A compound drug for treating osimertinib-resistant non-small cell lung cancer

Abstract

The present invention provides a composite drug for treating osimertinib-resistant non-small cell lung cancer, belonging to the field of biomedicine technology. Osimertinib combined with SR11302 inhibits the proliferation of drug-resistant cells and reduces the drug resistance of cells, so that the IC of cells to osimertinib is reduced. 50 The combined use of SR1302 and osimertinib decreased the expression of ERBB2, ERBB3, p-AKT, and p-ERK1 / 2; in mouse experiments, the combined use of osimertinib and SR11302 significantly inhibited tumor growth. This suggests that combined therapy may be an effective strategy to overcome osimertinib resistance and can provide more effective treatment options for NSCLC patients.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a composite drug for treating osimertinib-resistant non-small cell lung cancer. Background Art

[0002] Lung cancer is divided into small cell lung cancer and non-small cell lung cancer (including large cell lung cancer, squamous cell carcinoma and adenocarcinoma), of which 85% of patients are non-small cell lung cancer (NSCLC). Through genetic testing, it was found that more than 30% of patients had EGFR mutations, so the first and second generation epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs): gefitinib and erlotinib, etc., are used as therapeutic drugs to treat non-small cell lung cancer. However, after 10-15 months of treatment, patients developed acquired resistance, and the causes of resistance included MET amplification, HER2 amplification, EGFR re-mutation, etc., of which 49% had T790M mutation (Sequist, LV; Waltman, BA; Dias-Santagata, D.; Digumarthy, S.; Turke, AB; Fidias, P.; Bergethon, K.; Shaw, AT; Gettinger, S.; Cosper, AK; Akhavanfard, S.; Heist, RS; Temel, J.; Christensen, JG; Wain, JC; Lynch, TJ; Vernovsky, K.; Mark, EJ; Lanuti, M.; Iafrate, AJ; Mino-Kenudson, M.; Engelman, JA, Genotypic and Histological Evolution of Lung Cancers Acquiring Resistance to EGFR Inhibitors. Science Translational Medicine 2011,3(75),75ra26-75ra26.), a third-generation targeted drug osimertinib was developed for this mutation.Compared with traditional platinum-based chemotherapy or first- and second-generation targeted drugs, osimertinib significantly improves patient survival (Mok, TS; Wu, Y.-L.; Ahn, M.-J.; Garassino, MC; Kim, HR; Ramalingam, SS; Shepherd, FA; He, Y.; Akamatsu, H.; Theelen, WSME; Lee, CK; Sebastian, M.; Templeton, A.; Mann, H.; Marotti, M.; Ghiorghiu, S.; Papadimitrakopoulou, VA, Osimertinib or Platinum–Pemetrexed in EGFR T790M–Positive Lung Cancer. New England Journal of Medicine 2016,376(7),629-640.;Soria,J.-C.;Ohe,Y.;Vansteenkiste,J.;Reungwetwattana,T.;Chewaskulyong,B.;Lee,KH;Dechaphunkul,A.;Imamura,F.;Nogami,N.;Kurata,T.;O Kamoto, I.; Zhou, C.; Cho, BC; Cheng, Y.; Cho, EK; Voon, PJ; Planchard, D.; Su, W.-C.; Gray, JE; Lee, S.-M.; Hodge, R.; Marotti, M.; Rukazenkov, Y.; Ramalingam, SS, Osimertinib in Untreated EGFR-Mutated AdvancedNon–Small-CellLung Cancer.NewEnglandJournal ofMedicine 2017,378(2),113-125.), 10.1 months vs 4.4 months and 18.9 months vs 10.2 months respectively. However, osimertinib resistance is still not avoided. The resistance mechanisms of osimertinib that have been elucidated include EGFR re-mutation, EGFR-independent gene mutation, amplification and fusion. Currently, 50-60% of the resistance mechanisms have not been elucidated (Schmid, S.; Li, JJN; Leighl, NB, Mechanisms of osimertinib resistance and emerging treatment options. Lung Cancer 2020,147,123-129.).

