Application of tasilisib in preparation of product for overcoming drug resistance of non-small cell lung cancer

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to application of tasilimus in preparation of a product for overcoming drug resistance of non-small cell lung cancer. The invention finds that the combined use of the first generation (gefitinib) and the third generation (osimertinib) EGFR-TKI can significantly reduce the tolerance degree of EGFR-TKI drug-resistant cells to the two drugs. In a nude mouse model experiment, the curative effect of the combination of the tasilisib and the osimertinib is obviously superior to that of the single use of the osimertinib or the combination of the osimertinib and the LY294002, the obvious tumor growth inhibition effect is shown, and the drug-resistant relapse of tumors can be effectively delayed. Different from other PI3KCA inhibitors, the enhancement of the EGFR-TKI targeted therapy effect of the tasilimus does not depend on the inhibition of a PI3K / AKT pathway, but recovers the sensitivity of the drug-resistant cells to the EGFR-TKI by awakening the dormant state of the tumor, thereby providing a new thought for a therapeutic scheme.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to application of tasimelte in preparation of a product for overcoming non-small cell lung cancer drug resistance. BACKGROUND

[0002] EGFR gene encodes epidermal growth factor receptor (EGFR) tyrosine kinase and is widely expressed in normal tissues. Up to 50% of East Asian non-small cell lung cancer (NSCLC) patients can observe somatic activating mutations that cause changes in the EGFR kinase domain. And it is worth noting that the rate of EGFR activating mutations is higher in women and patients who have never smoked or are former light smokers. These genetic changes promote tumor cell survival and proliferation by causing ligand-independent constitutive receptor activation and persistent conduction of downstream signals. Among them, the classic activating mutations, exon 19 deletion (47%) and L858R point mutation (41%) are the main types of EGFR mutations and are clearly defined as sensitive mutations with good clinical response to EGFR tyrosine kinase inhibitors (EGFR-TKI). According to several reports on phase III clinical trials of first-generation EGFR-TKI (gefitinib and erlotinib) and second-generation EGFR-TKI (afatinib and dacomitinib), the progression-free survival (PFS) of patients with advanced NSCLC who are initially treated and EGFR-mutated and receive EGFR-TKI targeted therapy is significantly improved compared with platinum-based chemotherapy. In addition, the third-generation EGFR-TKI osimertinib shows excellent therapeutic efficacy, significantly improving PFS (18.9 months vs 10.2 months) and overall survival (OS, 38.6 months vs 31.8 months). This indicates that EGFR-TKI has significant antitumor activity for NSCLC patients carrying EGFR mutations, representing a major breakthrough in targeted therapy for NSCLC patients. However, this group of patients who respond to EGFR-TKI treatment inevitably develop EGFR-TKI resistance, ultimately leading to tumor recurrence. Therefore, overcoming the clinical problem of EGFR-TKI acquired resistance caused by EGFR-TKI targeted therapy is imminent for EGFR-mutated NSCLC patients.

[0003] However, the resistance mechanisms of osimertinib are significantly different from the previous generations of TKIs. Although EGFR resistance mutations (such as T790M) are common in patients treated with early TKIs, the proportion of resistance mutations is relatively low in osimertinib-resistant patients. Studies have shown that about 30% of drug-resistant tumor samples have EGFR-dependent mutations such as C797S, while the remaining about 70% of samples lack clear resistance mutations, making the study of resistance mechanisms more complex. Current research has focused on off-target resistance mechanisms, especially those mediated by bypass activation. For example, amplification or mutation of MET, HER2, and FGFR can be important factors contributing to drug resistance. Although certain combination therapy strategies, such as combining osimertinib with MET inhibitor savolitinib, have shown some efficacy in overcoming MET-amplified mEGFR NSCLC patients (response rate ORR 30-60%, progression-free survival PFS improved), but the overall efficacy is limited. This indicates that relying solely on single combination therapy strategies to overcome drug resistance cannot fundamentally solve the problem. In addition, the sensitivity of mEGFR tumor cells to TKIs, as well as the complexity and cross-regulation characteristics of EGFR downstream signaling networks (such as PI3K-AKT / mTOR and RAS-MAPK pathways), make it a major challenge to achieve comprehensive inhibition of bypass activation. The interactivity of this network makes it difficult to simply target a single signaling pathway, which is often affected by feedback mechanisms, limiting clinical efficacy. Therefore, future research may need to explore more systematic and comprehensive treatment strategies, combining multiple targeted drugs and treatments with different mechanisms of action, to effectively address tumor cell drug resistance and improve the chances of long-term survival for patients.

[0004] NSCLC patients who have been effectively treated with TKIs for months to years will often relapse after a period of progression-free survival. For example, in a group of advanced NSCLC patients who received osimertinib as first-line treatment, the average progression-free survival (PFS) after initial remission was 15-18 months, with a partial response rate (PR) of over 70%. However, these patients often relapse after effective treatment without the presence of treatment residual lesions being clearly detected by imaging methods, which can indicate that tumor cells have entered a dormant state. Tumor dormancy is defined as a non-proliferative state of tumor cells, but still has the potential to restore growth. Recent studies have emphasized that the persistence of dormant tumor cells is an important cause of drug resistance and recurrence, which severely limits the long-term remission of EGFR-TKI therapy. A study by Kurppa et al. showed that double inhibition of the EGFR and MEK pathways can lead to a senescent-like dormant state characterized by elevated YAP activity. In this state, YAP forms a complex with the TEAD transcription factor and inhibits the pro-apoptotic protein BMF by binding to the epithelial-mesenchymal transition (EMT) transcription factor SLUG, thereby limiting drug-induced apoptosis. This finding suggests that eliminating dormant tumor cells that survive initial targeted therapy can provide a new potential method for clinical treatment.

