A pharmaceutical composition for treating hepatocellular carcinoma and use thereof

Abstract

The present application relates to the technical field of biological medicine, and in particular to a pharmaceutical composition for treating hepatocellular carcinoma and application thereof, wherein the pharmaceutical composition comprises metformin or a pharmaceutically acceptable salt thereof and obeticholic acid or a pharmaceutically acceptable salt thereof, and the weight ratio of the metformin to the obeticholic acid is 1:0.05-0.2. It is found for the first time that the combination of metformin and farnesol X receptor agonist can regulate the AMPK / mTOR pathway and the immune recruitment mediated by CXCL16 in a synergistic manner, inhibit the proliferation of tumor cells, repair the recruitment function of NKT cells in the microenvironment of hepatocellular carcinoma, and realize the dual anti-tumor effect of metabolic inhibition and immune enhancement. Animal experiments show that the tumor inhibition rate of the combination drug group is significantly better than that of the single drug group, and the safety is good.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a pharmaceutical composition for treating hepatocellular carcinoma and its application. Background Technology

[0002] Hepatocellular carcinoma (HCC) is a malignant tumor with leading incidence and mortality rates worldwide. Treatment methods mainly include surgical resection, liver transplantation, local ablation, transarterial chemoembolization, and systemic drug therapy. However, the prognosis for patients with advanced HCC remains poor, with a five-year survival rate of less than 18%, necessitating the development of more effective treatment strategies.

[0003] In recent years, tumor metabolic reprogramming and immune microenvironment regulation have become hot topics in HCC treatment research. Metformin, a classic adenosine monophosphate-activated protein kinase (AMPK) activator, has been shown to have anti-HCC potential in preclinical studies and retrospective clinical data analysis by inhibiting the mammalian target of rapamycin (mTOR) pathway and inducing cell cycle arrest and apoptosis. However, recent studies have found that while metformin inhibits tumor cell proliferation, it may also inhibit the expression of the chemokine CXCL16 in hepatic sinusoidal endothelial cells through the AMPK / farnesine X receptor (FXR) signaling axis, thereby weakening the recruitment of natural killer T cells (NKT) to the tumor site and leading to immune microenvironment suppression. This immune side effect may partially offset its direct anti-tumor effect and limit its clinical efficacy.

[0004] On the other hand, farnesoid X receptor agonists such as obeticholic acid (OCA) have been shown to specifically upregulate CXCL16 expression, promoting NKT cell recruitment and activation, and have demonstrated good efficacy in cholestatic liver disease. However, their application in HCC treatment has not been fully explored, especially whether combining them with metformin can reverse the immunosuppression caused by the latter and achieve a synergistic effect of metabolic-immune dual regulation; there are currently no relevant reports. Existing combination therapy regimens mostly focus on the combination of metformin with chemotherapy drugs and targeted drugs, which, while improving efficacy to some extent, still fail to systematically solve the problem of immune microenvironment suppression. Therefore, developing a combination therapy regimen that can simultaneously exert both metabolic regulation and immune repair functions has become an important direction for improving the treatment effect of HCC. Summary of the Invention

[0005] The purpose of this invention is to overcome the immunosuppressive side effects of metformin monotherapy in hepatocellular carcinoma (HCC) in the prior art, and to provide a drug that can reverse these side effects while enhancing the direct anti-tumor effect. Through the synergistic effect of metformin and FXR agonists, such as obeticholic acid, the NKT cell recruitment function in the HCC microenvironment is restored, achieving a dual anti-tumor effect of metabolic inhibition and immune enhancement.

[0006] In a first aspect, the present invention provides a pharmaceutical composition for treating hepatocellular carcinoma, comprising metformin or a pharmaceutically acceptable salt thereof, and obeticholic acid or a pharmaceutically acceptable salt thereof, wherein the weight ratio of metformin to obeticholic acid is 1:0.05-0.2.

[0007] The pharmaceutical composition of the present invention is based on the rational design of metabolic-immune synergistic regulation. Metformin directly acts on tumor cell metabolism, namely the AMPK / mTOR pathway, while farnesol X receptor agonist targets the tumor microenvironment and upregulates CXCL16. The combination of the two can produce a synergistic anti-tumor effect that surpasses that of single drugs.

