Anti-glioma pharmaceutical composition and application thereof

Abstract

The invention discloses an anti-glioma pharmaceutical composition and an application thereof. The pharmaceutical composition comprises an STING agonist or a pharmaceutically acceptable salt thereof, temozolomide or a pharmaceutically acceptable salt thereof, and / or Echinomycin. Experiments prove that the pharmaceutical composition containing two or three active components can significantly inhibit the growth of glioma cells and promote the apoptosis of glioma, and a brand new strategy and thought are provided for treatment of glioma.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular, the present invention relates to a pharmaceutical composition for treating glioma. Background Art

[0002] Gliomas are tumors that occur in the neuroectoderm, so they are also called neuroectodermal tumors or neuroepithelial tumors. Tumors originate from neural interstitial cells, namely glial cells, ependymal cells, choroid plexus epithelial cells and neural parenchymal cells, namely neurons. Most tumors originate from different types of glial cells, but based on their histological origins and similar biological characteristics, various tumors that occur in the neuroectoderm are generally called gliomas. There are many ways to classify gliomas, and clinical workers often use the Kernohan classification method, which is relatively simple. Among all types of gliomas, astrocytomas are the most common, followed by glioblastomas, followed by medulloblastomas, ependymomas, oligodendrogliomas, pinealomas, mixed gliomas, choroid plexus papilloma, unclassified gliomas and neuronal tumors.

[0003] At present, the treatment of glioma is divided into surgery, radiotherapy, and chemotherapy according to the effect. Because glioma grows invasively and has unclear boundaries with surrounding tissues, even if a microscope is used during surgery, it is inevitable that a small amount of tumor cells will remain, leading to tumor recurrence. Although new progress has been made in chemotherapy, it is difficult for systemic chemotherapy to pass through the blood-brain barrier, and it is difficult to achieve effective drug concentrations in the local tumor. In addition, high-dose chemotherapy causes obvious systemic toxic side effects, such as bone marrow suppression, leukopenia, and pulmonary fibrosis. Although each treatment method has made significant progress, and the combined treatment of the three methods can prolong the patient's survival, glioma is still difficult to cure. In recent years, new therapies such as immunotherapy, anti-angiogenesis, and gene therapy have moved from the laboratory to the clinic. Temozolomide (TMZ) is currently the most important drug for the treatment of glioma. Researchers are looking for new drugs based on this drug or drugs that can be used in combination with it to exert a synergistic effect. Summary of the invention

[0004] In order to make up for the deficiencies of the prior art, the purpose of the present invention is to provide a pharmaceutical composition that can be used in combination with temozolomide to treat glioma.

[0005] In order to achieve the above object, the present invention adopts the following technical solution:

[0006] The first aspect of the present invention provides a pharmaceutical composition for treating glioma.

[0007] Furthermore, the pharmaceutical composition comprises a STING agonist or a pharmaceutically acceptable salt thereof, and temozolomide or a pharmaceutically acceptable salt thereof.

[0008] Furthermore, the STING agonist includes DMXAA, diABZI, MSA-2, SR-717 and / or C53.

[0009] Furthermore, the STING agonist is selected from DMXAA.

[0010] Furthermore, the pharmaceutically acceptable salts include alkali metal salts, alkaline earth metal salts and ammonium salts.

[0011] Furthermore, the alkali metal salt includes potassium salt and sodium salt.

[0012] Furthermore, the alkaline earth metal salt includes calcium salt and magnesium salt.

[0013] In the present invention, treatment refers to the improvement, prevention, or reversal of a disease or condition or at least one distinguishable symptom thereof. In certain specific embodiments, the treatment refers to the improvement, prevention, or reversal of at least one measurable physiological parameter associated with the disease or condition to be treated, and the parameter is not necessarily identifiable in mammals or recognized by mammals. In some embodiments, the treatment refers to the inhibition or mitigation of a disease or course of disease, and this inhibition or mitigation may be physical, such as certain distinguishable adverse symptoms. The "treatment" used in the present invention covers diseases in mammals, especially humans, including: (a) preventing the occurrence of a disease or condition in individuals who are susceptible to the disease but have not yet been diagnosed with the disease. (b) Inhibiting the disease, such as blocking the development of the disease. Or (c) Alleviating the disease, such as alleviating the symptoms associated with the disease.

[0014] In the present invention, the temozolomide may be temozolomide itself, or may be a hydrate, a solvate or a crystalline form of temozolomide (hereinafter also referred to as a compound).

[0015] In the present invention, the pharmaceutically acceptable salt refers to an acid salt formed with an inorganic and / or organic acid and a basic salt formed with an inorganic and / or organic base. In addition, when the compound contains a basic portion (such as but not limited to, pyridine or imidazole) and an acidic portion (such as but not limited to, carboxylic acid), zwitterions can be formed and the zwitterions are included in the pharmaceutically acceptable salts described in the present application. Pharmaceutically acceptable (i.e., non-toxic, physiologically acceptable) salts are preferred, but other salts are also useful. The pharmaceutically acceptable salt of the compound can be formed, for example, by reacting the compound with a certain amount of acid or base in a medium, such as a medium in which the salt is precipitated or an aqueous medium (freeze-dried after the reaction).

[0016] Specific pharmaceutically acceptable salts include those salts which are suitable for contact with the tissues of humans and lower animals without excessive toxicity, irritation, allergic reaction, etc., and which are commensurate with a reasonable benefit or risk ratio, within the scope of sound medical judgment. Pharmaceutically acceptable salts (pharmaceutically acceptable salts) are well known in the art. Pharmaceutically acceptable salts of the compounds described herein include salts derived from suitable inorganic and organic acids and inorganic and organic bases.

