WO2021162460A1 - Novel pharmaceutical composition for treating non-alcoholic liver disease - Google Patents

Abstract

The present invention relates to a novel composition for treating non-alcoholic liver disease, and more specifically, a pharmaceutical composition which is for treating non-alcoholic liver disease and includes, as an active ingredient: (a) GLP-1 or an analog thereof and the IL-10 protein; (b) a fusion protein in which GLP-1 or an analog thereof is linked to the IL-10 protein; or (c) a dual specificity dimer fusion protein in which GLP-1 or an analog thereof is linked to the N-terminus of an antibody Fc region, and the IL-10 protein is linked to the C-terminus of the antibody Fc region.

Description

Pharmaceutical composition for treatment of novel non-alcoholic liver disease

The present invention relates to a novel pharmaceutical composition, and more particularly, to a pharmaceutical composition for the treatment of non-alcoholic liver disease.

Among nonalcoholic liver diseases, non-alcoholic steatohepatitis (hereinafter, referred to as 'NASH') has a characteristic etiology depending on the stage of progression. That is, the etiology of insulin resistance, glucose/lipid regulation, starting from dysregulation, steatosis, inflammation and fibrosis, and apoptosis are gradually related to non-alcoholic fatty liver. disease, hereinafter abbreviated as 'NAFLD') to NASH (classified into F0, F1, F2, F3, and F4 stages) and further progresses to cirrhosis (compensated → decompensated).

Among non-alcoholic liver diseases, non-alcoholic fatty liver disease (hereinafter, abbreviated as 'NAFLD') is a liver disease that is rapidly increasing along with metabolic syndrome accompanied by obesity, diabetes, and hypertension. The incidence of non-alcoholic steatohepatitis (hereinafter referred to as 'NASH'), which can progress to liver cancer and is a severe inflammatory form of NAFLD, and NASH-related cirrhosis are rapidly increasing. Many studies are being conducted for

It is known that NASH has a characteristic etiology depending on the stage of progression. That is, the etiology of insulin resistance, glucose/lipid regulation, starting from dysregulation, steatosis, inflammation and fibrosis, and apoptosis are gradually related to non-alcoholic fatty liver. disease, hereinafter abbreviated as 'NAFLD') to NASH (classified into F0, F1, F2, F3, F4 stages) and further progresses to cirrhosis (compensated → decompensated).

However, unfortunately, there is no drug approved as a treatment for NASH so far, and considering the burden of the disease, it is a drug with high unmet needs, in which patients' needs for treatment options are not met. The basic pathophysiological mechanisms contributing to the development and progression of NAFLD and NASH are very complex, and these mechanisms of action have been confirmed in the development of various therapeutic agents for various targets currently being studied. To a large extent, drug development focuses on the modulation of metabolic pathways, inflammatory cascades, and mechanisms of action that influence fibrosis. Although the mechanism of action for the pathogenesis of NAFLD has been largely elucidated, there are many difficulties in developing therapeutic agents due to the complexity of clinical trial design. Candidates currently undergoing phase 3 clinical trials are showing good results in terms of reducing hepatic steatosis, necrotic inflammation, and fibrosis while suggesting potential as therapeutic agents. If long-term safety and efficacy are established through multiple cohort studies, it will help reduce potential morbidity and mortality.

In addition, attempts have been made to use the GLP-1 receptor as a therapeutic agent for NAFLD or NASH. In this regard, WO2016043533A1 discloses a therapeutic agent for nonalcoholic fatty liver comprising an oxyntomodulin derivative, which is a dual agonist of GLP-1/glucagon receptor with increased half-life as an active ingredient, and US9938335 also discloses dual action of glucagon receptor/GLP-1 receptor. Disclosed are a glucagon-like peptide used as an agent and a treatment method for NAFLD and NASH using the same.

Although some of the effects of the substances described in the prior art have been confirmed in animal models, there is still no substance that has finally passed clinical trials.

An object of the present invention is to solve various problems, including the above-mentioned problems, (a) GLP-1 or an analog thereof and IL-10 protein; (b) a fusion protein in which GLP-1 or an analog thereof is linked to an IL-10 protein; Or (c) GLP-1 or an analog thereof is linked to the N-terminus of the antibody Fc region, and a bispecific dimeric fusion protein linked to the C-terminus of the antibody Fc region with IL-10 protein as an active ingredient. To provide a pharmaceutical composition for the prevention or treatment of alcoholic liver disease (nonalcoholic liver disease).

In order to achieve the above object, the present invention provides (a) GLP-1 or an analog thereof and IL-10 protein; (b) a fusion protein in which GLP-1 or an analog thereof is linked to an IL-10 protein; Or (c) GLP-1 or an analog thereof is linked to the N-terminus of the antibody Fc region, and a bispecific dimeric fusion protein linked to the C-terminus of the antibody Fc region with IL-10 protein as an active ingredient. It provides a pharmaceutical composition for the prevention or treatment of alcoholic liver disease.

The present invention also provides a therapeutically effective amount of (a) GLP-1 or an analog thereof and an IL-10 protein; (b) a fusion protein in which GLP-1 or an analog thereof is linked to an IL-10 protein; or (c) administering a bispecific dimeric fusion protein in which GLP-1 or an analog thereof is linked to the N-terminus of the antibody Fc region and IL-10 protein is linked to the C-terminus of the antibody Fc region. It provides a method for treating non-alcoholic liver disease, comprising.

The composition according to the present invention can effectively inhibit lipid accumulation in the liver, relieve inflammation accompanying fatty liver, and can also effectively inhibit lipid toxicity caused by excessive lipids in the liver, preventing or preventing non-alcoholic liver disease or It can be usefully used for treatment.

1 is a bispecific dimer in which GLP-1 or an analog thereof is linked to the N-terminus of the antibody Fc region produced according to an embodiment of the present invention, and IL-10 protein is linked to the C-terminus of the antibody Fc region. This is a series of SDS-PAGE gel pictures showing the results of the generation of the fusion protein (GLP-1/IL-10 fusion protein; MD100). GLP-1-hyFc (left) and hyFc-IL-10V (center) were produced separately to confirm the combined effect:

left and center

1: Non-reducing conditions

2: Reduction conditions

Right (GLP-1-hyFc-IL-10V)

1: input - non-reducing condition

2: flow through - non-reducing conditions

3: elution - non-reducing conditions

4: input - reduction condition

5: flow through - reducing conditions

6: elution - reducing conditions

Figure 2 is two times for examining the in vitro activity of GLP-1 / IL-10 fusion protein (MD100), GLP-1-hyFc (GX-G6) and liraglutide prepared according to various embodiments of the present invention. It is a series of graphs showing the measurement results of cAMP inducibility.

Figure 3 is a GLP-1 / IL-10 fusion protein (MD100), recombinant human IL-10 protein and hyFc-IL-10V according to an embodiment of the present invention according to the concentration of each concentration treatment of TNFα secretion by ELISA analysis. It is a graph showing the comparison of the results.

Figure 4 is a GLP-1 / IL-10 fusion protein according to an embodiment of the present invention (MD100, right) and recombinant IL-10 (left) the binding force of IL-10R Octet Bio-layer interferometry (interferometry) It is a series of graphs that compare the results analyzed through

5 is a graph showing the results of analyzing the in vivo pharmacokinetics (PK) of the GLP-1 / IL-10 fusion protein (MD100) according to an embodiment of the present invention by ELISA using an Fc region-specific antibody; am.

