Anti-glioblastoma pharmaceutical composition and application thereof

Abstract

The invention discloses an anti-glioblastoma pharmaceutical composition and application thereof.The pharmaceutical composition comprises an SETD8 inhibitor and temozolomide or pharmaceutical salt thereof, it is found for the first time that the SETD8 inhibitor UNC0379 and temozolomide are combined to have a synergistic effect on treatment of glioblastoma; the invention provides an effective drug combination strategy for treatment of glioblastoma, and has a wide application prospect in the technical field of development of glioblastoma treatment drugs.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular, the present invention relates to an anti-glioblastoma pharmaceutical composition and application thereof. Background Art

[0002] Gliomas are the most common primary malignant tumors of the central nervous system, accounting for about 28% of primary intracranial tumors and 80% of primary intracranial malignant tumors. Among them, glioblastoma (GBM), the most malignant tumor, accounts for about 57% of gliomas. The clinical treatment effect of glioblastoma is poor, which has imposed a heavy burden on society and patients' families. At present, the standard treatment for primary glioblastoma is surgical resection combined with radiotherapy and temozolomide chemotherapy, but the median survival time of patients receiving standard treatment is only 14.6 months, and the 5-year survival rate is less than 10%.

[0003] In recent years, the great potential of immunotherapy in the field of tumors has brought a ray of hope to break the treatment dilemma of glioblastoma (GBM). However, the effect of immunotherapy in glioblastoma is not satisfactory. The reasons are that the lack of tumor antigens, the expression of immune checkpoint molecules, the infiltration of myeloid immunosuppressive cells, and the irreversibility of T cell exhaustion have limited the current application of immunotherapy in glioblastoma. Therefore, new treatment methods and more effective therapeutic drugs are urgently needed in the clinic to break the treatment dilemma of glioblastoma, prolong the survival time of glioblastoma patients, and improve the quality of life of glioblastoma patients. Summary of the invention

[0004] In order to make up for the deficiencies of the prior art, the object of the present invention is to provide an anti-glioblastoma pharmaceutical composition and application thereof.

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

[0006] The first object of the present invention is to provide a pharmaceutical composition for treating glioblastoma.

[0007] Furthermore, the pharmaceutical composition comprises a SETD8 inhibitor and temozolomide or a pharmaceutically acceptable salt thereof.

[0008] Furthermore, the SETD8 inhibitor is selected from UNC0379 or a pharmaceutically acceptable salt thereof.

[0009] Furthermore, the pharmaceutically acceptable salts include alkali metal salts, alkaline earth metal salts and ammonium salts.

[0010] Furthermore, the alkali metal salt includes potassium salt and sodium salt.

[0011] Furthermore, the alkaline earth metal salt includes calcium salt and magnesium salt.

[0012] The SETD8 inhibitor UNC0379 described in the present invention is a selective substrate-competitive lysine methyltransferase SETD8 inhibitor with an IC50 of 7.3 μM and a KD value of 18.3 μM. It inhibits the activity of the SETD8 enzyme, reduces the expression level of related proteins, and thus affects related biological processes, including DNA damage response, etc.

[0013] The temozolomide (TMZ) described in the present invention is a clinical first-line orphan drug with a broad anti-tumor spectrum, high activity, and good oral absorption. Temozolomide can quickly penetrate the blood-brain barrier, and the drug concentration in the cerebrospinal fluid is 30% of the plasma concentration, and the concentration in tumor cells is much higher than that in normal cells. Therefore, it can kill tumor cells while protecting normal tissues to the maximum extent.

[0014] In a specific embodiment of the present invention, the present invention has found through experiments that the combination of the SETD8 inhibitor UNC0379 and temozolomide can produce a synergistic effect in the treatment of glioblastoma and can significantly inhibit the proliferation of glioblastoma cells.

[0015] Furthermore, the concentration ratio of UNC0379 to temozolomide is 1:1000 to 1:15.625.

[0016] Furthermore, the concentration ratio of UNC0379 and temozolomide is (0.2 μM to 1.6 μM): (25 μM to 200 μM).

[0017] Furthermore, the concentration ratio of UNC0379 to temozolomide is 1:1000 to 1:62.5.

