduced (figure 17B); furthermore, the expression of FOXP3 and FOXP3⁺Account for CD4⁺Also significantly decreased in the ratio of (FIG. 18A), CD11b⁺Gr1⁺The number of cells was also significantly lower (fig. 18B), indicating that compound I was able to effectively reduce infiltration of M2 type macrophages and other suppressive immune cells in lung metastases, had a certain therapeutic effect on infiltration metastasis that is likely to be caused by TNBC late phase, and this effect may be related to improvement of immunosuppressive tumor microenvironment after compound I inhibited SMS2 activity.

To demonstrate that the therapeutic effect of compound I is specific to the tumor environment, the intraperitoneal macrophages of mice perfused for 14 days were extracted for flow cytometry analysis, and the expression of intraperitoneal macrophage surface proteins F4/80 and CD206 in mice in the compound I administration group and the control group were compared. The results are shown in FIG. 17A, F4/80 between groups⁺CD206⁺The percent of macrophages in the mouse were not significantly altered, indicating that the mouse macrophage phenotype in the normal physiological state was not affected by the SMS2 inhibitor. The effect of compound I on macrophage polarization is the interaction with the tumor microenvironmentAnd (4) raw.

Example 11: oral administration of compound I can effectively reduce the in situ growth of triple negative breast cancer in mouse models, change the infiltration ratio of suppressive macrophages and killer T lymphocytes in the tumor environment, and limit the maturation of blood vessels at the tumor site.

Experimental mice were first divided into two groups, compound I (20mg/kg/day) was first intragastrically administered to the administration group for two weeks, 4T1 cells were collected in the logarithmic growth phase of culture, and the cell suspension density was adjusted to 5X10 using precooled PBS⁶And (4) sucking 0.1mL of single cell suspension by using a 1mL insulin syringe, inoculating the single cell suspension under a second breast pad on the left side of a BALB/c mouse, establishing a breast cancer in-situ tumor model and continuously performing intragastric administration every day. Starting on the fifth day after inoculation, the length and width of the tumor body were measured with a vernier caliper every other day according to the formula: v (mm)³) Tumor size was calculated and recorded for three weeks to the end of dosing, i.e., L × W/2(V for volume, L for tumor length, and W for tumor width).

After the experiment was completed, mouse tumor tissue was isolated, weighed and tissue sections prepared. Then, inspecting the necrosis condition of the tumor tissue through H & E staining; TUNEL staining investigates apoptosis of cells in tumor tissue; masson staining examined the collagen fiber content in tumor tissue. As a result, as shown in FIG. 19, the tumor bodies were softer and lighter in weight in the group to which Compound I was administered (FIG. 19A), the number of tumor cells undergoing apoptosis increased (FIG. 19B), and the necrotic area of the tumor increased (FIG. 19C), as compared with the control group.

Immunohistochemical staining was also used to detect the levels of type 2 macrophages and killer T cells in the tumor environment. From the results (fig. 20), it can be seen that the expression of macrophage universal protein F4/80 was not significantly different between the two groups of tumors, but the expression of M2 type marker protein CD206 was significantly reduced in the compound I-administered group, and the ratio of CD206 to F4/80 was also significantly reduced, indicating that compound I did not affect the total number of macrophages infiltrated in the tumor tissue. But was effective in inhibiting infiltrating macrophages type M2 in tumor tissue (fig. 20B). We also detected killer CD8 in tumor tissues⁺The results of the T lymphocyte content in the tumor tissue of the treatment group are shown in FIG. 20A, CD8⁺The infiltration degree of T lymphocytes is much larger than that of the control group. Finally, we analyzed vascular endothelial cell marker protein CD31 and pericyte marker protein α -SMA (α -actin) in tumor tissues using immunofluorescence staining. As shown in fig. 21, the mature vascular luminal area was significantly reduced in the tumors of compound I treated group compared to the control group (fig. 21A), the pericyte marker protein α -SMA was significantly reduced, and the normal mature development of the vessels in the tumor tissue was restricted (fig. 21B).

According to the results, the oral administration of the compound I can effectively reduce the plasma SM level of mice, improve the tumor growth condition of TNBC, reduce the infiltration degree of M2 type macrophages in tumor tissues, increase killer immune cells, block the normal growth of blood vessels in tumors and play a role in treating TNBC.