Application of combination of benzisoselenazole compound and radiotherapy in treatment of diffuse midline glioma

Abstract

The invention discloses application of combination of a benzisoselenazole compound and radiotherapy in treatment of diffuse midline glioma, and creatively finds that the combination of the benzisoselenazole compound and radiotherapy has a remarkable synergistic treatment effect on the diffuse midline glioma for the first time. The invention provides an effective drug combination strategy for treatment of diffuse midline glioma, and has good clinical application prospects and important transformation significance in the technical field of research and development of diffuse midline glioma treatment drugs.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the application of a benzisoselenazole compound combined with radiotherapy in the treatment of diffuse midline glioma. Background Art

[0002] Diffuse midline gliomas (DMGs) are primary brain tumors. They are often located in midline structures such as the thalamus, brainstem, and spinal cord. The high incidence of histone H3K27M mutations is a key molecular biological basis for their occurrence. DMGs in the brainstem and thalamus are highly invasive, capable of diffusely invading surrounding normal brain tissue, making them one of the most lethal brain tumors. Due to the unique expression of H3K27 mutations in diffuse midline gliomas and their significant correlation with prognosis, the 2016 WHO Classification of Tumors of the Central Nervous System first classified them as WHO grade IV. Their pathological characteristics are characterized by histone H3K27M mutations, diffuse infiltrative growth, and extremely high malignancy.

[0003] However, the treatment and prognosis of DMG are not ideal. The median survival of DMG patients is less than 1 year, and there is currently no standard treatment for DMG. Single therapies such as surgical resection and conventional chemoradiotherapy are unlikely to improve the prognosis. At the surgical level, tumors are mostly located in key midline areas such as the thalamus and brainstem, growing in a diffuse and infiltrative manner with unclear boundaries from normal tissue, making complete resection impossible. Forced surgery may also damage nerve function and cause serious complications such as paralysis. Conventional chemoradiotherapy is also ineffective. Radiotherapy can shrink the tumor in the short term but cannot prevent recurrence. High doses can also damage normal brain tissue, leading to sequelae such as cognitive decline. Chemotherapy is hindered by the blood-brain barrier, and tumors are prone to drug resistance, making it difficult to achieve effective concentrations.

[0004] Therefore, developing effective combination therapy for DMG has become a key direction for conquering DMG. How to integrate the advantages of different therapies to synergistically overcome difficulties such as tumor invasion and improve the survival of DMG patients is one of the major technical challenges currently faced by those skilled in the art. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide the art with an application of a benzisoselenazole compound combined with radiotherapy in the treatment of diffuse midline glioma.

[0006] The present invention adopts the following technical solutions to achieve the above-mentioned invention objectives:

[0007] The first aspect of the present invention provides the use of a benzisoselenazole compound or a pharmaceutically acceptable salt thereof in combination with radiotherapy in the preparation of a medicament for treating and / or preventing diffuse midline glioma or spinal cord glioma, wherein the structural formula of the benzisoselenazole compound is shown in formula (I):

[0008]

[0009] Furthermore, the radiotherapy is X-ray irradiation, gamma ray irradiation, beta ray irradiation, proton beam irradiation and / or electron beam irradiation;

[0010] Optionally, the radiotherapy is X-ray irradiation.

[0011] Furthermore, the dosage ratio of the benzisoselenazole compound or a pharmaceutically acceptable salt thereof to radiotherapy is 0.5-1.5:1.5-0.5;

[0012] Optionally, the dosage ratio of the benzisoselenazole compound or a pharmaceutically acceptable salt thereof to radiotherapy is 1:1.

[0013] Furthermore, the dosage ratio of the benzisoselenazole compound or a pharmaceutically acceptable salt thereof to radiotherapy is: 1-15 μM: 1-15 Gy;

[0014] Optionally, the dosage ratio of the benzisoselenazole compound or a pharmaceutically acceptable salt thereof to radiotherapy is: 2-10 μM: 2-10 Gy.

[0015] In a specific embodiment of the present invention, the present invention discovered for the first time that the above-mentioned benzisoselenazole compound or a pharmaceutically acceptable salt thereof combined with radiotherapy has a synergistic effect in the treatment of diffuse midline glioma or spinal cord glioma. The present invention has demonstrated through in vitro cell experiments and in vivo animal experiments that the combination of the two can significantly inhibit the growth of diffuse midline glioma cells or spinal cord glioma cells, promote the apoptosis of diffuse midline glioma cells or spinal cord glioma cells, and inhibit the growth of diffuse midline glioma tissue or spinal cord glioma tissue, showing a significant synergistic killing effect.

[0016] In some embodiments, the pharmaceutically acceptable salt of the benzisoselenazole compound refers to a salt form obtained by salt modification of the benzisoselenazole compound, which is suitable for use in pharmaceutical formulations and clinical applications. Salts used in the salt modification process include, but are not limited to, inorganic acid salts (e.g., hydrochlorides, sulfates, phosphates) and organic acid salts (e.g., citrates, maleates, tartrates). These salts are suitable for contact with patients within the scope of reliable medical judgment and do not produce undue toxicity, irritation, allergic reactions, etc.

[0017] Specific pharmaceutically acceptable salts include those salts that are suitable for contact with the tissues of humans and lower animals without excessive toxicity, irritation, allergic reactions, etc., and 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 of the present invention include salts derived from suitable inorganic and organic acids and inorganic and organic bases.

[0018] In some embodiments, examples of pharmaceutically acceptable salts include, but are not limited to, salts with (as counter ions) alkali metal ions such as Na + 、Li + or K + or salts with alkaline earth metal ions such as Ca 2+ or Mg 2+ or any other pharmaceutically acceptable metal ion such as Zn 2+ or Al 3+ or a pharmaceutically acceptable salt formed with an organic base such as diethanolamine, ethanolamine, N-methylglucamine, triethanolamine or tromethamine.

[0019] In some embodiments, pharmaceutically acceptable base addition salts are formed with metals or amines, such as alkali metal and alkaline earth metal hydroxides or organic amines. Examples of metals used as cations include magnesium, potassium, sodium, calcium, and the like. Examples of suitable amines include N,N'-dibenzylethylenediamine, diethanolamine, chloroprocaine, choline, ethylenediamine, N-methylglucamine, or procaine.