[0017] The example of pharmaceutically acceptable nontoxic acid addition salt is the salt formed with inorganic acid, for example hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid, or the salt formed with organic acid, for example acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid. Also include the salt formed using the conventional methods in the art, for example, ion exchange method.Other pharmaceutically acceptable salts include: adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, gluconate, glycerophosphate, hemisulfate, enanthate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, mesylate. Pharmaceutically acceptable salts derived from suitable bases include alkali metal, alkaline earth metal, ammonium salts. Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium salts and the like.

[0018] In the present invention, a hydrate refers to a compound combined with water.

[0019] In the present invention, the solvate refers to a form of a compound or salt thereof that is combined with a solvent, usually formed by a solvolysis reaction. This physical association may include hydrogen bonding. Conventional solvents include methanol, ethanol, acetic acid, DMSO, THF, ether, etc. In some cases, the solvate will be able to separate, for example, when one or more solvent molecules are incorporated into the crystal lattice of the crystalline solid. Solvates include solvates in solution and separable solvates.

[0020] In the present invention, the crystalline form refers to the crystalline form of a compound with a specific crystal packing arrangement. Different crystalline forms usually have different X-ray diffraction patterns, infrared spectra, melting points, densities, hardnesses, crystal shapes, photoelectric properties, stability and solubility. Recrystallization solvents, crystallization rates, storage temperatures and other factors may lead to a crystalline form being dominant. Various polymorphs of a compound can be prepared by crystallization under different conditions. In the present invention, crystalline form also includes special crystalline states, such as amorphous, etc.

[0021] Furthermore, the concentration ratio of DMXAA to temozolomide in the pharmaceutical composition is 1:60 to 1:2.5.

[0022] Furthermore, the concentration ratio of DMXAA to temozolomide in the pharmaceutical composition is 1:15 to 1:2.5.

[0023] Preferably, the concentration ratio of DMXAA to temozolomide in the pharmaceutical composition is (2.5 μM-20 μM): (50 μM-150 μM).

[0024] More preferably, the concentration ratio of DMXAA to temozolomide in the pharmaceutical composition is (10 μM-20 μM): (50 μM-150 μM).

[0025] Furthermore, the pharmaceutical composition also includes Echinomycin.

[0026] Furthermore, when the pharmaceutical composition further includes Echinomycin, the concentration ratio of DMXAA, temozolomide and Echinomycin in the pharmaceutical composition is (2.5 μM~20 μM): (50 μM~300 μM): (0.5 nM~5 nM).

[0027] Furthermore, the pharmaceutical composition also includes a pharmaceutically acceptable carrier and / or excipient.

[0028] Furthermore, the pharmaceutically acceptable excipients include but are not limited to diluents, excipients, fillers, binders, wetting agents, disintegrants, emulsifiers, cosolvents, solubilizers, osmotic pressure regulators, lubricants, surfactants, coating materials, colorants, pH regulators, antioxidants or antibacterial agents.

[0029] Further, the diluent includes but is not limited to lactose, sodium chloride, glucose, urea, starch, water, etc. The binder includes but is not limited to starch, pregelatinized starch, dextrin, maltodextrin, sucrose, gum arabic, gelatin, methylcellulose, carboxymethylcellulose, alginic acid and alginate, xanthan gum, hydroxypropyl cellulose, etc. The surfactant includes but is not limited to sodium lauryl sulfate, stearic acid monoglyceride, hexadecanol, etc. The lubricant includes but is not limited to zinc stearate, talc, calcium and magnesium stearate, polyethylene glycol, polyoxyethylene monostearate, monolauric sucrose acid ester, magnesium lauryl sulfate, etc. The filler includes but is not limited to mannitol, xylitol, sorbitol, maltose, glucose, lactose, sucrose, dextrin, starch, etc. The disintegrant includes but is not limited to cross-linked vinyl pyrrolidone, sodium carboxymethyl starch, low-substituted hydroxypropyl methyl, cross-linked sodium carboxymethyl cellulose, soybean polysaccharide, etc.

[0030] Furthermore, the pharmaceutical composition also includes other drugs for treating gliomas. These drugs are not particularly limited, and as long as they can produce a therapeutic effect on gliomas, they are within the protection scope of this application. These drugs include, but are not limited to, Acivicin, Aclarubicin, Acodazole Hydrochloride, AcrQnine, Adozelesin, Aldesleukin, Asparaginase, Asperlin, Azacitidine, Azetepa, Azotomycin, Batimastat, Benzodepa, Bicalutamide, Bisantrene Hydrochloride, Bisnafide Dimesylate, Bizelesin, Bleomycin Sulfate, Brequinar Sodium, and Bruganastat. Sodium), Bropirimine, Busulfan, Cactinomycin, Calusterone, Caracemide, Carbetimer, Carboplatin, Carmustine, Carubicin Hydrochloride, Carzelesin, Cedefingol, Chlorambucil, Cirolemycin, Cisplatin, Cladribine.

[0031] The pharmaceutical composition of the present invention can be used alone or in combination with surgery, radiotherapy, hormone therapy, chemotherapy and / or immune cell therapy.

