Drugs for treating fatty liver and nonalcoholic steatohepatitis

Abstract

The inventors of the present invention have found that HMGB1-derived peptides can be used as a therapeutic drug and / or a prophylactic drug for fatty liver, nonalcoholic steatohepatitis, and various symptoms associated with fatty liver. On the basis of this finding, the present application provides a usage of HMGB1-derived peptides that differs from previous usages.

Description

[Technical Field]

[0001] The present application relates to a pharmaceutical composition for the prevention and / or treatment of fatty liver and non-alcoholic steatohepatitis. [Background technology]

[0002] Fatty liver disease can be caused by viral infections, alcohol, or other reasons. Fatty liver disease caused by factors other than viruses and alcohol is often caused by diabetes, dyslipidemia, obesity, or metabolic syndrome. Fatty liver disease that is not caused by viruses or alcohol and the resulting liver diseases are called non-alcoholic fatty liver disease (NAFLD). Generally, fatty liver disease is considered non-alcoholic if the patient's alcohol intake is less than 30g / day for men and 20g / day for women.

[0003] NAFLD, when fatty liver disease is accompanied by inflammation, is called nonalcoholic steatohepatitis (NASH). This inflammation can sometimes be accompanied by fibrosis. As this fibrosis progresses, it can lead to cirrhosis. In Japan, 20-30% of adults who undergo health checkups are said to have NAFLD, and 5-20% of these are said to progress to NASH. Currently, there are many drugs for treating NASH, but most of them are drugs that suppress liver inflammation and fibrosis, and there are no effective drugs that are effective against fatty liver itself. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] WO 2012 / 147470 [Non-patent literature]

[0005] [Non-Patent Document 1] Younossi ZM, Koenig AB, Abdelatif D, Fazel Y, Henry L and Wymer M. Global epidemiology of nonalcoholic fatty liver disease-Meta-analytic assessment of prevalence, incidence, and outcomes. Hepatology. 2016;64:73-84. Summary of the Invention [Problem to be solved by the invention]

[0006] The present application aims to provide an agent for treating and / or preventing fatty liver and non-alcoholic steatohepatitis. [Means for solving the problem]

[0007] The present inventors have discovered that peptides derived from the high mobility group box 1 (HMGB1) protein can not only ameliorate liver inflammation and fibrosis, but also ameliorate fatty liver itself.

[0008] That is, the present application provides the following: [1] A pharmaceutical composition for preventing and / or treating fatty liver, comprising any one of the following substances (a) to (c): (a) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1; (b) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 in which 1 to 4 amino acids have been substituted, deleted, inserted, or added; and (c) A peptide consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 1. [2] The pharmaceutical composition according to [1] above, which also improves blood lipid levels. [3] The pharmaceutical composition according to [1] or [2] above, which is administered repeatedly at least once a week for two or more consecutive weeks. [4] The pharmaceutical composition according to any one of [1] to [3] above, wherein the fatty liver is a fatty liver of fatty liver disease. [5] The pharmaceutical composition according to [4] above, wherein the fatty liver disease is one or more selected from the group consisting of non-alcoholic fatty liver disease, alcoholic fatty liver disease, nutritional fatty liver disease, starvation fatty liver disease, obesity fatty liver disease, and diabetic fatty liver disease. [6] A pharmaceutical composition for the prevention and / or treatment of non-alcoholic steatohepatitis, comprising any one of the following substances (a) to (c): (a) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1; (b) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 in which 1 to 4 amino acids have been substituted, deleted, inserted, or added; and (c) A peptide consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 1.

[0009] [7] A method for preventing and / or treating fatty liver, comprising the step of administering to a subject in need thereof a therapeutically effective amount of a substance selected from any one of the following (a) to (c): (a) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1; (b) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 in which 1 to 4 amino acids have been substituted, deleted, inserted, or added; and (c) A peptide consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 1. [8] A substance according to any one of (a) to (c) below for use in the prevention and / or treatment of fatty liver: (a) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1; (b) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 in which 1 to 4 amino acids have been substituted, deleted, inserted, or added; and (c) A peptide consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 1. [9] Use of a substance according to any one of (a) to (c) below in the manufacture of a medicament for the prevention and / or treatment of fatty liver: (a) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1; (b) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 in which 1 to 4 amino acids have been substituted, deleted, inserted, or added; and (c) A peptide consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 1. [Brief explanation of the drawings]

