CN115364222A

CN115364222A - Use of EP4 receptor inhibitors for the treatment of liver fibrosis

Info

Publication number: CN115364222A
Application number: CN202210892136.2A
Authority: CN
Inventors: 朱鏐娈; 曹颖; 闫杰
Original assignee: Beijing Ditan Hospital
Current assignee: Beijing Ditan Hospital
Priority date: 2021-09-09
Filing date: 2022-07-27
Publication date: 2022-11-22

Abstract

The invention discloses an application of a selective inhibitor of prostaglandin E2 (PGE 2) receptor EP4, in particular to an application of E7046 in preparing a medicament for treating hepatic fibrosis. E7046 can inhibit PGE2 recombinant protein to induce the expression of hepatic fibrosis related gene and autophagy related gene, thus inhibit autophagy and activation of hepatic stellate cells, obviously reduce liver fat accumulation, lighten liver collagen deposition, have apparent effects of anti-hepatic fibrosis.

Description

Use of EP4 receptor inhibitors for the treatment of liver fibrosis

Technical Field

The invention relates to the field of medicines, in particular to an application of a selective inhibitor of prostaglandin E2 (PGE 2) receptor EP4, especially E7046, in the preparation of a medicine for treating hepatic fibrosis.

Background

Hepatic fibrosis refers to excessive deposition of diffuse extracellular matrix in the liver, and is a repair response of the body to chronic liver injury. Hepatic fibrosis is a common pathological change in chronic liver damage caused by various reasons and is also an indispensable link for the development of chronic liver diseases to cirrhosis. Liver cancer risk is greatly increased in patients with liver diseases once they have entered the cirrhosis stage from liver fibrosis. Therefore, how to reverse liver fibrosis is the key to delay and block the progression of cirrhosis and liver cancer. Activation and transdifferentiation of hepatic stellate cells into myofibroblasts, resulting in excessive extracellular collagen deposition, is a hallmark feature of hepatic fibrosis.

At present, no medicine which is approved by the State food and drug administration for improving the symptoms of the hepatic fibrosis exists. Therefore, there is an urgent need to develop effective therapeutic drugs or functional health products for hepatic fibrosis.

Prostaglandin E2 (PGE 2) is the major eicosanoid expressed under inflammatory conditions. PGE2 is also involved in a variety of physiological and/or pathological conditions such as hyperalgesia, uterine contractions, digestive peristalsis, wakefulness, inhibition of gastric acid secretion, blood pressure, platelet function, bone metabolism, angiogenesis, and cancer cell growth, invasion, and metastasis. There are four PGE2 receptor subtypes, namely EP1, EP2, EP3 and EP4, which exhibit different pharmacological properties. The EP4 receptor subtype belongs to the subfamily of G protein-coupled receptors and is referred to as a receptor with seven transmembrane domains. Thus, EP4 plays an important role in biological events by stimulating cAMP signaling-mediated functions. The prior art has demonstrated the role of EP4 receptors in cancer, and the growth of colon, breast, stomach, lung, prostate cancer is inhibited and/or metastasized in animal tumor models using EP4 receptor antagonists. However, no effect on hepatic fibrosis has been found with respect to EP4 receptor inhibitors.

Disclosure of Invention

The object of the present application is to provide a novel use of prostaglandin E2 (PGE 2) EP4 receptor inhibitors for liver fibrosis.

As one aspect of the present application, the present application provides the use of an EP4 receptor inhibitor for the manufacture of a medicament for preventing, alleviating, treating and/or reversing liver fibrosis.

Further, the liver fibrosis includes, but is not limited to, fibrosis caused by non-alcoholic fatty liver and drug-induced liver fibrosis.

Further, the use of the EP4 receptor inhibitor for inhibiting hepatic stellate cell activation and/or transformation into myofibroblasts.

Further, the use of said EP4 receptor inhibitor for reducing hepatic stellate cell autophagy.

Further, the EP4 receptor inhibitor can be used for inhibiting the expression of hepatic fibrosis related genes Actb2, col1a1 and Col3a 1.

