Use of the pnp inhibitor forodesine in the treatment of brain tumors

Abstract

This invention belongs to the fields of chemical pharmaceuticals, neurosurgery, and oncology therapeutics, specifically relating to the application of purine nucleoside phosphorylase inhibitors (PNP inhibitors) such as Forodesine or its pharmaceutically acceptable salts in the treatment of brain tumors, including the use of Forodesine or its pharmaceutically acceptable salts in inhibiting glioma growth and promoting anti-tumor immunity. It also relates to a novel treatment method for gliomas. Through extensive exploratory experiments, the applicant of this invention has for the first time discovered that Forodesine or its pharmaceutically acceptable salts can inhibit glioma growth by enhancing anti-tumor immunity, exhibiting inhibitory effects on glioma cells. Therefore, Forodesine or its pharmaceutically acceptable salts can serve as potential drugs for the treatment of brain tumors.

Description

Technical Field

[0001] This invention belongs to the fields of chemical drugs, neurosurgery, and oncology therapeutics, specifically relating to the use of purine nucleoside phosphorylase inhibitors (PNP inhibitors) forodesine or pharmaceutically acceptable salts thereof in the treatment of brain tumors, such as the use of forodesine or pharmaceutically acceptable salts thereof in inhibiting glioma growth, in promoting anti-tumor immunity, etc., and also relates to a new treatment method for gliomas. Background Technology

[0002] Brain tumors are tumors that occur in the brain, and there are many types, among which gliomas are a specific type of brain tumor. Gliomas are tumors originating from glial cells in the nervous system and are the most common primary malignant tumors in the brain, accounting for approximately 40% to 50% of all intracranial tumors. Depending on the cell type, gliomas can be classified into several types, including astrocytomas, glioblastomas (GBM), and oligodendrogliomas.

[0003] The World Health Organization (WHO) classification of central nervous system tumors divides gliomas into grades I to IV. Grades I and II astrocytomas and oligodendrogliomas are classified as low-grade gliomas, while grades III and IV astrocytomas and oligodendrogliomas are classified as high-grade gliomas. Mixed oligodendrogliomas are classified as mixed gliomas. Grade IV gliomas, also known as glioblastomas (GBM), account for 40%–50% of all malignant brain tumors and are the most malignant. Due to their high malignancy, even with aggressive treatment, GBM has a low survival rate; some studies have found that the 5-year survival rate is typically less than 6%, and the survival time is generally less than 15 months. The older the age of onset, the worse the prognosis; survival time is significantly shorter in the elderly. Currently, the main treatment for GBM is maximal safe resection of the tumor, combined with postoperative temozolomide (TMZ) concurrent chemoradiotherapy and adjuvant chemotherapy, electric field therapy, etc., but the results are still not ideal.

[0004] Infiltrative spread is a characteristic of GBM (globulin-tumor leukoma). GBM can rapidly spread to surrounding brain tissue, resulting in unclear boundaries between the tumor and normal brain tissue. Therefore, surgery cannot completely remove the tumor, leading to a high recurrence rate. Furthermore, the blood-brain barrier (BBB) ​​– a tight barrier that isolates brain tissue from harmful substances and protects vulnerable neural tissue from systemic circulatory factors – prevents many chemotherapy drugs from reaching the lesion area, significantly reducing the effectiveness of chemotherapy. Unfortunately, the recurrence rate of GBM is as high as 100%, and once recurrence occurs, treatment options become extremely limited. Currently, after GBM recurrence, the only way to prolong the patient's survival is through existing medical methods, but the results are not ideal. To improve the survival prognosis of clinical patients, researchers and medical professionals in various fields have been actively researching and exploring new targets or approaches to provide new treatment options for gliomas. Summary of the Invention

[0005] One object of the present invention is to provide a new treatment option for treating brain tumors.

[0006] Another objective of this invention is to provide a novel treatment option to inhibit the growth of gliomas.

[0007] Another objective of this invention is to provide a novel treatment option for glioma by enhancing anti-tumor immunotherapy.

[0008] To achieve the above objectives, the present invention provides, in one aspect, the use of a purine nucleoside phosphorylase inhibitor (PNP inhibitor) forodesine or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the treatment of brain tumors.

[0009] In some embodiments, the brain tumor may be a glioma, and more particularly, a glioblastoma.

[0010] In other embodiments, the pharmaceutically acceptable salt of Forodesine includes several types. The selection of the salt form is generally based on factors such as the solubility, stability, and bioavailability of the corresponding drug, with Forodesine hydrochloride being preferred.

[0011] On the other hand, the present invention also provides a treatment method for patients with brain tumors using the PNP inhibitor Forodesine or a pharmaceutically acceptable salt thereof, the method comprising the step of administering a therapeutically effective amount of Forodesine or a pharmaceutically acceptable salt thereof to the patient with the brain tumor.

[0012] In some embodiments, the brain tumor patient includes, but is not limited to, mammals, such as humans, mice, rats, monkeys, dogs, etc.; the method of administering Forodesine or its pharmaceutically acceptable salts to the patient (i.e., the administration method) includes, but is not limited to, intravenous administration, oral administration, intramuscular injection, intracavitary injection, arterial injection, intrathecal administration, rectal administration, skin administration, inhalation administration, intratumoral injection, etc. The preferred administration method of the present invention is oral administration.

[0013] In some embodiments, the therapeutically effective dose is 8-35 mg / kg body weight, preferably 10-30 mg / kg body weight.

[0014] In other embodiments, the treatment of brain tumors by the forodesine or its pharmaceutically acceptable salts manifests as inhibition of brain tumor growth / prolongation of survival in mice bearing or transplanting brain tumors. The inhibition of brain tumor growth includes, but is not limited to, inhibiting the growth of brain tumor cells, inhibiting the self-renewal capacity of brain tumor cells, inhibiting the viability of brain tumor cells, inhibiting the tumorigenic capacity of brain tumor cells, and promoting apoptosis of brain tumor cells.

[0015] In some other embodiments, the brain tumor may be a glioma, more specifically a glioblastoma, or a tumor induced by glioma stem cells or glioma cells. In embodiments of the present invention, the glioma stem cells may be 4121GSC, 387GSC, 456GSC, 3832GSC, 3691GSC, etc., and the glioma cells may be G27, etc.

[0016] Furthermore, this invention provides the application of the PNP inhibitor Forodesine or a pharmaceutically acceptable salt thereof in antitumor immunity, specifically including the following steps:

[0017] 1) Activate T cells in the tumor microenvironment; and

[0018] 2) Promotes the killing of brain tumor cells by T cells.

