Application of N-trans-feruloyltyramine sensitized dorafenib in treatment of liver cancer

Abstract

The invention relates to the technical field of biological medicines, and particularly discloses application of N-trans-feruloyltyramine sensitized dorafenib to treatment of liver cancer. The invention further relates to a preparation method of the N-trans-feruloyltyramine sensitized dorafenib. By combining the N-trans-feruloyltyramine, the growth inhibition effect and invasion inhibition effect of dorafenib-mediated hepatoma carcinoma cells can be remarkably enhanced, and the inhibition effect of dorafenib-mediated hepatoma carcinoma cell metabolism (including glycometabolism and fatty acid metabolism) can be effectively enhanced. In different experimental models, the Q value of the combination scheme is greater than or equal to 1.15, and the stable synergistic anti-liver cancer effect is shown. According to the present invention, the N-trans-feruloyltyramine can enhance the anti-liver cancer effect of the dorafenib through the triple mechanism of improving the intracellular concentration of the targeting drug, blocking the metabolic compensation pathway and enhancing the target sensitivity, such that the clinical drug dose of the dorafenib is reduced, the toxic-side effect is reduced, and the new effective scheme is provided for the liver cancer treatment.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of biological medicines, and particularly discloses application of N-trans-feruloyltyramine in sensitizing donafenib in treatment of liver cancer. BACKGROUND

[0002] Liver cancer is a malignant tumor with high morbidity and mortality worldwide, and hepatocellular carcinoma accounts for 70-85%. The occurrence and development of liver cancer are regulated by multiple factors such as viral infection (e.g. hepatitis B virus, hepatitis C virus), alcoholic liver disease, non-alcoholic fatty liver disease, etc. Liver cancer has the characteristics of insidious onset, rapid progression and poor prognosis. Because the early symptoms of liver cancer are insidious, most patients are diagnosed at an advanced stage. Current clinical treatments for liver cancer include surgical resection, liver transplantation, interventional therapy, targeted therapy and immunotherapy, etc. However, for patients with advanced liver cancer, targeted therapy is still one of the main treatment methods.

[0003] Donafenib is an oral multi-target tyrosine kinase inhibitor (TKI). Its target points include VEGFR, PDGFR, FGFR and other angiogenesis-related kinases, as well as Raf kinase and other tumor cell proliferation-related signal molecules. It plays a dual role of anti-angiogenesis and inhibition of tumor cell proliferation by inhibiting tumor angiogenesis and tumor cell proliferation. It has been approved for use in the first-line treatment of unresectable hepatocellular carcinoma. However, in clinical application, it is found that some patients may be accompanied by adverse reactions such as hand-foot skin reaction, diarrhea, hypertension / hypertension, and decreased platelet count after using donafenib, which limits the further improvement of its clinical therapeutic effect. Therefore, the development of a combined treatment regimen that can enhance the anti-liver cancer activity of donafenib and reduce its toxic side effects has become a research hotspot in the field of targeted therapy for liver cancer.

[0004] In recent years, traditional Chinese medicine has attracted widespread attention in the application of adjuvant therapy for tumors due to its wide range of sources, diverse biological activities, and lower toxicity. Studies have shown that traditional Chinese medicine can regulate the physiological functions of tumor cells through multiple targets and multiple pathways, and can achieve the effect of "synergistic effect and reduced toxicity" when combined with chemotherapy drugs or targeted drugs.

[0005] N-trans-feruloyltyramine (trans-feruloyltyramine) is a phenolic amine compound isolated and purified from natural plants (such as wolfberry and angelica), which is widely distributed in medicinal plants such as wolfberry, angelica and chuanxiong. Its structural formula is as follows:

[0006] Molecular formula: C 18 H 19NO4, the chemical structure of which is: the amino group of 4-hydroxyphenethylamine is connected to the carboxyl group of trans-ferulic acid through an amide bond, that is, trans-4-hydroxy-3-methoxycinnamoyl-4'-hydroxyphenethylamine.

[0007] The existing research shows that it has anti-inflammatory, antioxidant, neuroprotective, anti-tumor and other biological activities, and can play an anticancer effect through inhibiting tumor cell proliferation, inducing cell apoptosis and other pathways. However, there is no relevant research on the combination of N-trans-feruloyltyramine and donafenib for liver cancer treatment and the enhancement of the anti-liver cancer activity of donafenib, and the synergistic mechanism of the two is not clear. SUMMARY

[0008] The purpose of the present application is to provide the combined application of N-trans-feruloyltyramine and donafenib in order to solve the problems of limited efficacy, obvious side effects and the like in the existing donafenib treatment of liver cancer, and to provide a new effective solution for liver cancer treatment by enhancing the killing effect of N-trans-feruloyltyramine on liver cancer cells, reducing the dosage of donafenib, and reducing the side effects of donafenib.