[0032] In the present invention, chemotherapy refers to the administration of chemotherapeutic agents to treat tumors. Chemotherapeutic agents refer to compounds or derivatives thereof that can interact with cancer cells, thereby reducing the proliferation state of cells and / or killing cells, for example, by impairing cell division or DNA synthesis, or by effectively targeting rapidly dividing cells by damaging DNA. Examples of chemotherapeutic agents include, but are not limited to, alkylating agents (e.g., cyclophosphamide, ifosfamide); metabolic antagonists (e.g., methotrexate (MTX), 5-fluorouracil or its derivatives); substituted nucleotides; substituted nucleosides; DNA demethylating agents (also known as antimetabolites), such as azacitidine); antitumor antibiotics (e.g., mitomycin, doxorubicin); plant-derived antitumor agents (e.g., vincristine, vindesine, paclitaxel, Abraxane); cisplatin; carboplatin; etoposide. These agents may further include, but are not limited to, the anticancer agents trimethoprim-butyl ester (TMTX); temozolomide; raltitrexed; S-(4-nitrobenzyl)-6-thioinosine (NBMPR); 6-benzylguanidine (6-BG); nitrosoureas (rabinopyranosyl-N-methyl-N-nitrosourea (Aranose), carmustine (BCNU, BiCNU), chloramphenicol, ethylnitrosourea (ENU), fordomustine, lomustine (CCNU), nimustine, N-nitroso-N-methylurea (NMU), ranimustine (MCNU), semustine, streptozotocin (streptozotocin)); cytarabine; camptothecin; and any therapeutic derivatives thereof.

[0033] In the present invention, immunotherapy includes but is not limited to chimeric antigen receptor T cell immunotherapy, chimeric antigen receptor NK cell immunotherapy, and tumor infiltrating lymphocyte therapy.

[0034] The second aspect of the present invention provides an in vitro non-therapeutic method for inhibiting the growth of glioma cells and promoting the apoptosis of glioma cells.

[0035] Furthermore, the method comprises treating glioma cells with the pharmaceutical composition described in the first aspect of the present invention.

[0036] Furthermore, the glioma cells include U87-MG, TBD0220 and / or GL261.

[0037] The third aspect of the present invention provides any of the following applications:

[0038] (1) Use of a STING agonist or a pharmaceutically acceptable salt thereof and temozolomide or a pharmaceutically acceptable salt thereof in combination in the preparation of a product for treating and / or preventing glioma;

[0039] (2) Use of a STING agonist or a pharmaceutically acceptable salt thereof and temozolomide or a pharmaceutically acceptable salt thereof in combination in the preparation of an agent for inhibiting glioma cell proliferation and promoting glioma cell apoptosis in vitro;

[0040] (3) Use of a STING agonist or a pharmaceutically acceptable salt thereof in the preparation of a drug for improving the therapeutic effect of temozolomide in treating glioma and / or increasing the sensitivity of temozolomide;

[0041] (4) Use of a STING agonist or a pharmaceutically acceptable salt thereof in combination with Echinomycin in the preparation of a drug for improving the therapeutic effect of temozolomide in treating glioma and / or increasing the sensitivity of temozolomide;

[0042] (5) Use of a STING agonist or a pharmaceutically acceptable salt thereof, temozolomide or a pharmaceutically acceptable salt thereof, and echinomycin in combination for the preparation of a product for treating and / or preventing glioma;

[0043] (6) Use of a STING agonist or a pharmaceutically acceptable salt thereof, temozolomide or a pharmaceutically acceptable salt thereof, and echinomycin in combination for the preparation of an agent for inhibiting glioma cell proliferation and promoting glioma cell apoptosis in vitro;

[0044] (7) Use of a STING agonist or a pharmaceutically acceptable salt thereof, temozolomide or a pharmaceutically acceptable salt thereof, and echinomycin in combination for the preparation of a reagent for increasing the expression of proteins related to the cGAS-STING signaling pathway;

[0045] (8) Use of a STING agonist or a pharmaceutically acceptable salt thereof, temozolomide or a pharmaceutically acceptable salt thereof, and echinomycin in combination for the preparation of a reagent for increasing the expression of proteins related to the cGAS-STING signaling pathway;

[0046] (9) Use of a STING agonist or a pharmaceutically acceptable salt thereof, temozolomide or a pharmaceutically acceptable salt thereof, and echinomycin in combination for reversing and / or inhibiting the transformation of M1 macrophages to M2 macrophages.

[0047] Furthermore, the STING agonist includes DMXAA, diABZI, MSA-2, SR-717 and / or C53.

[0048] Furthermore, the STING agonist is selected from DMXAA.

[0049] Furthermore, the products include anti-glioma drug preparations and anti-glioma combination drugs.

[0050] Furthermore, the cGAS-STING signaling pathway related proteins include TBK1, P-TBK1, P65 and / or P-P65.

[0051] In the present invention, improving refers to allowing a subject or tumor cell to increase its ability to respond to the treatment disclosed herein. For example, the improved response can include at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% or more of the responsiveness. For example, the improved response can refer to the total percentage of subjects who respond to the treatment, wherein the percentage is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% or more.

