KR20020095553A - Pharmaceutical composition for prevention or treatment of obesity, hyperlipidemia or fatty liver containing inhibitors of isocitrate dehydrogenase activity - Google Patents

Abstract

PURPOSE: A preventing or therapeutic agent for obesity, hyperlipidemia or fatty liver containing an isocitrate dehydrogenase activity inhibitor as an effective ingredient is provided which has excellent effects for reducing body weight and suppressing obesity and biosynthesis of neutral lipid and cholesterol. CONSTITUTION: This prophylactic or treating agent for obesity, hyperlipidemia or fatty liver contains oxalomalic acid, methylisocitric acid, oxaloacetate and glyoxylate as an isocitrate dehydrogenase activity inhibitor. This agent is administered to a patient at dosages in a preferred range of about 7mg to 70g per day as adult patient(70kg).

Description

Pharmaceutical composition for prevention or treatment of obesity, hyperlipidemia or fatty liver containing inhibitors of isocitrate dehydrogenase activity}

The present invention relates to an agent for treating obesity, hyperlipidemia or fatty liver, comprising an isocitrate dehydrogenase activity inhibitor as an active ingredient, and more particularly, oxaloomalic acid, methyl isocitric acid The present invention relates to an agent for preventing or treating obesity, hyperlipidemia and fatty liver, including an isocitric acid dehydrogenase activity inhibitor including methylisocitric acid as an active ingredient.

Isocitric acid dehydrogenase is a major enzyme in the tricarboxilic acid (TCA) cycle for energy metabolism. It produces α-ketoglutaric acid and NADH or NADPH by oxidatively decarbonizing citric acid. It is known.

The isocitric acid dehydrogenase of higher animals is mitochondrial NAD ⁺ -specific isocitric acid dehydrogenase (hereinafter abbreviated as 'IDH'), cytoplasm, depending on the type of cofactor and location in the cell. NADP ⁺ - specific isocyanatomethyl trick acid dehydrogenation enzyme (hereinafter abbreviated as 'IDPc') and mitochondrial NADP ⁺ - 3 kinds of specific isocyanatomethyl trick acid dehydrogenation enzyme (hereinafter abbreviated as 'IDPm') of It is divided into isozymes.

Among them, IDH produces NADH and α-chitoglutarate by decarboxylating isocitric acid in the TCA cycle, which not only uses energy metabolism through electron transfer system, but also amino acid biosynthesis such as glutamic acid, glutamine, arginine and proline or Since it is an intermediate metabolite that synthesizes other biological metabolites, it acts as an important regulator of the TCA cycle for energy metabolism, protein biosynthesis and nitrogen metabolism.

IDPm and IDPc show strong tissue specificity in enzymatic activity. In cardiac tissue, more than 90% of the total enzymatic activity of NADP ⁺ -specific isocitric acid dehydrogenase is present in the mitochondria and about 10% is in the cytoplasm. In contrast, only 3% of liver tissues are known to be present in the mitochondria and the remaining 97% in the cytoplasm (Plaut, GWE, Current Topics in Cell Regulation , 2, 1-27, 1983).

The isocitric acid dehydrogenase isoenzymes have only been revealed in their structural features, but little is known about their function. In particular, the exact mechanism of action of IDPc and IDPm has not been studied.

