Application of SRC inhibitor combined with KRAS-G12C inhibitor in preparation of medicine for treating KRAS-G12C mutant lung cancer and medicine composition

Abstract

The invention provides application of an SRC inhibitor combined with a KRAS-G12C inhibitor to preparation of a medicine for treating KRAS-G12C mutant lung cancer and a medicine composition, and relates to the technical field of biological medicine, compared with independent use of the KRAS-G12C inhibitor, the scheme of the SRC inhibitor combined with the KRAS-G12C inhibitor can effectively inhibit proliferation and survival of KRAS-G12C mutant lung cancer cells, and the application of the SRC inhibitor combined with the KRAS-G12C inhibitor to preparation of the medicine for treating the KRAS-G12C mutant lung cancer can be used for preparing the medicine for treating the KRAS-G12C mutant lung cancer. The compound can effectively inhibit the activation of a KRAS-MARK pathway and an SRC-FAK pathway, the combination of the compound and the compound has a synergistic anti-tumor effect, and the compound and the compound can be directly combined for medication, can also be prepared into a combined preparation for treating KRAS mutant lung cancer, and have a good clinical transformation prospect.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an application of an SRC inhibitor combined with a KRAS-G12C inhibitor in preparing a drug for treating KRAS-G12C mutant lung cancer and a pharmaceutical composition. Background Art

[0002] The treatment of KRAS mutant tumors has always been a hot topic and difficulty in the field of cancer research. On May 29, 2021, the U.S. FDA announced the accelerated approval of Sotorasib (Lumakras, AMG510) developed by Amgen for the treatment of patients with locally advanced or metastatic non-small cell lung cancer carrying KRAS-G12C mutations. The drug can irreversibly lock the KRAS-G12C mutant in an inactive GDP-bound state, inhibit the conduction of downstream signaling pathways, and thus inhibit the proliferation of KRAS-G12C mutant lung cancer cells. However, like the difficulties faced by most targeted drugs, clinical results show that the total response rate of patients treated with Sotorasib is 36% (95% CI: 28-45), the median duration of response is 10 months, some lung cancer patients will have primary drug resistance, and some patients will still have tumor progression after a certain degree of remission. Therefore, there is an urgent need to provide a new treatment strategy for patients with KRAS-G12C mutant lung cancer. Summary of the invention

[0003] One of the purposes of the present invention is to provide a use of a SRC inhibitor in combination with a KRAS-G12C inhibitor for preparing a drug for treating KRAS-G12C mutant lung cancer.

[0004] A second object of the present invention is to provide a pharmaceutical composition.

[0005] To achieve the above object, the present invention provides the following technical solutions: In a first aspect, the present invention provides the use of a SRC inhibitor in combination with a KRAS-G12C inhibitor for preparing a medicament for treating KRAS-G12C mutant lung cancer.

[0006] According to a preferred embodiment, the SRC inhibitor includes dasatinib or saracatinib, and the KRAS-G12C inhibitor includes sotorasib.

[0007] According to a preferred embodiment, the molar ratio of Sotorasib to Dasatinib is 1:2 or 5:1, and the molar ratio of Sotorasib to Saracatinib is 1:5 or 2.5:1.

[0008] In a second aspect, the present application also provides a pharmaceutical composition for preparing a drug for treating KRAS-G12C mutant lung cancer, wherein the pharmaceutical composition comprises a SRC inhibitor and a KRAS-G12C inhibitor.

[0009] According to a preferred embodiment, the SRC inhibitor includes dasatinib or saracatinib, and the KRAS-G12C inhibitor includes sotorasib.

[0010] According to a preferred embodiment, the molar ratio of Sotorasib to Dasatinib is 1:2 or 5:1, and the molar ratio of Sotorasib to Saracatinib is 1:5 or 2.5:1.

