Application of glutathione in reversing drug resistance of osimertinib

Abstract

The invention discloses application of a preparation capable of improving glutathione concentration in reversing osimertinib drug resistance. The invention also discloses a pharmaceutical composition for treating the drug-resistant lung cancer, and the pharmaceutical composition comprises a preparation capable of improving the concentration of glutathione (GSH) and osimertinib (AZD9291). The invention finds that the combination of GSH and AZD9291 has a synergistic effect, the combination of 3mM GSH and 1 mu M osimertinib can inhibit the proliferation of non-small cell lung cancer, the inhibition rate is significantly higher than that of the single use of AZD9291, and the combination of GSH and osimertinib has a synergistic inhibition effect on AZD9291 drug-resistant NSCLC cells. Meanwhile, the intracellular lipid peroxidation level of lung cancer cells is remarkably improved through GSH treatment, and the treatment effect of AZD9291 on NSCLC is enhanced.

Description

Technical Field

[0001] The present invention belongs to the field of anti-tumor drugs, and in particular relates to the application of a preparation capable of increasing glutathione concentration in reversing osimertinib resistance. Background Art

[0002] Lung cancer is a malignant tumor with high morbidity and mortality worldwide, and is one of the biggest threats to human health. Non-small cell lung cancer (NSCLC) accounts for more than 80% of all lung cancers. Nearly two-thirds of NSCLC patients carry carcinogenic gene mutations, and about half of them have lesions that can be targeted for treatment. Epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR TKI) is one of the most commonly used clinical targeted treatment drugs for NSCLC.

[0003] The first and second generation EGFR TKIs will not significantly inhibit EGFR signal transduction after prolonged treatment, leading to drug resistance in patients. Osimertinib (AZD9291) is the first third generation EGFR-TKI approved by the US FDA for marketing. It can selectively bind to sensitive mutant and T790M resistant mutant EGFR irreversibly, blocking downstream phosphorylation signal transduction, thereby overcoming tumor resistance.

[0004] However, during clinical treatment, patients still develop acquired resistance to osimertinib, leading to treatment failure and reduced survival. The mechanisms of acquired resistance to EGFR TKIs are divided into EGFR-dependent mechanisms and EGFR-independent mechanisms. EGFR-dependent mechanisms include secondary kinase mutations in various EGFR-mutant NSCLCs, such as the G1202R mutation in ALK rearrangement, the G2032R mutation in ROS1, and the D1228 and Y1230 residue mutations in MET. EGFR-independent mechanisms include activation of bypass receptor tyrosine kinase (RTK) signals and activation of acquired fusion proteins in oncogenic signaling pathways.

[0005] In recent years, the clinical treatment of advanced NSCLC has developed rapidly. The discovery of activating EGFR, ALK, ROS1, MET, RET, NTRK, and BRAF genomic alterations and the approval of progressive and effective systemic targeted therapies have changed the treatment status of these NSCLC subgroups. At the same time, multiple other types of targeted inhibitors have been developed. Unfortunately, these therapeutic advances are still insufficient to improve the overall survival of advanced non-small cell lung cancer, and metastatic disease remains incurable. Drug reversal agents with higher safety and better efficacy need to be further explored and discovered. Summary of the invention

[0006] Glutathione (GSH) is a tripeptide containing a γ-amide bond and a sulfhydryl group. It is composed of glutamic acid, cysteine ​​and glycine. It exists in almost every cell in the body. Its structural formula is as follows:

[0007]

[0008] The idea of ​​the present invention is to investigate the inhibitory effect of glutathione on the proliferation and survival of non-small cell lung cancer (NSCLC) resistant tumor cell lines through in vitro efficacy evaluation, and to investigate the drug resistance improvement effect of glutathione on resistant tumor cells. The non-small cell lung cancer cell lines H1975 (osimertinib-sensitive strain) and H1975AR (osimertinib-resistant strain) were used for in vitro efficacy evaluation.

[0009] The first aspect of the present invention is to provide use of a preparation capable of increasing glutathione concentration in the preparation of a drug for reversing osimertinib resistance.

[0010] The second aspect of the present invention is to provide a pharmaceutical composition for treating lung cancer, wherein the pharmaceutical composition comprises a preparation capable of increasing the concentration of glutathione (GSH) and osimertinib (AZD9291).