[0010] [Figure 1] FIG. 1 is a diagram illustrating the experimental scheme for creating NASH model mice and administering HMGB1 fragment peptides to them. [Figure 2] FIG. 2 is a graph showing the body weight, liver weight, liver-to-body weight ratio, and body weight change of NASH model mice. [Figure 3] Figure 3 is a graph showing the levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), albumin (Alb), total bilirubin (T-bil), total cholesterol (T-Cho), and triglycerides (TG) in the serum of NASH model mice. [Figure 4] Figure 4 shows micrographs of liver tissue from NASH model mice (all magnifications 100x). From top to bottom, the results are shown for hematoxylin and eosin (HE) staining, F4 / 80 immunohistochemical staining, and Sirius Red staining. [Figure 5]FIG. 5 is a graph showing the quantification of the results of Sirius Red staining, hydroxyproline content, and F4 / 80 immunostaining in liver tissue from NASH model mice. [Figure 6] FIG. 6 is a graph showing lobular inflammation in liver tissue, hepatocyte ballooning, and NAFLD Activity Score (NAS) in NASH model mice. DETAILED DESCRIPTION OF THE INVENTION

[0011] The present application provides a pharmaceutical composition for preventing and / or treating fatty liver, comprising a substance described in any one of (a) to (c) below (hereinafter, sometimes referred to as the "peptide of the present application"): (a) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1; (b) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 in which 1 to 4 amino acids have been substituted, deleted, inserted, or added; and (c) A peptide consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 1.

[0012] The present application provides a pharmaceutical composition for the prevention and / or treatment of non-alcoholic steatohepatitis, comprising the peptide of the present application.

[0013] The present application provides a method for preventing and / or treating fatty liver, comprising administering a therapeutically effective amount of a peptide of the present application to a subject in need of such prevention and / or treatment. In another embodiment, the present application provides a substance comprising the peptide of the present application for use in the prevention and / or treatment of fatty liver. In yet another embodiment, the present application provides use of a substance comprising the peptide of the present application in the manufacture of a medicament for the prevention and / or treatment of fatty liver.

[0014] The invention of the present application described above is based on experimental results using an animal model in which NASH is induced by feeding a high-fat diet, for example, NASH mice in which NASH is induced by feeding a high-fat diet to melanocortin type 4 receptor knockout mice.

[0015] The "high-fat diet" in this application refers to a diet that preferably contains 20% or more of fat in terms of energy ratio. The energy content of such a diet is preferably 400 kcal / 100 g or more. Examples of fats contained in such high-fat diets include animal fats such as beef tallow, lard, and milk fat, and vegetable oils such as rapeseed oil, corn oil, and soybean oil. The components other than fat in such high-fat diets may be normal dietary components, and may include proteins, carbohydrates, minerals, etc.

[0016] In this application, the term "pharmaceutical composition for prevention and / or treatment" is used interchangeably with, for example, "pharmaceutical composition for prevention and / or treatment" or "pharmaceutical composition for prevention and / or treatment." The term "pharmaceutical composition" is used interchangeably with "medicine," "drug," or "pharmaceutical composition."

[0017] In this application, fatty liver refers to a state in which droplets of neutral fat accumulate in liver cells. These droplets of neutral fat are called lipid droplets, and pathologically, a state in which lipid droplets are formed in 5% or more of liver cells is called fatty liver. Therefore, if the proportion of such lipid droplets decreases, it can be said that fatty liver has improved. Furthermore, the act of reducing the proportion of lipid droplets is included in the treatment of fatty liver in this application.

[0018] Diagnostic criteria for fatty liver are well known, and prevention and treatment of fatty liver can be evaluated based on the well-known diagnostic criteria. For example, as reported in the following document, Controlled Attenuation Parameter (CAP) was measured using Fibroscan (registered trademark). TM ) can be used to measure the degree of fatty liver non-invasively. Newsome PN, Sasso M, Deeks JJ, Paredes A, Boursier J, Chan WK, Yilmaz Y, Czernichow S, Zheng MH, Wong VW, Allison M, Tsochatzis E, Anstee QM, Sheridan DA, Eddowes PJ, Guha IN, Cobbold JF, Paradis V, Bedossa P, Miette V, Fournier-Poizat C, Sandrin L, Harrison SA. FibroScan-AST (FAST) score for the non-invasive identification of patients with non-alcoholic steatohepatitis with significant activity and fibrosis: a prospective derivation and global validation study. Lancet Gastroenterol Hepatol. 2020 Apr;5(4):362-373.