[0005] Taselisib (GDC-0032) is a potent small molecule inhibitor developed by Roche, which specifically targets the class I phosphatidylinositol 3-kinase (PI3K) alpha isoform (PIK3CA). Studies have shown that taselisib exhibits significant selectivity and potency in inhibiting different PI3K isoforms, with an inhibition constant (Ki) of 0.12 nM for PI3K delta, 0.29 nM for PI3K alpha, 0.97 nM for PI3K gamma, and 9.1 nM for PI3K beta. Its chemical name is 2-(4-(2-(1-isopropyl-3-methyl-1H-1,2,4-triazol-5-yl)-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepin-9-yl)-1H-pyrazol-1-yl)-2-methylpropanamide, with a molecular formula of C 24 H 28 N8O2, a molecular weight of 460.53, a PubChem CID of 51001932, and a CAS number of 1282512-48-4.

[0006] The existing literature reports that the main clinical trials of taselisib focus on PIK3CA mutant breast cancer patients. Among them, Richard D Baird et al. evaluated the safety of taselisib in PIK3CA mutant and wild-type estrogen receptor (ER) positive metastatic breast cancer patients, and preliminarily observed the antitumor activity of taselisib combined with tamoxifen treatment. The recommended dose of taselisib in the clinical phase 2 trial is 4 mg / day. In addition, for PIK3CA mutant, ER positive, and human epidermal growth factor receptor 2 (HER2) negative advanced breast cancer patients, studies have shown that the triple therapy of palbociclib, taselisib and fulvestrant may bring significant benefits. Some PIK3CA mutant triple-negative breast cancer patients benefit from the dual therapy of palbociclib and taselisib, which suggests a new strategy for potential molecular targeted therapy for PIK3CA mutant triple-negative breast cancer patients. In a recent III phase randomized study, the efficacy of taselisib combined with fulvestrant and placebo combined with fulvestrant in ER positive, PIK3CA mutant and HER2 negative advanced breast cancer patients was compared, and the results showed that taselisib combined with fulvestrant significantly improved the progression-free survival of PIK3CA mutant tumor patients. In summary, taselisib shows good antitumor activity, especially in breast cancer patients with PIK3CA mutation, providing new hope and direction for future targeted therapy.

[0007] However, so far, there has been no report on the use of taselisib (taselisib) combined with EGFR-TKIs to treat EGFR mutant NSCLC. Although taselisib is a PI3KCA inhibitor, its substrate and mechanism may suggest its potential to overcome EGFR-TKIs resistance, but it is not yet clear whether PI3KCA inhibitors generally have the ability to awaken tumor dormant phenotypes and thus improve the efficacy of EGFR-TKIs combination therapy. In addition, current clinical trials of PI3KCA combined with EGFR-TKIs have not made breakthrough progress. Therefore, it is urgent to carry out systematic research to explore whether taselisib and EGFR-TKIs combination therapy has a synergistic effect in EGFR mutant NSCLC, and to provide a scientific basis for improving treatment efficacy. SUMMARY

[0008] In view of the problem of gradual drug resistance of EGFR-TKIs in the treatment of EGFR mutant NSCLC, the present application proposes the application of taselisib in drug resistance reversal, including the following purposes: The first aspect of the present application aims to provide the use of taselisib in the preparation of a product for overcoming drug resistance of non-small cell lung cancer.

[0009] The second aspect of the present application aims to provide the use of taselisib and an EGFR tyrosine kinase inhibitor in combination in the manufacture of a product for treating non-small cell lung cancer.

[0010] The third aspect of the present application aims to provide a pharmaceutical composition.

[0011] To achieve the above-mentioned objects of the present application, the technical solutions adopted by the present application are as follows: The first aspect of the present application provides the use of taselisib in the manufacture of a product for overcoming drug resistance of non-small cell lung cancer.

[0012] The second aspect of the present application provides the use of taselisib and an EGFR tyrosine kinase inhibitor in combination in the manufacture of a product for treating non-small cell lung cancer.

[0013] In the first and second aspects of the present application, the chemical name of the taselisib is 2-(4-(2-(1-isopropyl-3-methyl-1H-1,2,4-triazol-5-yl)-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepin-9-yl)-1H-pyrazol-1-yl)-2-methylpropanamide, the molecular formula is C 24 H 28 N8O2, the molecular weight is 460.53, the PubChem CID is 51001932, and the CAS number is 1282512-48-4.

[0014] In some embodiments of the present application, the drug resistance of non-small cell lung cancer includes EGFR tyrosine kinase inhibitor resistance.

[0015] In some embodiments of the present application, the EGFR tyrosine kinase inhibitor is selected from at least one of a first-generation EGFR tyrosine kinase inhibitor, a second-generation EGFR tyrosine kinase inhibitor, and a third-generation EGFR tyrosine kinase inhibitor. In some embodiments of the present application, the first-generation EGFR tyrosine kinase inhibitor includes but is not limited to gefitinib or erlotinib.

[0016] In some embodiments of the present application, the second-generation EGFR tyrosine kinase inhibitor includes but is not limited to afatinib or dacomitinib.

[0017] In some embodiments of the present application, the third-generation EGFR tyrosine kinase inhibitor includes but is not limited to osimertinib, almonertinib, or furmonertinib.

[0018] In some embodiments of the present application, the non-small cell lung cancer is EGFR mutant non-small cell lung cancer.

