Application of gefitinib combined with AC480 in preparation of medicine for treating non-small cell lung cancer
Through the combined use of gefitinib and AC480, the drug resistance of advanced non-small cell lung cancer cells to gefitinib is solved, and effective inhibition and efficacy improvement of drug-resistant cells are achieved.
Patent Information
- Application Number
- CN202510370706.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-27
- Publication Date
- 2025-05-09
AI Technical Summary
When traditional single drugs treat advanced non-small cell lung cancer, the tumor is prone to drug resistance, resulting in a decrease in efficacy. It is difficult to effectively solve the drug resistance problem of existing targeted drugs and immunotherapy.
By combining gefitinib with AC480, suitable drug combination ratios were screened out and their synergistic efficacy in inhibiting the proliferation of drug-resistant non-small cell lung cancer cells was experimentally verified.
The combined use of gefitinib and AC480 can significantly reduce the acquired resistance of gefitinib to gefitinib by drug-resistant PC9/GR cells and enhance its inhibitory effect on tumor cells.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the use of gefitinib (Gefitinib) combined with AC480 (BMS599626) in preparing a drug for treating drug-resistant non-small cell lung cancer. Background Art
[0002] Lung cancer poses a serious threat to human health. Non-small cell lung cancer (NSCLC) is a common type of lung cancer, accounting for more than 85%. Traditional single-drug therapy has limited effect in advanced NSCLC, and tumors are prone to drug resistance and recurrence. Single chemotherapy has large toxic and side effects, and it is easy for cancer cells to develop adaptive drug resistance, and the disease is still prone to progression after treatment. Although targeted drugs are accurate, many patients will develop drug resistance after using them for a period of time, and the efficacy is greatly reduced. Immunotherapy is also only effective for some patients. Based on this, drug combination therapy has become a research hotspot. Through the rational combination of chemotherapy, targeted, immune and other drugs, tumors can be inhibited from multiple pathways, overcoming the limitations of single medication, improving efficacy, improving patients' quality of life, and prolonging survival.
[0003] Gefitinib is a first-generation oral epidermal growth factor receptor tyrosine kinase (EGFR-TK) inhibitor, suitable for the treatment of locally advanced or metastatic non-small cell lung cancer that has previously received chemotherapy. Gefitinib is designated as the standard first-line treatment for patients with EGFR mutation NSCLC in various countries. However, patients will acquire drug resistance after taking it for a period of time. Therefore, we constructed a cell line with acquired resistance to Gefitinib and explored overcoming its resistance through drug combination.
[0004] In summary, developing new drug compositions for lung cancer and expanding treatment options are of great significance in the field of lung cancer treatment. Summary of the invention
[0005] The present invention provides a use of gefitinib combined with AC480 in preparing a drug for treating gefitinib-resistant non-small cell lung cancer. AC480 can improve the drug sensitivity of gefitinib-resistant non-small cell lung cancer cells to gefitinib.
[0006] The drug ingredients (or active ingredients) described in the present invention are Gefitinib and AC480, and one or more pharmaceutically acceptable excipients may be added to improve the absorption effect of the drug or facilitate use, such as capsules or pills, powders, tablets, granules, oral liquids and injections, etc., that is, suitable dosage forms are prepared.
[0007] The chemical structure of Gefitinib is as follows: ; The chemical structure of AC480 is as follows: .
[0008] The purpose of the present invention is achieved through the following technical solutions: 1. The present invention uses a concentration gradient method to screen a cell line PC9 / GR resistant to Gefitinib, wherein the resistance index (RI) is 100, RI>5, and a resistant strain PC9 / GR is obtained; 2. PC9 cell line and PC9 / GR cell line were treated with the same concentration of Gefitinib for the same period of time. Compared with PC9 cells, the sensitivity of the resistant strain PC9 / GR to Gefitinib was significantly reduced, the expression of proteins p-AKT, p-ERK, p-EGFR, and p-HER2 in the cells could not be completely inhibited, and the downstream signaling pathways continued to be activated; 3. Under the condition of AC480 combined with Gefitinib, according to the IC values of the two drugs against the drug-resistant strain PC9 / GR 50 Value, select Gefitinib:AC480=1:3, Gefitinib:AC480=1:1, Gefitinib:AC480=3:1 drug combination ratio for experiments, to explore the combined effect of the two drugs in PC9 / GR cell line. According to the CI index of several combination ratios, we selected Gefitinib:AC480=1:3 combination ratio for subsequent experiments; PC9 / GR cells were treated with Gefitinib and AC480 alone or in combination, and through clone formation experiments, it was found that the combination group could reduce the acquired resistance of resistant PC9 / GR cells to Gefitinib compared with single drug use.
