A composition for treating non-small cell lung cancer and its application in preparing medicine

Abstract

The present invention provides a composition for treating non-small cell lung cancer and its application in preparing drugs, and the present invention belongs to the field of biomedicine technology. The composition provided by the present invention is an EGFR inhibitor and an MTH1 inhibitor. The present invention uses the EGFR inhibitor and the MTH1 inhibitor for combined use, and the inhibition rate of non-small cell lung cancer PC-9 cell proliferation is significantly higher than that of single use, especially the synergistic index CI value of the two is less than 1, indicating that the combined use has a synergistic inhibitory effect on non-small cell lung cancer cells PC-9, and greatly reduces the drug resistance of non-small cell lung cancer PC-9OR caused by osimertinib.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a composition for treating non-small cell lung cancer and an application thereof in preparing medicines. Background Art

[0002] Non-small cell lung cancer (NSCLC) accounts for about 85% of all lung cancer patients. According to its histopathological type, NSCLC is divided into three subtypes: squamous cell carcinoma, adenocarcinoma, and large cell carcinoma. Epidermal growth factor receptor (EGFR) is a key factor in controlling the growth and survival of epithelial cells. Its abnormal activation caused by high expression or mutation is closely related to the progression and drug resistance of various tumors including NSCLC. Therefore, EGFR is one of the earliest discovered and most intensively studied anti-tumor targets. The EGFR mutation rate in NSCLC is as high as 30%, and EGFR inhibitors are the first-line targeted treatment for NSCLC.

[0003] EGFR inhibitors are small molecule inhibitors that compete with ATP for the intracellular tyrosine kinase catalytic site of EGFR to block the kinase activity of EGFR, thereby inhibiting the malignant proliferation of lung cancer cells. However, a large amount of clinical and experimental data show that after treatment with EGFR inhibitors, most patients will develop acquired drug resistance.

[0004] Studies have shown that acquired T790M mutation is the most common cause of resistance in patients with advanced EGFR mutations. Most patients develop T790M mutations after first-line use of first-generation EGRF inhibitors (such as gefitinib). If T790M resistance occurs, it can be treated with the third-generation EGFR-TKI (osimertinib). Unfortunately, however, patients still develop resistance after several months of treatment with osimertinib. Patients who become resistant to third-generation EGRF inhibitors have to choose chemotherapy regimens with greater toxicity and side effects, resulting in a significant decrease in the quality of life and survival of patients.

[0005] At present, the application of chemotherapy to treat advanced NSCLC has reached a plateau. In order to further improve the treatment effect of advanced NSCLC, a number of randomized clinical trials have evaluated the efficacy and safety of molecular targeted drugs combined with chemotherapy as the first-line treatment for advanced NSCLC. Among various targeted drugs for the treatment of lung cancer, the results of randomized controlled studies of EGFR inhibitors gefitinib or erlotinib combined with chemotherapy as the first-line treatment for advanced NSCLC were negative, and it was not confirmed that chemotherapy combined with EGFR inhibitors can improve survival. Therefore, it is urgent to develop drugs that can be combined with EGFR inhibitors to treat non-small cell lung cancer. Summary of the invention

[0006] The object of the present invention is to provide a composition which has a high inhibition rate on non-small cell lung cancer, has a synergistic effect and greatly reduces drug resistance.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a composition for treating non-small cell lung cancer, which comprises an EGFR inhibitor and an MTH1 inhibitor.

[0009] Preferably, the EGFR inhibitor includes gefitinib, icotinib, afatinib, dacomitinib or osimertinib.

[0010] Preferably, the MTH1 inhibitors include TH588 and (S)-crizotinib.

[0011] The present invention also provides an application of the above composition in preparing a drug for treating non-small cell lung cancer.

[0012] The present invention also provides an application of the above composition in preparing a drug for treating drug-resistant non-small cell lung cancer.

