Application of 1, 2, 3, 4, 6-O-pentagalloylglucose in preparation of anti-cholestatic liver disease medicine

Abstract

The invention relates to application of 1, 2, 3, 4, 6-O-pentagalloylglucose in preparation of a medicine for resisting cholestatic liver diseases, and belongs to the technical field of medicinal chemistry. The 1, 2, 3, 4, 6-O-pentagalloylglucose provided by the invention can be used for promoting bile acid excretion by reducing WDR6 protein expression. In-vivo and in-vitro experiments prove that the 1, 2, 3, 4, 6-O-pentagalloylglucose can be used for remarkably relieving the symptom of the cholestatic liver disease. In addition, the 1, 2, 3, 4, 6-O-pentagalloylglucose has no obvious toxic or side effect, and has no obvious influence on the liver function and the kidney function. The 1, 2, 3, 4, 6-O-pentagalloylglucose has a wide anti-cholestatic liver disease spectrum, and is suitable for various cholestatic liver diseases, such as primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC) and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of medicinal chemistry, and in particular to application of 1,2,3,4,6-O-pentagalloylglucose in the preparation of an anti-cholestatic liver disease drug. Background Art

[0002] Cholestatic liver disease (CLD) is a condition characterized by liver damage and fibrosis due to bile stasis, exemplified by primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC). Cholestasis is a pathological condition caused by impaired bile acid metabolism, resulting in impaired bile production, secretion, and / or flow, leading to bile acid accumulation in the liver and even reflux into the bloodstream. It often leads to progressive hepatobiliary damage, and untreated, it can lead to severe conditions such as liver fibrosis, cirrhosis, and even liver failure. In recent years, the prevalence of cholestatic liver disease in my country has been increasing. Ursodeoxycholic acid is the only approved drug for its treatment. However, 40% of patients still experience unsatisfactory treatment responses, necessitating the urgent need to identify and develop new drugs to treat cholestatic liver disease.

[0003] 1,2,3,4,6-O-pentagalloylglucose (PGG, CAS: 14937-32-7) belongs to the gallotannin family and is a hydrolyzable tannin. Composed of five galloyl groups, PGG has a glucose core, a structural characteristic that confers high bioavailability. PGG can be obtained as a byproduct from various medicinal plants, such as gallnut, tree peony, and herbaceous peony. Its main functional properties include antibacterial, anti-inflammatory, anti-cancer, anti-diabetic, and antioxidant properties. 1,2,3,4,6-O-pentagalloylglucose has been shown to play a certain role in many malignant tumors such as breast cancer, prostate cancer, and liver cancer, but there have been no reports on its role in cholestatic liver disease. Summary of the Invention

[0004] In view of the problem that 1,2,3,4,6-O-pentagalloylglucose has not yet been used in the preparation of drugs for cholestatic liver diseases, the present invention provides an application of 1,2,3,4,6-O-pentagalloylglucose in the preparation of anti-cholestatic liver disease drugs to solve the above problem.

[0005] The technical solutions of the present invention are as follows: The present invention provides an application of 1,2,3,4,6-O-pentagalloylglucose in the preparation of an anti-cholestatic liver disease drug.

[0006] The present invention discovered that WDR6 protein expression is increased in the liver tissue of patients with cholestasis. WDR6 plays an important role in regulating bile acid metabolism. Increased WDR6 protein expression inhibits the expression of its downstream molecule, the bile acid receptor TGR5, and in turn inhibits the expression of the bile acid transporters ABCC3 and ABCC4, causing bile acids to accumulate in hepatocytes and prevent their excretion, leading to cholestasis. Through small molecule drug screening, we found that 1,2,3,4,6-O-pentagalloylglucose binds to the WDR6 protein and can reduce its expression. 1,2,3,4,6-O-pentagalloylglucose (PGG) inhibits WDR6 protein expression, activates the expression of the downstream regulatory molecule TGR5, and then promotes bile acid excretion by upregulating the expression of the bile acid transporters ABCC3 and ABCC4, alleviating cholestasis.

[0007] Furthermore, the cholestatic liver disease includes primary biliary cholangitis and primary sclerosing cholangitis.

[0008] Furthermore, the 1,2,3,4,6-O-pentagalloylglucose is the only active ingredient in the drug.

[0009] Furthermore, the effective concentration of the 1,2,3,4,6-O-pentagalloylglucose for treating cholestatic liver disease is 5-10 μg / kg.

[0010] Furthermore, the effective concentration of the 1,2,3,4,6-O-pentagalloylglucose for preventing cholestatic liver disease is 10 μg / kg.

[0011] Furthermore, the anti-cholestatic liver disease drug includes pharmaceutically acceptable excipients.

