Application of stiripentanol in preparation of medicine for resisting diffuse midline glioma

Abstract

The invention relates to application of stiripentol in preparation of a medicine for treating diffuse midline glioma, and belongs to the technical field of biological medicine. It is found for the first time that the medicine stiripentanol for treating pediatric epilepsy can effectively inhibit tumor growth of mice with diffuse midline glioma and remarkably prolong the lifetime of the mice with the tumor, and meanwhile, the medicine stiripentanol can activate the tumor immune microenvironment and enhance the anti-tumor immune response; in addition, when the stiripentanol and the chemotherapeutic drug are combined for use, the DNA damage effect of the chemotherapeutic drug on tumor cells can be synergistically enhanced, and the curative effect of chemotherapy is remarkably improved. The invention discloses the new application of the stiripentanol in the treatment of the diffuse midline glioma for the first time, expands the clinical adaptation disease range of the stiripentanol, and provides an innovative strategy for developing an efficient and low-toxicity combined treatment scheme based on the known clinical safety and low-toxicity characteristics of the stiripentanol.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and specifically to the application of stiripentol in the preparation of a medicament for treating diffuse midline glioma. Background Art

[0002] Diffuse midline glioma (DMG) is a highly malignant pediatric central nervous system tumor characterized by H3K27M mutation at the epigenetic level. It commonly occurs in key functional areas such as the brainstem, thalamus, and spinal cord, accounting for approximately 10%-15% of pediatric brain tumors and 75% of pediatric brainstem tumors. Due to its deep anatomical location and strong invasiveness, the risk of surgical resection is extremely high, and radical resection cannot be achieved in more than 90% of cases. Although traditional radiotherapy and chemotherapy can briefly relieve symptoms, tumor cells rapidly develop resistance to treatment, and the median survival of patients is only 9-15 months, with a two-year survival rate of less than 10%. There is an urgent clinical need for breakthrough treatment strategies. In recent years, studies have found that the malignant phenotype of DMG is closely related to epigenetic dysregulation and metabolic reprogramming. For example, the H3K27M mutation leads to the abnormal activation of oncogenes by inhibiting histone H3K27 trimethylation (H3K27me3), and at the same time, tumor cells rely on glycolysis to produce a large amount of lactic acid to maintain the global hypomethylation state of the genome. In response to these mechanisms, epigenetic drugs (such as histone deacetylase inhibitors) and metabolic intervention methods (such as isocitrate dehydrogenase 1 inhibitors) have become research hotspots, but their single-agent efficacy is limited and lacks clinical translation verification.

[0003] Stiripentol, a clinically safe drug approved for the treatment of epilepsy, in addition to regulating neurotransmitters, recent studies have revealed that it can significantly reduce the intracellular lactic acid level in tumor cells by inhibiting the activity of lactate dehydrogenase (LDH), thereby interfering with the lactylation modification of the key DNA damage repair protein NBS1 and reversing chemotherapy resistance. This discovery provides a theoretical basis for the repositioning of stiripentol in tumor treatment. It is worth noting that our research has found that the commonly existing H3K27M mutation in DMG is closely related to abnormal high lactic acid metabolism, and its drug resistance may rely on lactate-mediated epigenetic remodeling and enhanced DNA repair. However, the therapeutic potential of stiripentol for DMG has not been reported in the prior art, nor has its synergistic effect with standard chemotherapy or radiotherapy been explored. Therefore, developing new uses of stiripentol in the treatment of DMG, especially combination treatment regimens based on the lactate metabolism-epigenetic regulation axis, is expected to break through the existing treatment bottleneck and provide an innovative solution for improving the survival prognosis of patients. Summary of the Invention

[0004] In view of the deficiencies of the prior art, the present invention provides a new application of stiripentol in the preparation of drugs for treating diffuse midline glioma. By developing a new indication of stiripentol, a drug clinically approved for the treatment of epilepsy, in the treatment of diffuse midline glioma, the bottlenecks of strong tumor drug resistance, low chemotherapy sensitivity, and poor patient survival prognosis in existing clinical treatments are to be broken through.

[0005] To achieve the object of the present invention, the technical solutions adopted are as follows:

[0006] The present invention for the first time confirms through research that stiripentol can significantly inhibit the tumor growth of mice with diffuse midline glioma and prolong the survival period of tumor-bearing mice. Through in vivo experiments, it is verified that after stiripentol treatment, the tumor volume is significantly reduced compared with the control group, and the median survival period of tumor-bearing mice is significantly prolonged. At the same time, stiripentol can activate the tumor immune microenvironment, specifically manifested as promoting the infiltration of T cells and natural killer (NK) cells into the tumor tissue, and up-regulating the expression of tumor-killing effector molecules such as IFNγ in the above cells, thereby synergistically enhancing the anti-tumor immune response. Therefore, in the first aspect, the present invention proposes an application of stiripentol in the preparation of drugs for treating diffuse midline glioma.