On the other hand, the accumulation of fat in higher animals is performed according to the following process. In other words, excessive energy in the body promotes the differentiation of fat cells, which increase the number and size of white adipose tissue, thereby increasing the accumulation of fat, thereby increasing the expression of ob gene in white adipose tissue. Increasing leptin levels in the body leads to altered hormonal action in the brain, thereby lowering appetite, and on the other hand, the mechanism of dissipating excess calories at body temperature by UCP (uncoupler proteins). Going through. In white adipose tissue, gene expression such as PPARγ (Peroxisome Proliferator-Activated Receptor γ), C / EBPa and ADD1 / SREBP1, which are known as master transcription factors for adipocyte differentiation, are promoted to promote adipocyte differentiation and fat accumulation. This increases the amount of energy stored in the body in the form of fat, which regulates the energy balance in the body (Hu, E. et al., Proc. Natl. Acad. Sci. USA , 1995, 92, 9856-9860 Keller, H. et al., Proc. Natl. Acad. Sci. USA , 1993, 20, 9856-9860; Freytag.SO et al., Genes Dev ., 1994, 8, 1654-1663; Tontonoz, P. et al., Mol. Cell. Biol. , 1993, 13, 4753-4759; Spiegelman, BM, Cell , 1996, 87, 377-389). Substances that act as ligands in vivo for activation of PPARγ, a major transcription factor for adipocyte differentiation, are linoleic acid, docosahexaenoic acid (DHA), and arachidonic acid ( polyunsaturated fatty acids such as arachidonic acid (Krey, G. et al., Mol. Endocrinol. , 1997, 11, 779-791; Yu et al., J. Biol. Chem. , 1995, 270, 23975-23983 And prostaglandin J2 (Forman BM et al., Cell , 1995, 83, 803-812; Kliewer. SA et al., Cell , 1995, 83, 813-819).

The present inventors have found that the gene expression of isocitric acid dehydrogenase and the increase of enzymes and the increase of NADPH, which is a product of enzymatic reaction, increase fat accumulation and cause obesity and fatty liver, and at the same time neutral lipid in blood ( triglyceride) and cholesterol (cholesterol) was also confirmed to increase the amount (Korean Patent Application 2000-0061962). Therefore, the present inventors have been administered to oxalo malic acid and methyl iso citric acid, the inhibitors of isocitric acid dehydrogenase activity to adipocytes and mice, the weight loss effect, obesity inhibitory effect, biotriglyceridemia and cholesterol lowering effect is excellent The present invention has been completed by confirming that the isotactic acid dehydrogenase inhibitor can be used as an agent for preventing or treating obesity, hyperlipidemia or fatty liver.

An object of the present invention is to provide an agent for preventing or treating obesity, hyperlipidemia or fatty liver, which is excellent in weight loss effect, obesity inhibitory effect, biotriglyceridemic and cholesterol lowering effect, including isocitric acid dehydrogenase activity inhibitor.

1 is a graph showing the degree of enzyme activity inhibition in adipocytes treated with oxalo malic acid and methyl isocitric acid as inhibitors of isocitric acid dehydrogenase activity,

◆: 0.5mM oxalo malic acid, ■: 0.5mM methylisocitric acid,

Figure 2 is a graph showing the inhibitory effect of fat accumulation in adipocytes treated with oxalo malic acid and methyl isocitric acid, which are inhibitors of isocitric acid dehydrogenase activity,

A of FIG. 3 is a photograph comparing a result of the accumulation of fat in the back neck region of a mouse (right) administering to the oxaloacetic malic acid isocyanatomethyl trick acid dehydrogenation enzyme activity-inhibiting substance with normal mice (left);

B of FIG. 3 is a photograph comparing a result of the accumulation of abdominal fat in the trick isocyanic acid dehydrogenation enzyme activity inhibitor of oxaloacetic treated with malic acid mouse (right) and normal mice (left);

C of FIG. 3 is a photograph result of taking off the fatty tissue of the abdomen in a mouse (right) administering to the oxaloacetic malic acid isocyanatomethyl trick acid dehydrogenation enzyme activity-inhibiting substance: comparing normal mice (left) and size,

D in Figure 3 is the result of comparing the degree of accumulation of fat in the subcutaneous tissue of the mouse (right) administering to the oxaloacetic malic acid isocyanatomethyl trick acid dehydrogenation enzyme activity-inhibiting substance with normal mice (left) pictures,

Figure 4 is a bar graph showing the effect of inhibiting the increase in body weight in mice administered oxalo malic acid, an isocitric acid dehydrogenase activity inhibitor,

5 is a bar graph showing the effect of lowering blood triglycerides (triglycerides) in mice administered with oxalo malic acid, which is an isocitric acid dehydrogenase inhibitor,

6 is a bar graph showing the effect of lowering blood cholesterol in mice to which isocitric acid dehydrogenase activity inhibitors were administered.