Further, the EP4 receptor inhibitor can be used for inhibiting the expression of autophagy-related genes Atg5, atg7 and Becn 1.

Further, the EP4 receptor inhibitor is useful for inhibiting liver fat accumulation and/or liver collagen deposition and/or liver dysfunction and/or liver histopathological structural changes.

Further, the EP4 receptor inhibitor is selected from any one or more of AH23848, E7046, AAT-008, ONO-AE3-208, MF498, CJ-023423, L-161982, GW627368X, BGC20-1531, CJ-42794, ASP7657 and CJ-42794. Preferably E7046.

As another aspect of the present invention, the present invention provides a pharmaceutical composition characterized in that the composition comprises an effective amount of an EP4 receptor inhibitor and a pharmaceutically acceptable carrier.

In the present invention, "effective amount" means a nontoxic but sufficient amount of a drug or medicament to provide the desired effect, and "effective amount" of an ingredient or formulation unit means an amount of the ingredient or formulation unit effective to provide the desired effect when used in combination with other ingredients. The "effective amount" will vary from subject to subject, depending on age and general condition of the individual, the particular active agent, and the like. Thus, an exact "effective amount" may not always be possible, however, a suitable "effective amount" in any individual case may be determined by one of ordinary skill in the art using routine experimentation.

The invention has the following beneficial effects:

the research of the invention shows that the EP4 receptor inhibitor E7046 can inhibit the expression of PGE2 recombinant protein induced liver fibrosis related genes and autophagy related genes, thereby inhibiting the autophagy and activation of hepatic stellate cells, obviously reducing liver fat accumulation, relieving liver collagen deposition and having obvious anti-liver fibrosis effect.

In addition, the EP4 receptor inhibitor E7046 can obviously improve symptoms such as liver function abnormality, liver tissue pathological structure change, collagen deposition and the like related to liver fibrosis.

Drawings

FIG. 1 is the protein expression levels of hepatic fibrosis and autophagy marker molecules in the hepatic stellate cells of each group of example 1.

FIG. 2 is a photograph of immunofluorescent staining of liver tissue of each group of mice in example 2.

FIG. 3 is a photograph of the pathological section staining of liver tissue of each group of mice in example 2.

FIG. 4 is a graph showing serum ALT and AST levels of each group of mice in example 3.

FIG. 5 is a photograph of pathological staining of liver tissues of mice of each group in example 3.

FIG. 6 shows the liver alpha-SMA protein expression level and relative intensity of each group of mice in example 3; among them, fig. 6A: the expression levels of α -SMA protein in each group of mice, fig. 6B: relative strength of α -SMA protein in each group of mice.

FIG. 7 shows the liver Acta2 and Col1a1 mRNA expression levels of various groups of mice of example 3; among them, fig. 7A: acta2 mRNA expression levels, fig. 7B: col1a1 mRNA expression level.

Detailed Description

The present invention is further illustrated by the following specific examples. It is to be understood that the invention is not limited to those precise embodiments. It will be recognized by those skilled in the art that the present invention encompasses all alternatives, modifications, and equivalents as may be included within the scope of the claims and that such equivalents are encompassed within the scope of the invention as claimed.

Example 1 E7046 cell assay for inhibiting hepatic fibrosis

1 preparation of the medicament

E7046 is available from Shanghai blue Wood chemical Co., ltd, china (cat. No. S6649). E7046 was dissolved in DMSO and added to the cell culture medium at a final concentration of 100 nM.

2 preparation of hepatic stellate cell activation model

Test materials: human hepatic stellate cell line LX-2, RPMI-1640 medium, fetal Bovine Serum (FBS), PGE2 recombinant protein (Peprotech, inc., U.S.A.), E7046.

The treatment method comprises the following steps: LX-2 cells were inoculated into 10% FBS-containing RPMI-1640 medium and cultured at 37 ℃ under 5% CO2 conditions. Adding E7046 into a cell culture medium, adding PGE2 recombinant protein to 100nM after 1h and collecting cells after 100nM of the final concentration, extracting total RNA for real-time PCR detection, and extracting total protein for Western blot detection.

