Use of dihydroartemisinin as a YAP inhibitor in intrahepatic cholangiocarcinoma

Abstract

This application discloses the use of dihydroartemisinin as a YAP inhibitor in intrahepatic cholangiocarcinoma. Dihydroartemisinin can target YAP in the Hippo pathway, not only directly inhibiting tumor growth but also relieving the inhibitory effect of YAP on CD8+ T cells, enhancing the efficacy of tumor immunotherapy, and significantly inhibiting intrahepatic cholangiocarcinoma.

Description

Technical Field

[0001] This article relates to the pharmaceutical field, particularly the use of dihydroartemisinin in the preparation of Hippo pathway inhibitors and YAP inhibitors for intrahepatic cholangiocarcinoma. Background Technology

[0002] Intrahepatic cholangiocarcinoma (ICC) is the second most common primary liver cancer after hepatocellular carcinoma (HCC). It is highly malignant and currently has no effective treatment, accounting for 20% of all liver malignancies and 3% of gastrointestinal malignancies. In recent years, the incidence of ICC has been on the rise, with an extremely poor prognosis and high mortality rate; the five-year mortality rate is approximately 9%. However, the exact cause of ICC remains unclear. Identified risk factors for ICC include polycystic fibrosis (e.g., choledochal cysts), primary sclerosing cholangitis, liver stones, cirrhosis, viral hepatitis, parasitic infections (e.g., Clonorchis sinensis, Clonorchis sinensis), obesity-related steatohepatitis, diabetes, and exposure to carcinogens (e.g., nitrosamines, sedexstat), as well as at least four known genetic disorders, including Lynch syndrome, BRCA-associated protein-1 (BAP-1) tumor susceptibility syndrome, cystic fibrosis, and biliary neoplasm. Radical resection is the only potentially curative treatment, but patients are often too late for surgery at diagnosis. For the past decade, doublet chemotherapy with gemcitabine and cisplatin has been considered the most effective first-line treatment, but recent results with triple therapy and even immunotherapy may change this paradigm. Therefore, finding effective drugs to treat ICC is an urgent need.

[0003] The Hippo signaling pathway was first discovered in fruit flies and is named for the excessive growth of their tissues and the formation of large, wrinkled epidermal structures resembling those of a hippopotamus due to mutations in its core members. It translates signals into transcriptional regulation by sensing the physical environment surrounding the cell, precisely balancing proliferation, differentiation, and apoptosis. YAP (Yes-associated protein) is a key effector molecule in the Hippo signaling pathway, responsible for executing the "cell proliferation" command. Its overactivation is a key cause of cancer and organ overgrowth. In various solid tumors, the intrinsic YAP signaling pathway in tumor cells is abnormally activated, evading immune surveillance by remodeling the tumor microenvironment. The main mechanisms include: inhibiting antigen presentation to evade immune recognition, reprogramming the extracellular matrix, inducing T cell dysfunction, and inducing T cell exhaustion via exosomes. In these tumor cells, YAP is the "driver" of immune escape. Approximately 67% of intrahepatic cholangiocarcinomas show high YAP expression; YAP is one of the key driving factors in intrahepatic cholangiocarcinoma and is directly associated with poorer patient prognosis. In animal models, simple activation of YAP is sufficient to directly transform normal hepatocytes or bile duct cells into intrahepatic cholangiocarcinoma. YAP not only promotes cancer cell proliferation but also suppresses the immune system, reducing the number of cytotoxic T cells within the tumor and leading to poor efficacy of immunotherapy.

[0004] Dihydroartemisinin (DHA) is the active metabolite of artemisinin and its derivatives. It is an effective clinical drug widely used to treat malaria. Compared with artemisinin, DHA has better water solubility and stronger antimalarial activity. Summary of the Invention

[0005] The inventors discovered that dihydroartemisinin can target YAP in the Hippo pathway, not only directly inhibiting tumor growth but also relieving the inhibitory effect of YAP on CD8+ T cells, enhancing the efficacy of tumor immunotherapy, and significantly inhibiting intrahepatic cholangiocarcinoma. Furthermore, dihydroartemisinin shows a significantly reduced therapeutic effect on intrahepatic cholangiocarcinoma with the YAP gene knocked out; therefore, dihydroartemisinin is particularly suitable for treating intrahepatic cholangiocarcinoma with high YAP expression and / or low CD8+ T cell infiltration.

[0006] In a first aspect of this application, embodiments of this application provide the use of dihydroartemisinin or a pharmaceutically acceptable salt, solvate or hydrate thereof in the preparation of Hippo pathway inhibitors in intrahepatic cholangiocarcinoma.

[0007] In a second aspect of this application, embodiments of this application provide the use of dihydroartemisinin or a pharmaceutically acceptable salt, solvate or hydrate thereof in the preparation of YAP inhibitors for intrahepatic cholangiocarcinoma.

[0008] In a third aspect of this application, embodiments of this application provide the use of dihydroartemisinin or a pharmaceutically acceptable salt, solvate or hydrate thereof in the preparation of a reagent for inhibiting the Hippo pathway in intrahepatic cholangiocarcinoma cells and / or tissues, said use excluding the diagnosis and treatment of diseases.

[0009] In a fourth aspect of this application, embodiments of this application describe the use of dihydroartemisinin or a pharmaceutically acceptable salt, solvate, or hydrate thereof in the preparation of a reagent for reducing the expression level of YAP in intrahepatic cholangiocarcinoma cells and / or tissues, wherein the use does not include the diagnosis and treatment of diseases.

[0010] In a fifth aspect of this application, embodiments of this application provide the use of dihydroartemisinin or a pharmaceutically acceptable salt, solvate or hydrate thereof in the preparation of a reagent for promoting CD8+ T cell infiltration in the tumor microenvironment of intrahepatic cholangiocarcinoma, said use excluding the diagnosis and treatment of the disease.

