Pharmaceutical composition for treating non-small cell lung cancer

Abstract

The invention provides a pharmaceutical composition for treating non-small cell lung cancer, and belongs to the technical field of biological medicine, osimertinib is combined with SR11302 to inhibit proliferation of drug-resistant cells and reduce drug resistance of the cells, so that IC50 of the cells to osimertinib is reduced; a Westernblot experiment shows that the expression of the ERBB2, the expression of the ERBB3, the expression of the p-AKT and the expression of the p-ERK1 / 2 are reduced by jointly using the SR1302 and the osimertinib; in mouse experiments, the combined use of osimertinib and SR11302 significantly inhibits the growth of tumors. The result shows that the combined treatment may be an effective strategy for overcoming the drug resistance of osimertinib; and a more effective treatment scheme can be provided for NSCLC patients.

Description

[0001] This invention is a divisional application of the application with the filing date of June 25, 2024, application number 202410827063.8, and invention title "A Composite Drug for Treating Osimertinib-Resistant Non-Small Cell Lung Cancer". Technical Field

[0002] This invention belongs to the field of biomedical technology, and particularly relates to a pharmaceutical composition for treating non-small cell lung cancer. Background Art

[0003] 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), and 85% of patients are non-small cell lung cancer (NSCLC). Through genetic testing, it is found that more than 30% of patients have EGFR mutations. Therefore, the first- and second-generation epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs): gefitinib and erlotinib, etc., are used as therapeutic drugs for the treatment of non-small cell lung cancer. However, after 10-15 months of medication, patients develop acquired drug resistance. The reasons for drug resistance include MET amplification, HER2 amplification, EGFR re-mutation, etc., and 49% of them have the T790M mutation (Sequist, L.V.; Waltman, B.A.; Dias-Santagata, D.; Digumarthy, S.; Turke, A.B.; Fidias, P.; Bergethon, K.; Shaw, A.T.; Gettinger, S.; Cosper, A.K.; Akhavanfard, S.; Heist, R.S.; Temel, J.; Christensen, J.G.; Wain, J.C.; Lynch, T.J.; Vernovsky, K.; Mark, E.J.; Lanuti, M.; Iafrate, A.J.; Mino-Kenudson, M.; Engelman, J.A., Genotypic and Histological Evolution of Lung Cancers Acquiring Resistance to EGFR Inhibitors. Science Translational Medicine 2011, 3(75), 75ra26-75ra26.), and the third-generation targeted drug osimertinib has emerged for this mutation.Compared with traditional platinum-based chemotherapy drugs or first- and second-generation targeted therapies, osimertinib significantly improves the survival of patients (Mok, T.S.; Wu, Y.-L.; Ahn, M.-J.; Garassino, M.C.; Kim, H.R.; Ramalingam, S.S.; Shepherd, F.A.; He, Y.; Akamatsu, H.; Theelen, W.S.M.E.; Lee, C.K.; Sebastian, M.; Templeton, A.; Mann, H.; Marotti, M.; Ghiorghiu, S.; Papadimitrakopoulou, V.A., 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, K.H.; Dechaphunkul, A.; Imamura, F.; Nogami, N.; Kurata, T.; Okamoto, I.; Zhou, C.; Cho, B.C.; Cheng, Y.; Cho, E.K.; Voon, P.J.; Planchard, D.; Su, W.-C.; Gray, J.E.; Lee, S.-M.; Hodge, R.; Marotti, M.; Rukazenkov, Y.; Ramalingam, S.S., Osimertinib in Untreated EGFR-Mutated Advanced Non–Small-Cell Lung Cancer. New England Journal of Medicine 2017, 378(2), 113-125.), which are 10.1 months vs 4.4 months and 18.9 months vs 10.2 months respectively. However, the resistance of osimertinib still cannot be avoided. The elucidated resistance mechanisms of osimertinib include EGFR re-mutation, EGFR-independent gene mutations, amplifications, and fusions, etc. Currently, 50-60% of the resistance mechanisms remain unelucidated (Schmid, S.; Li, J.J.N.; Leighl, N.B., Mechanisms of osimertinib resistance and emerging treatment options. Lung Cancer 2020, 147, 123-129.).

