Application of PNP inhibitor Forodesine in treatment of brain tumor

Abstract

The invention belongs to the fields of chemical drugs, neurosurgery and oncotherapy, and particularly relates to application of a purine nucleoside phosphorylase inhibitor (PNP inhibitor) Forodesine or a pharmaceutically acceptable salt thereof in treatment of brain tumors, such as application of Forodesine or a pharmaceutically acceptable salt thereof in inhibition of glioma growth, application of Forodesine in promotion of anti-tumor immunity and the like, and particularly relates to application of a purine nucleoside phosphorylase inhibitor (PNP inhibitor) Forodesine or a pharmaceutically acceptable salt thereof in treatment of brain tumors. The invention also relates to a novel glioma treatment method. Through a large number of exploratory experiments, the applicant of the invention finds for the first time that Forodesine or pharmaceutically acceptable salt thereof can inhibit the growth of glioma by enhancing anti-tumor immunity and has an inhibition effect on glioma cells. Therefore, Forodesine or the pharmaceutically acceptable salt thereof can be used as a potential drug for treating brain tumors.

Description

Technical Field

[0001] The present invention belongs to the fields of chemical medicine, neurosurgery and tumor therapeutics, and specifically relates to the use of purine nucleoside phosphorylase inhibitor (PNP inhibitor) Forodesine or its pharmaceutically acceptable salt in the treatment of brain tumors, such as the use of Forodesine or its pharmaceutically acceptable salt in inhibiting the growth of gliomas, in promoting anti-tumor immunity, etc., and also relates to a new method for treating gliomas. Background Art

[0002] Brain tumors are tumors that occur in the brain and include many types, among which gliomas are a specific type of brain tumor. Glioma is a tumor that originates from glial cells in the nervous system. It is the most common primary malignant tumor in the brain, accounting for about 40% to 50% of the total number of intracranial tumors. Depending on the type of cell, gliomas can be divided into astrocytomas, glioblastomas (GBM), oligodendrogliomas and other types.

[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 into low-grade gliomas, grades III and IV astrocytomas and oligodendrogliomas into high-grade gliomas, and mixed oligoastrocytomas into mixed gliomas. Among them, grade IV glioma, also known as glioblastoma (GBM), accounts for 40% to 50% of all malignant brain tumors and is the most malignant brain tumor. Due to the high malignancy of GBM, even after active treatment, the survival rate is low. Some studies have found that the 5-year survival rate of GBM is usually less than 6%, and the survival time is generally less than 15 months. The older the age of onset, the worse the prognosis, and the survival time of the elderly is usually significantly shortened. At present, the main treatment method for GBM is to safely remove the tumor to the maximum extent, combined with postoperative temozolomide (TMZ) synchronous chemoradiotherapy and adjuvant chemotherapy, electric field therapy, etc., but the effect is still not ideal.

[0004] Infiltrative spread is one of the characteristics of GBM. GBM can spread rapidly to the surrounding brain tissue, resulting in unclear boundaries between the tumor and normal brain tissue. Therefore, surgery cannot completely remove the tumor, and the probability of recurrence in patients remains high. In addition, the brain has a blood-brain barrier (BBB) ​​- a barrier that tightly connects the brain tissue to isolate harmful substances, protecting the fragile neural tissue from the influence of systemic circulation factors, so that many chemotherapy drugs cannot reach the lesion area, which greatly reduces the therapeutic effect of chemotherapy drugs. Unfortunately, the recurrence rate of GBM is as high as 100%. Once recurring, the choice of treatment will become very limited. At present, after GBM relapses, the patient's survival time can only be extended by existing medical means, but the effect is not ideal. In order to improve the survival prognosis of clinical patients, scientific researchers or medical workers in different fields have been actively studying and exploring new targets or ideas to provide new treatment options for glioma treatment. Summary of the invention

[0005] An object of the present invention is to provide a new therapeutic regimen for treating brain tumors.

[0006] Another object of the present invention is to provide a new therapeutic regimen to inhibit the growth of glioma.

[0007] Another object of the present invention is to provide a new glioma treatment regimen by enhancing anti-tumor immunotherapy.

[0008] In order to achieve the above-mentioned object, the present invention provides, on one hand, the use of a purine nucleoside phosphorylase inhibitor (PNP inhibitor) Forodesine or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating brain tumors.

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

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

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

[0012] In some embodiments, the brain tumor patient includes but is not limited to mammals, and the mammal can be humans, mice, rats, monkeys, dogs, etc.; the manner of administering Forodesine or a pharmaceutically acceptable salt thereof to the patient (i.e., the mode of administration) includes but is not limited to intravenous administration, oral administration, intramuscular injection, intracavitary injection, arterial injection, intrathecal administration, rectal administration, skin administration, inhalation administration, intratumor injection, etc., and the mode of administration of the present invention is preferably oral administration.

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

[0014] In other embodiments, the treatment of brain tumors in patients by Forodesine or a pharmaceutically acceptable salt thereof is manifested in inhibiting the growth of brain tumors / prolonging the survival period of brain tumor-bearing or transplanted tumor mice, and the inhibition of brain tumor growth includes but is not limited to inhibiting the growth of brain tumor cells, inhibiting the self-renewal ability of brain tumor cells, inhibiting the vitality of brain tumor cells, inhibiting the tumorigenic ability of brain tumor cells, promoting the apoptosis of brain tumor cells, etc.

