A compound for increasing sensitivity of anti-hepatoma drugs, composition and application thereof

Abstract

This invention discloses a compound, composition, and application for increasing the sensitivity of anti-liver cancer drugs. The compound is a biguanide compound with the following structural formula: R1 is a C4 saturated alkyl group, R2 is a para-substituted aromatic cyclic group, and the substituent is trifluoromethoxy. The composition comprises the above-mentioned biguanide compound and lenvatinib. The compound and composition of this application can improve the sensitivity of anti-liver cancer drugs and have a significant anti-liver cancer effect.

Description

Technical Field

[0001] This invention relates to a compound, composition, and application for increasing the sensitivity of anti-liver cancer drugs, belonging to the field of biomedical technology. Background Technology

[0002] Hepatocellular carcinoma (HCC), a major type of primary liver cancer, is the sixth most common malignant tumor worldwide and has an extremely high mortality rate. Surgical resection and liver transplantation are the main radical treatments for early-stage HCC, but the vast majority of liver cancer patients are diagnosed at an intermediate or advanced stage, losing the opportunity for surgical treatment. Commonly used chemotherapy drugs such as cisplatin, fluorouracil, and capecitabine generally suffer from problems such as tumor resistance and significant toxic side effects, resulting in extremely limited overall survival benefits for patients. Compared to traditional chemotherapy, targeted therapy can achieve precise intervention on tumor cells and reduce damage to normal tissues. With its advantages in efficacy and relatively controllable safety, it has become an important treatment direction for intermediate and advanced-stage HCC.

[0003] Lenvatinib is a first-line targeted therapy for the clinical treatment of intermediate-to-advanced hepatocellular carcinoma (HCC). It is an oral multi-target kinase inhibitor that exerts its anti-tumor effects by targeting VEGFR1-3, FGFR1-4, PDGFRα, RET, and KIT. Global multicenter phase III clinical trial data showed that, compared to sorafenib, lenvatinib increased the objective response rate in HCC from 9.2% to 24.1%, demonstrating a significant improvement in treatment efficacy. Despite lenvatinib's clear clinical advantages in treating advanced HCC, 75%-80% of patients still do not respond to its treatment, and insufficient drug sensitivity severely limits its clinical application. Therefore, improving the sensitivity of HCC cells to lenvatinib is a key approach to overcoming current treatment bottlenecks and improving the clinical prognosis of HCC.

[0004] Combination therapy is a core strategy in current cancer treatment to overcome the limitations of monotherapy and drug resistance. It leverages the synergistic effects of different drugs' mechanisms of action to improve overall treatment efficacy by enhancing tumor-killing efficiency, delaying the onset of drug resistance, and expanding treatment indications. However, combination therapy also carries the risk of cumulative side effects. Achieving both high efficiency and low toxicity is a crucial consideration in the development of clinical combination regimens. Existing studies have shown that phenformin can increase the sensitivity of liver cancer cells to lenvatinib, but its own anti-liver cancer activity still has room for improvement, and the effective dose when used in combination with lenvatinib is relatively high, requiring further optimization to reduce drug-related risks. Therefore, developing novel, highly effective, and low-toxicity combination therapy strategies to significantly improve the drug sensitivity of liver cancer to lenvatinib has become a critical clinical need to be met in the field of targeted therapy for hepatocellular carcinoma.

[0005] Based on the aforementioned clinical problems and previous research, there is an urgent need for a targeted solution to address the technical challenge of insufficient sensitivity of hepatocellular carcinoma to anti-hepatocellular carcinoma drugs. Summary of the Invention

[0006] In view of the shortcomings of the prior art, the present invention aims to provide a composition and its application for increasing the sensitivity of hepatocellular carcinoma to anti-hepatocellular carcinoma drugs, specifically addressing the technical problem of insufficient sensitivity of hepatocellular carcinoma to anti-hepatocellular carcinoma drugs, and having significant clinical significance and application value for improving the treatment effect of mid-to-late stage hepatocellular carcinoma.