[0009] The present application finds that when N-trans-feruloyltyramine is combined with the tyrosine kinase inhibitor donafenib, it can significantly enhance the growth inhibition, invasion inhibition and metabolic inhibition of donafenib on liver cancer cells, and the two produce a synergistic anticancer effect, thereby solving the problem of dose-related adverse reactions in the monotherapy of donafenib for liver cancer.

[0010] The present application is realized by the following technical scheme: In a first aspect, the present application provides an application of N-trans-feruloyltyramine in sensitizing donafenib in the preparation of a drug for treating liver cancer.

[0011] In a second aspect, the present application provides an application of N-trans-feruloyltyramine in sensitizing donafenib in the preparation of a drug for inhibiting the growth, invasion, sugar metabolism and fatty acid metabolism of liver cancer cells. Specifically, it includes inhibiting the growth of liver cancer cells, inhibiting the invasion ability of metastatic liver cancer cells, inhibiting the sugar metabolism (reducing lactic acid and glucose consumption) and fatty acid metabolism (reducing fatty acid synthesis and release) of liver cancer cells.

[0012] In the present application, the liver cancer cells include one or more of liver cancer cells HepG2, Huh7, SMMC-7721, MHCC97 and Bel-7402. Preferably, the liver cancer cells are Huh7.

[0013] In a third aspect, the present application provides a pharmaceutical composition comprising N-trans-feruloyltyramine or a pharmaceutically acceptable salt thereof and a tyrosine kinase inhibitor comprising donafenib or a pharmaceutically acceptable salt thereof.

[0014] Preferably, the molar ratio of N-trans-feruloyltyramine or a pharmaceutically acceptable salt thereof to donafenib or a pharmaceutically acceptable salt thereof is 1-50:1.

[0015] Further preferably, the molar ratio of N-trans-feruloyltyramine or a pharmaceutically acceptable salt thereof to donafenib or a pharmaceutically acceptable salt thereof is 20-50:1.

[0016] Most preferably, the molar ratio of N-trans-feruloyltyramine or a pharmaceutically acceptable salt thereof to donafenib or a pharmaceutically acceptable salt thereof is 20:1.

[0017] Preferably, the preparation form of the pharmaceutical composition is an oral preparation or an injection preparation.

[0018] Preferably, the oral preparation comprises one or more of tablets, capsules, granules and suspensions; and the injection preparation comprises one or more of intravenous injection and intraperitoneal injection.

[0019] In a fourth aspect, the present application provides a kit comprising N-trans-feruloyltyramine or a pharmaceutically acceptable salt thereof and donafenib or a pharmaceutically acceptable salt thereof packaged separately.

[0020] In the present application, the CAS registry number of N-trans-feruloyltyramine is 66648-43-9; and the CAS registry number of donafenib is 1130115-44-4.

[0021] In the present application, the term "pharmaceutically acceptable salt" refers to a salt that retains the biological activity of the compound and has no adverse effects in biological or other aspects, including pharmaceutically acceptable base addition salts and acid addition salts. The pharmaceutically acceptable base addition salts can be prepared from inorganic bases (such as hydroxides or carbonates of sodium, potassium, lithium, calcium, magnesium, etc. metals) or organic bases (such as isopropylamine, trimethylamine, ethanolamine, piperazine, morpholine, etc.); and the pharmaceutically acceptable acid addition salts can be prepared from inorganic acids (such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc.) or organic acids (such as acetic acid, propionic acid, citric acid, tartaric acid, methanesulfonic acid, p-toluenesulfonic acid, etc.).

[0022] In specific embodiments, the pharmaceutical or pharmaceutical composition of the present application further comprises a pharmaceutically acceptable carrier or excipient, which is a non-toxic, inert solid, semi-solid or liquid material, including but not limited to: fillers or extenders such as lactose, dextrose, sucrose, starches, mannitol, silicic acid, and the like; binders such as carboxymethylcellulose, alginic acid, gelatin, polyvinylpyrrolidone, acacia, and the like; humectants such as glycerol, polyethylene glycol, and the like; disintegrating agents such as agar-agar, calcium carbonate, alginic acid, crospovidone, and the like; lubricants such as talc, magnesium stearate, sodium lauryl sulfate, and the like; solvents such as water, ethanol, propylene glycol, 1,3-butanediol, vegetable oils (such as sesame oil, corn oil), and the like, as well as coloring agents, releasing agents, coating agents, sweetening agents, flavoring agents, and perfuming agents, and the like.