[0007] As a preferred embodiment of the application of the present invention, the stiripentol exerts anti-tumor effects through at least one of the following methods: (a) inhibiting the proliferation of diffuse midline glioma cells, reducing the tumor volume and prolonging the survival period of tumor-bearing individuals; (b) activating the tumor immune microenvironment and promoting the polarization of tumor-associated macrophages to an anti-tumor phenotype; (c) combined with chemotherapy drugs to synergistically enhance the DNA damage effect of chemotherapy drugs on tumor cells.

[0008] As a preferred embodiment of the application of the present invention, the tumor is diffuse midline glioma, and the diffuse midline glioma is pediatric midline glioma carrying H3K27M mutation.

[0009] As a preferred embodiment of the application of the present invention, the effective dose of stiripentol is 50 - 300 mg / kg / day. The most preferred effective dose is 150 mg / kg / day.

[0010] In the second aspect, the present invention discloses a pharmaceutical composition for treating diffuse midline glioma, comprising stiripentol as an active ingredient and a pharmaceutically acceptable carrier or excipient.

[0011] As a preferred embodiment of the application of the present invention, the dosage form of the pharmaceutical composition is an oral preparation or an injection preparation. The oral preparation includes tablets, capsules or suspensions, and the injection preparation includes lyophilized powder for injection or injection solution.

[0012] The present invention discovers through research that when stiripentol is combined with chemotherapy drugs cisplatin or etoposide, it can significantly reduce the drug resistance of primary cells of diffuse midline glioma derived from patients to chemotherapy drugs. It is confirmed by immunofluorescence detection of γ-H2AX foci (DNA damage markers) that the formation of γ-H2AX foci in cells of the stiripentol combined with cisplatin or etoposide group is significantly increased compared with the group using cisplatin or etoposide alone; flow cytometry further shows that the γ-H2AX positive cells in the combination drug group are significantly increased compared with the single drug group. The above results indicate that stiripentol reverses chemotherapy drug resistance by interfering with the DNA damage repair ability of tumor cells, thereby producing a synergistic anti-tumor effect with chemotherapy drugs. Therefore, in the third aspect, the present invention provides a combined drug composition for treating diffuse midline glioma.

[0013] As a preferred embodiment of the application of the present invention, the stiripentol is used in combination with a chemotherapy drug, and the chemotherapy drug is selected from at least one of DNA damage drugs including platinum-based drugs or poly ADP-ribose polymerase inhibitors.

[0014] As a preferred embodiment of the application of the present invention, the chemotherapy drug is cisplatin or etoposide.

[0015] As a preferred embodiment of the application of the present invention, the stiripentol is used in combination with cisplatin, the molar concentration of cisplatin is 3 μmol / L, and the molar concentration of stiripentol is 125 μmol / L.

[0016] As a preferred embodiment of the application of the present invention, the stiripentol is used in combination with etoposide, the molar concentration of etoposide is 0.25 μmol / L, and the molar concentration of stiripentol is 125 μmol / L.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0018] The present invention first confirms that the marketed epilepsy treatment drug stiripentol used alone can effectively inhibit the tumorigenesis process of diffuse midline glioma, and significantly prolong the median survival period of tumor-bearing mice by inhibiting tumor cell proliferation and inducing immune microenvironment reprogramming; in addition, the present invention discovers through research that when stiripentol is combined with DNA damage chemotherapy drugs, it can reverse its inherent chemotherapy drug resistance by interfering with the DNA damage repair ability of tumor cells, and significantly enhance the DNA damage effect of chemotherapy drugs on tumor cells. The present invention first reveals a new indication of stiripentol in the treatment of diffuse midline glioma, expanding its clinical application scope from epilepsy treatment to the field of malignant tumors. Based on its proven clinical safety and tolerance characteristics, stiripentol used alone or in combination with DNA damage chemotherapy drugs can provide a new treatment strategy for patients with diffuse midline glioma. Brief Description of the Drawings

[0019] Figure 1 This is the result graph of the effect of stiripentol on the lactic acid content of patient-derived diffuse midline glioma cells in Example 1 of the present invention.

[0020] Figure 2 This is the result graph of the construction of a spontaneous tumor-bearing mouse model of diffuse midline glioma in Example 2 of the invention.

[0021] Figure 3 This is the result graph of the effect of stiripentol on the tumor formation size and survival outcome of mice with diffuse midline glioma in Example 2 of the present invention.

[0022] Among them, Figure A: The result of the effect of stiripentol on the tumor formation size of mice with diffuse midline glioma; Figure B: The statistical result graph of the effect of stiripentol on the tumor formation size of mice with diffuse midline glioma; Figure C: The statistical result of the effect of stiripentol on the survival time of mice with diffuse midline glioma.

[0023] Figure 4 This is the result graph of the effect of stiripentol on the tumor microenvironment of mice with diffuse midline glioma in Example 3 of the present invention.

[0024] Among them, Figure A: The result graph of the effect of stiripentol on tumor-infiltrating lymphocytes in mice with diffuse midline glioma; Figure B: The statistical result graph of the effect of stiripentol on tumor-infiltrating lymphocytes in mice with diffuse midline glioma.

[0025] Figure 5 This is the result of the effect of stiripentol alone or in combination with the chemotherapeutic drugs cisplatin or etoposide on the expression of the DNA damage marker γ-H2AX in patient-derived diffuse midline glioma cells in Example 4 of the present invention.