[0018] Furthermore, the concentration ratio of UNC0379 to temozolomide is (0.2 μM to 0.8 μM): (50 μM to 200 μM).

[0019] Furthermore, the concentration ratio of UNC0379 to temozolomide is 1:500.

[0020] Furthermore, the concentration ratio of UNC0379 and temozolomide is 0.2 μM:100 μM.

[0021] In a specific embodiment of the present invention, the present invention has experimentally verified that when the concentration ratio of the SETD8 inhibitor UNC0379 and temozolomide is (0.2μM~1.6μM): (25μM~200μM), the combination of the two can significantly inhibit the growth of glioblastoma cells and produce a synergistic effect. More specifically, the concentration ratio is (0.2μM~0.8μM): (50μM~200μM). In a preferred embodiment, the concentration ratio is 0.2μM: 100μM.

[0022] In some embodiments, a pharmaceutical composition comprising a combination of other SETD8 inhibitors other than UNC0379 and temozolomide is also included in the protection scope of the present invention, wherein the SETD8 inhibitor includes any agent capable of downregulating the expression or activity of SETD8, preferably, the agent includes but is not limited to: interfering molecules that inhibit SETD8 gene expression or transcription, agents that inhibit SETD8 protein activity, and small molecule inhibitors targeting SETD8.

[0023] In some embodiments, the interfering molecules that inhibit SETD8 expression or transcription include but are not limited to: at least one of siRNA, shRNA, dsRNA, microRNA, and antisense nucleic acid targeting SETD8, or a construct that can express or form the siRNA, shRNA, dsRNA, microRNA, and antisense nucleic acid; the reagents that inhibit SETD8 protein activity include but are not limited to: antibodies or fragments thereof targeting SETD8, polypeptides targeting SETD8, and SETD8 binding protein fragments; the small molecule inhibitors targeting SETD8 include but are not limited to: UNC0379, BVT948, Ryuvidine, and UNC0379TFA.

[0024] Furthermore, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient.

[0025] In some embodiments, the pharmaceutically acceptable carriers and / or excipients include, but are not limited to, diluents, binders, surfactants, wetting agents, adsorption carriers, lubricants, fillers, and disintegrants.

[0026] In some embodiments, the diluent includes, but is not limited to, lactose, sodium chloride, glucose, urea, starch, water, and the like.

[0027] In some embodiments, the binder includes but is not limited to starch, pregelatinized starch, dextrin, maltodextrin, sucrose, gum arabic, gelatin, methyl cellulose, carboxymethyl cellulose, ethyl cellulose, polyvinyl alcohol, polyethylene glycol, polyvinyl pyrrolidone, alginic acid and alginate, xanthan gum, hydroxypropyl cellulose and hydroxypropyl methylcellulose, etc.

[0028] In some embodiments, the surfactant includes, but is not limited to, polyoxyethylene sorbitan fatty acid ester, sodium lauryl sulfate, stearic acid monoglyceride, cetyl alcohol, and the like.

[0029] In some embodiments, the humectant includes but is not limited to glycerol, starch, and the like.

[0030] In some embodiments, the adsorption carrier includes, but is not limited to, starch, lactose, bentonite, silica gel, kaolin, and bentonite.

[0031] In some embodiments, the lubricant includes but is not limited to: zinc stearate, glyceryl monostearate, polyethylene glycol, talc, calcium and magnesium stearate, polyethylene glycol, boric acid powder, hydrogenated vegetable oil, sodium stearyl fumarate, polyoxyethylene monostearate, monolauric sucrose ester, sodium lauryl sulfate, magnesium lauryl sulfate, magnesium dodecyl sulfate, etc.

[0032] In some embodiments, the filler includes but is not limited to: mannitol (granular or powdered), xylitol, sorbitol, maltose, erythrose, microcrystalline cellulose, polymeric sugars, coupling sugars, glucose, lactose, sucrose, dextrin, starch, sodium alginate, laminarin powder, agar powder, calcium carbonate, sodium bicarbonate, etc.

[0033] In some embodiments, the disintegrant includes but is not limited to: cross-linked vinyl pyrrolidone, sodium carboxymethyl starch, low-substituted hydroxypropyl methyl, cross-linked sodium carboxymethyl cellulose, soybean polysaccharide, etc.

