WO2022145512A1 - Pharmaceutical composition for preventing or treating non-alcoholic steatohepatitis - Google Patents

Abstract

The present invention relates to a pharmaceutical composition comprising DPP-4 inhibitor for preventing or treating non-alcoholic fatty liver diseases. The pharmaceutical composition is expected, on basis of effectiveness in high-fat diet animal models, to be used in treating non-alcoholic fatty liver diseases, and especially preventing or treating non-alcoholic steatohepatitis.

Description

Pharmaceutical composition for preventing or treating steatohepatitis

The present invention relates to a pharmaceutical composition for preventing or treating steatohepatitis.

Nonalcoholic fatty liver disease (NAFLD) includes a variety of diseases ranging from simple steatosis to nonalcoholic steatohepatitis and/or cirrhosis. NAFLD is associated with insulin resistance, oxidative stress, inflammation, mitochondrial dysfunction and fibrosis. In particular, the function of mitochondria increases according to lipid overload of hepatocytes, but continuous lipid overload and accumulation can lead to impaired mitochondrial function. Autophagy is important for maintaining cellular function by removing unnecessary or dysfunctional cellular components.

DPP-4 (dipeptidyl peptidase-4) inhibitors are known to improve the control of blood sugar levels in type 2 diabetic patients as anti-diabetic therapeutics, which are mainly caused by DPP-4 glucagon-like peptide-1 (GLP-1) It prevents the decomposition of GLP-1 is an incretin hormone released from L-cells in the lower small intestine after a meal. GLP-1 stimulates glucose-dependent insulin secretion from pancreatic β cells and increases the pancreatic β cell population. DPP-4 can rapidly cleave and inactivate GLP-1.

In modern society, as the diet of fat is increasing, interest in non-alcoholic fatty liver disease is growing. However, there is no drug approved for nonalcoholic fatty liver disease itself, and research and experiments are ongoing. The pharmaceutical composition of the present invention has been effective in an animal model of non-alcoholic fatty liver disease, and it can be expected to be used as a therapeutic agent for non-alcoholic fatty liver disease in the future.

1. A pharmaceutical composition for preventing or treating non-alcoholic fatty liver disease, including a dipeptidyl peptidase-4 (DPP-4) inhibitor.

2. The pharmaceutical composition for the prevention or treatment of nonalcoholic fatty liver disease according to the above 1, wherein the DPP-4 inhibitor is ebogliptin.

3. The pharmaceutical composition for preventing or treating nonalcoholic fatty liver disease according to 1 above, wherein the nonalcoholic fatty liver disease is nonalcoholic simple fatty liver, nonalcoholic steatohepatitis or nonalcoholic cirrhosis.

4. The pharmaceutical composition for the prevention or treatment of nonalcoholic fatty liver disease according to 1 above, wherein the nonalcoholic fatty liver disease is nonalcoholic steatohepatitis.

The present invention provides a pharmaceutical composition capable of preventing or treating non-alcoholic fatty liver disease, which can help research and experiments on therapeutic agents for non-alcoholic fatty liver disease for which there is currently no approved therapeutic agent.

1 is a graphical representation of body weight, epididymal fat pad weight, glucose level, food intake, serum insulin and liver enzyme (ALT, AST) levels between three groups.

Figure 2A shows the degree of liver lipid accumulation and liver damage through Oil-Red O staining for each group, and Figure 2B is a graph showing the expression levels of DGAT2 and Pnpla2 for each group.

3 is a graph showing H&E and MT staining results for each group and morphological changes in the liver. In the graph, the part where the x-axis is not indicated indicates the ND, HFD, and HFD+Evo groups in order.

4 shows the expression levels of autophagy markers for each group.

5 shows the expression levels of mitophagy markers for each group.

Figure 6 confirms the death of liver cells using TUNEL analysis.

Hereinafter, the present invention will be described in detail.

The present invention relates to a pharmaceutical composition for preventing or treating non-alcoholic fatty liver disease, comprising a dipeptidyl peptidase-4 (DPP-4) inhibitor.

DPP-4 (dipeptidyl peptidase-4) refers to an enzyme that degrades GLP-1, which lowers blood sugar. It acts to lower blood sugar.

The DPP-4 inhibitor may be used without limitation as long as it inhibits DPP-4 of an individual when administered to an individual, for example, evogliptin, vildagliptin, and sitagliptin may be used, but is not limited thereto.

Non-alcoholic fatty liver disease (NAFLD) refers to fatty liver disease not caused by alcohol among fatty liver diseases such as fatty liver and steatohepatitis. For example, it may be nonalcoholic simple fatty liver, nonalcoholic steatohepatitis or nonalcoholic cirrhosis.

In the present invention, the pharmaceutical composition may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and sterile injection solutions according to conventional methods, respectively. , but is not limited thereto.