3 results of the experiment

3.1 E7046 reduction of hepatic fibrosis-associated Gene expression in hepatic stellate cells

Extracting total RNA, and detecting liver fibrosis related genes Actb2, col1A1 and Col3A1 (respectively encoding Actb2, COL1A1 and COL3A 1) by adopting real-time PCR; extracting total protein, and detecting the expression level of hepatic fibrosis marker molecule alpha-SMA by using Western blot.

The Real-time PCR result is shown in Table 1, the expression of the PGE2 recombinant protein induced liver fibrosis related genes Actb2, col1a1 and Col3a1 is up-regulated, the gene expression is obviously reduced after E7046 treatment, and the difference has statistical significance.

TABLE 1 fibrosis marker gene mRNA expression levels

	Acta2	Col1a1	Col3a1
				Blank control group	0.92±0.08	1.01±0.12	1.07±0.17
PGE2 group	2.26±0.11***	3.59±0.77***	4.17±0.46***
				PGE2+ E7046 group	0.93±0.07 ^###	1.38±0.10 ^##	1.31±0.13 ^###

Note: * The group represented PGE2 was statistically different from the blank group by p <0.001.# represents the statistical difference between PGE2+ E7046 and PGE2, #, p <0.01; # #, p <0.001.

Western blot detection results are shown in figure 1 and table 2, the expression of the alpha-SMA is up-regulated by the PGE2 recombinant protein, and the expression of the alpha-SMA is remarkably reduced after E7046 treatment.

TABLE 2 expression level of hepatic fibrosis marker molecule alpha-SMA protein

	α-SMA
		Blank control group	0.22±0.02
PGE2 group	1.98±0.03***
		PGE2+ E7046 group	0.47±0.02 ^###

Note: the protein expression level is calculated by the average optical density of a Western blot detection band, and the relative expression intensity is calculated according to the internal reference beta-Actin. Densitometry analysis was performed using ImageJ image analysis software. * The group represented PGE2 was statistically different from the blank group by p <0.001.# represents the statistical difference between PGE2+ E7046 and PGE2, # # #, p <0.001.

The results of the above experimental examples show that E7046 can inhibit the activation of hepatic stellate cells and the transdifferentiation to myofibroblasts.

3.2 E7046 reducing hepatic stellate cell autophagy

Extracting total RNA, and detecting autophagy-related genes Atg5, atg7 and Becn1 (respectively encoding Atg5, atg7 and Beclin-1) by adopting real-time PCR; and extracting total protein, and detecting the expression level of autophagy marker molecules LC3B and the conversion rate of LC3B-I to LC3B-II by using Western blot.

The Real-time PCR result is shown in Table 3, the expression of the autophagy-related genes Atg5, atg7 and Becn1 induced by the PGE2 recombinant protein is up-regulated, the expression of the genes is obviously reduced after E7046 treatment, and the difference has statistical significance.

TABLE 3 expression levels of autophagy-related gene mRNA

	Atg5	Atg7	Becn1
				Blank control group	0.97±0.19	0.97±0.18	0.93±0.08
PGE2 group	2.50±0.22***	2.30±0.14***	2.81±0.09***
				PGE2+ E7046 group	1.24±0.15 ^##	1.06±0.09 ^###	1.62±0.04 ^###

Note: * The group representing PGE2 was statistically different from the blank control group by p <0.001.# represents the statistical difference between PGE2+ E7046 and PGE2, #, p <0.01; # #, p is less than 0.001.

Western blot detection results are shown in FIG. 1 and Table 4, PGE2 remarkably promotes the conversion from LC3B-I to LC3B-II, autophagy of cells is marked, and the conversion rate of LC3B-I/II is obviously reduced by E7046 treatment.