[0011] In a sixth aspect of this application, embodiments of this application provide the use of dihydroartemisinin or a pharmaceutically acceptable salt, solvate or hydrate thereof in the preparation of a medicament for inhibiting the Hippo pathway in intrahepatic cholangiocarcinoma cells and / or tissues and / or reducing the expression level of YAP in intrahepatic cholangiocarcinoma cells and / or tissues; wherein the intrahepatic cholangiocarcinoma is a YAP-high expressing type and / or a CD8+ T cell-low infiltrating type of intrahepatic cholangiocarcinoma.

[0012] In a seventh aspect of this application, embodiments of this application provide the use of dihydroartemisinin or a pharmaceutically acceptable salt, solvate or hydrate thereof in the preparation of a medicament for promoting CD8+ T cell infiltration in the tumor microenvironment of intrahepatic cholangiocarcinoma; wherein the intrahepatic cholangiocarcinoma is YAP-high expressing type and / or CD8+ T cell-low infiltrating type intrahepatic cholangiocarcinoma.

[0013] In an eighth aspect of this application, embodiments of this application provide dihydroartemisinin or pharmaceutically acceptable salts, solvates, or hydrates thereof for use as a medicine. In one exemplary embodiment, this application provides dihydroartemisinin or pharmaceutically acceptable salts, solvates, or hydrates thereof for the treatment and / or prevention of intrahepatic cholangiocarcinoma, wherein the intrahepatic cholangiocarcinoma is YAP-overexpressing and / or CD8+ T-cell-low invasive intrahepatic cholangiocarcinoma.

[0014] In a ninth aspect of this application, embodiments of this application provide a treatment method for intrahepatic cholangiocarcinoma, wherein the intrahepatic cholangiocarcinoma is YAP-high expressing and / or CD8+ T cell-low infiltrating intrahepatic cholangiocarcinoma, the method comprising administering a therapeutically effective amount of dihydroartemisinin or a pharmaceutically acceptable salt, solvate or hydrate thereof to an individual in need.

[0015] Other features and advantages of this application will be set forth in the following description, and will be apparent in part from the description, or may be learned by practicing the application. Other advantages of this application can be realized and obtained by means of the embodiments described in the description and the accompanying drawings. Attached Figure Description

[0016] The accompanying drawings are used to provide an understanding of the technical solutions of this application and constitute a part of the specification. They are used together with the embodiments of this application to explain the technical solutions of this application and do not constitute a limitation on the technical solutions of this application.

[0017] Figure 1 In Example 1 of this application, ICC cells were treated with different concentrations of dihydroartemisinin for 72 h, and cell viability was measured by CCK-8 assay, along with the inhibition rate curves and IC50 values ​​for each cell type.

[0018] Figures 2-3 This is the result of the experiment in Example 2 of this application on the inhibition of intrahepatic cholangiocarcinoma in situ proliferation in mice by dihydroartemisinin. Figure 2 In this study, A. Experimental design of the sgP19-KRAS in situ carcinoma model (n=6); B. Representative images of mouse livers from each group and HE staining results (N represents normal tissue area, T represents tumor area); D. Quantitative statistics of HE staining results; C. Statistical results of mouse liver weight (n=6). Figure 3 Immunohistochemical staining results of Ki67 and PCNA and statistical analysis of positive cell rates. *: P < 0.05; **: P < 0.01; ***: P < 0.001; ns: No statistically significant difference between the two groups. ±s, n=3.

[0019] Figures 4-5 This is the experimental design and research results of Example 3 of this application, which describes how dihydroartemisinin inhibits tumor development by inhibiting the Hippo pathway. Figure 4 In the sgP19 / KRAS in situ carcinoma model group, transcriptomic sequencing analysis was performed on liver tumor samples from normal liver tissue, control group, and dihydroartemisinin-treated group, and KEGG analysis was performed based on the sequencing results; B. GSEA analysis of the Hippo pathway was performed on the sequencing results; C. Western blotting analysis was used to detect the expression of enriched pathway proteins. Figure 5A. Schematic diagram of the construction of 4 sgYAP plasmids; B. Cell survival curves detected by CCK-8 assay after QBC-939 cells were transfected with sgYAP and sgGFP; C. Experimental design of the sgP19-KRAS subcutaneous tumor model transfected with sgYAP and sgGFP (n=6); D. Changes in subcutaneous tumor volume in each group during the experimental period; E. Photographs of subcutaneous tumors in each group after sampling; F. Weight analysis of subcutaneous tumors in each group; *: P<0.05; ns: No statistically significant difference between the two groups. ±s, n=6.

[0020] Figure 6 This document presents the experimental design and results of Example 4 of this application, which describes the indirect inhibition of tumor development by dihydroartemisinin via CD8 T cells. The results include: A. Immunohistochemical staining and quantitative analysis of CD3 and CD8; B. Experimental design of the sgP19-KRAS in situ carcinoma model in C57 mice (n=6); C. Photographic recording of liver tumors in each group; and D. Statistical analysis of liver weight in each group. *: P < 0.05; **: P < 0.01; ns: No statistically significant difference between the two groups. ±s, n=6. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of this application clearer, the embodiments of this application will be described in detail below. It should be noted that, unless otherwise specified, the embodiments and features described in these embodiments can be arbitrarily combined with each other.

[0022] In a first aspect of this application, embodiments of this application provide the use of dihydroartemisinin or a pharmaceutically acceptable salt, solvate or hydrate thereof in the preparation of Hippo pathway inhibitors in intrahepatic cholangiocarcinoma.

[0023] In a second aspect of this application, embodiments of this application provide the use of dihydroartemisinin or a pharmaceutically acceptable salt, solvate or hydrate thereof in the preparation of YAP inhibitors for intrahepatic cholangiocarcinoma.

[0024] In one exemplary embodiment, a YAP inhibitor may refer to the inhibition of the expression of total YAP protein and / or dephosphorylated YAP protein.

