CN113194940A - Composition comprising monoacetyldiacylglycerol compounds for the treatment of fatty liver - Google Patents

Abstract

A therapeutic lipid is disclosedA composition for treating fatty liver is provided. The composition comprises a monoacetyldiacylglycerol compound of formula 1 as an active ingredient for treating fatty liver disease. [ formula 1]

Wherein R1 and R2 are independently fatty acid residues of 14 to 22 carbon atoms.

Description

Composition comprising monoacetyldiacylglycerol compounds for the treatment of fatty liver

RELATED APPLICATIONS

This application requests the benefit of priority from U.S. provisional application No. 62/908,392 filed on 30/9/2019; korean patent application No. 10-2019-0026326, filed 3, 7.2019; and korean patent application No. 10-2019-0086967, filed on 2019, 7, 18, the entire contents of which are incorporated herein by reference.

Technical Field

The present invention relates to a composition comprising a monoacetyldiacylglycerol compound as an active ingredient for treating fatty liver diseases, and more particularly to a composition for preventing and/or alleviating symptoms of fatty liver diseases, comprising a monoacetyldiacylglycerol compound, for oral administration.

Background

The liver is one of the most important metabolic organs in human organs and has important functions such as secretion of bile, storage of nutrients, detoxification and the like. If the liver is in an abnormal state, metabolic abnormalities such as sugar metabolism (glucose metabolism), lipid metabolism, protein and nitrogen metabolism, amino acid metabolism, protein metabolism, and hepatic and cerebral diseases (hepatic disease), vitamin metabolism, malabsorption, etc. may occur. In addition, liver diseases may be exacerbated by infection, fatty liver, cirrhosis, and the like. The liver diseases known at present are related to acute hepatitis, alcoholic fatty liver, obesity, diabetes, nonalcoholic fatty liver caused by hyperlipidemia, acute hepatitis, chronic hepatitis, liver cirrhosis caused by viral infection, and the like.

Among them, non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease, associated with obesity, diabetes and hyperlipidemia, not associated with alcohol, and is characterized by the accumulation of excess lipids (mainly triglycerides) in the liver. Typically, in NAFLD, lipids make up more than 5% by weight of the total weight of the liver. NAFLD has the potential to develop into steatohepatitis, fibrosis, cirrhosis and even hepatocellular carcinoma.

There is much research on NAFLD, but relatively little is known about its association with type 1diabetes (T1D). Recent reports indicate that T1D may cause NAFLD, an independent risk factor for chronic diabetic complications.

Type 1diabetes (T1D) is an autoimmune disease in which the patient's own immune system attacks and destroys beta cells. The pathogenesis of fatty liver in type 1diabetes (T1D) is not clear. However, Regnell and Lernmark suggest that this may be caused by abnormalities in lipoproteins that regulate glucose metabolism and lipid synthesis, respectively, primarily through activation of blood and lymphocyte trafficking lipids and/or transcription factors, carbohydrate response element binding protein (ChREBP) and sterol regulatory element-binding protein 1C (SREBP-1C) (s.e.regnell, a.lernmark, hepatotic steatosis in type 1diabetes Rev Diabet Stud 8(2011) 454-.

Non-alcoholic steatohepatitis (NASH) is one of non-alcoholic fatty liver diseases (NAFLD). The root cause of NASH is not known. However, in patients with NASH analyzed, NASH is associated with obesity, type 2 diabetes, dyslipidemia, or metabolic syndrome (triglycerides with waist circumference above 90cm, greater than or equal to 150mg/dL, HDL less than or equal to 40mg/dL, fasting glucose of 100mg/dL or higher, systolic blood pressure of 130mmHg or higher). Therefore, they are considered to be factors causing nonalcoholic fatty liver disease (NAFLD), and are considered to be a major cause of an increase in nonalcoholic steatohepatitis (NASH) patients.

It is well known that non-alcoholic fatty liver disease (NAFLD) increases the risk of developing type 2 diabetes 2 by 2 to 4 fold compared to a control group not suffering from non-alcoholic fatty liver disease (NAFLD). In other words, nonalcoholic fatty liver disease (NAFLD) is closely associated with insulin resistance.

Lipoprotein lipase (LPL), which is a lipase for pancreas, liver and endothelium, is mainly distributed in skeletal muscle, heart and adipose tissues. Lipoprotein lipase (LPL) comprises Chylomicrons (CM) which are a very low density lipoprotein, hydrolyzable Triglycerides (TG), bound to ApoE (APO protein) and disperse triglycerides into muscle and adipose tissue. They play an important role in hydrolyzing Triglycerides (TG) and removing Triglycerides (TG) from peripheral tissues such as muscle and adipose tissues as energy or storage sources. Therefore, when lipoprotein lipase (LPL) is decreased (lost), Triglyceride (TG) is excessively circulated, which is a cause of hypertriglyceridemia.

Streptozotocin (STZ) is a selective pancreatic beta cytotoxin that induces rapid and irreversible necrosis of cells and is commonly used to induce diabetes. It is also a suitable candidate to mimic hepatic steatosis. STZ-induced diabetes is characterized by hyperglycemia, dyslipidemia, weight loss, and hepatic steatosis.

Therefore, new therapies for fatty liver disease are desired.

Disclosure of Invention

In one aspect, compositions and methods for treating fatty liver disease are provided, the compositions and methods comprising a monoacetyldiacylglycerol compound. In preferred methods and compositions, Triglyceride (TG) levels in hepatocytes and plasma can be reduced, and expression of lipoprotein lipase (LPL) can be increased

In another aspect, compositions and methods for treating fatty liver disease are provided, comprising a monoacetyldiacylglycerol compound as an active ingredient, which is non-toxic and alleviates fatty liver disease, including symptoms thereof.

More specifically, compositions and methods comprising monoacetyldiacylglycerol compounds of formula 1 for treating fatty liver diseases are provided.

[ formula 1]

Wherein R1 and R2 are independently a fatty acid residue of 14 to 22 carbon atoms, preferably a fatty acid residue of 15 to 20 carbon atoms.

In one embodiment, the monoacetyldiacylglycerol is a compound of formula 2 below:

[ formula 2]

The compound of formula 2 is 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol and corresponds to the compound of formula 1, wherein R1 and R2 of formula 1 are palmitoyl and linoleoyl, respectively. In the present disclosure, the compound of formula 2 is sometimes referred to as "PLAG" or "EC-18".

As discussed, the compounds of formulae I and 2 may be used to treat individuals suffering from or susceptible to fatty liver disease, including non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), fatty liver disease caused by hepatitis, fatty liver disease caused by obesity, fatty liver disease caused by diabetes, fatty liver disease caused by insulin resistance, fatty liver disease caused by hypertriglyceridemia, β -lipoprotein deficiency, glycogen storage disease, nodular non-suppurative panniculitis (Weber-Christian disease), wolman disease, acute fatty liver during pregnancy, and/or lipodystrophy.

In particular aspects, the compounds of formulae 1 and 2 are useful for treating an individual suffering from or susceptible to nonalcoholic steatohepatitis (NASH).

In another particular aspect, the compounds of formulae 1 and 2 are used to treat an individual suffering from or susceptible to non-alcoholic fatty liver disease (NAFLD).

In particular aspects, an individual will be identified and selected for treatment of the diseases or disorders disclosed herein, followed by administration of a compound of formula 1 or 2 to the identified and selected individual.

For example, a patient having nonalcoholic steatohepatitis (NASH) can be identified and selected, and a compound of formula 1 or 2 can be administered to a patient identified as having nonalcoholic steatohepatitis (NASH), thereby alleviating or treating nonalcoholic steatohepatitis (NASH). A patient having non-alcoholic fatty liver disease (NAFLD) may be identified and selected, and a compound of formula 1 or 2 may be administered to a patient identified as having non-alcoholic fatty liver disease (NAFLD), thereby alleviating or treating non-alcoholic fatty liver disease (NAFLD). A patient having liver fibrosis can be identified and selected, and a compound of formula 1 or 2 can be administered to a patient identified as having liver fibrosis, thereby alleviating or treating non-alcoholic fatty liver disease (NAFLD).

In certain aspects, one or more compounds of formula 1 or 2 or PLAG may be administered to an individual in combination or coordination with one or more liver fibrosis therapeutics other than the one or more compounds of formula 1 or 2 or PLAG. In certain aspects, the one or more different therapeutic agents for liver fibrosis co-administered or administered in combination with one or more compounds of formula 1 or 2 or PLAG may be obeticholic acid (OCA), elafinibrand (GFT505), selonsertib (GS-4997), ceriviroc (CVC), liraglutide (liraglutide), metadoxine (metadoxine), hydroxytyrosol (hydroxytyrosol) and vitamin E, NGM282(M70), BMS-986036, emrican (IDN-6556), aramchol, atorvastatin (atorvastatin) and/or L-carnitine (L carnitine), MGL-3196 (remeiro), volixibat (SHP626), sargraciltide-9674, semaglutide (semaglutidine), saxatilic, NCT 026616 (MSDc), mazetimid (MSDJ-76352), mazetimid (MSDc-76352), mazetimigron (MSDc-76352), sargragliptin (US), SAGIT-9674, SAGIT (MSD) (MSD-7695), SAGIT-GCR (SAGIT-W), SAGIT-G, and a, JKB-121, IMM-124E and/or ARI-3037MO, and pharmaceutically acceptable salts or acids thereof.

In certain preferred aspects, PLAG may be co-administered with obeticholic acid (OCA) to treat a subject, e.g., a human suffering from a fatty liver disease or disorder such as NASH or NAFLD. In certain further preferred aspects, PLAG may be co-administered with MGL-3196 to treat a subject, e.g., a human suffering from a fatty liver disease or disorder such as NASH or NAFLD. In certain further preferred aspects, PLAG may be co-administered with MGL-3196 and obeticholic acid (OCA) to treat a subject, e.g., a human suffering from a fatty liver disease or disorder such as NASH or NAFLD.