[0012] Furthermore, the acceptable excipients are selected from one or more of diluents, disintegrants, precipitation inhibitors, glidants, binders, dispersants, suspending agents, isotonic agents, thickeners, emulsifiers, preservatives, stabilizers, hydrating agents, ion exchangers, flavoring agents or antioxidants.

[0013] The beneficial effects of the present invention are: The 1,2,3,4,6-O-pentagalloylglucose provided by the present invention can promote bile acid excretion by reducing the expression of WDR6 protein. Through in vivo and in vitro experiments, it was found that 1,2,3,4,6-O-pentagalloylglucose can significantly alleviate the symptoms of cholestatic liver disease. In addition, 1,2,3,4,6-O-pentagalloylglucose has no obvious toxic side effects and has no significant effect on liver function and kidney function. 1,2,3,4,6-O-pentagalloylglucose has a wide spectrum of anti-cholestatic liver diseases and is suitable for a variety of cholestatic liver diseases, such as primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC).

[0014] In summary, 1,2,3,4,6-O-pentagalloylglucose has great application prospects in the treatment of cholestatic liver disease and will provide new ideas and methods for drug development based on the WDR6 protein target. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0016] Figure 1 is a schematic diagram of molecular screening according to Example 1 of the present invention; wherein Figure 1 A is a schematic diagram of the protein 3D structure, a is the N-terminal domain containing amino acids 1-329 of the WDR6 protein, b is the M domain containing amino acids 334-687, and c is the C-terminal domain containing amino acids 687-1121; Figure 1 B is a schematic diagram of molecular docking.

[0017] Figure 2 This is a diagram showing the results of immunoblotting experiments in Example 2 of the present invention to identify the inhibitory effect of 1,2,3,4,6-O-pentagalloylglucose (PGG) on WDR6 protein in hepatocytes.

[0018] Figure 3 This figure shows the results of serological assays for the therapeutic effect of 1,2,3,4,6-O-pentagalloylglucose (PGG) on cholestatic mice in Example 2 of the present invention. A is a bar graph comparing serum ALT (alanine aminotransferase); B is a bar graph comparing serum AST (aspartate aminotransferase); C is a bar graph comparing serum ALP (alkaline phosphatase); D is a bar graph comparing serum total bile acids; and E is a bar graph comparing liver total bile acids. Asterisks in the figure indicate statistically significant differences between groups (*p < 0.05; **p < 0.01; ***p < 0.001).

[0019] Figure 4 The results of immunohistochemical detection of the therapeutic effect of PGG on the liver tissue of cholestatic mice in Example 3 of the present invention are shown in FIG. In the figure, F is a HE staining image of the liver tissue; G is a picrosirius red staining image of the liver tissue. DETAILED DESCRIPTION

[0020] In order to enable those skilled in the art to better understand the technical solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts should fall within the scope of protection of the present invention.

[0021] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof.

[0022] In view of the current urgent need to research and explore new drugs for treating cholestatic liver diseases, the present invention proposes the use of 1,2,3,4,6-O-pentagalloylglucose in the preparation of drugs for treating cholestatic liver diseases.

[0023] In some embodiments of the present invention, there is provided the use of 1,2,3,4,6-O-pentagalloylglucose in the preparation of an anti-cholestatic liver disease drug.

[0024] Furthermore, the cholestatic liver disease includes primary biliary cholangitis and primary sclerosing cholangitis.

[0025] 1,2,3,4,6-O-pentagalloylglucose, molecular formula C 41 H 32 O 26 , CAS: 14937-32-7.

[0026] The present study discovered for the first time that 1,2,3,4,6-O-pentagalloylglucose can significantly alleviate the symptoms of cholestatic liver disease. Specifically, 1,2,3,4,6-O-pentagalloylglucose at a concentration of 5-10 μg / kg can significantly reduce the content of bile acids in serum caused by DDC (3,5-diethoxycarbonyl-1,4-dihydro-2,4.6-trimethylpyridine)-induced cholestasis.

[0027] The present invention also provides a pharmaceutical preparation, which comprises the above-mentioned 1,2,3,4,6-O-pentagalloylglucose and a pharmaceutically acceptable carrier or excipient.

[0028] In some examples of this embodiment, the pharmaceutically acceptable carrier or excipient is selected from one or more of a diluent, a disintegrant, a precipitation inhibitor, a glidant, a binder, a dispersant, a suspending agent, an isotonic agent, a thickener, an emulsifier, a preservative, a stabilizer, a hydrating agent, an ion exchanger, a flavoring agent or an antioxidant.