[0023] The pharmaceutical composition or drug of the present application can be administered by various routes, including oral, parenteral (subcutaneous injection, intravenous injection, intramuscular injection, etc.), topical administration, etc., preferably oral administration or intravenous injection.

[0024] Based on the different administration routes, the drug of the present application can be prepared into various dosage forms: Oral solid dosage forms include capsules, tablets, pills, granules, etc., in which the active ingredient is mixed with the above fillers, binders, disintegrating agents, etc., and then compressed or filled to prepare; oral liquid dosage forms include oral solutions, syrups, emulsions, etc., in which the active ingredient is dissolved or dispersed in solvents such as water, ethanol, propylene glycol, etc., and sweeteners, flavorings, etc. can be added; Injectables include sterile aqueous or oily suspensions, sterile solutions, etc., which are prepared using a solvent (such as physiological saline, 1,3-butanediol) free of pyrogens according to known techniques using appropriate dispersing agents or wetting agents and suspending agents; Topical administration dosage forms include ointments, lotions, gels, powders, solutions, sprays, etc., in which the active ingredient is mixed with a pharmaceutically acceptable carrier and any required preservatives or desired buffers under sterile conditions.

[0025] In the pharmaceutical composition, kit or drug of the present application, N-trans-feruloyltyramine or its pharmaceutically acceptable salt and tyrosine kinase inhibitor can be prepared as the same preparation (such as compound tablets, compound injections) for simultaneous administration, or as two independent preparations, which can have the same or different drug dosage forms, and can be administered simultaneously or sequentially.

[0026] In clinical applications, the administration dose and frequency of N-trans-feruloyltyramine and donafenib need to be adjusted according to factors such as the body weight, age, and severity of the patient's condition.

[0027] The drug or pharmaceutical composition of the present application can be prepared by a method known in the art, for example, N-trans-feruloyltyramine, donafenib (20:1 in molar ratio) is uniformly mixed with lactose (filler), sodium carboxymethyl cellulose (binder), magnesium stearate (lubricant), sieved through an 80-mesh sieve, granulated, dried, and then compressed into oral tablets. N-trans-feruloyltyramine hydrochloride, donafenib mesylate (20:1 in molar ratio) is dissolved in normal saline (containing 0.1% Tween 80), stirred uniformly, filtered through a 0.22 μm microporous filter to remove bacteria, and then sterilized to obtain an injection in an ampoule.

[0028] As a multi-target tyrosine kinase inhibitor (TKI), the core mechanism of action of donafenib revolves around the dual pathways of "anti-angiogenesis" and "inhibition of tumor cell proliferation". However, in clinical application, donafenib has the problems of limited efficacy and dose-dependent toxic side effects. Hepatocellular carcinoma cells can maintain energy supply by activating compensatory metabolic pathways (such as enhanced glycolysis and up-regulated fatty acid synthesis), which counteracts the inhibition of proliferation by donafenib. And high-dose donafenib will non-specifically inhibit the VEGFR and other kinases of normal cells, causing adverse reactions such as hand-foot skin reaction and hypertension, which limits the increase of its clinical dose.

[0029] As a phenolic amine compound isolated from natural plants such as Lycium barbarum, the mechanism of N-trans-feruloyltyramine in sensitizing donafenib is to block the compensatory pathway of hepatocellular carcinoma cells and enhance the sensitivity of donafenib targets. Specifically, N-trans-feruloyltyramine can down-regulate the expression of key glycolytic enzymes (such as hexokinase HK2 and lactate dehydrogenase LDHA), inhibit glucose uptake and lactic acid production. At the same time, N-trans-feruloyltyramine can directly bind to the active site of FASN, inhibit its catalytic activity, reduce fatty acid synthesis and release, and cut off the compensatory metabolic pathway of hepatocellular carcinoma cells, making the inhibitory effect of donafenib on proliferation more effective. N-trans-feruloyltyramine can regulate the activity of protein phosphatase in hepatocellular carcinoma cells, reduce the dephosphorylation level of VEGFR, Raf and other kinases, and make these kinases more easily bind to donafenib. In addition, N-trans-feruloyltyramine can increase the intracellular concentration of donafenib and enhance its inhibitory effect on targets.