[0004] FOSL1 (FOSL-like antigen 1, alias Fra1) is one of the components of the transcription complex AP-1. It has been reported that FOSL1 is highly expressed in colorectal cancer (Diesch, J.; Sanij, E.; Gilan, O.; Love, C.; Tran, H.; Fleming, N.I.; Ellul, J.; Amalia, M.; Haviv, I.; Pearson, R.B.; Tulchinsky, E.; Mariadason, J.M.; Sieber, O.M.; Hannan, R.D.; Dhillon, A.S., 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 the PN subtype to the MES subtype (Chen, Z.; Wang, S.; Li, H.L.; Luo, H.; Wu, X.; Lu, J.; Wang, H.W.; Chen, Y.; Chen, D.; Wu, W.T.; 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 radiotherapy resistance and higher invasiveness.FOSL1 is highly expressed in the tissues of patients with head and neck squamous cell carcinoma, and promotes tumor metastasis and occurrence through a super-enhancer-driven transcription program (Zhang, M.; Hoyle, R.G.; Ma, Z.; Sun, B.; Cai, W.; Cai, H.; Xie, N.; Zhang, Y.; Hou, J.; Liu, X.; Chen, D.; Kellogg, G.E.; 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.). Inhibitors of AP-1 can effectively inhibit tumor proliferation, but the mechanism of action in osimertinib resistance still needs to be further studied and reported. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a compound drug for treating osimertinib-resistant non-small cell lung cancer. The present invention has found that SR11302 can inhibit the proliferation of osimertinib-resistant cells and reduce the tumor growth rate in mice.

[0006] The present invention provides a compound drug for treating osimertinib-resistant non-small cell lung cancer, comprising osimertinib and SR11302.

[0007] Preferably, the dosage of osimertinib is 5-10 mg / kg / day, and the dosage of SR11302 is 25-35 mg / kg / day.

[0008] Preferably, the mass ratio of osimertinib to SR11302 for administration is 4:(13-17).

[0009] The present invention also provides the use of SR11302 in the preparation of a reagent for inhibiting the proliferation of osimertinib-resistant non-small cell lung cancer cell lines.

[0010] Preferably, the osimertinib-resistant non-small cell lung cancer cell line is the HCC827 / OR or PC-9 / OR cell line.

[0011] Preferably, the use concentration of SR11302 is 15-35 μM.

[0012] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a compound 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 drug resistance of osimertinib-resistant cells, making the IC 50 of the cells decrease for osimertinib; As shown by Western blot experiments, the combined use of SR1302 and osimertinib decreases the expression of ERBB2, ERBB3, p-AKT, and p-ERK1 / 2; In mouse experiments, the combined use of osimertinib and SR11302 significantly inhibits tumor growth. This indicates that combination therapy may be an effective strategy to overcome osimertinib resistance. By targeting multiple signaling pathways simultaneously, the possibility of tumor cells evading treatment can be reduced, thereby improving the treatment effect; It can provide a more effective treatment plan for NSCLC patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 IC 50 values of HCC827 and HCC827 / OR measured by CCK-8 assay;

[0014] Figure 2 IC 50 values of PC-9 and PC-9 / OR;

[0015] Figure 3 Colony formation ability of sensitive cells and resistant cells with and without drug treatment;

[0016] Figure 4 For Figure 3 result statistics, 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 < 0.01.