6 is a graph showing the metabolic-related effects of administration of GLP-1 + IL-10 combination (Combo) and GLP-1/IL-10 fusion protein (MD100) according to an embodiment of the present invention, and the body weight ( A), weight change (B), blood triglyceride content (TG, C), total cholesterol content (D), total bilirubin content (E), insulin resistance (G), glucose tolerance (G) and blood glucose level (H) Show a series of graphs representing the measurement results.

7 is a result of examining the effect on hepatic steatosis and inflammation according to the administration of GLP-1 + IL-10 combination (Combo) and GLP-1/IL-10 fusion protein (MD100) according to an embodiment of the present invention; A series of photographs (A) showing the results of Sirius Red staining of liver tissue slices or photographing livers extracted from each experimental group, the weight of the liver in each experimental group (B), the weight-to-weight ratio (C), and the content of liver triglycerides (D) ), ALT content (E), AST content (F), TNFα expression level (G), IL-6 expression level (H), and IL-1β expression level (I) are shown, respectively.

8 is a result of examining the effects of GLP-1 + IL-10 combination administration (Combo) according to an embodiment of the present invention on hepatic steatosis and inflammation in a high-fat diet-induced nonalcoholic steatohepatitis mouse model. A series of photographs showing the results of Sirius Red staining of excised livers or liver tissue sections (A), liver weight (B), weight-to-weight ratio (C), liver triglyceride content (D), ALT in each experimental group A series of graphs showing the content (E), the AST content (F), the TNFα expression level (G), the IL-6 expression level (H), and the IL-1β expression level (I) are shown, respectively.

9 is a result of examining the metabolic profile by administration of a GLP-1/IL-10 fusion protein (MD100) according to an embodiment of the present invention in a high-fat diet-induced nonalcoholic steatohepatitis mouse model by concentration, in each experimental group. A series of graphs showing the results of measuring body weight (A), body weight change (B), blood glucose change (C), and blood glucose level (D) are shown.

10 is a result of examining the effects of administration of GLP-1/IL-10 fusion protein (MD100) according to an embodiment of the present invention on hepatic steatosis and inflammation in a high-fat diet-induced nonalcoholic steatohepatitis mouse model. , A series of photographs showing the results of Sirius Red staining of liver tissue slices or photographing livers extracted from each experimental group (A), the weight of the liver in each experimental group (B), the weight-to-weight ratio (C), and the liver triglyceride content ( D), ALT content (E), AST content (F), TNFα expression level (G), IL-6 expression level (H) and IL-1β expression level (I) are a series of graphs, respectively.

11 is a result of examining the effect on liver fibrosis according to the administration of each concentration of GLP-1/IL-10 fusion protein (MD100) according to an embodiment in a high-fat diet-induced nonalcoholic steatohepatitis mouse model. A series of photographs (A) showing the results of Sirius Red staining for one liver tissue and a graph (B) showing the results of analysis of hydroxyproline are shown.

12 is a result of investigating the in vitro lipid toxicity using palmitic acid according to the treatment of GLP-1 + IL-10 combination (Combo) and GLP-1/IL-10 fusion protein (MD100) according to an embodiment of the present invention. , shows a series of graphs showing the results of measuring cell viability (A) and expression levels of various apoptosis markers (B) upon drug treatment.

13 is a result of in vitro analysis of the degree of inflammation improvement according to the treatment of GLP-1 + IL-10 combination (Combo) and GLP-1/IL-10 fusion protein (MD100) according to an embodiment of the present invention. As a result, after treatment with LPS-induced bone marrow-derived macrophages (BMDM), various inflammation-related markers such as phosphorylated NK-κB (A), TNF-α (B), IL-6 (C) and IL A series of graphs showing the results of measuring the expression level of -1β(D) are shown.

14 shows in vitro hepatic steatosis induced by oleic acid by treatment with GLP-1 + IL-10 combination (Combo) and GLP-1/IL-10 fusion protein (MD100) according to an embodiment of the present invention. ), a series of fluorescence micrographs showing the results confirmed through BODIPY staining (A) and a graph (B) quantifying the results of A are shown.

15 is a graph showing the effect of the treatment of GLP-1 + IL-10 combination (Combo) and GLP-1/IL-10 fusion protein (MD100) on the differentiation of preadipocytes into adipocytes according to an embodiment of the present invention; As the analysis results, a series of photographs showing the Sirius Red staining results in each experimental group (A), the triglyceride accumulation level in each experimental group (B), and the PPARγ expression level were confirmed by Western blot analysis (C), PPARγ ( D) shows a series of graphs showing the expression levels of C/EBPa(E) and aP2(F).

The term "GLP-1" used in the present invention is an abbreviation of "glucagon-like peptide-1", and is 30 or 31 amino acids induced by tissue-specific post-translational processing of proglucagon peptides. It is a peptide hormone of length. GLP-1 is produced and secreted by specific neurons in the enteroendocrine L-cells of the small intestine and the solitary tract nucleus of the brain stem during food intake. The initial product, GLP-1(1-37), is readily amidated and cleaved with two equivalent biological activities by cleavage (GLP-1(7-36) amide and GLP-1(7-37)). is converted to Active GLP-1 contains two alpha-helical regions at amino acid positions 13-20 and 24-35 and a linker region connecting the two alpha-helical regions. Since GLP-1 acts to lower blood sugar levels in a glucose-dependent manner, it has been developed and used as a treatment for type 2 diabetes. However, since GLP-1 is rapidly degraded by dipeptidyl peptidase-4 (DPP-4) in vivo, its in vivo half-life is only 2 minutes, so its effect is extremely limited as a natural peptide.

As used herein, the term "GLP-1 analog" refers to a protein that biologically performs the function of GLP-1 and can mediate downstream signaling by binding to the GLP-1/exendin-4 receptor.

As used herein, the term "fusion protein" refers to a recombinant protein in which two or more proteins or domains responsible for a specific function in the protein are linked so that each protein or domain takes on its original function.

As used herein, the term “antibody Fc region” refers to a crystallized fragment among fragments generated when an antibody is cleaved with papain, and refers to a cell surface receptor called Fc receptor and a complement system. It interacts with several proteins. The antibody Fc region exhibits a homodimeric structure in which fragments comprising the second and third constant regions (CH2 and CH3) of the heavy chain are linked by intermolecular disulfide bonds at the hinge region. The Fc region of IgG has a number of N-glycan attachment sites, which are known to play an important role in Fc receptor-mediated action, and IgG1, IgG2, IgG3, IgG4, and IgD may be used.

As used herein, the term "hybrid antibody Fc region" refers to an Fc region peptide produced by a combination of parts of various subtypes of Ig Fc region described in Korean Patent No. 897938, and The combination may show a difference from the wild-type antibody Fc region in binding capacity to Fc receptors and complement.

As used herein, the term "Exendin" is a peptide composed of 39 amino acids isolated from the poison of the lizard Heloderma suspectum. Exendin 4 is 50% identical in amino acid sequence to GLP-1, is a member of the glucagon peptide family, and is known to perform an equivalent role to GLP-1 as an agonist of the GLP-1 receptor. Exendin 4 is also called "extenatide". Exendin 3 is a mutant in which the second and third amino acids in Exendin 4 are substituted with serine and aspartic acid, respectively.