[0003] FOSL1 (FOSL-like antigen 1, also known as Fra1) is one of the components of the transcription complex AP-1. Studies have reported that FOSL1 is highly expressed in colorectal cancer (Diesch, J.; Sanij, E.; Gilan, O.; Love, C.; Tran, H.; Fleming, NI; Ellul, J.; Amalia, M.; Haviv, I.; Pearson, RB; Tulchinsky, E.; Mariadason, JM; Sieber, OM; Hannan, RD; Dhillon, AS, Widespread FRA1-dependent control of mesenchymal transdifferentiation programs in colorectal cancer cells. PLoS One 2014, 9(3), e88950.), regulates EMT (epithelial-mesenchymal transition) related genes, activates the RAS-ERK signaling pathway, and promotes tumor invasion. In glioblastoma (GBM), FOSL1 promotes the transformation of GBM from PN subtype to MES subtype (Chen, Z.; Wang, S.; Li, HL; Luo, H.; Wu, X.; Lu, J.; Wang, HW; Chen, Y.; Chen, D.; Wu, WT; Zhang, S.; He, Q.; Lu, D.; Liu, N.; You, Y.; Wu, W.; Wang, H., FOSL1 promotes proneural-to-mesenchymal transition of glioblastoma stem cells via UBC9 / CYLD / NF-kappaB axis. Mol Ther 2022, 30(7), 2568-2583.), leading to radioresistance and higher invasiveness.FOSL1 is highly expressed in tissues of patients with head and neck squamous cell carcinoma and promotes tumor metastasis and occurrence through super-enhancer-driven transcription program (Zhang, M.; Hoyle, RG; Ma, Z.; Sun, B.; Cai, W.; Cai, H.; Xie, N.; Zhang, Y.; Hou, J.; Liu, X.; Chen, D.; Kellogg, GE; Harada, H.; Sun, Y.; Wang, C.; Li, J., FOSL1 promotes metastasis of head and neck squamous cell carcinoma through super-enhancer-driven transcription program. Mol Ther 2021, 29(8), 2583-2600.). AP-1 inhibitors can effectively inhibit tumor proliferation, but its mechanism of action in osimertinib resistance still needs to be further studied and reported. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide a composite drug for treating osimertinib-resistant non-small cell lung cancer. The present invention found that SR11302 can inhibit the proliferation of osimertinib-resistant cells and reduce the growth rate of mouse tumors.

[0005] The present invention provides a composite medicine for treating osimertinib-resistant non-small cell lung cancer, comprising osimertinib and SR11302.

[0006] Preferably, the dosage of osimertinib is 5-10 mg / kg / day, and the dosage of SR11302 is 25-35 mg / kg / day.

[0007] Preferably, the drug mass ratio of osimertinib to SR11302 is 4:(13-17).

[0008] The present invention also provides the use of SR11302 in preparing a reagent for inhibiting the proliferation of osimertinib-resistant non-small cell lung cancer cell lines.

[0009] Preferably, the osimertinib-resistant non-small cell lung cancer cell line is HCC827 / OR or PC-9 / OR cell line.

[0010] Preferably, the SR11302 is used at a concentration of 15 to 35 μM.

[0011] Compared with the prior art, the present invention has the following beneficial effects: the present invention provides a composite drug for treating osimertinib-resistant non-small cell lung cancer, osimertinib combined with SR11302 inhibits the proliferation of resistant cells, SR11302 can significantly reduce the resistance of osimertinib-resistant cells, making the cells IC 50 The combined use of SR1302 and osimertinib decreased the expression of ERBB2, ERBB3, p-AKT, and p-ERK1 / 2; in mouse experiments, the combined use of osimertinib and SR11302 significantly inhibited tumor growth. This suggests that combined therapy may be an effective strategy to overcome osimertinib resistance. By targeting multiple signaling pathways at the same time, the possibility of tumor cells escaping treatment can be reduced, thereby improving the therapeutic effect; it can provide more effective treatment options for NSCLC patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 is the IC of HCC827 and HCC827 / OR determined by CCK-8 assay 50 value;

[0013] Figure 2 IC for PC-9 and PC-9 / OR 50 value;

[0014] Figure 3 The cloning ability of sensitive cells and resistant cells with and without drug addition;

[0015] Figure 4 For Figure 3 The results are statistically shown in Figure 2. The left side shows HCC827 and HCC817 / OR, and the right side shows PC-9 and PC-9 / OR. Statistical analysis was performed using two-way ANOVA. ** indicates p<0.01.