[0007] This invention provides a compound for increasing the sensitivity of anti-hepatocellular carcinoma drugs. The compound is a biguanide compound with the following structural formula: R1 is a C4 saturated alkyl group, R2 is a para-substituted aromatic cyclic group, and the substituent is trifluoromethoxy. This biguanide derivative exhibits excellent antitumor activity in ovarian and bladder cancer cell models. In studies on its anti-hepatocellular carcinoma activity, the inhibitory effect of the new biguanide derivative 4C on hepatocellular carcinoma cells was approximately 76 times higher than that of phenformin. Mechanistic studies confirmed that this biguanide derivative can also enhance the sensitivity of cancer cells to olaparib treatment by regulating cell cycle arrest.

[0008] As a further embodiment of the present invention, the structural formula of the compound is as follows: The above compounds were synthesized as follows: Commercially available compound 4-(trifluoromethoxyaniline) was first reacted with sodium dihydrodiimide at 80°C to give an intermediate. Subsequently, it was reacted with n-butylamine in tetrahydrofuran at 40°C until the intermediate was completely reacted. Finally, hydrochloric acid solution was added and stirred for 30 minutes. Then, ethylenediaminetetraacetic acid (EDTA) solution was added dropwise to the reaction mixture and filtered to obtain the above biguanide compound, also known as neobiguanide 4C.

[0009] The present invention also provides applications of the above-mentioned compounds, which are used to inhibit the proliferation of liver cancer cells.

[0010] Furthermore, the anticancer drug used in the application is lenvatinib.

[0011] The present invention also provides a composition for increasing the sensitivity of anti-liver cancer drugs, comprising the above-mentioned compounds.

[0012] Furthermore, the composition also includes lenvatinib.

[0013] Furthermore, the molar ratio of the biguanide compound to lenvatinib is (0.188-3):1.

[0014] Furthermore, the composition may be used by one or a combination of oral administration, intravenous injection, and intraperitoneal injection.

[0015] The present invention also provides the application of the above composition for increasing drug sensitivity against liver cancer.

[0016] Furthermore, the liver cancer is HepG2 cells.

[0017] Compared with the prior art, the technical solution of this application has the following beneficial effects: (1) The human equivalent doses of new biguanide 4C and lenvatinib were clarified. The human dose of new biguanide 4C was 0.486 mg / kg and the human dose of lenvatinib was 0.2592 mg / kg by accurate conversion using the body surface area method, thus avoiding safety risks or poor efficacy caused by improper dosage.

[0018] (2) Both in vitro and in vivo experiments have confirmed that the combination of Biguanide 4C and Lenvatinib has a significant synergistic anti-tumor effect: the in vitro MTT assay showed that the survival rate of liver cancer cells in the combination group was significantly lower than that in the Lenvatinib monotherapy group, and the combination index (CI) was about 0.5, indicating that the two drugs had a good synergistic effect; the cell clonal assay confirmed that the combination can further enhance the inhibitory ability of Lenvatinib on the formation of liver cancer cell colonies; the EdU assay showed that the combination can significantly reduce the number of EdU-positive cells in liver cancer cells and synergistically inhibit the proliferation of liver cancer cells; the in vivo nude mouse assay confirmed that the anti-tumor effect of the combination group was more significant than that of the monotherapy group, providing a practical and effective combination drug regimen for clinical tumor treatment.

[0019] (3) The combination of new biguanide 4C and lenvatinib is safe. In this invention, it is clear that the two drugs are relatively safe when administered to nude mice at the dosage (new biguanide 4C 6 mg / kg intraperitoneal injection and lenvatinib 3.2 mg / kg gavage, both 100 μL / mouse). Furthermore, the H&E staining results of liver and kidney tissues of nude mice further confirm the safety of the combination regimen, indicating that the regimen of this invention is feasible.