[0025] In a third aspect of this application, embodiments of this application provide the use of dihydroartemisinin or a pharmaceutically acceptable salt, solvate or hydrate thereof in the preparation of a reagent for inhibiting the Hippo pathway in intrahepatic cholangiocarcinoma cells and / or tissues, said use excluding the diagnosis and treatment of diseases.

[0026] In a fourth aspect of this application, embodiments of this application provide the use of dihydroartemisinin or a pharmaceutically acceptable salt, solvate or hydrate thereof in the preparation of a reagent for reducing the expression level of YAP in intrahepatic cholangiocarcinoma cells and / or tissues, said use excluding the diagnosis and treatment of diseases.

[0027] In one exemplary embodiment, YAP comprises total YAP protein and / or dephosphorylated YAP protein.

[0028] In a fifth aspect of this application, embodiments of this application provide the use of dihydroartemisinin or a pharmaceutically acceptable salt, solvate or hydrate thereof in the preparation of a reagent for promoting CD8+ T cell infiltration in the tumor microenvironment of intrahepatic cholangiocarcinoma, said use excluding the diagnosis and treatment of the disease.

[0029] In one exemplary embodiment, promoting CD8+ T cell infiltration in the tumor microenvironment of intrahepatic cholangiocarcinoma is achieved by inhibiting the expression or activity of YAP protein in the Hippo pathway.

[0030] According to the third to fifth aspects of this application, when dihydroartemisinin or its pharmaceutically acceptable salts, solvates or hydrates are used to treat intrahepatic cholangiocarcinoma cells, the concentration used, based on the original dihydroartemisinin drug, can be from 0 μM to 1000 mM, but excluding 0 μM, such as, but not limited to, 0.01 μM to 100 mM, 0.05 μM to 10 mM, 0.05 μM to 1 mM, 0.05 μM to 500 μM, 0.05 μM to 300 μM, 0.05 μM to 200 μM, 0.05 μM to 150 μM, 0.05 μM to 120 μM, 0.05 μM to 100 μM, 0.05 μM to 80 μM, 0.05 μM to 60 μM, 0.05 μM to 50 μM, 0.05 μM to 40 μM, 0.05 μM to 20 μM. μM, 0.05 μM to 10 μM, 0.05 μM to 8 μM, 0.05 μM to 6 μM, 0.05 μM to 4 μM, 0.05 μM to 3 μM, 0.05 μM to 2.5 μM, or 0.1 μM to 2.5 μM. When individuals are treated with dihydroartemisinin or its pharmaceutically acceptable salts, solvates, or hydrates, the concentrations used, based on the dihydroartemisinin parent drug, can be from 0.001 mg / kg to 500 mg / kg, such as, but not limited to, 0.01 mg / kg to 300 mg / kg, 0.1 mg / kg to 200 mg / kg, 10 mg / kg to 200 mg / kg, 20 mg / kg to 150 mg / kg, 30 mg / kg to 120 mg / kg, 40 mg / kg to 120 mg / kg, or 50 mg / kg to 100 mg / kg.

[0031] As used herein, the term "reagent" encompasses any substance or composition, alone or in combination, used to achieve a detection or research purpose. In one exemplary embodiment, "reagent" may refer to a non-therapeutic research reagent, which may be individually packaged dihydroartemisinin or a pharmaceutically acceptable salt, solvate, or hydrate thereof, or may be dihydroartemisinin or a pharmaceutically acceptable salt, solvate, or hydrate thereof as a component of a kit. For example, dihydroartemisinin or a pharmaceutically acceptable salt, solvate, or hydrate thereof may be used as a positive control to screen candidate compounds that inhibit intrahepatic cholangiocarcinoma via the Hippo / YAP pathway and / or regulate CD8+ T cell activity, or as a probe component for biomarker detection.

[0032] In a sixth aspect of this application, embodiments of this application provide the use of dihydroartemisinin or a pharmaceutically acceptable salt, solvate or hydrate thereof in the preparation of a medicament for inhibiting the Hippo pathway in intrahepatic cholangiocarcinoma cells and / or tissues and / or reducing the expression level of YAP in intrahepatic cholangiocarcinoma cells and / or tissues; wherein the intrahepatic cholangiocarcinoma is a YAP-high expressing type and / or a CD8+ T cell-low infiltrating type of intrahepatic cholangiocarcinoma.

[0033] In a seventh aspect of this application, embodiments of this application provide the use of dihydroartemisinin or a pharmaceutically acceptable salt, solvate or hydrate thereof in the preparation of a medicament for promoting CD8+ T cell infiltration in the tumor microenvironment of intrahepatic cholangiocarcinoma; wherein the intrahepatic cholangiocarcinoma is YAP-high expressing type and / or CD8+ T cell-low infiltrating type intrahepatic cholangiocarcinoma.

[0034] In an eighth aspect of this application, embodiments of this application provide dihydroartemisinin or pharmaceutically acceptable salts, solvates, or hydrates thereof for use as a medicine. In one exemplary embodiment, this application provides dihydroartemisinin or pharmaceutically acceptable salts, solvates, or hydrates thereof for the treatment and / or prevention of intrahepatic cholangiocarcinoma, wherein the intrahepatic cholangiocarcinoma is YAP-overexpressing and / or CD8+ T-cell-low invasive intrahepatic cholangiocarcinoma.

[0035] In a ninth aspect of this application, embodiments of this application provide a treatment method for intrahepatic cholangiocarcinoma, wherein the intrahepatic cholangiocarcinoma is YAP-high expressing and / or CD8+ T cell-low infiltrating intrahepatic cholangiocarcinoma, the method comprising administering a therapeutically effective amount of dihydroartemisinin or a pharmaceutically acceptable salt, solvate or hydrate thereof to an individual in need.