[0030] The synergistic effect of N-trans-feruloyltyramine and donafenib is not a simple superposition of effects, but is achieved through "complementary blocking" to enhance the therapeutic effect and reduce the toxicity and side effects. Donafenib mainly blocks the "angiogenesis-cell proliferation" pathway, and N-trans-feruloyltyramine blocks the "metabolic compensation-inflammation protection" pathway. The combined action of the two can comprehensively inhibit the survival and progression pathways of hepatocellular carcinoma cells, and avoid the compensatory drug resistance caused by blocking a single pathway.

[0031] The technical solution of the present application has at least the following advantages and beneficial effects: The present application provides the application of N-trans-feruloyltyramine in sensitizing donafenib in the treatment of hepatocellular carcinoma, and clarifies the synergistic mechanism of the two. Compared with donafenib monotherapy, the combined regimen significantly enhances the growth inhibition effect and invasion inhibition effect on Huh7 hepatocellular carcinoma cells, and significantly enhances the inhibition of glucose metabolism and fatty acid metabolism of hepatocellular carcinoma cells, achieving comprehensive inhibition of "proliferation-invasion-metabolism". In different experimental models, the Q value of the combined regimen is all greater than or equal to 1.15, showing stable synergistic anti-hepatocellular carcinoma effect. The present study found that N-trans-feruloyltyramine can enhance the anti-hepatocellular carcinoma effect of donafenib while reducing the clinical dosage of donafenib by improving the intracellular concentration of donafenib targeted drugs, blocking the metabolic compensation pathway, and enhancing the sensitivity of the target, thereby reducing the toxic and side effects of hand-foot skin reactions and hypertension caused by high doses of donafenib, and providing a new effective regimen for the treatment of hepatocellular carcinoma. BRIEF DESCRIPTION OF DRAWINGS

[0032] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following will briefly introduce the drawings needed to be used in the embodiments. It should be understood that the following drawings only show some embodiments of the present application, and therefore should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can also be obtained without creative labor on the basis of these drawings.

[0033] Figure 1 The schematic diagram of the results of the influence of different proportions of N-trans-feruloyltyramine and donafenib combined use on the proliferation inhibition rate of HuH-7 cells in Example 1 is shown in the figure. Figure 2 The schematic diagram of the results of N-trans-feruloyltyramine enhancing the growth inhibition effect of donafenib on Huh7 hepatocellular carcinoma cells in Example 2 is shown in the figure. Figure 3 The schematic diagram of the results of N-trans-feruloyltyramine enhancing the invasion inhibition effect of donafenib on Huh7 hepatocellular carcinoma cells in Example 3 is shown in the figure. Figure 4 Figure for the results of N-trans-feruloyltyramine enhancing the inhibition of Huh7 hepatoma cells' glucose metabolism by donafenib in Example 4; Figure 5 Figure for the results of N-trans-feruloyltyramine enhancing the inhibition of Huh7 hepatoma cells' fatty acid metabolism by donafenib in Example 5; Figure 6 Figure for the results of N-trans-feruloyltyramine affecting the intracellular concentration of donafenib in Example 6; Figure 7 Figure for the results of N-trans-feruloyltyramine affecting the expression of key enzymes (HK2, LDHA, FASN, ACC) of hepatoma cell metabolism in Example 7; Figure 8 Figure for the results of N-trans-feruloyltyramine affecting the phosphorylation level of donafenib targets (VEGFR2, C-Raf, ERK1 / 2) in Example 8.

[0034] In the above figures, ns represents P>0.05, * represents P<0.05, ** represents P<0.01, *** represents P<0.001, and **** represents P<0.0001. DETAILED DESCRIPTION

[0035] In order to make the purpose, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below. If specific conditions are not indicated in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. If the reagents or instruments used are not indicated by the manufacturer, they are all conventional products that can be purchased on the market.

[0036] Example 1: Effect of N-trans-feruloyltyramine combined with donafenib at different ratios on the inhibition rate of HuH-7 cell proliferation 1. Cell culture: Human hepatoma cell line HuH-7 was cultured in DMEM complete medium containing 10% fetal bovine serum and 1% penicillin-streptomycin double-antibiotic mixture in a constant temperature incubator at 37°C and 5% CO2. When the cell density was more than 1×10 6 5 6

[0037] 2. CCK-8 method for determining the survival rate of cells after drug action ​​​HuH-7 hepatocarcinoma cells in good growth condition in the logarithmic growth phase were inoculated in a 96-well plate at 5×10 3 cells per well in 100 μL complete culture medium, and cultured overnight in a 37°C, 5% CO2 incubator. After the cells adhered, the old culture medium was discarded, and 100 μL DMEM high-sugar culture medium containing different proportions of N-trans-feruloyltyramine (NTF) and donafenib combination drug groups (NTF:donafenib = 1:1, 10:1, 20:1, 30:1, 40:1, 50:1, wherein donafenib is 1 μM) were added to the HuH-7 hepatocarcinoma cells, and three replicate wells were set for each group. After 48 h of 37°C culture, 10 μL CCK-8 reagent was added to each well, and after 2 h of incubation in the dark, the OD value was detected at 450 nm wavelength using an enzyme-labeled instrument, and the cell proliferation inhibition rate was calculated.