[0020] (4) Compared with the prior art, the present invention uses biguanide compounds in combination with lenvatinib, which can effectively increase the sensitivity of anti-liver cancer drugs, thereby inhibiting the proliferation of liver cancer cells and effectively controlling liver cancer. Attached Figure Description

[0021] Figure 1 Graph showing the antitumor activity of the new biguanide 4C combined with lenvatinib and its combination index; Figure 2 Diagram illustrating the inhibition of colony formation in liver cancer cells by the combination of new biguanide 4C and lenvatinib; Figure 3 Comparison of the number of EdU-positive cells in liver cancer cells inhibited by the combination of new biguanide 4C and lenvatinib; Figure 4The embodiments of this application provide comparative figures on the changes in weight, volume, and body weight of nude mice after administration of new biguanide 4C, lenvatinib, and the combination of the two drugs to tumor-forming nude mice. Figure 5 The embodiments of this application provide comparative H&E staining images of liver and kidney tissues of nude mice after administration of new biguanide 4C, lenvatinib, and the combination of the two drugs; Figure 6 Immunohistochemical comparison of Ki67 protein expression in tumor tissues of nude mice after administration of new biguanide 4C, lenvatinib, and the combination of the two drugs, provided in the embodiments of this application; Figure 7 : Changes in the expression level of autophagy-related protein P62 caused by the combined use of lenvatinib and biguanide 4C. Detailed Implementation

[0022] To enable those skilled in the art to better understand the technical solutions in this application, the technical solutions in the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application. Unless otherwise specified, all raw materials, reagents, instruments, and equipment used in this invention can be purchased on the market or prepared by existing methods.

[0023] This invention discloses a compound for increasing the sensitivity of anti-liver cancer drugs. The compound is a biguanide compound with the following structural formula: wherein R1 is a C4 saturated alkyl group, R2 is a para-substituted aromatic cyclic group, and the substituent is trifluoromethoxy.

[0024] As a further embodiment of the present invention, the structural formula of the compound is as follows: The above compounds were synthesized as follows: Commercially available compound 4-(trifluoromethoxyaniline) was first reacted with sodium dihydrodiimide at 80°C to give an intermediate. Subsequently, it was reacted with n-butylamine in tetrahydrofuran at 40°C until the intermediate was completely reacted. Finally, hydrochloric acid solution was added and stirred for 30 minutes. Then, ethylenediaminetetraacetic acid (EDTA) solution was added dropwise to the reaction mixture and filtered to obtain the above biguanide compound, also known as neobiguanide 4C.

[0025] Embodiments of the present invention also provide applications of the above-mentioned compounds, which are used to inhibit the proliferation of liver cancer cells.

[0026] As a further embodiment of the present invention, the anticancer drug used in the application is lenvatinib, which was purchased from Shanghai Ruihui Chemical Technology Co., Ltd.

[0027] Embodiments of the present invention also provide a composition for increasing the sensitivity of anti-liver cancer drugs, comprising the above-described compounds.

[0028] As a further embodiment of the present invention, the composition further includes lenvatinib.

[0029] As a further embodiment of the present invention, the molar ratio of the biguanide compound to lenvatinib is (0.188-3):1.

[0030] As a further embodiment of the present invention, the composition is used by one or a combination of oral administration, intravenous injection, and intraperitoneal injection.

[0031] Embodiments of the present invention also provide applications of the above-described composition for increasing drug sensitivity against liver cancer.

[0032] As a further embodiment of the present invention, the liver cancer is HepG2 cells.

[0033] Example 1 In vitro antitumor efficacy test of biguanide 4C and lenvatinib against hepatocellular carcinoma cells: The MTT assay was used to test the in vitro antitumor effects of lenvatinib and biguanide 4C in the HepG2 hepatocellular carcinoma cell line, comparing the effects of single administration versus combined administration. Two concentrations of biguanide 4C and five concentrations of lenvatinib were selected, and CI values ​​were calculated using CompuSyn software.

[0034] The specific steps are as follows: S101. Prepare DMEM complete medium according to the ratio of DMEM medium: fetal bovine serum: penicillin-streptomycin-amphoteric acid = 89:10:1; S102. HepG2 cells were seeded into 96-well plates at a density of 6000 cells / well / 200 μL and incubated at 37°C in a 5% CO2 incubator for 24 h. S103. Dilute different concentrations of the drug with 1×PBS and add 20 μL to each well of a cell-containing culture plate. The combination therapy group consisted of 10 μL lenvatinib (concentrations of 2, 4, 8, 16, and 32 μM) + 10 μL biguanide 4C (concentrations of 3 and 6 μM). The lenvatinib monotherapy group consisted of 10 μL lenvatinib + 10 μL 1×PBS (lenvatinib concentrations of 0, 2, 4, 8, 16, and 32 μM). The biguanide 4C monotherapy group consisted of 10 μL biguanide 4C + 10 μL 1×PBS (biguanide 4C concentrations of 0, 3, and 6 μM). Each concentration had at least three replicates. A control group (cells added, but no drug) and a blank group (no cells or drug added, only culture medium) were also included.