[0026] Figure 6 This is the result of the effect of stiripentol alone or in combination with the chemotherapeutic drugs cisplatin or etoposide on the formation of DNA damage marker γ-H2AX foci in patient-derived diffuse midline glioma cells in Example 5 of the present invention.

[0027] Among them, Figure A: The result graph of the effect of stiripentol alone or in combination with the chemotherapeutic drugs cisplatin or etoposide on the formation of DNA damage marker γ-H2AX foci in patient-derived diffuse midline glioma cell line SU-DIPGXIII; Figure B: The statistical result of the effect of stiripentol alone or in combination with the chemotherapeutic drugs cisplatin or etoposide on the formation of DNA damage marker γ-H2AX foci in patient-derived diffuse midline glioma cell line SU-DIPGXVII.

[0028] Figure 7This is the result of the effect of stiripentol alone or in combination with the chemotherapeutic drugs cisplatin or etoposide on the expression of DNA damage marker γ-H2AX positive cells in patient-derived diffuse midline glioma cells in Example 5 of the present invention.

[0029] Among them, Figures A - B: Results of the effect of stiripentol alone or in combination with the chemotherapeutic drugs cisplatin or etoposide on the expression of DNA damage marker γ-H2AX positive cells in patient-derived diffuse midline glioma cells SU-DIPGXIII and SU-DIPGXVII; Figures C - D: Statistical results of the effect of stiripentol alone or in combination with the chemotherapeutic drugs cisplatin or etoposide on the expression of DNA damage marker γ-H2AX positive cells in patient-derived diffuse midline glioma cells SU-DIPGXIII and SU-DIPGXVII. Detailed implementation methods

[0030] The technical solutions, implementation effects and innovative advantages of the present invention are further clarified through the following specific examples. The experimental materials (including but not limited to reagents and detection kits) involved in the examples are all commercially available standard products or commercial reagents without special statements. It should be noted that the said examples are only used to exemplarily display the core inventive points of the present invention. Those skilled in the art can achieve equivalent technical effects by adjusting the dosage scheme, the types of combined drugs or the scope of indications without departing from the technical concept of the present invention.

[0031] Example 1 Effect of stiripentol on the lactic acid content of patient-derived diffuse midline glioma cells

[0032] In this example, the effects of stiripentol on the lactic acid content of diffuse midline glioma were verified by using patient-derived diffuse midline glioma cells SU-DIPGXIII and SU-DIPGXVII. The specific scheme is as follows:

[0033] I. Culture of patient-derived diffuse midline glioma cells

[0034] (1) The complete culture medium for patient-derived diffuse midline glioma cells is basal medium + growth factors. The basal medium includes Neurobasal medium, DMEM / F-12 medium, HEPES, sodium pyruvate solution, glutamine solution and non-essential amino acid solution. The growth factors include EGF, FGF, PDGF-AA and PDGF-BB.

[0035] (2) Medium change: Observe the cell state under the microscope. Under normal circumstances, the cells grow in suspension as spheres. Aspirate all the liquid in the culture dish into a 15 ml centrifuge tube, centrifuge at a speed of 1000 rpm for 3 min, discard the supernatant, and resuspend with cell culture medium and inoculate into the culture dish.

[0036] (3) Passage: Aspirate all the liquid in the culture dish into a 15-ml centrifuge tube, centrifuge at 1000 rpm for 3 min, discard the supernatant, add 1 ml of protease cell dissociation solution, gently resuspend, place in a 37-degree cell culture incubator for digestion. During this period, gently flick the centrifuge tube to disperse the cell clumps until no cell clumps are visible under the light, indicating sufficient digestion. Then add 10 ml of PBS to resuspend and mix well, and centrifuge at 1000 rpm for 3 min. Subsequently, resuspend the cells with PBS and add Dnase I solution with a final concentration of 0.1 mg / ml, incubate at room temperature for 15 min, centrifuge at 1000 rpm to remove the supernatant, and then resuspend the cells with complete medium and inoculate them into a culture dish.

[0037] (4) Cryopreservation: Resuspend the digested cells with cell cryopreservation solution, aspirate them into an explosion-proof cryopreservation tube, and place them in a cryopreservation box for gradient cryopreservation.

[0038] II. Determination of Cellular Lactate Level

[0039] (1) Normally culture diffuse midline glioma cells derived from patients, and inoculate 2*10 6 cells into a 6-cm culture dish.

[0040] (2) Prepare complete media containing different concentrations (0, 125 μmol) of stiripentol to culture SU-DIPG XIII and SU-DIPG XVII cells for 24 h respectively.

[0041] (3) Aspirate the treated cells into a centrifuge tube, centrifuge at 1000 rpm for 3 min, discard the supernatant, and use pre-cooled PBS to wash the cell pellet to discard the residual medium.

[0042] (4) Resuspend the obtained cell pellet with 200 μl of lactate assay solution.

[0043] (5) Use a pipette to aspirate and blow the cell pellet up and down to mix well, place it in a 4-degree tabletop high-speed centrifuge, and centrifuge at the maximum speed for 5 minutes.

