A pharmaceutical composition for reversing lenvatinib resistance and use thereof

Abstract

The present application relates to the field of biological medicine, and particularly relates to a drug composition for reversing lenvatinib drug resistance and application thereof. One object of the present application is to provide a drug composition containing cubenix and lenvatinib. Through the synergistic effect of cubenix and lenvatinib, the inhibitory effect on liver cancer lenvatinib drug-resistant cells is significantly improved, at the same time, the lenvatinib drug-resistant cell colony formation can be significantly inhibited, the lenvatinib drug-resistant cell apoptosis is promoted, and a better tumor inhibition effect is achieved in the liver cancer tumor-bearing mouse experiment.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a pharmaceutical composition for reversing lenvatinib resistance and its use. Background Technology

[0002] Liver cancer currently ranks fourth in incidence and second in mortality among cancers in my country, posing a serious threat to the health of the vast majority of the population. Globally, it ranks sixth among common malignant tumors. Although hepatitis B patients are receiving increasing attention worldwide, early diagnosis of liver cancer remains inadequate due to varying economic conditions and regional differences. Patients, especially in economically disadvantaged rural areas and remote mountainous regions, often only seek medical attention when clinical symptoms appear, by which time the cancer has often progressed to middle or late stages. Treating middle and late-stage liver cancer remains a significant challenge. Despite various treatment options, long-term survival is difficult to achieve. The advent of each new drug generates considerable buzz in the medical community, but also brings numerous challenges. Most critically, long-term use of almost every chemotherapy drug leads to drug resistance.

[0003] Lenvatinib (trade name: Lenvatinib), the active ingredient is lenvatinib mesylate, molecular formula: C 21 H 19 ClN4O4·CH4O3S, with a molecular weight of 522.96, can inhibit multiple tyrosine kinase receptors, including fibroblast growth factor receptor (FGFR) 1-4, vascular endothelial growth factor receptor (VEGFR) 1-4, platelet-derived growth factor receptor (PDGR) α, proto-oncogene RET, and c-KIT. Lenvatinib inhibits tumor angiogenesis by blocking VEGF receptors, and inhibits tumor cell proliferation by blocking FGFR, PDFR α, RET, and c-KIT, ultimately exhibiting a potent anti-tumor effect. This is mainly manifested in lenvatinib's ability to prolong overall survival and progression-free survival in patients with advanced liver cancer, and its ability to convert some unresectable liver cancers into resectable ones. Currently, lenvatinib has been approved by the FDA for first-line targeted therapy in patients with advanced liver cancer. However, in clinical practice, some liver cancer patients have shown poor response to lenvatinib or tumor progression after treatment. The reasons for this can be broadly categorized into two types: one is the natural resistance of tumors to lenvatinib; the other is the adaptive regulation of tumors to lenvatinib, allowing tumor cells to survive at high concentrations of lenvatinib. However, the mechanisms of lenvatinib resistance in liver cancer and salvage treatments after resistance remain crucial scientific questions that urgently need to be addressed. Summary of the Invention

[0004] This invention aims to at least partially solve one of the technical problems in related technologies. Therefore, one object of this invention is to provide a pharmaceutical composition and its use in reversing lenvatinib resistance in hepatocellular carcinoma. Through the synergistic effect of curpannice and lenvatinib, the composition significantly enhances the inhibitory effect on lenvatinib-resistant hepatocellular carcinoma cells. Simultaneously, it significantly inhibits the formation of lenvatinib-resistant hepatocellular carcinoma cell colonies, promotes apoptosis of lenvatinib-resistant hepatocellular carcinoma cells, and achieves good tumor-suppressive effects in hepatocellular carcinoma tumor-bearing mouse experiments.

[0005] A first aspect of the present invention provides a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition comprises cupanixic and lenvatinib.

[0006] The pharmaceutical composition provided by this invention contains cupanixinib, which can reverse lenvatinib resistance, significantly inhibit the proliferation and colony formation of lenvatinib-resistant liver cancer cells, and promote apoptosis in lenvatinib-resistant liver cancer cells, effectively reversing lenvatinib resistance in liver cancer. Compared with the use of cupanixinib alone, the combination of cupanixinib and lenvatinib exhibits higher anticancer activity against lenvatinib-resistant liver cancer cells.