[0036] In aspects six through nine of this application, dihydroartemisinin or a pharmaceutically acceptable salt, solvate, or hydrate thereof may be used alone or in the form of a pharmaceutical composition. In one exemplary embodiment, the pharmaceutical composition comprises dihydroartemisinin or a pharmaceutically acceptable salt, solvate, or hydrate thereof, and a pharmaceutically acceptable carrier. The pharmaceutical composition may be formulated into any clinically or pharmaceutically acceptable dosage form, such as tablets, capsules, pills, granules, solutions, suspensions, syrups, injections (including injection solutions, sterile powders for injection, and concentrated solutions for injection), suppositories, inhalers, or sprays. In one exemplary embodiment, dihydroartemisinin or a pharmaceutically acceptable salt, solvate, or hydrate thereof, or a pharmaceutical composition comprising it, may be administered orally, parenterally, rectally, or pulmonaryly to a patient or subject requiring such treatment. When intended for oral administration, the pharmaceutical composition can be formulated into oral preparations, such as conventional oral solid dosage forms like tablets, capsules, pills, and granules; or into oral liquid preparations like oral solutions, oral suspensions, and syrups. When formulating oral preparations, suitable fillers, binders, disintegrants, lubricants, etc., can be added. When intended for parenteral administration, the above-mentioned pharmaceutical preparations can also be formulated into injections, including injection solutions, sterile powders for injection, and concentrated solutions for injection. When formulating injections, conventional methods in the existing pharmaceutical field can be used. When preparing injections, excipients may not be added, or suitable excipients may be added depending on the properties of the drug. When intended for rectal administration, the pharmaceutical composition can be formulated into suppositories, etc. When intended for pulmonary administration, the pharmaceutical composition can be formulated into inhalers or sprays, etc.

[0037] As used herein, the term "individual" refers to mammals, primates (e.g., humans (male or female)), dogs, rabbits, guinea pigs, pigs, rats, and mice. In one exemplary embodiment, the subject is a primate. In another exemplary embodiment, the subject is a human.

[0038] As used herein, the term “treatment” for any disease or disorder means relief or improvement of the disease or disorder (i.e., slowing or halting the development of the disease or at least one of its clinical symptoms); or relief or improvement of at least one physical parameter or biomarker associated with the disease or disorder, including physical parameters or biomarkers that the patient may not be able to identify.

[0039] As used herein, the term “prevention” for any disease or disorder means preventive treatment of the disease or disorder; or delaying the onset or progression of the disease or disorder.

[0040] As used in this article, a subject is considered "needing" treatment if they or an individual would benefit from it biologically, medically, or in terms of quality of life.

[0041] As used herein, the term "therapeuticly effective amount" of a compound of this application refers to the amount of the compound of this application that will induce a biological or medical response in a subject (e.g., a reduction or inhibition of enzyme or protein activity, or improvement of symptoms, relief of symptoms, slowing or delaying disease progression, or prevention of disease, etc.). In an exemplary embodiment, the therapeutically effective amount, based on the prototype dihydroartemisinin, is from 0.001 mg / kg to 500 mg / kg, such as, but not limited to, 0.01 mg / kg to 300 mg / kg, 0.1 mg / kg to 200 mg / kg, 10 mg / kg to 200 mg / kg, 20 mg / kg to 150 mg / kg, 30 mg / kg to 120 mg / kg, 40 mg / kg to 120 mg / kg, or 50 mg / kg to 100 mg / kg.

[0042] As used herein, the term "pharmaceutically acceptable salt" means that the compounds of the present invention retain their biological efficacy and properties and are typically not biologically or otherwise undesirable. In many cases, the compounds of the present invention are capable of forming acid salts and / or base salts due to the presence of amino and / or carboxyl groups or similar groups; wherein pharmaceutically acceptable acid addition salts can be formed with inorganic and organic acids; inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc.; organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid, sulfosalicylic acid, etc.; pharmaceutically acceptable base addition salts can be formed with inorganic and organic bases; inorganic bases from which salts can be derived include, for example, ammonium salts and metals from columns I to XII of the periodic table. In some embodiments, the salt is derived from sodium, potassium, ammonium, calcium, magnesium, iron, silver, zinc, and copper; particularly suitable salts include ammonium salts, potassium salts, sodium salts, calcium salts, and magnesium salts; organic bases from which the salt can be derived include, for example, primary, secondary, and tertiary amines; substituted amines (including naturally occurring substituted amines); cyclic amines; basic ion exchange resins, etc. Some organic amines include isopropylamine, benzylamine, choline salts, diethanolamine, diethylamine, lysine, meglumine, piperazine, and tromethamine.

[0043] As used herein, the term “pharmaceutically acceptable carrier” means a substance that can be used in the preparation or use of a pharmaceutical composition, and includes, for example, suitable diluents, solvents, dispersion media, surfactants, antioxidants, preservatives, isotonic agents, buffers, emulsifiers, absorption delay agents, salts, pharmaceutical stabilizers, binders, excipients, disintegrants, lubricants, wetting agents, sweeteners, flavoring agents, dyes, and combinations thereof, as known to those skilled in the art (see, for example, Remington: The Science and Practice of Pharmacy, 22nd ed., Pharmaceutical Press, 2013, pp. 1049-1070).

[0044] Example The materials used in the embodiments are shown below: 1. Reagents and materials Dihydroartemisinin (S2290 Selleck), dimethyl sulfoxide (DMSO, D8371 Solarbio), CCK8 kit (C0038 Beyotime), PVP K30 (DR0565 Harveybio), Ki67 (#ab92742 Abcam), Active-YAP (29495S CST), YAP / TAZ (8418S CST), Lipo-293 TM Plus transfection reagent (C0522 Beyotime), Polybrene (G1803-1ML Servicebio), Puromtcin (P8230 Solarbio), Matrigel (354234 Corning), Anti-CD8 alpha Rabbit pAb (GB114196-100 Servicebio), Anti-CD3 Rabbit mAb (GB13014-50 Servicebio), Anti-mouse CD8α-InVivo (A2102 Selleck) 2. Instruments Multifunctional microplate reader (SPARK10M TECAN) Example 1. Dihydroartemisinin (DHA) inhibits the survival of intrahepatic cholangiocarcinoma cells in vitro. In this embodiment, the CCK8 reagent kit was used to detect the inhibitory effect of dihydroartemisinin on intrahepatic cholangiocarcinoma cells in vitro.

