Use of 1,2,3,4,6-o-pentagalloyl glucose in the preparation of a drug for resisting cholestatic liver disease

Abstract

The present application relates to a kind of 1,2,3,4,6-O-penta-galloyl glucose in the application of preparation anti-cholic stasis hepatopathy drug, belong to the technical field of medicinal chemistry.The 1,2,3,4,6-O-penta-galloyl glucose provided in the present application can reduce WDR6 protein expression, and then promote bile acid efflux.Through in vivo and in vitro experiment verification, it is found that 1,2,3,4,6-O-penta-galloyl glucose can significantly reduce the symptoms of cholestatic liver disease.In addition, 1,2,3,4,6-O-penta-galloyl glucose has no obvious toxic side effects, and has no obvious effect on liver function and kidney function.1,2,3,4,6-O-penta-galloyl glucose has a wide range of anti-cholic stasis hepatopathy spectrum, suitable for various cholestatic liver diseases, such as primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC) and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of pharmaceutical chemistry, and particularly relates to application of 1,2,3,4,6-O-pentagalloylglucose in preparation of a drug for resisting cholestatic liver disease. BACKGROUND

[0002] Cholestatic Liver Disease (CLD) is a disease caused by cholestasis leading to liver damage and fibrosis, represented by primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC). Cholestasis is a pathological state caused by bile acid metabolism disorder, which damages the production, secretion and / or flow of bile, leading to the accumulation of bile acids in the liver, and even reflux into the blood. It usually causes progressive hepatobiliary damage, and if not treated in time, it can even lead to liver fibrosis, cirrhosis and even liver failure and other serious diseases. In recent years, the prevalence of cholestatic liver disease in China has shown an increasing trend. In clinical practice, ursodeoxycholic acid is the only drug approved for treatment. However, 40% of patients have unsatisfactory treatment effects, so it is particularly urgent to screen and develop new drugs for treating cholestatic liver disease.

[0003] 1,2,3,4,6-O-pentagalloylglucose (PGG, CAS: 14937-32-7) belongs to the gallotannin family and is a hydrolysable tannin. 1,2,3,4,6-O-pentagalloylglucose is composed of five galloyl groups, and its core is glucose. Its structural characteristics endow it with high biological power. 1,2,3,4,6-O-pentagalloylglucose can be obtained as a byproduct from various medicinal plants (such as Chinese gall, peony and paeony). The main functional properties of 1,2,3,4,6-O-pentagalloylglucose include antibacterial, anti-inflammatory, anticancer, antidiabetic and antioxidant properties. 1,2,3,4,6-O-pentagalloylglucose has been proven to play a certain role in many malignant tumor diseases such as breast cancer, prostate cancer and liver cancer, but there is no relevant report on cholestatic liver disease. SUMMARY

[0004] In view of the problem that 1,2,3,4,6-O-pentagalloylglucose has not been applied to preparation of a drug for cholestatic liver disease, the present application provides application of 1,2,3,4,6-O-pentagalloylglucose in preparation of a drug for resisting cholestatic liver disease, so as to solve the above problem.

[0005] The technical scheme of the present application is as follows:

[0006] The application provides application of 1,2,3,4,6-O-penta-galloyl glucose in preparation of a drug for resisting cholestatic liver disease.

[0007] The application finds that the expression of WDR6 protein in liver tissue of a cholestatic patient is increased, and WDR6 plays an important role in regulating the process of bile acid metabolism. The enhanced expression of WDR6 protein inhibits the expression of downstream molecules, bile acid receptor TGR5, and further inhibits the expression of bile acid transporters ABCC3 and ABCC4, so that bile acids accumulate in hepatocytes and cannot be discharged, causing cholestasis. Through small molecule drug screening, it is found that 1,2,3,4,6-O-penta-galloyl glucose can bind to WDR6 protein and reduce its expression. 1,2,3,4,6-O-penta-galloyl glucose (PGG) can activate the expression of downstream regulatory molecules TGR5 by inhibiting the expression of WDR6 protein, and further promote the expression of bile acid transporters ABCC3 and ABCC4, promote the excretion of bile acids, and reduce cholestasis.