[0007] According to an embodiment of the present invention, the molar ratio of curpannicotinib to lenvatinib in the pharmaceutical composition is (1-20):10. This further inhibits the proliferation and colony formation of lenvatinib-resistant cells and promotes apoptosis in these cells.

[0008] A second aspect of the present invention provides the use of curpannicine in the preparation of lenvatinib resistance reversal agents.

[0009] A third aspect of the present invention provides the use of cupanixine in reversing lenvatinib resistance.

[0010] Cupanicine can not only reverse lenvatinib resistance, but also significantly reduce drug concentration and improve anti-tumor efficacy when used in combination with other drugs.

[0011] The fourth aspect of this invention provides the use of curpannice in inhibiting the proliferation and colony formation of lenvatinib-resistant liver cancer cells.

[0012] The fifth aspect of this invention provides the use of cupanixine in promoting apoptosis in lenvatinib-resistant liver cancer cells.

[0013] The sixth aspect of the present invention provides the use of the pharmaceutical composition described in the first aspect in the preparation of a medicament for treating liver cancer.

[0014] The pharmaceutical compositions of the present invention, particularly for tissues or individuals resistant to lenvatinib, can significantly inhibit the proliferation and colony formation of resistant cells and promote apoptosis of resistant cells when used in combination. Therefore, the pharmaceutical compositions of the present invention can be used to prepare drugs for the treatment of liver cancer.

[0015] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0016] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0017] Figure 1 The screening process and morphological changes of Huh7 LR cells are shown; Figure A shows the changes in lenvatinib concentration and time axis during the process of obtaining Huh7 LR cells after multiple rounds of lenvatinib induction; Figure B shows the morphological differences between Huh7 P cells and Huh7 LR cells.

[0018] Figure 2 The IC50 assay for Huh7 P cells and Huh7 LR cells showed proliferation. 50 Test results;

[0019] Figure 3 The results of the cell colony formation experiment show the comparison of the proliferation rate of Huh7 P cells and Huh7 LR cells. Figure A shows the comparison of the proliferation rate of Huh7 P cells and Huh7 LR cells, Figure B shows the colony formation results of Huh7 P and Huh7 LR cells after treatment with lenvatinib, and Figure C shows the quantitative comparison of colony formation using ImageJ software. ***P<0.001;

[0020] Figure 4 The effect of lenvatinib on apoptosis of Huh7 P and Huh7 LR cells was detected by flow cytometry. Figure A shows the apoptosis of Huh7 P and Huh7 LR cells after 48 hours of lenvatinib treatment, as detected by flow cytometry. Figure B shows the statistical analysis results of apoptosis in different groups. ***P<0.001;

[0021] Figure 5The effects of different concentrations of lenvatinib (represented as Len), ecrizotinib (represented as Ela), and the combination of the two on the proliferation of Huh7 LR cells were shown. Figure A shows the effect of different concentrations of the two drugs alone and in combination on the proliferative activity of drug-resistant cells as detected by MTT assay. Figure B shows the plot and CI values ​​of the quantitative method based on the combined action of Chou-Talalay drugs. ***P<0.001;

[0022] Figure 6 The effects of lenvatinib (10 μM), ecrizotinib (2.5 μM) alone and in combination on Huh7 LR cell colony formation were shown. Figure A shows the cell colonies in different groups, and Figure B shows the statistical analysis results of cell colonies after different treatment groups. ***P<0.001;

[0023] Figure 7 The effects of lenvatinib (20 μM), ecrizotinib (10 μM), alone, and in combination on apoptosis in Huh7 LR cells were shown. Figure A shows the morphology of Huh7 LR cells 72 hours after treatment with different drugs; Figure B shows the apoptosis of cells in different drug treatment groups as detected by flow cytometry, with early apoptotic cells located in the lower right quadrant and late apoptotic cells located in the upper right quadrant; Figure C shows the statistical analysis results of apoptosis rate, ***P<0.001;

[0024] Figure 8 The diagram shows the drug targets targeting the EGFR / PI3K pathway, where gefitinib targets EGFR and copantinib targets PI3K.