[0045] CCK8 Detection Principle: WST-8 (chemical name 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonic acid phenyl)-2H-tetrazole monosodium salt) can be reduced to orange-yellow formazan by certain dehydrogenases in the mitochondria in the presence of an electron coupling reagent. The more and faster the cell proliferation, the darker the color; the greater the cytotoxicity, the lighter the color. For the same number of cells, the color intensity is linearly related to the cell number.

[0046] Intrahepatic cholangiocarcinoma cell lines with high YAP expression: Human intrahepatic cholangiocarcinoma RBE cells were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences (Catalog No.: THu179), Hu-CCT1 cells were purchased from Zhejiang Meisen Cell Technology Co., Ltd. (Catalog No.: CTCC-003-0101), and QBC-939 and HCCC-9810 cells were kindly donated by Dr. Xu Lei of the Department of Gastroenterology, Nanjing Drum Tower Hospital.

[0047] Drug solution preparation: Weigh 14.2175 mg of dihydroartemisinin and dissolve it in 1 mL of DMSO to prepare a 50 mM stock solution. Aliquot the stock solution and store it at -20 ℃ for later use. When using, dilute it with RPMI 1640 (gibco C11875500BT) cell culture medium to a dihydroartemisinin concentration of 1 μM to 128 μM.

[0048] Experimental methods: RBE, Hu-CCT1, HCCC-9810, and QBC-939 cells in logarithmic growth phase were seeded into 96-well plates, with 100 μL of cell suspension seeded into each well, and the cell density per well was adjusted to 5 × 10⁶ cells / well. 3 Cells were cultured at 37 ℃ in a 5% CO2 incubator until adherence was achieved. Experimental groups (containing 1 μM to 128 μM dihydroartemisinin), blank control groups (containing no cells, but with the same volume of cell culture medium as the experimental groups), and cell control groups (containing the same volume of culture medium without dihydroartemisinin as the experimental groups) were set up, with 6 replicates per group, and cultured for 72 h. The supernatant was removed, 90 μL of fresh culture medium was added, and 10 μL of CCK8 was added to each well. Incubation continued for 2 h, and the absorbance at 450 nm (A450nm) was measured using a microplate reader to calculate cell viability. The blank control group was set up to subtract background values ​​when measuring absorbance OD values ​​(A450nm); the cell control group was set up to measure cell viability under normal culture conditions; and the experimental groups were used to determine the effect of different concentrations of dihydroartemisinin on cell viability. Calculation formula: Cell viability (%) = (Experimental group A450nm - Blank group A450nm) / (Cell control group A450nm - Blank group A450nm) × 100%.

[0049] Test results: like Figure 1 As shown, it indicates that dihydroartemisinin can significantly inhibit the survival of intrahepatic cholangiocarcinoma cells, showing concentration-dependence.

[0050] Example 2. Inhibition of the proliferation of intrahepatic cholangiocarcinoma in situ in mice by dihydroartemisinin Experimental animals: C57BL / 6N mice (wild type), male, 6 to 8 weeks old. Throughout the adaptation and study period, all animals were maintained on a 12 h light-dark cycle (21 °C ± 2 °C, relative humidity of 45% ± 10%) under specific pathogen-free conditions and had free access to food and water. All animal experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health of the United States and were approved by the Laboratory Animal Ethics Committee of Capital Medical University. The mice were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd., license number: SCXK (Beijing) 2021-0011.

[0051] Method for constructing an intrahepatic cholangiocarcinoma in situ model of sgP19 / KRAS The reference for constructing the sgP19-KRAS in situ model is Wang J, Xu Z, Xu L, et al. UncouplingMetastasis and Epithelial-to-Mesenchymal Transition in sgP19 / kRAS-DrivenSpontaneous Metastatic Liver Tumor Model. Adv Sci (Weinh). 2026 Feb;13(8):e14198. The sgP19-KRAS in situ model is a genetically engineered mouse model established based on CRISPR / Cas9 technology. By simultaneously delivering sgRNA targeting the p19 gene and the activated form of the KRAS mutant (KRAS G12D ), ICC tumors can be induced to form in the mouse liver. This model mimics the common oncogenic KRAS activation mutation and protein P19ARF deletion in human ICC, and can induce the formation of in situ tumors with typical ICC pathological characteristics in the mouse liver, better restoring the malignant progression process of ICC.

[0052] Plasmids were delivered to the livers of 6-8 week old male C57 mice using high-pressure hydrodynamic tail vein injection. The plasmid volume was 10% of the mouse's body weight. The plasmid ratio for the sgP19 / KRAS model was: sgP19 (10 μg), KRAS (25 μg), and SB (5 μg) (SB, kRASG12D transposon plasmid, and sgP19 plasmid were provided by L. Zender of the University of Tübingen, Germany). The experimental procedures included: 1) Pre-experimental preparation: Measuring mouse weight and ear-tagging mice with ear-tag pliers, adjusting the plasmid injection volume according to mouse weight; 2) Irradiating mice under an infrared lamp for 30 minutes to dilate the tail vein, fixing the mice, and exposing the tail; wiping the mouse tail with an alcohol wipe, the tail vein was clearly visible; 3) Slowly injecting a small amount of plasmid one-third of the way down the tail tip. After no obvious resistance was observed, it indicated that the needle had entered the tail vein. The remaining plasmid was injected within 6-8 seconds.

