Use of tafamidis in activation of RXR activity

Abstract

Disclosed is the use of a compound as shown in formula (I) or a tautomer, stereoisomer, solvate, or pharmaceutically acceptable salt thereof in the preparation of a drug, wherein the drug is used for preventing and / or treating related diseases mediated by a retinoid X receptor (RXR). The compound as shown in formula (I) can bind to RXR and effectively regulate RXR activity, thereby effectively preventing and / or treating RXR-related diseases.

Description

Use of clomazone in activating RXR activity TECHNICAL FIELD

[0001] The present application belongs to the technical field of biopharmaceuticals, and particularly relates to use of clomazone in modulating RXR activity, more particularly to use of clomazone in preventing and / or treating RXR-related diseases, use of clomazone in modulating RXR activity, a method for modulating RXR activity, a combination drug or a kit and use thereof, and a method for preventing and / or treating RXR-related diseases. BACKGROUND

[0002] RXRα has a special position in the nuclear receptor family because it can form heterodimers with multiple members of the nuclear receptor family, and in this way RXRα participates in numerous nuclear receptor-mediated biological activities. The biological activities of RXRα are mainly divided into genotypic and non-genotypic activities. The genotypic activity of RXRα mainly manifests in its binding to DNA and thereby regulating the transcription of target genes. In the absence of ligands or inhibitory ligands, transcriptional repressors (such as N-CoR and SMRT) bind to RXRα, inhibiting the transcriptional activity of RXRα; when an activating ligand (such as 9-cis-RA) binds to RXRα, it induces a conformational change in RXRα, thereby causing the transcriptional repressors to dissociate from RXRα and promoting the recruitment of transcriptional activators (such as p300 / CBP and HAT) to RXRα, activating the transcriptional activity of RXRα. In addition to its genotypic function in the nucleus, RXRα also has extensive non-genotypic activity. For example, RXRα can bind to TR3 (also known as Nur77, NGFI-B or NR4A1), and under the stimulation of an apoptosis-inducing factor, it is transferred from the nucleus to the cytoplasm, and by binding to Bcl-2, it is localized on the mitochondria and induces a conformational change in Bcl-2. This conformational change causes Bcl-2 to change from an apoptosis-inhibiting molecule to an apoptosis-inducing molecule, inducing cell apoptosis.

[0003] The important difference between nuclear receptors and other transcription factors is that the activity of nuclear receptors can be regulated by specific small molecule ligands. A number of endogenous, natural and artificially synthesized RXRa ligands have been identified, such as 9-cis-RA, DHA (docosahexaenoic acid), CD3254 and UVI3003, and the like. According to the function of regulating the transcriptional activity of RXRa, the small molecule ligands are mainly divided into activating ligands and inhibiting ligands, and these small molecule ligands mainly bind to the LBP of the LBD domain of the RXRa protein. The small molecule ligand interacts with the side chain of the amino acid residue in the LBP through ionic bond, hydrogen bond and van der Waals force, and then induces the displacement of these amino acid residues, resulting in the conformational change of RXRa. The ligand induces the conformational change of RXRa, and then regulates the interaction of RXRa with proteins or nucleic acids. Ligand binding can also regulate the modification of RXRa, such as phosphorylation, ubiquitination, acetylation and sumoylation, and then regulate the stability and subcellular localization of RXRa. In summary, the ligand will affect the genotypic and non-genotypic activity of RXRa by binding.

[0004] Therefore, RXRa can be used as a target of a drug, and it is very important to develop new small molecule ligands targeting RXRa. SUMMARY

[0005] The present application aims to solve at least one of the technical problems in the prior art to some extent. To this end, the present application provides a use of tafamidis in regulating the activity of RXRa, which can target RXRa, regulate the activity of RXRa, and be used for preventing and / or treating RXRa-related diseases.

[0006] The present application is based on the following findings of the inventors:

[0007] Targretin is a selective agonist of RXRa, which has been approved for marketing as a drug for treating psoriasis, acne, Kaposi's sarcoma and cutaneous T-cell lymphoma, and the like. In addition, the agonist 9cUAB of RXRa has also carried out clinical trials for preventing cancer. It can be seen that RXRa can be used as a target of a drug, and small molecule ligands can play a therapeutic role in diseases by targeting RXRa, so it is very important to develop new small molecule ligands targeting RXRa.

[0008] Based on this, the inventors first found that tafamidis can bind to RXR (especially RXRa) and regulate the activity of RXR by screening small molecule ligands targeting RXR (such as RXRa, RXRβ, RXRγ). It is further verified that tafamidis has a good therapeutic effect on a variety of RXR-related diseases.

[0009] In a first aspect of the present application, the present application provides a use of a compound represented by Formula (I) or a tautomer, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing and / or treating an RXR-related disease;

[0010] According to embodiments of the present application, the compound represented by Formula (I) or a tautomer, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof can target RXR and modulate the activity of RXR. Thus, the medicament prepared from the compound represented by Formula (I) or a tautomer, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof can effectively prevent and / or treat an RXR-related disease.

[0011] According to embodiments of the present application, the RXR-related disease includes an RXRα-related disease, an RXRβ-related disease, and an RXRγ-related disease.

[0012] According to embodiments of the present application, the RXR-related disease includes a metabolic disease, inflammation, a neurodegenerative disease, a tumor, or a cancer.

[0013] According to embodiments of the present application, the RXRα-related disease includes alcoholic fatty liver disease, non-alcoholic fatty liver disease, hepatitis, liver fibrosis, liver cirrhosis, liver cancer, Alzheimer's disease, Parkinson's disease, neuroinflammation, a neural tumor, psoriasis, acne, psoriasis, lupus erythematosus, skin fibrosis, Kaposi's sarcoma, cutaneous T-cell lymphoma, nasopharyngitis, bronchitis, pneumonia, pulmonary fibrosis, a lung tumor, enteritis, intestinal fibrosis, an intestinal tumor, gastritis, a gastric tumor, urethritis, nephritis, renal fibrosis, a renal tumor, cholestasis, leukemia, diabetes, osteoporosis, arthritis, spondylitis, cholangitis, cholangiocarcinoma, atherosclerosis, myocarditis, hyperlipidemia, liver damage, fatty liver, lung cancer, colorectal cancer.

[0014] In a second aspect of the present application, the present application provides a use of a compound represented by Formula (I) or a tautomer, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof in the prevention and / or treatment of an RXRα-related disease;

[0015] According to embodiments of the present application, the compound represented by Formula (I) or a tautomer, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof can target RXR and modulate the activity of RXR, and can effectively prevent and / or treat an RXR-related disease.

[0016] According to embodiments of the present application, the RXR-related disease includes an RXRα-related disease, an RXRβ-related disease, and an RXRγ-related disease.

[0017] According to embodiments of the present application, the RXR-related disease includes a metabolic disease, inflammation, a neurodegenerative disease, a tumor, or a cancer.

[0018] According to embodiments of the present application, the RXR-related disease includes alcoholic fatty liver disease, non-alcoholic fatty liver disease, hepatitis, liver fibrosis, liver cirrhosis, liver cancer, Alzheimer's disease, Parkinson's disease, neuroinflammation, a neural tumor, psoriasis, acne, psoriasis, lupus erythematosus, skin fibrosis, Kaposi's sarcoma, cutaneous T-cell lymphoma, nasopharyngitis, bronchitis, pneumonia, pulmonary fibrosis, a lung tumor, enteritis, intestinal fibrosis, intestinal tumor, gastritis, gastric tumor, urethritis, nephritis, renal fibrosis, renal tumor, cholestasis, leukemia, diabetes, osteoporosis, arthritis, spondylitis, cholangitis, cholangiocarcinoma, atherosclerosis, myocarditis, hyperlipidemia, liver damage, fatty liver, lung cancer, colorectal cancer.

[0019] In a third aspect of the present application, the present application provides a compound represented by formula (I) or a tautomer, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof for use in the prevention and / or treatment of an RXR-related disease.

[0020] According to embodiments of the present application, the compound represented by formula (I) or a tautomer, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof can target RXR and modulate RXR activity, and can be effective in the prevention and / or treatment of an RXR-related disease.

[0021] According to embodiments of the present application, the RXR-related disease includes an RXRα-related disease, an RXRβ-related disease, or an RXRγ-related disease.

[0022] According to embodiments of the present application, the RXR-related disease includes a metabolic disease, inflammation, a neurodegenerative disease, a tumor, or a cancer.

[0023] According to embodiments of the present application, the RXR-related disease includes alcoholic fatty liver disease, non-alcoholic fatty liver disease, hepatitis, liver fibrosis, liver cirrhosis, liver cancer, Alzheimer's disease, Parkinson's disease, neuroinflammation, a neural tumor, psoriasis, acne, psoriasis, lupus erythematosus, skin fibrosis, Kaposi's sarcoma, cutaneous T-cell lymphoma, nasopharyngitis, bronchitis, pneumonia, pulmonary fibrosis, a lung tumor, enteritis, intestinal fibrosis, intestinal tumor, gastritis, gastric tumor, urethritis, nephritis, renal fibrosis, renal tumor, cholestasis, leukemia, diabetes, osteoporosis, arthritis, spondylitis, cholangitis, cholangiocarcinoma, atherosclerosis, myocarditis, hyperlipidemia, liver damage, fatty liver, lung cancer, colorectal cancer.

[0024] In a fourth aspect of the present application, the present application provides a use of a compound of Formula (I) or a tautomer, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof in the preparation of a reagent for modulating RXR activity or modulating activity of a dimer containing RXR;

[0025] According to embodiments of the present application, the compound of Formula (I) or a tautomer, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof can target RXR and modulate RXR activity. Thus, the reagent prepared by using the compound of Formula (I) or a tautomer, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof can effectively modulate RXR activity or modulate activity of a dimer containing RXR, especially for modulating activity of RXR or a dimer containing RXR in vitro.

[0026] According to embodiments of the present application, the dimer containing RXR includes a RXR homodimer, a heterodimer containing RXR.

[0027] According to embodiments of the present application, the dimer containing RXR includes a dimer containing RXRα, a dimer containing RXRβ, a dimer containing RXRγ.

[0028] According to embodiments of the present application, the heterodimer containing RXR is selected from RXR / RAR, RXR / PPAR, RXR / LXR, RXR / Nur77, RXR / VDR, RXR / TR, RXR / FXR, RXR / CAR, RXR / PXR, RXR / Nurr1, RXR / Nor1.

[0029] In a fifth aspect of the present application, the present application provides a use of a compound of Formula (I) or a tautomer, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof in modulating RXR activity or modulating activity of a dimer containing RXR;

[0030] According to embodiments of the present application, the compound of Formula (I) or a tautomer, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof can target RXR, modulate RXR activity, and modulate activity of a dimer containing RXR.

[0031] According to embodiments of the present application, the dimer containing RXR includes a RXR homodimer, a heterodimer containing RXR.

[0032] According to embodiments of the present application, the dimer containing RXR includes a dimer containing RXRα, a dimer containing RXRβ, a dimer containing RXRγ.

[0033] According to embodiments of the present application, the heterodimer comprising RXR is selected from the group consisting of RXR / RAR, RXR / PPAR, RXR / LXR, RXR / Nur77, RXR / VDR, RXR / TR, RXR / FXR, RXR / CAR, RXR / PXR, RXR / Nurr1, RXR / Nor1.

[0034] In a sixth aspect of the present application, the present application provides a method for modulating RXR activity or modulating activity of a dimer comprising RXR. According to embodiments of the present application, the method comprises: contacting a compound represented by formula (I) or a tautomer, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof with a cell expressing RXR;

[0035] According to embodiments of the present application, the compound represented by formula (I) or a tautomer, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof can target RXR, can modulate RXR activity, and can modulate activity of a dimer comprising RXR.

[0036] According to embodiments of the present application, the dimer comprising RXR comprises a homodimer of RXR, a heterodimer comprising RXR.

[0037] According to embodiments of the present application, the dimer comprising RXR comprises a dimer comprising RXRα, a dimer comprising RXRβ, a dimer comprising RXRγ.

[0038] According to embodiments of the present application, the heterodimer comprising RXR is selected from the group consisting of RXR / RAR, RXR / PPAR, RXR / LXR, RXR / Nur77, RXR / VDR, RXR / TR, RXR / FXR, RXR / CAR, RXR / PXR, RXR / Nurr1, RXR / Nor1.

[0039] In a seventh aspect of the present application, the present application provides a combination drug or a kit. According to embodiments of the present application, the combination drug or the kit comprises: a compound represented by formula (I) or a tautomer, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof as a first active ingredient; a ligand L as a second active ingredient, the ligand L being used for modulating activity of a nuclear receptor forming a dimer with RXRα;

[0040] According to embodiments of the present application, the compound represented by the above formula (I) or a tautomer, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof can target RXR and modulate RXR activity. Thus, the reagent and the ligand L prepared by using the compound represented by the above formula (I) or a tautomer, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof in combination can effectively modulate RXR activity or modulate the activity of a dimer containing RXR, and can effectively prevent and / or treat an RXR-related disease.

[0041] According to embodiments of the present application, the RXR includes RXRα, RXRβ, and RXRγ.

[0042] According to embodiments of the present application, the ligand L is selected from the group consisting of a ligand of RAR, a ligand of PPAR, a ligand of LXR, a ligand of TR, a ligand of VDR, a ligand of Nur77, a ligand of FXR, a ligand of CAR, a ligand of Nurr1, and a ligand of Nor1.

[0043] According to embodiments of the present application, the ligand L is selected from at least one of all-trans retinoic acid (ATRA), rosiglitazone, T0901317, fenofibrate, calcitriol, thyroid hormone, bile acid, androstane, and pregnane.

[0044] In an eighth aspect of the present application, the present application provides use of the combination drug or the kit of the seventh aspect in the preparation of a medicament for preventing and / or treating an RXR-related disease. As described above, the combination drug or the kit can effectively modulate RXR activity or modulate the activity of a dimer containing RXR, and can effectively prevent and / or treat an RXR-related disease.

[0045] According to embodiments of the present application, the RXR-related disease includes an RXRα-related disease, an RXRβ-related disease, and an RXRγ-related disease.

[0046] According to embodiments of the present application, the RXR-related disease includes a metabolic disease, inflammation, a neurodegenerative disease, a tumor, or a cancer.

[0047] According to embodiments of the present application, the RXR-related disease includes alcoholic fatty liver disease, non-alcoholic fatty liver disease, hepatitis, liver fibrosis, liver cirrhosis, liver cancer, Alzheimer's disease, Parkinson's disease, neuroinflammation, neurotumor, psoriasis, acne, psoriasis, lupus erythematosus, skin fibrosis, Kaposi's sarcoma, cutaneous T-cell lymphoma, nasopharyngitis, bronchitis, pneumonia, pulmonary fibrosis, lung tumor, enteritis, intestinal fibrosis, intestinal tumor, gastritis, gastric tumor, urethritis, nephritis, renal fibrosis, renal tumor, cholestasis, leukemia, diabetes, osteoporosis, arthritis, spondylitis, cholangitis, cholangiocarcinoma, atherosclerosis, myocarditis, hyperlipidemia, liver damage, fatty liver, lung cancer, colorectal cancer.

[0048] In a ninth aspect, the present application provides a method for preventing and / or treating an RXR-related disease. According to embodiments of the present application, the method comprises administering to a subject a pharmaceutically acceptable dose of a compound represented by Formula (I), or a tautomer, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof, or the combination drug or kit of the seventh aspect;

[0049] According to embodiments of the present application, the RXR-related disease includes an RXRα-related disease, an RXRβ-related disease, an RXRγ-related disease.

[0050] According to embodiments of the present application, the RXR-related disease includes a metabolic disease, inflammation, a neurodegenerative disease, a tumor, or a cancer.

[0051] According to embodiments of the present application, the RXR-related disease includes alcoholic fatty liver disease, non-alcoholic fatty liver disease, hepatitis, liver fibrosis, liver cirrhosis, liver cancer, Alzheimer's disease, Parkinson's disease, neuroinflammation, neurotumor, psoriasis, acne, psoriasis, lupus erythematosus, skin fibrosis, Kaposi's sarcoma, cutaneous T-cell lymphoma, nasopharyngitis, bronchitis, pneumonia, pulmonary fibrosis, lung tumor, enteritis, intestinal fibrosis, intestinal tumor, gastritis, gastric tumor, urethritis, nephritis, renal fibrosis, renal tumor, cholestasis, leukemia, diabetes, osteoporosis, arthritis, spondylitis, cholangitis, cholangiocarcinoma, atherosclerosis, myocarditis, hyperlipidemia, liver damage, fatty liver, lung cancer, colorectal cancer.

[0052] Additional aspects and advantages of the present application will be made apparent from the following description of embodiments of the present application, which will be given for the purpose of illustration and not limitation. BRIEF DESCRIPTION OF DRAWINGS

[0053] The above and / or additional aspects and advantages of the present application will become apparent and be readily appreciated from the following description of embodiments of the present application, which will be given for the purpose of illustration and not limitation, taken in conjunction with the accompanying drawings:

[0054] Figure 1A shows the activation of Gal4 / DBD-RXRα / LBD fusion protein transcriptional activity by Tafamidis (chlorobanase) as measured by mammalian one-hybrid assay in Example 1 of the present application, ns not significant, ** p < 0.01, **** p < 0.0001, n > 3.

[0055] Figure 1B shows the activation of RXRα homodimer transcriptional activity by Tafamidis (chlorobanase) as measured by reporter assay in Example 1 of the present application, ns not significant, **** p < 0.0001, n > 3.

[0056] Figure 2A shows the activation of RXRα / LXRα heterodimer transcriptional activity by Tafamidis (chlorobanase) as measured by reporter assay in Example 1 of the present application, ns not significant, * p < 0.05, *** p < 0.001, **** p < 0.0001, n > 3.

[0057] Figure 2B shows the activation of RXRα / PPARγ heterodimer transcriptional activity by Tafamidis (chlorobanase) as measured by reporter assay in Example 1 of the present application, ns not significant, * p < 0.05, *** p < 0.001, **** p < 0.0001, n > 3.

[0058] Figure 2C shows the activation of RXRα / RARα heterodimer transcriptional activity by Tafamidis (chlorobanase) as measured by reporter assay in Example 1 of the present application, ns not significant, * p < 0.05, **** p < 0.0001, n > 3.

[0059] Figure 3A shows the activation of Gal4 / DBD-RARα / LBD fusion protein transcriptional activity by Tafamidis (chlorobanase) as measured by mammalian one-hybrid assay in Example 1 of the present application, ns not significant, **** p < 0.0001, n > 3.

[0060] Figure 3B shows the activation of Gal4 / DBD-RARγ / LBD fusion protein transcriptional activity by Tafamidis (chlorobanase) as measured by mammalian one-hybrid assay in Example 1 of the present application, ns not significant, **** p < 0.0001, n > 3.

[0061] Figure 3C shows the activation of Gal4 / DBD-LXRα / LBD fusion protein transcriptional activity by Tafamidis (chlorobanase) as measured by mammalian one-hybrid assay in Example 1 of the present application, ns not significant, **** p < 0.0001, n > 3.

[0062] Figure 3D is the activation of Gal4 / DBD-LXRβ / LBD fusion protein transcriptional activity by Tafamidis (chlorobutanil) measured by mammalian one-hybrid assay in Example 1 of the present application, ns, not statistically significant, ****p<0.0001, n≥3.

[0063] Figure 3E is the activation of Gal4 / DBD-PPARα / LBD fusion protein transcriptional activity by Tafamidis (chlorobutanil) measured by mammalian one-hybrid assay in Example 1 of the present application, ns, not statistically significant, ****p<0.0001, n≥3.

[0064] Figure 3F is the activation of Gal4 / DBD-PPARγ / LBD fusion protein transcriptional activity by Tafamidis (chlorobutanil) measured by mammalian one-hybrid assay in Example 1 of the present application, ns, not statistically significant, ****p<0.0001, n≥3.

[0065] Figure 3G is the activation of Gal4 / DBD-ERα / LBD fusion protein transcriptional activity by Tafamidis (chlorobutanil) measured by mammalian one-hybrid assay in Example 1 of the present application, ns, not statistically significant, ****p<0.0001, n≥3.

[0066] Figure 3H is the activation of Gal4 / DBD-ERβ / LBD fusion protein transcriptional activity by Tafamidis (chlorobutanil) measured by mammalian one-hybrid assay in Example 1 of the present application, ns, not statistically significant, ****p<0.0001, n≥3.

[0067] Figure 4A is the binding of Tafamidis (chlorobutanil) to RXRα ligand binding domain (LBD) protein measured by differential scanning calorimetry in Example 2 of the present application; after mixing RXRα / LBD (20 μM) protein with Tafamidis (100 μM), the thermal change of the protein during the heating process was recorded by differential scanning calorimeter, and the plot was made by Origin software compared with the thermal change of RXRα / LBD (20 μM) protein itself.

[0068] Figure 4B is the binding of Tafamidis to RXRα / LBD protein measured by surface plasmon resonance in Example 2 of the present application; different concentrations of Tafamidis solution were passed through the CM5 chip coupled with RXRα / LBD protein in turn, the response value was obtained, and the K d value of Tafamidis and RXRα / LBD binding was calculated.