Carriers, excipients and diluents that may be contained in the pharmaceutical composition of the present invention include lactose, dextrose, sucrose, dextrin, maltodextrin, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil; It is not limited. In the case of formulation, it may be prepared using usually used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants, but is not limited thereto.

Solid preparations for oral administration include, but are not limited to, tablets, pills, powders, granules, capsules, etc., but these solid preparations include at least one excipient in the compound, for example, starch, calcium carbonate , sucrose or lactose, gelatin, etc. are mixed and prepared. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used, but the present invention is not limited thereto.

Liquid formulations for oral use include suspensions, solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients, for example, wetting agents, sweeteners, fragrances, preservatives, etc. may be included. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, etc. may be used, but the present invention is not limited thereto.

The preferred dosage of the pharmaceutical composition of the present invention varies depending on the absorption of the active ingredient in the body, the age, sex, and the degree of obesity of the patient, but may be appropriately selected by those skilled in the art. However, for a desirable effect, in the case of oral administration, the composition of the present invention is generally administered at an amount of 0.0001 to 500 mg/kg, preferably 0.001 to 500 mg/kg of the composition of the present invention per 1 kg of body weight to adults per day. can Administration may be administered once a day, or may be administered in several divided doses. The above dosage and administration method do not limit the scope of the present invention in any way.

Hereinafter, examples will be given to describe the present invention in detail.

experimental method

1. Ethics Statement

This experiment was approved by Gyeongsang National University's Animal Protection Ethics Committee (GNU-160804-M0034), and all animal experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

2. Experimental animals

Four-week-old male C57BL/6J mice (Koatech Inc., Peongtaek, Korea) were bred by alternating light and dark cycles of 12 hours. Mice were divided into 3 groups, and diets were different for each group from 4 weeks of age to 16 weeks as follows.

(1) Normal diet (ND, 2018S, 3.1 kcal/g, Harlan Laboratories, Inc., Indianapolis, IN, USA) group; (2) high fat diet (HFD, 45% fat, Research Diets, Inc., New Brunswick, NJ, USA) group; and (3) the HFD (HFD+Evo) group administered ebogliptin.

According to a previous study, Evo (Evogliptin, 250mg/kg ^-1 /day ^-1 , Dong-A ST, Seoul) was administered to mice daily on a high-fat-fed (HFD) diet. All mice were weighed weekly and sacrificed at 20 weeks. Epididymal fat pads were weighed and blood and live tissue were collected. Below is a table comparing nutrients of a normal diet (ND) and a high-fat diet (HFD) (Table 1).

[Table 1]

3. Histopathology

Mice (n=10 per group) were anesthetized with zoletil (5 mg/kg, Virbac Laboratories, Carros, France). Livers were fixed and processed for paraffin embedding. Then, for histopathological analysis, a 5 μm-thick section was cut and stained with H&E, and fibrosis was confirmed by MT (Massion's trichrome) staining. In addition, the cross-sections were visualized with an optical microscope and the digital images were analyzed using NIS Elements BR3.2 (Nikon, Tokyo, Japan). Based on previous studies, scoring for NAFLD was performed by a pathologist through histological analysis of H&E staining. Scores were quantified by summing the scores for lobular inflammation (0-2), giant vesicular and microvesicular steatosis (0-3), and hepatocellular expansion (0-3). Randomly 5 fields, sections and animals (n=5 per group) were used for quantification.

4. Oil Red O staining and MT (Masson's Trichrome) staining

To confirm hepatic lipid accumulation, frozen liver sections were stained with 0.5% Oil Red O (Sigma) for 10 min, washed, and counterstained with Mayer's hematoxylin (Sigma) for 45 sec. The cross-sections were then visualized under an optical microscope and digital images were captured and documented.

5. End-Deoxynucleotidyl Transferase dUTP Nick End-Labeling Assay

Apoptosis was assessed semi-quantitatively by TUNEL analysis (Roche, Indianapolis, IN, USA). TUNEL-positive cells were counted at 400x magnification in at least 10 random fields per slide and the mean was calculated. They were enumerated by single-blind observers using NIS Elements BR 3.2 (Nikon, Tokyo, Japan) software.

