CN114870021B - Antitumor pharmaceutical composition containing EZH2 inhibitor and polyunsaturated fatty acid inhibitor and application thereof - Google Patents

Antitumor pharmaceutical composition containing EZH2 inhibitor and polyunsaturated fatty acid inhibitor and application thereof Download PDF

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CN114870021B
CN114870021B CN202210682634.4A CN202210682634A CN114870021B CN 114870021 B CN114870021 B CN 114870021B CN 202210682634 A CN202210682634 A CN 202210682634A CN 114870021 B CN114870021 B CN 114870021B
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inhibitor
fatty acid
ezh2
polyunsaturated fatty
cancer
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CN114870021A (en
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薛丽香
霍霄
郭正阳
宋佳桂
宋颖
张腾瑞
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Peking University Third Hospital Peking University Third Clinical Medical College
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Abstract

The invention provides a pharmaceutical composition for enhancing anti-tumor effect of an EZH2 inhibitor, which comprises the EZH2 inhibitor and a polyunsaturated fatty acid inhibitor, wherein the polyunsaturated fatty acid inhibitor enhances the anti-lipid metabolism abnormal solid tumor effect of the EZH2 inhibitor. The invention also provides a related preparation of the pharmaceutical composition and application thereof in preparing antitumor drugs. The invention provides a new idea for safely and effectively using the EZH2 inhibitor to treat solid tumors, especially ovarian cancer in clinic, and has good clinical application prospect.

Description

Antitumor pharmaceutical composition containing EZH2 inhibitor and polyunsaturated fatty acid inhibitor and application thereof
Technical Field
The invention belongs to the field of medicines, and in particular relates to an anti-tumor pharmaceutical composition containing an EZH2 inhibitor and a polyunsaturated fatty acid inhibitor and application thereof in preparing an anti-tumor medicine.
Background
Disorders of epigenetic regulation have long been recognized as key factors affecting tumorigenesis and maintenance. Histone methyltransferase EZH2 (Enhancer of Zeste Homolog 2) is a catalytic subunit of epigenetic regulatory factor multi-comb inhibitory complex 2 (Polycomb Repressive Complex 2, prc 2), and can inhibit transcription of target genes by performing trimethylation (H3K 27me 3) on lysine at 27 th position of histone H3 through its histone methyltransferase activity, and participate in regulating physiological or pathological processes such as cell cycle, cell senescence, cell differentiation, cancer, and the like. Abnormal expression of EZH2 is closely related to malignant progression of tumor and poor prognosis of patients.
Many highly potent and selective EZH2 catalytic inhibitors have been obtained by high throughput screening, such as EPZ005687, EI1, GSK343, GSK126, etc., with almost all of their structures bearing 2-pyridone groups. Many EZH2 inhibitors have been developed as potential anti-cancer agents. Among them, CPI1205 (Lirametostat) has been tested by clinical trials, and EPZ-6438 (Tazemet) was approved by the FDA in 2020 for the treatment of epithelioid sarcoma. However, in solid tumors in which EZH2 is overexpressed, EZH2 inhibitors do not work well, such as the simultaneous mutation of the Ras pathway and SWI/SNF in gliomas and melanomas is able to escape the anti-tumor effects of EZH2 inhibitors. Accordingly, researchers have attempted to improve the efficacy of EZH2 inhibitors by employing therapeutic strategies that combine multiple drugs or multiple anti-tumor therapies (see, e.g., zhang Tengrui, et al Symphony of epigenetic and metabolic regulation-interaction between the histone methyltransferase EZH2 and metabolism of tumor, clinical Epigenetics,2020, 12:72).
Previous work by the present inventors showed that epigenetic regulation and metabolic alterations mediated by EZH2 show synergy in cancer cells. The inventors have initially found that poor therapeutic efficacy of EZH2 inhibitors may be due to a disorder of lipid metabolism. And on the basis, the combination of the EZH2 inhibitor and the SCD1 inhibitor can obviously enhance the curative effect of the EZH2 inhibitor on treating solid tumors such as liver cancer, melanoma and the like (see CN 202111545151.1).