TABLE 4 levels of autophagy in cells

	LC3B-II/LC3B-I
		Blank control group	0.40±0.02
PGE2 group	1.45±0.23**
		PGE2+ E7046 group	0.49±0.06 ^##

Note: the level of autophagy was calculated as the conversion of LC3B-I to LC 3B-II. The protein expression level is calculated by the average optical density of a Western blot detection band, and the optical density analysis adopts ImageJ image analysis software. * Represents the statistical difference between the PGE2 group and the blank control group, { p } <0.01.# represents the statistical difference between PGE2+ E7046 and PGE2, #, p <0.01.

The results of the above experimental examples show that E7046 can inhibit autophagy-mediated activation of hepatic stellate cells.

Example 2 E7046 inhibition of liver fibrosis in animals

1 preparation of the medicament

E7046 was purchased from Shanghai Hainan chemical Co., ltd, china (cargo number S6649). E7046 was dissolved in 0.5% methylcellulose and used as is. The oral gavage is carried out to the mice according to 150mg/kg, and the volume of the single gavage is not more than 200ul per mouse.

2 preparation of hepatic fibrosis mouse model

Methionine-choline deficiency (MCD) diet is the most common method for preparing mouse models of non-alcoholic fatty liver-related liver fibrosis. Research shows that when mice are fed MCD diet for 3-4 weeks, the liver presents obvious inflammation; after the mice are fed with MCD for 7-8 weeks, hepatic lobule structure is disordered, hepatic cells are degenerated by fat and become necrotic, and a large amount of collagen fiber deposits appear in hepatic sinus and a manifold area, so that typical hepatic fibrosis symptoms are presented. 24C 57BL/6 mice (male, 8-10 weeks, body weight 22 + -2 g) were selected for this experiment. The diet was randomized to normal diet, methionine-choline deficient (MCD) diet, and MCD + E7046 groups of 8 individuals. And (3) anesthetizing and killing the mice the next day after 7 weeks of model building, taking fresh liver tissues, and detecting the hepatic fibrosis level and the hepatic stellate cell autophagy and activation level of the mice by using pathological staining and immunofluorescence staining.

The mouse healthy diet feed and the MCD feed were purchased from Beijing Huafukang Biotech GmbH. The MCD feed (cat H10401) formulation was referenced to Harlan TD.90262. And (3) intragastrically feeding 150mg/kg of E7046 to MCD 7046 in MCD 7046 group mice every other day from the 5 th week of MCD diet, continuously taking materials from the 7 th week of MCD diet, and observing whether the E7046 can effectively relieve the hepatic fibrosis of NAFLD mice.

3 results of the experiment

3.1 E7046 reducing the expression of hepatic fibrosis related genes of non-alcoholic fatty liver mice

Mice were sacrificed under anesthesia 7 weeks after MCD or healthy diet, fresh liver tissue was taken, fixed with 10% formalin, paraffin embedded, sectioned and immunofluorescent stained. The hepatic fibrosis marker molecule alpha-SMA is marked by green fluorescent protein, the autophagy marker molecule LC3B is marked by red fluorescent protein, and the cell nucleus is marked by DNA dye 4', 6-diamidino-2-phenylindole (DAPI). The immunofluorescence results are shown in fig. 2, compared with MCD diet mice, the expression and co-localization degree of alpha-SMA and LC3B in liver tissues after E7046 treatment (MCD + E7046 group) are remarkably reduced, which indicates that E7046 inhibits the liver stellate cell autophagy and liver fibrosis related gene expression of mice.

3.2 E7046 improves the hepatic steatosis and the collagen fiber deposition of the non-alcoholic fatty liver disease mice

Taking paraffin sections of liver tissues for oil red staining and sirius red staining, wherein the results are shown in figure 3, and the oil red staining shows that a large amount of lipid droplets are accumulated in the liver tissues of MCD (micro-dermal disc) diet mice compared with a healthy diet group; whereas E7046 treatment (MCD + E7046 group) significantly improved hepatic lipid droplet accumulation. The dyeing result of sirius red shows that a great amount of collagen fibers are obviously deposited near the central vein and the junction area of liver tissues in an MCD group, and fibrous intervals are formed among liver lobules; the E7046 treatment (MCD + E7046 group) significantly improved collagen fiber deposition.

