Use of tenapanor in the preparation of a medicament for inhibiting brain metastasis of a tumor

Abstract

The application discloses application of tenapanol in preparation of a medicine for inhibiting tumor brain metastasis. The application targets neutrophils which are recruited in brain metastasis foci and promote tumor growth, screens out an inhibitor tenapanol for a neutrophil chemotactic receptor FPR2, inhibits the neutrophil recruitment to the brain and the subsequent promotion of tumor cell clonal growth by inhibiting the combination of the receptor and SAA1 which is specifically up-regulated in metastatic tumor cells, and thus the therapeutic effect of inhibiting the progression of intracranial lesions is achieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical applications, and more specifically, relates to the use of Tenapanol in the preparation of drugs for inhibiting tumor brain metastasis. Background Art

[0002] Brain metastases develop in approximately 9-50% of malignant tumor patients during their disease course. They are a common neurological complication of malignant tumors, a major contributor to decreased patient survival, and a significant therapeutic challenge. The vast majority of brain metastases arise from lung cancer, breast cancer, and melanoma, with incidences of 40-50%, 15-30%, and 5-20%, respectively. For example, lung cancer, the most common source of brain metastases, is found in 25-30% of patients with lung adenocarcinoma at diagnosis, and 50% of patients will eventually develop brain metastases. The median time from lung tumor discovery to confirmed brain metastasis is only 11 months, the shortest among all solid tumors. Increased intracranial pressure and specific neurological symptoms caused by brain metastases can severely reduce patient survival and quality of life, resulting in a generally poor prognosis, with a median survival of only 4-7 months. Current treatments for brain metastases include comprehensive management (surgery and radiotherapy) in addition to systemic therapy (chemotherapy, molecularly targeted therapy, and immunotherapy). However, while these treatment options can prolong patient survival, they still suffer from limited efficacy and specificity. The response rate and duration of systemic treatment for brain metastases are lower than those for primary lesions. In addition, the control rate of targeted local treatment is low, the recurrence and progression rate is high, and the adverse effects on neurocognitive function are severe. These problems urgently require effective solutions.

[0003] Studies have shown that neutrophils, absent from the normal brain, are significantly increased in brain metastases. These cells may promote brain metastasis by establishing a premetastatic niche, enhancing tumor cell adhesion to metastatic organs, selecting and expanding tumor subpopulations with high tumorigenic potential, suppressing anti-tumor immunity, and increasing colonization. Neutrophil counts in metastatic lesions have been reported to be associated with poor survival, and the neutrophil-to-lymphocyte ratio (NLR) in peripheral blood has become an increasingly important diagnostic indicator for brain metastases in cancer patients. Targeted neutrophil depletion can effectively inhibit brain metastasis, but this approach has only been partially validated in animals and has yet to be clinically applied. Importantly, existing approaches to targeting neutrophils typically involve the use of antibodies to deplete neutrophils. However, due to the blood-brain barrier, antibodies often have difficulty reaching brain metastases to exert their therapeutic effects and control intracranial lesions. Therefore, the discovery of compounds that specifically target neutrophils in brain metastases could potentially lead to the development of more effective and specific drugs to inhibit brain metastasis, a key challenge in the current research and treatment of brain metastasis. Summary of the Invention

[0004] In response to the above-mentioned existing technical problems, the primary purpose of the present invention is to provide the use of tenapanol in the preparation of a drug for inhibiting brain metastasis. Tenapanol inhibits the binding of the neutrophil chemotactic receptor formyl peptide receptor 2 (FPR2) to serum amyloid 1 (SAA1) secreted by tumor cells in brain metastases, thereby preventing the recruitment of neutrophils to brain metastases and the subsequent promotion of tumor growth.

[0005] The second object of the present invention is to provide the use of tenapanol in the preparation of inhibitors of formyl peptide receptor 2.

[0006] The third object of the present invention is to provide a drug containing a therapeutically effective amount of Tenapanol.

[0007] In order to achieve the above object, the present invention is implemented through the following technical solutions:

[0008] Tenapanor is an NHE3 (sodium hydrogen exchanger 3) inhibitor used to treat constipation-predominant irritable bowel syndrome. Domestic clinical studies have found that Tenapanor can effectively relieve kidney disease-related hyperphosphatemia.

[0009] The present invention provides the use of tenapanol in preparing a medicine for inhibiting tumor brain metastasis.