[0011] According to the present invention, the agent capable of increasing the concentration of glutathione is selected from: an inhibitor of glutathione S-transferase, an inhibitor of glutathione peroxidase, or glutathione.

[0012] According to the present invention, the preparation capable of increasing glutathione concentration and osimertinib can be prepared into a single preparation, or prepared into preparations independent of each other but provided in one kit. The pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0013] According to the present invention, the lung cancer is non-small cell lung cancer. Preferably, the lung cancer is non-small cell lung cancer resistant to osimertinib.

[0014] The third aspect of the present invention is to provide a use of a combined preparation comprising a preparation that can increase glutathione concentration and osimertinib in the preparation of a drug for treating lung cancer, and the use of a preparation that can increase glutathione concentration in the preparation of a drug for combined treatment of lung cancer with osimertinib. Preferably, the lung cancer is non-small cell lung cancer. More preferably, the lung cancer is non-small cell lung cancer resistant to osimertinib.

[0015] A fourth aspect of the present invention is to provide a method for treating lung cancer, the method comprising administering to a subject in need thereof a therapeutically effective amount of a combination preparation comprising a preparation that can increase glutathione concentration and osimertinib. Preferably, the two can be administered simultaneously or sequentially. Preferably, the lung cancer is non-small cell lung cancer. More preferably, the lung cancer is non-small cell lung cancer that is resistant to osimertinib.

[0016] According to the present invention, "pharmaceutically acceptable excipients" include, but are not limited to, any and all solvents, dispersion media or other liquid carriers, dispersion or suspension aids, diluents, isotonic agents, preservatives, colorants, sweeteners or flavoring agents, stabilizers, antioxidants, antimicrobial or antifungal agents, osmotic pressure concentration regulators, pH regulators, buffers, chelating agents, cryoprotectants and / or fillers, such as those suitable for the desired specific dosage form. Various excipients for preparing pharmaceutical compositions and techniques for preparing compositions are known in the art. Exemplary antimicrobial or antifungal agents include, but are not limited to, benzalkonium chloride, benzethonium chloride, methylparaben, ethylparaben, propylparaben, butylparaben, benzoic acid, hydroxybenzoic acid, potassium or sodium benzoate, potassium or sodium sorbate, sodium propionate, sorbic acid, etc., and combinations thereof. Exemplary preservatives include, but are not limited to, vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, ascorbic acid, etc., and combinations thereof. Exemplary buffers for controlling pH may include, but are not limited to, sodium phosphate, sodium citrate, sodium succinate, histidine (or histidine-HCl), sodium malate, sodium carbonate, etc., and / or combinations thereof. Exemplary cryoprotectants include, but are not limited to, mannitol, sucrose, trehalose, lactose, glycerol, dextrose, etc., and combinations thereof. Exemplary diluents include, for example, lactose, starch, cellulose derivatives, inorganic calcium salts, sorbitol, etc. Exemplary binders include, for example, starch, gelatin, sodium carboxymethyl cellulose, polyvinyl pyrrolidone, etc. Exemplary antioxidants include, for example, vitamin E, sodium bisulfite, sodium sulfite, butylated hydroxyanisole, etc. Exemplary lubricants include, for example, magnesium stearate, micropowdered silica gel, talc, etc. Exemplary disintegrants include, for example, starch, methyl cellulose, xanthan gum, cross-linked sodium carboxymethyl cellulose, etc.

[0017] The dosage form of the drug or combination preparation of the present invention can be an oral dosage form, such as tablets, capsules, pills, powders, granules, suspensions, syrups, etc.; it can also be an injectable dosage form, such as injections, powder injections, etc., through intravenous, intraperitoneal, subcutaneous or intramuscular routes. All dosage forms used are well known to ordinary technicians in the pharmaceutical field.

[0018] The drugs or combined preparations of the present invention can be administered to subjects by routes known in the art, including but not limited to oral, parenteral, subcutaneous, intramuscular, intravenous, intraperitoneal, intrahepatic, intramyocardial, intrarenal, vaginal, rectal, buccal, sublingual, intranasal, transdermal, etc.