[0025] Figure 9 The study showed the effects of different concentrations of lenvatinib, gefitinib, and curapanib alone, as well as the combination of lenvatinib and gefitinib, and the combination of lenvatinib and curapanib on the proliferation of Huh7 LR cells. Figure A shows the effect of MTT assay on the proliferation activity of drug-resistant Huh7 LR cells under different drug combinations and concentration gradients. The five bars in the bar chart, from left to right, represent the lenvatinib, gefitinib, lenvatinib + gefitinib, curapanib, and lenvatinib + curapanib groups. Figure B shows the plot and CI values ​​generated using a quantitative method based on the Chou-Talalay drug combination therapy. ***P<0.001;

[0026] Figure 10This study demonstrates the effects of lenvatinib, gefitinib, and cupanixeira alone, as well as the combination of lenvatinib and gefitinib, and the combination of lenvatinib and cupanixeira on apoptosis in Huh7 LR cells. Figure A shows the morphology of Huh7 LR cells 72 hours after treatment with different drugs; Figure B shows the proportion of apoptotic cells in different drug treatment groups as detected by flow cytometry, with early apoptotic cells located in the lower right quadrant and late apoptotic cells in the upper right quadrant; Figure C shows the statistical analysis results of the apoptosis rate, ***P<0.001.

[0027] Figure 11 The therapeutic effects of the solvent group, gefitinib monotherapy group, ectelide monotherapy group, lenvatinib monotherapy group, lenvatinib + gefitinib group, and lenvatinib + ectelide group on lenvatinib-resistant BALB / c mice were evaluated. Figure A shows the changes in tumor volume in different treatment groups, and Figure B shows the changes in mouse body weight over time. ***P<0.001. In the figures, Veh: solvent group (5‰ sodium carboxymethyl cellulose); Gef: gefitinib monotherapy group. Non-Tenet group (80 mg / kg / d); Ela: Eclita group (80 mg / kg / d); Len: Lenvatinib group (5 mg / kg / d); Len+Gef: Lenvatinib + Gefitinib group (Lenvatinib 5 mg / kg / d, Gefitinib 80 mg / kg / d); Len+Ela: Lenvatinib + Eclita group (Lenvatinib 5 mg / kg / d, Eclita 80 mg / kg / d);

[0028] Figure 12 The images show the therapeutic effects and tumor weight measurements of lenvatinib-resistant BALB / c mice in the solvent group, gefitinib monotherapy group, ectelida monotherapy group, lenvatinib monotherapy group, lenvatinib + gefitinib group, and lenvatinib + ectelida group. Figure A shows the size and morphology of tumors in different treatment groups, and Figure B shows the tumor weight. ***P<0.001. Detailed Implementation

[0029] The present invention will now be described with reference to specific embodiments. It should be noted that these embodiments are merely descriptive and do not limit the present invention in any way.

[0030] Unless otherwise specified, all reagents used in the experiments of the examples are commercially available.

[0031] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this disclosure, "a plurality of" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0032] According to a specific embodiment of the present invention, a pharmaceutical composition comprising cupanixic and lenvatinib is provided. Compared with the use of the drugs alone, the combination therapy significantly inhibits the proliferation of liver cancer tumor cells and reverses tumor drug resistance.

[0033] Elacridar is an inhibitor of both MDR1 and BCRP dual pumps. Current in vitro, preclinical, and clinical studies have shown that combined use of Elacridar can reverse MDR1 and / or BCRP-mediated chemotherapy drug resistance. Simultaneously, Elacridar can increase the blood concentration of antitumor drugs by inhibiting drug efflux pumps.

[0034] Gefitinib: This selectively inhibits the EGFR receptor and was initially used in patients with non-small cell lung cancer who had failed other treatments. Subsequently, it has been used as a monotherapy or in combination with other drugs to treat other cancers. Regarding its application in liver cancer, gefitinib can inhibit the growth of liver cancer cells, promote apoptosis, and induce cell cycle arrest. Furthermore, gefitinib has shown an inhibitory effect on the formation of liver cancer nodules in rat models.

[0035] Copanlisib is a PI3K inhibitor, initially used for relapsed follicular lymphoma, and subsequently for other advanced and relapsed solid tumors. Regarding its role in liver cancer, in vitro studies have shown that the combination of copanlisib and sorafenib can promote apoptosis in liver cancer cells.

[0036] Gefitinib and cupanixinib are both FDA-approved drugs.

[0037] According to a specific embodiment of the present invention, the present invention provides a pharmaceutical composition containing curpannicine and lenvatinib.

