Application of multi-target anti-tumor drugs combined with RAF / MEK dual-target inhibitors in the preparation of drugs for treating liver cancer and pharmaceutical compositions

Abstract

The present application provides an application of a multi-target anti-tumor drug combined with a RAF / MEK dual-target inhibitor for the preparation of a drug for the treatment of liver cancer and a pharmaceutical composition. It relates to the field of biomedicine technology. The multi-target anti-tumor drug provided by the present invention is combined with a RAF / MEK dual-target inhibitor for the preparation of a drug for the treatment of liver cancer. The multi-target anti-tumor drug includes sorafenib, and the RAF / MEK dual-target inhibitor includes VS-6766. Sorafenib is combined with VS-6766 for the treatment of liver cancer. Compared with the use of sorafenib or VS-6766 alone, the multi-target anti-tumor drug sorafenib combined with the RAF / MEK dual-target inhibitor VS-6766 can effectively inhibit the proliferation and survival of liver cancer cells, and more effectively and continuously inhibit the MAPK signaling pathway. The two have a synergistic anti-tumor effect. The two can be used in combination directly, or they can be made into a combined preparation for the treatment of liver cancer, which has good prospects for clinical transformation.

Description

Technical Field

[0001] The present application belongs to the field of biomedicine technology, and in particular relates to the use of a multi-target anti-tumor drug combined with a RAF / MEK dual-target inhibitor in the preparation of a drug for treating liver cancer and a pharmaceutical composition. Background Art

[0002] Hepatocellular carcinoma (HCC) is the sixth most common malignant tumor in the world and the third leading cause of cancer-related death worldwide. Most HCC patients are diagnosed in the advanced stage, and due to limited treatment options, the five-year survival rate is less than 20%. Sorafenib is the first systemic targeted drug approved for patients with advanced HCC. In recent years, new therapies such as donafenib, lenvatinib, regorafenib and immune checkpoint inhibitors have also shown promising clinical benefits, providing new strategies for the treatment of patients with advanced HCC. Despite the emergence of new therapies, sorafenib remains the main first-line therapy for patients with advanced HCC. However, sorafenib is only effective in 30% of HCC patients, and acquired resistance usually occurs within 6 months. Sorafenib was originally designed to target and inhibit BRAF and CRAF kinases. Subsequent studies have shown that sorafenib is a multi-target tyrosine kinase inhibitor that inhibits PDGFR-β, VEGFR2 and c-KIT, and plays an anti-proliferative and anti-angiogenic role in HCC.

[0003] Mitogen-activated protein kinase (MAPK), namely the RAF-MEK-ERK signaling pathway, is crucial for the development of HCC. The MAPK signaling pathway is activated through a series of cascade kinase phosphorylation pathways. Growth factors activate receptor tyrosine kinases, leading to the activation of RAS-GTPase. This activation converts RAS-GDP (inactive) to RAS-GTP (active), thereby activating downstream RAF kinases. RAF kinases include three isozymes: ARAF, BRAF and CRAF, among which BRAF has the highest kinase activity, followed by CRAF and ARAF. These three RAF isoforms dimerize in the form of homo- or heterodimers, and then phosphorylate and activate downstream MEK and ERK kinases. After activation, ERK enters the cell nucleus and regulates transcription factors such as c-Myc and c-Jun, thereby affecting important physiological processes such as cell proliferation, survival, differentiation and apoptosis. Resistance to RAS-MAPK pathway inhibitors is often associated with the reactivation of the MAPK signaling pathway. The RAF-MEK-ERK pathway is mainly driven by cascade phosphorylation and is complexly regulated by multiple feedback mechanisms and other signaling pathways. The dynamic effects of sorafenib on MAPK signaling may largely lead to drug resistance, but its exact mechanism remains unclear. Therefore, there is an urgent need to further elucidate the mechanism of sorafenib resistance and develop new combination therapies to combat its resistance. Summary of the invention

[0004] The purpose of the present application is to provide an application of a multi-target anti-tumor drug combined with a RAF / MEK dual-target inhibitor for the preparation of a drug for treating liver cancer and a pharmaceutical composition, aiming to solve the problem that drugs for treating liver cancer in the prior art have a certain drug resistance.

[0005] In order to achieve the above application purpose, the technical solution adopted in this application is as follows:

[0006] In a first aspect, the present application provides an application of a multi-target anti-tumor drug combined with a RAF / MEK dual-target inhibitor for preparing a drug for treating liver cancer, wherein the multi-target anti-tumor drug includes sorafenib, and the RAF / MEK dual-target inhibitor includes VS-6766.

[0007] In one or more embodiments of the present application, the structural formula of sorafenib is , the structural formula of VS-6766 is .

[0008] In one or more embodiments of the present application, the molar ratio of the sorafenib to the VS-6766 in the drug is (1-20): (0.3-5).

[0009] In a second aspect, the present application also provides a pharmaceutical composition for treating liver cancer, comprising a multi-target anti-tumor drug and a RAF / MEK dual-target inhibitor, wherein the multi-target anti-tumor drug comprises sorafenib, and the RAF / MEK dual-target inhibitor comprises VS-6766.

[0010] In one or more embodiments of the present application, the structural formula of sorafenib is , the structural formula of VS-6766 is .

[0011] In one or more embodiments of the present application, the molar ratio of sorafenib to VS-6766 in the pharmaceutical composition is (1-20): (0.3-5).

