AU2020231261A1 - Composition comprising monoacetyldiacylglycerol compound for treating fatty liver disease - Google Patents

Abstract

Disclosed is a composition for treating fatty liver disease. The composition comprises a monoacetyldiacylglycerol compound of Formula 1 as an active ingredient for treating fatty liver disease wherein R1 and R2 are independently a fatty acid residue of 14 to 22 carbon atoms.

Description

COMPOSITION COMPRISING MONOACETYLDIACYLGL Y CEROL COMPOUND FOR TREATING FATTY LIVER DISEASE

Cross-reference to Related Application

[001] This application claims the benefit of U.S. Provisional Application No. 62/908,392 filed September 30, 2019; Korean Patent Application No. 10-2019- 0026326 filed on March 07, 2019; and Korean Patent Application No. 10-2019- 0086967 filed on July 18, 2019, the entire contents of which are incorporated herein by reference.

FIELD

[002] This invention relates to a composition comprising a monoacetyldiacylglycerol compound as an active ingredient for treating fatty liver disease and more particularly to a composition comprising a monoacetyldiacylglycerol compound for oral administration which prevents and/or alleviates symptoms of fatty liver disease.

BACKGROUND

[003] The liver is one of the most important metabolic organs in the body's organs and performs important functions such as bile secretion, nutrient storage, detoxification and so on. If the liver is in an abnormal state, metabolic disorders such as glucose metabolism, lipid metabolism, protein and nitrogen metabolism, amino acid metabolism, protein metabolism and hepatic brain disease, vitamin metabolism, poor absorption, may occur. In addition, hepatic diseases may be exacerbated by infection, fatty liver, cirrhosis, and the like. Hepatic diseases known to date are associated with acute hepatitis caused by drugs, alcoholic fatty liver, obesity, diabetes, non-alcoholic fatty liver caused by hyperlipidemia, acute hepatitis caused by virus infection, chronic hepatitis, cirrhosis, and so on.

[004] Among them, the non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease that is associated with obesity, diabetes, and hyperlipidemia, irrespective of alcohol, and is characterized by excessive lipid (mainly triglycerides) accumulation in the liver. Generally, in the NAFLD, more than 5 weight% of the total liver weight is the lipid. The NAFLD has potential to develop into steatohepatitis, fibrosis, cirrhosis, and even hepatocellular carcinoma. [005] There are numerous studies on NAFLD, but relatively little is known about its association with type 1 diabetes(TlD). Recent reports have pointed out that T1D may could cause NAFLD, which is as an independent risk factor for chronic diabetic complications.

[006] The type 1 diabetes (T1D) is an autoimmune disease in which the patient's own immune system attacks and destroys beta cells. The pathogenesis of fatty liver in the type 1 diabetes(TlD) is not yet clear. However, Regnell and Lemmark suggested that it may be caused by abnormalities of lipoproteins, which mainly serve to transport lipids through the bloodstream and lymphocytes and/or activation of the transcription factors, carbohydrate response element-binding protein(ChREBP) and sterol regulatory element-binding protein lc(SREBP-lC), which regulate glucose metabolism and lipid synthesis, respectively (S.E. Regnell, A. Lemmark, Hepatic steatosis in type 1 diabetes Rev Diabet Stud 8 (2011) 454-467).

[007] Non-alcoholic steatohepatitis (NASH) is one of the non-alcoholic fatty liver diseases (NAFLD). The root cause of NASH is unknown. However, upon analyzing the patients having NASH, NASH is associated with obesity, type 2 diabetes, dyslipidemia, or metabolic syndrome (waist circumference of above 90cm, triglyceride of 150 mg/dL or more, HDL of 40 mg/dL or less, fasting blood glucose of 100 mg/dL or more, systolic blood pressure of 130 mmHg or more). Therefore, they are regarded as factors for causing non-alcoholic fatty liver disease (NAFLD) and are recognized as the main cause of the increase in non-alcoholic steatohepatitis (NASH) patients.

[008] It is also known that the non-alcoholic fatty liver disease (NAFLD) increases the risk of developing type 2 diabetes 2 to 4 times compared with the control group not having the non-alcoholic fatty liver disease (NAFLD). In other words, non-alcoholic fatty liver disease (NAFLD) would be closely related to an insulin resistance.

[009] Lipoprotein lipase (LPL) acts as a pancreatic, hepatic, and endothelial lipase, and is mainly distributed in skeletal muscle, heart and adipose tissue. The lipoprotein lipase (LPL) includes a chylomicron (CM) that binds to ApoE (APO protein) and distributes triglycerides to muscle and adipose tissue, and is very low-density lipoprotein, which hydrolyze triglycerides (triglycerides, TG). These play an important role in hydrolyzing triglyceride (TG) and removing triglyceride (TG) from peripheral tissues such as muscle and fat tissue for energy or storage sources. Therefore, when the lipoprotein lipase (LPL) is reduced(lost), triglyceride (TG) is excessively circulated, which is a cause of hypertriglyceridemia. [010] Streptozotocin (STZ) is a selective pancreatic b cell toxin that induces rapid and irreversible necrosis of cells and is often used to induce diabetes. It is also a suitable candidate for modeling liver steatosis. STZ-induced diabetes is characterized by hyperglycemia, dyslipidemia, weight loss, and hepatic steatosis.

[011] It thus would be desirable to have new therapies for fatty liver disease.

SUMMARY

[012] In one aspect, compositions and methods are provided for treating fatty liver diseases 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 improved

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

[014] More particularly, compositions and methods are provided comprising a monoacetyldiacylglycerol compound of Formula 1 for treating fatty liver disease.

[015] [Formula 1]

[016] 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.

[017] In one embodiment, the monoacetyldiacylglycerol is a compound of the following Formula 2:

[018] [Formula 2]

[019] The compound of Formula 2 is 1 -palmitoyl -2 -linoleoyl-3 -acetyl -rac-glycerol and corresponds to the compound of Formula 1 in which R₁ and R₂ of Formula 1 are palmitoyl and linoleoyl, respectively. The compound of Formula 2 is sometimes referred as“PLAG” or“EC-18” in this disclosure.

[020] As discussed, compounds of Formulae I and 2 may be used to treat a subject suffering from or susceptible to a fatty liver disease including a non-alcoholic fatty acid liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), fatty liver disease resulting from hepatitis, fatty liver disease resulting from obesity, fatty liver disease resulting from diabetes, fatty liver disease resulting from insulin resistance, fatty liver disease resulting from hypertriglyceridemia, Abetalipoproteinemia, glycogen storage diseases, Weber-Christian disease, Wolmans disease, acute fatty liver of pregnancy, and/or lipodystrophy.

[021] In a particular aspect, a compound of Formula 1 and 2 is used to treat a subject suffering from or susceptible to non-alcoholic steatohepatitis (NASH).

[022] In a further particular aspect, a compound of Formula 1 and 2 is used to treat a subject suffering from or susceptible to non-alcoholic fatty acid liver disease (NAFLD).

[023] In particular aspects, a subject will be identified and selected for treatment of a disease or disorder as disclosed herein, and then a compound of Formula 1 or 2 will be administered to the identified and selected subject.

[024] For instance, a patient may be identified and selected as suffering from non- alcoholic steatohepatitis (NASH) and that patient identified as suffering from non- alcoholic steatohepatitis (NASH) may be administered a compound of Formula 1 or 2 to thereby alleviate or treat the non-alcoholic steatohepatitis (NASH). A patient may be identified and selected as suffering from non-alcoholic fatty acid liver disease (NAFLD) and that patient identified as suffering from non-alcoholic fatty acid liver disease (NAFLD) may be administered a compound of Formula 1 or 2 to thereby alleviate or treat the non-alcoholic fatty acid liver disease (NAFLD). A patient may be identified and selected as suffering from liver fibrosis and that patient identified as suffering from liver fibrosis may be administered a compound of Formula 1 or 2 to thereby alleviate or treat the non-alcoholic fatty acid liver disease (NAFLD).

[025] In certain aspects, one or more compounds of Formula 1 or 2, or PLAG may be administered to a subject in combination or coordination with one or more liver fibrosis treatment agents that are distinct from the one or more compounds of Formula 1 or 2, or PLAG. In certain aspects, the one or more distinct liver fibrosis treatment agents that are co-administered or administered in combination with one or more compounds of Formula 1 or 2, or PLAG may be 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 L carnitine, MGL-3196 (Resmetirom), volixibat (SHP626), GS-9674, semaglutide, saroglitazar, agents in NCT02605616 (Mayo Clinic, Rochester, MN, USA), LMB763, IVA337, LJN452, CF102, MT-3995, pioglitazone, MN-001 (tipelukast), MSDC-0602K, JKB-121, IMM-124E, and/or ARI-3037MO, and pharmaceutically acceptable salts or acids thereof.

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

[027] As referred to herein a liver fibrosis treatment agent will be distinct from a compound of Formula 1 or 2, or PLAG where the liver fibrosis treatment agent differs in chemical structure from a compound of Formula 1 or 2, or PLAG. For instance, a distinct liver fibrosis treatment may not comprise a monoacetyldiacylglycerol structure, or may not comprise a diacylglycerol structure compound, or may not comprise a glycerol structure. An administered distinct liver fibrosis treatment agent also may differ (either higher or lower) in molecular weight from the co-administered compound of Formula 1 or 2, or PLAG by at least 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90 or 100 percent.