Ovarian cancer is the gynaecological malignancy with the highest mortality rate, and is the fifth leading cause of cancer death in women. Accounting for 4.4% of cancer-related deaths worldwide, there are about 24 tens of thousands of new cases annually worldwide. The overall 5-year survival of ovarian cancer is 45% with only 25% of the 5-year survival in advanced patients, the primary reason for this being the lack of obvious symptoms in the early stages of ovarian cancer and the effective screening, most patients (about 75%) being diagnosed as advanced, spreading to the abdominal cavity and distant metastases. Standard treatments for advanced ovarian cancer include tumor cytoreduction and normative chemotherapy. However, most patients relapse within 2-3 years after a first-line chemotherapy regimen and die of chemotherapy resistance. Finding new therapeutic strategies to improve the prognosis of ovarian cancer has been an important topic of ovarian cancer research.
Previous research results of the inventor show that EZH2 plays a promoting role in the occurrence and development of ovarian cancer and in the treatment of drug resistance, so that the EZH2 can be targeted to be inhibited or can be used for the treatment of ovarian cancer. However, the effect of GSK126 on proliferation of ovarian cancer cells SKOV3, IC was examined by CCK-8 assay 50 The value was 15.32. Mu.M. Therefore, there is still room for improvement in clinical application of EZH2 inhibitors in solid tumors, especially ovarian cancer, and combination therapy provides the possibility for clinical use thereof.
Furthermore, the inventors found polyunsaturated fatty acid synthesis-related genes directly regulated by EZH2, long chain fatty acid elongase ELOVL2, by analyzing ChIP-seq and RNA-seq data in earlier studies. RNA-seq data showed that the biosynthetic pathway of polyunsaturated fatty acids was significantly activated and that the expression of ELOVL2 and fatty acid desaturase 2 (FADS 2) isogenes was elevated in SKOV3 cells knocked out of EZH 2. The ChIP-seq data shows that the level of the ELOVL2 promoter regulatory region H3K27me3 is reduced in EZH2 knocked out cells. Inhibition of histone methyltransferase activity of EZH2 can increase expression of ELOVL2. That is, the present inventors found that EZH2 directly regulates polyunsaturated fatty acid synthesis-related gene ELOVL2, and that EZH2 inhibitors induce disorders of polyunsaturated fatty acid ester metabolism in ovarian cancer cells, which may be an important factor affecting the efficacy of treatment of ovarian cancer. The research of taking polyunsaturated fatty acid synthesis related gene ELOVL2 as an ovarian cancer treatment target is expected to open up a new idea for preventing and treating tumors.
In view of the above research background, the inventors speculate that the combined polyunsaturated fatty acid inhibitor may have an important role in combined therapy for some antitumor drugs related to lipid metabolism (especially for ovarian cancer), which is likely to provide a new thought for solving the poor treatment effect of the EZH2 inhibitor, and simultaneously provide a new medication strategy scheme for the treatment of EZH2 high-expression solid tumors, which is expected to provide a new thought and a new target point for the treatment of ovarian cancer, has important theoretical significance and application value for improving prognosis of ovarian cancer patients, and has potential social effect and economic value.
Disclosure of Invention
Aiming at the problem that the EZH2 inhibitor has poor activity on solid tumors, the invention provides a pharmaceutical composition containing the EZH2 inhibitor and a polyunsaturated fatty acid inhibitor, which can enhance the anti-tumor effect of the EZH2 inhibitor, and provides a new idea for safely and effectively using the EZH2 inhibitor to treat solid tumors (especially ovarian cancer) clinically.
Specifically, the invention is realized through the following technical schemes:
in a first aspect, the present invention provides a pharmaceutical composition for enhancing the antitumor effect of an EZH2 inhibitor, which is characterized in that: the pharmaceutical composition comprises an EZH2 inhibitor and a polyunsaturated fatty acid inhibitor, wherein the polyunsaturated fatty acid inhibitor enhances the effect of the EZH2 inhibitor against a solid tumor with abnormal lipid metabolism.