[0012] Based on the above technical solution, the application of a multi-target anti-tumor drug combined with a RAF / MEK dual-target inhibitor for preparing a drug for treating liver cancer and the pharmaceutical composition provided in the present application have at least the following beneficial technical effects:

[0013] The invention provides a multi-target anti-tumor drug combined with a RAF / MEK dual-target inhibitor for use in preparing a drug for treating liver cancer. The multi-target anti-tumor drug includes sorafenib, and the RAF / MEK dual-target inhibitor includes VS-6766. Sorafenib combined with VS-6766 is used to treat liver cancer. Compared with the use of multi-target anti-tumor drug sorafenib or RAF / MEK dual-target inhibitor VS-6766 alone, the multi-target anti-tumor drug sorafenib combined with RAF / MEK dual-target inhibitor VS-6766 can effectively inhibit the proliferation and survival of liver cancer cells, more effectively and continuously inhibit the MAPK signaling pathway, and the two have a synergistic anti-tumor effect. The two can be used in combination directly or made into a combined preparation for the treatment of liver cancer, which has a good prospect for clinical transformation. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0015] Figure 1 It is a schematic diagram of the structural formula of sorafenib and VS-6766, where: Figure 1 A is a schematic diagram of the structural formula of sorafenib. Figure 1 B is a schematic diagram of the structural formula of VS-6766.

[0016] Figure 2Schematic diagram of the synergistic mechanism of sorafenib and VS-6766.

[0017] Figure 3 This is the result of the CCK8 cell proliferation experiment of sorafenib combined with VS-6766.

[0018] Figure 4 This is the result of the clone formation experiment of sorafenib combined with VS-6766.

[0019] Figure 5 This is the result of Western blotting experiment of short-term and long-term sorafenib alone, among which, Figure 5 A is the results of a protein immunoblotting experiment in Huh7.5.1, SK-Hep1 and PLC / PRF5 liver cancer cells, when sorafenib was used alone for 24 hours. Figure 5 B is the results of a protein immunoblotting experiment in Huh7.5.1, SK-Hep1 and PLC / PRF5 liver cancer cells, in which sorafenib was used alone for 48 hours. Figure 5 C is the results of a protein immunoblotting experiment in Huh7.5.1, SK-Hep1 and PLC / PRF5 liver cancer cells treated with sorafenib alone for 0 to 120 hours.

[0020] Figure 6 This is the result of a protein immunoblotting experiment of VS-6766 alone.

[0021] Figure 7 This is the result of protein immunoblotting experiment of sorafenib combined with VS-6766.

[0022] Figure 8 This is the result of the cell apoptosis experiment of sorafenib combined with VS-6766.

[0023] Fig. 9 This is the in vivo experimental result of sorafenib combined with VS-6766 in treating HCC subcutaneous transplanted tumors in mice, among which, Fig. 9 A is a schematic diagram of tumor volume changes. Fig. 9 B is a schematic diagram of the weight changes of mice.

[0024] Fig.10 This is a comparison chart of the cell proliferation test results of Sorafenib combined with VS-6766, Trametinib, LY3009120 or BI3406. Fig.10 A is the result of the cell proliferation experiment of Sorafenib combined with Trametinib. Fig.10 B is the result of the cell proliferation experiment of sorafenib combined with LY3009120. Fig.10 C is the result of the cell proliferation experiment of sorafenib combined with BI3406. Fig.10D is the average synergy index graph of three types of liver cancer cells treated with sorafenib combined with VS-6766, Trametinib, LY3009120 or BI3406, respectively.

[0025] Fig.11 This is a comparison chart of the results of the clone formation experiment of sorafenib combined with VS-6766, LY3009120 or BI3406. DETAILED DESCRIPTION

[0026] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present application more clearly understood, the present application is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0027] In this application, the term "and / or" describes the association relationship of associated objects, indicating that there may be three relationships. For example, A and / or B can mean: A exists alone, A and B exist at the same time, and B exists alone. A and B can be singular or plural. The character " / " generally indicates that the associated objects are in an "or" relationship.

[0028] In this application, "at least one" means one or more, and "plurality" means two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single items or plural items. For example, "at least one of a, b, or c", or "at least one of a, b, and c" can all mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, and c can be single or multiple.

[0029] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean the order of execution, some or all of the steps can be executed in parallel or sequentially, and the execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0030] The terms used in the embodiments of the present application are only for the purpose of describing specific embodiments, and are not intended to limit the present application. The singular forms "a", "said" and "the" used in the embodiments of the present application and the appended claims are also intended to include plural forms, unless the context clearly indicates other meanings.

[0031] The weight of the relevant components mentioned in the embodiment description of the present application can not only refer to the specific content of each component, but also indicate the proportional relationship between the weights of the components. Therefore, as long as the content of the relevant components is proportionally enlarged or reduced according to the embodiment description of the present application, it is within the scope disclosed in the embodiment description of the present application. Specifically, the mass described in the embodiment description of the present application can be a mass unit known in the chemical industry such as µg, mg, g, kg, etc.

[0032] As a first-line targeted drug for liver cancer treatment, sorafenib (its structural formula is as follows Figure 1 A) was originally developed as a BRAF and CRAF kinase inhibitor, but its dynamic effect on MAPK signaling and specific regulatory mechanism are not clear. During the research process, the applicant found that short-term sorafenib can effectively inhibit the pERK signal of liver cancer cells, but long-term action will cause pERK signal reactivation, which may be an important reason for its drug resistance; pERK signal reactivation is accompanied by significant activation of upstream targets such as KRAS, CRAF and MEK. Therefore, the applicant speculates that combined targeting of upstream targets such as KRAS, CRAF and MEK may reverse long-term sorafenib-induced pERK signal reactivation. The applicant found through the study of a variety of RAS-MAPK signaling pathway inhibitors that the RAF / MEK dual-target inhibitor VS-6766 can effectively reverse long-term sorafenib-induced pERK signal reactivation, and the two have a synergistic anti-tumor effect.

