Use of SRC inhibitor in combination with KRAS-G12C inhibitor for the preparation of a medicament for treating KRAS-G12C mutant lung cancer and pharmaceutical composition

Abstract

The present invention provides an application of an SRC inhibitor combined with a KRAS-G12C inhibitor in the preparation of a medicament for treating KRAS-G12C mutant lung cancer and a pharmaceutical composition, relating to the technical field of biomedicine. Compared with the use of the KRAS-G12C inhibitor alone, the combination of the SRC inhibitor and 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 directly used in combination or made into a combined preparation for treating KRAS mutant lung cancer, and has good clinical transformation prospects.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to the use of an SRC inhibitor in combination with a KRAS-G12C inhibitor for the preparation of a medicament 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 and difficult point in the field of cancer research. On May 29, 2021, the US 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 the KRAS-G12C mutation. This drug can specifically and irreversibly lock the KRAS-G12C mutant in the inactive GDP-bound state, inhibit the downstream signal pathway transduction, and thus inhibit the proliferation of lung cancer cells with the KRAS-G12C mutation. However, like most targeted drugs, clinical results show that the overall 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 show primary drug resistance, and some patients will still experience 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 object of the present invention is to provide the use of an SRC inhibitor in combination with a KRAS-G12C inhibitor for the preparation of a medicament for treating KRAS-G12C mutant lung cancer.

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

[0005] To achieve the above objects, the present invention provides the following technical solutions:

[0006] In the first aspect, the present invention provides the use of an SRC inhibitor in combination with a KRAS-G12C inhibitor for the preparation of a medicament for treating KRAS-G12C mutant lung cancer.

[0007] According to a preferred embodiment, the SRC inhibitor includes dasatinib or seracatinib, and the KRAS-G12C inhibitor includes Sotorasib.

[0008] 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 seracatinib is 1:5 or 2.5:1.

[0009] In a second aspect, the present application also provides a pharmaceutical composition for preparing a medicament for treating KRAS-G12C mutant lung cancer, the pharmaceutical composition comprising an SRC inhibitor and a KRAS-G12C inhibitor.

[0010] According to a preferred embodiment, the SRC inhibitor comprises dasatinib or seracatinib, and the KRAS-G12C inhibitor comprises Sotorasib.

[0011] 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 seracatinib is 1:5 or 2.5:1.

[0012] Based on the above technical solutions, the application of the SRC inhibitor in combination with the KRAS-G12C inhibitor provided by the present application for preparing a medicament for treating KRAS-G12C mutant lung cancer and the pharmaceutical composition have at least the following technical effects:

[0013] The application of the SRC inhibitor in combination with the KRAS-G12C inhibitor provided by the present application for preparing a medicament for treating KRAS-G12C mutant lung cancer, compared with the use of the KRAS-G12C inhibitor alone, the combination of the SRC inhibitor and the KRAS-G12C inhibitor can effectively inhibit the proliferation and survival of KRAS-G12C mutant lung cancer cells, more effectively inhibit the activation of the KRAS-MARK pathway and the SRC-FAK pathway, and the combination of the two has a synergistic anti-tumor effect. The two can be directly combined for drug use or can be made into a combined preparation for treating KRAS mutant lung cancer, and has good clinical transformation prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0015] Figure 1 are the WB experimental result diagrams of AMG510 treating KRAS-G12C mutant lung cancer cells Calu-1 and NCI-H2030 respectively, wherein Figure 1 Figure A of is the result diagram of AMG510 treating cell Calu-1 at different concentrations, Figure 1 Figure B of is the result diagram of AMG510 treating cell Calu-1 at different times at 5 μM, Figure 1Figure C shows the results of treating cells NCI-H2030 with AMG510 at different concentrations. Figure 1 Figure D shows the results of treating cells NCI-H2030 with AMG510 for different times at 5 μM.

[0016] Figure 2 These are the results of Western blot experiments of treating cells Calu-1 and NCI-H2030 with different concentrations of AMG510 respectively.