[0019] In some embodiments of the present application, the EGFR mutant non-small cell lung cancer comprises at least one of E746-A750 deletion mutant, T790M / L858R double mutant.

[0020] In some embodiments of the present application, the E746-A750 deletion mutant refers to deletion of 5 amino acids from N-terminal to C-terminal of 746~750 of EGFR protein.

[0021] In some embodiments of the present application, the T790M / L858R double mutant refers to mutation of T to M at 790th amino acid and mutation of L to R at 858th amino acid of EGFR protein.

[0022] In some embodiments of the present application, the tasiplicib comprises a pharmaceutically acceptable salt.

[0023] In some embodiments of the present application, the pharmaceutically acceptable salt comprises at least one of metal salt, ammonium salt, salt with organic base, salt with basic amino acid.

[0024] In some embodiments of the present application, the metal salt comprises at least one of alkali metal salt, alkaline earth metal salt.

[0025] In some embodiments of the present application, the alkali metal salt comprises at least one of sodium salt, potassium salt.

[0026] In some embodiments of the present application, the alkaline earth metal salt comprises at least one of calcium salt, magnesium salt, barium salt, aluminum salt.

[0027] In some embodiments of the present application, the salt with organic base comprises at least one of salt with trimethylamine, triethylamine, pyridine, methylpyridine, 2,6-dimethylpyridine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, N,N'-dibenzylethylenediamine.

[0028] In some embodiments of the present application, the salt with basic amino acid comprises at least one of salt with arginine, lysine, ornithine.

[0029] In some embodiments of the present application, the product comprises a drug.

[0030] In some embodiments of the present application, the drug comprises a pharmaceutically acceptable excipient, and / or any one or more other active ingredients.

[0031] In some embodiments of the present application, the pharmaceutically acceptable excipient includes at least one of a solvent, a propellant, a solubilizer, a co-solvent, an emulsifier, a colorant, a binder, a disintegrant, a filler, a lubricant, a wetting agent, an osmotic pressure adjusting agent, a stabilizer, a glidant, a flavoring agent, a preservative, a suspending agent, a coating material, an aromatic agent, an anti-adhesion agent, an integrating agent, a penetration enhancer, a pH adjusting agent, a buffer, a plasticizer, a surfactant, a foaming agent, an antifoaming agent, a thickening agent, an inclusion agent, a humectant, an absorbent, a diluent, a flocculating agent and a deflocculating agent, a filtering aid, a release retardant, a carrier.

[0032] The above pharmaceutically acceptable excipient is generally recognized for this purpose and is a non-active ingredient of a medicament. A compilation of pharmaceutically acceptable excipients can be found in Handbook of Pharmaceutical Excipients, 2ndEdition, edited by A. Wade and P. J. Weller; published by American Pharmaceutical Association, Washington and The Pharmaceutical Press, London, 1994; the Pharmacopoeia of the People's Republic of China - Pharmaceutical Excipients; and the like. In some embodiments of the present application, In some embodiments of the present application, the dosage form of the product includes a gastrointestinal administration dosage form, or a non-gastrointestinal administration dosage form.

[0033] In some embodiments of the present application, the gastrointestinal administration dosage form includes at least one of a powder, a tablet, a granule, a capsule, a sustained release, a solution, a dry suspension, an effervescent tablet, an emulsion, a suspension, a syrup, a drop, a chewable tablet.

[0034] Further, the gastrointestinal administration dosage form includes, but is not limited to, an enteric tablet, a coated tablet, a film-coated tablet, a sugar-coated tablet, a dispersible tablet, a sucking tablet, a chewable tablet, an effervescent tablet, a scored tablet, a sustained release controlled release dosage form sustained release tablet, a sustained release coated tablet, a controlled release tablet, an oral disintegrating tablet, a buccal tablet, an oral patch, and the like.

[0035] In some embodiments of the present application, the non-gastrointestinal administration dosage form includes at least one of an injection administration dosage form, a respiratory administration dosage form, a skin administration dosage form, a mucosa administration dosage form, a cavity administration dosage form.

[0036] Further, the injection administration dosage form includes, but is not limited to, an injection solution, an injection solution, an injection solution for intravenous drip, an injection suspension, a sterile powder for injection, an intravenous injection needle, a water needle, an injection emulsion, a powder injection, a needle injection, a sterile powder needle, a freeze-dried powder needle, and the like.

[0037] In some embodiments of the present application, the subject of administration of the product is a mammal.

[0038] In some embodiments of the present application, the mammal includes a human.

[0039] In a third aspect of the present application, a composition is provided, comprising taselisib and an EGFR tyrosine kinase inhibitor.

[0040] In some embodiments of the present application, the EGFR tyrosine kinase inhibitor is selected from at least one of a first-generation EGFR tyrosine kinase inhibitor, a second-generation EGFR tyrosine kinase inhibitor, and a third-generation EGFR tyrosine kinase inhibitor. In some embodiments of the present application, the first-generation EGFR tyrosine kinase inhibitor includes, but is not limited to, gefitinib or erlotinib.

[0041] In some embodiments of the present application, the second-generation EGFR tyrosine kinase inhibitor includes, but is not limited to, afatinib or dacomitinib.

[0042] In some embodiments of the present application, the third-generation EGFR tyrosine kinase inhibitor includes, but is not limited to, osimertinib, almonertinib, or furmonertinib.

[0043] The beneficial effects of the present application are: 1. New discovery of combination therapy: The present application first discloses the important role of taselisib (GDC-0032) in overcoming EGFR-TKI resistance, and the combination of a first-generation (gefitinib) and a third-generation (osimertinib) EGFR-TKI can significantly reduce the tolerance of EGFR-TKI-resistant cells to these two drugs.