[0053] Drug preparation: Dissolve dihydroartemisinin in 15% polyvinylpyrrolidone (PVP) solution to prepare 50 mg / mL and 100 mg / mL solutions, and store at 4 ℃ for later use. Establishment of an sgP19 / KRAS intrahepatic cholangiocarcinoma in situ model and the therapeutic effect of dihydroartemisinin: Eighteen experimental mice were acclimatized for one week and randomly divided into three groups of six mice each. A mouse model of intrahepatic cholangiocarcinoma in situ was established using the high-pressure tail vein injection technique described above. Two groups were set up: a model group (Vehicle) and a model group + dihydroartemisinin administration group (50 mg / kg and 100 mg / kg). The model group was administered an equal volume of physiological saline by gavage, while the model group + administration group was administered different doses of dihydroartemisinin by gavage. After four weeks of administration, liver weight was recorded at the predetermined experimental endpoint, and the tissue was subjected to histopathological staining, including HE staining, Ki67 staining, and PCNA staining. Positive Ki67 and PCNA staining indicates active cell proliferation; under pathological conditions, an increase in Ki67-positive and PCNA-positive cells may indicate rapid tissue or tumor growth.

[0054] Result: As Figure 2 As shown, dihydroartemisinin was observed to have a significant inhibitory effect on a mouse model of intrahepatic cholangiocarcinoma in situ. Figure 3 Ki67 and PCNA staining results also showed that dihydroartemisinin can inhibit the proliferation of intrahepatic cholangiocarcinoma.

[0055] Example 3. Dihydroartemisinin inhibits tumor development by suppressing the Hippo pathway. (I) RNA transcriptomics analysis and Western blot analysis RNA transcriptomics sequencing was performed on tissue samples from the sgP19 / KRAS orthotopic carcinoma model. The steps included: (1) Sample preparation Immediately after the mice were euthanized by cervical dislocation at the end of Example 2, tissue samples were collected. The tissues were washed three times with physiological saline, and non-tumor parts such as connective tissue and adipose tissue were removed. The collected tumor samples were placed in 1.5 ml RNase-free EP tubes, flash-frozen on dry ice, and then temporarily stored at -80 ℃.

[0056] (2) Library construction and sequencing Following the manufacturer's instructions (Illumina Inc., CA, USA), sequencing libraries were prepared using the Illumina TruSeqstranded total RNA with Ribo-Zero gold kit. First, the rRNA-free RNA was fragmented, and a cDNA library was constructed using the Tru Seq RNA sample Prep Kit (Illumina, San Diego, CA, USA). Procedures included end repair, adapter ligation, PCR amplification, PCR product purification, library enrichment, and library quality control. Following the instructions, the library was sequenced on the Illumina Hi seq 2500 platform (Illumina, San Diego, CA, USA). The raw fluorescence imaging data obtained through the Hi seq high-throughput sequencing platform was converted into raw sequencing data (Raw data saved in Fast Q format). The Raw data underwent data preprocessing to filter out rRNA, tRNA sequences, and low-quality reads, obtaining Clean reads for subsequent data analysis.

[0057] (3) Data Analysis Data quality was assessed, and adapters and low-quality sequences were removed. Sequence alignment was then performed, and gene expression was quantified. Differential expression analysis and functional enrichment analysis, such as KEGG enrichment analysis and GSEA analysis, were performed using R packages. Finally, visualizations were generated using the R package ggplot2. The R packages included were: (DESeq2, 1.46.0), (clusterProfiler, 4.14.6), (enrichplot, 1.26.6), (org.Mm.eg.db, 3.20.0), (ggplot2, 4.0.2), (dplyr, 1.1.4), (tidyr, 1.3.1), (stringr, 1.5.1), (msigdbr, 24.1.0), (biomaRt, 2.62.1), and (data.table, 1.16.4). The p-value threshold for KEGG enrichment analysis was p < 0.05. GSEA analysis uses the h.all or c2.cp.kegg statistical method of a specific version (e.g., v7.4) in the MSigDB database as the default weighted enrichment analysis, with a significance threshold (p < 0.05, NES > 1.5). Western blotting analysis was used to detect the expression of Hippo pathway proteins.

[0058] result: like Figure 4 As shown in Figure A, DHA can regulate multiple pathways, including the Hippo pathway, and it has an inhibitory effect on the Hippo pathway. Figure 4 (B) Figure 4 In the C, NL represents normal liver tissue, YAP refers to the sum of all YAP proteins in the cell, and Active-YAP specifically refers to dephosphorylated, biologically active YAP. After entering the cell nucleus, Active-YAP binds to transcription factors such as TEAD, initiating downstream gene programs that promote proliferation and inhibit apoptosis, directly driving cancer progression. Figure 4 Western blotting analysis of the C-cells showed that the expression of Active-YAP and YAP was reduced compared to the control group.

[0059] (II) Inhibitory effect of dihydroartemisinin on in vitro ICC cells after YAP knockout using CRISPR / Cas9 To verify the key role of YAP in the anti-intrahepatic cholangiocarcinoma effect of dihydroartemisinin as demonstrated in the above studies, this study utilized CRISPR / Cas9 technology to knock out the YAP gene, constructing four sgYAP plasmids. The constructed plasmids were transfected into the QBC-939 cell line. After screening for stably transfected cells, the inhibitory effect of DHA on ICC cells after YAP knockout was detected using CCK-8 assay. Specifically, the experimental methods are as follows: (1) Design of YAP-targeting sgRNA and construction of plasmids: Table 1. Target sequence of sgYAP plasmid (2) Cell transfection and selection of stable cell lines: Transfection was performed using the lentivirus packaging method, and the transfection reagents used were as follows: (1) Lentiviral packaging 1) 293T cells were seeded into six-well plates at a concentration of 2 × 10⁻⁶. 5 Cells per well. When the cell confluence reaches 60%-70%, begin the transfection process.

[0060] 2) Discard the old culture medium and replace it with 2 ml of fresh culture medium (containing 10% serum and no antibiotics).

[0061] 3) Prepare the transfection system according to the table below. 293T cell lentivirus transfection system After mixing the separately prepared solution A and solution B thoroughly, let them stand at room temperature for 15 minutes.