[0034] In some embodiments, the pharmaceutically acceptable carrier and / or excipient is used to help the stability of the formulation or to help improve its activity or its biological effectiveness or to produce an acceptable taste or smell in the case of oral administration as needed. The pharmaceutical composition thus prepared can be administered by any appropriate administration method known to those skilled in the art as needed. When the pharmaceutical composition is used, a safe and effective amount of the pharmaceutical composition of the present invention is administered to a human.

[0035] In some embodiments, the suitable dosage of the pharmaceutical composition described in the present invention can be prescribed in a variety of ways according to factors such as 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 dosage that is effective for treatment.

[0036] Furthermore, the pharmaceutical composition of the present invention may also include additives such as stabilizers, bactericides, buffers, isotonic agents, chelating agents, pH control agents and surfactants.

[0037] Further, the pharmaceutical composition described in the present invention can also be used in combination with other drugs for treating glioblastoma, and other therapeutic compounds can be administered simultaneously with the main active ingredient, or even administered simultaneously in the same composition. Other therapeutic compounds can also be administered separately in a separate composition or in a dosage form different from the main active ingredient. Partial doses of the main ingredient can be administered simultaneously with other therapeutic compounds, while other doses can be administered separately. During the treatment process, the dosage of the pharmaceutical composition of the present invention can be adjusted according to the severity of the symptoms, the frequency of recurrence, and the physiological response of the treatment regimen.

[0038] Furthermore, the pharmaceutical composition is a single compound preparation or a combination of two separate single preparations.

[0039] Furthermore, the compound preparation is a compound preparation comprising a SETD8 inhibitor and temozolomide.

[0040] Furthermore, the combination of single-ingredient preparations is a combination of a single-ingredient preparation comprising a SETD8 inhibitor and a single-ingredient preparation comprising temozolomide.

[0041] Furthermore, the two single-ingredient preparations in the combination of single-ingredient preparations are administered simultaneously or sequentially.

[0042] In some embodiments, the compound preparation refers to a preparation made of two or more active pharmaceutical ingredients. For example, when the pharmaceutical composition of the present invention is a compound preparation, it may mean that it contains both the SETD8 inhibitor UNC0379 and temozolomide.

[0043] In some embodiments, the single preparation refers to a preparation made with a single active pharmaceutical ingredient. For example, when the pharmaceutical composition of the present invention is a combination of single preparations, it may represent a combination of a single preparation containing the SETD8 inhibitor UNC0379 and a single preparation containing temozolomide.

[0044] In some embodiments, the administration of the two single preparations in the combination of single preparations includes: simultaneous administration, sequential administration. When the administration of the two single preparations in the combination of single preparations is sequential administration, the administration includes: first administering the single preparation containing UNC0379, then administering the single preparation containing temozolomide; first administering the single preparation containing temozolomide, then administering the single preparation containing UNC0379.

[0045] Furthermore, the dosage form of the pharmaceutical composition includes a parenteral dosage form and / or a parenteral dosage form.

[0046] Furthermore, the non-intestinal administration dosage form includes an injection dosage form, a cavity administration dosage form, a mucosal administration dosage form and / or a skin administration dosage form.

[0047] Furthermore, the dosage forms for administration via the gastrointestinal tract include tablets, granules, capsules, solutions, powders, sustained-release preparations, emulsions, suspensions, syrups and / or drops.

[0048] In some embodiments, the dosage form of the pharmaceutical composition of the present invention is a dosage form that is prepared by conventional methods and is conducive to administration, including but not limited to: parenteral dosage forms, gastrointestinal dosage forms, and specific examples include but are not limited to: aqueous solution injection, powder injection, pills, powders, tablets, patches, suppositories, emulsions, creams, gels, granules, capsules, aerosols, sprays, powder sprays, sustained-release agents and controlled-release agents, etc.

[0049] In some embodiments, the injection dosage forms include, but are not limited to, intravenous injections, intramuscular injections, subcutaneous injections, intradermal injections, intracavity injections and other injections.