As used herein, the term "Lixisenatide" is one of the GLP-1 receptor agonists, manufactured by Sanofi, under the trade name Lyxumia in Europe and Adlyxin in the United States as a daily administration injection for the treatment of type 2 diabetes. It is a drug marketed as

The term "Albiglutide" used in the present invention is one of the GLP-1 receptor agonists sold under the trade name of Eperzan in Europe and Tanzeum in the United States as a treatment for type 2 diabetes by GSK.

As used herein, the term "Liraglutide" is a subcutaneously injectable GLP-1 receptor agonist marketed as a treatment for type 2 diabetes and obesity under the trade name "Victoza" by Novo Nordisk.

As used herein, the term "Taspoglutide" is a GLP-1 receptor agonist jointly developed by Ipsen and Roche, which is a therapeutic agent for type 2 diabetes, which is the 8th and 35th amino acids of the GLP-1(7-36) peptide. It is a GLP-1 derivative in which alanine is methylated and the last amino acid is amidated.

As used herein, the term "XTEN" is an unstructured low immunogenic peptide containing 6 amino acids added to improve the in vivo half-life of a protein drug developed by Amunix, usually 144 aa. It is composed of multiple amino acids as a unit (US20100239554A1).

As used herein, the term "linker peptide" refers to an unstructured peptide used to prepare a fusion protein by linking two or more proteins or peptides having different biological activities.

As used herein, the term “fusion protein” refers to a protein in which polypeptides or domains having two or more different biological functions are linked directly or by a linker peptide to simultaneously exert two biological functions.

As used herein, the term "dual-specific dimer fusion protein" refers to a protein having a homodimeric structure by intermolecular interaction among the fusion proteins.

According to one aspect of the present invention, (a) GLP-1 or an analog thereof and IL-10 protein; (b) a fusion protein in which GLP-1 or an analog thereof is linked to an IL-10 protein; Or (c) GLP-1 or an analog thereof is linked to the N-terminus of the antibody Fc region, and a bispecific dimeric fusion protein linked to the C-terminus of the antibody Fc region with IL-10 protein as an active ingredient. A pharmaceutical composition for preventing or treating alcoholic liver disease is provided.

The non-alcoholic liver disease may be a chronic metabolic liver disease, and preferably may be any one or more selected from the group consisting of non-alcoholic fatty liver, non-alcoholic steatohepatitis, non-alcoholic cirrhosis and non-alcoholic liver cancer. it is not

In the composition, the GLP-1 analog is GLP-1, Exendin 3 (Exendin 3), Exendin 4 (Exendin 4), GLP-1/Exendin 4 hybrid peptide, GLP-1-XTEN, Exendin 4-XTEN , Lixisenatide, Albiglutide, Liraglutide, or Taspoglutide.

In the composition, the GLP-1 may include the amino acid sequence of SEQ ID NO: 6 or 7.

In the composition, Exendin 3 may include the amino acid sequence of SEQ ID NO: 8.

In the composition, Exendin 4 may include the amino acid sequence of SEQ ID NO: 9.

In the composition, the GLP-1/Exendin 4 hybrid may include the amino acid sequence of SEQ ID NO: 1.

In the composition, the Lixisenatide may include the amino acid sequence of SEQ ID NO: 10.

In the composition, the Exendin 4-XTEN may include the amino acid sequence of SEQ ID NO: 11.

In the composition, the Albiglutide may include the amino acid sequence of SEQ ID NO: 12.

In the composition, the Liraglutide may include the amino acid sequence of SEQ ID NO: 13.

In the composition, the Taspoglutide may include the amino acid sequence of SEQ ID NO: 14.

In the composition, the fusion protein may include an antibody Fc region between the GLP-1 or GLP-1 analogue and the IL-10 protein, wherein the antibody Fc region is IgG1, IgG2, IgG3, IgG4, or IgD. It may be an Fc region or a hybrid antibody Fc region in which Fc regions of two or more isotypes are mixed.

In the composition, the antibody Fc region may include an amino acid sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 15 to SEQ ID NO: 19.

The composition may include an pharmaceutically acceptable carrier, and may additionally include a pharmaceutically acceptable adjuvant, excipient or diluent in addition to the carrier.

As used herein, the term “pharmaceutically acceptable” refers to a composition that is physiologically acceptable and does not normally cause gastrointestinal disorders, allergic reactions such as dizziness or similar reactions when administered to humans. Examples of such carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In addition, fillers, anti-agglomeration agents, lubricants, wetting agents, fragrances, emulsifiers and preservatives may be further included.

In addition, the pharmaceutical composition according to an embodiment of the present invention may be formulated using a method known in the art to enable rapid, sustained or delayed release of the active ingredient when administered to a mammal. Formulations include powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, and sterile powder forms.

The composition according to an embodiment of the present invention may be administered by various routes, for example, oral, parenteral, for example, suppository, transdermal, intravenous, intraperitoneal, intramuscular, intralesional, nasal, intravertebral administration may be administered, and may also be administered using an implantable device for sustained release or continuous or repeated release. The number of administration may be administered once a day or divided into several times within a desired range, and the administration period is not particularly limited.

The composition according to an embodiment of the present invention may be formulated in a suitable form together with a commonly used pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include, for example, carriers for parenteral administration such as water, suitable oils, saline, aqueous glucose and glycol, and may further include stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite or ascorbic acid. Suitable preservatives are benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. In addition, the composition according to the present invention can be used as a suspending agent, solubilizing agent, stabilizer, isotonic agent, preservative, adsorption inhibitor, surfactant, diluent, excipient, pH adjuster, analgesic agent, buffer, Antioxidants and the like may be included as appropriate. Pharmaceutically acceptable carriers and agents suitable for the present invention, including those exemplified above, are described in detail in Remington's Pharmaceutical Sciences, latest edition.

The dosage of the composition to a patient will depend on many factors, including the patient's height, body surface area, age, the particular compound being administered, sex, time and route of administration, general health, and other drugs being administered concurrently. The pharmaceutically active protein may be administered in an amount of 100 ng/body weight (kg) - 10 mg/body weight (kg), more preferably 1 to 500 μg/kg (body weight), and most Preferably, it may be administered at 5 to 50 μg/kg (body weight), and the dosage may be adjusted in consideration of the above factors.

According to another aspect of the present invention, a therapeutically effective amount of (a) GLP-1 or an analog thereof and IL-10 protein; (b) a fusion protein in which GLP-1 or an analog thereof is linked to an IL-10 protein; Or (c) GLP-1 or an analog thereof is linked to the N-terminus of the Fc region, and the C-terminus of the Fc region is linked to a bispecific dimer fusion protein comprising the step of administering to the subject a dimeric fusion protein , to provide a method for treating non-alcoholic liver disease.

The non-alcoholic liver disease may be a chronic metabolic liver disease, and preferably may be any one or more selected from the group consisting of non-alcoholic fatty liver, non-alcoholic steatohepatitis, non-alcoholic cirrhosis and non-alcoholic liver cancer. it is not

As used herein, the term "therapeutically effective amount" refers to an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level depends on the type and severity of the subject. , age, sex, drug activity, sensitivity to drug, administration time, administration route and excretion rate, duration of treatment, factors including concomitant drugs, and other factors well known in the medical field. The therapeutically effective amount of the composition of the present invention may be 0.1 mg/kg to 1 g/kg, more preferably 1 mg/kg to 500 mg/kg, but the effective dosage may vary depending on the age, sex and condition of the patient. can be appropriately adjusted.