[0020] In some embodiments, base addition salts of acidic compounds can be prepared by contacting the free acid form with a sufficient amount of the desired base in a conventional manner to form the salt. The free acid can be regenerated by contacting the salt form with an acid and isolating the free acid in a conventional manner.

[0021] In some embodiments, pharmaceutically acceptable salts may include cations based on alkali and alkaline earth metals, such as sodium, potassium, calcium, lithium, magnesium, and the like, as well as non-toxic ammonium, quaternary ammonium, and amine cations, including but not limited to ammonium, tetramethylammonium, methylamine, dimethylamine, tetraethylammonium, trimethylamine, triethylamine, ethylamine, and the like. Salts of amino acids, such as gluconate, arginate, galacturonate, and the like are also contemplated.

[0022] In the present invention, the technical solutions related to the use of the above-mentioned benzisoselenazole compound or its pharmaceutically acceptable salt or its hydrate, enantiomer, diastereomer, solvate or crystalline form in combination with radiotherapy in the preparation of a drug for treating and / or preventing diffuse midline glioma or spinal cord glioma all fall within the scope of protection of the present invention.

[0023] In some embodiments, the hydrate refers to a compound (specifically the above-mentioned benzisoselenazole compound) that is combined with water. Those skilled in the art will understand that an organic compound can form a complex with a solvent in which it reacts or precipitates or crystallizes. These complexes are called solvates. When the solvent is water, the complex is called a hydrate.

[0024] In some embodiments, the solvate refers to a compound (specifically the above-mentioned benzisoselenazole compound) or a salt thereof that is combined with a solvent, typically formed by a solvolysis reaction. This physical association may include hydrogen bonding. Conventional solvents include water, methanol, ethanol, acetic acid, DMSO, THF, ether, and the like. 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.

[0025] In some embodiments, the crystalline form refers to a crystalline form of a compound (specifically, the above-mentioned benzisoselenazole compound) with a specific crystal packing arrangement. Different crystalline forms generally 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 can lead to a dominant crystalline form. Various polymorphs of a compound can be prepared by crystallization under different conditions.

[0026] In the present invention, the term "treating and / or preventing" refers to delaying the development of a disease (specifically, diffuse midline glioma or spinal cord glioma), preventing the development of a disease, and / or reducing the severity of symptoms that will develop or are expected to develop. Thus, these terms include ameliorating existing disease symptoms, preventing additional symptoms, ameliorating or preventing potential metabolic causes of symptoms, inhibiting the disorder or disease, e.g., arresting the development of the disorder or disease, alleviating the disorder or disease, causing the disorder or disease to regress, alleviating symptoms caused by the disease or disorder, or halting symptoms of the disease or disorder.

[0027] In some embodiments, the dosage ratio of the benzisoselenazole compound or a pharmaceutically acceptable salt thereof to radiotherapy is any one of 2 μM:2 Gy, 4 μM:4 Gy, 6 μM:6 Gy, 8 μM:8 Gy, and 10 μM:10 Gy.

[0028] In some embodiments, the ratio of the amount of the benzisoselenazole compound or a pharmaceutically acceptable salt thereof to radiotherapy is: 30-180 mg / kg / time: 2-15 Gy.

[0029] The second aspect of the present invention provides a combined pharmaceutical composition.

[0030] Furthermore, the pharmaceutical composition comprises the benzisoselenazole compound described in the first aspect of the present invention and radiation.

[0031] Furthermore, the radiation is X-rays, gamma rays, beta rays, proton beams and / or electron beams;

[0032] Optionally, the radiation is X-rays;

[0033] Optionally, the dose ratio of the benzisoselenazole compound or a pharmaceutically acceptable salt thereof to the radiation is 0.5-1.5:1.5-0.5;

[0034] Optionally, the dose ratio of the benzisoselenazole compound or a pharmaceutically acceptable salt thereof to the radiation is 1:1;

[0035] Optionally, the ratio of the amount of the benzisoselenazole compound or a pharmaceutically acceptable salt thereof to the radiation is: 1-15 μM: 1-15 Gy;

[0036] Optionally, the dosage ratio of the benzisoselenazole compound or a pharmaceutically acceptable salt thereof to radiation is: 2-10 μM: 2-10 Gy.

[0037] In some embodiments, the administration of the benzisoselenazole compound in the pharmaceutical composition and the irradiation with radiation can be performed simultaneously, separately or sequentially.

[0038] Wherein, simultaneously refers to the simultaneous application of benzisoselenazole compound and the irradiation of radiation.If not simultaneously, then carry out successively within a time range so that the two can work therapeutically within the same time range.Therefore, successively can allow 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours or several hours after application of benzisoselenazole compound or radiation irradiation to carry out radiation irradiation or apply benzisoselenazole compound.The time delay of the application of benzisoselenazole compound and the irradiation of radiation will change according to the nature of the component, the interaction therebetween, and the drug half-life.With simultaneously or successively different, refer to respectively that the interval between the application of benzisoselenazole compound and the irradiation of radiation is significant.

[0039] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0040] In some embodiments, the pharmaceutically acceptable excipients include, but are not limited to, diluents, binders, surfactants, wetting agents, adsorption carriers, lubricants, fillers, and disintegrants.

[0041] In some embodiments, the diluent includes, but is not limited to, lactose, sodium chloride, glucose, urea, starch, water, and the like. The binder includes, but is not limited to, starch, pregelatinized starch, dextrin, maltodextrin, sucrose, gum arabic, gelatin, methylcellulose, carboxymethylcellulose, alginic acid and alginates, xanthan gum, hydroxypropyl cellulose, and the like. The surfactant includes, but is not limited to, sodium lauryl sulfate, monoglyceride stearate, cetyl alcohol, and the like. The humectant includes, but is not limited to, glycerol, starch, and the like. The adsorbent includes, but is not limited to, starch, lactose, bentonite, bentonite, and the like. The lubricant includes, but is not limited to, zinc stearate, talc, calcium and magnesium stearate, polyethylene glycol, polyoxyethylene monostearate, monolauric sucrose, magnesium lauryl sulfate, and the like. The filler includes, but is not limited to, mannitol, xylitol, sorbitol, maltose, glucose, lactose, sucrose, dextrin, starch, and the like. The disintegrants include but are not limited to: cross-linked vinyl pyrrolidone, sodium carboxymethyl starch, low-substituted hydroxypropyl methyl, cross-linked sodium carboxymethyl cellulose, soybean polysaccharides, etc.