In order to achieve the above object, the present invention provides an agent for the prevention or treatment of obesity, hyperlipidemia, or fatty liver, using an isocitric acid dehydrogenase activity inhibitor as an active ingredient.

The present invention also provides an agent for preventing or treating obesity, hyperlipidemia or fatty liver, which comprises oxalo malic acid or methyl isocitric acid as an active ingredient.

Hereinafter, the present invention will be described in detail.

The present invention provides a prophylactic or therapeutic agent for obesity, hyperlipidemia, or fatty liver, using an isocitric acid dehydrogenase activity inhibitor as an active ingredient.

The present inventors have found that the expression of NADPH-producing isocitric acid dehydrogenase gene in the cytoplasm, including the IDPc gene isozyme of isocitric acid dehydrogenase, and the enzymatic activity and the increase of NADPH are involved in lipid and cholesterol biosynthesis In addition, it promotes various enzyme reactions, and also increases the synthesis of lipoproteins through the increase of lipid components, thereby increasing the amount of biocholesterolemic complexes present in the form of lipoproteins (Korea Patent Application 2000- 0061962).

The present invention also provides an agent for preventing or treating obesity, hyperlipidemia or fatty liver, which comprises oxalo malic acid or methyl isocitric acid as an active ingredient.

Oxalo malic acid is produced in vivo by the aldol condensation reaction of oxalo acetate, an intermediate of the TCA cycle, and glyoxylate, an intermediate of the glyoxylate cycle, specifically inhibiting the activity of isocitric acid dehydrogenase and Isocitric acid strongly inhibits isocitric acid dehydrogenase, competing with isocitric acid as a substrate.

When oxalo malic acid and methyl isocitric acid are added to adipocytes, isocitric acid dehydrogenase activity is reduced by 50% in 30 minutes and 1 hour, respectively ( FIG. 1 ). In addition, differentiation into adipocytes is suppressed when treated on preadipocytes, and the amount of fat accumulation in adipocytes is also inhibited ( FIG. 2 ). In this case, the concentration of oxalo malic acid and methyl isocitric acid is suitably in the range of 0.1 μM to 100 mM, and preferably in the range of 10 μM to 5 mM.

It was confirmed through mouse experiments that isotactic acid dehydrogenase inhibitors showed a high effect on obesity inhibition and triglyceride and cholesterol lowering. This can be confirmed through the measurement of several indicators such as the increase of abdominal fat and the measurement of lipid accumulation in the body.

We administered oxalo malic acid and methylisocitric acid intraperitoneally to mice three times per week at 25 mg / kg body weight for 14 weeks. As a result, the body weight was reduced by more than 10% compared to the control mice intraperitoneally administered saline solution ( FIG. 4 ). When the mice were dissected, it was confirmed that the size of the abdominal fat tissue (epididymal fat pad) was very small and the weight of the fat tissue was reduced by about five times. In addition, after removing the outer skin, the dorsal appearance was also significantly reduced in the amount of obese tissue of mice treated with isocitric acid dehydrogenase inhibitor ( FIG. 3 ). In addition, the abdominal subcutaneous tissue was cut into the cross section to explore the degree of fat accumulation in the subcutaneous tissue, and then the tissues were embedded in paraffin after normal fixation and dehydration. Trick acid dehydrogenase inhibitors can be seen to play an important role in reducing weight and the accumulation of intracellular fat ( Fig. 3 ).

The present inventors confirmed how the above-described inhibitors of isocitric acid dehydrogenase activity inhibitors, such as oxalo malic acid and methyl isocitric acid, affect lipids and cholesterol in the body. Serum of mice treated with oxalo malic acid and methyl isocitric acid, which are the inhibitors of isocitric acid dehydrogenase activity, was isolated and measured for the amount of neutral lipid and total cholesterol, respectively. % And 30% decreased ( FIGS. 5 and 6 ).

