Application of secondary bile acid in preparation of antitumor drug and antitumor drug
By using secondary bile acids to activate macrophages and combined with immune checkpoint inhibitors, the problem of poor effect of hepatocellular carcinoma on existing immunotherapy is solved, significantly inhibiting tumor growth and migration, improving the immune microenvironment, and enhancing anti-tumor Effectiveness.
Patent Information
- Application Number
- CN202411358139.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-26
- Publication Date
- 2024-12-13
AI Technical Summary
Hepatocellular carcinoma (HCC) has low efficacy on existing immune checkpoint inhibitors, and tumor cells avoid immune surveillance through the immunosuppressive microenvironment, resulting in poor immunotherapy effects. New therapeutic strategies need to be explored to improve tumor immune microenvironment and Enhance the efficacy.
Secondary bile acids such as glycolite, taurite, and glycoldeoxycholic acid are used as antitumor drugs, and immunochemosolite, combined with immune checkpoint inhibitors by activating macrophage-mediated antitumor immunity effects , such as anti-PD-1 antibodies, synergistically anti-tumor.
Secondary bile acids can inhibit tumor cell growth, reduce migration ability, promote apoptosis, and significantly enhance the anti-tumor effect of immune checkpoint inhibitors by improving the immune microenvironment, significantly inhibit the growth of liver cancer tumors, and prolong survival time.
Smart Images
Figure CN119112910A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biomedicine technology, and in particular to the application of secondary bile acid in the preparation of anti-tumor drugs and the anti-tumor drugs. Background Art
[0002] Hepatocellular carcinoma (HCC) is a malignant tumor with chronic liver damage as the background. It has no obvious symptoms in the early stage, but it is highly malignant and progresses rapidly. It is the leading cause of cancer-related deaths worldwide. The surgical treatment, postoperative adjuvant therapy and local treatment (mainly including ablation, hepatic artery intervention and local radiotherapy) recommended by the latest version of the 2023 NCCN (National Comprehensive Cancer Network) guidelines for liver cancer can only benefit a small number of liver cancer patients, namely those with early liver cancer. Most HCC patients are already in the middle and late stages when diagnosed. For patients with advanced liver cancer, effective systemic treatment can reduce tumor burden and prolong survival time.
[0003] New immunotherapies, represented by blocking immune checkpoints, have been shown to show significant efficacy in some tumor patients and have made significant progress in clinical applications. Immune checkpoint inhibitors (ICBs) improve the body's anti-tumor immunity by blocking natural immune regulatory factors such as programmed cell death 1 antigen (PD-1). Although immune checkpoint blockade has achieved encouraging results in HCC patients, the remission rate is below 20%. Currently, the 5-year survival rate of HCC is still less than 16%. In addition, there are significant differences in the efficacy of immune checkpoint inhibitors among different individuals, and some patients show drug resistance. The reason may be that tumor cells have evolved to acquire multiple mechanisms to escape immune surveillance, forming an immunosuppressive microenvironment, resulting in the loss of anti-tumor immune response of immune cells. The failure of immune surveillance is one of the key features in the occurrence and evolution of HCC, inducing tumor proliferation and recurrence, and affecting the efficacy of immunotherapy. Therefore, it is urgent to explore therapeutic strategies that can effectively improve the tumor immune microenvironment, promote the response of tumor patients to immune checkpoint inhibitors, and increase the efficacy.
[0004] Bile acids are endogenous metabolites produced by the liver and are an important component of bile. They are synthesized by oxidation of cholesterol in the liver and form up to fifty different bile acids through enterohepatic circulation and under the joint action of the host and intestinal microorganisms. Bile acids are divided into primary bile acids and secondary bile acids. Primary bile acids are defined as bile acids synthesized directly by liver enzymes in hepatocytes, while secondary bile acids are formed by primary bile acids under intestinal microbial metabolism.
