Application of retinoic acid compound in preparation of medicine for treating and / or preventing glioblastoma

Abstract

The invention relates to application of a retinoic acid compound in preparation of a medicine for treating and / or preventing glioblastoma. Compared with the prior art, the retinoic acid compound (1uM, 5uM) group has the advantages that the tumor inhibition rate of a glioblastoma organ is higher than that of an apatinib (1uM, 5uM) group, and is higher than that of an apatinib (1uM, 5uM) group. The subcutaneous tumor inhibition rate of glioblastoma cells of a retinoic acid compound (0.5 mg / kg and 5mg / kg) group is higher than that of an apatinib (32.5 mg / kg) group, and is higher than that of the apatinib (32.5 mg / kg) group in tumor pathological blood vessel inhibition rate. Therefore, the medicine (retinoic acid compound) for treating and (or) preventing the glioblastoma has a remarkable treatment and (or) preventing effect on the glioblastoma.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to application of a retinoic acid compound in preparing a medicine for treating and / or preventing glioblastoma. Background Art

[0002] Glioblastoma Multiforme (GBM) is one of the most common and most malignant primary brain tumors in the central nervous system, characterized by extremely high invasiveness, rapid growth and extremely poor prognosis. Currently, standard treatment includes surgical resection, radiotherapy and chemotherapy, but despite multimodal comprehensive treatment, the median survival of patients is still less than 15 months. The high recurrence rate and drug resistance of glioblastoma are the main challenges facing treatment.

[0003] Pathological angiogenesis plays an important role in the occurrence and development of glioblastoma. Overexpression of angiogenic factors and their related signaling pathways not only promotes the formation of pathological angiogenesis, but also provides support for the proliferation and invasion of tumor cells.

[0004] Currently, among the therapeutic drugs for glioblastoma, targeted drugs that inhibit VEGF, such as bevacizumab and apatinib, have been shown to have a certain inhibitory effect on pathological blood vessels, but there are still significant limitations in clinical applications, including limited efficacy, drug resistance, and insufficient direct inhibition of tumor cells. Therefore, the development of dual-targeted drugs that simultaneously target tumor cells and pathological blood vessels is a key direction to improve the treatment effect of glioblastoma.

[0005] Retinoic acid is a class of compounds derived from vitamin A metabolism, mainly including all-trans retinoic acid (ATRA) and 9-cis retinoic acid (9-Cis Retinoic Acid). Retinoic acid drugs regulate gene transcription by binding to retinoic acid receptors (RAR) and retinoic acid X receptors (RXR), thus playing an important role in cell differentiation, proliferation and apoptosis. Due to its ability to regulate multiple tumor-related signaling pathways, retinoids have shown potential application value in the treatment of various malignant tumors.

[0006] However, existing retinoid drugs for treating glioblastoma also lack effective compounds that can target both tumor cells and pathological blood vessels simultaneously.

[0007] Chinese patent CN115974860B discloses that retinoic acid compounds have good inhibitory activity on tumor stem cells, providing a new treatment method for the treatment of patients with refractory, metastatic and drug-resistant tumors. However, GBM, as the most common primary malignant brain tumor, has stronger invasiveness and higher treatment difficulty compared with other common solid tumors. The tumor microenvironment of this cancer is highly complex, not only showing significant tumor cell heterogeneity, but also accompanied by abnormal pathological angiogenesis. Although the blood-brain barrier (BBB) ​​in the core area of ​​GBM tumors is destroyed to a certain extent, allowing some drugs to enter the tumor tissue, the BBB in the tumor infiltration area is still relatively intact, limiting the delivery of drugs to this area. At the same time, the highly active tumor stem cell population in GBM and its microenvironment adaptability further enhance the drug resistance of the tumor, making it difficult for existing therapies to effectively control the progression of the disease. Therefore, the development of new drugs that can simultaneously inhibit GBM cell proliferation and pathological angiogenesis has important clinical application value. Summary of the invention

[0008] In view of the fact that there is a lack of compounds that have inhibitory effects on both tumor cells and pathological blood vessels of glioblastoma in the prior art, the present invention provides a use of a retinoic acid compound in the preparation of a drug for treating and / or preventing glioblastoma.