Alternatively, in the above pharmaceutical composition, the mass ratio of the EZH2 inhibitor to the polyunsaturated fatty acid inhibitor is 1:1-1: the ratio of the amount of the EZH2 inhibitor to the polyunsaturated fatty acid inhibitor in the pharmaceutical composition may be determined by a clinician according to clinical experience based on the type of cancer in the patient.
Preferably, the mass ratio of the EZH2 inhibitor to the polyunsaturated fatty acid inhibitor is selected from 1: 1. 1: 2. 1: 3. 1: 4. 1: 5. 1: 6. 1: 7. 1: 8. 1:9 or 1:10.
alternatively, in the above pharmaceutical composition, the solid tumor is selected from the group consisting of: breast cancer, prostate cancer, melanoma, pancreatic cancer, lung cancer, bladder cancer, colon cancer, liver cancer, ovarian cancer, cervical cancer, gastric cancer, renal cancer, head and neck tumor, esophageal cancer, testicular cancer, or thyroid cancer.
Alternatively, in the above pharmaceutical composition, the solid tumor is selected from the group consisting of: breast cancer, ovarian cancer, melanoma, lung cancer, colon cancer, liver cancer, stomach cancer or prostate cancer.
Preferably, the solid tumor is ovarian cancer.
More preferably, the solid tumor is high grade serous ovarian cancer.
Alternatively, in the above pharmaceutical composition, the EZH2 inhibitor is selected from the group consisting of: tazemetostat (EPZ-6438), GSK126, liramimetostat (CPI-1205), SHR2554 or PF-06821497; the polyunsaturated fatty acid inhibitor is selected from the group consisting of a Δ6 desaturase inhibitor, a selective Δ5 desaturase inhibitor, an omega-3 fatty acid agent, an ELOVL6 inhibitor, or an ELOVL1 inhibitor, preferably the Δ6 desaturase inhibitor is SC-26196, the selective Δ5 desaturase inhibitor is compound-326, the omega-3 fatty acid agent is eicosapentaenoic acid ethyl ester, the ELOVL6 inhibitor is ELOVL6-IN-2, and the ELOVL1 inhibitor is ELOVL1-IN-1.
Preferably, the polyunsaturated fatty acid inhibitor is a Δ6 desaturase inhibitor.
Preferably, the Δ6 desaturase inhibitor is SC-26196.
Alternatively, in the above pharmaceutical composition, the EZH2 inhibitor is GSK126 and the polyunsaturated fatty acid inhibitor is SC-26196.
In a second aspect, the present invention provides a pharmaceutical formulation for enhancing the anti-tumour effect of an EZH2 inhibitor, the pharmaceutical formulation being prepared from a therapeutically effective amount of a pharmaceutical composition according to the first aspect described above and a pharmaceutically acceptable carrier.
Alternatively, in the above pharmaceutical preparation, the pharmaceutical preparation is an oral preparation.
Preferably, the oral preparation is an oral liquid, a tablet, a powder, a capsule or a granule.
In a third aspect, the present invention provides the use of a pharmaceutical composition as described in the first aspect above or a pharmaceutical formulation as described in the second aspect above in the manufacture of an anti-tumour medicament.
Alternatively, in the above use, the tumor is a solid tumor with abnormal lipid metabolism.
Alternatively, in the above use, the solid tumor is selected from: breast cancer, prostate cancer, melanoma, pancreatic cancer, lung cancer, bladder cancer, colon cancer, liver cancer, ovarian cancer, cervical cancer, gastric cancer, renal cancer, head and neck tumor, esophageal cancer, testicular cancer, or thyroid cancer.
Alternatively, in the above use, the solid tumor is selected from: breast cancer, ovarian cancer, melanoma, lung cancer, colon cancer, liver cancer, stomach cancer or prostate cancer.
Preferably, the solid tumor is ovarian cancer.
More preferably, the solid tumor is high grade serous ovarian cancer.
In a fourth aspect, the invention provides the use of a polyunsaturated fatty acid inhibitor in the manufacture of a medicament for enhancing the efficacy of an EZH2 inhibitor against solid tumors.