[0042] A third aspect of the present invention provides a pharmaceutical preparation.

[0043] Furthermore, the pharmaceutical preparation comprises the pharmaceutical composition according to the second aspect of the present invention;

[0044] Optionally, the dosage form of the pharmaceutical preparation includes an injection dosage form, an intrathecal administration dosage form under lumbar puncture, an oral administration dosage form, a respiratory tract administration dosage form, a cavity administration dosage form, a mucosal administration dosage form or a skin administration dosage form.

[0045] In some embodiments, the dosage form of the pharmaceutical preparation includes a dosage form for enteral administration and a dosage form for parenteral administration.

[0046] Illustratively, the dosage form for administration through the gastrointestinal tract includes tablets, granules, capsules, solutions, dry suspensions, powders, sustained-release preparations, effervescent preparations, emulsions, suspensions, syrups, drops or chewable tablets.

[0047] Illustratively, the non-gastrointestinal administration dosage form includes an injection dosage form, a respiratory tract administration dosage form, a cavity administration dosage form, a mucosal administration dosage form or a skin administration dosage form.

[0048] In some embodiments, the injection dosage forms include but are not limited to: intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection and intracavitary injection and other injections; the respiratory tract administration dosage forms include but are not limited to: sprays, aerosols, powder aerosols, etc.; the cavity administration dosage forms include but are not limited to: suppositories, aerosols, effervescent tablets, drops, pills, etc.; the mucosal administration dosage forms include but are not limited to: eye drops, nasal drops, eye ointments, gargles, sublingual tablets, adhesive tablets, patches, etc.; the skin administration dosage forms include but are not limited to: solutions, etc.

[0049] In some embodiments, the pharmaceutical composition or pharmaceutical preparation may further contain other drugs or agents that can be used to treat and / or prevent, or assist in the treatment and / or prevention of diffuse midline glioma or spinal cord glioma. The other drugs or agents are not particularly limited. As long as they can produce the expected treatment and / or prevention, or assist in the treatment and / or prevention effect on diffuse midline glioma or spinal cord glioma, they are within the scope of protection of the present invention.

[0050] In some embodiments, the other drugs or agents that can be used for the treatment and / or prevention, or adjuvant treatment and / or prevention of diffuse midline glioma or spinal cord glioma include but are not limited to: chemotherapy drugs, specifically, the chemotherapy drugs include but are not limited to: taxanes (paclitaxel, docetaxel, cabazitaxel), antibiotics (doxorubicin, epirubicin, daunorubicin, pirarubicin, etoposide, irinotecan, mitoxantrone), antimetabolites (cisplatin, carboplatin, lobaplatin, nedaplatin, oxaliplatin, gemcitabine, 5-fluorouracil), alkylating agents (cyclophosphamide, ifosfamide).

[0051] In some embodiments, the suitable dosage of the pharmaceutical composition or pharmaceutical preparation described in the present invention can be prescribed in a variety of ways according to factors such as the formulation method, administration method, patient's age, weight, gender, morbidity, diet, administration time, administration route, excretion rate and reaction sensitivity. A skilled physician can usually easily determine the prescription and the desired effective dosage for treatment. As long as it can produce the expected therapeutic and / or preventive effect on diffuse midline glioma or spinal cord glioma, such dosage is within the scope of protection of the present invention.

[0052] The fourth aspect of the present invention provides an in vitro method for inhibiting the growth of glioma cells and promoting the apoptosis of glioma cells for non-therapeutic purposes.

[0053] Furthermore, the method comprises: treating in vitro glioma cells with the benzisoselenazole compound described in the second aspect of the present invention in combination with radiation;

[0054] Optionally, the glioma cells are diffuse midline glioma cells or spinal cord glioma cells.

[0055] A fifth aspect of the present invention provides the application of any of the following aspects:

[0056] (1) Use of the benzisoselenazole compound or a pharmaceutically acceptable salt thereof described in the first aspect of the present invention in combination with radiotherapy in the preparation of a pharmaceutical preparation for treating and / or preventing diffuse midline glioma or spinal cord glioma;

[0057] (2) Use of the benzisoselenazole compound or a pharmaceutically acceptable salt thereof described in the first aspect of the present invention in the preparation of an anti-inflammatory drug.

[0058] Furthermore, the anti-inflammatory drug is a drug that reduces the increased levels of IL-6 and TNF-α caused by radiotherapy.

[0059] In addition, the present invention also provides a method for treating and / or preventing diffuse midline glioma or spinal cord glioma, the method comprising: administering a therapeutically and / or preventively effective amount of the pharmaceutical composition described in the second aspect of the present invention or the pharmaceutical preparation described in the third aspect of the present invention to a subject in need.

[0060] In the present invention, the subject includes mammals and non-mammals. Examples of mammals include, but are not limited to, any member of the class Mammalia: humans, non-human primates such as chimpanzees and other apes and monkeys; farm animals such as cattle, horses, sheep, goats, and pigs; domestic animals such as rabbits, dogs, and cats; and laboratory animals, including rodents such as rats, mice, and guinea pigs. Examples of non-mammals include, but are not limited to, birds and fish. In one embodiment of the present invention, the subject is a human. The subject includes patients diagnosed with diffuse midline glioma or spinal cord glioma.

[0061] In the present invention, the effective amount refers to the amount of the compound that effectively produces the desired preventive, allergic, or therapeutic effect. The amount of the pharmaceutical composition or pharmaceutical preparation of the present invention required to achieve the effective amount will vary depending on factors such as the compound, the symptoms and their severity, and the age of the mammal being treated. However, the specific amount can be routinely determined by a person of ordinary skill in the art based on their knowledge in the field and the disclosure herein. Any dosage that can produce the aforementioned effects is within the scope of this invention.

