Application of glucaric acid 1, 4-lactone and lenvatinib composition in preparation of STAT3 phosphorylation inhibitor

Abstract

The invention discloses an application of a glucaric acid 1, 4-lactone and lenvatinib composition in preparation of an STAT3 phosphorylation inhibitor, the pharmaceutical composition comprises an active component A and an active component B, the active component A is D-glucaric acid-1, 4-lactone, and the active component B is lenvatinib or a pharmaceutically acceptable salt thereof; the mass ratio of the D-glucaric acid-1, 4-lactone to the lenvatinib is 1 to (0.1 to 50). The pharmaceutical composition can be used for preparing STAT3 inhibitors, anti-liver cancer drugs and drugs for inhibiting immune escape of liver cancer cells. According to the pharmaceutical composition, the anti-tumor effect of lenvatinib is enhanced by inhibiting STAT3 phosphorylation and PD-L1 protein expression, and a new scheme is provided for solving the drug resistance problem of lenvatinib.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to the application of a glucose diacid 1,4-lactone and lenvatinib composition in the preparation of STAT3 pathway inhibitors. BACKGROUND

[0002] Hepatocellular carcinoma (HCC) is one of the main causes of cancer-related deaths worldwide. According to GLOBOCAN statistics, there were more than 900,000 new cases of liver cancer worldwide in 2020, and 830,000 deaths, with a particularly high incidence in East Asia. The pathogenic factors of HCC are complex, including chronic HBV / HCV infection, alcoholic liver disease, and metabolic syndrome, etc. Patients are often in the middle and advanced stages when diagnosed, and the traditional treatment methods such as surgical resection, local ablation or transarterial chemoembolization (TACE) have limited efficacy.

[0003] In recent years, the molecular targeted drug lenvatinib has become a first-line treatment for unresectable HCC. It delays tumor progression by inhibiting VEGFR, FGFR and other multi-targets, but clinical data shows that the objective response rate is only 18.8%, and it easily induces high expression of PD-L1 in tumor cells, promotes immune escape through the PD-1 / PD-L1 immune checkpoint pathway, leading to drug resistance and recurrence. Studies have shown that abnormal activation of the STAT3 signaling pathway is a key mechanism driving PD-L1 transcription. After STAT3 phosphorylation (Tyr705 site), a dimer is formed and enters the nucleus, directly binds to the PD-L1 promoter region, upregulates its expression and inhibits T cell anti-tumor activity. Therefore, targeting the STAT3-PD-L1 axis is an important direction to break through the bottleneck of HCC treatment. SUMMARY

[0004] In view of the limited efficacy of lenvatinib monotherapy and the drug resistance problem caused by immune escape in the treatment of hepatocellular carcinoma (HCC), the present application proposes a combination drug regimen of lenvatinib combined with natural compound 1,4-GL. By inhibiting STAT3 phosphorylation (P-STAT3 / STAT3 ratio decreased by more than 35%) and PD-L1 expression (mRNA decreased by 50%, protein decreased by 40%), the tumor immune suppression is reversed, and the efficacy of lenvatinib is enhanced, providing a new solution to the drug resistance problem of lenvatinib.

[0005] D-glucaric acid-1,4-lactone (1,4-GL) is a natural beta-glucuronidase inhibitor. Previous studies have shown that 1,4-GL can play a role in anti-HCC through regulating intestinal flora and inhibiting oxidative stress. For example, animal experiments have shown that 1,4-GL can significantly reduce the serum alpha-fetal protein content of rats induced by diethyl nitrosamine (DEN), but the synergistic effect and mechanism of 1,4-GL with targeted drugs have not been clarified. In the prior art, although there are studies on the combination of immune checkpoint inhibitors (such as PD-1 monoclonal antibody) and lenvatinib, there are defects such as high immune-related adverse reaction rate and unstable efficacy. In addition, the combination of natural compounds and targeted drugs focuses on direct cytotoxicity, and few attention is paid to the synergistic mechanism of immune microenvironment regulation.

[0006] Based on this, the present application first proposes the combination of 1,4-GL and lenvatinib, which reverses tumor immune escape by targeting the phosphorylation of STAT3 and the expression of PD-L1. Experiments have shown that 1,4-GL can significantly reduce the PD-L1 protein level (40% reduction) under lenvatinib monotherapy, and inhibit the phosphorylation of STAT3 (P-STAT3 / STAT3 ratio reduced by 45%), while up-regulating the serum IFN-γ level and remodeling the CD4⁺ / CD8⁺ T cell balance. This synergistic mechanism not only avoids the toxicity problem of traditional immunotherapy, but also provides an innovative strategy of "targeting-immune" double regulation for HCC treatment.

[0007] A pharmaceutical composition for use as a STAT3 phosphorylation inhibitor, comprising active ingredient A and active ingredient B, wherein the active ingredient A is D-glucaric acid-1,4-lactone, and the active ingredient B is lenvatinib or a pharmaceutically acceptable salt thereof; and the mass ratio of the active ingredient A to the active ingredient B is 1:0.1-50.

[0008] Optionally, the mass ratio of the active ingredient A to the active ingredient B is 1:0.1-20.

[0009] Optionally, the mass ratio of the active ingredient A to the active ingredient B is 1:0.1-10.

[0010] Further, the mass ratio of the active ingredient A to the active ingredient B is 1:0.2-1.

[0011] The present application also provides an application of a pharmaceutical composition in preparing a STAT3 phosphorylation inhibitor.

[0012] The present application also provides a kit for use as a STAT3 phosphorylation inhibitor, comprising: a preparation A comprising active ingredient A, wherein the active ingredient A is D-glucaric acid-1,4-lactone; The preparation B comprises an active ingredient B, which is lenvatinib or a pharmaceutically acceptable salt thereof.

[0013] Optionally, the preparation A and the preparation B are oral or injection.

[0014] Optionally, the preparation A and the preparation B are administered in a mass ratio of the active ingredient A to the active ingredient B of 1:0.1-50. Further, the active ingredient A and the active ingredient B are administered in a mass ratio of 1:0.1-20, further, the active ingredient A and the active ingredient B are administered in a mass ratio of 1:0.1-10; further, the active ingredient A and the active ingredient B are administered in a mass ratio of 1:0.2-1.

[0015] The combination of the active ingredient A and the active ingredient B in the present application can be prepared into a mixed preparation or a kit with each being independently packaged. For the kit, the two active ingredients can be administered simultaneously or at intervals, and the interval is not more than 2 hours. The combination can be administered orally or by injection.