Alternatively, in the above use, the tumor is a solid tumor with abnormal lipid metabolism.
Alternatively, in the above use, the solid tumor is selected from: breast cancer, prostate cancer, melanoma, pancreatic cancer, lung cancer, bladder cancer, colon cancer, liver cancer, ovarian cancer, cervical cancer, gastric cancer, renal cancer, head and neck tumor, esophageal cancer, testicular cancer, or thyroid cancer.
Alternatively, in the above use, the solid tumor is selected from: breast cancer, ovarian cancer, melanoma, lung cancer, colon cancer, liver cancer, stomach cancer or prostate cancer.
Preferably, the solid tumor is ovarian cancer.
More preferably, the solid tumor is high grade serous ovarian cancer.
Alternatively, in the above use, the EZH2 inhibitor is selected from: tazemetostat (EPZ-6438), GSK126, liramimetostat (CPI-1205), SHR2554 or PF-06821497; the polyunsaturated fatty acid inhibitor is selected from the group consisting of a Δ6 desaturase inhibitor, a selective Δ5 desaturase inhibitor, an omega-3 fatty acid agent, an ELOVL6 inhibitor, or an ELOVL1 inhibitor, preferably the Δ6 desaturase inhibitor is SC-26196, the selective Δ5 desaturase inhibitor is compound-326, the omega-3 fatty acid agent is eicosapentaenoic acid ethyl ester, the ELOVL6 inhibitor is ELOVL6-IN-2, and the ELOVL1 inhibitor is ELOVL1-IN-1.
Preferably, the polyunsaturated fatty acid inhibitor is a Δ6 desaturase inhibitor.
Preferably, the Δ6 desaturase inhibitor is SC-26196.
Alternatively, in the above use, the EZH2 inhibitor is GSK126 and the polyunsaturated fatty acid inhibitor is SC-26196.
It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. Is limited to a space and will not be described in detail herein.
Compared with the prior art, the invention has the following beneficial effects:
(1) The pharmaceutical composition containing the EZH2 inhibitor and the polyunsaturated fatty acid inhibitor, which can enhance the anti-tumor effect of the EZH2 inhibitor, provides a new thought for solving the problem of poor treatment effect of the EZH2 inhibitor, and provides a new medication strategy scheme for treating EZH2 high-expression solid tumors.
(2) Provides a new thought and a new target point for the treatment of ovarian cancer, has important theoretical significance and application value for improving the prognosis of ovarian cancer patients, and has potential social effect and economic value.
Drawings
Fig. 1: inhibition of histone methyltransferase activity of EZH2 can increase expression of ELOVL2. Upregulation of polyunsaturated fatty acid biosynthetic pathways following knockout of EZH2 in skov3 cells; b. GSEA analysis of polyunsaturated fatty acid biosynthesis pathway differential genes and heat maps of differential gene expression; IGV plots of H3K27me3 ChIP-seq data of ELOVL2 before and after knockout of EZH2 in skov3 cells; real-time qPCR results of elovl2 in WT and EZH2 knocked out cells; westernblot results of elovl2 in WT and EZH2 knockout cells; chIP-PCR results for ELOVL2. * p <0.05, < p <0.01, < p <0.001.
Fig. 2: analysis of metabolism of skov3 cells after DMSO or GSK126 treatment, DHA and EPA elevation was significantly elevated in GSK126 treated cells. b. TG levels were elevated in serum from tumor-bearing mice in the control and GSK126 groups. Mean ± SEM shows (n=5).
Fig. 3: the combined SC-26196 inhibitor can enhance the inhibition of the GSK126 on SKOV3 cells. Cck-8 kit to detect cell viability (n=5) of GSK126 and SC-26196 inhibitor treated SKOV3 cells; b. c, detecting migration and invasion capacity (n=5) of GSK126 and SC-26196 inhibitor treated SKOV3 cells through the IncuCyte S3; the cck-8 kit detects cell viability (n=3) of GSK126 and SC-26196 inhibitor treated ID8 cells; e. tumor weights of mice bearing ID8 were measured every 2 days with vernier calipers for small tumor volumes. * p <0.05, < p <0.01, < p <0.0001.