[0019] The fatty liver in the present application includes, for example, fatty liver of fatty liver disease. The fatty liver disease is one or more selected from the group consisting of non-alcoholic fatty liver disease, alcoholic fatty liver disease, nutritional fatty liver disease, starvation fatty liver disease, obesity fatty liver disease, and diabetic fatty liver disease. The pharmaceutical composition of the present invention is effective against such various fatty liver diseases.

[0020] Nonalcoholic steatohepatitis (NASH) is a condition in which inflammation occurs in fatty liver that is not viral or alcoholic. Strictly speaking, any type of steatohepatitis other than viral, alcoholic, autoimmune, drug-induced liver injury, or steatohepatitis caused by genetic diseases is classified as NASH. The inflammation associated with NASH can be accompanied by fibrosis. Fatty liver, inflammation, and fibrosis are the three major symptoms of NASH. In liver tissue with NASH, pathological conditions typically include the deposition of fat in hepatocytes (lipid droplets), balloon-like swelling of hepatocytes (balloon cells), infiltration of inflammatory cells, and fibrosis surrounding the hepatocytes.

[0021] Patients with NASH may have high blood levels of lipids such as cholesterol and triglycerides. Patients with NASH may also experience liver dysfunction, resulting in high blood levels of indicators of liver dysfunction such as bilirubin (Bil). Furthermore, patients with NASH may experience hepatocellular damage, resulting in high blood levels of indicators of hepatocellular damage such as AST and ALT.

[0022] Therefore, the pharmaceutical composition of the present application also preferably improves the blood lipid levels of a subject. The lipid levels referred to here include, for example, total cholesterol levels and triglyceride levels. The improvement of blood lipid levels referred to here means that, upon administration of the pharmaceutical composition of the present application, the total cholesterol level is reduced by preferably 20 mg / dL or more, more preferably 50 mg / dL or more, or the triglyceride level is reduced by preferably 20 mg / dL or more, more preferably 50 mg / dL or more.

[0023] In this application, the term "subject" is used interchangeably with "patient," "individual," or "animal."

[0024] In the present application, the peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 is a peptide consisting of amino acid residues 1-44 of human-derived HMGB1 protein.

[0025] In the present application, examples of HMGB1 proteins include, but are not limited to, proteins comprising the amino acid sequence set forth in SEQ ID NO: 2 and proteins encoded by DNA comprising the base sequence set forth in SEQ ID NO: 3.

[0026] In the pharmaceutical composition of the present application, the following peptides may be used in place of or in addition to the peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1: A) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 in which one or more amino acid residues have been modified (substituted, deleted, inserted or added); B) A peptide consisting of an amino acid sequence in which one or more amino acid residues in the amino acid sequence set forth in SEQ ID NO: 1 have been modified (substituted, deleted, inserted or added), and which is functionally equivalent to the peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1.

[0027] Examples of such peptides include, but are not limited to, the peptides described below as i) to ii), and peptides described below as i) to ii) that are functionally equivalent to the peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1: i) a peptide consisting of the amino acid sequence of SEQ ID NO: 1 in which one or more (for example, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 or 2) amino acids have been substituted, deleted, inserted or added; ii) A peptide consisting of an amino acid sequence having about 80% or more, for example about 85% or more, about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, or about 99% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 1.

[0028] In the present application, peptides functionally equivalent to the peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 include, but are not limited to, peptides having the following activities: · Stimulating activity of mesenchymal stem cell migration; Mobilizing mesenchymal stem cells from bone marrow into the blood; · Anti-inflammatory activity; fibrotic-modifying activity; or ·Tissue regenerating activity.

[0029] The amino acid length of the peptide in the present application may be, for example, in the range of 25 to 35 amino acids, 20 to 40 amino acids, 10 to 50 amino acids, 10 to 70 amino acids, or 10 to 100 amino acids, but is not limited to these.

[0030] A therapeutically effective amount of the peptide of the present application or a pharmaceutical composition containing it (hereinafter, sometimes referred to as the "peptide of the present application, etc.") is administered to a subject for the treatment or prevention of the diseases or symptoms described in this specification.