[0052] Advantages and beneficial effects of the present invention: The present invention experimentally proves that the STING agonist DMXAA combined with temozolomide has a synergistic effect in the treatment of glioma, and proves that DMXAA combined with Echinomycin can also enhance the therapeutic effect of TMZ, providing a new strategy for the treatment of glioma. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 Shows the IC50 of TMZ and TMZ+DMXAA in GBM cell lines U87-MG, TBD0220, and GL261;

[0054] Figure 2 Shows the drug synergistic effect score combination matrix (ZIP model) of cells treated with different concentrations of TMZ and DMXAA for 48 hours; a is the U87-MG cell line; b is the TBD0220 cell line;

[0055] Figure 3 Shows the cell viability of GBM cell lines U87-MG, TBD0220, and GL261 treated with different drug combinations for 48 hours;

[0056] Figure 4 Show the effects of different drug combinations on the clone-forming ability of U87-MG and TBD0220 cells and the statistical analysis of the number of cell clones;

[0057] Figure 5 The drug synergy score combination matrix (ZIP model) of TMZ and DMXAA with Echinomycin is shown;

[0058] Figure 6 Displays the expression of cGAS-STING signaling pathway-related proteins in different drug combinations;

[0059] Figure 7It shows that DMXAA combined with Echinomycin can enhance the therapeutic effect of TMZ; a is the experimental flow chart of constructing the GL261-PTRFov glioma orthotopic transplantation tumor model; b is the representative imaging of bioluminescent mice in vivo and the quantitative analysis of bioluminescent intensity of mice in vivo imaging; c is the curve of mouse weight change;

[0060] Figure 8 H&E staining and Ki-67 immunohistochemical staining analysis of tumor tissues of mice in each group are shown; a is a representative image of H&E staining of mouse brains in each group; b is a representative immunohistochemical staining of Ki-67 in each group, scale bar, 10 μm; c is a semi-quantitative analysis of Ki-67 immunohistochemical staining in each group;

[0061] Fig. 9 Shown are CD34 immunohistochemical staining analysis and MVD values ​​of tumor tissues of mice in each group, scale bar, 10 μm;

[0062] Fig.10 Shows the immunohistochemical staining and semi-quantitative analysis of MHCII and CD163 in tumor tissues of mice in each group, scale bar, 10 μm;

[0063] Fig.11 H&E staining of important organs and tissues of mice in each group is shown, scale bar, 200 μm. DETAILED DESCRIPTION

[0064] The above disclosure generally describes the present invention. A more complete understanding can be obtained by referring to the following specific examples. The purpose of describing these examples is only for illustration and is not intended to limit the scope of the present invention. Form changes and equivalent substitutions are considered to be circumstances that may suggest or confer convenience. Although specific terms are used in this article, the purpose of these terms is descriptive rather than limiting. The experimental methods in the following examples that do not specify specific conditions are usually tested under conventional conditions or under conditions recommended by the manufacturer. The raw materials and reagents used in the following examples are all commercially available products, or can be prepared by known methods.

[0065] The experiments used in the following examples of the present invention are as follows:

[0066] 1. CCK-8 experiment

[0067] (1) Cell plating: When the cells are growing well and have grown to about 80% of the bottom of the dish, digest the cells with trypsin, centrifuge and resuspend for counting. Dilute the cells to the concentrations of U87-MG: 30,000 cells / mL, TBD0220: 30,000 cells / mL, and GL261: 15,000 cells / mL. Add 100 μL of cell suspension to each well. In order to reduce errors, the principle of two-speed aspiration and one-speed addition is adopted. To prevent evaporation, PBS needs to be added to the surrounding wells, and then placed in a cell culture incubator for culture.

[0068] (2) Drug treatment: When the cells are attached and the confluence reaches about 60%, add drugs to the 96-well plate. Different concentrations of drugs can be prepared in advance, and the medium in the 96-well plate can be aspirated by changing the medium. Be careful to be gentle and clean, then add 100 μL of fresh medium containing drugs, gently shake the 96-well plate, and place it in the cell culture incubator for the required time.

[0069] (3) Add CCK-8 solution: After the incubation time is reached, take out the 96-well plate and add 10 μL CCK-8 to each well under light-proof conditions. Place the 96-well plate in a cell culture incubator for incubation, and observe the color changes during the incubation period, which takes about 1-4 hours.

[0070] (4) Measure absorbance and calculate cell viability: Select 450 nm wavelength on the microplate reader to measure absorbance, calculate the data, and draw a curve using GraphPad software.

[0071] 2. Plate colony formation experiment

[0072] (1) Plating: When the cells are in good growth condition and have grown to about 80% of the bottom of the dish, digest the cells with trypsin and centrifuge to make a single cell suspension for counting. Dilute the cells to 500 cells / mL for U87-MG and 500 cells / mL for TBD0220, respectively, and inoculate them into 6-well plates, with 3 replicates in each group. Mix well using the "cross method" and place in an incubator for culture.

[0073] (2) After the cells adhere to the wall, replace the medium and add drugs. Use a negative pressure aspirator in the clean bench to suck away the old culture medium and add the pre-diluted drugs, 2 mL of complete culture medium per well.

[0074] (3) Observe the cell status during the period. When obvious clones appear in the control group, use a negative pressure aspirator to remove the culture medium, wash twice with PBS, and then remove the PBS.

[0075] (4) Add tissue fixative to the wells to fix the cells and let them stand at room temperature for about 15 minutes. Then discard the fixative, wash gently with PBS three times, and aspirate the PBS.

[0076] (5) Add 2 mL of crystal violet dye to the wells for about 1 hour. Turn on the tap and adjust the water flow to a slow trickle to wash away the dye. Do not flush directly onto the cells. Finally, turn the 6-well plate upside down to allow it to dry naturally.

[0077] (6) Count the number of cloned cells and take photos to record.

[0078] 3. Western Blot

[0079] 3.1 Total cell protein extraction

[0080] (1) Add RIPA:PMSF:protein phosphatase inhibitor in a ratio of 100:1:1, invert to mix, and place on ice until ready to use.