[0008] Further, the cholestatic liver disease includes primary biliary cholangitis and primary sclerosing cholangitis.

[0009] Further, the 1,2,3,4,6-O-penta-galloyl glucose is the only active ingredient in the drug.

[0010] Further, the effective concentration of the 1,2,3,4,6-O-penta-galloyl glucose for resisting cholestatic liver disease is 5-10 μg / kg.

[0011] Further, the effective concentration of the 1,2,3,4,6-O-penta-galloyl glucose for resisting cholestatic liver disease is 10 μg / kg.

[0012] Further, the drug for resisting cholestatic liver disease includes pharmaceutically acceptable excipients.

[0013] Further, the acceptable excipients are selected from one or more of diluents, disintegrants, precipitation inhibitors, glidants, binders, dispersants, suspending agents, isotonic agents, thickening agents, emulsifiers, preservatives, stabilizers, hydrating agents, ion exchange agents, flavoring agents or antioxidants.

[0014] The application has the following beneficial effects:

[0015] The 1,2,3,4,6-O-penta-O-galloyl-glucose provided by the present application can reduce the expression of WDR6 protein, and further promote the bile acid efflux. Through in vivo and in vitro experiments, it is found that the 1,2,3,4,6-O-penta-O-galloyl-glucose can significantly alleviate the symptoms of cholestatic liver disease. In addition, the 1,2,3,4,6-O-penta-O-galloyl-glucose has no obvious toxic and side effects, and has no obvious effect on liver function and kidney function. The 1,2,3,4,6-O-penta-O-galloyl-glucose has a wide spectrum of anti-cholestatic liver disease, and is suitable for various cholestatic liver diseases, such as primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC).

[0016] In summary, the 1,2,3,4,6-O-penta-O-galloyl-glucose has good application prospect in the field of cholestatic liver disease treatment, and will provide new ideas and methods for drug development based on WDR6 protein target. BRIEF DESCRIPTION OF DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. Obviously, for those skilled in the art, other drawings can also be obtained without creative labor based on these drawings.

[0018] Figure 1 is a molecular screening schematic diagram of embodiment 1 of the present application; wherein Figure 1 A is a protein 3D structure schematic diagram, a is an N-terminal domain containing amino acids 1-329 of WDR6 protein, b is an M domain containing amino acids 334-687, and c is a C-terminal domain containing amino acids 687-1121; Figure 1 B is a molecular docking schematic diagram.

[0019] Figure 2 is a result diagram of immunoblotting experiment in embodiment 2 of the present application to identify the inhibitory effect of 1,2,3,4,6-O-penta-O-galloyl-glucose (PGG) on WDR6 protein in liver cells.

[0020] Figure 3 is a result diagram of serological index detection of the therapeutic effect of 1,2,3,4,6-O-penta-O-galloyl-glucose (PGG) on cholestatic mice in embodiment 2 of the present application. In the figure, A is an ALT (alanine aminotransferase) comparison column chart in serum; B is an AST (glutamic-oxaloacetic transaminase) comparison column chart in serum; C is an ALP (alkaline phosphatase) comparison column chart in serum; D is a total bile acid comparison column chart in serum; E is a total bile acid comparison column chart in liver. The asterisk mark in the figure indicates the statistical significance of the difference between groups (*p<0.05; **p<0.01; ***p<0.001).

[0021] Figure 4 Figure is the result chart of immunohistochemical detection of PGG on the treatment effect of cholestasis mouse liver tissue in embodiment 3 of the present application. In the figure, F is the HE staining chart of liver tissue; G is the picrosirius staining chart of liver tissue. DETAILED DESCRIPTION

[0022] In order for those skilled in the art to better understand the technical solutions in the present application, the technical solutions in the embodiments of the present application will be described clearly and completely below in combination with the drawings in the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor should fall within the scope of protection of the present application.