[0044] (6) Collect the supernatant into a clean EP tube, add perchloric acid with a final concentration of 1 M, and vortex to mix well to neutralize endogenous lactate dehydrogenase.

[0045] (7) Incubate the vortexed sample on ice for 5 minutes, and then place it in a 4-degree tabletop high-speed centrifuge and centrifuge at 13000 g for 2 minutes.

[0046] (8) Collect the supernatant after centrifugation into a clean EP tube, add 2 M potassium hydroxide solution with a pre-cooled volume of 34% and vortex to mix well.

[0047] (9) Adjust the pH of the sample to between 6.5 and 8 using 0.1 M potassium hydroxide solution or 0.1 M perchloric acid.

[0048] (10) Place the sample with adjusted pH in a tabletop high-speed centrifuge at 4 °C and centrifuge at 13,000 g for 15 min. Collect the supernatant solution after centrifugation into a clean EP tube.

[0049] (11) Pipette 50 μl of the collected supernatant solution into a 96-well black transparent plate, and set 4 replicates for each sample. Add 50 μl of lactic acid experimental solution to the blank well.

[0050] (12) Add 50 μl of lactic acid detection working solution to the blank well and the sample wells, mix well, and place in the dark at room temperature for 30 minutes for reaction.

[0051] (13) After the reaction, place the 96-well plate in an enzyme-linked immunosorbent assay (ELISA) reader and detect the emission wavelength at 590 nm under an excitation wavelength of 540 nm (Ex / Em = 540 / 590 nm).

[0052] (14) After statistically analyzing the lactic acid level in the sample, obtain Figure 1 .

[0053] The experimental results showed that: compared with the control group, treating SU-DIPG XIII and SU-DIPG XVII cells with stiripentol for 24 h could effectively inhibit the production of intracellular lactic acid level ( Figure 1 ).

[0054] Example 2 Effect of stiripentol on the tumor formation of diffuse midline glioma

[0055] In this example, the effect of stiripentol on the tumor formation of diffuse midline glioma was verified by using mice with spontaneous diffuse midline glioma tumors. The specific protocol was as follows:

[0056] I. Construction and treatment of a mouse model with spontaneous diffuse midline glioma tumors

[0057] (1) Infect DF1 cells with the obtained RCAS plasmids respectively, and perform transfection using PEI. Add 5 μg of plasmid and 25 μl of PEI to a 10 cm culture dish, and change the culture medium 24 hours after infection.

[0058] (2) Two weeks after infection, digest the DF1 cells, mix various plasmids in a 1:1 ratio, and ensure the final concentration is 1×10 5 / μl.

[0059] (3) Assemble and check the stereotaxic equipment and anesthesia machine.

[0060] (4) Anesthetize the experimental mice: Anesthetize the mice in the Chamber with isoflurane for 10 s. After the operation of the previous mouse is completed, then direct the isoflurane to the Chamber. Pay attention to observing the state of the mice at all times to avoid death due to over-anesthesia.

[0061] (5) Fix the experimental mice: Fix the upper jaw teeth of the experimental mice in the holes, and pull backward to confirm the fixation; while pulling the mouse tail, push the anesthesia Mask inward and tighten the fixing knob; support the mouse body with the right hand below, and push the ear rods on both sides with the left hand to fix the head. After tightening, gently press with fingers to confirm whether the back of the mouse skull is an un-inclined plane. If it is inclined, adjust the positions of the small rods fixed on both sides.

[0062] (6) In-situ injection: Disinfect the incision area with iodophor, and scrape the hair around the incision with a blade; lift the skin with a curved forceps, make a small incision in the midline with a straight scissors, and expand the incision to both sides with the blunt end of a cotton swab; gently press the bone pieces on both the left and right sides with a curved forceps to confirm the position of the anterior fontanelle; fix the microinjector, manually move the microinjector needle to directly above the posterior fontanelle, and set the X and Y axis values to 0; use the knobs at the back and on the side to adjust the X and Y axis values to -0.80 and -0.50 (i.e., 0.5 mm behind and 0.8 mm on the right side). At this time, the needle should be directly above the target site, and mark the drilling position with a marker pen; remove the microinjector and drill a hole with a drill: Hold the drill with the right hand and support the front end with the left hand. Keep the drill as perpendicular to the skull plane as possible and stop immediately when there is a sense of falling through. Use a 200 μl pipette to pipette and mix the cell suspension evenly, and aspirate 1+(1 - 2) μl of the suspension with the microinjector; fix the microinjector, confirm that the piston handle is fixed well, confirm that there are no bubbles in the syringe liquid and there is liquid flowing out of the needle; use the upper knob to move the needle to the tip entering the drilled hole and set the Z axis value to 0; use the upper knob to adjust the Z axis value to -5.20, that is, insert the needle 5.2 mm, and then adjust the value to -5.00, that is, retract the needle 0.2 mm to 5.0 mm; set the injection speed to 1 μl / min; seal the area around the needle entry with bone wax and start the injection. After the injection is completed, slowly and evenly retract the needle. For every 1 mm of needle retraction, let it stand for 1 - 2 minutes, and pay attention to observing whether there is suspension overflow; fill the bone wax, release the fixation, suture the wound and observe the recovery.