As described above, the isocitric acid dehydrogenase activity inhibitor can effectively reduce fat accumulation in the body as well as the effect of weight loss and inhibit the biosynthesis of cholesterol, thereby reducing the amount of biocholesterol complex production.

The effective dosage of the prophylactic or therapeutic agent for obesity, hyperlipidemia or fatty liver, which uses the isotactic acid dehydrogenase activity inhibitor of the present invention as an active ingredient, is usually about 7 mg to 70 g per day for an average adult patient (70 kg). Range.

Specific doses of the prophylactic or therapeutic agent for obesity, hyperlipidemia or fatty liver using the isocitric acid dehydrogenase activity inhibitor of the present invention according to age, weight, symptoms, diseases to be treated, administration route, treatment period, etc. Is determined. That is, the doctor will determine the best dose for each patient, and the dose can vary depending on the age, weight, symptoms and the disease to be treated. Therefore, the dosage may be smaller or larger than the above-mentioned range, and all such dosages are within the scope of the present invention.

Although the agent for preventing or treating obesity, hyperlipidemia or fatty liver using the isotactic acid dehydrogenase activity inhibitor of the present invention as an active ingredient may be administered alone, it is generally a pharmaceutical selected according to the intended route of administration and standard pharmaceutical practice. It is administered in admixture with an acceptable carrier or diluent. The administration of the prophylactic or therapeutic agent for obesity, hyperlipidemia or fatty liver of the present invention may be in an oral dosage form such as a solid dosage form, a liquid dosage form or other dosage form, or a parenteral dosage form such as an injection solution, a coating agent or a suppository. In addition, the prophylactic or therapeutic agent for obesity, hyperlipidemia or fatty liver may be administered once to several times a day.

A prophylactic or therapeutic agent for obesity, hyperlipidemia or fatty liver, which uses the isocitric acid dehydrogenase activity inhibitor of the present invention as an active ingredient, can be administered systemically or locally through oral or parenteral administration. For example, it is orally administered in the form of tablets, pills, granules, capsules, liquids, emulsions, suspensions, syrups and the like. When administered parenterally, it may be injected intravenously, intramuscularly or subcutaneously, for example in the case of injections. For example, it may be applied to the skin in the form of patches, ointments, or external preparations for transdermal administration, and may be administered intrarectally in the form of suppositories. Typical oral solid dosage forms include powders, tablets, pills, granules, capsulants, and the like, wherein the active ingredients are combined with one or more inert diluents commonly used in the art (eg, starch, cellulose, lactose, kaolin, etc.). May be mixed and mixed with any lubricant, binder, disintegrant, stabilizer, and the like. Typical oral liquid formulations include pharmaceutically acceptable liquids, emulsions, suspensions, syrups, elixirs, and the like, wherein the active ingredients are included in one or more inert diluents commonly used in the art (eg, purified water, ethanol, etc.). And may be mixed with any sweetener, fragrance, preservative, or the like. Typical parenteral injectables include sterile solutions, emulsions, suspensions, etc., wherein the active ingredients are one or more inert aqueous diluents (e.g., distilled water for injection, physiological saline, etc.) commonly used in the art or inert ratios. It can be mixed with an aqueous diluent (eg, polyethylene glycol, propylene glycol, ethynol, etc.) and can be mixed with any isotonic, buffering, preservative, stabilizer and the like. The forms of the formulations exemplified above may be prepared by known methods.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Determination of isocitric acid dehydrogenase activity in adipocytes treated with isocitric acid dehydrogenase inhibitor