[0017] Figure 3 These are the combination index graphs of AMG510 combined with Dasatinib and Saracatinib to treat cells Calu-1 and NCI-H2030 respectively; among them Figure 3 Figures A and B show the combination index graphs of AMG510 combined with Dasatinib to treat cells Calu-1 and NCI-H2030 respectively. Figure 3 Figures C and D show the combination index graphs of AMG510 combined with Saracatinib to treat cells Calu-1 and NCI-H2030 respectively.

[0018] Figure 4 These are the graphs showing the effects of combined targeting of SRC and KRAS on the growth and survival of KRAS-G12C mutant lung cancer cells. Among them, Figure 4 Figures A and B show the CCK8 results of treating cells Calu-1 and NCI-H2030 with Dasatinib combined with AMG510. Figure 4 Figure C shows the results of colony formation experiments of treating cells Calu-1 and NCI-H2030 with Dasatinib combined with AMG510. Figure 4 Figures D and E show the CCK8 results of treating cells Calu-1 and NCI-H2030 with Saracatinib combined with AMG510. Figure 4 Figure F shows the results of colony formation experiments of treating cells Calu-1 and NCI-H2030 with Saracatinib combined with AMG510. Figure 4 Figure G shows the WB results of treating NCI-H2030 cells with AMG510 combined with Dasatinib and Saracatinib respectively. Detailed implementation manners

[0019] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions of the present invention will be described in detail below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other implementation manners obtained by those of ordinary skill in the art without creative efforts shall fall within the scope protected by the present invention.

[0020] The applicant found in the research that: in KRAS-G12C mutant lung cancer cells, treatment with Sotorasib (Lumakras, AMG510) 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 will mediate the activation of FAK, which will further lead to downstream signal transduction, resulting in the rebound of the SRC pathway when treated with Sotorasib alone.

[0021] Currently, there are already a variety of drugs targeting SRC being used clinically. Dasatinib and Saracatinib are inhibitors of SRC. Among them, Dasatinib is a novel and effective multi-target inhibitor that can act on Abl, Src, and c-Kit, and can effectively inhibit the activity of Src kinase. Clinically, it is mainly used for patients with chronic myeloid leukemia after imatinib treatment and acute myeloid leukemia with Philadelphia chromosome positive (Ph+ ALL). Dasatinib shows moderate clinical activity as a single drug in patients with non-small cell lung cancer. Kras mutation cannot predict the response to Dasatinib, and some studies have also shown that Dasatinib has little efficacy in patients with advanced non-small cell lung cancer. Saracatinib is also an effective SRC inhibitor, and the FDA has granted orphan drug status to Saracatinib for the treatment of idiopathic pulmonary fibrosis (IPF). The efficacy of Saracatinib as a single drug in the treatment of non-small cell lung cancer is also very limited. There are currently no reports on the application of Dasatinib and Saracatinib in KRAC-G12C mutant lung cancer.

[0022] In the treatment of KRAS mutant lung cancer, how to achieve persistent and effective inhibition of tumor development remains an important scientific issue. Based on this, the present invention provides an application of an SRC inhibitor combined with a KRAS-G12C inhibitor in the preparation of a drug for the treatment of KRAS-G12C mutant lung cancer.

[0023] Example 1

[0024] Dynamic effect of AMG510 on the MAPK pathway in KRAS-G12C mutant lung cancer cells.

[0025] 1.1 Tumor cells and drugs involved.

[0026] KRAS-G12C mutant lung cancer cells Calu-1, NCI-H2030.

[0027] KRAS-G12C inhibitor: AMG510.

[0028] Among them, the lung cancer cells Calu-1 and NCI-H2030 were ordered from ATCC. AMG510 was ordered from Selleck. This drug is a chiral compound and an effective covalent inhibitor of KRAS-G12C, with potential anti-tumor activity. In the following examples, the same source of KRAS-G12C mutant lung cancer cells and AMG510 were used.

[0029] 1.2 Experimental methods:

[0030] Experimental grouping:

[0031] ① Control group: The cells were treated with dimethyl sulfoxide (DMSO, Sigma-aldrich, a commonly used polar solvent).

[0032] ② AMG510 group: The cells were treated with AMG510 alone.