[0005] Lithocholic acid (LCA) and deoxycholic acid (DCA) and their combined forms, glycolithocholic acid (GLCA), taurolithocholic acid (TLCA), glycodeoxycholic acid (GDCA) and taurodeoxycholic acid (TDCA), are the most important secondary bile acids in the human body. Among them, LCA is formed by 7α-dehydroxylation of the primary bile acid chenodeoxycholic acid (CDCA), and DCA is formed by 7α-dehydroxylation of the primary bile acid cholic acid (CA). LCA and DCA are further combined with glycine (G) or taurine (T) in the form of N-acylamidation to form GLCA, TLCA, GDCA, and TDCA.
[0006] The biological activity of bile acid molecules is closely related to their chemical properties. The number and direction of hydroxyl groups or whether they are conjugated directly affect hydrophilicity, receptor activation efficacy and transporter affinity, thereby affecting their metabolism and biological functions such as immune regulation, anti-aging, and tumor suppression. The molecular structure of LCA lacks a hydroxyl group at the C-7 position and is not conjugated with taurine or amino acids, which makes it the most hydrophobic bile acid, limiting its ability to emulsify and promote fat absorption to a certain extent. LCA is an effective activator of human PXR, while DCA cannot effectively activate PXR. LCA and GLCA are effective activators of VDR, but TLCA, DCA, and TDCA do not have this effect. At the same time, different bile acids have different affinities for transporters. Unconjugated bile acids such as LCA and DCA are mainly transported by OATPs (organic anion transporting polypeptides), while conjugated bile acids such as GLCA, TLCA, GDCA, and TDCA are mainly transported by NTCP (sodium / taurocholic acid co-transporting polypeptide). Bile acids may exhibit different biological functions through different effects on these intracellular proteins. LCA reduces intracellular glutathione through TGR5 signaling, thereby inhibiting dendritic cell activation, while TLCA has no such effect. Compared with DCA, LCA can significantly extend the lifespan of chronologically aged yeast through the cAMP / PKA pathway. However, the role of some bile acids in liver cancer remains unclear, and whether they can combine with immune checkpoint inhibitors to exert synergistic anti-tumor effects remains to be studied. Summary of the invention
[0007] To solve the above problems, in a first aspect, the present invention provides the use of secondary bile acid in the preparation of anti-tumor drugs, which have the effects of inhibiting tumor cell growth and migration and promoting tumor cell apoptosis, and can be used in the treatment of tumors.
[0008] The technical solution of the present invention to solve the above problems is as follows:
[0009] The invention provides application of secondary bile acid in preparing anti-tumor medicine.
[0010] The present invention provides application of secondary bile acid in preparing medicine for treating hepatocellular carcinoma.
[0011] The invention provides application of glycolithocholic acid, taurolithocholic acid and taurodeoxycholic acid in preparing medicine for treating hepatocellular carcinoma.
[0012] Preferably, the drug is taurolithocholic acid, glycolithocholic acid, or taurodeoxycholic acid alone.
[0013] Preferably, the drug is an oral drug.
[0014] The present invention provides the use of taurolithocholic acid / glycolithocholic acid in preparing a drug for inhibiting the growth of liver cancer cells, reducing the migration ability of liver cancer cells and promoting the apoptosis of liver cancer cells.
[0015] The present invention provides application of glycodeoxycholic acid / taurodeoxycholic acid in preparing a drug for improving tumor immune microenvironment.
[0016] The invention provides application of taurodeoxycholic acid in preparing medicine for inhibiting tumor growth.
[0017] The present invention provides the use of glycodeoxycholic acid / taurodeoxycholic acid combined with an immune checkpoint inhibitor in the preparation of an anti-tumor drug.
[0018] Preferably, the drug activates macrophage-mediated anti-tumor immunity to cooperate with immune checkpoint inhibitors for synergistic anti-tumor effects.
[0019] Preferably, the immune checkpoint inhibitor is selected from one or more of anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, anti-LAG-3 antibody, anti-TIGIT antibody, anti-TIM-3 antibody, anti-CD47 or anti-VISTA antibody.
