Pharmaceutical composition for treating osimertinib drug-resistant non-small cell lung cancer and application thereof

Abstract

The invention provides a pharmaceutical composition for treating osimertinib-resistant non-small cell lung cancer and application of the pharmaceutical composition, and belongs to the technical field of biological medicines. According to the present invention, the ORY1001 and the osimertinib are combined to provide the synergistic inhibition effect in the third generation EGRF inhibitor osimertinib drug-resistant non-small cell lung cancer PC9-OR and H1975-OR cells, and the drug resistance of the non-small cell lung cancer PC9-OR and H1975-OR caused by the osimertinib can be significantly reduced.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and in particular, to a pharmaceutical composition for treating osimertinib-resistant non-small cell lung cancer and its application. Background Art

[0002] Non-small cell lung cancer (NSCLC) accounts for approximately 80% of the total number of lung cancer patients. According to different tissue structures, it is divided into three main subtypes: squamous cell carcinoma, adenocarcinoma, and large cell carcinoma. Epidermal growth factor receptor (EGFR) is a key factor controlling the growth and survival of epithelial cells, and its abnormal activation caused by high expression or mutation is related to tumor progression and treatment resistance of various cancers including non-small cell lung cancer (NSCLC). Therefore, EGFR inhibitors (EGFRi) are often used for targeted therapy of epithelial malignancies.

[0003] Osimertinib is a third-generation EGFR inhibitor and is often used for the treatment of non-small cell lung cancer patients. Although most patients have a good initial response, drug resistance inevitably occurs after about 2 years. For patients who develop resistance to the third-generation EGRF inhibitor, they have to choose chemotherapy regimens with greater toxicity and side effects, resulting in a significant decline in the quality of life and survival period of the patients. Therefore, providing a medication regimen for patients with osimertinib resistance is of great significance for improving the quality of life of patients and prolonging the survival period. Summary of the Invention

[0004] The purpose of the present invention is to provide a pharmaceutical composition for treating osimertinib-resistant non-small cell lung cancer and its application, and the combined use of ORY1001 and osimertinib has a synergistic inhibitory effect on osimertinib-resistant non-small cell lung cancer.

[0005] The present invention provides a pharmaceutical composition for treating osimertinib-resistant non-small cell lung cancer, and the composition is an EGFR inhibitor and an LSD1 inhibitor.

[0006] Preferably, the EGFR inhibitor is Osimertinib, and the LSD1 inhibitor is ORY1001.

[0007] The present invention also provides an application of the above pharmaceutical composition in the preparation of a drug for treating osimertinib-resistant non-small cell lung cancer.

[0008] Preferably, the cell lines of osimertinib-resistant non-small cell lung cancer are H1975-OR and PC9-OR.

[0009] Preferably, the drug further comprises a pharmaceutically acceptable excipient.

[0010] In the present invention, the combined use of ORY1001 and osimertinib has a significantly enhanced inhibitory effect on non-small cell lung cancer PC9-OR and H1975-OR cells resistant to the third-generation EGRF inhibitor osimertinib. The proliferation inhibition rate of the combined drug is significantly higher than that of the single drug, and the synergy index CI value of the two is less than 1. It can be seen that the combined use of ORY1001 and osimertinib in the present invention has a synergistic effect in the treatment of osimertinib-resistant non-small cell lung cancer, and can significantly reduce the drug resistance of non-small cell lung cancer PC9-OR and H1975-OR caused by osimertinib. Description of the Drawings

[0011] Figure 1 In [the figure], A is the inhibition rate of different concentrations of osimertinib on H1975 cells and H1975-OR cells in Example 1, and B is the inhibition rate of different concentrations of osimertinib on PC9 cells and PC9-OR cells in Example 1;

[0012] Figure 2 is the inhibitory effect of different concentrations of ORY1001 alone and different concentrations of ORY1001 combined with osimertinib on PC9-OR cells (A) and H1975-OR cells (B) in Example 2;

[0013] Figure 3 is the microscopic result diagram of the colony formation assay of PC9-OR (A) and H1975-OR (B) when ORY1001, osimertinib are used alone and in combination in Example 3;

[0014] Figure 4 is the statistical result diagram of the number of cell colonies formed in the colony formation assay of PC9-OR (A) and H1975-OR (B) when ORY1001, osimertinib are used alone and in combination in Example 3;

[0015] Figure 5 is the anti-tumor effect diagram of ORY1001 and osimertinib in Example 4, where A is the volume of the tumor under different treatment regimens, B is the body weight of the mice under different treatment regimens, C is the morphology of the tumor under different treatment regimens, and D is the weight of the tumor under different treatment regimens. Detailed Embodiments