Hereinafter, the present invention will be described in more detail through examples. These examples are for explaining the present invention in more detail, and the scope of the present invention is not limited to these examples.

Example 1. Research materials and methods

1.1. laboratory animal

C57BL6/J mice [20-25 g, 5 weeks old (CD-HFD) or 9 weeks old (CDA-HFD), male) were purchased through the central laboratory animal, and the animals were supplied with food and drinking water ad libitum, temperature and light They were acclimatized 7 days before the start of the experiment in a controlled aviary (23±1°C, 12:12h light/dark cycle with lights turned on at 08:00).

1.2. Diet-induced nonalcoholic steatohepatitis animal model and drug dosing

① CD-HFD-induced NASH model : 6-week-old mice were fed a CD-HFD (choline-deficient high fat diet, 45% kcal fat) diet for 17 weeks. Fructose (23.1 g/L) + Glucose (18.9 g/L) drinking water was supplied for about 4.5 weeks from 5.5 to 10 weeks. After 13 weeks of CD-HFD diet, mice were randomly divided into 4 groups [CD-HFD, GLP-1, GLP-1+IL-10 (Combo), GLP-1-hyFc-IL-10V (MD100)], In the Chow group and CD-HFD group, saline, GLP-1 (5 nmol/kg), Combo (5+5 nmol/kg), and MD100 (5 nmol/kg) were administered subcutaneously every 3 days for 4 weeks. measured.

② CDA-HFD-induced NASH model : 10-week-old mice were fed a CDA-HFD (choline-deficient, L-amino acid-restricted high fat diet, 60 kcal% fat) diet for 8 weeks. After 5 weeks of CDA-HFD diet, mice were randomly divided into 7 groups (CDA-HFD, GLP-1, IL-10, Combo, 10 nmol MD100, 20 nmol MD100, 40 nmol MD100). -HFD (hyFc M1, backbone), GLP-1 (10 nmol/kg), IL-10 (10 nmol/kg), Combo (10+10 nmol/kg), MD100 (10, 20, 40 nmol/kg) was administered subcutaneously every 2 days for 3 weeks, and body weight was measured weekly.

1.3. Abdominal glucose tolerance test (IPGTT) and insulin resistance test (ITT)

① IPGTT : Mice were fasted for 6 hours, and 2 g/kg D-glucose was administered intraperitoneally. Using a blood glucose meter, blood glucose levels were measured at intervals of 10, 20, 30, 45, 60, 90, and 120 minutes.

② ITT: Mice were fasted for 4 hours, and 0.5 U/kg insulin was administered intraperitoneally. Using a blood glucose meter, blood glucose levels were measured at intervals of 10, 20, 30, 45, 60, 90, and 120 minutes.

1.4. blood chemistry analysis

Serum was separated from blood collected from mice using a centrifuge. The content of triglyceride (TG), total cholesterol (TCHO), total bilirubin (TBIL), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) in 10 μl of serum was measured using an automated clinical chemistry analyzer (FUJI). DRI-CHEM 4000i, Japan) was used.

1.5. histological analysis

① Hematoxylin and eosin (H&E) staining : After fixing mouse liver tissue in 10% formalin for 24 hours, it was embedded in paraffin to make a paraffin block. Tissues were cut to a thickness of 5 μm and stained with H&E.

② Sirius Red Staining : Sirius Red staining was performed using the Picrosirius Red Stain Kit (#24901A-250, #24901B-250, Polysciences Inc., Warrington), and the method was performed according to the manufacturer's instructions.

1.6. liver triglycerides

After homogenizing 30 mg of liver tissue, the liver triglyceride content was measured using the EnzyChrom Triglyceride Assay Kit (#ETGA-200, Bioassay), and the specific method was performed according to the manufacturer's instructions.

1.7. Hydroxyproline analysis

After homogenizing 10 mg of liver tissue, the hydroxyproline content was measured using a Hydroxyproline Colorimetric Assay Kit (#K555, Biovision), and the specific method was performed according to the manufacturer's instructions.

1.8. cell culture

① AML12 cells were stored in DMEM containing 10% fetal bovine serum (FBS, Hyclone, HS3243.01), 1% Insulin-Transferrin-Selenium-A (ITS, Gibco, 51300-044), and 1% penicillin-streptomycin at temperature 37 ℃, 5% CO ₂ Incubated in the supplied incubator.

② 3T3-L1 cells were cultured in DMEM containing 10% calf serum and 1% penicillin-streptomycin at a temperature of 37° C. and 5% CO ₂ supplied in an incubator.

1.9 3T3-L1 Cell Differentiation

6x10 ⁴ 3T3-L1 preadipocytes were inoculated in a 60 mm culture dish. The culture medium was replaced every 2 days, and on the day after 2 days after saturation, DMEM medium containing 10% FBS, 520 μM IBMX, 1 μg/ml insulin, and 1 μM dexamethasone was replaced. And once every 2 days, the culture medium was replaced in the following order. 1) DMEM medium containing 10% FBS and 1 μg/ml insulin, 2) DMEM medium containing only 10% FBS, and 3) DMEM medium containing only 10% FBS, and cultured for 2 more days.

1.10. Palmitic acid (PA) and oleic acid (OA) treatment

① PA treatment: 160 mM palmitic acid (Sigma Aldrich, P0500) was mixed to 500 μM in DMEM culture medium containing 1% BSA.

② OA treatment method: 1 M oleic acid (Sigma Aldrich, O1008) was mixed with DMEM culture medium containing 1% BSA to 200 μM.

1.11. Molecular and cell biological analysis

① Immunoblot analysis: Prepared cells were treated with 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride (PMSF; Sigma Aldrich, 10837091001), 10% glycerol, 0.1% Triton X-100 ( Sigma Aldrich, T8787), 1 mM ethylenediaminetetraacetate (EDTA), 0.5% ^IGEPAL® CA-630 (Sigma Aldrich, 56741), 10 mM β-phosphoglyceride, 1 mM Na ₃ V0 ₄ , 5 mM NaF, 1 mg/ml of aprotinin (Sigma Aldrich, 10236624001) and leupeptin (Sigma Aldrich, L8511). was used to dissolve it. The prepared lysate was electrophoresed using an SDS-PAGE gel to separate the proteins by size, and then the proteins were transcribed into a polyvinylidene difluoride membrane (PVDF; Merk Millipore, L-IPVH 00010). Then, in order to examine the expression of the protein, the primary antibody was reacted overnight at 4 ℃. Then, the HRP-conjugated secondary antibody was reacted at room temperature for 1 hour. Then, the expression of the protein was confirmed using a chemiluminescence kit (Thermo Fisher Scientific, 34580). The antibodies used were as follows. anti-β-actin antibody (Santa Cruz Biotechnology, sc-47778); Anti-cleavable caspase-3 antibody (Cell Signaling Technology, 9661S), anti-cleavable PARP antibody (Cell Signaling Technology, 9544S), anti-p-NF-kB antibody (Cell Signaling Technology, 3033S), anti-NF -kB antibody (Cell Signaling Technology, 8242S), anti-PPARγ antibody (Cell Signaling Technology, 2430S).