As referred to herein, a therapeutic agent for liver fibrosis will be different from a compound of formula 1 or 2 or PLAG, wherein the therapeutic agent for liver fibrosis is chemically different from a compound of formula 1 or 2 or PLAG. For example, different liver fibrosis treatments may not contain monoacetyldiacylglycerol structures, or may not contain diacylglycerol structural compounds, or may not contain glycerol structures. The molecular weight of the different liver fibrosis therapeutic agent administered may also be different (higher or lower) by at least 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90 or 100 percent from the molecular weight of the co-administered compound of formula 1 or 2 or PLAG.

In certain aspects, the present methods of treatment are not related to treating an individual having hepatitis. In this regard, individuals who have hepatitis and/or are seeking hepatitis treatment will be excluded from the present treatment method. In a related aspect, individuals seeking treatment who have been identified as having hepatitis will be excluded from the present treatment method.

In yet another aspect, the present treatment methods are not related to the treatment of individuals with diabetes. In this regard, individuals who have diabetes and/or are seeking treatment for diabetes would be excluded from the present treatment method.

In yet another aspect, the present methods of treatment are not associated with an individual having a wound or injured tissue. In this regard, individuals who have and/or are seeking treatment for a wound or wounded tissue would be excluded from the present treatment method. In a related aspect, individuals seeking treatment involving tissue repair or regeneration would be excluded from the present treatment method.

In another aspect, there is provided a pharmaceutical composition comprising a compound of formula 1 or 2 as described above. The composition may suitably comprise one or more pharmaceutically acceptable carriers. In preferred embodiments, the compositions may be formulated or otherwise suitable for use in the treatment of fatty liver diseases disclosed herein. In a preferred aspect, the composition may be suitable for oral administration as a tablet or capsule.

In yet another aspect, a kit for treating or preventing a fatty liver disease disclosed herein is provided. The kits of the invention may suitably comprise 1) one or more compounds of formula 1 or 2; and 2) instructions for using one or more compounds as disclosed herein to treat or prevent fatty liver disease. Preferably, the kit will comprise a therapeutically effective amount of one or more compounds of formula 1 or 2. The instructions may suitably be in written form, including as product labels.

The present invention also provides a health functional food composition comprising the monoacetyldiacylglycerol compound of formula 1 for alleviating or preventing fatty liver diseases.

The compositions of the invention can reduce the expression of apo B protein, including ApoB48, in the portal vein, are non-toxic and can treat and/or alleviate fatty liver disease.

Other aspects of the invention are disclosed below.

Drawings

This patent or application document contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the office upon request and payment of the necessary fee.

FIG. 1 shows an experimental protocol of the present invention.

Fig. 2 (comprising fig. 2A and 2B) shows a graph and chart showing the change in body weight (fig. 2A) and a chart showing the change in liver weight (fig. 2B) when a composition according to the invention is administered.

Figure 3 shows photomicrographs of H & E and ORO stained liver sections when using a composition according to the invention.

Fig. 4 shows an exemplary mechanism by which fat absorbed in the small intestine affects the liver.

Fig. 5 (including fig. 5A and 5B) shows graphs showing TG levels and contents in liver and plasma (fig. 5A) and a graph showing expression of apoB48 protein in portal plasma (fig. 5B) when the composition according to the present invention was used.

Fig. 6 (including fig. 6A to 6C) shows a graph showing TG content in muscle tissue (fig. 6A), a graph showing relative mRNA expression of LPL in muscle tissue (fig. 6B), and a picture showing representative immunohistochemical images of staining of LPL in muscle sections (fig. 6C) when using the composition according to the present invention.

Fig. 7 (including fig. 7A to 7C) shows graphs showing the weight of muscle specimen and skeletal muscle of mice (fig. 7A), and the results of relative mRNA expression of caveolin (caveolin)3 in muscle tissues including gastrocnemius and quadriceps (fig. 7B) and caveolin 3 and myogenin in myoblasts and myotubes (fig. 7C).

Fig. 8 (including fig. 8A to 8D) shows the structural chemistry of PLAG and PLG (fig. 8A) and a graph showing weight change (fig. 8B) and a picture of Gross (Gross) liver specimens from each group (fig. 8C) and a picture of H & E stained liver sections (fig. 8D).

FIG. 9 shows another experimental protocol of the present invention.

Fig. 10 is a graph showing the change in body weight when the composition according to the present invention was used.

Figure 11, including figures 11A to 11C, shows a graph showing weight (figure 11A), liver weight (figure 11B) and the ratio of liver weight to weight (figure 11C) when using a composition according to the invention.

Fig. 12, including fig. 12A to 12C, shows a graph showing plasma ALT (fig. 12A) and plasma AST (fig. 12B) and the ALT/AST ratio (fig. 12C) when using a composition according to the invention.

Figure 13 shows a micrograph of a liver section stained with H & E and ORO when using a composition according to the invention.

Fig. 14 (including fig. 14A to 14D) shows a diagram of liver sections stained with H & E and ORO when using a composition according to the invention.

Fig. 15 shows a micrograph of a liver section stained with Sirius red-stabilized when using a composition according to the invention.

Fig. 16 shows a graph of the fibrous part of the liver tissue (sirius red positive area) when using the composition according to the invention.

Figure 17 shows a graph of liver triglyceride levels for groups 1 to 6 in example 12.

Fig. 18 shows a graph of plasma CK-18 in groups 1 to 6 in example 12.

FIG. 19 shows representative photomicrographs of F4/80 immunostained liver sections from groups 1 through 6 in example 12.

Fig. 20 shows a graph of the area of inflammation (F4/80 positive area%) for groups 1 to 6 in example 12.

FIG. 21 shows the relative gene expression measured by quantitative RT-PCR in groups 1 to 6 in example 12.

Detailed Description

The composition for treating fatty liver disease according to the present invention comprises the monoacetyldiacylglycerol compound of formula 1 as an active ingredient.

[ formula 1]

In the present invention, the term "monoacetyldiacylglycerol compound" refers to a glycerol derivative containing an acetyl group and two acyl groups, also referred to as Monoacetyldiacylglycerol (MADG).

In formula 1, R1 and R2 are independently a fatty acid residue of 14 to 22 carbon atoms, preferably a fatty acid residue of 15 to 20 carbon atoms. By fatty acid residue is meant the remainder of the fatty acid in which the-OH group is excluded from its carboxyl group. Non-limiting examples of R1 and R2 in formula 1 thus include palmitoyl, oleoyl, linoleoyl, linolenoyl, stearoyl, myristoyl, arachidonoyl, and the like. Preferred combinations of R1 and R2 include oleoyl/palmitoyl, palmitoyl/oleoyl, palmitoyl/linoleoyl, palmitoyl/linolenoyl, palmitoyl/arachidonoyl, palmitoyl/stearoyl, palmitoyl/palmitoyl, oleoyl/stearoyl, linoleoyl/palmitoyl, linoleoyl/stearoyl, stearoyl/linoleoyl, stearoyl/oleoyl, myristoyl/linoleoyl, myristoyl/oleoyl, and the like. A more preferred combination of R1 and R2 is palmitoyl/linoleoyl. In terms of optical activity, the monoacetyldiacylglycerol derivative of formula 1 may be in the (R) -form, (S) -form or a racemic mixture, preferably a racemic mixture, and may include its iso-stereoisomers.

In one embodiment, the monoacetyldiacylglycerol is a compound of formula 2 below:

[ formula 2]

The compound of formula 2 is 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol and corresponds to the compound of formula 1, wherein R1 and R2 of formula 1 are palmitoyl and linoleoyl, respectively. In the present disclosure, the compound of formula 2 is sometimes referred to as "PLAG" or "EC-18".

The monoacetyldiacylglycerol compound may be isolated and extracted from natural velvet antler (antler), or may be produced by a known organic synthesis method (korean patent No. 10-0789323). More specifically, cornu Cervi Pantotrichum is extracted with hexane, and then the residue is extracted with chloroform and the chloroform is removed to provide a chloroform extract. The amount of solvent extracted is just enough to immerse the deer antler. Generally, about 4 to 5 liters of hexane and/or chloroform is used for 1kg of velvet antler, but is not limited thereto. The extract obtained by the method is further fractionated and purified by a series of silica gel column chromatography and TLC method to obtain the monoacetyldiacylglycerol compound of the present invention. The extraction solvent is selected from chloroform/methanol, hexane/ethyl acetate/acetic acid, but not limited thereto.

A chemical synthesis method for preparing a monoacetyldiacylglycerol compound is shown in korean patent No. 10-0789323. Specifically, the method comprises (a) a step of preparing 1-R1-3-protecting group-glycerol by introducing a protecting group at the 3-position of 1-R1-glycerol; (b) a step of introducing R2 at the 2-position of 1-R1-3-protecting group-glycerol to prepare 1-R1-2-R2-3-protecting group-glycerol; (c) the deprotection reaction and acetylation reaction of 1-R1-3-protecting group-glycerol are simultaneously carried out to prepare the desired monoacetyldiacylglycerol compound. The monoacetyldiacylglycerol compound can be further purified, if desired. Alternatively, the monoacetyldiacylglycerol compound may be prepared by acid decomposition (acetylation) of phosphatidylcholine, but is not limited thereto. Stereoisomers of the compounds of formula 1 are also within the scope of the invention.

The monoacetyldiacylglycerol compounds of the present invention can be effectively used for treating and/or alleviating fatty liver diseases. The term "fatty liver disease" is a disease characterized by the accumulation of excess fat (primarily triglycerides) in the liver. Generally, the term "fatty liver disease" refers to a condition in which the amount of fat in the liver exceeds 5% by weight of the total weight of the liver.