[0016] The present application also provides a use of a pharmaceutical composition in the preparation of an anti-liver cancer drug.

[0017] Optionally, the anti-liver cancer drug is a drug for inhibiting immune escape of liver cancer cells.

[0018] The present application also provides a use of a pharmaceutical composition in the preparation of a drug for inhibiting immune escape of liver cancer cells, which can solve the problem of inhibiting immune escape of liver cancer cells.

[0019] The present application also provides a use of D-glucaric acid-1,4-lactone in the preparation of a synergist of an anti-liver cancer drug, which is lenvatinib or a pharmaceutically acceptable salt thereof.

[0020] The present application also provides an anti-liver cancer drug, which comprises a therapeutically effective amount of the pharmaceutical composition and a pharmaceutically acceptable carrier.

[0021] The present application finds that the use of 1,4-GL or lenvatinib alone has no regulatory effect on PD-L1 / STAT3 / P-STAT3, and the combination of the two drugs can enhance the anti-tumor effect by inhibiting STAT3 phosphorylation and increasing the expression of PD-L1 protein, wherein the combination of the two drugs can reduce the ratio of P-STAT3 / STAT3 by more than 35%, reduce the expression of PD-L1 mRNA by more than 40%, and reduce the expression of PD-L1 protein in tumor tissue to less than 70% of that in the single-drug group.

[0022] Optionally, the administration dose of the pharmaceutical composition is: 1,4-GL: 10 mg / kg / day to 200 mg / kg / day; Lenvatinib: 5 mg / kg / day to 50 mg / kg / day.

[0023] Compared with the prior art, the present application has the following beneficial effects: The present application found that 1,4-GL can enhance the anti-cancer effect of lenvatinib on H22 tumor-bearing mice, and explored the changes of some cytokines and genes. The results showed that 1,4-GL can significantly increase TNF-α when using lenvatinib alone, and there is a trend change in the cytokine indicators such as AFP, MDA, SOD, etc. with the increase of 1,4-GL concentration; 1,4-GL can significantly change the relative abundance of CD4 + T cells and CD8 + T cells; 1,4-GL can significantly reduce the IC 50 of lenvatinib.

[0024] In addition, the present application also found that 1,4-GL promotes the inhibitory effect of lenvatinib on HCC by inhibiting the PD-L1 / STST3 / P-STAT3 signaling pathway, enhances the anti-liver cancer effect of lenvatinib, solves the drug resistance problem of lenvatinib, and promotes the understanding of 1,4-GL as a potential treatment for hepatocellular carcinoma. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 Figure A is the tumor volume and weight change curve of H22 tumor-bearing mice in Example 1 (A is the change curve of mouse body weight with time, B is the change curve of mouse tumor volume with time, C is the tumor picture of each group, D is the tumor weight of mice, compared with the model group * p < 0.01).

[0026] Figure 2 Figure A is the tumor volume and weight change curve of H22 tumor-bearing mice in Example 1 (A is the change curve of mouse body weight with time, B is the change curve of mouse tumor volume with time, C is the tumor picture of each group, D is the tumor weight of mice, compared with the model group * p < 0.05, ** p < 0.01, *** p < 0.001).

[0027] Figure 3 Figure A is the tumor volume and weight change curve of H22 tumor-bearing mice in Example 1 (A is the change curve of mouse body weight with time, B is the change curve of mouse tumor volume with time, C is the tumor picture of each group, D is the tumor weight of mice, compared with the model group * p < 0.05, ** p < 0.01,*** p < 0.001).

[0028] Figure 4 Figure 2 is a graph of the protein changes in H22 tumor-bearing mouse tumor tissue (A is a photograph of the related proteins in H22 tumor-bearing mouse tumor tissue detected by western blotting; B-E are the relative expressions of PD-L1, STAT3, P-STAT3, and P-STAT3 / STAT3 in H22 tumor-bearing mouse tumor tissue detected by western blotting, respectively; * p < 0.05, ** p < 0.01, *** p < 0.001, Figure 5 Figure 4 is a graph of the changes in PD-L1, STAT3, and pSTAT3 mRNA before and after the combination of lenvatinib and 1,4-GL (A is the change in PD-L1 mRNA in H22 tumor-bearing mouse tumor tissue, B is the change in PD-L1 mRNA in Huh7 cells, and C is the change in PD-L1 mRNA in HepG2 cells. * p < 0.05, ** p < 0.01, *** p < 0.001, ns no statistical significance).

[0029] Figure 6 Figure 5 is a graph of the changes in PD-L1, STAT3, and pSTAT3 protein in Huh7 cells and HepG2 cells before and after the combination of lenvatinib and 1,4-GL (A-D are photographs of the protein expression of each gene in Huh7 and HepG2 cells under different dosing conditions, E-H are protein expression analysis of each gene in Huh7 cells under different dosing conditions, and I-L are protein expression analysis of each gene in HepG2 cells under different dosing conditions. * p < 0.05, ** p < 0.01, * * * p < 0.001, ns no statistical difference. Figure 7 Figure 6 is a graph of the changes in IC50 values in in vitro cell experiments (A-D are cell viability in Huh7 cells under different concentrations of 1,4-GL combined with lenvatinib, and E-H are cell viability in HepG2 cells under different concentrations of 1,4-GL combined with lenvatinib).

[0030] Figure 8Results of scratch test and cell clone test to evaluate the effect of 1,4-GL on the migration and proliferation of lenvatinib on Huh7 cells and HepG2 cells (A) Representative images of cell migration of each administration group at 0h, 18h, 36h in Huh7 cells and HepG2 cells (scale bar = 200pm), (B-C) Migration rate analysis of Huh7 cells and HepG2 cells at 18h and 36h, (D) Colony formation after different methods of treatment in Huh7 cells and HepG2 cells, (E-F) Colony formation analysis after different methods of treatment in Huh7 cells and HepG2 cells, * p<0.05, * * p<0.01, * * * p<0.001, ns no statistical significance). DETAILED DESCRIPTION The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present application belongs. The terminology used in the description herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application.

[0032] Test materials: 1, 1,4-GL (D-glucaric acid-1,4-lactone) was purchased from LGC, England; Lenvatinib was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China).

[0033] The structure of 1,4-GL (D-glucaric acid-1,4-lactone) is as follows: .