[0009] The purpose of the present invention can be achieved by the following technical solutions:

[0010] The present invention provides an application of a retinoic acid compound in preparing a medicine for treating and / or preventing glioblastoma, wherein the retinoic acid compound is used as an active ingredient of the medicine.

[0011] In one embodiment of the present invention, a retinoid compound is used in the preparation of a drug for treating glioblastoma by dually inhibiting glioblastoma cell proliferation and pathological angiogenesis.

[0012] In one embodiment of the present invention, a retinoic acid compound is used in the preparation of a drug for treating glioblastoma by inhibiting the Hedgehog pathway to achieve dual inhibition of glioblastoma cell proliferation and pathological angiogenesis.

[0013] In one embodiment of the present invention, the retinoic acid compound includes a compound represented by Formula I, an enantiomer, a diastereomer or a pharmaceutically acceptable salt thereof:

[0014]

[0015] Wherein, X is O, S, S=O, or O=S=O;

[0016] R1, R2, and R3 are independently selected from hydrogen, C1-C6 alkyl, C2-C6 alkenyl, halogen-substituted C1-C6 alkyl, C1-C6 alkoxy, C1-C6 acyl, C6-C 10 The aryl group or the heteroatom is an oxygen, sulfur or nitrogen atom, and the number of heteroatoms is 1 to 2 C3-C6 heteroaryl groups.

[0017] In one embodiment of the present invention, the C1-C6 alkyl group includes a linear or branched alkyl group selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl or hexyl. Preferably, the C1-C6 alkyl group is methyl or ethyl.

[0018] In one embodiment of the present invention, the C2-C6 alkenyl group is a straight chain or branched alkenyl group containing at least one carbon-carbon unsaturated double bond, such as vinyl, propenyl, 1-pentenyl. Preferably, the C2-C6 alkenyl group is vinyl or propenyl.

[0019] In one embodiment of the present invention, the halogen-substituted C1-C6 alkyl group refers to a C1-C6 alkyl group in which at least one hydrogen atom is replaced by a halogen atom, and the halogen atom includes fluorine, chlorine, bromine, and iodine. Preferably, the halogen-substituted C1-C6 alkyl group is a trifluoromethyl group.

[0020] In one embodiment of the present invention, the C1-C6 alkoxy group is methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, pentyloxy or hexyloxy.

[0021] In one embodiment of the present invention, the C1-C6 acyl group is a group obtained by removing the hydroxyl group from a C1-C6 monocarboxylic acid. Preferably, the C1-C6 acyl group is an acetyl group or a formyl group.

[0022] In one embodiment of the present invention, the C6-C 10 Aryl is a monovalent aromatic carbocyclic ring system having at least one aromatic ring or a polycondensed ring in which at least one ring is an aromatic ring, such as phenyl, naphthyl, biphenyl or indanyl. 10 Aryl is phenyl.

[0023] In one embodiment of the present invention, the C3-C6 heteroaryl is an aromatic group containing heteroatoms, including an aromatic 5-6-membered monocyclic ring containing 1, 2 or 3 atoms selected from nitrogen, oxygen and / or sulfur, such as furanyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, thienyl, oxazolyl, diazolyl, imidazolyl, pyrrolyl, pyrazolyl, triazolyl, thiazolyl, isothiazolyl. Preferably, the C3-C6 heteroaryl is pyridyl or pyrimidinyl.

[0024] In one embodiment of the present invention, the retinoic acid compound is a compound represented by formula (II), an enantiomer, a diastereomer or a pharmaceutically acceptable salt thereof:

[0025]

[0026] In the formula, X is S or S=O.

