A pharmaceutical composition for treating intrahepatic cholangiocarcinoma

Abstract

The application provides application of ursodeoxycholic acid or stereoisomers, prodrugs, crystal forms, pharmaceutically acceptable salts, pharmaceutically acceptable esters or pharmaceutically acceptable solvates thereof in preparation of a medicine for treating intrahepatic cholangiocarcinoma and a medicine composition for treating intrahepatic cholangiocarcinoma. The application provides application of ursodeoxycholic acid in treatment of intrahepatic cholangiocarcinoma, and application can significantly inhibit growth of a tumor and significantly prolong survival time of a subject animal. The application also provides a medicine composition for treating intrahepatic cholangiocarcinoma and containing ursodeoxycholic acid and an immune checkpoint inhibitor, solves technical problems such as lack of a precise treatment method for reversing CD8+ T cell exhaustion in intrahepatic cholangiocarcinoma, large side effects and limited effects of an existing treatment method and the like in the prior art, provides a new strategy for treating intrahepatic cholangiocarcinoma, and has high clinical application value and wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to a pharmaceutical composition for treating intrahepatic cholangiocarcinoma. Background Technology

[0002] Cholangiocarcinoma is a malignant tumor originating from the bile duct epithelium. Anatomically, it is divided into intrahepatic cholangiocarcinoma (ICC), which originates from the intrahepatic bile duct tree, and extrahepatic cholangiocarcinoma, which is located outside the liver parenchyma. Intrahepatic cholangiocarcinoma is the second most common primary liver malignancy, accounting for approximately 10%-15% of all primary liver cancers. It is characterized by its occult nature, high invasiveness, difficulty in treatment, and poor prognosis. Systemic therapy is a crucial treatment for 70%-80% of patients with advanced, unresectable metastatic ICC, including systemic chemotherapy, targeted therapy, and immunotherapy.

[0003] In recent years, immune checkpoint inhibitors (ICIs) have shown significant efficacy in patients with ischemic lung cancer (ICC), but they still have obvious limitations. First, the overall response rate of ICIs is low. Clinical studies have shown that the response rate of ICIs in ICC patients using ICIs alone is usually less than 20%, meaning that most patients do not benefit from monotherapy. Furthermore, ICIs can trigger a series of immune-related adverse events when activating the immune response. These adverse events involve multiple systems, such as skin toxicity (rash, itching), endocrine toxicity (thyroid dysfunction, diabetes), hepatotoxicity, and pulmonary toxicity. These side effects can severely impact patients' quality of life and even lead to treatment discontinuation. Most importantly, clinical observations have shown that some patients respond to ICIs during initial treatment, but may develop resistance over time. ICI resistance is the biggest obstacle to the long-term benefit of immunotherapy for cancer patients; therefore, there is an urgent clinical need for new methods to enhance the efficacy of ICIs in ICC patients.

[0004] Currently, immunotherapy for advanced ICC mainly involves chemotherapy combined with immunotherapy. However, this approach has serious side effects due to long-term use. Chemotherapy combined with immune checkpoint inhibitors for cholangiocarcinoma presents severe side effects, including autoimmune reactions such as colitis, hepatitis, and pneumonia; bone marrow suppression; gastrointestinal reactions; and abnormal liver function. Furthermore, it cannot fundamentally improve the immune response or significantly prolong patient survival. Moreover, treatment efficacy varies from person to person; some ICC patients respond poorly to this therapy and do not benefit from it.

[0005] Furthermore, current treatments cannot reverse the depletion of CD8+ T cells, and depleted CD8+ T cells respond poorly to ICIs, preventing some ICC patients from benefiting from immunotherapy. In addition, depleted CD8+ T cells are difficult to differentiate into long-term surviving memory T cells, limiting the durability of immunotherapy. Tumor cells may survive and reproliferate under immune stress, leading to tumor recurrence. Summary of the Invention

[0006] The present invention aims to at least solve one of the aforementioned technical problems existing in the prior art. Therefore, the object of the present invention is to provide the use of ursodeoxycholic acid in the preparation of a medicament for treating intrahepatic cholangiocarcinoma, and a pharmaceutical composition for treating intrahepatic cholangiocarcinoma.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The first aspect of this invention provides the use of ursodeoxycholic acid or its stereoisomers, prodrugs, crystal forms, pharmaceutically acceptable salts, pharmaceutically acceptable esters or pharmaceutically acceptable solvates in the preparation of a medicament for treating intrahepatic cholangiocarcinoma.