[0034] 2, Liver function and oxidative stress detection kit: aspartate aminotransferase (AST) kit, alanine aminotransferase (ALT) kit, malondialdehyde (MDA) kit (TBA method), total superoxide dismutase (T-SOD) detection kit (hydroxylamine method), TNF-a, IL-2 and IFN-g detection kit were purchased from Nanjing Jiancheng Biological Engineering Institute (Nanjing, China).

[0035] 3. Flow cytometry antibodies: purified anti-mouse CD16 / 32 antibody, PE-labeled anti-mouse CD3 antibody, FITC-labeled anti-mouse CD4 antibody, and APC-labeled anti-mouse CD8a antibody were purchased from Elabscience.

[0036] 4. Immunoblotting antibodies PD-L1 antibody, GAPDH monoclonal antibody, HRP-labeled goat anti-rabbit IgG (H+L), and HRP-labeled goat anti-mouse IgG (H+L) were purchased from Proteintech (Wuhan, China). Anti-phosphorylated STAT3 (Y705) antibody and anti-STAT3 antibody were purchased from Huawenbio (Hangzhou, China).

[0037] 5. H22 and Huh7 cell lines were provided by Wuhan Ponsay Life Science Co., Ltd. (Wuhan, China). The HepG2 cell line was a gift from Shanghai East Hospital and was also commercially available (e.g., Pishuang (Shanghai) Biotechnology Co., Ltd.). H22 cells were cultured in 1640 medium containing 10% FBS, and Huh7 and HepG2 cells were cultured in DMEM medium containing 10% FBS. All cell culture media were supplemented with 100 U / mL penicillin and 100 μg / mL streptomycin mixed antibiotics.

[0038] 6. KM mice and SD rats were purchased from Zhejiang Vantoll Life-Science Experimental Animals Co., Ltd. (License No. SCXK(Zhe)2024-0001). All animal experiments were conducted after being approved by the Animal Ethics Committee of Jiaxing University. The animals were housed in a SPF room with a relative humidity of 40-60%, a temperature of 20-22°C, and a 12-hour light-dark cycle. They were given free access to water and food.

[0039] All other reagents were routine laboratory reagents and were commercially available.

[0040] Example 1: Anti-tumor effect in vivo A H22 hepatocellular carcinoma subcutaneous tumor model was established in KM mice to verify the therapeutic effect of the drug: Thirty-six tumor-bearing mice were randomly divided into six groups (6 mice per group): blank control (BLANK), model (Model), lenvatinib (Letb), lenvatinib combined with low-concentration 1,4-GL (LLGL), lenvatinib combined with medium-concentration 1,4-GL (LMGL), and lenvatinib combined with high-concentration 1,4-GL (LHGL).

[0041] The BLANK group was given normal diet, and the remaining 30 mice were injected subcutaneously with H22 cells for modeling. When the subcutaneous tumor reached 100 mm 3After the left and right lumps, randomly grouped and administered treatment, letb, LLGL, LMGL, LHGL group according to the following grouping agents and doses were gavaged, the model group was gavaged with the same amount of normal saline, once a day, for 15 days.

[0042] Letb group (0.15% CMC-Na): gavage dose was 20 mg / kg / d; LLGL: mixed agent gavage dose was Letb: 20 mg / kg / d and 1,4-GL: 25 mg / kg / d; LMGL: gavage dose: Letb: 20 mg / kg / d and 1,4-GL: 50 mg / kg / d; LHGL: gavage dose: Letb: 20 mg / kg / d and 1,4-GL: 100 mg / kg / d.

[0043] Combined drug group, first gavage Letb, interval of about half an hour gavage 1,4-GL.

[0044] After administration, the body weight was weighed every two days and the tumor size was recorded, and on the 15th day, all the blood, tumor, liver, spleen, thymus were taken, the supernatant was taken by centrifugation, and all the samples were stored at -80℃.

[0045] The formula for calculating the tumor volume is: V = 0.5 × L × W², L and W are the long diameter (maximum diameter) and short diameter (perpendicular to the maximum diameter), respectively.

[0046] The formula for calculating the tumor inhibition rate is: tumor inhibition rate = (W0-W1) / W0 × 100%; W0 is the tumor weight of the Model group; W1 is the tumor weight of the corresponding treatment group.

[0047] The results of H22 mouse body weight and tumor change are shown in Figure 1 , Figure 1 A is the mouse body weight change curve, B is the mouse tumor volume change curve, C is the tumor picture of each group, D is the mouse tumor weight, compared with the model group * * p < 0.01. From the results Figure 1 It can be seen that before inoculation of H22 cells, the body weights of mice in each group were similar and had no difference, all the rats had smooth fur, good mental state and normal excretion. After successful modeling, during the administration period, the body weights of each group had no significant difference and all increased steadily with time (A), the tumor of each group changed with time (B), the tumor weight of each group was significantly lower than that of the model group (C), and the tumor inhibition rate of each group was significantly higher than that of the model group (D). Figure 1 Figure 1 ​The middle B) shows that the tumor growth of the model group is the most significant, the lenvatinib group is the second, and the growth of each 1,4-GL combined group is obviously decreased, and the LMGL of the middle dose group is the least. During the administration, the tumor size of each 1,4-GL combined group is obviously reduced Figure 1 The middle C) shows that the LMGL of the dose group is more obviously reduced.

[0048] The tumor weight and inhibition rate of each treatment group are shown in Table 1. The tumor weight of each 1,4-GL combined group is obviously reduced relative to the lenvatinib single use group, and the tumor inhibition rate is significantly improved. The inhibition effect of the middle dose group LMGL is the best, and the inhibition rate is as high as 89.97%, which is significantly better than the single drug group (60.26%, Table 1). It is shown that 1,4-GL has a more obvious enhancement on the liver cancer treatment effect of lenvatinib, and the combined treatment group (LMGL) shows significant anti-tumor activity.

[0049] Table 1 Tumor weight and inhibition rate of H22 mice

[0050] Blood biochemical index detection and T lymphocyte subpopulation proportion distribution analysis: The serum sample was taken to determine TNF-a, AFP, MDA, SOD, ALT, AST, IL-2, and IFN-g (according to the respective kit instructions).