[0017] Figure 5 DNA replication ability of sensitive cells and resistant cells with and without drug treatment detected by EDU labeling assay;

[0018] Figure 6 For Figure 5 result statistics, 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;

[0019] Figure 7 Comparison of invasion ability of sensitive cells and resistant cells with and without drug treatment;

[0020] Figure 8For the Figure 7 result statistics, HCC827 and HCC817 / OR are on the left, and PC-9 and PC-9 / OR are on the right; statistical analysis was performed by two-way ANOVA, *** indicates p < 0.001;

[0021] Figure 9 It is a comparison of the migration ability of sensitive cells and drug-resistant cells with and without drug addition;

[0022] Figure 10 For the Figure 9 result statistics, HCC827 and HCC817 / OR are on the left, and PC-9 and PC-9 / OR are on the right. Statistical analysis was performed by two-way ANOVA, ** indicates p < 0.01, and *** indicates p < 0.001;

[0023] Figure 11 It is to detect the effect of SR11302 alone and the combination of osimertinib and SR11302 on the proliferation of HCC827 / OR by CCK-8;

[0024] Figure 12 It is to determine the osimertinib IC 50 value of HCC827 / OR by measuring with 20 μM or 30 μM SR11302;

[0025] Figure 13 It is to detect the effect of SR11302 alone and the combination of osimertinib and SR11302 on the proliferation of PC-9 / OR by CCK-8;

[0026] Figure 14 It is to detect the difference in the protein levels of ERBB2, ERBB3, p-AKT and p-ERK1 / 2 by Western blot for SR11302 alone and in combination with osimertinib;

[0027] Figure 15 It is a photo of the tumor size of different treatment groups in Example 3;

[0028] Figure 16 It is the tumor growth curve of different treatment groups in Example 3;

[0029] Figure 17 It is the tumor WB test result of different treatment groups in Example 3. Detailed implementation mode

[0030] The present invention provides a composite drug for treating osimertinib-resistant non-small cell lung cancer, comprising osimertinib and SR11302.

[0031] In the present invention, the dosage of osimertinib is preferably 5-10 mg / kg / day, more preferably 6-9 mg / kg / day, and even more 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 even more preferably 30 mg / kg / day.

[0032] The present invention provides the use of SR11302 in the preparation of a reagent for inhibiting the proliferation of osimertinib-resistant cells.

[0033] In the present invention, the osimertinib-resistant non-small cell lung cancer cell line is the HCC827 cell line or the PC-9 cell line; the use concentration of SR11302 is 15-35 μM, more preferably 20-30 μM; SR11302 is preferably used in combination with osimertinib, and the use concentration of osimertinib is preferably 450-550 nM, more preferably 500 nM.

[0034] 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.

[0035] Example 1

[0036] Use osimertinib to induce the non-small cell lung cancer cell lines HCC827 and PC-9 to establish stable resistant cell lines HCC827 / OR and PC-9 / OR.

[0037] The human non-small cell lung cancer cell lines HCC827 and PC-9 were purchased from the Cell Bank of the Chinese Academy of Sciences.

[0038] Cell resuscitation: Take 1 cryopreserved cell from the liquid nitrogen tank, quickly place it in a 37°C water bath to melt, then transfer it to a centrifuge tube, add 3 ml of RPMI-1640 medium, blow it evenly, and centrifuge at 1000 rpm for 5 min. Discard the supernatant, resuspend the cells with 1 mL of RPMI-1640 medium, transfer them to a culture dish, add RPMI-1640 medium, mix well and place them in an incubator. The cells were cultured in a 5% CO 2 , 37°C constant temperature incubator, and the medium contained 10% FBS and 1% penicillin / streptomycin. The HCC827 cells and PC-9 cells were cultured in a medium containing a certain concentration of osimertinib. HCC827 and PC-9 were respectively cultured at 0.2 μM and 0.06 μM, that is, the IC of sensitive cells 50is the initial concentration. Gradually increase the concentration of osimertinib. After it can proliferate normally at the initial concentration, increase the concentration until drug-resistant cell lines are established when they can stably proliferate at 500 nM and 100 nM respectively, named HCC827 / OR and PC-9 / OR. Thereafter, maintain the drug-resistant cells in a medium containing osimertinib for culture.

