KR20240061958A - Pharmaceutical composition for preventing and treating fatty liver disease comprising VER-246608 - Google Patents

Abstract

The present invention relates to a composition for preventing, improving or treating fatty liver disease containing VER-246608. More specifically, VER-246608 can effectively inhibit fat accumulation in hepatocytes at low concentrations without cytotoxicity, It can be widely used in the fields of medicine, food, cosmetics, and feed to prevent, improve, or treat fatty liver disease.

Description

Pharmaceutical composition for preventing or treating fatty liver disease comprising VER-246608 {Pharmaceutical composition for preventing and treating fatty liver disease comprising VER-246608}

The present invention relates to a composition for preventing, improving or treating fatty liver disease comprising VER-246608 or a pharmaceutically acceptable salt thereof.

Fatty liver disease is a disease caused by excessive accumulation of fat in the liver. It refers to cases where more than 5% of the liver weight is fat, especially neutral fat. It is known to be caused by excessive alcohol consumption or drug use, hepatitis virus, some genetic diseases, and metabolic diseases such as obesity or type 2 diabetes.

Among these, those caused by alcohol consumption are classified as alcoholic fatty liver, and those caused by alcohol consumption are classified as non-alcoholic fatty liver. It is a disease that starts from simple steatosis and progresses to steatohepatitis, liver cirrhosis, etc.

There is no clear treatment for fatty liver other than improving lifestyle habits such as diet and exercise. Diabetes drugs (thiazolidinediones, metformin, GLP-1 agonist, DPP4 inhibitor), antioxidants and hepatocyte protectors (vitamin E, ursodeoxycholic acid), and non-steroidal anti-inflammatory drugs. (NSAID) etc. are used in a limited manner.

Currently, the development of treatments for fatty liver/steatohepatitis using various new molecular targets is active, with Phase 3 clinical trials underway for Elafibranor, a PPARα/δ ligand, Cenicriviroc, a CCR2/5 inhibitor, Obeticholic acid, an FXR agonist, and Selonsertib, an ASK-1 inhibitor. It is progressing ( Nature Medicine , 2018, 24(7):908-922).

Recent research has shown that the metabolism of pyruvate, a product of glycolysis, is important for fat accumulation in liver cells, and methods for inhibiting MPC (mitochondrial pyruvate carrier), which delivers pyruvate to mitochondria ( Cellular and molecular gastroenterology and hepatology , 7(2), 275-284.) and a method of activating pyruvate dehydrogenase (PDH), an enzyme that converts pyruvate to Acetyl-CoA ( ACS Pharmacol. Transl. Sci . 2021, 4(2): 582-588) etc. were presented.

It is known that the activity of PDH in metabolic diseases is mainly inhibited by PDK2/4, an enzyme that phosphorylates PDH. Increased expression of this enzyme was confirmed in animal models of fatty liver, and improvement of fatty liver was confirmed by inhibition of PDK2/4. ( Diabetes , 2016, 65(10):2876-2887; Biochem Biophys Res Commun , 2018, 495(1):582-586).

However, even for drugs that inhibit PDK activity, the effect of alleviating liver fat accumulation may vary from drug to drug, so individual verification is required to determine whether fatty liver disease is improved.

VER-246608 is an inhibitor active against all four PDK isoforms and has been reported to have anticancer activity by inhibiting sugar metabolism in cancer cells ( Oncotarget , 2014, 5(24):12862-12876), but has an excellent PDK inhibition effect. Despite this, the effect of inducing actual cell death was reported to be very weak (BMB Rep, 2021, 54(11):563-568).

However, to date, no studies have been reported on other physiological activities of VER-246608 other than its anti-cancer activity, and no studies have been reported on its effect in suppressing fatty liver disease.

Accordingly, the inventors of the present invention confirmed that VER-246608 can effectively inhibit fat accumulation in hepatocytes at low concentrations without cytotoxicity, and focused on its applicability in preventing, improving, or treating fatty liver, and developed the present invention. It has reached completion.