[0011] Based on the above technical solution, the use of the SRC inhibitor combined with the KRAS-G12C inhibitor provided in the present application for preparing a drug for treating KRAS-G12C mutant lung cancer and the pharmaceutical composition have at least the following technical effects: The application provides an application of an SRC inhibitor combined with a KRAS-G12C inhibitor for the preparation of a drug for treating KRAS-G12C mutant lung cancer. Compared with the use of a KRAS-G12C inhibitor alone, the combination of an SRC inhibitor and a KRAS-G12C inhibitor can effectively inhibit the proliferation and survival of KRAS-G12C mutant lung cancer cells, and more effectively inhibit the activation of the KRAS-MARK pathway and the SRC-FAK pathway. The combination of the two has a synergistic anti-tumor effect. The two can be used directly in combination or can be prepared into a combined preparation for the treatment of KRAS mutant lung cancer, which has good prospects for clinical transformation. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0013] Figure 1 The WB results of AMG510 treatment of KRAS-G12C mutant lung cancer cells Calu-1 and NCI-H2030, respectively, are shown in Figure 1. Figure 1 Figure A shows the results of treating Calu-1 cells with AMG510 at different concentrations. Figure 1 Figure B shows the results of treating Calu-1 cells with AMG510 at 5 μM for different time periods. Figure 1 Figure C shows the results of treating NCI-H2030 cells with AMG510 at different concentrations. Figure 1Figure D shows the results of treating NCI-H2030 cells with 5 μM AMG510 for different time periods.

[0014] Figure 2 The figure shows the results of protein immunoblotting experiments in which Calu-1 and NCI-H2030 cells were treated with different concentrations of AMG510.

[0015] Figure 3 It is the synergy index diagram of AMG510 combined with Dasatinib and Saracatinib to treat Calu-1 and NCI-H2030 cells respectively; Figure 3 Figure A and Figure B are the synergy index diagrams of AMG510 combined with Dasatinib to treat Calu-1 and NCI-H2030 cells respectively; Figure 3 Figure C and Figure D are the synergy index graphs of AMG510 combined with Saracatinib to treat Calu-1 and NCI-H2030 cells respectively.

[0016] Figure 4 This is a graph showing the effect of combined targeting of SRC and KRAS on the growth and survival of KRAS-G12C mutant lung cancer cells, where: Figure 4 Figures A and B are the CCK8 results of Dasatinib combined with AMG510 in treating Calu-1 and NCI-H2030 cells. Figure 4 Figure C shows the colony formation experiment results of Calu-1 and NCI-H2030 cells treated with Dasatinib combined with AMG510. Figure 4 Figures D and E are the CCK8 results of Saracatinib combined with AMG510 in treating Calu-1 and NCI-H2030 cells. Figure 4 Figure F shows the results of the colony formation experiment of Calu-1 and NCI-H2030 cells treated with Saracatinib combined with AMG510. Figure 4 Figure G shows the WB results of NCI-H2030 cells treated with AMG510 combined with Dasatinib and Saracatinib, respectively. DETAILED DESCRIPTION

[0017] To make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described in detail below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other implementation methods obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention.

[0018] The applicant found in the study that in KRAS-G12C mutant lung cancer cells, Sotorasib (Lumakras, AMG510) treatment can effectively inhibit the activity of the KRAS-MAPK pathway, but at the same time it will cause a significant increase in the phosphorylation level of the non-receptor kinase FAK. As a non-receptor tyrosine kinase, the activation of SRC kinase mediates the activation of FAK, which in turn leads to downstream signal transduction, so that Sotorasib treatment alone will lead to a rebound of the SRC pathway.

[0019] Currently, there are many drugs targeting SRC in clinical use. Dasatinib and Saracatinib are inhibitors of SRC. Among them, Dasatinib is a new and effective multi-target inhibitor that can act on Abl, Src and c-Kit, and can effectively inhibit Src kinase activity. It is mainly used in patients with chronic myeloid leukemia and Philadelphia chromosome-positive acute myeloid leukemia (Ph+ ALL) after imatinib treatment. Dasatinib as a single drug shows moderate clinical activity in patients with non-small cell lung cancer. Kras mutations do not predict response to dasatinib. Some studies have also shown that dasatinib has almost no effect in patients with advanced non-small cell lung cancer. Saracatinib is also an effective SRC inhibitor. The FDA has granted Saracatinib orphan drug status for the treatment of idiopathic pulmonary fibrosis (IPF). The efficacy of Saracatinib alone in the treatment of non-small cell lung cancer is also very limited. There are currently no reports on the use of dasatinib and saracutinib in KRAC-G12C mutant lung cancer.

[0020] In the treatment of KRAS mutant lung cancer, how to achieve long-lasting and effective inhibition of tumor development remains an important scientific issue. Based on this, the present invention provides an application of a SRC inhibitor combined with a KRAS-G12C inhibitor in the preparation of a drug for treating KRAS-G12C mutant lung cancer.

[0021] Example 1

[0022] Dynamic effects of AMG510 on the MAPK pathway in KRAS-G12C mutant lung cancer cells.

[0023] 1.1 Tumor cells and drugs involved.

[0024] KRAS-G12C mutant lung cancer cells Calu-1 and NCI-H2030.