[0033] VS-6766 (its structural formula is as follows Figure 1 B) can directly bind to MEK kinase, causing MEK kinase to be in an inhibitory conformation, and then form a stable, difficult-to-phosphorylate RAF / MEK / VS-6766 inhibitory complex with RAF kinase. Based on its unique pharmacological mechanism, previous studies have shown that VS-6766 can effectively inhibit the activity of pMEK and pERK at the same time, and may break the pMEK reactivation induced by the negative feedback of ERK signal, thereby continuously inhibiting MAPK signaling. However, the applicant found in the course of the study that VS-6766 can effectively inhibit the activity of pERK in a concentration-dependent manner; in some liver cancer cells, low concentrations of VS-6766 will paradoxically activate pMEK (similar to the RAF single-target inhibitor reported in previous studies), and is accompanied by significant activation of the upstream kinase CRAF kinase. The activation of MEK and CRAF kinases upstream of pERK may affect the inhibitory effect of VS-6766 alone in liver cancer cells.

[0034] The MAPK signaling pathway controls the proliferation and differentiation of tumor cells and is a key pathway for carcinogenic signal transduction. How to achieve long-term and effective inhibition of its downstream MAPK signaling pathway is an important scientific issue. Based on this, the present invention provides a multi-target anti-tumor drug combined with a RAF / MEK dual-target inhibitor for use in the preparation of a drug for treating liver cancer. The multi-target anti-tumor drug includes sorafenib, and the RAF / MEK dual-target inhibitor includes VS-6766. The synergistic mode of sorafenib and VS-6766 is as follows Figure 2 As shown, short-term sorafenib can effectively inhibit pERK signaling; however, under the action of sorafenib for a long time, under the regulation of pathways such as ERK negative feedback, upstream KRAS, CRAF, MEK, etc. are reactivated, eventually leading to a rebound in pERK signaling; and sorafenib combined with VS-6766 can continuously inhibit ERK signaling by vertically blocking multiple targets of the MAPK pathway, thereby exerting a synergistic anti-tumor effect.

[0035] The technical solution of the present application is described in detail below in conjunction with embodiments.

[0036] Example 1

[0037] Sorafenib combined with VS-6766 can more effectively inhibit liver cancer growth in both in vivo and in vitro models.

[0038] (1) Tumor cells and drugs involved:

[0039] Human hepatoma cells: Huh7.5.1, SK-Hep1, and PLC / PRF5 (National Key Laboratory of Biotherapy, West China Hospital, Sichuan University).

[0040] Sorafenib (Sorafenib, S7397, Selleckchem, USA).

[0041] RAF / MEK dual-target inhibitor Avutometinib (VS-6766, S7170, Selleckchem, USA).

[0042] (2) Experimental methods:

[0043] Experimental groups:

[0044] ①Control group: DMSO (dimethyl sulfoxide, D4540, Sigma-Aldrich, USA)

[0045] Treat cells;

[0046] ② Sorafenib group: cells were treated with sorafenib alone;

[0047] ③VS-6766 group: cells were treated with VS-6766 alone;

[0048] ④ Sorafenib combined with VS-6766 group: Sorafenib and VS-6766 were used to treat cells at the same time. Sorafenib and VS-6766 were prepared into solutions with DMSO respectively, and the combined group used two solutions to treat cells at the same time.

[0049] 2.1 CCK8 cell proliferation assay.

[0050] Prepare fresh complete medium for cell culture: DMEM medium (DMEM, Gibico, USA) + 10% fetal bovine serum (FBS, Cytiva, USA) + 1% penicillin-streptomycin (Cytiva, USA). Hepatoma cells Huh7.5.1, SK-Hep1 and PLC / PRF5 in the logarithmic growth phase were evenly inoculated in 96-well plates, with 3 replicate wells for each drug concentration. Different drugs were added after the cells adhered. The specific groups were:

[0051] ①Control group: add DMSO;

[0052] ② Sorafenib group: For Huh7.5.1 cells, 1μM, 2μM, 3μM,

[0053] 4 μM and 5 μM sorafenib; for SK-Hep1 and PLC / PRF5 cells, 3 μM, 5 μM, 10 μM, 15 μM, and 20 μM sorafenib were added, respectively;

[0054] ③VS-6766 group: VS-6766 was added at concentrations of 0.3μM, 1μM, 3μM, and 5μM, respectively;

[0055] ④ Sorafenib + VS-6766 group: For Huh7.5.1 cells, sorafenib was added at concentrations of 1μM, 2μM, 3μM, 4μM and 5μM, and VS-6766 was combined with concentrations of 0.3μM, 1μM, 3μM and 5μM, respectively; for SK-Hep1 and PLC / PRF5 cells, sorafenib was added at concentrations of 3μM, 5μM, 10μM, 15μM and 20μM, and VS-6766 was combined with concentrations of 0.3μM, 1μM, 3μM and 5μM, respectively.

[0056] Each group of drugs was prepared with fresh complete medium, and the final volume of the reaction system in each well was the same (100 μL). After continuing to culture for 72 h, CCK8 reagent (Target Mol, USA) was added, and the absorbance (450 nm) was measured with a microplate reader. CCK8 reagent can quickly and sensitively detect cell proliferation and cytotoxicity, and indirectly reflect the number of living cells through OD value. The Loewe model on the SynergyFinder (https: / / synergyfinder.org / ) platform was used to evaluate the synergistic potential of the two-drug combination. Because the evaluation of the synergistic effect of combined drug use is highly dependent on the specific situation, there is no recognized threshold to define a good synergistic score. According to experience: a synergistic score of less than -10 points indicates that the interaction between the two drugs is antagonistic; a synergistic score between -10 and 10 points indicates that the interaction between the two drugs is additive; a synergistic score greater than 10 points indicates that the interaction between the two drugs is synergistic.