[0013] The composition for treating non-small cell lung cancer provided by the present invention is a combination of an EGFR inhibitor and an MTH1 inhibitor, and the proliferation inhibition rate of non-small cell lung cancer PC-9 cells is significantly higher than that of single drug use, especially the synergy index CI value of the two is less than 1, indicating that the combination has a synergistic inhibitory effect on non-small cell lung cancer PC-9 cells. Moreover, in non-small cell lung cancer PC-9OR cells resistant to the third-generation EGRF inhibitor osimertinib, the proliferation inhibition rate of the combination of EGFR inhibitor and MTH1 inhibitor is significantly higher than that of single drug use, especially the synergy index CI value of the two is less than 1. It shows that the combination of the EGFR inhibitor and the MTH1 inhibitor of the present invention has a synergistic effect in the treatment of non-small cell lung cancer, and greatly reduces the drug resistance of non-small cell lung cancer PC-9OR caused by osimertinib. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The figures are scatter plots of the combination index CI of different concentrations of TH588 and EGFR inhibitors in non-small cell lung cancer parental cells PC-9. From left to right, the first figure is TH588 combined with Gefitinib, the second figure is TH588 combined with Icotinib, the third figure is TH588 combined with Afatinib, the fourth figure is TH588 combined with Dacomitinib, and the fifth figure is TH588 combined with Osimertinib.

[0015] Figure 2is the combination index CI of different concentrations of TH588 and different concentrations of osimertinib in osimertinib-resistant non-small cell lung cancer PC-9OR cells.

[0016] Figure 3 This is the inhibition rate curve of different concentrations of TH588 combined with different concentrations of osimertinib in osimertinib-resistant non-small cell lung cancer PC-9OR cells. DETAILED DESCRIPTION

[0017] 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.

[0018] Example 1

[0019] The survival rate and CI drug combination index of the parental cell line PC-9 cells or osimertinib-resistant cell line PC-9OR cells were calculated by combining different concentrations of EGFR inhibitors with different concentrations of MTH1 inhibitors.

[0020] 1. Experimental materials

[0021] (1) PC-9 cells were provided by the Stem Cell Bank of the Chinese Academy of Sciences. PC-9 osimertinib-resistant cells (PC-9OR) were constructed by our laboratory using the concentration gradient method. The specific operation method was as follows: the IC of parental PC-9 cells was determined by the CCK8 method. 50 , and select IC 50 The parental PC-9 cells were cultured with half of the concentration as the starting concentration, the drug was added to the culture medium (RPMI-1640 medium + 10% South American fetal bovine serum), the PC-9 cells were placed in the above culture medium containing the drug, and cultured in a cell culture incubator. After culturing for 24 hours, the culture medium without drug addition (RPMI-1640 medium + 10% South American fetal bovine serum) was resumed for culture, and the cells were cultured until the confluence of about 80% was reached using the same concentration (IC 50The cells were treated with a drug (half the concentration) for multiple times until they could stably grow under the drug concentration treatment, and they were used as the first generation of drug-resistant cells. The above steps were repeated with gradually increasing drug concentrations. The administration concentrations of osimertinib were 50nM, 100nM, 150nM, 200nM, 250nM, 300nM, 350nM, 400nM, 450nM, 500nM, 600nM, 700nM, 800nM, 900nM, 1000nM, 1100nM, 1200nM, 1300nM, 1400nM, 1500nM, 1600nM, 1700nM, 1800nM, 1900nM, 2000nM, 2100nM, 2200nM, 2300nM, 2400nM and 2500nM, and the experiment was carried out using the constructed 30th generation of drug-resistant cells.

[0022] (2) Gefitinib, icotinib, afatinib, dacomitinib, osimertinib and TH588 were purchased from Shanghai TargetMol Biotechnology Co., Ltd.

[0023] (3) CCK8 detection kit was purchased from Shanghai TargetMol Company.

[0024] (4) South American fetal bovine serum, brand SORFA (#SX1500), was purchased from Zhengzhou Xinsaimei Biotechnology Co., Ltd.

[0025] (5) RPMI-1640 medium (#L220KJ) (500 mL) was purchased from Shanghai Yuanpei Biotechnology Co., Ltd.

[0026] 2. Cell culture medium

[0027] 500 mL of RPMI-1640 culture medium was divided into 45 mL portions in a clean bench, 5 mL of fetal bovine serum was added to each portion, and the portions were stored in a 4°C refrigerator for later use.