[0016] Figure 5 To detect the DNA replication ability of sensitive cells and resistant cells with and without drug addition through EdU labeling experiment;

[0017] Figure 6 For Figure 5 Results: HCC827 and HCC817 / OR on the left, PC-9 and PC-9 / OR on the right; statistical analysis was performed by two-way ANOVA, *** indicates p<0.001;

[0018] Figure 7 Comparison of the invasion ability of sensitive cells and resistant cells with and without drug addition;

[0019] Figure 8For Figure 7 Results: HCC827 and HCC817 / OR on the left, PC-9 and PC-9 / OR on the right; statistical analysis was performed by two-way ANOVA, *** indicates p<0.001;

[0020] Fig. 9 Comparison of the migration ability of sensitive cells and resistant cells with and without drug addition;

[0021] Fig.10 For Fig. 9 Results: HCC827 and HCC817 / OR on the left, PC-9 and PC-9 / OR on the right, statistical analysis by two-way ANOVA, ** indicates p<0.01, *** indicates p<0.001;

[0022] Fig.11 To detect the effects of SR11302 alone and the combination of osimertinib and SR11302 on the proliferation of HCC827 / OR by CCK-8;

[0023] Fig.12 Determination of osimertinib IC for HCC827 / OR in combination with 20 μM or 30 μM SR11302 50 value;

[0024] Fig.13 To detect the effects of SR11302 alone and the combination of osimertinib and SR11302 on PC-9 / OR proliferation using CCK-8;

[0025] Fig.14 Western blot was used to detect the differences in ERBB2, ERBB3, p-AKT and p-ERK1 / 2 protein levels between SR11302 alone and combined with osimertinib;

[0026] Fig.15 The photos of tumor sizes in different treatment groups in Example 3;

[0027] Fig.16 The tumor growth curves of different treatment groups in Example 3;

[0028] Fig.17 These are the WB detection results of tumors in different treatment groups in Example 3. DETAILED DESCRIPTION

[0029] The present invention provides a composite medicine for treating osimertinib-resistant non-small cell lung cancer, comprising osimertinib and SR11302.

[0030] In the present invention, the dosage of osimertinib is preferably 5-10 mg / kg / day, more preferably 6-9 mg / kg / day, and further preferably 7-8 mg / kg / day; the dosage of SR11302 is preferably 25-35 mg / kg / day, more preferably 28-32 mg / kg / day, and further preferably 30 mg / kg / day.

[0031] The present invention provides the use of SR11302 in preparing a reagent for inhibiting the proliferation of osimertinib-resistant cells.

[0032] In the present invention, the osimertinib-resistant non-small cell lung cancer cell line is HCC827 cell line or PC-9 cell line; the SR11302 is used at a concentration of 15 to 35 μM, more preferably 20 to 30 μM; the SR11302 is preferably used in combination with osimertinib, and the osimertinib is preferably used at a concentration of 450 to 550 nM, more preferably 500 nM.

[0033] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0034] Example 1

[0035] Osimertinib was used to induce the non-small cell lung cancer cell lines HCC827 and PC-9 to establish stable drug-resistant cell lines HCC827 / OR and PC-9 / OR.

[0036] Human non-small cell lung cancer cell lines HCC827 and PC-9 were purchased from the Cell Bank of the Chinese Academy of Sciences.