[0019] The dosage of the drug or combination preparation of the present invention will depend on the age, health and weight of the recipient, the type of the combined drug, the frequency of treatment, the route of administration, etc. The drug can be administered in a single daily dose, or the total daily dose can be administered in divided doses twice, three times or four times a day. The drug can be administered before, during or after surgery. The dose can be administered once or multiple times, and the administration time can range from a single day to several months or longer. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The validation results of the acquired drug resistance cell model H1975AR of osimertinib (AZD9291) in the non-small cell lung cancer cell line H1975. Figure 1 A: Cell viability was detected by MTT assay, the results are the mean ± SD of n samples, n = 6, *** represents p < 0.01; Figure 1 B: SDS-PAGE electrophoresis was used to detect the phosphorylation levels of EGFR and ERK proteins in the cells, as well as the expression level of BCL-2 protein (GAPDH was used as an internal reference).

[0021] Figure 2 Changes in protein expression in H1975AR cell line. Figure 2 A and Figure 2 B: KEGG analysis results; Figure 2 C: Fluorescence quantitative PCR detection of mRNA levels of glutathione synthesis-related genes, the results are the mean ± standard deviation of n samples, n = 3, * represents p < 0.05; Figure 2 D: Western blotting was used to detect the expression levels of glutathione synthesis-related proteins.

[0022] Figure 3 Results of GSH and GSSG content detection in H1975 cells and H1975AR cells and culture medium. Among them, osimertinib (AZD9291) treatment can significantly increase the level of GSH in cells, but the content of GSH in H1975AR cells is significantly lower than that in H1975 cells ( Figure 3A), the GSSG content in H1975AR cells was also significantly lower than that in H1975 cells ( Figure 3 B), the content of GSSG in the culture medium of H1975AR cells was significantly higher than that in the culture medium of H1975 cells ( Figure 3 C), the ratio of GSH / GSSG in H1975AR cells was significantly higher than that in H1975 cells ( Figure 3 D). The results in the figure are the mean ± standard deviation of n samples, n = 3, * represents p < 0.05, ** represents p < 0.01.

[0023] Figure 4 Effect of GSH on the proliferation activity of H1975 cells and H1975AR cells treated with osimertinib (AZD9291). The results in the figure are the mean ± standard deviation of n samples, n=6.

[0024] Figure 5 Effect of GSH on lipid peroxidation levels in cells treated with osimertinib (AZD9291). The results in the figure are the mean ± standard deviation of n samples, n = 3, * represents p < 0.05. DETAILED DESCRIPTION

[0025] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content recorded in the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the present invention equally.

[0026] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods. The experimental methods are conventional molecular biology methods in the art and can be performed with reference to the instructions of molecular biology experimental manuals in the art or kit product instructions.

[0027] Material Description

[0028] Human non-small cell lung cancer cell line H1975 was purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences;

[0029] Osimertinib (AZD9291) was purchased from Sigma-Aldrich;

[0030] Glutathione (GSH) was purchased from Aladdin, USA;

[0031] Fetal bovine serum was purchased from Suzhou Yikesai Biotechnology Co., Ltd., China;

[0032] RPMI1640 culture medium and penicillin-streptomycin (100X) were purchased from Gibco, USA;

[0033] MTT detection kit, GSH detection kit, and lipid peroxidation detection kit were purchased from Beyotime Biotech Co., Ltd.

[0034] Example 1 Cultivation of drug-resistant non-small cell lung cancer cell line H1975AR

[0035] 1. Cell Recovery

[0036] Immediately disinfect the cryovials with alcohol after taking them out of the liquid nitrogen tank, stir them quickly in a 37℃ water bath to melt them quickly, and immediately transfer them to a 15mL centrifuge tube, add no less than 9 times the volume of the culture medium and mix them well. After centrifugation at 1000rpm for 3 minutes, discard the supernatant, add 1mL of fresh culture medium preheated at 37℃ to resuspend, mix well, inoculate into a new culture dish, and place it in a 37℃, 5.0% CO2 incubator for static culture. Change the medium no more than 72h during the culture process.

[0037] 2. Cell Passaging

[0038] Before subculturing, preheat the culture medium, PBS and trypsin in a 37°C water bath for 30 minutes. Turn on the UV irradiation in the biosafety cabinet for at least 30 minutes. Use a vacuum filter pump to suck away the old culture medium and wash twice with PBS to remove the residual culture medium. After removing the PBS, add 0.25% trypsin to digest the cells for 1-3 minutes until the cells begin to detach from the bottom of the culture dish. Add normal culture medium and blow evenly to terminate the digestion. Transfer the cells to a 15mL centrifuge tube, centrifuge at 1000rpm for 3 minutes, discard the supernatant, re-add fresh culture medium and mix evenly. Inoculate the cells into a new culture dish according to the proportion, place it in a 37°C, 5.0% CO2 incubator for static culture, and change the medium no more than 72 hours during the culture process.