[0008] In some embodiments, the drug also contains an immune checkpoint inhibitor.

[0009] In some embodiments, the immune checkpoint inhibitor includes at least one of anti-PD-1 antibody, anti-PD-L1 antibody, and anti-CTLA-4 antibody.

[0010] A second aspect of the present invention provides a pharmaceutical composition for treating intrahepatic cholangiocarcinoma, comprising a first component and a second component. The first component is an immune checkpoint inhibitor. The second component is ursodeoxycholic acid or its stereoisomer, prodrug, crystal form, pharmaceutically acceptable salt, pharmaceutically acceptable ester, or pharmaceutically acceptable solvate.

[0011] In some embodiments, the immune checkpoint inhibitor includes at least one of anti-PD-1 antibody, anti-PD-L1 antibody, and anti-CTLA-4 antibody.

[0012] In some embodiments, the first component and the second component are in the same formulation unit.

[0013] In some embodiments, the first component and the second component are in different formulation units.

[0014] In some embodiments, the first component and the second component are administered simultaneously, separately, or sequentially.

[0015] In some embodiments, the pharmaceutical composition further comprises pharmaceutically acceptable excipients.

[0016] In some embodiments, the pharmaceutical composition is in the form of a gastrointestinal or non-gastrointestinal dosage form.

[0017] The beneficial effects of this invention are: This invention provides the application of ursodeoxycholic acid in the treatment of intracranial cholestasis of liver disease (ICC). Its administration can significantly inhibit tumor growth and significantly prolong the survival time of test animals. Furthermore, ursodeoxycholic acid, as an approved clinical drug, has been approved for the treatment of cholestatic liver disease and has good safety and tolerability. This invention proposes to use it for the treatment of ICC, which can accelerate the entry into the clinical stage and reduce research and development costs and time.

[0018] This invention also provides a pharmaceutical composition for treating ICC containing ursodeoxycholic acid and an immune checkpoint inhibitor. By regulating the FXR signaling pathway through ursodeoxycholic acid, it can reverse the depletion state of CD8+ T cells, enhance the proliferation, killing and migration functions of CD8+ T cells, thereby enhancing immunotherapy, inhibiting the progression of ICC, and avoiding the serious side effects of chemotherapy. It solves the technical problems of the lack of precise treatment methods to reverse CD8+ T cell depletion in ICC and the large side effects and limited efficacy of existing treatment methods in the prior art. It provides a new strategy for treating ICC and has high clinical application value and broad application prospects. Attached Figure Description

[0019] Figure 1 The study demonstrates the combination of UDCA and anti-PD-L1 monoclonal antibody for the treatment of ICC. In this study, A shows the animal experimental procedure in Example 1, B shows a gross image of the mouse liver, C shows the liver weight ratio of the mouse, and D shows the survival time of the mouse.

[0020] Figure 2 The study demonstrated the PDTF system for validating the efficacy of UDCA combined with anti-PD-L1 monoclonal antibody therapy. Specifically, A showed the experimental procedure for validating the efficacy of UDCA combined with anti-PD-L1 monoclonal antibody therapy using a tumor tissue block culture model derived from human ICC patients; B showed the TUNEL staining results; and C showed the Annexin V / PI staining results.

[0021] Figure 3The therapeutic effect of UDCA on ICC was demonstrated. A shows the animal experiment procedure in Example 3. B shows the mRNA expression level of Nr1h4 in sorted liver tumor-infiltrating CD8+ T cells detected by qPCR. C shows a gross image of the mouse liver. D shows the liver weight ratio of the mouse. E shows the survival time of the mouse. FI shows the proportion of activated CD8+ T cells, Tpex cells, activated CD8+ T cells secreting IFN-γ, and activated CD8+ T cells secreting TNF-α in isolated tumor tissue lymphocytes. Detailed Implementation

[0022] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments can be obtained from conventional commercial sources or by existing technical methods. Unless otherwise specified, the experimental or testing methods are conventional methods in the art.