② Quantitative RT-PCR analysis method

RNA was extracted by treating the prepared cells with Ribonuclease Inhibitor (Sigma Aldrich, R1158) ^{with TRIzol ㄾ reagent (MRC, TR 118).} For reverse transcription of the extracted RNA (1 μg), cDNA was synthesized using a random hexamer primer and TAKARA cDNA synthesis kit (TaKaRa, RR036A). The synthesized cDNA was ^{subjected to quantitative PCR analysis using SYBR ㄾ} Green (ABI, 4367659) and mouse gene-specific primers listed in Table 1 below.

gene	Primer direction	Nucleic Acid Sequence (SEQ ID NO:)
TNF-α	forward	5'-GCCACCACGCTCTTCTG-3' (20)
	Reverse	5'-GGTGTGGGTGAGGAGCA-3' (21)
IL-6	forward	5'-ACAACCACGGCCTTCCCTAC-3' (22)
	Reverse	5'-CACGATTTCCCAGAGAACATGTG-3' (23)
IL-1β	forward	5'-AACCTGCTGGTGTGTGACGTTC-3' (24)
	Reverse	5'-CAGCACGAGGCTTTTTTGTTGT-3' (25)
PPARγ	forward	5'-CTCTCAGCTGTTCGCCAA-3' (26)
	Reverse	5'-CACGTGCTCTGTGACGATCT-3' (27)
CEBPα	forward	5'-CTGGCTCTGGGTCTGGAA-3' (28)
	Reverse	5'-AGCCACAGGGGTGTGTGT-3' (29)
aP2	forward	5'-CCGCAGACGACAGGAAGG-3' (30)
	Reverse	5'-AGGGCCCCGCCATCT-3' (31)
18S	forward	5'-CGCTCCCAAGATCCAACTAC-3' (32)
	Reverse	5'-CTGAGAAACGGCTACCACATC-3' (33)

1.12. BODIPY staining

The prepared cells were fixed in 4% paraformaldehyde at room temperature for 15 minutes, and then stained with BODIPY staining reagent (1 μg/ml, Thermo Fisher Scientific, USA) for 20 minutes at room temperature, and then fat was observed with a confocal microscope.

1.13. Oil red O dyeing

A working solution of Oil red O (ORO) was prepared by mixing 0.5% ORO dissolved in isopropanol and deionized water in a ratio of 3:2. The prepared ORO working solution was fixed with 4% formaldehyde for 15 minutes, 1 ml per culture dish (60 mm standard) was added to the prepared cells, and then reacted at room temperature for 2 hours. After 2 hours, they were washed with deionized water and observed under a microscope. Then, after adding 1 ml of isopropanol and reacting for 5 minutes, absorbance was measured at 500 nm to quantitatively measure the degree of accumulation of triglyceride (TG).

Example 2. Design of a dual specificity fusion protein

We devised a gene construct encoding GLP-1-hyFc-IL-10V protein in which GLP-1 or GLP-1 analogues are linked by an IL-10 variant (IL-10V) by a hyFc region.

Specifically, the GLP-1 / Exendin 4 hybrid having the amino acid sequence shown in SEQ ID NO: 1, the hyFc region shown in SEQ ID NO: 2, and IL-10V shown in SEQ ID NO: 3 amino acid sequences shown in SEQ ID NOs: 4 and 5, respectively After designing a bispecific fusion protein to be linked by a linker peptide having And after subcloning into IL-10V sub-vector and backbone vector (pBispecific vector, Genexine, Inc.), it was reacted in one tube to prepare a final vector construct. In order to confirm the combined effect, GLP-1-hyFc and hyFc-IL-10V were separately designed and used. Transient expression of the vector constructs prepared as described above was performed using Thermo Fisher's ExpiCHO kit. Specifically, the ExpiCHO-S cell was mixed with the vector construct prepared as above and the ExpiFectamine reagent included in the kit, and then _{cultured for 1 day in an incubator with 8% CO 2} and 37°C conditions, and then the temperature was set to 32°C. It was lowered and cultured until the 7th day.

The supernatant obtained through the above culture was used as a 4X LDS sample for a fusion protein purified through a Protein A column and a secondary column (referred to as 'GLP-1-hyFc', 'hyFc-IL-10V', and 'MD100', respectively). It was diluted appropriately with buffer and water for injection to prepare a final 3-10 µg/20 µL. In the case of reducing condition samples, each material to be analyzed, 4X LDS sample buffer, 10X reducing agent, and water for injection were appropriately diluted to make a final 3-10 μg/20 μL, and heated in a heating block at 70° C. for 10 minutes. 20 μL of the prepared sample was loaded into each well of the gel fixed in the pre-installed electrophoresis equipment. For size markers, 3-5 μL/well were loaded. After setting the power supply to 120 V, 90 minutes, electrophoresis was performed. After the electrophoresis was completed, the gel was separated and stained using a staining solution and a de-staining solution, and the results were analyzed. As a result, as shown in FIG. 1 , it was confirmed that the dual specificity fusion protein according to an embodiment of the present invention was normally expressed. Thereafter, the gene construct encoding the obtained bispecific fusion protein was inserted into the pAD15 expression vector (WO2015/009052A), and then transfected into CHO DG44 (from Dr. Chasin, Columbia University, USA) cells, A cell line stably expressing the dual specificity fusion protein was constructed.

Example 3. In vitro GLP-1 activity assay

In order to confirm whether the fusion protein purified in Example 2 normally exhibits GLP-1 activity, the present inventors analyzed the GLP-1 in vitro activity of the dual specificity fusion protein prepared in Example 2 with cAMP. was investigated by analysis. Specifically, in order to evaluate the degree of cAMP induction by GLP-1 specific reaction, cAMP Hunter ^TM eXpress GLP1R CHO-K1 GPCR Assay kit (DiscoverX, Cat# 95-0062E2CP2M) was used. cAMP Hunter ^TM eXpress Assay cells were inoculated into 96-well plates and cultured for 16 hours, followed by MD100 and GLP-1-hyFc and liraglutide at a concentration of 0.1 to 10 nM and incubated for 30 minutes. _{The biological activity (EC 50} ) of the drug was evaluated by measuring the luminescent signal 1 hour after adding the anti-cAMP antibody and the cAMP detection reagent.

As a result, as confirmed in FIG. 2 and Table 2 (GLP-1 activity analysis of the dual specificity fusion protein), the MD100 prepared according to an embodiment of the present invention is GLP almost equivalent to Liraglutide, a commercially available GLP-1 analogue. -1 activity. The same results were also found in GLP-1-hyFc, which is a result of proving that GLP-1 included in the fusion protein according to an embodiment of the present invention functions normally.

		GLP-1-hyFc (GX-G6)	Liraglutide	MD100 (GLP-1-hyFc-IL-10V)
Primary	EC 50 (nM) (relative activity versus GLP-1-hyFc)	0.257 (100%)	0.230 (112%)	0.268 (95.9%)
	R 2	0.998	0.997	0.998
Secondary	EC 50 (nM) (relative activity versus GLP-1-hyFc)	0.303 (100%)	0.250 (121%)	0.314 (96.5%)
	R 2	0.993	0.939	0.995
Average EC 50 (nM)		0.280±0.03	0.240±0.01	0.291±0.03
relative activity		100%	116%	96.2%

Example 4. In Vitro IL-10 Activity Assay

The present inventors then analyzed IL-10 activity under in vitro conditions to investigate whether the fusion protein prepared according to an embodiment of the present invention can properly function as an IL-10 protein.

