Medicine containing elemene and kinase inhibitor

Abstract

The invention relates to a combined medicine for treating cancer, the combined medicine contains beta elemene, the combined medicine is a single compound preparation or a combination of two single preparations, and the combined medicine effectively realizes the synergism of medicines.

Description

Technical Field

[0001] The present invention relates to a pharmaceutical composition with a synergistic effect for treating cancer, and belongs to the field of medicine. Background Art

[0002] Tumor is a major problem in medical research in the world today, seriously threatening human health and life safety. Nearly ten million people die of cancer every year in the world, and about one million people die of malignant tumors in China every year. At present, the main treatments for tumors are surgery, chemotherapy and radiotherapy. However, a large number of chemotherapy drugs currently used clinically still have problems such as large systemic toxic side effects, drug resistance and weak selectivity. In recent years, a large number of clinical and experimental studies have proved that traditional Chinese medicine and combined drug therapies have special effects in the prevention, treatment and rehabilitation of tumors, and have gradually become the focus of attention.

[0003] β-elemene is a naturally occurring sesquiterpene and the main component of zedoary oil and citronella oil. It has been proven to be used for broad-spectrum treatment of various tumors and has broad application prospects and market demands. In order to meet the treatment needs of cancer patients as soon as possible, it is urgent to develop more combined drug dosage forms and administration methods with clinical value. Summary of the Invention

[0004] The present invention provides a combined drug for treating cancer, the combined drug comprising β-elemene and a second drug, and the second drug is a kinase inhibitor. Preferably, the kinase inhibitor is selected from one or more of TKI inhibitors, KRAS inhibitors, IDH1 inhibitors, and mTOR inhibitors.

[0005] Further, the TKI inhibitor is anlotinib.

[0006] Further, the molar ratio of anlotinib to β-elemene is 1:0.1 - 1:2000, preferably 1:1 - 1:2000, preferably 1:1 - 1:1500, preferably 1:1 - 1:1282, preferably 1:10 - 1:1282, preferably 1:20 - 1:321, preferably 1:40 - 1:321, preferably 1:130 - 1:321.

[0007] Further, the molar ratio of anlotinib to β-elemene is 1:0.1, 1:1, 1:10, 1:20, 1:40, 1:130, 1:321, 1:1282, 1:1500, 1:2000. Preferably, the molar ratio of anlotinib to β-elemene is 1:40, 1:130, 1:321.

[0008] Further, the TKI inhibitor is icotinib.

[0009] Further, the molar ratio of icotinib to β-elemene is 1:25 - 1:1000, preferably 1:100 - 1:1000, preferably 1:25 - 1:400, preferably 1:100 - 1:400, preferably 1:100 - 1:200.

[0010] Further, the molar ratio of icotinib to β-elemene is 1:25, 1:50, 1:100, 1:200, 1:400, 1:1000, and preferably the molar ratio of icotinib to β-elemene is 1:100, 1:200.

[0011] Further, the TKI inhibitor is ceritinib.

[0012] Further, the molar ratio of ceritinib to β-elemene is 1:7 - 1:222, preferably 1:28 - 1:111.

[0013] Further, the molar ratio of ceritinib to β-elemene is 1:7, 1:14, 1:28, 1:56, 1:111, 1:222, 1:500, and preferably the molar ratio of ceritinib to β-elemene is 1:28, 1:56, 1:111.

[0014] Further, the TKI inhibitor is larotrectinib.

[0015] Further, the molar ratio of larotrectinib to β-elemene is 1:62.5 - 1:2000, preferably 1:125 - 1:1000, preferably 1:125 - 1:500, preferably 1:250 - 1:500.

[0016] Further, the molar ratio of larotrectinib to β-elemene is 1:62.5, 1:125, 1:250, 1:500, 1:1000, 1:2000, and preferably the molar ratio of larotrectinib to β-elemene is 1:250, 1:500.

[0017] Further, the TKI inhibitor is capmatinib.

[0018] Further, the molar ratio of capmatinib to β-elemene is 1:0.5 - 1:20, preferably 1:0.5 - 1:5, preferably 1:1.25 - 1:20, preferably 1:1.25 - 1:5, preferably 1:2.5 - 1:5.

[0019] Further, the molar ratio of capmatinib to β-elemene is 1:0.5, 1:1.25, 1:2.5, 1:5, 1:20, and preferably the molar ratio of capmatinib to β-elemene is 1:2.5, 1:5.

[0020] Further, the TKI inhibitor is dacomitinib.

[0021] Further, the molar ratio of dacomitinib to β-elemene is 1:1 - 1:2000, preferably 1:5 - 1:2000, preferably 1:5 - 1:500, preferably 1:5 - 1:250, preferably 1:5 - 1:125, preferably 1:15.63 - 1:2000, preferably 1:15.63 - 1:500, preferably 1:62.5 - 1:125.

[0022] Further, the molar ratio of dacomitinib to β-elemene is 1:5, 1:15.63, 1:62.5, 1:125, 1:250, 1:500, 1:2000.

[0023] Further, the TKI inhibitor is apatinib.

[0024] Further, the molar ratio of apatinib to β-elemene is 1:0.63 - 1:40, preferably 1:1.25 - 1:20, preferably 1:1.25 - 1:10, preferably 1:1.25 - 1:5, preferably 1:1.25 - 1:2.5.

[0025] Further, the molar ratio of apatinib to β-elemene is 1:0.63, 1:1.25, 1:2.5, 1:5, 1:10, 1:20, 1:40, and preferably the molar ratio of apatinib to β-elemene is 1:1.25, 1:2.5.

[0026] Further, the second drug is regorafenib.

[0027] Further, the molar ratio of regorafenib to β-elemene is 1:12.5 - 1:1000, preferably 1:25 - 1:500, preferably 1:62.5 - 1:1000, preferably 1:125 - 1:500, preferably 1:250 - 1:500, preferably 1:12.5 - 1:200, preferably 1:25 - 1:100.

[0028] Further, the molar ratio of regorafenib to β-elemene is 1:12.5, 1:25, 1:62.5, 1:100, 1:125, 1:200, 1:250, 1:500, 1:1000.

[0029] Further, the TKI inhibitor is gilteritinib.

[0030] Further, the molar ratio of gilteritinib to β-elemene is 1:3.47 - 1:444.44, preferably 1:27.78.

[0031] Further, the TKI inhibitor is neratinib.

[0032] Further, the molar ratio of neratinib to β-elemene is 1:156.25 - 1:20000, preferably 1:5000.

[0033] Further, the KRAS inhibitor is sotorasib.

[0034] Further, the molar ratio of sotorasib to β-elemene is 1:10 - 1:1000, preferably 1:10 - 1:500, preferably 1:10 - 1:160, preferably 1:80 - 1:1000, preferably 1:80 - 1:500, preferably 1:80 - 1:160.

[0035] Further, the molar ratio of sotorasib to β-elemene is 1:10, 1:40, 1:60, 1:80, 1:160, 1:500, 1:1000.

[0036] Further, the IDH1 inhibitor is ivosidenib.

[0037] Further, the molar ratio of ivosidenib to β-elemene is 1:31.25 - 1:4000, preferably 1:250.

[0038] Further, the mTOR inhibitor is temsirolimus.

[0039] Further, the molar ratio of temsirolimus to β-elemene is 1:1250 - 1:10000, preferably 1:2500 - 1:5000, preferably 1:2500, 1:5000.

[0040] Further, the combined drug is a pharmaceutical composition or a single compound preparation or a combination of two separate preparations. Preferably, the active ingredients of the pharmaceutical composition are composed of β-elemene and a second drug.

[0041] Further, the pharmaceutical composition contains pharmaceutically acceptable excipients, and the excipients include but are not limited to injection oils, carriers, fillers, disintegrants, binders, lyoprotectants, pH regulators, preservatives, antioxidants, wetting agents, etc. The pharmaceutical composition can be prepared into a preparation, and the preparation can be one or more of tablets, capsules, pills, mixtures, gels, ointments, granules, powders, plasters, infusions, pills for oral administration, dripping pills, injections or any other dosage forms.

[0042] Further, the present invention provides the use of a drug in the preparation of a drug for treating cancer.