The present invention was conceived in consideration of the above problems, and the object of the present invention is to provide a composition for preventing, improving or treating fatty liver disease containing VER-246608 or a pharmaceutically acceptable salt thereof as an active ingredient. will be.

The problem to be solved by the present invention is not limited to the problem(s) mentioned above, and other problem(s) not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating fatty liver disease containing VER-246608 or a pharmaceutically acceptable salt thereof as an active ingredient.

The VER-246608 may inhibit fat accumulation in hepatocytes.

The fat accumulation may be induced by free fatty acids.

The fatty liver disease may be one or more types selected from the group consisting of alcoholic fatty liver disease, non-alcoholic simple fatty liver disease, non-alcoholic steatohepatitis, non-alcoholic cirrhosis, and end-stage fibrotic liver disease.

The concentration of VER-246608 or a pharmaceutically acceptable salt thereof in the pharmaceutical composition may be 0.1 to 100 μM.

Additionally, the present invention provides a health functional food composition for preventing or improving fatty liver disease, comprising VER-246608 or a pharmaceutically acceptable salt thereof as an active ingredient.

Additionally, the present invention provides a feed composition for preventing or improving fatty liver disease comprising VER-246608 or a pharmaceutically acceptable salt thereof as an active ingredient.

Additionally, the present invention provides a cosmetic composition for preventing or improving fatty liver disease, comprising VER-246608 or a pharmaceutically acceptable salt thereof as an active ingredient.

Additionally, the present invention provides a pharmaceutical composition for inhibiting fat accumulation in hepatocytes containing VER-246608 or a pharmaceutically acceptable salt thereof as an active ingredient.

The pharmaceutical composition may prevent or treat one or more diseases selected from the group consisting of obesity, non-alcoholic fatty liver disease, diabetes, and hyperlipidemia, which are caused by fat accumulation in hepatocytes.

According to the present invention, VER-246608 can effectively inhibit fat accumulation in hepatocytes at a low concentration that does not show cytotoxicity, so it can be widely used in the fields of medicine, food, cosmetics, and feed for preventing, improving, or treating fatty liver. there is.

The effects of the present invention are not limited to the effects described above, and should be understood to include all effects that can be inferred from the configuration of the invention described in the detailed description or claims of the present invention.

Figure 1 is (A) a graph of MTT assay results and (B) a phase contrast microscope image confirming the survival rate of mouse hepatocytes (AML12) according to palmitic acid treatment.
Figure 2 is (B) a BODIFY 493/503 staining image showing the degree of fat accumulation in mouse hepatocytes (AML12) according to palmitic acid treatment, and (A) a graph quantifying this.
Figure 3 is a Western blot image confirming the expression of pyruvate dehydrogenase phosphorylation (p-PDHA1), pyruvate dehydrogenase kinase (PDKs), and lactate dehydrogenase (LDHA) in mouse hepatocytes (AML12) following palmitic acid treatment.
Figure 4 is a graph of MTT assay results confirming cytotoxicity according to the concentration of VER-246608 in mouse hepatocytes (AML12) (A), and the degree of fat accumulation according to treatment of VER-246608 in mouse hepatocytes (AML12) in which fat accumulation was induced. Shown are (C) a BODIFY 493/503 staining image and (B) a graph quantifying it, and (D) an Oil Red O staining image and (E) a graph quantifying it.
Figure 5 is a graph showing the results of the MTT assay confirming cytotoxicity according to the concentration of DCA in mouse hepatocytes (AML12) (A), and (C) showing the degree of fat accumulation according to the treatment of DCA in mouse hepatocytes (AML12) in which fat accumulation was induced. BODIFY 493/503 staining image and (B) a graph quantifying it.
Figure 6 is a graph showing the results of the MTT assay confirming cytotoxicity according to the concentration of AZD7545 in mouse hepatocytes (AML12) (A), and (C) showing the degree of fat accumulation according to the treatment of AZD7545 in mouse hepatocytes (AML12) in which fat accumulation was induced. BODIFY 493/503 staining image and (B) a graph quantifying it.