[0044] 2. Low concentration and high efficiency: The drug concentration of taselisib used is significantly lower than the half growth inhibition concentration required for its single treatment of drug-resistant strains, indicating that it can significantly restore the sensitivity to EGFR-TKI without affecting the growth and apoptosis of drug-resistant cells. This provides a new strategy for developing efficient and low-toxicity combination therapy.

[0045] 3. Significant effect in animal models: In the nude mouse model experiment, the efficacy of taselisib combined with osimertinib is significantly better than that of osimertinib alone or osimertinib combined with pan-PI3K inhibitor LY294002, showing a significant tumor growth inhibition effect and effectively delaying the recurrence of drug resistance of the tumor.

[0046] 4. Unique mechanism of action: Unlike other PI3KCA inhibitors, the promotion of EGFR-TKI targeted therapy by taselisib does not depend on the inhibition of the PI3K / AKT pathway, but rather on the reactivation of the dormant state of the tumor and the restoration of the sensitivity of drug-resistant cells to EGFR-TKI, thus providing a new idea for treatment programs.

[0047] These advantages indicate the potential application value of the present application in the field of tumor treatment, and fully demonstrate its innovation and clinical prospect. BRIEF DESCRIPTION OF DRAWINGS

[0048] The present application will be further described below in combination with the drawings and examples, in which: Figure 1 Results of construction and verification of drug-resistant cell models, in which A is a flowchart for constructing stable drug-resistant (GR / OR) cell models of gefitinib and osimertinib; B is IC 50 The experiment detected the half-inhibitory concentration of HCC827-gefitinib stable drug-resistant cell lines (HCC827-GR) and H1975-osimertinib stable drug-resistant cell lines (H1975-OR) to gefitinib and osimertinib, respectively.

[0049] Figure 2 Results of flow cytometry detection of cell cycle, in which A is the flow cytometry analysis result of the cell cycle of HCC827-gefitinib stable drug-resistant cell lines (HCC827-GR); B is the flow cytometry analysis result of the cell cycle of H1975-osimertinib stable drug-resistant cell lines (H1975-OR).

[0050] Figure 3 Results of half-inhibitory concentration of taselisib alone in treating gefitinib or osimertinib drug-resistant strains, in which A is IC 50 The experiment detected the half-inhibitory concentration of HCC827-gefitinib stable drug-resistant cell lines to taselisib; B is IC 50 The experiment detected the half-inhibitory concentration of H1975-osimertinib stable drug-resistant cell lines to taselisib.

[0051] Figure 4 Influence of taselisib on tumor dormant phenotype, in which A is the Ki-67 immunofluorescence staining result of HCC827-gefitinib stable drug-resistant cell lines (HCC827-GR); B is the Ki-67 immunofluorescence staining result of H1975-osimertinib stable drug-resistant cell lines (H1975-OR).

[0052] Figure 5Results of half growth inhibition concentration of resistant strains treated with 200 nM pan-PI3K inhibitor LY294002 or Taselisib combined with Gefitinib or Osimertinib, wherein: A is IC 50 Results of half growth inhibition concentration of HCC827-Gefitinib stable resistant cell strain to Gefitinib combined with LY294002 or Taselisib; B is IC 50 Results of half growth inhibition concentration of H1975-Osimertinib stable resistant cell strain to Osimertinib combined with LY294002 or Taselisib.

[0053] Figure 6 Effects of pan-PI3K inhibitor LY294002 or Taselisib combined with Gefitinib or Osimertinib on the clonal growth ability of resistant cell strains, wherein A is the effect of LY294002 or Taselisib combined with Gefitinib on the clonal growth ability of HCC827-Gefitinib stable resistant cell strains detected by clonogenicity experiment; B is the effect of LY294002 or Taselisib combined with Gefitinib on the clonal growth ability of H1975-Osimertinib stable resistant cell strains detected by clonogenicity experiment.

[0054] Figure 7 Effects of pan-PI3K inhibitor LY294002 or Taselisib combined with Osimertinib on tumor resistance and recurrence detected by subcutaneous tumor formation experiment in nude mice, wherein A is that nude mice inoculated with H1975-PR cells are randomly divided into groups, and are treated with Osimertinib, Taselisib, LY294002 alone or in combination, respectively, and the administration is stopped after 39 days, until the end of the experiment (60 days), and the volume change of subcutaneous tumor growth is continuously monitored during the experiment; B is the volume change of subcutaneous tumors in nude mice at 39 days after treatment with Osimertinib, Osimertinib combined with Taselisib, and Osimertinib combined with LY294002, compared with the baseline level. DETAILED DESCRIPTION

[0055] The concept and technical effects of the present application will be described below in combination with examples, so as to fully understand the purpose, features and effects of the present application. Obviously, the described examples are only part of the examples of the present application, but not all examples. Based on the examples of the present application, other examples obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0056] Example 1: Establishment and verification of Gefitinib and Osimertinib stable resistant non-small cell lung cancer cell models 1. Experimental materials First generation EGFR-TKI drug Gefitinib (HY-50895A, MedChemExpress) and third generation EGFR-TKI drug Osimertinib (S7297, Selleck).

[0057] 2. Experimental subjects HCC827 (ΔE746-A750) and H1975 (T790M / L858R) cells with EGFR mutations were purchased from Shanghai Cell Biology Research Institute of Chinese Academy of Sciences, and were identified correctly by STR (Short Tandem Repeat) method of Forensic Identification Center of Sun Yat-sen University. All cells were cultured in DMEM medium (12800017, Gibco) containing 1% penicillin-streptomycin double-antibiotic solution (30-002-CI, cellgro) and 10% FBS.

