Pharmaceutical composition for anti-glioma and use thereof

Abstract

The application discloses an anti-glioma pharmaceutical composition and application thereof, and the pharmaceutical composition comprises a STING agonist or a pharmaceutically acceptable salt thereof, and temozolomide or a pharmaceutically acceptable salt thereof, and / or echinomycin. The application proves through experiments that the pharmaceutical composition containing two or three active ingredients can significantly inhibit the growth of glioma cells and promote the apoptosis of glioma, and provides a brand-new strategy and thought for the treatment of glioma.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a pharmaceutical composition for treating glioma. BACKGROUND

[0002] Glioma is a tumor occurring in neuroectoderm, and is also called neuroectodermal tumor or neural epithelial tumor. The tumor originates from neural interstitial cells, i.e. neuroglia, ependyma, choroid plexus epithelium and neural parenchymal cells, i.e. neurons. Most tumors originate from different types of neuroglia, but according to the histogenetic origin and similar biological characteristics, various tumors occurring in neuroectoderm are generally called glioma. There are many classification methods for glioma, and clinical workers often use the simple Kernohan classification method. Among various types of glioma, the most common is astrocytoma, followed by glioblastoma, then medulloblastoma, ependymoma, oligodendroglioma, pineal tumor, mixed glioma, choroid plexus papilloma, unclassified glioma and neuronal tumor.

[0003] At present, the treatment of glioma is surgery, radiotherapy and chemotherapy in order of effect. Due to the invasive growth of glioma and unclear boundary with the surrounding tissue, although a microscope is used in surgery, a small amount of tumor cells will inevitably 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 concentration in the local tumor, and high-dose chemotherapy causes obvious systemic toxic side effects, such as bone marrow suppression, reduction of white blood cells and platelets, and pulmonary fibrosis. Although each treatment method has made obvious progress, and the comprehensive treatment of the three methods can prolong the survival period of patients, glioma is still difficult to cure. In recent years, new therapies such as immunotherapy, anti-angiogenesis and gene therapy have gone from the laboratory to the clinic. Temozolomide (TMZ) is the most important drug for treating glioma at present, and researchers are looking for new drugs based on this drug or drugs that can be used in combination to produce a synergistic effect. SUMMARY

[0004] In order to make up for the deficiencies of the prior art, the purpose of the present application is to provide a pharmaceutical composition capable of being used in combination with temozolomide for treating glioma.

[0005] In order to achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0006] The present application provides a pharmaceutical composition for treating glioma.

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

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

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

[0010] Further, the pharmaceutically acceptable salt includes alkali metal salts, alkaline earth metal salts, and ammonium salts.

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

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

[0013] In the present application, treatment refers to the improvement, prevention, or reversal of a disease or disorder or at least one discernible 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 disorder to be treated, which parameter need not necessarily be discernible or recognized by the mammal. In some embodiments, the treatment refers to the inhibition or slowing of a disease or disease progression, which inhibition or slowing can be physical, such as the alleviation of some discernible adverse symptoms. As used herein, "treatment" covers the treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease or disorder from occurring in an individual that is predisposed or does not yet experience the disease but that is at risk of getting the disease due to one or more factors; (b) inhibiting the disease, e.g., arresting its development; or (c) relieving the disease, e.g., causing regression of the symptoms associated with the disease.

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

[0015] In the present application, the pharmaceutically acceptable salt refers to acid salts formed with inorganic and / or organic acids and base salts formed with inorganic and / or organic bases. In addition, when the compound contains a basic moiety (for example, but not limited to, pyridine or imidazole) and an acidic moiety (for example, but not limited to, carboxylic acid), a zwitterion can be formed and the zwitterion is included in the pharmaceutically acceptable salts described in the present application. A pharmaceutically acceptable salt is preferably a salt that is pharmaceutically acceptable (i.e., non-toxic, physiologically acceptable) but other salts are also useful. The pharmaceutically acceptable salts of the compound can be formed, for example, by reacting the compound in a medium with a sufficient amount of an acid or a base, such as a medium in which the salt precipitates or an aqueous medium (which is lyophilized after the reaction).