The fatty liver disease includes non-alcoholic fatty liver disease (NAFLD). Nonalcoholic fatty liver disease (NAFLD) is an excessive accumulation of fat in the liver due to reasons other than drinking.

Specifically, non-alcoholic fatty liver disease (NAFLD) is associated with overweight, obesity, insulin resistance, diabetes (type 1diabetes (T1D), type 2 diabetes, etc.), dyslipidemia (hyperlipidemia), metabolic syndrome (e.g., hypertension, hyperglycemia, abdominal obesity, low HDL cholesterol and high triglyceride-induced metabolic syndrome), and the like. NAFLD is caused by accumulation of excessive fat (mainly triglycerides) in the liver due to reasons other than drinking, and generally means that the accumulation of fat accounts for 5% by weight or more of the total weight of the liver. In the present disclosure, NAFLD includes diseases caused by NAFLD.

The non-alcoholic fatty liver disease (NAFLD) includes not only non-alcoholic fatty liver where fat is simply accumulated in the liver without damaging hepatocytes, but also non-alcoholic steatohepatitis (NASH), hepatic fibrosis, cirrhosis, and even hepatocellular carcinoma. Specifically, non-alcoholic fatty liver disease (NAFLD) can be classified into types 1 to 4. Specifically, type 1 refers to simple fatty liver with only steatosis and no liver cell damage. Type 2 refers to steatohepatitis in which the lobules develop an inflammatory response due to severe and persistent steatosis and hepatocyte injury. Type 3 refers to a state in which hepatocytes show balloon-like changes and steatosis and fat necrosis. Type 4 refers to a state in which Mallory bodies (Mallory bodies) or fibrosis occurs with type 3 symptoms. Liver cirrhosis may occur in type 3 and type 4.

Approximately 10% of all fatty liver disease patients are known as steatohepatitis. The nonalcoholic steatohepatitis (NASH) is caused by an increase in triglyceride and a decrease in fat metabolism caused by hyperinsulinemia (hyperinsulinemia). If non-alcoholic fatty liver disease and non-alcoholic steatohepatitis are not treated, liver function may deteriorate, and liver cirrhosis may occur through liver fibrosis.

Fibrosis is a wound healing process in liver tissue. When liver tissue is damaged by hepatitis virus, non-alcoholic fatty liver, medicine, etc., the vascular structure in the liver lobule collapses, and the liver cells are continuously damaged and regenerated. As a result, the nonfunctional regenerative nodules take over and the liver undergoes a fibrotic response and may cause cirrhosis.

Damaged hepatocytes secrete free radicals and inflammatory substances to activate Cooper cells and inflammatory cells, resulting in activation of hepatic stellate cells. Hepatic stellate cells are normally the reservoir of vitamin a, but are activated when liver injury occurs, and can synthesize and secrete various extracellular matrices such as collagen, causing hepatic fibrosis. When liver injury is repeated chronically, the damaged hepatocytes are no longer regenerated and gradually replaced with extracellular matrix such as collagen, resulting in liver fibrosis and cirrhosis.

That is, the non-alcoholic fatty liver disease (NAFLD) is a persistent disease that progresses from simple non-alcoholic fatty liver through fatty liver disease and hepatic fibrosis to cirrhosis.

The monoacetyldiacylglycerol compound according to the present invention can be effectively used for treating nonalcoholic fatty liver disease (NAFLD), including nonalcoholic fatty liver, nonalcoholic steatohepatitis (NASH), and liver fibrosis. The term "treating" includes delaying or alleviating the symptoms of non-alcoholic fatty liver, non-alcoholic steatohepatitis (NASH) and liver fibrosis, as well as partially or completely eliminating or preventing the symptoms, by administering the composition of the present invention.

Combination therapy

As discussed, one or more compounds of formula 1 or 2 or PLAG and one or more liver fibrosis therapeutics may be administered in combination to treat individuals, including humans with fatty liver disease, including NASH.

As used herein, the term "combination" in the context of administering a therapy to an individual refers to the use of more than one therapy for therapeutic benefit. The term "combination" in the context of administration may also refer to the prophylactic use of a therapy on an individual when used together with at least one additional therapy. The use of the term "combination" does not limit the order in which therapies (e.g., first and second therapies) are administered to an individual. Treatment a second therapy can be administered to a subject in need of treatment as disclosed herein at (e.g., 1 minute, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), at the same time or after (e.g., 1 minute, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after). The treatments are administered to the individual in a sequence and at intervals such that the treatments can work together. In a particular embodiment, the treatments are administered to the individual in a sequence and within a time interval such that they provide increased benefit over when administered otherwise. Any additional treatments may be performed in any order with other additional treatments.

Administration of a compound (e.g., a compound of formula 1 or 2, or PLAG) and one or more liver fibrosis therapeutics may be carried out in a suitable manner that results in concentrations of the therapeutic in combination with other components effective to ameliorate, reduce or stabilize a fatty liver disease, such as non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), liver fibrosis, a fatty liver disease caused by hepatitis, a fatty liver disease caused by obesity, a fatty liver disease caused by diabetes, a fatty liver disease caused by insulin resistance, a fatty liver disease caused by hypertriglyceridemia, a β -lipoprotein deficiency, glycogen storage disease, nodular non-suppurative panniculitis, walman's disease, acute fatty liver during pregnancy, and/or a fat metabolism disorder.

The compound (e.g., a compound of formula 1 or 2, or PLAG) and one or more different therapeutic agents for liver fibrosis can be administered simultaneously or sequentially. In some embodiments, liver fibrosis treatment is an established therapy for the disease indication, and such treatment improves the therapeutic benefit to the patient by adding a compound (e.g., a compound of formula 1 or 2, or PLAG). This improvement can be measured as an increase in response on a per patient basis or in a patient population. Combination therapy may also provide improved response at lower or less frequent therapeutic agent doses, resulting in a more tolerable treatment regimen. As noted, combination therapy of a compound (e.g., a compound of formula 1 or 2, or PLAG) and one or more different therapeutic agents for liver fibrosis may provide enhanced clinical activity by various mechanisms, e.g., the mechanism by which fat is absorbed small intestine affects liver (e.g., increased dietary fat transport from small intestine (gut) to liver due to increased intake or dysregulation of gut physiology and microbiota); 2) increased inflow of free fatty acids from non-esterification pools (e.g., from white adipose tissue); 3) increased hepatic de novo lipogenesis due to excess carbohydrate (and/or hyperinsulinemia due to insulin resistance in adipose tissue), and the like.

In some embodiments, the methods (e.g., combination therapy for fatty liver disease) may comprise administering a second therapeutic agent (e.g., one or more liver fibrosis therapeutic agents other than a compound of formula 1 or 2 or PLAG) or treatment for a second therapy of liver fibrosis (e.g., a therapeutic agent or therapy standard in the art).

In some embodiments, the methods (e.g., combination therapy for fatty liver disease) may comprise administering a second therapeutic agent (e.g., one or more liver fibrosis therapeutic agents other than a compound of formula 1 or 2 or PLAG) or treating NAFLD and/or NASH with a second therapy (e.g., a therapeutic agent or therapy standard in the art).

Exemplary therapeutic agents include one or more liver fibrosis therapeutic agents. "liver fibrosis therapeutic agent" is a compound useful for the treatment of fatty liver diseases, for example, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), liver fibrosis, fatty liver disease caused by hepatitis, fatty liver disease caused by obesity, fatty liver disease caused by diabetes, fatty liver disease caused by insulin resistance, fatty liver disease caused by hypertriglyceridemia, β -lipoprotein deficiency, glycogen storage disease, nodular non-suppurative panniculitis, Wallman's disease, acute fatty liver during pregnancy, and/or lipodystrophy.

Examples of liver fibrosis therapeutics for use in the present methods, compositions and kits include obeticholic acid (OCA), elafibranor (GFT505), selonsertib (GS-4997), cenicriviroc (CVC), liraglutide, metadoxine, hydroxytyrosol and vitamin E, NGM282(M70), BMS-986036, emricasan (IDN-6556), aramchol, atorvastatin and/or levocarnitine, MGL-3196, volixia (SHP62667-s), GS-9674, semaglutide, saroglitazar, agents in NCT 05616 (Meito center, Rochester, Minnesota, USA), LMB763, IVA337, LJN452, CF102, MT-95, pioglitazone, MN-001(tipelukast), MSDC-0602K, JKB-060121, IMM-026124, and/or ARMO 303I, and pharmaceutically acceptable salts thereof. As discussed, certain preferred methods, kits, and compositions may comprise one or more liver fibrosis therapeutics comprising MGL-3196 (remeirom) and/or obeticholic acid (OCA), including treating an individual, e.g., a human having a fatty liver disease or disorder such as NASH or NAFLD.

Furthermore, examples of therapeutic agents for liver fibrosis include peroxisome proliferator-activator receptor (PPARs alpha, beta/delta and/or gamma) agonists (e.g., GW501516, elafibranor, thiazolidinediones (thiazolidinediones), pioglitazone or saroglitazar), FXR-bile acid axis (FXR-bile acid axis) (e.g., obeticholic acid, FGF-19, NGM-282, agents in NCT02548351, agents in NCT02443116, etc.), lipid altering agents (e.g., stearoyl-CoA desaturase (SCD) inhibitors, aramchol), drugs for incretin-based therapy (e.g., exenatide, liraglutide, sitagliptin, etc.), drugs for inflammation, cell injury or apoptosis and oxidative stress (e.g., drugs in vitamin E, ENCORE-NF; NCT 762, hexitone, 86762, and the like), drugs for intestinal tract related therapies such as theobromine, IMM-124e, orlistat, the agents in NCT02510599, etc.) and/or agents for anti-fibrotic therapy (e.g., simtuzumab, GR-MD-02, etc.).