[0058] 3. Experimental methods In this example, first generation EGFR-TKI drug Gefitinib and third generation EGFR-TKI drug Osimertinib (S7297, Selleck) were used to treat HCC827 and H1975 cells respectively by concentration gradient increasing method to construct Gefitinib-resistant cell line (HCC827-GR) and Osimertinib-resistant cell line (H1975-OR).

[0059] Cell construction: First, HCC827 cells were treated with Gefitinib at half inhibitory concentration (IC 50 , 30nM), and H1975 cells were treated with Osimertinib at IC 50 (10nM). During the drug induction treatment, the survival of the cells was observed every day. In order to avoid the influence of cell metabolites and cell debris on living cells, the culture medium was discarded in time after 24-48 h of drug treatment, and the cells were washed with 1xPBS solution, and then replaced with fresh culture medium containing the same concentration of Gefitinib or Osimertinib for continuous culture. After continuous treatment with the same drug concentration for 7 days, if the cells can still grow stably at this concentration, and the density on the 10 cm plate reaches 90%, then the cells are treated with 2 times the drug concentration, and then the above treatment is repeated for six months. The construction process is shown in Fig. 1A. Figure 1

[0060] Detection of IC 50 of stable drug-resistant strains treated with Gefitinib or Osimertinib Experimental grouping: negative control wells (Control) with Gefitinib or Osimertinib concentration of 0, and 10-11 consecutive dilution wells with increasing concentrations of Gefitinib or Osimertinib.

[0061] ​The following steps are performed: (1) After the above cell counting, seed 5000-8000 cells per well in a 96-well plate at a density of 5000-8000 cells per well, with a volume of 200 μl per well, and repeat 3 times; (2) Place in a constant temperature and pressure cell incubator at 37°C, 5% CO2 for 24 h, and after the cells adhere, treat the above cells according to the above experimental grouping. (3) After 48 h of TKI drug exposure culture, add 20 μl MTT (5 mg / ml) and incubate for 4 h. (4) Remove the complete culture medium in the well and add 150 μl DMSO, and place in a shaking bed for 10-15 s to fully dissolve the crystals. (5) Measure the absorbance value (OD 490 nm) of each well on an enzyme-linked immunoassay instrument, record and save the results. (6) Analyze the data: take the drug concentration as the horizontal coordinate and the cell survival (%) as the vertical coordinate to draw the IC 50 curve, and calculate the inhibition rate. The calculation formula is as follows: cell viability= (OD TKI ) / (OD Control )*100%, cell inhibition=1-cell viability.

[0062] Flow verification verifies the cell cycle: Cell cycle detection kit (Kaiji Biotechnology Co., Ltd., Jiangsu).

[0063] The following steps are performed: (1) Seed the above parent and drug-resistant cells, and after the cells adhere (about 4-6 h), replace with DMEM basic medium and starve for 12 h or more; (2) Discard the DMEM basic medium and replace with DMEM complete medium for continuous culture for 8-12 h; (3) Trypsinize the cells, wash the cells once with 1×PBS, and centrifuge at 1000 rpm for 5 min; (4) Resuspend the cell pellet using 1×PBS, count 1×106 cells in a new 1.5 ml EP tube; (5) Centrifuge the above cell suspension to remove the supernatant, add 500 μl of 70% ice ethanol, mix gently, and fix at 4°C for 2 h; (6) Centrifuge at 1000 rpm for 5 min to collect the cells, carefully aspirate the supernatant, and wash the cells once with 1×PBS; (7) Centrifuge at 1000 rpm for 5 min to collect the cells, prepare a working dye solution by mixing RNase A and PI at a ratio of 1:9, resuspend the cells with 1 ml of the above working dye solution, and filter through a 200-400 mesh filter into a flow tube to filter the cells into single cells; (8) Stain at 4°C for 30 min in the dark, and mix gently during the period; (9) Detect red fluorescence at an excitation wavelength of 488 nm using a flow cytometer, and detect light scattering at the same time; (10) Perform cell DNA content analysis using FlowJo analysis software.

[0064] 4. Experimental results In this embodiment, NSCLC parental (PR) cell lines HCC827-PR (E746-A750 deletion) and H1975-PR (EGFR T790M / L858R mutation) were selected to construct Gefitinib resistant (GR) and Osimertinib resistant (OR) cell lines HCC827-GR and H1975-OR. The construction of in vitro drug-resistant cell lines used the method of EGFR-TKIs drug concentration gradient multiplication (A) Figure 1 , starting from the dose of half maximal inhibitory concentration (IC 50 ) of HCC827-PR and H1975-OR initial drugs to treat cells. During drug treatment, the complete medium containing EGFR-TKIs was replaced every 2-3 days. When the surviving cell population showed signs of proliferation and the density on the 10 cm plate reached 90%, the cells were passaged and treated with 2-fold drug concentration, and this culture process was repeated for six months. Finally, the IC 50 of the established drug-resistant cell lines was detected, and the results showed that the IC 50 of HCC827-GR cell line was 19.444 μM, and the IC 50 of H1975-OR cell line was 13.447 μM (B) Figure 1 ). Further, flow cytometry detection of cell cycle found that compared with parental cells, most drug-resistant cells were in G0 / G1 phase (A, B) Figure 2 , suggesting that drug-resistant cells enter the dormant state of cell cycle arrest. The above results suggest that the dormant drug-resistant cell line is successfully constructed.