The experimental results show that E7046 obviously improves the hepatic fibrosis of the non-alcoholic fatty liver disease mouse.

Example 3 E7046 improves CCl ₄ Experiment of liver fibrosis model mouse serum liver function and liver tissue pathological injury

1 method of experiment

1.1 CCl ₄ Preparation of hepatic fibrosis mouse model

5% CCl was prepared ₄ Olive oil solution (volume ratio 1.

30C 57BL/6 mice, clean grade, 6-8 weeks old, male, weight 20g + -2 g, purchased from Beijing Huafukang Biotech GmbH, license number: SCXK (Jing) 2019-0008. Adaptive feeding for 1 week before experiment. The temperature is 20-25 ℃ and the time is 12 h. Fasting for 12h before molding, and drinking water freely.

30C 57BL/6J mice were randomly divided into 3 groups, i.e., control group, CCl ₄ Group sum CCl ₄ + E7046 groups of 10 individuals each. CCl ₄ Mice were injected intraperitoneally with 5% CCl4 oil solution (15 mL/kg), 3d/1 times, 300 ul/mouse, for 27 consecutive days. CCl ₄ + E7046 group, intragastric administration 30mg/ml E7046 blocking agent, 3d/1 times, 100 ul/one, continuous 27 days, intragastric injection 5% CCl4 oil solution into abdominal cavity at the same time of intragastric administration, the method is the same as CCl ₄ Group (d); control group was injected intraperitoneally with 5% olive oil; control group and CCl ₄ The mice in the group were gavaged with equal volume of 0.5% methylcellulose. Each group of mice was anesthetized to kill the mice 12 hours after the last intraperitoneal injection. Liver and serum samples were collected immediately after mice were sacrificed.

1.2 preparation of the medicament

E7046 was purchased from Shanghai Baphicans chemical Co., ltd., china (product number S6649), dissolved in 0.5% methyl cellulose, and prepared to 30mg/ml, ready to use. The oral gavage is carried out to the mice according to 150mg/kg, and the volume of the single gavage is not more than 200ul per mouse.

2 results of the experiment

2.1 E7046 pairs of CCl ₄ Effect of serum liver function level in mice

CCl ₄ The mice were kept for 27 days, and 12 hours after the last administration, the mice were sacrificed under anesthesia, and serum samples were immediately collected. After the mice were bled, the samples were centrifuged at 2000 rpm for 15 minutes with a radius r =186. Serum was retained and ALT (alanine aminotransferase) and AST (aspartate aminotransferase) levels were measured using an automated biochemical analyzer (Hitachi, japan).

The results are shown in FIG. 4. As shown in FIG. 4, CCl was compared with the control group ₄ The serum ALT activity of the group mice is obviously improved, and the CCl ₄ + E7046 group mice were more active than CCl ₄ Compared with the group, the content of the compound is obviously reduced, the difference was statistically significant (F =111.06, P<0.001；****P<0.001 ); mouse serum AST Activity, CCl compared to control group ₄ Significantly increased group activity, CCl ₄ + E7046 group mice were more active than CCl ₄ Group comparison, significantly decreased, and the difference was statistically significant (F =174.2<0.001；****P<0.001)。

2.2 E7046 pairs of CCl ₄ Pathological damage to liver tissue and effects of collagen deposition in mice

The liver tissue specimen was stained with paraffin fixed section, and the result is shown in FIG. 5. As shown in fig. 5, h.e staining results of mouse liver tissues showed that the liver hepatocyte structure of the control group was normal, the lobular structure of the liver was normal, and no damage was observed; CCl ₄ Severe damage of liver lobules, structural disorder, obvious swelling and deformation, sheet necrosis and inflammatory cell infiltration (yellow arrow); the degree of inflammation and necrotic area in group E7046 were significantly reduced compared to the model group, as indicated by a lack of significant edema improvement, a reduction in necrotic foci, and a reduction in inflammatory cell infiltration. The results of Masson staining and sirius red staining of mouse liver tissues show that the control group liver tissues have no collagen deposition; CCl ₄ Group mass collagen deposition (black and blue arrows); collagen fibers were significantly reduced in group E7046, but still partially visible, with CCl ₄ Compared with mice, the composition is obviously improved. It shows that E7046 obviously improves CCl ₄ The degree of hepatic fibrosis in the mice (Note: the length of the black scale is 100 μm, and the length of the blue scale is 20 μm).