[0027] In one embodiment of the present invention, the retinoid compound is in the form of a pharmaceutical composition comprising the retinoid compound, and the retinoid compound serves as the sole active ingredient of the pharmaceutical composition.

[0028] In one embodiment of the present invention, the medicament further comprises a pharmaceutically acceptable carrier.

[0029] In one embodiment of the present invention, the pharmaceutically acceptable carrier is a pharmaceutically acceptable pharmaceutical excipient.

[0030] In one embodiment of the present invention, the retinoid compound is in the form of a kit containing the retinoid compound, and the kit also includes a drug for treating a disease associated with glioblastoma.

[0031] In one embodiment of the present invention, the drug is an injection.

[0032] The present invention proposes the use of retinoic acid compounds in the preparation of drugs for treating and / or preventing glioblastoma. The retinoic acid compounds show significant anti-tumor effects in both GBM in vitro organoid models and mouse subcutaneous tumor models. The retinoic acid compounds can not only significantly inhibit the proliferation of glioblastoma cells, but also have excellent inhibitory effects on pathological angiogenesis, and can reduce the formation of tumor pathological blood vessels, thereby achieving a dual inhibitory effect.

[0033] Compared with the prior art, the compounds of the present invention have obvious advantages in terms of efficacy, selectivity and safety, and provide a new strategy for the treatment of glioblastoma.

[0034] The compounds of the present invention exhibit significant anti-tumor activity and can effectively inhibit the proliferation, growth, metastasis and recurrence of glioblastoma. In addition, the compounds of the present invention also have excellent inhibitory effects on pathological blood vessels of glioblastoma, providing a novel and efficient solution for the treatment of glioblastoma.

[0035] Experiments have shown that the glioblastoma organoid tumor inhibition rate of the retinoic acid compound (1uM, 5uM) group of the present invention is higher than that of the apatinib (1uM, 5uM) group, and is higher than its intratumor pathological blood vessel inhibition rate. The subcutaneous tumor inhibition rate of glioblastoma cells in the retinoic acid compound (0.5mg / kg, 5mg / kg) group of the present invention is higher than that of the apatinib (32.5mg / kg) group, and is higher than its intratumor pathological blood vessel inhibition rate. It shows that the drug (retinoic acid compound) for treating and (or) preventing glioblastoma of the present invention has a significant treatment and (or) prevention effect on glioblastoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Other features, objects and advantages of the present invention will become more apparent from a reading of the detailed description of non-limiting embodiments made with reference to the following accompanying drawings:

[0037] Figure 1 Schematic diagram of ZSH-512 inhibition of glioblastoma organoids.

[0038] Figure 2 Schematic diagram of ZSH-512 inhibiting pathological vascularization in glioblastoma organoids.

[0039] Figure 3 Schematic diagram of ZSH-512 inhibiting endothelial cell vascular lumen formation.

[0040] Figure 4 Schematic diagram of ZSH-512 inhibiting endothelial cell migration.

[0041] Figure 5 Tumor volume and weight of subcutaneous tumors formed in ZSH-512 mice injected with 500,000 glioblastoma cells.

[0042] Figure 6 Schematic diagram of ZSH-512 inhibiting intratumor angiogenesis and cell proliferation. DETAILED DESCRIPTION

[0043] In order to make the purpose, technical solution and advantages of the embodiment of the present invention clearer, the technical solution of the embodiment of the present invention will be clearly and completely described below in conjunction with the accompanying drawings of the embodiment of the present invention. Obviously, the described embodiment is a part of the embodiment of the present invention, not all of the embodiments. Based on the described embodiment of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0044] Unless otherwise defined, the technical or scientific terms used herein shall have the common meanings understood by persons with ordinary skills in the field to which the present invention belongs. The words "first", "second" and similar words used in the patent application specification and claims of the present invention do not indicate any order, quantity or importance, but are only used to distinguish different components. Similarly, words such as "one" or "a" do not indicate a quantity limitation, but indicate the existence of at least one.