[0050] In some embodiments, the cavity administration dosage form includes, but is not limited to, suppositories, aerosols, effervescent tablets, drops, pills, etc., and is used in the rectum, vagina, urethra, nasal cavity, ear canal, etc.

[0051] In some embodiments, the mucosal administration dosage form includes, but is not limited to, eye drops, nasal drops, eye ointments, gargles, sublingual tablets, adhesive tablets, patches, and the like.

[0052] In some embodiments, the skin administration dosage form includes, but is not limited to, topical solutions, lotions, liniments, ointments, plasters, pastes, patches, and the like.

[0053] In some embodiments, the pharmaceutical composition contains pharmaceutical excipients, which can be conventionally used in various preparations, including but not limited to isotonic agents, buffers, flavoring agents, excipients, fillers, adhesives, disintegrants and lubricants, etc.; or selected for use in order to adapt to the substance, including but not limited to emulsifiers, solubilizers, antibacterial agents, analgesics and antioxidants, etc. Such excipients can effectively improve the stability and solubility of the active ingredients contained in the composition or change the release rate and absorption rate of the active ingredients, thereby improving the metabolism of various active ingredients in the body, thereby enhancing the administration effect of the composition. In addition, excipients used to achieve specific administration purposes or methods, such as sustained-release administration, controlled-release administration and pulse administration, etc., include but are not limited to gelatin, albumin, chitosan, polyether and polyester polymer materials (for example: polyethylene glycol, polyurethane, polycarbonate and its copolymers, etc.). The main manifestations of the said advantages in administration are: improving the therapeutic effect, improving the bioavailability, reducing toxic side effects and improving patient compliance, etc.

[0054] The second aspect of the present invention provides an in vitro method for inhibiting the proliferation of glioblastoma cells and promoting the apoptosis of glioblastoma cells for non-therapeutic purposes.

[0055] Furthermore, the method comprises the following steps: treating glioblastoma cells in vitro using the pharmaceutical composition described in the first aspect of the present invention.

[0056] In a specific embodiment of the present invention, the present invention has experimentally discovered for the first time that the combination of the SETD8 inhibitor UNC0379 and temozolomide can produce a synergistic effect on the treatment of glioblastoma, significantly inhibiting the proliferation of glioblastoma cells. Therefore, the pharmaceutical composition can be used as an inhibitor for inhibiting the growth and proliferation of glioblastoma cells for non-therapeutic purposes, and can be used in the field of scientific research, such as further studying the growth and metabolic mechanism or behavior of glioblastoma cells, screening potential drugs for treating glioblastoma, etc.

[0057] In a specific embodiment of the present invention, the present invention further verifies the synergistic effect of the combination of the SETD8 inhibitor UNC0379 and temozolomide by the combined drug CI index. The combined drug CI (combination index, CI) index is a method for evaluating the combined effect of drugs, which is based on the dose-effect curve of the drug, and is calculated by calculating the ratio of the actual effect to the theoretical effect during combined drug use (the specific calculation method is a method for calculating the combined drug index CI using CalcuSyn software known to those skilled in the art) to determine whether the combined drug has a synergistic effect, an additive effect, or an antagonistic effect.

[0058] The third aspect of the present invention provides any one of the following applications.

[0059] Furthermore, the application includes:

[0060] (1) Use of a SETD8 inhibitor and temozolomide or a pharmaceutically acceptable salt thereof in combination in the preparation of a pharmaceutical composition for treating and / or preventing glioblastoma;

[0061] (2) Use of a SETD8 inhibitor and temozolomide or a pharmaceutically acceptable salt thereof in combination for the preparation of a pharmaceutical preparation for the treatment and / or prevention of glioblastoma;

[0062] (3) Use of SETD8 inhibitors in the preparation of drugs for improving the therapeutic effect of temozolomide in treating brain gliomas;

[0063] (4) Use of the pharmaceutical composition described in the first aspect of the present invention in the preparation of an agent for inhibiting the proliferation of glioblastoma cells and promoting the apoptosis of glioblastoma cells in vitro.

[0064] Furthermore, the SETD8 inhibitor is selected from UNC0379 or a pharmaceutically acceptable salt thereof.