[0019] In some embodiments, the brain tumor may be a glioma, more specifically a glioblastoma, or a tumor arising from glioma stem cells or glioma cells. In an embodiment of the present invention, the glioma cells are GL261.

[0020] The applicant of this invention, through extensive exploratory experiments, has for the first time discovered that forodesine or a pharmaceutically acceptable salt thereof can inhibit the growth of gliomas by enhancing anti-tumor immunity, and has an inhibitory effect on glioma cells. Therefore, forodesine or a pharmaceutically acceptable salt thereof may serve as a potential drug for the treatment of brain tumors. Attached Figure Description

[0021] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following figures are provided:

[0022] Figure 1 Figure A shows the line graphs of different concentrations of Forodesine inhibiting the viability of tumor cells in Example 1. Figure A is a line graph of different concentrations of Forodesine inhibiting the viability of 4121GSCs cells.

[0023] Figure B is a line graph showing the inhibition of G27 cell viability by different concentrations of forodesine;

[0024] Figure C is a line graph showing the inhibition of 387GSCs cell viability by different concentrations of forodesine;

[0025] Figure D is a line graph showing the inhibition of 456GSCs cell viability by different concentrations of forodesine;

[0026] Figure E is a line graph showing the inhibition of G7 cell viability by different concentrations of forodesine;

[0027] Figure F is a line graph showing the inhibition of G19 cell viability by different concentrations of forodesine;

[0028] Figure G is a line graph showing the inhibition of G29 cell viability by different concentrations of forodesine;

[0029] Figure H is a line graph showing the inhibition of NHA cell (human astrocyte) viability by different concentrations of forodesine.

[0030] Figure 2 The graph shows the half-inhibitory concentration (WIC) curves of Forodesine against different tumor cells (glioma stem cells or glioma cells) and NHA cells in Example 1.

[0031] Figure 3 The graph shows the flow spectroscopy results of different concentrations of forodesine in Example 2 on promoting apoptosis of glioma stem cells 4121GSCs.

[0032] Figure 4 Figure A shows the results of Forodesine inhibiting the glycolysis level of tumor cells G27 in Example 3. Figure A shows the continuous monitoring results of the overall glycolysis level of the Forodesine group and the control group by the seahorse experiment.

[0033] Figure B shows the statistical analysis results of the experimental results in Figure A.

[0034] Figure 5This is a graph showing the results of Forodesine inhibiting tumor growth and prolonging the survival of tumor-bearing mice in Example 4.

[0035] Figure A is a flowchart of the experiment;

[0036] Figure B shows the tumor growth of GBM xenografts under real-time dynamic monitoring using in vivo imaging.

[0037] Figure C shows the fluorescence values ​​of GBM xenografts under real-time dynamic monitoring of tumor growth using in vivo imaging, and the results are quantified and statistically analyzed. Figure D shows the Kaplan-Meier survival curve analysis of the survival period of tumor-bearing mice in each group of G27.

[0038] Figure 6 This is a graph showing the concentration detection results of forodesine in mouse brain and blood samples in Example 5.

[0039] Figure A shows the peak diagram of Forodesine in mass spectrometry detection;

[0040] Figure B is a bar chart showing the calculated concentrations of forodesine in brain and blood samples obtained by mass spectrometry.

[0041] Figure 7 Figure A shows the results of different concentrations of Forodesine promoting the killing of tumor cells by T cells. Figure A is a flow analysis result of Forodesine promoting the killing of GL261 glioma cells by T cells.

[0042] Figure B shows the statistical analysis results of the experimental results in Figure A.

[0043] Figure 8 The image shows the tumor size results on day 27 in mice given different doses of Forodesine in Example 7.

[0044] Figure 9 The graph shows the tumor growth volume curves in mice given different doses of Forodesine in Example 7.

[0045] Figure 10 This is a graph showing the proportion of T cells that play a killing role in mouse tumors treated with different doses of Forodesine in Example 7. Detailed Implementation

[0046] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following embodiments. All technical solutions implemented based on the above-described content of the present invention fall within the scope of the present invention.

[0047] The chemical structure of Forodesine (BCX-1777) used in the above-described technical solution of this invention is shown below. It is a highly effective orally active purine nucleoside phosphorylase inhibitor (PNP inhibitor). Forodesine is an effective inhibitor of human lymphocyte proliferation and can induce apoptosis in leukemia cells by increasing dGTP levels.

[0048]

[0049] Forodesine received orphan drug designation in the United States in 2004 for the treatment of T-cell non-Hodgkin's lymphoma and chronic myeloid leukemia. It received orphan drug designation in the European Union in 2006, 2007, and 2010, respectively, for the treatment of acute lymphoblastic leukemia, cutaneous T-cell lymphoma, and chronic lymphocytic leukemia. In 2017, Forodesine was approved for marketing in Japan for the treatment of relapsed / refractory peripheral T-cell lymphoma. As a novel nucleoside metabolism inhibitor, Forodesine is a valuable drug for the treatment of relapsed / refractory peripheral T-cell lymphoma.

[0050] The brain tumor may be a mammalian brain tumor. The brain tumor cells, the glioma cells, and the glioma stem cells may all be mammalian cells. The mammal may be a human or a mouse. In one embodiment of the invention, the mouse is an immunodeficient mouse, Balb / c nude, also known as a BALB / c-nu athymic nude mouse. In another embodiment of the invention, the mouse is an immunocompetent mouse, C57BL / 6J.

[0051] All raw materials used in the specific embodiments of the present invention are known products, obtained by purchasing commercially available products.

[0052] The forodesine used in the following examples was provided by TargetMol, catalog number T15337, with a purity of 99.88%.

[0053] The 387GSC, 4121GSC, and 456GSC used in the following embodiments are all used in the literature (Hypoxic Induction of Vasorin Regulates Notch 1 Turnover to Maintain Glioma Stem-like Cells, Man et al., 2018, Cell Stem Cell. https: / / doi.org / 10.1016 / j.stem.2017.10.005) and are publicly available from the applicant of this invention. G27, G7, and G29 are tumor cells isolated by the applicant of this invention from GBM patient samples provided by the First Medical Center of the Chinese People's Liberation Army. G19 is a tumor cell isolated by the applicant of this invention from GBM patient samples provided by Beijing Fengtai Hospital. NHA cells (human astrocytes) were purchased from Beijing Bena Biotechnology Research Institute.

[0054] psPAX2 plasmid: Addgene, catalog number #12260.

[0055] pCI-VSVG plasmid: Addgene, catalog number #1733.

[0056] pLenti CMV V5-LUC Blast plasmid, Addgene, catalog number #21474.