[0062] In some embodiments, administration of the pharmaceutical composition or pharmaceutical preparation can be carried out in any form, including but not limited to: oral, topical, parenteral, intramuscular, intravenous, subcutaneous, transdermal (which can include a penetration enhancer), and other routes of administration. In one embodiment of the present application, the active compound or combination of compounds as previously described is provided in a solution dosage form as is known in the art. The most effective dosage form will depend on the bioavailability or pharmacokinetics of the particular agent selected and the severity of the patient's disease.

[0063] The present application has the following advantages and beneficial effects relative to the prior art:

[0064] The present application first creatively discovers that the combination of benzisoselenazol compounds and radiotherapy has a significant synergistic therapeutic effect on diffuse midline glioma, and the therapeutic effect of the combined drug composition on diffuse midline glioma is significantly better than that of benzisoselenazol compounds or radiotherapy alone, which has a significant advantage in the treatment of diffuse midline glioma. The present application provides an effective drug combination strategy for the treatment of diffuse midline glioma, and has good clinical application prospect and important transformation significance in the technical field of research and development of diffuse midline glioma treatment drugs. BRIEF DESCRIPTION OF DRAWINGS

[0065] Figure 1 : Cell survival rate of different doses of BS1801, radiotherapy and combined treatment, blue fold line is the single radiotherapy group, green fold line is the BS1801 single drug treatment group, red fold line is the combined treatment group. The horizontal coordinate is the dose of radiotherapy and BS1801, and the vertical coordinate is the relative cell survival rate. The statistical method of each point is t test, and the comparison group is the single radiotherapy group. Each dose point has 3 parallel repeats;

[0066] Figure 2 : Synergistic effect of BS1801 and radiotherapy Fa-CI curve, combined treatment group 0 μM+0 Gy, 2 μM+2 Gy, 4 μM+4 Gy, 6 μM+6 Gy, 8 μM+8 Gy, 10 μM+10 Gy, and the combined effect of the single drug and single radiotherapy group. The points in the figure represent each combination point, and the dose increases from right to left; CI=1 is considered equivalent, CI>1 represents that at this combination dose, the two treatment measures are antagonistic and do not have synergistic effect; CI<1 represents that at this combination dose, the two treatment measures have synergistic effect and have synergistic effect;

[0067] Figure 3Figure 6: Tumor volume changes of different treatment groups during treatment, wherein, Control: control group; IR: radiotherapy alone group; BSL: BS1801 low-dose group (30 mg / kg); BSM: BS1801 medium-dose group (90 mg / kg); BSH: BS1801 high-dose group (180 mg / kg); BSM+IR: BS1801 medium-dose group (90 mg / kg) + radiotherapy combined treatment group; the abscissa is the measurement time point after treatment, and the ordinate is the tumor volume;

[0068] Figure 4 Figure 6: Tumor volume changes of different treatment groups during treatment, wherein, Control: control group; IR: radiotherapy alone group; BSL: BS1801 low-dose group (30 mg / kg); BSM: BS1801 medium-dose group (90 mg / kg); BSH: BS1801 high-dose group (180 mg / kg); BSM+IR: BS1801 medium-dose group (90 mg / kg) + radiotherapy combined treatment group; the abscissa is the measurement time point after treatment, and the ordinate is the tumor volume;

[0069] Figure 5 Figure 6: Tumor volume changes of different treatment groups during treatment, wherein, Control: control group; IR: radiotherapy alone group; BSL: BS1801 low-dose group (30 mg / kg); BSM: BS1801 medium-dose group (90 mg / kg); BSH: BS1801 high-dose group (180 mg / kg); BSM+IR: BS1801 medium-dose group (90 mg / kg) + radiotherapy combined treatment group; the abscissa is the measurement time point after treatment, and the ordinate is the tumor volume;

[0070] Figure 6 Figure 6: Tumor volume changes of different treatment groups during treatment, wherein, Control: control group; IR: radiotherapy alone group; BSL: BS1801 low-dose group (30 mg / kg); BSM: BS1801 medium-dose group (90 mg / kg); BSH: BS1801 high-dose group (180 mg / kg); BSM+IR: BS1801 medium-dose group (90 mg / kg) + radiotherapy combined treatment group; the abscissa is the measurement time point after treatment, and the ordinate is the tumor volume;

[0071] Figure 7: Immunohistochemistry results of paraffin sections of tumor tissues in different treatment groups, where Control: control group; IR: radiotherapy alone group; BSH: BS1801 high-dose group (180 mg / kg); BSM+IR: BS1801 medium-dose group (90 mg / kg) plus radiotherapy combined treatment group; the left side is a schematic diagram of typical results, scale bar is 100 μm, blue represents cell nuclei, tan represents CHOP-positive cells, and the right side shows the number of CHOP-positive cells in each group calculated using ImageJ software; *** and **** represent p < 0.001 and p < 0.0001, respectively;

[0072] Figure 8 : The levels of IL-6 and TNF-α in the serum of mice in different treatment groups, among which, Control: control group; IR: radiotherapy alone group; BSL: BS1801 low-dose group (30 mg / kg); BSM: BS1801 medium-dose group (90 mg / kg); BSH: BS1801 high-dose group (180 mg / kg); BSM+IR: BS1801 medium-dose group (90 mg / kg) + radiotherapy combined treatment group. DETAILED DESCRIPTION

[0073] The present invention will be further described below in conjunction with specific examples. The following specific examples are only used to explain the present invention and are not to be construed as limiting the present invention. Those skilled in the art will appreciate that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and purpose of the present invention, and that the scope of the present invention is defined by the claims and their equivalents. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.

[0074] The reagents, raw materials, and experimental consumables used in the present invention are readily available to those of ordinary skill in the art and, unless otherwise specified, can be obtained commercially. Experimental methods for which specific conditions are not specified in the present invention are generally performed under conventional conditions or as recommended by the manufacturer. In particular, the following examples are intended only to illustrate the present invention and should not limit the scope of the present invention in any way. It should be noted that the experimental conditions and results described in the following examples are intended only to illustrate the present invention and should not, and will not, limit the present invention described in detail in the claims.