[0065] In the present invention, the treatment refers to preventing and reducing the occurrence or development of the disease, so that the development or aggravation of the disease course can be inhibited, contained, alleviated, improved, slowed down, stopped, delayed or reversed. The various indicators of the disease, disorder or pathological state during maintenance and / or medication include alleviating or reducing symptoms or complications, or curing or eliminating the disease, disorder or condition. In a specific embodiment of the present invention, the disease is preferably glioblastoma.

[0066] In the present invention, prevention refers to various means or measures used to prevent the occurrence or development of a disease before the disease is recognized by clinical standards, including medical, physical or chemical methods, to prevent and reduce the occurrence or development of various symptoms of the disease. In a specific embodiment of the present invention, the disease is preferably glioblastoma.

[0067] In addition, the present invention also provides a method for treating glioblastoma, comprising the following steps: administering a therapeutically effective amount of the pharmaceutical composition of the first aspect of the present invention or the pharmaceutical preparation of the second aspect of the present invention to a subject in need.

[0068] In the present invention, the effective amount refers to the amount of the compound that effectively produces the desired preventive, alleviating or therapeutic effect. Depending on the compound, the symptoms and their severity, the age of the treated mammal and other factors, the amount of the pharmaceutical composition or pharmaceutical preparation of the present invention to achieve the effective amount will also vary accordingly, but the specific amount can be routinely determined by a person of ordinary skill in the art based on the knowledge in the art mastered by him / her and the contents disclosed in the present invention.

[0069] In some embodiments, the pharmaceutical composition or pharmaceutical preparation can be administered to the subject by injection, topical administration or oral administration. For example, the method may include administering the pharmaceutical composition or pharmaceutical preparation to the subject three times a day, once a day, once every two days, etc. In some embodiments, injection administration may include subcutaneous injection, intramuscular injection, intravenous injection, etc. In some embodiments, injection administration may include injecting the pharmaceutical composition directly into the lesion or in an area near the lesion, and in some embodiments, topical administration may include rectal administration, nasal administration, ear administration, intramedullary administration, intraarticular administration, intrapleural administration, etc., or any combination thereof. In some embodiments, the pharmaceutical composition or pharmaceutical preparation may be administered to the subject via a combination of different administration methods.

[0070] In some embodiments, the subject is an animal, preferably a mammal (human and non-human animals), including but not limited to: humans, non-human primates (particularly higher primates, such as macaques, crab-eating macaques, short-tailed macaques, bear monkeys, flat-topped monkeys, golden monkeys and tree shrews), sheep, dogs, rodents (such as mice or rats), guinea pigs, goats, pigs, cats, rabbits, cows, any livestock or pets, etc. In a preferred embodiment, the subject is a human.

[0071] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0072] The present invention discovers for the first time that the combined use of the SETD8 inhibitor UNC0379 and temozolomide exerts a synergistic inhibitory effect on the proliferation of glioblastoma cells, that is, the combination of the two has a significant synergistic effect, and the inhibitory effect of the pharmaceutical composition on glioblastoma cells is significantly better than that of a single UNC0379 or temozolomide. The present invention provides an effective drug combination strategy for the treatment of glioblastoma, and has broad application scenarios in the technical field of development of glioblastoma therapeutic drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] Figure 1 Flowchart for discovering epigenetic target SETD8; Figure 1 A is the screening flow chart of target genes. Figure 1 B is the result diagram of differentially expressed genes between temozolomide-sensitive and temozolomide-resistant glioblastoma cell lines. Figure 1 C is a result diagram of the intersection of the differentially expressed gene sets between glioblastoma cell lines that are sensitive to temozolomide and those that are resistant to temozolomide, the gene sets related to the prognosis of glioblastoma, and the gene sets involved in epigenetic regulation;

[0074] Figure 2 The expression characteristics of SETD8 in glioma and its relationship with patient survival; Figure 2 A is the expression characteristic diagram of SETD8 in glioma. Figure 2 B. Figure 2 C. Figure 2 D are the Kaplan-Meier survival analysis results of astrocytoma IDH mutant (mut-noncodel), oligodendroglioma IDH mutation and 1p / 19q co-deletion (mut-codel) and glioblastoma IDH wild type (wt) in the Chinese Glioma Genome Atlas (CGGA) database;