[0063] (7) On the 14th day after stereotactic injection, inject normal saline or 30 mg / kg stiripentol into the abdominal cavity of the mice, and continue until the 26th day after stereotactic injection.

[0064] (8) Record the survival time of the mice; in addition, when symptoms such as epilepsy appear in the control group (the group injected with normal saline), take 3 mice from the control group and 3 mice from the drug-administered group at the same time for section staining to observe the size of tumor formation.

[0065] II. Perfuse the mice to obtain brain sections

[0066] (1) Anesthetize the mice and place them in a sterile laminar flow hood. Fix the mice's bodies, cut open the chest skin to expose the heart, make a small incision at the right auricle, and inject pre-cooled PBS from the apex of the left ventricle using a disposable venous blood collection needle to drain the blood. Stop the PBS perfusion when the mouse's liver turns white.

[0067] (2) Subsequently, perfuse pre-cooled 4% PFA along the same position and stop the perfusion when the mouse's body becomes rigid.

[0068] (3) Cut open the mouse's scalp to expose the skull, carefully isolate the mouse's brain tissue, and place it in a centrifuge tube containing 4% PFA solution for fixation for 24 hours.

[0069] (4) After fixation, transfer the mouse's brain tissue to 30% sucrose for dehydration, and replace the fresh sucrose solution every 12 hours until the mouse's brain tissue precipitates to the bottom of the centrifuge tube.

[0070] (5) Air-dry the dehydrated mouse brain tissue and embed it in OCT for sectioning. The section thickness is 10 μm.

[0071] (6) Let the frozen sections air-dry at room temperature overnight and store them in a -80°C refrigerator.

[0072] III. H&E Staining

[0073] (1) Take out the frozen sections and let them warm up to room temperature, then place the sections in a 37°C constant temperature drying oven for 30 minutes.

[0074] (2) Place the warmed-up sections in hematoxylin for nuclear staining for 10 minutes.

[0075] (3) Wash with ultrapure water 2 times, 5 minutes each time.

[0076] (4) Place the sections in the differentiating solution to wash away the excess hematoxylin, and soak for 20 seconds.

[0077] (5) Wash with ultrapure water 2 times, 5 minutes each time.

[0078] (6) Place the sections in the bluing solution for bluing and soak for 4 minutes.

[0079] (7) Wash with ultrapure water 2 times, 5 minutes each time.

[0080] (8) Stain the cytoplasm with 30% eosin staining solution and soak for 30 seconds.

[0081] (9) Subsequently, dehydrate with gradient ethanol: soak in 80% ethanol for 5 seconds, 95% ethanol for 1 minute, 100% ethanol for 1 minute, and 100% ethanol for 1 minute.

[0082] (10) The sections were then immersed in xylene for 5 minutes and immersed twice.

[0083] (11) After mounting with neutral resin, the sections were placed in an oven at 65 °C overnight and then used for imaging.

[0084] The experimental results showed that after treatment with stiripentol on the mice with spontaneous tumors of diffuse midline glioma ( Figure 2 ), compared with the control group of mice, the tumor volume of the treatment group of mice was significantly reduced ( Figure 3 A - B), their survival period was significantly prolonged, and the prognosis was significantly improved ( Figure 3 C).

[0085] Example 3 Effects of stiripentol on the tumor microenvironment of mice with diffuse midline glioma

[0086] In this example, flow cytometry was used to verify the effects of stiripentol on the tumor microenvironment of mice with spontaneous tumors of diffuse midline glioma. The specific protocol was as follows:

[0087] I. Preparation of tumor - infiltrating immune cells in mice

[0088] (1) On the 32nd day after stereotactic injection, flow cytometry analysis was performed on the mice with the same drug - administration treatment conditions as in Example 2: After anesthesia, the mice were placed in a sterile laminar flow hood, their bodies were fixed, the chest skin was incised to expose the heart, a small incision was made at the right auricle, and pre - cooled lactated Ringer's solution was injected from the apex of the left ventricle with a disposable intravenous blood - taking needle to drain the blood. Perfusion was terminated when the mouse liver turned white.

[0089] (2) The scalp of the mouse was incised to expose the skull, the mouse brain tissue was carefully separated, and the intracranial tumor tissue was further separated with surgical instruments and cut into pieces.

[0090] (3) The tumor pieces were placed in an EP tube containing 2 ml of tissue digestion solution and digested in a 37 °C cell incubator for 30 minutes, and shaken and mixed every 10 minutes.

[0091] (4) 2 ml of culture medium (DMEM medium + 10% fetal bovine serum) was added to terminate digestion. The digested tumor pieces were ground in a 70 - μm cell sieve, and during the grinding process, flow solution (PBS + 2% fetal bovine serum) was added to keep the tissue moist and the cells were flushed into a prepared 50 - ml centrifuge tube.

[0092] (5) The ground cells were centrifuged in a refrigerated centrifuge at a speed of 2000 rpm for 5 minutes.

[0093] (6) The supernatant was discarded, 5 ml of red blood cell lysate was added to the centrifuge tube to resuspend the cell pellet, and it was left standing at room temperature for 5 minutes.