[0008] Metformin or its pharmaceutically acceptable salts are among the core active ingredients of a pharmaceutical composition. Metformin is a biguanide insulin sensitizer, and its salt forms, such as hydrochloride, can improve solubility or stability.

[0009] Farnesol X receptor agonists or their pharmaceutically acceptable salts are among the core active ingredients of a pharmaceutical composition. They are compounds that can specifically activate FXR nuclear receptors, such as obeticholic acid, and their salt forms are also chosen for pharmaceutical performance considerations.

[0010] Obeticholic acid is a potent and selective FXR agonist approved for clinical use. The combination of metformin and obeticholic acid significantly reduces development risks and improves the feasibility and clinical translation prospects of this invention. It provides a concrete target for subsequent dosage form development, dosage exploration, and efficacy validation, enhancing the clarity and reproducibility of the invention.

[0011] The aforementioned ratio range, derived through in vivo pharmacodynamic experiments in animals, is crucial for achieving synergistic effects. This ratio range aims to ensure that the two drugs reach effective blood concentrations in vivo, maximizing their individual pharmacological effects while maximizing synergistic effects, and avoiding reduced efficacy or increased side effects due to improper ratios.

[0012] When the FXR agonist is obeticholic acid, the preferred weight ratio of metformin to obeticholic acid is 1:0.05-0.2. This ratio is derived from specific animal experimental verification data and is an effective ratio to achieve the dual effects of synergistic tumor suppression and NKT recruitment repair.

[0013] Preferably, the pharmaceutical composition is an oral formulation, including any one of tablets, capsules, granules, powders, and oral liquids. Oral formulations have high patient compliance and are suitable for long-term or adjuvant treatment of chronic diseases such as hepatocellular carcinoma.

[0014] Preferably, the oral formulation further comprises one or more of a pharmaceutically acceptable carrier, diluent, disintegrant, lubricant, binder, and surfactant.

[0015] In a second aspect, the present invention provides the use of the above-described pharmaceutical composition in the preparation of a medicament for treating hepatocellular carcinoma.

[0016] Preferably, the drug achieves a synergistic anti-tumor effect by upregulating the expression of CXCL16 in the hepatocellular carcinoma microenvironment to restore or enhance NKT cell recruitment. Specifically, it repairs the tumor immune microenvironment by reversing the downregulation of the CXCL16 / NKT axis by metformin through an FXR agonist.

[0017] Preferably, the drug is also used to activate the AMPK pathway and inhibit the mTOR pathway, inhibit tumor cell proliferation, and induce one or more of autophagy and apoptosis.

[0018] Preferably, the pharmaceutically acceptable salt of metformin is metformin hydrochloride, and the obeticholic acid or its pharmaceutically acceptable salt includes sodium, potassium, calcium, or magnesium salts.

[0019] In this invention, a pharmaceutically acceptable salt refers to a salt suitable for contact with human and animal tissues without excessive toxicity, irritation, or allergic reactions. For metformin, a pharmaceutically acceptable salt is typically an acid addition salt, such as hydrochloride, sulfate, phosphate, fumarate, lactate, malate, maleate, etc., preferably metformin hydrochloride.

[0020] In a preferred embodiment, the pharmaceutically acceptable salt of the metformin is metformin hydrochloride, and for FXR agonists (such as obeticholic acid), the pharmaceutically acceptable salt can be a base addition salt. The pharmaceutically acceptable salt of the farnesoid X receptor agonist is a sodium salt, potassium salt, calcium salt, or magnesium salt.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0022] 1. This invention provides a pharmaceutical composition for treating hepatocellular carcinoma, which achieves synergistic treatment of hepatocellular carcinoma by combining metformin with a farnesoid X receptor agonist, and its efficacy is superior to that of monotherapy.

[0023] 2. This invention provides a pharmaceutical composition for treating hepatocellular carcinoma, which achieves a dual anti-tumor mechanism of metabolic inhibition and immune repair by simultaneously targeting tumor cell metabolism (AMPK / mTOR pathway) and tumor immune microenvironment (FXR / CXCL16 axis), overcoming the limitations of single-target therapy.