[0045] In the following examples, the drug ZSH-512 used has the following structural formula:

[0046]

[0047] The synthesis method thereof is disclosed in patent CN115974860B, and reference may be made to the synthesis method of compound ZSH-512 described in Example 2 of patent CN115974860B.

[0048] In the examples of the present application, the synthesis method of the compound ZSH-512 used is as follows:

[0049]

[0050] Specific preparation steps: Add the prepared ZSH-510 (155 mg, 0.42 mmol) to a flask, add 5 mL of dry dichloromethane, cool to 0 ° C in an ice-water bath, add m-CPBA (86 mg, 0.5 mmol), react in an ice-water bath for 1 hour, then move to room temperature for 2 hours, and track by TLC. After the reaction is completed, quench with sodium thiosulfate solution, dilute with ethyl acetate, wash with saturated sodium bicarbonate, wash with saturated sodium chloride, dry the organic phase with anhydrous sodium sulfate, filter, spin dry, and flash column chromatography (DCM: MeOH = 20: 1) to obtain the product ZSH-512 (105 mg, yield 65%). Among them, the preparation of ZSH-510 can refer to the synthesis method of compound ZSH-510 recorded in Example 1 of patent CN115974860B.

[0051] The H NMR spectrum data of the product ZSH-512 obtained in this example are as follows:

[0052] 1H NMR (500MHz, DMSO-d6) δ8.75(s,1H),8.24(s,1H),7.85(s,1H),7.74(d,J=8.0Hz,2H),7.62(d,J=8.0Hz,1H),4.32(q,J=7.1Hz,2H) ,3.33-3.37(m,1H),3.03-3.08(m,1H),2.33-2.28(m,1H),1.81-1.86(m,1H),1.42(s,3H),1.33(t,J=7.1Hz,3H),1.31(s,3H).13C NMR(126MHz,DMSO-d6)δ165.53,162.32,159.34,154.19,145.98,140.96,132.27,130.66,123.84,103.86, 90.02,85.95,61.45,42.69,34.71,31.17,30.84,29.34,14.50.ESI(+)-MS:384.1[M+1]+.HRMS(ESI):calcd for C20H22N3O3S[M+H]+,found:384.1372.

[0053] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0054] Example 1

[0055] Experimental study on the inhibition of glioblastoma organoid proliferation by ZSH-512

[0056] refer to Figure 1 , immerse the glioblastoma tissue in tissue washing buffer to keep it moist and remove contaminants and non-cellular debris. After repeated multiple rinses and thorough cleaning, place it in a culture dish for mechanical shearing. The obtained microtissue blocks are resuspended in culture medium and cultured. After the primary organoids are formed, they are processed into single cells by mechanical dissociation or digestion and subcultured to construct a glioblastoma organoid model to simulate the in vivo growth environment of the tumor. After the organoids are stably formed, they are divided into a control group (0.1% DMSO), a ZSH-512 treatment group (1μM and 5μM), and an Apatinib group (1μM and 5μM). On the first, fifth, and 15th days of organoid culture, ZSH-512 and apatinib (Selleck) were added for treatment, respectively, and then observed and photographed by a Leica inverted microscope (DMI-6000B), and the relative volume of each group of organoid spheres was calculated.

[0057] The relative volume of glioblastoma organoids = the volume of organoids in the experimental group / the volume of organoids in the control group.

[0058] The experimental results showed that ZSH-512 could significantly inhibit the growth of organoids through comparison of organoid volume at the endpoint of drug administration, and the inhibition rate increased with increasing concentration.