For example, peroxisome proliferator-activated receptors (PPARs) comprise a group of nuclear receptors expressed in the liver, adipose tissue, heart, skeletal muscle and kidney and transcriptionally regulate a variety of metabolic processes including B-oxidation, lipid transport and gluconeogenesis.

Additional liver fibrosis agents that may be used in the present methods, compositions, and kits include, for example, soluble guanylate cyclase (sGC) stimulators disclosed in international patent publication No. WO 2017/136309; and cericiviroc as disclosed in U.S. patent publication No. 2018/0360846.

As discussed, 1) a compound of formula 1 or 2 or PLAG and 2) one or more different therapeutic agents for liver fibrosis may be "co-administered," i.e., administered together in a coordinated manner to an individual, either as separate pharmaceutical compositions or mixed in a single pharmaceutical composition. By "co-administration", one or more additional different therapeutic agents for liver fibrosis may also be administered simultaneously with the compound of formula 1 or 2, or separately from the compound of formula 1 or 2, including at different times and different frequencies. The one or more different therapeutic agents for liver fibrosis may be administered by any suitable route of administration, e.g., oral, intravenous, subcutaneous, intramuscular, intranasal, etc.; and the therapeutic agent may also be administered by any conventional route. In at least certain embodiments, one or more different liver fibrosis agents can be administered orally.

In some embodiments, a compound (e.g., a compound of formula 1 or 2, or PLAG) and/or one or more different therapeutic agents for liver fibrosis may be administered daily, e.g., every 24 hours, or continuously or several times daily, e.g., every 1 hour, every 2 hours, every 3 hours, every 4 hours, every 5 hours, every 6 hours, every 7 hours, every 8 hours, every 9 hours, every 10 hours, every 11 hours, or once every 12 hours.

Exemplary effective daily doses of different liver fibrosis therapeutics include between 0.1 μ g/kg and 100 μ g/kg body weight, e.g., 0.1, 0.3, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 99 μ g/kg body weight.

Alternatively, the different liver fibrosis therapeutics are administered about once per week, for example about once every 7 days. Alternatively, the different liver fibrosis therapeutics are administered twice a week, three times a week, four times a week, five times a week, six times a week, or seven times a week. Exemplary weekly effective doses of a therapeutic agent for liver fibrosis include between 0.0001mg/kg and 4mg/kg body weight, e.g., 0.001, 0.003, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1,2, 3, or 4mg/kg body weight. For example, effective weekly doses of different liver fibrosis therapeutics are between 0.1 μ g/kg body weight and 400 μ g/kg body weight.

In the examples of the present disclosure, when the monoacetyldiacylglycerol compound is used, (1) it is shown that the decreased body weight of Streptozotocin (STZ) is restored and (2) it is shown that the increased liver weight is decreased due to excessive accumulation of Triglyceride (TG) in the liver (example 1). Thus, it can be seen that administration of the monoacetyldiacylglycerol compound improves fatty liver.

Triglycerides (TG, neutral fat) are absorbed in the small intestine as exogenous fat and include apolipoproteins of ApoB 48. Triglycerides (TG) recombine with Chylomicrons (CM) and ApoB 48. Triglyceride-rich CM is transported into the blood via the lymphatic system while circulating in the body, and Triglyceride (TG) is absorbed into surrounding tissues, including muscle, adipose tissue and monocytes, for energy or storage. Triglyceride (TG) contained in CM is then hydrolyzed by lipoprotein lipase (LPL) and transported to the liver via the portal vein, producing smaller residual particles of CM. Therefore, when lipoprotein lipase (LPL) is decreased, unused CM in peripheral tissues including muscle, adipose tissue and immune cells is transported to the liver, resulting in excessive circulation of Triglycerides (TG) into the liver. This can lead to hypertriglyceridemia and to fatty liver.

ApoB48 is a lipoprotein that transports cholesterol and triglycerides in the blood. When ApoB48 is too much, it means that Triglyceride (TG) is high, which may cause dyslipidemia. Thus, the uptake of Triglycerides (TG) in the liver can promote fat accumulation in hepatocytes, resulting in fatty liver (see fig. 4).

In other embodiments of the disclosure, the livers of the experimental groups were observed, vesicle fat was imaged using hematoxylin and eosin (H & E), and oil red O (ORO, Sigma Aldrich, st. The results show that: (1) reduced Triglyceride (TG) concentrations in hepatocytes and plasma (examples 2, 3), (2) increased lipoprotein lipase (LPL) expression (example 4), (3) reduced expression of apolipoproteins including ApoB48 in the portal vein (example 3). It was confirmed that functional improvement of peripheral tissues producing and secreting lipoprotein lipase (LPL) was achieved due to the monoacetyldiacylglycerol compound. These results also indicate that the monoacetyldiacylglycerol compound is effective for treating fatty liver.

In other examples of the present disclosure, when monoacetyldiacylglycerol compounds are used, NAFLD Activity Scores (NAS) for non-alcoholic fatty liver disease (NAFLD), steatosis, lobular inflammation and decreased hepatocyte ballooning are indicated (example 10), and (2) fibrosis of liver tissue is partially decreased (example 11).

The pharmaceutical composition comprising the monoacetyldiacylglycerol compound of the present invention may include a conventional pharmaceutically acceptable carrier, excipient or diluent. The content of monoacetyldiglyceride in the pharmaceutical composition may vary widely, and is not particularly limited, and specifically 0.0001 to 100% by weight, preferably 0.001 to 90% by weight, and for example, the content of monoacetyldiglyceride may be 70 to 80% by weight, relative to the total amount of the composition.

The pharmaceutical composition of the present invention may further comprise other active ingredients having the effect of treating fatty liver diseases. The pharmaceutical compositions may be formulated for oral or non-oral administration in the form of a solid, liquid, gel or suspension, for example, tablets, boluses, powders, granules, capsules such as hard or soft gelatin capsules, emulsions, suspensions, syrups, emulsion concentrates, sterile aqueous solutions, non-aqueous solutions, lyophilized formulations and the like. In formulating the composition, conventional excipients or diluents such as fillers, binders, wetting agents, disintegrants and surfactants may be used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules and the like, and can be prepared by mixing one or more active ingredients with at least one excipient such as starch, calcium carbonate, sucrose, lactose, gelatin and the like. In addition to excipients, lubricants such as magnesium stearate and talc may be used. Liquid preparations for oral administration include emulsions, suspensions, syrups and the like, and may include conventional diluents such as water and liquid paraffin, or may include various excipients such as wetting agents, sweeteners, flavoring agents and preservatives. Non-oral formulations include sterile aqueous solutions, non-aqueous solutions, lyophilized formulations, suppositories, and the like, and the solvents for such solutions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, esters for syringe injection such as ethyl oleate. The base material of the suppository may include witepsol, polyethylene glycol, tween 61, cocoa butter, glycerol laurate and glycerogelatin.

The monoacetyldiacylglycerol compound may be administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" is used to refer to an amount sufficient to achieve a desired result in medical treatment. The "pharmaceutically effective amount" may be determined according to the category, age, sex, severity and type of disease, activity of the drug, sensitivity to the drug, administration time, administration route, excretion rate, etc., of the individual. The compositions of the present invention may be administered alone or sequentially or simultaneously with other therapeutic agents. The composition of the invention may be administered one or more times. The preferred amount of the composition of the present invention may vary depending on the condition and body weight of the patient, the severity of the disease, the dosage form of the drug, the route of administration and the treatment time. An appropriate total amount administered may be determined by a physician per day, and is generally from about 0.001 to about 5,000mg/kg, preferably from about 0.05 to 1,000mg/kg, once per day, or may be administered in divided doses several times per day. The composition of the present invention may be administered to any subject in need of prevention or treatment of fatty liver disease. For example, the composition of the present invention can be administered not only to humans but also to non-human animals (particularly mammals), such as monkeys, dogs, cats, rabbits, guinea pigs, rats, mice, cows, sheep, pigs, goats, and the like.

In some embodiments, the present invention provides a healthy functional food composition for preventing, alleviating or improving fatty liver disease, comprising monoacetyldiacylglycerol of formula 1 as an active ingredient.

The monoacetyldiacylglycerol compound according to the present invention may be included in a healthy functional food composition to improve fatty liver disease in an individual. Monoacetyldiacylglycerol compounds and fatty liver diseases are described above. When the compound of the present invention is included in a health functional food composition, the amount of monoacetyldiacylglycerol in the health functional food composition may be appropriately determined depending on the intended use. Generally, when monoacetyldiacylglycerol is contained in a food or beverage, the amount of monoacetyldiacylglycerol is preferably 0.01 to less than 15% by weight relative to the total amount of the health functional food composition. However, the amount of monoacetyldiacylglycerol may be increased or decreased. In the case of long-term use for the purpose of health control and hygiene, the amount of monoacetyldiacylglycerol may be less than the above range. The monoacetyldiacylglycerol may be used in an amount greater than the above range since there is no problem in safety. The food to which the compound of the present invention can be added is not limited and includes various foods such as meats, sausages, breads, chocolates, candies, snacks, pizzas, noodles, chewing gums, dairy products such as ice cream, soups, drinks, teas, beverages, alcoholic beverages, multivitamins and any health functional foods.