[0057] 2.2 Clone formation experiment.

[0058] Clone formation is an experiment to evaluate cell survival, which is used to evaluate the ability of a single cell to proliferate and form clones after drug treatment. Liver cancer cells in the logarithmic growth phase are evenly inoculated in a 12-well plate, and the cells are cultured for 3-4 days after attachment, and then different groups of drugs are added for treatment. The specific groups are:

[0059] ①Control group: add DMSO.

[0060] ②Sorafenib group: Sorafenib was added at a concentration of 5 μM.

[0061] ③VS-6766 group: VS-6766 was added at a concentration of 2 μM.

[0062] ④ Sorafenib + VS-6766 group: 5 μM sorafenib and 2 μM VS-6766 were added.

[0063] Prepare each group of drugs with fresh complete medium, and the final volume of the reaction system in each well is the same (1mL). After adding the corresponding drugs, continue to culture for 5-10 days (replace fresh medium and drugs every 72 hours during this period). After colonies are formed, fix and stain, and observe the number of cell clones.

[0064] 2.3 Protein immunoblotting experiment.

[0065] Hepatoma cells Huh7.5.1, SK-Hep1 and PLC / PRF5 in the logarithmic growth phase were evenly inoculated in 6-well plates, and different drugs were added for treatment after the cells adhered to the wall. The specific groups are:

[0066] 2.3.1 Sorafenib concentration gradient experiment:

[0067] ①Control group: add DMSO;

[0068] ② Sorafenib group: 5μM, 10μM or 15μM sorafenib was added for 24h or 48h;

[0069] Each group of drugs was prepared with fresh complete medium, and the final volume of the reaction system in each well was the same (2 mL). Total cell protein was extracted 24 h or 48 h after adding the drug, and western blotting was performed to detect the expression levels of phospho-ERK1 (pT202 / pY204) / ERK2 (pT185 / pY187) (ab76299, Abcam, USA); ERK1 / 2 (A4782, ABclonal, China); Tubulin (TA347064, ZSGB-BIO, China) proteins.

[0070] 2.3.2 Sorafenib single action time gradient experiment:

[0071] ①Control group: add DMSO and act for 120h;

[0072] ② Sorafenib group: For Huh7.5.1 cells, 5 μM sorafenib was added and acted for 24h, 48h, 72h, 96h or 120h; for SK-Hep1 and PLC / PRF5 cells, 10 μM sorafenib was added and acted for 24h, 48h, 72h, 96h or 120h. Each group of drugs was prepared with fresh complete medium, and the final volume of the reaction system in each well was the same (2mL). Fresh medium and drugs were replaced every 72h. Drugs were added at the corresponding time points, and after treatment for 24h, 48h, 72h, 96h or 120h, total cell protein was extracted and western blotting was performed to detect the expression levels of phospho-CRAF (pS338, #9427, Cell Signaling Technology, USA); phospho-MEK (pS221, #2338, Cell Signaling Technology, USA); phospho-ERK1 (pT202 / pY204) / ERK2 (pT185 / pY187) (ab76299, Abcam, USA); ERK1 / 2 (A4782, ABclonal, China); Tubulin (TA347064, ZSGB-BIO, China) proteins.

[0073] 2.3.3 VS-6766 single action concentration gradient experiment:

[0074] Hepatoma cells Huh7.5.1, SK-Hep1 and PLC / PRF5 in the logarithmic growth phase were evenly inoculated in 6-well plates, and different drugs were added for treatment after the cells adhered to the wall. The specific groups are:

[0075] ①Control group: add DMSO;

[0076] ②VS-6766 group: 0.5μM, 1μM or 2μM VS-6766 was added for 24h;

[0077] Each group of drugs was prepared with fresh complete medium, and the final volume of the reaction system in each well was the same (2 mL). Total cell protein was extracted 24 hours after adding the drug, and western blotting was performed to detect the expression levels of phospho-CRAF (pS338, #9427, Cell Signaling Technology, USA); phospho-MEK (pS221, #2338, Cell Signaling Technology, USA); phospho-ERK1 (pT202 / pY204) / ERK2 (pT185 / pY187) (ab76299, Abcam, USA); Tubulin (TA347064, ZSGB-BIO, China) proteins.

[0078] 2.3.4 Western blotting experiment of sorafenib combined with VS-6766:

[0079] ①Control group: add DMSO and act for 120h;

[0080] ② Sorafenib group: For Huh7.5.1 cells, 5 μM sorafenib was added and acted for 120 h; for SK-Hep1 and PLC / PRF5 cells, 10 μM sorafenib was added and acted for 120 h;

[0081] ③VS-6766 group: VS-6766 was added at a concentration of 2 μM for 48 h;

[0082] ④ Sorafenib + VS-6766 group: For Huh7.5.1 cells, 5 μM sorafenib was added (120 h), and 2 μM VS-6766 was added (48 h); for SK-Hep1 and PLC / PRF5 cells, 10 μM sorafenib was added (120 h), and 2 μM VS-6766 was added (48 h);

[0083] Each group of drugs was prepared with fresh complete culture medium. The final volume of the reaction system in each well was the same (2 mL). Fresh culture medium and drugs were replaced every 72 h. Drugs were added at the corresponding time points, and after treatment for 48 h or 120 h, total cell protein was extracted and western blotting was performed to detect the expression levels of phospho-CRAF (pS338, #9427, Cell Signaling Technology, USA); phospho-MEK (pS221, #2338, Cell Signaling Technology, USA); phospho-ERK1 (pT202 / pY204) / ERK2 (pT185 / pY187) (ab76299, Abcam, USA); ERK1 / 2 (A4782, ABclonal, China); CyclinD1 (ab16663, Abcam, USA); Bcl-XL (ab32370, Abcam, USA); Tubulin (TA347064, ZSGB-BIO, China) and other proteins.