[0081] (2) Take the cells out of the incubator and observe the density to determine how much cell protein lysis buffer to add (① If the cell density is particularly high, add 1 mL; ② If the cell density is 80%, add 800 μL; ③ If the cell density is 70%, add 700 μL).

[0082] (3) Remove the culture medium in a clean bench, rinse with PBS three times, and add protein lysis buffer. Shake the culture dish to allow the cell lysis buffer to fully contact the cells in the dish, then place the dish on ice for 30 minutes, shaking it every 5 minutes to fully lyse the cells.

[0083] (4) Precool the centrifuge at 4°C, clean the cell scraper with double distilled water, scrape the cells from the culture dish and transfer them to a marked EP tube, place it in a centrifuge at 4°C, set the speed to 13,000 rpm, and the time to 15 minutes. Pay attention to balancing.

[0084] (5) After centrifugation, transfer the cell lysis supernatant to a new EP tube and record the amount of aspirate.

[0085] 3.2 BCA protein concentration determination

[0086] (1) Prepare BCA assay working solution at a ratio of 50:1 for BCA reagent and 50:1 for Cu reagent. Use a vortexer to oscillate the solution to mix thoroughly.

[0087] (2) First add 45 μL PBS to the set mixing well, then add 5 μL of the sample to be tested to mix them thoroughly, and then transfer 20 μL of the mixed sample to the sample well.

[0088] (3) Add 200 μL of the pre-prepared BCA working solution to each well containing 20 μL of the sample to be tested. After adding, carefully move the 96-well plate to a 37°C incubator and react for 30 minutes.

[0089] (4) After the reaction is completed, remove the 96-well plate and move it steadily to the microplate reader in a light-proof condition. Set the microplate reader to 562 nm, measure the absorbance of the sample, and calculate its protein concentration.

[0090] (5) Add 1 / 5 volume of loading buffer to the protein supernatant and boil at 100°C for 10 minutes to denature the protein. Store at -20°C.

[0091] (6) After observing that the remaining separation gel in the gel tube has solidified, pour out the distilled water and absorb the remaining liquid between the glass plates for later use. At the same time, prepare the comb for later use.

[0092] (7) Prepare the concentrated gel according to the proportion and slowly inject it into the glass plate using a 200 μL pipette. To prevent the concentrated gel from solidifying, immediately and gently insert the comb diagonally, taking care to prevent the formation of bubbles.

[0093] (8) After waiting for about 30 minutes, the concentrated gel solidifies and the next step of electrophoresis can be carried out. The routine operations of electrophoresis, membrane transfer, antibody incubation and luminescence imaging are as follows.

[0094] 4. Construction of orthotopic transplanted tumor model

[0095] (1) Routinely culture TBD0220, U87-MG, GL261, and CT2A cell lines stably transfected with PTRF-overexpressing virus in advance.

[0096] (2) Digest the cells routinely to make a single-cell suspension. Use a disposable counting plate to count the cells. After centrifugation, discard the supernatant. Use pre-cooled PBS to wash the serum and adjust the concentration to 3-5×10 5 / 3μL;

[0097] (3) The model mice were conventionally anesthetized, the scalp was incised to expose the skull, a hole was drilled 2 mm to the right of the midline connecting the anterior and posterior bregma, and the cell suspension was aspirated with a 10 μL microneedle. The mouse was fixed with the assistance of a stereotaxic apparatus, the needle was inserted to a depth of 3 mm, the needle was withdrawn 1 mm, and a microinjection of 3 μL was performed. After the needle was stopped for 1 minute, it was removed, the exposed bone surface was wiped with a sterile cotton swab, and the skin was sutured;

[0098] (4) It takes about one week for tumors to form and bioluminescence imaging (isofluorane inhalation anesthesia) is performed. The substrate is diluted to 1× and injected intraperitoneally at 200 μL / mouse. After about 8 minutes, the luminometer is used to image the tumor and monitor tumor growth.

[0099] 5. Immunohistochemical staining experiment

[0100] (1) Dewaxing and rehydration: Dewaxing and rehydrating the paraffin sections according to the following process: soak in xylene I and xylene II for 30 minutes respectively, and soak in anhydrous ethanol I, anhydrous ethanol II, 95% ethanol, 85% ethanol, and distilled water for 5 minutes respectively. During this period, be sure to shake the sections several times to prevent insufficient dewaxing and rehydration.

[0101] (2) EDTA antigen repair: Prepare antigen repair solution with EDTA and distilled water at a ratio of 1:50. Microwave repair: high heat, 20-30 minutes, and heat a bottle of repair solution at the same time for each addition. Allow to cool naturally to room temperature and rinse with tap water. Note: Put the slices in the boiling state of the repair solution, and do not let the slices dry during the whole process; because the repair solution evaporates quickly in the later stage, observe frequently around the 13th minute to prevent the slices from drying out.

[0102] (3) Add endogenous peroxidase blocker (be careful to allow the droplets to accumulate on the tissue; failure to block properly may result in nonspecific background), incubate at 37°C for 10 minutes, and rinse with PBS 3 times for 3 minutes.

[0103] (4) Prepare the primary antibody at an appropriate concentration according to the antibody instructions. After adding the primary antibody, incubate at 4°C overnight or at 37°C for 2 hours. Note that the slide should not be too wet or too dry when wiping. Too wet will dilute the concentration of the primary antibody. Too dry will not form droplets. The primary antibody will flow to the edge and cannot gather on the tissue, which may cause uneven antibody incubation.