[0035] S104. After incubating at 37°C in a 5% CO2 incubator for 72 h, add 50 μL of MTT solution (2 mg / mL) to each well and continue incubation for 6 h.

[0036] S105. Remove the culture medium, add 150 μL of dimethyl sulfoxide, and mix thoroughly by shaking in the dark for 15 min. Measure the OD value of each well at a detection wavelength of 490 nm using a microplate reader. Calculate the cell viability based on the measured OD values ​​and calculate the IC50 using Grapad Prism 8. Calculate the CI value using CompuSyn software, where CI < 1 indicates synergistic effect, CI = 1 indicates additive effect, and CI > 1 indicates antagonistic effect.

[0037] The results showed that lenvatinib and biguanide 4C had a strong synergistic inhibitory effect on liver cancer cells, with a CI value around 0.5, indicating a strong synergistic effect. The results for the combination group with a biguanide 4C concentration of 6 μM are shown in Table 1. Figure 1 .

[0038] Table 1. Relevant data for 5 key proportion nodes in the MTT results. Example 2 Using a clonogenic assay, the antitumor effects of biguanide 4C and lenvatinib alone and in combination were compared in the HepG2 liver cancer cell line.

[0039] The specific steps are as follows: S201. Spread cells evenly in a 24-well plate at a density of 2000 cells / well, mix with 0.8 mL of culture medium, and set up 3 parallel replicates for each group.

[0040] S202. After the cells adhered (24 h), the drug was diluted to a certain concentration with complete culture medium and added to the culture plate at a volume of 200 μL / well. The combination group consisted of 100 μL lenvatinib (concentration: 4 μM) + 100 μL biguanide 4C (concentration: 2 μM), the lenvatinib monotherapy group consisted of 100 μL drug solution + 100 μL culture medium (lenvatinib concentration: 4 μM), the biguanide 4C monotherapy group consisted of 100 μL drug solution + 100 μL culture medium (biguanide 4C concentration: 2 μM), and the control group consisted of 200 μL culture medium.

[0041] S203. Incubate at 37℃ in a 5% CO2 incubator for about 10 days. When the clones grow to a diameter of 1-2 mm, terminate the culture.

[0042] S204. Discard the cell solution and wash twice with PBS.

[0043] S205. Add 1 mL of 10% paraformaldehyde to each well and fix for 15 min.

[0044] S206. Discard the fixative and add 500 μL of 1% crystal violet solution to each well for 30 min.

[0045] S207. Rinse slowly with tap water to remove residual dye solution and air dry at room temperature.

[0046] S208. Take a picture and measure the absorbance using a microplate reader with a wavelength of 550 nm.

[0047] The results showed that, compared with monotherapy, the combination of the two drugs further inhibited tumor cell proliferation, and the effect was significant. See results below. Figure 2 . Figure 2 The combination of the novel biguanide 4C and lenvatinib resulted in a significant reduction in tumor cells, demonstrating remarkable efficacy.

[0048] Example 3 The EdU assay was used to compare the antitumor proliferative effects of lenvatinib and biguanide 4C alone and in combination in the HepG2 liver cancer cell line.

[0049] The specific steps are as follows: S301. Spread cells evenly in a 96-well plate at a density of 6000 cells / well, mix with 200 μL of culture medium, and set up 3 parallel replicates for each group.