[0016] Specific pharmaceutically acceptable salts include those that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, and the like, and are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts (salts thereof which are pharmaceutically acceptable) 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] Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or the salts of organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid. Also included are salts formed when an acidic proton present in the parent compound is replaced by a cation process such as ion exchange procedures. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, besylate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate. Pharmaceutically acceptable salts derived from suitable bases include alkali metal, alkaline earth metal, ammonium salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like.

[0018] In the present application, hydrate means a compound combined with water.

[0019] In the present application, solvate means a compound or its salt form combined with a solvent, usually formed by solvolysis. This physical association can include hydrogen bonding. Conventional solvents include, but are not limited to, methanol, ethanol, acetic acid, DMSO, THF, ether, and the like. In some instances, the solvate will be capable of isolation, for example, where one or more solvent molecules are incorporated in the crystal lattice of the solid state form of the compound. Solvates include solution-phase solvates and isolatable solvates.

[0020] In the present application, crystalline form means a 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, density, hardness, crystal shape, optical and electrical properties, stability, and solubility. The crystallization solvent, rate of crystallization, storage temperature, and other factors can result in one crystalline form being preferred. Various polymorphs of a compound can be prepared by crystallization under different conditions. In the present application, crystalline form also includes special crystal state, such as amorphous, etc.

[0021] Further, the concentration ratio of DMXAA and temozolomide in the pharmaceutical composition is 1:60~1:2.5.

[0022] Further, the concentration ratio of DMXAA and temozolomide in the pharmaceutical composition is 1:15~1:2.5.

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

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

[0025] Further, the pharmaceutical composition further comprises Echinomycin.

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

[0027] Further, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient.

[0028] Further, the pharmaceutically acceptable excipient includes, but is not limited to, a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an emulsifier, a co-solvent, a solubilizer, an osmotic pressure regulator, a lubricant, a surfactant, a coating material, a coloring agent, a pH regulator, an antioxidant or a bacteriostatic agent.

[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 alginic acid salt, xanthan gum, hydroxypropylcellulose, etc. The surfactant includes, but is not limited to, sodium dodecyl sulfate, stearic acid monoglyceride, cetyl alcohol, etc. The lubricant includes, but is not limited to, zinc stearate, talc, calcium and magnesium stearate, polyethylene glycol, polyoxyethylene monostearate, monolauryl sucrose acid ester, magnesium dodecyl 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 vinylpyrrolidone, sodium carboxymethyl starch, low-substituted hydroxypropyl methyl, cross-linked sodium carboxymethyl cellulose, soybean polysaccharide, etc.

[0030] Further, the pharmaceutical composition can further include other drugs for treating glioma. These drugs are not particularly limited as long as they can produce a therapeutic effect on glioma, and are within the scope of the present application. These drugs include, but are not limited to, acivicin, aclarubicin, acodazole hydrochloride, acronine, adozelesin, aldesleukin, asparaginase, asperlin, azacitidine, azetepa, azotomycin, batimastat, benzodepa, bicalutamide, bisantrene hydrochloride, bisnafide dimesylate, bizelesin, bleomycin sulfate, brequinar sodium, bropirimine, busulfan, cactinomycin, calusterone, caracemide, carbetimer, carboplatin, carmustine, carubicin hydrochloride, carzelesin, cedefingol, chlorambucil, cirolemycin, cisplatin, cladribine.

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

[0032] In the present application, chemotherapy refers to the treatment of a tumor by administration of a chemotherapeutic agent. A chemotherapeutic agent refers to a compound or derivative thereof that can interact with cancer cells, thereby reducing the proliferative state of the cells and / or killing the 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 derivatives thereof); substituted nucleotides; substituted nucleosides; DNA demethylating agents (also known as antimetabolites), such as azacitidine); antitumor antibiotics (e.g., mitomycin, doxorubicin); antitumor agents of plant origin (e.g., vincristine, vindesine, paclitaxel, Abraxane); cisplatin; carboplatin; etoposide. These agents can further include, but are not limited to, the anticancer agent trimethoxybenzoic acid ester (TMTX); temozolomide; raltitrexed; S-(4-nitrobenzyl)-6-thioinosine (NBMPR); 6-benzylguanine (6-BG); nitrosoureas (rabinopyranosyl-N-methyl-N-nitrosourea (Aranose), carmustine (BCNU, BiCNU), chlorozotocin, ethylnitrosourea (ENU), fotemustine, lomustine (CCNU), nimustine, N-nitroso-N-methylurea (NMU), ranimustine (MCNU), semustine, streptozocin (streptozotocin)); cytarabine; camptothecin; and therapeutic derivatives of any of the foregoing.