[0024] 3. As shown in the embodiments of the present invention, in a mouse orthotopic liver cancer model, the combined drug group achieved a tumor inhibition rate of 52.3%, significantly superior to the metformin monotherapy group (23.5%) and the obeticholic acid monotherapy group (18.7%). Furthermore, it simultaneously restored the proportion of NKT cells in the tumor microenvironment suppressed by metformin to above normal levels. This fully demonstrates its outstanding synergistic effect and its remarkable ability to repair the immune microenvironment. Attached Figure Description

[0025] Figure 1 The drug administration time is shown in the graph for mice in different experimental groups; Figure 2 These are gross photographs of orthotopic liver cancer specimens from each group of mice at the end of the experiment. Figure 3 A bar chart comparing the average tumor weight of each group based on tumor weight; Figure 4 A tumor growth curve monitored by in vivo imaging or ultrasound in small animals; Figure 5 H&E stained pathological section of tumor tissue; Figure 6 A statistical graph showing the proportion of immune cell infiltration in the tumor microenvironment as analyzed by flow cytometry. Figure 7 Representative electrophoretic images for Western Blot detection of the expression of key signaling pathway proteins such as AMPK, p-AMPK, mTOR, p-mTOR, DEPTOR, Bax, and Bcl-2 in tumor tissues; Figure 8 Box plots showing the concentrations of chemokine CXCL16 protein and cytokine IFN-γ protein in liver tissue as detected by ELISA. Figure 9 Box plots showing the concentrations of chemokine CXCL16 protein and cytokine IFN-γ protein in tumor tissue as detected by ELISA. Figure 10 Box plots showing the concentrations of serum chemokine CXCL16 protein and cytokine IFN-γ protein as detected by ELISA. Figure 11 The bar chart shows the results of measuring the levels of liver function indicators (ALT, AST) and kidney function indicators (CRE) in the serum of mice in each group.

[0026] Figure 12 A volcano plot showing differentially expressed genes between human liver cancer tissues and their paired adjacent normal tissues.

[0027] Figure 13 Volcano plot of differentially expressed genes between the CXCL16 high expression group and the low expression group.

[0028] Figure 14 A heatmap showing the expression of key genes related to anti-tumor immunity in the CXCL16 high / low expression groups.

[0029] Figure 15 This is a graph showing the correlation between CXCL16 and the expression levels of various anti-tumor immune-related genes. Detailed Implementation

[0030] The present invention will now be described in further detail with reference to specific embodiments. However, this should not be construed as limiting the scope of the present invention to the following embodiments; all technologies implemented based on the content of the present invention fall within the scope of the present invention.

[0031] Example 1 To verify the synergistic therapeutic effect of metformin combined with obeticholic acid on a mouse model of orthotopic hepatocellular carcinoma. 1. Experimental Objective To verify the inhibitory effect of the combination of metformin (MET) and obeticholic acid (OCA) on the growth of hepatocellular carcinoma (HCC) and to evaluate its impact on the tumor immune microenvironment.

[0032] 2. Experimental Materials Laboratory animals: 6-8 week old male BALB / c mice, SPF grade, purchased from Chengdu Dashuo Laboratory Animal Co., Ltd. All animal experiments were approved by the Animal Management Committee of the Western Theater Command General Hospital. Animals weighed 18-22 g and were housed in cages of 6, with free access to food and water. All cages were maintained at a constant temperature of 20℃-24℃ and a relative humidity of 50%.

[0033] Cell line: Mouse hepatocellular carcinoma cell line H22, purchased from Matson Cell Biologics. H22 cells were cultured in Roswell Park Memorial Institute-1640 (RPMI-1640) medium containing 1% penicillin-streptomycin and 10% fetal bovine serum (FBS) at 37°C and 5% CO2.

[0034] Drugs and reagents: Metformin hydrochloride (purity ≥97%), obeticholic acid (purity ≥99%), and Tween 20 were all commercially available analytical grade reagents; isoflurane; cell culture related reagents; Flow cytometry antibodies (anti-mouse CD3, CD4, CD8, NK1.1, CD69, etc.); Enzyme-linked immunosorbent assay (ELISA) kit (mouse CXCL16, IFN-γ, CXCL9, CXCL10); Western Blot-related antibodies (p-AMPKα, AMPKα, p-FXR, FXR, p-mTOR, mTOR, etc.); Biochemical assay kit: alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (CRE).