When monoacetyldiacylglycerol is used in a beverage product, the beverage product can include sweeteners, flavoring agents, or carbohydrates. Examples of the carbohydrate include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. The amount of carbohydrate in the beverage composition may vary widely and is not particularly limited, preferably from 0.01 to 0.04 grams, more preferably from 0.02 to 0.03 grams per 100 milliliters of beverage. Examples of sweeteners include natural sweeteners such as soxhlet horse sweet (thaumatin) and stevia extract, and artificial sweeteners such as saccharin and aspartame. In addition to the above, the health functional food composition of the present invention may include various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salt, alginic acid and its salt, organic acids, protective colloid thickener, pH controlling agent, stabilizer, preservative, glycerin, alcohol, carbonation used in carbonated beverage, and the like. In addition, the health functional food composition of the present invention may include fruits for preparing natural fruit juice and fruit juice beverage and vegetable beverage.

The invention provides a method for treating fatty liver diseases, which comprises the step of administering the pharmaceutical composition to an individual suspected of being suffering from the fatty liver diseases. Fatty liver disease is effectively treated by administering the composition to a patient suspected of having fatty liver disease. The term "suspected fatty liver patient" refers to those who have developed or are likely to develop fatty liver. Fatty liver disease can be treated or prevented by administering an effective amount of the compound to a patient in need thereof. The kind of the monoacetyldiacylglycerol compound and the dosage of the monoacetyldiacylglycerol compound and fatty liver disease are as described above. The term "administering" refers to introducing a pharmaceutical composition of the invention into a patient in need thereof by any suitable method. The route of administration may be any one or more routes, oral or non-oral, as long as the target tissue can be reached, and for example, oral administration, intraperitoneal administration, transdermal administration (topical application, etc.), intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, intranasal administration, rectal administration, intranasal administration, intraperitoneal administration, etc. may be used, but is not limited thereto.

Examples

The following examples are provided to better understand the present invention. However, the present invention is not limited to these examples.

To demonstrate the efficacy of 1-palmitoyl-2-linoleoyl-3-acetyl glycerol (EC-18 or PLAG) in the treatment of fatty liver disease, a Streptozotocin (STZ) -induced fatty liver model was used in the experiment.

Experimental example 1 preparation of control group and Experimental group to confirm therapeutic Effect on non-alcoholic fatty liver disease (NAFLD)

FIG. 1 shows an experimental protocol of the present invention. As shown in fig. 1, mice were randomly divided into a control group and a Streptozotocin (STZ) -treated group. All STZ-treated groups were injected intraperitoneally with STZ dissolved in sodium citrate buffer (pH 4.5) at a dose of 200mg/kg Body Weight (BW) (1 day).

The following day after Streptozotocin (STZ) administration, diabetes induction and induction of acute diabetes were confirmed by a glucometer (ACCU-CHEK, Rochediagnostics Inc.). All mice with fasting blood glucose levels >200mg/dL were considered to have acute diabetes. After confirmation of induction, experimental mice were further randomized into three groups: STZ-only treatment group and low and high dose PLAG plus STZ treatment group. 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol represented by formula 2(PLAG) was used as an active ingredient.

Mice received PLAG for 3 days by feeding through the feeding tube. The expected oral doses in mice were 250 and 50mg/kg BW, respectively. The control group and STZ-treated group alone were orally administered with the same PBS for 3 days, and mice were sacrificed on the fourth day.

Example 1 body and relative liver gravimetric analysis

The body weights of all mice of the control group, streptozotocin alone (STZ), Streptozotocin (STZ) plus low dose PLAG treated group, Streptozotocin (STZ) plus high dose PLAG treated group prepared in the above experiment were measured and recorded at the same time every day, and the results are shown in fig. 2A. In addition, the relative liver weights of the control group and the experimental group were calculated using the following equation, and the results are shown in fig. 2B.

(equation 1)

(Absolute liver weight)/(sacrificial daily body weight) X100

As shown in FIG. 2A, all STZ-treated mice showed a statistically significant reduction in BW compared to the control group. However, the high dose PLAG group showed less BW loss compared to mice receiving STZ alone. There was no difference in BW between STZ group and low dose PLAG group. This indicates that oral PLAG administration reduces the weight loss caused by diabetes.

As shown in FIG. 2-B, the relative liver weight (per gram BW) was significantly higher in the STZ group alone than in the control group. However, the mean liver weight was lower in the PLAG treated group than in the STZ group, indicating that PLAG supplementation improved liver steatosis.

Example 2 histopathological analysis

At the end of the 4-day experimental period, mice were fasted for 6 hours and then sacrificed. Figure 3 is a picture of H & E and ORO stained liver sections. Specifically, in other aspects of measuring the degree of liver fat accumulation, livers of the control group and the experimental group were observed and imaged using hematoxylin and eosin (H & E) and oil red O. Liver specimens were immediately fixed with 10% formalin buffer at room temperature, then paraffin-embedded, sectioned, and stained with hematoxylin and eosin (H & E). Frozen liver sections were fixed with 4% paraformaldehyde for 20 min, followed by ORO staining for 15 min at room temperature. The samples were then counterstained with Mayer hematoxylin for 10 seconds and fixed. Images were obtained under an optical microscope (Olympus, Tokyo, Japan).

As shown in FIG. 3, H & E staining, as expected, showed significant evidence of hepatic steatosis in the STZ group alone, as evidenced by an increased number of empty fatty bubbles (both macrovesicular and microvesicle steatosis). However, PLAG at a dose of 250 mg/kg/day significantly reduced microbubble steatosis. Groups of mice treated with low dose PLAG also showed a reduction, although to a different extent.

ORO staining showed higher hepatic TG content in STZ group alone, and this was reduced by PLAG. These results indicate that PLAG reduces hepatic steatosis in a dose-dependent manner.

Example 3 analysis of liver and plasma TG levels

To measure the concentration of Triglyceride (TG) in plasma of the control group and the experimental group, blood was collected by ocular hemorrhage, and plasma was separated by centrifugation at 3,000rpm for 10 minutes. Plasma samples were kept frozen until assayed.

Fig. 4 is an example of the mechanism of the effect of fat absorbed by the small intestine on the liver. FIG. 5 is a graph showing the levels and amounts of TG in the liver and plasma when using the composition of the present invention (FIG. 5A) and a graph showing the expression of apoB48 protein in portal plasma (FIG. 5B). Specifically, in addition to measuring the Triglyceride (TG) content of hepatocytes, liver tissue (20mg) was homogenized in isopropanol using a tissue grinder. The homogenate was centrifuged at 5,000rpm for 10 minutes, and the supernatant was collected. The TG content of the supernatant was measured with a triglyceride H kit (Wako Diagnostics, Richmond, Va., USA). To confirm hepatic TG uptake, we measured ApoB48 expression in portal blood as a marker for TG-rich CM transport and uptake in vivo.

As shown in fig. 5, the level of ApoB48 was increased in STZ-treated mice compared to the control group. However, PLAG treatment dose-dependently reduced ApoB48 levels, suggesting that PLAG improves lipid metabolism by enhancing peripheral tissue or overall TG uptake.

Example 4 immunohistochemical analysis

Fig. 6 is a graph of TG content in muscle tissue (fig. 6A), a graph of relative mRNA expression of LPL in muscle tissue (fig. 6B) and a picture showing immunohistochemical representations of LPL staining in muscle sections (fig. 6C) when using a composition according to the invention. In particular, lipoprotein lipase (LPL) is the rate-limiting enzyme for TG clearance, controlling the catabolism of TG-rich lipoproteins, including CM. To investigate TG clearance by peripheral tissues after PLAG treatment, we examined LPL protein and mRNA levels in muscle tissue.

As shown in FIG. 6, there was no significant difference in the muscle TG content between the groups (FIG. 6A). The RT-PCR results showed a decrease in LPL mRNA expression in the STZ group and an increase in LPL mRNA expression in the high dose PLAG group compared to the control group (FIG. 6B).

Immunohistochemistry showed abundant expression in the control group, while expression was reduced in STZ-treated animals. However, the expression of LPL protein in the PLAG-treated group was similar to the control group. Overall, mRNA and protein findings indicate that PLAG induces TG clearance by restoring this function in peripheral tissues (fig. 6C).

Example 5 Western blotting

Fig. 7 is a graph showing the results of the weight of muscle specimen and skeletal muscle from mice and the relative mRNA expression of caveolin 3 and myogenin in muscle tissue (fig. 7A), including gastrocnemius and quadriceps (fig. 7B) and myoblasts and myotubes (fig. 7C). Specifically, the relative amount of apolipoprotein B48(ApoB48) in portal plasma was assessed by western blotting. Constant volume plasma was separated by electrophoresis on a 5% sodium dodecyl sulfate-polyacrylamide gel. Protein extracts were immunoblotted with ApoB48 antibody (1:500, Abcam, Cambridge, UK). Detection was performed using Immobilon Western chemiluminescence HRP substrate (Millipore Corporation, Billerica, MA, USA).

As shown in fig. 7, muscle atrophy is defined as the loss of muscle mass, occurring in the case of STZ-induced diabetes. Like BW, STZ treatment also significantly reduced muscle mass. Although the PLAG treatment slightly improved muscle mass, there was no significant difference between the STZ and PLAG groups alone (FIG. 7A).

This investigated the effect of PLAG on muscle function by measuring differentiation of myoblasts into myotubes. This conversion causes changes in morphological and muscle specific gene expression. We found that CAV3 and myogenin mRNA levels were reduced in STZ-treated C2C12 cells, while PLAG treatment restored normal levels (fig. 5C). This is consistent with the in vivo results for muscle tissue, including the quadriceps femoris and gastrocnemius muscles (fig. 5B). These results indicate that STZ impairs normal myoblast differentiation, while PLAG may help normalize muscle function.