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

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

[0035] Further, the method comprises treating the glioma cells with the pharmaceutical composition according to the first aspect of the present application.

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

[0037] The third aspect of the present application provides the use of any of the following:

[0038] (1) the 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 the treatment and / or prevention of glioma;

[0039] (2) The use of the STING agonist or a pharmaceutically acceptable salt thereof and the temozolomide or a pharmaceutically acceptable salt thereof in combination in the preparation of a reagent for inhibiting the proliferation of glioma cells and promoting the apoptosis of glioma cells in vitro;

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

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

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

[0043] (6) The use of the STING agonist or a pharmaceutically acceptable salt thereof and the temozolomide or a pharmaceutically acceptable salt thereof and the Echinomycin in combination in the preparation of a reagent for inhibiting the proliferation of glioma cells and promoting the apoptosis of glioma cells in vitro;

[0044] (7) The use of the STING agonist or a pharmaceutically acceptable salt thereof and the temozolomide or a pharmaceutically acceptable salt thereof and the Echinomycin in combination in the preparation of a reagent for increasing the expression amount of a protein related to the cGAS-STING signaling pathway;

[0045] (8) The use of the STING agonist or a pharmaceutically acceptable salt thereof and the temozolomide or a pharmaceutically acceptable salt thereof and the Echinomycin in combination in the preparation of a reagent for increasing the expression amount of a protein related to the cGAS-STING signaling pathway;

[0046] (9) The use of the STING agonist or a pharmaceutically acceptable salt thereof and the temozolomide or a pharmaceutically acceptable salt thereof and the Echinomycin in combination in reversing and / or inhibiting the transformation of M1 type macrophages into M2 type macrophages.

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

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

[0049] Further, the product includes an anti-glioma drug preparation, an anti-glioma combination drug.

[0050] Further, the protein related to the cGAS-STING signaling pathway includes TBK1, P-TBK1, P65 and / or P-P65.

[0051] In the present application, to improve means to allow the subject or tumor cell to enhance 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 response ability. 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] The present application has the advantages and beneficial effects: the present application proves by experiments that the STING agonist DMXAA combined with temozolomide has a synergistic effect on the treatment of glioma, and proves that DMXAA combined with Echinomycin can also enhance the treatment effect of TMZ, providing a new strategy for the treatment of glioma. BRIEF DESCRIPTION OF DRAWINGS

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

[0054] Figure 2 Drug synergy score combination matrix (ZIP model) of different concentrations of TMZ and DMXAA treated cells for 48 hours were shown; wherein, a is U87-MG cell line; b is TBD0220 cell line;

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

[0056] Figure 4 The effect of different drug combinations on the clonogenic ability of U87-MG, TBD0220 cells and statistical analysis of the number of cell clones were shown;

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

[0058] Figure 6 Expression of cGAS-STING signaling pathway related proteins in different drug combinations were shown;

[0059] Figure 7Figure 6 shows that DMXAA combined with Echinomycin can enhance the therapeutic effect of TMZ; wherein a is the experimental flow chart of constructing GL261-PTRFov glioma orthotopic tumor model; b is representative imaging of bioluminescent mice and quantitative analysis of bioluminescent intensity of mice in vivo imaging; c is the curve of body weight change of mice;

[0060] Figure 8 Figure 7 shows H&E staining and Ki-67 immunohistochemical staining analysis of tumor tissues of mice in each group; wherein a is a representative image of H&E staining of brain of each group of mice; 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] Figure 9 Figure 8 shows CD34 immunohistochemical staining analysis and MVD value of tumor tissues of mice in each group, scale bar, 10 μm;

[0062] Figure 10 Figure 9 shows MHCII, CD163 immunohistochemical staining and its semi-quantitative analysis of tumor tissues of mice in each group, scale bar, 10 μm;

[0063] Figure 11 Figure 10 shows H&E staining of important organ tissues of mice in each group, scale bar, 200 μm. DETAILED DESCRIPTION