[0029] Example 1 Screening of small molecule compounds targeting WDR6 protein interactions The 1,2,3,4,6-O-pentagalloylglucose used in the examples of the present invention was purchased from Shanghai MedChemExpress Company, with the product number: HY-N0527.

[0030] High-throughput virtual screening 1. Protein Preparation The 3D structure of the human WDR6 protein (AF-Q9NNW5-F1-v4) was downloaded from the AlphaFold Protein Structure Database. The protein was structurally optimized and energetically optimized using the Protein Preparation Wizard module of Maestro 11.4 software (OPLS2005 force field, RMSD of 0.30 Å). A grid file (centered on SER352, THR355, PRO400, SER442, CYS496, VAL575, GLY618, and SER659) was generated using the ReceptorGrid Generation module, with a box size of 20 Å × 20 Å × 20 Å.

[0031] 2. Compound Preparation The 2D format of the small molecules in the L4000-Targetmol-Bioactive Compound Library was processed by the Schrödinger software LigPrep Module for hydrogenation and energy optimization, and the 3D structure was output for virtual screening.

[0032] 3. Molecular Docking Virtual screening was performed using the Virtual Screening Workflow module, and the prepared compounds were imported and molecular docking was performed using the Glide module. First, the small molecule compounds prepared in the L4000-Targetmol-Bioactive Compound Library were screened using the High Throughput Screening (HTVS) mode within the Glide module. The top 10% of the scoring small molecule compounds were selected for a second round of screening using the Standard (SP) mode. Subsequently, the top 10% of the scoring small molecule compounds were selected for a third round of screening using the High Precision (XP) mode to obtain a ranking of the small molecule compounds.

[0033] Finally, 1,2,3,4,6-O-pentagalloylglucose (PGG, PubChem CID: 65238) was screened out as having good WDR6 protein binding ability.

[0034] Figure 1 The 3D structure of the protein in A shows that WDR6 is composed of three relatively independent domains (a, b, c), namely the N-terminal domain containing amino acids 1-329 of the WDR6 protein (a); the M domain containing amino acids 334-687 (b); and the C-terminal domain containing amino acids 687-1121 (c); among them, 1,2,3,4,6-O-pentagalloylglucose (red arrow) can be completely bound to the pocket of b (gray). Figure 1 Molecular docking in B showed that 1,2,3,4,6-O-pentagalloylglucose can form multiple hydrogen bond interactions with leucine 356 (LEU), threonine 355 (THR), glutamate 397 (GLU), tryptophan 443 (TRP), valine 572 and 575 (VAL), arginine 587 (ARG), and histidine 657 (HIS) of WDR6 protein.

[0035] The chemical formula of 1,2,3,4,6-O-pentagalloylglucose is as follows: .

[0036] Example 2 1,2,3,4,6-O-pentagalloylglucose binds to WDR6 in hepatocytes and inhibits its expression 1. Cell Culture AML12 cells were cultured in DMEM / F12 high-glucose medium containing 10% serum, 1% ITS, 40 ng / ml dexamethasone, 1% penicillin and streptomycin. They were incubated at 37°C in a 5% CO2 incubator. At a cell density of 80%–90%, cells were trypsinized, centrifuged, and resuspended in fresh medium for passage.

[0037] 2. Treating cells with drugs Observe the cell growth status. When the cells maintain good growth and reach 90% confluency, digest and passage them. Seed the cells at 40% confluency in 12-well plates. Twenty-four hours after seeding, treat with different concentrations of 1,2,3,4,6-O-pentagalloylglucose (0, 2.5 μmol / L, 5 μmol / L, 10 μmol / L, 20 μmol / L, and 40 μmol / L). Incubate for an additional 24 hours and analyze WDR6 protein expression by Western blot. Repeat the experiment three times.

[0038] Experimental group: DMSO (dimethyl sulfoxide) was used as solvent to prepare different concentrations of 1,2,3,4,6-O-pentagalloylglucose.

[0039] 3. Results: Figure 2 As shown, using Gapdh (glyceraldehyde-3-phosphate dehydrogenase) as the normalized internal control for the experiment, the WDR6 protein level gradually decreased with the increase of 1,2,3,4,6-O-pentagalloylglucose concentration, indicating that 1,2,3,4,6-O-pentagalloylglucose significantly inhibited the expression of WDR6 protein in cells.

[0040] Example 3 1,2,3,4,6-O-pentagalloylglucose alleviates serum bile acid levels caused by DDC-induced cholestasis DDC (3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine) is a chemical inducer commonly used to establish animal models of cholestatic liver disease. Its mechanism of action and scientific research applications are mainly reflected in the following aspects: (1) Mechanism of action: Induces cholestasis. DDC interferes with the function of bile duct epithelial cells, leading to bile excretion disorders and intrahepatic cholestasis. This process is accompanied by hepatocyte damage, reactive bile duct hyperplasia, and inflammatory cell infiltration in the portal vein area.