Example 6 specificity of PLAG

Fig. 8 is a graph (fig. 8B) and structural chemistry of PLAG and PLG showing the picture of weight change and gross liver specimens from each group (fig. 8C) and H & E stained liver sections. In particular, the selectivity of PLAG was investigated by comparing the effects of PLAG and PLG in a STZ-induced mouse model. PLG is the prototype of diglyceride and PLAG is one of the acetylated diglycerides. As shown in figure 8 below, PLAG-treated mice showed less BW loss compared to mice receiving STZ alone. There was no difference in BW between STZ group and PLG treated mice (fig. 8B). In addition, the results indicate that PLAG can reduce hepatic steatosis. In contrast, PLG (C & D) did not reduce liver damage. These results indicate that PLAG plays a specific role in protecting the liver from STZ-induced hepatic steatosis.

Example 7 RNA isolation and RT-PCR

Analysis total RNA was isolated from muscle of individual mice using TRIzol reagent (Favorgen Biotech, Taiwan). cDNA was synthesized using M-MLV reverse transcriptase. Gene expression was analyzed in duplicate for each sample and normalized against the internal control gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Primer sequences for GAPDH, LPL, caveolin 3, and myogenin are shown in table 1 below.

[ Table 1]

Experimental example 2 preparation of experimental group to examine the role of EC-18 in STAM model of non-alcoholic steatohepatitis

To determine the efficacy of 1-palmitoyl-2-linoleoyl-3-acetyl glycerol (EC-18 or PLAG) for the treatment of non-alcoholic steatohepatitis and liver fibrosis, experiments were performed using models derived from Streptozotocin (STZ) and High Fat Diet (HFD).

FIG. 9 is another experimental protocol of the present invention, as shown in FIG. 9, all mice (C57BL/6J) were injected subcutaneously at birth with 200 μ g Streptozotocin (STZ) solution and fed with a High Fat Diet (HFD) after 4 weeks of age. Groups of mice were further randomized into 6 groups: control group (PBS), MGL-3196 treated group, obeticholic acid (OCA) treated group, EC-18 low group, EC-18 medium group, and EC-18 high group. EC-18 uses 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol represented by formula 2, MGL-3196 uses 2- (3,5-dichloro-4- ((5-isopropyl-6-oxo-1, 6-dihydropyridazin-3-its) oxy) phenyl) -3,5-dioxo-2,3,4,5-tetrahydro-1,2, 4-triaza-benzene-6-carbonitrile (2- (3,5-dichloro-4- ((5-isoprox-6-oxo-1, 6-dihydropyridozin-3-yl) oxy) phenyl) -3,5-dioxo-2,3,4, 5-tetrahydrogen-1, 2,4-triazine-6-carbonitrile (remeirom), and obeticholic acid (OCA) using 6 α -ethyl-chenodeoxycholic acid (6 α -ethyl-chenodeoxycholic acid, 6-ECDCA).

Example 8 body and relative liver gravimetric analysis

FIG. 10 is a graph showing the change in body weight when the composition of the present invention is used. Fig. 11 is a graph showing weight (fig. 11A), liver weight (fig. 11B), and liver weight versus weight (fig. 11C) when using a composition according to the present invention. Specifically, the mice of experimental example 2 were recorded for body weight before treatment, body weight was measured every day at the same time, and body weight change was recorded. Mice were observed about 60 minutes after each administration for significant clinical symptoms of toxicity, moribund and death. One mouse in each of the OCA and EC-18 groups died during the treatment period. Mice were sacrificed at week 9. The relative liver weights and average body weights of the mice are shown in table 2 below.

[ Table 2]

As shown in FIGS. 10 and 11 and Table 2, there was no significant difference in mean body weight at the sacrifice day between the control group and the treatment group. The average liver weight was significantly reduced in the MGL-3196 and EC-18 low groups compared to the control group. The mean liver weight of the group in EC-18 tended to decrease compared to the control group. The liver weight ratio of the OCA group tended to increase compared to the vehicle group, and the liver weight ratio of the MGL-3196 group tended to decrease compared to the control group.

Example 9 measurement of plasma biochemistry

Figure 12 is a graph showing plasma ALT (a) and plasma AST (b) and ALT/AST ratio (C) when using a composition according to the invention. Specifically, non-fasting blood was collected in polypropylene tubes containing an anticoagulant (Novo-Heparin) and centrifuged at 1,000Xg for 15 minutes at 4 ℃. Plasma alanine Aminotransferase (ALT) and plasma aspartate Aminotransferase (AST) were measured by FUJI DRI-CHEM 7000. The ALT/AST ratio was calculated and is shown in Table 3 below.

Plasma ALT is an enzyme contained in large quantities in hepatocytes. When the liver is damaged, plasma ALT activity is elevated. Plasma ALT is an indicator of liver disease. Plasma ALT is also distributed in erythrocytes, skeletal muscle, but not in hepatocytes, and is released into the blood upon cell death/destruction.

[ Table 3]

As shown in FIG. 12 and Table 3, there was no significant difference in plasma ALT levels between the control and treated groups. There was no significant difference in plasma AST levels between the control and other treatment groups (FIG. 12A). Plasma AST levels tended to decrease in OCA group compared to control group (fig. 12B). There was no significant difference in ALT/AST ratios between the control and other treatment groups. The ALT/AST ratio tended to increase in the OCA group compared to the control group (FIG. 12C).

Example 10 histological analysis

Fig. 13 is a picture of liver sections stained with H & E and ORO when using a composition according to the invention. Fig. 14 is a diagram showing liver sections stained with H & E and ORO when using a composition according to the present invention. Specifically, for NAFLD Activity Score (NAS), statistical analysis was performed using Dunnett's multiple comparison test, as shown in table 4 below. In addition, the results of statistical analysis of NAS in each treatment group are shown in table 5.

Among the values of NAS, a P value <0.05 is considered statistically significant. For other data, statistical analysis was performed using Bonferroni multiple comparison test. P values <0.05 were considered statistically significant. When the single tail t-test returns a P-value <0.1, a trend or trend is assumed. All results are expressed as mean ± SD. Fractional ranges of NAS values are shown in table 6 below.

[ Table 4]

[ Table 5]

[ Table 6]

As shown in fig. 13 and 14 and table 4, liver sections from the control group showed microbubble and large-bubble fat deposition, hepatocyte gas spheroidisation and inflammatory cell infiltration. The OCA and EC-18 high group showed a significant decrease in NAS compared to the control group. NAS tended to decrease in the MGL-3196, EC-18 low and EC-18 middle groups compared to the control group.

Example 11: histological analysis to determine the efficacy of liver fibrosis

The same was applied to the hepatocytes of the mouse used in Experimental example 1. Fig. 15 is a photomicrograph of HE stained liver sections. Specifically, to visualize collagen deposition, fixed liver sections of Bouin were stained using sirius red solution. For quantitative analysis of the fibrotic region, bright field images of sirius red stained sections were randomly captured around the central vein at 200 x magnification using a digital camera.

FIG. 16 is a diagram showing a liver tissue fibrous part (lupus erythematosus-positive region) when the composition of the present invention is used. Specifically, in liver sections stained with sirius red solution, fibrosis sites were observed, and the values of the sites are shown in table 7 below.

[ Table 7]

As shown in fig. 15 and table 7, liver sections of the control group showed increased collagen deposition in the central peripheral region of the liver lobules. The OCA and EC-18 high group showed a significant reduction in the fibrotic region (sirius red positive region) compared to the control group. The fibrosis area of MGL-3196 and EC-18 low groups tended to decrease compared to the vector group. There was no significant difference in the area of fibrosis between the vehicle group and the EC-18 intermediate group.

As shown in the examples, EC-18 low and high showed statistical reduction in NAS and fibrotic regions, and in EC-18, NAS. In conclusion, EC-18 is characterized by an anti-NASH and anti-fibrotic effect.

Example 12 in vivo efficacy Studies of EC-18 in a STATM mouse model of non-alcoholic steatohepatitis

In addition, in vivo efficacy studies of EC-18 in the STAM model of nonalcoholic steatohepatitis were performed.

Glossary

CK-18: cytokeratin 18

MCP: monocyte chemotactic protein

NASH: non-alcoholic steatohepatitis

OCA: obeticholic acid

PBS: phosphate buffered saline

SD: standard deviation of

SMA: smooth muscle actin

STAM: stelic animal model

TGF: transforming growth factor

And (3) TIMP: tissue inhibitors of metalloproteinases

TNF: tumor necrosis factor

Materials and methods

Sample (I)

Liver and plasma samples from the following groups were used.

Group 1: vector set

Eight NASH mice were orally administered vehicle group [ PBS ] in a volume of 5mL/kg once a day during the 6 to 9 week period.

Group 2: MGL-3196

Eight NASH mice were orally supplemented with MGL-3196 [ 2% Klucel LF, 0.1% Tween 80 in water ] once a day during the 6 to 9 week age at a dose of 3mg/kg (volume 5 mL/kg).

Group 3: OCA

Seven NASH mice were orally supplemented with OCA once a day at a dose of 30mg/kg [ 1% methylcellulose ] (volume of 5mL/kg) over a period of 6 to 9 weeks of age.

Group 4: EC-1830 mg/kg

Eight NASH mice were orally supplemented with EC-18 in a vehicle group at a dose of 30mg/kg (volume 5mL/kg) once a day over a period of 6 to 9 weeks of age

mg/kg (volume 5mL/kg)

Group 5: EC-18100 mg/kg

Seven NASH mice were orally supplemented with EC-18 in a vehicle group at a dose of 100mg/kg (volume 5mL/kg) once a day over a period of 6 to 9 weeks of age.