[0043] Further, the cancer is lung cancer, breast cancer, renal cell carcinoma, ovarian cancer, small cell lung cancer, non-small cell lung cancer, malignant melanoma, B-lymphocyte tumors (including chronic lymphocytic leukemia / small lymphocytic lymphoma, mantle cell lymphoma, Waldenström macroglobulinemia, follicular lymphoma, diffuse large B-cell lymphoma of the non-germinal center subtype, etc.), leukemia, colorectal cancer, malignant pleural mesothelioma, pancreatic cancer, head and neck cancer, gastric cancer, bladder cancer, bone cancer, esophageal cancer, brain tumor, liver cancer, cervical cancer, cancerous pleural effusion and ascites. Preferably, the cancer is lung cancer, liver cancer, colorectal cancer, leukemia, breast cancer, renal cell carcinoma. More preferably, the cancer is lung cancer, breast cancer, liver cancer, renal cell carcinoma. More preferably, it is lung cancer, breast cancer, renal cell carcinoma. More preferably, it is lung cancer, liver cancer. The lung cancer is preferably squamous cell lung cancer or large cell lung cancer.

[0044] Specific implementation examples

[0045] The technical solutions of the present invention will be further elaborated in detail below in conjunction with the specification and specific implementation examples, but the implementation of the present invention is not limited thereto. Equivalent substitutions, combinations, improvements or modifications made by those skilled in the art to the technical solutions of the present invention according to the description of the present invention should all be covered by the protection scope of the present invention.

[0046] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field. Unless otherwise specified, the reagents and materials used in the following examples are all commercially available products.

[0047] The following examples are calculated in the following manner:

[0048] 1) Calculate the in vitro tumor growth inhibition rate

[0049] Tumor cell growth inhibition rate IR(%) = (1 - (OD compound - OD blank control) / (OD solvent control - OD blank control)) * 100%

[0050] 2) Judgment of the effect of combined use of two drugs:

[0051] The Jin Zhengjun q-value method is used for judgment (see non-patent literature: Dai Tijun, "Additivity in combined drug use", Acta Pharmacologica Sinica, December 1980, 1(2) 70-76). q = P (A+B) / (P A +P B -P A ×P B ). In the formula, P A , P B and P (A+B)They are the inhibition rates of the β-elemene drug group, the second drug group, and the combination of the two drugs, respectively. q < 0.85 indicates antagonism after the combination of the two drugs; q > 1.15 indicates synergy after the combination of the two drugs, and 0.85 ≥ q ≥ 1.15 indicates an additive effect after the combination of the two drugs.

[0052] 3) Calculate the in vivo tumor growth inhibition rate:

[0053] Tumor growth inhibition rate TGI (%) = [(1 - (average bioluminescence signal at the end of drug administration in a certain treatment group - average bioluminescence signal at the start of drug administration in that treatment group)) / (average bioluminescence signal at the end of treatment in the vehicle control group - average bioluminescence signal at the start of treatment in the vehicle control group)] * 100%. The data in this application all conform to statistical laws.

[0054] Example 1

[0055] Divide β-elemene and anlotinib into:

[0056] Group 1: 2000 uM β-elemene, Group 2: 500 uM β-elemene, Group 3: 62.5 uM β-elemene, Group 4: 31.25 uM β-elemene; Group 5: 15.625 uM β-elemene, Group 6: 1.56 uM anlotinib;

[0057] Group 7: 2000 uM β-elemene + 1.56 uM anlotinib, Group 8: 500 uM β-elemene + 1.56 uM anlotinib, Group 9: 62.5 uM β-elemene + 1.56 uM anlotinib, Group 10: 31.25 uM β-elemene + 1.56 uM anlotinib, Group 11: 15.625 uM β-elemene + 1.56 uM anlotinib.

[0058] The above groups all use 0.25% DMSO and 0.25% absolute ethanol as the vehicle.

[0059] Cell culture: NCI-H2170 cells were inoculated into a sterile cell culture flask, and an appropriate amount of 1640 + 10% FBS medium was added. The cells were cultured in an incubator at 37°C, 5% CO2, and saturated humidity. The cells grew adherently in a monolayer and were passaged once every 2 - 3 days. When passaging, the cultured cells were digested with 0.25% trypsin + 0.02% EDTA for 2 - 4 minutes, and the cell suspension was made by pipetting the culture medium to blow and beat the cells. Then, the cells were inoculated according to the required concentration. Logarithmic growth phase NCI-H2170 cells were made into a single-cell suspension according to the passaging method, counted, and the cell concentration was adjusted to: 3.5*10^4 cells / mL. The above cell suspension was inoculated into a 96-well cell culture plate, and 100 μL of the cell suspension was added to each well. Wells without cell culture medium were taken as blank controls for zero adjustment. After the culture plate was placed in an incubator at 37°C and 5% CO2 for adherent culture for 12 hours, the culture medium in the wells was removed. 100 μL of medium (containing 2.5 μL of β-elemene at different concentrations + 2.5 μL of anlotinib) was added to groups seven to eleven, 100 μL of medium (containing 2.5 μL of β-elemene at different concentrations + 2.5 μL of DMSO) was added to groups one to five, 100 μL of medium (containing 2.5 μL of absolute ethanol + 2.5 μL of anlotinib) was added to group six, and the vehicle control was 100 μL of medium (containing 2.5 μL of DMSO and 2.5 μL of absolute ethanol). Each group was set with 3 replicate wells. After culturing for 72 hours respectively, the culture medium was removed, 10% CCK8 working solution was added, and the mixture was incubated at 37°C for 1 hour. Then, the OD value was measured at 450 nM using a full-automatic microplate reader. The inhibition rate was calculated according to the aforementioned formula. The results showed that each group had a certain inhibitory effect on the cells. The q value was calculated based on the inhibition rate, and the synergistic effect is shown in Table 1:

[0060] Table 1

[0061]

[0062]

[0063] Example 2

[0064] β-elemene and icotinib were divided into:

[0065] Group 1: 2000 uM β-elemene, Group 2: 1000 uM β-elemene, Group 3: 500 uM β-elemene, Group 4: 250 uM β-elemene, Group 5: 125 uM β-elemene; Group 6: 5 uM icotinib;

[0066] Group 7: 2000 uM β-elemene + 5 uM icotinib, Group 8: 1000 uM β-elemene + 5 uM icotinib, Group 9: 500 uM β-elemene + 5 uM icotinib, Group 10: 250 uM β-elemene + 5 uM icotinib, Group 11: 125 uM β-elemene + 5 uM icotinib.

[0067] The above groups all used 0.25% DMSO and 0.25% absolute ethanol as solvents.

[0068] Cell culture: NCI-H2170 cells were inoculated into a sterile cell culture flask, and an appropriate amount of 1640 + 10% FBS medium was added. The cells were cultured in an incubator at 37°C, 5% CO2, and saturated humidity. The cells grew adherently in a monolayer and were passaged once every 2 - 3 days. When passaging, the cultured cells were digested with 0.25% trypsin + 0.02% EDTA for 2 - 4 minutes, and the cell suspension was made by pipetting the culture medium to blow and beat the cells. Then, the cells were inoculated according to the required concentration. Logarithmic growth phase NCI-H2170 cells were made into a single-cell suspension according to the passaging method, counted, and the cell concentration was adjusted to: 3.5*10^4 cells / mL. The above cell suspension was inoculated into a 96-well cell culture plate, and 100 μL of the cell suspension was added to each well. The wells without cell culture medium were used as blank zero adjustments. After the culture plate was placed in a 37°C, 5% CO2 incubator and adhered for 12 hours, the culture medium in the wells was removed. 100 μL of medium (containing 2.5 μL of β-elemene at different concentrations + 2.5 μL of icotinib) was added to groups seven to eleven, 100 μL of medium (containing 2.5 μL of β-elemene at different concentrations + 2.5 μL of DMSO) was added to groups one to five, 100 μL of medium (containing 2.5 μL of absolute ethanol + 2.5 μL of icotinib) was added to group six, and the solvent control was added with 100 μL of medium (containing 2.5 μL of DMSO and 2.5 μL of absolute ethanol). Each group was set with 3 replicate wells. After culturing for 72 hours respectively, the culture medium was removed, 10% CCK8 working solution was added, and it was incubated at 37°C for 1 hour. Then, the OD value was measured at 450 nM using a full-automatic microplate reader. The inhibition rate was calculated according to the aforementioned formula. The results showed that each group had a certain inhibitory effect on the cells. The q value was calculated based on the inhibition rate, and the synergistic effect is shown in Table 2:

[0069] Table 2

[0070] Group Icotinib:β - elemene molar ratio Q value Group Seven 1:400 1.03 Group Eight 1:200 1.28 Group Nine 1:100 1.35 Group Ten 1:50 0.98 Group Eleven 1:25 0.93

[0071] Example 3

[0072] β-elemene and ceritinib were divided into:

[0073] Group 1: 2000 uM β-elemene, Group 2: 1000 uM β-elemene, Group 3: 500 uM β-elemene, Group 4: 250 uM β-elemene, Group 5: 125 uM β-elemene; Group 6: 62.5 uM β-elemene, Group 7: 9 uM ceritinib;

[0074] Group VIII: 2000 uM β-elemene + 9 uM ceritinib, Group IX: 1000 uM β-elemene + 9 uM ceritinib, Group X: 500 uM β-elemene + 9 uM ceritinib, Group XI: 250 uM β-elemene + 9 uM ceritinib, Group XII: 125 uM β-elemene + 9 uM ceritinib, Group XIII: 62.5 uM β-elemene + 9 uM ceritinib.

[0075] For the above groups, 0.25% DMSO and 0.25% absolute ethanol were used as solvents.

[0076] Cell culture: NCI-H2170 cells were inoculated into a sterile cell culture flask, and an appropriate amount of 1640 + 10% FBS medium was added. The cells were cultured in an incubator at 37°C, 5% CO2 and saturated humidity. The cells grew adherently in a monolayer and were passaged once every 2 - 3 days. When passaging, the cultured cells were digested with 0.25% trypsin + 0.02% EDTA for 2 - 4 minutes, and the cell suspension was made by pipetting the culture medium to blow and beat the cells. Then, the cells were inoculated according to the required concentration. Logarithmic growth phase NCI-H2170 cells were made into a single-cell suspension according to the passaging method, counted, and the cell concentration was adjusted to: 3.5*10^4 cells / mL. The above cell suspension was inoculated into a 96-well cell culture plate, and 100 μL of the cell suspension was added to each well. Wells without cell culture medium were taken as blank controls for zero adjustment. After the culture plate was placed in an incubator at 37°C, 5% CO2 for adherent culture for 12 hours, the culture medium in the wells was removed. 100 μL of medium (containing 2.5 μL of β-elemene at different concentrations + 2.5 μL of ceritinib) was added to Groups VIII to XIII, 100 μL of medium (containing 2.5 μL of β-elemene at different concentrations + 2.5 μL of DMSO) was added to Groups I to VI, 100 μL of medium (containing 2.5 μL of absolute ethanol + 2.5 μL of ceritinib) was added to Group VII, and the solvent control was 100 μL of medium (containing 2.5 μL of DMSO and 2.5 μL of absolute ethanol). Each group was set with 3 replicate wells. After culturing for 72 hours respectively, the culture medium was removed, 10% CCK8 working solution was added, and the mixture was incubated at 37°C for 1 hour. Then, the OD value was measured at 450 nM using a fully automatic microplate reader. The inhibition rate was calculated according to the aforementioned formula. The results showed that each group had a certain inhibitory effect on the cells. The q value was calculated based on the inhibition rate, and the synergistic effect is shown in Table 3:

[0077] Table 3

[0078] Group Ceritinib:β - elemene molar ratio Q value Group Eight 1:222 1.06 Group Nine 1:111 1.27 Group Ten 1:56 1.26 Group Eleven 1:28 1.24 Group Twelve 1:14 1.06 Group Thirteen 1:7 0.94

[0079] Example 4

[0080] β-elemene and sotorasib were divided into:

[0081] Group 1: 2000 uM β-elemene, Group 2: 1000 uM β-elemene, Group 3: 500 uM β-elemene, Group 4: 125 uM β-elemene; Group 5: 12.5 uM sotorasib;

[0082] Group 6: 2000 uM β-elemene + 12.5 uM sotorasib, Group 7: 1000 uM β-elemene + 12.5 uM sotorasib, Group 8: 500 uM β-elemene + 12.5 uM sotorasib, Group 9: 125 uM β-elemene + 12.5 uM sotorasib.

[0083] For the above groups, 0.25% DMSO and 0.25% absolute ethanol were used as solvents.

[0084] Cell culture: NCI-H2170 cells were inoculated into a sterile cell culture flask, and an appropriate amount of 1640 + 10% FBS medium was added. The cells were cultured in an incubator at 37°C, 5% CO2 and saturated humidity. The cells grew adherently in a monolayer and were passaged once every 2 - 3 days. When passaging, the cultured cells were digested with 0.25% trypsin + 0.02% EDTA for 2 - 4 minutes, and the cell suspension was made by pipetting the culture medium to blow and beat the cells. Then, the cells were inoculated according to the required concentration. Logarithmic growth phase NCI-H2170 cells were made into a single cell suspension according to the passaging method, counted, and the cell concentration was adjusted to: 3.5*10^4 cells / mL. The above cell suspension was inoculated into a 96-well cell culture plate, and 100 μL of the cell suspension was added to each well. Wells without cell culture medium were taken as blank controls for zero adjustment. After the culture plate was placed in an incubator at 37°C and 5% CO2 for adherent culture for 12 hours, the culture medium in the wells was removed. For groups 6 to 9, 100 μL of medium (containing 2.5 μL of β-elemene at different concentrations + 2.5 μL of sotorasib) was added respectively. For groups 1 to 4, 100 μL of medium (containing 2.5 μL of β-elemene at different concentrations + 2.5 μL of DMSO) was added respectively. For group 5, 100 μL of medium (containing 2.5 μL of absolute ethanol + 2.5 μL of sotorasib) was added. The solvent control was added with 100 μL of medium (containing 2.5 μL of DMSO and 2.5 μL of absolute ethanol). Each group was set with 3 replicate wells. After culturing for 72 hours respectively, the culture medium was removed, 10% CCK8 working solution was added, and the mixture was incubated at 37°C for 1 hour. Then, the OD value was measured at 450 nM using a fully automatic microplate reader. The inhibition rate was calculated according to the aforementioned formula. The results showed that each group had a certain inhibitory effect on the cells. The q value was calculated based on the inhibition rate. The synergistic effect is shown in Table 4:

[0085] Table 4

[0086] Group Sotorasib:β - elemene molar ratio Q value Group Six 1:160 1.17 Group Seven 1:80 1.65 Group Eight 1:40 0.77 Group Nine 1:10 0.67

[0087] Example 5

[0088] Beta-elemene and larotrectinib are divided into:

[0089] Group 1: 2000 μM β-elemene, Group 2: 1000 μM β-elemene, Group 3: 500 μM β-elemene, Group 4: 250 μM β-elemene, Group 5: 125 μM β-elemene; Group 6: 62.5 μM β-elemene, Group 7: 1 μM larotrectinib;

[0090] Group eight: 2000μM β-elemene + 1μM larotrectinib, group nine: 1000μM β-elemene + 1μM larotrectinib, group ten: 500μM β-elemene + 1μM larotrectinib, group eleven: 250μM β-elemene + 1μM larotrectinib, group twelve: 125μM β-elemene + 1μM larotrectinib, group thirteen: 62.5μM β-elemene + 1μM larotrectinib.

[0091] The above groups all used 0.25% DMSO and 0.25% anhydrous ethanol as solvents.