[0038] Cell proliferation inhibition rate (%) = [1-(OD experimental group-OD blank group) / (OD control group-OD blank group)] x 100%.

[0039] The results, as shown in Figure 1 Table 1, using donafenib combined with N-trans-feruloyltyramine to treat the HuH-7 cell line for 48 h, each proportion combination group had obvious anti-hepatocarcinoma cell strain HuH-7 proliferation effect, and the cell proliferation inhibition rate was significantly higher than that of other groups when the molar ratio was 20:1. Therefore, 20:1 was determined as the best combination ratio for subsequent experiments.

[0040] Example 2: N-trans-feruloyltyramine enhances the growth inhibition effect of donafenib on hepatocarcinoma cells Huh7 1. Cell culture The human hepatocarcinoma cell line Huh7 was cultured in DMEM high-sugar culture medium containing 10% fetal bovine serum and 1% penicillin-streptomycin double-antibiotic mixture, and placed in a 37°C, 5% CO2 constant-temperature incubator for culture, with cell passage every 2-3 days, and the cells in the logarithmic growth phase were used for experiments.

[0041] 2. Experimental setup Four groups were set: a control group (Cont): the same volume of culture medium; an NTF group (single-drug group): N-trans-caffeoyltyramine (20 μM); a donafenib group (single-drug group): donafenib (1 μM); and a combination group (Comb): N-trans-caffeoyltyramine (20 μM) + donafenib (1 μM).

[0042] 3. MTS method for detecting cell anchorage-dependent growth Huh7 cells were inoculated at 5×103 Huh7 cells were seeded in 96-well plates at a density of 500 cells / well. After the cells adhered, the old medium was discarded, and 100 μL of medium containing different treatment groups was added to the Huh7 cells. After 48 hours of continuous culture, 20 μL of MTS solution was added to each well, and the cells were incubated at 37°C for 2 hours. The absorbance value (OD value) of each well was measured at 490 nm using a microplate reader, and the cell growth inhibition rate was calculated. Cell growth inhibition rate (%) = [1 - (OD experimental group - OD blank group) / (OD control group - OD blank group)] x 100%.

[0043] 4. Soft agar colony formation assay for detecting cell anchorage-independent growth Preparation of lower agarose: 2% agarose was mixed with DMEM medium containing 20% fetal bovine serum at a ratio of 1:1 to prepare a 1% agarose solution. 100 μL of the solution was added to each well of a 96-well plate, and it was allowed to solidify at 4°C for 15 minutes. Preparation of upper agarose: Huh7 cells (1 x 10 3 cells / well) were mixed with 2% agarose and DMEM medium containing 20% fetal bovine serum at a ratio of 1:1:2 to prepare a 0.5% agarose cell suspension. 100 μL of the suspension was added to the lower agarose in each well, and it was incubated at 37°C for 30 minutes to solidify. After solidification, the cells were dosed according to the grouping, with 3 replicate wells in each group. The cells were cultured for 14 days, the culture medium was discarded, and the cells were washed twice with PBS. The cells were fixed with 4% paraformaldehyde for 30 minutes, the fixing solution was discarded, 1 ml of 0.1% crystal violet staining solution (prepared with PBS) was added, and the cells were stained for 15 minutes. The crystal violet was eluted with distilled water, and the OD value was measured at 570 nm using a microplate reader. The colony formation rate was calculated.

[0044] Colony formation inhibition rate (%) = [1 - (OD experimental group - OD blank group) / (OD control group - OD blank group)] x 100%.

[0045] 5. Evaluation of synergistic effect The synergistic effect of N-trans-feruloyltyramine and donafenib was evaluated using Jin Zhengjun's Q value method. The Q value calculation formula is: Q = E a+b / (E a +E b -E a x E b ). Wherein, E a+b is the inhibition rate (%) of the combination group, E a is the inhibition rate (%) of the N-trans-feruloyltyramine single drug group, and E b is the inhibition rate (%) of the donafenib single drug group. Q < 0.85 is antagonistic effect; 0.85 ≤ Q < 1.15 is additive effect; Q ≥ 1.15 is synergistic effect.