[0010] The structural formula of the tenapanol is shown in the following formula (I):

[0011]

[0012] Through research, the inventors discovered that compared with primary tumor lesions and normal brain tissue, the number of neutrophils in brain metastases is significantly increased, and they play an important role in the development of brain metastatic tumor cells. FPR2 is a neutrophil membrane surface receptor. Single-cell data analysis results show that it can recruit neutrophils to the brain and promote tumor cell colonization and growth by binding to the chemoattractant protein SAA1, which is specifically upregulated by tumor cells in brain metastases. The present invention conducted drug screening by molecular docking with the FPR2 extracellular domain and comparing its binding ability with known FPR2 small molecule inhibitors WRW4 and WKYMVm. Ultimately, the drug Tenapano was discovered to effectively inhibit the binding of SAA1 to neutrophil FPR2 in vitro, thereby preventing neutrophil recruitment and promoting tumor growth. In an in vivo experiment, a Balb / c nude mouse model of brain metastasis was constructed using lung adenocarcinoma cells A549 that highly expressed SAA1. Tenapano was injected intraperitoneally into mice with brain metastasis, and it was found that it could effectively inhibit the development of brain metastasis, thus confirming the good therapeutic effect of Tenapano on tumor brain metastases.

[0013] Preferably, the tumor brain metastasis refers to brain metastasis of lung adenocarcinoma, breast cancer or melanoma.

[0014] Preferably, the tenapanor inhibits tumor brain metastasis by preventing the recruitment and growth of neutrophils in brain metastases.

[0015] Preferably, Tenapano inhibits tumor brain metastasis by preventing the secretory protein SAA1 from binding to the neutrophil membrane surface receptor.

[0016] Preferably, the neutrophil membrane surface receptor is formyl peptide receptor 2.

[0017] Preferably, the tenapanol inhibits tumor brain metastasis by binding to neutrophil membrane surface receptors.

[0018] Preferably, the cancer cells of the tumor are one or more of A549 lung adenocarcinoma cells, H1975 lung adenocarcinoma cells, MDA-MB-231 breast cancer cells, MCF7 breast cancer cells, and A375 melanoma cells.

[0019] Furthermore, the present invention seeks to protect the use of Tenapanol in the preparation of inhibitors of formyl peptide receptor 2.

[0020] Furthermore, the present invention claims protection for a drug comprising a therapeutically effective amount of the above-mentioned Tenapanol.

[0021] Furthermore, the present invention also claims a method for inhibiting tumor brain metastasis, comprising administering a therapeutically effective amount of the above-mentioned compound Tenapanol to a subject.

[0022] In the present invention, the drug can be administered in the following forms: orally in the form of capsules, tablets, pills, powders, sustained release injections (such as sterile solutions, suspensions or emulsions); by topical treatment forms such as pastes, creams or ointments; or by suppositories such as suppositories; or by inhalation or insufflation.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The present invention utilizes the function of neutrophils in brain metastases to recruit and promote tumor cell growth, and screens out Tenapano, an inhibitor of the neutrophil chemotactic receptor FPR2. By inhibiting the recruitment of neutrophils to brain metastases and their subsequent promotion of tumor cell growth, it exerts an effect of inhibiting the progression of tumor brain metastasis. By binding to the neutrophil FPR2 receptor, Tenapano can inhibit the interaction of SAA1, which is specifically upregulated and secreted by metastatic tumor cells, with this receptor, thereby preventing the recruitment of neutrophils in the brain and promoting tumor growth, resulting in the therapeutic effect of inhibiting the progression of intracranial lesions. The present invention overcomes the limitations of existing tumor brain metastasis treatment options and solves the problem of poor efficacy of conventional treatment methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Figure 2 shows the differential gene expression between brain metastases and primary tumor cells in the GSE123902 single-cell dataset.

[0026] Figure 2 This is the verification of the efficiency of stable and high expression of SAA1 mRNA levels in five cell lines.

[0027] Figure 3 Twenty-eight days after the A549 cell line expressing normal or high SAA1 was injected into the ventricle of nude mice, in vivo imaging was used to monitor the metastasis of tumor cells.

[0028] Figure 4 These are the H&E staining results of neutrophils in the peripheral blood of tumor patients and paired brain metastases.

[0029] Figure 5 The H&E staining results of HL-60 cells after induction with 1.25% DMSO for different days.

[0030] Figure 6 The effect of Tenapanor on tumor cell survival in an in vitro system with or without co-culture with dHL-60 cells.

[0031] Figure 7 The half-maximal inhibitory concentration of Tenapanor in a cell line that highly expresses SAA1 in an in vitro system co-cultured with dHL-60 cells.

[0032] Figure 8 Schematic diagram of the in vivo experiment testing the inhibitory effect of Tenapanor on brain metastasis of the A549 tumor cell line that overexpresses SAA1. DETAILED DESCRIPTION

[0033] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0034] The phrase "therapeutically effective amount" refers to that amount of an active compound or pharmaceutical agent that will elicit the biological or medical response in a tissue, system, animal, individual or human that is being sought by a researcher, veterinarian, medical doctor or other clinician.