[0020] Preferably, the tumor includes hepatocellular carcinoma, gastric cancer, colorectal cancer, cervical cancer, ovarian cancer, breast cancer, lung cancer, esophageal cancer, testicular cancer, bone cancer, pancreatic cancer, thyroid cancer, and melanoma.
[0021] Preferably, the glycodeoxycholic acid / taurodeoxycholic acid is used as a potentiator of immune checkpoint inhibitors.
[0022] In a second aspect, the present invention further provides an anti-tumor drug, which comprises an immune checkpoint inhibitor and the above-mentioned glycodeoxycholic acid / taurodeoxycholic acid.
[0023] Preferably, the immune checkpoint inhibitor is selected from one or more of anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, anti-LAG-3 antibody, anti-TIGIT antibody, anti-TIM-3 antibody, anti-CD47 or anti-VISTA antibody.
[0024] The present invention has the following beneficial effects:
[0025] The invention provides the use of secondary bile acids in the preparation of anti-tumor drugs; the secondary bile acids include taurolithocholic acid, glycolithocholic acid, glycodeoxycholic acid and taurodeoxycholic acid. Among them, taurolithocholic acid and glycolithocholic acid can inhibit the growth of tumor cells, reduce the migration ability of tumor cells, and promote the apoptosis of tumor cells. Glycodeoxycholic acid and taurodeoxycholic acid can effectively promote the infiltration of M1 macrophages in the immune microenvironment of tumor tissue, so that tumor cells are recognized and cleared by M1 macrophages and the tumor is inhibited. Based on the immunoregulatory effect of glycodeoxycholic acid and taurodeoxycholic acid on the tumor microenvironment, the present invention also provides a combined anti-tumor scheme of glycodeoxycholic acid / taurodeoxycholic acid and immune checkpoint inhibitors. Compared with the use of immune checkpoint inhibitors alone, the combined treatment scheme of glycodeoxycholic acid / taurodeoxycholic acid and immune checkpoint inhibitors can significantly inhibit the growth of liver cancer tumors, has the effect of promoting anti-tumor immunity and thus synergistically inhibiting tumor growth, and can be applied to the treatment of tumors. Therefore, combining glycodeoxycholic acid / taurodeoxycholic acid with immune checkpoint inhibitors such as PD-1 monoclonal antibody has considerable clinical significance and economic value. BRIEF DESCRIPTION OF THE DRAWINGS
[0026] Figure 1 1 is a graph showing the growth inhibition effect of taurocholic acid (TCA), taurochenodeoxycholic acid (TCDCA) and tauroursodeoxycholic acid (TUDCA) on liver cancer cell lines in Example 1 of the present invention; wherein, Figure 1 A is a graph showing the test results of the growth inhibition of taurocholic acid on Huh7 human liver cancer cell line; Figure 1 B is a graph showing the test results of the growth inhibition of taurochenodeoxycholic acid on Huh7 human liver cancer cell line; Figure 1 C is a graph showing the test results of the growth inhibition of tauroursodeoxycholic acid on Huh7 human liver cancer cell line;