[0015] In some other embodiments, the brain tumor may be a glioma, further may be a glioblastoma, may be a tumor caused by a glioma stem cell or a glioma cell. In an embodiment of the present invention, the glioma stem cell may be 4121GSC, 387GSC, 456GSC, 3832GSC, 3691GSC, etc., and the glioma cell may be G27, etc.

[0016] In another aspect, the present invention provides a use of a PNP inhibitor Forodesine or a pharmaceutically acceptable salt thereof in anti-tumor immunity, which specifically comprises the following steps:

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

[0018] 2) Promote T cells to kill brain tumor cells.

[0019] In some embodiments, the brain tumor may be a glioma, further may be a glioblastoma, may be a tumor induced by a glioma stem cell or a glioma cell. In an embodiment of the present invention, the glioma cell is GL261.

[0020] The applicant of the present invention has discovered for the first time through a large number of exploratory experiments 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 can be used as a potential drug for treating brain tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to make the purpose, technical solution and beneficial effects of the present invention clearer, the present invention provides the following drawings:

[0022] Figure 1 It is a line graph of different concentrations of Forodesine inhibiting tumor cell viability in Example 1, wherein Figure A is a line graph of different concentrations of Forodesine inhibiting 4121GSCs cell viability;

[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) activity by different concentrations of Forodesine.

[0030] Figure 2 This is a half-inhibitory concentration curve of Forodesine in Example 1 on different tumor cells (glioma stem cells or glioma cells) and NHA cells.

[0031] Figure 3 This is a graph showing the results of Flow cytometry analysis of different concentrations of Forodesine in Example 2 to promote apoptosis of glioma stem cells 4121GSCs.

[0032] Figure 4 The results of Forodesine inhibiting the glycolysis level of tumor cells G27 in Example 3 are shown in Figure A, wherein Figure A is the continuous monitoring result of the overall glycolysis level of the Forodesine group and the control group in the seahorse experiment;

[0033] Figure B is the statistical analysis result 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 period of tumor-bearing mice in Example 4, wherein:

[0035] Figure A is the experimental flow chart;

[0036] Figure B is a diagram showing the real-time dynamic monitoring of GBM transplant tumor growth using in vivo imaging;

[0037] Figure C shows the fluorescence value of GBM transplanted tumor growth monitored in real time by in vivo imaging, and quantitative statistics; Figure D shows the Kaplan-Meier survival curve analysis of the survival period of G27 tumor-bearing mice in each group.

[0038] Figure 6 This is a graph showing the concentration of Forodesine in mouse brain and blood samples in Example 5, wherein:

[0039] Figure A is 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 detected by mass spectrometry.

[0041] Figure 7 The figures are the results of different concentrations of Forodesine promoting the killing of tumor cells by T cells in Example 6, wherein Figure A is the result of Flow cytometry analysis of Forodesine promoting the killing of glioma cells GL261 by T cells;

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

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

[0044] Fig. 9 This is a curve diagram of tumor growth volume of mice given different doses of Forodesine in Example 7.

[0045] Fig.10 This is a graph showing the proportion of T cells that exert a killing effect in tumors of mice given different doses of Forodesine in Example 7. DETAILED DESCRIPTION

[0046] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following embodiments. All technical solutions implemented based on the above contents of the present invention belong to the scope of the present invention.

[0047] The chemical structure of Forodesine (BCX-1777) used in the above technical solution of the present 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 of leukemia cells by increasing dGTP levels.

[0048]

[0049] Forodesine received orphan drug status in the United States in 2004 for the treatment of T-cell non-Hodgkin's lymphoma and chronic myeloid leukemia; it received orphan drug status 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 new type of nucleoside metabolic inhibitor, Forodesine is a very 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 present invention, the mouse is an immunodeficient mouse Balb / c nude, which is also called a BALB / c-nu athymic nude mouse. In another embodiment of the present invention, the mouse is an immune normal mouse C57BL / 6J.

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

[0052] Forodesine used in the following examples was provided by Target Mol, with a product number of T15337 and a purity of 99.88%.

[0053] The 387GSC, 4121GSC, and 456GSC used in the following examples are all used in the literature (Hypoxic Induction of Vasorin Regulates Notch1 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 the public can obtain them from the applicant of the present invention. G27, G7, and G29 are tumor cells isolated from GBM patient samples by the applicant of the present invention, and the GBM patient samples are provided by the First Medical Center of the Chinese People's Liberation Army. G19 is a tumor cell isolated from GBM patient samples by the applicant of the present invention, and the GBM patient samples are provided by Beijing Fengtai Hospital. NHA cells (human astrocytes) were purchased from Beijing Benachuanglian 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: Experiments at the cellular level

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

[0059] The above 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. The cells to be tested were inoculated in a 96-well plate, with 2000 cells per well, and divided into five groups, namely, control group (DMSO), Forodesine-10μM treatment group, Forodesine-20μM treatment group, Forodesine-50μM treatment group, Forodesine-100μM treatment group, with 5 replicates in each group, and 200μL NBM complete medium was added to each well of the tumor cells, wherein the above-mentioned NBM complete medium was added with 5mL of penicillin-streptomycin (Zhongke Maichen Technology Co., Ltd., catalog number CC004), 10mL of B27-supplement serum substitute (ThermoFisher, catalog number 12580-010), 20ng / mL EGF (R&D Company, catalog number Cat#236-EG), and 20ng / mL b-FGF (R&D Company, Cat#4114-TC), 5 mL of L-glutamine (China Science and Technology Co., Ltd., Cat#CC009); 200 μL of DMEM complete medium per well of NHA cells, wherein the above DMEM complete medium is prepared by adding 5 mL of penicillin-streptomycin (China Science and Technology Co., Ltd., Cat#CM004) and 50 mL of fetal bovine serum (EXCELL Company, Cat#FSD500) to 500 mL of basal medium DMEM (China Science and Technology Co., Ltd., Cat#CM10013);