[0023] application The first aspect of this invention provides the use of ursodeoxycholic acid or its stereoisomers, prodrugs, crystal forms, pharmaceutically acceptable salts, pharmaceutically acceptable esters or pharmaceutically acceptable solvates in the preparation of a medicament for treating intrahepatic cholangiocarcinoma.

[0024] Ursodeoxycholic acid (UDCA) is an FXR inhibitor that has been clinically approved for use in cholestatic liver disease. This invention provides a novel application of the FXR inhibitor UDCA in the treatment of ICC. In a spontaneous mouse model of ICC, UDCA effectively inhibited tumor progression and prolonged survival in mice.

[0025] In this invention, the term "prodrug" refers to a derivative that can be hydrolyzed, oxidized, or otherwise reacted under biological conditions (in vitro or in vivo) to provide the compounds of this invention. Prodrugs become active compounds only after undergoing this reaction under biological conditions, or they do not have or only have low activity in their unreacted forms. Prodrugs can generally be prepared using well-known methods, such as those described in Burger's Medicinal Chemistry and Drug Discovery (1995) 172-178, 949-982 (Manfred E. Wolff, 5th edition).

[0026] In this invention, the term "crystal form" refers to the crystal structure of a substance. During crystallization, various factors alter the intramolecular or intermolecular bonding patterns, resulting in different arrangements of molecules or atoms in the crystal lattice, thus forming different crystal structures. The compounds of this invention can exist in one crystal structure or multiple crystal structures, i.e., they possess "polymorphism." The compounds of this invention can exist in different crystal forms.

[0027] In this invention, the term "pharmaceutically acceptable salt" refers to (i) a salt formed by an acidic functional group (e.g., -COOH) present in the compounds provided by this invention and a suitable inorganic or organic cation (base), including but not limited to, alkali metal salts such as sodium salts, potassium salts, lithium salts, etc.; alkaline earth metal salts such as calcium salts, magnesium salts, etc.; other metal salts such as aluminum salts, iron salts, zinc salts, copper salts, nickel salts, cobalt salts, etc.; inorganic base salts such as ammonium salts; organic base salts such as tert-octylamine salts, dibenzylamine salts, morpholine salts, glucosamine salts, phenylglycine alkyl ester salts, ethylenediamine salts, N-methylglucosamine salts, guanidine salts, diethylamine salts, triethylamine salts, dicyclohexylamine salts, N,N'-dibenzylethylenediamine salts, chloroprocaine salts, procaine salts, diethanolamine salts, N-benzyl-phenylethylamine salts, piperazine salts, tetramethylamine salts, and tris(hydroxymethyl)aminomethane salts. And (ii) the salts formed by the basic functional group (e.g., -NH2) present in the compounds provided by the present invention and suitable inorganic or organic anions (acids), including but not limited to, hydrohalides, such as hydrofluoric acid salts, hydrochlorides, hydrobromic acid salts, hydroiodides, etc.; inorganic acid salts, such as nitrates, perchlorates, sulfates, phosphates, etc.; lower alkyl sulfonates, such as methanesulfonates, trifluoromethanesulfonates, ethanesulfonates, etc.; aryl sulfonates, such as benzenesulfonates, p-benzenesulfonates, etc.; organic acid salts, such as acetates, malates, fumarates, succinates, citrates, tartrates, oxalates, maleates, etc.; amino acid salts, such as glycine salts, trimethylglycine salts, arginine salts, ornithine salts, glutamate salts, aspartate salts, etc.

[0028] In this invention, the term "pharmaceutically acceptable ester" refers to an ester formed by the -COOH group present in the compounds provided by this invention and a suitable alcohol, or an ester formed by the -OH group present in the compounds provided by this invention and a suitable acid (e.g., a carboxylic acid or an oxy-containing inorganic acid). Suitable ester groups include, but are not limited to, formate, acetate, propionate, butyrate, acrylate, ethyl succinate, stearate, or palmitate. Esters can undergo hydrolysis in the presence of acid or base to produce the corresponding acid or alcohol.