[0033] After treating the KRAS-G12C mutant lung cancer cells Calu-1 and NCI-H2030 with AMG510 at concentration gradients of 0.5, 1, and 5 μM / mL for 24 hours, the total cellular proteins were extracted, and then the changes in key molecules of the intracellular MAPK pathway were detected by WB experiment. Specifically:

[0034] The KRAS-G12C mutant lung cancer cells Calu-1 and NCI-H2030 were each divided into three groups, and different amounts of AMG510 were added to each group to form concentration gradients of 0.5, 1, and 5 μM / mL. After culturing for 24 hours, the culture medium was discarded, and the cells were washed once with pre-cooled PBS. According to the cell number, cell lysis buffer was added. The cells were scraped off with a cell scraper and collected into 1.5 mL EP tubes, and lysed on ice for 30 min, vortexing every 5 min to accelerate lysis. After the lysis was completed, the samples were centrifuged at 4°C and 12,000 rpm for 10 min in a refrigerated centrifuge.

[0035] According to the cell number, Loading Buffer was added, and polyacrylamide gel electrophoresis was performed. After electrophoresis, the proteins were transferred to a PVDF membrane, blocked with 5% skim milk for 1 h, incubated with the corresponding primary and secondary antibodies, and finally developed with an infrared fluorescence scanner to obtain Figure 1 the bands shown.

[0036] As Figure 1 shown, Figure 1 Figure A shows the results of treating cells Calu-1 with AMG510 at different concentrations, Figure 1 Figure B shows the results of treating cells Calu-1 with AMG510 at 5 μM for different times, Figure 1Figure C shows the results of treating cells NCI-H2030 with AMG510 at different concentrations. Figure 1 Figure D shows the results of treating cells NCI-H2030 with AMG510 at 5 μM for different times. The results show that as the dose of AMG510 increases, the expression of p-CRAF-S338 and p-ERK 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 the key protein activities in the KRAS downstream pathway of KRAS-G12C mutant lung cancer cells. AMG510 at a concentration of 1 μM can almost completely inhibit the activity of the intracellular MAPK pathway. In the experiment, we also found that the protein band of KRAS showed an obvious upward shift in the WB results of the cells after AMG510 treatment. This is because AMG510 mainly inhibits its activity by covalently modifying the KRAS-G12C protein, and the molecular weight of the modified KRAS-G12C protein will become larger, resulting in an upward shift of its protein band in the WB results. However, with the increase of the treatment time, especially for cells NCI-H2030, there is a tendency of rebound in p-CRAF-S338 and p-ERK AMG510.

[0037] Example 2

[0038] Dynamic effect of KRAS-G12C inhibitor AMG510 on the SRC-FAK pathway in KRAS-G12C mutant lung cancer cells.

[0039] 2.1 Tumor cells and drugs involved.

[0040] KRAS-G12C mutant lung cancer cells Calu-1, NCI-H2030;

[0041] KRAS-G12C inhibitor: AMG510.

[0042] 2.2 Experimental method:

[0043] Experimental grouping:

[0044] ① Control group: Treat cells with DMSO;

[0045] ② AMG510 group: Treat cells with AMG510 alone.

[0046] Treat KRAS-G12C mutant lung cancer cells Calu-1 and NCI-H2030 with different concentration gradients of AMG510 for 24 hours, extract the total cell protein, and then detect the changes of key molecules in the intracellular SRC-FAK pathway.

[0047] As Figure 2 shown, Figure 2The figure shows the results of a protein immunoblotting experiment in which cells Calu-1 and NCI-H2030 were treated with different concentrations of AMG510. The effects of treatment with different concentrations of AMG510 on the expression of P-SRC-Y419 and P-FAK-Y397 in lung cancer cells with KRAS-G12C mutation were examined. The results showed that treatment with AMG510 significantly promoted the expression levels of P-SRC and P-FAK. Combining with the results of Example 1, it was shown that after covalently modifying and acting on the KRAS-G12C protein, AMG510 could continuously inhibit the kinase activity of its downstream CRAF, while the expression of P-FAK and P-SRC increased significantly in a short time, suggesting that the FAK-SRC pathway might be rebound-activated at the early stage of the action of AMG510, and then mediated the tolerance and resistance of lung cancer cells to AMG510.