[0027] Figure 2 1 is a graph showing the test results of the growth inhibition of lithocholic acid, taurolithocholic acid and glycolithocholic acid on normal liver cell lines and liver cancer cell lines in Example 2 of the present invention; wherein, Figure 2 A is the test result of the inhibitory effect of lithocholic acid, taurolithocholic acid and glycolithocholic acid on the growth of LO2 human normal liver cell line; Figure 2 B is the test result of the growth inhibition of SUN449 human liver cancer cell line by lithocholic acid, taurolithocholic acid and glycolithocholic acid; Figure 2 C is a graph showing the test results of the growth inhibition of Huh7 human liver cancer cell line by lithocholic acid, taurolithocholic acid and glycolithocholic acid; Figure 2D is the test result of the growth inhibition of Hep3B human liver cancer cell line by lithocholic acid, taurolithocholic acid and glycolithocholic acid;
[0028] Figure 3 3 is a graph showing the test results of the inhibitory effects of lithocholic acid, taurolithocholic acid and glycolithocholic acid on the migration of liver cancer cells in Example 3 of the present invention; wherein, Figure 3 A is the test result of the inhibitory effect of lithocholic acid on the migration of SUN449 and Hep3B human liver cancer cell lines; Figure 3 B is the test result of the inhibitory effect of taurolithocholic acid on the migration of SUN449 and Hep3B human liver cancer cell lines; Figure 3 C is a graph showing the test results of the inhibitory effect of glycolithocholic acid on the migration of SUN449 and Hep3B human liver cancer cell lines;
[0029] Figure 4 4 is a graph showing the test results of the effect of lithocholic acid, taurolithocholic acid and glycolithocholic acid on promoting apoptosis of liver cancer cell lines in Example 4 of the present invention; wherein, Figure 4 A is the test result of the effect of lithocholic acid on promoting apoptosis of SUN449 and Hep3B human liver cancer cell lines; Figure 4 B is the test result of the effect of glycolithocholic acid on the apoptosis promotion of SUN449 and Hep3B human liver cancer cell lines; Figure 4 C is the test result of the effect of taurolithocholic acid on the apoptosis promotion of SUN449 and Hep3B human liver cancer cell lines;
[0030] Figure 5 This is a graph showing the test results of glycodeoxycholic acid (GDCA) promoting macrophage M1 polarization in vitro in Example 5 of the present invention; wherein, Figure 5 A is THP1 (monocyte), (PMA-induced differentiation of non-polarized macrophages) and after GDCA treatment morphological changes; Figure 5 B: QT-PCR was used to detect the changes in mRNA expression of CD14 and CD68 in THP1 after PMA differentiation; Figure 5 C is the expression changes of CD68 in THP1 after PMA differentiation detected by flow cytometry; Figure 5 D is flow cytometry detection of GDCA-free and GDCA-treated cells Expression of CD86, CD80 and HLAⅡ on the cell surface; Figure 5 E is the detection of GDCA-free / GDCA-treated cells by QT-PCR mRNA expression levels of cell M1 macrophage markers;
[0031] Figure 6This is a test result diagram of glycodeoxycholic acid (GDCA) inhibiting liver cancer cell proliferation and migration by promoting macrophage M1 polarization in Example 6 of the present invention; wherein, Figure 6 A is a schematic diagram of the preparation of conditioned medium; Figure 6 B is a graph showing that the conditioned medium of macrophages treated with different concentrations of GDCA inhibited the proliferation of Hep3B, HepG2, and Huh7 liver cancer cells; Figure 6 C is a graph showing that the conditioned medium of macrophages treated with GDCA inhibited the migration of Hep3B, HepG2, and Huh7 liver cancer cells.