[0038] According to a specific embodiment of the present invention, in addition to cupanixic and lenvatinib as active ingredients, the pharmaceutical composition may also contain other excipients known in the art that can be added to pharmaceuticals.

[0039] According to a specific embodiment of the present invention, the pharmaceutical composition of the present invention includes a mixture of cupanixinib and lenvatinib to form a mixed drug, or cupanixinib and lenvatinib to form two drugs independently, but are administered in combination.

[0040] According to a specific embodiment of the present invention, the pharmaceutical composition contains curpannicotinib and lenvatinib, wherein the molar ratio of curpannicotinib to lenvatinib is (1-20):10.

[0041] According to a specific embodiment of the present invention, in the pharmaceutical composition, for example, the concentration of curpannicine is 3 μM and the concentration of lenvatinib is 30 μM, or the concentration of curpannicine is 12.5 μM and the concentration of lenvatinib is 10 μM, or the concentration of curpannicine is 25 μM and the concentration of lenvatinib is 20 μM.

[0042] In this invention, "Huh7 LR cells" refers to Huh7 Lenvatinib resistance (Huh7 LR), i.e., lenvatinib-resistant cells.

[0043] The present disclosure will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be construed as limiting the scope of the disclosure. Where specific techniques or conditions are not specified in the embodiments, they are performed in accordance with the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0044] Example 1: Preparation of the lenvatinib-resistant hepatocellular carcinoma cell line Huh7 LR

[0045] 1. Induction of lenvatinib-resistant Huh7 LR hepatocellular carcinoma cell lines

[0046] After 10 months of gradually increasing lenvatinib dosage induction, Huh7 lenvatinib-resistant (Huh7 LR) cells were able to tolerate a 20 μM lenvatinib concentration. Subsequently, Huh7 LR cells were maintained in a 20 μM lenvatinib-containing medium. The Huh7 LR cell selection process and drug dosage process are as follows: Figure 1 As shown in Figure A. Compared to the original cells (Huh7 P), Huh7 LR cells exhibit morphological changes, including smaller size, aggregated cell growth, and a cell morphology as shown in Figure A. Figure 1 As shown in Figure B.

[0047] 2. Detection of drug resistance in Huh7 LR cells

[0048] (1) Drug resistance of Huh7 LR cells was detected by MTT assay. 96-hour MTT results showed that lenvatinib IC50 in Huh7 P cells was significantly reduced. 50 The value was 5.34±1.07 μM, while the IC50 value of lenvatinib in Huh7 LR cells was 5.34±1.07 μM. 50 Values ​​greater than 20 μM. Both IC values... 50 The difference was significant (P < 0.05). Figure 2 Half-maximal inhibitory concentration (IC50) of a drug.50 () refers to the drug concentration that reduces cell activity to 50%.

[0049] The cell proliferation experiment procedure is as follows:

[0050] Cells in the logarithmic growth phase were collected, digested with trypsin, and after digestion was terminated, the cells were collected by centrifugation and prepared into a single-cell suspension. After counting using a cell counting chamber, the cell density was adjusted to 4 × 10⁶ cells / year. 4 Cells / ml. After preparing the cell suspension, mix well and add 100 μl to each well of a 96-well plate (4000 cells / well). Incubate overnight in a cell culture incubator (37℃, 5% CO2). During this process, do not add cells to the edge wells of the 96-well plate; instead, add PBS to reduce the impact of liquid evaporation on cell proliferation. The next day, discard the culture medium and add different concentration gradients of the drug, and continue incubation for 96 hours. After 96 hours, discard the culture medium and add MTT working solution (final working concentration 5 mg / ml), and continue incubation for 4 hours. After 4 hours, discard the liquid and add 100 μl of DMSO to each well. Incubate at 37℃ for 10 minutes to fully dissolve the bottom crystals. Detect the OD value using a multi-functional microplate reader at a wavelength of 490 nm. Throughout the process, the blank control group (without cells) undergoes the same operation, with ≥3 replicates per gradient well. Calculate cell viability (%) = (OD value of experimental group - OD value of blank control group) / (OD value of no drug group - OD value of blank control group).