[0051] Tumor HE staining: This experiment was performed on the day of the end of the experiment. First, the fresh spleen was ground and crushed, and the sieve was washed with PBS. The basic washing was 50 ml, 300 g centrifugation for 5 min, and the supernatant was discarded. 2 ml of red blood cell lysis solution was added to resuspend the cells, and the cells were lysed at room temperature for 3 min. 10 ml of PBS was added, and the cells were centrifuged at 300 g for 5 min. The supernatant was discarded. 1 ml of cell staining buffer (PBS+1% BSA) was used to resuspend the cells, and the cell suspension was again passed through a 200 mesh sieve, and the cell number was adjusted to 10 7 / ml. 100 μL of cell suspension was taken, 3 μL of FC receptor (CD16 / 32 antibody) was added, and the mixture was incubated at room temperature for 10 min. The appropriate sample was selected as a blank tube and a single dye tube. The blank tube did not add antibody, and the single dye tube added corresponding antibody. The sample to be tested added CD3, CD4 and CD8 antibodies, and was mixed and incubated at 4°C for 30 min in the dark. Then the supernatant was removed, 200 μL of buffer was added, and the detection was carried out. The proportion of T lymphocyte subpopulation was evaluated by flow cytometry.

[0052] The determination results are shown in Figure 2In the figure, (AH) represents the effect of 1,4-GL on serum TNF-α, AFP, MDA, SOD, ALT, AST, IL-2, and IFN-γ, respectively, and (I) represents the tumor HE staining results (200×, scale bar = 100 μm). The results showed that serum IFN-γ levels were significantly elevated in the combined groups (LMGL, LMGL, LHGL). Figure 2 The presence of AFP (a type of protein) suggests an enhanced immune response; AFP levels decreased to 8.34 ± 1.49 ng / mL (close to the normal range). Figure 2 The presence of B in the combined group indicates a decrease in tumor malignancy. Oxidative stress markers showed that the combined group had a lower MDA level (32.90 ± 4.41 nmol / mL) compared to the model group. Figure 2 The levels of C and SOD (55.95 ± 2.33 U / mL) increased (p<0.01). Figure 2 (Middle D), indicating a reduction in oxidative damage. ALT (in all combined groups) Figure 2 (E) and AST ( Figure 2 The levels of F1 and FN-γ were significantly reduced. Figure 2 The levels of IL-2 in the LMGL and LHGL groups have increased. Figure 2 The H value has increased.

[0053] HE staining ( Figure 3 The results showed that the model group had dense tumor cells with a high nucleus-cytoplasm ratio and no obvious necrosis; while the combined groups (LMGL, LMGL, LHGL) showed extensive necrosis of tumor tissue, a significant reduction in cell number, and obvious nuclear condensation, with the LMGL treatment group showing the most obvious necrosis.

[0054] To further investigate the effects of 1,4-GL on the immune function of tumor-bearing mice, flow cytometry was used to determine the distribution and proportion of T cell subsets in spleen cells. The results are shown below. Figure 3 As shown in Figures A through D, compared to the Model group, the Letb group showed a higher CD4 count after using lenvatinib. + T cells and CD8 + T cells increased in both A and B. Lenvatinib, in combination with 1,4-GL, increased CD4 counts. + T cells were significantly increased (A and B), CD8 + T cell counts decreased (A and C), and the CD4⁺ / CD8⁺ T cell ratio in the combination group increased by 30% compared to the monotherapy group (D), indicating that 1,4-GL can enhance the antitumor effect of lenvatinib by regulating the balance of immune cells.

[0055] The liver, spleen, and thymus were weighed, and the organ index was calculated as organ weight / body weight. The results are shown below. Figure 4As shown in FIGS. 1G, 1H and 1I, the thymus index (E), the spleen index (F) and the liver index (G) were determined. Compared with the model group, the thymus index of the Letb group was increased, and the three combined groups showed an increasing trend compared with the Letb group, indicating that 1,4-GL had a certain protective effect on the thymus. The spleen index of the Letb group had no significant difference with the model group, but had a downward trend, and LLGL, LMGL and LHGL had obvious differences with the model group, indicating that 1,4-GL could enhance the immune function of H22 tumor-bearing mice. The liver index of each administration group increased very close, and had no significant difference, indicating that 1,4-GL combined with lenvatinib had no hepatotoxicity.

[0056] Example 2: In vivo mechanism verification (1) The H22 tumor-bearing mouse model of Example 1 was used, and the protein and mRNA expression levels of PD-L1, STAT3 and phosphorylated STAT3 (p-STAT3) in the tumor tissues of different treatment groups were detected by Western blot and RT-qPCR.

[0057] The total protein of the sample was extracted and quantified by BCA assay kit. After all the protein was denatured, it was separated by polyacrylamide gel and then transferred to a PVDF membrane. After blocking, the membrane was incubated with PD-L1, STAT3, P-STAT3 and GAPDH antibodies at 4°C overnight. The membrane was washed with TBST and then incubated with corresponding mouse or rabbit secondary antibody at room temperature for 2 hours. Finally, enhanced chemiluminescence (ECL) reagent was used to observe the protein on BIO-RAD Gel Doc™ XR+ imaging system (BIO-RAD, HercμLes, CA, USA).

[0058] Total RNA was extracted using VeZol Reagent (R411, Vazyme Biotech Co., Ltd., Nanjing, China), cDNA was generated using HiScript IV All-in-One Ultra RT SuperMix for qPCR (R433, Vazyme Biotech Co., Ltd., Nanjing, China), and then amplified using Taq Pro Universal SYBR qPCR Master Mix (Q712, Vazyme Biotech Co., Ltd., Nanjing, China). Real-time quantitative PCR detection was performed using ABI 7500 real-time fluorescent quantitative PCR system (Applied Biosystems, America).

[0059] The protein change results of tumor tissues of H22 tumor-bearing mice are shown in Figure 5 , in which (A) is a photograph of related proteins of H22 tumor-bearing mice tumor tissues detected by western blotting, (B-E) are relative expressions of PD-L1, STAT3, P-STAT3, P-STAT3 / STAT3 in H22 tumor-bearing mice tumor tissues detected by western blotting, respectively.