[0075] Example 1 Cell experiments demonstrate that the combination of BS1801 and radiotherapy has a synergistic therapeutic effect on the treatment of diffuse midline glioma

[0076] 1. Experimental Materials

[0077] (1) Matrigel: high concentration low factor phenol red-free matrigel; lamboride company; Beijing; MG2263; the substrate gel polymerizes to form a three-dimensional matrix with biological activity at room temperature, simulating the structure, composition, physical properties and function of the body cell basement membrane, which is conducive to the culture and differentiation of cells in vitro, and the study of cell morphology, biochemical function, migration, invasion and gene expression.

[0078] (2) Cell Counting Kit 8: Cell Counting Kit-8 cell proliferation toxicity detection kit; Dojindo company; Japan; CK04; the kit uses a new type of water-soluble tetrazolium salt WST-8 formazan dye as a chromogenic substrate, which is reduced by dehydrogenase in cells to produce water-soluble formazan dye. By directly measuring the absorbance of formazan dye (450 nm), the number of living cells can be measured, and the number of cells is proportional to the formazan dye.

[0079] (3) Irradiator: PRECISION X-Rad160XL is used to irradiate cells; PRECISION company; USA; X-Rad160XL; the machine uses X-ray for external irradiation, which can be used for radiotherapy of cells or small animals.

[0080] 2. Experimental method

[0081] (1) Add 400 μL of matrigel to 50 mL of PBS solution and mix well to prepare working solution, add 100 μL of matrigel working solution to each well of 96-well plate, and place the plate in a 37℃, 5% CO2 incubator for 1h for coating. Take out the coated 96-well plate, discard the matrigel working solution, add 100 μL of PBS solution to each well, and prepare for use.

[0082] (2) SCA-S02 cells in logarithmic growth phase (the laboratory independently constructed human diffuse midline glioma cells carrying H3K27M mutation, which are patient-derived spinal cord glioma cells, also known as human diffuse midline glioma cells, which are preserved in China General Microbiological Culture Collection Center, with the preservation number of CGMCC NO.45809. The relevant information is disclosed in the invention patent application with the application number of 202411765890.5 and the invention name of a mutant spinal cord glioma cell line) are washed with PBS, then 1 mL of Accutase solution is added, and the cells are digested at 37℃, 5% CO2 for 5 min. Centrifuge at 1000 rpm for 5 min, resuspend with 1 mL of medium. Take 10 μL of cell suspension, add 190 μL of PBS and mix well, then take 20 μL of cell solution and add it to the cell counting plate for cell counting.

[0083] (3) Take 3×10 5 Place cells in a 15 mL centrifuge tube, add culture medium to 6 mL, and mix thoroughly. Seed the cells in a 96-well plate at a density of 5,000 cells per well, using 100 μL of cell solution per well, and incubate overnight at 37°C in a 5% CO2 incubator.

[0084] (4) Prepare BS1801 (benzisoselenazole compound, the corresponding structural formula of which is shown in Formula (I), and its relevant information is clearly disclosed in the invention patent application number 202211460109.4, entitled "Use of a Benzisoselenazole Compound in the Preparation of a Medicament for Treating Spinal Cord Glioma") stock solutions of 0 μM, 2 μM, 4 μM, 6 μM, 8 μM, and 10 μM, with three replicate wells for each concentration. Discard the culture medium in the 96-well plate, add 100 μL of the corresponding drug-containing culture medium according to the above grouping, and incubate in a 37°C, 5% CO2 incubator for 24 h.

[0085]

[0086] (5) The doses of the radiotherapy group (X-ray) were: 0 Gy, 2 Gy, 4 Gy, 6 Gy, 8 Gy, and 10 Gy, with 3 auxiliary holes for each dose.

[0087] (6) Combined treatment group: The dose ratio of BS1801 to radiotherapy was 1:1, i.e., 0 μM + 0 Gy, 2 μM + 2 Gy, 4 μM + 4 Gy, 6 μM + 6 Gy, 8 μM + 8 Gy, and 10 μM + 10 Gy. The radiotherapy and combined treatment groups were treated with radiotherapy using an irradiator at the prescribed dose, followed by incubation at 37°C, 5% CO2 for 24 hours. In the combined treatment group, the drug was administered first, followed by immediate radiotherapy. BS1801 was added to a 96-well plate at the prescribed dose, and the 96-well plate was immediately placed on the radiotherapy table for radiotherapy at the indicated dose.

[0088] (7) Prepare CCK8 working solution: Add 10 μL of CCK8 stock solution to 100 μL of culture medium per well to prepare an appropriate volume of working solution. Store in a dark place. Discard the culture medium and add 100 μL of CCK8 working solution to each well. Shake the 96-well plate and incubate in a 37°C, 5% CO2 incubator for 2 h.

[0089] (8) Place the 96-well plate in a microplate reader and shake for 10 seconds. Measure the absorbance of each well at 450 nm and 630 nm. Calculate the relative cell viability (%): absorbance A = OD450 – OD630; relative cell viability = experimental group A / control group A × 100%.

[0090] (9) Use GraphPad software to fit the curve and calculate IC 50CompuSyn software was used to calculate the coupling coefficient of BS1801 and radiotherapy and fit the Fa-CI curve.

[0091] 3. Experimental results

[0092] The results of cell survival rates of different doses of BS1801, radiotherapy and combined treatment are shown in the figure below. Figure 1 As shown, the blue line is the radiotherapy group, the green line is the BS1801 monotherapy group, and the red line is the combined therapy group. The horizontal axis is the dose of radiotherapy and BS1801, and the vertical axis is the relative cell survival rate. It can be seen that with the increase of the dose, the cell survival rate decreased significantly. The dotted line is the median lethal dose, i.e. IC 50 The statistical method for each point was t-test, and the control group was the radiotherapy group alone. Each dose point was repeated three times.