The isocitric acid dehydrogenase activity was measured by incubating for 10 minutes, 30 minutes, and 1 hour of treatment with 0.5 mM oxalolic acid and methyl isocitric acid in the NIH3T3-L1 preadipocyte cell line, respectively. 3 × 10 ⁷ cells were washed twice with 1 × phosphate buffer and then disrupted with sucrose buffer (0.32 M sucrose, 0.01 M Tris-Cl, pH 7.4). Cell debris was removed by centrifugation at 1,000 g, followed by centrifugation at 15,000 g to separate the cytoplasmic portion of the supernatant and the mitochondria, which are precipitates. The phosphate buffer containing 0.1% Triton X-100 was added to the supernatant, which is cytoplasmic nucleus, and quantified by Bradford assay. The enzyme activity was measured at 25 ° C. The reaction was performed in a buffer solution (50 mM MOPS, pH 7.2, 35.5 mM triethanolamine, pH 7.2, 2 mM NADP ⁺ , 2 mM MgCl ₂ , 5 mM isocitrate, 1 μg / ml rotenone). After completion of the reaction, the change in absorbance was measured on the colorimeter at 340 nm for 2 minutes, and the activity of the enzyme was shown as 1 unit of NADPH generating 1 μM for 1 minute.

As a result, control cells showed no change in isocitric acid dehydrogenase activity for 1 hour, whereas cells treated with oxalic acid and methyl isocitric acid decreased about 50% of isocitric acid dehydrogenase activity. Was confirmed ( Table 1 , Figure 1 ).

Enzyme Inhibition Degree (%) Processing time Oxalo malic acid Methylisocitric acid 0 min 100% 100% 10 minutes 59.1% 54.1% 30 minutes 50% 48.6% 60 minutes 59.1% 44.5%

<Example 2> Confirmation of fat synthesis amount in adipocytes treated with isocitric acid dehydrogenase inhibitor

To determine the effect of isocitric acid dehydrogenase inhibitors on fat synthesis, NIH3T3-L1 cell lines were inoculated at 5 μg / ml in DMEM containing 10% fetal bovine serum (FBS). 50,000 0.5 liter 3-isobutyl-1-methylxanthine (IBMX, Sigma), 1 μM dexamethasone (DEX), penicillin-streptomycin (Gibco BRL) each Incubation was carried out by U to promote differentiation of adipocytes for two days until the cell density reached about 3 × 10 ⁴ cells / cm 2. After that, the medium was changed every two days using the medium from which IBMX and DEX were removed from the medium, and the culture was continued for 10 days. Cell culture was performed in 5% CO ₂ , 37 ° C. in a CO ₂ incubator. After the incubation, oil red O, which specifically stains the fat formed in the adipocytes, was treated to observe the number of fat granules formed in the adipocytes and the degree of fat accumulation.

Oil red o staining discards the medium from the plate in which the cells are incubated and adds 10 ml of fixed solution (cacodylate buffer, 90 mM cacodylate, pH 7.2, 2% formaldehtde, 2.5% glutarardehyde, 0.025% CaCl ₂ , 5% sucrose). Then, the mixture was allowed to stand at 4 ° C. for 1 hour, then removed, and 5 ml of oil red o solution dissolved in 40% isopropanol was added thereto, mixed slowly for 1 hour, and then washed with 40% isopropanol.

As a result, as shown in Figure 2 , in the cells treated with isocitric acid dehydrogenase inhibitors it can be confirmed that the production of fat is reduced compared to the control ( Fig. 2 ). The results indicate that isotactic acid dehydrogenase activity inhibitors play an important role in reducing the accumulation of fat in cells.

Example 3 Investigation of Body Weight, Body Lipids and Cholesterol Increase in Mice by Isocitric Acid Dehydrogenase Inhibitors

<3-1> Change of Body Weight by Isocitric Acid Dehydrogenase Inhibitor

Mice were administered oxalo malic acid and methylisocitric acid by intraperitoneal injection three times a week at 25 mg / kg body weight for 14 weeks. As a result, the body weight was reduced by more than 10% compared with the control mice intraperitoneally administered saline, and the anatomy of the abdominal adipose tissue (epididymal fat pad) was very small. About 5 times reduction. In addition, after removal of the outer skin, the dorsal appearance was significantly reduced in the amount of obese tissue of mice treated with isocitric acid dehydrogenase inhibitor. To explore the degree of fat accumulation in the subcutaneous tissue, the abdominal subcutaneous tissue was cut in cross section, fixed with 10% neutral formalin solution, and the tissue was embedded with paraffin after the usual fixation procedure and dehydration. Embeded tissue was sectioned with a thickness of 4 μm and stained with hematoxylin and eosin and observed under an optical microscope to compare the degree of subcutaneous invasion with the control ( FIG. 3 ).