[0060] Figure 5 The B results show that, compared with the Model group, there is no obvious change in PD-L1 protein expression after using lenvatinib; compared with the Letb group, the PD-L1 protein expression in the LLGL group, the LMGL group and the LHGL group after using 1,4-GL is more obviously decreased, indicating that the enhancement of the anticancer effect of lenvatinib may be related to the decrease of PD-L1 protein expression. Further detection of the protein expression of STAT3 and P-STAT3 genes (C-E) shows that, compared with the Model group, there is no significant change in STAT3 protein, and the P-STAT3 protein is not obviously changed when using lenvatinib alone, but the expression gradually decreases after using 1,4-GL in combination with different concentrations, and the P-STAT3 / STAT3 also shows a downward trend. Figure 5

[0061] In addition, it is found by RT-qPCR that the mRNA also changes similarly to the protein (A), compared with the Model group, the PD-L1 mRNA in the Letb group has no obvious change; compared with the Letb group, the PD-L1 mRNA in the LLGL group, the LMGL group and the LHGL group all obviously decreases, and the downward trend is similar to the trend of tumor size change. It is shown that the effect of 1,4-GL in enhancing the effect of lenvatinib is related to PD-L1, and the decrease of PD-L1 gene expression is related to P-STAT3 / STAT3 gene. Figure 5 The in-vivo mechanism research shows that 1.4-GL combined with lenvatinib has a good inhibitory effect on hepatocellular carcinoma cells, and the effect is significantly higher than that of using lenvatinib alone. The in-vivo experimental results show that the PD-L1 protein expression in the tumor tissues of the combination group (LMGL) is significantly lower than that of the single-drug group (Letb) (p<0.05), and the p-STAT3 / STAT3 ratio is reduced by 35%-45%, suggesting that the STAT3 phosphorylation is inhibited. RT-qPCR shows that the PD-L1 mRNA is synchronously down-regulated, indicating transcriptional level regulation.

[0062]

[0063] ​​1,4-GL can significantly reduce the PD-L1 protein level under lenvatinib monotherapy, and inhibit STAT3 phosphorylation, and reshape the CD4⁺ / CD8⁺ T cell balance. 1.4-GL and lenvatinib can be used in combination or can be made into a combined preparation for the treatment of liver cancer.

[0064] The above results show that Letb alone has no regulatory effect on PD-L1 / STAT3 / P-STAT3, and after combined use of 1,4-GL, 1,4-GL inhibits STAT3 phosphorylation, blocks its nuclear translocation and transcriptional activation of PD-L1, reverses tumor immune escape, thereby enhancing the efficacy of lenvatinib and solving the problem of lenvatinib resistance.

[0065] Example 3: In vitro synergistic effect (1) Using Huh7 and HepG2 liver cancer cell lines, the cytotoxicity of 1,4-GL and the change of 1,4-GL on the cytotoxicity of lenvatinib were determined by in vitro experiment, and verified on Huh7 and HepG2 cells.

[0066] Logarithmic growth phase Huh7 cells and HepG2 cells were selected, and when the density exceeded 80%, trypsin digestion and centrifugation were performed, the supernatant was discarded, and the cells were resuspended and counted by adding culture medium. According to the experimental grouping, 1×104 cells per well were evenly plated in a 96-well plate, and PBS was added to the outer circle of the 96-well plate to prevent excessive evaporation of the culture medium. After the cells adhered, drug administration was started, and 1,4-GL low, medium and high concentrations were 200-4000 μmol / L, all of which were diluted with DMEM complete culture medium.

[0067] In vitro experiments showed that the survival rate of Huh7 and HepG2 cells using 1,4-GL below 4000 μmol / L was above 90%, indicating that 1,4-GL at this concentration had no killing ability to the cells.

[0068] (2) Using Huh7 and HepG2 liver cancer cell lines, after IFN-γ (20 ng / mL) induced PD-L1 expression, the in vitro experiment evaluated the regulatory effect of 1,4-GL combined with lenvatinib on STAT3 signaling pathway and PD-L1.

[0069] Studies have shown that among the many cytokines that affect PD-L1 expression, interferon-γ (IFN-γ) is the strongest inducing cytokine. Therefore, in this study, IFN-γ was used to up-regulate the expression of PD-L1 protein in hepatoma cells Huh7 and HepG2. In the pre-experiment, it was found that when using 20 ng / mL concentration of IFN-γ, the expression of PD-L1 in cells was more obvious. Therefore, this concentration of IFN-γ was used to induce the determination of PD-L1 protein expression in the subsequent in vitro experiments.

[0070] The specific test process is as follows: When the cells grow to 70% density, add interferon-γ (IFN-γ, 20 ng / mL) to induce for 12 hours to enhance PD-L1 expression, and remove IFN-γ after 12 hours, and then give drugs according to the grouping.

[0071] Drug grouping: Model group, 1,4-GL group, Letb group, LLGL group, LMGL group, and LHGL group. The Model group is the no-drug group, the 1,4-GL group is the single use of 1,4-GL, the Letb group is the single use of Letb, the LLGL is the Letb combined with low concentration of 1,4-GL group, the LMGL is the Letb combined with medium concentration of 1,4-GL group, and the LHGL is the Letb combined with high concentration of 1,4-GL group.

[0072] The final concentration of Letb added to different cells in each group is 25 μmol / L (Huh7 cells) and 40 μmol / L (HepG2 cells), and the final concentration of 1,4-GL added to different cells in each group is 200, 400, and 600 μmol / L for low, medium, and high, respectively. The final concentration of 1,4-GL added to the single use of 1,4-GL group is 400 μmol / L.

[0073] In the combination drug group, Letb was first administered intragastrically, and 1,4-GL was administered intragastrically about half an hour later.

[0074] In vitro experiments used Huh7 and HepG2 hepatoma cell lines to verify the specific mechanism of protein and mRNA. The protein and mRNA expression levels of PD-L1, STAT3, and phosphorylated STAT3 (p-STAT3) in Huh7 and HepG2 hepatoma cells were detected by Western blot and RT-qPCR. The specific operation process is described in Example 2.

[0075] The verification results of mRNA expression levels are shown in Figure 5 In the mRNA determination of Huh7 and HepG2, 1,4-GL had no significant change in the expression level of PD-L1 mRNA. In Huh7 cells (Fig. 6B) and HepG2 cells (Fig. 6C), the expression of PD-L1 mRNA was not significantly changed after the addition of 1,4-GL. Figure 5PD-L1 mRNA had no significant change but a downward trend compared with the Model group, but PD-L1 mRNA had no significant change compared with the Letb group, and PD-L1 mRNA in the LHGL group was significantly decreased compared with the Letb group in Huh7 cells (Fig. 2B) and HepG2 cells (Fig. 2C). Figure 6 PD-L1 mRNA had no significant change compared with the Model group, but PD-L1 mRNA in the LHGL group was significantly decreased compared with the Letb group in Huh7 cells (Fig. 2B) and HepG2 cells (Fig. 2C).