[0028] 3. CCK8 method to determine cell survival rate or proliferation inhibition rate

[0029] PC-9 cells in the logarithmic growth phase were taken and digested, terminated, centrifuged, and resuspended in the above cell culture medium to a cell suspension density of about 4 × 10 4 100 μL of culture medium was added to each well of a 96-well plate, about 4×10 3 The cells were placed in a cell culture incubator and cultured overnight to allow the cells in the 96-well plate to adhere to the wall. The conditions of the cell culture incubator were a constant temperature of 37° C., 5% CO 2 , and saturated humidity of 95%.

[0030] Experimental and control group settings

[0031] Experimental group 1 was to add different concentrations of EGFR inhibitors: such as 0.0, 2.0, 4.0, 6.0, 8.0, 10.0 μmol of Gefitinib or Icotinib per L of cell system; such as 0.0, 0.15625, 0.3125, 0.625, 1.25, 2.5 μmol of Afatinib or Dacomitinib or Osimertinib per L of cell system.

[0032] Experimental group 2 was added with different concentrations of TH588, which were 0.0, 2.0, 4.0, 6.0, 8.0, and 10.0 μmol of TH588 per L of cell system.

[0033] Experimental group 3 added different concentrations of combined drugs, EGFR inhibitor + TH588; the culture medium containing DMSO (concentration 0.1%) was used as the control group; the culture medium was used as the blank group; such as Gefitinib + TH588 or Icotinib + TH588, the groups were set to 2.0μM + 2.0μM, 4.0μM + 4.0μM, 6.0μM + 6.0μM, 8.0μM + 8.0μM, 10.0μM + 10.0μM; such as Afatinib + TH588 or Dacomitinib + TH588 or Osimertinib + TH588, the groups were set to 0.15625μM + 2.0μM, 0.3125μM + 4.0μM, 0.625μM + 6.0μM, 1.25μM + 8.0μM, 2.5μM + 10.0μM.

[0034] After 72 hours of action, 20 μL CCK8 was added to each well and placed in an incubator (the conditions of the cell incubator were a constant temperature of 37°C, 5% CO2, and 95% saturated humidity) for 90 minutes. The 96-well plate was taken out and the light absorption value (OD value) was measured at 450nm using an ELISA reader. The survival rate of cell proliferation was 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 was calculated according to the following formula: inhibition rate = 1-survival rate.

[0035] 4. Calcusyn software calculates CI combination index

[0036] Calcusyn software was used to calculate the combination index CI using the inhibition rate. In the software operation interface, the gradient concentration of different drugs, the inhibition rate of the drug alone, the specific ratio of the two drugs, and the inhibition rate of the drug combination were input, and the drug combination index was calculated using the Chou-Talay formula built into the software. 0.9≤CI≤1.1 is an additive effect; CI≤0.9 is considered to have a potential synergistic effect between the two drugs.

[0037] 5. Experimental results

[0038] Table 1. CI combination index of parental PC-9 cells at different concentrations of EGFR inhibitors and different concentrations of TH588 (n=3)

[0039]

[0040] Figure 1 The scatter plot of the combination index CI of different concentrations of TH588 and EGFR inhibitor in non-small cell lung cancer parental cells PC-9 was prepared using the data in Table 1. Figure 1 It can be seen that in the parental cell line of non-small cell lung cancer PC-9, after 72 hours of combined action of Gefitinib, one of the EGFR inhibitors, and TH588, the combination index CI was between 0.428 and 0.622, both less than 0.9, so the combination of Gefitinib, one of the EGFR inhibitors, and TH588 had a synergistic effect; after 72 hours of combined action of Icotinib, one of the EGFR inhibitors, and TH588, the combination index CI was between 0.228 and 0.366, both less than 0.9, so the combination of Icotinib, one of the EGFR inhibitors, and TH588 had a synergistic effect; after 72 hours of combined action of Afatinib, one of the EGFR inhibitors, and TH588, the combination index CI was between 0.4 The CI of the combination of Afatinib, one of the EGFR inhibitors, and TH588 was between 0.31 and 0.530, both less than 0.9, so the combination of Afatinib, one of the EGFR inhibitors, and TH588 had a synergistic effect; after 72 hours of combined action of Dacomitinib, one of the EGFR inhibitors, and TH588, the CI of the combination was between 0.524 and 0.713, both less than 0.9, so the combination of Dacomitinib, one of the EGFR inhibitors, and TH588 had a synergistic effect; after 72 hours of combined action of Osimertinib, one of the EGFR inhibitors, and TH588, the CI of the combination was between 0.479 and 0.609, both less than 0.9, so the combination of Osimertinib, one of the EGFR inhibitors, and TH588 had a synergistic effect. The above data show that the combination of EGFR inhibitors and TH588 has a synergistic inhibitory effect on non-small cell lung cancer parental cells PC-9.