[0057] Example 1: Cell-level experiments

[0058] Tumor cells to be tested: 4121GSC and G27.

[0059] The cells to be tested were treated with different concentrations of forodesine, and then the cell viability was detected at different time points.

[0060] The specific implementation steps are as follows:

[0061] 1. Seed the cells to be tested into 96-well plates, 2000 cells per well, and divided into five groups: control group (DMSO), Forodesine-10μM treatment group, Forodesine-20μM treatment group, Forodesine-50μM treatment group, and Forodesine-100μM treatment group, with 5 replicates per group. Each well contained 200μL of NBM complete medium for tumor cells. The NBM complete medium was prepared by adding 5mL of penicillin-streptomycin (Zhongke Maichen Technology Co., Ltd., catalog number CC004), 10mL of B27-supplement serum substitute (Thermo Fisher Scientific, catalog number 12580-010), and 20ng / mL EGF (R&D Company, catalog number Cat#236-EG) to 500mL of Neurobasal Mediμm (Thermo Fisher Scientific, catalog number 12348-017). b-FGF (R&D Company, Catalog No. Cat#4114-TC) and 5 mL of L-glutamine (Zhongke Maichen Technology Co., Ltd., Catalog No. CC009) were used to prepare NHA cells. Each well contained 200 μL of DMEM complete medium, which was prepared by adding 5 mL of penicillin-streptomycin (Zhongke Maichen Technology Co., Ltd., Catalog No. CC004) and 50 mL of fetal bovine serum (EXCELL, Catalog No. FSD500) to 500 mL of basal DMEM medium (Zhongke Maichen Technology Co., Ltd., Catalog No. CM10013).

[0062] 2. Add Forodesine to each well of the Forodesine-10μM treatment group, the Forodesine-20μM treatment group, the Forodesine-50μM treatment group, and the Forodesine-100μM treatment group, respectively. The concentrations of Forodesine in the system are 10μM, 20μM, 50μM, and 100μM, respectively.

[0063] 3. Place the seeded 96-well plate in a 37°C constant temperature carbon dioxide incubator (SANYO, catalog number: MCO-5AC) for incubation;

[0064] 4. Cell viability was measured in each well of the 96-well plate on days 0, 2, 4, and 6: First, 100 μL of CellTiter-Glo Luminescent Cell Viability Assay (Promega, catalog number: G7572) was added to each well. The cells and cell viability assay solution were thoroughly mixed by pipetting 10 times with a 200 μL eight-pipette. Then, 200 μL of the mixture was transferred from each well to a 96-well cell culture plate with a black transparent bottom (Beyotime, catalog number: FCP965-48) for cell viability assay. The 96-well cell culture plate was placed on a horizontal shaker in the dark and shaken at 200 rpm for 10 minutes. The fluorescence value was then measured using a microplate reader.

[0065] 5. Since the intensity of the fluorescence signal is directly related to the amount of ATP, and the amount of ATP is directly related to the number of viable cells, the number of viable cells can be indirectly assessed by measuring the intensity of the generated light signal, thus reflecting cell viability. The average fluorescence value of each group of cells on day 0 was taken as the fold increase in cell viability. The ratios of the fluorescence values ​​on days 2, 4, and 6 to the average fluorescence value on day 0 were used as the fold increase in cell viability. See the line graph for the experimental results. Figure 1 .

[0066] 6. Based on the cell viability results, calculate the half-maximal inhibitory concentration (WMC) of Forodesine for tumor cells and normal cells. Taking the calculation of the WMC on day 6 after Forodesine treatment as an example, the average fluorescence value of the control group (DMSO) on day 6 for each cell group was used. The WMC was calculated by the ratio of the average fluorescence value of the Forodesine-10μM, Forodesine-20μM, Forodesine-50μM, and Forodesine-100μM treatment groups on day 6 to the average fluorescence value of the control group (DMSO) on day 6. The calculation results are shown in [link to calculation]. Figure 2 .

[0067] Experimental conclusions: Forodesine can significantly inhibit the cell viability of glioma stem cells or glioma cells. The inhibitory effect of Forodesine on tumor cells increases with increasing Forodesine concentration (a direct dose-response relationship). However, Forodesine has a lower inhibitory effect on the cell viability of NHA cells, which reflects that Forodesine causes less damage to normal cells and has fewer side effects when used to treat gliomas.

[0068] Example 2: Cell-level experiments

[0069] Tumor cells to be tested: 4121GSC.

[0070] The tumor cells were treated with different concentrations of forodesine, and the apoptosis rate of the tumor cells was detected 48 hours after drug administration.

[0071] The specific implementation steps are as follows:

[0072] 1. Seed the cells to be tested into 6-well plates, 3 x 10 cells per well. 5 Tumor cells were divided into three groups: control group (DMSO), Forodesine-50μM treatment group, and Forodesine-100μM treatment group. Each well contained 1.5mL of NBM complete medium. The NBM complete medium was prepared by adding 20ng / mL EGF (R&D, Cat#236-EG) and 20ng / mL bFGF (R&D, Cat#4114-TC), 10mL B27-supplement serum substitute (ThermoFisher, Cat#12580-010), 5mL penicillin-streptomycin (Zhongke Maichen Technology Co., Ltd., Cat#CC004), and 5mL L-glutamine (Zhongke Maichen Technology Co., Ltd., Cat#CC009) to 500mL of Neurobasal Mediμm (ThermoFisher, Catalog No. 12348-017).

[0073] 2. Add Forodesine to each well of the Forodesine-50μM treatment group and the Forodesine-100μM treatment group, respectively. The concentrations of Forodesine in the system are 50μM and 100μM, respectively.

[0074] 3. Place the seeded 6-well plate in a 37°C constant temperature carbon dioxide incubator (SANYO, product number: MCO-5AC) for incubation;

[0075] 4. The apoptosis rate in each well of a 6-well plate was determined 48 hours after the addition of forodesine: Tumor cells were first digested into single cells with trypsin Accutase (Sigma, catalog number: A6964); after centrifugation at 800 rpm for 3 minutes, the supernatant was removed, and the cells were washed 3 times with PBS (Zhongke Maichen Technology Co., Ltd., catalog number CC008) to remove as much trypsin residue as possible. The experiment was carried out according to the steps provided by the apoptosis detection kit (Pulley, catalog number: P04D03).

[0076] 5. After the previous step, flow cytometry was used to detect the apoptosis rate in different treatment groups. FlowJo software was used to process the data and plot the graphs. See [link to results]. Figure 3 .