[0092] Cell culture: Cells were inoculated in sterile cell culture flasks, and an appropriate amount of 1640+10% FBS medium was added. The cells were cultured in an incubator at 37°C, 5% CO2 and saturated humidity. The cells grew as monolayers and were passaged once every 2 to 3 days. When passaged, cells were first digested with 2mL 0.25% trypsin for 2 to 4min, then neutralized with 2mL 1640+10% FBS, and the culture medium was pipetted to blow the cells into single cells, and then passaged at a density of 2 to 3*10^6 / bottle. Take NCI-H2170 cells in the logarithmic growth phase, make a single cell suspension according to the passage method, count, and adjust the cell concentration to: 3.5*10^4 / mL. The above cell suspension was inoculated in a 96-well cell culture plate, and 100μL of cell suspension was added to each well. Take the blank culture solution as the blank zero well. After the culture plate was placed in a 37°C, 5% CO2 incubator for 24 hours, the culture medium in the well was removed, and 100 μL of culture medium (containing 0.25 μL of different concentrations of β-elemene + 0.25 μL of larotrectinib) was added to groups 8 to 13, 100 μL of culture medium (containing 0.25 μL of different concentrations of β-elemene + 0.25 μL of DMSO) was added to groups 1 to 6, and 100 μL of culture medium (containing 0.25 μL of anhydrous ethanol + 0.25 μL of larotrectinib) was added to group 7. The solvent control was to add 100 μL of culture medium (containing 0.25 μL of anhydrous ethanol and 0.25 μL of DMSO). Three replicate wells were set for each group. After 72 hours of culture, the culture medium in the well was removed, 10% of CCK8 working solution was added, and the culture was incubated at 37°C for 1 to 3 hours. The OD value was measured at 450 nM using an automatic microplate reader. The inhibition rate was calculated according to the above formula. The results showed that each group had a certain inhibitory effect on the cells. The q value was calculated according to the inhibition rate. The synergistic effect is shown in Table 5:

[0093] Table 5

[0094] Group Larotrectinib and β - elemene molar ratio Q value Group Eight 1:2000 0.89 Group Nine 1:1000 0.98 Group Ten 1:500 3.69 Group Eleven 1:250 2.45 Group Twelve 1:125 1.10 Group Thirteen 1:62.5 0.74

[0095] Example 6

[0096] Divide β-elemene and capmatinib into:

[0097] Group 1: 2000 μM β-elemene, Group 2: 500 μM β-elemene, Group 3: 250 μM β-elemene, Group 4: 125 μM β-elemene; Group 5: 100 μM capmatinib;

[0098] Group 6: 2000 μM β-elemene + 100 μM capmatinib, Group 7: 500 μM β-elemene + 100 μM capmatinib, Group 8: 250 μM β-elemene + 100 μM capmatinib, Group 9: 125 μM β-elemene + 100 μM capmatinib.

[0099] The above groups all use 0.25% DMSO and 0.25% absolute ethanol as solvents.

[0100] Cell culture: NCI-H2170 cells were inoculated into a sterile cell culture flask, and an appropriate amount of 1640 + 10% FBS medium was added. The cells were cultured in an incubator at 37°C, 5% CO2, and saturated humidity. The cells grew adherently in a monolayer and were passaged once every 2 - 3 days. When passaging the cells, first digest them with 2 mL of 0.25% trypsin for 2 - 4 minutes, then add 2 mL of 1640 + 10% FBS to neutralize. Use a pipette to aspirate the culture medium and blow the cells into single cells, and then passage them at a density of 2 - 3×10^6 cells per flask. Take NCI-H2170 cells in the logarithmic growth phase, prepare a single-cell suspension according to the passaging method, count, and adjust the cell concentration to 3.5×10^4 cells / mL. Inoculate the above cell suspension into a 96-well cell culture plate, and add 100 μL of the cell suspension to each well. Take the blank culture medium as the blank zero-adjustment well. After culturing the culture plate adherently in an incubator at 37°C, 5% CO2 for 24 hours, remove the culture medium in the wells. Add 100 μL of the medium (containing 0.25 μL of β-elemene at different concentrations + 0.25 μL of capmatinib) to groups six to nine respectively, add 100 μL of the medium (containing 0.25 μL of β-elemene at different concentrations + 0.25 μL of DMSO) to groups one to four respectively, add 100 μL of the medium (containing 0.25 μL of absolute ethanol + 0.25 μL of capmatinib) to group five, and the vehicle control is to add 100 μL of the medium (containing 0.25 μL of absolute ethanol and 0.25 μL of DMSO). Set 3 replicate wells for each group. After culturing for 72 hours, remove the culture medium in the wells, add 10% CCK8 working solution, incubate at 37°C for 1 - 3 hours, and measure the OD value at 450 nM using a fully automatic microplate reader. Calculate the inhibition rate according to the aforementioned formula. The results show that each group has a certain inhibitory effect on the cells. Calculate the q value according to the inhibition rate, and the synergistic effect is shown in Table 6:

[0101] Table 6

[0102] Group Capmatinib and β - elemene molar ratio Q value Group Six 1:20 0.88 Group Seven 1:5 1.89 Group Eight 1:2.5 1.62 Group Nine 1:1.25 1.04

[0103] Example 7

[0104] Divide β-elemene and dacomitinib into:

[0105] Group 1: 2000 μM β-elemene, Group 2: 500 μM β-elemene, Group 3: 250 μM β-elemene, Group 4: 125 μM β-elemene; Group 5: 62.5 μM β-elemene, Group 6: 15.625 μM β-elemene, Group 7: 1 μM dacomitinib;

[0106] Group VIII: 2000 μM β-elemene + 1 μM dacomitinib, Group IX: 500 μM β-elemene + 1 μM dacomitinib, Group X: 250 μM β-elemene + 1 μM dacomitinib, Group XI: 125 μM β-elemene + 1 μM dacomitinib, Group XII: 62.5 μM β-elemene + 1 μM dacomitinib, Group XIII: 15.625 μM β-elemene + 1 μM dacomitinib.

[0107] The above groups all used 0.25% DMSO and 0.25% absolute ethanol as solvents.

[0108] Cell culture: NCI-H2170 cells were inoculated into sterile cell culture flasks, and an appropriate amount of 1640 + 10% FBS medium was added. The cells were cultured in an incubator at 37 °C, 5% CO2 and saturated humidity. The cells grew adherently in a monolayer and were passaged once every 2 - 3 days. When passaging the cells, first digest with 2 mL of 0.25% trypsin for 2 - 4 min, then add 2 mL of 1640 + 10% FBS to neutralize. Use a pipette to aspirate the culture medium and blow the cells into single cells, and then passage at a density of 2 - 3×10^6 cells / flask. Take NCI-H2170 cells in the logarithmic growth phase, prepare a single cell suspension according to the passaging method, count, and adjust the cell concentration to: 3.5×10^4 cells / mL. Inoculate the above cell suspension into a 96-well cell culture plate, and add 100 μL of the cell suspension to each well. Take the wells without cell culture medium as blank controls for zero adjustment. After culturing the plate adherently in an incubator at 37 °C, 5% CO2 for 12 hours, remove the culture medium in the wells. Group VIII to Group XIII were respectively added with 100 μL of medium (containing 0.25 μL of β-elemene at different concentrations + 0.25 μL of dacomitinib), Group I to Group VII were respectively added with 100 μL of medium (containing 0.25 μL of β-elemene at different concentrations + 0.25 μL of DMSO), Group VIII was added with 100 μL of medium (containing 0.25 μL of absolute ethanol + 0.25 μL of dacomitinib), and the solvent control was added with 100 μL of medium (containing 0.25 μL of absolute ethanol and 0.25 μL of DMSO). Each group was set with 3 replicates. After culturing for 72 h respectively, remove the culture medium, add 10% CCK8 working solution, incubate at 37 °C for 1 h, and then measure the OD value at 450 nM using a full-automatic microplate reader. Calculate the inhibition rate according to the aforementioned formula. The results showed that each group had a certain inhibitory effect on the cells. Calculate the q value according to the inhibition rate. The synergistic effect is shown in Table 7:

[0109] Table 7

[0110] Group Dacomitinib and β - elemene molar ratio Q value Group Eight 1:2000 0.99 Group Nine 1:500 1.04 Group Ten 1:250 1.06 Group Eleven 1:125 1.12 Group Twelve 1:62.5 1.10 Group Thirteen 1:15.63 1.08

[0111] Example 8

[0112] β-elemene and apatinib were divided into:

[0113] Group 1: 2000 uM β-elemene, Group 2: 1000 uM β-elemene, Group 3: 500 uM β-elemene, Group 4: 250 uM β-elemene, Group 5: 125 uM β-elemene; Group 6: 62.5 uM β-elemene, Group 7: 31.25 uM β-elemene, Group 8: 50 uM apatinib;

[0114] Group 9: 2000 uM β-elemene + 50 uM apatinib, Group 10: 1000 uM β-elemene + 50 uM apatinib, Group 11: 500 uM β-elemene + 50 uM apatinib, Group 12: 250 uM β-elemene + 50 uM apatinib, Group 13: 125 uM β-elemene + 50 uM apatinib, Group 14: 62.5 uM β-elemene + 50 uM apatinib, Group 15: 31.25 uM β-elemene + 50 uM apatinib;

[0115] For the above groups, 0.5% DMSO and 1% absolute ethanol were used as solvents.