[0041] The osimertinib-resistant PC-9OR cells were tested according to the above experimental steps.

[0042] Experimental and control group settings

[0043] Experimental group 4 added different concentrations of osimertinib: 0.0, 0.15625, 0.3125, 0.625, 1.25, and 2.5 μmol of osimertinib per L of cell system.

[0044] Experimental group 5 added different concentrations of TH588, which were 0.0, 2.0, 4.0, 6.0, 8.0, and 10.0 μmol of TH588 per L of cell system.

[0045] Experimental group 6 added different concentrations of combined drugs, Osimertinib + TH588, and the groups were set as 0.15625μM + 2.0μM, 0.3125μM + 4.0μM, 0.625μM + 6.0μM, 1.25μM + 8.0μM, and 2.5μM + 10.0μM.

[0046] Calculate the survival rate. Figure 2 As shown, after Osimertinib and TH588 were used alone in the Osimertinib-resistant cell line PC-9OR for 72 hours, the cell survival rate decreased with the increase of drug concentration, and the inhibitory effect on cell proliferation was significantly enhanced; the inhibition rate of the group with combined action of Osimertinib and TH588 was significantly higher than that of the corresponding group with single drug use.

[0047] The calculated CI values ​​are shown in Table 2. Figure 3 It is a scatter plot of CI values ​​based on Table 2.

[0048] Table 2 CI combination index of different concentrations of osimertinib and different concentrations of TH588 in osimertinib-resistant PC-9OR cells (n=1)

[0049]

[0050] From Table 2 and Figure 3 It can be seen that in the Osimertinib-resistant cell line PC-9OR of non-small cell lung cancer, after 72 hours of combined action of Osimertinib, one of the EGFR inhibitors, and TH588, the combination index CI was between 0.445 and 0.611, both less than 0.9. Therefore, the combination of Osimertinib, one of the EGFR inhibitors, and TH588 still has a synergistic effect in Osimertinib-resistant cells PC-9OR.

[0051] The above data show that the combination of EGFR inhibitors Osimertinib and TH588 has a synergistic inhibitory effect on Osimertinib-resistant PC-9OR cells of non-small cell lung cancer.

[0052] Example 2

[0053] The cell inhibition rate of the parental cell line PC-9 cells was calculated by combining a fixed concentration combination of EGFR inhibitors with different concentrations of TH588.

[0054] 1. Experimental materials

[0055] (1) PC-9 cells were provided by the Stem Cell Bank of the Chinese Academy of Sciences. Cells were cultured in a drug-free culture medium (RPMI-1640 medium + 10% South American fetal bovine serum).

[0056] (2) Gefitinib, Icotinib, Afatinib, Dacomitinib, Osimertinib and TH588 were purchased from Shanghai TargetMol Co., Ltd.

[0057] (3) CCK8 detection kit was purchased from Shanghai TargetMol Company.

[0058] (4) South American fetal bovine serum, brand SORFA (#SX1500), was purchased from Zhengzhou Xinsaimei Biotechnology Co., Ltd.

[0059] (5) RPMI-1640 medium (#L220KJ) (500 mL) was purchased from Shanghai Yuanpei Biotechnology Co., Ltd.

[0060] 2. Cell culture medium

[0061] 500 mL of RPMI-1640 culture medium was divided into 45 mL portions in a clean bench, 5 mL of fetal bovine serum was added to each portion, and the portions were stored in a 4°C refrigerator for later use.