[0084] 2.4 Cell apoptosis experiment.

[0085] Hepatoma cells Huh7.5.1, SK-Hep1 and PLC / PRF5 in the logarithmic growth phase were evenly inoculated in 6-well plates, and different drugs were added for treatment after the cells adhered to the wall. The specific groups are:

[0086] ①Control group: add DMSO and act for 48 hours.

[0087] ② Sorafenib group: For Huh7.5.1 cells, 2 μM sorafenib was added and acted for 48 h; for SK-Hep1 and PLC / PRF5 cells, 5 μM sorafenib was added and acted for 48 h.

[0088] ③VS-6766 group: For Huh7.5.1 cells, 1 μM VS-6766 was added and acted for 48 h; for SK-Hep1 and PLC / PRF5 cells, 3 μM VS-6766 was added and acted for 48 h.

[0089] ④ Sorafenib + VS-6766 group: For Huh7.5.1 cells, 2 μM sorafenib and 1 μM VS-6766 were added and acted for 48 h; for SK-Hep1 and PLC / PRF5 cells, 5 μM sorafenib and 3 μM VS-6766 were added and acted for 48 h.

[0090] Annexin V-FITC apoptosis detection was performed by flow cytometry. All experimental procedures were strictly carried out according to the instructions provided by the kit (ABclonal, China). The main steps included: digestion with EDTA-free trypsin (BioFroxx, China) and centrifugation to collect cells (centrifugation at 1000 rpm / min for 5 min), resuspending and centrifuging with pre-cooled PBS buffer (Biosharp, China) and washing twice, adding 500 μl of binding buffer to resuspend the cells (adjust the cell concentration to 5 × 10 5 / ml); add 1.5ul Annexin-V and 1.5ul PI to each sample, mix gently by pipetting, and incubate at room temperature in the dark for 15min. Detect by flow cytometry within 1h.

[0091] 2.5 Mouse subcutaneous tumor transplantation experiment.

[0092] Male NOD / SCID mice aged 5 to 6 weeks were purchased from Huafukang Biotechnology Co., Ltd. (Beijing, China). 1 × 10 6 Huh7.5.1 cells were injected subcutaneously in the right shoulder of mice. After the tumor became palpable, the mice were randomly divided into four groups ( n =6): control group (vehicle), sorafenib group (10 mg / kg), VS-6766 group (2 mg / kg) and sorafenib (10 mg / kg) and VS-6766 (2 mg / kg) combined group. The solvent was 5% DMSO (Sigma-Aldrich, USA) + 30% PEG300 (Selleckchem, USA) + 5% Tween 80 (Sangon Biotech, China) + 60% ddH 2 O. The drug was administered once every two days (all by oral gavage), and the mouse body weight and tumor volume were recorded. The tumor volume was expressed in mm 3 The tumor volume was calculated by the following formula: tumor volume = (length × width 2 ) / 2.

[0093] The experimental results are as follows:

[0094] Figure 3 The results of the CCK8 cell proliferation experiment of sorafenib combined with VS-6766 are shown in Figure 2. The synergy index (sngergy score) is calculated by the Loewe model to determine the synergy between the two drugs. If the synergy index is greater than 10, the interaction between the two drugs is synergistic; if it is less than -10, the interaction between the two drugs is antagonistic; if it is between -10 and 10, the interaction between the two drugs is additive.

[0095] Depend on Figure 3 It can be seen that both the single-drug sorafenib group and the single-drug VS-6766 group can effectively inhibit the proliferation of liver cancer cells, and the relative cell proliferation ratio of the sorafenib + VS-6766 group is significantly lower than that of the single-drug sorafenib group and the single-drug VS-6766 group. In addition, in the three types of liver cancer cells, the synergistic index is greater than 10, indicating that the combination of sorafenib and VS-6766 has a synergistic effect in inhibiting the proliferation of liver cancer cells.

[0096] Figure 4 This is the result of the clone formation experiment of Sorafenib combined with VS-6766. Figure 4 It can be seen that both the single-drug sorafenib group and the single-drug VS-6766 group can effectively inhibit the proliferation and survival of liver cancer cells, especially the single-drug VS-6766 group has a stronger inhibitory effect on the clone formation of liver cancer cells, while the number of cell clones in the sorafenib + VS-6766 group is significantly less than that in the single-drug sorafenib group and the single-drug VS-6766 group. This shows that compared with the single-drug sorafenib group and the single-drug VS-6766 group, the combination of sorafenib + VS-6766 can more effectively inhibit the proliferation and survival of liver cancer cells.

[0097] Figure 5 The following is the results of Western blotting experiments of short-term and long-term sorafenib alone. Figure 5 It can be seen that in Huh7.5.1, SK-Hep1 and PLC / PRF5 liver cancer cells, Figure 5 A and Figure 5 B. Short-term (24h or 48h) sorafenib can effectively inhibit pERK activity in a concentration-dependent manner; however, Figure 5 C Under long-term (≥72h) sorafenib treatment, pERK activity rebounded, accompanied by significant activation of pCRAF S338 and pMEK S221 kinases upstream of the RAS-MAPK pathway. This result suggests that the reactivation of ERK signal induced by long-term sorafenib treatment may be related to the activation of upstream RAF and MEK kinases, and simultaneous targeting of upstream RAF and MEK kinases may reverse the reactivation of ERK signal induced by long-term sorafenib treatment.