[0041] (2) Promote fibrosis and inflammation: Long-term DDC exposure can activate signaling pathways such as NF-κB, induce liver inflammatory response and collagen deposition, and ultimately lead to portal vein fibrosis.

[0042] (3) Impact on liver cell regeneration: DDC-induced damage triggers bile duct cells to reprogram into hepatic progenitor-like cells (LPLCs), which participate in liver regeneration and repair, but excessive damage may inhibit the proliferation ability of liver cells.

[0043] 1. Establishment of a mouse model of cholestasis: C57BL / 6 mice were purchased and housed conventionally. After acclimatization for 7 days, the mice were randomly divided into 4 groups, with 6 mice in each group. Experimental group 1: normal diet CD group; Experimental group 2: normal diet and CD+PGG administration group; Experimental group 3: diet-induced cholestasis DDC group; Experimental group 4: Diet-induced administration of DDC+PGG group.

[0044] First, experimental group 1 gave mice a normal diet for 4 days; experimental group 2 gave mice a normal diet for 4 days; experimental group 3 gave mice a DDC diet for 4 days to induce; experimental group 4 gave mice a DDC diet for 4 days to induce.

[0045] Then, experimental groups 2 and 4 were treated with PGG (10 mg / kg) by intraperitoneal injection, while experimental groups 1 and 3 were injected with an equal volume of DMSO. The injections were repeated every other day for 2 consecutive weeks.

[0046] Specimen Collection and Testing: Two weeks after administration, mice were sacrificed and serum and liver tissue were obtained. Serum levels of ALT (alanine aminotransferase), AST (aspartate aminotransferase), ALP (alkaline phosphatase), total bile acid, and liver bile acid were measured using a fully automated biochemical analyzer (Mindray BS-830). Liver tissue morphology was assessed using HE staining, and liver damage was assessed using picrosirius red staining.

[0047] 3. Results: Figure 3 As shown in the results of serum and liver tissue tests, after DDC induced hepatic cholestasis, ALT (alanine aminotransferase), AST (aspartate aminotransferase), ALP (alkaline phosphatase) and bile acid levels increased significantly, and liver bile acid content also increased significantly. After PGG treatment, serum AST and ALP levels decreased significantly, and bile acid levels in serum and liver also decreased significantly. Figure 4As shown in the figure, HE staining and picrosirius red staining results showed that the DDC diet caused damage to liver tissue morphology and severe fibrosis in the liver tissue. After PGG treatment, the tissue morphology was partially restored and the degree of fibrosis was alleviated. These results indicate that PGG treatment alleviates DDC-induced liver damage and reduces bile acid content.

[0048] Although the present invention has been described in detail with reference to the accompanying drawings and in conjunction with preferred embodiments, the present invention is not limited thereto. Without departing from the spirit and essence of the present invention, persons of ordinary skill in the art may make various equivalent modifications or substitutions to the embodiments of the present invention, and such modifications or substitutions shall be within the scope of the present invention. Any changes or substitutions that can be easily conceived by persons skilled in the art within the technical scope disclosed in the present invention shall be within the scope of protection of the present invention.

Claims

1. Use of 1,2,3,4,6-O-pentagalloylglucose in the preparation of an anti-cholestatic liver disease drug.

2. The use according to claim 1, characterized in that Cholestatic liver diseases include primary biliary cholangitis and primary sclerosing cholangitis.

3. The use according to claim 1, characterized in that The 1,2,3,4,6-O-pentagalloyl glucose is the only active ingredient in the anti-cholestatic liver disease drug.

4. The use according to claim 1, wherein The effective concentration of the 1,2,3,4,6-O-pentagalloylglucose for preventing cholestatic liver disease is 5-10 μg / kg.

5. The use according to claim 4, wherein The effective concentration of the 1,2,3,4,6-O-pentagalloylglucose for preventing cholestatic liver disease is 10 μg / kg.

6. The use according to claim 1, wherein The anti-cholestatic liver disease drug comprises pharmaceutically acceptable excipients.

7. The use according to claim 6, characterized in that The pharmaceutically acceptable excipient is selected from at least one of a diluent, a disintegrant, a precipitation inhibitor, a glidant, a binder, a dispersant, a suspending agent, an isotonic agent, a thickener, an emulsifier, a preservative, a stabilizer, a hydrating agent, an ion exchanger, a flavoring agent or an antioxidant.

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