[0062] 3. CCK8 method to determine cell proliferation inhibition rate

[0063] Take PC-9 or PC-9OR cells in the logarithmic growth phase, and digest, terminate, centrifuge, and resuspend them to make the cell suspension density about 4×10 4 100 μL of culture medium was added to each well of a 96-well plate, about 4×10 3 The cells were placed in a cell culture incubator and cultured overnight to allow the cells in the 96-well plate to adhere to the wall. The conditions of the cell culture incubator were a constant temperature of 37° C., 5% CO 2 , and saturated humidity of 95%.

[0064] Experimental and control group settings

[0065] Experimental group 1 was to add different concentrations of EGFR inhibitors: for example, each L of cell system contained 0.0, 0.01953, 0.07813, 0.3125, 1.25, 5.0 μmol of Gefitinib or Osimertinib; each L of cell system contained 0.0, 0.07813, 0.3125, 1.25, 5.0, 20.0 μmol of Icotinib; each L of cell system contained 0.0, 0.00488, 0.01953, 0.07813, 0.3125, 1.25 μmol of Afatinib or Dacomitinib.

[0066] Experimental group 2 was added with a fixed concentration of TH588, containing 4.0 μmol of TH588 per L of cell system.

[0067] Experimental group 3 was added with different concentrations of combined drugs, EGFR inhibitor + TH588; the culture medium containing DMSO (concentration 0.1%) was used as the control group, and the culture medium was used as the blank group: such as Gefitinib + TH588 or Osimertinib + TH588, the groups were set to 0.01953μM + 4.0μM, 0.07813μM + 4.0μM, 0.3125μM + 4.0μM, 1.25μM + 4.0μM, 5.0μM + 4.0μM; such as Icotinib + TH58 8. Set the groups to 0.07813μM+4.0μM, 0.3125μM+4.0μM, 1.25μM+4.0μM, 5.0μM+4.0μM, and 20.0μM+4.0μM; such as Afatinib+TH588 or Dacomitinib+TH588, set the groups to 0.00488μM+4.0μM, 0.01953μM+4.0μM, 0.07813μM+4.0μM, 0.3125μM+4.0μM, and 1.25μM+4.0μM.

[0068] After 72 hours of action, 20 μL CCK8 was added to each well and placed in an incubator (the conditions of the cell incubator were a constant temperature of 37°C, 5% CO2, and 95% saturated humidity) for 90 minutes. The 96-well plate was taken out and the light absorption value (OD value) was measured at 450nm using an ELISA reader. The survival rate of cell proliferation was 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 was calculated according to the following formula: inhibition rate = 1-survival rate, and the results are shown in Table 3.

[0069] Table 3 The inhibition rate of PC-9 cell proliferation by a fixed concentration of TH588 combined with different concentrations of EGFR inhibitors (n=1)

[0070]

[0071] According to the inhibition rate values ​​in Table 3, in this experiment: the inhibition rate of TH588 on non-small cell lung cancer cell line PC-9 was 7.51%. In different EGFR groups, the effect of inhibiting cell proliferation gradually increased with the increase of drug concentration, and the inhibition rate of the combined action group of EGFR and TH588 was significantly higher than that of the corresponding single drug group.

[0072] The above data indicate that EGFR inhibitors and TH588 have a synergistic inhibitory effect on the non-small cell lung cancer cell line PC-9, and the same concentration of TH588 also has a relatively synergistic inhibitory effect on different concentrations of EGFR inhibitors.

[0073] From the above examples, it can be seen that the present invention provides a composition, and the synergistic range of TH588 is 2.0-10 μmol / L, which has a synergistic inhibitory effect on 0.01953-10 μM Gefitinib, 0.07813-10 μM Icotinib, 0.00488-2.5 μM Afatinib / Dacomitinib, and 0.01953-5.0 μM Osimertinib. It also has a synergistic inhibitory effect on drug-resistant lung cancer cells.

[0074] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Use of a composition in the preparation of a drug for treating osimertinib-resistant non-small cell lung cancer, It is characterized in that The composition comprises an EGFR inhibitor and an MTH1 inhibitor; The EGFR inhibitor is osimertinib; The MTH1 inhibitor is TH588.

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