[0098] Figure 6 This is the result of Western blotting experiment of VS-6766 alone. Figure 6It can be seen that in Huh7.5.1, PLC / PRF5 and SK-Hep1 cells, VS-6766 can significantly inhibit p-ERK1 / 2 activity after 24 hours of action, and it is concentration-dependent. However, in Huh7.5.1 cells, VS-6766 activates p-MEK S221; in PLC / PRF5 cells, low concentrations (0.5μM) of VS-6766 activate p-MEK S221, and higher concentrations (≥1μM) of VS-6766 can inhibit p-MEK S221; in SK-Hep1 cells, 0.5μM VS-6766 can effectively inhibit p-MEK S221 activity. In the above three types of liver cancer cells, 0.5μM, 1μM or 2μM VS-6766 can significantly activate p-CRAF S338. The above results show that, similar to the RAF single-target inhibitors reported in previous studies, the RAF / MEK dual-target inhibitor VS-6766 also paradoxically activates p-MEK at low drug concentrations when used alone, and is accompanied by significant activation of the upstream kinase p-CRAF S338. The activation of MEK and CRAF kinases upstream of ERK may affect the inhibitory effect of VS-6766 alone in liver cancer cells.

[0099] Figure 7 The figure shows the results of Western blotting of sorafenib combined with VS-6766. Figure 7 It can be seen that compared with the control group (DMSO), the pERK activity could not be effectively inhibited when sorafenib was used alone for a long time (120h), indicating that the inhibitory effect of sorafenib on pERK activity had been significantly weakened when sorafenib was used alone for a long time; compared with the control group, VS-6766 alone (48h) could still effectively inhibit pERK activity; and compared with the sorafenib single-agent group and the VS-6766 single-agent group, the sorafenib + VS-6766 combination group could more effectively inhibit pERK activity, indicating that VS-6766 could reverse the rebound of pERK activity induced by long-term sorafenib alone. In addition, compared with the single-drug sorafenib group and the single-drug VS-6766 group, the combination of sorafenib + VS-6766 can more effectively inhibit the activity of pMEK S221, partially reverse the activation of pCRAF S338 kinase, and more significantly inhibit the expression of CyclinD1 protein (proliferation) and Bcl-xL protein (anti-apoptosis) (especially in Hub7.5.1 cells). This shows that compared with the single-drug group, the combination of sorafenib + VS-6766 can more effectively inhibit the activity of the MAPK pathway and more effectively inhibit the proliferation of liver cancer cells. The two have a synergistic anti-tumor effect. This is consistent with the results of CCK8 and clone formation experiments.

[0100] Figure 8 This is the result of the cell apoptosis experiment of Sorafenib combined with VS-6766. Figure 8 It can be seen that compared with the sorafenib single-drug group and the VS-6766 single-drug group, the sorafenib + VS-6766 combination group can more effectively induce apoptosis of liver cancer cells.

[0101] Fig. 9 This is the in vivo experimental result of sorafenib combined with VS-6766 in treating HCC subcutaneous transplanted tumors in mice. Fig. 9 It can be seen that compared with the sorafenib monotherapy group, the sorafenib + VS-6766 combination group can more effectively inhibit the growth of subcutaneous transplanted liver cancer models. Fig. 9 A, indicating that VS-6766 can significantly enhance the inhibitory effect of sorafenib on liver cancer. However, at this dose (10 mg / kg sorafenib + 2 mg / kg VS-6766), the body weight of mice in the sorafenib + VS-6766 combination group showed a downward trend, although there was no significant difference in the body weight of mice in the sorafenib + VS-6766 combination group compared with the control group, as shown in Figure 2. Fig. 9 B.

[0102] From the above results, it can be seen that compared with the use of sorafenib alone or VS-6766 alone, the combination of sorafenib and VS-6766 can effectively inhibit the proliferation and survival of liver cancer cells, and more effectively and continuously inhibit the signal transduction of the MAPK pathway. The two have a synergistic anti-tumor effect.

[0103] Example 2

[0104] Comparison of the effects of sorafenib combined with VS-6766, Trametinib, LY3009120 or BI3406 in inhibiting the proliferation and survival of liver cancer cells.

[0105] (1) Tumor cells and drugs involved:

[0106] Human hepatoma cells: Huh7.5.1, SK-Hep1, and PLC / PRF5 (National Key Laboratory of Biotherapy, West China Hospital, Sichuan University).

[0107] Sorafenib (Sorafenib, S7397, Selleckchem, USA);

[0108] RAF / MEK dual-target inhibitor: Avutometinib (VS-6766, S7170, Selleckchem, USA);

[0109] RAF dimer inhibitor: LY3009120 (S7842, Selleckchem);

[0110] MEK inhibitor: Trametinib (GSK1120212, S2673, Selleckchem);

[0111] KRAS activity inhibitor: BI3406 (HY-125817, MedChemExpress, USA).