[0051] (2) Assess the colony-forming ability of Huh7 LR cells

[0052] The real-time proliferation rate of drug-resistant cells was detected using a real-time cell activity counter (RTCA). The results showed that Huh7LR cells proliferated faster than Huh7P cells in lenvatinib-containing medium (20 μM). Figure 3 (See Figure A). Furthermore, colony formation assays were used to compare the colony-forming abilities of primitive cells and drug-resistant cells. The results showed that, under the same cell inoculation and culture conditions, Huh7 LR cells formed significantly more colonies than Huh7 cells in medium containing lenvatinib (20 μM). Figure 3 (See Figures B and C). The differences were statistically significant (P < 0.001). The colony formation assay results showed that Huh7 LR cells had a significantly stronger colony formation ability than Huh7 P cells.

[0053] The experimental method for colony formation is as follows:

[0054] Cells in the logarithmic growth phase were digested with trypsin, centrifuged, and resuspended to form a cell suspension. To compare the colony-forming ability of drug-resistant cells and virgin cells, 1000 cells were seeded per well in a 6-well plate, and medium containing lenvatinib (20 μM) was added. To compare the colony-forming ability of cells treated with different drug combinations, 2000 cells were added to each well in a 6-well plate and cultured overnight in a cell culture incubator (37°C, 5% CO2). The next day, the medium was discarded and replaced with medium containing different drug concentrations, and cultured for another 72 hours. From 72 hours to two weeks later, the medium was replaced with drug-free medium, with a cycle of 3 days. After two weeks of either treatment, colony formation was visible to the naked eye. The medium was discarded, the cells were washed twice with PBS, fixed with methanol for 20 minutes, stained with 0.1% crystal violet for 10 minutes, photographed, and colony counting was performed using ImageJ software.

[0055] (3) Flow cytometry was used to assess the anti-apoptotic ability of Huh7 LR cells.

[0056] Flow cytometry was used to detect the anti-apoptotic capacity of cells. Huh7 cells and Huh7 LR cells were treated with lenvatinib (20 μM). After 48 hours, the apoptosis rate of Huh7 cells was 11.74%, while that of Huh7 LR cells was 3.27%; the difference was statistically significant (P < 0.001). Figure 4 (Figures A and B in the middle).

[0057] The flow cytometry assay for apoptosis is as follows:

[0058] Cells in the logarithmic growth phase were harvested, discarded from the culture medium, washed with PBS, digested with trypsin to terminate the process, and then the cells were counted after preparation of a cell suspension. Cells were seeded in 6-well plates at a density of 200,000 cells / well and cultured overnight. The next day, the culture medium was replaced with medium containing different drug concentrations, and incubation continued for 72 hours. Subsequent steps are as follows:

[0059] ① After culture, wash the cells with PBS, digest them with trypsin without EDTA and collect the cells in a flow cytometry tube at 1500g / min for 5 minutes; wash once with PBS and repeat the centrifugation once more.

[0060] ② Discard the supernatant and gently resuspend the cells in 195 μl of Annexin V-FITC binding solution;

[0061] ③ Add 5 μl Annexin V FITC and mix gently;

[0062] ④ Add 10 μl of propidium iodide (PI) staining solution and mix gently;

[0063] ⑤ Incubate at room temperature in the dark for 15 minutes, then perform flow cytometry analysis, with at least 50,000 cells analyzed for each sample.

[0064] Example 2: Eclipta-methyl and Lenvatinib synergistically inhibit Huh7 LR cell proliferation

[0065] 1. Cell proliferation experimental methods

[0066] Huh7 LR cells in the logarithmic growth phase were harvested, digested with trypsin, and collected by centrifugation after digestion to prepare a single-cell suspension. Cells were counted using a cell counting chamber, and the cell density was adjusted to 4 × 10⁻⁶ cells / cell. 4 Cells / ml. After preparing the cell suspension, mix well and add 100 μl to each well of a 96-well plate (4000 cells / well). Incubate overnight in a cell culture incubator (37℃, 5% CO2). During this process, do not add cells to the edge wells of the 96-well plate; instead, add PBS to reduce the impact of liquid evaporation on cell proliferation. The next day, discard the culture medium and add different concentration gradients of the drug, and continue incubation for 96 hours. After 96 hours, discard the culture medium and add MTT working solution (final working concentration 5 mg / ml), and continue incubation for 4 hours. After 4 hours, discard the liquid and add 100 μl of DMSO to each well. Incubate at 37℃ for 10 minutes to fully dissolve the bottom crystals. Detect the OD value using a multi-functional microplate reader at a wavelength of 490 nm. Throughout the process, the blank control group (without cells) undergoes the same operation, with ≥3 replicates per gradient well. Calculate cell viability (%) = (OD value of experimental group - OD value of blank control group) / (OD value of no drug group - OD value of blank control group).