[0039] 3. Screening of drug-resistant cell lines

[0040] The H1975AR drug-resistant cell line was established using the concentration-increasing method, which involves treating cells with increasing concentrations of the drug and culturing them in the presence of the drug for a period of time to screen for drug-resistant cells.

[0041] 1) H1975 cells with good growth status were selected, and different concentrations of AZD9291 were used to treat the cells when the cells were in the logarithmic growth phase. The proliferation inhibition rate was determined by MTT, and the concentration with a proliferation inhibition rate of 10% to 20% was selected as the initial AZD9291 treatment concentration.

[0042] 2) Prepare a culture medium containing AZD9291 according to the starting concentration for culturing cells, and the initial cell density is about 20%. When the cell density reaches 90%, subculture at a ratio of 1 / 3, and the culture medium contains 1.5 to 2 times the starting concentration of AZD9291. When the cell density reaches 90%, the concentration of AZD9291 is also increased by 1.5 to 2 times at a ratio of 1 / 3. Continue to subculture for two months. When the proliferation and survival of AZD9291-treated cells show significant differences relative to normal H1975, it indicates that the drug-resistant cell line has been established.

[0043] 4. MTT assay for cell viability

[0044] After the treatment is completed according to the predetermined experimental steps, the culture medium is washed off, and the cells are washed twice with PBS. After the PBS is sucked off, a serum-free culture medium containing 5 mg / mL of MTT is added, and the cells are placed in a 37°C, 5.0% CO2 incubator for incubation for 4 hours. After the incubation is completed, the MTT culture medium is sucked off and the cells are washed twice with PBS. 100 μL DMSO is added to each well and shaken at a low speed for 1 minute to fully dissolve the crystals. The absorbance value at 490 nm is detected with an enzyme marker. Cell survival rate (%) = (absorbance of the experimental group - absorbance of the blank group) / (absorbance of the control group - absorbance of the blank group).

[0045] 5. Protein sample preparation and protein expression level detection by western blotting

[0046] The cells were discarded from the culture medium, rinsed twice with PBS, discarded from the PBS, and then added with cell lysis solution (containing 100 μM PMSF), and placed on ice for lysis for 30 minutes. The cells were scraped, and the cell lysate was transferred to a pre-cooled 1.5 mL centrifuge tube, and centrifuged at 12000 rpm, 4°C for 10 minutes. The supernatant was quantified for protein concentration using the BCA quantitative method.

[0047] SDS-PAGE electrophoresis: (1) Heat the protein sample in a metal bath at 100°C for 5 minutes. After cooling, add the same volume of the different treated protein samples to the wells of the concentrated gel and start electrophoresis. After the electrophoresis, transfer the cellulose acetate membrane (NC membrane) using the transfer buffer by wet transfer. The transfer current is 300mA and the time is 90 minutes. The transfer time can be extended or shortened according to the molecular weight of the protein to be tested. After the transfer, place the NC membrane in a blocking solution of 5% skim milk and block it at room temperature for 1 hour. Wash the NC membrane twice with TBST. Remove the TBST, add the diluted primary antibody, and incubate it on a shaker at 4°C with slow shaking overnight. Wash the NC membrane with TBST 3 times, 10 minutes each time. After washing, incubate the NC membrane with the diluted secondary antibody at room temperature for 2 hours. After the secondary antibody incubation, remove the secondary antibody and wash the NC membrane 3 times with TBST. Mix equal volumes of ECL solution A and solution B, soak the NC membrane in the solution for 1 minute, and develop and collect signals in a chemiluminescence instrument.

[0048] The results are as follows Figure 1 As shown, in Figure 1 In A, treatment of H1975 cells with 1 μM AZD9291 can inhibit their proliferation activity by 50%. However, even treatment of H1975AR cells with 5 μM AZD9291 still had no significant inhibitory effect on their proliferation activity. Figure 1 In B, AZD9291 can significantly downregulate the phosphorylation levels of EGFR and ERK proteins in H1975 cells, indicating that AZD9291 can normally inhibit the phosphorylation of tyrosine kinases, while the level of phosphorylated proteins in H1975AR cells is already at a low level. AZD9291 treatment can significantly downregulate the expression of anti-apoptotic protein BCL-2 in H1975 cells, while the expression level of BCL-2 in H1975AR cells is significantly higher than that in H1975 cells, and AZD9291 treatment has no significant effect on the expression of BCL-2 in H1975AR cells. The above results indicate that H1975AR cells have developed resistance to AZD9291, that is, the resistant cell line was successfully constructed.