[0116] Cell culture: HuH-7 cells were inoculated into a sterile cell culture flask, and an appropriate amount of DMEM + 10% FBS medium was added. The cells were cultured in an incubator at 37°C, 5% CO2 and saturated humidity. The cells grew adherently in a monolayer and were passaged once every 2 - 3 days. When passaging the cells, first digest with 2 mL of 0.25% trypsin for 2 - 4 minutes, then add 2 mL of DMEM + 10% FBS to neutralize. Use a pipette to aspirate the culture medium and blow the cells into single cells, and then passage at a density of 2 - 3×10^6 cells per flask. Take HuH-7 cells in the logarithmic growth phase, prepare a single-cell suspension according to the passaging method, count, and adjust the cell concentration to 3.5×10^4 cells / mL. Inoculate the above cell suspension into a 96-well cell culture plate, add 100 μL of the cell suspension to each well. Take the blank culture medium as the blank zero-adjustment well. After culturing the plate adherently in an incubator at 37°C and 5% CO2 for 24 hours, remove the culture medium in the wells. For Groups 9 to 15, add 100 μL of the medium (containing 1 μL of β-elemene at different concentrations + 0.5 μL of apatinib) respectively, for Groups 1 to 7, add 100 μL of the medium (containing 1 μL of β-elemene at different concentrations + 0.5 μL of DMSO) respectively, for Group 8, add 100 μL of the medium (containing 1 μL of absolute ethanol + 0.5 μL of apatinib), and the solvent control is to add 100 μL of the medium (containing 1 μL of absolute ethanol and 0.5 μL of DMSO). Set 3 replicate wells for each group. After culturing for 72 hours, remove the culture medium in the wells, add 10% CCK8 working solution, incubate at 37°C for 1 - 3 hours, and measure the OD value at 450 nM using a fully automatic microplate reader. Calculate the inhibition rate according to the aforementioned formula. The results show that each group has a certain inhibitory effect on the cells. Calculate the q value according to the inhibition rate. The synergistic effect is shown in Table 8:

[0117] Table 8

[0118] Group Apatinib and β - elemene molar ratio Q value Group Nine 1：40.00 0.99 Group Ten 1：20.00 1.00 Group Eleven 1：10.00 1.05 Group Twelve 1：5.00 1.15 Group Thirteen 1：2.50 1.38 Group Fourteen 1：1.25 1.34 Group Fifteen 1：0.63 0.87

[0119] Example 9

[0120] Divide β-elemene and regorafenib into:

[0121] Group 1: 2000 uM β-elemene, Group 2: 1000 uM β-elemene, Group 3: 250 uM β-elemene, Group 4: 125 uM β-elemene; Group 5: 10 uM regorafenib;

[0122] Group 6: 2000 uM β-elemene + 10 uM regorafenib, Group 7: 1000 uM β-elemene + 10 uM regorafenib, Group 8: 250 uM β-elemene + 10 uM regorafenib, Group 9: 125 uM β-elemene + 10 uM regorafenib.

[0123] For the above groups, 0.5% DMSO and 1% absolute ethanol are used as solvents.

[0124] Cell culture: HuH-7 cells are inoculated into a sterile cell culture flask, and an appropriate amount of DMEM + 10% FBS medium is added. The cells are cultured in an incubator at 37°C, 5% CO2 and saturated humidity. The cells grow adherently in a monolayer and are passaged once every 2 - 3 days. When passaging the cells, first digest with 2 mL of 0.25% trypsin for 2 - 4 minutes, then add 2 mL of DMEM + 10% FBS to neutralize. Use a pipette to aspirate the culture medium and pipette the cells into single cells, and then passage at a density of 2 - 3×10^6 cells / flask. Take HuH-7 cells in the logarithmic growth phase, prepare a single-cell suspension according to the passaging method, count, and adjust the cell concentration to: 4.5×10^4 cells / mL. Inoculate the above cell suspension into a 96-well cell culture plate, and add 100 μL of the cell suspension to each well. Take the blank culture medium as the blank zero-adjustment well. After culturing the culture plate in a 37°C, 5% CO2 incubator for 24 hours, remove the culture medium in the wells. Add 100 μL of medium (containing 1 μL of β-elemene at different concentrations + 0.5 μL of regorafenib) to Groups 6 to 9, add 100 μL of medium (containing 1 μL of β-elemene at different concentrations + 0.5 μL of DMSO) to Groups 1 to 4, add 100 μL of medium (containing 1 μL of absolute ethanol + 0.5 μL of regorafenib) to Group 5, and the solvent control is to add 100 μL of medium (containing 0.5 μL of DMSO and 1 μL of absolute ethanol). Set 3 replicate wells for each group. After culturing for 72 hours, remove the culture medium in the wells, add 10% CCK8 working solution, incubate at 37°C for 1 - 3 hours, and measure the OD value at 450 nM using a fully automatic microplate reader. Calculate the inhibition rate according to the aforementioned formula. The results show that each group has a certain inhibitory effect on the cells. Calculate the q value according to the inhibition rate. The synergistic effect is shown in Table 9:

[0125] Table 9

[0126] Group Regorafenib and β - elemene molar ratio Q value Group Six 1：200.00 0.97 Group Seven 1：100.00 1.12 Group Eight 1：25.00 1.05 Group Nine 1：12.50 0.95

[0127] Example 10

[0128] Divide β-elemene and regorafenib into:

[0129] Group 1: 2000 uM β-elemene, Group 2: 1000 uM β-elemene, Group 3: 500 uM β-elemene, Group 4: 250 uM β-elemene, Group 5: 125 uM β-elemene; Group 6: 2 uM regorafenib;

[0130] Group 7: 2000 uM β-elemene + 2 uM regorafenib, Group 8: 1000 uM β-elemene + 2 uM regorafenib, Group 9: 500 uM β-elemene + 2 uM regorafenib, Group 10: 250 uM β-elemene + 2 uM regorafenib, Group 11: 125 uM β-elemene + 2 uM regorafenib.

[0131] Use 0.5% DMSO and 1% absolute ethanol as solvents for the above groups.

[0132] Cell culture: HCT-15 cells were inoculated into a sterile cell culture flask, and an appropriate amount of 1640 + 10% FBS medium was added. The cells were cultured in an incubator at 37°C, 5% CO2 and saturated humidity. The cells grew adherently in a monolayer and were passaged once every 2 - 3 days. When passaging the cells, first digest them with 2 mL of 0.25% trypsin for 2 - 4 minutes, then add 2 mL of DMEM + 10% FBS to neutralize. Use a pipette to aspirate the culture medium and blow the cells into single cells, and then passage them at a density of 3 - 4×10^6 cells per flask. Take HCT-15 cells in the logarithmic growth phase, prepare a single-cell suspension according to the passaging method, count the cells, and adjust the cell concentration to: 3.5×10^4 cells / mL. Inoculate the above cell suspension into a 96-well cell culture plate, and add 100 μL of the cell suspension to each well. Take the wells without cell culture medium as the blank control for zero adjustment. After culturing the plate adherently in an incubator at 37°C, 5% CO2 for 12 hours, remove the culture medium in the wells. Add 100 μL of medium (containing 1 μL of β-elemene at different concentrations + 0.5 μL of regorafenib) to groups seven to eleven, add 100 μL of medium (containing 1 μL of β-elemene at different concentrations + 0.5 μL of DMSO) to groups one to five, add 100 μL of medium (containing 1 μL of absolute ethanol + 0.5 μL of regorafenib) to group six, and the vehicle control was added with 100 μL of medium (containing 0.5 μL of DMSO and 1 μL of absolute ethanol). Each group was set with 3 replicate wells. After culturing for 72 hours respectively, remove the culture medium, add 10% CCK8 working solution, incubate at 37°C for 1 hour, and then measure the OD value at 450 nM using a fully automatic microplate reader. Calculate the inhibition rate according to the aforementioned formula. The results showed that each group had a certain inhibitory effect on the cells. Calculate the q value according to the inhibition rate, and the synergistic effect is shown in Table 10:

[0133] Table 10

[0134]

[0135]

[0136] Example 11

[0137] Divide β-elemene and gilteritinib into:

[0138] Group 1: 2000 μM β-elemene, Group 2: 1000 μM β-elemene, Group 3: 500 μM β-elemene, Group 4: 250 μM β-elemene, Group 5: 125 μM β-elemene; Group 6: 62.5 μM β-elemene, Group 7: 31.25 μM β-elemene, Group 8: 15.625 μM β-elemene, Group 9: 4.5 μM gilteritinib;

[0139] Group ten: 2000 μM β-elemene + 4.5 μM gilteritinib, Group eleven: 1000 μM β-elemene + 4.5 μM gilteritinib, Group twelve: 500 μM β-elemene + 4.5 μM gilteritinib, Group thirteen: 250 μM β-elemene + 4.5 μM gilteritinib, Group fourteen: 125 μM β-elemene + 4.5 μM gilteritinib, Group fifteen: 62.5 μM β-elemene + 4.5 μM gilteritinib, Group sixteen: 31.25 μM β-elemene + 4.5 μM gilteritinib; Group seventeen: 31.25 μM β-elemene + 4.5 μM gilteritinib;

[0140] For the above groups, 1% absolute ethanol and 0.25% DMSO were used as solvents.

[0141] Cell culture: K562 cells were inoculated into cell culture dishes, and an appropriate amount of 1640 + 10% FBS medium was added. The cells grew in suspension and were passaged once every 2 days. When passaging, the cells were pipetted and mixed evenly with a pipette tip, 2 / 3 volume of the cell suspension was aspirated, and the same volume of fresh medium was added to the cell culture dish, and then placed in an incubator at 37°C, 5% CO2 and saturated humidity for culture. K562 cells in the logarithmic growth phase were made into a single-cell suspension according to the passaging method, counted, and the cell concentration was adjusted to: 3×105 cells / mL. The above cell suspension was inoculated into a 96-well cell culture plate, 100 μL of the cell suspension was added to each well, and blank culture medium was taken as the blank zero-adjustment well. The cell culture plate was placed in an incubator at 37°C and 5% CO2 for standby. The culture supernatant in the well plate was not removed, 100 μL of the drug-containing medium of each group was added to the well again, and at the same time, the pipette tip was used to pipette several times in the well to make the drug evenly mixed in the well to obtain the required final test concentration. Then, for groups ten to seventeen, 200 μL of medium (containing 2 μL of β-elemene at different concentrations + 0.5 μL of gilteritinib) was added to each well, for groups one to eight, 200 μL of medium (containing 2 μL of β-elemene at different concentrations + 0.5 μL of DMSO) was added to each well, for group nine, 200 μL of medium (containing 2 μL of absolute ethanol + 0.5 μL of gilteritinib) was added to each well, and for the solvent control, 200 μL of medium (containing 2 μL of absolute ethanol and 0.5 μL of DMSO) was added to each well. Each group was set with 3 replicate wells. After culturing for 72 h, 20 μL of the CCK8 detection reagent stock solution was added to each well, incubated at 37°C for 2.5 h, and then the OD value was measured at 450 nM using a fully automatic microplate reader. The inhibition rate was calculated according to the aforementioned formula. The results showed that each group had a certain inhibitory effect on the cells. The q value was calculated based on the inhibition rate, and the synergistic effect is shown in Table 11:

[0142] Table 11

[0143] Group Gilteritinib and β - elemene molar ratio Q value Group Ten 1：444.44 0.98 Group Eleven 1：222.22 0.98 Group Twelve 1：111.11 0.98 Group Thirteen 1：55.56 0.98 Group Fourteen 1：27.78 1.14 Group Fifteen 1：13.89 1.00 Group Sixteen 1：6.94 1.04 Group Seventeen 1：3.47 1.00

[0144] Example 12

[0145] β-elemene and ivosidenib were divided into:

[0146] Group 1: 2000 μM β-elemene, Group 2: 1000 μM β-elemene, Group 3: 500 μM β-elemene, Group 4: 250 μM β-elemene, Group 5: 125 μM β-elemene; Group 6: 62.5 μM β-elemene, Group 7: 31.25 μM β-elemene; Group 8: 15.625 μM β-elemene, Group 9: 500 nM ivosidenib;

[0147] Group 10: 2000 μM β-elemene + 500 nM ivosidenib, Group 11: 1000 μM β-elemene + 500 nM ivosidenib, Group 12: 500 μM β-elemene + 500 nM ivosidenib, Group 13: 250 μM β-elemene + 500 nM ivosidenib, Group 14: 125 μM β-elemene + 500 nM ivosidenib, Group 15: 62.5 μM β-elemene + 500 nM ivosidenib, Group 16: 31.25 μM β-elemene + 500 nM ivosidenib, Group 17: 15.625 μM β-elemene + 500 nM ivosidenib.

[0148] 1% absolute ethanol and 0.25% DMSO were used as solvents for the above groups.

[0149] Cell culture: K562 cells were inoculated into cell culture dishes, and an appropriate amount of 1640 + 10% FBS medium was added. The cells grew in suspension and were passaged once every 2 days. During passage, the cells were pipetted and mixed evenly with a pipette tip, 2 / 3 volume of the cell suspension was aspirated, and the same volume of fresh medium was added to the cell culture dish. Then it was placed in an incubator at 37°C, 5% CO2 and saturated humidity for culture. K562 cells in the logarithmic growth phase were made into a single-cell suspension according to the passage method, counted, and the cell concentration was adjusted to: 3×105 cells / mL. The above cell suspension was inoculated into a 96-well cell culture plate, 100 μL of the cell suspension was added to each well, and blank culture medium was taken as the blank zero-adjustment well. The cell culture plate was placed in an incubator at 37°C and 5% CO2 for standby. The culture supernatant in the well plate was not removed. 100 μL of the drug-containing medium of each group was added to the well again, and at the same time, the pipette tip was used to pipette several times in the well to make the drug evenly mixed in the well to obtain the required final test concentration. Then, for groups ten to seventeen, 200 μL of medium (containing 2 μL of β-elemene at different concentrations + 0.5 μL of ivosidenib) was added to each well, for groups one to eight, 200 μL of medium (containing 2 μL of β-elemene at different concentrations + 0.5 μL of DMSO) was added to each well, for group nine, 200 μL of medium (containing 2 μL of absolute ethanol + 0.5 μL of ivosidenib) was added to each well, and for the solvent control, 200 μL of medium (containing 2 μL of absolute ethanol and 0.5 μL of DMSO) was added to each well. Three replicate wells were set in each group. After culturing for 72 h, 20 μL of the CCK8 detection reagent stock solution was added to each well and incubated at 37°C for 2.5 h. Then the OD value was measured at 450 nM using a fully automatic microplate reader. The inhibition rate was calculated according to the aforementioned formula. The results showed that each group had a certain inhibitory effect on the cells. The q value was calculated based on the inhibition rate, and the synergistic effect is shown in Table 12:

[0150] Table 12

[0151] Group Ivosidenib and β - elemene molar ratio Q value Group Ten 1：4000 1.00 Group Eleven 1：2000 1.00 Group Twelve 1：1000 1.00 Group Thirteen 1：500 1.00 Group Fourteen 1：250 1.41 Group Fifteen 1：125 0.94 Group Sixteen 1：62.5 0.62 Group Seventeen 1：31.25 0.42

[0152] Example 13

[0153] β-elemene and neratinib were divided into:

[0154] Group 1: 2000 uM β-elemene, Group 2: 1000 uM β-elemene, Group 3: 500 uM β-elemene; Group 4: 62.5 uM β-elemene, Group 5: 31.25 uM β-elemene, Group 6: 15.625 uM β-elemene, Group 7: 0.1 uM neratinib;

[0155] Group VIII: 2000uM β-elemene + 0.1uM neratinib, Group IX: 1000uM β-elemene + 0.1uM neratinib, Group X: 500uM β-elemene + 0.1uM neratinib, Group XI: 62.5uM β-elemene + 0.1uM neratinib, Group XII: 31.25uM β-elemene + 0.1uM neratinib; Group XIII: 15.625uM β-elemene + 0.1uM neratinib.