[0067] 2. Experimental group design and results

[0068] The MTT assay was used to detect the cell proliferation activity of different treatment groups, such as... Figure 5 The results showed that: ① Cell viability was almost unaffected by the use of lenvatinib (10 μM, denoted as Len) or ectelida (2.5 μM, denoted as Ela) alone, but decreased to 71.70% when lenvatinib (10 μM) and ectelida (2.5 μM) were used in combination; ② Cell viability was 76.23% when lenvatinib (20 μM) was used alone, and 92.49% when ectelida (5 μM) was used alone, but decreased to 39.67% when lenvatinib (20 μM) and ectelida (5 μM) were used in combination. Figure 5(See Figure A). Based on the cell proliferation rates under the above two concentration gradients, the combination index (CI) was further calculated using the quantitative method for the combined effect of Chou-Talalay drugs: lenvatinib 10 μM combined with ectodiclofenac 2.5 μM, CI = 0.57; lenvatinib 20 μM combined with ectodiclofenac 5.0 μM, CI = 0.720. Given that all the above CI values ​​are less than 1, it indicates that lenvatinib and ectodiclofenac can synergistically inhibit the proliferation of Huh7LR cells (…). Figure 5 (Figure B in the middle)

[0069] Example 3: Effect of the combination of ecrigrade and lenvatinib on the colony-forming ability of Huh7 LR

[0070] 1. Colony Formation Experimental Methods

[0071] Logarithmically growing Huh7 LR cells were digested with trypsin, centrifuged, and resuspended to form a cell suspension. To compare the colony-forming ability of drug-resistant cells versus virgin cells, 1000 cells were seeded per well in 6-well plates, and medium containing lenvatinib (20 μM) was added. To compare the colony-forming ability of cells treated with different drug combinations, 2000 cells were added to each well in 6-well plates and cultured overnight in a cell culture incubator (37°C, 5% CO2). The next day, the medium was discarded and replaced with medium containing different drug concentrations, and cultured for another 72 hours. From 72 hours to two weeks later, the medium was replaced with drug-free medium, with a cycle of 3 days. After two weeks of both treatments, colony formation was visible to the naked eye. The medium was discarded, the cells were washed twice with PBS, fixed with methanol for 20 minutes, stained with 0.1% crystal violet for 10 minutes, photographed, and colony counting was performed using ImageJ software.

[0072] 2. Experimental Groups and Results

[0073] Tumor stem cells possess abnormal differentiation and self-renewal potential characteristic of stem cells and are associated with resistance to chemotherapy drugs in various tumors. A colony formation assay was conducted to assess the effects of drugs on tumor cell stem cell proliferation. Cells were divided into four groups: control group, lenvatinib group (10 μM), ectotrione group (2.5 μM), and lenvatinib (10 μM) + ectotrione (2.5 μM) group. The results are as follows: Figure 6 As shown, lenvatinib monotherapy can inhibit Huh7 LR cell colony formation, and lenvatinib combined with ecrizotinib can further inhibit Huh7 LR cell colony formation. Figure 6 (Figure A); further statistical analysis showed that the difference was statistically significant (P<0.001). Figure 6 (Figure B in the middle)

[0074] Example 4: Effect of combination of ectodiclofenac and lenvatinib on apoptosis in Huh7 LR cells

[0075] 1. Experimental design for flow cytometry detection of apoptosis

[0076] Huh7 LR cells in the logarithmic growth phase were harvested, the culture medium was discarded, and the cells were washed with PBS. After digestion with trypsin to terminate the process, the cells were suspended and counted. Cells were seeded in 6-well plates at a density of 200,000 cells / well and cultured overnight.

[0077] The following day, the culture medium was replaced with one containing different drug concentrations, and incubation continued for another 72 hours. Subsequent steps are as follows:

[0078] ① After culture, wash the cells with PBS, digest them with trypsin without EDTA and collect the cells in a flow cytometry tube at 1500g / min for 5 minutes; wash once with PBS and repeat the centrifugation once more.