[0049] Example 2 Protein changes in H1975AR cell line

[0050] 1. Protein Extraction and Alkylation

[0051] After extracting total cell protein with Urea, add DTT (10mM), IAA (15mM) and DTT (20mM) respectively, and shake on a shaker for 1 hour each. After the alkylation reaction is completed, add at least 4 times the volume of -20℃ pre-cooled protein precipitation buffer (acetone: ethanol: acetic acid = 50:50:0.1) to the protein solution, mix well and place at -20℃ for precipitation overnight. Centrifuge at 20000g, 4℃ for 10 minutes, remove the supernatant and retain the precipitate. Wash the precipitate once with -20℃ pre-cooled acetone and 75% ethanol respectively, and retain the precipitate. Leave it at room temperature for 5 to 10 minutes to evaporate the ethanol. Add 200μL 8mol / L urea (100mmol / LNH3HCO3, pH=7.8) to the protein precipitate, and slowly shake on a shaker to dissolve the precipitate.

[0052] 2. Trypsin Digestion

[0053] Use 100mmol / L ammonium bicarbonate to dilute the Urea in the sample to 1mol / L. Use Bradford to quantify the protein, add trypsin at a ratio of protein:trypsin = 50:1, mix well, and then shake at 37℃ for 18 hours. After the end, add 0.1% TFA, mix well and set aside.

[0054] 3.TiO2 enrichment of phosphorylated peptides

[0055] After desalting the peptide sample with a C18 column, add 150 μL of buffer B to the dried peptide sample and sonicate for 30 minutes to completely dissolve the peptide. Balance the TiO2 adsorption column. Add the sample to the TiO2 adsorption column, centrifuge at 1000g for 10 minutes, and repeat this step once. Wash the TiO2 adsorption column once with 20 μL of buffer B, then wash it once with 20 μL of buffer A, and discard the flow-through liquid. Add 100 μL of elution buffer to the TiO2 adsorption column and centrifuge at 1000g for 5 minutes. Concentrate and dry the eluate using a vacuum freeze-spin concentrator, and the resulting solid powder is the dried phosphorylated peptide.

[0056] 4. Mass spectrometry detection

[0057] The samples were ionized by the electrospray ion source and then entered the mass spectrometer. The mass spectrometer data was collected using an Agilent triple quadrupole mass spectrometer. The capillary temperature was 320°C and the electrospray voltage was 1.85 kV. The PRM mode was used to collect data. The information of the ions to be detected was imported into Exclusion, including charge-to-mass ratio, detection time, charge, etc., and then data was collected.

[0058] 5. Fluorescence quantitative PCR

[0059] Extraction of total RNA from cells: discard the culture medium from the cells, wash twice with pre-cooled PBS to remove the residual culture medium, and add an appropriate amount of Trizol to lyse the cells. Add chloroform (1 mL Trizol plus 0.2 mL chloroform) to the centrifuge tube in proportion, shake vigorously for 30 seconds to mix it thoroughly, and let it stand at room temperature for 10 minutes. Centrifuge the sample at 12000g and 4℃ for 15 minutes. Use a pipette to transfer the top aqueous phase to a new RNase-free centrifuge tube in batches. Add isopropanol with an equal volume to the aqueous phase, gently invert and mix 8 to 10 times, and let it stand at room temperature for 10 minutes. Centrifuge the sample at 12000g and 4℃ for 15 minutes, and discard the supernatant. Add 1 mL of pre-cooled 75% ethanol, and gently invert and invert 8 to 10 times. Centrifuge the sample at 12000g and 4℃ for 10 minutes, aspirate the supernatant, and leave the centrifuge tube open for about 5 minutes until the precipitate is observed to dry. 20 μL of RNase-free water was added to dissolve the precipitate, and the RNA concentration was determined by a spectrophotometer before reverse transcription and fluorescence quantitative PCR reactions. The amounts of reagents and reaction conditions in the reactions were carried out in accordance with the instructions of the commercial kit.