[0062] 4) Add 250 μL of the solution obtained in the above steps to a 6-well plate slowly and evenly.

[0063] 5) After culturing for 48 h, collect the supernatant, store it in a 4 °C refrigerator for later use, and add fresh culture medium (containing 10% serum and no antibiotics) to the 6-well plate.

[0064] 6) After culturing for 72 h, collect the supernatant, mix it with the supernatant collected for 48 h, filter it through a 0.45 μm filter membrane, aliquot it and store it in a -80 °C freezer for later use.

[0065] (2) Lentiviral transfection of target cells 1) Seed the target cells into six-well plates at a seeding density of 2 × 10⁶ cells / well. 5 Cells / well

[0066] 2) Mix the virus solution and culture medium at a ratio of 1:1, add polybrene (10 mg / ml), and prepare a solution with a final concentration of 8 mg / ml.

[0067] 3) Discard the culture medium in the six-well plate and replace it with the solution obtained in the previous step.

[0068] 4) 72 h after transfection, digest the cells and seed them into new culture dishes. After the cells adhere, select the target cells using culture medium containing puromycin (2 μg / ml).

[0069] 5) When the cell density reaches 80%-90%, collect the cells and verify the knockout effect using Western blotting. Continue adding cells for further selection to prevent a decrease in the editing effect of the mutated gene during passage.

[0070] (3) CCK-8 assay for cell proliferation inhibition: Logarithmic growth phase sgYAP and sgGFP cells were seeded into 96-well plates, with 100 μL of cell suspension in each well, and the cell density per well was adjusted to 5 × 10⁶ cells / well. 3 Cells were cultured at 37 ℃ in a 5% CO2 incubator until adherence was achieved. Experimental groups (containing 1 μM to 128 μM dihydroartemisinin), blank control groups (containing no cells, but the same volume of cell culture medium as the experimental groups), and cell control groups (containing the same volume of culture medium without dihydroartemisinin as the experimental groups) were set up, with 6 replicates per group, and cultured for 72 h. The supernatant was removed, and 90 μL of fresh culture medium was added to each well, along with 10 μL of CCK8. Incubation continued for 2 h, and the absorbance at 450 nm (A450nm) was measured using a microplate reader to calculate cell viability. The blank control group was set up to subtract background values ​​when measuring absorbance OD values ​​(A450nm); the cell control group was set up to measure cell viability under normal culture conditions; and the experimental groups were used to determine the effect of different concentrations of dihydroartemisinin on cell viability. Calculation formula: Cell viability (%) = (Experimental group A450nm - Blank group A450nm) / (Cell control group A450nm - Blank group A450nm) × 100%.

[0071] result: like Figure 5 As shown in Figure B, knocking out YAP increased the half-maximal inhibitory concentration (ICC) of dihydroartemisinin in ICC cells to varying degrees. This result confirms that YAP plays a crucial role in the in vitro anti-ICC effect of DHA.

[0072] (III) Inhibitory effect of dihydroartemisinin on in vivo ICC subcutaneous tumors after YAP knockout using CRISPR / Cas9 To better verify the role of YAP, further in vivo studies were conducted. The sgYAP2 plasmid was transfected into primary sgP19 / KRAS cells using lentiviral packaging, following the transfection method described in (II).

[0073] Constructing a subcutaneous tumor model: (1) Cell preparation 1) Collect cells in the logarithmic growth phase, ideally with a cell density of approximately 80%-90%. Replace the culture medium with fresh one the night before collecting the cells.

[0074] 2) Cell pretreatment: Cells were collected by washing, digestion, and centrifugation. After centrifugation, the cells were washed twice with PBS to obtain a cell pellet, which was then resuspended in pre-cooled PBS. After mixing, the cells were counted.

[0075] 3) Resuspend the cells in PBS buffer containing matrix gel at a 1:1 ratio to adjust the cell density to 2.5 × 10⁶ cells / year. 5 200 μl of each cell was prepared. The prepared cell solution was aliquoted and placed on ice for later use.

[0076] (2) Animal preparation Tumor cells can be injected into 5-6 week old C57 mice. The specific injection steps are as follows: 1) Before the experiment, the back of the mouse needs to be prepared so that the injection area is free of fur to facilitate the injection of tumor cells, and the mouse should be ear-tagged.

[0077] 2) Keep the mouse with its back facing up, and pinch the skin on the back of the mouse's neck with your thumb and forefinger.

[0078] 3) The injection needle is inserted from the tail of the mouse toward the head, and inserted into the skin on the back of the neck to form a triangular area.

[0079] 4) The needle can be gently moved left and right. If it moves easily, it means that it has entered the subcutaneous tissue. Then gently aspirate. After observing that there is no blood return, you can slowly push the liquid in.

[0080] 5) To prevent leakage, after insertion, allow the needle to remain subcutaneously for a few seconds; when removing the needle, hold the insertion site firmly with your thumb and forefinger for a moment.

[0081] 6) After the injection, put the mouse back in its cage and observe its activity level.

[0082] (3) Grouping and administration On day 4 after inoculation with primary cells, the subcutaneous tumor volume in mice reached 30-40 mm. 3 Around 10:00. At this time, the subcutaneous tumor model mice were randomly divided into two groups: (1) saline control group, which was given an equal volume of saline by gavage every day. (2) DHA administration group, which was given DHA at a concentration of 100 mg / kg by gavage every day, 6 times a week. During the administration period, the length and width of the subcutaneous tumor were measured with calipers every 2 days, the volume of the subcutaneous tumor was calculated, and the weight of the mice was measured once. After 15 days of administration, the mice were sacrificed for sampling (experimental procedure as follows). Figure 5 As shown in Figure C, the subcutaneous tumor tissue was photographed and weighed.

[0083] result: like Figure 5 As shown in the DE curve, there was no significant difference in the size and weight of the subcutaneous tumors compared to the control group after drug administration. The growth curves of the mouse subcutaneous tumors showed no significant difference in tumor growth rate between the drug-treated group and the control group. These results indicate that YAP also plays a crucial role in DHA's anti-ICC response in vivo.