[0069] Figure 4C is the binding of Tafamidis to RXRα / LBD protein measured by fluorescence titration in Example 2 of the present application; different concentrations of Tafamidis solution were added to the purified RXRα / LBD protein solution, and the emission spectrum of RXRα / LBD protein excited by 280 nm excitation light was detected, and the experimental data were calculated by Origin software and the formula "y = C x (x + 5 + K d ) / 2 - C x sqrt(x + 5 + K d ) 2 -4 x 5x) / 2" to obtain the K d value of the binding of Tafamidis and RXRα / LBD.

[0070] Figure 5 is the binding of Tafamidis to RXRα protein measured by cell thermal shift assay in Example 2 of the present application; after Tafamidis was treated at a concentration of 20 μM for 3 hours, the cell lysate was incubated at different temperatures, and after centrifugation, the supernatant was detected by immunoblotting method to detect the change of RXRα protein content. After the obtained protein band was quantitatively analyzed by ImageJ, the relative value of RXRα protein content at each temperature (percentage of RXRα protein amount at the lowest temperature) was calculated, and the protein content change curve was drawn by GraphPad software.

[0071] Figure 6A is the morphological change and quantitative statistics of Tafamidis on fibrotic cells under a general microscope in Example 3 of the present application; after CFSC rat hepatic stellate cells were treated with TGFβ (5 ng / mL) or co-treated with Tafamidis (10 μM) for 24 hours, microphotographs were taken, and the scale is 50 μm. The length-width ratio of 10 cells in each field was counted by ImageJ, and **p < 0.01.

[0072] Figure 6B is the change and quantitative statistics of Tafamidis on lipid droplets of fibrotic cells under a general microscope in Example 3 of the present application; after CFSC rat hepatic stellate cells were treated with TGFβ (5 ng / mL) or co-treated with Tafamidis (10 μM) for 24 hours, oil red O staining was performed, and microphotographs were taken, and the scale is 50 μm. The oil red O staining intensity was counted by ImageJ software, *p < 0.05, **p < 0.01, n = 6.

[0073] Figure 7A is the down-regulation of Tafamidis on COL1A1 and α-SMA proteins in the fibrotic cell model measured by immunoblotting experiment in Example 3 of the present application; rat hepatic stellate cells CFSC were treated with TGFβ (5 ng / mL) or co-treated with different concentrations of Tafamidis for 24 hours, and the expression of COL1A1 and α-SMA proteins was detected by immunoblotting.

[0074] Figure 7B is the down-regulation of COL1A1 and a-SMA protein by Tafamidis in a cell fibrosis model as measured by Western blotting in Example 3 of the present application; CFSC rat hepatic stellate cells were treated with TGF (5 ng / mL) or co-treated with Tafamidis (10 mM) for 24 hours or 36 hours, and the expression of COL1A1 and a-SMA protein was detected by Western blotting.

[0075] Figure 7C is the down-regulation of a-SMA, COL1A1 and CTGF mRNA levels by Tafamidis in a fibrotic cell model as measured by qRT-PCR in Example 3 of the present application; CFSC rat hepatic stellate cells were treated with TGF (5 ng / mL) or co-treated with Tafamidis (10 mM) for 24 hours, and the mRNA expression levels of stellate cell activation markers were detected by qRT-PCR, with β-actin as the internal reference gene and normalized to the control, ***p < 0.001, ****p < 0.0001, n = 3.

[0076] Figure 8 is the change in fluorescence intensity of a-SMA, a cell fibrosis marker, and statistics of Tafamidis as measured by confocal microscopy in Example 3 of the present application; CFSC rat hepatic stellate cells were treated with TGF (5 ng / mL) or co-treated with Tafamidis (10 mM) for 24 hours, and the expression of a-SMA was observed by immunofluorescence staining, with the scale bar being 18.4 μm. The fluorescence staining intensity was statistically analyzed using ImageJ, ***p < 0.001, n > 3.

[0077] Figure 9A is the down-regulation of COL1A1 protein by Tafamidis in a cell fibrosis model as measured by Western blotting in Example 3 of the present application; human hepatic stellate cells LX2 were treated with TGF (5 ng / mL) or co-treated with different concentrations of Tafamidis for 24 hours, and the expression of COL1A1 and protein was detected by Western blotting.

[0078] Figure 9B is the down-regulation of COL4A1 and COL1A1 mRNA levels by Tafamidis in a cell fibrosis model as measured by qRT-PCR in Example 3 of the present application; LX2 human hepatic stellate cells were treated with TGF (5 ng / mL) or co-treated with Tafamidis (10 mM) for 24 hours, and the mRNA expression levels of stellate cell activation markers were detected by qRT-PCR, with β-actin as the internal reference gene and normalized to the control, ***p < 0.001, ****p < 0.0001, n = 3.

[0079] Figures 10A-B are the results of immunoblotting experiments showing that Tafamidis down-regulates COL1A1 and a-SMA protein in a RXRa-dependent manner in a cell fibrosis model; LX2 human hepatic stellate cells (A) and CFSC rat hepatic stellate cells (B) with stable interference of RXRa expression were treated with TGF (5 ng / mL) or co-treated with Tafamidis (10 μM) for 24 hours, and the activation of stellate cells was detected by immunoblotting.

[0080] Figure 11A is the effect of Tafamidis on the morphology of liver tissue in a CCl4 liver fibrosis model in mice according to Example 4 of the present application; C57BL / 6 mice were injected intraperitoneally with CCl4 (2 mL / kg, 25% corn oil solution) three times a week, and after three weeks, the mice were administered solvent or Tafamidis (5 mg / kg and 20 mg / kg) by gavage for two weeks, with 6 mice in each group. After the experiment, the livers were collected and photographed to record the appearance of the livers in vitro, and the scale bar is 0.5 cm.

[0081] Figure 11B is the effect of Tafamidis on liver function in a CCl4 liver fibrosis model in mice according to Example 4 of the present application; After blood was collected from the mice, serum was collected by centrifugation, and the serum liver function alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were detected, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, n=6.

[0082] Figure 12 is the effect of Tafamidis on fibrosis in a CCl4 liver fibrosis model in mice according to Example 4 of the present application; C57BL / 6 mice were injected intraperitoneally with CCl4 (2 mL / kg, corn oil solution) three times a week, and after three weeks, the mice were administered solvent or Tafamidis (5 mg / kg and 20 mg / kg) by gavage for two weeks. The livers of the mice were paraffin sectioned and stained with HE (A) and Sirius red (B), and the scale bar is 100 μm. ImageJ software was used to calculate the fibrosis area represented by Sirius red staining, **p<0.01, ***p<0.001, ****p<0.0001, n=6.

[0083] Figure 13A is the effect of Tafamidis on fibrosis in a mouse CCl4 liver fibrosis model according to Example 4 of the present application; C57BL / 6 mice were injected intraperitoneally with CCl4(2 mL / kg, corn oil as solvent) three times a week for three weeks, and then were administered solvent or Tafamidis (5 mg / kg and 20 mg / kg) by gavage for two weeks. After the liver tissue was extracted for RNA, the mRNA levels of liver fibrosis-related indicators a-SMA, COL1A1, and TIMP1 were detected by qRT-PCR, with GAPDH as an internal reference gene and normalized to the control. **p<0.01, ***p<0.001, n=6.

[0084] Figure 13B is the effect of Tafamidis on fibrosis in a mouse CCl4 liver fibrosis model according to Example 4 of the present application; after the liver tissue was lysed and the protein was extracted, the expression levels of a-SMA and COL1A1 were detected by immunoblotting. The results of the immunoblotting bands were quantitatively analyzed using ImageJ software. ns, no statistical significance; *p<0.05, **p<0.01, ***p<0.001.

[0085] Figure 14 is the effect of Tafamidis on the inhibition of the activation of hepatic stellate cells in a mouse CCl4 liver fibrosis model according to Example 4 of the present application; C57BL / 6 mice were injected intraperitoneally with CCl4(2 mL / kg, corn oil as solvent) three times a week for three weeks, and then were administered solvent or Tafamidis (5 mg / kg and 20 mg / kg) by gavage for two weeks. After the mouse primary hepatic stellate cells were extracted and the protein lysate was obtained, the expression levels of liver fibrosis-related proteins a-SMA and COL1A1 in the primary hepatic stellate cells were detected by immunoblotting. The results of the bands obtained by immunoblotting were quantitatively analyzed using ImageJ software. *p<0.05, **p<0.01.

[0086] Figure 15 is the effect of Tafamidis on the inhibition of liver inflammatory cell infiltration in a mouse CCl4 liver fibrosis model according to Example 4 of the present application; the above-mentioned mouse liver paraffin sections were stained by F4 / 80 immunohistochemistry, and the scale in the figure is 100 μm. The positive area of the immunohistochemical staining results was statistically analyzed using ImageJ software. ns, no statistical significance; **p<0.01, ***p<0.001, n=6.

[0087] Figure 16A is the effect of Tafamidis on liver tissue morphology measured in a mouse MCD non-alcoholic fatty liver model in Example 5 of the present application; C57BL / 6 male mice, after one week of adapting to the model feed, were fed with methionine choline deficient (MCD) feed for 3 weeks, after 3 weeks, the model group was randomly divided into solvent group, Tafamidis 5 mg / kg group and Tafamidis 20 mg / kg group, daily administration and MCD feeding, after three weeks, the ex vivo liver appearance was recorded by taking pictures, the ruler in the figure is 0.5 cm.

[0088] Figure 16B is the effect of Tafamidis on liver function measured by biochemical detection kit in a mouse MCD non-alcoholic fatty liver model in Example 5 of the present application; after taking blood from the above mice, serum was collected by centrifugation, serum liver function ALT, AST, total bilirubin (TBIL) indicators were detected, ns no statistical significance, *p<0.05, **p<0.01, ****p<0.0001, n=6.

[0089] Figures 17A-C are the effect of Tafamidis on fibrosis measured in a mouse MCD non-alcoholic fatty liver model in Example 5 of the present application; paraffin sections of mouse liver were stained with HE (A) and Sirius red (B), the ruler in the figure is 100 μm. C. The fibrous area represented by Sirius red staining was counted using ImageJ software, **p<0.01, ***p<0.001, ****p<0.0001, n=6.

[0090] Figure 17D is the effect of Tafamidis on liver fat accumulation measured in a mouse MCD non-alcoholic fatty liver model in Example 5 of the present application; after liver lysis, the triglyceride content was detected, ns no statistical significance, *p<0.05, ****p<0.0001, n=6.

[0091] Figure 18A is the effect of Tafamidis on liver fibrosis measured by qRT-PCR in a mouse MCD non-alcoholic fatty liver model in Example 5 of the present application; C57BL / 6 male mice, after one week of adapting to the model feed, were fed with MCD feed for 3 weeks, after 3 weeks, the model group was randomly divided into solvent group, Tafamidis 5 mg / kg group and Tafamidis 20 mg / kg group, daily administration and MCD feeding, after three weeks, the liver tissue was taken to extract RNA, and the transcription levels of a-SMA, COL1A1, TIMP1 and TNFα were detected by qRT-PCR, GAPDH was used as an internal reference gene and normalized with the control, ns no statistical significance, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, n=6.

[0092] Figure 18B is the effect of Tafamidis on fibrosis in a mouse MCD non-alcoholic fatty liver model in Example 5 of the present application, as measured by immunoblotting; the mice were sacrificed and the liver tissue was taken for immunoblotting to detect the expression level of a-SMA. The bands obtained from the immunoblotting were quantitatively analyzed using ImageJ software, ns, not statistically significant, *p < 0.05.

[0093] Figure 19 is the effect of Tafamidis on inhibiting liver inflammatory cell infiltration in a mouse MCD non-alcoholic fatty liver model in Example 5 of the present application, as measured by immunohistochemistry; the liver paraffin sections of the mice were stained by F4 / 80 immunohistochemistry, and the scale bar is 100 pm. The positive area of the immunohistochemistry staining results was statistically analyzed using ImageJ software, ***p < 0.001, ****p < 0.0001, n = 6.

[0094] Figure 20A is the effect of 10 pM and 20 pM Tafamidis on ABCA1 in microglial cells in Example 6 of the present application, as measured by immunoblotting; the BV2 mouse microglial cells were uniformly plated in a 12-well plate at a density of about 60%, and after the cells adhered, the medium was replaced with 1% serum DMEM medium, and different concentrations of Tafamidis were added to make the final concentration 10 pM and 20 pM. The samples were collected after 12 h, and the expression of ABCA1 was detected by immunoblotting. The bands obtained from the immunoblotting were quantitatively analyzed using ImageJ software, ****p < 0.0001.

[0095] Figure 20B is the effect of Tafamidis on a BV2 cell neuroinflammation model induced by lipopolysaccharide LPS (1 pg / mL) in Example 6 of the present application; the BV2 cells were uniformly plated in a 12-well plate at a density of about 60%, and after the cells adhered, the medium was replaced with 1% serum DMEM medium, and 10 pM Tafamidis was added. After 12 h, lipopolysaccharide LPS (1 pg / mL) was added, and the samples were collected after 12 h of induction. The expression level of iNOS was detected by immunoblotting, and the bands obtained from the immunoblotting were quantitatively analyzed using ImageJ software, ****p < 0.0001.

[0096] Figure 21A is the effect of Tafamidis on the body weight of mice in a model of cholestatic liver injury and cholangitis in Example 22 of the present application. C57BL / 6 mice were subjected to bile duct ligation (BDL) surgery, and after 3 days, they were randomly divided into a solvent group and a Tafamidis (5 mg / kg) group. The mice were administered the drug once a day by gavage, and the body weight was recorded every other day. The samples were collected after 2 weeks.

[0097] Figure 21B is the effect of Tafamidis on the appearance and morphology of the liver in a mouse model of cholestatic liver injury and cholangitis in Example 22 of the present application. The scale bar is 1 cm.

[0098] Figure 21C shows the effect of Tafamidis on liver function in the mouse cholestatic liver injury and cholangitis model in Example 22. The mice were taken blood from the orbital vein, and the serum was collected by centrifugation. The serum liver function indicators ALT, AST, and TBIL were detected. ns, no statistical significance; *p < 0.05; **p < 0.01; ***p < 0.001; n = 6.

[0099] Figure 21D shows the effect of Tafamidis on liver fibrosis in the mouse cholestatic liver injury and cholangitis model in Example 22. After the liver tissue was taken and RNA was extracted, the transcription levels of a-SMA, COL1A1, and TIMP1 were detected by qRT-PCR. GAPDH was used as an internal reference gene and normalized with the control. ns, no statistical significance; *p < 0.05; **p < 0.01; ***p < 0.001; n = 6.

[0100] Figure 21E shows the effect of Tafamidis on fibrosis, cholangitis inflammation, and liver lobule bile duct compensatory hyperplasia in the mouse cholestatic liver injury and cholangitis model in Example 22. The mouse liver was paraffin sectioned, HE stained, and Sirius red stained. The scale bar is 100 pm. The fibrous area represented by Sirius red staining was counted using ImageJ software. **p < 0.01; ****p < 0.0001; n = 6.

[0101] Figure 21F shows the effect of Tafamidis on collagen content in the mouse cholestatic liver injury model in Example 22. The mouse liver was taken and hydrolyzed with hydrochloric acid, and the content of hydroxyproline (HYP) in the collagen was detected. **p < 0.01; ****p < 0.0001; n = 6. The results are shown in Figure 21F.

[0102] Figure 22A shows the experimental operation process of acetaminophen (APAP)-induced acute liver injury in mice in Example 23. ICR mice were randomly divided into 3 groups, and were given solvent or Tafamidis (20 mg / kg) by gavage 3 days in advance. Twelve hours after the last gavage, APAP (300 mg / kg) was injected intraperitoneally, and solvent or Tafamidis was given by gavage again 12 hours later. The samples were collected 12 hours later.

[0103] Figure 22B shows the effect of Tafamidis on the appearance and morphology of the liver in the mouse APAP-induced acute liver injury model in Example 23. The scale bar is 1 cm.

[0104] Figure 22C is a biochemical detection kit measured Tafamidis on liver function in the mouse APAP-induced acute liver injury model in Example 23 of the application; the mice were taken out of the eye blood after centrifugation to collect serum, serum liver function ALT, AST, TBIL index, ns no statistical significance, *p<0.05, n=6.

[0105] Figure 22D is the effect of Tafamidis on the central vein around the liver parenchymal cell necrosis in the mouse APAP-induced acute liver injury model in Example 23 of the application; the mouse liver was paraffin section HE staining, the scale in the figure is 100 μm. The area of necrotic region was counted using ImageJ software, ** p<0.01, n=6.

[0106] Figure 23A is the effect of Tafamidis on CCl4-induced HepG2 cell damage in Example 24 of the application; the saturated solution of CCl4 and Tafamidis (10 μM) were treated together with HepG2 hepatoma cells, and the culture medium was collected after 7.5 hours. The content of AST in the culture medium was detected by biochemical detection kit.

[0107] Figure 23B is the experimental operation process of CCl4-induced acute liver injury in mice in Example 24 of the application. C57BL / 6 mice were randomly divided into two groups, and were given solvent or Tafamidis (20 mg / kg) by gavage 7 days in advance. The last gavage was injected intraperitoneally with CCl4 24 hours later, and the solvent or Tafamidis was given again 24 hours later. The sample was collected 24 hours later.

[0108] Figure 23C is the effect of Tafamidis on liver function in the mouse CCl4-induced acute liver injury model measured by biochemical detection kit in Example 24 of the application; the mice were taken out of the eye blood after centrifugation to collect serum, serum liver function AST index, * p<0.05, n=6.

[0109] Figure 23D is the effect of Tafamidis on the central vein around the liver parenchymal cell necrosis in the mouse CCl4-induced acute liver injury model in Example 24 of the application; the mouse liver was paraffin section HE staining, the scale in the figure is 100 μm. The area of necrotic region was counted using ImageJ software, * p<0.05, n=6.

[0110] Figure 24A is the effect of Tafamidis on serum total cholesterol (T-CHO) in a mouse model of high-fat diet-induced fatty liver, as measured by a biochemical detection kit. C57BL / 6 mice were fed with high-fat diet (HFD) and normal diet (ctr). After 5 months, the mice fed with high-fat diet were randomly divided into a model group and a drug administration group. The mice were then administered vehicle or Tafamidis (10 mg / kg) by gavage every day. After four weeks, the mice were enucleated to collect blood, which was then centrifuged to collect serum. The serum T-CHO index was detected, * p<0.05, **** p<0.0001, n=6.

[0111] Figure 24B is the effect of Tafamidis on liver steatosis and ballooning in a mouse model of high-fat diet-induced fatty liver. The livers of the above mice were paraffin sectioned and HE stained. The scale in the figure is 100 μm. ImageJ software was used to calculate the areas of steatosis and ballooning. *** p<0.001, **** p<0.0001, n=6.

[0112] Figure 25 is the effect of Tafamidis on OA-induced lipid droplet accumulation in HepG2 cells. HepG2 cells were co-treated with OA (100 μM) and Tafamidis (2.5 μM) in 10% serum MEM medium. After 24 hours, oil red O staining was performed, and microscopic photographs were taken. The scale in the figure is 112.5 μm. ImageJ software was used to calculate the oil red O positive area. * p<0.05, ** p<0.01, n=3.

[0113] Figure 26 is the effect of Tafamidis on LPS-induced iNOS expression in mouse alveolar macrophage MH-S cells. MH-S cells were co-treated with LPS (100 ng / mL) and Tafamidis (2.5 μM) in 1% serum 1640 medium for 24 hours. Immunoblotting was used to detect iNOS levels. ImageJ software was used to calculate the gray value of the band. GAPDH was used as an internal reference and was normalized. *** p<0.001, **** p<0.0001, n=3.

[0114] Figure 27 is the inhibitory effect of Tafamidis on human acute promyelocytic leukemia cells NB4. After Tafamidis (5 μM) treated NB4 cells for 48 hours, the cell survival rate was detected by CCK-8.* p<0.05, n=6.

[0115] Figure 28 is the effect of Tafamidis on the Okadaic acid (OA)-induced SH-SY5Y cell TAU protein hyperphosphorylation model in Example 6 of the present application; SH-SY5Y human neuroblastoma cells were evenly plated in 12-well plates at a density of about 60%, and after the cells adhered, the medium was replaced with 1% serum DMEM medium, 10 μΜ of Tafamidis was added, and 12 h later, Okadaic acid (OA) (20 nM) was added. The sample was collected 12 h later, and the expression level of p-TAU was detected by immunoblotting. The results of the immunoblotting bands were quantitatively analyzed using ImageJ software, ***p<0.001, ****p<0.0001.