[0039] When the cell confluence is 80%-90%, discard the medium and wash twice with PBS. Add 1 mL of 0.25% trypsin to a 10-cm culture dish and digest for 1 minute. Discard the trypsin, add 1 mL of medium to resuspend the cells, take 10 μL and place it on a counting plate, and count with an automatic cell counter. Plate at a concentration of 5000 cells / 100 μL per well.

[0040] To verify the establishment of the drug-resistant cell model, the IC 50 values of HCC827, PC-9 sensitive cells and drug-resistant cells were measured using CCK-8, and at the same time, colony formation assay, EDU proliferation assay, invasion assay and scratch assay were carried out.

[0041] CCK-8 proliferation assay

[0042] When the cell confluence is 80%-90%, discard the medium and wash twice with PBS. Add 1 mL of 0.25% trypsin to a 10-cm culture dish and digest for 1 minute. Discard the trypsin, add 1 mL of medium to resuspend the cells, take 10 μL and place it on a counting plate, and count with an automatic cell counter. Plate at a concentration of 5000 cells / 100 μL per well.

[0043] IC 50 determination: Take 1 96-well plate, plate 5000 cells per well in the 96-well plate, and place it in the incubator overnight. Incubate with osimertinib at 0, 0.01, 0.1, 1, 5, 10 μM for 72 h respectively. Discard the medium, add 100 μL of CCK8 reaction solution and incubate in the incubator for 2 h. Measure the OD value with an enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 450 nm, and set 3 replicates for each concentration.

[0044] Colony formation assay

[0045] When the cell confluence reaches 80%-90%, discard the culture medium and wash the cells twice with PBS. Add 1 mL of 0.25% trypsin to a 10-cm culture dish and digest for 1 minute. Discard the trypsin, add 1 mL of culture medium to resuspend the cells, take 10 μL and place it on a counting plate, and count the cells using an automatic cell counter. After cell counting, evenly seed 700 cells per well in a 6-well plate, add 0 or 500 nM osimertinib to the culture medium and incubate. Change the medium every 3 days and culture for a total of 10 days. After 10 days, discard the culture medium, wash twice with PBS, fix with 4% paraformaldehyde for 20 min, wash once with PBS, stain with 1% crystal violet for 10 min, gently wash with water to remove the crystal violet, air dry and then take pictures with a camera, and use ImageJ to count the number of clones in the pictures. Perform parallel determinations 3 times.

[0046] EDU proliferation assay

[0047] 1) When the cell confluence reaches 80%-90%, discard the culture medium and wash the cells twice with PBS. Add 1 mL of 0.25% trypsin to a 10-cm culture dish and digest for 1 minute. Discard the trypsin, add 1 mL of culture medium to resuspend the cells, take 10 μL and place it on a counting plate, and count the cells using an automatic cell counter.

[0048] 2) After cell counting, seed 5000 cells per well in a 96-well plate and place it in an incubator overnight. Add 0 or 500 nM osimertinib to the culture medium and incubate for 48 h. There are 3 replicate wells for each concentration.

[0049] 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 diluted EdU solution to each well and incubate for 2 h, then wash twice with PBS, 5 min for each wash.

[0050] 4) Add 50 μL of 4% paraformaldehyde to each well to fix the cells for 30 min, discard the fixing solution; add 50 μL of 2 mg / ml glycine to neutralize the excess aldehyde groups to ensure the staining system, incubate on a shaker for 5 min, discard the glycine solution, add 100 μL of PBS and wash for 5 min, discard the PBS.

[0051] 5) Add 100 μL of 0.5% TritonX-100 permeabilization solution and incubate on a shaker for 10 min, then wash with PBS for 5 min.

[0052] 6) Add 100 μL of 1× Apollo staining solution to each well, incubate in the dark on a shaker for 30 min, and discard the staining solution.

[0053] 7) Then wash twice with 0.5% TritonX-100 permeabilization solution on a shaker, 10 min each time, and discard the permeabilization solution.

[0054] 8) Preparation of 1× Hoechst 33342 reaction solution: Add 100 μL to each well (deionized water: reagent F = 100:1), incubate in the dark on a shaker for 30 min, wash twice with PBS, and add 100 μL of PBS to each well for storage in the dark.