[0060] The results are as follows Figure 2 As shown in Figure 2, the most significant signaling pathway in H1975AR cells compared to H1975 cells was the glutathione (GSH) synthesis pathway ( Figure 2 A and Figure 2 B). Fluorescence quantitative PCR ( Figure 2 C) The test results showed that the mRNA expression of glutathione synthesis-related genes such as GCLC, GCLM, GSS, and GSR was significantly downregulated, while the mRNA expression of glutathione metabolism-related genes such as GPX3 was significantly upregulated. Protein immunoblotting detection ( Figure 2 D) The test results showed that the expression of glutathione metabolism-related proteins such as GPX4, GSTM2 and GPX1 in H1975AR cells was significantly upregulated, while the expression of glutathione synthesis-related protein GSR was significantly downregulated, suggesting that reduced glutathione synthesis may be the cause of H1975AR cell resistance.

[0061] Example 3 GSH and GSSG content determination

[0062] The GSH and GSSG detection kit (PMK1879) of PUMEC was used to measure the GSH / GSSG content of H1975 cells and H1975AR cells. 6For each cell, first wash the cells twice with cold PBS (resuspend the cells with PBS, centrifuge at 4°C 600g for 10min), add 1mL of pre-cooled extract to resuspend the cells or bacteria, and disrupt them in an ice bath with ultrasonic waves for 5min (power 20% or 200W, ultrasonic for 3s, interval 7s, repeat 30 times). After ultrasonication, centrifuge the sample at 8000g at 4°C for 10min, take the supernatant, and place it on ice for testing. GSH / GSSG determination: preheat the microplate reader or visible light spectrophotometer for more than 30min, adjust the wavelength to 412nm, and zero the visible light spectrophotometer with deionized water. Add the sample to a 96-well plate, add the relevant test kit, mix well, incubate at 37°C in the dark for 10min, and record the absorbance Ablank, Astandard, and Atest at 10min. Calculate ΔAtest=Atest-Ablank, ΔAstandard=Astandard-Ablank. Calculation of GSH content: GSH (nmol / 10 4 cells) = y × V sample ÷ (500 × V sample ÷ V sample total) × n = y ÷ 500 × n = 0.002 × y × n. GSSG content calculation: GSSG (nmol / 10 4 cells)=y×Vsample÷(500×Vsample÷Vsampletotal)×10×n=0.02y×n, Vsample: the volume of sample added in the reaction, 0.002mL, converted according to the ratio 10μL×(21÷105)=2μL; W: sample mass, g; Vsampletotal: the volume of extract added, 1mL; 10: the dilution factor of sample detection, (10+90)÷10; 2: the dilution factor of liquid sample preparation, adding an equal volume of extract means diluting the sample by 2 times; n: the dilution factor of the sample for further dilution; 500: the number of cells, 5 million.

[0063] The results are as follows Figure 3 As shown in Figure 2, AZD9291 treatment can increase the level of GSH, and the content of GSH in H1975AR cells is significantly lower than that in H1975 cells ( Figure 3 A). Similarly, AZD9291 treatment can increase the level of GSSG, and the content of GSSG in H1975AR cells is also significantly lower than that in H1975 cells ( Figure 3 B). At the same time, the content of GSSG in the culture medium of H1975AR cells was significantly higher than that in H1975 cells ( Figure 3 C), which ultimately resulted in a significantly higher GSH / GSSG ratio in H1975AR cells than in H1975 cells ( Figure 3 D) Since GSSG is a metabolite of GSH, it means that the decrease in GSH content in H1975AR cells is due to more GSH being metabolized and then excreted out of the cells.

[0064] Example 4 GSH reversal of drug resistance experiment

[0065] Take H1975 cells and corresponding H1975AR cells in the logarithmic growth phase and digest them with trypsin at 37°C for 30s. After digestion, add complete culture medium containing 10% fetal bovine serum to terminate digestion, collect cells and count them with a cell counting plate. After determining the number of cells, evenly spread them into a 96-well plate at 5000 cells / well. After culturing overnight until the cells adhere to the wall, add the corresponding concentrations of GSH and AZD9291 according to the experimental design, and continue to culture for 72h. After the culture is completed, wash off the culture medium, add MTT working solution, incubate at 37°C for 4h, discard MTT, add 100μL DMSO to each space, mix thoroughly, use an enzyme reader to measure the absorbance at 450nm and calculate the cell proliferation activity.