[0016] The technical solutions provided by the present invention will be described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0017] 1. Experimental raw materials

[0018] (1) PC9 cells were provided by the Stem Cell Bank of the Chinese Academy of Sciences. PC9 osimertinib-resistant cells (PC9-OR) were constructed by this laboratory according to the method of increasing concentration gradient. The specific operation method is as follows: The IC 50 of parental PC9 cells was determined by the CCK8 method, and about half of the IC 50 concentration was selected as the starting concentration to culture parental PC9 cells. The drug was added to the culture medium (RPMI-1640 medium + 10% South American fetal bovine serum), and PC9 cells were placed in the above culture medium containing the drug and cultured in a cell incubator. After culturing for about 72 h, the cells were restored to be cultured with the drug-free culture medium (RPMI-1640 medium + 10% South American fetal bovine serum). When the cells grew to about 80% confluence, the cells were treated multiple times with the same concentration (half of the IC 50 concentration) of the drug until the cells could grow stably under this drug concentration, and they were used as the first-generation resistant cells. The above steps were repeated by gradually increasing the drug concentration. The dosing concentrations of osimertinib were successively: 50 nM, 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 350 nM, 400 nM, 450 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, 1000 nM, 1100 nM, 1200 nM, 1300 nM, 1400 nM, 1500 nM, 1600 nM, 1700 nM, 1800 nM, 1900 nM, 2000 nM, 2100 nM, 2200 nM, 2300 nM, 2400 nM, 2500 nM, and the 30th-generation resistant cells constructed were used for experiments.

[0019] (2) H1975 cells were provided by the Stem Cell Bank of the Chinese Academy of Sciences. H1975 osimertinib-resistant cells (H1975-OR) were constructed by this laboratory according to the method of increasing concentration gradient. The dosing concentrations of osimertinib were successively: 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, 1000 nM, 1100 nM, 1200 nM, 1300 nM, 1400 nM, 1600 nM, 1800 nM, 2000 nM, 2200 nM, 2400 nM, 2600 nM, 2800 nM, 3000 nM, 3200 nM, 3400 nM, 3600 nM, 4000 nM, 4400 nM, 4800 nM, 5200 nM, 5600 nM, 6000 nM. The construction method was the same as that shown for PC9-OR above, and the 30th-generation resistant cells constructed were used for experiments.

[0020] (3) Osimertinib and ORY1001 were purchased from TargetMol (Shanghai, China).

[0021] (4) The CCK8 assay kit was purchased from TargetMol (Shanghai, China).

[0022] (5) Fetal bovine serum (SORFA, #SX1500) was purchased from Zhengzhou Xinsemei Biotechnology Co., Ltd.

[0023] (6) RPMI-1640 medium (500 mL, #L220KJ) was purchased from Shanghai Yuanpei Biotechnology Co., Ltd.

[0024] 2. Cell culture medium

[0025] The 500 mL of RPMI-1640 medium was aliquoted into 45 mL per portion in a laminar flow hood, and 5 mL of fetal bovine serum was added to each portion. Then it was stored at 4 °C in the refrigerator for later use.

[0026] 3. Determination of cell viability or proliferation inhibition rate by CCK8 method

[0027] PC9 cells in the logarithmic growth phase were taken. After cell digestion, termination, centrifugation and other steps, they were resuspended with the above cell culture medium to make the cell suspension density about 4×10 4 cells / mL. 100 μL of the culture solution containing about 4×10 3 cells was added to each well of a 96-well plate and cultured overnight in a cell culture incubator to allow the cells in the 96-well plate to adhere. The conditions of the cell culture incubator were a constant temperature of 37 °C, 5% CO2, and 95% saturated humidity.

[0028] Example 1

[0029] 1. Take the H1975 cells and H1975 osimertinib-resistant cells (H1975-OR) inoculated in 96-well plates as the experimental groups, and add drugs of different concentrations of Osimertinib (80.00 μM, 26.67 μM, 8.89 μM, 2.96 μM, 0.99 μM, 0.33 μM, 0.11 μM, 0.011 μM, 0.0011 μM, 0.00011 μM, 0.000011 μM, 0.0000011 μM) respectively. Use the medium containing DMSO (concentration 0.1%) as the control group; the medium is used as the blank group. After acting for 72 h, add 20 μL of CCK8 to each well and place it in an incubator (the conditions of the cell incubator are a constant temperature of 37 °C, 5% CO2, and 95% saturated humidity) for 90 min. Take out the 96-well plate and measure the light absorption value (OD value) at 450 nm with an enzyme-labeled instrument. Calculate the IC50 and cell survival rate. The cell survival rate is calculated according to the following formula: Survival rate = (OD value of the drug-added group - OD value of the blank group) / (OD value of the control group - OD value of the blank group) × 100%. The inhibition rate of cell proliferation is calculated according to the following formula: Inhibition rate = 1 - survival rate. The results are as Figure 1 shown in A of