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

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

[0064] 4. Measure the cell viability of the cells in each well of the 96-well plate on day 0, day 2, day 4, and day 6: first, add 100 μL of cell viability detection solution CellTiter-Glo Luminescent Cell Viability Assay (Promega, catalog number: G7572) to each well, pipette 10 times with a 200 μL eight-row pipette to fully mix the cells and the cell viability detection solution, then transfer 200 μL of the mixture from each well to a black transparent bottom 96-well cell culture plate (Beyotime, catalog number: FCP965-48) for cell viability determination, place the black transparent bottom 96-well cell culture plate on a horizontal shaker in the dark, shake at 200 rpm for 10 minutes, and place the above-mentioned well plate on an ELISA reader to detect the fluorescence value;

[0065] 5. Since the intensity of the fluorescence signal is directly related to the amount of ATP, which in turn is directly related to the number of living cells, the number of living cells can be indirectly assessed by measuring the intensity of the generated light signal, thereby reflecting the cell viability. The fluorescence value of each group of cells on day 0 was averaged, and the ratio of the fluorescence value on day 2, day 4, and day 6 to the average fluorescence value on day 0 was used as the cell viability growth multiple. For a line graph of the experimental results, see Figure 1 .

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

[0067] Experimental conclusion: Forodesine can significantly inhibit the cell viability of glioma stem cells or glioma cells. The inhibitory effect of Forodesine on tumor cells increases with the increase of Forodesine concentration (directly proportional dose-effect relationship); while Forodesine has a lower inhibitory effect on the cell viability of NHA cells, which also reflects that Forodesine causes less damage to normal cells when used to treat gliomas and has lower side effects of treatment.

[0068] Example 2: Experiments at the cellular level

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

[0070] The above tumor cells were treated with different concentrations of Forodesine, and then the apoptosis ratio of the tumor cells was detected 48 hours after the addition of Forodesine.

[0071] The specific implementation steps are as follows:

[0072] 1. Inoculate the cells to be tested in a 6-well plate, 3x10 5 cells, and divided them into 3 groups, namely, a control group (DMSO), a Forodesine-50μM treatment group, and a Forodesine-100μM treatment group, with 1.5mL NBM complete medium for each well of tumor cells, wherein the NBM complete medium is prepared by adding 20ng / mL EGF (R&D Company, Cat#236-EG) and 20ng / mL bFGF (R&D Company, Cat#4114-TC), 10mL B27-supplement serum replacement (ThermoFisher Company, 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 basal medium Neurobasal Mediμm (Thermo Fisher Company, Cat#12348-017);

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

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

[0075] 4. At 48 hours after adding Forodesine, the apoptotic ratio of cells in each well of the 6-well plate was determined: first, the tumor cells were digested into single cells with trypsin Accutase (Sigma, Catalog No.: 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 No. CC008) to remove the trypsin residue as much as possible, and the experiment was carried out according to the steps provided by the cell apoptosis detection kit (Pulile, Catalog No.: P04D03);

[0076] 5. After the previous step, flow cytometry was used to detect the apoptotic ratio of cells in different treatment groups. FlowJo software was used to process the data and plot the graphs. The experimental results are shown in Figure 3 .

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

[0078] Example 3: Experiments at the cellular level

[0079] Tumor cells to be tested: G27.

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

[0081] The specific implementation steps are as follows:

[0082] 1. Hydration probe plate:

[0083] (1) Take 20 mL of hydration solution (Agilent, Catalog No.: 100840-000) in a 50 mL centrifuge tube and place it in the Pre Station next to the Seahorse XFe96 equipment. Preheat it to 37°C for use.

[0084] (2) Open the sensor cartridge set (Agilent, catalog number: 102416-100), remove the two plates on the top for drug addition (to be used on the second day), then pick up the lid and the 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 card, gently insert the probe card into the plate with the added liquid, cover the lid and mark the identification information (name, time, etc.), and after inspection, place it in an open 37°C CO2-free incubator (Pre Station) and incubate for 2 hours. Check whether there are bubbles in the probe hole. If there are bubbles, gently move the probe card up and down to remove the bubbles. Incubate the probe card overnight in the Pre Station (more than 12 hours).

[0086] 2. Cell culture:

[0087] (1) Before the test, the tumor cells G27 to be tested are prepared in the laboratory in advance and checked to see whether they have been cultured to a confluence of about 80%.

[0088] (2) Take out the blue-packaged cell culture plate XFe96 Cell Culture Microplates (Nunc, catalog number: 165306), mark the lid, and determine the position of each experimental group. The cell G27 tested in this example is a suspension cell, so the cell culture plate needs to be treated with glue to assist the cell adhesion. The experimental glue is Matrigel (Corning, catalog number: 354277). Add 50 μL of Matrigel to each well and place the cell plate in a 37°C constant temperature CO2 incubator for 1 hour.