[0065] Example 2 Half maximal growth inhibition concentration of Gefitinib-resistant strain treated with Taselisib alone and its effect on tumor dormancy 1. Experimental materials Taselisib (HY-13898, MedChem Express), thiazolyl blue (MTT, JT343-250MG, Genview), dimethyl sulfoxide (DMSO, D4540-1L, Sigma), Ki-67 antibody (CST, 9129).

[0066] 2. Experimental method Cells: HCC827 Gefitinib-resistant strain (HCC827-GR) and H1975 Osimertinib-resistant strain (H1975-OR).

[0067] Half maximal inhibitory concentration (IC50) determination: Experimental grouping: negative control wells (Control) with no Taselisib and 8-10 wells with serial dilutions of Taselisib.

[0068] Experimental procedure: (1) After cell counting, 5000-8000 cells per well were seeded into 96-well plates in a volume of 200 μl, with 3 replicates. (2) The plates were incubated in a 37°C, 5% CO2 incubator for 24 h. After the cells adhered, the cells were treated according to the experimental grouping described above. (3) After 48 h of Taselisib exposure, 20 μl of MTT (5 mg / ml) was added and incubated for 4 h. (4) The complete medium was removed and 150 μl of DMSO was added. The plates were shaken for 10-15 s to dissolve the crystals. (5) The absorbance of each well was measured at 490 nm using an enzyme-linked immunoassay instrument. The results were recorded and saved. (6) Data analysis: the drug concentration was used as the horizontal coordinate and the cell viability (%) was used as the vertical coordinate. The IC50 curve was plotted and the inhibition rate was calculated. The formula was as follows: cell viability= (OD 50 ) / (OD TKI ) * 100%, cell inhibition=1-cell viability. Control

[0069] Tumor dormancy determination: Cell grouping: HCC827 gefitinib-resistant strain (HCC827-GR) and H1975 osimertinib-resistant strain (H1975-OR) were treated with DMSO and Taselisib for 7 d.

[0070] Experimental procedure: (1) The cover slip was irradiated with ultraviolet light for 30 min and then placed in a 24-well plate. 3x10 4 ​The cells were cultured on cover slips overnight; (2) the next day, the culture medium was discarded, and the cells were washed once with 500 μl of 1x PBS per well; (3) 200-300 μl of 4% paraformaldehyde was added to each well to fix the cells, and the cells were allowed to stand at room temperature for 1.5 min; (4) the paraformaldehyde was discarded, and 200-300 μl of pre-cooled methanol was added to each well to fix the cells, and the cells were allowed to stand at -20°C for 15 min; (5) the methanol was discarded, and the cells were washed with 1x PBS, and the cells were placed on a horizontal shaker and gently shaken at room temperature for 10 min, and the washing was repeated three times; (6) the 1x PBS was discarded, and 200-300 μl of 0.5% Triton X-100 solution was added to each well to increase the permeability of the cell membrane, and the cells were allowed to stand at room temperature for 5 min; (7) the Triton X-100 solution was discarded, and the washing of step (5) was repeated; (8) the 1x PBS was discarded, and 200-300 μl of 10% BSA solution was added to each well, and the cells were allowed to stand on a horizontal shaker for blocking incubation for 30 min; (9) the 10% BSA blocking solution was discarded, and 200 μl of Ki 67 primary antibody solution diluted with 0.1% PBST was added to each well, and the cells were allowed to stand on a 4°C chromatography cabinet shaker for incubation overnight; (10) the next day, the primary antibody solution was recovered, and the cells were washed with 0.1% PBST three times for 10 min each time; (11) the secondary antibody solution was diluted with 0.1% PBST, and 200 μl was added to each well, and the cells were allowed to stand on a horizontal shaker for incubation at room temperature for 1 h in the dark; (12) the secondary antibody solution was recovered, and the cells were washed with 0.1% PBST three times for 10 min each time in the dark; (13) DAPI powder was dissolved in 1x PBS, and after mixing, 200 μl of DAPI solution was added to each well for staining in the dark for about 30 s; (14) the cells were washed once with 1x PBS, and the cells were washed on a horizontal shaker at room temperature for 5-10 min; (15) the cells were dried in the dark overnight, and after the slide was dried, the slide was sealed and fixed with a mounting medium, and then the slide was observed and photographed under a fluorescence microscope.

[0071] 3. Experimental results To investigate the effect of taselisib on the dormant phenotype of tumors, the present example first evaluated the half growth inhibition concentration of taselisib on dormant drug-resistant cells. IC 50 The experimental results show that the IC 50 values of taselisib on HCC827-GR and H1975-OR cells are 3.297 μM and 3.060 μM, respectively. Figure 3HCC827-GR and H1975-OR were treated with Taselisib at a lower dose (200 nM) for 7 days to avoid the killing effect of Taselisib monotherapy on dormant drug-resistant cells. Ki-67 immunofluorescence staining was used to evaluate the dormancy of the above cells. The results showed that the number of Ki-67 positive cells in drug-resistant cells treated with Taselisib was significantly increased compared with the solvent control (Fig. 2A and B), suggesting that Taselisib treatment can wake up dormant drug-resistant cells and make drug-resistant cells exit the dormant phenotype. Figure 4 HCC827-GR and H1975-OR were treated with Taselisib at a lower dose (200 nM) for 7 days to avoid the killing effect of Taselisib monotherapy on dormant drug-resistant cells. Ki-67 immunofluorescence staining was used to evaluate the dormancy of the above cells. The results showed that the number of Ki-67 positive cells in drug-resistant cells treated with Taselisib was significantly increased compared with the solvent control (Fig. 2A and B), suggesting that Taselisib treatment can wake up dormant drug-resistant cells and make drug-resistant cells exit the dormant phenotype.