[0076] The changes in PD-L1, STAT3, and pSTAT3 protein in Huh7 cells and HepG2 cells before and after the combination of lenvatinib and 1,4-GL are shown in Fig. 2D. Figure 6 Fig. 2D shows the changes in PD-L1, STAT3, and pSTAT3 protein in Huh7 cells and HepG2 cells before and after the combination of lenvatinib and 1,4-GL. Figs. 2E-2H show the protein expression analysis of each gene in Huh7 cells under different administration conditions. Figs. 2I-2L show the protein expression analysis of each gene in HepG2 cells under different administration conditions. The results show that in the protein expression determination (Figs. 2E-2H) Figure 6 In Huh7 cells, the PD-L1 protein expression in the 1,4-GL group was slightly increased compared with the Model group, but in HepG2 cells, the PD-L1 protein expression in the 1,4-GL group had no significant change compared with the Model group. In Huh7 and HepG2 cells, STAT3, P-STAT3, and P-STAT3 / STAT3 had no significant change compared with the Model group in the 1,4-GL group, indicating that 1,4-GL alone had no regulatory effect on PD-L1 / STAT3 / P-STAT3. In Huh7 cells, the relative expression of PD-L1, STAT3, and P-STAT3 in the Letb group had no significant change compared with the Model group (Figs. 2E-2H). Figure 7 In Huh7 cells, the relative expression of PD-L1, STAT3, and P-STAT3 in the Letb group had no significant change compared with the Model group (Figs. 2E-2H).

[0077] In the in vitro test, Western blot and RT-qPCR results showed that 1,4-GL alone or Letb alone had no regulatory effect on PD-L1 / STAT3 / P-STAT3, but the combination significantly down-regulated the PD-L1 protein expression (40% reduction in Huh7 cells, p< 0.01 50% reduction in HepG2 cells, p<0.01 and inhibited STAT3 tyrosine phosphorylation (P-STAT3 / STAT3 ratio decreased by 35.88%-45.59%), indicating that the two had a synergistic effect and the synergistic effect was derived from the inhibition of the STAT3-PD-L1 signaling pathway. (3) The in vitro antitumor activity of 1,4-GL combined with lenvatinib was evaluated using two hepatocellular carcinoma cell lines, Huh7 and HepG2.

[0078] The experiment was divided into a single-drug group (lenvatinib) and a combination group (lenvatinib + different concentrations of 1,4-GL).

[0079] Lenvatinib concentration gradients were 200, 100, 50, 25, 12.5, 6.25, and 3.125 μmol / L, and 1,4-GL low, medium, and high concentrations were 200, 400, and 600 μmol / L, respectively. All were diluted using complete DMEM medium. Lenvatinib alone was designated the Letb group, lenvatinib combined with a low concentration of 1,4-GL was designated the LLGL group, lenvatinib combined with a medium concentration of 1,4-GL was designated the LMGL group, and lenvatinib combined with a high concentration of 1,4-GL was designated the LHGL group. Cell viability was determined using the CCK-8 assay, and the half-maximal inhibitory concentration (IC50) of lenvatinib was calculated. 50 ).

[0080] CCK-8 method for determining IC 50 Huh7 and HepG2 cells were seeded into 96-well plates with 100 μL of culture medium per well and cultured at 37°C for 24 hours. Then, cells were treated with different concentrations of lenvatinib and 1,4-GL as needed. After incubation for 24-72 hours, each well was treated with 100 μL of 1×CCK-8 (MCE, USA). After incubation for 2-4 hours, absorbance was measured at 450 nm using a microplate reader. The IC50 was then calculated by determining the concentration of lenvatinib at each concentration of 1,4-GL. 50 .

[0081] CCK-8 method for determining IC 50 See the value results Figure 7 , Figure 7 The AD value represents the cell viability of Huh7 cells at different concentrations of 1,4-GL combined with lenvatinib, and the IC50 of lenvatinib in the Letb group, LLGL group, LMGL group, and LHGL group. 50 The concentrations were 32.32 μmol / L, 21.34 μmol / L, 21.75 μmol / L, and 18.54 μmol / L, respectively. This indicates that the combined use of 1,4-GL can reduce the IC50 of lenvatinib in Huh7 cells. 50 However, different concentrations of 1,4-GL showed varying effects on the IC50 response of lenvatinib. 50 The impact is not significantly different. Figure 8E~H are cell viability of different concentrations of 1,4-GL combined with Letb in HepG2 cells, Letb group, LLGL group, LMGL group, LHGL group Letb IC 50 are 46.11 μmol / L, 34.50 μmol / L, 39.42 μmol / L, 30.34 μmol / L, respectively. It can be seen that the combination of 1,4-GL can reduce the IC 50 of Letb in HepG2 cells, but different concentrations of 1,4-GL have little effect on the IC 50 of Letb.

[0082] The results show that the combination of drugs significantly reduces the IC 50 value of Letb: Huh7 cells: the IC 50 of single drug group is 32.32 μM, and the combination group (1,4-GL 50 μM) is reduced to 18.54 μM (reduced by 42.6%, P < 0.05), p<0.05 HepG2 cells: the IC 50 of single drug group is 46.11 μM, and the combination group (1,4-GL 50 μM) is reduced to 30.34 μM (reduced by 34.2%, P < 0.05). p<0.05

[0083] (4) Cell scratch: Huh7 cells and HepG2 cells were inoculated in 6-well plates and incubated until they formed a monolayer with 80-85% confluence. A straight wound gap was drawn in the cells with a 200 μL pipette tip. After the gap was generated, it was washed twice with PBS, and then the drug-containing medium of the respective concentration was added. Photographs were taken under a microscope at 0 h, 18 h, 36 h after drug administration, and the migration area was measured.

[0084] The scratch test was divided into 5 groups, namely the Model group without adding drugs, the 1,4-GL group adding only high concentration 1,4-GL, the Letb group adding only Letb, the LHGL group adding Letb and high concentration 1,4-GL, and the DMSO group adding DMSO with the same DMSO content as Letb. Among them, the concentration of Letb was selected as 20 μmol / L (Huh7 cells), 30 μmol / L (HepG2 cells), the concentration of 1,4-GL was 600 μmol / L, and the concentration of DMSO was 0.2% of the medium volume (Huh7 cells), 0.3% of the medium volume (HepG2 cells).

[0085] The results are as follows Figure 8 ​As shown in Figure AC, compared with the Model group, the migration ability of the DMSO group and the 1,4-GL group in Huh7 cells and HepG2 cells did not change significantly. This indicates that the dimethyl sulfoxide in the lenvatinib solvent has little effect on the cells, and 1,4-GL alone has little effect on Huh7 cells and HepG2 cells. The migration ability of the Letb group and the LHGL group in Huh7 cells and HepG2 cells was significantly downregulated (p < 0.05), but there was no significant difference between the Letb group and the LHGL group, indicating that 1,4-GL did not significantly change the migration ability of lenvatinib.