[0059] At the same time, sections (4 μm thick) were prepared by paraffin embedding. After sectioning, the organoids were stained using the H&E staining method, and the cell morphology, proliferation and tissue structure changes were observed under a microscope. H&E staining can intuitively display cell density, cell arrangement and cell apoptosis. The experimental results showed that in the control group, the organoids proliferated actively, the cell density was high, and the morphology was complete. In the ZSH-512-treated group, the volume of the organoids was significantly reduced, the cell density was significantly reduced, the cells were irregularly arranged, and cell apoptosis occurred in some areas. By quantitatively analyzing the volume and cell density of the organoids, it was found that ZSH-512 could significantly inhibit the proliferation of glioblastoma organoids, and the inhibitory effect was dose-dependent. This result verifies the inhibitory effect of ZSH-512 on the proliferation of glioblastoma cells. For related data, see Figure 1 .

[0060] Example 2

[0061] Experimental study on the inhibition of pathological blood vessels in glioblastoma by ZSH-512

[0062] The organoids treated in Example 1 above were fixed and sliced, and a staining machine (Leica, bond III / bond MAX) was used to operate dewaxing, antigen repair and staining steps. The antigen repair conditions were ER1 / ER2 antigen repair solution (Leica, AR9961-CN, AR9640-CN) at 100 degrees for 20 minutes. Then incubate with hydrogen peroxide for 10 minutes and block with blocking solution for 10 minutes. After blocking, wash with TBST 3-5 times. Antibody diluent / blocking solution (Yuanxi Biology) dilutes the endothelial cell marker CD31 (CST: 77699S) antibody according to the concentration and incubate at room temperature for 45 minutes. Wash with TBST 3-5 times. Subsequently, the Leica DS9800 staining system secondary antibody (Leica goat anti-rabbit poly-HRP / Leica goat anti-rabbit poly-HRP) was incubated at room temperature for 60 minutes. Wash with TBST 3-5 times. . Then use DAB colorimetric method (Leica) and hematoxylin counterstaining nuclei for staining. After dehydration with gradient ethanol, xylene transparency, and neutral gum sealing, the immunohistochemically stained tissue section images were taken using a Leica DMI-6000B inverted microscope, and three high-power fields of view were randomly selected for analysis to ensure the representativeness of the data. The IHC section images taken by the microscope were opened in the ImageJ software, and the "Color Deconvolution" tool was used to separate the DAB positive signal (CD31 brown staining). The "Threshold" tool was selected to optimize the contrast between the background and the target area to make the positive signal clearly visible. The "Analyze Particles" function was used to calculate the proportion of the positive signal area to the entire field of view (i.e., the proportion of positive cells), and the data was recorded to calculate the IHC-score, which is defined as the proportion of CD31 positive cells in each field of view, ranging from 0 to 1. This result shows that ZSH-512 can effectively inhibit pathological angiogenesis. For related data, see Figure 2 .

[0063] Example 3

[0064] ZSH-512 inhibits endothelial cell vascular lumen formation experiment

[0065] The supernatant of each group of glioblastoma organoids constructed and cultured in Example 1 was collected into a sterile 15 ml centrifuge tube (Corning) as a processing solution for the experiment. The collected supernatant was filtered to remove residual cell impurities to ensure its purity, and then used for the next experiment.

[0066] Prepare an endothelial cell line (human umbilical vein endothelial cells HUVECs) and culture them to the logarithmic growth phase using ECM medium (containing basal medium, 5% fetal bovine serum, 1% endothelial cell growth supplement ECGS and 1% penicillin / streptomycin solution). After counting the endothelial cells, inoculate them in a 24-well plate with approximately 2×104 cells per well and culture them to 80% confluence. At this point, prepare to start the tube formation experiment.

[0067] The organoid supernatants of the control group (0.1% DMSO), ZSH-512 treatment group (1 μM and 5 μM) and Apatinib (1 μM and 5 μM) were added to endothelial cells and cultured for 3-6 hours, and the lumen formation of endothelial cells under different organoid supernatant incubation conditions was observed.