[0077] Experimental conclusions: Forodesine treatment leads to an increase in the proportion of apoptotic cells, and this proportion increases with increasing Forodesine concentration, showing a Forodesine concentration-dependent trend. For 4121GSC cells, 48 ​​hours after Forodesine treatment, the proportions of apoptotic cells in the control group (DMSO), the Forodesine-50μM treatment group, and the Forodesine-100μM treatment group were 4.58%, 11.9%, and 26.1%, respectively.

[0078] Example 3: Cell-level experiments

[0079] Tumor cells to be tested: G27.

[0080] The tumor cells were treated with different concentrations of forodesine, and the glycolysis level of the cells was detected 12 hours after drug administration.

[0081] The specific implementation steps are as follows:

[0082] 1. Hydration probe plate:

[0083] (1) Take 20 mL of hydration solution (Agilent, product number: 100840-000) in advance using a 50 mL centrifuge tube and place it in the Pre Station next to the Seahorse XFe96 equipment, preheat it to 37°C and wait for use.

[0084] (2) Open the probe plate kit (Agilent Technologies, part number: 102416-100), remove the two plates on top for adding the drug (to be used on the second day), and then pick up the lid and probe plate (Sensor Cartridge) and place them upside down on the table (be careful not to touch the probe during operation).

[0085] (3) Add 200 μL / well of hydration solution to the groove below the probe plate, gently insert the probe plate into the plate with the added liquid, cover it and label it with identification information (name, time, etc.), after checking, put it in an open 37℃ CO2-free incubator (Pre Station) and incubate for 2 hours. Check the probe well for air bubbles. If there are air bubbles, move the probe plate up and down slightly to remove them. Incubate the probe plate overnight in the Pre Station (more than 12 hours).

[0086] 2. Cell culture:

[0087] (1) Before testing, prepare the G27 tumor cells to be tested in the laboratory and check whether they have been cultured to about 80% confluence.

[0088] (2) Take out the blue-packaged XFe96 Cell Culture Microplates (Nunc, catalog number: 165306), mark the cap, and determine the position of each experimental group. In this example, the G27 cells tested are suspension cells, so the cell culture plates need to be coated with Matrigel (Corning, catalog number: 354277) to help the cells adhere. Add 50 μL of Matrigel to each well and incubate the cell culture plates in a 37°C CO2 incubator for 1 hour.

[0089] (3) G27 cells were digested into single cells with trypsin, added to culture medium to prepare a single-cell suspension, and the cells were counted using a cell counter. 1 x 102 cells were seeded in each well. 4 80 μL of culture medium was added to each of the four background wells. The cells were divided into two groups: Forodesine-0 μM group and Forodesine-50 μM group, with four replicates for each group. Forodesine was added to the corresponding wells at final concentrations of 0 μM and 50 μM.

[0090] (4) Place the cell well plate in a 37°C CO2 incubator overnight to allow the cells to adhere fully.

[0091] 3. Preparation of test culture medium and test reagents:

[0092] (1) Preparation of XF Glycolysis Stress Test Assay Medium: Add 0.5 mL of 200 mM glutamine to 100 mL of preheated basal culture medium (Agilent Technologies, catalog number: 102353-100), and adjust the pH of the solution to 7.4 ± 0.05 with 1 N NaOH. After preparation, filter the solution through a 0.22 μm microfilter into 50 mL centrifuge tubes, wrap them with aluminum foil, and preheat them in a 37 °C water bath for 1 h.

[0093] (2) Reagent preparation: Prepare an 8× glucose solution, the default value is 80 mM, i.e., add 96 μL of 2.5 M glucose to 3 mL of glycolysis culture medium. Prepare a 9× oligomycin solution, add 10.8 μL of oligomycin stock solution to 3 mL of culture medium to prepare an 18 μM working solution, i.e., a final concentration of 2 μM. 2-Deoxy-D-glucose (2-DG) solution does not need to be prepared; preheat to 37℃ to thaw and use directly, with a final working concentration of 50 mM.

[0094] 4. Change the cell culture medium:

[0095] (1) Remove the Seahorse cell culture plate and aspirate 40 μL / well of culture medium.

[0096] (2) Dilute with 160 μL / well of experimental culture medium, then aspirate 160 μL / well. Repeat 2-3 times.

[0097] (3) Add 135 μL / well of experimental culture medium, so that the final volume of each well is 175 μL.

[0098] (4) After the medium is changed, place it in a 37°C CO2-free incubator (Pre Station) for 1 hour.

[0099] 5. Add reagents and run the test:

[0100] (1) Prepare the drugs, sample container, 100μL multichannel pipette and 200μL pipette tip. This step can be performed during the waiting time after changing the cell medium. Place the drug delivery plate (four wells A, B, C and D) on the probe plate corresponding to the well positions, add 25μL / well of the corresponding concentration of Forodesine to each well (remember to change the delivery plate during the process), and then place it in a 37℃ CO2-free incubator.

[0101] (2) The general order of the glycolysis experiment is: A: glucose, B: oligomycin, C: 2-DG.

[0102] (3) All wells with injection settings must be filled with 80 μL of PBS.

[0103] (4) After setting the relevant parameters, perform probe calibration. After 25-30 minutes, the probe calibration will be completed. Click "OK" to bring up the hydration plate (Utility Plate). Then place the cell plate with the cap removed and click "I'm Ready" to start the experiment.

[0104] (5) After 1.5-2 hours, the program will finish running. Click "OK" to pop up the tray. Remove the tray and click again to put the tray back in. View and process the data. See the experimental results below. Figure 4 .

[0105] Example 4: Animal-level experiments

[0106] Laboratory animals: Immunodeficient mice Balb / c nude (Beijing Vital River Laboratory Animal Technology Co., Ltd., 4 weeks old)

[0107] Tumor cells to be tested: G27.

[0108] The specific implementation steps are as follows:

[0109] 1. Lentiviral packaging

[0110] 3×10 6 HEK293TN cells were seeded in 10cm culture dishes and cultured in DMEM complete medium. The DMEM complete medium was prepared by adding 5mL of penicillin-streptomycin (Zhongke Maichen Technology Co., Ltd., catalog number CC004) and 50mL of fetal bovine serum (EXCELL, catalog number FSD500) to 500mL of basal DMEM medium (Zhongke Maichen Technology Co., Ltd., catalog number: CM10013). Lentiviral packaging plasmids psPAX2 (5μg), pCI-VSVG (5μg), and pLenti CMV V5-LUCBlast plasmid (5μg) were transfected into HEK293TN cells using a calcium phosphate transfection kit (Zhongke Maichen Technology Co., Ltd., catalog number: Ctk001). After 5 hours of transfection, the DMEM complete medium was replaced. After 60 hours of cell culture, viral supernatant was collected and centrifuged at 2500 rpm for 5 minutes. The viral supernatant was filtered through a 0.45 μm filter to remove cell debris and other impurities. The filtered virus was collected in a 15 mL centrifuge tube, and 5x PEG lentiviral concentrate (Best Inc., catalog number: C103-05) was added to concentrate the virus. The centrifuge tube was placed in a 4°C freezer for 12 hours for concentration. The centrifuge tube was then centrifuged at 7,000 rpm for 10 minutes at 4°C to remove the supernatant. The virus at the bottom of the tube was washed with 1 mL of PBS, mixed, and transferred to a 1.5 mL EP tube. The tube was centrifuged at 15,000 rpm for 15 minutes at 4°C to remove residual serum and other impurities. The supernatant containing pLenti CMVV5-LUC Blast virus was stored at -80°C for later use.