Hereinafter, embodiments of the present invention will be described in more detail with reference to the attached drawings. The embodiments of the present invention can be modified in various forms, and the scope of the present invention should not be construed as being limited to the following embodiments. This example is provided to more completely explain the present invention to those skilled in the art. Therefore, the shapes of elements in the drawings are exaggerated to emphasize clearer explanation.

The present invention provides a pharmaceutical composition for preventing or treating fatty liver disease, comprising VER-246608 or a pharmaceutically acceptable salt thereof as an active ingredient.

The VER-246608 may be a compound represented by the following formula (1). Additionally, in the present invention, VER-246608 may include various derivatives thereof, but the scope of the present invention is not limited thereto. In the present invention, VER-246608 and its derivatives can be used as compounds isolated from nature, chemically synthesized compounds, or commercially sold.

[Formula 1]

The VER-246608 (CAS No. 1684386-71-7) is an inhibitor active against all four PDK isoforms and has been reported to have anticancer activity by inhibiting sugar metabolism in cancer cells. However, despite its excellent PDK inhibitory effect, The actual effect of inducing cell death was reported to be very weak. However, to date, no studies have been reported on other physiological activities of VER-246608 other than its anti-cancer activity, and no studies have been reported on its effect in suppressing fatty liver disease.

In the present invention, it was confirmed that VER-246608 efficiently inhibits fat accumulation in normal hepatocytes at a low concentration compared to other PDK inhibitors, and a composition for inhibiting fat accumulation in hepatocytes containing VER-246608 as an active ingredient or VER-246608 was used. It is intended to be applied as a composition for preventing, improving or treating fatty liver disease containing it as an active ingredient.

According to one embodiment of the present invention, the VER-246608 may inhibit fat accumulation in hepatocytes. Additionally, the fat accumulation may be induced by free fatty acid. Free fatty acids are also called non-ester fatty acids, and their main components include palmitic acid, stearic acid, oleic acid, and linoleic acid, and can bind to albumin in the blood and be transported to liver tissue. When adipocyte dysfunction occurs due to unbalanced diet and obesity, free fatty acids are abnormally accumulated in liver tissue due to lipolysis, which may increase the risk of fatty liver disease.

According to one embodiment of the present invention, the fatty liver disease may be one or more types selected from the group consisting of alcoholic fatty liver disease, non-alcoholic simple fatty liver disease, non-alcoholic steatohepatitis, non-alcoholic cirrhosis, and end-stage fibrotic liver disease.

In the present invention, the term “fatty liver disease” refers to a phenomenon in which neutral fat appears to be abnormally deposited within liver cells, unlike normal cases. A normal liver is composed of approximately 5% of fatty tissue, and neutral fat, fatty acids, phospholipids, cholesterol, and cholesterol esters are the main components of fat, but once fatty liver occurs, most of the components are replaced by neutral fat, and the amount of neutral fat decreases. If it is more than 5% of the liver weight, it is diagnosed as fatty liver. Additionally, fatty liver disease can be largely divided into non-alcoholic fatty liver disease caused by obesity, diabetes, hyperlipidemia, drugs, etc., and alcoholic fatty liver disease caused by excessive drinking.

The above-mentioned “non-alcoholic fatty liver disease” refers to a case of fatty liver disease without a history of alcohol consumption, and is known to be related to metabolic diseases such as obesity, diabetes, and hyperlipidemia. Non-alcoholic fatty liver disease includes not only fat accumulation within the liver, but also non-alcoholic steatohepatitis or end-stage fibrotic liver disease.