[0156] For the above groups, 0.5% DMSO and 1% absolute ethanol were used as solvents.

[0157] Cell culture: MCF-7 cells were inoculated into a sterile cell culture flask, and an appropriate amount of DMEM + 10% FBS medium was added. The cells were cultured in an incubator at 37°C, 5% CO2 and saturated humidity. The cells grew adherently in a monolayer and were passaged once every 2 - 3 days. When passaging the cells, first digest with 2 mL of 0.25% trypsin for 2 - 4 minutes, then add 2 mL of DMEM + 10% FBS to neutralize. Use a pipette to aspirate the culture medium and blow the cells into single cells, and then passage at a density of 2 - 3×10^6 cells per flask. Take MCF-7 cells in the logarithmic growth phase, prepare a single-cell suspension according to the passage method, count, and adjust the cell concentration to: 3.5×10^4 cells / mL. Inoculate the above cell suspension into a 96-well cell culture plate, add 100 μL of the cell suspension to each well. Take the blank culture medium as the blank zero-adjustment well. After culturing the culture plate adherently in an incubator at 37°C, 5% CO2 for 24 hours, remove the culture medium in the wells. Add 100 μL of medium (containing 1 μL of β-elemene at different concentrations + 0.5 μL of neratinib) to Groups VIII to XIII, add 100 μL of medium (containing 1 μL of β-elemene at different concentrations + 0.5 μL of DMSO) to Groups I to VI, add 100 μL of medium (containing 1 μL of absolute ethanol + 0.5 μL of neratinib) to Group VII, and the solvent control was added with 100 μL of medium (containing 1 μL of absolute ethanol and 0.5 μL of DMSO). Each group was set with 3 replicate wells. After culturing for 72 hours, remove the culture medium in the wells, add 10% CCK8 working solution, incubate at 37°C for 1 - 3 hours, and measure the OD value at 450 nM using a fully automatic microplate reader. Calculate the inhibition rate according to the aforementioned formula. The results showed that each group had a certain inhibitory effect on the cells. Calculate the q value according to the inhibition rate. The synergistic effect is shown in Table 13:

[0158] Table 13

[0159] Group Neratinib:β - elemene molar ratio Q value Group Eight 1:20000 0.93 Group Nine 1:10000 0.94 Group Ten 1:5000 1.18 Group Eleven 1:625 0.86 Group Twelve 1:312.5 0.81 Group Thirteen 1:156.25 0.79

[0160] Example 14

[0161] β-elemene and temsirolimus were divided into:

[0162] Group 1: 500 uM β-elemene; Group 2: 250 uM β-elemene; Group 3: 125 uM β-elemene; Group 4: 62.5 uM β-elemene; Group 5: 50 nM temsirolimus;

[0163] Group 6: 500 uM β-elemene + 50 nM temsirolimus; Group 7: 250 uM β-elemene + 50 nM temsirolimus; Group 8: 125 uM β-elemene + 50 nM temsirolimus; Group 9: 62.5 uM β-elemene + 50 nM temsirolimus.

[0164] For the above groups, 0.5% DMSO and 1% absolute ethanol were used as solvents.

[0165] Cell culture: 786-o cells were inoculated into a sterile cell culture flask, and an appropriate amount of DMEM + 10% FBS medium was added. The cells were cultured in an incubator at 37°C, 5% CO2 and saturated humidity. The cells grew adherently in a monolayer and were passaged once every 2 - 3 days. When passaging the cells, first digest them with 2 mL of 0.25% trypsin for 2 - 4 minutes, then add 2 mL of DMEM + 10% FBS to neutralize. Use a pipette to aspirate the culture medium and pipette the cells into single cells, and then passage them at a density of 2 - 3×10^6 cells per flask. Take HuH-7 cells in the logarithmic growth phase, prepare a single-cell suspension according to the passage method, count, and adjust the cell concentration to: 4.5×10^4 cells / mL. Inoculate the above cell suspension into a 96-well cell culture plate, add 100 μL of the cell suspension to each well. Take the blank culture medium as the blank zero-adjustment well. After culturing the culture plate in an incubator at 37°C, 5% CO2 for 24 hours, remove the culture medium in the wells. For groups 6 to 9, add 100 μL of medium (containing 1 μL of β-elemene at different concentrations + 0.5 μL of temsirolimus) respectively; for groups 1 to 4, add 100 μL of medium (containing 1 μL of β-elemene at different concentrations + 0.5 μL of DMSO) respectively; for group 5, add 100 μL of medium (containing 1 μL of absolute ethanol + 0.5 μL of temsirolimus). The solvent control was to add 100 μL of medium (containing 0.5 μL of DMSO and 1 μL of absolute ethanol). Each group was set with 3 replicate wells. After culturing for 72 hours, remove the culture medium in the wells, add 10% CCK8 working solution, incubate at 37°C for 1 - 3 hours, and measure the OD value at 450 nM using a fully automatic microplate reader. Calculate the inhibition rate according to the aforementioned formula. The results showed that each group had a certain inhibitory effect on the cells. Calculate the q value according to the inhibition rate, and the synergistic effect is shown in Table 14:

[0166] Table 14

[0167]

[0168] Example 15

[0169] Cell culture: Human lung cancer NCI-H460-luc (NCI-H460, HTB-177) cells were cultured in a monolayer in vitro. The culture conditions were RPMI1640 medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin solution, and cultured in an incubator at 37°C with 5% CO2. Routine digestion and passage were performed using trypsin-EDTA. When the cell confluence reached 80%-90%, the cell viability was 97.90%, and the cell number reached the required amount, the cells were harvested, counted, and inoculated.

[0170] Cell inoculation and modeling: Female BALB / c nude mice were anesthetized with 1.25% avertin. After determining that the animals were in a deep anesthesia state, they were fixed. The left upper limb of the mice was disinfected with 75% ethanol, the surface position corresponding to the lungs of the mice was located, and a 1-2 cm incision was made with surgical scissors. A 20 μL cell suspension (2x106 NCI-H460-luc cells and 50% Matrigel) was inoculated into the lung lobe of the mice. After checking to ensure that there was no leakage at the injection site, the skin was sutured surgically, and the animals were kept warm and observed until they fully recovered. To relieve the pain of the animals, meloxicam was administered to the animals on the day after surgery and at 24 h and 48 h after surgery.

[0171] Animal grouping: After inoculation, the mice were intraperitoneally injected with 150 mg / kg luciferin according to their body weight (10 μL / g). After 10 minutes, the mice were pre-anesthetized with a mixed gas of oxygen and isoflurane. After the mice were anesthetized, they were transferred to the imaging chamber of IVIS (Lumina II) to start detecting bioluminescence signals. The bioluminescence signals and the generated image information in the animals were detected and recorded. Six days after the NCI-H460-luc model was inoculated, when the average luminescence value reached 2.21E+07, the animals were grouped for drug administration, with 4 mice in each group. There were a total of four groups:

[0172] Group 1) Normal saline,

[0173] Group 2) Anlotinib tablets (commercially available: Zhengda Tianqing Pharmaceutical Co., Ltd.)

[0174] Group 3) β-Elemene albumin preparation (Take 2.0 g of β-elemene and 4.8 g of soybean oil to obtain the oil phase. Measure 30 g of commercially available 20% human albumin, add an appropriate amount of injection water to dilute to 30 ml to obtain the water phase. Add the oil phase to the water phase, shear it with an online shearer at 12,000 rpm to prepare the primary emulsion. The primary emulsion is homogenized at 18,000 psi by high-pressure microfluidics to obtain the homogenized emulsion. Add 25 g of commercially available 40% trehalose solution to the homogenized emulsion, make up the injection water to a total volume of 100 g, stir evenly, and filter through a 0.22 μm microporous membrane to obtain the final emulsion. The final emulsion is freeze-dried by the freeze-drying process to obtain the injectable β-elemene albumin preparation),

[0175] Group 4) Anlotinib tablets + β-elemene albumin preparation (where the molar ratio of anlotinib to β-elemene is 1:130).