Fig. 4: the combined SC-26196 inhibitor can enhance the activity of GSK126 for inhibiting the ovarian cancer organoid cells and reduce the growth of the ovarian cancer organoid cells. * P <0.001.
Detailed Description
Based on the previous studies, the present inventors further found that the therapeutic effect of EZH2 inhibitors on treatment of abnormal lipid metabolism solid tumors (particularly ovarian cancer) can be significantly enhanced by combining the EZH2 inhibitors with polyunsaturated fatty acid inhibitors through a large number of screening in the intensive studies on the mechanism of action of EZH2 on lipid metabolism regulation in tumor cells and the mechanism of action of EZH2 inhibitors on tumor resistance. The present invention has been completed on the basis of this finding.
As used herein, the EZH2 inhibitor and the polyunsaturated fatty acid inhibitor in the pharmaceutical composition of the present invention may be administered in the same pharmaceutical formulation or may be administered in different pharmaceutical formulations. In the case of administration in different pharmaceutical formulations, the dosage forms of the EZH2 inhibitor and the polyunsaturated fatty acid inhibitor may be the same or different. Also, the EZH2 inhibitor and the polyunsaturated fatty acid inhibitor may be administered simultaneously or sequentially.
As used herein, the dosage form of the pharmaceutical formulation of the present invention is a tablet, capsule, granule, oral liquid or inhalant. Preferably, the dosage form of the present invention is a tablet or capsule.
As used herein, the "pharmaceutically acceptable carrier" of the present invention refers to a pharmaceutical carrier conventional in the pharmaceutical formulation field, and is selected from one or more of fillers, binders, disintegrants, lubricants, suspending agents, wetting agents, pigments, flavoring agents, solvents, and surfactants.
Fillers of the present invention include, but are not limited to, starch, microcrystalline cellulose, sucrose, dextrin, lactose, powdered sugar, dextrose, and the like; such lubricants include, but are not limited to, magnesium stearate, stearic acid, sodium chloride, sodium oleate, sodium lauryl sulfate, poloxamers, and the like; such binders include, but are not limited to, water, ethanol, starch slurry, syrup, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, sodium alginate, polyvinylpyrrolidone, and the like; such disintegrants include, but are not limited to, starch effervescent mixtures, i.e., sodium bicarbonate and citric acid, tartaric acid, low-substituted hydroxypropyl cellulose, and the like; such suspending agents include, but are not limited to, polysaccharides such as acacia, agar, alginic acid, cellulose ethers, carboxymethyl chitin and the like; such solvents include, but are not limited to, water, balanced salt solutions, and the like.
Preferably, the medicament of the present invention can be prepared into various solid oral preparations, liquid oral preparations, etc. Pharmaceutically acceptable solid formulations of oral agents are: common tablet, dispersible tablet, enteric coated tablet, granule, capsule, dripping pill, powder, etc., and oral liquid preparation comprises oral liquid, emulsion, etc. Alternatively, the medicament of the present invention may be formulated into a topical application form such as an inhalant.
The various formulations described above may be prepared according to conventional techniques in the pharmaceutical formulation arts.
In the above-described pharmaceutical compositions, pharmaceutical preparations and medical uses, the administration time, the administration number and the administration frequency of the "EZH2 inhibitor" and the "polyunsaturated fatty acid inhibitor" and the like are required depending on the specific diagnosis result of the condition, and are within the technical scope of the person skilled in the art.
The invention will be further illustrated with reference to specific examples. It should be understood that the detailed description and specific examples are intended for purposes of illustration only and are not intended to limit the scope of the invention.
The specific techniques or conditions are not identified in the examples and are described in the literature in this field or are carried out in accordance with the product specifications. The reagents or equipment used were conventional products available for purchase through regular channels, with no manufacturer noted.
The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the examples described below, unless otherwise specified, are all commercially available products.
Unless otherwise indicated, the percentages and parts referred to in the present invention are weight percentages and parts.