[0031] As used herein, a therapeutically effective amount refers to an amount sufficient to treat or prevent a disease or condition as described herein. As used herein, treating includes, but is not limited to, reducing, delaying, preventing, ameliorating, remission, curing, and complete recovery. As used herein, preventing includes, but is not limited to, reducing, delaying, preventing, and the like.

[0032] The subject of the present application is not particularly limited, and examples thereof include mammals, birds, fish, etc. Mammals include humans and non-human animals, such as, but not limited to, humans, mice, rats, monkeys, pigs, dogs, rabbits, hamsters, guinea pigs, horses, sheep, and whales.

[0033] There are no limitations on the site of administration of the peptides, etc. of the present application, and the peptides, etc. of the present application can exert their effects regardless of the site of administration, such as the site where a lesion appears or its vicinity, a site different from the site (site other than the site), a site away from the site where a lesion appears, a site distal to the site where a lesion appears, or a site distal to and ectopic to the site where a lesion appears.

[0034] Furthermore, the peptides and the like of the present application can exert their effects even when administered to the liver, tissues other than the liver, tissues distant from the liver, tissues distal to the liver, or tissues distal and ectopic to the liver.

[0035] Methods for administering the peptides and the like of the present application include oral administration and parenteral administration, and parenteral administration methods include, but are not limited to, intravascular administration (intra-arterial administration, intravenous administration, etc.), intramuscular administration, subcutaneous administration, intradermal administration, intraperitoneal administration, nasal administration, pulmonary administration, transdermal administration, etc. Furthermore, the peptides and the like of the present application can be administered systemically or locally (e.g., subcutaneously, intradermally, to the skin surface, the eyeball or palpebral conjunctiva, nasal mucosa, oral and gastrointestinal mucosa, vaginal or intrauterine mucosa, or a lesion site, etc.) by injection, for example, intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, etc.

[0036] The administration schedule of the peptides and the like of the present application is, for example, preferably at least once a week for two or more consecutive weeks, and more preferably at least twice a week for four or more consecutive weeks.

[0037] Furthermore, instead of the peptides of the present application, cells that secrete the peptides of the present application, vectors for gene therapy into which DNA encoding the peptides has been inserted, and pharmaceutical compositions containing these can also be used.

[0038] The peptides of the present application can be obtained as recombinants by incorporating DNA encoding the peptides into an appropriate expression system.

[0039] The peptides in this application can also be artificially synthesized. In the peptide synthesis method in this application, peptides can be chemically synthesized by methods such as peptide liquid phase synthesis and peptide solid phase synthesis. Peptide solid phase synthesis is one of the methods commonly used when chemically synthesizing peptides. Polystyrene polymer gel beads with a diameter of about 0.1 mm, whose surface is modified with amino groups, are used as the solid phase, and the amino acid chain is extended one by one from here by a dehydration reaction. Once the desired peptide sequence is completed, it is cut from the solid phase surface to obtain the desired substance. Solid phase synthesis can be used to synthesize ribosomal peptides, which are difficult to synthesize in bacteria, and to synthesize peptides using D-isomers and stable isotopes ( 2 H, 13 C. 15 It is also possible to introduce unnatural amino acids such as N-substituted amino acids, heavy atom substitutes (e.g., selenoamino acids such as selenomethionine), and modifications of peptide and protein backbones. When synthesizing long peptide chains exceeding 70 to 100 amino acids using the solid-phase method, native chemical ligation can be used to join two peptide chains.

[0040] The peptide of the present application may be in the form of a pharmaceutically acceptable salt of the peptide. Examples of pharmaceutically acceptable salts include, but are not limited to, hydrochloride, acetate, trifluoroacetate, etc. The peptide of the present application may be in the form of a solvate of the peptide or a solvate of a pharmaceutically acceptable salt of the peptide. A solvate refers to a compound in which any number of solvent molecules are coordinated to a solute molecule, and includes, but is not limited to, a hydrate.

[0041] Furthermore, the administration method can be selected appropriately depending on the age and symptoms of the subject. When the peptide of the present application is administered to a human, for example, the dosage can be selected, for example, from a range of 0.5 mg to 25 mg per kg of body weight per administration. Alternatively, the dosage can be selected, for example, from a range of 25 to 2500 mg per patient. When cells secreting the peptide of the present application or a gene therapy vector into which DNA encoding the peptide has been inserted are administered, the amount of the peptide can also be administered so as to fall within the above range. However, the pharmaceutical composition of the present application is not limited to these dosages.