[0050] S302. After the cells adhered (24 h), the drug was diluted to a certain concentration with 1×PBS and added to the culture plate at a volume of 20 μL / well. The combination group was 10 μL lenvatinib (concentration: 8 μM) + 10 μL biguanide 4C (concentration: 8 μM), the lenvatinib monotherapy group was 10 μL drug solution + 10 μL 1×PBS (lenvatinib concentration: 8 μM), the biguanide 4C monotherapy group was 10 μL drug solution + 10 μL 1×PBS (biguanide 4C concentration: 8 μM), and the control group was 20 μL 1×PBS.

[0051] S303. After incubating at 37℃ in a 5% CO2 incubator for 36 hours, discard the original culture medium, wash twice with 1×PBS, add 40 μL of 4% paraformaldehyde to each well, and incubate at room temperature for 20 minutes.

[0052] S304. Discard the 4% paraformaldehyde, add 50 μL of 2 mg / mL glycine solution to each well, and incubate at room temperature for 5 min.

[0053] S305. Discard the glycine solution, add 100 μL of 1×PBS containing 3% BSA to each well, and wash the cells twice. Add 100 μL of 0.5% Triton X-100 to each well and permeate at room temperature for 15 min.

[0054] S306. Discard 0.5% Triton X-100, add 100 μL of 1×PBS solution containing 3% BSA to each well, and wash the cells twice.

[0055] S307. Prepare the Click-iT reaction mixture according to the EdU kit instructions. Add 100 μL of Click-iT reaction mixture to each well and incubate at room temperature in the dark for 30 min.

[0056] S308. Discard the Click-iT reaction mixture. Add 100 μL of 1×PBS solution containing 3% BSA to each well and wash the cells 3 times.

[0057] S309. Add 100 μL of 5ug / mL nuclear staining solution (Hoechst33342) to each well and incubate at room temperature in the dark for 20 min.

[0058] S310. Discard the staining solution, wash twice with 1×PBS, and take a fluorescence photograph using an inverted fluorescence microscope.

[0059] See results Figure 3 Biguanide 4C alone has a certain effect on inhibiting liver cancer cells, while the combination of biguanide 4C and lenvatinib significantly reduces the number of EdU-positive cells, showing the best effect.

[0060] Example 4 Animal model experiments were conducted, and a HepG2 liver cancer nude mouse model was constructed to explore the anti-tumor effect of lenvatinib combined with biguanide 4C at the in vivo level.

[0061] The specific steps are as follows: S401. Collect HepG2 cells in good growth condition by digestion and centrifugation, resuspend in PBS and count the cells, control the cell density to 1×105 cells / μL, and place on ice.

[0062] S402. After mixing the cells, 100 μL of HepG2 cell suspension was injected subcutaneously into the axilla of female BALB / c nude mice (purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.).

[0063] S403. Wait until the subcutaneous tissue of the nude mouse has grown to 60-70mm. 3 Dosing was initiated when the tumor reached a certain size (approximately 10 days). The treatment groups were: control group; lenvatinib group (64 μg / mouse); biguanide 4C group (120 μg / mouse); and combination group (lenvatinib 64 μg / mouse + biguanide 4C 120 μg / mouse). Lenvatinib was prepared as a stock solution of 96 mg / mL using DMSO, and biguanide 4C was prepared as a stock solution of 120 mg / mL using DMSO. Certain amounts of the lenvatinib and biguanide 4C stock solutions were diluted to the corresponding dosing concentrations with 0.5% sodium carboxymethyl cellulose and 1×PBS, respectively. The drugs were prepared and used immediately.

[0064] S404. Lenvatinib was administered via gavage, and biguanide 4C was administered via intraperitoneal injection. Dosing was once daily for 14 days. The weight and tumor size of the nude mice were measured and recorded daily. The mice were sacrificed after 14 days of administration. The tumors were weighed and photographed; tumors, liver, and kidney tissues were fixed with tissue fixative. The tumor volume was calculated using the formula: Tumor volume = 1 / 2 (length × width²), and a tumor growth curve was plotted.

[0065] The results showed that the combination of lenvatinib and biguanide 4C significantly inhibited tumor growth in nude mice. (See results below.) Figure 4 The combined use of the two drugs resulted in the smallest tumor volume, the smallest weight change, and the smallest change in body weight in nude mice.