Example 1: inhibition of histone methyltransferase activity of EZH2 can increase expression of ELOVL2
The ChIP-seq and RNA-seq data from the earlier study (PMID: 33163485) were analyzed to find lipid metabolism related genes directly regulated by EZH2, such as ELOVL2, FADS2.
RNA-seq data shows that the biosynthesis pathway of polyunsaturated fatty acids is obviously changed, the expression of genes such as ELOVL2 and FADS2 in EZH2-KO SKOV3 cells is increased, the level of an ELOVL2 promoter regulatory region H3K27me3 in the EZH2-KO cells is reduced, and the expression of ELOVL2 is up-regulated.
The specific experimental results are shown in fig. 1.
Example 2: GSK126 can affect polyunsaturated fatty acid metabolism in ovarian cancer cells
The SKOV3 cell metabolism spectrum after GSK126 or DMSO (Ctrl) treatment is analyzed by using an ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) system, and DHA and EPA are increased after GSK126 treatment. GSK126 treated ID8 cell tumor bearing mice (see section 3.4 of example 3 for specific methods of tumor vaccination) also had elevated blood Triglyceride (TG) levels (as shown in FIG. 2). These results suggest that GSK126 can affect ovarian cancer cell polyunsaturated fatty acid metabolism.
Specific experimental methods for metabolic profiling were as follows, cells were collected after DMSO or GSK126 (15 μmol/L) treatment for 48h, mixed with 10 pre-chilled zirconia beads and 20 μl deionized water in eppendorf safelock microcentrifuge tubes. The sample was homogenized for 3min and 150. Mu.L of the extract metabolite containing the internal standard methanol was added. The mixture was homogenized for 3min, centrifuged at 18000g for 20min, and the supernatant was transferred to a 96-well plate. The following operations were performed on a Biomek 4000 workstation. mu.L of freshly prepared derivative reagent was added to each well. The plates were then derivatised at 30℃for 60min, evaporated for 2h after derivatisation and samples reconstituted by adding 330. Mu.L ice-cold 50% methanol solution. Preserving at-20 ℃ for 20min, centrifuging at 4 ℃ for 30min at 4000g, transferring 135 mu L of supernatant to a new 96-well plate, and carrying out 10 mu L of internal standard in each well. Serial dilutions of the derivatized stock standard were added to the left well. And finally, performing LC-MS analysis on the sealing plate. All standards were from Sigma-Aldrich, steraloids inc. (Newport, RI, USA) and TRC Chemicals (Toronto, ON, canada). The project uses an ultra high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) system (ACQUITY UPLC-Xevo TQ-S, waters Corp., milford, mass., USA) to quantify all target metabolites.
Example 3: the combination of SC-26196 can enhance the inhibition of GSK126 on SKOV3 cells
3.1 cell proliferation assay
Cells were seeded in 96-well plates with approximately 5000 cells per well. After 24 hours, treatment with GSK126 and/or SC-26196 was performed, and the drug concentration and the number of repetitions were as shown in the figure. Then on the IncuCyte S3 platform (Sartorius,germany) using phase contrast channels. Four sets of phase contrast images were taken from different areas of each well every 3 hours using a 10X objective. Cell edges were detected by setting the incuCyte S3 image analysis software to determine the percent cell confluency.
The CCK-8 method is to measure the cell viability by adding 10. Mu.L of CCK-8 (Dojindo Laboratories, rockville, mass., USA) to each well after culturing the cells in a 96-well plate for 48 hours, and measuring the absorbance at 450nm by using an enzyme-labeled instrument after 1 hour.
3.2 cell migration experiments
Cells were seeded in 96-well plates and cultured using serum-free medium. Adhering overnight, and allowing cell growth to reach 90-100% confluence, usingThe WoundMaker scratcher tool precisely scratches within a single layer of cells.PBS was washed once and the medium was replaced with serum-free GSK126 and/or SC-26196 treatment medium, and the drug concentration and the number of repetitions were shown in the figure. Cells were then imaged using the phase contrast channel at the incuCyte S3 platform. Every 3 hours, two sets of phase contrast images were taken from different areas of each well using a 10X objective. Scratch areas were measured, calculated and analyzed using the incuCyte S3 scratch block software and Image J software.