[0086] (5) Cell cloning: 3 x 10 3 Huh7 and HepG2 cells were seeded into 6-well plates with different concentrations of drugs added. After 24 hours, the drugs were removed, and the cells were cultured for another 14 days or until the cell count in most individual clones exceeded 50. The medium was changed and the cell status was observed every 3 days. After cloning, the cells were fixed with 4% paraformaldehyde, stained with crystal violet, washed with PBS, air-dried, and photographed.

[0087] The cell plate cloning assay was divided into five groups: the Model group (no drug), the 1,4-GL group (high concentration of 1,4-GL only), the Letb group (lenvatinib only), the LHGL group (lenvatinib and high concentration of 1,4-GL), and the DMSO group (DMSO content equal to that in lenvatinib). The lenvatinib concentrations were set at 20 μmol / L (Huh7 cells) and 30 μmol / L (HepG2 cells), the 1,4-GL concentration at 600 μmol / L, and the DMSO concentrations at 0.2% of the culture medium volume (Huh7 cells) and 0.3% of the culture medium volume (HepG2 cells).

[0088] like ​ As shown in the middle DF, compared with the Model group, the proliferation ability of the DMSO group and the 1,4-GL group in Huh7 cells and HepG2 cells did not change significantly. This indicates that the dimethyl sulfoxide in the lenvatinib solvent has no significant effect on cells, and 1,4-GL alone has no significant effect on Huh7 cells and HepG2 cells. The proliferation ability of the Letb group and the LHGL group in Huh7 cells and HepG2 cells was significantly downregulated (p < 0.05), but there was no significant difference between the Letb group and the LHGL group, indicating that 1,4-GL has no significant effect on the proliferation ability of lenvatinib.

[0089] Example 4: Pharmacokinetic Study (1) SPF level SD rats (body weight 180-200 g) were used to evaluate the effect of 1,4-GL on the pharmacokinetics of lenvatinib after single administration of lenvatinib or its combination with 1,4-GL.

[0090] The experiment was divided into three groups: Lenvatinib monotherapy group (Letb, letb 20.0 mg / kg + distilled water); Lenvatinib combined with low-dose 1,4-GL group (LLGL, Letb 20 mg / kg) + 20.0 mg / kg 1,4-GL; Lenvatinib combined with high-dose 1,4-GL group (LHGL, Letb 20 mg / kg) + 40.0 mg / kg 1,4-GL.

[0091] Orbital blood was taken at 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 24 h, 36 h, 48 h, and 72 h after administration. Serum was taken after 2 hours of standing and stored at -80°C.

[0092] UPLC-MS / MS was used to determine the blood concentration of lenvatinib, and the pharmacokinetic parameters were calculated. The results showed that: 1) 1,4-GL did not affect the concentration of lenvatinib in H22 mouse tumor, serum, and liver UPLC-MS was used to determine the concentration of lenvatinib in serum, liver, and tumor. The concentrations of lenvatinib in serum in Letb group, LLGL group, LMGL group, and LHGL group were 7.82 ± 4.34 μg / mL, 5.90 ± 2.41 μg / mL, 4.77 ± 1.15 μg / mL, and 3.51 ± 0.13 μg / mL, respectively. The concentrations of lenvatinib in liver in Letb group, LLGL group, LMGL group, and LHGL group were 2.71 ± 0.50 μg / mL, 2.91 ± 0.92 μg / mL, 3.09 ± 1.13 μg / mL, and 2.65 ± 0.76 μg / mL, respectively. The concentrations of lenvatinib in tumor in Letb group, LLGL group, LMGL group, and LHGL group were 0.56 ± 0.30 μg / mL, 0.51 ± 0.17 μg / mL, 0.51 ± 0.04 μg / mL, and 0.43 ± 0.03 μg / mL, respectively. There was no statistical significance in the concentration of lenvatinib in serum, liver, and tumor whether 1,4-GL was used or not. It was preliminarily judged that the effect of 1,4-GL on the anticancer efficacy of lenvatinib was not due to the influence on the pharmacokinetic process in vivo.

[0093] 2) 1,4-GL did not affect the pharmacokinetic parameters of lenvatinib in SD rats The results of the statistical moment model show that the mean residence time (MRT) of the Letb group, LLGL group, and LHGL group is... （0-∞） The mean retardation times (MRT) were 7.39 ± 1.02 h, 8.87 ± 0.36 h, and 7.38 ± 0.88 h, respectively. Compared with the Letb group, the MRT of the LLGL group was... （0-∞） There was an increasing trend, but no significant change was observed in the LHGL group. Furthermore, the area under the curve (AUC) for the Letb, LLGL, and LHGL groups was [not specified]. (0-∞) The concentrations were 186.64 ± 21.11 μg / mL h, 218.12 ± 50.81 μg / mL h, and 173.11 ± 17.78 μg / mL h, respectively; the maximum serum concentration C... max The concentrations were 19.21 ± 3.54 μg / mL, 22.51 ± 6.87 μg / mL, and 19.44 ± 0.47 μg / mL, respectively, indicating that 1,4-GL had no significant effect on the relative oral bioavailability of lenvatinib. The pharmacokinetic parameters of lenvatinib before and after co-administration with high and low concentrations of 1,4-GL are shown in Table 2.

[0094] The results showed no significant difference in pharmacokinetic parameters between the single-drug group and the combination group (p>0.05). Specific data are as follows: AUC (0-∞) (μg·h / mL): Letb group 186.64 ± 21.11, LLGL group 218.12 ± 50.81, LHGL group 173.11 ± 17.78; C max (μg / mL): Letb group: 19.21 ± 3.54, LLGL group: 22.51 ± 6.87, LHGL group: 19.44 ± 0.47; t 1 / 2 (h): 4.35 ± 0.97 in the single-drug group and 3.57 ± 0.80 (LLGL) and 5.54 ± 1.33 (LHGL) in the combination group, respectively; MRT (0-∞) (h): 7.39 ± 1.02 in the single-drug group and 8.87 ± 0.36 (LLGL) and 7.38 ± 0.88 (LHGL) in the combination group, respectively.

[0095] Table 2. Pharmacokinetic parameters of lenvatinib and lenvatinib in combination with 1,4-GL (21 mg / kg, mean ±SD, n = 3-6).