[0116] Figure 29 is the effect of Tafamidis on neuroinflammation in the BV2 mouse microglial neuroinflammatory model in Example 6 of the present application; BV2 control cells (sgCON) and RXRα-knockout cells (sgRXRα) were evenly plated in 12-well plates at a density of about 60%, and after adhering, the medium was replaced with serum-free DMEM for 0.5 h, Tafamidis was added to a final concentration of 10 μΜ, and the time was 0.5 h, and then LPS (1 μg / mL) was added. The sample was collected 5.5 h later, and the relative expression levels of the mRNA of inflammation-related indicators COX-2, TNF-α, IL-1β, and IL-6 were detected by qRT-PCR. β-actin was used as an internal control gene and was normalized with the control, ****p<0.0001, n=3.

[0117] Figure 30 is a schematic diagram of brain stereotaxic injection to construct a neuroinflammation model and the effect of Tafamidis on neuroinflammation measured by qRT-PCR in Example 6 of the present application; we gave C57BL / 6J mice Tafamidis (10 mg / kg) by gavage for 7 consecutive days, and then injected LPS into the substantia nigra using brain stereotaxic injection technology, wherein the injection coordinates of the substantia nigra compact part were: anteroposterior axis -3.3 mm (relative to the bregma), medial-lateral axis ±1.2 mm, and dorsal-ventral axis -4.6 mm (relative to the brain surface). Using a microsyringe pump, LPS (2 μg / μL dissolved in sterile PBS) was injected into the bilateral substantia nigra compact part at a speed of 0.1 μL / min, and the injection volume was 2 μL. The control (vehicle) mice were injected with the same volume of sterile PBS at the same coordinates, and the samples were collected after 2 days. Figure 30A is a schematic diagram of the experimental process. After collecting the mouse hippocampus, RNA extraction was performed, and the transcription levels of IL-6, TNF-α, and IL-1β were detected by qRT-PCR method, with β-actin as the internal reference gene and normalized with the control, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0118] Figure 31 is a schematic diagram of brain stereotaxic injection to construct a neuroinflammation model and the effect of Tafamidis on neuroinjury measured by qRT-PCR in Example 6 of the present application; we gave C57BL / 6J mice Tafamidis (10 mg / kg) by gavage for 7 consecutive days, and then injected LPS into the substantia nigra using brain stereotaxic injection technology, wherein the injection coordinates of the substantia nigra compact part were: anteroposterior axis -3.3 mm (relative to the bregma), medial-lateral axis ±1.2 mm, and dorsal-ventral axis -4.6 mm (relative to the brain surface). Using a microsyringe pump, LPS (2 μg / μL dissolved in sterile PBS) was injected into the bilateral substantia nigra compact part at a speed of 0.1 μL / min, and the injection volume was 2 μL. The control (vehicle) mice were injected with the same volume of sterile PBS at the same coordinates, and the samples were collected after 2 days. After collecting the mouse hippocampus, RNA extraction was performed, and the transcription levels of Synaptophysin and PSD-95 were detected by qRT-PCR method, with β-actin as the internal reference gene and normalized with the control, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0119] Figure 32 shows the effect of Tafamidis on lung fibrosis measured in Example 29 of the present application, human lung fibroblast WI-38 was treated with TGF (10 ng / mL) alone or with Tafamidis at different concentrations (2.5 μΜ, 5 μΜ, 10 μΜ, 20 μΜ) for 48 hours, and the expression of COL1A1 and a-SMA protein was detected by immunoblotting.

[0120] Figure 33 shows the effect of Tafamidis on lung tumor measured in Example 30 of the present application, human lung tumor cell HCC827 was plated in 96-well plates and treated with DMSO or Tafamidis (10 μΜ and 20 μΜ), after 24h incubation, cell survival rate was detected by CCK8.

[0121] Figure 34 shows the killing effect of Tafamidis on human colon cancer cells (HCT116) in Example 31 of the present application; cell survival rate was detected by CCK-8 after Tafamidis treatment of HCT116 cells for 24 hours, *p<0.05, ***p<0.001, ****p<0.0001, n=3. DETAILED DESCRIPTION

[0122] In this text, the term "comprising" or "including" is an open expression, i.e. including the specified content of the present application, but not excluding other aspects.

[0123] In this text, the term "optionally", "optional" or "optional" generally means that the event or condition described subsequently can, but need not, occur, and the description includes situations where the event or condition occurs, as well as situations where it does not.

[0124] In this text, the term "pharmaceutically acceptable" or "pharmaceutically acceptable" means that the substance or composition must be chemically and / or toxicologically compatible with the other ingredients of the formulation and / or the mammal being treated therewith. Preferably, "pharmaceutically acceptable" according to the present application means approved or approved by a federal regulatory agency or national government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and particularly in humans.

[0125] In the present text, the term "salt" or "pharmaceutically acceptable salt" includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts. The term "pharmaceutically acceptable acid addition salt" denotes those salts which retain the biological effectiveness and non-toxicity of the free bases, and which are, in turn, not deleterious to the recipients thereof. The "pharmaceutically acceptable base addition salts" are those salts which retain the biological effectiveness and non-toxicity of the free acids, and which are, in turn, not deleterious to the recipients thereof. In addition to pharmaceutically acceptable salts, other salts are also contemplated, for example, in the isolation or purification of a compound of the present application. They can serve as intermediates in the purification of the compounds of the application or in the preparation of other pharmaceutically acceptable salts.

[0126] In the present text, the term "amine salt" refers to the product obtained by neutralizing a primary, secondary or tertiary alkylamine with an acid. The acids include the inorganic or organic acids described in the present application.

[0127] In the present text, the term "stereoisomer" refers to isomers that have the same molecular formula but different structures, resulting from a difference in the arrangement of atoms in space. This includes enantiomers and diastereomers.

[0128] Depending on the choice of starting materials and methods, the compounds of the present application can be present in the form of one of the possible isomers or as a mixture of them, for example, as a racemic mixture or as a mixture with other diastereomers. When describing compounds having optical activity, the prefixes D and L or R and S are used to denote the absolute configuration of the molecule about its chiral center(s). The prefixes D and L or (+) and (-) are used to designate the rotational orientation of an optical isomer about its chiral center(s) when placed in plane-polarized light. A compound that is (+) or D is dextrorotary.

[0129] When bonds to a chiral carbon in the formula of the present application are depicted as straight lines, it is understood that both the (R) and (S) configurations of the chiral carbon and the enantiomerically pure compounds and mixtures resulting therefrom are included within the scope of the general formula. The graphical representation of racemates or enantiomerically pure compounds herein is from Maehr, J. Chem. Ed. 1985, 62: 114-120. Wedge and dash bonds are used to represent the absolute configuration of one stereogenic center.

[0130] As used herein, the term "tautomer" refers to isomers of a functional group that result from the rapid movement of an atom in a molecule between two positions. The compounds of the present application can exhibit tautomerism. Compounds that tautomerize can exist in two or more interconvertible forms. Proton-shift tautomers result from the migration of a hydrogen atom covalently bonded between two atoms. Tautomers generally exist in equilibrium, and attempts to isolate a single tautomer usually result in a mixture whose physical and chemical properties are consistent with a mixture of compounds. The position of equilibrium depends on the chemical properties within the molecule. For example, in many aliphatic aldehydes and ketones, such as acetaldehyde, the keto form predominates; in phenols, the enol form predominates. The present application encompasses all tautomeric forms of the compounds.

[0131] As used herein, the term "solvate" refers to a compound of the present application or a salt thereof, including stoichiometric or non-stoichiometric amounts of a solvent, when the solvent is water, then a hydrate.

[0132] As used herein, the term "treatment" refers to obtaining a desired pharmacologic and / or physiologic effect. The effect can be prophylactic in terms of completely or partially preventing a disease or symptom thereof and / or can be therapeutic in terms of a partial or complete cure for a disease and / or adverse effect attributable to the disease. "Treatment" as used herein covers the treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease or condition from occurring in an individual which can be predisposed to the disease but has not yet developed the disease; (b) inhibiting the disease, i.e., arresting its development; or (c) relieving the disease, i.e., causing the partial or complete regression of the disease. As used herein, "treatment" covers any administration of a drug to an individual to treat, cure, heal, alleviate, relieve, or prevent a disease in the individual, including but not limited to the administration of a drug described herein to an individual in need thereof.

[0133] As used herein, the terms "cancer" or "tumor" can be any unregulated cell growth. Exemplary can be colon cancer, rectal cancer, kidney cancer, skin cancer (especially cutaneous T-cell lymphoma), and the like.

[0134] Detailed description of the use of tarenflurbil in activating RXRa activity

[0135] The present application provides a use of tarenflurbil in preventing and / or treating RXR-related diseases, a use of tarenflurbil in modulating RXR activity, a method of modulating RXR activity, a combination or a kit and uses thereof, a method of preventing and / or treating RXR-related diseases, which will be described in detail respectively.

[0136] Use

[0137] In a first aspect, the present application provides a use of a compound of Formula (I) or a tautomer, stereoisomer, solvate, or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for preventing and / or treating an RXR-related disease;

[0138] In the course of studying novel ligands for the nuclear receptor RXR, the inventors surprisingly found that Tafamidis (chlorbenzoxazoles, i.e. a compound of Formula (I)) can target RXR and modulate RXR activity (e.g. significantly activate or enhance the activation of the homodimeric and heterodimeric (e.g. RXRα / RARα, RXRα / PPARγ or RXRα / LXRα) transcriptional activity of RXRα). Further experiments have shown that Tafamidis can target RXR to treat RXR-related diseases, including but not limited to liver fibrosis (both in vitro cell experiments and mouse CCl4 liver fibrosis model), liver injury, cholestasis, non-alcoholic steatohepatitis (mouse MCD model), neuroinflammation and Alzheimer's disease.

[0139] In the present application, the term "RXR-related disease" should be interpreted broadly, and includes RXR-mediated related diseases, and also includes non-RXR-mediated related diseases, but diseases that can be affected by RXR activity (e.g. diseases that can be treated by targeting RXR and modulating RXR activity), and the specific type of disease is not limited as long as it is related to RXR.

[0140] In some embodiments of the present application, Tafamidis is used to treat related diseases by modulating the physiological functions of the corresponding nuclear receptors through the modulation of RXRα (the dimeric transcriptional activity, etc.).

[0141] According to embodiments of the present application, the RXR-related disease includes an RXRα-related disease, an RXRβ-related disease, and an RXRγ-related disease.

[0142] According to embodiments of the present application, the RXR-related disease includes a metabolic disease, inflammation, a neurodegenerative disease, a tumor or cancer.

[0143] According to embodiments of the present application, the RXR-related diseases include alcoholic fatty liver disease, non-alcoholic fatty liver disease, hepatitis, liver fibrosis, liver cirrhosis, liver cancer, Alzheimer's disease, Parkinson's disease, neuroinflammation, neurotumor, psoriasis, acne, psoriasis, lupus erythematosus, skin fibrosis, Kaposi's sarcoma, cutaneous T-cell lymphoma, nasopharyngitis, bronchitis, pneumonia, pulmonary fibrosis, lung tumor, enteritis, intestinal fibrosis, intestinal tumor, gastritis, gastric tumor, urethritis, nephritis, renal fibrosis, renal tumor, cholestasis, leukemia, diabetes, osteoporosis, arthritis, spondylitis, cholangitis, cholangiocarcinoma, atherosclerosis, myocarditis, hyperlipidemia, liver damage, fatty liver, lung cancer, colorectal cancer.

[0144] According to embodiments of the present application, the metabolic diseases include alcoholic fatty liver disease, non-alcoholic fatty liver disease, cholestasis, liver damage, diabetes, hyperlipidemia, fatty liver, osteoporosis, atherosclerosis. Exemplarily, the compounds described in the present application can significantly inhibit OA-induced accumulation of lipid droplets in HepG2, and treat metabolic diseases, as described in Example 26 of the present application.

[0145] According to embodiments of the present application, the inflammation includes hepatitis, psoriasis, acne, psoriasis, lupus erythematosus, pneumonia, enteritis, nephritis, cholangitis, liver damage, arthritis, spondylitis, urethritis, gastritis, nasopharyngitis, bronchitis, neuroinflammation, fibrosis. Exemplarily, the compounds described in the present application can inhibit LPS-induced upregulation of iNOS in microglia cells, inhibit LPS-induced expression of inflammatory factors in BV2 cells, and inhibit LPS-induced expression of inflammatory factors in mouse hippocampus, thereby treating inflammation, as described in Example 6 of the present application.

[0146] According to embodiments of the present application, the liver damage includes, but is not limited to, acute liver damage and chronic liver damage.

[0147] According to embodiments of the present application, the fibrosis includes skin fibrosis, intestinal fibrosis, liver fibrosis, liver cirrhosis (advanced liver fibrosis), pulmonary fibrosis, and renal fibrosis.

[0148] According to embodiments of the present application, the neurodegenerative diseases include Parkinson's disease and Alzheimer's disease. Exemplarily, the compounds described in the present application can promote the expression of ABCA1 in microglial cells, increase the clearance of AD pathogenic protein Aβ by microglial cells, and inhibit the phosphorylation of TAU protein in nerve cells induced by okadaic acid, thereby treating Alzheimer's disease; the compounds described in the present application can inhibit the function of LPS-induced nerve cell damage, thereby treating neurodegenerative diseases, as described in Example 6 of the present application.

[0149] According to an embodiment of the present application, the tumor or cancer includes gastric tumor, cholangiocarcinoma, neural tumor, Kaposi's sarcoma, cutaneous T-cell lymphoma, liver cancer, lung cancer, colorectal cancer, renal tumor, leukemia, lung tumor, intestinal tumor. Among them, the tumor of benign or unclear nature includes Kaposi's sarcoma, cutaneous T-cell lymphoma; the malignant tumor includes liver cancer, lung cancer, colorectal cancer, renal tumor, leukemia, lung tumor, intestinal tumor, gastric tumor, cholangiocarcinoma, neural tumor.

[0150] According to an embodiment of the present application, the administration amount of the compound represented by formula (I) or a tautomer, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof is about 1.0 mg to 100.0 mg, for example, 1.0 mg, 5.0 mg, 10.0 mg, 15.0 mg, 20.0 mg, 25.0 mg, 30.0 mg, 35.0 mg, 40.0 mg, 45.0 mg, 50.0 mg, 55.0 mg, 60.0 mg, 65.0 mg, 70.0 mg, 75.0 mg, 80.0 mg, 85.0 mg, 90.0 mg, 95.0 mg, 100.0 mg, or a range value between any two of them as end values, for example, 20.0 mg to 100.0 mg.

[0151] In this document, the term "about" or "approximately" means an acceptable limit of error for a particular value as determined by one of ordinary skill in the art depending in part on how the value is measured or determined. In certain embodiments, the term "about" or "approximately" means within 1, 2, 3, or 4 standard deviations. In certain embodiments, the term "about" or "approximately" means within 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, or 0.05% of a given value or range.

[0152] In this document, the administration amount of the compound represented by formula (I) or a tautomer, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof is about 20.0 mg to 100.0 mg, and the administration frequency can be adjusted according to actual needs, including but not limited to once a week, once every two days, once a day, twice a day (i.e. BID), three times a day, etc., and the administration amount is set based on the administration amount for a person with normal body weight, which can be adjusted according to the body weight and condition of different people, and all of them are within the protection scope of the present application.

[0153] In a second aspect of the present application, the present application provides a use of a compound represented by formula (I) or a tautomer, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof in preventing and / or treating RXR related diseases;

[0154] According to embodiments of the present application, the compound represented by the above formula (I) or a tautomer, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof can target RXR and regulate RXR activity, and can be effective in preventing and / or treating an RXR-related disease.

[0155] According to embodiments of the present application, the RXR-related disease includes an RXRα-related disease, an RXRβ-related disease, and an RXRγ-related disease.

[0156] According to embodiments of the present application, the RXR-related disease includes a metabolic disease, inflammation, a neurodegenerative disease, a tumor, or a cancer.

[0157] According to embodiments of the present application, the RXR-related disease includes alcoholic fatty liver disease, non-alcoholic fatty liver disease, hepatitis, liver fibrosis, liver cirrhosis, liver cancer, Alzheimer's disease, Parkinson's disease, neuroinflammation, a neural tumor, psoriasis, acne, psoriasis, lupus erythematosus, skin fibrosis, Kaposi's sarcoma, cutaneous T-cell lymphoma, nasopharyngitis, bronchitis, pneumonia, pulmonary fibrosis, a pulmonary tumor, enteritis, intestinal fibrosis, an intestinal tumor, gastritis, a gastric tumor, urethritis, nephritis, renal fibrosis, a renal tumor, cholestasis, leukemia, diabetes, osteoporosis, arthritis, spondylitis, cholangitis, cholangiocarcinoma, atherosclerosis, myocarditis, hyperlipidemia, liver damage, fatty liver, lung cancer, colorectal cancer.

[0158] According to embodiments of the present application, the compound represented by the above formula (I) or a tautomer, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof is administered in an amount of about 1.0 mg to 100.0 mg, and exemplarily in an amount of 20.0 mg to 100.0 mg.

[0159] In a third aspect of the present application, the present application provides a compound represented by the above formula (I) or a tautomer, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof for use in preventing and / or treating an RXR-related disease.

[0160] According to embodiments of the present application, the compound represented by the above formula (I) or a tautomer, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof can target RXR and regulate RXR activity, and can be effective in preventing and / or treating an RXR-related disease.

[0161] According to embodiments of the present application, the RXR-related disease includes an RXRα-related disease, an RXRβ-related disease, and an RXRγ-related disease.

[0162] According to embodiments of the present application, the RXR-related disease includes a metabolic disease, inflammation, a neurodegenerative disease, a tumor, or a cancer.

[0163] According to embodiments of the present application, the RXR-related diseases include alcoholic fatty liver disease, non-alcoholic fatty liver disease, hepatitis, liver fibrosis, liver cirrhosis, liver cancer, Alzheimer's disease, Parkinson's disease, neuroinflammation, neurotumor, psoriasis, acne, psoriasis, lupus erythematosus, skin fibrosis, Kaposi's sarcoma, cutaneous T-cell lymphoma, nasopharyngitis, bronchitis, pneumonia, pulmonary fibrosis, lung tumor, enteritis, intestinal fibrosis, intestinal tumor, gastritis, gastric tumor, urethritis, nephritis, renal fibrosis, renal tumor, cholestasis, leukemia, diabetes, osteoporosis, arthritis, spondylitis, cholangitis, cholangiocarcinoma, atherosclerosis, myocarditis, hyperlipidemia, liver damage, fatty liver, lung cancer, colorectal cancer.

[0164] According to embodiments of the present application, the compound of formula (I) or a tautomer, stereoisomer, solvate, or pharmaceutically acceptable salt thereof is administered in an amount of about 1.0 mg to 100.0 mg, for example, 20.0 mg to 100.0 mg.

[0165] In a fourth aspect of the present application, the present application provides a use of a compound of formula (I) or a tautomer, stereoisomer, solvate, or pharmaceutically acceptable salt thereof in the preparation of a reagent for modulating RXR activity or modulating the activity of a dimer containing RXR;

[0166] According to embodiments of the present application, the compound of formula (I) or a tautomer, stereoisomer, solvate, or pharmaceutically acceptable salt thereof can target RXR and modulate RXR activity. Thus, the reagent prepared by using the compound of formula (I) or a tautomer, stereoisomer, solvate, or pharmaceutically acceptable salt thereof can effectively modulate RXR activity or modulate the activity of a dimer containing RXR, and is particularly suitable for in vitro regulation of the activity of RXR or a dimer containing RXR.

[0167] In an alternative embodiment of the present application, modulating RXR activity includes, but is not limited to, activating the transcriptional activity of RXR, activating or inhibiting the binding activity of RXR to DNA and RNA, activating or inhibiting the binding activity of RXR to proteins, activating or inhibiting the binding activity of RXR to lipids, and activating or inhibiting the localization activity of RXR in cells.

[0168] According to embodiments of the present application, the dimer containing RXR includes RXR homodimers and heterodimers containing RXR.

[0169] According to embodiments of the present application, the RXR-containing dimer includes RXRα-containing dimer, RXRβ-containing dimer, RXRγ-containing dimer. According to embodiments of the present application, the RXR-containing heterodimer is selected from the group consisting of RXR / RAR, RXR / PPAR, RXR / LXR, RXR / Nur77, RXR / VDR, RXR / TR, RXR / FXR, RXR / CAR, RXR / Nurr1, RXR / Nor1.

[0170] In an alternative embodiment of the present application, the RXR-containing heterodimer is selected from the group consisting of RXRα / RARα, RXRα / PPARγ, and RXRα / LXRα, RXRα / Nur77, RXRα / VDR, RXRα / TR, RXRα / FXR, RXRα / CAR, RXRα / PXR, RXRα / Nurr1, RXRα / Nor1.