[0030] 2. Take the PC9 cells and PC9 osimertinib-resistant cells (PC9-OR) inoculated in 96-well plates as the experimental groups, and add drugs of different concentrations of Osimertinib (80.00 μM, 26.67 μM, 8.89 μM, 2.96 μM, 0.99 μM, 0.33 μM, 0.11 μM, 0.011 μM, 0.0011 μM, 0.00011 μM, 0.000011 μM, 0.0000011 μM) respectively. Use the medium containing DMSO (concentration 0.1%) as the control group; the medium is used as the blank group. After acting for 72 h, add 20 μL of CCK8 to each well and place it in an incubator (the conditions of the cell incubator are a constant temperature of 37 °C, 5% CO2, and 95% saturated humidity) for 90 min. Take out the 96-well plate and measure the light absorption value (OD value) at 450 nm with an enzyme-labeled instrument. Calculate the IC 50 50 and cell inhibition rate. The cell survival rate is calculated according to the following formula: Survival rate = (OD value of the drug-added group - OD value of the blank group) / (OD value of the control group - OD value of the blank group) × 100%. The inhibition rate of cell proliferation is calculated according to the following formula: Inhibition rate = 1 - survival rate. The results are as Figure 1 shown in B of

[0031] It can be Figure 1 seen that the IC 50Compared with H1975, it was significantly increased from 0.378 μM to 4.725 μM. The sensitivity of drug-resistant cells to the drug decreased by 12.61 times. The IC of osimertinib against PC9-OR 50 Compared with PC9, it was also significantly increased from 0.124 μM to 1.477 μM. The sensitivity of drug-resistant cells to the drug decreased by 11.91 times. It can be seen that the inhibitory effect of the drug on drug-resistant cells was significantly reduced.

[0032] Example 2

[0033] 1. Take PC9 osimertinib-resistant cells (PC9-OR) inoculated in 96-well plates, and add different concentrations of ORY1001 (160.00 μM, 80.00 μM, 40.00 μM, 20.00 μM, 10.00 μM, 5.00 μM, 2.50 μM, 1.25 μM) and different concentrations of ORY1001 (160.00 μM, 80.00 μM, 40.00 μM, 20.00 μM, 10.00 μM, 5.00 μM, 2.50 μM, 1.25 μM) + Osimertinib (3.00 μM) drugs. Use the medium containing DMSO (concentration 0.1%) as the control group; the medium is used as the blank group. After acting for 72 h, add 20 μL of CCK8 to each well and place it in an incubator (the conditions of the cell incubator are a constant temperature of 37 °C, 5% CO2, and 95% saturated humidity) for 90 min. Take out the 96-well plate and measure the light absorption value (OD value) at 450 nm with an enzyme-labeled instrument. Measure the IC 50 value and calculate the cell inhibition rate, and use Calcusyn software to calculate the CI combination index. The results are as shown in Figure 2 A shown below.

[0034] 2. Take H1975 osimertinib-resistant cells (H1975-OR) inoculated in 96-well plates, and add different concentrations of ORY1001 (160.00 μM, 80.00 μM, 40.00 μM, 20.00 μM, 10.00 μM, 5.00 μM, 2.50 μM, 1.25 μM) and different concentrations of ORY1001 (160.00 μM, 80.00 μM, 40.00 μM, 20.00 μM, 10.00 μM, 5.00 μM, 2.50 μM, 1.25 μM) + Osimertinib (3.00 μM) drugs. Use the medium containing DMSO (concentration 0.1%) as the control group; the medium is used as the blank group. After acting for 72 h, add 20 μL of CCK8 to each well and place it in an incubator (the conditions of the cell incubator are a constant temperature of 37 °C, 5% CO2, and 95% saturated humidity) for 90 min. Take out the 96-well plate and measure the light absorption value (OD value) at 450 nm with an enzyme-labeled instrument. Measure the IC 50Values were calculated and the cell inhibition rate was determined. The combination index (CI) was calculated using Calcusyn software. The results are shown as Figure 2 shown in B below.