[0055] 9) Take pictures with a fluorescence microscope at 550 nm and 350 nm. Perform statistics using Image J.

[0056] Invasion assay

[0057] 1) Coating with Matrigel: Take out Matrigel from -20 °C and place it at 4 °C overnight one day before the experiment. The next day, pre-cool the pipette tips in the refrigerator, mix Matrigel and basal medium in a ratio of 1:1, take 60 μL and place it in the transwell chamber, and dry it in the incubator for 2 h.

[0058] 2) When the cell confluence reaches 80%-90%, discard the culture medium, wash twice with PBS, add 1 mL of 0.25% trypsin to a 10 cm culture dish and digest for 1 minute, discard the trypsin, add 1 mL of culture medium to resuspend the cells, take 10 μL and place it on a counting plate, and count the cells using an automatic cell counter.

[0059] 3) After cell counting, take 2×10 5 cells, supplement to 100 μL with basal medium, put them into the chamber, add 500 μL of complete medium to the lower chamber, and incubate in the incubator for 36 h.

[0060] 4) Fixation of the chamber: Discard the culture medium in the chamber, add 500 μL of PBS to the lower chamber, wash 3 times, 5 min each time.

[0061] 5) Discard the PBS, add 500 μL of methanol to the lower chamber and fix at room temperature for 30 min, wash 3 times with PBS.

[0062] 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 all the obtained solution through a 0.45 μm filter. Take 500 μL and place it in the lower chamber, stain at 37 °C for 30 min; wash twice with PBS.

[0063] 7) Photography: Gently wipe the inner side of the chamber with a cotton swab dipped in water, wipe clean and dry, then use it to take pictures under a 20× microscope, and take 5 views for each chamber. Use ImageJ to count the number of invaded cells.

[0064] Scratch assay

[0065] 1) When the cell confluence reaches 80%-90%, discard the culture medium, wash twice with PBS, add 1 mL of 0.25% trypsin to a 10 cm culture dish and digest for 1 minute, discard the trypsin, add 1 mL of culture medium to resuspend the cells, take 10 μL and place it on a counting plate, and count the cells using an automatic cell counter.

[0066] 2) After cell counting, take 2×10 5 cells and make up to 180 μL. Take 80 μL each and add to both sides of the scratch insert. Incubate overnight in the incubator.

[0067] 3) Observe under the microscope the next day. After the cells are roughly confluent, remove the insert, gently wash away a small amount of floating cells with PBS, add 1 mL of medium containing 0.05% FBS, and take a photo under a 10× microscope. This is the area at 0 h.

[0068] 4) Take a photo again under a 10× microscope after 24 h. Use ImageJ to statistically analyze the scratch area and calculate the healing rate. Healing rate = (area at 0 h - area at 12 h) / area at 0 h * 100%.

[0069] Experimental results:

[0070] IC 50 The measurement results of the Figure 1 and 2 values are shown as 50 follows. The IC Figure 1 values of HCC827 and HCC827 / OR are 0.1597 μM and 5.454 μM respectively ( 50 ), and the drug resistance index is 34.15. The IC Figure 2 values of PC-9 and PC-9 / OR are 0.0177 μM and 1.508 μM respectively (

[0071] ) and the drug resistance index is 85.19. The results of the colony formation assay showed that there was no significant difference in the colony formation ability between HCC827 and HCC827 / OR, and the same was true for PC-9 and PC-9 / OR, without the effect of osimertinib. However, under the action of 500 nM osimertinib, the colony formation ability of the sensitive cell HCC827 was significantly weaker than that of the drug-resistant cell HCC827 / OR ( Figure 3 ), and under the action of 100 nM osimertinib, the colony formation ability of the sensitive cell PC-9 was significantly weaker than that of the drug-resistant cell PC-9 / OR ( Figure 4 ). The EDU proliferation assay showed that after adding the drug, the DNA replication ability of the drug-resistant cells was also significantly stronger than that of the sensitive cells ( Figure 5 and Figure 6 ). The results of the invasion assay and the scratch assay showed that the invasion ( Figure 7 and Figure 8 ) and migration ability ( Figure 9 and Figure 10 ) of the drug-resistant cells were also stronger than those of the sensitive cells. Therefore, a drug-resistant cell line of osimertinib was successfully established.