[0066] Example 5 The hepatic toxicity of nude mice in different control groups was detected by hematoxylin-eosin staining.

[0067] The specific steps are as follows: S501. Nude mouse liver and kidney tissues were labeled according to their groups and then embedded and sectioned.

[0068] S502. Dewaxing: Place the sections on a metal rack in sequence, immerse them in xylene I and xylene II for 10 min, and then place them on a shaker to shake slowly.

[0069] S503. After soaking, soak the slices in anhydrous ethanol, 95% alcohol and 75% alcohol for 5 minutes each. Then place the slices in a 1000 mL beaker and rinse slowly with running water for 5 minutes, and then rinse twice with distilled water.

[0070] S504. Stain the sections with hematoxylin for 5 min, rinse the sections with running water, place the sections in hematoxylin for 20 s, rinse with tap water for 1 min, and then place the sections at 45℃ for 7 min to re-blue.

[0071] S505. Immerse the slide in eosin for 2 minutes, then rinse with tap water.

[0072] S506. Soak the slices in 75% alcohol, 95% alcohol and anhydrous ethanol for 3 minutes each, and in xylene II and xylene I for 5 minutes each. Then place them in a fume hood to air dry slightly.

[0073] S507. Add neutral resin to the slide, cover with a coverslip, and then place the slide under an inverted microscope to collect sample images.

[0074] The results are as follows Figure 5 The results showed no obvious pathological damage to the liver and kidney tissues of nude mice. Combined with the standardized staining procedure, this further confirms that the new metformin 4C, lenvatinib monotherapy and combination therapy are relatively safe at this dosage and did not cause significant toxicity to the liver and kidney tissues of nude mice. The combination therapy is relatively safe.

[0075] Example 6 Immunohistochemistry was used to detect the expression level of Ki-67 in tumor tissues of nude mice in different control groups.

[0076] The specific steps are as follows: S601. Nude mouse liver and kidney tissues were labeled according to their groups and then sent to Hubei Bios Biotechnology Co., Ltd. for embedding and sectioning.

[0077] S602. Dewaxing: Place the sections in a 50℃ oven and bake for 60 minutes. Once the sections produce droplets of paraffin, quickly immerse them in xylene I for 10 minutes, then immerse them in xylene II for 10 minutes. During the immersion process, a shaker should be placed on the oven and the sections should be shaken slowly.

[0078] S603. Soak the slices in anhydrous ethanol, 95% ethanol, and 75% ethanol in sequence for 5 minutes each. Then place the slices in a 1000 mL beaker, rinse slowly with running water for 5 minutes, and then rinse twice with distilled water.

[0079] S604. Inactivation: Immerse the sections in hydrogen peroxide solution for 15 min, then remove the sections and rinse slowly under running water for 5 min. Then rinse the sections twice with distilled water, 2 min each time.

[0080] S605. Repair: Prepare 10× antigen repair solution according to the ratio of double distilled water: EDTA: Tris = 1000 mL: 3.7 g: 12.1 g. Then dilute the 10× repair solution with distilled water, pour it into a pressure cooker and boil for 10 min. After cooling to room temperature, take it out and rinse it with distilled water 3 times, 5 min each time.

[0081] S606. Blocking: Place the slices in a humidified chamber, add 5% skim milk, and block at room temperature for 1 hour.

[0082] S607. Primary antibody incubation: Wash the tissue sections with PBST three times, 5 min each time. Dilute the primary antibody to a certain ratio and add it to the tissue. Then incubate the tissue in a humidified chamber at 4°C for about 15 h.

[0083] S608. Secondary antibody incubation: After primary antibody incubation, wash three times with PBST, 5 min each time. After removing excess PBST, add diluted secondary antibody and incubate at room temperature for 60 min.

[0084] S609. Staining: After secondary antibody incubation, wash three times with PBST, 5 min each time. Add DAB chromogenic solution (DAB substrate solution: DAB solution = 1:30 ratio) to the slides for staining, and observe the color development under a microscope in real time (usually 1–5 min). When a brownish-yellow precipitate appears in the positive areas, immediately rinse with distilled water to stop the reaction. Stain the slides with hematoxylin for 5 min, then slowly rinse with tap water to remove excess stain. Immerse the slides in hematoxylin for 10 s, rinse with tap water for 1 min, and then place the slides in 45℃ warm water for 7 min to re-blue.