The above-mentioned “alcoholic fatty liver disease” refers to a condition that occurs when excessive alcohol intake promotes fat synthesis in the liver and prevents normal energy metabolism. Alcohol cannot be stored in the body and is completely eliminated through oxidation in the liver. Specifically, in the liver, alcohol is largely consumed by the alcohol dehydrogenase (ADH) pathway and the microsomal alcohol oxidizing system (MEOS). ) pathway and the catalase pathway, it is metabolized into acetaldehyde, which is then metabolized into acetate by dehydrogenase (aldehyde dehydrogenase: ALDH). At this time, acetaldehyde is toxic and can damage liver cells, and as a result of the metabolism of alcohol, a large amount of fatty acids are produced, causing fat to accumulate in the liver. Fatty liver accumulated in the liver due to the above causes is called alcoholic fatty liver.

According to one embodiment of the present invention, the concentration of VER-246608 or a pharmaceutically acceptable salt thereof may be 0.1 to 100 μM, 1 to 50 μM, 1 to 20 μM, 1 to 10 μM, or 10 to 50 μM. , but is not limited to this. Within the above concentration range, not only is the effect of VER-246608 excellent in suppressing fat accumulation in hepatocytes, but there is no or very low cytotoxicity to hepatocytes.

In addition, the present invention includes the step of contacting hepatocytes induced with fatty liver or hepatocytes induced with fat accumulation with VER-246608 or a pharmaceutically acceptable salt thereof in vitro or ex vivo. Provides a method of inhibiting fat accumulation in liver cells.

Additionally, the present invention provides a method for preventing or treating fatty liver disease, comprising administering VER-246608 or a pharmaceutically acceptable salt thereof to a subject.

Additionally, the present invention provides a method for inhibiting fat accumulation in hepatocytes, comprising administering VER-246608 or a pharmaceutically acceptable salt thereof to a subject.

In the method for inhibiting fat accumulation in hepatocytes or preventing or treating fatty liver disease, VER-246608 (or a pharmaceutically acceptable salt thereof) is contacted with fatty liver-induced hepatocytes or hepatocytes with induced fat accumulation, or administered to an individual. ) The concentration may be 0.1 to 100 μM, 1 to 50 μM, 1 to 20 μM, 1 to 10 μM, or 10 to 50 μM, but is not limited thereto. Within the above range, not only is the effect of VER-246608 excellent in suppressing fat accumulation in hepatocytes, but there is no or very low cytotoxicity to hepatocytes.

The term “prevention” used in the present invention refers to any action that inhibits fat accumulation in hepatocytes by administering the composition of the present invention, or thereby delays the progression of fatty liver disease.

The term "treatment" used in the present invention refers to any action that suppresses fat accumulation in hepatocytes by administering the composition of the present invention, or improves or beneficially changes fatty liver disease, and useful results including clinical results. Or it means an attempt to achieve a desirable result. A useful or desirable clinical outcome may be alleviation or improvement of one or more symptoms or conditions, reduction of the extent of the disease, stabilization of the disease state, inhibition of the development of the disease, inhibition of the spread of the disease, or delay or slowing of the progression of the disease, whether detectable or not. , may include, but is not necessarily limited to, delaying or delaying the onset of a disease, improving or alleviating a disease state, and reducing (partial or total). Additionally, “treatment” can mean prolonging the patient's survival beyond what would be expected in the absence of treatment. Additionally, “treatment” may refer to inhibiting the progression of the disease, temporarily slowing the progression of the disease, or more preferably permanently halting the progression of the disease. In the present invention, it may mean improving the survival of patients by improving the treatment of fatty liver disease, especially non-alcoholic fatty liver disease.

The pharmaceutical composition of the present invention can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, and sterile injection solutions according to conventional methods. there is. Carriers, excipients, and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, and cellulose. , methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations include the compound of the present invention with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, It is prepared by mixing gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, oral solutions, emulsions, syrups, etc. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. . Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable esters such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao, laurin, glycerogenatin, etc. can be used.