[0066] The results are as follows Figure 4 As shown in the figure, AZD9291 can reduce the cell viability of H1975 cells by about 50%, but H1975AR cells show strong resistance to AZD9291 and can still maintain 90% cell viability. When GSH is combined with AZD9291, AZD9291 can reduce the cell viability of H1975AR cells to 58% of the original, which is close to that of H1975 cells, indicating that the combination of GSH and AZD9291 reverses the drug resistance of H1975AR cells.

[0067] Example 5 Detection of lipid peroxidation level after GSH treatment

[0068] Plant the cells in a 6-well plate, remove the culture medium, and wash the cells once with PBS or other appropriate solutions if necessary according to the specific experiment. The experiment uses the lipid peroxidation detection kit (BODIPY 581 / 591C11), and 1ml of BODIPY 581 / 591C11 staining working solution is added to the 6-well plate. Incubate at 37℃ in a cell culture incubator for 30 minutes. After the incubation at 37℃, remove the supernatant and wash twice with PBS. Add 2ml PBS and observe and photograph under a laser confocal microscope.

[0069] The results are as follows Figure 5As shown in the figure, in H1975AR cells, AZD9291 treatment alone and GSH treatment alone had no significant effect on the lipid peroxidation level of cells, but the combined treatment of GSH and AZD9291 could significantly increase the lipid peroxidation level of H1975AR cells. Since lipid peroxidation is the process of ROS oxidation of biological membranes after enhanced oxidative stress, excessive production of ROS can cause cell membrane damage, DNA oxidative damage, protein oxidation, etc., ultimately leading to decreased cell viability and survival rate. It can be inferred that the combined use of GSH and AZD9291 in the above results can significantly reduce the viability and survival rate of H1975AR cells, which may be caused by the increase in the intracellular lipid peroxidation level when GSH and AZD9291 are combined, that is, GSH reverses the drug resistance of H1975AR cells by regulating intracellular lipid peroxidation.

[0070] In summary, the present invention shows that the GSH synthesis of osimertinib (AZD9291) resistant cells is significantly reduced compared with sensitive cells, suggesting that GSH may play an important role in the process of osimertinib resistance. The present invention first discovered that GSH combined with AZD9291 has a synergistic effect. 3mM GSH combined with 1μM osimertinib can inhibit the proliferation of non-small cell lung cancer, and the inhibition rate is significantly higher than that of AZD9291 alone. The combination of the two has a synergistic inhibitory effect on AZD9291-resistant NSCLC cells. At the same time, GSH treatment significantly increases the level of lipid peroxidation in lung cancer cells, enhancing the therapeutic effect of AZD9291 on NSCLC.

[0071] The above is an explanation of the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Use of a preparation that can increase glutathione concentration in the preparation of a drug for reversing osimertinib resistance.

2. Use of a combination of a preparation capable of increasing glutathione concentration and osimertinib in the preparation of a drug for treating lung cancer.

3. Use of a preparation capable of increasing glutathione concentration in the preparation of a drug for combined treatment of lung cancer with osimertinib.

4. The use according to claim 2 or 3, characterized in that: The lung cancer is non-small cell lung cancer. Preferably, the non-small cell lung cancer is osimertinib-resistant non-small cell lung cancer.

5. The use according to any one of claims 1 to 4, characterized in that: The agent capable of increasing the glutathione concentration is selected from: an inhibitor of glutathione S-transferase, an inhibitor of glutathione peroxidase, or glutathione.

6. A pharmaceutical composition for treating lung cancer, characterized in that: The pharmaceutical composition comprises a preparation capable of increasing glutathione concentration and osimertinib.

7. The pharmaceutical composition according to claim 6, characterized in that The agent capable of increasing the glutathione concentration is selected from: an inhibitor of glutathione S-transferase, an inhibitor of glutathione peroxidase, or glutathione.

8. The pharmaceutical composition according to claim 6, characterized in that The pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

9. A method for treating lung cancer, characterized in that: The method comprises administering to a subject in need thereof a therapeutically effective amount of a combination formulation comprising an agent that increases glutathione concentration and osimertinib.

10. The method according to claim 9, characterized in that The lung cancer is non-small cell lung cancer resistant to osimertinib, and the preparation capable of increasing the concentration of glutathione is selected from: an inhibitor of glutathione S-transferase, an inhibitor of glutathione peroxidase, or glutathione.

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