Application of trinapalnol in preparation of medicine for inhibiting tumor brain metastasis

Abstract

The invention discloses an application of trinaparnol in preparation of a medicine for inhibiting tumor brain metastasis. According to the application disclosed by the invention, neutrophile granulocytes for collecting and promoting tumor growth in brain metastatic lesions are targeted, an inhibitor Turbalo for a chemotactic receptor FPR2 of the neutrophile granulocytes is screened out, and the effect of collecting the neutrophile granulocytes to the brain and subsequently promoting the clonal growth of tumor cells is inhibited by inhibiting the combination of the receptor and SAA1 specifically up-regulated by metastatic tumor cells. The curative effect of inhibiting the progress 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 application of Tenapano in the preparation of drugs for inhibiting tumor brain metastasis. Background Art

[0002] Brain metastasis occurs in about 9-50% of patients with malignant tumors during the course of the disease. It is a common neurological complication of malignant tumors and is also one of the main reasons for the decline in patient survival and an important treatment challenge. The vast majority of brain metastases are caused by lung cancer, breast cancer, and melanoma, with incidences of 40-50%, 15-30%, and 5-20%, respectively. Taking lung cancer as an example, as the most common source of brain metastasis, 25-30% of patients with lung adenocarcinoma have brain metastases at diagnosis, 50% of patients will eventually develop brain metastases, and the median time from the discovery of lung tumors to the diagnosis of brain metastases is only 11 months, which is the shortest among all solid tumors. Due to the increased intracranial pressure and specific neurological symptoms caused by brain metastasis, the patient's prognosis is generally poor, with a median survival of only 4-7 months. At present, the treatment of brain metastasis of tumors includes comprehensive treatment of brain metastases (surgery and radiotherapy) on the basis of systemic treatment (chemotherapy, molecular targeted therapy, and immunotherapy). However, although the above treatment regimens can prolong the survival of patients, they still have the disadvantages of poor 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 serious. These problems urgently need effective solutions.

[0003] Studies have shown that neutrophils, which do not exist in the normal brain, increase significantly in tumor brain metastases and may promote tumor brain metastasis by constructing pre-metastatic niches, enhancing tumor cell adhesion to metastatic organs, selecting and expanding tumor subpopulations with high tumorigenic potential, inhibiting anti-tumor immunity, and increasing colonization. Neutrophils in metastatic lesions have been reported to be associated with poor survival prognosis in patients, and the neutrophil / lymphocyte ratio (NLR) in peripheral blood has gradually become a diagnostic indicator for brain metastasis in tumor patients. Targeted elimination of neutrophils can effectively inhibit tumor brain metastasis, but this treatment method has only been partially verified in animals and has not yet been clinically applied. More importantly, the existing means of targeting neutrophils generally use antibodies to deplete neutrophils, and due to the presence of the blood-brain barrier, antibodies usually have difficulty reaching brain metastases to exert therapeutic effects to control intracranial lesions. Therefore, exploring and discovering targeted compounds that can specifically target neutrophils in brain metastases will likely develop more effective and specific drugs to inhibit tumor brain metastasis, which is also a key difficulty in the current research and treatment of tumor brain metastasis. Summary of the invention

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

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

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

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

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

[0011]

[0012] The inventors found through research that compared with the primary tumor and normal brain tissue, the number of neutrophils in brain metastases increased significantly, and played an important role in the development of brain metastatic tumor cells. FPR2 is a neutrophil membrane surface receptor. The results of single-cell data analysis show that it can bind to the chemotactic protein SAA1, which is specifically upregulated in tumor cells in brain metastases, to recruit neutrophils to the brain and promote tumor cell colonization and growth. The present invention conducts drug screening by molecular docking with the extracellular domain of FPR2 and comparing the binding ability with the known FPR2 small molecule inhibitors WRW4 and WKYMVm, and finally discovered the drug Tenapano, which can effectively inhibit the binding of SAA1 to neutrophil FPR2 in vitro, thereby preventing neutrophil recruitment and promoting tumor growth. In the 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 tenapanol inhibits tumor brain metastasis by preventing the recruitment and growth of neutrophils in brain metastases.

[0015] Preferably, the tenapanol 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 claims the use of tenapano 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 Tenapano.

[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 Tenapano to a subject.

[0022] In the present invention, the drug can be administered in the following forms: oral administration 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 recruiting and promoting tumor cell growth in brain metastases, and screens out Tenapano, an inhibitor of the neutrophil chemotactic receptor FPR2, to inhibit the recruitment of neutrophils to brain metastases and the subsequent promotion of tumor cell growth, thereby inhibiting the progression of tumor brain metastasis. Tenapano can inhibit the interaction between SAA1 specifically upregulated and secreted by metastatic tumor cells and the receptor by binding to the neutrophil FPR2 receptor, thereby preventing the recruitment of neutrophils in the brain and promoting tumor growth, producing the therapeutic effect of inhibiting the progression of intracranial lesions. The present invention overcomes the limitations of existing treatment options for brain metastases of tumors and solves the problem of poor efficacy of conventional treatment methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The differential gene expression of brain metastatic lesions vs. primary lesion tumor cells in the GSE123902 single-cell dataset.

[0026] Figure 2 This is to verify 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 in vivo.