Group 6: EC-18250 mg/kg

Eight NASH mice were orally supplemented with EC-18 in a vehicle group at a dose of 250mg/kg (volume 5mL/kg) once a day over a period of 6 to 9 weeks of age

Determination of hepatic triglyceride content

The total lipid extract from the liver was obtained by the method of Folch (Folch J. et al, J.biol.chem.1957; 226: 497). Liver samples were homogenized in chloroform-methanol (2:1, v/v) and incubated overnight at room temperature. After washing with chloroform-methanol-water (8:4:3, v/v/v), the extract was evaporated to dryness and dissolved in isopropanol. The hepatic triglyceride content was measured by the triglyceride E-test kit (FUJIFILM Wako Pure Chemical Corporation, Japan).

Measurement of plasma CK-18 levels

Plasma CK-18 levels were quantified by the mouse cytokeratin 18-M30 ELISA kit (Cusabio Biotech Co., Ltd, China).

Histological analysis

For immunohistochemistry, sections were cut from frozen liver Tissue embedded in Tissue-Tek o.c.t. The compound was immobilized in acetone. Endogenous peroxidase activity was blocked with 0.03% H2O2 for 5 minutes, followed by incubation with Block Ace (DS Pharma Biomedical co., ltd., Japan) for 10 minutes. Sections were incubated with anti-F4/80 antibody for 1 hour at room temperature. The sections were then incubated with biotin-conjugated secondary antibodies (VECTASTAIN Elite ABC kit, Vector labs, inc. usa) for 30 minutes at room temperature, and then with ABC reagents, respectively. The enzyme-substrate reaction was performed using a3, 3' -diaminobenzidine/H2O 2 solution (Nichirei Bioscience inc., Japan). For quantitative analysis of the inflammatory area, bright field images of F4/80 immunostained sections were captured around the central vein at 200-fold magnification using a digital camera (DFC 295; Leica), and measurements were made using 5 fields per positive area of the section. ImageJ software (National Institute of Health). The profiles of primary and secondary antibodies are shown in table 8.

[ Table 8]

Quantitative RT-PCR

Total RNA was extracted from liver samples using RNAiso (Takara Bio, Japan) according to the manufacturer's instructions. Mu.g of RNA was reverse transcribed (Promega), 5 Xfirst Strand buffer (Promega), 10mM dithiothreitol (Invitrogen, USA) and 200U MMLV-RT (Invitrogen) using a reaction mixture containing 4.4mM MgCl2(F.Hoffmann-La Roche, Switzerland), 40U RNase inhibitor (Toyobo, Japan), 0.5mM dNTP (Promega, USA), 6.28. mu.M random hexamer, to a final volume of 20. mu.L. The reaction was carried out at 37 ℃ for 1 hour and then at 99 ℃ for 5 minutes. Real-time PCR was performed using real-time PCR DICE and TB GreenTM Premix Ex TaqTM II (Takara Bio). To calculate the relative mRNA expression levels, the expression of each gene was normalized to the expression of the reference gene 36B4 (gene symbol: Rplp 0). Information on the PCR-primer sets and plate arrangements are described in tables 9 to 10.

[ Table 9] PCR-primers

36B 4: ribosomal protein, Large, PO (Rplp0)

MCP-1: chemokine (C-C motif) ligand 2(ccl2)

TNF- α: tumor necrosis factor (Tnf)

TGF-. beta.s: transforming growth factor, beta 1(Tgfb1)

TIMP-1: tissue inhibitors of metalloproteinase 1 (Timp1)

alpha-SMA: actin, α 2, smooth muscle, aorta (Acta2)

TABLE 10 PCR-plate

Statistical test

Statistical analysis was performed on GraphPad Prism 6(GraphPad software inc., USA) using Bonferroni multiple comparison test. P values <0.05 were considered statistically significant. When the single tail t-test returns a P-value <0.1, a trend or trend is assumed. Results are expressed as mean ± SD.

Results

Biochemistry

Liver triglycerides (FIG. 17 and Table 11)

The liver triglyceride content was significantly reduced in the MGL-3196 group compared to the vehicle group. The hepatic triglyceride content tended to decrease in the EC-18100 mg/kg and EC-18250 mg/kg groups compared to the vehicle group. There was no significant difference in hepatic triglyceride content between the vehicle group and the other treatment groups.

Plasma CK-18 (FIG. 18 and Table 11)

The EC-1830 mg/kg, EC-18100 mg/kg and EC-18250 mg/kg groups showed a significant decrease in plasma CK-18 levels compared to the vector group. Plasma CK-18 levels tended to be reduced in the OCA group compared to the vehicle group. There was no significant difference in plasma CK-18 levels between the vector group and MGL-3196 group.

[ Table 11]

F4/80 immunostaining and inflammatory area (FIGS. 19-20 and Table 12)

A representative micrograph of the F4/80 immunostained section is shown in FIG. 2.1. F4/80 immunostaining of liver sections from the vector group demonstrated the accumulation of F4/80+ cells in liver lobules. The OCA and EC-18250 mg/kg groups showed a significant reduction in the inflammatory zone (F4/80 positive zone) compared to the vehicle group. The areas of inflammation tended to decrease in the EC-1830 mg/kg and EC-18100 mg/kg groups compared to the vehicle group.

[ Table 12] histological analysis

Gene expression analysis (FIG. 21 and Table 13)

MCP-1

The expression level of MCP-1mRNA in MGL-3196 group tends to be down-regulated compared to the vector group. There was no significant difference in MCP-1mRNA expression levels between the vector group and the other treatment groups.

TNF-α

There was no significant difference in the level of expression of TNF-. alpha.mRNA between the vector group and any of the treatment groups.

TGF-β

The TGF-. beta.mRNA expression levels of the MGL-3196 group tended to be down-regulated compared to the vector group. There was no significant difference in TGF- β mRNA expression levels between the vector group and the other treatment groups.

TIMP-1

There was no significant difference in TIMP-1mRNA expression levels between the vector group and any of the treatment groups.

α-SMA

There was no significant difference in the expression level of α -SMA mRNA between the vector group and any of the treatment groups.

[ Table 13] analysis of Gene expression

Summary of the invention

MGL-3196

MGL-3196 treatment showed a significant reduction in hepatic triglyceride levels and a downward trend in MCP-1 and TGF-beta mRNA expression levels compared to the vector group.

OCA

OCA treatment showed a significant reduction in the inflammatory zone and a downward trend in plasma CK-18 levels compared to vehicle group.

EC-18

Treatment with EC-18 at a dose of 30mg/kg showed a significant decrease in plasma CK-18 levels and a downward trend in the inflammatory zone compared to the vehicle group. Treatment with EC-18 at a dose of 100mg/kg showed a significant reduction in plasma CK-18 levels and a trend towards a reduction in hepatic triglyceride content and inflammatory area compared to the vehicle group. Treatment with EC-18 at a dose of 250mg/kg showed a significant reduction in plasma CK-18 levels and inflammatory areas, with a trend toward a decrease in hepatic triglyceride levels, compared to the vehicle group.

All documents mentioned herein are fully incorporated by reference herein.

Claims (55)

1. A composition for treating fatty liver disease comprising a compound of formula 1,

[ formula 1]

Wherein R1 and R2 are independently fatty acid residues of 14 to 22 carbon atoms.

2. The composition of claim 1, wherein R1 and R2 are independently selected from the group consisting of palmitoyl, oleoyl, linoleoyl, linolenoyl, stearoyl, myristoyl, and arachidonoyl.

3. The composition of claim 1, wherein the compound of formula 1 is a compound of formula 2:

[ formula 2]

4. The composition of any one of claims 1 to 3, wherein the fatty liver disease is non-alcoholic fatty liver disease (NAFLD).

5. The composition of claim 4, wherein the non-alcoholic fatty liver disease (NAFLD) is non-alcoholic steatohepatitis (NASH).

6. The composition of claim 4, wherein the non-alcoholic fatty liver disease (NAFLD) is liver fibrosis.

7. The composition of any one of claims 1 to 6, wherein the fatty liver disease is induced by type 1diabetes (T1D).

8. The composition according to any one of claims 1 to 7, wherein the compound of formula 1 reduces the concentration of Triglycerides (TG) in hepatocytes and plasma.

9. The composition of any one of claims 1 to 8, wherein the compound of formula 1 improves expression of lipoprotein lipase (LPL).

10. The composition of any one of claims 1 to 9, wherein the compound of formula 1 reduces the expression of apolipoproteins including ApoB48 in the portal vein.

11. The composition of any one of claims 1 to 10, wherein the composition comprises one or more compounds of formula 1 in an amount of 0.001 to 100% by weight of the composition.

12. A method of treating fatty liver disease comprising administering to an individual a composition according to any one of claims 1 to 11.

13. A health functional food composition for alleviating or preventing fatty liver disease, comprising a monoacetyldiacylglycerol compound of formula 1,

[ formula 1]

Wherein R1 and R2 are independently fatty acid residues of 14 to 22 carbon atoms.

14. The health functional food composition of claim 13, wherein R1 and R2 are independently selected from the group consisting of palmitoyl, oleoyl, linoleoyl, linolenoyl, stearoyl, myristoyl, and arachidonyl.

15. A method of treating an individual suffering from or susceptible to fatty liver disease, comprising:

administering to the individual an effective amount of a compound of formula 1:

[ formula 1]

Wherein R1 and R2 are independently fatty acid residues of 14 to 22 carbon atoms.

16. The method of claim 15, wherein R1 and R2 are independently selected from the group consisting of palmitoyl, oleoyl, linoleoyl, linolenoyl, stearoyl, myristoyl, and arachidonoyl.

17. The method of claim 15, wherein the compound of formula 1 is a compound of formula 2:

[ formula 2]

18. The method of any one of claims 15 to 17, wherein the individual has or is predisposed to non-alcoholic fatty liver disease (NAFLD).

19. The method of any one of claims 15 to 18, wherein the individual is suffering from or susceptible to nonalcoholic steatohepatitis (NASH).