6. Quantitative Real-Time PCR

Transcriptional levels of lipid metabolism-related factors were determined by quantitative real-time (q)PCR. Liver tissue was resuspended in 1 mL of TRIzol Reagent (Invitrogen Life Technologies, Carlsbad, CA, USA), and total RNA was extracted. Purified RNA was reverse transcribed into cDNA using the iScript cDNA synthesis kit (Bio-Rad Laboratories, Hercules, CA, USA) and oligo-dT primers. After reverse transcription, total DNA was diluted with ddHO for qPCR. Quantitative cDNA amplification was performed using a ViiA 7 Real-Time System (Applied Biosystems Inc., Foster City, CA, USA). Each reaction mixture contained 9 μL of template cDNA, 10 μL of Taqman Universal Master MixII, UNG (Applied Biosystems, Foster City, CA, USA) and diglyceride acyltransferase 2 (Dgat2) containing protein 2 (Pnpla2, ID: Mm00503040_m1). , ID: Mm00499536_m1) and 20 TaqMan gene expression assay mix for 1 μL of patatin-like phospholipase domain, the total volume was 20 μL. The temperature cycle conditions are as follows; Denaturation at 95°C for 3 minutes, 40 cycles denaturation at 95°C for 15 seconds, annealing and extension at 60°C for 60 seconds. GAPDH (ID: Mm99999915_g1) was used as an internal control for normalization of RNA amount, and the relative gene expression level of each sample was quantified using the 2DDCt method.

7. Protein Preparation and Western Blotting

Tissues were homogenized in lysis buffer and protein (50 μg) was loaded and electroblotted. The blots were anti-Ambra1 (ab69501, Abcam, Cambridge, MA, USA), anti-Beclin-1 (sc-48381), p62 (sc-48373), Pink1 (sc-33796), Parkin (sc-30130), BNIP. -3 (sc-56167) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-LC3B (#2775), p53 (#9282) and pAkt (#9271) (Cell Signaling Technology, Danvers, MA, USA) was irradiated overnight at 4°C with a polyclonal primary antibody against Primary antibody was visualized by adding secondary antibody and performing electroluminescence analysis (Amersham Pharmacia Biotech, Piscataway, NJ, USA).

8. Immunohistochemistry

After deparaffinization, the sections were polyclonal anti-LC3B (#2775, Cell Signaling Technology. Danvers, MA, USA), LAMP-1 (sc-17768, Santa Cruz Biotechnology, Santa Cruz, CA, USA), collagen-III (sc-271249, Santa Cruz Biotechnology, Santa Cruz, CA, USA), 8-OHdG (ab62623, Abcam, Cambridge, MA, USA) and MDA (MDA11-S, Alpha Diagnostic International. San Antonio, TX, USA) was incubated with the primary antibody against Then, biotin-conjugated secondary IgG (1:200 dillution, Vector Laboratories, Burlingame, CA, USA), avidin-biotin-peroxidase complex (ABC Elite Kit; Vector Laboratories) and DAB were added. Finally, the cross-sections were visualized by light microscopy and digital images were analyzed (Elements BR3.2, Nikon, Japan).

9. Statistical Analysis

Statistical analysis was performed using GraphPad Prism software (v. 8.0; GraphPad Software Inc., La Jolla, CA, USA). Data were assessed using one-way ANOVA analysis with Tukey's multiple comparison test (for comparison of all groups). A p-value <0.05 was used to reflect statistical significance. Values mean the standard error of the mean.

Experiment result

1. Effect of evogliptin on body weight, epididymal fat pad weight, glucose level, food intake, serum insulin and liver enzymes in HFD mice

There was no difference in body weight between the three groups at the initial baseline. The body weight of the HFD group increased compared to the ND group, and the HFD+Evo group showed a significantly lower body weight than the HFD group, but at 16 weeks after the HFD diet, the body weight was similar to that of the ND group ( FIG. 1A ). Epididymal fat weight was significantly increased in the HFD group compared to the ND group, and decreased to a level similar to the ND group in the HFD+Evo group (Fig. 1B).

At baseline, there was no significant difference in glucose levels between the three groups. Glucose levels were significantly increased in the HFD group compared to the ND group at 16 weeks after the HFD diet. The level of the HFD+Evo group was lower than that of the HFD group, but this was not significant (Fig. 1C).

Overall, the HFD and HFD+Evo groups consumed more energy per day than the ND group, whereas the energy consumption did not show a significant difference between the HFD and HFD+Evo groups (Fig. 1D). Insulin levels were significantly increased in the HFD group, and it can be seen that evogliptin improved hyperinsulinemia (FIG. 1E). Serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were increased in the HFD group and significantly decreased in the HFD+Evo group ( FIGS. 1E and F ).

2. Effect of evogliptin on steatohepatitis in HFD mice

In the ND group, liver morphology was normal, and there was no Oil red O positive staining. However, in the HFD group, Oil Red O-positive lipid droplets were distributed throughout the liver and corresponded to the pathological features of steatohepatitis including steatosis and lipogranuloma. The HFD+Evo group showed reduced hepatic lipid accumulation and impaired liver parenchyma compared to the HFD group ( FIG. 2A ). The expression of DGAT2, which catalyzes the last step of triacylglycerol (TG) synthesis, was further increased in the HFD group compared to the ND group, and decreased in the HFD+Evo group ( FIG. 2B ). The expression of patatin-like phospholipase domain-containing 2 (Pnpla2), which catalyzes the intracellular hydrolysis of stored triglycerides, was significantly down-regulated in the HFD and HFD+Evo groups compared to the ND group (Fig. 2B). Therefore, it can be seen that Evo can improve HFD-induced steatohepatitis by regulating the expression of lipid metabolism-related genes.