[0023] It should be noted that the terms used herein are only intended to describe specific embodiments, and are not intended to limit the exemplary embodiments according to the present application. As used herein, the singular form is intended to include the plural form unless the context clearly indicates otherwise, and it should also be understood that when the terms "comprise" and / or "include" are used in the specification, there is a presence of the features, steps, operations, devices, components and / or combinations thereof.

[0024] In view of the urgent need to research and explore new drugs for anti-cholestatic liver disease, the present application proposes an application of 1,2,3,4,6-O-penta-O-galloyl-glucose in the preparation of anti-cholestatic liver disease drugs.

[0025] In some embodiments of the present application, an application of 1,2,3,4,6-O-penta-O-galloyl-glucose in the preparation of anti-cholestatic liver disease drugs is provided.

[0026] Further, the cholestatic liver disease includes primary biliary cholangitis and primary sclerosing cholangitis.

[0027] 1,2,3,4,6-O-penta-O-galloyl-glucose, molecular formula C 41 H 32 O 26 , CAS: 14937-32-7.

[0028] The present application first found that 1,2,3,4,6-O-penta-O-galloyl-glucose can significantly alleviate the symptoms of cholestatic liver disease, wherein 1,2,3,4,6-O-penta-O-galloyl-glucose at a concentration of 5-10 μg / kg can significantly reduce the content of bile acid in serum caused by DDC (3,5-diethoxycarbonyl-1,4-dihydro-2,4.6-trimethylpyridine) induced cholestasis.

[0029] The present application also provides a pharmaceutical preparation comprising the above-mentioned 1,2,3,4,6-O-penta-O-galloyl-glucose and a pharmaceutically acceptable carrier or excipient.

[0030] In some embodiments of the 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 thickening agent, an emulsifying agent, a preservative, a stabilizer, a hydrating agent, an ion exchanger, a flavoring agent, or an antioxidant.

[0031] Example 1

[0032] Screening of small molecule compounds with targeting WDR6 protein interaction

[0033] The 1,2,3,4,6-O-penta-O-galloyl-glucose used in the embodiments of the present application is purchased from Shanghai MedChemExpress Company, item number: HY-N0527.

[0034] High-throughput virtual screening

[0035] 1. Protein preparation

[0036] The 3D structure of human WDR6 protein was downloaded from the AlphaFold Protein Structure Database (AF-Q9NNW5-F1-v4). The Protein Preparation Wizard module of Maestro 11.4 software was used to optimize the structure and energy of the protein (OPLS2005 force field, RMSD 0.30 Å). The processed WDR6 protein was used to make a grid file (centered on SER352, THR355, PRO400, SER442, CYS496, VAL575, GLY618, SER659) using the ReceptorGrid Generation module, and the box size was set to 20 Å × 20 Å × 20 Å.

[0037] 2. Compound preparation

[0038] The 2D format of L4000-Targetmol-Bioactive Compound Library compound library small molecules was processed by hydrogenation, energy optimization, etc. through Schrödinger software LigPrep Module, and the 3D structure was output for virtual screening.

[0039] 3. Molecular docking

[0040] Virtual screening was performed using the Virtual Screening Workflow module, and the prepared compounds were imported. Molecular docking was performed using the Glide module. First, the prepared small molecule compounds in L4000-Targetmol-Bioactive Compound Library were screened using the high-throughput screening (HTVS) mode in the Glide module, and the top 10% of small molecule compounds were selected for the second round of screening using the standard (SP) mode. Then, the top 10% of the scoring values were selected for the third round of screening using the high-precision (XP) mode, and the ranking of the small molecule compounds was obtained.

[0041] Finally, 1,2,3,4,6-O-pentagalloylglucose (abbreviated as PGG, PubChem CID: 65238) was screened out as having good WDR6 protein binding ability.