The dosage of the pharmaceutical composition of the present invention will vary depending on the age, gender, and weight of the subject to be treated, the specific disease or pathological state to be treated, the severity of the disease or pathological state, the route of administration, and the judgment of the prescriber. Dosage determinations based on these factors are within the level of one skilled in the art, and dosages generally range from 0.01 mg/kg/day to approximately 2000 mg/kg/day. A more preferred dosage is 0.1 mg/kg/day to 1000 mg/kg/day. Administration may be administered once a day, or may be administered several times. The above dosage does not limit the scope of the present invention in any way.

The pharmaceutical composition of the present invention can be administered to mammals such as rats, livestock, and humans through various routes. All modes of administration are contemplated, for example, oral, rectal or by intravenous, intramuscular, subcutaneous, intrathecal or intracerebrovascular injection.

In the present invention, the pharmaceutical composition for preventing or treating fatty liver disease includes, in addition to the active ingredient, any compound or natural extract known to have activity whose safety has already been verified in order to increase and reinforce the effect of inhibiting fat accumulation in hepatocytes. It can be included as .

Additionally, the present invention provides a food composition or health functional food composition for preventing or improving fatty liver disease, comprising VER-246608 or a pharmaceutically acceptable salt thereof as an active ingredient.

The food or health functional food composition may further include an additive selected from the group consisting of flavoring agents, flavors, colorants, fillers, stabilizers, natural carbohydrates, nutrients, vitamins, thickeners, pH adjusters, preservatives, and mixtures thereof. You can.

The food composition of the present invention includes all types of functional foods, nutritional supplements, health foods, and food additives. Food compositions of this type can be prepared in various forms according to conventional methods known in the art.

For example, as a health food, the composition itself can be prepared and consumed in the form of tea, juice, and drinks, or can be consumed by granulating, encapsulating, and powdering. In addition, functional foods include beverages (including alcoholic beverages), fruits and their processed foods (e.g. canned fruit, bottled foods, jam, marmalades, etc.), fish, meat, and their processed foods (e.g. ham, sausages, corned beef, etc.), Bread and noodles (e.g. udon, buckwheat noodles, ramen, spaghetti, macaroni, etc.), fruit juice, various drinks, cookies, taffy, dairy products (e.g. butter, cheese, etc.), edible vegetable oil, margarine, vegetable protein, retort food, It can be manufactured by adding extracts to frozen foods and various seasonings (e.g. soybean paste, soy sauce, sauce, etc.). Additionally, in order to use the composition of the present invention in the form of a food additive, it can be prepared and used in the form of a powder or concentrate.

The preferred content of VER-246608 in the food composition of the present invention may be 0.001 to 50%, preferably 0.01 to 30%, based on the total weight of the food composition.

In one embodiment of the present invention, the health functional food composition of the present invention can be manufactured in general dosage forms such as tablets, pills, granules, powders, liquids, hard capsules, soft capsules, etc., and can be used in porridge, bread, beverages, bars, It can be manufactured in any form such as chocolate, cookies, tea, drinks, vitamin complex, meat, sausage, candy, noodles, jelly, etc.

In order to manufacture various dosage forms or forms as described above, food-acceptable carriers or additives such as the above-mentioned excipients can be used, and it is known in the art that they can be used in the production of the desired dosage form or form. Any carrier or additive may be used.

Additionally, the present invention provides a feed composition for preventing or improving fatty liver disease comprising VER-246608 or a pharmaceutically acceptable salt thereof as an active ingredient.

When VER-246608 of the present invention is provided in the form of a feed composition, the feed composition may additionally include known feed supplements, food additives or feed additives, and may be provided in the form of fermented feed, compounded feed, pellet form, silage, etc. can be manufactured.

Additionally, the present invention provides a cosmetic composition for preventing or improving fatty liver disease, comprising VER-246608 or a pharmaceutically acceptable salt thereof as an active ingredient.