[0035] Example 1 Single Cell Data Analysis

[0036] (1) The published single-cell sequencing data GSE123902 includes 8 primary tumor lesions and 3 brain metastases, with a total cell number of 21,795 and 4,305, respectively. Using the inferCNV algorithm, tumor cells were identified, with 819 primary tumor cells and 851 brain metastases.

[0037] (2) The identified tumor cells were analyzed for differentially expressed genes using the FindMarker function of the R package Seurat. The results are shown in Figure 2. Figure 1 As shown, it can be seen that compared with the primary lesion, brain metastatic tumor cells specifically upregulate SAA1 (serum amyloid protein 1).

[0038] Example 2 Construction of a tumor cell line stably expressing SAA1

[0039] (1) HEK293T cells were transfected according to the instructions of the lipo3000 kit purchased from Invitrogen. Virus suspensions were collected 24, 48, and 72 hours after transfection and added to 8 mg / ml polybrene solution at a ratio of 1:1000. The cells were infected with fluorescently labeled A549 lung adenocarcinoma cell line, H1975 lung adenocarcinoma cell line, MDA-MB-231 breast cancer cell line, MCF7 breast cancer cell line, and A375 melanoma cell line, respectively. Each infection lasted 24 hours. Three days after infection, cells with low infection efficiency were removed using antibiotics, and resistant cells were collected. Grouping: ①A549-Vector (pSin lentiviral vector, purchased from Addgene); ②A549-SAA1; ③H1975-Vector; ④H1975-SAA1; ⑤MDA-MB-231-Vector; ⑥MDA-MB-231-SAA1; ⑦MCF7-Vector; ⑧MCF7-SAA1; ⑨A375-Vector; ⑩A375-SAA1.

[0040] (2) Efficiency detection of stable high-expression cell lines. Discard the supernatant of the 10 cell lines in (1), wash three times with PBS, collect the cells with 1ml Trizol and put them into 1.5ml enzyme-free EP tubes, add 200ul chloroform to each tube, shake for 20s, centrifuge at 12000rpm and 4℃ for 15 minutes, transfer the supernatant to a new 1.5ml enzyme-free EP tube, add pre-cooled isopropanol in a 1:1 ratio, mix thoroughly and let it stand at -40℃ for 1h. Centrifuge at 12000rpm and 4℃ for 15 minutes, discard the supernatant, add 1ml pre-cooled enzyme-free 75% ethanol solution, shake and centrifuge at 12000rpm and 4℃ for 5 minutes, at which time a white precipitate is obvious. Discard the supernatant, wait for the ethanol solution to evaporate, dissolve the RNA in enzyme-free water and quantify it. According to the instructions of the reverse transcription and qPCR reagents of Promega products, the collected RNA was verified by qRT-PCR method, and the results are as follows. Figure 2 As shown, SAA1 was stably and highly expressed in five tumor cell lines compared with Vector cells.

[0041] Example 3 Verification of SAA1 Promoting Brain Metastasis in Vivo

[0042] 6-8 week old female Balb / c mice were purchased from Beijing Weitonglihua Biotechnology Co., Ltd. and injected with 5*10^5 / 100ul / mouse of A549-Vector and A549-SAA1 cell lines by intraventricular injection. The metastasis of tumor cells in the mice was monitored by in vivo imaging once a week. Figure 3 As shown, at the fourth week after injection, the brain metastasis rate of mice injected with tumor cells that highly expressed SAA1 increased compared with the Vector group, and this result indicated that SAA1 promoted brain metastasis in a T cell immunity-independent manner.

[0043] Example 4 Induction of dHL-60 cells

[0044] (1) HL-60 is an acute myeloid lymphoblastic leukemia cell line cultured in RPMI-1640 medium at 37°C and 5% CO2. Under the induction of 1.25% DMSO (dimethyl sulfoxide), it gradually differentiates into a granulocyte line (dHL-60), which is used as an in vitro neutrophil model. In order to better simulate the neutrophil microenvironment of brain metastasis in vitro, we collected brain metastasis tissue and peripheral blood from a tumor patient. Using the CD66b-labeled magnetic bead pull-down method, we collected neutrophils from the patient's brain metastasis and blood, and observed the morphology of neutrophils in different parts by H&E staining. The specific method is as follows: resuspend the cells in 0.5ml PBS, take 2-3 drops of cell suspension and add them to the slide, spread it and dry it; add 95% ethanol solution to fix it for 20 minutes, then dry it with absorbent paper; then add hematoxylin solution to stain for 3 minutes, immerse in water and wash; stain with eosin solution for 10 seconds and then immerse in water and wash; after the slide is dried, observe it under the microscope. Figure 4 As shown in the figure, peripheral blood neutrophils mostly showed mature nuclear lobes, while neutrophils in brain metastases mainly showed immature unlobed nuclear states.