[0064] The above disclosure describes the present application in general terms. A more complete understanding can be obtained by reference to the following specific examples. The purpose of describing these examples is merely to illustrate and not intended to limit the scope of the present application. Form changes and equivalent replacements are considered as circumstances may suggest or confer convenience. Although specific terms are used herein, the purpose of these terms is descriptive rather than limiting. The experimental methods in the following examples, unless otherwise specified, are generally carried out according to conventional conditions or according to the conditions recommended by the manufacturer. The raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

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

[0066] 1. CCK-8 experiment

[0067] (1) Cell plating: under the microscope, when the cells grow well and reach about 80% of the bottom of the dish, the cells are trypsinized, centrifuged and resuspended for counting. The cells are diluted to U87-MG: 30000 cells / mL, TBD0220: 30000 cells / mL, GL261: 15000 cells / mL, respectively. Add 100 μL of cell suspension to each well. To reduce errors, use the principle of two-speed suction and one-speed addition. To prevent evaporation, add PBS around the hole, and then place it in the cell culture incubator.

[0068] (2) Drug treatment: when the cells adhere and the confluence reaches about 60%, add drugs to the 96-well plate. Prepare different concentrations of drugs in advance. Replace the culture medium in the 96-well plate with fresh medium containing drugs, be careful to move gently and suck clean, then add 100 μL of fresh medium containing drugs, shake the 96-well plate gently, and place it in the cell culture incubator for the required time.

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

[0070] (4) Measure the absorbance to calculate the cell survival rate: select 450 nm wavelength on the enzyme marker to measure the absorbance, calculate the data, and draw the curve with GraphPad software.

[0071] 2. Plate colony formation experiment

[0072] (1) Plating: under the microscope, when the cells grow well and reach about 80% of the bottom of the dish, the cells are trypsinized, centrifuged and resuspended for counting. The cells are diluted to U87-MG: 500 cells / mL, TBD0220: 500 cells / mL, respectively, and inoculated in 6-well plates, 3 replicates per group. Mix well with the "cross method" and place in the incubator.

[0073] (2) After the cells adhere, use a negative pressure aspirator to remove the old culture medium in a clean bench, and add the drug diluted in advance, 2 mL of complete medium per well.

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

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

[0076] (5) Add 2 mL of crystal violet dye to the hole for dyeing, and the dyeing time is about 1 hour. Open the faucet and adjust to a slow water flow to wash off the dye, do not directly flush the cells. Finally, invert the 6-well plate and let it dry naturally.

[0077] (6) Calculate the number of cloned cells and take a photo.

[0078] 3. Western blot

[0079] 3.1 Total protein extraction

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

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

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

[0083] (4) Pre-cool the 4°C centrifuge, wash the cell scraper with distilled water, and then transfer the cells from the culture dish to the labeled EP tube, and place it in the 4°C centrifuge, set the speed to 13000 rpm, and the time to 15 minutes, and make sure it is balanced.

[0084] (5) After centrifugation, transfer the cell lysate supernatant to a new EP tube and record the volume.

[0085] 3.2 BCA protein concentration determination

[0086] (1) Prepare the BCA detection working solution with a ratio of BCA reagent:Cu reagent = 50:1, and use a vortex to mix thoroughly.

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

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

[0089] (4) After the reaction is completed, take out the 96-well plate, and move it to the enzyme marker under the condition of avoiding light. Set the enzyme marker to 562 nm, measure the absorbance of the sample, and calculate the protein concentration.

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

[0091] (6) After observing the remaining separation gel in the glue pipe, pour off the distilled water, and absorb the remaining liquid between the glass plates for standby, and prepare a comb for standby.

[0092] (7) Prepare the concentrated gel according to the proportion, and slowly inject 200 μL of the concentrated gel into the glass plate with a pipette. To prevent the concentrated gel from solidifying, immediately insert the comb obliquely and gently, and pay attention to prevent air bubbles from being generated.

[0093] (8) After waiting for about 30 minutes, the concentrated gel solidifies, and the next step of electrophoresis can be performed. The electrophoresis, membrane transfer blocking, antibody incubation, and luminescence imaging are performed according to the conventional operation.