[0035] As can be seen from Figure 2 the results, for the PC9 osimertinib-resistant cells (PC9-OR), the IC 50 value of ORY1001 + osimertinib combination was significantly lower than that of ORY1001 alone. The combination index of ORY1001 + osimertinib was 0.5 < 0.9, indicating a synergistic effect between the two. For the H1975 osimertinib-resistant cells (H1975-OR), the IC 50 value of ORY1001 + osimertinib combination was 10.75 μM, which was significantly lower than that of ORY1001 alone. The combination index of ORY1001 + osimertinib was 0.495 < 0.9, indicating a synergistic effect between the two.

[0036] Example 3

[0037] PC9-OR cells and H1975-OR cells in the logarithmic growth phase were digested with trypsin and then resuspended in cell culture medium (RPMI-1640 medium + 10% fetal bovine serum) to form cell suspensions. The cell suspensions were seeded into 24-well plates, with 100 cells seeded in each well. The cells were divided into a control group (added with normal saline), an ORY1001 group (added with 1.00 μM ORY1001), an osimertinib group (added with 1.00 μM osimertinib), and an ORY1001 + osimertinib group (added with 1.00 μM ORY1001 and 1.00 μM osimertinib simultaneously), with 3 replicates in each group. The plates were placed in a CO2 incubator and cultured at 37 °C, 5% CO2, and saturated humidity for 14 days. After the culture was completed, the culture medium was discarded, and the cells were washed twice with PBS. 1 mL of 4% paraformaldehyde was added to each well to fix the cells for 30 min, and then the cells were washed once with PBS. 1 mL of crystal violet staining solution was added to stain the cells for 15 min. After washing the cells three times with PBS, the plates were air-dried, and the number of colonies formed was counted by photographing under a microscope. The results are shown as Figure 3 and 4 shown below.

[0038] As can be seen from Figure 3 and 4It can be seen that both ORY1001 and Osimertinib alone can inhibit the cloning of PC9-OR cells and H1975-OR cells to a certain extent. When used alone, Osimertinib has a stronger inhibitory effect on PC9-OR cells, and ORY1001 has a stronger inhibitory effect on H1975-OR cells. However, when ORY1001 + Osimertinib are used in combination, they have a very significant inhibitory effect on both PC9-OR cells and H1975-OR cells, and the number of cell clones formed is significantly reduced.

[0039] Example 4

[0040] A cell line xenograft model was constructed. The cultured drug-resistant cells PC9-OR were inoculated into nude mice with an inoculation volume of 0.20 mL (i.e., 10 million cells in 0.20 mL of PBS). The inoculated mice were divided into four groups. One group was given normal saline as a control, and the other three groups were given ORY1001 (administration frequency once a week, dose 350.00 μg / kg), Osimertinib (administration frequency once a day, dose 2.00 mg / kg), and ORY1001 + Osimertinib (where ORY1001 administration frequency is once a week, dose 350.00 μg / kg; Osimertinib administration frequency is once a day, dose 2.00 mg / kg), respectively. During this period, the morphological changes of the tumors and the body weights of the mice were observed daily. After 40 days, the tumor tissues were taken to observe the morphology and weigh the tumor weights. The results are as Figure 5 shown.

[0041] As Figure 5 shown in A, the tumor volume of the mice in the ORY1001 + Osimertinib combination treatment group increased the slowest, while the tumors in the other three groups increased significantly. It can be seen that the combination treatment can significantly inhibit the tumors of the mice. As shown in B, all the treatment groups will cause a certain degree of decrease in the body weight of the mice, but in the ORY1001 + Osimertinib combination treatment regimen, the body weight of the mice will gradually recover after the 20th day. It can be seen that the combination treatment regimen also has a relatively small impact on the body weight of the mice. As shown in C, compared with the control group, each treatment group can reduce the size of the tumor to a certain extent, and the combination treatment group has the best effect, extremely significantly inhibiting the growth of the tumor. As shown in D, compared with the control group, the weights of the tumors in each treatment group have decreased, and the combination treatment group (COM) has the best effect. In summary, the combination of ORY1001 + Osimertinib has a synergistic inhibitory effect on the osimertinib-resistant cells PC9-OR.

[0042] 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 osimertinib-resistant non-small cell lung cancer, characterized in that, The composition is an EGFR inhibitor and an LSD1 inhibitor.

2. The pharmaceutical composition according to claim 1, wherein The EGFR inhibitor is Osimertinib, and the LSD1 inhibitor is ORY1001.

3. Use of the pharmaceutical composition according to claim 1 or 2 in the preparation of a medicament for treating osimertinib-resistant non-small cell lung cancer.

4. The application according to claim 3, characterized in that, The cell lines of osimertinib-resistant non-small cell lung cancer are H1975-OR and PC9-OR.

5. The application according to claim 4, characterized in that, The medicament further comprises a pharmaceutically acceptable excipient.

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