[0042] The pharmaceutical compositions of the present application can be formulated according to conventional methods (e.g., Remington's Pharmaceutical Science, latest edition, Mark Publishing Company, Easton, USA) and may contain pharmaceutically acceptable carriers and additives. Examples of such carriers include, but are not limited to, surfactants, excipients, colorants, flavorings, preservatives, stabilizers, buffers, suspending agents, isotonicity agents, binders, disintegrants, lubricants, flow enhancers, and flavoring agents. Other commonly used carriers can also be used as appropriate. Specific examples of such carriers include light anhydrous silicic acid, lactose, crystalline cellulose, mannitol, starch, carmellose calcium, carmellose sodium, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl acetal diethylaminoacetate, polyvinylpyrrolidone, gelatin, medium-chain fatty acid triglycerides, polyoxyethylene hydrogenated castor oil 60, sucrose, carboxymethylcellulose, corn starch, and inorganic salts.

[0043] All prior art documents cited in this specification are hereby incorporated by reference.

[0044] The present invention is further illustrated by, but not limited to, the following examples. [Example]

[0045] Example 1 Evaluation of the efficacy of HMGB1 fragment peptides in NASH model mice (1) Materials and Methods i) Drug Preparation A peptide consisting of amino acid residues 1-44 (SEQ ID NO: 1) of human HMGB1 protein was chemically synthesized by solid-phase synthesis. Hereinafter, this HMGB1 fragment peptide will be referred to simply as the HMGB1 peptide and will be abbreviated as "HMGB1" in the figures. The control group described below will be abbreviated as "Control" in the figures.

[0046] ii) Creation of NASH model mice Melanocortin receptor type 4 knockout mice (hereinafter also referred to as "MC4R-KO mice"; genetic background: C57BL / 6J) were provided by Joel K Elmquist at the University of Texas Medical Center. Next, NASH was induced in MC4R-KO mice by feeding them a high-fat diet, as detailed in the following literature. Specifically, as shown in Figure 1, MC4R-KO mice were fed a normal diet (ND) until they were 8 weeks old. Then, the diet was switched from the normal diet to a high-fat diet (HFD), and the high-fat diet was continued for 20 weeks. This allowed the mice to develop NASH. Itoh M, Suganami T, Nakagawa N, et al. Melanocortin 4 receptor-deficient mice as a novel mouse model of nonalcoholic steatohepatitis. Am J Pathol 2011;179:2454-63.

[0047] The normal diet used was CE-2 (manufactured by CLEA Japan), and the high-fat diet used was Western Diet (manufactured by EPS Ekishin Co., Ltd., containing 0.21% cholesterol and 41% kcal milk fat).

[0048] iii) Administration of HMGB1 peptide The mice were divided into two groups: an HMGB1 peptide-treated group (HMGB1-treated group) and a control group (saline-treated; NS-treated; CTRL-treated groups). The HMGB1-treated group received HMGB1 peptide at 5 mg / kg twice weekly via tail vein injections for 4 weeks, from 16 to 20 weeks after the start of the high-fat diet. The CTRL-treated group received saline at 5 ml / kg twice weekly via tail vein injections for 4 weeks, from 16 to 20 weeks after the start of the high-fat diet.

[0049] iv) Evaluation of administration effects Twenty weeks after starting the high-fat diet, the mice were euthanized, and arterial blood was collected from the hearts of the mice. Furthermore, the livers of the mice were also collected. The collected blood and livers were then subjected to the following analyses. In the following statistical analyses, significance was determined using the t-test.

[0050] a) Blood analysis The serum concentrations of aspartate aminotransferase (AST), alanine aminotransferase (ALT), albumin (Alb), total bilirubin (T-bil), total cholesterol (T-Cho), and triglycerides (TG) in the collected arterial blood were measured by Oriental Yeast Co., Ltd. Nagahama LSL (Nagahama, Japan).

[0051] b) Liver tissue analysis b-1) HE staining and Sirius Red staining The harvested livers were fixed in 10% formalin and cut into 4-μm-thick sections. These sections were then stained with hematoxylin and eosin (HE) and Sirius Red. The liver tissue sections were stained with HE and examined under a microscope to confirm the presence and proportion of lipid droplets. Ten randomly selected micrographs were taken per mouse, and the area stained with Sirius Red was measured using image analysis software.