When VER-246608 of the present invention is provided as a cosmetic composition, the cosmetic composition may include commonly accepted ingredients in addition to the above active ingredients without limitation, such as antioxidants, stabilizers, solubilizers, vitamins, pigments and fragrances. It may include common auxiliaries such as, and carriers. The cosmetic composition can be interpreted to include various materials for skin health without limitation.

Additionally, the present invention provides an adjuvant for the treatment of fatty liver disease containing VER-246608 or a pharmaceutically acceptable salt thereof as an active ingredient.

As used herein, the term "adjuvant for the treatment of fatty liver disease" refers to a substance that synergistically increases the therapeutic effect of fatty liver disease by preventing side effects caused by the pharmaceutical composition when applied in combination with treatment with the pharmaceutical composition according to the present invention. means composition. Therefore, the adjuvant for treating fatty liver disease can be administered together with the pharmaceutical composition, simultaneously or sequentially.

Additionally, the present invention provides a pharmaceutical composition for inhibiting fat accumulation in hepatocytes containing VER-246608 or a pharmaceutically acceptable salt thereof as an active ingredient. The pharmaceutical composition for inhibiting fat accumulation in hepatocytes can prevent, improve, or treat one or more metabolic diseases among obesity, non-alcoholic fatty liver disease, diabetes, and hyperlipidemia caused by excessive fat accumulation in hepatocytes.

The above description explains the technical idea of the present invention using one embodiment, and those skilled in the art will be able to make various modifications and variations without departing from the essential characteristics of the present invention. Accordingly, the embodiments described in the present invention are not intended to limit the technical idea of the present invention, but are for illustrative purposes, and the scope of the technical idea of the present invention is not limited by these embodiments. The scope of protection of the present invention should be interpreted in accordance with the claims, and all technical ideas within the equivalent scope should be interpreted as being included in the scope of rights of the present invention.

Hereinafter, the present invention will be described in more detail through examples.

Example 1. Confirmation of hepatocyte toxicity by palmitic acid

Palmitic acid (PA) bound to BSA (bovine serum albumin) was treated with mouse hepatocyte AML12 cells at various concentrations (10, 20, 50, 100, 200, and 500 μM) and cultured for 24 hours. The survival rate of the cells was measured by MTT. It was observed through assay (Figure 1A) and phase contrast microscopy (Figure 1B).

Referring to Figures 1A and 1B, it can be seen that PA does not show significant cytotoxicity to hepatocytes up to 100 μM, but shows significant cytotoxicity at concentrations higher than that. ***p < 0.001 (vs control)

Example 2. Confirmation of fat accumulation in hepatocytes by palmitic acid

After treating AML12 mouse hepatocytes with palmitic acid (PA) conjugated to BSA at various concentrations (20, 50, 100 μM) and culturing for 24 hours, accumulation of intracellular fat was examined by BODIFY 493/503 staining (Figure 2A- 2B) was used to confirm. At this time, in Figure 2B, Hoechest 33342 stains the cell nucleus and is used to confirm the number of living cells. ** p < 0.01, *** p < 0.001 (vs control)

Referring to Figures 2A and 2B, it was found that the treatment of PA in hepatocytes increased fat accumulation in a concentration-dependent manner, thereby establishing in vitro experimental conditions that could confirm fat accumulation in hepatocytes using PA.

Example 3. Confirmation of phosphorylation of pyruvate dehydrogenase (p-PDHA1), expression of pyruvate dehydrogenase phosphorylase (PDKs) and lactate dehydrogenase (LDHA) by palmitic acid.

Palmitic acid (PA) bound to BSA was treated at various concentrations in mouse liver cells, AML12, and cultured for 24 hours. The cells were recovered, the protein was extracted, and the amount of protein was measured by electrophoresis and Western blot using antibody reaction. , the results are shown in Figure 3.