[028] In certain aspects, the present therapeutic methods are not associated with treatment of a subject suffering from hepatitis. In this aspect, subjects that are suffering from hepatitis and/or are seeking treatment for hepatitis would be excluded from the present therapeutic methods. In a related aspect, subjects seeking treatment that have been identified as having hepatitis would be excluded from the present therapeutic methods. [029] In further aspects, the present therapeutic methods are not associated with treatment of a subject suffering from diabetes. In this aspect, subjects that are suffering from diabetes and/or are seeking treatment for diabetes would be excluded from the present therapeutic methods.

[030] In yet additional aspects, the present therapeutic methods are not associated with a subject suffering from a wound or injured tissue. In this aspect, subjects that are suffering from a wound or injured tissue and/or are seeking treatment for a wound or injured tissue would be excluded from the present therapeutic methods. In a related aspect, subjects seeking treatment involving tissue repair or regeneration would be excluded from the present therapeutic methods.

[031] In a further aspect, pharmaceutical compositions are provided comprising a compound of Formula 1 or 2 as set forth above. The compositions suitably may comprise one or more pharmaceutically acceptable carriers. In preferred embodiments, the compositions may be formulated or otherwise adapted for treatment of fatty liver disease as disclosed herein. In preferred aspects, the composition may be adapted for oral administration as a tablet or capsule.

[032] In a yet further aspect, kits are provided for use to treat or prevent a fatty liver disease as disclosed herein. Kits of the invention suitably may comprise 1) one or more compounds of Formulae 1 or 2; and 2) instructions for using the one or more compounds for treating or preventing fatty liver disease as disclosed herein. Preferably, a kit will comprise a therapeutically effective amount of one or more compounds of Formulae 1 or 2. The instructions suitably may be in written form, including as a product label.

[033] The present invention also provides a health functional food composition comprising a monoacetyldiacylglycerol compound of the Formula 1 for alleviating or preventing fatty liver disease.

[034] The composition of the present invention can reduce the expression of apo B protein including ApoB48 in the portal vein, is non-toxic and treats and/or alleviates fatty liver disease.

[035] Other aspects of the invention are disclosed infra.

BRIEF DESCRIPTION OF THE DRAWINGS The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[036] FIG. 1 shows an experimental protocol of the present invention.

[037] FIG. 2 (includes FIGS. 2A and 2B) shows a graph and chart (FIG. 2A) showing the body weight change and the chart (FIG. 2B) showing the liver weight change, when administering the composition according to the present invention.

[038] FIG. 3 shows photomicrographs of liver sections stained with H&E and ORO, when using the composition according to the present invention.

[039] FIG. 4 shows an exemplary mechanism in which fat absorbed in the small intestine affects the liver.

[040] FIG. 5 (includes FIGS. 5A and 5B) shows a chart (FIG. 5A) showing the TG levels and contents in the liver and plasma and a chart (FIG. 5B) showing the apoB48 protein expression in portal vein plasma, when using the composition according to the present invention.

[041] FIG. 6 (includes FIGS. 6A through 6C) shows a chart (FIG. 6A) showing the TG content in muscle tissue, a chart (FIG. 6B) showing the relative mRNA expression of LPL in muscle tissue and pictures (FIG. 6C) showing the representative immunohistochemical images of LPL staining in muscle sections, when using the composition according to the present invention.

[042] FIG. 7 (includes FIGS. 7A through 7C) shows a chart (FIG. 7A) showing the weight of muscle specimens and skeletal muscle from mice, and the result of the relative mRNA expression of caveolin3 in muscle tissue, including the gastrocnemius and quadriceps (FIG. 7B) and the result of the relative mRNA expression of caveolin3 and myogenin in myoblasts and myotubes (FIG. 7C).

[043] FIG. 8 (includes FIGS. 8A through 8D) shows structural chemical formulas of PLAG and PLG (FIG. 8A) and a chart (FIG. 8B) showing the body weight change and the picture of the Gross liver specimens from each group (FIG. 8C) and pictures (FIG. 8D) of the H&E-stained liver sections.

[044] FIG. 9 shows another experimental protocol of the present invention.

[045] FIG. 10 is a graph showing the body weight change, when using the composition according to the present invention.

[046] FIG. 11 (includes FIGS. 11A through 11C) is a graph showing the weight (FIG. 11A), the liver weight (FIG. 11B) and the ratio of liver weight to weight (FIG. 11C), when using the composition according to the present invention.

[047] FIG. 12 (includes FIGS. 12A through 12C) is a graph showing the plasma ALT (FIG. 12A) and the plasma AST (FIG. 12B) and the ALT/AST ratio (FIG. 12C), when using the composition according to the present invention.

[048] FIG. 13 shows photomicrographs of liver sections stained with H&E and ORO, when using the composition according to the present invention.

[049] FIG. 14 (includes FIGS. 14A through 14D) shows graphs showing of liver sections stained with H&E and ORO, when using the composition according to the present invention.

[050] FIG. 15 shows photomicrographs of Sirius red-stained liver sections, when using the composition according to the present invention.

[051] FIG. 16 shows a graph showing the fibrous part of liver tissue (sirius red-positive area), when using the composition according to the present inventi on.

[052] FIG. 17 shows a graph of level of liver triglyceride in Groups 1-6 in Example 12.

[053] FIG. 18 shows a graph of plasma CK-18 in Groups 1-6 in Example 1 2

[054] FIG. 19 shows representative photomicrographs of F4/80-immunostained liver sections in Groups 1-6 in Example 12.

[055] FIG. 20 shows a graph of inflammation area (F4/80-positive area %) in Groups 1-6 in Example 12.

[056] FIG. 21 shows relative gene expression measured by quantitative RT-P CR in Groups 1-6 in Example 12.

DETAILED DESCRIPTION

[057] The composition for treating fatty liver disease according to the present invention comprises a monoacetyldiacylglycerol compound of Formula 1 as an active ingredient.

[058] [Formula 1]

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

[060] 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. The fatty acid residue means the remaining portion of the fatty acid in which the -OH group is excluded from its carboxyl group. In the Formula 1, non-limiting examples of R₁ and R2 thus include palmitoyl, oleoyl, linoleoyl, linolenoyl, stearoyl, myristoyl, arachidonoyl, and so on. Preferable 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 so on. More preferable combination of R₁ and R2 is palmitoyl/linoleoyl. In optical activity, the monoacetyldiacylglycerol derivatives of Formula 1 can be (R)-form, (S)-form or a racemic mixture, preferably a racemic mixture, and may include their stereoisomers.

[061] In one embodiment, the monoacetyldiacylglycerol is a compound of the following Formula 2:

[062] [Formula 2]

[063] The compound of Formula 2 is 1 -palmitoyl -2 -linoleoyl-3 -acetyl -rac-glycerol and corresponds to the compound of Formula 1 in which R₁ and R₂ of Formula 1 are palmitoyl and linoleoyl, respectively. The compound of Formula 2 is sometimes referred as“PLAG” or“EC-18” in this disclosure. [064] The monoacetyldiacylglycerol compounds can be separated and extracted from the natural deer antler or can be produced by known organic synthesis methods (Korean Patent No. 10-0789323). More specifically, deer antler is extracted with hexane, followed by extracting the residue with chloroform and removing the chloroform to provide chloroform extracts. The volume of the solvents for this extraction is just enough to immerse the deer antler. In general, about 4-5 liters of hexane and/or chloroform for 1kg of deer antler is used, but not limited thereto. The extracts obtained by this method is further fractionated and purified using series of silica gel column chromatograph and TLC method to obtain the monoacetyldiacylglycerol compound of the present invention. A solvent for the extraction is selected among chloroform/methanol, hexane/ethylacetate/acetic acid, but not limited thereto.

[065] A chemical synthetic method for the preparation of the monoacetyldiacylglycerol compounds 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 in the position 3 of 1-R1-glycerol; (b) a step of preparing 1- R1 -2 -R2 -3 -protecting group-glycerol by introducing R2 in the position 2 of the 1-R1-3-protecting group-glycerol; and (c) a step of preparing the desired monoacetyldiacylglycerol compound by performing a deprotection reaction and the acetylation reaction of the 1-R1 -3 -protecting group-glycerol at the same time. The monoacetyldiacylglycerol compound may be further purified if necessary. Alternatively, monoacetyldiacylglycerol compounds can be prepared by acid decomposition of phosphatidylcholine(acetolysis) but is not limited thereto. Stereoisomers of the compounds of formula 1 are also within the scope of the invention.

[066] The monoacetyldiacylglycerol compound of the present invention can be effectively used for the treatment and/or alleviation of fatty liver disease. The term "fatty liver disease" is a disease characterized by excessive fat (mainly triglycerides) accumulated in the liver. Generally, the term "fatty liver disease" means a state that the amount of fat in the liver is more than 5 weight% of the total liver weight.