[0032] Figure 7 This is a diagram showing the effects of glycodeoxycholic acid (GDCA) and taurodeoxycholic acid (TDCA) alone and their combination with PD-1 monoclonal antibody on the development of liver cancer in chemically induced liver cancer mice in Example 7 of the present invention; wherein, Figure 7 A is a schematic diagram of the experimental scheme. Figure 7 B is the picture of mouse liver in each experimental group. Figure 7 C is the weight of mice in each experimental group. Figure 7 D is the number of tumors in each experimental group, Figure 7 E is the maximum tumor size of each experimental group, Figure 7 F is the tumor burden of each experimental group;
[0033] Figure 8 This is a graph showing the effect of glycodeoxycholic acid (GDCA) and taurodeoxycholic acid (TDCA) alone and their combination with PD-1 monoclonal antibody on M1 macrophage infiltration in the tumor microenvironment of liver cancer in Example 8 of the present invention; wherein, Figure 8 A is the gating strategy for mouse liver flow cytometry; Figure 8 B is the flow cytometry results of the proportion of M1 macrophages and M2 macrophages in the tumor immune microenvironment of mice after treatment with different drug groups; Figure 8 C is the flow cytometry results of the proportion of M1 macrophages and M2 macrophages in the resident Kupffer cells (KCs) in the mouse liver after treatment with different drug groups; Figure 8 D is the flow cytometry results of the proportion of M1 macrophages and M2 macrophages in monocyte-derived macrophages (MoMFs) in mouse liver after treatment with different drug groups. DETAILED DESCRIPTION
[0034] In order to make the purpose, technical scheme and advantages of the present invention more clear, the present invention is further described in detail in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0035] Example 1: Test on the growth inhibition effect of taurocholic acid, taurochenodeoxycholic acid and tauroursodeoxycholic acid on liver cancer cell lines
[0036] Experimental methods: Huh7 human liver cancer cells were cultured to a density of about 90% by CCK8 method, and the cells were counted. They were inoculated in 96-well culture plates according to the experimental groups, with 3000 cells per well, and 3 replicates per group. After inoculation, they were cultured in a 37°C, 5% CO2 incubator for 24 hours. Taurocholic acid, taurochenodeoxycholic acid and tauroursodeoxycholic acid were added to the drug administration groups at final concentrations of 0, 3.9, 7.8, 15.6, 31.25, 62.5, 125, 250, and 500 μmol / L, respectively. After 120 hours of culture, 10 μL of CCK8 solution was added, and the cells were incubated in a 37°C incubator for 4 hours. The OD value at 450 nm was measured on an ELISA reader. Data analysis was performed, and cell viability = (OD value of the experimental group - OD value of the blank group) / (OD value of the control group - OD value of the blank group) × 100%. SPSS15.0 statistical software was used for statistical analysis, and the means between the two groups were compared using t-test.
[0037] Experimental results: The results are as follows Figure 1 As shown, taurocholic acid, taurochenodeoxycholic acid and tauroursodeoxycholic acid had no inhibitory effect on the growth of human hepatoma cell line Huh7.
[0038] Example 2: Test of the growth inhibition effect of lithocholic acid, taurolithocholic acid and glycolithocholic acid on liver cancer cell lines
[0039] Experimental methods: LO2 normal liver cells and three human liver cancer cells, SUN449, Hep3B, and Huh7, were cultured to a density of about 90% by CCK8 method, and the cells were counted and inoculated in 96-well culture plates according to the experimental groups, with 3000 cells per well, and 3 replicates per group. After inoculation, they were placed in a 37°C, 5% CO2 incubator for 24 hours. Lithocholic acid, taurolithocholic acid, or glycolithocholic acid at a final concentration of 0, 0.8, 1.6, 3.1, 6.3, 12.5, 25, 50, and 100 μmol / L were added to the drug administration groups, respectively. After 120 hours of culture, 10 μL of CCK8 solution was added, and the cells were placed in a 37°C incubator for 4 hours. The OD value at 450 nm was measured on an ELISA instrument. Data analysis was performed, cell viability = (OD value of experimental group - OD value of blank group) / (OD value of control group - OD value of blank group) × 100%. Statistical analysis was performed using SPSS 15.0 statistical software, and the mean comparison between the two groups was performed using t-test.
[0040] Experimental results: The results are as follows Figure 2 As shown in the figure, lithocholic acid, taurolithocholic acid and glycolithocholic acid all inhibited the growth of human liver cancer cell lines SUN449, Hep3B and Huh7, and the inhibitory effect increased with the increase of lithocholic acid, taurolithocholic acid and glycolithocholic acid concentration. At the same time, it had no toxic effect on normal human liver cells LO2.