[0089] (3) Digest G27 cells with trypsin to make single cells, add culture medium to make single cell suspension, and count the cells with a cell counter. Seed 1x10 cells per well. 4 cells / 80μL, 80μL of culture medium was added to each of the four background wells. The cells were divided into two groups: Forodesine-0μM group and Forodesine-50μM group, with 4 replicate wells in each group. Forodesine with final concentrations of 0μM and 50μM was added to the corresponding wells.

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

[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 glutamine with an initial concentration of 200 mM to 100 mL of preheated basal medium (Agilent, Catalog No.: 102353-100), and adjust the pH of the solution to 7.4±0.05 with 1N NaOH. After preparation, filter the solution into a 50 mL centrifuge tube using a 0.22 μm small filter, wrap it with tin foil, and preheat it in a 37°C water bath for 1 h.

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

[0094] 4. Replace cell culture medium:

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

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

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

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

[0099] 5. Add reagents to the machine:

[0100] (1) Prepare the drug, sample loading tank, 100μL sample gun and 200μL pipette tip. This step can be performed while waiting after the cell solution is replaced. Place the drug loading plate corresponding to the port on the probe plate (A, B, C, D), add 25μL / well of the corresponding concentration of Forodesine to each well (remember to change the loading plate during the process), and then place it in a 37℃ CO2-free incubator.

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

[0102] (3) All wells set for injection must not be left empty and must be filled with 80 μL of PBS.

[0103] (4) After setting the corresponding parameter information, perform probe calibration. After 25-30 minutes, the probe calibration is completed. Click "OK" to pop up the hydration plate (Utility Plate) below. Then put in the cell plate with the lid removed and click "I'm Ready" to start the experiment.

[0104] (5) After 1.5-2 hours, the program ends. Click "OK" to eject the board. After removing the board, click again to store the tray. Check and process the data. For the experimental results, see Figure 4 .

[0105] Example 4: Experiments at the animal level

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

[0107] Tumor cells to be tested: G27.

[0108] The specific implementation steps are as follows:

[0109] 1. Lentivirus packaging

[0110] 3×10 6 HEK293TN cells were seeded in a 10 cm culture dish and cultured with DMEM complete medium, wherein the DMEM complete medium was 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 basal medium DMEM (Zhongke Maichen Technology Co., Ltd., catalog number CM10013); lentiviral packaging plasmids psPAX2 (5 μg) and pCI-VSVG (5 μg) and pLenti CMV V5-LUCBlast plasmid (5 μg) were transfected into HEK293TN cells respectively using a calcium phosphate transfection kit (Zhongke Maichen Technology Co., Ltd., catalog number: Ctk001), and new DMEM complete medium was replaced 5 h after transfection. After 60 hours of cell culture, the virus supernatant was collected and centrifuged at 2500rpm for 5 minutes. The virus supernatant was filtered with a 0.45μm filter to remove impurities such as cell debris. The filtered virus was collected in a 15mL centrifuge tube, and 5xPEG lentivirus concentrate (Best Innovation Company, Catalog No.: C103-05) was added to concentrate the virus. The centrifuge tube was placed in a 4℃ refrigerator for concentration for 12 hours. The centrifuge tube was centrifuged at 7,000rpm for 10 minutes at 4℃, the supernatant was removed, and the virus at the bottom of the tube was washed with 1mL of PBS and mixed and transferred to a 1.5mL EP tube. It was centrifuged at 15,000rpm at 4℃ for 15 minutes to remove impurities such as residual serum. The supernatant containing pLenti CMVV5-LUC Blast virus after centrifugation was stored in a -80℃ refrigerator for use.

[0111] 2. Infect G27 with lentivirus to obtain G27-luciferase-blasticidin cells

[0112] 2×10 6G27 cells were inoculated in 10 cm culture dishes, with 8 mL of NBM complete medium in each dish, wherein the NBM complete medium was prepared by adding 20 ng / mL EGF (R&D, Cat#236-EG), 20 ng / mL bFGF (R&D, Cat#4114-TC), 10 mL B27-supplement serum replacement (ThermoFisher, Cat#12580-010), 5 mL penicillin-streptomycin (Zhongke Maichen Technology Co., Ltd., Cat#CC004), and 5 mL of L-glutamine (Zhongke Maichen Technology Co., Ltd., Cat#CC009) to 500 mL of basal medium Neurobasal Medium (Thermo Fisher, Cat#12348-017); 200 μL of concentrated lentivirus containing pLenti CMV V5-LUC Blast was added, the cells were shaken and placed in a 37°C constant temperature carbon dioxide incubator for 48 h. The cultured cells were digested into single cells using trypsin Accutase, re-seeded in a 10 cm dish with NBM complete medium, and the antibiotic blasticidin (final concentration in the system was 10 μg / mL) was added for cell screening.

[0113] 3. Identification of luciferase expression in G27

[0114] The G27 infected with lentivirus was placed in a 96-well plate, and 1 μL of luciferin (substrate of luciferase) was added to the plate (protected from light). After the substrate reacted with the cells for 3 minutes, the plate was placed in a living imager (PerkinElmer) to detect the cell fluorescence value. The G27 positive cells infected with lentivirus had strong fluorescence.