2.3 E7046 pairs of CCl ₄ Effect of hepatic fibrosis-related Gene expression in mice

Taking a liver tissue specimen to extract total protein for Western blot detection, and extracting total RNA for real-time PCR analysis. Alpha smooth muscle actin (alpha-SMA) and type I collagen (Col 1a 1) are marker molecules for hepatic stellate cell activation. Western blot results show that CCl compared to the control group ₄ The liver alpha-SMA expression of the mice is increased; and CCl ₄ Group comparison, CCl ₄ The expression level of α -SMA in group + E7046 was decreased (fig. 6A). Relative intensity analysis of protein bands was performed using ImageJ software, with alpha-SMA/Actin in control, CCl ₄ Group sum CCl ₄ The relative expression levels in the + E7046 group were 0.634. + -. 0.024, 0.970. + -. 0.035 and 0.578. + -. 0.043, respectively. CCl compared to control group ₄ The expression of histone is obviously increased; and CCl ₄ Group comparison, CCl ₄ + E7046 histone expression was significantly reduced, with statistical significance for the difference (F =73.32,. Star. P)<0.001，****P<0.001 (FIG. 6B).

The mRNA expression levels of the alpha-SMA encoding gene Acta2 and the encoding gene Col1a1 were further detected by real-time PCR, and the results are shown in FIG. 7. The results of Acta2 gene expression are shown in FIG. 7A, comparing CCl with the control group ₄ Liver Acta2 of the group mice is obviously up-regulated; and CCl ₄ Group comparison, CCl ₄ The liver Acta2 expression was reduced in the + E7046 group of mice, with statistical differences (F =14.50,. P = 0.002;. P = 0.008); the results of Col1a1 gene expression are shown in FIG. 7B, where CCl is compared with the control group ₄ Livers Col1a1 of the mice in the group are obviously up-regulated; and CCl ₄ Group comparison, CCl ₄ + E7046 group mice had decreased expression of Col1a1 in liver, the difference was statistically significant (F =41.50, P<0.001；****P<0.001)。

In conclusion, the selective inhibitor of EP 4E 7046 significantly reduces CCl ₄ The mouse liver tissue damage reduces the liver collagen deposition, and has obvious anti-hepatic fibrosis effect.

Claims

Use of an EP4 receptor inhibitor for the preparation of a medicament for the prevention, alleviation, treatment and/or reversal of liver fibrosis.
2. The use of claim 1, wherein the liver fibrosis comprises fibrosis caused by non-alcoholic fatty liver disease and drug-induced liver fibrosis.
3. Use according to claim 1, wherein the EP4 receptor inhibitor inhibits hepatic stellate cell activation and/or transformation into myofibroblasts.
4. The use of claim 1, wherein the EP4 receptor inhibitor is used to reduce autophagy in hepatic stellate cells.
5. Use according to claim 1, wherein the EP4 receptor inhibitor inhibits the expression of the liver fibrosis related genes Actb2, col1a1 and Col3a 1.
6. Use according to claim 1, wherein the EP4 receptor inhibitor inhibits the expression of autophagy-related genes Atg5, atg7, becn 1.
7. Use according to claim 1, wherein the EP4 receptor inhibitor is used to inhibit liver fat accumulation and/or liver collagen deposition and/or liver dysfunction and/or changes in the pathological structure of the liver tissue.
8. The use according to any one of claims 1 to 7, wherein the EP4 receptor inhibitor is E7046.
9. A pharmaceutical composition for preventing, ameliorating, treating and/or reversing liver fibrosis, comprising an effective amount of an EP4 receptor inhibitor and a pharmaceutically acceptable carrier.
10. The pharmaceutical composition of claim 9, wherein the EP4 receptor inhibitor is E7046.