Referring to Figure 3, it was confirmed that the phosphorylation of PDHA1 increased and the expression of PDK2, PDK4, and LDHA increased due to treatment with PA. Therefore, it is consistent with existing research that promoting the conversion of pyruvate to Acetyl-CoA by inhibiting the phosphorylation of PDH through inhibition of PDK2/4 can be a way to inhibit fat accumulation in liver cells caused by a high-fat diet. You can check it.

Example 4. Confirmation of the inhibitory effect on hepatocellular toxicity and fat accumulation by VER-246608

VER-246608, a PDK inhibitor, was treated with mouse hepatocyte AML12 cells at various concentrations (1, 5, 10, 20, 50 μM) and cultured for 24 hours, and cytotoxicity was confirmed by MTT assay (Figure 4A). As a result, more than 80% of VER246608 cells survived even at 50 μM, the highest concentration tested, and therefore its toxicity to AML12 was relatively weak. * p < 0.05, ** p < 0.01, *** p < 0.001 (vs control).

In addition, under experimental conditions in which fat accumulates in AML12 mouse hepatocytes (PA 50 μM), the PDK inhibitor VER-246608 was treated at different concentrations (10, 20, and 50 μM), and fat accumulation was observed 24 hours later using BODOPY 493/503. (Figure 4C) and Oil Red O (Figure 4D), and the staining intensity was quantified and shown in Figure 4B (BODOPY 493/503) and Figure 4E (Oil Red O), respectively. ***p < 0.001. Referring to Figures 4B to 4D, it can be seen that VER-246608 effectively inhibits fat accumulation caused by PA without showing significant toxicity to hepatocytes.

Comparative Example 1. Confirmation of the inhibitory effect on hepatocellular toxicity and fat accumulation by DCA

DCA is a drug used in the clinical treatment of congenital lactic acidosis for over 30 years. It is previously known as a PDK inhibitor and has been reported to increase the activity of PDH by effectively inhibiting phosphorylation of PDH. Therefore, it received attention as a new anticancer agent as it was discovered that it induces apoptosis of cancer cells due to an increase in reactive oxygen species by inducing a metabolic switch from glycolysis to oxidative oxidation process, and its blood sugar reducing function was reported in diabetic patients and diabetic animal models. There is a bar.

In Comparative Example 1, first, to confirm the cytotoxicity of DCA on mouse hepatocytes, AML12, DCA was treated at different concentrations (1, 5, 10, 20, 50 mM), cultured for 24 hours, and then cells were analyzed through MTT assay. The survival rate was confirmed (Figure 5A). As a result, DCA showed cytotoxicity to AML12 cells in a concentration-dependent manner, but the degree of cytotoxicity was relatively weak. ns: no significant, * p < 0.001.

In addition, AML12 mouse hepatocytes were simultaneously treated with DCA at an active concentration (10, 50 mM) that inhibits PDK under experimental conditions (PA 50 μM) in which fat accumulates, and then 24 hours later, fat accumulation was detected with BODIPY 493/503. It was stained and observed through fluorescent photography (Figure 5C), and the degree of localization was quantified and displayed in a graph (Figure 5B). * p < 0.05, * p < 0.001. Referring to Figures 5B and 5C, DCA appears to slightly inhibit PA-induced fat accumulation in hepatocytes at a low concentration (10mM), but not at all at a high concentration (50mM), and its activity concentration is in millimolar units. It has a very high disadvantage.

From the above, it was confirmed that DCA, a well-known PDK inhibitor, has weak toxicity to AML12 mouse hepatocytes, but does not effectively inhibit hepatocyte fat accumulation caused by PA.

Comparative Example 2. Confirmation of the inhibitory effect on hepatocellular toxicity and fat accumulation by AZD7545

AZD7545 is a low-molecular-weight substance known to be a selective inhibitor of PDK2. It also has the function of inhibiting PDK1 and 3 at some high concentrations, and has been developed as an oral drug by a pharmaceutical company (AstraZeneca) to treat type II diabetes. It has been reported that it effectively inhibits the activity of PDK by binding to the lipoyl-binding pocket of PDK, but it has little anticancer effect, and there are not many reports on other biological actions.