[0079] ② Discard the supernatant and gently resuspend the cells in 195 μl of Annexin V-FITC binding solution;

[0080] ③ Add 5 μl Annexin V FITC and mix gently;

[0081] ④ Add 10 μl of propidium iodide (PI) staining solution and mix gently;

[0082] ⑤ Incubate at room temperature in the dark for 15 minutes, then perform flow cytometry analysis, with at least 50,000 cells analyzed for each sample.

[0083] 2. Experimental Groups and Results

[0084] Huh7 LR cells were treated with different drugs (treatment time 72 hours): control group, lenvatinib group (20 μM), ecrizotinib group (10 μM), and lenvatinib (20 μM) + ecrizotinib (10 μM) group. Figure 7 (Figure A). Then, Annexin V-FITC / PI staining was used to detect the proportion of apoptosis (early apoptosis + late apoptosis) in different treatment groups by flow cytometry. Figure 7 Figure B in the middle section was further analyzed statistically. Figure 7 (Figure C). The results showed that, compared with monotherapy, the combination of ecrifatinib and lenvatinib significantly promoted apoptosis in Huh7 LR cells, and the difference was statistically significant (P < 0.001).

[0085] Example 5: Synergistic inhibition of Huh7LR cell proliferation by EGFR / PI3K inhibitors and lenvatinib

[0086] Activation of the EGFR signaling pathway and its downstream PI3K / AKT pathway plays a crucial role in mediating drug resistance in malignant tumors. Lenvatinib acts on tyrosine kinase receptors on the cell membrane surface, including VEGFR, PDGFR, FGFR, c-KIT, and RET, but not EGFR. Based on this, the inventors hypothesized that EGFR pathway activation might be involved in mediating lenvatinib resistance. Therefore, FDA-approved drugs gefitinib (targeting EGFR) and curapannixi (targeting PI3K) were selected for further research. Figure 8 A schematic diagram of drug targets targeting the EGFR / PI3K pathway is shown.

[0087] The same cell proliferation assay method as in Example 2 was used, and MTT assay was applied to detect cell proliferation activity. Figure 9 As shown in Figure A, the results indicate that: ① when lenvatinib alone at 10 μM (Len) and 20 μM, cell viability was 98.01% and 76.30%, respectively; when gefitinib alone at 5 μM and 10 μM (Gef), cell viability was 86.93% and 77.23%, respectively; and when cupanixinib alone at 12.5 nM and 25 nM (Cop), cell viability was 64.77% and 58.20%, respectively; ② when lenvatinib (10 μM) + gefitinib (5 μM) and lenvatinib (20 μM) + gefitinib (10 μM), cell viability was 60.04% and 48.20%, respectively; and when lenvatinib (10 μM) + cupanixinib (12.5 nM) and lenvatinib (20 μM) + gefitinib (25 nM), cell viability was 46.74% and 31.8%, respectively. Using the quantitative method for the combined effects of Chou-Talalay drugs, the combination index (CI) was calculated: lenvatinib 10 μM combined with gefitinib 5.0 μM, CI = 0.611; lenvatinib 20 μM combined with gefitinib 10.0 μM, CI = 0.967; lenvatinib 10 μM combined with cupanixine 12.5 nM, CI = 0.542; lenvatinib 20 μM combined with cupanixine 25.0 nM, CI = 0.700. Given that all the above CI values ​​are less than 1, this indicates that lenvatinib and gefitinib (or cupanixine) have a synergistic effect in inhibiting the proliferation of Huh7 LR cells. Figure 9 (Figure B in the middle)

[0088] Example 6: EGFR / PI3K inhibitor combined with lenvatinib promotes apoptosis in Huh7 LR cells

[0089] To further evaluate the effect of the combination of EGFR / PI3K pathway inhibitors and lenvatinib on apoptosis, flow cytometry was used to detect apoptosis. The same flow cytometry design for apoptosis detection as in Example 4 was employed.

[0090] First, Huh7 LR cells were treated with different drugs (for 72 hours): control group, lenvatinib group (30 μM), gefitinib group (30 μM), curapanixic group (3 μM), lenvatinib (30 μM) + gefitinib (30 μM) group, and lenvatinib (30 μM) + curapanixic group (3 μM). Figure 10 (Figure A). Then, Annexin V-FITC / PI staining was used, and flow cytometry was applied to detect the proportion of apoptosis (early apoptosis + late apoptosis) in different treatment groups. Figure 10 Figure B in the middle section was further analyzed statistically. Figure 10 (Figure C). The results showed that, compared with lenvatinib monotherapy, the combination of lenvatinib + gefitinib or lenvatinib + curpannice significantly promoted apoptosis in Huh7 LR cells (P<0.001).

