WO2018095056A1

WO2018095056A1 - Applications of fumarates in preparing medicament for treating liver diseases

Info

Publication number: WO2018095056A1
Application number: PCT/CN2017/093253
Authority: WO
Inventors: 陈永恒; 刘霆; 严璐; 陈娅琦; 张桂英; 陈主初
Original assignee: 中南大学湘雅医院
Priority date: 2016-11-23
Filing date: 2017-07-18
Publication date: 2018-05-31
Also published as: CN107088190A

Abstract

Disclosed are applications of fumarates in preparing a medicament for treating liver diseases. The fumarates comprise dimethyl fumarate (DMF) and monomethyl fumarate (MMF). The liver diseases comprise fatty liver disease, drug-induced liver disease, cholestatic liver disease, viral hepatitis, cirrhosis, and liver cancer.

Description

Application of fumarate in the preparation of medicine for treating liver diseases

Technical field

The invention relates to the field of chemical medicine. Specifically, it relates to the use of fumarate in the preparation of a medicament for treating various liver diseases.

Background technique

Oxidative stress means that when the body suffers from various harmful stimuli, high activity molecules such as reactive oxygen species (ROS) and reactive nitrogen radicals (RNS) are produced in the body, and the degree of oxidation exceeds the removal of oxides, resulting in tissue damage. . The liver, as an important metabolic organ, is often exposed to higher concentrations of exogenous substances and other chemicals. Although the liver has a range of mechanisms to protect against harmful chemicals and their metabolites, it is still susceptible to oxidative damage caused by intermediate active substances. Oxidative stress is a common pathogenesis of many liver diseases. Oxidative damage is closely related to most liver diseases, including: fatty liver disease, drug-induced liver disease, cholestatic liver injury, viral hepatitis, cirrhosis, and even liver cancer, so antioxidant Treatment plays an important role in the prevention and treatment of liver diseases.

Nrf2 is a newly discovered nuclear transcription factor mediated by oxidative stress. The Keap1-Nrf2-ARE signaling pathway mediates the transcription of phase II detoxification enzymes and antioxidant genes. It is considered to be the most antioxidant mechanism in cells. Important pathway. The activation and expression of Nrf2 and its downstream protective genes can induce the expression of a series of detoxification enzymes, biotransformases, and exogenous extracellular transporters, and increase the body's antioxidant capacity. Studies have shown that activation of Nrf2 plays an important role in combating oxidative stress damage in the liver. Robert H. and other studies have found that both DMF and MMF can enhance the protection of cells by activating the intracellular Nrf2 signaling pathway, so that they can better tolerate oxidative stress.

DMF, an inhibitor of NF-Kappa B activation, plays an immunomodulatory and neuroprotective role and is clinically used to treat multiple sclerosis (MS). Clinical studies have shown that DMF can play a role in reducing cellular inflammation and oxidative stress by activating the Nrf2 pathway. After oral administration, DMF can be rapidly hydrolyzed by the esterase in the body to the most biologically active metabolite, MMF, which is then safely absorbed in the small intestine. The MMF plasma concentration peaked at DMF for 5-6 hours; the DMF half-life was 12 minutes, and the MMF half-life was 36 hours, and the MMF biological effect lasted longer.

Although DMF and MMF have the function of activating Nrf2 pathway to reduce cellular inflammation and oxidative stress, and Nrf2 is important in combating oxidative stress in liver, DMF and MMF are used in the treatment of liver diseases associated with oxidative stress injury. Whether or not there is a role has not been reported, and no one has yet to conduct in-depth research in the world.

Summary of the invention

The present invention relates to the effects of dimethyl fumarate (DMF) and monomethyl fumarate (MMF) on Nrf2 signaling pathway in hepatocytes, and the liver associated with oxidative stress damage In-depth study of the treatment of the disease. Studies have shown that fumarate including DMF and MMF can be used to prepare drugs for treating various liver diseases including fatty liver disease, drug-induced liver disease, cholestatic liver disease, viral hepatitis, liver cirrhosis, liver cancer and the like. The researchers have confirmed through a large number of scientific studies that the two drugs can activate the Nrf2 signaling pathway in a variety of human hepatoma cells and mouse hepatocytes, and can also prevent liver damage caused by oxidative stress in a mouse liver disease model. , has a good liver protection. The human liver cancer cells are HepG2 cells and Huh7 cells, and the mouse liver cells are AML-12 cells.

Preferably, the dosage form of the medicament is a powder. The administration route of the drug includes topical application, intravenous and intraperitoneal injection, and the like.