3.3 cell invasion assay
Cells were seeded in 96-well plates and cultured using serum-free medium. Adhering overnight, and allowing cell growth to reach 90-100% confluence, usingThe WoundMaker scratcher tool precisely scratches within a single layer of cells. The wells were washed once with PBS. Matrigel (356231, BD) was thawed by being placed one night in advance at 4 ℃. The culture was diluted in ice-cold medium at a ratio of 1:3 and prepared as-is and stored on ice. The remaining cell culture medium, PBS and scraped cells were removed using a vacuum pump, 30. Mu.L of matrigel diluent was added to each well, and the mixture was placed in a 37℃cell incubator for 30min until the matrigel diluent solidified. The addition medium was serum-free GSK126 and/or SC-26196 treatment medium, and the drug concentration and the repetition number are shown in the figure. Cells were then imaged using the phase contrast channel at the incuCyte S3 platform. Every 3 hours, two sets of phase contrast images were taken from different areas of each well using a 10X objective. Scratch areas were measured, calculated and analyzed using the incuCyte S3 scratch block software and Image J software.3.4 mouse subcutaneous tumor experiments:
(1) Preparing mice: female C57BL/6J mice of 6-7 weeks old were purchased from the department of animal science, university of Beijing, and were randomly divided into four groups, and were numbered by cutting the toes. The number of each group is 6.
(2) Preparing cells: culturing target cells according to the required cell quantity, digesting the centrifuged cells when the cell confluence reaches about 80%, re-suspending the cell sediment by using PBS buffer solution, centrifuging, washing the cells twice by using PBS, sucking the PBS buffer solution, re-suspending the cells by using a small amount of PBS buffer solution, blowing the cells into single cell suspension, counting, and diluting the cells to the target concentration by using the PBS buffer solution;
(3) Subcutaneous injection of tumor cells: the cell suspension was slowly injected subcutaneously into mice (100. Mu.L of each mouse, containing 1X 10 cells in number) using a 1mL syringe 7 Personal), care was taken to avoid leakage of cell suspension from the pinholes;
(4) The subcutaneous tumor formation of the mice was observed once every two days, the tumor volume was measured with a vernier caliper and recorded, and the drug intervention was performed on the sixth day, as follows:
a first group: control group. Injecting physiological saline;
second group: GSK126 group, GSK126 was injected as body weight: 50mg/kg twice every three days;
third group: SC-26196 group, SC-26196 was injected by weight: 30mg/kg once every three days;
fourth group: gsk126+sc-26196 group. The two are combined.
(5) The length and width of the mouse subcutaneous tumor mass were measured once every two days using vernier calipers, and the tumor size was calculated according to the calculation rule of tumor size = length x width 2/2.
(6) After the drug intervention is finished, removing eyeballs of the mice on seventeenth day to collect peripheral blood of the mice, centrifuging to obtain serum, and then placing the serum in a refrigerator at-80 ℃ for freezing storage for later use; killing the mice by adopting a cervical dislocation method, taking out subcutaneous tumors, weighing tumor blocks, and photographing;
(7) Paraffin-embedded sections were prepared after fixing the tumor with formalin for subsequent immunohistochemistry.
3.5 experimental results
As shown in FIG. 3, the combination of GSK126 and SC-26196 treated SKOV3 cells, and the combination of SC-26196 was found to significantly enhance the effect of inhibiting cell proliferation. Migration and invasion experiments were performed in SKOV3 cells. Compared with the cell proliferation experiment, the two medicines are combined to obviously and effectively inhibit cell migration and invasion compared with the control group. Combination therapy with GSK126 and SC-26196 reduced the growth of ID8 cells in mice.