[0009] Advantages and technical effects of the present invention: The present invention uses Gefitinib combined with AC480 to treat non-small cell lung cancer. Compared with the use of Gefitinib alone or AC480 alone, the combined use of AC480 and Gefitinib plays a synergistic role in inhibiting the proliferation of non-small cell lung cancer cells and can reverse the drug resistance of non-small cell lung cancer cells to Gefitinib. Therefore, the combined use of Gefitinib and AC480 for the treatment of non-small cell lung cancer has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS
[0010] Figure 1 IC50 value of wild-type PC9 cells for Gefitinib Figure 2The IC50 value of Gefitinib in the Gefitinib-acquired drug-resistant cell PC9 / GR; Figure 3 Western blotting was used to detect the protein expression results in PC9 cells or PC9 / GR cells after Gefitinib treatment; Figure 4 This is a graph showing the results of the cytotoxicity experiment of PC9 / GR treated with AC480 detected by CCK-8 method; Figure 5 This is a diagram showing the pharmacodynamic effect of Gefitinib combined with AC480 (Gefitinib:AC480=1:3, Gefitinib:AC480=1:1, Gefitinib:AC480=3:1) in the treatment of non-small cell lung cancer using the CCK-8 method, where Figure A is a dose-effect relationship diagram for the combination of AC480 and gefitinib, Figure B is a CI index curve for the combination of AC480 and gefitinib, and Figure C is a diagram showing the synergistic effect of the drug combination; Figure 6 The results of the plate clone formation experiment of Gefitinib and AC480 used alone and in combination are shown in the figure above, which is a schematic diagram of the plate, and the figure below is a diagram of the clone statistics result; Figure 7 The results of cell apoptosis experiments with Gefitinib and AC480 used alone or in combination are shown in Figure 2. Figure 8 The results of cell cycle experiments of Gefitinib and AC480 used alone or in combination are shown in Figure 2. Fig. 9 This is the result of western blotting experiment to detect PC9 / GR cells treated with Gefitinib and AC480 alone or in combination. DETAILED DESCRIPTION
[0011] The substantive content of the present invention is further described below in conjunction with the accompanying drawings and examples. The examples are only for a better understanding of the present invention but are not limited to the scope of the present invention. The methods in the examples are conventional methods unless otherwise specified, and the reagents used are conventional commercially available reagents or reagents prepared according to conventional methods unless otherwise specified; Example 1: Constructing a Gefitinib-resistant PC9 cell line (PC9 / GR) and screening the resistant cell line using the concentration gradient method. The specific steps are as follows: 1. PC9 cells in the logarithmic growth phase were selected and placed in 1640 complete medium containing 2.5 μmol / L Gefitinib. After culturing at 37°C and 5% CO2 for 24 hours, the cells were washed with PBS and then cultured in 1640 complete medium without drugs at 37°C and 5% CO2. When the cell confluence reached 80-90%, they were digested and passaged. On the second day of passage, the cells were cultured in 1640 complete medium containing 2.5 μmol / L Gefitinib for 24 hours. The above process was repeated twice to adapt the cells to the drug environment at this concentration. 2. After the cells have adapted to the initial drug concentration, gradually increase the drug concentration by 1 μmol / L each time until it reaches 20 μmol / L, and repeat each concentration 2-3 times. After changing the concentration, the process is the same as step 1; 3. On the basis of 20μmol / L, increase the drug concentration by 5μmol / L each time until it reaches 50μmol / L. Repeat each concentration 6-8 times. After changing the concentration, the process is the same as step 1. During the screening period, the drug resistance index of the drug-resistant cell line was calculated. When the drug resistance index was greater than 5, the drug-resistant strain PC9 / GR was successfully constructed. The results are as follows: Figure 1-2 As shown, the drug resistance index RI = drug-resistant cell line IC 50 / Parental cell line IC 50 , RI=100 (RI>5 indicates that the PC9 / GR cell line was successfully constructed).
[0012] Example 2: Using Western Blotting to Detect Protein Expression Levels in PC9 Cells and Drug-Resistant PC9 / GR Cells after Gefitinib Treatment After PC9 cells and drug-resistant PC9 / GR cell lines were counted in a cell counting plate, the cells were evenly plated in a six-well plate according to the cell plating method and cultured at 37°C and 5% CO2 for 24 hours. When the cells grew to 60%, PC9 and PC9 / GR cells were treated with 0μM, 0.5μM, and 1μM Gefitinib for 48 hours, respectively. The cell culture plate was removed, the culture medium was discarded, and an appropriate amount of RIPA lysis buffer was immediately added to lyse the cells, and the protein was extracted for western blotting experiments. The results are shown in Figure 2. Figure 3 As shown, after treatment with the same concentration of Gefitinib, compared with PC-9 cells, the expression of p-AKT, p-ERK, p-EGFR, and p-HER2 in PC9 / GR cell lines could not be completely inhibited, and the downstream signaling pathways continued to be activated.