[0041] Example 3: Test of the inhibitory effect of lithocholic acid, taurolithocholic acid and glycolithocholic acid on the migration ability of liver cancer cell lines
[0042] Experimental method: SUN449 and Hep3B cells were cultured in DMEM high-glucose medium (supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin) and placed in a cell culture incubator at 37°C and 5% CO2. When the cells grew to 70% to 80%, they were digested with trypsin containing EDTA. SUN449 and Hep3B human liver cancer cells in the logarithmic growth phase were inoculated into 6-well plates, and 1×10 4 cells / well, 2mL culture medium / well. Incubate in a cell thermostat at 37°C and 5% CO2 for 24h. The cell monolayer covers the bottom wall, and a 20μL pipette tip is used to evenly draw lines on the bottom of the culture dish. Wash the cells 3 times with PBS, treat the experimental group cells with serum-free medium containing 50μM lithocholic acid, taurolithocholic acid, and glycolithocholic acid, and treat the control group cells with serum-free medium containing DMSO. Use an inverted microscope to take samples and take pictures at 0 and 72h.
[0043] Experimental results: The results are as follows Figure 3 As shown in the figure, it can be seen that lithocholic acid, taurolithocholic acid and glycolithocholic acid all have inhibitory effects on the migration of liver cancer cell lines SUN449 and Hep3B.
[0044] Example 4: Test on the induction effect of lithocholic acid, taurolithocholic acid and glycolithocholic acid on apoptosis of liver cancer cell lines
[0045] Experimental method: SUN449 and Hep3B cells were cultured in DMEM high-glucose medium (supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin) and placed in a cell culture incubator at 37°C and 5% CO2. When the cells grew to 70% to 80%, they were digested with trypsin containing EDTA. SUN449 and Hep3B human liver cancer cells in the logarithmic growth phase were inoculated into 6-well plates, and 1×10 4 cells / well, 2mL culture medium / well. The final concentration of lithocholic acid treatment was 50μM, the final concentration of taurolithocholic acid and glycolithocholic acid treatment was 100μM, and the control group was treated with the same amount of DMSO solvent. After incubation in a cell constant temperature incubator at 37°C and 5% CO2 for 120h, the cells were digested and centrifuged with EDTA-free trypsin, washed twice with PBS, and collected to prepare a single cell suspension. SUN449 and Hep3B cells were stained with PI and AnnexinV (Yishen, China) and analyzed by flow cytometry to investigate the effects of lithocholic acid, taurolithocholic acid and glycolithocholic acid on the apoptosis of liver cancer cells.
[0046] Experimental results: The results are as follows Figure 4 As shown in the figure, the apoptosis ratio of liver cancer cells increased after treatment with lithocholic acid, taurolithocholic acid and glycolithocholic acid. Lithocholic acid, taurolithocholic acid and glycolithocholic acid induced apoptosis and necrosis of liver cancer cells, and the apoptosis ratio of different liver cancer cells was different.
[0047] Example 5: Glycodeoxycholic acid (GDCA) promotes macrophage M1 polarization test in vitro
[0048] Experimental methods: After incubating THP-1 cells with 160nmol / L PMA in a six-well plate for 48h, THP-1 cells were induced into adherent M0 macrophages. After adding 500μM GDCA or DMSO solvent for 72h, trypsin digestion and centrifugation were performed, RNA was extracted after washing with PBS, and reverse transcription was performed. The mRNA expression of CD86, TNFα, IL1β and COX2 was detected using the PikoReal Real-Time PCR System (Thermo, Waltham, MA). After washing, the single cell suspension was blocked with Fc receptor blocker for 10min, flow antibody staining was performed for 30min, and the expression of CD86, CD80 and HLAII proteins on the cell surface was detected using the Cytoflex instrument (Beckman, USA).
[0049] Experimental results: The results are as follows Figure 5 As shown, Figure 5 A shows the human monocytic cell line THP-1 differentiated into unpolarized macrophages by PMA, which showed a morphological change from small, round cells in suspension to larger, irregular cells attached to the wall. Figure 5 B and Figure 5 C indicates changes in the expression of CD14 and CD68, which can also prove the differentiation from monocytes to macrophages, specifically manifested by decreased CD14 expression and increased CD68 expression. Figure 5 DE indicates that GDCA can promote macrophage M1 polarization, which is mainly manifested by morphological cell elongation and increased expression of M1 macrophage markers, including CD86, CD80, HLAII, TNFA, IL1B and COX2.