[0042] Figure 1 The 3D structure of protein A shows that WDR6 is composed of three relatively independent domains (a, b, c), which are the N-terminal domain containing amino acids 1-329 of 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 completely bind to the pocket of b (gray). Figure 1 The molecular docking in B shows that 1,2,3,4,6-O-pentagalloylglucose can form multiple hydrogen bond interactions with the 356th leucine (LEU), the 355th threonine (THR), the 397th glutamic acid (GLU), the 443rd tryptophan (TRP), the 572nd and 575th valine (VAL), the 587th arginine (ARG), and the 657th histidine (HIS) of WDR6 protein.

[0043] The chemical formula of 1,2,3,4,6-O-pentagalloylglucose is as follows:

[0044] .

[0045] Example 2

[0046] 1,2,3,4,6-O-pentagalloyl glucose binds to WDR6 in hepatocytes and inhibits its expression

[0047] 1. Cell culture

[0048] AML12 cells were cultured in DMEM / F12 high glucose medium containing 10% serum, 1% ITS, 40 ng / ml dexamethasone, 1% penicillin and streptomycin double antibody. They were placed in a 37°C incubator containing 5% CO2, and when the cell density was 80%-90%, the cells were resuspended after trypsin digestion and centrifugation, and then subcultured by adding new culture medium.

[0049] 2. Drug treatment of cells

[0050] After observing the growth state of the cells, which were in good condition and had a cell confluence of 90%, the cells were digested and subcultured, and then inoculated in a 12-well plate at a confluence of 40%. After 24 hours, different concentrations of 1,2,3,4,6-O-pentagalloyl glucose (0, 2.5 μmol / L, 5 μmol / L, 10 μmol / L, 20 μmol / L, 40 μmol / L) were added for treatment, and the cells were cultured for another 24 hours. Western Blot was used to detect the changes in the expression of WDR6 protein. The experiment was repeated three times.

[0051] Experimental group: DMSO (dimethyl sulfoxide) was used as the solvent to prepare different concentrations of 1,2,3,4,6-O-pentagalloyl glucose.

[0052] 3. Results: As shown in Figure 2 , Gapdh (glyceraldehyde-3-phosphate dehydrogenase) was used as the standardized internal control for the experiment. The level of WDR6 protein gradually decreased with increasing concentrations of 1,2,3,4,6-O-pentagalloyl glucose, indicating that 1,2,3,4,6-O-pentagalloyl glucose significantly inhibited the expression of WDR6 protein in cells.

[0053] Example 3

[0054] 1,2,3,4,6-O-pentagalloyl glucose reduces the content of bile acids in serum caused by DDC-induced cholestasis

[0055] DDC (3,5-diethoxycarbonyl-1,4-dihydro-2,4.6-trimethylpyridine) is a chemical inducer commonly used to construct animal models of cholestatic liver disease. Its mechanism of action and research applications mainly include the following aspects:

[0056] (1) Mechanism of action: Inducing cholestasis. DDC causes bile excretion disorder by interfering with the function of bile duct epithelial cells, leading to intrahepatic cholestasis. This process is accompanied by hepatocyte damage, bile duct reactive hyperplasia and inflammatory cell infiltration in the portal area.

[0057] (2) Promoting fibrosis and inflammation: Long-term DDC exposure can activate signaling pathways such as NF-κB, inducing liver inflammatory response and collagen deposition, ultimately leading to fibrosis in the portal area.

[0058] (3) Affecting hepatocyte regeneration: DDC-induced damage triggers bile duct cell reprogramming into liver progenitor-like cells (LPLC), participating in liver regeneration and repair, but excessive damage may inhibit the proliferative capacity of hepatocytes.

[0059] 1. Constructing a mouse model of cholestasis: After purchasing C57BL / 6 mice, they were routinely fed and adapted to the environment for 7 days. The mice were randomly divided into 4 groups, 6 in each group, including:

[0060] Group 1: normal diet CD group;

[0061] Group 2: normal diet and drug administration CD+PGG group;

[0062] Group 3: diet-induced cholestasis DDC group;

[0063] Group 4: diet-induced and drug administration DDC+PGG group.