[0093] The results of the combined effect of 0μM+0Gy, 2μM+2Gy, 4μM+4Gy, 6μM+6Gy, 8μM+8Gy, and 10μM+10Gy compared with the single drug and radiotherapy groups are shown in the figure below. Figure 2 As shown, the dots in the figure represent each combination point, with increasing doses from right to left. A CI of 1 is considered equivalent, a CI > 1 indicates antagonism between the two treatments at that combination dose, with no synergistic effect, and a CI < 1 indicates synergistic effect between the two treatments at that combination dose. These results demonstrate that BS1801 exhibits a synergistic effect with radiotherapy starting at the lowest combined dose of 2 μM + 2 Gy, demonstrating that combination therapy can enhance the effects of either treatment alone. Specifically, the combination of BS1801 and radiotherapy has a synergistic therapeutic effect in the treatment of diffuse midline gliomas.

[0094] Example 2 Animal experiments demonstrate that the combination of BS1801 and radiotherapy has a synergistic therapeutic effect on the treatment of diffuse midline glioma

[0095] 1. Experimental Materials

[0096] (1) Experimental animals: 50 SPF-grade female 6-week-old BALB / c nude mice, weighing approximately 20 g, were purchased from Beijing Weitonglihua Company.

[0097] (2) Tribromoethanol: 2,2,2-tribromoethanol; purchased from Sigma, USA; T48402; used as an anesthetic for rodents.

[0098] 2. Experimental methods

[0099] (1) Fifty SPF female 6-week-old BALB / c nude mice were housed in the animal room for 3 days to allow the mice to adapt to the environment.

[0100] (2) After amplification of SCA-S02 cells, the cells were collected in a 1.5 mL EP tube and counted to make a 1×10 6 50 μL of normal saline was added to the suspension to reduce the error of the solution volume, and the cell suspension was placed on ice for inoculation.

[0101] (3) Pick up the mouse with your left hand in a standard mouse-holding posture, disinfect the mouse's armpit skin with an alcohol cotton ball, insert the syringe needle 5 mm into the mouse's armpit and withdraw the needle 2 mm, and slowly inject 10 μL of cell suspension. After the injection is completed, stabilize the needle for 20 seconds to prevent the cell fluid from flowing out, then remove the syringe and inoculate the next mouse.

[0102] (4) 14 days after inoculation, the subcutaneous tumor volume was measured using a vernier caliper and the mice were weighed. 36 mice with similar tumor volume and body weight were divided equally into 6 groups, with 6 mice in each group. Treatments were performed as shown in Table 1 below.

[0103] Table 1 Treatment

[0104]

[0105] (5) The BS1801 treatment group received a single oral administration with a volume of 100 μL per day for 15 consecutive days. The start time of administration was D1, the second day of administration was D2, and so on.

[0106] (6) The radiotherapy group received a single dose of 1 Gy per day for 15 consecutive days, with a total dose of 15 Gy. Radiotherapy was performed using a small animal irradiator. Before irradiation, anesthetics were first prepared. Tribromoethanol was diluted with PBS at a ratio of 1:40, mixed and protected from light. 350 μL of anesthetic was injected intraperitoneally into the mouse to anesthetize it. The mice in the radiotherapy group and the combined treatment group were shielded with lead plates, and only the tumor-inoculated site in the armpit was exposed for irradiation. Among them, the combined treatment group was first administered with medication and then immediately radiotherapy. 100 μL of BS1801 was first orally administered, and then the mouse was immediately placed on the radiotherapy table for radiotherapy at a dose of 1 Gy / day.

[0107] (7) During the treatment period, the mice were monitored daily. The long and short diameters of the axillary tumors were measured every 2 days using a vernier caliper, and the tumor volume was calculated using the following formula: tumor volume (mm 3 ) = 0.5236 × major diameter × minor diameter × minor diameter. The mice were weighed on a body scale. 18 hours after the last treatment, all mice were bled from their eyeballs and euthanized. The tumor volume was measured and weighed once.

[0108] (8) Use Excel and GraphPad software for data processing and statistical analysis and draw charts.

[0109] 3. Experimental results

[0110] The gross images of the tumors in the 6 groups of mice after 15 days of treatment are as follows Figure 3 The results show that the BSM+IR group had a significantly better therapeutic effect than the other groups.

[0111] Tumor volume change curves of the 6 groups of mice during treatment are shown in Figure 2. Figure 4 The results show that the BSM+IR group had a significantly better therapeutic effect than the other groups.

[0112] The tumor volumes and tumor inhibition rates at the end of treatment in the 6 groups of mice were as follows: Figure 5 The results are shown in the table below: Control: Control group; IR: Radiotherapy alone; BSL: Low-dose BS1801 (30 mg / kg); BSM: Medium-dose BS1801 (90 mg / kg); BSH: High-dose BS1801 (180 mg / kg); and BSM+IR: Combined treatment with medium-dose BS1801 (90 mg / kg) and radiotherapy. The results showed that the combination therapy group had the best therapeutic effect, demonstrating that BS1801 and radiotherapy can exert a synergistic effect.

[0113] The weight change curves of the 6 groups of mice during treatment are as follows Figure 6 The results are shown in the figure below, where Control: Control group; IR: Radiotherapy alone group; BSL: Low-dose BS1801 group (30 mg / kg); BSM: Medium-dose BS1801 group (90 mg / kg); BSH: High-dose BS1801 group (180 mg / kg); and BSM+IR: Combined treatment group with medium-dose BS1801 (90 mg / kg) plus radiotherapy. The results showed that mice in the radiotherapy alone group experienced a significant weight loss, exceeding 10%, while the weight of mice in the other groups increased. This suggests that radiotherapy has severe toxic side effects, while BS1801 has a good safety profile and that combined radiotherapy can mitigate these toxic side effects.

[0114] Example 3 Immunohistochemical detection of CHOP in tumor tissues of mice in different treatment groups

[0115] 1. Experimental Materials

[0116] (1) Antigen retrieval solution: Immunohistochemistry antigen retrieval buffer (EDTA antigen retrieval solution pH 8.0); Zhongshan Jinqiao Company; Beijing; ZLI-9067; used for antigen retrieval before immunohistochemical staining, the main components include disodium ethylenediaminetetraacetic acid, tris(hydroxymethyl)aminomethane, etc.