[0072] Example 2

[0073] Effect of SR11302 on the proliferation of osimertinib-resistant cells

[0074] Take 5 96-well plates, seed 5000 resistant cells HCC827 / OR in each well of the 96-well plates, and place them in the incubator overnight. Set up six groups: without adding drugs, using osimertinib alone at 500 nM, using 20 μM SR11302 alone, using 30 μM SR11302 alone, combining osimertinib at 500 nM and 20 μM SR11302, and combining osimertinib at 500 nM and 30 μM SR11302. Set the above 6 groups on each plate, with 3 replicates for each group; measure the OD values of the 5 plates at 0, 24 h, 48 h, 72 h, and 96 h respectively. Discard the culture medium before measurement, add 100 μL of CCK8 reaction solution and incubate in the incubator for 2 h, then measure the OD value with an enzyme-labeling instrument at a wavelength of 450 nm, and set 3 replicates for each concentration.

[0075] For the resistant cells PC-9 / OR, the concentration of osimertinib used alone is 100 nM, and the concentration of osimertinib in combination is 100 nM; other conditions are the same as above.

[0076] IC 50 The measurement method of the value is the same as that in Example 1.

[0077] Western blot experiment (HCC827 / OR resistant cell line):

[0078] 1) Cell counting and plating: When the cell confluence is 80%-90%, discard the culture medium, wash twice with PBS, add 1 mL of 0.25% trypsin to a 10 cm culture dish and digest for 1 minute, discard the trypsin, add 1 mL of culture medium to resuspend the cells, take 10 μL and place it in a counting plate, and count with an automatic cell counter. After cell counting, take 2×10 5 cells and seed them in a 6-well plate, add 2 mL of complete culture medium, and culture overnight.

[0079] 2) Cell stimulation: The next day, according to the experimental needs, add 500 nM osimertinib alone, 20 μM SR11302, or combine osimertinib and SR11302, and incubate in the incubator for 72 h.

[0080] 3) Cell lysis: After 72 h, take out from the incubator, discard the culture medium in the 6-well plate, wash twice with PBS to remove floating cells, add 200 μL of cell lysis buffer and 2 μL of phosphatase inhibitor to each well, and lyse on an ice shaker for 30 min.

[0081] 4) Pre-cool the centrifuge at 4 °C, transfer the cell lysis solution in the six-well plate to a 1.5 mL EP tube. Centrifuge at 12000 rpm for 10 min at 4 °C. Transfer the supernatant to a new EP tube. Store it on ice for later use.

[0082] 5) Take 10 μL and mix with 90 μL PBS to use as diluent for concentration determination.

[0083] 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.

[0084] 7) Draw a standard curve and calculate the protein sample concentration. The standard sample configuration is shown in Table 1:

[0085] Table 1 Standard product configuration

[0086]

[0087]

[0088] 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.

[0089] 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.

[0090] Table 2 Preparation of lower layer glue

[0091] 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 1M Tris (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

[0092] 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.

[0093] Table 3 Preparation of upper layer glue

[0094]

[0095]

[0096] 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.

[0097] 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.

[0098] 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.

[0099] 12) Blocking: Prepare 5% skimmed milk powder with TBST and incubate the PDVF membrane on a shaker for 90 minutes.

[0100] 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)

[0101] 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)

[0102] 15) Wash three times with TBST with rapid shaking, 10 min each time.

[0103] 16) Luminescent reagents A and B (Affinity TM ECL kit (femtogram)-#KF8003) was configured in 1:1 and exposed.