[0171] According to embodiments of the present application, the final concentration of the compound of Formula (I) or a tautomer, stereoisomer, solvate, or pharmaceutically acceptable salt thereof used is about 0.1 μM to about 50.0 μM, for example, 0.1 μM, 0.5 μM, 1 μM, 2.0 μM, 2.5 μM, 3.0 μM, 3.5 μM, 4.0 μM, 4.5 μM, 5.0 μM, 5.5 μM, 6.0 μM, 7.0 μM, 8.0 μM, 9.0 μM, 10.0 μM, 11.0 μM, 12.0 μM, 13.0 μM, 14.0 μM, 15.0 μM, 16.0 μM, 17.0 μM, 18.0 μM, 19.0 μM, 20.0 μM, 21.0 μM, 22.0 μM, 23.0 μM, 24.0 μM, 25.0 μM, 30.0 μM, 35.0 μM, 40.0 μM, 45.0 μM, 50.0 μM, or a range between any two of these values, for example, 2.0 μM to 50.0 μM.

[0172] In this context, the term "final concentration used" refers to the final concentration added for the purpose of modulating RXRα activity or modulating the activity of RXRα-containing dimers. Illustratively, when cells are cultured in vitro, the final concentration used is the final concentration in the cell culture medium.

[0173] According to embodiments of the present application, the final concentration of the compound of Formula (I) or a tautomer, stereoisomer, solvate, or pharmaceutically acceptable salt thereof used is about 2.5 μM to about 20.0 μM.

[0174] In a fifth aspect, the present application provides a use of a compound of Formula (I) or a tautomer, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof in modulating RXR activity or modulating activity of a dimer containing RXR.

[0175] According to an embodiment of the present application, the compound of Formula (I) or a tautomer, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof can target RXR, modulate RXR activity, and modulate activity of a dimer containing RXR.

[0176] According to an embodiment of the present application, the dimer containing RXR includes a RXR homodimer, a heterodimer containing RXR.

[0177] According to an embodiment of the present application, the dimer containing RXR includes a RXR homodimer, a heterodimer containing RXR.

[0178] According to an embodiment of the present application, the heterodimer containing RXR is selected from RXR / RAR, RXR / PPAR, RXR / LXR, RXR / Nur77, RXR / VDR, RXR / TR, RXR / FXR, RXR / CAR, RXR / PXR, RXR / Nurr1, RXR / Nor1.

[0179] In an alternative embodiment of the present application, the heterodimer containing RXR is selected from RXRα / RARα, RXRα / PPARγ, RXRα / LXRα, RXRα / Nur77, RXRα / VDR, RXRα / TR, RXRα / FXR, RXRα / CAR, RXRα / PXR, RXRα / Nurr1, RXRα / Nor1.

[0180] In an alternative embodiment of the present application, when used for modulating RXR activity or modulating activity of a dimer containing RXR in vitro, the compound of Formula (I) or a tautomer, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof has a final concentration of about 0.1 μM to about 50.0 μM.

[0181] In an alternative embodiment of the present application, when used for activating RXRα activity or activating activity of a dimer containing RXRα in vitro, the compound of Formula (I) or a tautomer, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof has a final concentration of about 2.5 μM to about 20.0 μM.

[0182] Method for modulating RXR activity or modulating activity of a dimer containing RXR

[0183] In a sixth aspect, the present application provides a method of modulating RXR activity or modulating activity of a dimer containing RXR. According to embodiments of the present application, the method comprises contacting a compound represented by Formula (I) or a tautomer, stereoisomer, solvate, or pharmaceutically acceptable salt thereof with a cell expressing RXR;

[0184] According to embodiments of the present application, the compound represented by Formula (I) or a tautomer, stereoisomer, solvate, or pharmaceutically acceptable salt thereof can target RXR, modulate RXR activity, and further modulate activity of a dimer containing RXR. According to embodiments of the present application, the dimer containing RXR includes RXR homodimers, RXR containing heterodimers.

[0185] According to embodiments of the present application, the dimer containing RXR includes RXRα containing dimers, RXRβ containing dimers, RXRγ containing dimers.

[0186] According to embodiments of the present application, the RXR containing heterodimers are selected from the group consisting of RXR / RAR, RXR / PPAR, RXR / LXR, RXR / Nur77, RXR / VDR, RXR / TR, RXR / FXR, RXR / CAR, RXR / PXR, RXR / Nurr1, RXR / Nor1.

[0187] In an alternative embodiment of the present application, the RXR containing heterodimers are selected from the group consisting of RXRα / RARα, RXRα / PPARγ, RXRα / LXRα, RXRα / Nur77, RXRα / VDR, RXRα / TR, RXRα / FXR, RXRα / CAR, RXRα / PXR, RXRα / Nurr1, RXRα / Nor1.

[0188] In an alternative embodiment of the present application, the RXR containing heterodimers are selected from the group consisting of RXRα / RARα, RXRα / VDR, or RXRα / TR, and the method further comprises contacting a ligand of RARα, a ligand of VDR, or a ligand of TR with the compound represented by Formula (I) or a tautomer, stereoisomer, solvate, or pharmaceutically acceptable salt thereof and the cell.

[0189] In an alternative embodiment of the present application, the RXR containing heterodimers are selected from the group consisting of RXRα / RARα, and the method further comprises contacting all-trans retinoic acid (ATRA) with the compound represented by Formula (I) or a tautomer, stereoisomer, solvate, or pharmaceutically acceptable salt thereof and the cell.

[0190] It should be noted that when "contacting all-trans retinoic acid ATRA, a compound represented by formula (I) or a tautomer, stereoisomer, solvate, or pharmaceutically acceptable salt thereof with cells", all-trans retinoic acid ATRA and the compound represented by formula (I) or a tautomer, stereoisomer, solvate, or pharmaceutically acceptable salt thereof can be added simultaneously or separately (the time difference between the former and the latter is not more than 24 h, preferably not more than 18 h, 12 h, 6 h, 1 h, preferably not more than 0.5 h, more preferably not more than 10 min), and the specific type is not limited and can be adjusted according to the actual situation.

[0191] In an optional embodiment of the present application, all-trans retinoic acid ATRA, a compound represented by formula (I) or a tautomer, stereoisomer, solvate, or pharmaceutically acceptable salt thereof are simultaneously contacted with cells.

[0192] According to an embodiment of the present application, when used for in vitro modulation of RXR activity or modulation of the activity of a dimer containing RXR, the final concentration of the compound represented by formula (I) or a tautomer, stereoisomer, solvate, or pharmaceutically acceptable salt thereof is about 0.1 μM to 50.0 μM.

[0193] According to an embodiment of the present application, when used for in vitro modulation of RXRα activity or modulation of the activity of a dimer containing RXRα, the final concentration of the compound represented by formula (I) or a tautomer, stereoisomer, solvate, or pharmaceutically acceptable salt thereof is about 2.5 μM to 20.0 μM.

[0194] Combination drug or kit, and use thereof

[0195] In a seventh aspect of the present application, a combination drug or kit is provided. According to an embodiment of the present application, the combination drug or kit comprises: a compound represented by formula (I) or a tautomer, stereoisomer, solvate, or pharmaceutically acceptable salt thereof as a first active ingredient; and a ligand L as a second active ingredient, wherein the ligand L is used for modulating the activity of a nuclear receptor forming a dimer with RXRα.

[0196] According to an embodiment of the present application, the compound represented by formula (I) or a tautomer, stereoisomer, solvate, or pharmaceutically acceptable salt thereof can target RXR and modulate the activity of RXR. Therefore, the reagent prepared from the compound represented by formula (I) or a tautomer, stereoisomer, solvate, or pharmaceutically acceptable salt thereof and the ligand L can effectively modulate the activity of RXR or the activity of a dimer containing RXR, and can effectively prevent and / or treat diseases related to RXR.

[0197] According to an embodiment of the present application, the ligand L is selected from the group consisting of a ligand of RAR, a ligand of PPAR, a ligand of LXR, a ligand of TR, a ligand of VDR, a ligand of Nurse77, a ligand of FXR, a ligand of CAR, a ligand of PXR, a ligand of Nurr1, a ligand of Nor1.

[0198] According to an embodiment of the present application, the ligand L includes, but is not limited to, a ligand of RARa, a ligand of PPARy and a ligand of LXRa, a ligand of Nur77, a ligand of TR, a ligand of VDR, a ligand of FXR, a ligand of CAR, a ligand of PXR, a ligand of Nurr1, a ligand of Nor1.

[0199] According to an embodiment of the present application, the ligand L is selected from at least one of all-trans retinoic acid ATRA, rosiglitazone, T0901317 fenofibrate, calcitriol, thyroid hormone, bile acid, androstane, and progesterone.

[0200] According to an embodiment of the present application, the molar ratio of the ligand L and the compound of formula (I) or a tautomer, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof is about (0.01-10.0):(0.1-50.0).

[0201] According to an embodiment of the present application, the molar ratio of the ligand L and the compound of formula (I) or a tautomer, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof is about (0.01-10.0):(0.1-20.0).

[0202] According to an embodiment of the present application, the molar ratio of the ligand L and the compound of formula (I) or a tautomer, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof is about (0.1-1.0):(0.1-50.0).

[0203] In an alternative embodiment of the present application, the ligand L is selected from all-trans retinoic acid ATRA, and the molar ratio of the ligand L and the compound of formula (I) or a tautomer, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof is about (0.1-1.0):(2.5-20.0), preferably (0.1-0.5):(2.5-20.0), more preferably 0.1:(2.5-20.0).

[0204] In an alternative embodiment of the present application, the ligand L is selected from rosiglitazone, and the molar ratio of the ligand L and the compound of formula (I) or a tautomer, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof is about (0.1-1.0):(2.5-20.0), preferably (0.5-1.0):(2.5-20.0), more preferably 1:(2.5-20.0).

[0205] In an alternative embodiment of the present application, the ligand L is selected from T0901317, and the molar ratio of the ligand L and the compound of formula (I) or a tautomer, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof is about (0.1-1.0):(2.5-20.0), preferably (0.5-1.0):(2.5-20.0), more preferably 1:(2.5-20.0).

[0206] In an eighth aspect of the present application, the present application provides use of the combination drug or kit of the seventh aspect in the preparation of a medicament for preventing and / or treating RXR-related diseases. As known from the foregoing, the combination drug or kit can effectively regulate RXR activity or regulate the activity of a dimer containing RXR, and can effectively prevent and / or treat RXR-related diseases.

[0207] According to embodiments of the present application, the RXR-related diseases include RXRα-related diseases, RXRβ-related diseases, RXRγ-related diseases

[0208] According to embodiments of the present application, the RXR-related diseases include metabolic diseases, inflammation, neurodegenerative diseases, tumors or cancers.

[0209] According to embodiments of the present application, the RXR-related diseases include alcoholic fatty liver disease, non-alcoholic fatty liver disease, hepatitis, liver fibrosis, liver cirrhosis, liver cancer, Alzheimer's disease, Parkinson's disease, neuroinflammation, neurotumor, psoriasis, acne, psoriasis, lupus erythematosus, skin fibrosis, Kaposi's sarcoma, cutaneous T-cell lymphoma, nasopharyngitis, bronchitis, pneumonia, pulmonary fibrosis, lung tumor, enteritis, intestinal fibrosis, intestinal tumor, gastritis, gastric tumor, urethritis, nephritis, renal fibrosis, renal tumor, cholestasis, leukemia, diabetes, osteoporosis, arthritis, spondylitis, cholangitis, cholangiocarcinoma, atherosclerosis, myocarditis, hyperlipidemia, liver damage, fatty liver, lung cancer, colorectal cancer.

[0210] According to embodiments of the present application, the molar ratio of the ligand L and the compound of formula (I) or a tautomer, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof is about (0.01-10.0):(0.1-50.0).

[0211] According to embodiments of the present application, the molar ratio of the ligand L and the compound of formula (I) or a tautomer, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof is about (0.01-10.0):(0.1-20.0).

[0212] According to embodiments of the present application, the molar ratio of the ligand L and the compound of formula (I) or a tautomer, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof is about (0.1-1.0):(0.1-50.0).

[0213] In an alternative embodiment of the present application, the ligand L is selected from all-trans retinoic acid ATRA, and the molar ratio of the ligand L and the compound of formula (I) or a tautomer, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof is about (0.1-1.0):(2.5-20.0), preferably (0.1-0.5):(2.5-20.0), more preferably 0.1:(2.5-20.0).

[0214] In an alternative embodiment of the present application, the ligand L is selected from rosiglitazone, and the molar ratio of the ligand L and the compound of formula (I) or a tautomer, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof is about (0.1-1.0):(2.5-20.0), preferably (0.5-1.0):(2.5-20.0), more preferably 1:(2.5-20.0).

[0215] In an alternative embodiment of the present application, the ligand L is selected from T0901317, and the molar ratio of the ligand L and the compound of formula (I) or a tautomer, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof is about (0.1-1.0):(2.5-20.0), preferably (0.5-1.0):(2.5-20.0), more preferably 1:(2.5-20.0).

[0216] Method for preventing and / or treating RXR related diseases

[0217] In a ninth aspect, the present application provides a method for preventing and / or treating RXR related diseases. According to embodiments of the present application, the method comprises administering to a subject a pharmaceutically acceptable dose of the compound of formula (I) or a tautomer, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof, or the combination drug or kit of the seventh aspect.

[0218] According to embodiments of the present application, the RXR related diseases include RXRα related diseases, RXRβ related diseases, RXRγ related diseases.

[0219] According to embodiments of the present application, the RXR related diseases include metabolic diseases, inflammation, neurodegenerative diseases, tumors or cancers.

[0220] According to an embodiment of the present application, the RXR-related disease includes alcoholic fatty liver disease, non-alcoholic fatty liver disease, hepatitis, liver fibrosis, liver cirrhosis, liver cancer, Alzheimer's disease, Parkinson's disease, neuroinflammation, neurotumor, psoriasis, acne, psoriasis, lupus erythematosus, skin fibrosis, Kaposi's sarcoma, cutaneous T-cell lymphoma, nasopharyngitis, bronchitis, pneumonia, pulmonary fibrosis, lung tumor, enteritis, intestinal fibrosis, intestinal tumor, gastritis, gastric tumor, urethritis, nephritis, renal fibrosis, renal tumor, cholestasis, leukemia, diabetes, osteoporosis, arthritis, spondylitis, cholangitis, cholangiocarcinoma, atherosclerosis, myocarditis, hyperlipidemia, liver damage, fatty liver, lung cancer, colorectal cancer.

[0221] According to an embodiment of the present application, the compound of formula (I) or a tautomer, stereoisomer, solvate, or pharmaceutically acceptable salt thereof is administered in an amount of about 1.0 mg to 100.0 mg, illustratively 20.0 mg to 100.0 mg.

[0222] The present application will be explained in detail with reference to the following examples. Those skilled in the art will appreciate that the following examples are intended to be illustrative only and are not intended to limit the scope of the present application. Unless otherwise indicated, the techniques and conditions used in the examples are those described in the literature or as specified by the manufacturer of the product. Unless otherwise indicated, the reagents and instruments used in the examples are conventional products available on the market.

[0223] Herein, the pCMV-myc-RXRα plasmid refers to a pCMV plasmid encoding an RXRα protein with a myc tag.

[0224] Herein, the pCMV-myc-RARα plasmid refers to a pCMV plasmid encoding an RARα protein with a myc tag.

[0225] Herein, the pCMV-myc-PPARγ plasmid refers to a pCMV plasmid encoding a PPARγ protein with a myc tag.

[0226] Herein, the pCMV-myc-LXRα plasmid refers to a pCMV plasmid encoding an LXRα protein with a myc tag.

[0227] Herein, the pBIND-RXRα LBD plasmid refers to a pBIND plasmid encoding a Gal4 / DBD-RXRα / LBD fusion protein.

[0228] Herein, the pBIND-RARα LBD plasmid refers to a pBIND plasmid encoding a Gal4 / DBD-RARα / LBD fusion protein.

[0229] As used herein, the pBIND-RARγLBD plasmid refers to a pBIND plasmid encoding a Gal4 / DBD-RARγ / LBD fusion protein.

[0230] As used herein, the pBIND-LXRαLBD plasmid refers to a pBIND plasmid encoding a Gal4 / DBD-LXRα / LBD fusion protein.

[0231] As used herein, the pBIND-LXRβLBD plasmid refers to a pBIND plasmid encoding a Gal4 / DBD-LXRβ / LBD fusion protein.

[0232] As used herein, the pBIND-PPARαLBD plasmid refers to a pBIND plasmid encoding a Gal4 / DBD-PPARα / LBD fusion protein.

[0233] As used herein, the pBIND-PPARγLBD plasmid refers to a pBIND plasmid encoding a Gal4 / DBD-PPARγ / LBD fusion protein.

[0234] As used herein, the pBIND-ERαLBD plasmid refers to a pBIND plasmid encoding a Gal4 / DBD-ERα / LBD fusion protein.

[0235] As used herein, the pBIND-ERβLBD plasmid refers to a pBIND plasmid encoding a Gal4 / DBD-ERβ / LBD fusion protein.

[0236] As used herein, the pGL6-TA-RXRE-luciferase plasmid refers to a reporter plasmid responsive to RXR / RXR homodimer, wherein RXRE refers to the DNA binding element for RXR / RXR homodimer, for detecting the transcriptional activity of RXR / RXR homodimer.

[0237] As used herein, the pGL6-TA-PPRE-luciferase plasmid refers to a reporter plasmid responsive to RXR / PPAR heterodimer, wherein PPRE refers to the DNA binding element for RXR / PPAR heterodimer.

[0238] As used herein, the pGL6-TA-LXRE-luciferase plasmid refers to a reporter plasmid responsive to RXR / LXR heterodimer, wherein LXRE refers to the DNA binding element for RXR / LXR heterodimer.

[0239] As used herein, the pGL6-TA-RARE-luciferase plasmid refers to a reporter plasmid responsive to RXR / RAR heterodimer, wherein RARE refers to the DNA binding element for RXR / RAR heterodimer.

[0240] Herein, the pG5-luciferase plasmid refers to a reporter gene plasmid of a corresponding Gal4 / DBD fusion protein.

[0241] Herein, the renilla-luciferase plasmid refers to a sea cucumber luciferase plasmid, which is an internal reference for a reporter gene experiment.

[0242] The Gene ID of the nucleotide sequence in the embodiment herein is shown in the following table:

[0243] The amino acid sequence or nucleotide sequence in the embodiment herein is shown in the following table:

[0244] Note: N in the above table is a random nucleotide, which can be A, C, G or T.

[0245] The materials and methods used in the following embodiments are as follows:

[0246] 1. Experimental materials

[0247] 1.1 Tafamidis is derived from Macrin.

[0248] 1.2 The experimental animals are C57BL / 6 and ICR male mice, 6-8 weeks old, SPF level, purchased from Hangzhou Medical College, and raised in the SPF environment laboratory of the Experimental Animal Center of Xiamen University.

[0249] 2. Experimental methods

[0250] 2.1 Cell transfection

[0251] (1) When the cell density reaches about 60%, replace the cell culture medium with serum-free basic medium (DMEM or Opti-MEM) 1 hour before transfection.

[0252] (2) Mix an appropriate amount of plasmid DNA to be transfected with an appropriate volume of Opti-MEM medium, then add an appropriate amount of Lipo2000 liposome transfection reagent, mix thoroughly, and stand at room temperature for 15 minutes. The specific amount of each reagent is shown in the following table:

[0253] (3) After standing for 15 minutes, add the mixture to the cell culture dish and place it in a CO2 incubator at 37°C for culture. After 6 hours, replace the fresh culture medium containing serum, and perform subsequent experiments after 12-24 hours.

[0254] 2.2 In the embodiment of the application, the qRT-PCR primer sequence is shown in the following table:

[0255] 2.3 Data statistics and analysis

[0256] All data are expressed as mean ± standard error (SEM) and analyzed using GraphPad Prism software. Statistical analysis was performed using t-test or one-way ANOVA, p < 0.05 (*) is statistically significant, p < 0.01 (**) is highly significant, p < 0.001 (***), and p < 0.0001 (****) is extremely significant, ns is not significant.

[0257] Example 1: Determination of the effect of Tafamidis on the transcriptional activity of RXRa and its dimers

[0258] 1. Mammalian one-hybrid assay to determine the effect of Tafamidis on the transcriptional activity of RXRa

[0259] HEK 293T cells were transfected with pG5-luciferase and pBIND-RXRaLBD plasmids for 24 hours, followed by treatment with DMSO (as a negative control group), 0.5 mM of Tafamidis, 1 mM of Tafamidis, 2.5 mM of Tafamidis, 5 mM of Tafamidis, 10 mM of Tafamidis, 20 mM of Tafamidis, or 0.1 mM of CD3254 (as a positive control group) for 12 hours, and the reporter gene activity was determined using the firefly and sea pansy dual luciferase system. The results are shown in Figure 1A.

[0260] The results show that Tafamidis significantly activates the transcriptional activity of Gal4 / DBD-RXRa / LBD.

[0261] 2. Reporter gene assay to determine the effect of Tafamidis on the transcriptional activity of RXRa homodimers

[0262] HEK 293T cells were co-transfected with pGL6-TA-RXRE-luciferase, renilla-luciferase, and pCMV-myc-RXRa plasmids for 24 hours, followed by treatment with DMSO (as a negative control group), 0.5 mM of Tafamidis, 1 mM of Tafamidis, 2.5 mM of Tafamidis, 5 mM of Tafamidis, 10 mM of Tafamidis, 20 mM of Tafamidis, or 0.1 mM of 9-cis-retinoic acid (9-c-RA) (as a positive control group) for 12 hours, and the reporter gene activity was determined using the firefly and sea pansy dual luciferase system. The results are shown in Figure 1B.