Specifically, using mouse bone marrow-derived mast cells (BMMC), dinitrophenyl (DNp)-BSA-specific antibody IgE (anti-DNP IgE) 1 μg/ml was administered at various concentrations (0, 10, 100 and 1000). pM) to induce TNF-α secretion by treatment with the fusion protein (MD100) according to an embodiment of the present invention, and 1 μg of the antigen (DNp-BSA) (Sigma #A6681, USA) after 1 day has elapsed After 1 day of treatment with /ml, TNF-α secretion inhibitory ability was analyzed by ELISA using an anti-TNF-α antibody (abchem, USA) in cell culture medium. As a control, commercially available rhIL-10 (Peprotech, USA) and hyFc-IL-10V prepared in Example 1 were used. As a result, as confirmed in FIG. 3 , the fusion protein according to an embodiment of the present invention exhibited an ability to inhibit TNF-α secretion equivalent to that of commercially available rhIL-10 or hyFc-IL-10V, and at a low concentration, it was relatively It showed a better TNF-α secretion inhibitory ability. This is to prove that the fusion protein according to an embodiment of the present invention exerts the normal IL-10 function.

Example 5. IL-10R receptor binding affinity evaluation

The present inventors used the Octet Assay System (Octet K2, ForteBio, USA) to confirm the binding affinity to the IL-10 receptor for the dual specificity fusion protein (MD100) according to an embodiment of the present invention - a bio-layer interference method ( Binding kinetics were measured using Bio-layer Interferometry (BLI). To this end, specifically, the IL-10 receptor is immobilized on an amine-responsive second-generation (AR2G) biosensor (Fortebio, USA) and loaded into the Octet Assay System device, and a fusion protein (MD100) according to an embodiment of the present invention The association constant (K _a ) and dissociation rate constant (K _d ) were calculated to calculate the association constant (K _D ), and a commercially available recombinant human IL-10 (rhIL-10; Peprotech, USA) was used as a control. did.

As a result, as confirmed in FIGS. 4 and 3 (analysis of binding affinity of the fusion protein with the IL-10 receptor), the binding rate constant (Ka) of the fusion protein according to an embodiment of the present invention is that of recombinant human IL-10. On the other hand, the dissociation rate constant (Kd) was higher than that of recombinant human IL-10. _{However, when viewed as a whole, the binding constant (K D} ) of the fusion protein according to an embodiment of the present invention to the IL-10 receptor was confirmed to be almost equivalent to that of recombinant human IL-10.

sample	rhIL-10	MD100
Mean K D (nM)	46.5	61.35
Average K a (1/Ms)	56500±1050	12850±495
Average K d (1/s)	0.0026±0.0002	0.0008±0.0001
average R 2	0.95	0.98

Example 6. Pharmacokinetics analysis

The present inventors performed pharmacokinetic analysis to investigate whether the fusion protein according to an embodiment of the present invention stays for a long time in vivo.

Specifically, the fusion protein (MD100) according to an embodiment of the present invention was subcutaneously administered to wild-type rats at doses of 0.5 and 1.5 mg/kg, respectively (n=3). And before administration and 1, 5, 10, 24, 48, 72, 120, and 168 hours after administration, the concentration of MD100 in the serum was measured by ELISA analysis using a hyFc-specific antibody, and then using Winnolin S/W to calculate the PK parameters. As a result, as confirmed in Figure 5 and Table 4 (pharmacokinetic profile of fusion protein), C _max is 2530.3 ~ 5533.3 ng / mL, half-life (t _1/2 ) is sufficient in vivo exposure to 21 ~ 98 hr and durability were confirmed.

Dosage	C max (ng/mL)	T max (hr)	C last (ng/mL)	t 1/2 (hr)	AUC last (ng×hr/mL)
0.5 mg/kg	2530.27 ± 208.62	48.00 ± 0.00	445.03 ± 10.47	21.3 ± 4.03	408186.33 ± 14791.41
1.5 mg/kg	5533.29 ± 901.50	56.00 ± 13.86	1066.11 ± 130.01	98.4 ± 18.6	1001119.24 ± 75675.59

Example 7. Single Toxicity Assay

In order to analyze the single-time toxicity of the fusion protein according to an embodiment of the present invention, the present inventors subcutaneously administer the fusion protein (MD100) according to an embodiment of the present invention at various doses to wild-type rats once, thereby maximally tolerated administration Amount and serological changes and morphological changes of target organs were investigated.

As a result of the test, as shown in Table 5 (one-time toxicity evaluation experimental group setting), no drug administration-related changes were observed in clinical symptoms, visual observation, and serological examination. In the case of body weight, most showed a decrease compared to before drug administration on the 2nd day after administration, and this was accompanied by a decrease in feed intake. This is a pharmacological action of GLP-1 contained in the fusion protein according to an embodiment of the present invention, which is determined to be weight loss due to decreased appetite, and has no significant toxicological significance.

As a result of organ weight analysis, an increase in the weight of the spleen and a decrease in the weight of the thymus were confirmed in the male high-dose group compared to the control group, which is consistent with the results observed at doses of 2 mg/kg or more in the known toxicity test of IL-10. . In the case of spleen enlargement, it is considered that the pharmacological action is due to an immunological response due to a heterologous cytokine such as human recombinant IL-10. In the case of the thymus, as a histopathological change reported, cortical apoptosis occurred, and this change was observed in the repeated administration of 8 mg/kg for 2 weeks, but was not observed in the long-term study of 3 months or longer. In addition, it was judged to be a temporary phenomenon, judging from the fact that it was not related to the number of lymphocytes and did not affect the function and was also found in the control group. In conclusion, in the fusion protein (MD100) according to an embodiment of the present invention, no toxicological effect was observed up to the highest dose of 50 mg/kg in a test in which 0.5, 5, and 50 mg/kg were administered as a single dose, so maximum tolerated administration The dose is judged to be 50 mg/kg or more.

experimental group	matter	Concentration (mg/kg)	route of administration	Dosage	Number of experimental heads (male/female)
One	Control (Formulation Buffer)	0	subcutaneous (s.c)	5 ml/kg	6/6
2	MD100	0.5			6/6
3	MD100	5			6/6
4	MD100	50			6/6

Example 8. Effects of GLP-1+IL-10 (Combo) and a dual specificity fusion protein according to an embodiment of the present invention on body weight, fat and glucose homeostasis in a CD-HFD-induced NASH mouse model

In order to examine the effect of GLP-1+IL-10 (Combo) and the fusion protein (MD100) according to an embodiment of the present invention on energy metabolism in a CD-HFD-induced NASH mouse model, the present inventors conducted CD for a total of 17 weeks. -HFD was fed and GLP-1, Combo, or MD100 was subcutaneously administered to mice for 4 weeks from 13 to 17 weeks. All of the GLP-1, Combo, and MD100 groups significantly decreased body weight during 4 weeks of treatment compared to the CD-HFD group (FIG. 6A), and after 2 weeks of treatment, a decrease in body weight was observed in the Combo and MD100 groups (FIG. 6B). . Blood triglyceride (TG) was statistically decreased in the Combo group compared with the CD-HFD group ( FIG. 6C ). However, the total cholesterol (TCHO) in the blood did not show a significant decrease in the Combo group compared to the CD-HFD group (FIG. 6D), and the total bilirubin (TBIL) level showed a tendency to increase (FIG. 6E). . In addition, to investigate the roles of Combo and MD100 on glucose homeostasis, the present inventors performed insulin resistance and glucose tolerance experiments. As a result, compared to the CD-HFD group, it was possible to improve insulin resistance and glucose load in the Combo-administered group and the MD100-administered group ( FIGS. 6F and 6G ). In addition, it was confirmed that the blood glucose of the mice after fasting was improved in the GLP-1 administration group and the Combo administration group (FIG. 6H). Taken together, these data suggest that Combo and MD100 have potential therapeutic effects on weight and glycemic control.