[067] The fatty liver disease includes non-alcoholic fatty liver disease (NAFLD). The non-alcoholic fatty liver disease (NAFLD) is excessive fat build-up in the liver due to causes other than alcohol use.

[068] Specifically, the non-alcoholic fatty liver disease (NAFLD) is a chronic liver disease that is associated with overweight, obese, insulin resistance, diabetes (type 1 diabetes(TID), type 2 diabetes, etc.), dyslipidemia(hyperlipidemia), metabolic syndrome (for example, hypertension, high blood sugar, abdominal obesity, metabolic syndrome caused by low HDL cholesterol and high triglycerides), and so on. The NAFLD is caused by the accumulation of excessive fat (mainly, triglycerides) in the liver due to causes other than alcohol use, and usually refers to the accumulation of more than 5 weight% of fat with respect to the total liver weight. In this disclosure, the NAFLD includes the diseases resulting from the NAFLD.

[069] The non-alcoholic fatty liver disease (NAFLD) includes not only non-alcoholic fatty liver in which fat is simply accumulated in the liver without damaging hepatocytes, but also non-alcoholic steatohepatitis (NASH), liver fibrosis, cirrhosis, and even hepatocellular carcinoma. Specifically, the non-alcoholic fatty liver disease (NAFLD) can be classified into Type l to Type 4. Specifically, Type 1 means a simple fatty liver with only steatosis, without hepatocytes damage. Type 2 means a steatohepatitis with inflammatory reactions in the lobules, due to severe and persistent steatosis and hepatocytes damage. Type 3 means a state in which balloon-shaped alteration of hepatocytes and steatosis and fat necrosis appear. Type 4 means a state in which mallory body or fibrosis appear with the symptoms of Type 3. Cirrhosis may occur in Type 3 and Type 4.

[070] About 10% of all fatty liver disease patients are known as steatohepatitis. The non-alcoholic steatohepatitis (NASH) is caused by increased triglycerides and decreased fat metabolism by hyperinsulinemia. If the non-alcoholic fatty liver and non- alcoholic fatty hepatitis are not treated, liver function may be deteriorated, and liver cirrhosis may occur through liver fibrosis.

[071] The fibrosis is atype of wound healing process in liver tissue. When livertissue is damaged by hepatitis virus, non-alcoholic fatty liver, drugs, etc., the vascular structure collapses in the hepatic lobules, as the hepatocytes are continuously damaged and regenerated. As a result, regenerative nodules lost function take their place, and a fibrosis reaction occurs in the liver, and may lead to cirrhosis of the liver.

[072] The damaged hepatocytes secrete free radicals and inflammatory substances to activate Cooper cells and inflammatory cells, which leads to activation of hepatic stellate cells. Hepatic stellate cells are a reservoir of vitamin A in normal condition, but are activated when liver damage occurs, and can synthesize and secrete various extracellular substrates such as collagen to cause liver fibrosis. When liver damage is chronically repeated, damaged hepatocytes can no longer be regenerated and gradually replaced with extracellular matrix such as collagen, which can lead to liver fibrosis and cirrhosis.

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

[074] The monoacetyldiacylglycerol compound according to the present invention can be effectively used for the treatment of non-alcoholic fatty liver disease (NAFLD) including non-alcoholic fatty liver, non-alcoholic steatohepatitis (NASH) and liver fibrosis. The term“treatment” includes delay or reduction of symptoms, as well as partial or complete elimination or prevention of symptoms, of non-alcoholic fatty liver, non-alcoholic steatohepatitis (NASH) and liver fibrosis by administering the composition of the present invention.

[075] Combination therapy

[076] As discussed, one or more compounds of Formula 1 or 2, or PLAG and the one or more liver fibrosis treatment agents can be administered in combination such as to treat a subject including a human suffering from a fatty liver disease including NASH.

[077] As used herein, the term“in combination” in the context of the administration of a therapy to a subject refers to the use of more than one therapy for therapeutic benefit. The term“in combination” in the context of the administration can also refer to the prophylactic use of a therapy to a subject when used with at least one additional therapy. The use of the term“in combination” does not restrict the order in which the therapies (e.g., a first and second therapy) are administered to a subject. A therapy can be administered prior to (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), concomitantly with, or subsequent to (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 administration of a second therapy to a subject in need of treatment as disclosed herein. The therapies are administered to a subject in a sequence and within a time interval such that the therapies can act together. In a particular embodiment, the therapies are administered to a subject in a sequence and within a time interval such that they provide an increased benefit than if they were administered otherwise. Any additional therapy can be administered in any order with the other additional therapy.

[078] The administration of the compounds (e.g., compounds of Formula 1 or 2, or PLAG) and the one or more liver fibrosis treatment agents may be by suitable means that results in a concentration of the therapeutic that, combined with other components, is effective in ameliorating, reducing, or stabilizing a fat liver disease, e.g. non- alcoholic fatty acid liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), liver fibrosis, fatty liver disease resulting from hepatitis, fatty liver disease resulting from obesity, fatty liver disease resulting from diabetes, fatty liver disease resulting from insulin resistance, fatty liver disease resulting from hypertriglyceridemia, Abetalipoproteinemia, glycogen storage diseases, Weber-Christian disease, Wolmans disease, acute fatty liver of pregnancy, and/or lipodystrophy.

[079] The compound (e.g., compounds of Formula 1 or 2, or PLAG) and the one or more distinct liver fibrosis treatment agents may be administered simultaneously or sequentially. In some embodiments, the liver fibrosis treatment is an established therapy for the disease indication and by addition of compounds (e.g., compounds of Formula 1 or 2, or PLAG), such treatment improves the therapeutic benefit to the patients. Such improvement could be measured as increased responses on a per patient basis or increased responses in the patient population. Combination therapy could also provide improved responses at lower or less frequent doses of the therapeutic agents resulting in a better tolerated treatment regimen. As indicated, the combined therapy of the compound (e.g., compounds of Formula 1 or 2, or PLAG) and the one or more distinct liver fibrosis treatment agents could provide enhances clinical activity through various mechanisms, for example, mechanisms in which fat absorbed in the small intestine affects the liver (e.g., increased dietary fat delivery to the liver from the small intestine (gut), either due to increased intake or a dysregulated gut physiology and microbiome); 2) increased influx of free fatty acids from the non-esterified pool (e.g., from white adipose tissue); 3) increased de novo hepatic lipogenesis from excess carbohydrates (and or hyperinsulinemia from adipose tissue insulin resistance), and the like.

[080] In some embodiments, the methods (e.g., combination therapy for fat liver disease) may include administration of second therapeutic agent (e.g., one or more liver fibrosis treatment agents that are distinct from a compound of Formulae 1 or 2 or PLAG) or treatment with a second therapy (e.g., a therapeutic agent or therapy that is standard in the art) for liver fibrosis.

[081] In some embodiments, the methods (e.g., combination therapy for fat liver disease) may include administration of second therapeutic agent (e.g., one or more liver fibrosis treatment agents that are distinct from a compound of Formulae 1 or 2 or PLAG) or treatment with a second therapy (e.g., a therapeutic agent or therapy that is standard in the art) for treatment of NAFLD and/or NASH.

[082] Exemplary therapeutic agents include one or more liver fibrosis treatment agents. A“liver fibrosis treatment agent” is a chemical compound useful in the treatment of a fatty liver disease, for example, non-alcoholic fatty acid liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), liver fibrosis, fatty liver disease resulting from hepatitis, fatty liver disease resulting from obesity, fatty liver disease resulting from diabetes, fatty liver disease resulting from insulin resistance, fatty liver disease resulting from hypertriglyceridemia, Abetalipoproteinemia, glycogen storage diseases, Weber-Christian disease, Wolmans disease, acute fatty liver of pregnancy, and/or lipodystrophy.

[083] Examples of liver fibrosis treatment agents for use in the present methodsi, compositons 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 L carnitine, MGL-3196, volixibat (SHP626), GS-9674, semaglutide, saroglitazar, agents in NCT02605616 (Mayo Clinic, Rochester, MN, USA), LMB763, IVA337, LJN452, CF102, MT-3995, pioglitazone, MN-001 (tipelukast), MSDC-0602K, JKB-121, IMM-124E, and/or ARI-3037MO, and pharmaceutically acceptable salts or acids thereof. As discussed, certain preferred methods, kits and composition may comprise one or more liver fibrosis treatments agents that comprise MGL-3196 (Resmetirom) and/or obeticholic acid (OCA) including to treat a subject e.g. human suffering from a fatty liver disease or disorder such as NASH or NAFLD.

[084] In addition, examples of liver fibrosis treatment agents include peroxisome proliferator-activator receptors (PPARs a, b/d, and/or g) agonists (e.g., GW501516, elafibranor, thiazolidinediones, pioglitazone, or saroglitazar), FXR-bile acid axis (e.g., obeticholic acid, FGF-19, NGM-282, agents in NCT02548351, agents in NCT02443116, or the like), lipid-altering agents (e.g., stearoyl-CoA desaturase (SCD) inhibitor, aramchol,), agents for incretin-based therapies (e.g., exenatide, liraglutide, sitagliptin, or the like), agents targeting inflammation, cell injury or death (apoptosis) and oxidative stress (e.g., vitamin E, agents in ENCORE-NF; NCT02686762, pentoxifylline, or the like), agent used in gut and microbiome related therapies (e.g., IMM-124e, orlistat, agents in NCT02510599, or the like) and/or agents for antifibrotic therapies (e.g., simtuzumab, GR-MD-02, or the like).