[0013] Example 3: Cytotoxicity experiment of AC480 treating drug-resistant PC9 / GR cells The drug-resistant PC9 / GR cells of non-small cell lung cancer in the logarithmic growth phase were collected and evenly inoculated in a 96-well plate. After the cells were attached to the wall, different concentrations of the drug AC480 were added for treatment. There were 3 replicate wells for each drug concentration. The concentrations of AC480 were 0, 0.001, 0.01, 1, 10, 100, 500, 1000, 5000, and 10000 μM. After drug treatment for 48 hours, CCK-8 was added. After incubation for 1 hour at 37°C and 5% CO2, the OD values were measured at 450 and 630 nm with an enzyme reader, and the cell proliferation curve was drawn. The results are shown in the figure. Figure 4 As shown, the IC of AC480 on PC9 / GR cells 50 The value is 5.589 μM.
[0014] Example 4: Cell proliferation experiment of PC-9 cells and PC9 / GR cells treated with Gefitinib combined with AC480 The IC of the two drugs after acting on the cells was counted by the above CCK-8 method. 50 The constant combination ratio (Gefitinib:AC480=3:1, Gefitinib:AC480=1:1, Gefitinib:AC480=1:3) was designed by diagonal constant combination for concentration ratio. The compatibility data points were calculated using 1 / 4× (IC 50 )、1 / 2×(IC 50 )、1×IC 50 , 2×IC 50 and 4×IC 50 Count the combined index. Add 100 μL of complete medium and 3,000 PC-9 or PC9 / GR cells to each well. After adherence, add AC480 and Gefitinib at a concentration of 1 / 4× (IC 50 )、1 / 2×(IC 50 )、1×IC 50 , 2×IC 50 and 4×IC 50; After 48 hours of culture, CCK-8 reagent was added, and the OD values were measured at 450 and 630 nm after incubation at 37°C and 5% CO2 for 1 hour. The combination index was counted using Calcusyn software to determine whether AC480 and Gefitinib had a synergistic effect when used in combination. The combined use of drugs can maximize the efficacy of the drugs. When using multiple drugs, drug dosage is particularly important. The combination index (CI) of drugs judges the combined effect of drugs in a quantitative way: when the CI value is less than 1, it has a synergistic effect; when the CI value is equal to 1, it has an additive effect; when the CI value is greater than 1, it has an antagonistic effect; the CI value decreases with the increase of the ED value, indicating that the high-dose combination effect is better; the CI value increases with the increase of the ED value (such as ED 50 When CI = 0.5, ED 90 CI = 1.2), indicating that high-dose combination may lose synergy.
[0015] The results of the combined effects of different drug ratios are as follows Figure 5 As shown, the CI values of cells at ED50, ED75, ED90, and ED95 of drugs in different ratios are shown in the following table:
[0016] The results showed that the CI value decreased with the increase of ED value, indicating that the high-dose combination effect was better. Under the action of effective concentration ED95, the CI value was less than 1 when AC480 and Gefitinib were used in combination at 3:1, 1:1 and 1:3, indicating that AC480 and Gefitinib had a combined effect at the ratios of 3:1, 1:1 and 1:3, and when AC480 and Gefitinib were used in combination at 3:1, the CI value was <0.1, indicating that the two drugs had a strong synergistic effect when used in a 3:1 ratio.
[0017] Example 5: Experiment on the formation of drug-resistant PC9 / GR cell clones after Gefitinib and AC480 alone or in combination PC9 / GR cells were treated with 3μM AC480, 1μM Gefitinib, and 1μM Gefitinib + 3μM AC480 (1:3) mixed drugs, and no drug was added as a blank control. The results are as follows Figure 6The results showed that both the AC480 single-drug group and the Gefitinib single-drug group could effectively inhibit the proliferation and survival of Gefitinib-resistant lung cancer cells; the number of cell clones in the AC480+Gefitinib group was significantly less than that in the AC480 single-drug group and the Gefitinib single-drug group, indicating that the combination of AC480 and Gefitinib can more effectively inhibit the proliferation and survival of Gefitinib-resistant lung cancer cells; the results showed that the combination of AC480 and Gefitinib can reduce the acquired resistance of PC9 / GR cells to Gefitinib.