[0029] In this invention, the term "solvent" refers to a compound existing in combination with a solvent molecule. This combination may include a stoichiometric amount of a solvent, such as a monohydrate or dihydrate, or may include any amount of water; for example, methanol or ethanol may form an "alcohol," which may be stoichiometric or non-stoichiometric. As used herein, the term "solvent" refers to a solid form, i.e., a compound in a solution of a solvent, although it may be solvated, is not a solvate as used herein.

[0030] In this invention, the term "treatment" generally refers to achieving the desired pharmacological and / or physiological effect. This effect may be preventative based on the complete or partial prevention of the disease or its symptoms; and / or therapeutic based on the partial or complete stabilization or cure of the disease and / or side effects resulting from the disease. As used herein, "treatment" encompasses any treatment of a patient's disease, including: (a) prevention of disease or symptoms occurring in a patient who is susceptible to the disease or its symptoms but has not yet been diagnosed with the disease; (b) suppression of the symptoms of the disease, i.e., prevention of its progression; or (c) relief of the symptoms of the disease, i.e., causing the disease or its symptoms to regress.

[0031] In some embodiments, the drug also contains an immune checkpoint inhibitor.

[0032] This invention has discovered a novel use of the FXR inhibitor UDCA in improving the immune response in ICC. In a spontaneous mouse model of ICC, the combination of UDCA and an immune checkpoint inhibitor effectively inhibited tumor progression and prolonged survival time in mice compared to the immune checkpoint inhibitor monotherapy group.

[0033] In some embodiments, the immune checkpoint inhibitor includes at least one of anti-PD-1 antibody, anti-PD-L1 antibody, and anti-CTLA-4 antibody.

[0034] In some embodiments, the anti-PD-1 antibody is selected from at least one of xindilimumab, toripalimab, camrelizumab, tislelizumab, pembrolizumab, or nivolumab.

[0035] In some embodiments, the anti-PD-L1 antibody is selected from at least one of durvalumab or atezolizumab.

[0036] Pharmaceutical Composition A second aspect of the present invention provides a pharmaceutical composition for treating intrahepatic cholangiocarcinoma, comprising a first component and a second component. The first component is an immune checkpoint inhibitor. The second component is ursodeoxycholic acid or its stereoisomer, prodrug, crystal form, pharmaceutically acceptable salt, pharmaceutically acceptable ester, or pharmaceutically acceptable solvate.

[0037] In some embodiments, the immune checkpoint inhibitor includes at least one of anti-PD-1 antibody, anti-PD-L1 antibody, and anti-CTLA-4 antibody.

[0038] In some embodiments, the anti-PD-1 antibody is selected from at least one of xindilimumab, toripalimab, camrelizumab, tislelizumab, pembrolizumab, or nivolumab.

[0039] In some embodiments, the anti-PD-L1 antibody is selected from at least one of durvalumab or atezolizumab.

[0040] In some embodiments, the pharmaceutical composition contains effective amounts of the first component and the second component.

[0041] In this invention, "effective amount" refers to the amount that effectively achieves the desired therapeutic effect at the necessary dosage and time. The "effective amount" of the substance / molecule of this invention can vary depending on factors such as an individual's disease state, age, sex, weight, and the ability of the substance / molecule to elicit the desired response in the individual. Effective amount also encompasses the amount in which the beneficial therapeutic effect of the substance / molecule outweighs any toxic or harmful consequences.

[0042] In some embodiments, the first component and the second component are in the same formulation unit.

[0043] In some embodiments, the first component and the second component are in different formulation units.

[0044] In some embodiments, the pharmaceutical composition is in the form of a gastrointestinal or non-gastrointestinal dosage form.

[0045] In some embodiments, the gastrointestinal dosage form includes at least one of powder, tablet, granule, capsule, sustained-release, solution, dry suspension, effervescent tablet, emulsion, suspension, syrup, drops, and chewable tablet.

[0046] In some embodiments, the non-gastrointestinal dosage form includes at least one of injection dosage form, respiratory dosage form, skin dosage form, mucosal dosage form, and cavity dosage form.

[0047] In some embodiments, the dosage form of the pharmaceutical composition is any one of powder, granules, tablets, capsules, gels, suspensions, drops, pills, injections, suppositories, aerosols, oral liquids, ointments, emulsions, and irrigants.