[0037] Cell recovery: Take one frozen cell from the liquid nitrogen tank, quickly thaw it in a 37°C water bath, transfer it to a centrifuge tube, add 3 ml of RPMI-1640 medium, blow and mix the medium evenly, and centrifuge at 1000 rpm for 5 minutes. Discard the supernatant, resuspend the cells in 1 mL of RPMI-1640 medium, transfer them to a culture dish, add RPMI-1640 medium, mix well, and place in an incubator. Incubate the cells at 5% CO 2 , cultured in a 37°C incubator, the culture medium contained 10% FBS and 1% penicillin / streptomycin. HCC827 cells and PC-9 cells were cultured in culture medium containing a certain concentration of osimertinib, HCC827 and PC-9 were cultured at 0.2μM and 0.06μM, respectively, which are the IC values ​​of sensitive cells. 50The concentration of osimertinib was gradually increased. When the cells could proliferate normally at the initial concentration, the concentration was increased until the cell lines were established when the cells could proliferate stably at 500 nm and 100 nm, respectively. The cell lines were named HCC827 / OR and PC-9 / OR. Thereafter, the resistant cells were maintained in a culture medium containing osimertinib.

[0038] When the cell confluence is 80%-90%, discard the culture medium and wash twice with PBS, add 1mL 0.25% trypsin to a 10cm culture dish for digestion for 1 minute, discard the trypsin, add 1mL culture medium to resuspend the cells, take 10μL in a counting plate, count with an automatic cell counter, and plate at a concentration of 5000 cells / 100μL per well.

[0039] To verify the establishment of the drug-resistant cell model, CCK-8 was used to determine the IC of HCC827, PC-9 sensitive cells and drug-resistant cells. 50 Values ​​were used and clone formation assay, EdU proliferation assay, invasion assay and scratch assay were performed simultaneously.

[0040] CCK-8 proliferation assay

[0041] When the cell confluence is 80%-90%, discard the culture medium and wash twice with PBS, add 1mL 0.25% trypsin to a 10cm culture dish for digestion for 1 minute, discard the trypsin, add 1mL culture medium to resuspend the cells, take 10μL in a counting plate, count with an automatic cell counter, and plate at a concentration of 5000 cells / 100μL per well.

[0042] IC 50 Determination: Take a 96-well plate, plate 5000 cells per well in the 96-well plate, and place it in an incubator overnight. Incubate with 0, 0.01, 0.1, 1, 5, and 10 μM osimertinib for 72 hours. Discard the culture medium, add 100 μL CCK8 reaction solution and incubate in the incubator for 2 hours. Use an enzyme marker to measure the OD value at a wavelength of 450 nm, and set up 3 replicates for each concentration.

[0043] Colony formation assay

[0044] When the cell confluence is 80%-90%, discard the culture medium and wash twice with PBS. Add 1mL0.25% trypsin to a 10cm culture dish for digestion for 1 minute, discard the trypsin, add 1mL culture medium to resuspend the cells, take 10μL in a counting plate, and count with an automatic cell counter. After cell counting, 700 cells per well are evenly spread in a 6-well plate, 0 or 500nM osimertinib is added to the culture medium for incubation, and the medium is changed every 3 days for a total of 10 days. After 10 days, discard the culture medium, wash twice with PBS, fix with 4% paraformaldehyde for 20min, wash once with PBS, stain with 1% crystal violet for 10min, gently wash with water to remove crystal violet, dry and take pictures with a camera, and count the number of clones in the pictures using imageJ. Parallel determination 3 times.

[0045] EdU proliferation assay

[0046] 1) When the cell confluence is 80%-90%, discard the culture medium and wash twice with PBS. Add 1 mL of 0.25% trypsin to a 10 cm culture dish for digestion for 1 minute. Discard the trypsin, add 1 mL of culture medium to resuspend the cells, take 10 μL in a counting plate, and count using an automatic cell counter.

[0047] 2) After cell counting, 5000 cells per well were plated in a 96-well plate and placed in an incubator overnight. 0 or 500 nM osimertinib was added to the culture medium and incubated for 48 h. Each concentration was replicated in triplicate.

[0048] 3) Dilute the EdU solution with complete culture medium at a ratio of 1000:1. After discarding the culture medium in the 96-well plate, add 100 μL of the EdU dilution solution to each well and incubate for 2 h. Then wash twice with PBS, each time for 5 min.

[0049] 4) Add 50 μL of 4% paraformaldehyde to each well to fix the cells for 30 min, and discard the fixative; add 50 μL of 2 mg / ml glycine to neutralize excess aldehyde groups to ensure the staining system, incubate on a shaker for 5 min, discard the glycine solution, add 100 μL PBS to wash for 5 min, and discard the PBS.