[0111] 2. G27 cells were obtained by lentiviral infection of G27 cells.

[0112] 2×10 6One G27 cell was seeded into a 10cm culture dish, with 8mL of NBM complete medium per dish. The NBM complete medium was prepared by adding 20ng / mL EGF (R&D, Cat#236-EG), 20ng / mL bFGF (R&D, Cat#4114-TC), 10mL B27-supplement serum substitute (ThermoFisher, 12580-010), 5mL penicillin-streptomycin (Zhongke Maichen Technology Co., Ltd., CC004), and 5mL L-glutamine (Zhongke Maichen Technology Co., Ltd., CC009) to 500mL of Neurobasal Medium (ThermoFisher, catalog number 12348-017). 200μL of concentrated lentivirus containing pLenti CMV V5-LUC Blast was added. After the cells were shaken well, they were incubated in a 37°C CO2 incubator for 48h. The cultured cells were digested into single cells using the trypsin enzyme Accutase, and then re-seeded in 10 cm dishes with NBM complete medium. Cell selection was performed by adding the antibiotic blasticidin (final concentration of 10 μg / mL).

[0113] 3. Identification of luciferase expression in G27

[0114] Lentivirally infected G27 cells were placed in 96-well plates, and 1 μL of luciferin (a substrate of luciferase) was added to the wells (operated in the dark). After the substrate reacted with the cells for 3 minutes, the wells were placed in a live imaging system (PerkinElmer) to detect the cell fluorescence value. Lentivirally infected G27 positive cells showed strong fluorescence.

[0115] 4. After resuspending the G27 cells stably expressing luciferase in the previous step in NBM basal medium (Neurobasal Medium, Thermo Fisher Scientific, catalog number 12348-017), perform cell counting and dilute to 5 x 10⁻⁶ cells / mL. 4 5 × 10⁵ cells / 20 μL were injected orally into the right frontal lobe of the brain of Balb / c nude immunodeficient mice to construct a GBM xenograft model. Each mouse received 5 × 10⁵ cells / 20 μL. 4 Each mouse was injected with cells, and tumor growth was monitored weekly using an in vivo imaging system. On day 8, after in vivo imaging, mice were randomly divided into two groups based on tumor size: a control group and a forodesine-treated group, with eight mice in each group. The following treatments were administered to each group:

[0116] Control group (Ctrl): 100 μL of purified water was administered by gavage every other day.

[0117] Forodesine administration group: 100 μL of Forodesine was administered by gavage every other day (based on a dose of 30 mg / kg after weighing the mice).

[0118] Tumor growth was observed in real time using in vivo imaging, and the survival time of tumor-bearing mice was recorded. The experimental results are shown in [link to experimental results]. Figure 5 .

[0119] Experimental conclusion:

[0120] Analysis of the experimental results revealed that, compared to the control group (treated with purified water), the tumor growth in mice treated with Forodesine was significantly slower, and the survival time of the mice was also prolonged. After the average grouping on day 8, there was no significant difference in tumor size. Subsequently, the control group received purified water, while the experimental group continued to receive Forodesine treatment. In vivo imaging results on days 15 and 22 showed that the tumor size in the treatment group was significantly smaller than that in the control group, indicating that Forodesine has an inhibitory effect on tumor growth.

[0121] Therefore, forodesine can inhibit the growth of GBM xenografts, which has important implications for the clinical treatment of glioma patients. The applicant of this invention is the first to discover that forodesine has an inhibitory effect on glioma cells and can inhibit glioma growth. Therefore, forodesine or a pharmaceutically acceptable salt thereof may be a potential drug for the treatment of brain tumors.

[0122] Example 5: Animal-level experiments

[0123] Laboratory animals: Immunocompetent mice C57BL / 6J (Beijing Vital River Laboratory Animal Technology Co., Ltd., 4 weeks old)

[0124] Experimental objective: To detect whether forodesine can cross the blood-brain barrier.

[0125] The specific implementation steps are as follows:

[0126] 1. Lentiviral packaging

[0127] 3×10 6HEK293TN cells were seeded in 10cm culture dishes and cultured in DMEM complete medium. The DMEM complete medium was prepared by adding 5mL of penicillin-streptomycin (Zhongke Maichen Technology Co., Ltd., catalog number CC004) and 50mL of fetal bovine serum (EXCELL, catalog number FSD500) to 500mL of basal DMEM medium (Zhongke Maichen Technology Co., Ltd., catalog number: CM10013). Lentiviral packaging plasmids psPAX2 (5μg), pCI-VSVG (5μg), and pLenti CMV V5-LUCBlast plasmid (5μg) were transfected into HEK293TN cells using a calcium phosphate transfection kit (Zhongke Maichen Technology Co., Ltd., catalog number: Ctk001). After 5 hours of transfection, the DMEM complete medium was replaced. After 60 hours of cell culture, viral supernatant was collected and centrifuged at 2500 rpm for 5 minutes. The viral supernatant was filtered through a 0.45 μm filter to remove cell debris and other impurities. The filtered virus was collected in a 15 mL centrifuge tube, and 5x PEG lentiviral concentrate (Best Inc., catalog number: C103-05) was added to concentrate the virus. The centrifuge tube was placed in a 4°C freezer for 12 hours for concentration. The centrifuge tube was then centrifuged at 7,000 rpm for 10 minutes at 4°C to remove the supernatant. The virus at the bottom of the tube was washed with 1 mL of PBS, mixed, and transferred to a 1.5 mL EP tube. The tube was centrifuged at 15,000 rpm for 15 minutes at 4°C to remove residual serum and other impurities. The supernatant containing pLenti CMVV5-LUC Blast virus was stored at -80°C for later use.