[0046] The experimental results are shown in Figure 2 Table 3. The combination group had a significantly higher inhibition rate on the growth of liver cancer cells than the single drug group (P<0.001) in the MTS method and soft agar colony formation experiment. N-trans-feruloyltyramine can enhance the inhibitory effect of donafenib on the growth of liver cancer cells. The Q value calculation results: Q=1.17 in the MTS method, Q=1.19 in the soft agar colony formation experiment, both ≥1.15, indicating that the combination of N-trans-feruloyltyramine and donafenib has a significant synergistic inhibitory effect on the anchorage-dependent and anchorage-independent growth of liver cancer cells.

[0047] Example 3: N-trans-feruloyltyramine enhances the invasion inhibitory effect of donafenib on liver cancer cells Huh7 1. Experimental method: Huh7 cells were starved with serum-free DMEM medium for 24 hours to synchronize the cell cycle. Matrigel matrix glue was evenly spread on the bottom of the upper chamber of the Transwell chamber, and the matrix glue was diluted with serum-free DMEM medium at a ratio of 1:8, 50 μL was added to each well, and it was incubated at 37°C for 1 hour to make it coagulate, forming a gel membrane to simulate the basement membrane environment of tumor cell invasion in vivo. The starved Huh7 cells were adjusted to a concentration of 1×10 5 6 / mL, 100 μL was inoculated in the upper chamber of the Transwell chamber; 600 μL of DMEM medium containing 20% fetal bovine serum was added to the lower chamber as a chemotactic factor. In the transwell chamber system, the drugs were administered according to the grouping of Example 2, 3 replicates per group, and incubated at 37°C, 5% CO2 in an incubator for 24 hours. After incubation, the cells in the upper chamber that did not penetrate the membrane were gently wiped off with a cotton swab, the chamber was placed in 4% paraformaldehyde for 30 minutes, 0.1% crystal violet was used for staining for 20 minutes, and the excess dye was washed with PBS. After air-drying, the OD value was measured at 570 nm wavelength using a microplate reader, and the invasion cell rate was calculated: Invasion inhibition rate (%) = [1-(OD experimental group-OD blank group) / (OD control group-OD blank group)]x100%.

[0048] The Jin Zhengjun (Jin Zhengjun) Q value method was used to judge the synergistic effect of N-trans-feruloyltyramine and donafenib. The Q value calculation formula is: Q=E a+b / (E a +E b -E a x E b ). Wherein, E a+b is the cell invasion inhibition rate (%) of the combination group, E aThe cell invasion inhibition rate (%) of the N-trans-feruloyltyramine monotherapy group, E b The cell invasion inhibition rate (%) in the donafenib monotherapy group is given. Q < 0.85 indicates antagonistic effect; 0.85 ≤ Q < 1.15 indicates additive effect; Q ≥ 1.15 indicates synergistic effect.

[0049] Experimental results are as follows Figure 3 As shown, the inhibition rate of hepatocellular carcinoma cell invasion in the combination therapy group was significantly higher than that in the single-drug group (P<0.001), indicating that N-trans-feruloyltyramine enhanced the inhibitory effect of donafenib on hepatocellular carcinoma cell invasion. The Q value was calculated to be 1.20, indicating that the combination of N-trans-feruloyltyramine and donafenib had a significant synergistic inhibitory effect on hepatocellular carcinoma cell invasion.

[0050] Example 4: N-trans-feruloyltyramine enhances the inhibitory effect of donafenib on glucose metabolism in Huh7 liver cancer cells. 1. Lactic acid content detection Huh7 cells were fed at a rate of 2 × 10 5 Cells were seeded per well in 6-well plates and cultured for 24 hours until adherence. Then, the cells were administered the drug according to the grouping method in Example 2, with 3 replicates per group, and cultured for another 24 hours. The cell culture supernatant from each group was collected, and the lactate content was detected using a lactate assay kit (Nanjing Jiancheng Bioengineering Institute). The OD value was measured at 530 nm using a microplate reader, and the lactate inhibition rate was calculated.

[0051] Lactic acid inhibition rate (%) = [1 - (OD experimental group - OD blank group) / (OD control group - OD blank group)] × 100%.