[0064] First, the mice in group 1 were given normal diet for 4 days; the mice in group 2 were given normal diet for 4 days; the mice in group 3 were given DDC diet for 4 days for induction; the mice in group 4 were given DDC diet for 4 days for induction.

[0065] Then, groups 2 and 4 were treated with PGG (10 mg / kg) by intraperitoneal injection, and groups 1 and 3 were injected with the same volume of DMSO. Injection was performed every other day for 2 weeks.

[0066] 2. Sample collection and detection: After 2 weeks of administration, the mice were sacrificed, and the serum and liver tissue of the mice were taken. The content of ALT (alanine aminotransferase), AST (glutamic-oxalacetic transaminase), ALP (alkaline phosphatase), total bile acid in serum and bile acid in liver were detected by using automatic biochemical analyzer (Mindray BS-830). The morphology of liver tissue was detected by HE staining, and the liver damage was observed by picrosirius staining.

[0067] 3. Results: As Figure 3As shown, the results of serum and liver tissue detection showed that after DDC induced liver cholestasis, the contents of ALT (alanine aminotransferase), AST (glutamic-oxaloacetic transaminase), ALP (alkaline phosphatase) and bile acid in serum were significantly increased, and the content of bile acid in liver was also significantly increased. After PGG treatment, the contents of AST and ALP in serum were significantly decreased, and the contents of bile acid in serum and liver were significantly decreased. For example Figure 4 As shown, the results of HE staining and picrosirius staining showed that DDC diet caused damage to the morphology of liver tissue, and the liver tissue appeared to be severely fibrotic. After PGG treatment, the tissue morphology was partially restored, and the degree of fibrosis was reduced. The above results all showed that PGG treatment reduced DDC-induced liver injury and reduced the content of bile acid.

[0068] Although the present application has been described in detail with reference to the preferred embodiments, the application is not limited to the preferred embodiments. Various equivalent modifications or replacements can be made to the embodiments of the present application by those skilled in the art without departing from the spirit and essence of the present application, and these modifications or replacements should be within the scope of the present application. Any person skilled in the art can easily think of changes or replacements within the technical scope disclosed by the present application, and these changes or replacements should be within the protection scope of the present application.

Claims

1. Use of 1,2,3,4,6-O-pentagalloyl glucose in the manufacture of a medicament for the treatment of cholestatic liver disease, characterized in that, The 1,2,3,4,6-O-pentagalloyl-glucose is the only active ingredient in the drug for treating cholestatic liver disease; the cholestatic liver disease includes primary biliary cholangitis and primary sclerosing cholangitis; the effective concentration of the 1,2,3,4,6-O-pentagalloyl-glucose for treating cholestatic liver disease is 5-10 μg / kg.

2. Use according to claim 1, characterized in that, The effective concentration of the 1,2,3,4,6-O-pentagalloyl-glucose for treating cholestatic liver disease is 10 μg / kg.

3. The use according to claim 1, wherein The drug for treating cholestatic liver disease comprises a pharmaceutically acceptable excipient.

4. The use according to claim 3, wherein the compound is ###0002### The pharmaceutically acceptable excipient is selected from at least one of diluents, disintegrants, precipitation inhibitors, glidants, binders, dispersants, suspending agents, isotonic agents, thickening agents, emulsifiers, preservatives, stabilizers, hydrating agents, ion exchange agents, flavoring agents, or antioxidants. The pharmaceutically acceptable excipient is selected from at least one of diluents, disintegrants, precipitation inhibitors, glidants, binders, dispersants, suspending agents, isotonic agents, thickening agents, emulsifiers, preservatives, stabilizers, hydrating agents, ion exchange agents, flavoring agents, or antioxidants.

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