[0094] 4. Constructing an orthotopic tumor model

[0095] (1) The TBD0220, U87-MG, GL261, and CT2A cell lines stably transfected with the overexpressed PTRF virus were routinely cultured in advance.

[0096] (2) The cells were routinely digested to form a single cell suspension, and the cells were counted using a disposable counting plate. After centrifugation, the supernatant was discarded, the serum was washed with pre-cooled PBS, and the concentration was adjusted to 3-5 x 10 5 per 3 μL;

[0097] (3) The model mouse was routinely anesthetized, the scalp was incised to expose the skull, and the right side was opened 2 mm from the midline between the anterior fontanel and the posterior fontanel. Drill a hole, use a 10 μL micropipette to suck the cell suspension, use a brain stereotactic instrument to assist fixation, and the needle depth is 3 mm. Retract the needle by 1 mm, inject 3 μL, stop the needle for 1 minute after the end, and remove it. Wipe the exposed bone surface with a sterile cotton swab, and suture the skin;

[0098] (4) Tumors can be formed in about 1 week, and bioluminescence imaging (isoflurane inhalation anesthesia) is performed. Dilute the substrate to 1 x, inject it intraperitoneally, and 200 μL per mouse. Imaging is performed after about 8 minutes using a luminescence instrument to detect tumor growth.

[0099] 5. Immunohistochemical staining experiment

[0100] (1) Dewaxing and rehydration: The paraffin sections are dewaxed and rehydrated according to the following procedure: xylene I, xylene II, 100% ethanol, 100% ethanol, 95% ethanol, 85% ethanol, and distilled water, each for 30 minutes. During the process, shake the sections several times to prevent incomplete dewaxing and rehydration.

[0101] (2) EDTA antigen retrieval: Prepare an antigen retrieval solution by mixing EDTA and distilled water at a ratio of 1:50. Microwave for 20-30 minutes, and heat a bottle of the solution for each addition. Allow the solution to cool to room temperature naturally, and then rinse with tap water. Note: The solution should be added to the sections while boiling, and the sections should not be dried during the entire process. Since the solution evaporates quickly, observe the sections frequently after 13 minutes to prevent drying.

[0102] (3) Add endogenous peroxidase blocking agent dropwise (make sure the drops are on the tissue; if the blocking is not complete, it may result in non-specific background), 37°C, 10 minutes, and rinse with PBS 3 times for 3 minutes each.

[0103] (4) Prepare the primary antibody according to the manufacturer's instructions. After adding the primary antibody, incubate at 4°C overnight or at 37°C for 2 hours. Note: Do not make the sections too wet or too dry when wiping them; if they are too wet, the primary antibody concentration will be diluted, and if they are too dry, the primary antibody drops will flow to the edges and not gather on the tissue, which may result in uneven antibody incubation.

[0104] (5) To prevent the sections from falling off, first place the wet box containing the slides at room temperature and allow them to warm up for 30 minutes. After warming, rinse the sections with PBS 3 times for 3 minutes each.

[0105] (6) Add biotin-labeled goat anti-rabbit IgG dropwise, 37°C, 20 minutes, and rinse with PBS 3 times for 3 minutes each.

[0106] (7) DAB color development: Mix 1 mL of DAB substrate solution with 1 drop of DAB concentrate, and use it immediately. Note: Avoid light. Incubate at room temperature for 6 minutes. Rinse with tap water, but do not let the water reach the tissue.

[0107] (8) Hematoxylin counterstaining: Incubate at room temperature for about 3 minutes and 30 seconds. The cytoplasmic protein may be incubated for a little longer, while the nuclear protein may be incubated for a little shorter. After rinsing with tap water, immerse the sections in water.

[0108] (9) 1% hydrochloric acid alcohol differentiation: Place the sections in hydrochloric acid alcohol for a few seconds (the action should be fast), then remove them and rinse with water (prepare the water in advance). This determines the degree of nuclear staining.

[0109] (10) Ammonia blue return: About 2 seconds, depending on the blue return.

[0110] (11) Dehydration, transparency: 85% ethanol (1 minute) → 95% ethanol (1 minute) → anhydrous ethanol II (1 minute) → anhydrous ethanol I (5 minutes, note that at this point, take out the slide rack to control it before putting it into the next staining cylinder) → xylene II (1 minute) → xylene I (10 minutes).