[0052] 2. Glucose consumption detection The cell culture supernatants from each group were collected, and the glucose content was detected using a glucose detection kit (Beyotime Biotechnology Research Institute). The OD value was measured at a wavelength of 505 nm using an ELISA reader, and the glucose consumption inhibition rate was calculated.

[0053] Glucose consumption inhibition rate (%) = [1 - (OD blank group - OD experimental group) / (OD blank group - OD control group)] × 100%.

[0054] Experimental results are as follows Figure 4As shown, the inhibition rates of lactate and glucose consumption in the combination therapy group were significantly higher than those in the single-drug group (P<0.001), indicating that N-trans-feruloyltyramine enhanced the inhibitory effect of donafenib on glucose metabolism in hepatocellular carcinoma cells. The Q-values ​​for lactate inhibition and glucose consumption inhibition were 1.20 and 1.22, respectively, both ≥1.15, indicating that the combination of N-trans-feruloyltyramine and donafenib has a significant synergistic inhibitory effect on glucose metabolism in Huh7 cells.

[0055] Example 5: N-trans-feruloyltyramine enhances the inhibitory effect of donafenib on fatty acid metabolism in Huh7 liver cancer cells. Experimental method: Huh7 cells were cultured at a rate of 2 × 10⁻⁶. 5 Cells were seeded per well in 6-well plates and cultured for 24 hours until adherence. Then, the cells were administered the drug according to the grouping method in Example 2, with 3 replicates per group, and cultured for another 24 hours. The cell culture supernatant from each group was collected, and the amount of fatty acid released was detected using a fatty acid assay kit (Nanjing Jiancheng Bioengineering Institute). The OD value was measured at 550 nm using a microplate reader, and the fatty acid release inhibition rate was calculated.

[0056] Fatty acid release inhibition rate (%) = [1 - (OD experimental group - OD blank group) / (OD control group - OD blank group)] × 100%.

[0057] Experimental results are as follows Figure 5 As shown, the inhibition rate of fatty acids in the combination group was significantly higher than that in the single-drug group (P<0.001), indicating that N-trans-feruloyltyramine enhanced the inhibitory effect of donafenib on fatty acid metabolism in hepatocellular carcinoma cells. The calculated Q value was 1.18, ≥1.15, indicating that the combination of N-trans-feruloyltyramine and donafenib had a significant synergistic inhibitory effect on fatty acid metabolism in Huh7 cells.

[0058] Example 6: Effect of N-trans-feruloyltyramine on intracellular concentration of donafenib 1. Cell culture: Human hepatocellular carcinoma cells Huh7 were cultured in DMEM complete medium containing 10% fetal bovine serum and 1% penicillin-streptomycin mixed solution, and were placed in a constant temperature incubator at 37℃ and 5% CO2. Cells in the logarithmic growth phase were used for experiments.

[0059] 2. Experimental groups: Control group (Cont): Huh7 cells + equal volume of culture medium; Donafenib monotherapy group: Huh7 cells + 1 μM donafenib; Combination group (Comb): Huh7 cells + 1 μM danofenib + 20 μM NTF.

[0060] 3. Intracellular concentration detection: After 24 h of culture, the cells in each group were collected, lysed in ice bath for 30 min with cell lysis solution containing protease inhibitors, and centrifuged at 12,000 rpm for 15 min to obtain the supernatant. The supernatant was filtered through a 0.22 μm filter membrane, and the concentration of danofenib was detected by HPLC (chromatographic column: C18 column; mobile phase: methanol-water = 70:30; detection wavelength: 254 nm), and the average intracellular concentration of danofenib in each group was calculated.

[0061] The experimental results are shown in Table 1. Figure 6 As shown in Table 1, the intracellular concentration of the danofenib single-drug group was 22.31 ± 1.19 ng / mg protein, and the intracellular concentration of the combination group was 41.87 ± 1.57 ng / mg protein, which was significantly higher than that of the single-drug group (P < 0.0001), indicating that N-trans-feruloyltyramine can significantly increase the intracellular concentration of danofenib in Huh7 cells, providing a concentration basis for enhancing the efficacy of danofenib.

[0062] Example 7: Effect of N-trans-feruloyltyramine on the expression of key enzymes of liver cancer cell metabolism 1. Experimental grouping: The same as in Example 6.

[0063] 2. Experimental steps: After 48 h of culture, the total protein was extracted, the protein concentration was determined by BCA method, and an equal amount of protein was subjected to SDS-PAGE electrophoresis, transferred to a PVDF membrane, blocked, and then added with a primary antibody (HK2, LDHA, FASN, and ACC, all of which are rabbit anti-human monoclonal antibodies) for overnight incubation at 4°C, added with a HRP-labeled secondary antibody for 1 h of incubation at room temperature, developed by ECL chemiluminescence, and the gray value of the band was quantified by ImageJ software, with β-actin as an internal reference, and the relative expression amount of each protein was calculated.