[0115] 4. Resuspend the G27 cells that stably express luciferase in the previous step with NBM basal medium (Neurobasal Medium, Thermo Fisher, Cat. No. 12348-017), count the cells, and dilute to 5x10 4 The cells were injected orally into the right frontal lobe of the brain of immunodeficient Balb / c nude mice using a 100 mL insulin injection syringe (BD, catalog number: 320310) to construct a GBM transplant tumor model. Each mouse was injected with 5×10 4 After the injection of cells, the growth of mouse tumors was detected by in vivo imaging every other week. After in vivo imaging on the 8th day, the mice were evenly divided into two groups according to the tumor size: a control group and a Forodesine-treated group, with 8 mice in each group. Each group was treated as follows:

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

[0117] Forodesine administration group: 100 μL of Forodesine was administered by gavage every other day (the mice were weighed and administered at 30 mg / kg).

[0118] Live imaging was used to observe tumor growth in real time and record the survival of tumor-bearing mice. The experimental results can be found in Figure 5 .

[0119] Experimental conclusion:

[0120] After collating and analyzing the experimental results, it was found that compared with the control group (pure water), the tumor growth of mice in the Forodesine-treated group was significantly slowed down, and the survival period of mice was also extended to a certain extent. After the average grouping on the 8th day, there was no significant difference in the size of the mouse tumors. After that, the control group was given pure water, and the experimental group continued to be treated with Forodesine. From the experimental results of in vivo imaging on the 15th and 22nd days, the tumor size of the drug-treated group was significantly lower than that of the control group, which shows that Forodesine has the effect of inhibiting tumor growth.

[0121] It can be seen that Forodesine can inhibit the growth of GBM transplanted tumors, which has important implications for the clinical treatment of glioma patients. The applicant of the present invention first discovered that Forodesine has an inhibitory effect on glioma cells and can inhibit the growth of gliomas. Therefore, Forodesine or a pharmaceutically acceptable salt thereof can be used as a potential drug for the treatment of brain tumors.

[0122] Example 5: Experiments at the animal level

[0123] Experimental animals: Immunized normal mice C57BL / 6J (Beijing Weitonglihua Experimental Animal Technology Co., Ltd., 4 weeks old)

[0124] Experimental purpose: To test whether Forodesine can enter the blood-brain barrier

[0125] The specific implementation steps are as follows:

[0126] 1. Lentivirus packaging

[0127] 3×10 6HEK293TN cells were seeded in a 10 cm culture dish and cultured with DMEM complete medium, wherein the DMEM complete medium was 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 basal medium DMEM (Zhongke Maichen Technology Co., Ltd., catalog number CM10013); lentiviral packaging plasmids psPAX2 (5 μg) and pCI-VSVG (5 μg) and pLenti CMV V5-LUCBlast plasmid (5 μg) were transfected into HEK293TN cells respectively using a calcium phosphate transfection kit (Zhongke Maichen Technology Co., Ltd., catalog number: Ctk001), and new DMEM complete medium was replaced 5 h after transfection. After 60 hours of cell culture, the virus supernatant was collected and centrifuged at 2500rpm for 5 minutes. The virus supernatant was filtered with a 0.45μm filter to remove impurities such as cell debris. The filtered virus was collected in a 15mL centrifuge tube, and 5xPEG lentivirus concentrate (Best Innovation Company, Catalog No.: C103-05) was added to concentrate the virus. The centrifuge tube was placed in a 4℃ refrigerator for concentration for 12 hours. The centrifuge tube was centrifuged at 7,000rpm for 10 minutes at 4℃, the supernatant was removed, and the virus at the bottom of the tube was washed with 1mL of PBS and mixed and transferred to a 1.5mL EP tube. It was centrifuged at 15,000rpm at 4℃ for 15 minutes to remove impurities such as residual serum. The supernatant containing pLenti CMVV5-LUC Blast virus after centrifugation was stored in a -80℃ refrigerator for use.

[0128] 2. Infect GL261 with lentivirus to obtain GL261-luciferase-blasticidin cells

[0129] 2×10 6 GL261 cells were inoculated in 10 cm culture dishes, with 8 mL of DMEM complete medium per dish, wherein the above DMEM complete medium was prepared by adding 5 mL of penicillin-streptomycin (Zhongke Maichen Technology Co., Ltd., item number CC004) and 50 mL of fetal bovine serum (EXCELL, item number FSD500) to 500 mL of basal medium DMEM (Zhongke Maichen Technology Co., Ltd., item number CM10013); 200 μL of concentrated lentivirus containing pLenti CMV V5-LUC Blast was added, and the cells were shaken and placed in a 37°C constant temperature carbon dioxide incubator for 48 h. Digested into single cells with trypsin (Zhongke Maichen Company, item number CC012), re-inoculated in a 10 cm dish with DMEM complete medium, and added with antibiotic blasticidin (final concentration in the system was 10 μg / mL) for cell screening.

[0130] 3. Identification of luciferase expression in GL261

[0131] GL261 infected with lentivirus was placed in a 96-well plate, and 1 μL of luciferin (substrate of luciferase) was added to the plate (protected from light). After the substrate reacted with the cells for 3 minutes, the plate was placed in a living imager (PerkinElmer) to detect the cell fluorescence value. GL261 positive cells infected with lentivirus had strong fluorescence.