[0128] 2. Lentiviral infection of GL261 cells to obtain GL261-luciferase-blasticidin cells

[0129] 2×10 6 One GL261 cell was seeded into 10cm culture dishes, each containing 8mL of DMEM complete medium. The DMEM complete medium was prepared by adding 5mL of penicillin-streptomycin (Zhongke Maichen Technology Co., Ltd., catalog number CC004) and 50mL of fetal bovine serum (EXCELL, catalog number FSD500) to 500mL of basal DMEM medium (Zhongke Maichen Technology Co., Ltd., catalog number CM10013). 200μL of concentrated lentivirus containing pLenti CMV V5-LUC Blast was added. After mixing the cells, they were incubated at 37℃ for 48h in a CO2 incubator. Single cells were then digested with trypsin (Zhongke Maichen Technology Co., Ltd., catalog number CC012), re-seeded into 10cm dishes with DMEM complete medium, and the antibiotic blasticidin (final concentration 10μg / mL) was added for cell selection.

[0130] 3. Identification of luciferase expression in GL261

[0131] Lentivirally infected GL261 cells were placed in 96-well plates, and 1 μL of luciferin (a substrate of luciferase) was added to the wells (in the dark). After the substrate reacted with the cells for 3 minutes, the wells were placed in a live imaging system (PerkinElmer) to detect the cell fluorescence value. Lentivirally infected GL261 positive cells showed strong fluorescence.

[0132] 4. Sample Collection

[0133] After resuspending the GL261 cells stably expressing luciferase in DMEM basal medium (Zhongke Maichen Technology Co., Ltd., catalog number CM10013), cell counting was performed, and the cells were diluted to 8x10⁻⁶. 4 8 × 10⁸ cells / 20 μL were injected orally into the right frontal lobe of C57BL / 6J immunocompetent mice using a 100 mL insulin syringe (BD, catalog number: 320310) to construct a GBM xenograft model. 4 Cells were injected, and on day 10 post-injection, tumor growth in mice was assessed using in vivo imaging. Mice were divided into three groups of seven each: Ctrl group (no tumor formation, no drug administration), Blank + forodesine group (no tumor formation, drug administration), and Tumor + forodesine group (tumor formation, drug administration). Each group received the following treatments:

[0134] Ctrl group (no tumor formation, no drug administration): Blood was collected from the orbital cavity of mice, 300 μL per mouse. After blood collection, the mice underwent cardiac perfusion, and then the brain was removed.

[0135] Blank+forodesine group (non-tumor-forming administration): 100 μL of forodesine was administered by gavage (based on a dose of 30 mg / kg after weighing the mice). Four hours later, 300 μL of blood was collected from the orbital cavity of each mouse. After blood collection, the mice underwent cardiac perfusion, and then the brain was harvested.

[0136] Tumor+forodesine group (tumor-induced administration): Mice with tumors detected by in vivo imaging were administered 100 μL of Forodesine by gavage (administered at 30 mg / kg after weighing the mice). Four hours later, 300 μL of blood was collected from the orbital cavity of each mouse. After blood collection, the mice underwent cardiac perfusion, and then the brain was harvested.

[0137] 5. Sample pretreatment and detection

[0138] (1) Blood sample processing: Centrifuge the blood sample at 12000rpm for 10min at 4℃, take the supernatant sample and transfer it to a new 1.5mL EP tube, and store it in a -80℃ freezer for later use.

[0139] (2) Brain sample processing: The brain samples were weighed for subsequent concentration calculation. After weighing, the samples were cut into small pieces and frozen in liquid nitrogen for 10 min. Then, grinding beads were added to the tubes and the samples were ground for 10 min until they were powdered. Each sample was resuspended in 1 mL of 80% methanol (v / v), placed in a -80℃ freezer for 1 h, and centrifuged at 14000 rpm for 20 min at 4℃. The supernatant sample was transferred to a new 1.5 mL EP tube and stored in a -80℃ freezer for later use.

[0140] The pretreated samples were sent to the Pharmaceutical Technology Center of Tsinghua University for mass spectrometry analysis. The experimental results are shown below. Figure 6 .

[0141] Experimental conclusion:

[0142] Analysis of the experimental results revealed that, compared to the Ctrl group, mice in both the Blank+forodesine and Tumor+forodesine groups showed significant accumulation of forodesine in their brains, with the forodesine concentration in the Tumor+forodesine group being significantly higher than that in the Blank+forodesine group. Similarly, a certain concentration of forodesine was detected in the blood of both the Blank+forodesine and Tumor+forodesine groups, indicating that after administration via gavage, forodesine was absorbed into the bloodstream and entered the brain, reaching the tumor area.

[0143] This demonstrates that after being absorbed into the bloodstream in mice, Forodesine reaches the brain and can cross the blood-brain barrier to reach the tumor area, confirming the reliability of Forodesine in the treatment of glioma.

[0144] Example 6: Cell-level experiments

[0145] Tumor cells to be tested: GL261.

[0146] Experimental mice: Immunocompetent mice C57BL / 6J (Beijing Vital River Laboratory Animal Technology Co., Ltd., 6 weeks old).

[0147] The tumor cells to be tested were treated with different concentrations of forodesine, and then the killing ratio of T cells to tumor cells after co-incubation of tumor cells and T cells was detected.

[0148] The specific implementation steps are as follows:

[0149] 1. Isolation and activation of T cells from mouse spleen: One C57BL / 6J mouse (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., 6-week-old female) was euthanized by cervical dislocation. The mouse body was disinfected by spraying with 75% ethanol (v / v). The spleen was removed from the cadaver and immersed in a 1.5 mL EP tube containing double antibiotics to avoid contamination. The spleen was rinsed once with physiological saline in a clean bench. A 70 μm filter (Biosharp, catalog number: BS-70-XBS) was placed on a 50 mL centrifuge tube. The filter was moistened with 2 mL of TIB solution (PBS + 1% BSA + 2 mM EDTA). The spleen was placed on the filter. Using a 2 mL syringe, the stopper was removed, and the spleen was crushed by squeezing it up and down with the rubber head of the stopper. The cells were rinsed with TIB solution and transferred to the centrifuge tube. The tube was centrifuged at 1,500 rpm for 3 min, and the supernatant was discarded. Add 1 mL of ACK lysing buffer (Thermo Fisher Scientific, catalog number: A1049201) to a centrifuge tube and lyse erythrocytes for 1 min. Neutralize with 4 mL of TIB solution, filter through a 40 μm filter (Biosharp, catalog number: BS-40-XBS), rinse the filter for residual cells with TIB solution, and centrifuge at 1,500 rpm for 3 min. Discard the supernatant. Resuspend the cell pellet in 100 μL of TIB. (The following steps should be performed in the dark.) Add 10 μL of Biotiu-Antibody coektail (Mitentin Biotech, catalog number: 130-104-075), pipette to mix, and incubate at room temperature for 5 min. Add 30 μL of TIB and 20 μL of Anti-Biotin microbeads (Mitentin Biotech, catalog number: 130-104-075), pipette to mix, and incubate at room temperature for 10 min. Install the LS column (Mitentech, catalog number: 130-042-401), moisten with 3 mL of TIB, and pass the cell suspension through the column. Collect CD8+ T cells in a 15 mL centrifuge tube below the column. The liquid flow should be continuous after adding TIB. Finally, rinse the centrifuge tube with TIB and pass the liquid through the column again. After the liquid has completely drained, remove the LS column. Do not apply pressure during the entire column passing process. Centrifuge the collected CD8+ T cell suspension at 1,500 rpm for 3 min and discard the supernatant. Resuspend the cell pellet in RPMI 1640 complete medium. The RPMI 1640 complete medium is prepared by adding 5 mL of penicillin-streptomycin (Zhongke Maichen Technology Co., Ltd., catalog number CC004) and 50 mL of fetal bovine serum (EXCELL, catalog number FSD500) to 500 mL of RPMI 1640 (Thermo Fisher Scientific, catalog number 21870076).