[085] For instance, the peroxisome proliferator-activator receptors (PPARs) include a group of nuclear receptors that are expressed in the liver, adipose tissue, heart, skeletal muscle and kidney and transcriptionally regulate multiple metabolic processes including B-oxidation, lipid transport and gluconeogenesis.

[086] Additional liver fibrosis agents that may be used in the present methods, compositions and kits include soluble guanylate cyclase (sGC) stimulators such as disclosed in WO2017/136309; and cenicriviroc as disclosed in US2018/0360846.

[087] As discussed, 1) a compound of Formula 1 or 2, or PLAG and 2) one or more distinct liver fibrosis treatment agents may be“co-administered”, i.e, administered together in a coordinated fashion to a subject, either as separate pharmaceutical compositions or admixed in a single pharmaceutical composition. By “co- administered”, the one or more additional distinct liver fibrosis treatment agents may also be administered simultaneously with a compound of Formula 1 or 2, or be administered separately with compound of Formula 1 or 2, including at different times and with different frequencies. The one or more distinct liver fibrosis treatment agents may be administered by any appropriate route for the agent(s), such as orally, intravenously, subcutaneously, intramuscularly, nasally, and the like; and the therapeutic agent may also be administered by any conventional route. In at least certain embodiments, at the one or more distinct liver fibrosis agents may be administered orally.

[088] In some embodiments, the compounds (e.g., compounds of Formula 1 or 2, or PLAG) and/or the one or more distinct liver fibrosis treatment agents may be administered daily, e.g., every 24 hours, or, continuously or several times per day, 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 every 12 hours.

[089] Exemplary effective daily doses of the distinct liver fibrosis treatment agent(s) include between 0.1 mg/kg and 100 mg/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 mg/kg body weight.

[090] Alternatively, the distinct liver fibrosis treatment agent is administered about once per week, e.g., about once every 7 days. Or, the distinct liver fibrosis treatment agent is administered twice per week, three times per week, four times per week, five times per week, six times per week, or seven times per week. Exemplary effective weekly doses of the liver fibrosis treatment agents include between 0.0001 mg/kg and 4 mg/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 4 mg/kg body weight. For example, an effective weekly dose of the distinct liver fibrosis treatment agent is between 0.1 mg/kg body weight and 400 mg/kg body weight.

[091] In the examples of the present disclosure, when using the monoacetyldiacylglycerol compound, (1) it is shown that the body weight reduced by streptozotocin (STZ) is recovered and (2) it is shown that the liver weight, which is increased due to excessive accumulation of triglycerides(TG) in the liver, is reduced(Example 1). Thus, it can be seen that the administration of the monoacetyldiacylglycerol compound improves fatty liver.

[092] The triglyceride (TG, neutral fat) is absorbed in the small intestine as exogenous fat and includes the apolipoprotein of ApoB48. The triglyceride (TG) is recombined with chylomicron (CM) along with ApoB48. The triglyceride -rich CM is transported into the blood through the lymphatic system, while circulating throughout the body, and triglycerides (TG) are absorbed into peripheral tissues, including muscle, adipose tissue and monocytes, for energy or storage. And then, the triglycerides (TG) contained in the CM are hydrolyzed by the lipoprotein lipase (LPL) in order that it can be transported to the liver through the portal vein, resulting in smaller CM residual particles. Therefore, when the lipoprotein lipase (LPL) is reduced, 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. It can cause hypertriglyceridemia and lead to fatty liver disease.

[093] The ApoB48 is a lipoprotein that transports cholesterol in the blood and triglycerides. When the ApoB48 is excessive, it means that triglyceride (TG) is high, which contributes to dyslipidemia. Because of this, the uptake of triglycerides (TG) in the liver may promote fat accumulation of hepatocytes causing fatty liver (See FIG. 4).

[094] In other examples of the present disclosure, the liver of the experimental group was observed, hematoxylin and eosin(H&E) was used to image the vesicular fat, and oil red O(ORO, Sigma Aldrich, St. Louis, MO, USA) was used to measure the degree of accumulation of liver fat. It is shown that (1) the concentration of triglyceride(TG) in hepatocytes and plasma was reduced (Example 2, 3), (2) the expression of lipoprotein lipase(LPL) was improved(Example 4), and (3) the expression of apolipoprotein including ApoB48 in the portal vein was reduced (Example 3). It was confirmed that due to the monoacetyldiacylglycerol compounds, a functional improvement of peripheral tissues producing and secreting lipoprotein lipase (LPL) was achieved. These results also show that the monoacetyldiacylglycerol compound is effective for treating fatty liver.

[095] In other examples of the present disclosure, when using the monoacetyldiacylglycerol compound, it is shown that the NAFLD Activity score(NAS), steatosis, lobular inflammation and hepatocyte ballooning for non-alcoholic fatty liver disease(NAFLD) were reduced (Example 10), and (2) the fibrotic portion of the liver tissue was reduced (Example 11).

[096] The pharmaceutical composition comprising the monoacetyldiacylglycerol compound of the present invention may include conventional pharmaceutically acceptable carriers, excipients, or diluents. The amount of monoacetyldiacylglycerol in the pharmaceutical composition can be widely varied without specific limitation, and is specifically 0.0001 to 100 weight%, preferably, 0.001 to 90 weight%, for example, the monoacetyldiacylglycerol may be contained in 70 to 80 weight%, with respect to the total amount of the composition.

[097] The pharmaceutical composition of the present invention may further include other active ingredients having a therapeutic effect of fatty liver disease. The pharmaceutical composition may be formulated into solid, liquid, gel or suspension form for oral or non-oral administration, for example, tablet, bolus, powder, granule, capsule such as hard or soft gelatin capsule, emulsion, suspension, syrup, emulsifiable concentrate, sterilized aqueous solution, non-aqueous solution, freeze-dried formulation, and so on. In formulating the composition, conventional excipients or diluents such as fillers, bulking agents, binders, wetting agents, disintegrating agents, and surfactants can be used. The solid formulation for oral administration includes tablet, bolus, powder, granule, capsule and so on, and the solid formulation can be prepared by mixing one or more of the active components and at least one excipient such as starch, calcium carbonate, sucrose, lactose, gelatin, and so on. Besides the excipient, a lubricant such as Magnesium stearate and talc can also be used. The liquid formulation for oral administration includes emulsion, suspension, syrup, and so on, and may include conventional diluents such as water and liquid paraffin or may include various excipients such as wetting agents, sweeting agents, flavoring agents, and preserving agents. The formulation for non-oral administration includes sterilized aqueous solution, non-aqueous solution, freeze-dried formulation, suppository, and so on, and solvent for such solution may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and ester for syringe injection such as ethyl oleate. Base materials of the suppository may include witepsol, macrogol, tween 61, cacao butter, Laurin and glycerogelatin.

[098] The monoacetyldiacylglycerol compound can be administered in a pharmaceutically effective amount. The term“pharmaceutically effective amount” is used to refer to an amount that is sufficient to achieve a desired result in a medical treatment. The“pharmaceutically effective amount” can be determined according to the subject’s category, age, sex, severity and type of disease, activity of drug, sensitivity to drug, administration time, administration route, excretion rate, and so forth. The composition of the present invention can be administered alone or with other therapeutic agents sequentially or simultaneously. The composition of the present invention can be administered once or multiple times. The preferable amount of the composition of the present invention can be varied according to the condition and weight of patient, severity of disease, formulation type of drug, administration route and period of treatment. An appropriate total amount of administration per 1 day can be determined by a physician and is generally about 0.001 to about 5,000 mg/kg, preferably about 0.05 to 1,000 mg/kg, once a day or can be administered in divided doses multiple times daily. The composition of the present invention can be administered to any subject that requires the prevention or treatment of fatty liver disease. For example, the composition of the present invention can be administered to not only human but also non-human animal (specifically mammals) such as monkey, dog, cat, rabbit, guinea pig, rat, mouse, cow, sheep, pig, goat, and so on.

[099] In some embodiments, the present invention provides a health functional food composition for preventing, alleviating or improving fatty liver disease, which comprises a monoacetyldiacylglycerol of formula 1 as an active ingredient.