Example 9. Analysis of the effect of Combo and fusion protein according to an embodiment of the present invention on hepatic steatosis and inflammation in a CD-HFD-induced NASH mouse model

The present inventors investigated the effect of the combined administration of GLP-1 and IL-10 (Combo) and the fusion protein (MD100) according to an embodiment of the present invention on lipid accumulation in the liver in a CD-HFD-induced NASH mouse model For this purpose, the fat content in liver tissue was investigated. As a result, as shown in FIG. 7 , the liver of the CD-HFD group was larger than that of the Chow group (negative control group fed normal Chow feed), and the color was white due to fat, but in the CD-HFD group, In comparison, the size of the liver was reduced in the GLP-1, Combo, and MD100 administration groups, and the color was also observed to be deep red. As a result of performing H&E staining, it was confirmed that a decrease in fat droplets was observed in the GLP-1 administration group, the Combo administration group, and the MD100 administration group compared with the CD-HFD model ( FIG. 7A ). In addition, liver weight (FIG. 7B) and liver to body weight ratio (FIG. 7C) were also significantly decreased in the GLP-1 administration group, Combo administration group, and MD100 administration group compared to the CD-HFD group. Hepatic TG content was also decreased in the GLP-1 administration group, Combo administration group, and MD100 administration group ( FIG. 7D ). In the case of serum ALT level, an indicator of liver inflammation, the GLP-1 administration group did not change compared to the CD-HFD group, but the Combo administration group and the MD100 administration group significantly decreased ( FIG. 7E ). On the other hand, there was no difference in AST levels between groups (FIG. 7F). In addition, the present inventors performed a qRT-PCR experiment using mouse liver tissue to examine the anti-inflammatory effects of the GLP-1 administration group and the Combo administration group in a CD-HFD-induced NASH mouse model. As a result, no significant expression inhibitory effect of the inflammatory target gene was observed in the Combo-administered group and the MD100-administered group ( FIGS. 7G-7I ). Based on the above results, it was found that the Combo-administered group and the MD100-administered group were effective in reducing lipid accumulation in the liver tissue of CD-HFD-induced NASH mice.

Example 10. Effect of combined administration (Combo) of GLP-1 and IL-10 on hepatic steatosis and inflammation in a CDA-HFD-induced NASH mouse model

The present inventors investigated the fat content in liver tissue to examine the effect of the combined administration of GLP-1 and IL-10 (Combo) on TG and fibrosis in the liver in a CDA-HFD-induced NASH mouse model. Compared to the Chow group, the liver of the CDA-HFD group was larger and the color was white due to fat. Compared to the CDA-HFD group, the liver size of the GLP-1 administration group and the Combo administration group was smaller, and the color of the liver was observed. (Fig. 8A, top). Corresponding to the above results, as a result of performing H&E staining, it was observed that fat droplets were reduced in both the GLP-1 administration group and the Combo administration group (Fig. 8A, bottom). Liver weight (Fig. 8B) and liver-to-body weight ratio (Fig. 8C) were significantly decreased in the GLP-1 administration group and Combo administration group compared to the CDA-HFD group. The TG content of the liver was significantly increased in the CDA-HFD group compared to the Chow group, but there was no improvement effect in the GLP-1 administration group and the Combo administration group ( FIG. 8D ). Serum ALT level, which is an indicator of liver inflammation, was significantly decreased in the GLP-1 administration group and the Combo administration group compared to the CDA-HFD group ( FIG. 8E ). The AST level showed an improvement effect only in the Combo administration group (FIG. 8F). In order to investigate the anti-inflammatory effect of the Combo-administered group in the CDA-HFD-induced NASH mouse model, a qRT-PCR experiment using mouse liver tissue was performed. It was observed that there is an expression inhibitory effect (FIGS. 8G-8I). These data suggest that the Combo-administered group is effective in reducing hepatic steatosis and inflammation in the NASH model induced by CDA-HFD.

Example 11. Effect of MD100 on Metabolic Profile in CDA-HFD Induced NASH Mouse Model

In order to investigate the change in the metabolic profile according to the dose of MD100 in the CDA-HFD-induced NASH mouse model, the present inventors fed CDA-HFD for a total of 8 weeks, and from 5 weeks to 8 weeks, various concentrations of the present invention for 3 weeks. The fusion protein according to an embodiment (10, 20, 40 nmol/kg, MD100) was subcutaneously administered to mice. As a result, in all MD100-administered groups, 1 and 2 weeks were significantly reduced compared to the CDA-HFD group, but after 3 weeks of administration, 20 nmol MD100 regained weight similarly to the CDA-HFD group ( FIGS. 9A and 9B ). When comparing before and after drug administration, no difference in blood glucose level was observed in the Chow, CDA-HFD, and 20 nmol MD100 administration groups, but the 10 nmol MD100 administration group increased blood glucose levels after administration, and the 40 nmol MD100 administration group showed blood glucose levels after administration. was decreased (Fig. 9C). However, compared with the CDA-HFD group, blood glucose levels were decreased in all experimental groups after drug administration ( FIG. 9D ).

Example 12. Effect of MD100 on hepatic steatosis and inflammation in a mouse model of CDA-HFD induced NASH

To investigate the dose-dependent effect of MD100 on lipid accumulation and fibrosis in the liver in a CDA-HFD-induced NASH mouse model, the present inventors investigated the fat content in liver tissue. Compared to the Chow group, the liver of the CDA-HFD group was larger and had a white color due to fat, but compared to the CDA-HFD group, the liver size and color slightly darkened in the 40 nmol MD100 administration group were observed (Fig. 10A, top). However, as a result of H&E staining, fat droplets were not reduced in the groups administered with 10, 20 and 40 nmol MD100 (Fig. 10A, bottom). The liver weight was significantly decreased in both the 10 nmol administration group and the 40 nmol MD100 administration group compared with the CDA-HFD group ( FIG. 10B ), but the liver to body weight ratio increased in all groups compared to the Chow group ( FIG. 10C ). The TG content of the liver was significantly increased in the CDA-HFD group compared with the Chow group, but no improvement effect was observed in the 10 nmol and 40 nmol MD100 administration groups, but rather increased in the 20 nmol MD100 administration group ( FIG. 10D ). Serum ALT level, an indicator of liver inflammation, was significantly decreased in the 40 nmol MD100 administration group compared to the CD-HFD group (FIG. 10E), and the AST level was also improved in the 40 nmol MD100 administration group (FIG. 10F). In order to investigate the anti-inflammatory effect according to the dose of MD100 in the CDA-HFD-induced NASH mouse model, a qRT-PCR experiment using mouse liver tissue was performed. could be observed ( FIGS. 10G-10I ). These data suggest that MD100 is effective in reducing hepatic steatosis and inflammation in a model of NASH induced by CDA-HFD.