[0150] 2. After counting the isolated CD8+ T cells, seed them into 6-well plates (pre-coated with CD3 antibody + CD28 antibody, CD3: 2.92 μg / mL, CD28: 2.82 μg / mL), with 2 x 10 cells seeded per well. 6 To promote T cell proliferation, 1 / 1000 of mercaptoethanol and 10 ng / mL IL-2 were added to each well.

[0151] 3. Seed the GL261 tumor cells to be tested into 10cm culture dishes, 2 x 10 cells per dish. 6 The cells were divided into three groups: control group (DMSO), forodesine-50μM treatment group, and forodesine-100μM treatment group. Each plate of tumor cells contained 8mL of DMEM complete medium. The DMEM complete medium was prepared by adding 5mL of penicillin-streptomycin (Zhongke Maichen Technology Co., Ltd., catalog number CC004) and 50mL of fetal bovine serum (EXCELL, catalog number FSD500) to 500mL of basal DMEM medium (Zhongke Maichen Technology Co., Ltd., catalog number CM10013).

[0152] 4. Forodesine was added to each cell in the Forodesine-50μM treatment group and the Forodesine-100μM treatment group, with the concentrations of Forodesine in the system being 50μM and 100μM, respectively. An equal amount of DMSO was added to the control group.

[0153] 5. Place the culture dish containing the seeded cells in a 37°C constant temperature carbon dioxide incubator (SANYO, product number: MCO-5AC) for incubation.

[0154] 6. After culturing cells for 48 hours, digest them into single cells using trypsin (Zhongke Maichen Company, catalog number: CC012). Wash once with PBS, resuspend in DMEM complete medium, count the cells, and seed them into 24-well plates at 5 x 10⁶ cells per well. 4 One cell was cultured in a 37°C constant temperature carbon dioxide incubator overnight.

[0155] 7. The activated T cells were co-incubated with tumor cells treated with Forodesine. The experiment was divided into 6 groups: GL261+Forodesine-0μM group, GL261+Forodesine-50μM group, GL261+Forodesine-100μM group, GL261+Forodesine-0μM+T cell group, GL261+Forodesine-50μM+T cell group, and GL261+Forodesine-100μM+T cell group. Each group was set up with 3 replicates. 2.5 x 10 μM of Forodesine was added to the wells of the GL261+Forodesine-0μM+T cell group, GL261+Forodesine-50μM+T cell group, and GL261+Forodesine-100μM+T cell group, respectively. 5 The cells were divided into three groups: GL261+Forodesine-0μM, GL261+Forodesine-50μM, and GL261+Forodesine-100μM. The same volume of RPMI 1640 complete medium was added to each group. The 24-well plates with the cells were placed in a 37°C constant temperature carbon dioxide incubator for culture. The T cells and GL261 tumor cells were co-incubated for 24 hours.

[0156] 8. Cells were digested from the wells of the plate using Accutase trypsin (Sigma, catalog number: A6964), washed three times with PBS, and stained. T cells were labeled with CD8-APC and stained at room temperature for 15 min. The cells were then centrifuged at 1500 rpm for 3 min, and the supernatant was discarded. Dead tumor cells were labeled with Annexin V-FITC and stained at room temperature for 10 min. Each sample was supplemented with 200 μL of PBS before flow cytometry analysis. The experimental results are shown in [link to results]. Figure 7 .

[0157] Experimental conclusion:

[0158] Forodesine treatment leads to an increase in the proportion of apoptotic tumor cells, and this proportion increases with increasing forodesine concentration (a direct dose-response relationship), meaning the proportion of apoptotic cells shows a forodesine concentration-dependent trend. The proportion of T cells killing tumor cells also increases with increasing forodesine concentration used to treat tumor cells, indicating a forodesine concentration-dependent trend in T cell killing of tumor cells.

[0159] Example 7: Animal-level experiments

[0160] Laboratory animals: Immunocompetent mice C57BL / 6J (Beijing Vital River Laboratory Animal Technology Co., Ltd., 4 weeks old)

[0161] Tumor cells to be tested: GL261.

[0162] The specific implementation steps are as follows:

[0163] 1. Construction of subcutaneous tumorigenic mice: GL261 tumor cells to be tested were expanded to at least 2.4 x 10⁻⁶. 7 The tumor cells to be tested were resuspended in PBS and counted, then diluted to 8 x 10⁶ cells. 5 Cells / 100 μL were injected into the subcutaneous area of ​​the right abdomen of immunocompetent C57BL / 6J mice using a 1 mL ordinary syringe (Sinopharm, catalog number: 92311609) to construct a mouse subcutaneous xenograft model. Each mouse was injected with 8 x 10 cells / 100 μL. 5 Cells were injected, and the tumor size was measured on day 9 after tumor formation. Tumor size was then measured every other day thereafter, using electronic calipers to measure both the long and short diameters of the tumor. The tumor volume was calculated using the formula: long diameter x short diameter. 2 ÷2, based on tumor size, mice were divided into three groups: a control group (water group), a forodesine-10mg / kg administration group, and a forodesine-30mg / kg administration group, with 10 mice in each group. Each group underwent the following treatments:

[0164] Control group: 100 μL of purified water was administered by gavage every other day;

[0165] Forodesine-10mg / kg administration group: 100μL of Forodesine was administered by gavage every other day (the mice were weighed and administered according to a dose of 10mg / kg);

[0166] Forodesine-30mg / kg administration group: 100μL of Forodesine was administered by gavage every other day (the mice were weighed and administered according to the dosage of 30mg / kg);

[0167] On day 27, after the tumors reached a certain size, the mice were euthanized by cervical dislocation. Subcutaneous tumors were removed, photographed, and their sizes were compared between different experimental groups. The data on changes in tumor volume were plotted as a curve. The experimental results are shown in [link to experimental results]. Figure 8 , Figure 9 .