20. The method of any one of claims 15 to 19, wherein the individual is suffering from or susceptible to liver fibrosis.

21. A method of treating an individual suffering from or susceptible to non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), liver fibrosis, fatty liver disease caused by hepatitis, fatty liver disease caused by obesity, fatty liver disease caused by diabetes, fatty liver disease caused by insulin resistance, fatty liver disease caused by hypertriglyceridemia, β -lipoprotein deficiency, glycogen storage disease, nodular non-suppurative panniculitis, walmann's disease, acute fatty liver during pregnancy, and/or lipodystrophy, the method comprising:

administering to the individual an effective amount of a compound of formula 1:

[ formula 1]

Wherein R1 and R2 are independently fatty acid residues of 14 to 22 carbon atoms.

22. The method of claim 21, wherein R1 and R2 are independently selected from the group consisting of palmitoyl, oleoyl, linoleoyl, linolenoyl, stearoyl, myristoyl, and arachidonoyl.

23. The method of claim 21, wherein the compound of formula 1 is a compound of formula 2:

[ formula 2]

24. The method of any one of claims 17 to 23, wherein:

the subject is identified as having or susceptible to non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), liver fibrosis, fatty liver disease caused by hepatitis, fatty liver disease caused by obesity, fatty liver disease caused by diabetes, fatty liver disease caused by insulin resistance, fatty liver disease caused by hypertriglyceridemia, beta-lipoprotein deficiency, glycogen storage disease, nodular non-suppurative panniculitis, Wolman's disease, acute fatty liver and/or lipodystrophy during pregnancy, and

administering said compound of formula 1 to said identified individual.

25. The method of claim 24, wherein said individual is identified as having nonalcoholic fatty liver disease (NAFLD), and said compound of formula I is administered to said identified individual.

26. The method of claim 24, wherein said individual is identified as having nonalcoholic steatohepatitis (NASH), and said compound of formula I is administered to said identified individual.

27. The method of claim 24, wherein the individual is identified as having liver fibrosis, and the compound of formula I is administered to the identified individual.

28. The method of any one of claims 12 or 15-27, wherein the subject is a human.

29. A kit, comprising:

(a) a compound or composition according to any one of claims 1 to 11;

(b) instructions for using the compound to treat or prevent fatty liver disease in an individual.

30. A kit, comprising:

(a) l-palmitoyl-2-linoleoyl-3-acetyl glycerol (PLAG);

(b) instructions for using the PLAG to treat or prevent fatty liver disease.

31. The kit of claim 31, wherein the kit comprises a therapeutically effective amount of PLAG.

32. The kit of any one of claims 29 to 31, wherein the kit comprises written instructions for using the compound or PLAG.

33. The kit of any one of claims 29 to 32, wherein the kit comprises written instructions for using the compound or PLAG to treat non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), liver fibrosis, fatty liver disease caused by hepatitis, fatty liver disease caused by obesity, fatty liver disease caused by diabetes, fatty liver disease caused by insulin resistance, fatty liver disease caused by hypertriglyceridemia, β -lipoprotein deficiency, glycogen storage disease, nodular non-suppurative panniculitis, walmann's disease, acute fatty liver of pregnancy, and/or lipodystrophy.

34. The kit of any one of claims 29 to 33, wherein the instructions are a product label.

35. A method of treating an individual suffering from or susceptible to a fatty liver disease, comprising administering to the individual i) an effective amount of a compound of formula 1, and ii) one or more additional liver fibrosis therapeutic agents different from the compound of formula 1:

[ formula 1]

Wherein R1 and R2 are independently fatty acid residues of 14 to 22 carbon atoms.

36. The method of claim 34, wherein R1 and R2 are independently selected from the group consisting of palmitoyl, oleoyl, linoleoyl, linolenoyl, stearoyl, myristoyl, and arachidonoyl.

37. The method of claim 34, wherein the compound of formula 1 is a compound of formula 2:

[ formula 2]

38. The method of any one of claims 34 to 36, wherein the individual is suffering from or susceptible to non-alcoholic fatty liver disease (NAFLD).

39. The method of any one of claims 34 to 37, wherein the individual has or is predisposed to nonalcoholic steatohepatitis (NASH).

40. The method of any one of claims 34 to 38, wherein the individual is suffering from or susceptible to liver fibrosis.

41. The method of any one of claims 34 to 39, wherein the one or more different fibrosis therapeutics comprises one or more agents selected from the group consisting of obeticholic acid (OCA), elafibranor (GFT505), selonsertib (GS-4997), cericiriorc (CVC), liraglutide, metadoxine, hydroxytyrosol and vitamin E, NGM282(M70), BMS-986036, emricasan (IDN-6556), aramchol, atorvastatin and/or levocarnitine, MGL-3196, volixibat (SHP62667-s), GS-9674, semaglutide, saroglitazar, NCT02605616 (Meito medical center, Rochester, Minn., USA), LMB763, IVA337, LJN452, CF102, MT-3995, pioglitazone, MN-001 (tipelikast), MSDC-0602K, JKB-121, IMM-124E and/or ARI-3037MO, and pharmaceutically acceptable salts or acids thereof.

42. A method of treating an individual suffering from or susceptible to non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), liver fibrosis, fatty liver disease caused by hepatitis, fatty liver disease caused by obesity, fatty liver disease caused by diabetes, fatty liver disease caused by insulin resistance, fatty liver disease caused by hypertriglyceridemia, β -lipoprotein deficiency, glycogen storage disease, nodular non-suppurative panniculitis, walman's disease, acute fatty liver during pregnancy, and/or lipodystrophy, the method comprising administering to the individual i) an effective amount of a compound of formula 1, and ii) an effective amount of one or more liver fibrosis therapeutics other than one or more compounds of formula 1:

[ formula 1]

Wherein R1 and R2 are independently fatty acid residues of 14 to 22 carbon atoms.

43. The method of claim 41, wherein R1 and R2 are independently selected from the group consisting of palmitoyl, oleoyl, linoleoyl, linolenoyl, stearoyl, myristoyl, and arachidonoyl.

44. The method of claim 41, wherein the compound of formula 1 is a compound of formula 2:

[ formula 2]

45. The method of any one of claims 41 to 43, wherein the individual is suffering from or susceptible to non-alcoholic fatty liver disease (NAFLD).

46. The method of any one of claims 41 to 44, wherein the individual is suffering from or susceptible to nonalcoholic steatohepatitis (NASH).

47. The method of any one of claims 41 to 45, wherein the individual is suffering from or susceptible to liver fibrosis.

48. The method of any one of claims 41 to 46, wherein the one or more different fibrosis therapeutic agents comprises one or more agents selected from the group consisting of obeticholic acid (OCA), elafibranor (GFT505), selonsertib (GS-4997), cericiriorc (CVC), liraglutide, metadoxine, hydroxytyrosol and vitamin E, NGM282(M70), BMS-986036, emricasan (IDN-6556), aramchol, atorvastatin and/or levocarnitine, MGL-3196, volixibat (SHP62667-s), GS-9674, semaglutide, saroglitazar, NCT02605616 (Meito medical center, Rochester, Minn., USA), LMB763, IVA337, LJN452, CF102, MT-3995, pioglitazone, MN-001 (tipelikast), MSDC-0602K, JKB-121, IMM-124E and/or ARI-3037MO, and pharmaceutically acceptable salts or acids thereof.

49. A kit, comprising:

(a) a compound or composition according to any one of claims 1 to 11;

(b) one or more therapeutic agents for liver fibrosis other than a compound or composition according to any one of claims 1 to 11; and

(c) instructions for using the compound and one or more different therapeutic agents for liver fibrosis to treat or prevent a fatty liver disease in an individual.

50. A kit, comprising:

(a) l-palmitoyl-2-linoleoyl-3-acetyl glycerol (PLAG);

(b) one or more liver fibrosis therapeutic agents other than the PLAG; and

(b) instructions for using the PLAG and one or more additional therapeutic agents for liver fibrosis to treat or prevent fatty liver disease.

51. The kit of claim 49, wherein the kit comprises a therapeutically effective amount of PLAG.

52. The kit of any one of claims 48 to 50, wherein the kit comprises written instructions for using the compound or PLAG and the one or more different therapeutic agents for liver fibrosis.

53. The kit of any one of claims 48 to 51, wherein the kit comprises written instructions for using i) the compound or the PLAG and ii) the one or more additional liver fibrosis therapeutics to treat non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), liver fibrosis, hepatitis-induced fatty liver disease, obesity-induced fatty liver disease, diabetes-induced fatty liver disease, insulin resistance-induced fatty liver disease, hypertriglyceridemia-induced fatty liver disease, beta-lipoprotein deficiency, glycogen storage disease, nodular non-suppurative steatohepatitis, Wolman's disease, acute fatty liver of pregnancy, and/or lipodystrophy.

54. The kit of any one of claims 48 to 52, wherein the instructions are a product label.

55. The kit of any one of claims 48 to 53, wherein the one or more different fibrosis therapeutics comprises one or more of obeticholic acid (OCA), elafinibant (GFT505), selonsertib (GS-4997), cericivir (CVC), liraglutide, metadoxine, hydroxytyrosol and vitamin E, NGM282(M70), BMS-986036, emricasan (IDN-6556), aramchol, atorvastatin and/or levocarnitine, MGL-3196, volixibat (SHP62667-s), GS-9674, semaglutide, saroglitazar, medicament in NCT02605616 (Meioer center of medicine, Rochester, Minn., USA), LMB763, IVA337, LJN452, CF102, MT-3995, pioglitazone, MN-001 (tipelikast), MSDC-0602K, JKB-121, IMM-124E and/or ARI-3037MO, and one or more pharmaceutically acceptable salts or acids thereof.

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