[0068] The tube formation experiment uses a Matrigel matrix layer to simulate the microenvironment of cells in vivo. The specific steps are: preheat Matrigel matrix gel (Corning) to 37°C and evenly apply it on the bottom of a 24-well plate, add 125μL Matrigel matrix gel to each well, and cover it with culture medium. After the Matrigel matrix gel solidifies, add the treated endothelial cell suspension to the Matrigel matrix gel cover layer and continue to culture for 3-6 hours. Then, observe the tube formation of the cells under an inverted microscope. The tube structure formed by endothelial cells in Matrigel matrix gel can be clearly seen under a microscope.

[0069] After the experiment, ImageJ software was used to quantitatively analyze the lumen structures formed by endothelial cells. Specific analysis indicators included the number of lumens, average lumen length and morphological characteristics. Three fields of view were randomly selected for image acquisition, and the "Analyze Skeleton" plug-in was used in ImageJ for vascular network analysis. The inhibitory effects of ZSH-512 and apatinib on endothelial cell lumen formation were evaluated by measuring the branch structure in each well and the average length of each lumen. The experimental results showed that in the control group, endothelial cells formed a complete vascular network in Matrigel, with obvious and evenly distributed lumen structures. In the ZSH-512-treated group, the number of lumens formed by endothelial cells was significantly reduced, the lumen structure was incomplete, and the lumen length was short, showing a significant inhibitory effect of ZSH-512 on endothelial cell angiogenesis. With the increase of drug concentration, the inhibitory effect became more obvious and dose-dependent. For related data, see Figure 3 .

[0070] Example 4

[0071] Experiment on the inhibition of endothelial cell transwell migration by ZSH-512

[0072] This example evaluates the inhibitory effect of ZSH-512 on endothelial cell migration, and the Transwell migration assay is used for detection. The culture supernatant of each group of organoids treated in Example 1 was collected into a 15 ml sterile centrifuge tube (Corning), and the cell residues were filtered to obtain a pure organoid supernatant for subsequent experiments.

[0073] HUVECs were first cultured under conventional culture conditions (containing endothelial cell growth supplement ECGS) until 80% confluence, and then transferred to a 24-well Transwell chamber (Corning). Endothelial cell suspension (2×10 cells / well) was added to the upper chamber of the Transwell chamber. 4 800ul of organoid culture supernatant was added to the lower chamber as a chemotactic factor, and the Transwell chamber was placed in an incubator at 37°C and 5% CO2 for 24 hours to promote endothelial cell migration. After the culture, the upper non-migrating cells in the chamber were washed with PBS buffer, the endothelial cells on the membrane were fixed with 4% paraformaldehyde, and Giemsa staining was used for cell staining. After staining, the migrating cells in the lower chamber were observed under a microscope and three fields of view were randomly selected to photograph.

[0074] The image analysis software ImageJ was used to quantitatively analyze the number of endothelial cells that passed through the filter membrane in the Transwell migration experiment. The "Threshold" tool was used to optimize the contrast, and then the "Analyze Particles" function was used to automatically identify and count the number of cells that passed through the filter membrane. The experimental results showed that compared with the control group, the number of endothelial cells that passed through the filter membrane in the ZSH-512-treated group was significantly reduced, indicating that ZSH-512 can inhibit the migration ability of endothelial cells. This result further supports the mechanism of action of ZSH-512 in intervening in the glioblastoma microenvironment by inhibiting pathological angiogenesis. Related data can be found in Figure 4 .

[0075] Example 5

[0076] Inhibitory effect of ZSH-512 on tumorigenicity of glioblastoma

[0077] In Example 5, the inhibitory effects of ZSH-512 and apatinib on the tumorigenicity of glioblastoma cells were evaluated, and the experiment was conducted using a mouse subcutaneous tumor model.

[0078] First, cells of the glioblastoma cell line U87 were collected during the logarithmic growth phase, digested with a solution containing 0.25% trypsin, and resuspended in PBS to adjust the cell concentration to 1×10 7 / mL. Subsequently, 50 μL of the cell suspension (containing about 5×105 cells) was taken and inoculated under the armpit of the nude mouse by subcutaneous injection.