[0035] 3. Experimental Methods 3.1 Construction of an orthotopic model of liver cancer H22 cells in logarithmic growth phase were taken and resuspended in PBS to a concentration of 5 × 10⁻⁶ cells / mL. 7 A cell suspension containing 1 × 10⁶ cells / mL was prepared. Mice were anesthetized with 2% isoflurane, routinely disinfected, and the left lobe of the liver was exposed via a midline abdominal incision. 20 μL of the cell suspension (containing 1 × 10⁶ cells / mL) was injected using a 31G insulin syringe. 6 (One cell) is slowly injected into the liver parenchyma over a period of about 15 seconds. After hemostasis by applying pressure to the puncture site, the layers are sutured one by one.

[0036] Dosing time during model construction process, such as Figure 1 As shown, mice were inoculated with H22 cells on day 0, and then treated with various drugs. Specific grouping and administration protocols are detailed in Section 3.2. H&E staining results are shown below. Figure 5 As shown, from Figure 5 The cancer cells observed varied in size, exhibiting large nucleoli and increased mitotic figures, indicating the successful establishment of the orthotopic liver cancer model. After 19 days of experimentation, representative liver images from four groups of mice were obtained after dissection. Figure 2 As shown.

[0037] On the 7th day after surgery, mice with uniform tumor volume (long diameter of about 2-3 mm) were selected using a small animal ultrasound imaging system for subsequent experiments.

[0038] 3.2 Animal grouping and administration Tumor-forming mice were randomly divided into 4 groups (n=10). Control group: Normal saline was administered by gavage at a volume of 50 mL / kg; Metformin monotherapy group (MET): 250 mg / kg metformin saline solution was administered by gavage at a volume of 10 mL / kg, and freshly prepared weekly.

[0039] Obeticholic acid monotherapy group (OCA): 20 mg / kg obeticholic acid was administered by gavage (obeticholic acid was dissolved in physiological saline containing 0.5% Tween 20 to prepare a suspension of 2 mg / mL, and the administration volume was 10 mL / kg). It was stored at 4°C protected from light and freshly prepared every 3 days.

[0040] The combination therapy group (MET+OCA) was administered by gavage with 250 mg / kg metformin and 20 mg / kg obeticholic acid (the two drugs were mixed in a saline solution containing Tween 20, and the administration volume was 10 mL / kg, so that the concentrations of metformin and obeticholic acid were 25 mg / mL and 2 mg / mL, respectively).

[0041] Starting from the 3rd day after surgery, the medication was administered once every 2 days for a total of 8 times, with an experimental period of 19 days. The patient was weighed before administration to adjust the dosage volume.

[0042] 3.3 Sample Collection and Testing Indicators Twenty-four hours after the last administration, mice were anesthetized, and serum, in situ tumor tissue, and adjacent liver tissue were collected.

[0043] The detection indicators and methods are as follows: Tumor growth evaluation: Completely dissect the tumor, weigh it, and calculate the tumor inhibition rate.

[0044] Tumor inhibition rate (%) = [(average tumor weight of control group - average tumor weight of experimental group) / average tumor weight of control group] ×100%.

[0045] Flow cytometry analysis of tumor immune infiltration: Analysis of NKT cells (CD3+) in tumor tissue + NK1.1 + ) and its activated subgroup (CD69) + The ratio of ) was used to prepare tumor single-cell suspensions, which were then stained and analyzed for CD4. + T cells, CD8 + T cells, NK cells, NKT cells (CD3) + NK1.1 + ) and CD69 + NKT cell ratio.

[0046] Cytokine and chemokine detection: The concentrations of CXCL16 and IFN-γ in tumor tissue homogenate and serum were detected. The concentrations of CXCL16 and IFN-γ in serum, supernatant of adjacent liver tissue homogenate, and supernatant of tumor tissue homogenate were detected by ELISA.