In Comparative Example 2, first, to confirm the cytotoxicity of AZD7545 on AML12 mouse hepatocytes, AML12 cells were treated at different concentrations (1, 5, 10, 20, 30 mM), cultured for 24 hours, and then tested using MTT assay. The survival rate of cells was measured (Figure 6A). As a result, AZD7545 showed cytotoxicity to AML12 cells in a concentration-dependent manner, but the degree of cytotoxicity was relatively weak. ** p < 0.01, *** p < 0.001.

In addition, AML12 mouse hepatocytes were simultaneously treated with AZD7545 at active concentrations (10 and 20 μM) that inhibit PDK under experimental conditions (PA 50 μM) in which fat accumulates in hepatocytes, and then 24 hours later, fat accumulation was treated with BODIPY 493/503. It was stained and observed through fluorescent photography (Figure 6C), and the degree of localization was quantified and displayed in a graph (Figure 6B). ***p < 0.001. Referring to Figures 6B and 6C, it can be seen that AZD7545, a known PDK inhibitor, does not inhibit fat accumulation in AML12 mouse hepatocytes induced by PA.

From the above, it was confirmed that among various compounds with PDK inhibitory activity, VER-246608 shows the lowest cytotoxicity against hepatocytes and is the drug that most effectively inhibits fat accumulation caused by PA.

Therefore, the composition containing VER-246608 or a pharmaceutically acceptable salt thereof according to the present invention can be applied to inhibit fat accumulation in hepatocytes or to prevent, improve, or treat fatty liver disease.

The above detailed description is illustrative of the present invention. Additionally, the foregoing is intended to illustrate preferred embodiments of the present invention, and the present invention can be used in various other combinations, modifications, and environments. That is, changes or modifications can be made within the scope of the inventive concept disclosed in this specification, a scope equivalent to the written disclosure, and/or within the scope of technology or knowledge in the art. The written examples illustrate the best state for implementing the technical idea of the present invention, and various changes required for specific application fields and uses of the present invention are also possible. Accordingly, the detailed description of the invention above is not intended to limit the invention to the disclosed embodiments. Additionally, the appended claims should be construed to include other embodiments as well.

Claims (10)

A pharmaceutical composition for preventing or treating fatty liver disease, comprising VER-246608 or a pharmaceutically acceptable salt thereof as an active ingredient. According to paragraph 1,
The VER-246608 is a pharmaceutical composition that inhibits fat accumulation in hepatocytes. According to paragraph 2,
A pharmaceutical composition wherein the fat accumulation is induced by free fatty acids. According to paragraph 1,
The pharmaceutical composition, wherein the fatty liver disease is at least one selected from the group consisting of alcoholic fatty liver disease, non-alcoholic simple fatty liver disease, non-alcoholic steatohepatitis, non-alcoholic cirrhosis, and end-stage fibrotic liver disease. According to paragraph 1,
A pharmaceutical composition wherein the concentration of VER-246608 or a pharmaceutically acceptable salt thereof is 0.1 to 100 μM. A health functional food composition for preventing or improving fatty liver disease, comprising VER-246608 or a pharmaceutically acceptable salt thereof as an active ingredient. A feed composition for preventing or improving fatty liver disease comprising VER-246608 or a pharmaceutically acceptable salt thereof as an active ingredient. A cosmetic composition for preventing or improving fatty liver disease comprising VER-246608 or a pharmaceutically acceptable salt thereof as an active ingredient. A pharmaceutical composition for inhibiting fat accumulation in hepatocytes, comprising VER-246608 or a pharmaceutically acceptable salt thereof as an active ingredient. According to clause 9,
The pharmaceutical composition is for preventing or treating one or more diseases selected from the group consisting of obesity, non-alcoholic fatty liver disease, diabetes, and hyperlipidemia caused by fat accumulation in hepatocytes.