[0117] (2) Immunohistochemistry pen: Immunohistochemistry pen; Zhongshan Jinqiao Company; Beijing; ZLI-9303; used for hydrophobic tissue sections.

[0118] (3) Endogenous peroxidase inhibitor, blocking sheep serum, enzyme-labeled sheep anti-mouse / rabbit IgG polymer, and DAB color development solution: purchased from Zhongshan Jinqiao Immunohistochemistry Kit; Beijing; PV-6000D; used to remove peroxidase from sections, block antigens, bind secondary antibodies after primary antibodies, and develop immunohistochemical staining.

[0119] (4) CHOP antibody: Anti-CHOP mouse monoclonal antibody; CST; USA; 2895S; dilution ratio 1:200; used for primary antibody labeling of tissue sections.

[0120] 2. Experimental methods

[0121] (1) Tissue embedding: The tumors of each group of mice were cut into pieces no larger than 5 mm. 3 The small pieces were embedded in embedding boxes and dehydrated overnight using a dehydrator.

[0122] (2) Place the dehydrated tissue on a 65°C operating table, place the tissue into a metal embedding box in turn, and pour in paraffin solution to avoid bubbles.

[0123] (3) Place the metal embedding box on a -20℃ operating table to cool and make a wax block.

[0124] (4) Use a microtome to cut the paraffin-embedded tissue into 5 μm thick slices, spread the slices in a 40°C warm water bath, fix the slices on a slide, and dewax in a 65°C oven for 3 h.

[0125] (5) Place the slices on a slice rack and sequentially place them in xylene I (10 min), xylene II (10 min), anhydrous ethanol I (10 min), anhydrous ethanol II (10 min), 95% alcohol I (10 min), 95% alcohol II (10 min), 75% alcohol (5 min), and rinse with ultrapure water twice, each time for 3 min.

[0126] (6) Place the slices in a humidified chamber and add 200 μL of 0.1% Triton X-100 solution to permeabilize the membrane for 15 min. Discard the liquid and rinse once with ultrapure water.

[0127] (7) Place the slice rack in a 1L beaker, dilute 10× sodium citrate antigen retrieval solution with ultrapure water to 500mL of 1× antigen retrieval solution, pour it into the beaker, and place it in a microwave oven.

[0128] (8) Heat on high heat for 3-5 minutes until the liquid boils, then adjust to medium-high heat. Continue heating for 15 minutes, then remove the beaker. After cooling naturally to room temperature, remove the slice rack and rinse it in PBS solution for 2 minutes.

[0129] (9) Add 200 μL of endogenous peroxidase blocker to the slices, place them in the dark at room temperature for 10 min, spin dry the liquid, and wash them with PBS three times, each time for 5 min.

[0130] (10) Add 200 μL of blocking sheep serum solution to the slices, place them in the dark at room temperature for 1 h, and shake off the liquid.

[0131] (11) Antibody diluent: Prepare the primary antibody solution according to the concentration in the instructions. Add 50 μL of CHOP antibody primary antibody solution to the slices and incubate in a humidified chamber at 4°C overnight.

[0132] (12) Prepare PBST solution by mixing Tween and PBS at a ratio of 1:1000. Wash the sections with PBST solution three times, 10 min each time, add 100 μL of secondary antibody solution, and incubate at room temperature in the dark for 1 h.

[0133] (13) Wash the sections three times with PBST solution, each time for 10 minutes. Prepare DAB colorimetric solution at a ratio of 1:20, shake off the PBST, and add 50 μL of DAB colorimetric solution to each section. Observe the sections under a microscope. When the tissue is stained brownish yellow, immediately soak the sections in PBST to terminate the color development.

[0134] (14) Stain the nucleus with hematoxylin for 10 seconds, wash the sections with PBST, and observe the degree of staining under a microscope. If the nucleus is darkly stained, differentiate it with 1% hydrochloric acid alcohol, and then rinse with tap water to return to blue for 15 minutes.

[0135] (15) Place the slices on a slice rack and sequentially place them in 75% alcohol (5 min), 95% alcohol I (10 min), 95% alcohol II (10 min), anhydrous ethanol I (10 min), anhydrous ethanol II (10 min), xylene I (10 min), and xylene II (10 min). Air-dry the dehydrated and transparent slices in a fume hood, add 100 μL of neutral gum to seal the slices, scan and photograph the slices with a Zeiss scanner, and calculate the proportion of CHOP-positive cells using ImageJ and GraphPad software and plot the graph.

[0136] 3. Experimental results

[0137] CHOP immunohistochemical staining of paraffin sections of tumor tissues of the four groups of mice after treatment Figure 7 Figure 2 shows the following: Control: control group; IR: radiotherapy alone; BSH: high-dose BS1801 (180 mg / kg); BSM+IR: combination of BS1801 (90 mg / kg) and radiotherapy. The left side shows a schematic diagram of typical results, with a scale bar of 100 μm. Blue represents cell nuclei, and tan represents CHOP-positive cells. The right side shows the number of CHOP-positive cells in each group, calculated using ImageJ software. CHOP is a marker for apoptosis; a higher number of positive cells indicates a higher number of apoptotic cells. The results demonstrate that the combination of BS1801 and radiotherapy significantly increases the number of apoptotic cells.

[0138] Example 4 Detection of IL-6 and TNF-α levels in the serum of mice in different treatment groups

[0139] 1. Experimental Materials

[0140] ELISA kit: Mouse IL-6, TNF-α double antibody sandwich ELISA detection kit; Proteintech; Wuhan; KE10007 and KE10002; used for quantitative detection of mouse IL-6 and TNF-α concentrations in serum, plasma and cell supernatant.

[0141] 2. Experimental methods

[0142] (1) After the mouse eyeball blood was coagulated at room temperature for 15 minutes, it was centrifuged at 1000 rpm for 10 minutes and the supernatant was collected into an EP tube.