[0048] Example 3

[0049] Synergistic effect of SRC kinase inhibitors Dasatinib or Saracatinib combined with AMG510.

[0050] 3.1 Tumor cells and drugs involved.

[0051] KRAS-G12C mutant lung cancer cells Calu-1, NCI-H2030 (ATCC);

[0052] KRAS-G12C inhibitor: AMG510 (Selleck);

[0053] SRC inhibitors: Dasatinib (Selleck) and Saracatinib (Selleck).

[0054] 3.2 Experimental methods:

[0055] Experimental grouping:

[0056] ① Control group: Cells were treated with DMSO;

[0057] ② AMG510 group: Cells were treated with AMG510 alone;

[0058] ③ Dasatinib group: Cells were treated with Dasatinib alone;

[0059] ④ Saracatinib group: Cells were treated with Saracatinib alone;

[0060] ⑤ AMG510 + Dasatinib group: Cells were treated with AMG510 and Dasatinib simultaneously;

[0061] ⑥AMG510 + Saracatinib group; cells were treated with AMG510 and Saracatinib simultaneously.

[0062] Cells in good growth state were digested and counted, and the cell suspension concentration was adjusted so that the cell concentration per well was 5×10 3 cells / 100 μL. The cells were seeded into 96-well plates and cultured at 37 °C for 24 h. Then the medium containing the drug was replaced and the culture was continued. After the culture was completed, the medium was aspirated, 45 μL of DMEM and 5 μL of CCK8 solution were added to each well, and the reaction was carried out at 37 °C for 1 h. The OD value of each well was detected at a wavelength of 450 nm in an enzyme-linked immunosorbent assay reader. Cell survival rate = (OD value of the experimental group - OD value of the blank well) / (OD value of the control group - OD value of the blank well)

[0063] In Cula1 cells, in the AMG510 group, AMG510 with concentrations of 0.04 μM, 0.2 μM, and 1 μM were added respectively; in the Dasatinib group, Dasatinib with concentrations of 0.08 μM, 0.4 μM, and 2 μM were added respectively; in the Saracatinib group, Saracatinib with concentrations of 0.2 μM, 1 μM, and 5 μM were added respectively; in the AMG510 + Dasatinib group (A + D), AMG510 with a concentration of 0.04 μM and Dasatinib with a concentration of 0.08 μM were added respectively, or AMG510 with a concentration of 0.2 μM and Dasatinib with a concentration of 0.4 μM were added respectively, or AMG510 with a concentration of 1 μM and Dasatinib with a concentration of 2 μM were added respectively (the molar ratio of AMG510 and Dasatinib was 1:2); in the AMG510 + Saracatinib group (A + S), AMG510 with a concentration of 0.04 μM and Saracatinib with a concentration of 0.2 μM were added respectively, or AMG510 with a concentration of 0.2 μM and Saracatinib with a concentration of 1 μM were added respectively, or AMG510 with a concentration of 1 μM and Saracatinib with a concentration of 5 μM were added respectively (the molar ratio of AMG510 and Saracatinib was 1:5).

[0064] In NCI-H2030 cells, for the AMG510 group: AMG510 with concentrations of 0.2 μM, 1 μM, and 5 μM were added respectively; for the Dasatinib group: Dasatinib with concentrations of 0.04 μM, 0.2 μM, and 1 μM were added respectively; for the Saracatinib group: Saracatinib with concentrations of 0.08 μM, 0.4 μM, and 2 μM were added respectively; for the AMG510+Dasatinib group (A+D): 0.2 μM of AMG510 and 0.04 μM of Dasatinib were added respectively, or 1 μM of AMG510 and 0.2 μM of Dasatinib were added respectively, or 5 μM of AMG510 and 1 μM of Dasatinib were added respectively (the molar ratio of AMG510 and Dasatinib was 5:1); for the AMG510+Saracatinib group (A+S): 0.2 μM of AMG510 and 0.08 μM of Saracatinib were added respectively, or 1 μM of AMG510 and 0.4 μM of Saracatinib were added respectively, or 5 μM of AMG510 and 2 μM of Saracatinib were added respectively (the molar ratio of AMG510 and Saracatinib was 2.5:1). The volume of the solution containing different concentrations of drugs added to each group was 100 μL.