[0052] b-2) Immunohistochemical staining The collected livers were fixed in 10% formalin and cut into 4-μm-thick sections. These sections were immunohistochemically stained using anti-F4 / 80 rabbit monoclonal antibody (Abcam, Cambridge, UK). Microscopic images were taken as described above, and the area stained with anti-F4 / 80 antibody was measured using image analysis software.

[0053] b-3) Hydroxyproline assay 20 mg of the collected liver tissue was homogenized, and the amount of hydroxyproline in the sample was measured using QuickZyme Hydroxyproline Assays (QuickZyme Bioscience, Zernikedreef, Netherlands).

[0054] b-4) Measurement of NAS The resulting stained liver tissue was observed under a microscope, and lobule inflammation and hepatocyte ballooning in the liver tissue were evaluated according to the criteria shown in Table 1 below. When hepatocytes are damaged, they swell and balloon. Therefore, a high number of ballooned hepatocytes indicates a high number of damaged hepatocytes.

[0055] [Table 1]

[0056] Furthermore, the NAFLD Activity Score (NAS) was calculated based on the following literature. NAS is an index showing the severity of NASH, with a higher value indicating a more advanced or worsening NASH condition. Kleiner DE, Brunt EM, Van Natta M, Behling C, Contos MJ, Cummings OW, Ferrell LD, Liu YC, Torbenson MS, Unalp-Arida A, Yeh M, McCullough AJ, Sanyal AJ; Nonalcoholic Steatohepatitis Clinical Research Network. Design and validation of a histological scoring system for nonalcoholic fatty liver disease. Hepatology. 2005 Jun;41(6):1313-21.

[0057] (2) Results i) Body weight, liver weight The upper left panel of Figure 2 shows the body weight of the mice 20 weeks after the initiation of the high-fat diet, the liver weight in the middle panel, and the liver-to-body weight ratio in the upper right panel. The lower panel also shows the changes in body weight of the mice from the start of HMGB1 peptide administration until the end of breeding. As shown in these figures, no significant differences were observed in body weight, liver weight, liver-to-body weight ratio, or body weight change between the HMGB1 peptide-administered and control groups (indicated by "ns" in the figures).

[0058] ii) Serum biochemical analysis The upper left panel of Figure 3 shows serum AST concentrations, the second from the left shows ALT concentrations, the second from the right shows Alb concentrations, and the right shows T-bil concentrations. The lower left panel of Figure 3 shows T-cho concentrations, and the right shows TG concentrations. Compared to the control group, the HMGB1 peptide administration group showed significant decreases (improvements) in AST, ALT, T-bil, and T-cho levels.

[0059] iii) Histological evaluation The top row of Figure 4 shows the results of HE staining, the middle row shows the results of F4 / 80 immunohistochemistry, and the bottom row shows the results of Sirius Red staining. The left row is a micrograph of the control group, and the right row is a micrograph of the HMGB1 peptide-treated group. HE staining revealed fewer lipid droplets in hepatocytes in the HMGB1 peptide-treated group than in the control group. In other words, a decrease in lipid droplets was confirmed in the HMGB1 peptide-treated group compared to the control group. F4 / 80 immunohistochemistry showed a smaller stained area and fewer F4 / 80-positive cells in the HMGB1 peptide-treated group compared to the control group. Sirius Red staining showed a smaller stained area in the HMGB1 peptide-treated group than in the control group.

[0060] iv) Liver tissue analysis results The upper left panel of Figure 5 shows the Sirius Red-positive area, the upper right panel shows the quantification results for hydroxyproline, and the lower panel shows the F4 / 80-positive area. The Sirius Red-positive area, hydroxyproline amount, and F4 / 80-positive area were all significantly lower in the HMGB1 peptide-administered group compared to the control group.

[0061] v) NAS analysis results The left side of Figure 6 shows the results of the evaluation of lobule inflammation, the middle side shows the results of the evaluation of hepatocyte ballooning, and the right side shows NAS. Compared to the control group, the HMGB1 peptide-administered group had significantly lower scores for lobule inflammation, hepatocyte ballooning, and NAS.