[0263] The results show that Tafamidis significantly activates the transcriptional activity of RXRα / RXRα homodimer.

[0264] 3. Reporter assay to determine the effect of Tafamidis on the transcriptional activity of RXRα heterodimers

[0265] 3.1 HEK 293T cells were cotransfected with pGL6-TA-LXRE-luciferase, renilla-luciferase, pCMV-myc-RXRα and pCMV-myc-LXRα plasmids for 24 hours, followed by treatment with DMSO, 0.1 μΜ of Tafamidis, 0.5 μΜ of Tafamidis, 2.5 μΜ of Tafamidis, 10 μΜ of Tafamidis and / or 1 μΜ T0901317 for 12 hours. Reporter activity was determined using the dual luciferase system. The results are shown in Figure 2A.

[0266] The results show that Tafamidis activates the transcriptional activity of RXRα / LXRα heterodimers both alone and significantly enhances the activation of the transcriptional activity of RXRα / LXRα heterodimers by T0901317.

[0267] 3.2 HEK 293T cells were cotransfected with pGL6-TA-PPRE-luciferase, renilla-luciferase, pCMV-myc-RXRα and pCMV-myc-PPARγ plasmids for 24 hours, followed by treatment with DMSO, 0.1 μΜ of Tafamidis, 0.5 μΜ of Tafamidis, 2.5 μΜ of Tafamidis, 10 μΜ of Tafamidis and / or 1 μΜ rosiglitazone (ROZ) for 12 hours. Reporter activity was determined using the dual luciferase system. The results are shown in Figure 2B.

[0268] The results show that Tafamidis activates the transcriptional activity of RXRα / PPARγ heterodimers both alone and significantly enhances the activation of the transcriptional activity of RXRα / PPARγ heterodimers by rosiglitazone.

[0269] 3.3 HEK 293T cells were cotransfected with pGL6-TA-RARE-luciferase, renilla-luciferase, pCMV-myc-RXRα and pCMV-myc-RARα plasmids for 24 hours, followed by treatment with DMSO, 0.1 μM, 0.5 μM, 2.5 μM, 10 μM of Tafamidis and / or 0.1 μM all-trans retinoic acid (ATRA) for 12 hours. Reporter gene activity was measured by the dual luciferase system of firefly and sea pansy. The results are shown in Figure 2C.

[0270] The results show that Tafamidis alone cannot activate the transcriptional activity of RXRα / RARα heterodimer, because RXR forms a non-permissive heterodimer with RAR, but Tafamidis can significantly enhance the activation of the transcriptional activity of RXRα / RARα heterodimer by ATRA.

[0271] 3.4 HEK 293T cells were cotransfected with VDRE-luciferase, renilla-luciferase, pCMV-myc-RXRα and VDR plasmids for 24 hours, followed by treatment with DMSO, 0.1 μM, 0.5 μM, 2.5 μM, 10 μM of Tafamidis and / or 0.1 μM calcipotriol for 12 hours. Reporter gene activity was measured by the dual luciferase system of firefly and sea pansy. The results show that Tafamidis can significantly enhance the activation of the transcriptional activity of RXRα / VDR heterodimer by calcipotriol.

[0272] 3.5 HEK 293T cells were cotransfected with CARE-luciferase, renilla-luciferase, pCMV-myc-RXRα and CAR plasmids for 24 hours, followed by treatment with DMSO, 0.1 μM, 0.5 μM, 2.5 μM, 10 μM of Tafamidis and / or 1 μM TCPOBOP for 12 hours. Reporter gene activity was measured by the dual luciferase system of firefly and sea pansy. The results show that Tafamidis can significantly enhance the activation of the transcriptional activity of RXRα / CAR heterodimer by TCPOBOP.

[0273] 3.6 HEK 293T cells were cotransfected with PXRE-luciferase, renilla-luciferase, pCMV-myc-RXRα and PXR plasmids for 24 hours, followed by treatment with DMSO, 0.1 μM, 0.5 μM, 2.5 μM, 10 μM of Tafamidis and / or 10 μM of rifampicin for 12 hours. Reporter gene activity was measured using the dual luciferase system. The results are shown in the figure. The results show that Tafamidis significantly enhances rifampicin activation of the transcriptional activity of RXRα / PXR heterodimers.

[0274] 3.7 HEK 293T cells were cotransfected with TRE-luciferase, renilla-luciferase, pCMV-myc-RXRα and TRα plasmids for 24 hours, followed by treatment with DMSO, 0.1 μM, 0.5 μM, 2.5 μM, 10 μM of Tafamidis and / or 1 μM of T3 for 12 hours. Reporter gene activity was measured using the dual luciferase system. The results are shown in the figure. The results show that Tafamidis significantly enhances T3 activation of the transcriptional activity of RXRα / TRα heterodimers.

[0275] 3.8 HEK 293T cells were cotransfected with NurRE-luciferase, renilla-luciferase, pCMV-myc-RXRα and Nur77 plasmids for 24 hours, followed by treatment with DMSO, 0.1 μM, 0.5 μM, 2.5 μM, 10 μM of Tafamidis and / or 1 μM of CsnB for 12 hours. Reporter gene activity was measured using the dual luciferase system. The results are shown in the figure. The results show that Tafamidis significantly enhances CsnB activation of the transcriptional activity of RXRα / Nur77 heterodimers.

[0276] 3.9 HEK 293T cells were cotransfected with FXRE-luciferase, renilla-luciferase, pCMV-myc-RXRα and FXR plasmids for 24 hours, followed by treatment with DMSO, 0.1 μM, 0.5 μM, 2.5 μM, 10 μM of Tafamidis and / or 1 μM of chenodeoxycholic acid for 12 hours. Reporter gene activity was measured using the dual luciferase system. The results are shown in the figure. The results show that Tafamidis significantly enhances chenodeoxycholic acid activation of the transcriptional activity of RXRα / FXR heterodimers.

[0277] 4. Mammalian two-hybrid assay to determine the effect of Tafamidis on the transcriptional activity of other nuclear receptors

[0278] HEK 293T cells were transfected with pG5-luciferase and pBIND-RARaLBD plasmid or pBIND-RARyLBD plasmid or pBIND-LXRaLBD plasmid or pBIND-LXRyLBD plasmid or pBIND-PPARaLBD plasmid or pBIND-PPARyLBD plasmid or pBIND-ERaLBD plasmid or pBIND-ERyLBD plasmid for 24 hours, followed by treatment with DMSO (as negative control group), 10 μM Tafamidis or 0.1 μM ATRA, 1 μM T0901317, 10 μM WY14643, 1 μM Rosiglitazone, 0.01 μM E2 (as positive control group) for 12 hours, and then luciferase activity was determined by dual luciferase reporter assay system, and the results are shown in Figure 3.

[0279] The results show that Tafamidis cannot activate the transcriptional activity of Gal4 / DBD-RARa / LBD, Gal4 / DBD-RARy / LBD, Gal4 / DBD-LXRa / LBD, Gal4 / DBD-LXRy / LBD, Gal4 / DBD-PPARa / LBD, Gal4 / DBD-PPARy / LBD, Gal4 / DBD-ERa / LBD, Gal4 / DBD-ERy / LBD.

[0280] Therefore, it can be explained that Tafamidis can specifically regulate RXRa, and can activate the transcriptional activity of RXRa or activate the transcriptional activity of RXRa homodimer or its heterodimer.

[0281] Example 2: Determination of the direct binding of Tafamidis to RXRa protein by binding experiment

[0282] 1. Differential scanning calorimetry to determine the binding of Tafamidis to RXRa protein

[0283] After mixing RXRa / LBD protein (20 μM, amino acid sequence shown in SEQ ID NO: 2) with Tafamidis (100 μM), the thermal changes of the protein during heating were recorded by differential scanning calorimetry, and compared with the thermal changes of RXRa / LBD (20 μM) protein alone. The protein Tm value was obtained by plotting with Origin software, and the results are shown in Figure 4A.

[0284] The results show that RXRa / LBD protein exhibits two Tm in thermal denaturation, and Tafamidis can significantly increase the two Tm values of RXRa / LBD, indicating that Tafamidis can stabilize RXRa / LBD protein, and also indicating that Tafamidis can directly bind to RXRa / LBD protein.

[0285] 2. Surface plasmon resonance (SPR) assay to determine the binding of Tafamidis to RXRα protein.

[0286] The extracted and purified RXRα / LBD protein was coupled to a CM5 chip compatible with Biacore T200. Tafamidis solutions of different concentration gradients were prepared (see Figure 4B for details), and the solutions were passed through the chip sequentially while the response values ​​were recorded. The results were plotted using Graphpad software, as shown in Figure 4B.

[0287] The results showed that the RXRα / LBD protein response value increased with the increase of Tafamidis concentration gradient, indicating that Tafamidis can bind to RXRα.

[0288] 3. Determination of the binding of Tafamidis to RXRα protein by fluorescence titration.

[0289] Different concentrations of Tafamidis (see the horizontal axis of Figure 4C) and purified 1 μM RXRα / LBD protein were incubated at room temperature for 2 minutes. The emission spectra of RXRα / LBD protein excited by 280 nm excitation light in the 300-450 nm range were detected, and the results were analyzed using Origin software and the formula "y=C×(x+5+K". d ) / 2-C×sqrt(x+5+K d ) 2 -4×5x) / 2” calculates the K of Tafamidis combined with RXRα d The value is shown in Figure 4C.

[0290] The results showed that Tafamidis can bind to RXRα, and the K-value of Tafamidis and RXRα binding is... d The value is 10.97±0.70μM.

[0291] 4. Determination of the binding of Tafamidis to RXRα protein using thermoplethysmography.

[0292] HeLa cells were treated with DMSO (equal volumes) and Tafamidis, respectively. Lysates were collected and incubated at progressively increasing temperatures. The supernatant from the centrifuged lysates was used for Western blotting to detect undenatured RXRα protein. The obtained protein bands were quantitatively analyzed using ImageJ software. The relative values ​​of RXRα protein content at each temperature (percentage of RXRα protein at the lowest temperature) were calculated, and protein content variation curves were plotted using Graphpad software. The results are shown in Figure 5.

[0293] The results show that the treatment of Tafamidis can significantly enhance the Tm of RXRa protein, indicating that Tafamidis can promote the thermal stability of RXRa protein by binding to RXRa protein.

[0294] Example 3: Effect of Tafamidis on fibrosis-related indicators in a cell fibrosis model

[0295] 1. Effect of Tafamidis on TGFβ-induced activation phenotype of hepatic stellate cells

[0296] Hepatic stellate cells in the resting state are polygonal and contain vitamin A-rich lipid droplets in the cytoplasm; after activation, they transform into myofibroblasts, which are spindle-shaped and have reduced or no lipid droplets. Rat hepatic stellate cells CFSC were treated with TGFβ (5 ng / mL) alone or with Tafamidis (10 μM) for 24 hours, and the cell morphology was observed under a microscope. The results are shown in Figure 6A. The results show that TGFβ induces the transformation of cells from polygonal to spindle-shaped, and Tafamidis inhibits the TGFβ-induced transformation of cell morphology.

[0297] Rat hepatic stellate cells CFSC were treated with TGFβ (5 ng / mL) alone or with Tafamidis (10 μM) for 24 hours, and then stained with oil red O. The lipid droplets were observed under a microscope. The results are shown in Figure 6B. The results show that TGFβ induces a decrease in lipid droplets in cells, and Tafamidis significantly restores the content of lipid droplets in cells.

[0298] 2. Effect of Tafamidis on TGFβ-induced activation gene expression of hepatic stellate cells

[0299] 2.1 Rat hepatic stellate CFSC cells were treated with TGFβ (5 ng / mL) alone or with different concentrations (2.5 μM, 5 μM, 10 μM) of Tafamidis for 24 hours, and the expression of COL1A1 and α-SMA proteins was detected by immunoblotting. The results are shown in Figure 7A. The results show that Tafamidis can inhibit the expression of COL1A1 and α-SMA proteins induced by TGFβ.

[0300] 2.2 CFSC cells were treated with TGFβ (5 ng / mL) alone or with Tafamidis (10 μM) for 24 hours or 36 hours, and the expression of COL1A1, fibronectin, and α-SMA proteins was detected by immunoblotting. The results are shown in Figure 7B. The results show that Tafamidis can inhibit the expression of COL1A1, fibronectin, and α-SMA proteins induced by TGFβ, and this inhibition occurs at both 24 hours and 36 hours.

[0301] 2.3 CFSC cells were treated with TGFβ (5 ng / mL) alone or with Tafamidis (10 μΜ) for 24 hours, qRT-PCR was used to detect the mRNA expression levels of the activation markers of stellate cells, α-SMA, COL1A1 and CTGF, with β-actin as the internal reference gene and normalized to the control. The results are shown in Figure 7C. The results show that Tafamidis can inhibit the expression of TGFβ-induced COL1A1, α-SMA and CTGF mRNA levels.

[0302] 2.4 After CFSC cells were treated with TGFβ (5 ng / mL) alone or with Tafamidis (10 μΜ) for 24 hours, immunofluorescence staining was used to observe the expression of α-SMA, and the scale in the figure is 18.4 μm. ImageJ was used to count the fluorescence staining intensity, and the results are shown in Figure 8. The results show that Tafamidis can inhibit the expression of the fibrosis marker α-SMA.

[0303] 2.5 In human hepatic stellate LX2 cells, TGFβ (5 ng / mL) alone or with different concentrations (2.5 μΜ, 5 μΜ, 10 μΜ) of Tafamidis were used for 24 hours, and immunoblotting was used to detect the expression of COL1A1 protein, and the results are shown in Figure 9A. The results show that Tafamidis can inhibit the expression of TGFβ-induced COL1A1 protein.

[0304] 2.6 LX2 cells were treated with TGFβ (5 ng / mL) alone or with Tafamidis (10 μΜ) for 24 hours, and qRT-PCR was used to detect the mRNA expression levels of the activation markers of stellate cells, COL1A1 and COL4A1, with β-actin as the internal reference gene and normalized to the control. The results are shown in Figure 9B. The results show that Tafamidis can inhibit the mRNA expression of TGFβ-induced COL1A1 and COL4A1.

[0305] 3. RXRα-dependent inhibition of Tafamidis on the expression of hepatic stellate cell activation genes

[0306] The expression of RXRα was inhibited by small hairpin RNA (shRNA) in LX2 cells (Figure 10A) and CFSC cells (Figure 10B), respectively, and treated with TGFβ (5 ng / mL) alone or with Tafamidis (10 μΜ) for 24 hours, and immunoblotting was used to detect the expression of the activation marker protein of hepatic stellate cells.

[0307] The results show that Tafamidis inhibits the expression of TGFβ-induced activated fibrosis genes COL1A1 or α-SMA, and this effect is RXRα-dependent.

[0308] Example 4: Effect of Tafamidis on fibrosis-related indicators in a CCl4 liver fibrosis model in mice

[0309] A CCl4 liver fibrosis model was constructed, in which SPF grade C57BL / 6 male mice of about 8 weeks of age were randomly divided into a control group and a model group. CCl4 was mixed with corn oil at a ratio of 1:3. During the modeling process, the model group mice were injected intraperitoneally with 25% CCl4 corn oil solution three times a week, and the control group mice were injected intraperitoneally with corn oil at a dose of 2 mL / kg. The modeling time was a total of five weeks. After three weeks of modeling, the mice were injected intraperitoneally with corn oil or 25% CCl4 corn oil solution while being gavaged with solvent or Tafamidis (5 mg / kg and 20 mg / kg) for two weeks, and the mice were sampled.

[0310] 1. The mouse liver was collected, and the liver appearance was observed. The results are shown in FIG. 11A. As shown in the figure, the liver of the control group was red and smooth in surface, and soft in texture; compared with the control group, the liver surface of the CCl4 model group mice had obvious granular feeling, reduced luster, and hard texture, and showed obvious liver fibrosis phenotype, and Tafamidis could improve the liver appearance of the model group mice, and make the liver appearance partially return to normal.

[0311] 2. After blood was collected from the CCl4 liver fibrosis model mice, the serum was collected by centrifugation, and the serum liver function ALT and AST indicators were detected. The results are shown in FIG. 11B. As shown in the figure, Tafamidis could dose-dependently down-regulate the ALT and AST contents in the serum of the model group mice, i.e., Tafamidis could improve the liver function damage induced by CCl4.

[0312] 3. Tafamidis could dose-dependently improve the morphological disorder and collagen deposition of the liver tissue of the CCl4-induced mice.

[0313] 3.1 The liver of the mouse was paraffin sectioned, and then HE staining was performed. The results are shown in FIG. 12A, in which the scale is 100 pm. As shown in the figure, the liver cells of the control group were arranged regularly, and were arranged radially around the central vein; the model group had a pseudolobular structure, the liver cells were arranged in disorder, the cord arrangement was not obvious, and there were obvious gaps. The liver cells around the pseudolobule showed obvious acidophilia. The treatment of Tafamidis could obviously improve the pathological changes of the above liver tissue.

[0314] 3.2 The paraffin section of the liver of the mouse was subjected to Sirius red staining, the scale in the figure is 100 pm, and the fiber area represented by Sirius red staining was counted using ImageJ software. The results are shown in FIG. 12B. The results show that, compared with the control group, the liver of the model group mice has obvious collagen deposition, and Tafamidis can dose-dependently inhibit the collagen deposition of the liver of the model group mice.

[0315] 4. Tafamidis can improve the expression of fibrosis-related genes in CCl4-induced mouse liver tissues.

[0316] The liver tissues of CCl4 liver fibrosis model mice were taken, and after RNA extraction, the transcription levels of a-SMA, COL1A1 and TIMP1 were detected by qRT-PCR method, GAPDH was used as an internal reference gene and normalized with the control. The results are shown in Figure 13A. The liver tissues of CCl4 liver fibrosis model mice were taken, and the protein expression levels of a-SMA and COL1A1 were detected by immunoblotting. The results are shown in Figure 13B.

[0317] The results show that Tafamidis can inhibit the expression of CCl4-induced liver fibrosis marker proteins such as a-SMA and COL1A1.

[0318] 5. Tafamidis can improve the activation of mouse hepatic stellate cells induced by CCl4.

[0319] Primary hepatic stellate cells were isolated from the livers of control and liver fibrosis model mice, and the protein expression levels of a-SMA and COL1A1 were detected by immunoblotting. The results are shown in Figure 14. The results show that compared with the control group, the expression of a-SMA and COL1A1 in the liver stellate cells of the model group mice increased significantly, indicating that the liver stellate cells were significantly activated, and the activation state of stellate cells in the liver of Tafamidis-treated model mice was significantly down-regulated.

[0320] 6. Tafamidis can improve the inflammation infiltration of CCl4-induced mouse liver.

[0321] The liver of the mouse was paraffin sectioned, then immunohistochemical staining F4 / 80 was performed, the results are shown in Figure 15, the scale is 100 μm, and the staining area was counted using ImageJ software. The results show that the macrophages in the liver tissues of the control group mice are evenly distributed, while the number of macrophages in the liver of the model group mice increases significantly and shows an aggregated distribution, indicating that there is a significant inflammatory response in the liver of the model group, and Tafamidis can dose-dependently inhibit this inflammatory response.

[0322] Example 5: Effect of Tafamidis on NASH and fibrosis-related indicators in MCD model mice.

[0323] A mouse choline-methionine deficiency (MCD) model was constructed, i.e. SPF level C57BL / 6 male mice at about 8 weeks of age were randomly divided into a control group and a model group. The control group was fed with ordinary feed. The model group was first adaptively fed with a ratio of control feed to MCD feed of 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, and 1:4 for one week, and then completely fed with MCD feed. After a total of 3 weeks of feeding, the model group was randomly divided into different administration groups. After 3 weeks, the MCD model group was randomly divided into a solvent group, a Tafamidis 5 mg / kg group, and a Tafamidis 20 mg / kg group, which were administered daily and fed with MCD feed. Samples were collected after three weeks.

[0324] 1. The mouse liver was collected, and the liver appearance was observed. The results are shown in FIG. 16A. As shown in the figure, the liver of the control group was red, smooth in surface, and soft in texture; compared with the control group, the liver of the MCD feed model group was yellowish in color, had a clear granular sensation on the surface, showed obvious fatty liver and fibrosis phenotype, and the appearance of the liver of the MCD model group was improved by Tafamidis treatment

[0325] 2. After blood was collected from the MCD model mice, the serum was collected by centrifugation, and the serum liver function ALT, AST, and T-BiL indicators were detected. The results are shown in FIG. 16B. As shown in the figure, Tafamidis can dose-dependently down-regulate the content of ALT and AST in the serum of the model group mice, and a low concentration of Tafamidis can significantly down-regulate the T-BiL indicator in the serum of the model group mice, i.e. Tafamidis can improve the liver function damage induced by MCD.