[0079] After inoculation, the mice were randomly divided into four groups: control group (no drug), ZSH-512 low concentration group (0.5 mg / kg / d), ZSH-512 high concentration group (5 mg / kg / d) and apatinib group (32.5 mg / kg / d). The drug was given by intraperitoneal injection once a day for 2 weeks. During the experiment, the weight of the mice was measured regularly with a precision scale and the size of the tumor was measured with a vernier caliper.

[0080] Tumor growth was monitored by measuring the long diameter (L) and short diameter (W) of the tumor. The tumor volume was recorded regularly (once every 2 days) and the tumor volume (V) was calculated using the following formula: V = (L × W 2 ) / 2.

[0081] At the end of the experiment, the mice were anesthetized and the tumors were removed for observation, weighing, and volume measurement. Comparison of tumor volume and weight showed that the tumor volume and weight of the ZSH-512-treated group were significantly smaller than those of the control group, and the inhibitory effect of ZSH-512 was dose-dependent. Figure 5 .

[0082] Example 6

[0083] Inhibitory effects of ZSH-512 on angiogenesis and cell proliferation in tumors

[0084] In Example 6, based on Example 5, a mouse subcutaneous tumor model was used to obtain samples for histological analysis. The removed mouse subcutaneous tumor tissue was immediately fixed in a 10% neutral formalin solution for 24 hours. Subsequently, the fixed tumor tissue was subjected to gradient dehydration and transparent treatment, and paraffin-embedded to prepare continuous sections with a thickness of 4 μm.

[0085] First, the sections were used for the evaluation of angiogenesis (CD31 staining), and the endothelial cells were labeled with anti-CD31 primary antibody (CST: 77699S), followed by detection with HRP-conjugated secondary antibody (Leica goat anti-rabbit poly-HRP / Leica goat anti-rabbit poly-HRP), and the vascular structure was visualized by DAB staining (Leica). The intratumoral vascular distribution was observed under a microscope, and the high vascular density area (hot spot) of the tumor was selected, and 5 high-power fields of view were randomly taken. The image analysis software ImageJ was used to quantitatively analyze the vascular density (the number of blood vessels per unit area) in each field of view. Secondly, the sections were evaluated for tumor cell proliferation (Ki67 staining), and the proliferating cell nuclei were labeled with anti-Ki67 primary antibody (Bai Ling: BX50040-C3), and the subsequent staining steps were similar to CD31 staining. Multiple random fields of view of tumor tissue sections were photographed under a microscope, and the proportion of Ki67-positive cells (the percentage of positive cells to the total number of cells) was counted using ImageJ to evaluate the inhibitory effect of ZSH-512 on tumor cell proliferation. Ki67 immunohistochemical staining showed that the proliferation of tumor cells in the ZSH-512-treated group was significantly reduced, and the number of Ki67-positive cells was significantly reduced, further confirming the inhibitory effect of ZSH-512 on tumor proliferation. Figure 6 .

[0086] The above examples demonstrate that the low and high concentrations of retinoic acid compounds are significantly better than the control group in inhibiting the proliferation of glioblastoma organoids, inhibiting pathological angiogenesis, and inhibiting subcutaneous tumorigenesis in mice. Retinoic acid compounds also show significant inhibitory effects in inhibiting endothelial cell migration and angiogenesis. The tumor volume and weight of the retinoic acid compound group were significantly reduced, and Ki67 staining results showed that tumor cell proliferation was significantly reduced.

[0087] In summary, the retinoic acid compounds of the present invention have significant therapeutic effects on glioblastoma, can inhibit tumor cell proliferation and angiogenesis through a dual mechanism, and have the potential to be used as anti-tumor drugs.