Example 4: the combined SC-26196 can enhance the inhibition of GSK126 on ovarian cancer organoid cells
4.1 Experimental methods
Ovarian cancer organoid culture: fresh surgical specimens (gynaecological and obstetrical surgery at third hospital of Beijing university, 2022, 5 months, 13 pm, 47 years, high-grade serous ovarian cancer IIIC) were obtained directly from the clinic, reserving tissues for protein, RNA extraction and immunohistochemical experiments. Tissue for organoid culture was placed in cold calcium magnesium ion free PBS, washed 6-8 times, transferred to a clean petri dish, added with 1mL of mild Cell dissociation reagent (Stem Cell), sheared with sterile scissors and transferred to a 15mL centrifuge tube, supplemented with mild Cell dissociation reagent to 10mL, and incubated on a shaker at 37 ℃ for 0.5-1h. After centrifugation, the supernatant was discarded. The pellet was resuspended in 1mL of DMEM/F12 medium containing 1% BSA and diabody and filtered through a 70 μm cell sieve into a new centrifuge tube. After cell counting, the mixture was mixed with matrigel in a ratio of 1:1, and added in an amount of 20. Mu.L per droplet to a 24-well plate incubated in a 37℃incubator. Placing the 24-well plate in a cell culture box for 10min, and adding an ovarian cancer organoid culture medium into each well for culture after the matrigel is solidified. Ovarian cancer organoids include DMEM/F12 (containing HEPES and penicillin-streptomycin-gentamicin), WNT3A, RSPO1, B27 (50×), hEGF, nicotinamide (1M), FGF10 (100 μg/mL), noggin, and the like. The liquid is changed once every 3-4 days according to the growth state of the ovarian cancer organoid, and the ovarian cancer organoid is cultured for about two weeks for passage.
Detection of viability of ovarian cancer organoid cells: after treatment of human ovarian carcinoma organoids with DMSO, GSK126 (20. Mu.L), SC-26196 (60. Mu.L), GSK126 (20. Mu.L) +SC-26196 (60. Mu.L) for 72h, cell viability was detected by the 3D Cell Titer Glo kit.
4.2 experimental results
As shown in fig. 4, the experimental results demonstrate that the combined SC-26196 significantly enhanced the effect of inhibiting cell proliferation (< p < 0.001) after treatment of ovarian cancer organoid cells with GSK126, SC-26196, and combined GSK126 and SC-26196. The two medicines are combined to obviously and effectively inhibit the cell viability of the ovarian cancer organoid. GSK126 and SC-26196 combination therapy can reduce the growth of ovarian cancer organoid cells.
It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.

Claims (5)

1. A pharmaceutical composition for enhancing the anti-tumor effect of an EZH2 inhibitor, which is characterized in that: the pharmaceutical composition consists of an EZH2 inhibitor and a polyunsaturated fatty acid inhibitor, wherein the polyunsaturated fatty acid inhibitor enhances the effect of the EZH2 inhibitor on resisting lipid metabolism abnormality solid tumors, and the mass ratio of the EZH2 inhibitor to the polyunsaturated fatty acid inhibitor is 1:3, the solid tumor is high grade serous ovarian cancer, the EZH2 inhibitor is GSK126, and the polyunsaturated fatty acid inhibitor is SC-26196.
2. A pharmaceutical formulation for enhancing the antitumor effect of an EZH2 inhibitor, characterized in that: the pharmaceutical formulation is made from a therapeutically effective amount of the pharmaceutical composition of claim 1 and a pharmaceutically acceptable carrier, the tumor being high grade serous ovarian cancer.
3. The pharmaceutical formulation according to claim 2, wherein: the pharmaceutical preparation is an oral preparation.
4. A pharmaceutical formulation according to claim 3, wherein: the oral preparation is oral liquid, tablet, powder, capsule or granule.
5. Use of a pharmaceutical composition according to claim 1 or a pharmaceutical formulation according to any one of claims 2-4 for the preparation of an antitumor drug, characterized in that: the tumor is high grade serous ovarian cancer.
CN202210682634.4A 2022-06-16 2022-06-16 Antitumor pharmaceutical composition containing EZH2 inhibitor and polyunsaturated fatty acid inhibitor and application thereof Active CN114870021B (en)

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Non-Patent Citations (1)

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