[0050] 5) Add 100 μL of 0.5% Triton X-100 permeabilization solution and incubate on a shaker for 10 min, then wash with PBS for 5 min.

[0051] 6) Add 100 μL of 1× Apollo staining solution to each well, incubate on a shaker in the dark for 30 min, and discard the staining solution.

[0052] 7) Wash the cells twice on a shaker with 0.5% Triton X-100 permeabilization solution, each time for 10 min, and discard the permeabilization solution.

[0053] 8) Prepare 1×Hoechst33342 reaction solution and add 100 μL to each well (deionized water: reagent F = 100: 1), incubate on a shaker in the dark for 30 min, wash twice with PBS, add 100 μL PBS to each well and store in the dark.

[0054] 9) Fluorescence microscopy was used to take pictures at 550nm and 350nm. Image J was used for statistics.

[0055] Invasion assay

[0056] 1) Laying Matrigel: The day before the experiment, take out the Matrigel from -20℃ and place it at 4℃ overnight. The next day, pre-cool the pipette tip in the refrigerator, mix the Matrigel and basal culture medium in a 1:1 ratio, take 60μL and place it in the transwell chamber, and place it in the incubator to dry for 2h.

[0057] 2) When the cell confluence is 80%-90%, discard the culture medium and wash twice with PBS, add 1 mL of 0.25% trypsin to a 10 cm culture dish for digestion for 1 minute, discard the trypsin, add 1 mL of culture medium to resuspend the cells, take 10 μL in a counting plate, and count using an automatic cell counter.

[0058] 3) After counting the cells, take 2×10 5 The cells were supplemented with basal culture medium to 100 μL and placed in the small chamber, and 500 μL of complete culture medium was added to the lower chamber and incubated in the incubator for 36 h.

[0059] 4) Fixation of the chamber: discard the culture medium in the chamber, add 500 μL of PBS to the lower chamber, and wash three times, each time for 5 minutes.

[0060] 5) Discard PBS, add 500 μL methanol to the lower chamber for fixation at room temperature for 30 min, and wash with PBS three times.

[0061] 6) Staining: Weigh 0.5 g of crystal violet and dissolve it in 50 ml of methanol to obtain a 1% crystal violet solution. Filter the obtained solution with a 0.45 μm filter. Take 500 μL and place it in the lower chamber, stain at 37°C for 30 minutes; wash twice with PBS.

[0062] 7) Photographing: Use a cotton swab to gently wipe the inside of the chamber, wipe it clean and dry it, and then use it to photograph under a 20× microscope. Take 5 viewing angles for each chamber. Use ImageJ to count the number of invasions.

[0063] Scratch test

[0064] 1) When the cell confluence is 80%-90%, discard the culture medium and wash twice with PBS. Add 1 mL of 0.25% trypsin to a 10 cm culture dish for digestion for 1 minute. Discard the trypsin, add 1 mL of culture medium to resuspend the cells, take 10 μL in a counting plate, and count using an automatic cell counter.

[0065] 2) After counting the cells, take 2×10 5 Add 80 μL of each cell to each side of the scratch insert and place in an incubator overnight.

[0066] 3) Observe under a microscope the next day. When the cells are almost full, remove the plug, gently wash off a small amount of floating cells with PBS, add 1 mL of culture medium containing 0.05% FBS, and take a picture under a 10× microscope. This is taken as the area at 0 h.

[0067] 4) After 24 hours, take pictures again under a 10× microscope. Use ImageJ to analyze the scar area and calculate the healing rate. Healing rate = (0 hour area - 12 hour area) / 0 hour area * 100%.

[0068] Experimental results:

[0069] IC 50 The results of the determination of the value are as follows Figure 1 and 2 As shown, the IC of HCC827 and HCC827 / OR 50 values, respectively, are 0.1597μM and 5.454μM ( Figure 1 ), the drug resistance index was 34.15. The IC 50 values, respectively, are 0.0177μM and 1.508μM ( Figure 2 ), the drug resistance index was 85.19.