[0062] (3) Discussion i) Liver inflammation and hepatocellular / liver dysfunction F4 / 80 is known to be a macrophage marker. As shown in the middle panel of Figure 4 and the bottom panel of Figure 5, the area stained by F4 / 80 immunohistochemistry was smaller in liver tissue from the HMGB1 peptide-treated group than in liver tissue from the control group, and the number of F4 / 80-positive cells was also lower. This suggests that administration of HMGB1 peptide reduced the number of inflammatory cells in NASH model mice. In addition, as shown in the left panel of Figure 6, histological evaluation also confirmed reduced inflammation in the HMGB1 peptide-treated group compared to the control group. Furthermore, as shown in the center panel of Figure 6, the hepatocyte ballooning score also significantly improved with HMGB1 peptide administration. Furthermore, as shown in the top left and second panel from the left of Figure 3, serum biochemical analysis revealed reduced AST and ALT levels. This indicates that hepatocellular injury was reduced in the HMGB1 peptide-treated group compared to the control group. In addition, as shown in the top right panel of Figure 3, serum biochemical analysis revealed a decrease in T-bil levels. These results indicate that liver dysfunction was also reduced in the HMGB1 peptide-administered group compared to the control group. These results suggest that HMGB1 peptide improves liver inflammation, hepatocellular damage, and liver dysfunction caused by NASH.

[0063] ii) Liver fibrosis Sirius Red is known to stain type I and type III collagen and is used as a marker for fibrosis. As shown in the bottom of Figure 4 and the top left of Figure 5, the area stained with Sirius Red was smaller in the liver tissue of the HMGB1 peptide-treated group compared to the liver tissue of the control group, suggesting that administration of HMGB1 peptide reduced the amount of fibrotic tissue in the NASH model mice. Hydroxyproline is also known to be a marker for fibrosis precursors. The decrease in hydroxyproline levels also suggests a reduction in liver fibrosis. These results suggest that HMGB1 peptide may ameliorate liver fibrosis caused by NASH.

[0064] iii) Fatty liver and blood lipids As shown in Figure 4, upper, the number of lipid droplets was significantly reduced in the HMGB1 peptide-administered group compared to the control group. These results indicate that administration of HMGB1 peptide improved fatty liver itself. Furthermore, as shown in the bottom left of Figure 3, T-Cho levels also decreased in the HMGB1 peptide-administered group compared to the control group. This suggests that administration of HMGB1 peptide also improves blood lipid levels. Many NASH treatments improve liver inflammation and fibrosis, but few improve fatty liver or blood lipid levels. Therefore, it can be said that HMGB1 peptide can provide effects that are difficult to achieve with other NASH treatments. [Industrial Applicability]

[0065] Pharmaceutical compositions containing the peptides of the present application are expected to be of great benefit as therapeutic and / or preventive agents for fatty liver, non-alcoholic steatohepatitis, and various symptoms associated with fatty liver, for which existing therapeutic agents are not sufficiently effective.

Claims

1. A pharmaceutical composition for preventing and / or treating fatty liver, comprising any one of the following substances (a) to (c): (a) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1; (b) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 in which 1 to 4 amino acids have been substituted, deleted, inserted or added; and (c) A peptide consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO:

1.

2. The pharmaceutical composition according to claim 1, which also improves blood lipid levels.

3. The pharmaceutical composition described in claim 2, wherein the lipid concentration is one or more selected from the group consisting of total cholesterol concentration and triglyceride concentration.

4. The pharmaceutical composition according to any one of claims 1 to 3, which is administered repeatedly at least once a week for two or more consecutive weeks.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the fatty liver is a fatty liver of fatty liver disease.

6. 6. The pharmaceutical composition according to claim 5, wherein the fatty liver disease is one or more selected from the group consisting of non-alcoholic fatty liver disease, alcoholic fatty liver disease, nutritional fatty liver disease, starvation fatty liver disease, obesity fatty liver disease, and diabetic fatty liver disease.

7. A pharmaceutical composition for preventing and / or treating non-alcoholic steatohepatitis, comprising any one of the following substances (a) to (c): (a) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1; (b) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 in which 1 to 4 amino acids have been substituted, deleted, inserted or added; and (c) A peptide consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO:

1.

8. The pharmaceutical composition described in claim 7, which also improves fatty liver in non-alcoholic steatohepatitis.

9. A pharmaceutical composition described in claim 7 or 8, which also improves blood lipid levels.

10. The pharmaceutical composition described in claim 9, wherein the lipid concentration is one or more selected from the group consisting of total cholesterol concentration and triglyceride concentration.

11. A pharmaceutical composition described in any one of claims 7 to 10, which is administered repeatedly at least once a week for two or more consecutive weeks.

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