[0143] (2) Prepare the washing solution at a dilution ratio of 1:20; prepare the detection antibody at a dilution ratio of 1:100; prepare the HRP-labeled streptavidin at a dilution ratio of 1:100.

[0144] (3) Dilute the mouse serum sample 1:2 with ultrapure water and set up zero well, standard well, and mouse serum sample well. Add 100 μL of sample diluent to the zero well, and add 100 μL / well of the serially diluted standard or mouse serum sample to be tested to the remaining wells, avoiding bubbles. Cover with sealing film and incubate at 37°C for 2 hours.

[0145] (4) Remove the sealing film, discard the liquid, and pat dry; wash the strips with washing solution, add 350-400 μL to each well, shake off the liquid after washing, and pat dry the strips. Repeat this step 4 times to prevent foreign matter from entering the plate wells and the strips from drying out.

[0146] (5) Add 100 μL of detection antibody to each well, cover with sealing film, and incubate at 37°C for 1 hour.

[0147] (6) Remove the sealing film, discard the liquid, and pat dry; wash the strips with washing solution, add 350-400 μL to each well, shake off the liquid and pat dry the strips after washing, and repeat this step 4 times.

[0148] (7) Add 100 μL of HRP-labeled streptavidin to each well, cover with a sealing film, and incubate at 37°C for 40 minutes.

[0149] (8) Remove the sealing film, discard the liquid, and pat dry; wash the strips with washing solution, add 350-400 μL to each well, shake off the liquid and pat dry the strips after washing, and repeat this step 4 times.

[0150] (9) Color development: Add 100 μL of TMB color development solution to each well and develop the color at 37°C in the dark for 15 minutes. Keep the color development substrate in the dark at all times.

[0151] (10) Stop: Add 100 μL of stop solution to each well. The blue color will turn yellow. The stop solution and TMB color development solution should be added in the same order.

[0152] (11) Reading: Measure the optical density (OD) of each well using a microplate reader at a wavelength of 450 nm, using 630 nm as the calibration wavelength. Read within 5 minutes after adding the stop solution.

[0153] (12) Data Analysis: The OD value of the zero well was subtracted from the OD value of each standard and mouse serum sample. Three replicate wells were set up, and the average value was calculated. Four-parameter fitting was performed using Origin software, with the concentration of the standard as the horizontal axis and the OD value as the vertical axis. The fitted concentration was calculated from the standard curve based on the OD value of the mouse serum sample and multiplied by 2 to obtain the measured concentration of the sample. Histograms were drawn using GraphPad.

[0154] 3. Experimental results

[0155] The corresponding results of the levels of pro-inflammatory cytokines IL-6 and TNF-α in the serum of the six groups of mice after treatment are as follows Figure 8The results show that the levels of proinflammatory cytokines IL-6 and TNF-α in the serum of mice treated with radiotherapy alone were significantly increased, while BS1801 treatment reduced their expression, demonstrating the potential anti-inflammatory effects of BS1801.

Claims

1. Use of a benzisoselenazole compound or a pharmaceutically acceptable salt thereof in combination with radiotherapy in the preparation of a medicament for treating and / or preventing diffuse midline glioma or spinal cord glioma, wherein the structural formula of the benzisoselenazole compound is shown in formula (I):

2. The use according to claim 1, characterized in that The radiotherapy is X-ray irradiation, gamma ray irradiation, beta ray irradiation, proton beam irradiation and / or electron beam irradiation; Optionally, the radiotherapy is X-ray irradiation.

3. The use according to claim 1, characterized in that The dosage ratio of the benzisoselenazole compound or a pharmaceutically acceptable salt thereof to radiotherapy is 0.5-1.5:1.5-0.5; Optionally, the dosage ratio of the benzisoselenazole compound or a pharmaceutically acceptable salt thereof to radiotherapy is 1:

1.

4. The use according to claim 1, characterized in that The ratio of the benzisoselenazole compound or its pharmaceutically acceptable salt to radiotherapy is: 1-15 μM: 1-15 Gy; Optionally, the dosage ratio of the benzisoselenazole compound or a pharmaceutically acceptable salt thereof to radiotherapy is: 2-10 μM: 2-10 Gy.

5. A combined pharmaceutical composition, characterized in that: The pharmaceutical composition comprises the benzisoselenazole compound according to claim 1 and radiation.

6. The pharmaceutical composition according to claim 5, characterized in that The radiation is X-rays, gamma rays, beta rays, proton beams and / or electron beams; Optionally, the radiation is X-rays; Optionally, the dose ratio of the benzisoselenazole compound or a pharmaceutically acceptable salt thereof to the radiation is 0.5-1.5:1.5-0.5; Optionally, the dose ratio of the benzisoselenazole compound or a pharmaceutically acceptable salt thereof to the radiation is 1:1; Optionally, the ratio of the amount of the benzisoselenazole compound or a pharmaceutically acceptable salt thereof to the radiation is: 1-15 μM: 1-15 Gy; Optionally, the dosage ratio of the benzisoselenazole compound or a pharmaceutically acceptable salt thereof to radiation is: 2-10 μM: 2-10 Gy.

7. A pharmaceutical preparation, characterized in that The pharmaceutical preparation comprises the pharmaceutical composition according to claim 5 or 6; Optionally, the dosage form of the pharmaceutical preparation includes an injection dosage form, an intrathecal administration dosage form under lumbar puncture, an oral administration dosage form, a respiratory tract administration dosage form, a cavity administration dosage form, a mucosal administration dosage form or a skin administration dosage form.

8. 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 in vitro glioma cells with the benzisoselenazole compound of claim 5 or 6 in combination with radiation; Optionally, the glioma cells are diffuse midline glioma cells or spinal cord glioma cells.

9. Any of the following applications: (1) Use of the benzisoselenazole compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4 in combination with radiotherapy in the preparation of a pharmaceutical preparation for treating and / or preventing diffuse midline glioma or spinal cord glioma; (2) Use of the benzisoselenazole compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4 in the preparation of anti-inflammatory drugs.

10. The use according to claim 9, characterized in that The anti-inflammatory drug is a drug that reduces the increased levels of IL-6 and TNF-α caused by radiotherapy.

Images