[0100] The monoacetyldiacylglycerol compound according to the present invention may be included into a health functional food composition to improve fatty liver disease in a subject. The monoacetyldiacylglycerol compound and fatty liver disease are as described above. When the compound of the present invention is included into the health functional food composition, the amount of monoacetyldiacylglycerol in the health food composition can be determined suitably according to the intended use. Generally, the amount of monoacetyldiacylglycerol is preferably from 0.01 to less than 15 weight%, with respect to the total amount of the health functional food composition when the monoacetyldiacylglycerol is included in food or beverages. However, the amount of monoacetyldiacylglycerol may be increased or decreased. In case of long term use for the purpose of the health control and hygiene, the amount of the monoacetyldiacylglycerol can be less than the above range. Since there is no problem in terms of safety, the monoacetyldiacylglycerol may be used in an amount greater than the above range. Foods to which the compound of the present invention can be added are not limited and include various foods, for example, meats, sausages, breads, chocolates, candies, snacks, pizzas, noodles, gums, daily products such as ice creams, soups, beverages, teas, drinks, alcoholic beverages, vitamin complexes and any health functional food.

[0101] When the monoacetyldiacylglycerol is used in the beverage product, the beverage product may include sweeting agents, flavoring agents or carbohydrates. Examples of carbohydrates 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 can be widely varied without specific limitation, and is preferably 0.01 to 0.04 g, more preferably, 0.02 to 0.03 g per 100 ml of the beverage. Examples of sweeting agents include natural sweeteners such as 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, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickening agents, pH controlling agents, stabilizing agents, preserving agents, glycerin, alcohol, carbonizing agents used in carbonated beverages and so on. Moreover, the health functional food composition of the present invention may include fruits, as used in preparing natural fruit juices and fruit juice beverages and vegetable beverages.

[0102] The present invention provides a method for treating fatty liver disease, comprising the step of administering the pharmaceutical composition to a suspected individual of fatty liver disease. By administering the composition to a suspected subject of fatty liver disease, the fatty liver disease is efficiently treated. The term “suspected subject of fatty liver disease” refer to those that have or are likely to develop fatty liver disease. The 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 dose of the monoacetyldiacylglycerol compound and the fatty liver disease are as described above. The term“administration” means introducing the pharmaceutical composition of the present invention to a patient in need by any suitable method. The route of administration may be any or a various routes, oral or non-oral, as long as the target tissue can be reached, 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 and the like may be used, but is not limited thereto.

Examples

[0103] The following examples are provided for better understanding of this invention. However, the present invention is not limited by the examples.

[0104] In order to confirm the efficacy of l-palmitoyl-2-linoleoyl-3-acetylglycerol (EC- 18 or PLAG) in the treatment of fatty liver disease, Streptozotocin (STZ)-induced fatty liver disease model were used in the experiments.

[0105] Experimental Example 1: Preparation of control groups and experimental groups to confirm treatment efficacy for nonalcoholic fatty liver disease (NAFLD)

[0106] FIG. 1 shows the experimental protocol of the present invention. As shown in FIG. 1, mice were randomly divided into control and streptozotocin (STZ)-treated groups. All STZ-treated groups received an intraperitoneal injection of STZ at a dose (1 day) of 200 mg/kg body weight (BW), which was dissolved in sodium citrate buffer (pH 4.5).

[0107] The day after streptozotocin (STZ) administration, diabetes induction was confirmed by a glucometer (ACCU-CHEK, Roche diagnostics Inc.) and induced acute diabetes. All mice with fasting blood glucose levels >200 mg/dF were considered to have acute diabetes. After confirming induction, mice in the experimental group were further randomized into three groups: STZ alone-treated group and low- and high-dose PFAG plus STZ treated groups. l-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol represented by the Formula 2(PLAG) was used as the active ingredient.

[0108] The mice received PLAG via oral gavage for 3 days. The intended oral dosage was 250 and 50 mg/kg BW, in mice respectively. Control and STZ alone treated groups were administrated orally with the same PBS for 3 days, and the mice were sacrificed on the fourth day.

[0109] Example 1: Body and relative liver weight analysis

[0110] The body weights of all mice in the control group prepared in the above experiment, streptozotocin(STZ) alone, streptozotocin(STZ) plus low dose PLAG treatment group, and streptozotocin (STZ) plus high dose PLAG treatment group were measured and recorded at the same time every day, and the results are shown in FIG. 2A. Also, relative liver weight of the control group and experimental group were calculated using the following equation, and the results are shown in FIG. 2B.

[0111] (Equation 1)

( absoluteliverweight)/(BWonsacrificeday)X100

[0112] As shown in FIG. 2A, all STZ-treated mice showed a statistically significant decrease in BW compared with the control group. However, the high-dose PLAG group showed less BW loss compared to mice that received STZ alone. There was no difference in BW between the STZ and low-dose PLAG groups. This suggests that oral PLAG administration attenuated weight loss due to diabetes.

[0113] As shown in FIG. 2-B, the relative liver weight (per g BW) in the STZ alone group was significantly higher than in the control group. However, the PLAG-treated group had a lower mean liver weight than the STZ group, indicating that PLAG supplementation improved hepatic steatosis.

[0114] Example 2: Histopathological analysis

[0115] At the end of the experimental period for 4 days, the mice were fasted for 6 h and then sacrificed. FIG. 3 is the picture of liver sections stained with H&E and ORO. Specifically, in other to measure the degree of accumulation of liver fat, the liver of control and experimental groups were observed, hematoxylin and eosin ( H&E) and Oil red O was used to image. The liver samples were immediately fixed in 10% buffered formalin at room temperature, and then the tissues were embedded in paraffin, sectioned, and stained with hematoxylin and eosin(H&E). Frozen liver sections were fixed for 20 min with 4% paraformaldehyde followed by ORO staining for 15 min at room temperature. Then, the samples were counterstained with Mayer’s hematoxylin for 10 s and mounted. The images were obtained under light microscopy (Olympus, Tokyo, Japan).

[0116] As shown in FIG. 3, as expected, H&E staining showed dramatic evidence of liver steatosis in the STZ alone group, as evidenced by the increased number of empty fat vacuoles (macro- and microvesicular steatosis). However, PLAG at a dosage of 250 mg/kg/day visibly reduced microvesicular steatosis. Low-dose PLAG-treated mice group also exhibited a decrease, albeit not to the same extent.

[0117] ORO staining revealed higher hepatic TG content in the STZ alone group, and this was reduced by PLAG. These results suggest that PLAG mitigates hepatic steatosis in a dose-dependent manner.

[0118] Example 3: Liver and plasma TG levels analysis

[0119] To measure the concentration of triglyceride (TG) contained in the plasma of the control and experimental groups, blood was collected via eye bleeding, and plasma was separated by centrifugation at 3,000 rpm for 10 min. Plasma samples were kept frozen until assay.

[0120] FIG. 4 is an example of the mechanism by which fat absorbed in the small intestine affects the liver, and FIG. 5 is the chart (FIG. 5A) showing the TG levels and contents in the liver and plasma and the chart (FIG. 5B) showing the apoB48 protein expression in portal vein plasma, when using the composition according to the present invention. Specifically, in other to measure the triglyceride (TG) content of hepatocytes, liver tissue (20 mg) was homogenized in isopropanol with a tissue grinder. The homogenate was centrifuged at 5,000 rpm for 10 min, and the supernatant was collected. 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 vein blood, as a marker of TG-rich CM transport and uptake in the body.

[0121] As shown in FIG. 5, ApoB48 levels of STZ -treated mice were increased compared to the control group. However, PLAG treatment dose-dependently reduced ApoB48 levels, indicating that PLAG improved lipid metabolism by enhancing TG uptake into peripheral tissue or overall. [0122] Example 4: Immunohistochemical analysis

[0123] Fig. 6 is the chart (FIG. 6A) showing the TG content in muscle tissue, the chart (FIG. 6B) showing the relative mRNA expression of LPL in muscle tissue and the picture (FIG. 6C) showing the representative immunohistochemical images of LPL staining in muscle sections, when using the composition according to the present invention. Specifically, lipoprotein lipase (LPL), the rate-limiting enzyme in TG clearance, controls catabolism of TG-rich lipoproteins including CM. To investigate TG clearance by peripheral tissue following PLAG treatment, we examined LPL protein and mRNA levels in muscle tissue.

[0124] As shown in FIG. 6, the muscle TG contents were not significantly different among the groups (FIG. 6A). The RT-PCR results showed that LPL mRNA expression was reduced in the STZ group compared with the control group, while it was elevated in the high-dose PLAG group (FIG. 6B).

[0125] Immunohistochemistry showed abundant expression in the control group, which was decreased in STZ-treated animals. However, LPL protein expression in the PLAG-treated groups was similar to control. Overall, the mRNA and protein findings suggest that PLAG induces TG clearance by restoring this function in peripheral tissue (FIG. 6C).

[0126] Example 5: Western blotting

[0127] FIG 7 is the chart (FIG. 7A) showing the weight of muscle specimens and skeletal muscle from mice, and the result of the relative mRNA expression of caveolin3 in muscle tissue, including the gastrocnemius and quadriceps (FIG. 7B) and the result of the relative mRNA expression of caveolin3 and myogenin in myoblasts and myotubes (FIG. 7C). Specifically, the relative amounts of apolipoprotein B48(ApoB48) in portal vein plasma were evaluated by western blot. Constant volumes of plasma were separated with 5% sodium dodecyl sulfate -polyacrylamide gel electrophoresis. The protein extracts were immunoblotted with ApoB48 antibody (1:500, Abeam, Cambridge, UK). Detection was conducted using Immobilon Western Chemiluminescent HRP Substrate (Millipore Corporation, Billerica, MA, USA).