[0085] S610. Dehydration: Soak the slices in 75% alcohol, 95% alcohol, and anhydrous ethanol for 3 minutes in sequence, and then soak them in xylene II and xylene I for 5 minutes each. After that, place them in a fume hood to air dry slightly.

[0086] S611. Mounting and observation: Add neutral resin to the slide, mount with a coverslip, and then place under an inverted microscope to collect sample images.

[0087] See results Figure 6The study clearly demonstrated the differences in the inhibitory effects of the new biguanide 4C group, the lenvatinib group, and the combination of the two drugs on the proliferation of tumor cells in nude mice. The Ki67 protein expression level in the 4C single-drug group was significantly lower than that in the single-drug group (lenvatinib group), and the Ki67 protein expression level in the combination group was significantly lower than that in the single-drug groups (new biguanide 4C group and lenvatinib group), which further confirms that the combination of the two drugs can synergistically inhibit the proliferation of tumor cells in nude mice.

[0088] Example 7 Western blot analysis was used to detect the expression of autophagy-related proteins. The effects of lenvatinib and biguanide 4C on the expression of the autophagy-related protein P62 were examined in HepG2 cells and nude mouse tumor tissues.

[0089] The specific steps are as follows: S701. Four groups were set up (lenvatinib, biguanide 4C, lenvatinib + biguanide 4C treatment group, and no drug treatment group as control group). The concentration of lenvatinib at the cellular level was 16 μM, and the concentration of biguanide 4C was 4 μM.

[0090] S702. After drug treatment for a certain period of time (24 h for cells, 2 weeks for animals), cells or animal tissues were collected to extract proteins, and the expression level of P62 protein was detected by Western Blot.

[0091] Figure 7 A represents the protein expression level of P62 in HepG2 cells. Figure 7 B represents the protein expression level of P62 in animal tumor tissues. The results showed that the new biguanide 4C reversed the downregulation of P62 protein induced by lenvatinib. Therefore, the P62 protein expression level in the combination group was significantly higher than that in the lenvatinib monotherapy group, indicating that the new biguanide 4C can block the lenvatinib-induced autophagy process. The combination of the two drugs indeed inhibited lenvatinib-induced autophagy (i.e., reversed the autophagy process), suggesting that the new biguanide 4C can enhance the antitumor effect of lenvatinib.

[0092] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A compound for increasing the sensitivity of anti-liver cancer drugs, characterized in that, The compound is a biguanide compound, and the biguanide compound has the following structural formula: wherein R1 is a C4 saturated alkyl group, R2 is a para-substituted aromatic cyclic group, and the substituent is trifluoromethoxy.

2. The compound for increasing the sensitivity of anti-liver cancer drugs according to claim 1, characterized in that: The structural formula of the compound is as follows: .

3. An application of the compound as described in claims 1-2, characterized in that: The compound is used to inhibit the proliferation of liver cancer cells.

4. The application of the compound according to claim 3, characterized in that: The anticancer drug used in the application is lenvatinib.

5. A composition for increasing the sensitivity of anti-liver cancer drugs, characterized in that, Includes the compounds as described in claims 1-2.

6. The composition for increasing the sensitivity of anti-hepatocellular carcinoma drugs according to claim 5, characterized in that: The composition also includes lenvatinib.

7. The composition for increasing the sensitivity of anti-hepatocellular carcinoma drugs according to claim 6, characterized in that: The molar ratio of the biguanide compound to lenvatinib is (0.188-3):

1.

8. The composition for increasing the sensitivity of anti-hepatocellular carcinoma drugs according to claim 7, characterized in that: The composition is used by one or a combination of oral, intravenous, and intraperitoneal administration.

9. An application of the composition as described in claims 5-8, characterized in that: The composition is used to increase the drug sensitivity to liver cancer.

10. The application of the composition according to claim 9, characterized in that: The liver cancer described was HepG2 cells.

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