[0104] The results are as follows Figures 11 - 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 ( Figure 11 ). 20μM and 30μM SR11302 were used in combination, respectively, and the IC 50 The results showed that SR11302 significantly reduced drug resistance ( Figure 12) As shown by Western blot experiments, the combined use of SR1302 and osimertinib decreased the expression of ERBB2, ERBB3, p-AKT, and p-ERK1 / 2. Figure 14 ) Moreover, this combined effect of inhibiting cell proliferation was also observed in PC-9 / OR cells. Figure 13 ) This further verified the combined inhibitory effect of SR11302 and osimertinib on cell proliferation.

[0105] Example 3

[0106] A cell line xenograft (CDX) model was constructed by inoculating the drug-resistant cell HCC827 / OR prepared in Example 1 into NCG immunodeficient mice. The growth rate and size of tumors in the drug administration group and the control group were used to verify the synergistic effect of osimertinib and SR11302.

[0107] The mice used in the experiment were of SPF grade, and the NCG immunodeficient mice were purchased from Jiangsu Jicui Yakang Biotechnology Co., Ltd.

[0108] Male NCG mice at 3 weeks of age were allowed to adapt to the environment for 1 week. Then, 8×10 6 drug-resistant cells HCC827 / OR were resuspended in 50 μL of pre-cooled PBS, and then 50 μL of Matrigel was added and mixed well (operated on ice). The drug-resistant cells HCC827 / OR were subcutaneously injected into the right axilla of the mice. Two months after inoculation, when the tumors in the mice 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), and the combination of osimertinib (8 mg / kg / day) and SR11302 (30 mg / kg / day). Drug treatment: An appropriate amount of osimertinib was weighed after grinding and added to normal saline for ultrasonic-assisted dissolution. It was intragastrically administered at a dose of 8 mg / kg with 200 μL per day. An appropriate amount of SR11302 was dissolved in a small amount of DMSO, and then added to a physiological saline solution of 1% carboxymethylcellulose sodium for ultrasonic-assisted dissolution to obtain a suspension. It was intraperitoneally injected at a dose of 30 mg / kg with 200 μL per day, and the final concentration of DMSO was less than 2%.

[0109] The body weight and tumor size were measured every day.

[0110] The results are as Figures 15 - 17As shown, compared with the normal saline group, the group treated with SR11302 alone significantly slowed down the growth of tumors, and the group treated with osimertinib alone also significantly inhibited tumor growth. The combination of osimertinib and SR11302 led to a significant reduction in tumor volume. Compared with the normal saline group, the use of osimertinib and SR11302 alone significantly decreased the protein expression levels of ERBB2, ERBB3, p-AKT, and p-ERK1 / 2. The combination of osimertinib and SR11302 resulted in a more significant downregulation of proteins compared to the use of osimertinib or SR11302 alone.

[0111] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A pharmaceutical composition for treating non-small cell lung cancer, characterized in that: Including osimertinib and SR11302; the drug mass ratio of osimertinib to SR11302 is 4:(13-17); The dosage of osimertinib is 6-9 mg / kg / day, and the dosage of SR11302 is 28-32 mg / kg / day; The non-small cell lung cancer is osimertinib-resistant non-small cell lung cancer.

2. The pharmaceutical composition according to claim 1, characterized in that The dosage of osimertinib is 8 mg / kg / day, and the dosage of SR11302 is 30 mg / kg / day.

3. The pharmaceutical composition according to claim 2, characterized in that The osimertinib is added with normal saline to assist ultrasonic dissolution; After the SR11302 is dissolved in DMSO, a 1% sodium carboxymethyl cellulose solution in physiological saline is added, and ultrasonic dissolution is performed to obtain a suspension, wherein the final concentration of DMSO in the suspension is less than 2%.

4. 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: When the non-small cell lung cancer cell line is HCC827 / OR, the concentration of SR11302 is 15-35 μM, and the concentration of osimertinib is 450-550 nM.

5. The use according to claim 4, characterized in that: When the non-small cell lung cancer cell line is PC-9 / OR, the concentration of SR11302 is 15-35 μM; the concentration of osimertinib is 100 nM.

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