[0112] (2) Experimental methods

[0113] Experimental groups:

[0114] ①Control group: DMSO (dimethyl sulfoxide, D4540, Sigma-Aldrich, USA)

[0115] Treat cells;

[0116] ② Sorafenib group: cells were treated with sorafenib alone;

[0117] ③VS-6766, Trametinib, LY3009120 or BI3406 group: cells were treated with VS-6766, Trametinib, LY3009120 or BI3406 alone;

[0118] ④ Sorafenib combined with VS-6766, Trametinib, LY3009120 or BI3406 group: Sorafenib and VS-6766, Trametinib, LY3009120 or BI3406 were used to treat cells at the same time. When sorafenib and VS-6766, Trametinib, LY3009120 or BI3406 were used in combination, DMSO was used to prepare solutions respectively, and the combination group used two solutions to treat cells at the same time.

[0119] 2.1 CCK8 cell proliferation assay.

[0120] Prepare fresh complete medium for cell culture: DMEM medium (DMEM, Gibico, USA) + 10% fetal bovine serum (FBS, Cytiva, USA) + 1% penicillin-streptomycin (Cytiva, USA). Hepatoma cells Huh7.5.1, SK-Hep1 and PLC / PRF5 in the logarithmic growth phase were evenly inoculated in 96-well plates, with 3 replicate wells for each drug concentration. Different drugs were added after the cells adhered. The specific groups were:

[0121] (1) Sorafenib combined with Trametinib (MEK inhibitor).

[0122] ①Control group: add DMSO;

[0123] ② Sorafenib group: For Huh7.5.1 cells, 1μM, 2μM, 3μM,

[0124] 4μM and 5μM Sorafenib; for PLC / PRF5 cells, 3μM, 5μM, 10μM, 15μM and 20μM Sorafenib were added; for SK-Hep1 cells, 3μM, 5μM, 15μM and 20μM Sorafenib were added;

[0125] ③Trametinib group: For Huh7.5.1 and PLC / PRF5 cells, Trametinib was added at concentrations of 0.1μM, 0.3μM, 1μM and 3μM, respectively; for SK-Hep1 cells, Trametinib was added at concentrations of 0.1μM, 0.3μM and 1μM, respectively;

[0126] ④ Sorafenib + Trametinib group: For Huh7.5.1 cells, Sorafenib was added at concentrations of 1μM, 2μM, 3μM, 4μM and 5μM, and combined with Trametinib at concentrations of 0.1μM, 0.3μM, 1μM and 3μM, respectively; for PLC / PRF5 cells, Sorafenib was added at concentrations of 3μM, 5μM, 10μM, 15μM and 20μM, and combined with Trametinib at concentrations of 0.1μM, 0.3μM, 1μM and 3μM, respectively; for SK-Hep1 cells, Sorafenib was added at concentrations of 3μM, 5μM, 15μM and 20μM, and combined with Trametinib at concentrations of 0.1μM, 0.3μM and 1μM, respectively.

[0127] (2) Sorafenib combined with LY3009120 (RAF dimer inhibitor).

[0128] ①Control group: add DMSO;

[0129] ② Sorafenib group: For Huh7.5.1 and SK-Hep1 cells, sorafenib was added at concentrations of 3μM, 5μM, 10μM, 15μM and 20μM, respectively; for PLC / PRF5 cells, sorafenib was added at concentrations of 3μM, 5μM and 10μM, respectively;

[0130] ③ LY3009120 group: LY3009120 was added at concentrations of 0.1μM, 0.3μM, 1μM and 3μM respectively;

[0131] ④ Sorafenib + LY3009120 group: For Huh7.5.1 and SK-Hep1 cells, sorafenib was added at concentrations of 3μM, 5μM, 10μM, 15μM and 20μM, and combined with LY3009120 at concentrations of 0.1μM, 0.3μM, 1μM and 3μM, respectively; for PLC / PRF5 cells, sorafenib was added at concentrations of 3μM, 5μM and 10μM, and combined with LY3009120 at concentrations of 0.1μM, 0.3μM, 1μM and 3μM, respectively.

[0132] (3) Sorafenib combined with BI3406 (KRAS activity inhibitor).

[0133] ①Control group: add DMSO;

[0134] ② Sorafenib group: Sorafenib was added at concentrations of 3μM, 5μM, 10μM, 15μM and 20μM respectively;

[0135] ③BI3406 group: BI3406 was added at concentrations of 3μM, 5μM, 10μM and 15μM respectively;

[0136] ④ Sorafenib + BI3406 group: Sorafenib was added at concentrations of 3μM, 5μM, 10μM, 15μM and 20μM, and combined with BI3406 at concentrations of 3μM, 5μM, 10μM and 15μM, respectively.

[0137] Each group of drugs was prepared with fresh complete medium, and the final volume of the reaction system in each well was the same (100 μL). After 72 hours of continuous culture, CCK8 reagent (Target Mol, USA) was added, and the absorbance (450 nm) was measured with a microplate reader. The Loewe model on the SynergyFinder platform was used to evaluate the synergistic potential of the two-drug combination. A synergistic score greater than 10 points indicated synergistic effects.

[0138] 2.2 Clone formation experiment.

[0139] Hepatoma cells in the logarithmic growth phase were uniformly inoculated into a 12-well plate, and the cells were cultured for 3-4 days after attachment, and then different groups of drugs were added for treatment.