[0104] (5) To prevent tissue detachment, first place the wet box containing the slides at room temperature and rewarm for 30 minutes. After rewarming, rinse with PBS 3 times for 3 minutes.

[0105] (6) Add biotin-labeled goat anti-rabbit IgG and incubate at 37°C for 20 minutes. Rinse with PBS three times for 3 minutes.

[0106] (7) DAB color development: 1 mL DAB substrate solution + 1 drop of DAB concentrate, mix well, prepare and use immediately, and avoid light. Incubate at room temperature for 6 minutes. Rinse with tap water, and be sure not to rinse the tissue.

[0107] (8) Hematoxylin counterstaining: Incubate at room temperature for about 3 minutes and 30 seconds. The time for cytoplasmic proteins can be longer, while the time for nuclear proteins should be shorter. Rinse with tap water and immerse in water.

[0108] (9) 1% hydrochloric acid and alcohol differentiation: Place in hydrochloric acid and alcohol for a few seconds (be quick) and then take out and rinse (prepare water in advance) to determine the degree of nuclear staining.

[0109] (10) Ammonia water turns blue: about 2 seconds, the time depends on the blue return situation.

[0110] (11) Dehydration and transparency: 85% ethanol (1 minute) → 95% ethanol (1 minute) → anhydrous ethanol II (1 minute) → anhydrous ethanol I (5 minutes. Note that when changing the staining jar here, you need to control the slice rack before putting it into the next staining jar) → xylene II (1 minute) → xylene I (10 minutes).

[0111] (12) Sealing: Dilute xylene and neutral gum in a ratio of 1:1 to make a sealing agent. After adding the sealing agent, cover with a coverslip, press to remove bubbles, and allow to dry naturally.

[0112] 6. Immunofluorescence double staining experiment

[0113] (1) Dewaxing and rehydrating: Dewaxing and rehydrating the paraffin sections according to the following process: soak in xylene I and xylene II for 30 minutes respectively, and soak in anhydrous ethanol I, anhydrous ethanol II, 95% ethanol, 85% ethanol, and distilled water for 5 minutes respectively. During this period, be sure to shake the sections several times to prevent insufficient dewaxing and rehydration.

[0114] (2) EDTA antigen repair: In the last 15 minutes of dewaxing and rehydration, boil the EDTA repair solution in advance. When boiling bubbles are observed, place the hydrated sections in the repair solution. Set the repair time according to the preliminary experiment, which is about 20-30 minutes. Note: The tissue must be kept in the repair solution throughout the process. Because the repair solution evaporates quickly in the later stage, observe frequently around the 13th minute to prevent the sections from drying out.

[0115] (3) Leave it at room temperature and allow it to cool naturally to room temperature. Rinse it back and forth in a basin filled with tap water and rinse it once with PBS.

[0116] (4) Gently wipe off the PBS solution around the tissue with a paper towel, being careful not to damage the tissue. Dilute Triton X-100 with PBS solution to a concentration of 0.5%. Use a clean 100 μl pipette tip to drop the solution onto the tissue. Incubate at room temperature for 15 minutes. After incubation, rinse with PBS three times, each time for about 3 minutes.

[0117] (5) After wiping off the PBS, add 10% FBS and block at room temperature for 60 minutes.

[0118] (6) Wash off the blocking solution, add the primary antibody, and keep in a 4°C refrigerator overnight. Double distilled water needs to be added to the wet box in advance to prevent the slides from drying out. (Dilution of the antibody with antibody diluent is done in multiples according to the antibody instructions.)

[0119] (7) Rinse with PBS 3 times for 3 minutes and wipe off the excess PBS. Add 1:200 fluorescent secondary antibody in a dark room and incubate at room temperature for 60 minutes in the dark.

[0120] (8) Rinse with PBS three times, 3 minutes each time, and mount the sections with mounting medium containing DAPI.

[0121] (9) After staining, place the sample in a dry dark box and store it at -20°C away from light for a long time.

[0122] (10) After the sealing medium is dry, use a pathology slide scanner to take photos and analyze them.

[0123] Example 1 DMXAA increases TMZ sensitivity in vitro

[0124] To verify whether DMXAA increases the sensitivity of TMZ in vitro, we used the CCK-8 method to detect the IC50 of TMZ and TMZ+DMXAA combined treatment in three GBM cell lines, U87-MG, TBD0220, and GL261. Figure 1 As shown, the IC50 of the TMZ+DMXAA combined treatment group in the three cell lines was lower than that of the TMZ alone group, suggesting that DMXAA can increase the sensitivity of TMZ.

[0125] Next, we tested the in vitro synergistic ability of TMZ and DMXAA in U87-MG and TBD0220 cell lines using CCK-8 assay. We chose to treat U87-MG and TBD0220 cells for 48 h using the concentrations of TMZ and / or DMXAA derived from the preliminary experimental results. Figure 2 As shown, the synergistic effect scores calculated by the ZIP model were all greater than 10, indicating that TMZ and DMXAA exhibited a high degree of synergism in vitro.