[0094] (7) Add 10 ml of flow solution to the centrifuge tube to terminate red blood cell lysis, and centrifuge it in a refrigerated centrifuge at a speed of 2000 rpm for 5 minutes.

[0095] (8) Discard the supernatant, and add 10 ml of flow solution to wash the cell pellet.

[0096] (9) Resuspend the cell pellet in 10 ml of 52% Percoll, and centrifuge it in a refrigerated centrifuge at a speed of 800 g for 20 minutes under the condition of 3 up and 1 down.

[0097] (10) After centrifugation, remove the upper layer of tumor cells as much as possible and discard the supernatant, retaining the cell pellet.

[0098] (11) Add 10 ml of 1640 medium to the centrifuge tube to wash the cell pellet, centrifuge it in a refrigerated centrifuge at a speed of 2000 rpm for 5 minutes, discard the supernatant, and the pellet is the enriched tumor-infiltrating immune cells.

[0099] II. Flow cytometry

[0100] a) Extracellular staining

[0101] (1) PMA / ionomycin stimulation: Count the enriched tumor-infiltrating immune cells, take 1×10 6 tumor-infiltrating immune cells, resuspend them in 100 μl of PMA / ionomycin stimulation solution, and place them in a 37-degree cell incubator for 4 hours for stimulation.

[0102] (2) Blocking: Take 1×10 6 tumor-infiltrating immune cells, resuspend them in 100 μl of flow solution, add anti-CD16 / 32 antibody (1:500), mix well, and place them at 4 degrees or on ice for 10 minutes for blocking.

[0103] (3) Staining: Then add 1 ml of flow solution to wash the sample, centrifuge it in a bench-top refrigerated high-speed centrifuge at a speed of 2000 rpm for 5 minutes, resuspend the cell pellet in the pre-prepared 100 μl of antibody (1:400), label different cell populations respectively, and place them on ice at 4 degrees or on ice for 30 minutes. In addition, prepare cell samples stained separately with each antibody fluorescence channel for compensation adjustment. After staining, directly add 1 ml of flow solution to the sample to wash the cells, centrifuge it in a bench-top refrigerated high-speed centrifuge at a speed of 2000 rpm for 5 minutes, and wash twice.

[0104] b) Intracellular staining

[0105] (1) Fixation: Resuspend the tumor-infiltrating immune cell pellet with 200 μl of BD fixative, and fix it at room temperature for 20 minutes;

[0106] (2) Permeabilization: After fixation, add 1 ml of BD permeabilization solution directly to the cells to wash them, mix well, and centrifuge at 800 g for 5 minutes in a desktop refrigerated high-speed centrifuge;

[0107] (3) Staining: Add the antibody to be tested (1:400) prepared in advance with BD permeabilization solution directly to the cell pellet after centrifugation and let stand at room temperature for 1 hour;

[0108] (4) Loading: After staining, add 1 mL of BD permeabilization solution to wash the cells, and centrifuge at 4000 rpm for 5 minutes at 4 degrees. Repeat the washing process 1-2 times, resuspend the washed cells in 300 μl of flow cytometry solution, and then load the cells for detection.

[0109] The experimental results showed that in the spontaneous tumor formation model mice of diffuse midline glioma, the experimental group treated with stiripentol showed significant changes in the tumor microenvironment compared with the untreated group: CD4 + and CD8 + The infiltration density of T lymphocytes increased significantly, and the levels of interferon gamma (IFNγ) and granzyme B (GzmB) secreted specifically by CD8+T cells increased statistically. The simultaneous enhancement of these immune indicators reflects that stiripentol treatment can effectively activate the body's specific immune response to tumors ( Figure 4 AB).

[0110] Example 4 Effect of stiripentol on the ability of chemotherapeutic drugs to kill diffuse midline gliomas.

[0111] This example verifies the effect of stiripentol on the expression of γ-H2AX, a DNA damage marker, in patient-derived diffuse midline glioma cells SU-DIPGXIII and SU-DIPGXVII by Western blotting. The specific scheme is as follows:

[0112] 1. Protein Immunoblotting

[0113] (1) Patient-derived diffuse midline glioma cells SU-DIPGXIII and SU-DIPGXVII were inoculated into 6 cm culture dishes, and complete culture medium containing 125 μmol stiripentol or 3 μmol cisplatin or 0.25 μmol etoposide or 125 μmol stiripentol + 3 μmol cisplatin or 125 μmol stiripentol + 0.25 μmol cisplatin was added thereto, respectively, for 24 hours.

[0114] (2) Cell lysis: Collect the processed cells into a 15 ml centrifuge tube, centrifuge at 1000 rpm for 3 minutes, discard the supernatant, wash the cell pellet once with pre-cooled PBS to remove residual medium, then centrifuge at 1000 rpm for 3 minutes. Add lysis buffer according to the cell quantity, collect the lysed mixture, add protein loading buffer, and denature at 100 °C for 10 minutes.

[0115] (3) SDS-PAGE electrophoresis: Prepare a 12% concentration gel according to the size of γ-H2AX, add protein samples in sequence, and add protein loading buffer to the two side wells. Keep the upper gel at a constant voltage of 80 V for about 20 minutes, then adjust the voltage to 120 V for about 90 minutes. Stop electrophoresis when the bromophenol blue runs to the bottom of the glass plate.