[0132] 4. Sample collection

[0133] The GL261 cells stably expressing luciferase in the previous step were resuspended in DMEM basal medium (Zhongke Maichen Technology Co., Ltd., Catalog No. CM10013) and the cells were counted and diluted to 8x10 4 The cells were injected orally into the right frontal lobe of the brain of immune-normal C57BL / 6J mice using a 100 mL insulin injection syringe (BD, catalog number: 320310) to construct a GBM transplant tumor model. Each mouse was injected with 8×10 4 The growth of the tumor in mice was detected by in vivo imaging on the 10th day after the injection of cells. The mice were divided into three groups, 7 mice in each group, as follows: Ctrl group (no tumor formation, no drug administration), Blank+forodesine group (no tumor formation, drug administration), Tumor+forodesine group (tumor formation, drug administration), and each group was treated as follows:

[0134] Ctrl group (no tumor formation, no medication): Orbital blood was collected from mice, 300 μL of blood was collected from each mouse, and after blood collection, the mice were perfused by heart, and then the brains were removed.

[0135] Blank+forodesine group (non-tumor-forming administration): 100 μL of Forodesine was administered by gavage (the mice were weighed and administered at 30 mg / kg). After 4 hours, blood was collected from the eye sockets of the mice, 300 μL of blood was collected from each mouse. After blood collection, the mice were perfused through the heart and the brains were removed.

[0136] Tumor+forodesine group (tumor administration): 100 μL of Forodesine was administered by gavage to mice with tumors detected by in vivo imaging (the mice were weighed and administered at 30 mg / kg). After 4 hours, blood was collected from the eye sockets of the mice, 300 μL of blood was collected from each mouse. After blood collection, the mice were perfused by heart and their brains were removed.

[0137] 5. Sample pretreatment and testing

[0138] (1) Blood sample processing: The blood samples were centrifuged at 12000 rpm for 10 min at 4°C. The supernatant samples were transferred to new 1.5 mL EP tubes and stored in a -80°C refrigerator 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 placed in liquid nitrogen for freezing for 10 min. Grinding beads were added to the tubes and ground for 10 min until they were ground into powder. Each tube of samples was resuspended in 1 mL of 80% methanol (v / v), placed in a -80°C refrigerator for 1 h, and centrifuged at 14,000 rpm for 20 min at 4°C. The supernatant samples were transferred to new 1.5 mL EP tubes and stored in a -80°C refrigerator for later use.

[0140] The samples after the above pretreatment were sent to the Pharmaceutical Technology Center of Tsinghua University for mass spectrometry detection. The experimental results are shown in Figure 6 .

[0141] Experimental conclusion:

[0142] After analyzing the experimental results, it was found that compared with the Ctrl group, the mice in the Blank+forodesine group and the Tumor+forodesine group had obvious accumulation of Forodesine in the brain, and the Forodesine concentration in the Tumor+forodesine group was significantly higher than that in the Blank+forodesine group. Similarly, a certain concentration of Forodesine was detected in the blood of the Blank+forodesine group and the Tumor+forodesine group, which shows that after oral administration to mice, Forodesine was absorbed into the blood and entered the brain to reach the tumor area.

[0143] This shows that after Forodesine is absorbed into the blood in mice, it reaches the brain and can penetrate the blood-brain barrier to reach the tumor area, which confirms the reliability of Forodesine in the treatment of brain glioma.

[0144] Example 6: Experiments at the cellular level

[0145] Tumor cells to be tested: GL261.

[0146] Experimental mice: immune normal mice C57BL / 6J (Beijing Weitonglihua Experimental Animal Technology Co., Ltd., 6 weeks old).

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

[0148] The specific implementation steps are as follows:

[0149] 1. Isolation and activation of T cells from mouse spleen: Take one C57BL / 6J mouse (purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., 6-week-old female), kill the mouse by cervical dislocation, spray the mouse body with 75% ethanol (v / v) for disinfection, dissect the body to remove the spleen, and soak the spleen in the double antibody of 1.5mL EP tube to avoid contamination. Soak and rinse the spleen with physiological saline in the clean bench, place a 70μm filter (Biosharp, catalog number: BS-70-XBS) on a 50mL centrifuge tube, wet the filter with 2mL TIB solution (PBS+1% BSA+2mM EDTA), place the spleen on the filter, use a 2mL syringe, pull out the stopper, squeeze the spleen up and down with the rubber head of the stopper to crush it, rinse the cells into the centrifuge tube with TIB solution, centrifuge at 1,500rpm for 3min, and discard the supernatant. Add 1mL ACK lysing buffer (Thermo Fisher Scientific, Catalog No.: A1049201) to the centrifuge tube to lyse the red blood cells for 1min, add 4mL TIB solution to neutralize, filter with a 40μm filter (Biosharp, Catalog No.: BS-40-XBS), rinse the residual cells on the filter into the tube with TIB solution, centrifuge at 1,500rpm for 3min, and discard the supernatant. Resuspend the cell pellet with 100μL TIB. (The following steps should be kept away from light) Add 10μL Biotiu-Antibody coektail (Miltenyi Biotec, Catalog No.: 130-104-075), mix with a pipette, and let stand at room temperature for 5min. Add 30μL TIB and 20μL Anti-Biotin microbeads (Miltenyi Biotec, Catalog No.: 130-104-075), mix with a pipette, and let stand at room temperature for 10min. Install the LS column (Miltenyi Biotec, catalog number: 130-042-401), wet it with 3 mL of TIB, pass the cell suspension through the column, and use a 15 mL centrifuge tube to receive CD8+T cells under the column. The liquid should not be interrupted after adding TIB to wet it. Finally, rinse the centrifuge tube with TIB to pass the liquid through the column. After the liquid is fully drained, remove the LS column. The entire column process is not pressurized. The collected CD8+T cell suspension is centrifuged at 1,500 rpm for 3 minutes, and the supernatant is discarded. Resuspend the cell pellet in RPMI1640 complete medium, wherein the above RPMI1640 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 basal medium RPMI1640 (Thermo Fisher Scientific, catalog number 21870076).