[0050] Example 6: Glycodeoxycholic acid (GDCA) inhibits the proliferation and migration of liver cancer cells
[0051] Experimental method: After incubating THP1 cells with 160nmol / L PMA in a six-well plate for 48h, THP-1 cells were induced into adherent M0 macrophages. After adding 500μM GDCA or DMSO solvent for 72h, the supernatant was centrifuged at 10000rpm for 5min to prepare conditioned medium. The conditioned medium obtained from macrophages treated differently was used to further treat Hep3B, HepG2, and Huh7 liver cancer cells for 48h, and the cell proliferation was detected using CCK8 reagent, and the cell migration was detected using scratch test.
[0052] Experimental results: The results are as follows Figure 6 As shown in the figure, GDCA-treated macrophages treated with conditioned medium to treat liver cancer cells can inhibit the proliferation and migration of liver cancer cells. These indicate that GDCA can inhibit liver cancer by promoting macrophage M1 polarization.
[0053] Example 7: Effects of GDCA and TDCA alone and their combination with PD-1 monoclonal antibody on the development and progression of liver cancer in mice with chemically induced liver cancer
[0054] Experimental methods: 2-week-old male C57BL / 6 mice were intraperitoneally injected with diethylnitrosamine (DEN) (25 mg / kg), and 10% carbon tetrachloride (CCl4) (5 ml / kg) was intraperitoneally injected weekly from 4 weeks of age to establish a liver cancer mouse model. At week 17, the mice were randomly divided into 6 groups: control group, GDCA group, TDCA group, PD-1 monoclonal antibody group, GDCA+PD-1 monoclonal antibody group, and TDCA+PD-1 monoclonal antibody group.
[0055] Control group: Chemically induced HCC mice were treated with standard diet and isotype control.
[0056] GDCA group: Chemically induced liver cancer mice were treated with feed supplemented with 0.3% GDCA and isotype control.
[0057] TDCA group: Chemically induced liver cancer mice were treated with feed supplemented with 0.3% TDCA and isotype control.
[0058] PD-1 monoclonal antibody group: Chemically induced liver cancer mice were treated with standard feed and PD-1 monoclonal antibody.
[0059] GDCA+PD-1 mAb group: Chemically induced liver cancer mice were treated with feed supplemented with 0.3% GDCA and PD-1 mAb.
[0060] TDCA+PD-1 mAb group: Chemically induced liver cancer mice were treated with feed supplemented with 0.3% TDCA and PD-1 mAb.
[0061] Mice were sacrificed at 25 weeks of age and livers were removed to count the number of tumors and measure tumor size. Tumor burden was the sum of all tumor sizes.
[0062] Experimental results: The results are as follows Figure 7 As shown, Figure 7 A is a schematic diagram of the experimental scheme. Figure 7 B is the picture of mouse liver in each experimental group. Figure 7 C is the weight of mice in each experimental group. Figure 7 D is the number of tumors in each experimental group, Figure 7 E is the maximum tumor size of each experimental group, Figure 7 F is the tumor burden of each experimental group. The results show that glycodeoxycholic acid or taurodeoxycholic acid alone can significantly inhibit the growth of liver cancer in chemically induced liver cancer mice, and compared with the use of glycodeoxycholic acid alone, taurodeoxycholic acid alone, or PD-1 monoclonal antibody alone, the combined treatment of glycodeoxycholic acid and PD-1 monoclonal antibody and the combined treatment of taurodeoxycholic acid and PD-1 monoclonal antibody can more significantly inhibit the growth of liver cancer tumors, and the two have a synergistic inhibitory effect on liver cancer. Example 8: Effects of glycodeoxycholic acid (GDCA), taurodeoxycholic acid (TDCA) alone and their combination with PD-1 monoclonal antibody on M1 macrophage infiltration in the tumor microenvironment of liver cancer.