[0025] KRAS-G12C inhibitor: AMG510.

[0026] The lung cancer cells Calu-1 and NCI-H2030 were ordered from ATCC. AMG510 was ordered from Selleck. The drug is a chiral compound and an effective KRAS-G12C covalent inhibitor with potential anti-tumor activity. All subsequent examples use KRAS-G12C mutant lung cancer cells and AMG510 from the same source.

[0027] 1.2 Experimental methods: Experimental groups: ① Control group: cells were treated with dimethyl sulfoxide (DMSO, Sigma-aldrich, a commonly used polar solvent).

[0028] ②AMG510 group: cells were treated with AMG510 alone.

[0029] KRAS-G12C mutant lung cancer cells Calu-1 and NCI-H2030 were treated with AMG510 at concentration gradients of 0.5, 1, and 5 μM / mL for 24 hours, and total cell protein was extracted. Then, WB experiments were performed to detect changes in key molecules of the intracellular MAPK pathway. Specifically: KRAS-G12C mutant lung cancer cells Calu-1 and NCI-H203 were divided into three groups, and different amounts of AMG510 were added to each group to form a concentration gradient of 0.5, 1, and 5 μM / mL. After 24 hours of culture, the culture medium was discarded, washed once with pre-cooled PBS, and cell lysis solution was added according to the number of cells. The cells were scraped off and collected into a 1.5 mL EP tube, placed on ice for 30 minutes, and vortexed every 5 minutes to accelerate lysis. After the lysis was completed, centrifuged at 4°C and 12000rpm in a refrigerated centrifuge for 10 minutes.

[0030] Loading Buffer was added according to the number of cells, and polyacrylamide gel electrophoresis was performed. After electrophoresis, the protein was transferred to a PVDF membrane, blocked with 5% skim milk for 1 hour, and then incubated with the corresponding primary and secondary antibodies. Finally, the membrane was developed using an infrared fluorescence scanner. Figure 1 The strips shown.

[0031] like Figure 1 As shown, Figure 1 Figure A shows the results of treating Calu-1 cells with AMG510 at different concentrations. Figure 1 Figure B shows the results of treating Calu-1 cells with AMG510 at 5 μM for different time periods. Figure 1 Figure C shows the results of treating NCI-H2030 cells with AMG510 at different concentrations. Figure 1Figure D shows the results of NCI-H2030 cells treated with AMG510 at 5 μM for different time periods. The results show that with the increase in the dose of AMG510, the expression of p-CRAF-S338 and p-ERK in the downstream of the KRAS pathway in KRAS-G12C mutant lung cancer cells gradually decreases. It can be seen that AMG510 treatment can effectively inhibit the expression of key protein activity in the downstream KRAS pathway in KRAS-G12C mutant lung cancer cells, and 1 μM concentration of AMG510 can almost completely inhibit the activity of the intracellular MAPK pathway. In the experiment, we also found that the KRAS protein band in the WB results of cells treated with AMG510 showed an obvious upward shift. This is because AMG510 mainly acts on the KRAS-G12C protein through covalent modification to inhibit its activity. The molecular weight of the modified KRAS-G12C protein will increase, and the WB result presented is that its protein band will shift upward. However, with the increase of treatment time, especially for NCI-H2030 cells, p-CRAF-S338 and p-ERKAMG510 showed a rebound trend.

[0032] Example 2

[0033] Dynamic effects of the KRAS-G12C inhibitor AMG510 on the SRC-FAK pathway in KRAS-G12C mutant lung cancer cells.

[0034] 2.1 Tumor cells and drugs involved.

[0035] KRAS-G12C mutant lung cancer cells Calu-1 and NCI-H2030; KRAS-G12C inhibitor: AMG510.

[0036] 2.2 Experimental methods: Experimental groups: ①Control group: cells were treated with DMSO; ②AMG510 group: cells were treated with AMG510 alone.

[0037] KRAS-G12C mutant lung cancer cells Calu-1 and NCI-H2030 were treated with AMG510 at different concentration gradients for 24 hours, and the total cell protein was extracted, and then the changes of key molecules of the intracellular SRC-FAK pathway were detected.