[0150] 2. Count the separated CD8+T cells and inoculate them into 6-well plates (pre-coated with CD3 antibody + CD28 antibody, CD3: 2.92μg / mL, CD28: 2.82μg / mL), inoculating 2x10 in each well. 6 1 / 1000 of mercaptoethanol and 10 ng / mL of IL-2 were added to each well to promote T cell proliferation.

[0151] 3. Inoculate the tumor cells GL261 to be tested in a 10 cm culture dish, 2 x 10 6 The cells were divided into three groups, namely, a control group (DMSO), a Forodesine-50 μM treatment group, and a Forodesine-100 μM treatment group. Each dish of tumor cells contained 8 mL of DMEM complete medium, wherein the DMEM complete medium was 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 basal medium DMEM (Zhongke Maichen Technology Co., Ltd., catalog number CM10013).

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

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

[0154] 6. After 48 hours of cell culture, the cells were digested into single cells with trypsin (Zhongke Maichen Company, catalog number: CC012), washed with PBS and resuspended with DMEM complete medium. After counting, the cells were inoculated into 24-well plates, with 5x10 cells per well. 4 The culture dish with the cells was placed in a 37°C constant temperature carbon dioxide incubator for overnight culture.

[0155] 7. Co-culture the activated T cells with Forodesine-treated tumor cells. 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, GL261+Forodesine-100μM+T cell group. Three duplicate wells were set up for each group. 2.5x10 5 T cells were added to make the ratio of T cells to tumor cells be 5:1, and the three groups, GL261+Forodesine-0μM group, GL261+Forodesine-50μM group, and GL261+Forodesine-100μM group, were supplemented with an equal volume of RPMI1640 complete medium, and the 24-well plates seeded with cells were placed in a 37°C constant temperature carbon dioxide incubator for culture, and T cells and tumor cells GL261 were co-incubated and cultured for 24 hours.

[0156] 8. Digest the cells in the well plate with Accutase trypsin (Sigma, catalog number: A6964), wash three times with PBS, stain the washed cells, label T cells with CD8-APC, stain at room temperature for 15 minutes, centrifuge at 1500 rpm for 3 minutes, and discard the supernatant. Label the dead tumor cells with Annexin V-FITC, stain at room temperature for 10 minutes, supplement each sample with 200 μL PBS and perform flow cytometry analysis. The experimental results are shown in Figure 7 .

[0157] Experimental conclusion:

[0158] Forodesine treatment will lead to an increase in the proportion of apoptotic tumor cells, and the proportion of apoptotic cells will increase with the increase of Forodesine concentration (direct proportional dose-effect relationship), that is, the proportion of apoptotic cells has a Forodesine concentration-dependent trend. The proportion of tumor cells killed by T cells will increase with the increase of Forodesine concentration used to treat tumor cells, that is, the proportion of tumor cells killed by T cells has a Forodesine concentration-dependent trend.

[0159] Example 7: Experiments at the animal level

[0160] Experimental animals: Immunized normal mice C57BL / 6J (Beijing Weitonglihua Experimental 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 tumor-bearing mice: Expand the tumor cells GL261 to be tested to at least 2.4x10 7 The tumor cells to be tested were resuspended in PBS and then the cells were counted and diluted to 8x10 5 The cells were injected into the subcutaneous part of the right abdomen of immune normal mice C57BL / 6J using a 1 mL common injection syringe (Sinopharm, Cat. No.: 92311609) to construct a mouse subcutaneous transplant tumor model. Each mouse was injected with 8x10 5 The tumor size was measured on the 9th day after the injection of cells, after the tumor was formed. The tumor size was measured every other day. The long diameter and short diameter of the tumor were measured with an electronic vernier caliper. The tumor volume was calculated as: long diameter x short diameter 2 ÷2, the mice were evenly divided into three groups according to the tumor size: control group (water group), Forodesine-10mg / kg group and Forodesine-30mg / kg group, 10 mice in each group, and each group was treated as follows:

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

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

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

[0167] On the 27th day, when the tumor grew to a certain size, the mice were killed by cervical dislocation. The subcutaneous tumors were removed and photographed to compare the tumor sizes of different experimental groups. The data of monitoring the changes in tumor volume size were plotted into a curve graph. The experimental results are shown in Figure 8 , Fig. 9 .

[0168] 2. After taking photos of the subcutaneous tumor, separate the tumor-infiltrating T cells and detect the activation ratio.

[0169] Prepare a 12-well plate, pour 1.5 mL of DMEM complete medium into each well, and temporarily store the removed subcutaneous tumor in the well plate to maintain cell activity. Transfer the tumor to a separation tube gentleMACS C Tubes (Miltenyi Biotec, catalog number: 130-093-237), add tumor separation buffer, and cut it into pieces with scissors to a size of 2-4 mm. The preparation steps of the above tumor separation 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, and 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 to each sample (wherein, the kit containing lysozyme D, lysozyme R, lysozyme A, and Buffer A is purchased from Miltenyi Biotec, catalog number: 130-096-730). The separation tube was placed on a tissue grinder (Miltenyi Biotec) and the program (37C_m_TDK_1) was set to automatically grind and digest for 40 minutes.