[0326] 3. Tafamidis can dose-dependently improve the fatty accumulation and collagen deposition of the liver tissue induced by MCD feed in mice.

[0327] The mouse liver was paraffin sectioned, and then HE staining was performed. The results are shown in FIG. 17A, in which the scale is 100 pm. As shown in the figure, the liver cells of the control group were arranged regularly, and were arranged radially around the central vein; the liver tissue lobule structure of the MCD feed group mice was destroyed, and a large number of large vacuolar ballooning cells appeared, showing fatty accumulation; the treatment of Tafamidis can obviously improve the pathological changes of the above liver tissue.

[0328] After the mouse liver tissue was lysed, the triglyceride content was detected. The results are shown in FIG. 17D. As shown in the figure, compared with the control group, the triglyceride content of the liver of the MCD model group mice was significantly increased, showing fatty accumulation; the treatment of Tafamidis can obviously improve the fatty accumulation of the liver.

[0329] The liver paraffin sections of mice were subjected to Sirius red staining, the scale in the figure is 100 pm, and the results are shown in Figure 17B; the fibrous area represented by Sirius red staining was counted using ImageJ software, and the results are shown in Figure 17C. The results show that the liver of the model group mice has obvious collagen deposition compared with the control group, and Tafamidis can dose-dependently inhibit the collagen deposition in the liver of the model group mice.

[0330] 4. Tafamidis can improve the expression of fibrosis-related genes in the liver tissue of MCD-fed mice.

[0331] The liver tissue of the MCD model mice was taken, RNA extraction was performed, and the transcription levels of a-SMA, COL1A1, TIMP1 and TNF a were detected by qRT-PCR method (qRT-PCR primer sequence, see step 4 in Example 4), GAPDH was used as an internal reference gene and normalized with the control, and the results are shown in Figure 18A. The liver tissue of the MCD model mice was taken, and the protein expression level of a-SMA was detected by immunoblotting, and the results are shown in Figure 18B.

[0332] The results show that Tafamidis can inhibit the expression of liver fibrosis marker proteins in MCD model mice.

[0333] 5. Tafamidis can improve the inflammatory infiltration of MCD diet-induced mouse liver.

[0334] The liver of the mice was paraffin sectioned, then subjected to immunohistochemical staining of F4 / 80, and the results are shown in Figure 19, the scale in the figure is 100 pm, and the stained area was counted using ImageJ software. The results show that the macrophages in the liver tissue of the control group mice are evenly distributed, while the number of macrophages in the liver of the model group mice is significantly increased and is in an aggregated distribution, indicating that there is a significant inflammatory response in the liver of the model group, and Tafamidis can dose-dependently inhibit this inflammatory response.

[0335] Example 6: Therapeutic effect of Tafamidis on neurodegenerative diseases

[0336] 1. Inducing effect of Tafamidis on ABCA1 protein of mouse microglial cells

[0337] The mouse microglial cells BV2 were treated with different concentrations (10 mM, 20 mM) of Tafamidis for 12 hours, and the protein expression level of ABCA1 was detected by immunoblotting, and the results are shown in Figure 20A. The results show that Tafamidis can induce ABCA1 protein expression in a concentration gradient, indicating that it may play a role in the process of clearing Aβ plaques. Aβ plaques are AD causative proteins, which indicates that Tafamidis may inhibit Alzheimer's disease by clearing Aβ plaques.

[0338] 2. Tafamidis inhibits LPS-induced inflammation in mouse microglia

[0339] Mouse microglia BV2 were co-treated with LPS (1 pg / mL) and Tafamidis 10 mM for 6 hours, and the protein expression level of iNOS was detected by immunoblotting. The results are shown in Figure 20B. The results showed that Tafamidis could not only reduce the protein expression of iNOS in cells themselves, but also reduce the protein expression of iNOS induced by LPS, indicating that it has the effect of inhibiting neuroinflammation.

[0340] 3. Tafamidis inhibits Tau protein phosphorylation

[0341] Human neuroblastoma cells SH-sy5y were first treated with okadaic acid OA (20 nM) for 12 hours to induce TAU protein phosphorylation, and then co-treated with Tafamidis 10 mM for 12 hours. Immunoblotting experiments revealed that Tafamidis could inhibit the expression of p-TAU protein, and the results are shown in Figure 28.

[0342] 4. Tafamidis improves cognition and clears Aβ plaques in AD model mice

[0343] Tafamidis (10 mg / Kg dose) was treated in 5xFAD, APP / PS1 two kinds of gene mice and WT mice for four weeks. In the Morris water maze, animal behavior experiments such as mouse nest building, it was found that Tafamidis could improve the cognitive ability of mice. The blood and cerebrospinal fluid of mice were collected, and it was found that Tafamidis significantly down-regulated the content of Aβ. The hippocampus and cortex of mouse brain tissue were separated, and a part of the hippocampus and cortex was subjected to protein immunoblotting, and it was found that Tafamidis could up-regulate the expression of ABCA1, ABCG1, ApoE and other proteins, but down-regulate the expression of Aβ and p-Tau. A part of the hippocampus and cortex was subjected to frozen section and immunostaining, and it was found that Tafamidis reduced the distribution of Aβ and p-Tau in the hippocampus and cortex.

[0344] 5. Tafamidis inhibits LPS-induced neuroinflammation

[0345] Mouse microglia BV2 were first co-treated with Tafamidis 10 mM for 12 hours, and then LPS (1 pg / mL) was added for 12 hours. qPCR detection found that Tafamidis inhibited the mRNA expression of inflammatory factors such as IL-6, TNF-a, IL-1β induced by LPS, and the results are shown in Figure 29.

[0346] Male C57BL / 6 mice were pretreated with Tafamidis at 10 mg / kg for 7 days, then treated with LPS at 4 μg for 2 days, and the hippocampus of the mice was extracted to detect the mRNA expression level of the related inflammatory factors, and the results are shown in Figure 30. The results show that Tafamidis inhibits the inflammatory response induced by LPS in the brain of mice;

[0347] Male C57BL / 6 mice were pretreated with Tafamidis at 10 mg / kg for 7 days, then treated with LPS at 4 μg for 2 days, and the hippocampus of the mice was extracted to detect the mRNA expression level of Synaptophysin and PSD95, and the results are shown in Figure 31. The results show that Tafamidis inhibits the down-regulation of Synaptophysin and PSD95 induced by LPS in the brain of mice, thereby improving the cognition of mice.

[0348] Male C57BL / 6 mice (4 weeks old, 15-20 g) were divided into 4 groups, with 7 to 10 mice in each group, and divided into a control group, an LPS-induced group, a single compound Tafamidis group, and an LPS+compound Tafamidis group. The compound Tafamidis was treated by gavage at a dose of 10 mg / Kg for a period of two weeks, and then the nerve inflammation model was induced by brain injection of LPS (750 μg / kg) for 4 days, and then detected. The animal behavior experiments of Morris water maze, mouse nesting experiment, mouse rotarod experiment, pole climbing, etc. revealed that Tafamidis could improve the cognitive ability of mice; the blood and cerebrospinal fluid of mice were collected, and it was found that Tafamidis could down-regulate inflammatory factors IL-6, TNF-α, IL-1β, etc.

[0349] From the above, it can be seen that Tafamidis can be used for the treatment of neurodegenerative diseases, such as AD and the like.

[0350] Example 7: Therapeutic effect of Tafamidis on cutaneous T-cell lymphoma

[0351] 1. CCK8 cell viability assay to determine the effect of Tafamidis on the proliferation of cutaneous T-cell lymphoma cells

[0352] Three strains of mature cutaneous T-cell lymphoma cells MJ, Hut78, and HH were respectively plated in 96-well plates, and the number of cells per well was 1×10 4MJ, Hut78, HH cells were plated in 6-well plates, and treated with 0, 0.1, 1, 10 μΜ Tafamidis for 24, 48, 72, 96 hours. Cells were collected and washed with cold PBS, and then fixed with cold 70% ethanol overnight. Next day, cells were washed with PBS, and stained with DAPI staining solution for 10 minutes at room temperature in the dark. Cell cycle distribution of different DNA contents was determined by flow cytometry. The results showed that Tafamidis had cycle arrest effect on the three strains of cutaneous T-cell lymphoma cells.

[0353] 2. Flow cytometry assay of the effect of Tafamidis on the cell cycle of cutaneous T-cell lymphoma

[0354] MJ, Hut78, HH cells were plated in 6-well plates, and treated with 0, 0.1, 1, 10 μΜ Tafamidis for 24, 48, 72, 96 hours. Cells were collected and washed with cold PBS, and then fixed with cold 70% ethanol overnight. Next day, cells were washed with PBS, and stained with DAPI staining solution for 10 minutes at room temperature in the dark. Cell cycle distribution of different DNA contents was determined by flow cytometry. The results showed that Tafamidis had cycle arrest effect on the three strains of cutaneous T-cell lymphoma cells.

[0355] 3. Flow cytometry assay of the effect of Tafamidis on the apoptosis of cutaneous T-cell lymphoma

[0356] MJ, Hut78, HH cells were plated in 6-well plates, and treated with 0, 0.1, 1, 10 μΜ Tafamidis for 24, 48, 72, 96 hours. Cells were collected and washed with cold PBS, and then resuspended with Binding Buffer to a final concentration of 10 6 100 μΐ^ of cell suspension was transferred to a culture tube, 5 μΐ^ of FITC Annexin V was added and incubated at room temperature in the dark for 5 minutes, and then 5 μΐ^ of PI staining solution was added and incubated at room temperature in the dark for 5 minutes. Subsequently, the cells were resuspended with 400 μΐ^ of Binding Buffer and analyzed for apoptosis by flow cytometry. The results showed that Tafamidis promoted the apoptosis of the three strains of cutaneous T-cell lymphoma cells.

[0357] 4. Western blot assay of the effect of Tafamidis on apoptosis-related proteins of cutaneous T-cell lymphoma

[0358] MJ, Hut78, and HH cells were plated in 12-well plates and treated with 0, 0.1, 1, or 10 μΜ Tafamidis for 24, 48, 72, or 96 hours. Cells were collected and lysed, and the expression of apoptosis-related proteins, such as PARP, caspase-3, Bcl-2, and survivin, was detected by immunoblotting. The results showed that Tafamidis promoted the expression of apoptosis-related proteins in the three strains of cutaneous T-cell lymphoma cells, indicating that Tafamidis promotes cell apoptosis.

[0359] Example 8: Therapeutic effect of Tafamidis on alcoholic fatty liver disease

[0360] 1. Effect of Tafamidis on indicators of steatosis and inflammation in a short-term alcoholic hepatitis model in mice

[0361] A short-term alcoholic hepatitis model in mice was constructed using 8-week-old SPF C57BL / 6 male mice, which were randomly divided into a control group and a model group. The model group was first given Lieber-DeCarli control liquid feed (35% fat calories) for 5 days. On the 6th day, the model group was changed to Lieber-DeCarli 5% (v / v) alcohol liquid feed. At 7-9 am on the 11th day, the model group was given 5 g / kg of ethanol by gavage, and the control group was given an equal volume of dextrin. After 9 hours, the samples were collected by sacrificing the mice. During the experiment, the mice were allowed to drink liquid feed freely, and the model group was randomly divided into a solvent group, a Tafamidis 5 mg / kg group, and a Tafamidis 20 mg / kg group, which were given gavage administration every day while the model was being established.

[0362] 1.1 After blood was taken from the mice, the serum was collected by centrifugation, and the serum liver function ALT and AST indicators were detected. As shown in the figure, Tafamidis can down-regulate the content of ALT and AST in the serum of the model group mice, i.e., Tafamidis can improve alcohol-induced liver function damage.

[0363] 1.2 The liver of the mouse was paraffin sectioned and then HE stained. As shown in the figure, the liver cells of the control group were arranged regularly, with a central vein as the center and arranged radially; the model group had a large area of lipid deposition in the liver tissue, with inflammatory cell infiltration and obvious water-like lesions in the hepatic portal area; the treatment of Tafamidis could significantly improve the lesions of the above liver tissue.

[0364] 1.3 The liver tissue of the mouse was taken, and after RNA extraction, the transcription level of liver cytokines IL-6 and CCL2 was detected by qRT-PCR method, with GAPDH as an internal reference gene and normalized to the control. The results showed that Tafamidis can inhibit the expression of inflammatory cytokines induced by alcohol.

[0365] 2. Effect of Tafamidis on related indicators of mouse short-term alcoholic liver fibrosis model

[0366] A mouse short-term alcoholic hepatitis model was constructed, that is, SPF C57BL / 6 male mice of about 8 weeks old were randomly divided into a control group and a model group. The control group was given Lieber-DeCarli control liquid feed. On days 1-2, the model group was given Lieber-DeCarli alcohol liquid feed containing 1% ethanol (v / v), and on days 3-4, the alcohol content was doubled. After the adaptation period, the mice were fed with 2% Lieber-DeCarli alcohol liquid feed for 2 weeks. During this period, the model group was injected intraperitoneally with carbon tetrachloride, and the control group was injected with corn oil, twice a week. During the experiment, the mice were allowed to drink liquid feed freely, and the model group was randomly divided into a solvent group, a Tafamidis 5 mg / kg group, and a Tafamidis 20 mg / kg group, which were administered orally at the same time every day.

[0367] 2.1 After the mice were bled, the serum was collected by centrifugation, and the serum liver function ALT and AST indicators were detected. As shown in the figure, Tafamidis can down-regulate the content of ALT and AST in the serum of the model group mice, that is, Tafamidis can improve alcohol-induced liver function damage.

[0368] 2.2 The liver of the mouse was paraffin sectioned, and then HE staining was performed. As shown in the figure, the liver cells of the control group were arranged regularly, and were arranged radially around the central vein; the model group had a large area of lipid deposition in the liver tissue, and inflammatory cell infiltration and obvious water-like lesions were observed in the hepatic portal area; the treatment of Tafamidis can significantly improve the lesions of the above liver tissue.

[0369] 2.3 The liver tissue of the mouse was taken, RNA was extracted, and then the transcription level of liver cytokines IL-6 and CCL2 was detected by qRT-PCR method, with GAPDH as an internal reference gene and normalized with the control. The results showed that Tafamidis can inhibit the expression of inflammatory cytokines induced by alcohol.

[0370] 2.4 The liver paraffin section of the mouse was subjected to Sirius red staining, and the fiber area represented by Sirius red staining was counted using ImageJ software. The results showed that, compared with the control group, the liver of the model group mice had obvious collagen deposition, and Tafamidis could dose-dependently inhibit the collagen deposition in the liver of the model group mice.

[0371] Example 9: Therapeutic effect of Tafamidis on Kaposi's sarcoma

[0372] 1. Tafamidis inhibits Kaposi's sarcoma cell proliferation

[0373] SK-RG cells are KSHV-infected SH-SY5Y cells; KMM cells are KSHV-transformed rat primary mesenchymal precursor cells. Both KSHV cell lines were plated in 96-well plates at 1 x 10 4 cells per well with 100 μL of culture medium. Cells were treated with 0, 0.1, 1, 10 μM of Tafamidis for 24, 48, 72, 96 hours, respectively, and then 20 μL of CCK8 reagent was added to each well. After incubation for 1 hour in the incubator, the absorbance value of each well was detected at 450 nm, and the cell viability was calculated. The results showed that Tafamidis could inhibit the proliferation of both KSHV cell lines.

[0374] 2. Tafamidis promotes apoptosis of KSHV cells

[0375] SK-RG and KMM cells were plated in 6-well plates and treated with 0, 0.1, 1, 10 μM of Tafamidis for 24, 48, 72, 96 hours, respectively. Cells were collected, washed with cold PBS, and resuspended with Binding Buffer at a final concentration of 10 6 cells / mL. 100 μL of cell suspension was transferred to a culture tube, 5 μL of FITC Annexin V was added and incubated at room temperature for 5 minutes in the dark, and then 5 μL of PI staining solution was added and incubated at room temperature for 5 minutes in the dark. Subsequently, the cells were resuspended with 400 μL of Binding Buffer and analyzed for apoptosis using a flow cytometer. The results showed that Tafamidis promoted the apoptosis of both KSHV cell lines.

[0376] 3. Tafamidis inhibits migration of KSHV cells

[0377] SK-RG and KMM cells were seeded into the lower chamber of a 24-well plate insert with FBS-free medium at a cell density of 1 x 10 5 cells per well. After 4 hours of Tafamidis treatment, 800 μL of medium containing 10% FBS was added to the lower chamber. After 36 hours of incubation, the cells were fixed in 4% paraformaldehyde for 20 minutes and stained in 0.1% crystal violet for 30 minutes. Six areas were randomly selected and the cells were counted using Image-J software. The results showed that Tafamidis inhibited the migration of both KSHV cell lines.

[0378] Example 10: Therapeutic effect of Tafamidis on acne

[0379] 1. A mouse inflammatory model induced by C. acnes was constructed. That is, 8-week-old male Kunming mice were injected intradermally with C. acnes (6 x 10 7C. difficile injection, the right ear of the same mouse was injected with 20 μL of C. difficile (CFU / 20 μL in PBS). The left ear of the same mouse was injected with an equal amount of PBS. 24 hours after C. difficile injection, Tafamidis (100 mg / mL) was soaked in a cotton swab and evenly applied to the surface of the right ear skin, and the model group was applied with PBS, for a total of 7 days of treatment.

[0380] 1.1 At the time of sample collection, the mouse ears were collected, and the thickness and weight were measured. The results showed that the Tafamidis-treated ears exhibited less erythema, thickness, and weight compared to the PBS-treated group.

[0381] 1.2 After homogenization in sterile saline, the ears were centrifuged at 3000 x g for 5 minutes. The supernatant was obtained after centrifugation, and the expression of IL-1β, IL-6, and TNF-α was determined by ELISA. The results showed that Tafamidis reduced the expression of inflammatory cytokines compared to the PBS-treated group.

[0382] 1.3 The mouse ears were paraffin-embedded and longitudinally sectioned, followed by HE staining. The results showed that C. difficile caused mouse ear edema and increased the number of inflammatory cells invading the dermis, while Tafamidis treatment significantly inhibited C. difficile-induced edema and inflammatory cell infiltration.

[0383] 2. Mouse macrophage RAW264.7 cells were seeded in a 12-well plate, and Tafamidis 10 μM or an equal volume of DMSO was used for pretreatment for 2 hours before adding LPS (1 μg / mL) for 24 hours. The serum was collected and used to determine the concentration of TNF-α, IL-1β, and IL-6 using an ELISA kit. The results showed that Tafamidis significantly inhibited LPS-induced expression of inflammatory cytokines compared to the control group.

[0384] Example 11: Therapeutic effect of Tafamidis on osteoporosis

[0385] 1. Tafamidis promotes mineralization of preosteoblast 3T3-L1 cells under differentiation agent-induced conditions

[0386] Preosteoblast MC3T3-E1 cells in the logarithmic growth phase were seeded in a 24-well plate and cultured in α-MEM medium containing Tafamidis and / or mineralization inducers (10 nM A A. and 50 μg / mL β-GP) for continuous culture for 14 days. At the time of sample collection, the cells were fixed for alizarin red staining, and after washing with distilled water, they were dried and observed. The results showed that Tafamidis can significantly enhance calcium deposition induced by differentiation agents, indicating that Tafamidis can enhance the mineralization effect induced by differentiation agents.

[0387] 2. Tafamidis inhibits differentiation of RAW264.7 cells into osteoclasts under differentiation agent-induced conditions

[0388] RAW264.7 cells were seeded into a six-well plate pre-coated with glass slides and cultured. After adhering, DME medium containing Tafamidis and / or RANKL (50 ng / mL) was added, and the cells were continuously cultured for 7 days. The medium was replaced every 2 days, and the cells were fixed for TRAP staining when the sample was collected. The results showed that the RAW264.7 cells under the control culture condition were regular round, small in size, and high in refractive index. After RANKL treatment, large and multinucleated cells appeared and were stained red by sodium tartrate, indicating that RANKL successfully induced the formation of osteoclasts, and Tafamidis inhibited the formation of multinucleated cells.

[0389] Example 12: Effect of Tafamidis treatment on primary biliary cholangitis

[0390] A mouse model of primary biliary cholangitis was constructed. That is, SPF C57BL / 6 male mice of about 8 weeks old were randomly divided into a control group and a model group. For the model group, 20A-BSA (2 mg / mL) was mixed with an equal volume of Complete Freund's adjuvant (CFA) for emulsion and then intraperitoneally injected (100 uL per mouse) for the first time. At 3 weeks and 5 weeks after the first intraperitoneal immunization, 20A-BSA was mixed with an equal volume of Incomplete Freund's adjuvant (IFA) for emulsion and then intraperitoneally injected (100 uL per mouse) for booster immunization. At the same time, from the third day of modeling, poly(I:C) (1 mg / mL) was additionally intraperitoneally injected at a dose of 5 mg / kg of mouse every three days, which lasted until the 12th week. From the 13th week, the model group was divided into groups, and Tafamidis was administered at a dose of 5 mg / kg and 20 mg / kg by gavage, and the model group was administered with solvent corn oil. The experiment was ended four weeks after administration, and the mouse samples were collected.