3. Effects of Evogliptin on Morphological Changes and Fibrosis of the Liver by HFD

Histological analysis by H&E staining demonstrates that hepatic steatosis (steatosis and hepatocellular expansion) was reduced in the HFD group treated with Evo (Fig. 3A). Accumulation of vascular collagen fibers by MT staining was observed in the HFD group, but Evo improved HFD-induced vascular fibrosis in the liver (Fig. 3B). To confirm the effect of Evo on HFD-induced liver fibrosis, staining of smooth muscle antibody (SMA) and collagen-III was performed. Positive signals of SMA and collagen-III were increased in the HFD group compared to the ND group, and significantly decreased in the HFD+Evo group ( FIGS. 3C and 3D ).

4. Effect of Evogliptin on Hepatic Autophagy in HFD Mice

Autophagy is associated with increased TG levels and oxidation inhibition. Therefore, as a result of examining the effect of Evo on autophagy in HFD mice, LC3, an autophagosome-related autophagy marker, was not expressed in the ND group. However, in the HFD group, a positive signal was detected in the cytoplasm of lipid-accumulating hepatocytes (black arrow, Fig. 4A). The number of positive signals in the HFD+Evo group was similar to that in the ND group. Immunohistochemical staining for LAMP-1, an autophagy lysosome-related marker, also showed an expression pattern similar to that of LC3 (FIG. 4A). In addition, to confirm the effect of Evo on autophagy in the liver by HFD, the expression of autophagy markers Beclin-1 and p62 was investigated. As a result, expression was increased in the HFD group and decreased in the HFD + Evo group (Fig. 4B).

5. Effect of Evogliptin on Hepatic Mitophagy and Oxidative Stress in HFD Mice

It has been reported that mitophagy alleviates hepatic steatosis, and its inhibition leads to exacerbation of NAFLD. The expression of mitophagy markers was significantly increased in the HFD group compared to the ND group. This expression was significantly lower in the HFD+Evo group compared to the HFD group ( FIG. 5A ). Mitophagy is associated with oxidative stress. Mitophagy is increased in the HFD group, where oxy-radical mediated DNA damage (production of 8-hydroxydeoxyguanosine (8-OHdG)) is more likely. Immunohistochemical staining showed that the signal of 8-OHdG-positive hepatocytes was increased in hepatocytes with small lipid droplets (#, microvesicular steatosis) as well as large lipid droplets (*, macrovesicular steatosis) in the HFD group compared to the ND group. and decreased in the HFD + Evo group (Fig. 5B). To confirm that Evo is involved in HFD-induced oxidative stress protection, malondialdehyde (MDA), a marker of oxidative stress in sections, was also investigated. The MDA-positive signal was mainly found in hepatocytes with large lipid droplets (*), and the signal was increased in the HFD group compared to the ND group and decreased in the HFD + Evo group (Fig. 5B).

6. Effect of Evogliptin on Hepatocellular Death by HFD

Apoptosis of liver cells was investigated using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay ( FIG. 6A ). The number of TUNEL-positive signals was significantly increased in the HFD group compared to the ND group, and most were detected in the nucleus of the damaged hepatocytes. However, the number of TUNEL positive signals was significantly reduced in the HFD + Evo group compared to the HFD group. The anti-apoptotic factor, Phospho-Akt (pAkt), was up-regulated in the HFD+Evo group compared to the HFD group, and the pro-apoptotic factor, p53, was down-regulated ( FIG. 6B ). These results indicate that Evo inhibits HFD-induced hepatocellular death by pAkt and p53-related mechanisms.

Claims (4)

1. A pharmaceutical composition for preventing or treating non-alcoholic fatty liver disease, comprising a dipeptidyl peptidase-4 (DPP-4) inhibitor.
2. The pharmaceutical composition for preventing or treating nonalcoholic fatty liver disease according to claim 1, wherein the DPP-4 inhibitor is evogliptin.
3. The pharmaceutical composition for preventing or treating nonalcoholic fatty liver disease according to claim 1, wherein the nonalcoholic fatty liver disease is nonalcoholic simple fatty liver, nonalcoholic steatohepatitis, or nonalcoholic cirrhosis.
4. The pharmaceutical composition for preventing or treating nonalcoholic fatty liver disease according to claim 1, wherein the nonalcoholic fatty liver disease is nonalcoholic steatohepatitis.

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