In summary, the present invention has been subjected to extensive experimental studies and found that both DMF and MMF can activate Nrf2 signaling pathways in various human hepatoma cells and mouse hepatocytes, and can also prevent liver damage caused by oxidative stress in a mouse liver disease model. The liver protection effect indicates that DMF and MMF can be used to prepare drugs for treating liver diseases. Therefore, the present invention has an important development and application prospect in the treatment of various liver diseases including fatty liver disease, drug-induced liver disease, cholestatic liver disease, viral hepatitis, liver cirrhosis, liver cancer and the like.

DRAWINGS

Figure 1: Effect of DMF on the expression of Nrf2 and its anti-oxidation target genes in human hepatoma HepG2 cells (10uM DMF treatment of HepG2 cells WB/PCR);

Figure 2: Effect of DMF on the expression of Nrf2 and its anti-oxidation target genes in human hepatoma Huh7 cells (DMB-treated Huh7 cells WB/PCR);

Figure 3: Effect of DMF on the expression of Nrf2 and its anti-oxidation target genes in mouse hepatocyte AML-12 cells (10uM DMF treatment of AML-12 cells WB/PCR);

Figure 4: Effect of MMF on the expression of Nrf2 and its anti-oxidation target genes in human hepatoma HepG2 cells (10uM MMF treatment of HepG2 cells WB/PCR);

Figure 5: Effect of MMF on the expression of Nrf2 and its anti-oxidation target genes in human hepatoma Huh7 cells (10uM MMF treatment of Huh7 cells WB/PCR);

Figure 6: Effect of MMF on the expression of Nrf2 and its anti-oxidation target genes in mouse hepatocyte AML-12 cells (10uM MMF treatment of AML-12 cells WB/PCR);

Figure 7: Performance of normal liver tissue in a blank control group under light microscopy (100x magnification on the left and 200x magnification on the right);

Figure 8: The performance of liver tissue of mice in the control group of alcoholic liver disease without DMF treatment under light microscope (100x magnification on the left and 200x magnification on the right);

Figure 9: Performance of liver tissue of a model mouse administered orally by small doses of DMF under light microscopy (left image magnification 100 Times, the picture on the right is magnified 200 times);

Figure 10: Performance of liver tissue of a model mouse administered orally by high dose of DMF under light microscopy (100x magnification on the left and 200x magnification on the right).

detailed description

Example 1

Pathway experiment of DMF in vitro activation of Nrf2 signaling

Selected cell lines include human liver cancer HepG2 cells, human liver cancer Huh7 cells, and mouse liver cells AML-12. The logarithmic growth phase cells were seeded in 6-well plates. The cells were added to DMF for 0, 2, 4, 6, 12, and 24 hours. The plates were removed and total cellular and total RNA were extracted. Western blot and QPCR were used. Techniques for detecting the expression of Nrf2 and its antioxidant target genes (GCLC, HO1) in cells.

Results As shown in Figure 1-3, DMF enhanced the expression of Nrf2 and its antioxidant target genes (GCLC, HO1) in hepatocytes.

Example 2

Pathway experiment of MMF in vitro activation of Nrf2 signaling

Selected cell lines include human liver cancer HepG2 cells, human liver cancer Huh7 cells, and mouse liver cells AML-12. The logarithmic growth phase cells were seeded in 6-well plates, and the cells were cultured (MMF) for 0, 2, 4, 6, 12, and 24 hours. The plates were removed and the total protein and total RNA were extracted. Western The expression of Nrf2 and its anti-oxidation target genes (GCLC, HO1) in cells was detected by blot and QPCR techniques.

Results As shown in Figure 4-6, MMF can enhance the expression of Nrf2 and its antioxidant target genes (GCLC, HO1) in hepatocytes.

Example 3

The role of DMF in the prevention of oxidative stress injury in mice with alcoholic liver disease

The mice selected for the experiment were C57BL/6 mice (male, 8 weeks old, and weighing 18-21 g) supplied by Shanghai Slack Company. They were randomly divided into 4 groups and housed in a common barrier environment. The model of alcoholic liver disease in mice was constructed by NIAAA method. After successful model construction, two groups of mice were randomly selected and given different doses of DMF orally, once a day for 10 days. After 10 days, the mice in the 4 groups were euthanized. The liver tissues were immersed in formalin. The liver tissues of each group were stained with HE and observed under 100 times and 200 light microscopes.

Results As shown in Figure 7-10, DMF can prevent liver damage caused by oxidative stress in a mouse liver disease model, and has a good liver protection effect.

Claims

The use of fumarate in the preparation of a medicament for treating liver diseases.
The use of claim 1 wherein the fumarate comprises DMF and MMF.
The use according to claim 1, wherein the liver disease comprises fatty liver disease, drug-induced liver disease, cholestatic liver damage, viral hepatitis, cirrhosis, and liver cancer.
The use according to claim 1 wherein the pharmaceutical dosage form is a powder.
The use according to claim 1, wherein the route of administration of the drug comprises topical application, intravenous and intraperitoneal injection.