[0116] (4) Blotting: Add pre-cooled transfer buffer into a rectangular container, stack the sponge, filter paper, gel, NC membrane, filter paper, and sponge together in sequence, use a glass rod to remove air bubbles, connect the transfer device, set the parameters to a constant current of 330 mA for 70 minutes, and perform it in an ice bath.

[0117] (5) Hybridization: Prepare a 3% BSA blocking solution using TBST, place the NC membrane in the blocking solution, and incubate at room temperature for 1 hour; transfer the NC membrane to 3% BSA containing the anti-γ-H2AX primary antibody, and incubate on a shaker at 4 °C overnight; wash the NC membrane with TBST, 5 minutes each time for 3 times; transfer the NC membrane to 3% BSA containing the secondary antibody, and incubate at room temperature for 1 hour; wash the NC membrane with TBST, 5 minutes each time for 3 times.

[0118] (6) Color development: Mix the luminescent solution in a ratio of 40:1, cover the surface of the NC membrane, and place it in the dark for 1 - 3 minutes; terminate color development with TBST or double-distilled water, and scan and develop using a gel imaging system.

[0119] The experimental results showed that when using stiripentol alone to treat SU-DIPG XIII and SU-DIPG XVII cells, no significant change was observed in the expression level of the DNA damage marker γ-H2AX; however, when stiripentol was combined with cisplatin or etoposide to form a combined treatment regimen, the cytotoxic effects of these two chemotherapeutic drugs on tumor cells could be significantly enhanced. In this combined treatment group, the expression level of γ-H2AX in tumor cells showed a significant upregulation, suggesting that stiripentol may play a synergistic anti-tumor role by enhancing the DNA damage mechanism induced by chemotherapy ( Figure 5 ).

[0120] Example 5 Effect of stiripentol on the ability of chemotherapeutic drugs to cause DNA damage in diffuse midline glioma.

[0121] In this example, immunofluorescence staining and flow cytometry were used to verify the effects of stiripentol on the formation of γ-H2AX foci, a DNA damage marker, and the proportion of γ-H2AX-positive cells in patient-derived diffuse midline glioma cells SU-DIPG XIII and SU-DIPG XVII induced by chemotherapeutic drugs cisplatin or etoposide. The specific protocol is as follows:

[0122] I. Immunofluorescence staining

[0123] (1) Patient-derived diffuse midline glioma cells SU-DIPG XIII and SU-DIPG XVII were seeded in 6-cm culture dishes and treated with complete medium containing 125 μmol stiripentol, 3 μmol cisplatin, 0.25 μmol etoposide, 125 μmol stiripentol + 3 μmol cisplatin, or 125 μmol stiripentol + 0.25 μmol cisplatin for 24 hours.

[0124] (2) The treated cells were digested with protease cell dissociation solution in a 37°C cell incubator for 1 minute, resuspended in 10 ml PBS, centrifuged at 1000 rpm for 3 minutes, and the supernatant was discarded. The cells were then washed once with PBS to remove residual protease cell dissociation solution, centrifuged at 1000 rpm for 3 minutes, and the cells were collected in an EP tube for subsequent experiments.

[0125] (3) The cells in the EP tube were resuspended and mixed with 100 μl of 4% PFA solution and fixed at room temperature for 10 minutes.

[0126] (4) The fixed cells were washed three times with 1 ml PBS (800 g for 5 minutes, and the subsequent PBS cell washes were all under the same centrifugation conditions). 100 μl of 0.1% Triton X-100 solution prepared with PBS was added to the EP tube and mixed for perforation at room temperature for 12 minutes.

[0127] (5) The perforated cells were washed three times with 1 ml PBS, and 100 μl of 3% BSA blocking solution prepared with PBS was added and mixed for blocking. The cells were blocked at room temperature in the dark for 1 hour (all subsequent steps were carried out in the dark).

[0128] (6) The cells were washed 3 times with 1 ml PBS, and γ-H2AX primary antibody prepared with the blocking solution was added and incubated overnight at 4°C.

[0129] (7) The cells were washed 3 times with 1 ml PBS, and fluorescent secondary antibody prepared with the blocking solution was added and incubated at room temperature for 1 hour.

[0130] (8) The cells were washed 3 times with 1 ml PBS, and 1:500 DAPI solution was added for nuclear staining at room temperature for 5 minutes.

[0131] (9) Wash 3 times with 1 ml of PBS. Discard the remaining supernatant in the last wash, leaving only about 20 μl of PBS. Resuspend the cells in PBS and drop them on an adhesion glass slide, and add 5 μl of water-soluble anti-quenching agent for mounting. After standing at room temperature for 15 minutes, perform confocal imaging.

[0132] The experimental results showed that in the two diffuse midline glioma cell models of SU-DIPGXIII and SU-DIPGXVII, single-agent treatment with stiripentol had no significant effect on the formation of DNA damage marker γ-H2AX foci; however, when combined with cisplatin or etoposide in combination treatment regimens, it could significantly enhance the killing efficiency of these two chemotherapeutic drugs on tumor cells: a significant increase in the number of γ-H2AX foci was detected in the cells of the combination treatment group compared with the single-agent group. These results indicate that stiripentol exerts a synergistic effect of chemotherapy sensitization by enhancing DNA damage induced by chemotherapeutic drugs ( Figure 6 ).