[0128] As shown in FIG. 7, muscle atrophy is defined as a loss of muscle mass and occurs in the setting of STZ-induced diabetes. Like BW, muscle weight was also significantly decreased by STZ treatment. While PLAG treatment slightly improved muscle mass, there was no significant difference between the STZ alone and PLAG groups (FIG. 7A).

[0129] It investigated the effect of PLAG on muscle function by measuring myoblast differentiation into myotubes. This transformation induces changes in morphology and muscle specific gene expression. We found that CAV3 and myogenin mRNA levels were decreased in STZ-treated C2C12 cells, while PLAG treatment restored levels to normal (FIG. 5C). This is consistent with the in vivo results from muscle tissue, including quadriceps and gastrocnemius (FIG. 5B). These results indicate that STZ impairs normal myoblast differentiation, and PLAG could help normalize muscle function.

[0130] Example 6: The specificity of PLAG

[0131] FIG. 8 is the structural chemical formulas of PLAG and PLG (FIG. 8A) and the chart (FIG. 8B) showing the change in body weight and the picture of the Gross liver specimens from each group (FIG. 8C) and the picture of the H&E-stained liver sections. Specifically, the selectivity of PLAG was investigated by comparing the effects of PLAG and PLG in the STZ-induced mouse model. PLG is a prototype of diacylglycerol, and PLAG is a type of acetylated diacylglycerol. As shown in FIG. 8 below, PLAG-treated mice showed less BW loss compared to mice that received STZ alone. There was no difference in BW between the STZ group and PLG-treated mice (FIG. 8B). In addition, results showed that PLAG attenuated hepatic steatosis. In contrast, liver damage was not decreased by PLG(C&D). These results suggest that PLAG plays a specific role in protecting the liver against STZ-induced hepatic steatosis.

[0132] Example 7: RNA isolation and RT-PCR analysis

[0133] Total RNA was isolated from muscles of individual mice using TRIzol reagent (Favorgen Biotech, Taiwan). cDNA was synthesized using M-MLV reverse transcriptase. Gene expression from each sample was analyzed in duplicate and normalized against the internal control gene glyceraldehyde 3 -phosphate dehydrogenase (GAPDH). The primer sequences of GAPDH, LPL, caveolin 3 and myogenin are shown in Table 1 below.

[0134] [Table 1]

[0135] Experimental Example 2: Preparation of experimental groups to examine effects of EC-18 in STAM model of non-alcoholic steatohepatitis

[0136] To determine the efficacy of l-palmitoyl-2-linoleoyl-3-acetylglycerol (EC- 18 or PLAG) for the treatment of non-alcoholic fatty hepatitis and liver fibrosis, and the experiment was performed using a model derived from the streptozotocin (STZ) and a high fat diet (HFD).

[0137] FIG. 9 is another experimental protocol of the present invention, as shown in FIG. 9, all mice(C57BF/6J) were injected subcutaneously with 200mg of streptozotocin (STZ) solution at birth and feeding with high fat diet (HFD) after 4 weeks of age. The mice groups were further randomized into 6 groups: control group (PBS), MGF-3196- treated group, obeticholic acid (OCA)-treated group, EC- 18 low group, EC- 18 middle group and EC- 18 high group. The EC- 18 used 1 -palmitoyl -2 -linoleoyl-3 -acetyl -rac- glycerol represented by the Formula 2, the MGL-3196 used 2-(3,5-dichloro-4-((5- isopropyl-6-oxo-1,6-dihydropyridazin-3-yl)oxy)phenyl)-3,5-dioxo-2,3,4,5-tetrahydro- 1,2, 4-triazine-6-carbonitrile(Resmetirom), and the obeticholic acid(OCA) used 6a- ethyl-chenodeoxy cholic acid(6-ECDCA).

[0138] Example 8: Body and relative liver weight analysis

[0139] FIG. 10 is the graph showing the change in body weight, when using the composition according to the present invention, and FIG. 11 is graph showing the weight (FIG. 11A), the liver weight (FIG. 11B) and the liver weight to weight (FIG. 11C), when using the composition according to the present invention. Specifically, the mice according to experimental Example 2 recorded the weight before treatment, measured the weight at the same time every day, and recorded the weight change. Mice were observed for significant clinical signs of toxicity, moribundity and mortality approximately 60 minutes after each administration. During the treatment period, it was dead one mouse in each of the OCA and EC-18 middle groups. The mice were sacrificed at 9 weeks. The relative liver weight and average body weight of the mice and are shown in Table 2 below

[0140] [Table 2]

[0141] As shown in FIG. 10 and 11 and Table 2, there were no significant differences in mean body weight on the day of sacrifice between the control group and the treatment groups. The MGL-3196 and EC- 18 low groups showed significant decreases in mean liver weight compared with the control group. Mean liver weight in the EC- 18 middle group tended to decrease compared with the control group. The liver-to-body weight ratio in the OCA group tended to increase compared with the Vehicle group, the liver- to-body weight ratio in the MGL-3196 group tended to decrease compared with the control group.

[0142] Example 9: Measurement of plasma biochemistry

[0143] FIG. 12 is the graph showing the plasma ALT(A) and the plasma AST(B) and the ALT/AST ratio(C), when using the composition according to the present invention. Specifically, non-fasting blood was collected in polypropylene tubes with anticoagulant (Novo-Heparin) and centrifuged at l,000xg for 15 minutes at 4°C. Plasma alanine aminotransferase (ALT) and plasma aspartate aminotransferase (AST) were measured by FUJI DRI-CHEM 7000. The ALT/AST ratio was calculated and shown in Table 3 below.

[0144] The plasma ALT is an enzyme contained in hepatocyte in a large amount. When the liver is damaged, blood plasma ALT activity is raised. The plasma ALT is an indicator of liver disease. The plasma ALT is also distributed in red blood cells, skeletal muscle, other than hepatocyte, and is released into the blood when cells die/destroy.

[0145] [Table 3]

[0146] As shown in FIG. 12 and Table 3, there were no significant differences in plasma ALT levels between the control group and the treatment groups. There were no significant differences in plasma AST levels between the control group and the other treatment groups (FIG. 12A). The plasma AST level in the OCA group tended to decrease compared with the control group (FIG. 12B). There were no significant differences in ALT/AST ratio between the control group and the other treatment groups. The ALT/AST ratio in the OCA group tended to increase compared with the control group (FIG. 12C).

[0147] Example 10: Histological analyses

[0148] FIG. 13 is the picture of liver sections stained with H&E and ORO, when using the composition according to the present invention. FIG. 14 is the graph showing of liver sections stained with H&E and ORO, when using the composition according to the present invention. Specifically, for NAFLD Activity score (NAS), statistical analyses were performed using Dunnetf s Multiple Comparison Test, and are shown in Table 4 below. In addition, the results of statistically analyzing NAS in each treatment group are shown in Table 5.

[0149] In the numerical values of the NAS, P values <0.05 were considered statistically significant. For other data, statistical analyses were performed using Bonferroni Multiple Comparison Test. P values <0.05 were considered statistically significant. A trend or tendency was assumed when a one-tailed t-test returned P values

<0.1. All results were expressed as mean ± SD. The ranges for the scores of the NAS values are shown in Table 6 below.

[0150] [Table 4]

[0151] [Table 5]

[0152] [Table 6]

[0153] As shown in FIGS. 13 and 14 and Table 4 above, the liver sections from the control group exhibited micro- and macrovesicular fat deposition, hepatocellular ballooning and inflammatory cell infiltration. The OCA and EC- 18 high groups showed significant decreases in NAS compared with the control group. NAS in the MGF-3196, EC- 18 low and EC- 18 middle groups tended to decrease compared with the control group.

[0154] Example 11: Histological Analysis to Determine Efficacy on Liver Fibrosis

[0155] The hepatocytes of the mice used in Experimental Example 1 were used in the same manner. FIG. 15 is the photomicrographs of HE-stained liver sections. Specifically, to visualize collagen deposition, Bouin’s fixed liver sections were stained using picro-Sirius red solution. For quantitative analysis of fibrosis area, bright field images of Sirius red-stained sections were randomly captured around the central vein using a digital camera at 200-fold magnification.

[0156] FIG. 16 is the graph showing the fibrous part of liver tissue (sirius red-positive area), when using the composition according to the present invention. Specifically, in the liver sections stained using the picro-Sirius red solution, the fibrosis site was observed, and the numerical value of the part was shown in Table 7 below.

[0157] [Table 7]

[0158] As shown in FIG. 15 and Table 7, the liver sections from the control group showed increased collagen deposition in the pericentral region of liver lobule. The OCA and EC- 18 high groups showed significant decreases in the fibrosis area (Sirius red- positive area) compared with the control group. The fibrosis area in the MGL-3196 and EC- 18 low groups tended to decrease compared with the Vehicle group. There was no significant difference in the fibrosis area between the Vehicle group and the EC- 18 middle group.