[0072] Example 3 Influence of Taselisib on drug-resistant strains is independent of the PI3K pathway 1. Experimental materials First-generation EGFR-TKI drug Gefitinib (HY-50895A, MedChemExpress), third-generation EGFR-TKI drug Osimertinib (S7297, Selleck), Taselisib (HY-13898, MedChem Express), thiazolyl blue (MTT, JT343-250MG, Genview), dimethyl sulfoxide (DMSO, D4540-1L, Sigma), methanol (Guangzhou Chemical Reagent Factory), crystal violet staining solution (C0121, Biyun Tian).

[0073] 2. Experimental methods Detection of the half growth inhibition concentration of drug-resistant strains treated with PI3K inhibitor LY294002 or Taselisib combined with Gefitinib or Osimertinib: Cells: HCC827 Gefitinib-resistant strain (HCC827-GR) and H1975 Osimertinib-resistant strain (H1975-OR).

[0074] Experimental grouping: negative control wells without Gefitinib or Osimertinib (Control) and 8-10 consecutive dilution wells containing Taselisib combined with Gefitinib or Osimertinib concentration gradient.

[0075] Experimental procedure: (1) After the above cell counting, seed 5000-8000 cells per well into a 96-well plate at a density of 5000-8000 cells per well, with a volume of 200 μl per well, and repeat 3 replicate wells; (2) Place in a constant temperature and pressure cell incubator at 37°C, 5% CO2, and incubate for 24 h. After the cells adhere, treat the above cells according to the above experimental grouping. (3) Add 20 μl MTT (5 mg / ml) after Taselisib combined drug exposure culture for 48 h, and incubate for 4 h. (4) Remove the complete culture medium in the wells and add 150 μl DMSO, and place in a shaking bed for 10-15 s to fully dissolve the crystals. (5) Measure the absorbance value (OD 490 nm) of each well on an enzyme-linked immunoassay instrument, record and save the results. (6) Analyze the data: take the drug concentration as the horizontal coordinate and the cell survival (%) as the vertical coordinate, draw the IC 50 curve, and calculate the inhibition rate. The calculation formula is as follows: cell viability = (OD TKI ) / (OD Control )*100%, and cell inhibition = 1-cell viability.

[0076] Clonogenic assay to detect the effect of PI3K inhibitor LY294002 or Taselisib combined with gefitinib or osimertinib on the clonal growth ability of drug-resistant strains: Experimental grouping: HCC827 gefitinib-resistant strain (HCC827-GR): DMSO solvent control group (Control), 15 μM gefitinib single-drug treatment group, 0.2 μM Taselisib single-drug treatment group, and 15 μM gefitinib combined with 0.2 μM Taselisib dual inhibitor treatment group.

[0077] H1975 osimertinib-resistant strain (H1975-OR): DMSO solvent control group (Control), 5 μM osimertinib single-drug treatment group, 0.1 μM Taselisib single-drug treatment group, and 5 μM osimertinib combined with 0.2 μM Taselisib dual inhibitor treatment group.

[0078] Experimental procedure: (1) After the above cell counting, seed 2-3x10 4Cell density of 1 x 105cells per well was added to 12-well plates. (2) Each well was supplemented with 1 ml of complete medium. (3) The 12-well plates were shaken according to the cross method, and the cells were evenly distributed before being placed in a constant-temperature and constant-pressure cell incubator at 37°C, 5% CO2. (4) The next day, the cells were treated according to the drug concentration of the experimental grouping design, and the culture was continued. (5) During the culture period, the medium was replaced every 3 days according to the drug concentration for continuous treatment. (6) After 7-10 days, the medium was discarded, and 1x PBS solution was used for 1-2 times of washing. (7) 100% methanol was used for room temperature fixation for 15-30 min. (8) The fixing solution was discarded, and crystal violet staining solution was used for staining for 30 min. (9) The crystal violet staining solution was recovered, and the 6-well plate was washed with tap water. (10) In the fume hood, it was inverted and dried.

[0079] 3. Experimental results: Taselisib as a PI3K inhibitor, its targeted inhibition of PI3K may be able to overcome EGFR-TKIs resistance, so this example parallelly used the pan-PI3K inhibitor LY294002 as a control to explore whether the role of Taselisib in overcoming EGFR-TKIs dormancy resistance is related to the inhibition of PI3K. When using 200 nM pan-PI3K inhibitor LY294002 or Taselisib combined with Gefitinib or Osimertinib to treat the dormancy-resistant cell lines, it was found that compared with Gefitinib or Osimertinib alone, the combination of 200 nM Taselisib could significantly reduce the sensitivity of the dormancy-resistant cells to EGFR-TKIs (HCC827-GR 25.51 μM vs 2.79 μM; H1975-OR 12.86 μM vs 0.56 μM) Figure 5 in A and B); while when 200 nM LY294002 was combined with Gefitinib or Osimertinib, the dormancy-resistant cells were still resistant to EGFR-TKIs (HCC827-GR 25.51 μM vs 17.62 μM; H1975-OR 12.86 μM vs 6.95 μM) Figure 5 in A and B). The clonogenic assay also obtained consistent results, that is, compared with the combination of solvent control or LY294002, the combination of 200 nM Taselisib and EGFR-TKIs could significantly eliminate the dormancy-resistant cells Figure 6 in A and B). The above results show that the mechanism of Taselisib in overcoming EGFR-TKIs resistance is not related to its targeted inhibition of the PI3K pathway.