[0170] After grinding and digestion, PBS with serum (PBS + 1% FBS) was added to dilute the cell suspension. 3 mL of PBS was added to each sample. The diluted cell suspension was filtered through a 70 μm filter (Biosharp, Catalog No.: BS-70-XBS), centrifuged at 1,500 rpm for 3 min at room temperature, and the supernatant was removed. 2 mL of red blood cell lysis buffer (Thermo Fisher Scientific, Catalog No.: A1049201) was added to each sample for lysis. The lysis was carried out at room temperature for 2 min. 5 mL of PBS with serum (PBS + 1% FBS) was added to each sample for dilution. The diluted cell suspension was filtered through a 40 μm filter (Biosharp, Catalog No.: BS-40-XBS), centrifuged at 1,500 rpm for 3 min at room temperature, and the supernatant was removed.

[0171] Resuspend the cells with an appropriate amount of PBS with serum (PBS + 1% FBS) according to the cell size, and take 5x10 6 5x10 cells were taken from each sample for subsequent protein secretion blocking treatment. 6 The cells were resuspended in 2 mL of RPMI1640 complete medium and inoculated into 12-well plates. 1 / 500 volume of culture medium protein secretion blocker (Thermo Fisher Scientific, catalog number: 00-4980-03) was added to each sample. The plate seeded with cells was placed in a 37°C constant temperature carbon dioxide incubator (SANYO, catalog number: MCO-5AC) for 4 h of culture.

[0172] The cells in the well plate were digested with Accutase trypsin (Sigma, Catalog No. A6964), washed three times with PBS, and the washed cells were stained with CD45-PerCP antibody (used at 1:200, Dakoway, Catalog No. 109828) to mark lymphocytes, CD8-FITC antibody (used at 1:200, Dakoway, Catalog No. 100706) to mark CD8+ T lymphocytes, and the fixable viability dye eFluor TM 780 (1:1000, Thermo Fisher Scientific, catalog number: 65-0865-18) was used to label dead cells. 100 μL of the staining system was added to each sample. The samples were stained at room temperature for 15 min, centrifuged at 1,500 rpm for 3 min, and the supernatant was discarded.

[0173] Cells were fixed with cell fixative (Thermo Fisher Scientific, Catalog No.: 00-5223-56) at room temperature for 1 hour, wherein the preparation steps of the cell fixative were as follows: 30 μL of fixative + 90 μL of buffer for each sample, centrifuged at 1,500 rpm for 3 minutes at room temperature, and the supernatant was removed. Cells were permeabilized with cell permeabilization solution (Thermo Fisher Scientific, Catalog No.: 00-8333-56) at room temperature for 10 minutes, wherein the preparation steps of the cell permeabilization solution were as follows: 10 μL of fixative + 90 μL of sterile water for each sample, centrifuged at 1,500 rpm for 3 minutes at room temperature, and the supernatant was removed. The intracellular IFNg and GZMB were stained. The IFNg in the cells was labeled with IFNg-PE antibody (used at 1:200, Dakoway, Catalog No.: 505808), and the GZMB in the cells was labeled with GZMB-PE-Cy7 antibody (used at 1:200, Dakoway, Catalog No.: 372214). 100 μL of the staining system was used for each sample. After staining at room temperature for 30 minutes, the samples were analyzed by flow cytometry. The experimental results are shown in Fig.10 .

[0174] Experimental conclusion:

[0175] After collating and analyzing the experimental results, it can be found that compared with the control group (water), the tumor growth of mice in the Forodesine-treated group was significantly slowed down, and the high-concentration Forodesine-treated group (30 mg / kg) had the best tumor inhibition effect. There was no significant difference in the size of the mouse tumors after average grouping on the 9th day. After that, the control group was given water, and the experimental group continued to be treated with different doses of Forodesine. From the tumor size results taken on the 27th day, the tumor size of the Forodesine-treated group was significantly lower than that of the control group, and the tumor size of the high-dose group (30 mg / kg) was lower than that of the low-dose group (10 mg / kg), indicating that the inhibitory effect of Forodesine on tumor growth is related to the dose of Forodesine. By collating the experimental results of flow analysis, it can be found that Forodesine administration promoted the increase in the proportion of IFNg+CD8+ T cells and the proportion of GZMB+CD8+ T cells, thereby making the anti-tumor immune effect better and inhibiting tumor growth.

[0176] It can be seen that Forodesine or a pharmaceutically acceptable salt thereof can inhibit the growth of glioma by enhancing anti-tumor immunity, and provides a new treatment method 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 treating brain tumors.

2. The use according to claim 1, wherein The brain tumor is a glioma.

3. The use according to claim 2, wherein The brain tumor is glioblastoma.

4. The use according to any one of claims 1 to 3, wherein The therapeutically effective amount of Forodesine or a pharmaceutically acceptable salt thereof is 8-35 mg / kg body weight.

5. The use according to claim 4, wherein The therapeutically effective amount is 10-30 mg / kg body weight.

6. The use according to any one of claims 1 to 3, wherein The Forodesine or its pharmaceutically acceptable salt is administered orally.

7. The use according to claim 4, wherein The pharmaceutically acceptable salt of Forodesine is Forodesine hydrochloride.

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