[0176] Using normal saline, the two preparations were configured into solutions. For anlotinib tablets: Take one tablet (12 mg / tablet), add normal saline to dissolve, stir with a magnetic stirrer, and make up the volume to 24.000 mL, then shake gently to obtain a 0.5 mg / mL solution. For β-elemene albumin injection: Dissolve β-elemene albumin injection (200 mg / vial) with normal saline and make up the volume to 16.667 mL, then shake gently to obtain a 12 mg / mL medicinal solution.

[0177] The dosing regimen is shown in Table 15. After the treatment ended, the tumor inhibition rate was calculated according to the aforementioned in vivo inhibition rate, and the q value was calculated. It was found that tumor inhibition effects were achieved in each group, and the q value was 1.26, indicating a synergistic effect. The results are shown in Table 16 below.

[0178] Table 15 Dosing Regimen

[0179] Group Dose (calculated by API) Administration method Administration frequency and cycle Group 1 / Injection Once a day, 18 days Group 2 3mg / kg Oral Once a day, 18 days Group 3 200mg / kg Injection Once a day, 18 days Group 4 3mg / kg + 200mg / kg Oral + Injection Once a day, 18 days

[0180] Table 16 Research Results

[0181]

Claims

1. A combined drug for treating cancer, characterized in that The combined drug comprises β-elemene and a second drug, and the second drug is a kinase inhibitor. Preferably, the kinase inhibitor is selected from one or more of TKI inhibitors, KRAS inhibitors, IDH1 inhibitors, and mTOR inhibitors.

2. The combination medicament according to claim 1, wherein The TKI inhibitor is anlotinib. Preferably, the molar ratio of anlotinib to β-elemene is 1:0.1 - 1:2000, preferably 1:1 - 1:2000, preferably 1:1 - 1:1500, preferably 1:1 - 1:1282, preferably 1:10 - 1:1282, preferably 1:20 - 1:321, preferably 1:40 - 1:321, preferably 1:130 - 1:

321. Preferably, the molar ratio of anlotinib to β-elemene is 1:40, 1:130, 1:

321.

3. The combined medicament according to claim 1, characterized in that The TKI inhibitor is icotinib. Preferably, the molar ratio of icotinib to β-elemene is 1:25 - 1:1000, preferably 1:100 - 1:1000, preferably 1:25 - 1:400, preferably 1:100 - 1:400, preferably 1:100 - 1:

200. Preferably, the molar ratio of icotinib to β-elemene is 1:100, 1:

200.

4. The combined medicament according to claim 1, wherein The TKI inhibitor is ceritinib. Preferably, the molar ratio of ceritinib to β-elemene is 1:28 - 1:

111. Preferably, the molar ratio of ceritinib to β-elemene is 1:28, 1:56, 1:

111.

5. The combined medicament according to claim 1, wherein The TKI inhibitor is larotrectinib. Preferably, the molar ratio of larotrectinib to β-elemene is 1:62.5 - 1:2000, preferably 1:125 - 1:1000, preferably 1:125 - 1:500, preferably 1:250 - 1:

500. Preferably, the molar ratio of larotrectinib to β-elemene is 1:250, 1:

500.

6. The combined medicine according to claim 1, wherein The TKI inhibitor is capmatinib. Preferably, the molar ratio of capmatinib to β-elemene is 1:0.5 - 1:20, preferably 1:0.5 - 1:5, preferably 1:1.25 - 1:20, preferably 1:1.25 - 1:5, preferably 1:2.5 - 1:

5. Preferably, the molar ratio of capmatinib to β-elemene is 1:2.5, 1:

5.

7. The combined medicine according to claim 1, characterized in that The TKI inhibitor is dacomitinib. Preferably, the molar ratio of dacomitinib to β-elemene is 1:1 - 1:2000, preferably 1:5 - 1:2000, preferably 1:5 - 1:500, preferably 1:5 - 1:250, preferably 1:5 - 1:125, preferably 1:15.63 - 1:2000, preferably 1:15.63 - 1:500, preferably 1:62.5 - 1:

125. Preferably, the molar ratio of dacomitinib to β-elemene is 1:62.5, 1:

125.

8. The combined medicine according to claim 1, wherein The TKI inhibitor is apatinib. Preferably, the molar ratio of apatinib to β-elemene is 1:0.63 - 1:40, preferably 1:1.25 - 1:20, preferably 1:1.25 - 1:10, preferably 1:1.25 - 1:5, preferably 1:1.25 - 1:2.

5. More preferably, the molar ratio of apatinib to β-elemene is 1:1.25 or 1:2.

5.

9. The combined medicament according to claim 1, wherein The TKI inhibitor is regorafenib. Preferably, the molar ratio of regorafenib to β-elemene is 1:12.5 - 1:1000, preferably 1:25 - 1:500, preferably 1:62.5 - 1:1000, preferably 1:125 - 1:500, preferably 1:250 - 1:500, preferably 1:12.5 - 1:200, preferably 1:25 - 1:

100. More preferably, the molar ratio of regorafenib to β-elemene is 1:25, 1:62.5, 1:100, 1:125, 1:200, 1:250, 1:500, or 1:1000.

10. The combined medicine according to claim 1, characterized in that The TKI inhibitor is gilteritinib. Preferably, the molar ratio of gilteritinib to β-elemene is 1:3.47 - 1:444.44, preferably 1:27.

78.

11. The combined medicament according to claim 1, wherein The TKI inhibitor is neratinib. Preferably, the molar ratio of neratinib to β-elemene is 1:156.25 - 1:20000, preferably 1:5000.

12. The combined medicine according to claim 1, characterized in that The KRAS inhibitor is sotorasib. Preferably, the molar ratio of sotorasib to β-elemene is 1:10 - 1:1000, preferably 1:10 - 1:500, preferably 1:10 - 1:160, preferably 1:80 - 1:1000, preferably 1:80 - 1:500, preferably 1:80 - 1:

160. More preferably, the molar ratio of sotorasib to β-elemene is 1:80 or 1:

160.

13. The combined medicament according to claim 1, characterized in that The IDH1 inhibitor is ivosidenib. Preferably, the molar ratio of ivosidenib to β-elemene is 1:31.25 - 1:4000, preferably 1:

250.

14. The combined medicine according to claim 1, wherein The mTOR inhibitor is temsirolimus. Preferably, the molar ratio of temsirolimus to β-elemene is 1:1250 - 1:10000, preferably 1:2500 - 1:5000. More preferably, the molar ratio of temsirolimus to β-elemene is 1:2500 or 1:5000.

15. The combination medicament according to any one of claims 1-14, characterized in that The combined drug is a pharmaceutical composition, or a single compound preparation, or a combination of two separate preparations. Preferably, the active ingredient of the pharmaceutical composition consists of β-elemene and a second drug. Preferably, the pharmaceutical composition contains pharmaceutically acceptable excipients.

16. Use of the drug according to any one of claims 1-15 in the preparation of a drug for treating cancer, preferably the cancer is lung cancer, breast cancer, renal cell carcinoma, ovarian cancer, small cell lung cancer, non-small cell lung cancer, malignant melanoma, B-lymphocyte tumor, leukemia, colorectal cancer, malignant pleural mesothelioma, pancreatic cancer, head and neck cancer, gastric cancer, bladder cancer, bone cancer, esophageal cancer, brain tumor, liver cancer, cervical cancer, cancerous pleural effusion and ascites, preferably the cancer is lung cancer, liver cancer, colorectal cancer, leukemia, breast cancer, renal cell carcinoma, preferably lung cancer, liver cancer, breast cancer, renal cell carcinoma, preferably lung cancer, breast cancer, renal cell carcinoma, and the lung cancer is preferably squamous cell lung cancer, large cell lung cancer.

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