In addition, in order to measure the weight of the mice and normal mice to which isocitric acid dehydrogenase activity inhibitor was administered, after adjusting the zero point of the weighing scale of the animal, the weight displayed after the mouse was placed upright was measured by the weight of the mouse. ( Table 2 , Figure 4 ).

Weight increase (g) Control 22.19 ± 1.11 g Oxalo Maleic Acid Treatment Group 17.17 ± 1.9 g

<3-2> Measurement of Neutral Lipid and Total Cholesterol Levels in Blood by Isocitric Acid Dehydrogenase Inhibitor

Neutral lipid and total cholesterol concentrations were measured by measuring kits from Asan Pharmaceutical Co., Ltd., respectively. Serum was obtained by centrifugation of blood from mice treated with isocitric acid dehydrogenase inhibitor and normal mice. 72 ml of 1.5 ml of neutral lipid measurement kit liquid (lipoprotein lipase 10800 U, glycerol kinase 5.4 U, peroxidase 13,5000 U, L-α-glycerophosphate oxidase 160 U) was added to 10 µl of the obtained serum. Solution dissolved in N, N-bis (2-hydroxyethyl) -2-aminomethanesulfonic acid buffer) or 1.5 ml cholesterol enzyme measurement kit solution (enzyme solution (cholesterol esterase 20.5 KU / L, cholesterol oxidase 10.7 KU) / l, sodium hydroxide 1.81 g / l) and buffer (a solution of 13.6 g / l potassium phosphate, 1.88 g / l) in a 1: 1 mixture, and then reacted at 37 ° C. for 5 minutes, followed by a microplate analyzer (microplate). In the reader, cholesterol was measured at 500 nm and neutral lipid was measured at 540 nm, respectively. Quantitative conversion of neutral lipids and cholesterol was carried out using standard concentration curves obtained after applying the same method to these standard solutions.

As a result, the weight of abdominal adipose tissue was reduced by about 5 times compared to the control ( Fig. 3 ). On the other hand, the amount of triglyceride and total cholesterol in the blood serum was reduced by 50% and 30%, respectively, compared to normal mice in mice treated with isocitric acid dehydrogenase inhibitors ( Table 3 , Figures 5, 6 ).

Triglycerides (mg / dl) Total Cholesterol Amount (mg / dl) Control 147.6 ± 8 214.23 ± 13.5 Oxalo Maleic Acid Treatment Group 70.07 ± 4.9 148.74 ± 6.6

As described above, the prophylactic or therapeutic agent for obesity, hyperlipidemia or fatty liver using the isocitric acid dehydrogenase activity inhibitor of the present invention as an active ingredient has a weight loss effect, an obesity inhibitory effect, biotriglyceridemia and cholesterol biosynthesis inhibitory effect. It is excellent and thus can be usefully used for the prevention or treatment of obesity, hyperlipidemia or fatty liver.

Claims (4)

A prophylactic or therapeutic agent for obesity, hyperlipidemia or fatty liver, containing an isocitric acid dehydrogenase activity inhibitor as an active ingredient. The prophylactic or therapeutic agent according to claim 1, comprising oxalo malic acid as an inhibitor of isocitric acid dehydrogenase activity. 2. The prophylactic or therapeutic agent according to claim 1, wherein the isocitric acid dehydrogenase activity inhibitor comprises methylisocitric acid. 2. The prophylactic or therapeutic agent according to claim 1, which comprises oxalo acetate and glyoxylate as inhibitors of isocitric acid dehydrogenase activity.