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

[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 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 to detect the inhibitory effect of Tenapanor on brain metastasis of A549 tumor cell line with high expression of SAA1. DETAILED DESCRIPTION

[0033] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. 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) In the published single-cell sequencing data GSE123902, the samples included 8 primary tumor lesions and 3 brain metastases, with a total number of cells of 21,795 and 4,305, respectively. Tumor cells were identified using the inferCNV algorithm, 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. Figure 1 As shown, it can be seen that compared with the primary lesions, brain metastatic tumor cells specifically upregulate SAA1 (serum amyloid protein 1).

[0038] Example 2 Construction of a tumor cell line that stably and highly expresses SAA1

[0039] (1) HEK293T cells were transfected according to the instructions of the lipo3000 kit purchased from Invitrogen Biotechnology Company. The virus solution was collected at 24, 48, and 72 hours after transfection, and 8 mg / ml polybrene solution was added at a ratio of 1:1000 to infect 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 was for 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 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, then dissolve the RNA in enzyme-free water and quantify it. According to the instructions for reverse transcription and qPCR reagents from Promega, the collected RNA was verified by qRT-PCR. 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 Weitong Lihua Biotechnology Co., Ltd. A549-Vector and A549-SAA1 cell lines were injected into the mice at 5*10^5 / 100ul / mouse 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% CO 2. Under the induction of 1.25% DMSO (dimethyl sulfoxide), it will gradually differentiate into the granulocyte lineage (dHL-60), which is used as an in vitro neutrophil model. In order to better simulate the neutrophils in the brain metastasis microenvironment 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: resuspend the cells with 0.5ml PBS, take 2-3 drops of cell suspension and add them to the slide, push it away and dry it; add 95% ethanol solution to fix it for 20 minutes, and then dry it with absorbent paper; then add hematoxylin stain for 3 minutes, and wash it with water; stain with eosin stain for 10 seconds and then wash it with water; observe it under a microscope after the slide is dry. The results are as follows Figure 4 As shown, 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 dHL-60 induction conditions suitable for studying the in vitro function of neutrophils. Figure 5 As shown, 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 chose HL-60 cells induced for one day 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 above five tumor cell lines were used and 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 scoring (affinity prediction) were added to the above four groups of systems (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 below. Figure 6 As shown, Tenapanor played an effective killing role in the co-culture system of SAA1 and dHL-60.

[0048] (2) IC50 test. The five tumor cell lines mentioned above were used in the SAA1+dHL-60 (co-culture) system, and 0.1, 1, 10, 100, 1000, and 10000 nM Tenapanor were 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 detection kit, and a curve was drawn to calculate the half inhibitory dose of Tenapanor. The results are shown in Figure 2. Figure 7 As shown, the IC50 of Tenapanor under the co-culture conditions of five cell lines and dHL-60 are A549 cells: 276.6nM; H1975 cells: 49.49nM; MDA-MB-231 cells: 326.3nM; MCF7 cells: 61.72nM; A375 cells: 192.9nM. It can be seen that Tenapanor has a good inhibitory effect on the five cell lines.

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

[0050] 6-8 week old female nude mice were purchased from Beijing Weitong Lihua Biological Co., Ltd. A549-Vector and A549-SAA1 cell lines were injected by ventricular injection at 5*10^5 / 100ul / mouse. Two weeks after the injection of tumor cells, the A549-SAA1 group was treated with Tenapanor. Tenapanor was purchased from MCE, and the solvent for in vivo experiments was: 10% DMSO, 40% PEG300, 5% Tween-80, 45% saline. The treatment dose was 200μg / mouse, intraperitoneally injected twice a week, and the metastasis of tumor cells in mice was monitored by live imaging once a week. The results are shown in the figure. Figure 8 As shown, at the fourth week after tumor cell injection, the brain metastasis rate of mice in the Tenapanor treatment group was significantly reduced compared with the group injected with high-expressing SAA1 tumor cells.

[0051] The foregoing examples are merely illustrative and are used to explain some features of the method of the present invention. The appended claims are intended to require the widest possible range that can be imagined, and the embodiments presented herein are demonstrated by the applicant's actual test results. Therefore, the applicant's intention is that the appended claims are not limited by the selection of examples that illustrate the features of the present invention. Some numerical ranges used in the claims also include sub-ranges therein, and changes in these ranges should also be interpreted as being covered by the appended claims where possible.

Claims

1. Application of Tenapano in the preparation of drugs for inhibiting tumor brain metastasis.

2. The application according to claim 1, characterized in that: The tumor brain metastasis refers to brain metastasis of lung adenocarcinoma, breast cancer or melanoma.

3. The application according to claim 1, characterized in that: The tenapanol inhibits tumor brain metastasis by preventing the recruitment and growth of neutrophils in brain metastases.

4. The use according to claim 1, characterized in that: The described tenapano inhibits tumor brain metastasis by preventing the secretory protein SAA1 from binding to the neutrophil membrane surface receptor.

5. The use according to claim 4, characterized in that: The neutrophil membrane surface receptor is formyl peptide receptor 2.

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

7. The use 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.

8. The use according to claim 1, characterized in that: The structural formula of the tenapano is shown in the following formula (I): 。 9. Use of tenapano in the preparation of inhibitors of formyl peptide receptor 2.

10. A drug, characterized in that Contains a therapeutically effective amount of tenapanol.

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