[0064] The experimental results are shown in Table 2. Figure 7 As shown in Table 2, compared with the control group, the expression of key enzymes in the danofenib single-drug group was significantly down-regulated (P < 0.001), and the relative expression amounts of HK2, LDHA, FASN, and ACC in the combination group were significantly lower than those in the danofenib single-drug group (P < 0.0001), indicating that N-trans-feruloyltyramine can significantly down-regulate the expression of key enzymes of glycolysis and fatty acid synthesis in liver cancer cells under the action of danofenib, block the metabolic compensation pathway, and enhance the inhibitory effect of danofenib.

[0065] Example 8: Effect of N-trans-feruloyltyramine on the phosphorylation level of the target of danofenib 1. Experimental grouping: The same as in Example 6.

[0066] 2. Experimental procedure: After 24h of cell culture, total protein was extracted to detect the phosphorylation levels of VEGFR2, C-Raf and ERK1 / 2 (primary antibody was rabbit anti-human phosphorylated monoclonal antibody), and the experimental procedure was the same as that of Example 7.

[0067] The experimental results are shown in Table 2. Figure 8 As shown in Table 2, the single-drug group of donafenib can significantly reduce the phosphorylation levels of VEGFR2, C-Raf and ERK1 / 2 (P<0.0001), and the phosphorylation levels of the combination group are significantly lower than those of the single-drug group (P<0.001), indicating that N-trans-feruloyltyramine can enhance the inhibitory effect of donafenib on the phosphorylation of its target kinase and improve the target sensitivity of donafenib.

[0068] The above example results show that N-trans-feruloyltyramine can significantly enhance the growth inhibition, invasion inhibition, glucose metabolism and fatty acid metabolism inhibition of donafenib on hepatoma cells, and the combination of the two shows a significant synergistic anti-hepatoma effect. N-trans-feruloyltyramine sensitizes the anti-hepatoma activity of donafenib by increasing the intracellular concentration of donafenib, blocking metabolic compensation pathways and enhancing target sensitivity, etc., reduces the clinical dosage of donafenib, and reduces the toxic and side effects such as hand-foot skin reaction and hypertension, thereby providing a new effective solution for the treatment of hepatoma.

[0069] The above are only preferred embodiments of the present application and are not used to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. Use of N-trans-feruloyltyramine sensitized lonafarnib in the preparation of a medicament for treating liver cancer.

2. Use of N-trans-feruloyltyramine sensitized lonafarnib in the preparation of a medicament for inhibiting the growth, invasion, glucose metabolism and fatty acid metabolism of liver cancer cells.

3. Use according to claim 2, characterized in that, The liver cancer cells include one or more of liver cancer cells HepG2, Huh7, SMMC-7721, MHCC97 and Bel-7402.

4. A pharmaceutical composition, characterized by, The medicament comprises N-trans-feruloyltyramine or a pharmaceutically acceptable salt thereof and a tyrosine kinase inhibitor, which comprises lonafarnib or a pharmaceutically acceptable salt thereof.

5. The pharmaceutical composition of claim 4, wherein, The molar ratio of N-trans-feruloyltyramine or a pharmaceutically acceptable salt thereof to lonafarnib or a pharmaceutically acceptable salt thereof is 1-50:

1.

6. The pharmaceutical composition of claim 4, wherein, The molar ratio of N-trans-feruloyltyramine or a pharmaceutically acceptable salt thereof to lonafarnib or a pharmaceutically acceptable salt thereof is 20-50:

1.

7. The pharmaceutical composition of claim 4, wherein, The molar ratio of N-trans-feruloyltyramine or a pharmaceutically acceptable salt thereof to lonafarnib or a pharmaceutically acceptable salt thereof is 20:

1.

8. The pharmaceutical composition according to any one of claims 4 to 7, characterized in that, The preparation form of the medicament composition is oral preparation or injection preparation.

9. The pharmaceutical composition of claim 8, wherein, The oral preparation comprises one or more of tablets, capsules, granules and suspensions; and the injection preparation comprises one or more of intravenous injection and intraperitoneal injection.

10. A kit characterized in that, The medicament comprises N-trans-feruloyltyramine or a pharmaceutically acceptable salt thereof and lonafarnib or a pharmaceutically acceptable salt thereof.

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