[0075] Figure 3This is the result diagram of the effect of down-regulating SETD8 on the proliferation of glioblastoma cell lines; Figure 3 A is the result of knocking down SETD8 in glioblastoma cell lines by si-SETD8. Figure 3 B is the proliferation curve of glioblastoma cell line after SETD8 knockdown. Figure 3 C is the result of cloning experiment of glioblastoma cell line after SETD8 knockdown;

[0076] Figure 4 This is a graph showing the inhibitory effect of SETD8 inhibitor combined with TMZ on glioblastoma cells; Figure 4 A is the Kaplan-Meier survival analysis result of SETD8 expression level and chemotherapy drug TMZ in patients with glioblastoma; Figure 4 B is a graph showing the inhibitory effect of UNC0379 on glioblastoma proliferation; Figure 4 C is a diagram showing the synergistic effect of UNC0379 combined with TMZ. DETAILED DESCRIPTION

[0077] The present invention is further described below in conjunction with specific examples, which are only used to explain the present invention and are not to be construed as limiting the present invention. It will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to these embodiments without departing from the principles and purposes of the present invention, and that the scope of the present invention is defined by the claims and their equivalents. The experimental methods for which specific conditions are not specified in the following examples are usually tested under conventional conditions or under conditions recommended by the manufacturer.

[0078] Example 1 Discovery of epigenetic target SETD8

[0079] 1. Experimental methods

[0080] The present invention searches for potential epigenetic therapeutic targets in glioblastoma by integrating and analyzing data. The differentially expressed gene sets between glioblastoma cell lines sensitive to temozolomide and glioblastoma cell lines resistant to temozolomide are screened out with the help of GDSC (Genomics of Drug Sensitivity in Cancer) and CCLE (Cancer Cell Line Encyclopedia) databases; then, the gene sets related to the prognosis of glioblastoma are screened out in the CGGA database; the above two gene sets are intersected with genes involved in epigenetic regulation to obtain the screened target genes.

[0081] 2. Experimental results

[0082] The screening process of the target gene of the present invention is as follows Figure 1The results of differentially expressed gene sets between temozolomide-sensitive and temozolomide-resistant glioblastoma cell lines are shown in Figure A. Figure 1 As shown in B; Figure 1 As shown in Figure C, after taking the intersection of the differentially expressed gene sets between temozolomide-sensitive and temozolomide-resistant glioblastoma cell lines, the gene sets related to glioblastoma prognosis, and the gene sets involved in epigenetic regulation, the only common gene SETD8 was obtained, among which gene set 1 refers to the differentially expressed gene sets between temozolomide-sensitive and temozolomide-resistant glioblastoma cell lines, gene set 2 refers to the gene sets related to glioblastoma prognosis in the CGGA database, and gene set 3 refers to the gene sets involved in epigenetic regulation.

[0083] Example 2 High expression of SETD8 is associated with poor prognosis in glioma patients

[0084] 1. Experimental methods

[0085] According to the fifth edition of the WHO Central Nervous System Tumors Classification in 2021, adult diffuse gliomas were divided into three groups: astrocytoma IDH mutant (mut-noncodel), oligodendroglioma IDH mutation and 1p / 19q co-deletion (mut-codel), and glioblastoma IDH wild-type (wt). Student's t test was used to compare the expression levels of SETD8 between different groups. Kaplan-Meier survival curves were used to compare the survival differences of brain glioma patients with different SETD8 expression levels. COX regression was used to analyze the correlation between SETD8 expression level and patient survival.

[0086] 2. Experimental results

[0087] like Figure 2 As shown in Figure A, SETD8 expression was significantly different in different types of gliomas and increased with the increase of tumor malignancy. It was highest in glioblastoma with the highest malignancy. The Kaplan-Meier survival analysis results of the mut-noncodel group, mut-codel group and wt group in the CGGA database are shown in Figure 2. Figure 2 B. Figure 2 C and Figure 2 As shown in D, the survival of patients with high SETD8 expression was significantly shortened. Further COX regression analysis was performed, and the results are shown in Table 1. The expression level of SETD8 is an independent risk factor independent of patient gender, age, radiotherapy, and chemotherapy, and can be used to predict patient prognosis.