[0048] In some embodiments, the first component and the second component are administered simultaneously, separately, or sequentially.

[0049] In some embodiments, the pharmaceutical composition further comprises pharmaceutically acceptable excipients.

[0050] In some embodiments, the excipients further include at least one of fillers, diluents, disintegrants, binders, lubricants, flow aids, surfactants, solvents, flavoring agents, and preservatives.

[0051] In some embodiments, the filler or diluent includes sugars such as lactose, sucrose, glucose, mannitol, sorbitol, and dextrin; starches such as starch, pregelatinized starch, α-starch, and dextrin; celluloses such as microcrystalline cellulose, gum arabic, and dextran; and inorganic salts such as calcium sulfate, calcium hydrogen phosphate, pharmaceutical-grade calcium carbonate, light anhydrous silicic acid, synthetic aluminum silicate, calcium silicate, and magnesium aluminosilicate.

[0052] In some embodiments, the lubricant, flow aid, or anti-sticking agent includes stearic acid; metal stearate salts such as calcium stearate or magnesium stearate; talc; colloidal silica; micronized silica gel; hydrogenated vegetable oil; polyethylene glycol; lauryl sulfate such as sodium lauryl sulfate or magnesium lauryl sulfate; silicates such as silicic anhydride or silicate hydrates, etc.

[0053] In some embodiments, the adhesive includes distilled water, ethanol of varying concentrations, starch paste, hydroxypropyl methylcellulose, hydroxypropyl cellulose, povidone, ethyl cellulose, sodium carboxymethyl cellulose, polyvinylpyrrolidone, polyethylene glycol, and compounds similar to the excipients described above.

[0054] In some embodiments, the disintegrant includes cellulose derivatives such as low-substituted hydroxypropyl cellulose, carboxymethyl cellulose, calcium carboxymethyl cellulose, or croscarmellose sodium; croscarmellose; and chemically modified starch / cellulose, such as carboxymethyl starch or sodium carboxymethyl starch.

[0055] In some embodiments, the surfactant includes Tween, sodium dodecyl sulfate, sodium stearate sulfonate, etc.

[0056] In some embodiments, the antioxidant includes sodium bisulfite, sodium metabisulfite, sodium sulfite, dried sodium sulfite, sodium thiosulfate, ascorbic acid, methionine, thiourea, phosphoric acid, citric acid, etc.

[0057] In some embodiments, the preservative or antibacterial agent includes benzoic acid and sodium benzoate, sorbic acid, ethanol, parabens, benzalkonium bromide, o-phenylphenol, benzyl alcohol, phenylethanol, sodium propionate, sorbic acid, eucalyptus oil, cinnamon oil, and peppermint oil.

[0058] In some embodiments, the flavoring agent includes sweeteners such as sodium saccharin, aspartame, syrup, steviol, mannitol, sorbitol, mannose, galactose, maltose, fructose, glucose, sucrose, etc.; acid flavoring agents such as citric acid, malic acid or tartaric acid; and aroma agents such as fennel oil, peppermint oil, menthol, peppermint water, cinnamon oil, lemon essence, lemon oil and various flavorings.

[0059] Example 1: UDCA combined with anti-PD-L1 monoclonal antibody for the treatment of ICC (I) Experimental Methods 1. UDCA combined with anti-PD-L1 monoclonal antibody administration like Figure 1 As shown in Figure A, 20 eight-week-old C57 mice were randomly divided into four groups. Following the method described in the reference (Fan B, Malato Y, Calvisi DF, Naqvi S, Razumilava N, Ribback S, et al. Cholangiocarcinomascan originate from hepatocytes in mice. J Clin Invest. 2012;122(8):2911-5.), a spontaneous ICC model was constructed. Starting from the third day of modeling, the mice were administered the drug every three days. After 28 days, the mice were anesthetized with isoflurane and sacrificed by spinal dislocation. The liver weight ratio of the mice was analyzed. Another batch of mice with the same treatment was used for survival analysis.