[0038] like Figure 2 As shown, Figure 2The results of protein immunoblotting experiments of Calu-1 and NCI-H2030 cells treated with different concentrations of AMG510 are shown. The effects of different concentrations of AMG510 treatment on the expression of P-SRC-Y419 and P-FAK-Y397 in lung cancer cells with KRAS-G12C mutations show that AMG510 treatment significantly promotes the expression levels of P-SRC and P-FAK. Combined with the results of Example 1, AMG510 covalently modifies KRAS-G12C protein and can continuously inhibit the kinase activity of its downstream CRAF, while the expression of P-FAK and P-SRC can show a significant increase in a short time, suggesting that the FAK-SRC pathway may rebound and activate in the early stage of AMG510 action, which then mediates the tolerance and resistance of lung cancer cells to AMG510.

[0039] Example 3

[0040] Synergistic effect of SRC kinase inhibitors Dasatinib or Saracatinib combined with AMG510.

[0041] 3.1 Tumor cells and drugs involved.

[0042] KRAS-G12C mutant lung cancer cells Calu-1 and NCI-H2030 (ATCC); KRAS-G12C inhibitor: AMG510 (Selleck); SRC inhibitors: Dasatinib (Selleck) and Saracatinib (Selleck).

[0043] 3.2 Experimental methods: Experimental groups: ①Control group: cells were treated with DMSO; ②AMG510 group: cells were treated with AMG510 alone; ③Dasatinib group: cells were treated with Dasatinib alone; ④Saracatinib group: cells were treated with Saracatinib alone; ⑤AMG510+Dasatinib group: cells were treated with AMG510 and Dasatinib at the same time; ⑥AMG510+Saracatinib group; cells were treated with AMG510 and Saracatinib at the same time.

[0044] Take cells with good growth status, count them after digestion, and adjust the concentration of cell suspension to make the cell concentration in each well 5×10 3Cells / 100μL, inoculated into 96-well plates, cultured at 37℃ for 24h. Then replace the medium containing drugs and continue to culture. After the culture is completed, aspirate the medium, add 45μL DMEM and 5μL CCK8 solution to each well, and react at 37℃ for 1h. In the microplate reader, the OD value of each well is detected at a wavelength of 450nm. Cell survival rate = (OD value of experimental group - OD value of blank well) / (OD value of control group - OD value of blank well) In Cula1 cells, AMG510 group: AMG510 was added at a concentration of 0.04μM, 0.2μM, and 1μM, respectively; Dasatinib group: Dasatinib was added at a concentration of 0.08μM, 0.4μM, and 2μM, respectively; Saracatinib group: Saracatinib was added at a concentration of 0.2μM, 1μM, and 5μM, respectively; AMG510+Dasatinib group (A+D): AMG510 was added at a concentration of 0.04μM and Dasatinib was added at a concentration of 0.08μM, or AMG510 was added at a concentration of 0.2μM and 0.4μM Dasatinib, or adding AMG510 at a concentration of 1μM and Dasatinib at 2μM (the molar ratio of AMG510 and Dasatinib is 1:2); AMG510+Saracatinib group (A+S): adding AMG510 at a concentration of 0.04μM and Saracatinib at 0.2μM, or adding AMG510 at a concentration of 0.2μM and Saracatinib at 1μM, or adding AMG510 at a concentration of 1μM and Saracatinib at 5μM (the molar ratio of AMG510 and Saracatinib is 1:5).

[0045] In NCI-H2030 cells, AMG510 group: AMG510 was added at a concentration of 0.2μM, 1μM, and 5μM, respectively; Dasatinib group: Dasatinib was added at a concentration of 0.04μM, 0.2μM, and 1μM, respectively; Saracatinib group: Saracatinib was added at a concentration of 0.08μM, 0.4μM, and 2μM, respectively; AMG510+Dasatinib group (A+D): AMG510 was added at a concentration of 0.2μM and Dasatinib was added at a concentration of 0.04μM, or AMG510 was added at a concentration of 1μM and Dasatinib was added at a concentration of 0.2μM, or AMG510 was added at a concentration of 5μM G510 and 1μM Dasatinib (the molar ratio of AMG510 to Dasatinib is 5:1); AMG510+Saracatinib group (A+S): 0.2μM AMG510 and 0.08μM Saracatinib, or 1μM AMG510 and 0.4μM Saracatinib, or 5μM AMG510 and 2μM Saracatinib (the molar ratio of AMG510 to Saracatinib is 2.5:1) were added. The volume of drugs dissolved in different concentrations in each group was 100μL.