[0070] The results of the clone formation experiment showed that in the absence of osimertinib, there was no significant difference in the clone formation ability between HCC827 and HCC827 / OR, and the same was true for PC-9 and PC-9 / OR. However, under the action of 500nM osimertinib, the clone formation ability of sensitive cells HCC827 was significantly weaker than that of resistant cells HCC827 / OR ( Figure 3 ), under the action of 100nM osimertinib, the clone formation ability of sensitive cells PC-9 was significantly weaker than that of resistant cells PC-9 / OR( Figure 4 ). EDU proliferation experiments showed that after drug addition, the DNA replication ability of drug-resistant cells was significantly stronger than that of sensitive cells ( Figure 5 and Figure 6 The results of invasion and scratch experiments showed that the invasion of drug-resistant cells ( Figure 7 and Figure 8 ) and migration capabilities ( Fig. 9 and Fig.10 ) were also stronger than sensitive cells. Therefore, osimertinib-resistant cell lines were successfully established.

[0071] Example 2

[0072] Effect of SR11302 on the proliferation of osimertinib-resistant cells

[0073] Take 5 96-well plates, and place 5000 drug-resistant HCC827 / OR cells in each well in the 96-well plate and place it in the incubator overnight. Set up six groups: no drug, osimertinib 500nM alone, 20μM SR11302 alone, 30μM SR11302 alone, osimertinib 500nM and 20μM SR11302 in combination, and osimertinib 500nM and 30μM SR11302 in combination. The above 6 groups are set on each plate, and each group is repeated 3 wells; the OD values ​​of the 5 plates are measured at 0, 24h, 48h, 72h and 96h, respectively. Before the measurement, discard the culture medium, add 100μL CCK8 reaction solution and incubate in the incubator for 2h, and use an enzyme reader to measure the OD value at a wavelength of 450nm, and set 3 replicates for each concentration.

[0074] For drug-resistant cells PC-9 / OR, the concentration of osimertinib alone was 100 nM, and the concentration of osimertinib combined was 100 nM; other conditions were the same as above.

[0075] IC 50 The method for determining the value is the same as in Example 1.

[0076] Western blot experiment (HCC827 / OR drug-resistant cell line):

[0077] 1) Cell counting and plating: When the cell confluence is 80%-90%, discard the culture medium and wash twice with PBS. Add 1 mL of 0.25% trypsin to a 10 cm culture dish for digestion for 1 minute, discard the trypsin, add 1 mL of culture medium to resuspend the cells, take 10 μL in a counting plate, and count with an automatic cell counter. After cell counting, take 2×10 5 The cells were plated in 6-well plates, 2 mL of complete medium was added, and cultured overnight.

[0078] 2) Cell stimulation: On the second day, according to experimental needs, 500 nM osimertinib, 20 μM SR11302, or a combination of osimertinib and SR11302 were added and incubated in an incubator for 72 h.

[0079] 3) Cell lysis: After 72 hours, the culture medium in the 6-well plate was removed from the incubator, and the floating cells were washed twice with PBS to remove them. 200 μL of cell lysis solution and 2 μL of phosphatase inhibitor were added to each well, and the cells were lysed on a shaker on ice for 30 minutes.

[0080] 4) Precool the centrifuge at 4℃ and transfer the cell lysate in the six-well plate to a 1.5mL EP tube. Centrifuge at 4℃, 12000rpm, for 10min. Transfer the supernatant to a new EP tube. Store on ice until use.

[0081] 5) Take 10 μL and mix with 90 μL PBS to use as diluent for concentration determination.

[0082] 6) Protein concentration determination (Biyuntian kit (P0010)): Prepare the reaction solution at a ratio of BCA reagent A: BCA reagent B = 50:1, add 200 μL of the reaction solution and 20 μL of the lysed cell diluent to a 96-well plate. Incubate in a metal bath at 37°C for 30 minutes, protected from light. Measure the OD value at 562 nm using an ELISA reader.

[0083] 7) Draw a standard curve and calculate the protein sample concentration. The standard sample configuration is shown in Table 1:

[0084] Table 1 Standard product configuration

[0085]

[0086]

[0087] 8) Sample preparation: After calculating the protein concentration, take the cell lysate, add 20 μL of 5× loading buffer, and then dilute to 100 μL with PBS to obtain a sample with a final concentration of 1 μg / μL. Boil the sample at 100°C for 10 min and set aside.