[0047] Western Blot: Detects the expression levels of related signaling pathway proteins in tumor tissues. Western Blot was used to detect the expression of p-AMPKα, AMPKα, p-FXR, FXR, p-mTOR, mTOR, DEPTOR, LC3B, Bax, and Bcl-2 proteins in tumor tissues.

[0048] Biochemical analysis: The levels of ALT, AST, and CRE in serum were detected using a fully automated biochemical analyzer to assess liver and kidney function.

[0049] 3.4 Statistical Analysis Data are expressed as mean ± standard deviation (Mean ± SD). One-way ANOVA was performed using SPSS 20.0 software. For pairwise comparisons between groups, the LSD test was used if the variances were homogeneous, and Tamhane's T2 test was used if the variances were unequal. A p-value < 0.05 was considered statistically significant.

[0050] 4. Experimental Results 4.1 Combined medication significantly inhibits tumor growth like Figure 3 As shown in Table 1, the combination therapy group (MET+OCA) showed the most significant inhibition of tumor growth. The tumor weight at the experimental endpoint was significantly lower than that of the control group and the two single-drug groups (P<0.01), with a tumor inhibition rate of 52.3%, significantly higher than that of the two single-drug groups (23.5% and 18.7%), suggesting a synergistic effect. The calculated tumor inhibition rates are shown in Table 1.

[0051] Table 1 summarizes the tumor weight and tumor inhibition rate of mice in each group.

[0052] like Figure 4 As shown, the dynamic changes in tumor volume of mice in each group were monitored by in vivo imaging or ultrasound in small animals. The results showed that from the 7th day of administration, the tumor growth rate of the combined drug group (MET+OCA) began to be lower than that of each single drug group. By the end of the experiment (day 19), the tumor volume of the combined drug group was significantly smaller than that of the control group, the MET group, and the OCA group (P<0.01), indicating that the combined drug can continuously and stably inhibit tumor growth.

[0053] The results of hematoxylin-eosin (H&E) staining pathological sections of tumor tissue are as follows: Figure 5As shown in the figure, the tumor tissue in the control group exhibited typical pathological features of hepatocellular carcinoma: cancer cells of varying sizes, large and deeply stained nuclei, prominent nucleoli, and readily visible pathological mitotic figures. The tumor cells were arranged in a trabecular or pseudoglandular pattern, with significant invasive growth. The tumor tissues in the MET monotherapy group and the OCA monotherapy group still showed the above malignant characteristics, but to a lesser degree. In contrast, the tumor tissue in the combination therapy group showed a significantly reduced tumor cell density, with large areas of necrosis and apoptotic cells. The remaining tumor cells exhibited degenerative changes, with frequent nuclear pyknosis and fragmentation, indicating that the combination therapy effectively induced tumor tissue necrosis and regression.

[0054] 4.2 Combined drug therapy to repair the tumor microenvironment and recruit NKT cells like Figure 6 As shown in the flow cytometry results, compared with the control group, MET monotherapy significantly reduced the proportion of NKT cells infiltrating the tumor (P<0.05), while OCA monotherapy showed an increasing trend. Importantly, the combination therapy completely reversed the MET-induced NKT cell reduction, restoring its proportion to levels exceeding those of the control group. Simultaneously, it activated NKT cells (CD69... + The proportion of ) was also significantly increased in the combined group (P<0.05).

[0055] ELISA results as follows Figure 8-10 The results showed that MET monotherapy reduced CXCL16 levels in tumors, liver, and serum, while OCA monotherapy and combination therapy significantly upregulated CXCL16. Intratumoral IFN-γ levels in the combination therapy group were also significantly higher than in any of the monotherapy groups.

[0056] 4.3 Synergistic Regulation of Key Signaling Pathways by Combined Drug Use Western Blot results are as follows Figure 7 As shown, MET activates AMPK and inhibits mTOR: the p-AMPKα / AMPKα ratio was significantly increased and the p-mTOR / mTOR ratio was decreased in both the MET group and the combination group.

[0057] OCA activates FXR: The p-FXR / FXR ratio increased in both the OCA group and the combined group.

[0058] Synergistic effect of combined drug therapy: The combined drug therapy activated AMPK while maintaining the activated state of FXR. The ratio of downstream autophagy marker protein LC3-II / I was significantly increased, and the ratio of pro-apoptotic / anti-apoptotic proteins (Bax / Bcl-2) was also significantly increased, indicating that the combined drug therapy enhanced both autophagy and apoptosis.