[0060] The dosing regimens for each group are as follows: Control group: PBS was injected intraperitoneally and the solvent (ddH2O + 1% anhydrous ethanol) was administered by gavage once every 3 days. UDCA monotherapy group: 30 mg / kg UDCA (Maclean, U820345) was administered by gavage every 3 days. Anti-PD-L1 monoclonal antibody treatment group: 300 μg of anti-PD-L1 monoclonal antibody (BioXCell, BE0101) was injected intraperitoneally every 3 days. The combination therapy group received 30 mg / kg UDCA (Maclean's, U820345) by gavage every 3 days, and 300 μg anti-PD-L1 monoclonal antibody (BioXCell, BE0101) by intraperitoneal injection every 3 days.

[0061] 2. Statistics All statistical analyses were performed using Prism 8. Data in figures are presented as mean ± SEM. Significance between two or more groups was assessed using a two-tailed, unpaired Student's t-test and one-way ANOVA. Statistical significance for all experiments was determined as p-value ≤ 0.05.

[0062] (II) Experimental Results like Figure 1 As shown in Figure B, the gross liver images of the four groups of mice on day 28 showed significant differences. From the gross liver images, the livers of mice treated with anti-PD-L1 monoclonal antibody alone and the control group showed diffuse vesicular lesions, namely translucent vesicles distributed throughout the liver (similar to "honeycomb" or "foam"), while the number of vesicles was significantly reduced in the UDCA monotherapy and combination therapy groups.

[0063] like Figure 1 As shown in Figure C, the liver weight ratio of mice treated with anti-PD-L1 monoclonal antibody alone was not different from that of the control group, indicating that it failed to show a significant anti-tumor effect. However, UDCA treatment alone significantly inhibited tumor growth. When UDCA was used in combination with anti-PD-L1 monoclonal antibody, the therapeutic effect was further improved, showing anti-tumor activity superior to UDCA or anti-PD-L1 monoclonal antibody alone.

[0064] like Figure 1 As shown in Figure D, survival analysis revealed that the survival time of mice in the combined treatment group was significantly prolonged.

[0065] These results suggest that UDCA combined with anti-PD-L1 monoclonal antibodies may provide a new treatment option for ICC patients who do not respond well to immune checkpoint blockade (ICB) therapy.

[0066] Example 2: Validation of the efficacy of UDCA combined with anti-PD-L1 monoclonal antibody therapy using patient-derived tumor fragment (PDTF) system. To directly evaluate the potential of UDCA-enhanced immune checkpoint blockade therapy in a clinically relevant setting, such as Figure 2 As shown in Figure A, a tumor tissue fragment (PDTF) culture model derived from human ICC patients was used to verify the efficacy of UDCA combined with anti-PD-L1 monoclonal antibody therapy. The tumor tissue fragments derived from human ICC patients used in this embodiment were provided by the Department of Hepatobiliary Surgery, The Third Affiliated Hospital of Sun Yat-sen University.

[0067] (I) Experimental Methods The method described in the reference (Voabil P, de Bruijn M, Roelofsen LM, Hendriks SH, Brokamp S, van den Braber M, et al. An ex vivo tumor fragment platform to dissect response to PD-1 blockade in cancer. Nat Med. 2021;27(7):1250-61.) verifies the efficacy of UDCA combined with anti-PD-L1 monoclonal antibody therapy using the PDTF system. The specific steps are as follows: Immediately after surgery, manually cut the patient's ICC tissue into 1-2 mm pieces on ice. 3 Small pieces of PDTF were then embedded in an artificial extracellular matrix. 40 μL of matrix gel was placed in each well of a pre-cooled 96-well plate as the bottom layer and cured at 37°C for 20–30 minutes. After rinsing the PDTF multiple times on a cell sieve in a 6-well plate (using DMEM + 10% FBS + 1% penicillin-dextrose antibody), a tissue piece was placed on the pre-cured matrix, and another 40 μL of matrix gel was added as a second covering layer. The 96-well plate was then incubated at 37°C for 20–30 minutes to cure. After curing, 120 μL of tumor culture medium was added to each well. The PDTF culture system was divided into four groups. Group 1 received 20 μM UDCA (Maclean's, U820345); Group 2 received 10 μg / mL anti-PD-L1 monoclonal antibody (BioXCell, BE0101); Group 3 received both 20 μM UDCA (Maclean's, U820345) and 10 μg / mL anti-PD-L1 monoclonal antibody (BioXCell, BE0101); and Group 4 served as the control group, receiving PBS buffer. The PDTF culture system was incubated at 37°C for 72 hours. After 72 hours of in vitro culture, the therapeutic effect of the combined treatment on PDTF was verified using the TUNEL assay (using a TUNEL kit (Thermo Fisher Scientific, C10617), method according to the kit instructions) and Annexin V / PI staining (using an Annexin V-FITC / PI apoptosis detection kit (Thermo Fisher Scientific, V13242), method according to the kit instructions).