[0091] Example 7 Inhibition of Apoptosis

[0092] 1. Tumor-bearing experiment in BALB / c nude mice

[0093] A lenvatinib resistance model was established using BALB / c nude mice. The steps and procedures are as follows:

[0094] ① Subcutaneous injection of 1×10⁻⁶ mice on the right dorsal side 7 Two Huh7 lenvatinib-resistant cells (cell suspension and Matrigel gel were mixed at a 1:1 ratio, totaling 200 μl);

[0095] ② When the tumor diameter reached approximately 6 mm, the animals were randomly divided into 6 groups: solvent group (5‰ sodium carboxymethyl cellulose solution), gefitinib group (80 mg / kg / d), ectotriazole group (80 mg / kg / d), lenvatinib group (5 mg / kg / d), lenvatinib (5 mg / kg / d) + gefitinib (80 mg / kg / d) group, and lenvatinib (5 mg / kg / d) + ectotriazole (80 mg / kg / d) group, and administered the drugs by gavage (5 times / week). In the ectotriazole combination therapy group, ectotriazole was administered 2 hours before lenvatinib.

[0096] ③During this process, the tumor volume and mouse weight were measured every 2-3 days. Tumor volume (mm) 3 = 0.5 × longest meridian of tumor × shortest meridian of tumor 2 .

[0097] 2. In vivo combined drug therapy to inhibit tumor growth

[0098] A tumor-bearing model was established in BALB / c nude mice using Huh7 lenvatinib-resistant cells to evaluate the in vivo antitumor effect of combined drug therapy. One week after subcutaneous inoculation of resistant cells, the tumor diameter was approximately 6 mm, and the mice were randomly divided into the 6 groups listed above, including the solvent group (…). Figure 11 (referred to as Veh in Chinese) Gefitinib monotherapy group, Ecleta monotherapy group, Lenvatinib monotherapy group, Lenvatinib + Gefitinib group, Lenvatinib + Ecleta group, were administered drugs according to the methods recorded above. Tumor volume and mouse weight were monitored. Two weeks after administration, the animals were euthanized, the tumors were harvested, photographed, and weighed. Figure 11 Figure A shows that, compared with the solvent group, gefitinib or ectocarboxamide alone did not inhibit tumor growth, but lenvatinib alone and lenvatinib combination therapy significantly inhibited tumor growth. Compared with lenvatinib alone, the lenvatinib combination therapy (lenvatinib + gefitinib, lenvatinib + ectocarboxamide) showed more significant inhibitory effects on tumor growth, with the lenvatinib + ectocarboxamide group showing the most significant inhibitory effect on tumor growth. Figure 11 (See Figure A). Meanwhile, during the medication process, the body weight of mice in different groups did not fluctuate significantly, indicating that the combined medication did not produce obvious side effects. Figure 11 (Figure B in the middle)

[0099] Observe the appearance and weight of tumors in different drug treatment groups, such as Figure 12 As shown in Figures A and B. Findings: ① The tumors in the solvent group, gefitinib monotherapy group, and ectenacin monotherapy group were red, highly vascularized tumors, while the tumors in the lenvatinib monotherapy group, lenvatinib + gefitinib group, and lenvatinib + ectenacin group were (light pink to white) poorly vascularized tumors. Figure 12 (Figure A); ② Further tumor weighing was performed, and the tumor weight showed a consistent trend with the tumor volume (i.e., the tumor weight was largest in the solvent application group, gefitinib monotherapy group, and ectelida monotherapy group, the tumor weight was in the middle in the lenvatinib monotherapy group, the tumor weight was smaller in the lenvatinib + gefitinib group, and the tumor weight was smallest in the lenvatinib + ectelida group). Figure 12 (Figure B in the middle)

[0100] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," "some implementations," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0101] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A pharmaceutical composition, characterized in that, It contains cupanixinib and lenvatinib, wherein the molar ratio of cupanixinib to lenvatinib is (1-20):

10.

2. Use of the pharmaceutical composition of claim 1 in the preparation of a medicament for treating liver cancer.

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