[0046] The results are as follows Figure 3 As shown, Figure 3 Figure A and Figure B are the synergy index diagrams of AMG510 combined with Dasatinib to treat Calu-1 and NCI-H2030 cells respectively; Figure 3 Figures C and D are the synergy index diagrams of AMG510 combined with Saracatinib to treat Calu-1 and NCI-H2030 cells, respectively. In Cula-1 and NCI-H2030 cells, AMG510 was combined with Dasatinib and Saracatinib, respectively. The cells were cultured for 3 days after adding the drugs, and then CCK8 was used to detect cell viability. The Chou-Talalay formula was used to calculate the synergy index (CI). The Chou-Talalay method is a commonly used quantitative analysis method for drug synergy. CI < 1 indicates that the two drugs have a synergistic effect. Figure 3 Figures A, B, C, and D all show that the CI index is below 1, indicating that AMG510 combined with Dasatinib and AMG510 combined with Saracatinib have strong synergistic anti-tumor activity and can effectively inhibit the growth of Calu-1 and NCI-H2030 cell lines. The efficacy of the two may vary depending on the cell type, and AMG510 combined with Dasatinib has a stronger synergistic inhibitory effect on the Calu1 cell line.

[0047] like Figure 4 As shown, Figure 4 This is a graph showing the effect of combined targeting of SRC and KRAS on the growth and survival of KRAS-G12C mutant lung cancer cells, where: Figure 4 Figures A and B are the CCK8 results of Dasatinib combined with AMG510 in treating Calu-1 and NCI-H2030 cells. Figure 4 Figure C shows the colony formation experiment results of Calu-1 and NCI-H2030 cells treated with Dasatinib combined with AMG510. Figure 4 Figures D and E are the CCK8 results of Saracatinib combined with AMG510 in treating Calu-1 and NCI-H2030 cells. Figure 4 Figure F shows the results of the colony formation experiment of Calu-1 and NCI-H2030 cells treated with Saracatinib combined with AMG510. Figure 4 Figure G shows the WB results of NCI-H2030 cells treated with AMG510 combined with Dasatinib and Saracatinib, respectively.

[0048] Figure 4 The CCK8 results in the AB graph show that Dasatinib combined with AMG510 can significantly inhibit the growth of Calu-1 and NCI-H2030 cells. Figure 4 C colony formation experiment showed that Dasatinib combined with AMG510 could significantly inhibit the survival of Calu-1 and NCI-H2030 cells. Figure 4 The CCK8 results of the DE graph showed that Saracatinib combined with AMG510 could significantly inhibit the growth of Calu-1 and NCI-H2030 cells. Figure 4 F colony formation assay showed that Saracatinib combined with AMG510 could significantly inhibit the survival of Calu-1 and NCI-H2030 cells. Figure 4 Figure G shows that WB results show that in NCI-H2030, AMG510 combined with Dasatinib and Saracatinib treatment can significantly inhibit the activity of the SRC-FAK pathway.

[0049] It can be seen that the SRC inhibitor combined with the KRAS-G12C inhibitor provided in the present application is used to prepare a regimen for treating KRAS-G12C mutant lung cancer. Compared with the use of the KRAS-G12C inhibitor alone, the regimen of the SRC inhibitor combined with the KRAS-G12C inhibitor can effectively inhibit the proliferation and survival of KRAS-G12C mutant lung cancer cells, and more effectively inhibit the activation of the KRAS-MARK pathway and the SRC-FAK pathway. The combination of the two has a synergistic anti-tumor effect. The two can be used directly in combination or prepared into a combined preparation for the treatment of KRAS mutant lung cancer, which has good prospects for clinical transformation.

[0050] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art who is familiar with the technical field can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. Use of SRC inhibitors combined with KRAS-G12C inhibitors for the preparation of drugs for the treatment of KRAS-G12C mutant lung cancer.

2. The use according to claim 1, characterized in that: The SRC inhibitor includes dasatinib or saracutinib, and the KRAS-G12C inhibitor includes sotorasib.

3. The use according to claim 2, characterized in that: The molar ratio of Sotorasib to Dasatinib is 1:2 or 5:1, and the molar ratio of Sotorasib to Saracatinib is 1:5 or 2.5:

1.

4. A pharmaceutical composition for treating KRAS-G12C mutant lung cancer, characterized in that: The pharmaceutical composition comprises a SRC inhibitor and a KRAS-G12C inhibitor.

5. The pharmaceutical composition according to claim 4, characterized in that The SRC inhibitor includes dasatinib or saracutinib, and the KRAS-G12C inhibitor includes sotorasib.

6. The pharmaceutical composition according to claim 5, characterized in that The molar ratio of Sotorasib to Dasatinib is 1:2 or 5:1, and the molar ratio of Sotorasib to Saracatinib is 1:5 or 2.5:1.

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