[0045] (2) Using the same method as (1), H&E staining was used to identify the dHL-60 induction conditions suitable for in vitro neutrophil function studies. Figure 5 As shown in the figure, the staining results showed that the state of HL-60 cells after one day of induction with 1.25% DMSO was closest to the neutrophils in brain metastases. Therefore, in subsequent in vitro research experiments, we selected HL-60 cells after one day of induction as the neutrophil model for in vitro brain metastasis research, recorded as dHL-60.

[0046] Example 5 Drug Screening

[0047] (1) Cell killing experiment. The five tumor cell lines mentioned above were divided into four groups: ① Vector; ② SAA1; ③ Vector + dHL-60 (co-culture); ④ SAA1 + dHL-60 (co-culture). Solvent control and 10 drugs from the FDA-approved drug library based on MOE score (affinity prediction) were added to the above four groups (each drug treatment concentration was 1 μM). After 48 hours, the dHL-60 cells were washed with PBS solution, and the fluorescence value in each group of cells was detected using the firefly luciferase activity single fluorescence detection kit. The results are shown as follows. Figure 6 As shown in the results, Tenapanor played an effective killing role in the co-culture system of SAA1 and dHL-60.

[0048] (2) IC50 assay. The five tumor cell lines mentioned above were used to culture the SAA1+dHL-60 (co-culture) system, and 0.1, 1, 10, 100, 1000, and 10,000 nM Tenapanor was added, respectively. After 48 hours, the dHL-60 cells were washed with PBS solution, and the fluorescence value in each group of cells was detected using a firefly luciferase activity single fluorescence assay kit. A curve was drawn to calculate the half-maximal inhibitory dose of Tenapanor. The results are shown in Figure 2. Figure 7 As shown, the IC50 values ​​of Tenapanor in co-culture conditions with dHL-60 cells are: A549 cells: 276.6 nM; H1975 cells: 49.49 nM; MDA-MB-231 cells: 326.3 nM; MCF7 cells: 61.72 nM; and A375 cells: 192.9 nM. This indicates that Tenapanor has a significant inhibitory effect on these five cell lines.

[0049] Example 6 Verification of the Inhibitory Effect of Tenapanor in Vivo

[0050] Six- to eight-week-old female nude mice were purchased from Beijing Weitonglihua Biotechnology Co., Ltd. and injected with 5*10^5 / 100ul / mouse of A549-Vector and A549-SAA1 cell lines via intraventricular injection. Two weeks after tumor cell injection, the A549-SAA1 group was treated with Tenapanor. Tenapanor was purchased from MCE and the solvent used for in vivo experiments was: 10% DMSO, 40% PEG300, 5% Tween-80, and 45% saline. The treatment dose was 200μg / mouse, injected intraperitoneally twice a week, and in vivo imaging was used to monitor tumor cell metastasis in mice once a week. The results are shown in Figure 2. Figure 8 As shown, at four weeks after tumor cell injection, the brain metastasis rate of mice in the Tenapanor-treated group was significantly reduced compared with the group injected with high-expressing SAA1 tumor cells.

[0051] The foregoing examples are merely illustrative, serving to illustrate some of the features of the method of the present invention. The appended claims are intended to claim the widest possible scope that can be envisioned, and the embodiments presented herein are demonstrated by the applicant's actual experimental results. Therefore, the applicant intends that the appended claims are not limited by the selection of examples illustrating the features of the present invention. Some numerical ranges used in the claims also include subranges therein, and variations in these ranges should also be interpreted as being covered by the appended claims where possible.

Claims

1. The use of Tenapano in the preparation of a drug for inhibiting brain metastasis of tumors, characterized in that: The tumor brain metastasis refers to brain metastasis of lung adenocarcinoma, breast cancer or melanoma; The structural formula of Tenapano is shown in the following formula (I): （I）。 2. The application according to claim 1, characterized in that Tenapano inhibits brain metastasis by preventing the recruitment and growth of neutrophils in brain metastases.

3. The application according to claim 1, characterized in that The described tenapanol inhibits tumor brain metastasis by preventing the secretory protein SAA1 from binding to the neutrophil membrane surface receptor.

4. The application according to claim 3, characterized in that The neutrophil membrane surface receptor is formyl peptide receptor 2.

5. The application according to claim 1, characterized in that: The tenapanol inhibits tumor brain metastasis by binding to neutrophil membrane surface receptors.

6. The application according to claim 1, characterized in that: The cancer cells of the tumor are one or more of A549 lung adenocarcinoma cells, H1975 lung adenocarcinoma cells, MDA-MB-231 breast cancer cells, MCF7 breast cancer cells, and A375 melanoma cells.

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