[0063] Experimental methods: 2-week-old male C57BL / 6 mice were intraperitoneally injected with diethylnitrosamine (DEN) (25 mg / kg), and 10% carbon tetrachloride (CCl4) (5 ml / kg) was intraperitoneally injected weekly from 4 weeks of age to establish a liver cancer mouse model. At week 17, the mice were randomly divided into 6 groups: control group, GDCA group, TDCA group, PD-1 monoclonal antibody group, GDCA+PD-1 monoclonal antibody group, and TDCA+PD-1 monoclonal antibody group.
[0064] Control group: Chemically induced HCC mice were treated with standard diet and isotype control.
[0065] GDCA group: Chemically induced liver cancer mice were treated with feed supplemented with 0.3% GDCA and isotype control.
[0066] TDCA group: Chemically induced liver cancer mice were treated with feed supplemented with 0.3% TDCA and isotype control.
[0067] PD-1 monoclonal antibody group: Chemically induced liver cancer mice were treated with standard feed and PD-1 monoclonal antibody.
[0068] GDCA+PD-1 mAb group: Chemically induced liver cancer mice were treated with feed supplemented with 0.3% GDCA and PD-1 mAb.
[0069] TDCA+PD-1 mAb group: Chemically induced liver cancer mice were treated with feed supplemented with 0.3% TDCA and PD-1 mAb.
[0070] Mice were killed at 25 weeks of age and their livers were removed. The livers were ground, digested, and prepared into single-cell suspensions. 36% percoll solution was used to extract immune cells and flow cytometry was used to detect F4 / 80 in the immune microenvironment. + CD11b + Ratio of M1 macrophages to M2 macrophages among macrophages, liver-resident Kupffer cells, and monocyte-derived macrophages.
[0071] Experimental results: The results are as follows Figure 8 As shown, GDCA and TDCA alone can promote the infiltration of M1 macrophages in the liver cancer microenvironment, and anti-PD-1 antibodies alone have no effect on M1 macrophages, but anti-PD-1 antibodies combined with GDCA or TDCA can enhance the promotion of macrophage M1 polarization, proving the synergistic effect of combined drug therapy on anti-tumor immunity. Among them, we found that anti-PD-1 antibodies combined with GDCA or TDCA mainly promote the polarization of liver-resident Kupffer cells M1, rather than monocyte-derived macrophages.
Claims
1. Application of secondary bile acids in the preparation of anti-tumor drugs.
2. Application of secondary bile acids in the preparation of drugs for the treatment of hepatocellular carcinoma.
3. Application of glycolithocholic acid, taurolithocholic acid and taurodeoxycholic acid in the preparation of drugs for the treatment of hepatocellular carcinoma.
4. The application of taurolithocholic acid / glycolithocholic acid in the preparation of drugs for inhibiting the growth of liver cancer cells, reducing the migration ability of liver cancer cells, and promoting the apoptosis of liver cancer cells.
5. Application of glycodeoxycholic acid / taurodeoxycholic acid in the preparation of drugs for improving tumor immune microenvironment.
6. Application of taurodeoxycholic acid in the preparation of drugs for inhibiting tumor growth.
7. Application of glycodeoxycholic acid / taurodeoxycholic acid combined with immune checkpoint inhibitors in the preparation of anti-tumor drugs.
8. The use according to claim 7, characterized in that: The immune checkpoint inhibitor is selected from one or more of anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, anti-LAG-3 antibody, anti-TIGIT antibody, anti-TIM-3 antibody, anti-CD47 or anti-VISTA antibody.
9. The use according to claim 7, characterized in that: The tumors include hepatocellular carcinoma, gastric cancer, colorectal cancer, cervical cancer, ovarian cancer, breast cancer, lung cancer, esophageal cancer, testicular cancer, bone cancer, pancreatic cancer, thyroid cancer, and melanoma.
10. An anti-tumor drug, characterized in that: Comprising an immune checkpoint inhibitor and the glycodeoxycholic acid / taurodeoxycholic acid of claim 7.