[0111] (12) Mounting: xylene and neutral gum are diluted as mounting agent at 1:1, drop the mounting agent, cover the cover glass, press to remove bubbles, and let it dry naturally.

[0112] 6. Immunofluorescence double staining experiment

[0113] (1) De-waxing and rehydrating: the paraffin sections are de-waxed and rehydrated according to the following process, xylene I and xylene II are soaked for 30 minutes respectively, anhydrous ethanol I, anhydrous ethanol II, 95% ethanol, 85% ethanol, and distilled water are soaked for 5 minutes respectively. Pay attention to shaking frequently during the process to prevent insufficient de-waxing and rehydrating.

[0114] (2) EDTA antigen repair: at the last 15 minutes of de-waxing and rehydrating, boil the EDTA repair solution in advance, and when boiling bubbles are observed, place the hydrated sections in the repair solution. The repair time is about 20-30 minutes according to the pre-experiment. Note that the tissue should be in the repair solution throughout the process, and observe frequently at about the 13th minute to prevent dry mounting due to rapid evaporation of the repair solution.

[0115] (3) Place at room temperature and cool naturally to room temperature. Rinse once in both directions in a basin filled with tap water, and rinse once with PBS.

[0116] (4) Gently wipe the PBS solution around the tissue with a paper towel, taking care not to damage the tissue. Dilute Triton X-100 with PBS to a concentration of 0.5%, and use a clean 100 μl syringe to drop the solution onto the tissue. Incubate at room temperature for 15 minutes, and then rinse with PBS for 3 times, each for about 3 minutes.

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

[0118] (6) Wash off the blocking solution and drop the primary antibody. Incubate in the refrigerator at 4°C overnight. Add double distilled water to the wet box in advance to prevent dry mounting. (According to the antibody instructions, dilute the antibody by the dilution ratio of the antibody diluent.)

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

[0120] (8) Rinse with PBS for 3 times, each for 3 minutes, and seal with mounting agent containing DAPI.

[0121] (9) After staining, place the slides in a dry dark box and store at -20°C in the dark for long-term storage.

[0122] (10) After the slides are dried, use a pathological slide scanner to take pictures and analyze them.

[0123] Example 1 DMXAA increases TMZ sensitivity in vitro

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

[0125] Next, we detected the in vitro synergy of TMZ and DMXAA in U87-MG and TBD0220 cell lines by CCK-8 experiment, and selected the concentration of TMZ and / or DMXAA obtained from the pre-experiment results to treat U87-MG and TBD0220 cells for 48 hours, as shown in Figure 2 , the synergy score calculated by ZIP model is greater than 10, indicating that TMZ and DMXAA show high synergy in vitro.

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

[0127] Encouraged by the strong effect of DMXAA in increasing TMZ sensitivity, and previous studies showing that Echinomycin can also increase TMZ sensitivity, we next evaluated the effect of this combination therapy in TMZ resistance, divided into the following four groups: DMSO group, TMZ group, TMZ+DMXAA group, TMZ+DMXAA+Echinomycin group, we selected the concentration of TMZ as 50μM, the concentration of DMXAA as 10μM, the concentration of Echinomycin as 2nM, according to the grouping to treat U87-MG, TBD0220 and GL261 cell lines for 48 hours, through CCK-8 experiment, further evaluate the effect of different drug combinations on cell viability, as shown in Figure 3 , the results found that in three cell lines compared with the control group, the number of surviving cells in the TMZ alone group was reduced, 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 reduced compared with the TMZ+DMXAA group.