[0133] II. Flow cytometry

[0134] (1) Seed the patient-derived diffuse midline glioma cells SU-DIPGXIII and SU-DIPGXVII in 6-cm culture dishes, and add complete medium containing 125 μmol of stiripentol or 3 μmol of cisplatin or 0.25 μmol of etoposide or 125 μmol of stiripentol + 3 μmol of cisplatin or 125 μmol of stiripentol + 0.25 μmol of cisplatin to each dish, and incubate for 24 hours.

[0135] (2) Digest the treated cells with protease cell dissociation solution in a 37°C cell incubator for 1 minute, add 10 ml of PBS to resuspend, centrifuge at 1000 rpm for 3 minutes, discard the supernatant, and then wash once with PBS to remove the remaining protease cell dissociation solution, and centrifuge at 1000 rpm for 3 minutes to collect the cells in an EP tube for subsequent experiments.

[0136] (3) Blocking: Resuspend the cells to be detected in 100 μl of flow cytometry solution, add anti-CD16 / 32 antibody (1:500), mix well, and incubate at 4°C or on ice for 10 minutes for blocking.

[0137] (4) Fixation: Resuspend the tumor-infiltrating immune cell precipitate with 200 μl of BD fixation solution and fix at room temperature for 20 minutes;

[0138] (5) Permeabilization: After fixation, directly add 1 ml of BD permeabilization solution to the cells to wash the cells, mix well, and centrifuge at 800 g in a tabletop refrigerated high-speed centrifuge for 5 minutes;

[0139] (6) Staining: Add the pre-prepared γ-H2AX antibody (1:400) with BD permeabilization solution directly to the cell pellet after centrifugation, and let it stand at room temperature for 1 hour;

[0140] (7) Instrumentation: After staining, add 1 mL of BD permeabilization solution to wash the cells, centrifuge at 4000 rpm for 5 minutes at 4°C. Repeat the washing 1-2 times. Resuspend the washed cells in 300 μL of flow cytometry solution and then perform instrumentation detection.

[0141] The experimental results showed that in the two diffuse midline glioma cell models of SU-DIPGXIII and SU-DIPGXVII, treatment with stiripentol alone did not significantly change the proportion of γ-H2AX positive cells; while when combined with cisplatin or etoposide, it could significantly enhance the anti-tumor activity of cisplatin or etoposide: the proportion of γ-H2AX positive cells in the combination treatment group was significantly higher than that in the single-agent chemotherapy group. The above results indicate that stiripentol produces a synergistic effect of chemotherapy sensitization by enhancing chemotherapy drug-mediated DNA damage ( Figure 7 ).

[0142] This specific embodiment is only an interpretation of the present application, and it does not limit the present application. Those skilled in the art can make modifications without creative contributions to this embodiment after reading this specification, but as long as it is within the scope of the claims of the present application, it is protected by the patent law.

Claims

1. Use of stiripentol in the preparation of a medicament for treating diffuse midline glioma.

2. The application according to claim 1, wherein The stiripentol exerts an anti-tumor effect through at least one of the following ways: (a) inhibiting the proliferation of diffuse midline glioma cells, reducing the tumor volume and prolonging the survival period of tumor-bearing individuals; (b) activating the tumor immune microenvironment and promoting the polarization of tumor-associated macrophages into an anti-tumor phenotype; (c) being used in combination with a chemotherapeutic drug to synergistically enhance the DNA damage effect of the chemotherapeutic drug on tumor cells.

3. The application according to claim 2, wherein The stiripentol is used in combination with a chemotherapeutic drug, and the chemotherapeutic drug is selected from DNA damage drugs including at least one of platinum-based drugs or poly ADP-ribose polymerase inhibitors.

4. The application according to claim 3, wherein The chemotherapeutic drug is cisplatin or etoposide.

5. The application according to claim 1, wherein The diffuse midline glioma is a pediatric midline glioma carrying an H3K27M mutation.

6. A pharmaceutical composition for treating diffuse midline glioma, characterized in that, It contains stiripentol as an active ingredient, and a pharmaceutically acceptable carrier or excipient.

7. The pharmaceutical composition according to claim 6, wherein, The dosage form of the pharmaceutical composition is an oral preparation or an injection preparation. The oral preparation includes tablets, capsules or suspensions, and the injection preparation includes freeze-dried powder injections or injections.

8. A combined drug composition for treating diffuse midline glioma, characterized in that, The combined medication composition includes stiripentol and cisplatin or etoposide.

9. The pharmaceutical composition according to claim 8, wherein The stiripentol is used in combination with cisplatin. The molar concentration of cisplatin is 3 μmol / L, and the molar concentration of stiripentol is 125 μmol / L.

10. The pharmaceutical composition according to claim 8, wherein The stiripentol is used in combination with etoposide. The molar concentration of etoposide is 0.25 μmol / L, and the molar concentration of stiripentol is 125 μmol / L.

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