[0168] 2. After photographing the subcutaneous tumor, the activation ratio of tumor-infiltrating T cells was detected.

[0169] Prepare a 12-well plate. Pour 1.5 mL of DMEM complete medium into each well. Place the extracted subcutaneous tumors into the wells for temporary preservation to maintain cell viability. Transfer the tumors to isolation tubes (Mittentech, catalog number: 130-093-237), add tumor isolation buffer, and mince them with scissors to a size of 2-4 mm. The preparation steps for the tumor isolation buffer are as follows: 3 mL of DMEM complete medium lysozyme D, 2.7 mL of DMEM complete medium lysozyme R, 1 mL of Buffer A lysozyme A. For each sample, add 2.5 mL of DMEM complete medium, 100 μL of enzyme D, 50 μL of enzyme R, and 12.5 μL of enzyme A (the kit containing lysozyme D, lysozyme R, lysozyme A, and Buffer A was purchased from Miltentech, catalog number: 130-096-730). Place the separation tube on a tissue homogenizer (Medtronic Corporation) and set the program (37C_m_TDK_1) for automatic homogenization and digestion for 40 minutes.

[0170] Dilute the cell suspension with serum-containing PBS (PBS + 1% FBS) after grinding and digestion. Add 3 mL of PBS to each sample. Filter the diluted cell suspension through a 70 μm filter (Biosharp, catalog number: BS-70-XBS), centrifuge at 1,500 rpm for 3 min at room temperature, and discard the supernatant. Add 2 mL of erythrocyte lysis buffer (Thermo Fisher Scientific, catalog number: A1049201) to each sample for lysis. Lyse at room temperature for 2 min. Dilute each sample with 5 mL of serum-containing PBS (PBS + 1% FBS). Filter the diluted cell suspension through a 40 μm filter (Biosharp, catalog number: BS-40-XBS), centrifuge at 1,500 rpm for 3 min at room temperature, and discard the supernatant.

[0171] Resuspend cells in an appropriate amount of serum-containing PBS (PBS + 1% FBS) according to cell mass, and after counting, take 5 x 10 cells from each sample. 6 Each cell was subjected to subsequent protein secretion blocking treatment. 5 x 10 cells were taken from each sample. 6 Cells were resuspended in 2 mL of RPMI 1640 complete medium and seeded into 12-well plates. 1 / 500 volume of protein secretion inhibitor (Thermo Fisher Scientific, catalog number: 00-4980-03) was added to each sample. The cells were then placed in a 37°C carbon dioxide incubator (SANYO, catalog number: MCO-5AC) for 4 h of incubation.

[0172] Cells were digested from the wells of the plate using Accutase trypsin (Sigma, catalog number: A6964), washed three times with PBS, and then stained. Lymphocytes were labeled with CD45-PerCP antibody (1:200, Dakota, catalog number: 109828), and CD8+ T lymphocytes were labeled with CD8-FITC antibody (1:200, Dakota, catalog number: 100706). The staining was performed using the viability-fixing dye eFluor. TM Dead cells were labeled with 780 (1:1000, Thermo Fisher Scientific, catalog number: 65-0865-18). Each sample was stained with 100 μL of the staining system. The staining was performed at room temperature for 15 min, followed by centrifugation at 1,500 rpm for 3 min. The supernatant was discarded.

[0173] Cells were fixed for 1 hour at room temperature using cell fixation solution (Thermo Fisher Scientific, catalog number: 00-5223-56). The preparation steps for the cell fixation solution are as follows: 30 μL fixation solution + 90 μL buffer per sample, centrifuged at 1,500 rpm for 3 minutes at room temperature, and the supernatant was discarded. Cells were then permeabilized for 10 minutes at room temperature using cell permeabilization solution (Thermo Fisher Scientific, catalog number: 00-8333-56). The preparation steps for the cell permeabilization solution are as follows: 10 μL fixation solution + 90 μL sterile water per sample, centrifuged at 1,500 rpm for 3 minutes at room temperature, and the supernatant was discarded. Intracellular IFNg and GZMB were stained. IFNg was labeled with IFNg-PE antibody (1:200, Dacron, catalog number: 505808), and GZMB was labeled with GZMB-PE-Cy7 antibody (1:200, Dacron, catalog number: 372214). Each sample was stained in a 100 μL staining system. After staining at room temperature for 30 min, the samples were analyzed by flow cytometry. Experimental results are shown below. Figure 10 .

[0174] Experimental conclusion:

[0175] Analysis of the experimental results revealed that, compared to the control group (water-treated), tumor growth in mice treated with Forodesine was significantly slowed, with the high-concentration Forodesine group (30 mg / kg) showing the best tumor-inhibiting effect. After averaging the group size on day 9, there was no significant difference in tumor size. Subsequently, the control group received water, while the experimental groups received continuous treatment with different doses of Forodesine. Tumor size measurements taken on day 27 showed that the tumor size in the Forodesine group was significantly smaller than that in the control group, and the high-dose group (30 mg / kg) had a smaller tumor size than the low-dose group (10 mg / kg), indicating that the inhibitory effect of Forodesine on tumor growth is related to the Forodesine dose. Analysis of the flow cytometry results showed that Forodesine administration promoted an increase in the proportions of IFNg+CD8+ T cells and GZMB+CD8+ T cells, thereby enhancing the anti-tumor immune effect and inhibiting tumor growth.

[0176] Therefore, it is evident that Forodesine or its pharmaceutically acceptable salts can inhibit glioma growth by enhancing anti-tumor immunity, providing a new treatment option for clinical immunotherapy of glioma patients.

Claims

1. Use of the PNP inhibitor Forodesine or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the treatment of glioblastoma.

2. The use according to claim 1, wherein, The therapeutically effective dose of the forodesine or its pharmaceutically acceptable salt is 8-35 mg / kg body weight.

3. The use according to claim 2, wherein, The effective therapeutic dose is 10-30 mg / kg body weight.

4. The use according to claim 1, wherein, The forodesine or its pharmaceutically acceptable salts are administered orally.

5. The use according to claim 2, wherein, The pharmaceutically acceptable salt of Forodesine is Forodesine hydrochloride.

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