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

[0129] Then, we detected the synergistic ability of TMZ and DMXAA and Echinomycin in the TBD0220 cell line by CCK-8 experiment, and the results are shown in Figure 5 The horizontal coordinate in the figure represents the renaming of different drug concentration combinations of TMZ and DMXAA used in combination, and the vertical coordinate represents different concentrations of Echinomycin; "0" refers to: TMZ 0 μM + DMXAA 0 μM combination; "0.5" refers to: TMZ 50 μM + DMXAA 2.5 μM combination; "2.5" refers to: TMZ 50 μM + DMXAA 20 μM combination; "3" refers to: TMZ 100 μM + DMXAA 2.5 μM combination; "5" refers to: TMZ 100 μM + DMXAA 20 μM combination; "5.5" refers to: TMZ 150 μM + DMXAA 2.5 μM combination; "7.5" refers to: TMZ 150 μM + DMXAA 20 μM combination; "8" refers to: TMZ 200 μM + DMXAA 2.5 μM combination; "10" refers to: TMZ 200 μM + DMXAA 20 μM combination; "10.5" refers to: TMZ 300 μM + DMXAA 2.5 μM combination; "12.5" refers to: TMZ 300 μM + DMXAA 20 μM combination, and the synergistic effect score calculated by ZIP model is 19.59, indicating that TMZ and DMXAA and Echinomycin show high synergistic effect.

[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 plays a role, we detected the expression of TBK1, p-TBK1, P65, p-p65 related proteins in the cGAS-STING signaling pathway, and we selected the same drug concentration and treatment time as above to act on the U87-MG cell line for Western Bolt experiment, as shown in Figure 6As shown, the expression of cGAS-STING signaling pathway-related proteins was significantly increased in the TMZ+DMXAA+Echinomycin combination treatment group, suggesting that this drug combination may increase TMZ sensitivity by activating the cGAS-STING pathway.

[0131] Our next step was to evaluate the antitumor activity of this combination therapy in vivo. We constructed an orthotopic xenograft model of glioma by stably transfecting GL261 cell lines overexpressing PTRF virus. The experimental procedure is as follows: Figure 7 As shown in Figure a, patients were treated with 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), respectively, for 5 days followed by a 2-day break. TMZ was administered orally, DMXAA was administered intraperitoneally, and Echinomycin was administered via tail vein injection. Figure 7 As shown in b, in vivo imaging results of bioluminescent small animals showed that both the TMZ group and the TMZ+DMXAA group inhibited tumor growth, but the TMZ+DMXAA+Echinomycin group had the lowest tumor burden in mice. Figure 7 As shown in Figure c, the mouse weight change curves show that, compared with the control group, the weight of mice in each treatment group exhibits a stable fluctuation, and there is no significant downward trend even in the later stages of treatment.

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

[0133] To further investigate the effects of different drug combinations on angiogenesis in vivo, we used immunohistochemical staining to detect the expression of CD34 in tumors of mice in each group and measured MVD. Figure 9 As shown, the TMZ+DMXAA+Echinomycin combined treatment group had the lowest MVD value. The DMSO and TMZ groups had intact vascular structures and significant endothelial cell angiogenesis, while the TMZ+DMXAA group had insignificant vascular lumen and vascular necrosis. The TMZ+DMXAA+Echinomycin group had no typical vascular structure, and vascular structure destruction and disintegration were visible, with a significant reduction in the number of microvessels.

[0134] MHCII is a marker of M1-TAMs, in order to further explore the effect of TMZ+DMXAA+Echinomycin combination therapy on tumor-associated macrophages, we used immunohistochemical staining method to detect the expression of MHCII, CD163 in tumor tissues of each group of mice, and the results are shown in Figure 10 Compared with the control group, M1-TAMs in TMZ+DMXAA group, TMZ+DMXAA+Echinomycin group increased, and M2-TAMs expression decreased significantly, while the simple TMZ group had no significant change. It is suggested that TMZ+DMXAA+Echinomycin combination therapy may reverse the transformation of M1-TAMs to M2-TAMs, and provide a new idea for the treatment of glioma.

[0135] Finally we evaluated the safety of the drug, H&E staining analysis of important organs of each group of mice, as shown in Figure 11 The lung of TMZ+DMXAA treatment group appeared slight congestion, and the liver of TMZ+DMXAA+Echinomycin treatment group showed slight edema, and other organs had no obvious damage.

[0136] The above description of the embodiments is only for the purpose of understanding the method of the present application and its core idea. It should be noted that for those skilled in the art, without departing from the principles of the present application, the present application can be improved and modified, and these improvements and modifications will also fall within the scope of protection of the claims of the present application.

Claims

1. A pharmaceutical composition for treating glioma, characterized by, The pharmaceutical composition comprises DMXAA or a pharmaceutically acceptable salt thereof and temozolomide or a pharmaceutically acceptable salt thereof. The concentration ratio of DMXAA and temozolomide in the pharmaceutical composition is 1:60-1:2.