[0088] Table 1 Results of multivariate COX regression analysis

[0089]

[0090] Example 3 Downregulation of SETD8 inhibits glioblastoma proliferation

[0091] 1. Experimental methods

[0092] Glioblastoma cell lines were cultured in 10 cm culture dishes, and si-SETD8 was transfected when the cell density reached 50%. During transfection, the transfection reagent was prepared first: 1.25 ml of opti-MEM medium (purchased from Gibco), 19 ul of lipo3000 transfer aid (purchased from Invitrogen) and 25 ul of si-SETD8 (purchased from Ruibo Bio) were put into a 1.5 ml sterile EP tube, mixed and left at room temperature for 15 minutes. After 15 minutes, the 10 cm culture dish to be transfected was taken out, the culture medium in the culture dish was removed, 9 ml of dual antibody-free culture medium was added, and all the prepared transfection reagents were transferred to the culture dish, gently shaken and mixed, and the culture dish was returned to the incubator. After 72 hours, the transfected cells were taken and plated for protein extraction, cell proliferation experiment and clone formation experiment. The specific operation steps are as follows: take the transfected cells, trypsinize, count the cells, take a 96-well plate for cell plating, add 100ul cell suspension to each well, which contains 3000 cells, set 3 replicates for each group at each time point, and use a microplate reader to detect the absorbance of each well at 24 hours, 48 ​​hours and 72 hours, respectively, and analyze the cell proliferation curve; take a 6cm cell culture dish, add a suspension containing 500 cells to each dish, add 4ml culture medium to mix, and culture in a cell culture incubator, set 3 replicates for each group, change the culture medium every 2-3 days, and after 2 weeks of culture, fix the cells with paraformaldehyde, stain with crystal violet, and observe the clone formation results; take a 10cm cell culture dish, add 200,000 cells and 10ml culture medium to each dish, culture in a cell culture incubator, and when the cell density reaches 80%, use Minute TM Protein extraction was performed using the Animal Cell / Tissue Total Protein Extraction Kit SD-001 / SN-002. The operation steps were performed according to the instructions, and the extracted protein was used for Western blot detection.

[0093] 2. Experimental results

[0094] Western blot results Figure 3 As shown in A, SETD8 protein expression was significantly inhibited, and SETD8 was successfully knocked down. CCK8 experiment was used to measure cell proliferation, and the results were shown in Figure 3 As shown in Figure B, downregulating SETD8 slowed down the proliferation of glioblastoma cell lines. Similarly, downregulating SETD8 expression can inhibit the formation of local clones in glioblastoma cell lines ( Figure 3 C). All the above results prove that SETD8 can promote the malignant proliferation of glioblastoma.

[0095] Example 4 Inhibitory effect of SETD8 inhibitor combined with TMZ on glioblastoma

[0096] 1. Experimental methods

[0097] According to the fifth edition of the WHO new classification, we collected sequencing data of tumor tissues from 223 glioblastoma patients, and divided the patients into four groups according to the expression level of SETD8 and whether they were treated with TMZ for Kaplan-Meier curve analysis; SETD8=H, Chemo=0 represents patients with high SETD8 expression and no TMZ treatment; SETD8=H, Chemo=1 represents patients with high SETD8 expression and TMZ treatment; SETD8=L, Chemo=0 represents patients with low SETD8 expression and no TMZ treatment; SETD8=L, Chemo=1 represents patients with low SETD8 expression and TMZ treatment.

[0098] Then, 0.2uM, 0.4uM, 0.8uM, 1.6uM and 3.2uM UNC0379 (SETD8 inhibitor, purchased from Selleck, catalog number S7570) solutions were prepared respectively, and their effects on the proliferation of glioblastoma (cell line purchased from Concordia Cell Bank) were determined by CCK8 experiment.