[0065] The results are as Figure 3 shown, where Figure 3 Figures A and B respectively are the synergy index diagrams of AMG510 combined with Dasatinib treating cells Calu-1 and NCI-H2030; Figure 3 Figures C and D respectively are the synergy index diagrams of AMG510 combined with Saracatinib treating cells Calu-1 and NCI-H2030. In cells Cula-1 and NCI-H2030 cells, AMG510 was combined with Dasatinib and Saracatinib respectively. After adding the drugs, the cells were cultured for 3 days, and then the cell viability was detected by CCK8, and the synergy index (CI) was calculated using the Chou-Talalay formula. 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 In Figures A, B, C, and D, it shows that the CI indexes are all below 1, indicating that both AMG510 combined with Dasatinib and AMG510 combined with Saracatinib have strong synergistic antitumor activities and can effectively inhibit the growth of Calu-1 and NCI-H2030 cell lines. The curative effects of the two may vary depending on the cell type. The synergistic inhibitory effect of AMG510 combined with Dasatinib on the Calu1 cell line is stronger.

[0066] As Figure 4 shown Figure 4 is the diagram of the effect of combined targeting of SRC and KRAS on the growth and survival of KRAS-G12C mutant lung cancer cells. Among them, Figure 4 Figures A and B of are the CCK8 result diagrams of Dasatinib combined with AMG510 treating cells Calu-1 and NCI-H2030, Figure 4 Figure C of is the colony formation experiment result diagram of Dasatinib combined with AMG510 treating cells Calu-1 and NCI-H2030, Figure 4 Figures D and E of are the CCK8 result diagrams of Saracatinib combined with AMG510 treating cells Calu-1 and NCI-H2030, Figure 4 Figure F of is the colony formation experiment result diagram of Saracatinib combined with AMG510 treating cells Calu-1 and NCI-H2030, Figure 4 Figure G of is the WB result diagram of AMG510 combined with Dasatinib and Saracatinib respectively treating NCI-H2030 cells.

[0067] Figure 4 The CCK8 results of Figures A-B show that Dasatinib combined with AMG510 can significantly inhibit the growth of Calu-1 and NCI-H2030 cells, Figure 4 The colony formation experiment of C shows that Dasatinib combined with AMG510 can significantly inhibit the survival of Calu-1 and NCI-H2030 cells. Figure 4 The CCK8 results of Figures D-E show that Saracatinib combined with AMG510 can significantly inhibit the growth of Calu-1 and NCI-H2030 cells, Figure 4 The colony formation experiment of F shows that Saracatinib combined with AMG510 can significantly inhibit the survival of Calu-1 and NCI-H2030 cells. Refer to Figure 4 Figure G of , the WB results show that in NCI-H2030, AMG510 combined with Dasatinib and Saracatinib treatments can both significantly inhibit the activity of the SRC-FAK pathway.

[0068] It can be seen that the solution of using an SRC inhibitor in combination with a KRAS-G12C inhibitor provided by the present application for preparing a medicament for treating KRAS-G12C mutant lung cancer, compared with the use of the KRAS-G12C inhibitor alone, the solution of the SRC inhibitor in combination 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 directly used in combination or made into a combined preparation for treating KRAS mutant lung cancer, and has good prospects for clinical transformation.

[0069] As described above, the above are only specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention can easily think of changes or substitutions, which should all be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

Claims

1. Use of salactinib combined with sotorasib for the preparation of a drug for treating KRAS-G12C mutant non-small cell lung cancer, characterized in that: The structural formula of Sotorasib is , the molar ratio of Sotorasib to Sacatinib is 1:5 or 2.5:

1.

2. A pharmaceutical composition for treating KRAS-G12C mutant non-small cell lung cancer, characterized in that: The pharmaceutical composition is Sotorasib and Saracatinib, wherein the structural formula of Sotorasib is , the molar ratio of Sotorasib to Sacatinib is 1:5 or 2.5:1.

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