[0088] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. It is obvious to those skilled in the art that the present invention is not limited to the details of the above exemplary embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or basic features of the present invention. Therefore, no matter from which point of view, the embodiments should be regarded as exemplary and non-restrictive. The scope of the present invention is defined by the attached claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims are included in the present invention. Any figure mark in the claims should not be regarded as limiting the claims involved.

[0089] Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the protection scope of the present invention.

Claims

1. Use of a retinoic acid compound in the preparation of a drug for treating and / or preventing glioblastoma, characterized in that: The retinoid compound is used as the active ingredient of the drug; Retinoic acid compounds include compounds represented by formula I, their enantiomers, diastereomers or pharmaceutically acceptable salts: Wherein, X is O, S, S=O, or O=S=O; R1, R2, and R3 are independently selected from hydrogen, C1-C6 alkyl, C2-C6 alkenyl, halogen-substituted C1-C6 alkyl, C1-C6 alkoxy, C1-C6 acyl, C6-C 10 The aryl group or the heteroatom is an oxygen, sulfur or nitrogen atom, and the number of heteroatoms is 1 to 2 C3-C6 heteroaryl groups.

2. The use according to claim 1, characterized in that: Application of retinoic acid compounds in the preparation of drugs for treating glioblastoma by exerting therapeutic effects through dual inhibition of glioblastoma cell proliferation and pathological angiogenesis.

3. The use according to claim 1, characterized in that: Application of retinoic acid compounds in the preparation of drugs for treating glioblastoma by inhibiting Hedgehog pathway to achieve dual inhibition of glioblastoma cell proliferation and pathological angiogenesis.

4. The use according to claim 1, characterized in that: The retinoid compound exists in the form of a pharmaceutical composition containing the retinoid compound, and the retinoid compound serves as the sole active ingredient of the pharmaceutical composition.

5. The use according to claim 1, characterized in that: The medicament further comprises a pharmaceutically acceptable carrier.

6. The use according to claim 1, characterized in that: The retinoic acid compound is in the form of a kit containing the retinoic acid compound, and the kit also includes a drug for treating diseases associated with glioblastoma.

7. The use according to claim 1, characterized in that: The medicine is an injection.

8. The use according to claim 1, characterized in that: The C1-C6 alkyl group is selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl or hexyl; The C2-C6 alkenyl group is a straight chain or branched alkenyl group containing at least one carbon-carbon unsaturated double bond; The halogen-substituted C1-C6 alkyl group refers to a C1-C6 alkyl group in which at least one hydrogen atom is substituted by a halogen atom, and the halogen atom includes fluorine, chlorine, bromine, and iodine; The C1-C6 alkoxy group is methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, pentyloxy or hexyloxy; The C1-C6 acyl group is a group formed by removing the hydroxyl group from a C1-C6 monocarboxylic acid; The C6-C 10 Aryl is a monovalent aromatic carbocyclic ring system having at least one aromatic ring or multiple fused rings in which at least one ring is aromatic; The C3-C6 heteroaryl group is an aromatic group containing heteroatoms, including an aromatic 5-6 membered monocyclic ring containing 1, 2 or 3 atoms selected from nitrogen, oxygen and / or sulfur, selected from furanyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, thienyl, oxazolyl, diazolyl, imidazolyl, pyrrolyl, pyrazolyl, triazolyl, thiazolyl, isothiazolyl.

9. The use according to claim 8, characterized in that: The C1-C6 alkyl group is a methyl group or an ethyl group; The C2-C6 alkenyl group is vinyl or propenyl; The halogen-substituted C1-C6 alkyl group is trifluoromethyl; The C1-C6 acyl group is an acetyl group or a formyl group; The C6-C 10 Aryl is phenyl; The C3-C6 heteroaryl group is a pyridyl group or a pyrimidinyl group.

10. The use according to claim 1, characterized in that: The retinoic acid compound is a compound represented by formula (II), its enantiomer, diastereomer or pharmaceutically acceptable salt: In the formula, X is S or S=O.

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