[0099] Next, glioblastoma cells in the logarithmic growth phase were plated in 96-well plates, and a series of concentrations of TMZ (purchased from Sigma-Aldrich, catalog number T2577) solutions (25uM, 50uM, 100uM, 200uM) and UNC0379 solutions (0.2uM, 0.4uM, 0.8uM, 1.6uM) were prepared for combined drug studies. Gradient-diluted TMZ and UNC0379 solutions and combinations were added and cultured in a cell culture incubator. The CCK8 experiment was used to determine the cell absorbance value, and the dose-effect data of the drug were analyzed and calculated, including the dose and effect value of each drug used alone, and the dose and effect value of the two drugs used in combination. These data were entered into the SynergyFinder website to calculate the drug combination index (CI) of UNC0379 and TMZ, and to evaluate the synergistic effect of the drugs. If the Synergy score is less than -10, it means that the two drugs are antagonistic; if the Synergy score is greater than 10, it means that the two drugs are synergistic; if the Synergy score is between -10 and 10, it means that the two drugs have no interaction.

[0100] 2. Experimental results

[0101] The results of Kaplan-Meier curve analysis are as follows Figure 4 As shown in A, there were significant differences in survival among the four groups of patients.

[0102] The results of the effects of different concentrations of UNC0379 (SETD8 inhibitor) solution on the proliferation of glioblastoma cells are as follows Figure 4 As shown in B, the results indicate that UNC0379 inhibits the growth of glioblastoma in a concentration-dependent manner.

[0103] The inhibitory results of UNC0379 and TMZ combined on glioblastoma Figure 4 As shown in Figure C, the combination of UNC0379 and TMZ has a significant synergistic effect in inhibiting the proliferation of glioblastoma (Synergy score = 15.60). The best synergistic concentration range of the two drugs is TMZ (50uM ~ 200uM), UNC0379 (0.2uM ~ 0.8uM); the best synergistic concentration of the two drugs is TMZ (100uM), UNC0379 (0.2uM).

[0104] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. A pharmaceutical composition for treating glioblastoma, characterized in that: The pharmaceutical composition comprises a SETD8 inhibitor and temozolomide or a pharmaceutically acceptable salt thereof; The SETD8 inhibitor is selected from UNC0379 or a pharmaceutically acceptable salt thereof.

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

3. The pharmaceutical composition according to claim 1, wherein the concentration ratio of UNC0379 to temozolomide is 1:1000 to 1:15.

625.

4. The pharmaceutical composition according to claim 3, wherein the concentration ratio of UNC0379 to temozolomide is (0.2 μM to 1.6 μM): (25 μM to 200 μM).

5. The pharmaceutical composition according to claim 3, wherein the concentration ratio of UNC0379 to temozolomide is 1:1000 to 1:62.

5. 6 . The pharmaceutical composition according to claim 5 , wherein the concentration ratio of UNC0379 to temozolomide is (0.2 μM to 0.8 μM): (50 μM to 200 μM).

7. The pharmaceutical composition according to claim 3, wherein the concentration ratio of UNC0379 to temozolomide is 1:

500. 8 . The pharmaceutical composition according to claim 7 , wherein the concentration ratio of UNC0379 to temozolomide is 0.2 μM:100 μM.

9. A method for inhibiting the proliferation of glioblastoma cells and promoting apoptosis of glioblastoma cells in vitro without therapeutic purpose, characterized in that: The method comprises the following steps: treating glioblastoma cells in vitro with the pharmaceutical composition according to any one of claims 1 to 8.

10. Any of the following applications, characterized in that: The applications include: (1) Use of a SETD8 inhibitor and temozolomide or a pharmaceutically acceptable salt thereof in combination in the preparation of a pharmaceutical composition for treating and / or preventing glioblastoma; (2) Use of a SETD8 inhibitor and temozolomide or a pharmaceutically acceptable salt thereof in combination for the preparation of a pharmaceutical preparation for the treatment and / or prevention of glioblastoma; (3) Use of SETD8 inhibitors in the preparation of drugs for improving the therapeutic effect of temozolomide in treating brain gliomas; (4) Use of the pharmaceutical composition according to any one of claims 1 to 8 in the preparation of an agent for inhibiting the proliferation of glioblastoma cells and promoting the apoptosis of glioblastoma cells in vitro; The SETD8 inhibitor is selected from UNC0379 or a pharmaceutically acceptable salt thereof.

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