[0018] Example 6: Apoptosis experiment of drug-resistant PC9 / GR cells treated with Gefitinib or AC480 alone or in combination Abbkine Annexin V-AbFluor™ 488 / PI double staining apoptosis detection kit was used to count PC9 / GR cells and evenly spread them in a six-well plate. After culturing at 37°C and 5% CO2 for 24 hours, the cells were divided into four groups when they grew to 60%. The first group was replaced with fresh culture medium, the second group was added with Gefitinib at a final concentration of 1μM, the third group was added with AC480 at a final concentration of 3μM, and the fourth group was added with Gefitinib and AC480 at final concentrations of 1μM and 3μM, respectively. PC9 / GR cells were treated for 24 hours, the cell culture plate was removed, the cells were digested with trypsin, and the cells were collected by centrifugation at 4°C and 300g for 5 minutes, washed twice with ice-cold PBS, and resuspended with 500μL 1×Annexin V Binding Buffer; 5μL Annexin V-AbFluor™488 and 2μL PI, gently mix; incubate at room temperature in the dark for 15 min; place on ice after incubation, and analyze cells by flow cytometry within 30 min after staining.
[0019] Results Figure 7 ,The results showed that the combination of AC480 and Gefitinib could induce apoptosis of PC9 / GR cells more effectively than single drug treatment.
[0020] Example 7: Detection of cell cycle arrest in drug-resistant PC9 / GR cells treated with Gefitinib, AC480 alone or in combination Using the Cell Cycle and Apoptosis Analysis Kit, PC9 / GR cells were counted and evenly spread in a six-well plate. When the cells grew to 60% at 37°C and 5% CO2 for 24 hours, they were divided into four groups. The first group was replaced with fresh culture medium, the second group was added with Gefitinib at a final concentration of 1μM, the third group was added with AC480 at a final concentration of 3μM, and the fourth group was added with Gefitinib and AC480 at a final concentration of 1μM and 3μM respectively. PC9 / GR cells were treated for 24 hours, the cell culture plate was removed, the cells were digested with trypsin, the collected cells were centrifuged at about 1000g for 3-5min, and the cells were precipitated; 1mL of pre-cooled PBS was added to resuspend the cells and transferred to a 1.5mL centrifuge tube. Centrifuge at 1000g for 3-5min again to precipitate the cells, and the supernatant was carefully aspirated and discarded, leaving about 50μL to avoid aspirating the cells. Gently tap the bottom of the centrifuge tube to properly disperse the cells and avoid cell agglomeration; add 1mL PBS to fully resuspend into single cells, gently vortex and slowly add 3mL pre-cooled anhydrous ethanol to a final concentration of 75%, and fix at 4℃ for 4h; centrifuge the fixed cell suspension at 1000g for 3-5min to precipitate the cells, and carefully aspirate the supernatant; add 1mL pre-cooled PBS to resuspend the cells, centrifuge at 1000g for 3-5min again to precipitate the cells, and carefully aspirate the supernatant; mix the staining buffer: propidium iodide PI staining solution (20X): RNase A (50X) = 100:5:2 to prepare the staining working solution; add 500μL of staining working solution to each tube of cell sample, gently and fully resuspend the cell pellet, and warm at 37℃ in the dark for 30min. After staining, store at 4℃ in the dark and then detect on the flow cytometer.
[0021] Results Figure 8 The results showed that compared with the blank control group, the combination of AC480 and Gefitinib and the single-drug treatment of AC480 and Gefitinib blocked the cell cycle progression of PCR / GR cells at the G1 phase, and the blocking effect of the combination of AC480 and Gefitinib was more obvious.
[0022] Example 8: Protein expression in drug-resistant PC9 / GR cells treated with Gefitinib, AC480 alone or in combination The PC9 / GR cells were counted and evenly spread in a six-well plate. They were cultured at 37°C and 5% CO2 for 24 hours. When the cells grew to 60%, they were divided into four groups. The first group used fresh culture medium to replace the medium, the second group was added with Gefitinib at a final concentration of 1 μM, the third group was added with AC480 at a final concentration of 3 μM, and the fourth group was added with Gefitinib and AC480 at final concentrations of 1 μM and 3 μM, respectively. PC9 / GR cells were treated for 48 hours, the cell culture plate was removed, the culture medium was discarded, and an appropriate amount of RIPA lysis buffer was immediately added to lyse the cells, the protein was extracted, and protein immunoblotting experiments were performed.
[0023] The results are as follows Fig. 9 As shown in the data, the combination of AC480 and Gefitinib can significantly inhibit the phosphorylation of HER2, EGFR, AKT and ERK, which is more obvious than when AC480 and Gefitinib are used alone. This shows that the combination of AC480 and Gefitinib can reverse the drug resistance of cells and restore the sensitivity of PC9 / GR to Gefitinib, which may be through downregulating PI3K / AKT and MAPK signaling pathways to inhibit cell proliferation.
Claims
1. Application of gefitinib combined with AC480 in the preparation of drugs for the treatment of drug-resistant non-small cell lung cancer; The chemical structural formula of the AC480 is as follows: 。 2. The use according to claim 1, characterized in that: AC480 can enhance the drug sensitivity of gefitinib-resistant non-small cell lung cancer cells to gefitinib.