[0059] 4.4 The combination therapy showed good safety. like Figure 11As shown, serum biochemical index detection indicated that there were no statistically significant differences in ALT, AST, and CRE levels between the mice in each treatment group and the control group (P>0.05), indicating that the combined drug administration did not cause significant damage to liver and kidney function at the stated dose and course of treatment, and the safety was good.

[0060] 5. Experimental Conclusions This embodiment demonstrates that the combined use of metformin (250 mg / kg) and obeticholic acid (20 mg / kg) produces a significant synergistic anti-hepatocellular carcinoma effect. The mechanism is as follows: metformin directly inhibits tumor cell metabolism (activating AMPK / inhibiting mTOR), while obeticholic acid upregulates CXCL16 by activating FXR, repairing the tumor intratumoral recruitment function of NKT cells suppressed by metformin, thereby reshaping the immune microenvironment and achieving a dual attack of metabolic inhibition and immune enhancement. This combined regimen has good safety at effective doses.

[0061] Example 2 Validation of the composition in human hepatocellular carcinoma tissue samples and bioinformatics To extend the findings of this invention from animal models to human diseases and to provide more robust evidence for clinical applications, we further validated them in human liver cancer tissue samples and the Totally Clinical and Genetic Database (TCGA).

[0062] 1. Validation of human liver cancer tissue samples Immunofluorescence staining was performed on a tissue microarray containing 60 pairs of human HCCs and paired adjacent normal tissues to detect LYVE1. + Expression level of CXCL16 on hepatic sinusoidal endothelial cells (LSECs).

[0063] Results: Compared with patients who did not take metformin, the expression level of CXCL16 on LSECs in the tumor tissue of liver cancer patients who took metformin long-term was significantly downregulated. This directly confirms the inhibitory effect of metformin on the key chemokine CXCL16 in human pathological samples, providing clinical evidence for the "immune side effects" problem that this invention aims to address.

[0064] 2. Bioinformatics Analysis To validate the role and clinical significance of CXCL16 in anti-tumor immunity at the systemic level, a bioinformatics analysis was performed based on the hepatocellular carcinoma (TCGA-LIHC) cohort.

[0065] (1) Differentially expressed gene analysis First, a differential gene expression profile analysis was performed on hepatocellular carcinoma tissues and paired adjacent normal tissues in the TCGA-LIHC cohort. For example... Figure 12As shown, the volcano plot illustrates the distribution of differentially expressed genes in hepatocellular carcinoma (HCC) tissues relative to adjacent normal tissues. The results showed that CXCL16 was significantly downregulated in HCC tissues (log2FC < -1, P < 0.05), and several immune-related genes, such as CD8A, GZMB, and PRF1, also exhibited a downregulated trend, suggesting the existence of an immunosuppressive microenvironment in HCC tissues.

[0066] Further, using the median CXCL16 expression level as the boundary, liver cancer samples were divided into a CXCL16 high-expression group and a low-expression group, and differentially expressed gene analysis was performed between the two groups. Figure 13 As shown, a large number of differentially expressed genes exist between the two groups. Gene set enrichment analysis revealed that the significantly enriched gene sets in the CXCL16 high-expression group mainly involve immune response, T cell activation, and cytokine signaling pathways, while the CXCL16 low-expression group is enriched in tumor proliferation-related pathways such as cell cycle and DNA replication. This indicates that the expression status of CXCL16 is closely related to the immune microenvironment characteristics of liver cancer.

[0067] (2) Expression characteristics of anti-tumor immune genes Figure 14 A heatmap of key anti-tumor immune genes expression in the CXCL16 high / low expression groups is presented. Each column in the figure represents a liver cancer sample, and each row represents an anti-tumor immune-related gene. The results show that the CXCL16 high expression group samples appear as a distinct red high expression area in the figure, while the low expression group is mainly represented by green low expression. Specifically, the following three types of genes are significantly highly expressed in the CXCL16 high expression group: T cell receptors and co-stimulatory molecules: CD3γ, CD3δ, CD3ε, CD28, ICOS, 4-1BB Cytotoxic effector molecules: perforin (PRF1), granzyme A (GZMA), granzyme B (GZMB) Death receptor ligands: FASLG, TNFSF10 These results confirm at the molecular level that high expression of CXCL16 is closely related to intact anti-tumor immune effector function.