[0088] 9) Glue preparation: Clean the glass plate and fix it, and use deionized water to check for leaks. Prepare 10% lower layer glue as shown in Table 2.

[0089] Table 2 Preparation of lower layer glue

[0090] 10% glue (ml) 5 10 15 20 30 50 Deionized water 1.3 2.7 4.0 5.3 8.0 13.3 30% AcrBic (29:1) 1.7 3.3 5 6.7 10.0 16.7 1MTris(PH8.8) 1.9 3.8 5.7 7.6 11.4 19.0 10% SDS 0.05 0.1 0.15 0.2 0.3 0.5 10% APS 0.05 0.1 0.15 0.2 0.3 0.5 TEMED 0.002 0.004 0.006 0.008 0.012 0.02

[0091] Add all ingredients except TEMED to a centrifuge tube, make sure the glass plate does not leak, then pour out the deionized water and absorb as much water as possible. Finally, add TEMED to the centrifuge tube and vortex to mix well. Immediately take 7mL and add it to the glass plate, and seal it with anhydrous ethanol. Solidify after 40 minutes. Then prepare 5% of the top layer glue, as shown in Table 3.

[0092] Table 3 Preparation of upper layer glue

[0093] 5% glue (ml) 2 3 4 6 8 10 Deionized water 1.4 2.1 2.7 4.1 5.5 6.8 30% AcrBic (29:1) 0.33 0.5 0.67 1.0 1.3 1.7 1MTris, PH6.8 0.25 0.38 0.5 0.75 1.0 1.25 10% SDS 0.02 0.03 0.04 0.06 0.08 0.1 10% APS 0.02 0.03 0.04 0.06 0.08 0.1 TEMED 0.002 0.003 0.004 0.006 0.008 0.01

[0094] After the lower layer of glue solidifies, pour out the anhydrous ethanol used for liquid sealing and let it dry. Add 3ml of 5% upper layer glue and quickly insert the 15-hole comb vertically. When inserting the comb, remove all bubbles to prevent bubbles from solidifying in the glue. Let it stand for 15 minutes and solidify for use.

[0095] 10) Electrophoresis: Pour electrophoresis liquid into the electrophoresis tank, fix the two sides of the pair of gel plates, then pour electrophoresis liquid into the gel plates, then slowly pull out the comb vertically, and add samples according to the pre-arranged loading order, first add the marker, then add the protein sample. (Note: when fixing the gel plates, the small glass plate faces inwards) Connect the electrophoresis tank and the electrophoresis instrument, positive against positive, negative against negative (i.e. red against red, black against black), adjust the voltage to 80V, and after about 60 minutes, the marker is completely separated, and the voltage is adjusted to 120V to continue electrophoresis for about 60 minutes.

[0096] 11) Transfer: Pre-cool 1× transfer solution in the refrigerator, cut the PVDF membrane to a size of about 8.5x4.5cm, and activate it in methanol for 1 minute before use. Negative electrode (black side) - positive electrode (white side): fiber pad, filter paper, gel, membrane, filter paper, fiber pad (membrane positive, gel negative). Cover the PVDF membrane on the SDS-PAGE gel, and make sure there are no bubbles between the PVDF membrane and the SDS-PAGE gel, otherwise it will affect the transfer. Clamp the transfer clamp and put it into the red transfer tank, fill the electrophoresis tank with pre-cooled transfer solution, and add an ice cube. Pay attention to the black side of the transfer clamp and the black side of the tank.

[0097] 12) Blocking: Prepare 5% skimmed milk powder with TBST and incubate the PDVF membrane on a shaker for 90 minutes.

[0098] 13) Primary antibody incubation: Wash once with TBST, cut the target band and place it in the primary antibody diluent, and incubate overnight in a shaker in a cold storage. (The diluent is a 5% BSA TBST solution filtered with a 0.45μm filter)

[0099] 14) Secondary antibody incubation: Wash three times with TBST, 10 minutes each time. Select mouse or rabbit secondary antibody according to the primary antibody, and incubate for 1 hour with slow shaking. (Mouse antibody 1:10000, rabbit antibody 1:5000, solvent is 1×TBST)

[0100] 15) Wash three times with TBST with rapid shaking, 10 min each time.