[0140] For Huh7.5.1 cells, the specific groups are:

[0141] ①Control group: add DMSO;

[0142] ② Sorafenib group: added with 2 μM sorafenib;

[0143] ③VS-6766 group: VS-6766 was added at a concentration of 1 μM;

[0144] ④ Sorafenib + VS-6766 group: 2 μM sorafenib and 1 μM VS-6766 were added;

[0145] ⑤LY3009120 group: LY3009120 was added at a concentration of 0.1 μM;

[0146] ⑥ Sorafenib + LY3009120 group: 2 μM sorafenib and 0.1 μM LY3009120 were added;

[0147] ⑦BI3406 group: BI3406 was added at a concentration of 3 μM;

[0148] ⑧Sorafenib + BI3406 group: 2 μM sorafenib and 3 μM BI3406 were added;

[0149] For PLC / PRF5 cells, the specific groups are:

[0150] ①Control group: add DMSO;

[0151] ② Sorafenib group: 3 μM sorafenib was added;

[0152] ③VS-6766 group: VS-6766 was added at a concentration of 3 μM;

[0153] ④ Sorafenib + VS-6766 group: 3 μM sorafenib and 3 μM VS-6766 were added;

[0154] ⑤LY3009120 group: LY3009120 was added at a concentration of 3 μM;

[0155] ⑥ Sorafenib + LY3009120 group: 3 μM sorafenib and 3 μM LY3009120 were added;

[0156] ⑦BI3406 group: BI3406 at a concentration of 10 μM was added;

[0157] ⑧ Sorafenib + BI3406 group: 3 μM sorafenib and 10 μM BI3406 were added;

[0158] For SK-Hep1 cells, the specific groups are:

[0159] ①Control group: add DMSO;

[0160] ② Sorafenib group: 5 μM sorafenib was added;

[0161] ③VS-6766 group: VS-6766 was added at a concentration of 3 μM;

[0162] ④ Sorafenib + VS-6766 group: 5 μM sorafenib and 3 μM VS-6766 were added;

[0163] ⑤LY3009120 group: LY3009120 was added at a concentration of 3 μM;

[0164] ⑥ Sorafenib + LY3009120 group: 5 μM sorafenib and 3 μM LY3009120 were added;

[0165] ⑦BI3406 group: BI3406 at a concentration of 10 μM was added;

[0166] ⑧ Sorafenib + BI3406 group: 3 μM sorafenib and 10 μM BI3406 were added;

[0167] Prepare each group of drugs with fresh complete medium, and the final volume of the reaction system in each well is the same (1mL). After adding the corresponding drugs, continue to culture for 5-10 days (replace fresh medium and drugs every 72 hours during this period). After colonies are formed, fix and stain, and observe the number of cell clones.

[0168] The experimental results are as follows:

[0169] Fig.10 The results of the cell proliferation experiment of sorafenib combined with VS-6766, trametinib, LY3009120 or BI3406 were compared. The synergy index of sorafenib combined with VS-6766 has been Figure 3 The average synergy index score was calculated based on SynergyFinder. A synergy score of less than -10 points indicates that the interaction between the two drugs is antagonistic; a synergy score between -10 and 10 points indicates that the interaction between the two drugs is additive; and a synergy score greater than 10 points indicates that the interaction between the two drugs is synergistic. Fig.10 It can be seen that the synergistic index of sorafenib + trametinib is greater than 10 in Huh7.5.1 and PLC / PRF5 cells; the synergistic index of sorafenib + LY3009120 is greater than 10 in PLC / PRF5 cells; the synergistic index of sorafenib + BI3406 is greater than 10 in PLC / PRF5 and SK-Hep1 cells; only sorafenib + VS-6766 has a synergistic index greater than 10 in all three liver cancer cells, indicating that the synergistic effect of the sorafenib + VS-6766 combination regimen is better than sorafenib + trametinib, sorafenib + LY3009120 or sorafenib + BI3406.

[0170] Fig.11The results of the clone formation experiment of sorafenib combined with VS-6766, LY3009120 or BI3406 were compared. Fig.11 It can be seen that among the three liver cancer cells tested, sorafenib combined with VS-6766, LY3009120, and BI3406 can more significantly inhibit the proliferation and survival of liver cancer cells compared with sorafenib alone. However, due to the different concentrations of the three drugs used in the cloning experiment (different effective concentrations), the effects of the three regimens can only be roughly compared.

[0171] The above results show that for liver cancer cells, in the sorafenib-based combination regimen, the synergistic effect of simultaneously targeting RAF and MEK targets (VS-6766) is better than targeting RAF target (LY3009120), MEK target (Trametinib) or KRAS target (BI3406) alone.

[0172] The present invention shows that the combined use of sorafenib and VS-6766 can effectively inhibit the proliferation and survival of liver cancer cells, and its effect is significantly better than that of sorafenib or VS-6766 alone, and the two have a synergistic anti-tumor effect. The two can be used in combination directly or made into a combined preparation for the treatment of liver cancer. Sorafenib is a first-line clinical drug for liver cancer patients that has been marketed, and VS-6766 is a "breakthrough drug" and "orphan drug" approved by the FDA. It has also shown reliable safety in related clinical trials. Therefore, the new combination of sorafenib + VS-6766 has good clinical transformation and application prospects, and it is worth further exploring its synergistic mechanism and verifying its synergistic anti-tumor effect in clinical trials.

[0173] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present application should be included in the protection scope of the present application.

Claims

1. Use of a multi-target anti-tumor drug combined with a RAF / MEK dual-target inhibitor for preparing a drug for treating liver cancer, characterized in that: The multi-target anti-tumor drug is sorafenib, the RAF / MEK dual-target inhibitor is VS-6766, and the structural formula of sorafenib is , the structural formula of VS-6766 is The molar ratio of sorafenib to VS-6766 in the drug is 1:0.3, 1:1, 1:3, 1:5, 2:0.3, 2:1, 2:3, 2:5, 3:1, 3:5, 4:0.3, 4:1, 4:3, 4:5, 5:0.3, 5:1, 5:3, 10:0.3, 10:1, 15:0.3, 15:1, 20:0.3 or 20:1.

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