[0068] All statistical analyses were performed using Prism 8. Data in figures are presented as mean ± SEM. Significance between two or more groups was assessed using a two-tailed, unpaired Student's t-test and one-way ANOVA. Statistical significance for all experiments was determined as p-value ≤ 0.05.

[0069] (II) Experimental Results Experimental results are as follows Figure 2 As shown in Figures B and C, in this in vitro model, using both TUNEL staining and flow cytometry-based apoptosis assays (Annexin V+), the combination of UDCA and an anti-PD-L1 antibody significantly increased the proportion of apoptotic tumor cells compared to monotherapy. This synergistic enhancement of tumor cell death clearly demonstrates that UDCA can enhance the efficacy of ICB therapy. These results suggest that UDCA combined with an anti-PD-L1 monoclonal antibody may provide a new treatment option for ICC patients who do not respond well to ICB.

[0070] Example 3: Therapeutic effect of UDCA on ICC (I) Experimental Methods like Figure 3 As shown in Figure A, 6-8 week old C57 mice were divided into two groups. Following the method described in the reference (Fan B, Malato Y, Calvisi DF, Naqvi S, Razumilava N, Ribback S, et al. Cholangiocarcinomas can originate from hepatocytes in mice. J Clin Invest. 2012;122(8):2911-5.), a spontaneous ICC model was constructed. Starting from day 3 of modeling, 30 mg / kg UDCA (Maclean's, U820345) or solvent control (ddH2O+1% anhydrous ethanol) was administered by gavage every 3 days. After 28 days, lymphocytes from tumor tissue were isolated. The expression level of FXR (encoded by the Nr1h4 gene) in CD8+ T cells infiltrating liver tumors was detected by PCR and analyzed by flow cytometry.

[0071] All statistical analyses were performed using Prism 8. Data in figures are presented as mean ± SEM. Significance between two or more groups was assessed using a two-tailed, unpaired Student's t-test and one-way ANOVA. Statistical significance for all experiments was determined as p-value ≤ 0.05.

[0072] (II) Experimental Results In the NICD / AKT ICC model, mice treated with UDCA by gavage showed a significantly lower expression level of FXR (encoded by the Nr1h4 gene) in liver tumor-infiltrating CD8+ T cells compared to the solvent control group. Figure 3 (Middle B). This intervention achieved remarkable therapeutic effects; the tumor burden in the UDCA-treated group of mice was significantly suppressed, and their survival was significantly prolonged. Figure 3(CE). Further flow cytometry analysis showed that UDCA treatment induced profound functional remodeling of intratumoral CD8+ T cells: it not only significantly increased the proportion of activated CD8+ T cells ( Figure 3 The F1 gene also promoted the expansion of precursor exhausted T cells (Tpex), which are crucial for maintaining a sustained anti-tumor immune response. Figure 3 The drug (L) effectively promoted the production and secretion of effector molecules (including IFN-γ and TNF-α), indicating that its cytotoxic killing ability was greatly enhanced (Figure H, I).

[0073] In summary, UDCA can effectively relieve the immune checkpoint inhibition of CD8+ T cells by antagonizing the FXR signaling pathway, thereby reversing T cell exhaustion and enhancing the stemness and cytotoxic function of CD8+ T cells.

[0074] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The use of ursodeoxycholic acid or a pharmaceutically acceptable salt thereof in the preparation of drugs for the treatment of intrahepatic cholangiocarcinoma; The drug also contains an anti-PD-L1 antibody; The intrahepatic cholangiocarcinoma mentioned refers to intrahepatic cholangiocarcinoma that does not respond well to immune checkpoint blockade therapy.

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