Example 13. Effect of MD100 on Liver Fibrosis in CDA-HFD Induced NASH Mouse Model

In order to examine whether MD100 exhibits an effect on improving liver fibrosis in a CDA-HFD-induced NASH mouse model, the present inventors performed Sirius Red staining and hydroxyproline analysis. The CDA-HFD, 10 nmol, and 20 nmol MD100 groups were observed to increase the amount of collagen compared to the Chow group, and it was confirmed that liver fibrosis was improved in the 40 nmol MD100 group ( FIG. 11A ). On the other hand, when quantitative analysis was performed through hydroxyproline analysis, the CDA-HFD group significantly increased the amount of collagen compared to the Chow group, but no inhibitory effect was observed in the 10, 20, and 40 nmol MD100 administration groups (FIG. 11B) .

Example 14. Effect of Combo and MD100 of GLP-1 and IL-10 in an in vitro lipotoxicity model using palmitic acid (PA)

Palmitic acid (PA) is known as the most abundant saturated fatty acid in human plasma, and promotes the production of free radicals to induce cell death. The induction of apoptosis by palmitic acid is referred to as “lipotoxicity”, which is known to be a major cause of increasing the pathogenesis of nonalcoholic steatohepatitis. Therefore, the present inventors measured cell viability after treatment with palmitic acid in cells to determine whether GLP-1 based candidates can inhibit lipotoxicity using an in vitro lipotoxicity model, while immunoblot analysis was performed. As a result, it was confirmed that the cell viability increased the most in the GLP-1 and IL-10 combination treatment group compared to the PA alone treatment group ( FIG. 12A ). In addition, consistent with the above results, it was confirmed that the expression of cleaved Caspase-3 and PARP, known as apoptosis markers, also decreased the most in the combined treatment group of GLP-1 and IL-10 ( FIG. 12B ). .

Example 15. Effect of Combo and MD100 in In Vitro Inflammation Model Using Bone Marrow-Derived Macrophages (BMDM)

It is known that nonalcoholic steatohepatitis is accompanied by inflammation, and the disease is accelerated by the continued inflammation. Therefore, the present inventors used an in vitro inflammation model in which BMDM was treated with LPS to induce inflammation. -The inflammatory effect was investigated through qPCR experiments and immunoblot analysis. As a result, it was confirmed that NF-kB signaling, a pro-inflammatory pathway, was inhibited in the combination treatment group (Combo) and MD100 treatment group of GLP-1 and IL-10 ( FIG. 13A ). Corresponding to these results, - It was confirmed that the expression of inflammatory target genes TNF-α (FIG. 13B), IL-6 (FIG. 13C) and IL-1β (FIG. 13D) is also reduced.

Example 16. Effect of Combo and MD100 in an in vitro hepatic steatosis model using oleic acid (OA)

The most characteristic feature of nonalcoholic fatty liver disease is the accumulation of lipids in the normal liver. Therefore, the present inventors induced lipid accumulation to investigate the lipid accumulation inhibitory effect of GLP-1-based candidates under in vitro conditions. A significant decrease was observed in the combination treatment group of -1 and IL-10, and the MD100 treatment group ( FIGS. 14A and 14B ).

Example 17. Identification of the effects of Combo and MD100 through in vitro lipogenesis model construction

It is known that metabolic syndrome such as obesity or insulin resistance is often accompanied when normal liver progresses to nonalcoholic fatty liver. In this process, more fatty acids are released from adipocytes, and thus excess fatty acids are introduced into the liver to induce fat synthesis in the liver. It is known that it induces excessive fat accumulation in liver cells to form fatty liver. Therefore, using the 3T3-L1 preadipocyte differentiation system, we investigated whether Combo and MD100 can reduce the amount of fatty acids released from adipocytes and flowing into the liver by inhibiting the adipogenesis process, the process in which adipocytes are formed. did. As a result, it was observed that the gene and lipid accumulation related to lipogenesis were most significantly decreased in the MD100 treatment group ( FIGS. 15A-15F ).

As described above, the co-administration of GLP-1 and IL-10 or the dual specificity fusion protein comprising GLP-1 and IL-10 according to an embodiment of the present invention effectively inhibits lipid accumulation in the liver as well as , it has been experimentally proven that it can relieve inflammation accompanying fatty liver and effectively suppress lipotoxicity caused by excessive lipids in the liver. Therefore, the composition for co-administration of GLP-1 and IL-10 and the dual specificity fusion protein according to an embodiment of the present invention are non-alcoholic fatty liver, non-alcoholic steatohepatitis and chronic metabolic liver disease such as liver fibrosis following the course of non-alcoholic fatty liver. It can be used effectively for the treatment of diseases.

Claims (15)

1. (a) GLP-1 or an analog thereof and an IL-10 protein;

   (b) a fusion protein in which GLP-1 or an analog thereof is linked to an IL-10 protein; or

   (c) non-alcoholic comprising as an active ingredient a bispecific dimer fusion protein in which GLP-1 or an analog thereof is linked to the N-terminus of the antibody Fc region and IL-10 protein is linked to the C-terminus of the antibody Fc region A pharmaceutical composition for the treatment of liver disease (nonalcoholic liver disease).
2. The method of claim 1,

   The GLP-1 analog is GLP-1, Exendin 3 (Exendin 3), Exendin 4 (Exendin 4), GLP-1 / Exendin 4 hybrid, GLP-1-EXTEN, Lixisenatide, Albiglutide, Liraglutide, Dulaglutide, Extenatide, Taspoglutide, or Lixisenatide, the composition.
3. 3. The method of claim 2,

   The GLP-1 is a composition comprising the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 7.
4. 3. The method of claim 2,

   The Exendin 3 is a composition comprising the amino acid sequence of SEQ ID NO: 8.
5. 3. The method of claim 2,

   The Exendin 4 is a composition comprising the amino acid sequence of SEQ ID NO: 9.
6. 3. The method of claim 2,

   The GLP-1 / Exendin 4 hybrid is a composition comprising the amino acid sequence of SEQ ID NO: 1.
7. 3. The method of claim 2,

   The Lixisenatide is a composition comprising the amino acid sequence of SEQ ID NO: 10.
8. 3. The method of claim 2,

   The Exendin 4-XTEN is a composition comprising the amino acid sequence of SEQ ID NO: 11.
9. 3. The method of claim 2,

   The Albiglutide is a composition comprising the amino acid sequence of SEQ ID NO: 12.
10. 3. The method of claim 2,

    The Liraglutide is a composition comprising the amino acid sequence of SEQ ID NO: 13.
11. 3. The method of claim 2,

    The Taspoglutide is a composition comprising the amino acid sequence of SEQ ID NO: 14.
12. The method of claim 1,

    The antibody Fc region is an Fc region or a hybrid antibody Fc region of IgG1, IgG2, IgG3, IgG4, or IgD.
13. 13. The method of claim 12,

    The hybrid antibody Fc region is an antibody Fc region in which at least two or more portions of two or more isotypes are mixed.
14. 13. The method of claim 12,

    The antibody Fc region is a composition comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 15 to SEQ ID NO: 19.
15. The method of claim 1,

    The non-alcoholic liver disease is any one or more selected from the group consisting of non-alcoholic fatty liver, non-alcoholic steatohepatitis, non-alcoholic cirrhosis and non-alcoholic liver cancer, the composition.

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