[0068] (3) Correlation analysis between CXCL16 and anti-tumor immune genes like Figure 15 As shown, we further performed a correlation analysis between CXCL16 and various anti-tumor immune-related genes. The results showed: CXCL16 showed a significant positive correlation with NKT cell-specific receptor CXCR6 (Pearson correlation coefficient > 0.6, P < 0.001), which is highly consistent with the mechanism of CXCL16-CXCR6 axis-mediated NKT cell recruitment observed in animal experiments of this invention.

[0069] CXCL16 showed a significant positive correlation with CD69, a marker of T cell activation, suggesting that high expression of CXCL16 is closely related to the activation status of T cells / NKT cells.

[0070] CXCL16 showed a significant positive correlation with key cytokines IFNG (interferon-γ) and IL12, indicating that tumor microenvironments with high CXCL16 expression have a stronger anti-tumor immune response.

[0071] Furthermore, high expression of CXCL16 is significantly positively correlated with the expression levels of several key anti-tumor immune genes, including: T cell activation and receptor-related genes (CD3γ, CD3δ, CD3ε, CD28, ICOS, 4-1BB), NKT cell-specific receptor (CXCR6), cytotoxic effector molecules (PRF1, GZMA, GZMB), and key cytokine signaling pathways (IFNG, IL12).

[0072] Gene set enrichment analysis (GSEA) further confirmed that in hepatocellular carcinoma samples with high CXCL16 expression, there was significant enrichment of genes related to the T cell receptor signaling pathway, cytotoxic T cell-related genes, interferon-γ response pathway, and NKT cell activation.

[0073] Prognostic analysis showed that patients with high CXCL16 expression and complete anti-tumor immune characteristics had significantly better overall survival than patients with low CXCL16 expression (P<0.05).

[0074] The above bioinformatics analysis results validate the scientific validity of the core mechanism of this invention from a systems biology perspective: CXCL16 exerts its immune surveillance function by recruiting and activating NKT cells and other anti-tumor immune cells, and its high expression is closely related to the good prognosis of liver cancer patients. This embodiment demonstrates, from the perspective of human samples and macro-population data, that the high expression of CXCL16 is closely related to intact anti-tumor immune gene characteristics and good patient prognosis. This provides strong clinical translational support for explaining the mechanism by which obeticholic acid in the composition of this invention exerts a synergistic anti-tumor effect by upregulating CXCL16 and repairing the immune microenvironment.

[0075] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A pharmaceutical composition for treating hepatocellular carcinoma, characterized in that, It comprises metformin or a pharmaceutically acceptable salt thereof, and obeticholic acid or a pharmaceutically acceptable salt thereof, wherein the weight ratio of metformin to obeticholic acid is 1:0.05-0.

2.

2. The pharmaceutical composition according to claim 1, characterized in that, The pharmaceutical composition is an oral preparation, including any one of tablets, capsules, granules, powders, and oral liquids.

3. The pharmaceutical composition according to claim 2, characterized in that, The oral formulation may also contain one or more of the following pharmaceutically acceptable carriers, diluents, disintegrants, lubricants, binders, and surfactants.

4. Use of the pharmaceutical composition according to any one of claims 1-3 in the preparation of a medicament for treating hepatocellular carcinoma.

5. The application according to claim 4, characterized in that, The drug achieves a synergistic anti-tumor effect by upregulating the expression of CXCL16 in the hepatocellular carcinoma microenvironment to restore or enhance the recruitment of NKT cells.

6. The application according to claim 4, characterized in that, The drug is also used to activate the AMPK pathway and inhibit the mTOR pathway, inhibit tumor cell proliferation, and induce one or more of autophagy and apoptosis.

7. The application according to claim 4, characterized in that, The pharmaceutically acceptable salt of metformin is metformin hydrochloride, and the obeticholic acid or its pharmaceutically acceptable salts include sodium, potassium, calcium, or magnesium salts.

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