[0159] As shown in Examples, EC- 18 low and high showed statistical reductions in NAS and fibrosis area, and EC- 18 middle showed statistical reductions in NAS compared with the control group. In conclusion, EC- 18 showed the feature of anti- NASH and anti -fibrosis effects.

[0160] Example 12: In Vivo Efficacy Study of EC-18 in STAM™ Mice Model of Non-alcoholic Steatohepatitis

[0161] In Vivo Efficacy Study of EC- 18 in STAM Model of Non-alcoholic Steatohepatitis were additionally conducted.

[0162] Glossary

CK-18 : Cytokeratin 18

MCP : Monocyte chemoattractant protein

NASH : Non-alcoholic steatohepatitis

OCA : Obeticholic acid

PBS : Phosphate buffered saline

SD : Standard deviation

SMA : Smooth muscle actin

STAM : Stelic animal model

TGF : Transforming growth factor

ΉMR : Tissue inhibitor of metalloproteinase TNF : Tumor necrosis factor

Materials and Methods

[0163] Samples

The liver and plasma samples from the following groups were used.

Group 1 : Vehicle

Eight NASH mice were orally administered vehicle [PBS] in a volume of 5 mL/kg once daily from 6 to 9 weeks of age.

Group 2: MGL-3196

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

Group 3 : OCA

Seven NASH mice were orally administered [1% methylcellulose] supplemented with OCA at a

dose of 30 mg/kg (in a volume of 5 mL/kg) once daily from 6 to 9 weeks of age.

Group 4: EC-18 30 mg/kg

Eight NASH mice were orally administered vehicle supplemented with EC- 18 at a dose of 30 mg/kg (in a volume of 5 mL/kg) once daily from 6 to 9 weeks of age.

Group 5: EC- 18 100 mg/kg

Seven NASH mice were orally administered vehicle supplemented with EC- 18 at a dose of 100

mg/kg (in a volume of 5 mL/kg) once daily from 6 to 9 weeks of age.

Group 6: EC- 18 250 mg/kg

Eight NASH mice were orally administered vehicle supplemented with EC- 18 at a dose of 250 mg/kg (in a volume of 5 mL/kg) once daily from 6 to 9 weeks of age. Measurement of liver triglyceride content

[0164] Liver total lipid-extracts were obtained by Folch’s method (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 extracts were evaporated to dryness, and dissolved in isopropanol. Liver triglyceride content was measured by Triglyceride E-test kit (FUJIFILM Wako Pure Chemical Corporation, Japan).

Measurement of plasma CK-18 level [0165] Plasma CK-18 level was quantified by Mouse Cytokeratin 18-M30 ELISA Kit (Cusabio Biotech Co., Ltd, China).

Histological analysis

[0166] Lor immunohistochemistry, sections were cut from frozen liver tissues embedded in Tissue-Tek O.C.T. compound and fixed in acetone. Endogenous peroxidase activity was blocked using 0.03% H202 for 5 minutes, followed by incubation with Block Ace (DS Pharma Biomedical Co., Ltd., Japan) for 10 minutes. The sections were incubated with anti-L4/80 antibody at room temperature for 1 hour. The sections were then incubated with biotin-conjugated secondary antibody (VECTASTAIN Elite ABC kit, Vector laboratories, Inc. USA) followed by ABC reagent each for 30 minutes at room temperature. Enzyme-substrate reactions were performed using 3, 3’-diaminobenzidine/H202 solution (Nichirei Bioscience Inc., Japan). For quantitative analysis of inflammation area, bright field images of F4/80- immunostained sections were captured around the central vein using a digital camera (DFC295; Leica) at 200-fold magnification, and the positive area in 5 fields/section were measured using ImageJ software (National Institute of Health). Profiles of primary and secondary antibodies are shown in Table 8.

[0167] [Table 8]

Quantitative RT-PCR

[0168] Total RNA was extracted from liver samples using RNAiso (Takara Bio, Japan) according to the manufacturer’s instructions. One mg of RNA was reverse- transcribed using a reaction mixture containing 4.4 mM MgC12 (F. Hoffmann-La Roche, Switzerland), 40 U RNase inhibitor (Toyobo, Japan), 0.5 mM dNTP (Promega, USA), 6.28 mM random hexamer (Promega), 5 x first strand buffer (Promega), 10 mM dithiothreitol (Invitrogen, USA) and 200 U MMLV-RT (Invitrogen) in a final volume of 20 mL. The reaction was carried out for 1 hour at 37°C, followed by 5 minutes at 99°C. 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 level, the expression of each gene was normalized to that of reference gene 36B4 (gene symbol: RplpO). Information of PCR-primer sets and the plate layout was described in

Tables 9-10.

Statistical tests [0171] Statistical analyses were performed using Bonferroni Multiple Comparison Test on GraphPad Prism 6 (GraphPad Software Inc., USA). P values <0.05 were considered statistically significant. A trend or tendency was assumed when a one-tailed t-test returned P values <0.1. Results were expressed as mean ± SD.

Results

Biochemistry

[0172] Liver triglyceride (Fig. 17 and Table 11)

The MGL-3196 group showed a significant decrease in liver triglyceride content compared with the Vehicle group. Liver triglyceride content in the EC- 18 100 mg/kg and EC-18 250 mg/kg groups tended to decrease compared with the Vehicle group. There were no significant differences in liver triglyceride content between the Vehicle group and the other treatment groups.

[0173] Plasma CK-18 (Fig. 18 and Table 11)

The EC-18 30 mg/kg, EC-18 100 mg/kg and EC-18 250 mg/kg groups showed significant decreases in plasma CK-18 levels compared with the Vehicle group. Plasma CK-18 level in the OCA group tended to decrease compared with the Vehicle group. There was no significant difference in plasma CK-18 level between the Vehicle group and the MGL-3196 group.

[0174] [Table 11]

[0175] F4/80 immunostaining and the inflammation area (Figs. 19-20 and Table 12) Representative photomicrographs of the F4/80-immunostained sections are shown in Figure 2.1. F4/80 immunostaining of liver sections from the Vehicle group demonstrated accumulation of F4/80+ cells in the liver lobule. The OCA and EC- 18 250 mg/kg groups showed significant decreases in the inflammation area (F4/80- positive area) compared with the Vehicle group. The inflammation area in the EC-18 30 mg/kg and EC- 18 100 mg/kg groups tended to decrease compared with the Vehicle group.

[0176] [Table 12] Histological Analysis

Gene expression analysis (Fig. 21 and Table 13)

[0177] MCP-1

MCP-1 mRNA expression level in the MGL-3196 group tended to down-regulate compared with the Vehicle group. There were no significant differences in MCP-1 mRNA expression levels between the Vehicle group and the other treatment groups.

[0178] TNF-a

There were no significant differences in TNF-a mRNA expression levels between the Vehicle group and any of the treatment groups.

[0179] TGF-b

TGF-b mRNA expression level in the MGL-3196 group tended to down-regulate compared with the Vehicle group. There were no significant differences in TGF-b mRNA expression levels between the Vehicle group and the other treatment groups.

[0180] TIMP-1

There were no significant differences in TIMP-1 mRNA expression levels between the Vehicle group and any of the treatment groups.

[0181] Alpha-SMA

There were no significant differences in a-SMA mRNA expression levels between the Vehicle group and any of the treatment groups. [0182] [Table 13] Gene expression analysis

Summary

[0183] MGL-3196

Treatment with MGL-3196 showed a significant decrease in liver triglyceride content, and decreasing trends in MCP-1 and TGF-b mRNA expression levels compared with the Vehicle group.

[0184] OCA

Treatment with OCA showed a significant decrease in the inflammation area, and a decreasing trend in plasma CK-18 level compared with the Vehicle group.

[0185] EC- 18

Treatment with EC- 18 at a dose of 30 mg/kg showed a significant decrease in plasma CK-18 level, and a decreasing trend in the inflammation area compared with the Vehicle group. Treatment with EC-18 at a dose of 100 mg/kg showed a significant decrease in plasma CK-18 level, and decreasing trends in liver triglyceride content and the inflammation area compared with the Vehicle group. Treatment with EC- 18 at a dose of 250 mg/kg showed significant decreases in plasma CK-18 level and the inflammation area, and a decreasing trend in liver triglyceride content compared with the Vehicle group.

[0186] All documents mentioned herein are fully incorporated herein by reference.

Claims (54)

Claims What is claimed is:

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

[Formula 1]

wherein R1 and R2 are independently a fatty acid residue of 14 to 22 carbon atoms.

2. The composition of claim 1 wherein R1 and R2 are independently selected from the group consisting ofpalmitoyl, 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 through 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 through 6 wherein the fatty liver disease is induced by type 1 diabetes (T1D).

8. The composition of any one of claims 1 through 7 wherein the compound of Formula 1 reduces a concentration of triglyceride (TG) in hepatocytes and plasma.

9. The composition of any one of claims 1 through 8 wherein the compound of Formula 1 improves an expression of lipoprotein lipase (LPL).

10. The composition of any one of claims 1 through 9 wherein the compound of Formula 1 reduces an expression of apolipoprotein including ApoB48 in a portal vein.

11. The composition of any one of claims 1 through 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 for treating fatty liver disease comprising administering to a subject the composition according to any one of claims 1 to 11.