[0080] Example 4. Effect of PI3K inhibitor or taselisib combined with osimertinib on tumor subcutaneous growth and drug resistance recurrence in nude mice subcutaneous tumor experiment 1. Experimental subjects H1975 tumor-bearing nude mice (BALB / c-nu / nu, 5-8 weeks old, 18-20 g). The experimental animals involved in this example are specific pathogen free (Specific Pathogen Free, SPF) level immunodeficient mice BALB / c-nu / nu (5-8 weeks old, 18-20 g) purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (SCXK (Jing) 2016-0011), and the experimental process conforms to the animal ethics and welfare guidelines.

[0081] 2. Experimental materials Osimertinib (S7297, Selleck), taselisib (HY-13898, MedChem Express).

[0082] 3. Experimental methods Experimental grouping: solvent control (Vehicle), taselisib (1 mg / kg / d), osimertinib (2 mg / kg / d) alone, and osimertinib (2 mg / kg / d) combined with taselisib (1 mg / kg / d) treatment groups.

[0083] Experimental steps: The effect of taselisib combined with osimertinib on tumor subcutaneous growth and drug resistance recurrence was verified by nude mouse subcutaneous tumor. H1975 cells were inoculated subcutaneously in nude mice, and when the tumor volume reached 200 mm 3 left, the tumor-bearing mice were randomly divided into three groups, and were treated with solvent control (Vehicle), taselisib (1 mg / kg / d), osimertinib (2 mg / kg / d) alone, and osimertinib (2 mg / kg / d) combined with taselisib (1 mg / kg / d). The change in tumor volume was continuously monitored within 39 days of treatment. Then the mouse gavage was stopped, and the tumor recurrence was observed. The long diameter and short diameter of the subcutaneous tumor were measured, and the subcutaneous tumor volume was calculated using the calculation method of "long diameter x short diameter x short diameter x 0.52". At the end of the experiment, the mice were anesthetized, and the nude mice were sacrificed by cervical dislocation, the complete tumor sample was taken out, weighed and measured, and then fixed in 4% paraformaldehyde solution.

[0084] 4. Experimental results This example evaluated the effect of Taselisib in combination with Osimertinib on tumor resistance and recurrence using a subcutaneous tumor formation experiment in nude mice. Nude mice inoculated with H1975-PR cells were randomly divided into groups and treated with Osimertinib, Taselisib, LY294002 alone or in combination for 39 days. Drug administration was stopped after 39 days until the end of the experiment (day 60). The change in subcutaneous tumor volume was continuously monitored during the experiment. The results showed that treatment of tumor-bearing mice with Taselisib or LY294002 alone did not affect the growth of subcutaneous tumors compared to the solvent control. In contrast, treatment with Osimertinib in combination with Taselisib significantly reduced tumor volume and significantly alleviated tumor recurrence after drug withdrawal compared to mice treated with Osimertinib alone or Osimertinib in combination with LY294002.

[0085] The embodiments of the application are described in detail above with reference to the accompanying drawings, but the application is not limited to the above-described embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the spirit of the application. Furthermore, the embodiments of the application and the features in the embodiments can be combined with each other without conflict.

Claims

1. Application of tacilisin in the preparation of products that overcome drug resistance in non-small cell lung cancer; The drug resistance is EGFR tyrosine kinase inhibitor resistance.

2. Application of the combined use of tacilix and EGFR tyrosine kinase inhibitors in the preparation of products for the treatment of non-small cell lung cancer.

3. The application according to any one of claims 1 to 2, characterized in that: The EGFR tyrosine kinase inhibitor is selected from at least one of the first-generation EGFR tyrosine kinase inhibitors, second-generation EGFR tyrosine kinase inhibitors, and third-generation EGFR tyrosine kinase inhibitors. Among them, the first-generation EGFR tyrosine kinase inhibitors include, but are not limited to, gefitinib or erlotinib; The second-generation EGFR tyrosine kinase inhibitors include, but are not limited to, afatinib or dacomitinib; The third-generation EGFR tyrosine kinase inhibitors include, but are not limited to, osimertinib, ametinib, or vometinib.

4. The application according to any one of claims 1 to 2, characterized in that: The non-small cell lung cancer mentioned is EGFR-mutant non-small cell lung cancer.

5. The application according to claim 4, characterized in that: The EGFR-mutant non-small cell lung cancer includes at least one of the E746-A750 deletion mutation and the T790M / L858R double mutation.

6. The application according to any one of claims 1 to 2, characterized in that: Tacilis includes pharmaceutically acceptable salts.

7. The application according to any one of claims 1 to 2, characterized in that: The products include pharmaceuticals.

8. The application according to claim 7, characterized in that: The drug includes pharmaceutically acceptable excipients.

9. The application according to claim 7, characterized in that: The dosage forms of the drug include those administered via the gastrointestinal tract or those administered outside the gastrointestinal tract.

10. A pharmaceutical composition comprising tacilisin and an EGFR tyrosine kinase inhibitor; The EGFR tyrosine kinase inhibitor is selected from at least one of the first-generation EGFR tyrosine kinase inhibitors, second-generation EGFR tyrosine kinase inhibitors, and third-generation EGFR tyrosine kinase inhibitors. in, The first-generation EGFR tyrosine kinase inhibitors include, but are not limited to, gefitinib or erlotinib; The second-generation EGFR tyrosine kinase inhibitors include, but are not limited to, afatinib or dacomitinib; The third-generation EGFR tyrosine kinase inhibitors include, but are not limited to, osimertinib, ametinib, or vometinib.

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