[0391] 1. After blood was taken from the mice, the serum was collected by centrifugation, and the serum liver function ALT, AST, and ALP indicators were detected. The results showed that Tafamidis could down-regulate the contents of ALT, AST, and ALP in the serum of the model mice, i.e., Tafamidis could improve the liver function damage of the mice with primary biliary cholangitis.

[0392] 2. The liver of the mouse was paraffin sectioned, and then HE staining was performed. The results showed that the liver cells of the control group were regularly arranged and radially arranged around the central vein; the liver tissue structure of the model group was abnormal, the bile duct in the hepatic portal area was obviously proliferated, the number of bile duct epithelial cells was increased, and multiple inflammatory cell infiltration foci were visible, and part of the liver cells were vacuolated; the treatment of Tafamidis could significantly improve the pathological changes of the above liver tissue.

[0393] Example 13: Effect of Tafamidis on atherosclerosis

[0394] 1. Tafamidis inhibits LPS-induced HUVEC cell inflammatory factor expression

[0395] Human umbilical vein endothelial cells (HUVECs) were treated with Tafamidis 10 μM and / or LPS (1 μg / mL), and the culture medium was collected to detect the concentration of cell inflammatory factors by ELISA. The results showed that Tafamidis can significantly inhibit LPS-induced HUVEC cell inflammatory factor expression.

[0396] 2. Tafamidis inhibits LPS-induced HUVEC cell atherosclerosis-related protein expression

[0397] Human umbilical vein endothelial cells (HUVECs) were treated with Tafamidis 10 μM and / or LPS (1 μg / mL), and the expression of iNOS, ICAM-1, and VCAM-1 in the cells was detected by immunoblotting after 24 hours of treatment. The results showed that Tafamidis significantly inhibited LPS-induced inflammatory and adhesion-related protein expression.

[0398] 3. Construction of a mouse atherosclerosis model, i.e., 8-week-old apoE - / - male mice were randomly divided into a control group and a model group. For the model group, the mice were fed with high-fat feed containing 21% fat + 0.15% cholesterol, and after 12 weeks, they were divided into a model group and a drug administration group. The drug administration group was fed with high-fat feed while being administered Tafamidis by gavage every day, and the model group was administered the corresponding solvent, corn oil, by gavage. The experiment was ended after 4 weeks of drug administration.

[0399] After the mice were bled, the serum was collected by centrifugation, and the levels of TC, TG, LDL-C, and HDL-C in the serum were detected, and the results are shown in the figure. As shown in the figure, Tafamidis can down-regulate the levels of TC, TG, and LDL-C in the serum of the model group mice and up-regulate the level of HDL-C, i.e., Tafamidis can improve the blood lipid levels of the atherosclerosis mice.

[0400] The aortas of the mice were frozen sectioned, and then HE staining was performed. The results showed that the aortic wall structure of the model group mice was arranged in disorder, the intima of each part was unevenly thickened, a fibrous cap and lipid necrosis were formed, and the lumen was convex; while the pathological conditions of the Tafamidis group mice were significantly reduced.

[0401] The aortas of mice were frozen sectioned and then subjected to oil red O staining. The results showed that the model group mice had fat droplets in the arterial lumen, and some areas were patchy distribution. The fat droplets in the aortic lumen of the Tafamidis group mice were significantly reduced, and the fat droplets were small and sparsely distributed, indicating that Tafamidis significantly reduced the lipid deposition in the aortic plaques of atherosclerotic mice.

[0402] Example 14: Effect of Tafamidis on fibrosis-related indicators in a CCl4 cirrhosis model in mice

[0403] A CCl4 cirrhosis model was constructed, i.e., SPF grade C57BL / 6 male mice about 8 weeks old were randomly divided into a control group and a model group. CCl4 and corn oil were mixed at a ratio of 1:3, and during the modeling process, the model group mice were injected intraperitoneally with 25% CCl4 corn oil solution three times a week, and the control group mice were injected intraperitoneally with corn oil at a dose of 2 mL / kg, and the modeling time was a total of ten weeks. Five weeks after modeling, the mice were intraperitoneally injected with CCl4 and gavaged with solvent or Tafamidis (5 mg / kg and 20 mg / kg) for five weeks, and the mice were sampled.

[0404] 1. The mouse livers were collected and the liver appearance was observed. The liver of the control group was red and smooth on the surface, and soft in texture; compared with the control group, the liver surface of the CCl4 model group mice had a obvious grainy feeling, reduced luster, and hard texture, showing obvious liver fibrosis phenotype, and Tafamidis could improve the above pathological conditions.

[0405] 2. After blood was collected from the CCl4 cirrhosis model mice, the serum was collected by centrifugation, and the serum liver function ALT and AST indicators were detected. Tafamidis could dose-dependently down-regulate the ALT and AST contents in the serum of the model group mice, i.e., Tafamidis could improve the liver function damage induced by CCl4.

[0406] 3. Sirius red staining was performed on the paraffin sections of the mouse livers, and the fibrous area represented by the Sirius red staining was counted using ImageJ software. Compared with the control group, the liver of the model group mice had obvious collagen deposition, and Tafamidis could dose-dependently inhibit the collagen deposition in the liver of the model group mice.

[0407] 4. Tafamidis could improve the expression of fibrosis-related genes in the liver tissue of the CCl4-induced cirrhosis model mice.

[0408] The liver tissues of the CCl4 liver fibrosis model mice were taken, RNA extraction was performed, and the transcription levels of a-SMA, COL1A1, and TIMP1 were detected by qRT-PCR method, GAPDH was used as an internal reference gene and normalized with the control. The liver tissues of the CCl4 liver fibrosis model mice were taken, and the protein expression levels of a-SMA and COL1A1 were detected by immunoblotting. Tafamidis can inhibit the expression of CCl4-induced liver fibrosis marker proteins such as a-SMA and COL1A1.

[0409] Example 15: Effect of Tafamidis on inflammatory factor-related indicators in an imiquimod-induced psoriasis model in mice

[0410] A psoriasis model induced by imiquimod (IMQ) was constructed, that is, 6-8 week-old BALB / c mice were selected, the back or ear skin of the mice was locally depilated, 5% imiquimod ointment 62.5 mg was locally applied to the back or ear depilated skin every day, and the solvent or Tafamidis (5 mg / kg and 20 mg / kg) was orally administered every day, and the mice were sampled after 5 days.

[0411] 1. PASI score, the inflammation of the back skin of the mice was scored according to the PASI score standard (including scales, erythema, and hypertrophy), and the cumulative score, body weight, and skin thickness changes were recorded. The control group treated with blank cream showed no inflammatory characteristics, the model group treated with IMQ showed a continuous increase in PASI score, a decrease in body weight, and an increase in skin thickness, and Tafamidis (5 mg / kg and 20 mg / kg) could significantly reduce the PASI score and reverse the skin lesions and increase in skin thickness.

[0412] 2. H&E staining was used to analyze the histopathological changes, the skin of the model mice showed erythema, scales, and thickening lesions, and inflammatory cell infiltration, and Tafamidis (5 mg / kg and 20 mg / kg) could inhibit the above pathological changes.

[0413] 3. After 5 days of IMQ treatment, the plasma was collected, the content of related cytokines was detected by ELISA, and the skin tissue was collected to detect the transcription level of related Marker genes by qRT-PCR. The results showed that IMQ treatment induced the production of inflammatory cytokines such as IL-6, IL-β, TNF-α, IFN-α, and CXCL1, and Tafamidis (5 mg / kg and 20 mg / kg) had a strong inhibitory effect.

[0414] 4. Detection of psoriasis biomarker IL-6, immunohistochemical results showed that the expression of IL-6 in the model group was increased, and Tafamidis reduced the expression of IL-6.

[0415] Example 16: Effect of Tafamidis on Hepatocarcinoma

[0416] 1. Hepatocarcinoma cells HepG2 and SK-HEP-1 were plated in 96-well plates at a density of 1 x 10 4 cells per well in 100 μL of medium. Cells were treated with 0, 1, 5, 10, 20 μM of Tafamidis for 24, 48, 72, 96 hours, and then 10 μL of CCK8 reagent was added to each well. After incubation for another 1 hour in the incubator, the absorbance value of each well was detected at 450 nm, and the cell viability was calculated. Tafamidis can significantly inhibit the proliferation of hepatocarcinoma cells.

[0417] 2. A mouse DEN / CCl4 hepatocarcinoma model was constructed. When the mice were fifteen days old, they were injected intraperitoneally with DEN (diluted with PBS, 25 mg / kg), and after 6 weeks, they were injected with CCl4 (diluted with corn oil, 0.5 mL / kg) intraperitoneally twice a week for a total of 17 weeks, and during the last four weeks, they were given solvent or Tafamidis (5 mg / kg and 20 mg / kg) by gavage every day for a total of 25 weeks.

[0418] 2.1 The size of the liver tumor was photographed and counted. Compared with the model group, the Tafamidis administration group significantly reduced the number and size of tumors.

[0419] 2.2 Immunohistochemical detection of cell proliferation marker Ki67 and apoptosis marker cleaved-caspase 3 showed that the number of Ki67-positive cells in the Tafamidis administration group was significantly reduced compared with the model group, while the number of cleaved-caspase 3-positive cells in the Tafamidis administration group was significantly increased compared with the model group.

[0420] 2.3 RT-PCR detection of inflammatory factors and proliferation markers in liver tissue found that the inflammatory factors IL-6, TNF-α and proliferation marker CyclinD1 in the Tafamidis administration group were significantly lower than in the model group.

[0421] Example 17: Effect of Tafamidis on Inflammatory Factor-Related Indicators in DSS-Induced Mouse Intestinal Inflammation Model

[0422] A DSS-induced mouse intestinal inflammation model was constructed by feeding the mice with 3% DSS in drinking water for five days, then changing to normal drinking water for four days to collect samples. During this period, the mice were given solvent or Tafamidis (5 mg / kg and 20 mg / kg) by gavage every day.

[0423] 1. The body weight of mice was measured every day and the state of mice was observed. The body weight of mice in the model group began to show a downward trend on the second day of DSS feeding, and the body weight still showed a downward trend in the recovery stage of feeding water, while the body weight of mice in the Tafamidis administration group showed no obvious downward trend in the DSS treatment stage, and the body weight rapidly recovered in the recovery stage, which could inhibit the damage induced by DSS.

[0424] 2. The H&E staining results showed that the mice in the Tafamidis administration group had less severe epithelial cell damage, crypts and ulcers compared with the mice in the model group.

[0425] 3. The serum of mice was collected, and the changes of IL-6 and TNF-α in the serum were detected by enzyme linked immunosorbent assay (ELISA), and it was found that the Tafamidis administration group could significantly reduce the expression of IL-6 and TNF-α.

[0426] 4. Immunohistochemical detection of proliferative proteins Ki67 and PCNA. The results showed that the number of positive cells of Ki-67 and PCNA in the intestinal crypts of mice in the Tafamidis administration group was more than that of mice in the model group.

[0427] 5. The intestinal tissue was subjected to qRT-PCR experiment to detect inflammatory factors IL-6, TNF-α and IL-11, and it was found that the Tafamidis administration group could significantly reduce the expression of IL-6, TNF-α and IL-11.

[0428] Example 18: Effect of Tafamidis on the treatment of intestinal cancer

[0429] 1. The colon cancer cells SW480, SW620 and HCT116 were plated in a 96-well plate, and the number of cells per well was 1 x 10 4 μL. The cells were treated with 0, 1, 5, 10 and 20 μM of Tafamidis for 24, 48, 72 and 96 hours, respectively, and then 10 μL of CCK8 reagent was added per well. After incubation in the incubator for another 1 hour, the absorbance value of each well was detected at 450 nm, and the cell viability was calculated. Tafamidis could significantly inhibit the proliferation of colon cancer cells.

[0430] 2. The AOM / DSS-induced mouse intestinal cancer model was constructed, and the mice were injected with AOM (10 mg / kg) intraperitoneally, and then fed with 2.5% DSS for seven days, followed by normal drinking water for 14 days. After three cycles, the mice were sacrificed at 120 days after AOM injection. During the first four weeks of AOM / DSS sampling, the mice were given Tafamidis (20 mg / kg) or the corresponding solvent by gavage every day.

[0431] 2.1 Photographs were taken and tumor sizes were measured when mice were collected, and it was found that the number of tumors in the Tafamidis administration group was significantly reduced compared to WT mice.

[0432] 2.2 Immunohistochemistry was performed on colon cancer tissues, and the results of Ki67 antibody group staining showed that Tafamidis can inhibit cell proliferation, while TUNEL detection of apoptosis showed that more cell apoptosis occurred in the Tafamidis administration group.

[0433] 2.3 RT-PCR analysis of colon cancer tissues found that Tafamidis administration group down-regulated the expression of cytokines IL-6, TNFα and IL-11.

[0434] 2.4 Western blot analysis of colon cancer tissues found that Tafamidis can inhibit the activation of STAT3 and the expression of CyclinD1.

[0435] Example 19: Therapeutic effect of Tafamidis on renal fibrosis

[0436] 1. A renal fibrosis cell model was constructed, and normal rat kidney fibroblasts NRK-49F were treated with TGFβ, and simultaneously treated with different concentrations (2.5 μM, 5 μM, 10 μM) of Tafamidis for 24 hours, and the renal fibrosis-related markers were detected.

[0437] 1.1 Western blotting was used to detect the expression of COL1A1 and α-SMA proteins, and it was found that Tafamidis can inhibit the expression of COL1A1 and α-SMA proteins activated by TGFβ.

[0438] 1.2 qRT-PCR was used to detect the mRNA expression levels of α-SMA, COL1A1 and CTGF, with β-actin as the internal reference gene and normalized with the control, and the results showed that Tafamidis can inhibit the expression of COL1A1, α-SMA and CTGF mRNA levels activated by TGFβ.

[0439] 1.3 Immunofluorescence staining was used to observe the expression of α-SMA, and the scale in the figure is 18.4 μm. ImageJ was used to analyze the fluorescence staining intensity. The results showed that Tafamidis can inhibit the expression of fibrosis marker α-SMA.

[0440] 2. A unilateral ureteral obstruction (UUO) induced renal fibrosis model was constructed, and the left ureter of an eight-week-old rat was cut off at the upper 1 / 3, and both ends were ligated. One week later, the rats were given Tafamidis (20 mg / kg) or the corresponding solvent by gavage, and the samples were collected two weeks after administration.

[0441] 2.1 Hematoxylin / eosin staining showed that the model group had inflammatory cell infiltration, edema, tubular dilation, structural disorder, tubular epithelial cell degeneration and necrosis. The degree of pathological changes in the Tafamidis administration group was lighter than that in the model group.

[0442] 2.2 After blood was taken from the rats, the serum was collected by centrifugation, and the serum renal function blood creatinine (Scr) and urea nitrogen (BUN) indicators were detected. Tafamidis can significantly down-regulate the content of Scr and BUN in the serum of the model group rats.

[0443] 2.3 Collagen I and collagen III were detected by picrosirius red staining and immunoblotting. It was found that Tafamidis inhibited the collagen deposition induced by UUO.

[0444] 2.4 After the kidney tissue was extracted, the transcription levels of kidney fibrosis related markers α-SMA, COL1A1, TIMP1 and inflammatory factors IL-6, TNFα and TGFβ were detected by qRT-PCR method, GAPDH was used as an internal reference gene and normalized with the control. The results showed that Tafamidis can inhibit the expression of kidney fibrosis marker proteins such as α-SMA, COL1A1 and TIMP1.

[0445] 2.5 The inflammatory cell marker F4 / 80 was detected by immunohistochemistry. The macrophage infiltration in the model group was significantly increased compared with the blank group, and Tafamidis can significantly inhibit the infiltration of macrophages.

[0446] Example 20: Effect of Tafamidis on related indicators of mouse diabetic model

[0447] The model of diabetes induced by high-fat diet and STZ (streptozotocin) is constructed. Male C57BL / 6 mice, 6-8 weeks old, are fed with normal feed in the blank group all the time. The rest of the mice are fed with 60% high-fat feed. After being fed with high-fat feed for a period of time, all the mice are fasted for 12 hours without water. The mice in the 60% high-fat group are injected intraperitoneally with 1.75% STZ citric acid-sodium citrate buffer at 35 mg / Kg. The mice in the blank group are injected intraperitoneally with the same dose of citric acid-sodium citrate buffer. After 72 hours of injection, all the mice are fasted for 12 hours without water. Blood is taken from the tail of the mice, and the fasting blood glucose (FBG) and 2-hour blood glucose (2hBG) after GTT are measured by a blood glucose meter. The mice with FBG≥7.8 mmol / l or blood glucose peak≥16.7 mmol / l are the successfully induced mice. The mice meeting the conditions are randomly divided into the blank group, the model group, and the administration group. The mice not meeting the conditions are excluded. The body weight of the mice in each group is measured every week, and the administration dose is adjusted according to the body weight. The random blood glucose is measured every two weeks. During this period, Tafamidis and the corresponding solvent are administered by gavage every day, and the administration is continuously performed for 6 weeks. The samples are collected.

[0448] 1. One week before sample collection, oral glucose tolerance test (OGTT) is performed to detect the blood glucose content of the mice at different times 0, 15, 30, 60, 120, and 180 minutes by One Touch Johnson blood glucose meter. The fasting insulin (FINS) is measured by enzyme-linked immunoassay. Tafamidis can effectively reduce the blood glucose increase induced by the model and improve insulin sensitivity.

[0449] 2. When the samples are collected, the blood of the mice is taken, and T-Cho, TG, HDL-C, LDL-C, CR, BUN, and ALT are analyzed and detected by a biochemical analyzer. It is found that Tafamidis can effectively reduce the serum content of triglyceride and low-density lipoprotein.

[0450] 3. The pancreas tissue is subjected to H&E staining. The islet cells in the blank group are normal, the cell nucleus is clear and round, the thin layer of fibrous tissue envelope is relatively complete, the lobulation is clear, and there is no thickening. No obvious pathological changes such as inflammatory cell infiltration and proliferation are observed. The islets in the model group are atrophic, irregular in shape, and some islet cells are degenerated, necrotic, and vacuolated. Nucleus pyknosis or dissolution is also observed. The Tafamidis administration group significantly repairs the damage of the pancreas tissue, and the inflammatory cell infiltration is reduced.

[0451] 4. The liver is subjected to H&E staining observation. The liver cords in the model group are arranged in order, the endothelial cells in the central vein are complete, and the hepatocyte morphology is normal. Many undyed round lipid droplets can be observed in the cytoplasm of the hepatocytes in the model group. In addition, the hepatocyte nucleus is swollen, and some hepatocytes are fatty degenerated. The Tafamidis administration group significantly improves the liver cell lesions caused by high-fat diet and STZ.

[0452] Example 21: Therapeutic effect of tafamidis on acute myeloid leukemia

[0453] 1. NB4 and HL60 were plated in 96-well plates at a cell number of 1 x 10 5 cells per well and 100 μL of medium. The cells were treated with 0, 1, 5, 10, 20 μM of tafamidis for 24, 48, 72 hours, respectively, and then 10 μL of CCK8 reagent was added to each well. After incubation in the incubator for another 1 hour, the absorbance value of each well was detected at 450 nm, and the cell viability was calculated. It was found that tafamidis could inhibit the proliferation of acute myeloid leukemia cells.

[0454] 2. A mouse model of acute myeloid leukemia was constructed. 7-8-week-old SCID (severe combined immunodeficiency) mice were randomly divided into a blank group, a model group and a drug administration group. The model group and the drug administration group were pretreated with intraperitoneal injection of cyclophosphamide at a dose of 100 mg / kg of body weight, once a day for 2 days, and on the third day, NB4 cell suspension was injected subcutaneously from a single point on the right flank of the mouse (1 x 10 7 cells per mouse). The blank control group was not treated. At 3 weeks after injection, the presence of tumor infiltration in the liver and spleen and the peripheral white blood cell count reaching 4.03 ± 1.92 x 10 9 / L were the criteria for tumor formation. At 3 weeks after injection of the NB4 cell suspension, the drug administration group was administered tafamidis (20 mg / kg) by gavage for 5 weeks, and the model group and the blank control group were administered the same volume of 0.9% sodium chloride injection by gavage for 5 weeks.

[0455] 2.1 At the end of the treatment, 0.1 mL of tail vein blood was taken, and the total number of white blood cells, red blood cells, neutrophils, lymphocytes and HL-60 cells was counted by manual counting. Tafamidis could significantly reduce the proportion of NB4 in the blood.

[0456] 2.2 NB4 in the blood and bone marrow of the mice was isolated and extracted, and the apoptosis and differentiation of the NB4 cells of the mice were detected. Tafamidis could promote the apoptosis and differentiation of the NB4 cells.