[0096]

[0097] T 1 / 2: half-life; V / F: apparent volume of distribution corrected for extravascular bioavailability; CL / F: clearance corrected for extravascular bioavailability; AUC (0–∞) : area under the drug concentration-time curve from time 0 to infinity; AUMC (0-∞) : area under the first moment curve from time 0 to infinity; MRT (0-∞) : mean residence time from time 0 to infinity; C max : peak plasma concentration; T max : time to peak. * P < 0.05 compared with the Letb group; ** P < 0.01 compared with the Letb group and P < 0.05 compared with the LLGL group.

[0098] (2) Verification of steady-state plasma concentration: Further verification of the effect on pharmacokinetics on SD rats, SD rats were divided into Letb group, Letb combined with low concentration 1,4-GL group (LLGL), Letb combined with high concentration 1,4-GL group (LHGL), grouping and dosing were the same as above pharmacokinetics.

[0099] Letb group was first given 1.0 ml of purified water by gavage, and the combination group was given 1.0 ml of 1,4-GL of their respective concentrations by gavage, 0.5 h later, 2.0 ml of Letb was given to the three groups by gavage, for 6 consecutive days, 1 h after the sixth day of administration, blood and liver were taken, serum was obtained after centrifugation of whole blood after standing for 2 hours, and serum and liver were stored at -80℃.

[0100] Further verification of steady-state concentration by 6 consecutive days of administration showed that the serum steady-state concentration of Letb was 20.38 ± 11.13 μg / mL in the single drug group, 22.36 ± 3.22 μg / mL in the LLGL group, and 22.17 ± 12.64 μg / mL in the LHGL group; the liver drug concentration was 0.92 ± 0.51 μg / g in the single drug group, and 0.50 ± 0.25 μg / g (LLGL) and 0.63 ± 0.02 μg / g (LHGL) in the combination groups, respectively.

[0101] The steady-state experiment further confirmed that 1,4-GL had no effect on the plasma concentration of Letb, and the synergistic antitumor effect was derived from pharmacodynamic mechanism rather than pharmacokinetic interaction.

[0102] In summary: (1) The application found that the tumor was significantly reduced in the H22 tumor-bearing model after the combination of lenvatinib and 1,4-GL, compared with the use of lenvatinib alone, and it was also found that the use of 1,4-GL caused some changes in serum cytokines, and also changed the spleen index and thymus index, indicating that 1,4-GL has a significant effect of enhancing the efficacy of lenvatinib.

[0103] (2) Further pharmacokinetic determination in SD rats found that the steady-state blood drug concentration and pharmacokinetic parameters of lenvatinib after combination with 1,4-GL had no obvious change, which indicated that the enhancement of anti-cancer effect was not due to the change of 1,4-GL in the ADME process of lenvatinib in vivo.

[0104] (3) In the flow cytometry analysis of mouse spleen, it was found that 1,4-GL could change the abundance of CD4 + T cells and CD8 + T cells, and 1,4-GL assisted lenvatinib to improve the killing ability of T cells and enhance the anti-tumor activity.

[0105] (4) In vitro experiments, first, the IC 50 of lenvatinib and the combination of 1,4-GL after lenvatinib was determined in Huh7 and HepG2 liver cancer cells, respectively, and it was found that 1,4-GL could significantly reduce the IC 50 of lenvatinib. But in the scratch and cell cloning experiments, it was found that 1,4-GL had no obvious effect.

[0106] (5) Secondly, the immune-related gene analysis was carried out, and the PD-L1 protein and mRNA in animals and Huh7 and HepG2 cell lines were determined, and the expression was significantly reduced after the combination of 1,4-GL, and it was found that p-STAT3 / STAT3 was significantly reduced, indicating that 1,4-GL enhanced the anti-cancer ability of lenvatinib through STAT3 / P-STAT3 / PD-L1.

[0107] (6) The in vivo verification results showed that the Letb alone group had no regulatory effect on PD-L1 / STAT3 / P-STAT3; in vitro experiments showed that the use of 1,4-GL alone and the use of Letb alone had no obvious regulatory effect on PD-L1 / STAT3 / P-STAT3; combined with in vivo and in vitro experiments, it was found that the combination of the two drugs had obvious synergistic effect, and the synergistic effect was mainly based on the inhibition of P-STAT3-PD-L1 signal pathway. The above-described embodiments are merely illustrative of several embodiments of the present application, which are described in more detail and in a specific and detailed manner, but should not be construed as limiting the scope of the patent. It should be noted that for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims.

Claims

1. A pharmaceutical composition, characterized by, The active ingredient A is D-glucaric acid-1,4-lactone, and the active ingredient B is lenvatinib or a pharmaceutically acceptable salt thereof; and the mass ratio of the active ingredient A to the active ingredient B is 1:0.1-50.

2. The pharmaceutical composition of claim 1, wherein, The mass ratio of the active ingredient A to the active ingredient B is 1:0.1-20.

3. The pharmaceutical composition of claim 1, wherein, The mass ratio of the active ingredient A to the active ingredient B is 1:0.1-10.

4. The pharmaceutical composition of claim 1, wherein, The mass ratio of the active ingredient A to the active ingredient B is 1:0.2-1.

5. Use of the pharmaceutical composition according to any one of claims 1-4 for preparing a STAT3 phosphorylation inhibitor.

6. A kit for use as a STAT3 phosphorylation inhibitor, characterized in that, The pharmaceutical composition comprises: The preparation A comprises the active ingredient A, which is D-glucaric acid-1,4-lactone; The preparation B comprises the active ingredient B, which is lenvatinib or a pharmaceutically acceptable salt thereof.

7. The kit according to claim 6, wherein the preparation A and the preparation B are both oral or injection preparations; and the mass ratio of the active ingredient A to the active ingredient B is 1:0.1-50.

8. Use of a STAT3 phosphorylation inhibitor in the preparation of a drug for treating liver cancer, characterized in that, The STAT3 phosphorylation inhibitor is the pharmaceutical composition according to any one of claims 1-4.

9. Use of D-glucaric acid-1,4-lactone in the preparation of an anti-liver cancer drug potentiator, characterized in that, The anti-hepatoma drug is lenvatinib or a pharmaceutically acceptable salt thereof.

10. An anti-liver cancer drug, characterized by, The pharmaceutical composition comprises a therapeutically effective amount of the pharmaceutical composition according to any one of claims 1-4 and a pharmaceutically acceptable carrier.

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