5.

2. The pharmaceutical composition of claim 1, wherein, The pharmaceutically acceptable salt comprises alkali metal salt, alkaline earth metal salt and ammonium salt.

3. The pharmaceutical composition of claim 2, wherein, The alkali metal salt comprises potassium salt and sodium salt.

4. The pharmaceutical composition of claim 2, wherein, The alkaline earth metal salt comprises calcium salt and magnesium salt.

5. The pharmaceutical composition of claim 1, wherein, The concentration ratio of DMXAA and temozolomide in the pharmaceutical composition is 1:15-1:2.

5.

6. The pharmaceutical composition according to any one of claims 1 to 5, characterized in that, The concentration ratio of DMXAA and temozolomide in the pharmaceutical composition is (2.5 μM-20 μM):(50 μM-150 μM).

7. The pharmaceutical composition of claim 6, wherein, The concentration ratio of DMXAA and temozolomide in the pharmaceutical composition is (10 μM-20 μM):(50 μM-150 μM).

8. The pharmaceutical composition of claim 1, wherein, The pharmaceutical composition further comprises Echinomycin. 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).

9. The pharmaceutical composition of claim 1, wherein, The pharmaceutical composition further comprises pharmaceutically acceptable excipient.

10. A method for inhibiting growth of glioma cells and promoting apoptosis of glioma cells for non-therapeutic purposes in vitro, characterized in that, The method comprises treating glioma cells with the pharmaceutical composition according to any one of claims 1-9.

11. The method of claim 10, wherein, The glioma cells comprise U87-MG, TBD0220 and / or GL261.

12. DMXAA or a pharmaceutically acceptable salt thereof and temozolomide or a pharmaceutically acceptable salt thereof are used in the preparation of a product for treating and / or preventing glioma; the concentration ratio of DMXAA and temozolomide is 1:60-1:2.

5.

13. Use according to claim 12, characterized in that, The product comprises anti-glioma pharmaceutical preparation.

14. The use according to claim 12, characterized in that, The product comprises anti-glioma combination drug.

15. DMXAA or a pharmaceutically acceptable salt thereof and temozolomide or a pharmaceutically acceptable salt thereof are used in the preparation of a reagent for inhibiting the proliferation of glioma cells and promoting the apoptosis of glioma cells in vitro; the concentration ratio of DMXAA and temozolomide is 1:60-1:2.

5.

16. DMXAA or a pharmaceutically acceptable salt thereof is used in the preparation of a drug for improving the therapeutic effect of temozolomide in treating glioma and / or increasing the sensitivity of temozolomide; the concentration ratio of DMXAA and temozolomide is 1:60-1:2.

5.

17. DMXAA or a pharmaceutically acceptable salt thereof and Echinomycin are used in the preparation of a drug for improving the therapeutic effect of temozolomide in treating glioma and / or increasing the sensitivity of temozolomide; the concentration ratio of DMXAA, temozolomide and Echinomycin is (2.5 μM-20 μM):(50 μM-300 μM):(0.5 nM-5 nM).

18. Use of DMXAA or a pharmaceutically acceptable salt thereof and temozolomide or a pharmaceutically acceptable salt thereof and Echinomycin in combination in the manufacture of a product for treating and / or preventing glioma; the concentration ratio of DMXAA, temozolomide and Echinomycin is (2.5 μM ~ 20 μM) : (50 μM ~ 300 μM) : (0.5 nM ~ 5 nM).

19. Use according to claim 18, characterized in that, The product comprises an anti-glioma pharmaceutical preparation.

20. The use according to claim 18, characterized in that, The product comprises an anti-glioma combination drug.

21. Use of DMXAA or a pharmaceutically acceptable salt thereof and temozolomide or a pharmaceutically acceptable salt thereof and Echinomycin in combination in the manufacture of a reagent for inhibiting the proliferation of glioma cells and promoting the apoptosis of glioma cells in vitro; the concentration ratio of DMXAA, temozolomide and Echinomycin is (2.5 μM ~ 20 μM) : (50 μM ~ 300 μM) : (0.5 nM ~ 5 nM).

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