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

[Formula 1]

wherein R1 and R2 are independently a fatty acid residue 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 arachidonoyl.

15. A method for treating a subject suffering from or susceptible to a fatty liver disease, comprising:

administering to the subject an effective amount of a compound of of Formula

1 :

[Formula 1]

wherein R1 and R2 are independently a fatty acid residue 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 the following Formula 2:

[Formula 2]

18. The method of any one of claims 15 through 17 wherein the subject is suffering from or susceptible to non-alcoholic fatty acid liver disease (NAFLD).

19. The method of any one of claims 15 through 18 wherein the subject is suffering from or susceptible to non-alcoholic steatohepatitis (NASH).

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

21. A method of treating a subject suffering from or susceptible to non-alcoholic fatty acid liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), liver fibrosis, fatty liver disease resulting from hepatitis, fatty liver disease resulting from obesity, fatty liver disease resulting from diabetes, fatty liver disease resulting from insulin resistance, fatty liver disease resulting from hypertriglyceridemia, Abetalipoproteinemia, glycogen storage diseases, Weber-Christian disease, Wolmans disease, acute fatty liver of pregnancy, and/or lipodystrophy, the method comprising: administering to the subject an effective amount of a compound of Formula 1:

[Formula 1]

wherein R1 and R2 are independently a fatty acid residue 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 l is a compound of the following Formula 2:

[Formula 2]

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

the subject is identified as suffering from non-alcoholic fatty acid liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), liver fibrosis, fatty liver disease resulting from hepatitis, fatty liver disease resulting from obesity, fatty liver disease resulting from diabetes, fatty liver disease resulting from insulin resistance, fatty liver disease resulting from hypertriglyceridemia, Abetalipoproteinemia, glycogen storage diseases, Weber-Christian disease, Wolman’s disease, acute fatty liver of pregnancy, and/or lipodystrophy; and

the compound of Formula 1 is administered to the identified subject.

25. The method of claim 24 wherein the subject is identified as suffering from non- alcoholic fatty acid liver disease (NAFLD) and the compound of Formula I is administered to the identified subject..

26. The method of claim 24 wherein the subject is identified as suffering from non- alcoholic fatty steatohepatitis (NASH) and the compound of Formula I is administered to the identified subject.

27. The method of claim 24 wherein the subject is identified as suffering from liver fibrosis and the compound of Formula I is administered to the identified subject.

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

29. A kit comprising:

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

(b) instructions for using the compound for treating or preventing a fatty liver disease in a subject.

30. A kit comprising:

(a) 1-palmitoyl-2-linoleoyl-3-acetylglycerol (PLAG);

(b) instructions for using the PLAG for treating or preventing a fatty liver disease.

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

32. A kit of any one of claims 29 through 31 wherein the kit comprises written instructions for use of the compound or PLAG.

33. A kit of any one of claims 29 through 32 wherein the kit comprises written instructions for use of the compound or the PLAG to treat non-alcoholic fatty acid liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), liver fibrosis, fatty liver disease resulting from hepatitis, fatty liver disease resulting from obesity, fatty liver disease resulting from diabetes, fatty liver disease resulting from insulin resistance, fatty liver disease resulting from hypertriglyceridemia, Abetalipoproteinemia, glycogen storage diseases, Weber-Christian disease, Wolmans disease, acute fatty liver of pregnancy, and/or lipodystrophy.

29. A kit of any one of claims 29 through 33 wherein the instructions are a product label.

34. A method of for treating a subject suffering from or susceptible to a fatty liver disease, comprising administering to the subject i) an effective amount of a compound of Formula 1 and ii) one or more additional liver fibrosis treatment agents that are distinct from the compound of Formula 1 :

[Formula 1]

wherein R1 and R2 are independently a fatty acid residue of 14 to 22 carbon atoms.

35. 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.

36. The method of claim 34 wherein the compound of Formula 1 is a compound of the following Formula 2:

[Formula 2]

37. The method of any one of claims 34 through 36 wherein the subject is suffering from or susceptible to non-alcoholic fatty acid liver disease (NAFLD).

38. The method of any one of claims 34 through 37 wherein the subject is suffering from or susceptible to non-alcoholic steatohepatitis (NASH).

39. The method of any one of claims 34 through 38 wherein the subject is suffering from or susceptible to liver fibrosis.

40. The method of any one of claims 34 through 39 wherein the one or more distinct fibrosis treatment agents comprise one or more selected from the group consisting of 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 L carnitine, MGL-3196, volixibat (SHP626), GS-9674, semaglutide, saroglitazar, agents in NCT02605616 (Mayo Clinic, Rochester, MN, USA), LMB763, IVA337, LJN452, CF102, MT-3995, pioglitazone, MN-001 (tipelukast), MSDC-0602K, JKB-121, IMM- 124E, and/or ARI-3037MO, and pharmaceutically acceptable salts or acids thereof.

41. A method of for treating a subject suffering from or susceptible to non-alcoholic fatty acid liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), liver fibrosis, fatty liver disease resulting from hepatitis, fatty liver disease resulting from obesity, fatty liver disease resulting from diabetes, fatty liver disease resulting from insulin resistance, fatty liver disease resulting from hypertriglyceridemia, Abetalipoproteinemia, glycogen storage diseases, Weber-Christian disease, Wolmans disease, acute fatty liver of pregnancy, and/or lipodystrophy, the method comprising administering to the subject i) an effective amount of a compound of Formula 1 and ii) an effective amount of one or more liver fibrosis treatment agents that are distinct from the compound one or more compounds of Formula 1 : [Formula 1]

wherein R1 and R2 are independently a fatty acid residue of 14 to 22 carbon atoms.

42. 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.

43. The method of claim 41 wherein the compound of Formula l is a compound of the following Formula 2:

[Formula 2]

44. The method of any one of claims 41 through 43 wherein the subject is suffering from or susceptible to non-alcoholic fatty acid liver disease (NAFLD).

45. The method of any one of claims 41 through 44 wherein the subject is suffering from or susceptible to non-alcoholic steatohepatitis (NASH).

46. The method of any one of claims 41 through 45 wherein the subject is suffering from or susceptible to liver fibrosis.

47. The method of any one of claims 41 through 46 wherein the one or more distinct liver fibrosis treatment agents comprise one or more selected from the group consisting of obeticholic acid (OCA), elafibranor (GFT505), selonsertib (GS-4997), cenicriviroc (CYC), liraglutide, metadoxine, hydroxytyrosol and vitamin E, NGM282 (M70), BMS-986036, emricasan (IDN-6556), aramchol, atorvastatin and/or L carnitine, MGL-3196, volixibat (SHP626), GS-9674, semaglutide, saroglitazar, agents in NCT02605616 (Mayo Clinic, Rochester, MN, USA), LMB763, IVA337, LJN452, CF102, MT-3995, pioglitazone, MN-001 (tipelukast), MSDC-0602K, JKB-121, IMM- 124E, and/or ARI-3037MO, and pharmaceutically acceptable salts or acids thereof.

48. A kit comprising:

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

(b) one or more liver fibrosis treatment agents distinct from the compound or composition of any one of claims 1 through 11 ; and

(c) instructions for using the compound and the one or more distinct liver fibrosis treatment agents for treating or preventing a fatty liver disease in a subject.

49. A kit comprising:

(a) 1-palmitoyl-2-linoleoyl-3-acetylglycerol (PLAG);

(b) one or more liver fibrosis treatment agents that are distinct form PLAG; and

(b) instructions for using the PLAG and the one or more distinct liver fibrosis treatment agents for treating or preventing a fatty liver disease.

50. A kit of claim 49 wherein the kit comprises a therapeutically effective amount of PLAG.

51. A kit of any one of claims 48 through 50 wherein the kit comprises written instructions for use of the compound or PLAG and the one or more distinct liver fibrosis treatment agents.

52. A kit of any one of claims 48 through 51 wherein the kit comprises written instructions for use of i) the compound or the PLAG and ii) the one or more additional liver fibrosis treatment agents, to treat non-alcoholic fatty acid liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), liver fibrosis, fatty liver disease resulting from hepatitis, fatty liver disease resulting from obesity, fatty liver disease resulting from diabetes, fatty liver disease resulting from insulin resistance, fatty liver disease resulting from hypertriglyceridemia, Abetalipoproteinemia, glycogen storage diseases, Weber- Christian disease, Wolmans disease, acute fatty liver of pregnancy, and/or lipodystrophy.

53. A kit of any one of claims 48 through 52 wherein the instructions are a product label.

54. The kit of any one of claims 48 through 53 wherein the one or more distinct fibrosis treatment agents comprise one or more s of 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 L carnitine, MGL-3196, volixibat (SHP626), GS-9674, semaglutide, saroglitazar, agents in NCT02605616 (Mayo Clinic, Rochester, MN, USA), LMB763, IVA337, LJN452, CF102, MT-3995, pioglitazone, MN-001 (tipelukast), MSDC-0602K, JKB-121, IMM-124E, and/or ARI-3037MO, and pharmaceutically acceptable salts or acids thereof.