[0126] Example 2 DMXAA combined with Echinomycin enhances the therapeutic effect of TMZ

[0127] Strongly encouraged by the fact that DMXAA increases TMZ sensitivity, and previous studies have confirmed that Echinomycin can also increase TMZ sensitivity, we next evaluated the effect of this combined treatment in TMZ resistance, divided into the following four groups: DMSO group, TMZ group, TMZ+DMXAA group, TMZ+DMXAA+Echinomycin group, we selected TMZ concentration of 50μM, DMXAA concentration of 10μM, Echinomycin concentration of 2nM, treated U87-MG, TBD0220 and GL261 cell lines for 48 hours according to the grouping, and further evaluated the effects of different drug combinations on cell viability through CCK-8 experiments, such as Figure 3 As shown, the results showed that in the three cell lines, the number of surviving cells in the TMZ alone group and the combination drug group was reduced compared with the control group. The number of surviving cells in the TMZ+DMXAA group was less than that in the TMZ alone group, and the number of surviving cells in the TMZ+DMXAA+Echinomycin group was significantly decreased compared with them.

[0128] Subsequently, we treated U87-MG and TBD0220 cell lines with the same grouping, dose, and treatment time, and confirmed by plate cloning, such as Figure 4 As shown, the results showed that compared with the control group, each treatment group had a certain degree of inhibition on the cell clone formation ability of the two cell lines, but the TMZ+DMXAA+Echinomycin group had the most obvious inhibitory effect on the cell clone formation ability.

[0129] Next, we tested the synergistic effect of TMZ with DMXAA and Echinomycin in TBD0220 cell line by CCK-8 assay. Figure 5 As shown in the figure, the horizontal axis represents the renaming of different drug concentration combinations of TMZ and DMXAA used in combination, and the vertical axis represents different concentrations of Echinomycin; "0" refers to: TMZ 0μM + DMXAA 0μM combination; "0.5" refers to: TMZ 50μM + DMXAA 2.5μM combination; "2.5" refers to: TMZ 50μM + DMXAA 20μM combination; "3" refers to: TMZ 100μM + DMXAA 2.5μM combination; "5" refers to: TMZ 100μM + DMXAA 20μM combination; "5.5" refers to: TMZ 150μM + DMXAA 2.5μM combination; "7.5" refers to: TMZ 150μM + DMXAA 20μM combination; "8" refers to: TMZ 200μM + DMXAA "10" refers to: TMZ 200μM + DMXAA 20μM combination; "10.5" refers to: TMZ 300μM + DMXAA 2.5μM combination; "12.5" refers to: TMZ 300μM + DMXAA 20μM combination. The synergistic effect score calculated by the ZIP model is 19.59, indicating that TMZ exhibits a high degree of synergism with DMXAA and Echinomycin.

[0130] The cGAS-STING signaling pathway is inhibited in glioma, leading to immunosuppression. In order to verify whether the combined treatment of TMZ+DMXAA+Echinomycin activates the cGAS-STING signaling pathway and exerts its effect, we detected the expression of TBK1, p-TBK1, P65, and p-p65, proteins related to the cGAS-STING signaling pathway. We selected the same drug concentration and treatment time as above to act on the U87-MG cell line for Western Bolt experiments, such as Figure 6As shown, the expression of cGAS-STING signaling pathway-related proteins in the TMZ+DMXAA+Echinomycin combined treatment group was significantly increased. The results suggested that this drug combination may increase the sensitivity of TMZ by activating the cGAS-STING pathway.

[0131] We next evaluated the antitumor activity of this combination therapy in vivo. We constructed an orthotopic glioma xenograft model using GL261 cells stably transfected with a virus overexpressing PTRF. Figure 7 As shown in a, DMSO, TMZ (5 mg / kg), TMZ (5 mg / kg) + DMXAA (12.5 mg / kg), and TMZ (5 mg / kg) + DMXAA (12.5 mg / kg) + Echinomycin (10 μg / kg) were given for treatment, with 5 days of treatment and 2 days of rest. TMZ was administered orally, DMXAA was injected intraperitoneally, and Echinomycin was injected into the tail vein. Figure 7 As shown in b, the results of bioluminescent small animal in vivo imaging showed that both the TMZ group and the TMZ+DMXAA group could inhibit tumor growth, but the tumor burden of mice in the TMZ+DMXAA+Echinomycin group was the lowest. Figure 7 As shown in Figure c, the weight change curve of mice showed that compared with the control group, the weight of mice in each treatment group showed a stable fluctuation state, and there was no obvious downward trend even in the late stage of treatment.

[0132] Subsequently, we performed H&E staining analysis on the tumor tissues of each group of mice. Figure 8 As shown in a, both the TMZ group and the TMZ+DMXAA group could inhibit tumor growth, while the TMZ+DMXAA+Echinomycin group had the best therapeutic effect and the smallest tumor burden. Next, we performed immunohistochemical staining on tumor tissues to detect the expression of Ki-67. Figure 8 As shown in b, the tumor proliferation expression in the TMZ+DMXAA+Echinomycin group was the lowest.

[0133] To further explore the effects of different drug combinations on angiogenesis in vivo, we used immunohistochemical staining to detect the expression of CD34 in the tumors of each group of mice and measured MVD, such as Fig. 9 As shown, the MVD value of the TMZ+DMXAA+Echinomycin combined treatment group was the lowest. The vascular structure was intact in the DMSO and TMZ groups, and endothelial cell vascular proliferation was significant, while the vascular cavity in the TMZ+DMXAA group was not significant, and vascular necrosis occurred. The TMZ+DMXAA+Echinomycin group had no typical vascular structure, and vascular structure destruction and disintegration were visible, and the number of microvessels was significantly reduced.