[0084] Example 4. Dihydroartemisinin indirectly inhibits tumor development via CD8+ T cells. CD8 T cells are "precision-guided killers" in the immune system responsible for directly eliminating cancer cells. They are born in the thymus, activated by antigen presentation and co-stimulatory signals, and precisely destroy targets using perforin / granzyme.

[0085] (I) Immunohistochemical staining To investigate how dihydroartemisinin suppresses tumor development through immunosuppression, this study fixed the tissues of the in situ carcinoma model from Example 2 and sent them to Seville Biosciences for immunohistochemical staining. The percentage of positive cells was statistically analyzed using Image J.

[0086] result: like Figure 6 The study showed that DHA treatment not only significantly increased the infiltration rate of total T cells (CD3+) in intrahepatic cholangiocarcinoma tissues, but more importantly, it specifically upregulated the relative abundance of cytotoxic T cells (CD8+) in the immune microenvironment. This suggests that dihydroartemisinin can reshape the tumor immunosuppressive microenvironment, polarizing it towards a cytotoxic phenotype that is favorable to the host's anti-tumor immunity.

[0087] (II) sgP19-KRAS in situ carcinoma model To further verify the role of CD8 T cells, an sgP19-KRAS in situ carcinoma model was constructed according to the method in Example 2. Mice were then randomly divided into four groups: (1) a saline control group, receiving an equal volume of saline via gavage daily; (2) a DHA administration group (DHA), receiving 100 mg / kg of DHA via gavage daily, 6 times a week for 4 weeks; (3) a CD8 clearance group (CD8), receiving 200 μg / μL of CD8 antibody (Anti-mouse CD8α-InVivo) intraperitoneally every 2 days for 2 weeks; and (4) a combined DHA and CD8 clearance group (DHA+CD8), receiving 200 μg / μL of CD8 antibody intraperitoneally every 2 days for 2 weeks, and receiving 100 mg / kg of DHA via gavage daily, 6 times a week for 4 weeks. Mice were sacrificed after administration for sample collection (experimental procedure as follows). Figure 6 (As shown in B), record the mouse's body weight and liver weight, and take a photo for record-keeping.

[0088] result: like Figure 6 As shown in the CD10 images, liver photographs after sampling revealed that tumors enlarged after CD8 removal, with the combined group showing larger tumors than the DHA-only group. Liver weight analysis showed that the combined group's DHA-induced tumor suppression effect was significantly lower than the DHA-only group. These results indicate that DHA partially exerts its effects through the immune system and can indirectly inhibit tumor development by activating CD8+ T cells.

[0089] In summary, this application experimentally demonstrates that dihydroartemisinin can inhibit the survival of intrahepatic cholangiocarcinoma cells; dihydroartemisinin has a certain inhibitory effect on a mouse model of intrahepatic cholangiocarcinoma in situ; and dihydroartemisinin targeting YAP in the Hippo pathway not only directly inhibits tumor growth but also relieves the inhibitory effect of YAP on CD8+ T cells, enhancing the efficacy of tumor immunotherapy. It has broad application prospects in the development of novel targeted drugs for the treatment of intrahepatic cholangiocarcinoma.

[0090] Although embodiments of this application have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting this application. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of this application.

Claims

1. Use of dihydroartemisinin or its pharmaceutically acceptable salts, solvates or hydrates in the preparation of Hippo pathway inhibitors in intrahepatic cholangiocarcinoma.

2. Use of dihydroartemisinin or its pharmaceutically acceptable salts, solvates or hydrates in the preparation of YAP inhibitors for intrahepatic cholangiocarcinoma.

3. The use according to claim 2, characterized in that, Dihydroartemisinin or its pharmaceutically acceptable salts, solvates or hydrates inhibit the expression of total YAP protein and / or dephosphorylated YAP protein.

4. Use of dihydroartemisinin or its pharmaceutically acceptable salts, solvates or hydrates in the preparation of reagents for inhibiting the Hippo pathway in intrahepatic cholangiocarcinoma cells and / or tissues, excluding the diagnosis and treatment of diseases.

5. Use of dihydroartemisinin or its pharmaceutically acceptable salts, solvates or hydrates in the preparation of reagents for reducing YAP expression levels in intrahepatic cholangiocarcinoma cells and / or tissues, wherein such use does not include the diagnosis and treatment of diseases; Optionally, the YAP includes total YAP protein and / or dephosphorylated YAP protein.

6. Use of dihydroartemisinin or its pharmaceutically acceptable salts, solvates or hydrates in the preparation of reagents for promoting CD8+ T cell infiltration in the tumor microenvironment of intrahepatic cholangiocarcinoma, wherein the use does not include the diagnosis and treatment of the disease.

7. The use according to claim 6, characterized in that, The promotion of CD8+ T cell infiltration in the tumor microenvironment of intrahepatic cholangiocarcinoma is achieved by inhibiting the expression or activity of YAP protein in the Hippo pathway.

8. The use of dihydroartemisinin or its pharmaceutically acceptable salts, solvates or hydrates in the preparation of medicaments for inhibiting the Hippo pathway and / or reducing YAP expression levels in intrahepatic cholangiocarcinoma cells and / or tissues; wherein, The intrahepatic cholangiocarcinoma is a YAP-high expression type and / or a CD8+ T cell-low infiltrative type of intrahepatic cholangiocarcinoma.

9. Use of dihydroartemisinin or its pharmaceutically acceptable salts, solvates or hydrates in the preparation of medicaments for promoting CD8+ T cell infiltration in the tumor microenvironment of intrahepatic cholangiocarcinoma; wherein, The intrahepatic cholangiocarcinoma is a YAP-high expression type and / or a CD8+ T cell-low infiltrative type of intrahepatic cholangiocarcinoma.

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