[0457] Example 22: Therapeutic effect of tafamidis on BDL-induced cholestatic liver injury and cholangitis

[0458] A BDL-induced cholestatic liver injury mouse model was constructed, which was suitable for cholangitis secondary to cholestasis. C57BL / 6 mice were subjected to common bile duct double ligation (BDL) surgery, and 3 days later, they were randomly divided into a sham control group (ctr), a model group (BDL), and a drug administration group (BDL+Tafamidis). The drug administration group was administered 5 mg / kg Tafamidis once a day by gavage, and the control group (ctr) and the model group (BDL) were administered the control solvent once a day by gavage. Body weight was recorded every other day, and samples were collected after 2 weeks for the detection of relevant injury indicators. Among them, those skilled in the art can understand that the BDL model is suitable for cholangitis indications, and the control group (ctr) is a sham operation group.

[0459] 1. This example calculates the body weight change from the start of administration to the end of the experiment, and the results are shown in Figure 21A. The results show that after 3 days of BDL surgery, the body weight of the mice was significantly lower than that of the control group, and as time went on, the body weight of the model group BDL mice gradually decreased, but the body weight of the drug administration group BDL mice decreased somewhat. The results show that Tafamidis can inhibit the down-regulation of mouse body weight caused by BDL.

[0460] 2. In this example, the mouse liver was collected and its appearance was observed, and the results are shown in Figure 21B. Compared with the control group, the liver of the model group mice was yellowish in color, and the bile in the gallbladder was severely cholestatic, but after administration, the liver color recovered somewhat and the gallbladder volume decreased. The results show that Tafamidis can improve the pathological morphology of the liver of BDL mice.

[0461] 3. In this example, the mice were enucleated to collect blood, and after centrifugation, the serum was collected to detect liver function-related indicators ALT, AST, and T-BiL, and the results are shown in Figure 21C. The results show that compared with the control group, the model group, the liver injury-related indicators ALT, AST, and T-BiL were significantly up-regulated, but after Tafamidis treatment, the relevant indicators were significantly down-regulated compared with the model group. The results show that Tafamidis can significantly inhibit liver injury caused by cholestasis.

[0462] 4. Tafamidis can significantly inhibit the expression of fibrosis-related genes in cholestatic liver injury. In this example, the mouse liver tissue was taken to extract RNA, and the expression level of fibrosis-related mRNA (COL1A1, a-SMA, Timp1) was detected by qRT-PCR technology. GAPDH was used as an internal reference and was normalized, and the results are shown in Figure 21D. The results show that the expression level of fibrosis-related genes in the model group was significantly up-regulated compared with the control group, but Tafamidis can significantly inhibit the expression of fibrosis-related genes induced by cholestasis.

[0463] 5. Tafamidis can significantly inhibit the bile ductular proliferation and liver fibrosis induced by cholestatic liver injury. In this example, the mouse liver tissues were fixed with paraformaldehyde, dehydrated, embedded with paraffin, sectioned, and the sections were stained with HE and Sirius red. The results are shown in Figure 21E. According to the results of HE and Sirius red staining, the model group showed significant bile ductular proliferation (a marker of cholestasis), inflammatory cell (small cells with nuclei around the bile duct) aggregation and collagen deposition in the portal area, but the bile ductular proliferation, inflammatory cell aggregation and collagen deposition were significantly improved after Tafamidis treatment.

[0464] 6. In order to further verify whether Tafamidis can inhibit liver fibrosis caused by cholestatic liver injury, in this example, the mouse liver tissues were detected for hydroxyproline. The results are shown in Figure 21F. The hydroxyproline content in the model group was significantly up-regulated, but it was significantly down-regulated after administration.

[0465] The above results show that Tafamidis has the effect of inhibiting cholestasis and cholangitis.

[0466] Example 23: Therapeutic effect of Tafamidis in paracetamol (APAP) induced acute liver injury

[0467] 1. Construct a paracetamol (APAP) induced acute liver injury mouse model, which is suitable for paracetamol (APAP) induced acute liver injury. Randomly divide ICR mice into control group (ctr), model group (APAP + corn oil) and administration group (APAP + Tafamidis). The model group mice were given corn oil solution by gavage 3 days in advance, and the administration group mice were given 20 mg / kg Tafamidis by gavage. After the last gavage, the model group and the administration group mice were injected with 300 mg / kg APAP intraperitoneally, and then gavaged again 12 hours after APAP injection. The samples were collected 24 hours after APAP injection. It is understood by those skilled in the art that the model group is suitable for paracetamol (APAP) induced acute liver injury.

[0468] 2. In this example, the mouse liver was collected and its appearance was observed. The results are shown in Figure 22B. Compared with the control group, the liver of the model group mice showed obvious congestion, indicating that it had suffered severe damage, but the appearance of the liver after administration was the same as that of the control group.

[0469] 3. The mice were enucleated and blood was collected. After centrifugation, the serum was collected to detect the liver function related indicators ALT, AST, T-BiL. The results are shown in Figure 22C. The results show that compared with the control group, the liver injury related indicators ALT, AST, T-BiL of the model group were significantly up-regulated, but the related indicators of Tafamidis treatment group were significantly down-regulated compared with the model group. This indicates that Tafamidis can significantly inhibit APAP-induced acute liver injury.

[0470] 4. Tafamidis can significantly inhibit APAP-induced central vein surrounding hepatocyte necrosis. In this embodiment, the mouse liver tissue was fixed with paraformaldehyde, dehydrated, paraffin-embedded, sectioned, and the sections were stained with HE. The results are shown in Figure 22D, where the scale bar is 100 μm. According to the results of HE staining, the model group showed significant central vein surrounding hepatocyte necrosis, but Tafamidis treatment basically completely reversed the damage.

[0471] The above results show that Tafamidis inhibits APAP-induced liver injury.

[0472] Example 24: Effect of Tafamidis on related indicators in CCl4-induced acute liver injury

[0473] 1. For the in vitro model, a CCl4-induced acute liver injury HepG2 cell model was constructed, divided into a control group (ctr), a model group (CCl4), and a drug administration group (CCl4+Tafamidis). The model group was treated with CCl4 (10% serum MEM CCl4 saturated solution) to treat HepG2 cells, and the drug administration group was co-treated with CCl4 and Tafamidis (10 μM) to treat HepG2 cells. The culture medium was collected after 7.5 hours to detect the content of AST, and the sample was collected after 24 hours of administration. It is understood by those skilled in the art that the model group is suitable for CCl4-induced acute liver injury. The results are shown in Figure 23A. The model group showed a significant up-regulation of AST compared with the control group, but the drug administration group could inhibit the up-regulation of AST induced by CCl4.

[0474] 2. For in vivo model, CCl4-induced acute liver injury mouse model was constructed, which was suitable for CCl4-induced acute liver injury, and C57BL / 6 mice were randomly divided into control group (ctr), model group (CCl4+corn oil) and drug administration group (CCl4+Tafamidis). One week in advance, 20 mg / kg Tafamidis and 20 mg / kg corn oil were used for gavage treatment, and after the gavage was completed, CCl4 (25% CCl4 in corn oil, 2 μL / g) was injected intraperitoneally once, and 24 hours later, gavage was given again. The sample was collected 24 hours after administration. It is understood by those skilled in the art that the model group is suitable for CCl4-induced acute liver injury, and the specific process diagram of the experiment is shown in FIG. 23B. In this embodiment, the mice were enucleated to collect blood, and the serum was collected after centrifugation to detect the liver function related index AST, and the results are shown in FIG. 23C. After Tafamidis treatment, AST was significantly down-regulated compared with the model group.

[0475] 3. Tafamidis can significantly inhibit APAP-induced central vein surrounding hepatocyte necrosis. In this embodiment, the mouse liver tissue was fixed with paraformaldehyde, dehydrated, paraffin-embedded, sectioned, and the section was stained with HE, and the results are shown in FIG. 23D, where the scale is 100 μm. According to the HE staining results, it can be seen that the model group has significant central vein surrounding hepatocyte necrosis, but the necrotic area is significantly reduced after Tafamidis treatment.

[0476] The above data reveals that Tafamidis has the effect of inhibiting CCl4-induced acute liver injury.

[0477] Example 25: Effect of Tafamidis on high-fat diet (HFD)-induced fatty liver related indicators

[0478] In this embodiment, a high-fat diet (HFD)-induced acute liver injury mouse model was constructed, which was suitable for high-fat diet-induced acute liver injury. C57BL / 6 mice were fed with high-fat feed and normal feed, and after 5 months, the high-fat feed-fed mice were randomly divided into model group (HFD+vehicle) and drug administration group (HFD+Tafamidis), and the normal feed-fed group was set as control group (ctr). Subsequently, the model group was gavaged with solvent (i.e. vehicle) every day, and the drug administration group was gavaged with 10 mg / kg Tafamidis every day. Four weeks later, the samples were collected for detection of related indicators; wherein, it is understood by those skilled in the art that the model group is suitable for high-fat diet (HFD)-induced fatty liver.

[0479] 1. The mice were enucleated and blood was collected. After centrifugation, the serum was collected to detect the total cholesterol (T-CHO) related to fatty liver. The results are shown in Figure 24A. The serum T-CHO of the model group was significantly up-regulated, but the content of total cholesterol in the serum could be reduced after Tafamidis treatment

[0480] 2. The mouse liver tissue was fixed with paraformaldehyde, dehydrated, paraffin-embedded, sectioned, and the sections were stained with HE. The results are shown in Figure 24B, where the scale bar is 100 μm. According to the results of HE staining, the model group showed significant steatosis and ballooning degeneration, but Tafamidis treatment could significantly inhibit the accumulation of fat in the liver induced by high-fat diet.

[0481] The above results reveal that Tafamidis has the effect of reducing blood lipids and inhibiting fatty liver.

[0482] Example 26: Effect of Tafamidis on oleic acid (OA)-induced lipid droplet accumulation in HepG2

[0483] The oleic acid (OA)-induced lipid droplet accumulation HepG2 cell model was constructed. The HepG2 cells were divided into a control group (ctr), a model group (OA), and a drug administration group (OA+Tafamidis). The model group was treated with 100 μM OA for 24 hours, and the drug administration group was co-treated with 100 μM OA and 2.5 μM Tafamidis for 24 hours. The content of lipid droplets in HepG2 cells was detected by oil red O, and the results are shown in Figure 25. Tafamidis can significantly inhibit the accumulation of lipid droplets in OA-induced HepG2, which suggests that Tafamidis may reduce blood lipids by inhibiting the accumulation of lipid droplets in OA-induced HepG2, and has an inhibitory effect on fatty liver and other diseases.

[0484] Example 27: Effect of Tafamidis on LPS (100 ng / mL)-induced inflammation of mouse alveolar macrophage (MH-S)

[0485] A model of LPS-induced inflammation of mouse alveolar macrophage (MH-S) was constructed, which was suitable for LPS-induced pneumonia. The MH-S cells were divided into a control group (ctr), a model group (LPS), and a drug administration group (LPS+Tafamidis). The model group was treated with 100 ng / mL LPS for 24 hours, and the drug administration group was treated with 100 ng / mL LPS and 2.5 μM Tafamidis for 24 hours. The protein expression level of iNOS was detected by immunoblotting, and the results are shown in Figure 26. The results showed that Tafamidis could reduce the expression of iNOS protein induced by LPS, indicating that Tafamidis had an inhibitory effect on pneumonia.

[0486] Example 28: Inhibitory effect of Tafamidis on the survival of leukemia cells NB4

[0487] Human acute promyelocytic leukemia cells NB4 were divided into a control group (ctr) and a drug administration group (Tafamidis). The drug administration group was treated with 5 μM Tafamidis for 48 hours. The cell proliferation was detected by CCK-8, and the results are shown in Figure 27. The results showed that Tafamidis could inhibit the proliferation of NB4 cells, indicating that Tafamidis might have a function of treating leukemia.

[0488] Example 29: Tafamidis inhibits TGFβ-activated lung fibroblasts

[0489] A model of TGFβ-activated lung fibroblasts was constructed, which was suitable for pulmonary fibrosis. The lung fibroblasts were divided into a control group (ctr), a model group (TGFβ), and a drug administration group (TGFβ+Tafamidis). The model group was activated with 10 ng / mL TGFβ for 24 hours, and the drug administration group was treated with 10 ng / mL TGFβ and 2.5 μM Tafamidis for 24 hours. The expression of fibrosis-related proteins COL1A1 and α-SMA was detected, and the results are shown in Figure 32.

[0490] The results showed that Tafamidis could inhibit the expression of COL1A1 and α-SMA proteins induced by TGFβ in lung fibroblasts.

[0491] Example 30: Tafamidis inhibits lung cancer cell survival

[0492] Lung cancer cells HCC827 were plated in a 96-well plate, with a cell number of 1 × 10 4HCT116 cells were plated in 96-well plates at 1 x 10

[0493] The results show that Tafamidis can significantly inhibit the survival of lung cancer cells.

[0494] Example 31: Tafamidis inhibits the survival of colon cancer cells

[0495] HCT116 cells were plated in 96-well plates at 1 x 10 4 HCT116 cells were plated in 96-well plates at 1 x 10

[0496] The results show that Tafamidis can significantly inhibit the survival of lung cancer cells.

[0497] In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "an example", "a specific example", or "some examples" and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the present specification, the illustrative description of the above terms is not necessarily directed to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any appropriate manner in any one or more embodiments or examples. In addition, different embodiments or examples described in the present specification and the features of different embodiments or examples can be combined and combined by those skilled in the art without contradiction.

[0498] Although the embodiments of the present application have been shown and described above, it is understood that the above-described embodiments are exemplary and are not to be construed as limiting the present application, and those skilled in the art can make changes, modifications, replacements and variations to the above-described embodiments within the scope of the present application.

Claims

1. Use of the compound of formula (I) or its tautomers, stereoisomers, solvates or pharmaceutically acceptable salts in the preparation of a medicament for the prevention and / or treatment of RXR-related diseases; 2. Use of a compound represented by Formula (I) or a tautomer, stereoisomer, solvate, or pharmaceutically acceptable salt thereof in the prevention and / or treatment of RXR-related diseases; 3. A compound represented by Formula (I) or a tautomer, stereoisomer, solvate, or pharmaceutically acceptable salt thereof for use in the prevention and / or treatment of RXR-related diseases; 4. Use according to any one of claims 1 to 3, characterized in that, The RXR-related disease includes an RXRα-related disease, an RXRβ-related disease, and an RXRγ-related disease.

5. Use according to any one of claims 1 to 3, characterized in that, The RXR-related disease includes a metabolic disease, inflammation, a neurodegenerative disease, a tumor, or a cancer.

6. Use according to any one of claims 1 to 3, characterized in that, The RXR-related disease includes alcoholic fatty liver disease, non-alcoholic fatty liver disease, hepatitis, liver fibrosis, liver cirrhosis, liver cancer, Alzheimer's disease, Parkinson's disease, neuroinflammation, a neural tumor, psoriasis, acne, psoriasis, lupus erythematosus, skin fibrosis, Kaposi's sarcoma, cutaneous T-cell lymphoma, nasopharyngitis, bronchitis, pneumonia, pulmonary fibrosis, a lung tumor, enteritis, intestinal fibrosis, an intestinal tumor, gastritis, a gastric tumor, urethritis, nephritis, renal fibrosis, a renal tumor, cholestasis, leukemia, diabetes, osteoporosis, arthritis, spondylitis, cholangitis, cholangiocarcinoma, atherosclerosis, myocarditis, hyperlipidemia, liver damage, fatty liver, lung cancer, colorectal cancer.

7. Use of a compound of Formula (I) or a tautomer, stereoisomer, solvate, or pharmaceutically acceptable salt thereof, in the manufacture of a reagent for modulating RXR activity, or modulating the activity of a dimer containing RXR; 8. Use of a compound of Formula (I) or a tautomer, stereoisomer, solvate, or pharmaceutically acceptable salt thereof, in modulating RXR activity or modulating the activity of a dimer containing RXR; 9. Use according to claim 7 or 8, characterized in that, The RXR-containing dimer includes an RXRα-containing dimer, an RXRβ-containing dimer, and an RXRγ-containing dimer.

10. Use according to claim 7 or 8, characterized in that, The RXR-containing dimer includes an RXR homodimer and an RXR-containing heterodimer.

11. Use according to claim 10, characterized in that, The RXR-containing heterodimer is selected from the group consisting of RXR / RAR, RXR / PPAR, RXR / LXR, RXR / Nur77, RXR / VDR, RXR / TR, RXR / FXR, RXR / CAR, RXR / PXR, RXR / Nurr1, and RXR / Nor1.

12. A method of activating RXR activity or activating activity of a dimer containing RXR, characterized by, including: contacting a compound of Formula (I) or a tautomer, stereoisomer, solvate, or pharmaceutically acceptable salt thereof with a cell that expresses RXR, or a dimer containing RXR; 13. The method of claim 12, wherein, The RXR-containing dimer includes an RXRα-containing dimer, an RXRβ-containing dimer, and an RXRγ-containing dimer.

14. The method of claim 12, wherein, The RXR-containing dimer includes an RXR homodimer and an RXR-containing heterodimer.

15. The method of claim 14, wherein, The RXR-containing heterodimer is selected from the group consisting of RXR / RAR, RXR / PPAR, RXR / LXR, RXR / Nur77, RXR / VDR, RXR / TR, RXR / FXR, RXR / CAR, RXR / PXR, RXR / Nurr1, and RXR / Nor1.

16. A combination or kit comprising, in combination, a compound of formula (I) or a pharmaceutically acceptable salt thereof, and a second therapeutic agent. including: the compound represented by Formula (I) or a tautomer, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof as a first active ingredient; as a second active ingredient, a ligand L for modulating the activity of a nuclear receptor forming a dimer with RXR; 17. The combination or kit according to claim 16, wherein The RXR includes RXRα, RXRβ, and RXRγ. The ligand L is selected from the group consisting of a ligand for RAR, a ligand for PPAR, a ligand for LXR, a ligand for VDR, a ligand for Nur77, a ligand for TR, a ligand for FXR, a ligand for CAR, a ligand for PXR, a ligand for Nurr1, and a ligand for Nor1.

18. The combination or kit according to claim 16, wherein The ligand L is selected from at least one of all-trans retinoic acid ATRA, rosiglitazone, T0901317, fenofibrate, calcitriol, a thyroid hormone, a bile acid, an androstane, and a pregnane.

19. Use of the combination drug or kit of any one of claims 16 to 18 for the manufacture of a medicament for the prevention and / or treatment of an RXR-related disease.

20. Use according to claim 19, characterized in that, The RXR-related disease includes an RXRa-related disease, an RXRb-related disease, and an RXRy-related disease.

21. The use according to claim 19, characterized in that, The RXR-related disease includes a metabolic disease, inflammation, a neurodegenerative disease, a tumor, or a cancer.

22. The use according to claim 19, characterized in that, The RXR-related disease includes alcoholic fatty liver disease, non-alcoholic fatty liver disease, hepatitis, liver fibrosis, liver cirrhosis, liver cancer, Alzheimer's disease, Parkinson's disease, neuroinflammation, neural tumor, psoriasis, acne, psoriasis, lupus erythematosus, skin fibrosis, Kaposi's sarcoma, cutaneous T-cell lymphoma, nasopharyngitis, bronchitis, pneumonia, pulmonary fibrosis, lung tumor, enteritis, intestinal fibrosis, intestinal tumor, gastritis, gastric tumor, urethritis, nephritis, renal fibrosis, renal tumor, cholestasis, leukemia, diabetes, osteoporosis, arthritis, spondylitis, cholangitis, cholangiocarcinoma, atherosclerosis, myocarditis, hyperlipidemia, liver damage, fatty liver, lung cancer, colorectal cancer.

23. A method of preventing and / or treating a RXR-related disease, comprising administering to a subject in need thereof a therapeutically effective amount of a compound of any one of claims 1-20, or a pharmaceutically acceptable salt thereof. The RXR-related disease includes an RXRa-related disease, an RXRb-related disease, and an RXRy-related disease. administering to the subject a pharmaceutically acceptable dose of a compound represented by Formula (I) or a tautomer, stereoisomer, solvate, or pharmaceutically acceptable salt thereof, or the combination drug or kit of any one of claims 16-18; 24. The method of claim 23, wherein, The RXR-related disease includes a metabolic disease, inflammation, a neurodegenerative disease, a tumor, or a cancer.

25. The method of claim 23, wherein, The RXR-related disease includes alcoholic fatty liver disease, non-alcoholic fatty liver disease, hepatitis, liver fibrosis, liver cirrhosis, liver cancer, Alzheimer's disease, Parkinson's disease, neuroinflammation, neural tumor, psoriasis, acne, psoriasis, lupus erythematosus, skin fibrosis, Kaposi's sarcoma, cutaneous T-cell lymphoma, nasopharyngitis, bronchitis, pneumonia, pulmonary fibrosis, lung tumor, enteritis, intestinal fibrosis, intestinal tumor, gastritis, gastric tumor, urethritis, nephritis, renal fibrosis, renal tumor, cholestasis, leukemia, diabetes, osteoporosis, arthritis, spondylitis, cholangitis, cholangiocarcinoma, atherosclerosis, myocarditis, hyperlipidemia, liver damage, fatty liver, lung cancer, colorectal cancer.

26. The method of claim 23, wherein, ​

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