[0101] 16) Luminescent reagents A and B (Affinity TM ECL kit (femtogram)-#KF8003) was configured in 1:1 and exposed.

[0102] The results are as follows Figures 11 to 14 As shown in the figure, 20 μM SR11302 alone could not inhibit the proliferation of drug-resistant cells, but combined with 500 nM osimertinib, cell proliferation was significantly reduced ( Fig.11 ). 20μM and 30μM SR11302 were used in combination, respectively, and the IC 50 The results showed that SR11302 significantly reduced drug resistance ( Fig.12 ), Western blot experiments showed that the combined use of SR1302 and osimertinib decreased the expression of ERBB2, ERBB3, p-AKT and p-ERK1 / 2 ( Fig.14 ). Moreover, this combination also inhibits cell proliferation in PC-9 / OR cells ( Fig.13 ). This once again verified that SR11302 and osimertinib combined to inhibit cell proliferation.

[0103] Example 3

[0104] A cell line transplant tumor (CDX) model was constructed, and the drug-resistant cells HCC827 / OR prepared in Example 1 were inoculated into NCG immunodeficient mice. The growth rate and size of the tumors in the drug-treated group and the control group were compared to verify the synergistic effect of osimertinib and SR11302.

[0105] The mice used in the experiment were of SPF grade, and NCG immunodeficient mice were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.

[0106] After acclimating to the environment for 1 week, 8×10 6 The drug-resistant HCC827 / OR cells were resuspended in 50 μL pre-cooled PBS, and then 50 μL of matrix gel was added and blown evenly (operated on ice). The drug-resistant HCC827 / OR cells were inoculated subcutaneously in the right armpit of the mouse. Two months after inoculation, the mouse tumor reached 100 mm 3 , the mice were evenly divided into four groups: normal saline, osimertinib alone (8 mg / kg / day), SR11302 alone (30 mg / kg / day), continuous osimertinib (8 mg / kg / day) and SR11302 (30 mg / kg / day). Drug treatment: After grinding osimertinib, weigh an appropriate amount and add normal saline to dissolve it with ultrasonic aid. Gavage 200 μL per day at a dose of 5 mg / kg. Weigh an appropriate amount of SR11302 and dissolve it in a small amount of DMSO, then add 1% sodium carboxymethyl cellulose in normal saline solution, and dissolve it with ultrasonic aid to obtain a suspension. At a dose of 30 mg / kg, inject 200 μL intraperitoneally every day, with the final concentration of DMSO being less than 2%.

[0107] Body weight and tumor size were measured daily.

[0108] The results are as follows Figures 15 to 17As shown, compared with the normal saline group, the SR11302 group alone significantly slowed down tumor growth, the osimertinib group alone also significantly inhibited tumor growth, and the combination of osimertinib and SR11302 significantly reduced tumor volume; compared with the normal saline group, osimertinib and SR11302 alone significantly decreased the protein expression of ERBB2, ERBB3, p-AKT and p-ERK1 / 2, and the combined use of osimertinib and SR11302 resulted in a more significant protein downregulation level than osimertinib or SR11302 alone.

[0109] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A pharmaceutical composition for treating osimertinib-resistant non-small cell lung cancer, characterized in that: Including osimertinib and SR11302; the drug mass ratio of osimertinib to SR11302 is 4:(13~17).

2. The pharmaceutical composition according to claim 1, characterized in that The dosage of osimertinib is 5~10mg / kg / day, and the dosage of SR11302 is 25~35mg / kg / day.

3. Use of SR11302 in combination with osimertinib in the preparation of an agent for inhibiting the proliferation of osimertinib-resistant non-small cell lung cancer cell lines, characterized in that: The concentration of SR11302 is 15-35 μM; the concentration of osimertinib is 450-550 nM.

4. The use according to claim 3, characterized in that: The osimertinib-resistant non-small cell lung cancer cell line is HCC827 / OR or PC-9 / OR.

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