[0134] MHCII is a marker of M1-TAMs. To further explore the effect of TMZ+DMXAA+Echinomycin combined treatment on tumor-associated macrophages, we used immunohistochemical staining to detect the expression of MHCII and CD163 in the tumor tissues of each group of mice. Fig.10 As shown in the figure, compared with the control group, the expression of M1-TAMs increased in the TMZ+DMXAA group and the TMZ+DMXAA+Echinomycin group, and the expression of M2-TAMs decreased significantly, while there was no significant change in the TMZ alone group. This suggests that the combined treatment of TMZ+DMXAA+Echinomycin may reverse the transformation of M1-TAMs to M2-TAMs, providing a new idea for the treatment of gliomas.

[0135] Finally, we evaluated the safety of the drug and performed H&E staining analysis on important organs of mice in each group, such as Fig.11 As shown, the lungs of the TMZ+DMXAA treatment group showed slight congestion, the livers of the TMZ+DMXAA+Echinomycin treatment group showed slight edema, and no obvious damage was found in other organs.

[0136] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. A pharmaceutical composition for treating glioma, characterized in that: The pharmaceutical composition comprises a STING agonist or a pharmaceutically acceptable salt thereof, and temozolomide or a pharmaceutically acceptable salt thereof; Preferably, the STING agonist comprises DMXAA, diABZI, MSA-2, SR-717 and / or C53; Preferably, the STING agonist is selected from DMXAA.

2. The pharmaceutical composition according to claim 1, characterized in that The pharmaceutically acceptable salts include alkali metal salts, alkaline earth metal salts and ammonium salts; Preferably, the alkali metal salt includes potassium salt and sodium salt; Preferably, the alkaline earth metal salts include calcium salts and magnesium salts.

3. The pharmaceutical composition according to claim 1, characterized in that The concentration ratio of DMXAA to temozolomide in the pharmaceutical composition is 1:60 to 1:2.5; Preferably, the concentration ratio of DMXAA to temozolomide in the pharmaceutical composition is 1:15 to 1:2.5; Preferably, the concentration ratio of DMXAA to temozolomide in the pharmaceutical composition is (2.5 μM-20 μM): (50 μM-150 μM); More preferably, the concentration ratio of DMXAA to temozolomide in the pharmaceutical composition is (10 μM-20 μM): (50 μM-150 μM).

4. The pharmaceutical composition according to claim 1, characterized in that The pharmaceutical composition further comprises Echinomycin; Preferably, when the pharmaceutical composition further comprises Echinomycin, the concentration ratio of DMXAA, temozolomide and Echinomycin in the pharmaceutical composition is (2.5 μM~20 μM): (50 μM~300 μM): (0.5 nM~5 nM).

5. The pharmaceutical composition according to claim 1, characterized in that The pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient; Preferably, the pharmaceutical composition also includes other drugs for treating glioma.

6. A method for inhibiting the growth of glioma cells and promoting apoptosis of glioma cells in vitro for non-therapeutic purposes, characterized in that: The method comprises treating glioma cells with the pharmaceutical composition according to any one of claims 1 to 5; Preferably, the glioma cells include U87-MG, TBD0220 and / or GL261.

7. Any of the following applications, characterized in that: The applications include: (1) Use of a STING agonist or a pharmaceutically acceptable salt thereof and temozolomide or a pharmaceutically acceptable salt thereof in combination in the preparation of a product for treating and / or preventing glioma; (2) Use of a STING agonist or a pharmaceutically acceptable salt thereof and temozolomide or a pharmaceutically acceptable salt thereof in combination in the preparation of an agent for inhibiting glioma cell proliferation and promoting glioma cell apoptosis in vitro; (3) Use of a STING agonist or a pharmaceutically acceptable salt thereof in the preparation of a drug for improving the therapeutic effect of temozolomide in treating glioma and / or increasing the sensitivity of temozolomide; (4) Use of a STING agonist or a pharmaceutically acceptable salt thereof in combination with Echinomycin in the preparation of a drug for improving the therapeutic effect of temozolomide in treating glioma and / or increasing the sensitivity of temozolomide; (5) Use of a STING agonist or a pharmaceutically acceptable salt thereof, temozolomide or a pharmaceutically acceptable salt thereof, and echinomycin in combination for the preparation of a product for treating and / or preventing glioma; (6) Use of a STING agonist or a pharmaceutically acceptable salt thereof, temozolomide or a pharmaceutically acceptable salt thereof, and echinomycin in combination for the preparation of an agent for inhibiting glioma cell proliferation and promoting glioma cell apoptosis in vitro; (7) Use of a STING agonist or a pharmaceutically acceptable salt thereof, temozolomide or a pharmaceutically acceptable salt thereof, and echinomycin in combination for the preparation of a reagent for increasing the expression of proteins related to the cGAS-STING signaling pathway; (8) Use of a STING agonist or a pharmaceutically acceptable salt thereof, temozolomide or a pharmaceutically acceptable salt thereof, and echinomycin in combination for the preparation of a reagent for increasing the expression of proteins related to the cGAS-STING signaling pathway; (9) Use of a STING agonist or a pharmaceutically acceptable salt thereof, temozolomide or a pharmaceutically acceptable salt thereof, and echinomycin in combination for reversing and / or inhibiting the transformation of M1 macrophages to M2 macrophages.

8. The use according to claim 7, characterized in that: The STING agonist includes DMXAA, diABZI, MSA-2, SR-717 and / or C53; Preferably, the STING agonist is selected from DMXAA.

9. The use according to claim 7, characterized in that: The products include anti-glioma drug preparations and anti-glioma combination drugs.

10. The use according to claim 7, characterized in that: The cGAS-STING signaling pathway related proteins include TBK1, P-TBK1, P65 and / or P-P65.

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