KR101068686B1 - Obesity prevention or treatment composition containing metadoxin as an active ingredient - Google Patents

Abstract

The present invention relates to a composition for the prevention or treatment of obesity or its complications containing metadoxin (pyridoxol L-2-pyrrolidone-5-carboxylate, metadoxine) as an active ingredient, and to the prevention or treatment of obesity or its complications using the same It is about.

Metadoxin, obesity treatment, pharmaceutical composition

Description

Composition for preventing or treating obesity containing metadoxin as an active ingredient {COMPOSITION COMPRISING METADOXINE FOR PREVENTING OR TREATING OBESITY}

The present invention relates to a composition for the prevention and treatment of obesity or its complications with metadoxin as an active ingredient.

The present invention is supported by the task of 'development of new drugs for the treatment of inflammation and cancer through the interdisciplinary technology convergence' of the Seoul technology base building (core) project.

Obesity is increasing in modern society. Obesity is a serious disease that causes diabetes, hyperlipidemia and cardiovascular disease, and thereby death, has emerged as a serious health and social problem in Korea as well as developed countries, including the United States. In the United States, about 6% of total health expenditures are paid for obesity treatment, and the domestic obesity treatment market is approaching 1 trillion won. Obesity treatment is considered as a representative quality of life (QOL) improvement agent along with erectile dysfunction treatment and alopecia treatment.

Obesity refers to a condition in which fat cells proliferate and differentiate due to metabolic disorders, resulting in excessive accumulation of fat. If the energy uptake increases relative to consumption, the mass and volume of fat cells increase, resulting in an increase in the mass of fat tissue (Otto et al., 2005). Obesity at the cellular level is understood as an increase in the number and volume of adipocytes due to the promotion of proliferation and differentiation of adipocytes (Hirsch et al, 1976).

Obesity is classified into abdominal obesity (upper body obesity, central obesity) and lower body obesity depending on the distribution of fat in the body. Abdominal obesity refers to a case where excessive fat is accumulated in the abdomen or abdominal cavity, which causes diabetes, hypertension, hyperlipidemia, and atherosclerotic cardiovascular disease (stroke, ischemic heart disease). Abdominal obesity is usually measured as the ratio of waist circumference to hip circumference (W / H). Normally 0.9 for men and 0.8 for women are considered obese, but the criteria vary slightly from country to country. Abdominal obesity is also measured using visceral fat / subcutaneous fat area ratio using computed tomography. Abdominal obesity includes the subcutaneous form, where fat accumulates under the skin of the abdominal abdomen, depending on the distribution of fat, and the visceral form, where fat builds up in the mesentery, which divides between the gastric periphery and the internal viscera of the abdominal cavity. As a cause of abdominal obesity If you are sitting in a chair for a long time, you may have a lack of exercise, alcohol (alcohol), or stress.

Obesity causes various complications associated with it, so prevention and treatment of obesity are very important. Complications related to obesity include, for example, metabolic syndrome (visceral fat syndrome, metabolic syndrome, etc.), hypertriglyceridemia, hypoHDLemia, angina pectoris, myocardial infarction, sexual dysfunction, sleep apnea, premenstrual syndrome, stress urinary Urinary incontinence including incontinence, hyperactivity disorder, chronic fatigue syndrome, osteoarthritis, cancer associated with weight gain, orthostatic hypotension, pulmonary hypertension, dysmenorrhea, diabetes, hypertension, impaired glucose tolerance, coronary thrombosis, stroke, depression, anxiety Psychosis, tardive dyskinesia, drug addiction, substance abuse, cognitive impairment, Alzheimer's disease, cerebral ischemia, obsessive compulsive behavior, panic attack, social phobia, bulimia, atherosclerosis, gallbladder disease such as cholelithiasis, anorexia, polycystic ovary disease and Reproductive disorders, infections, varicose veins, skin diseases such as epidermal growth and eczema, insulin resistance, chronic arterial obstruction, orthopedic injury, thromboembolism, heart Intestinal diseases, urinary diseases, lipid syndrome, hyperglycemia, stress, and the like, but are not limited thereto.

Currently, there is a drug-independent method that consumes excess energy through exercise, and obesity treatment by drugs suppresses appetite, restricts caloric intake, appetite suppressants, lipase inhibitors, bloating agents, and heat generation. Accelerators and the like are used. Sibutramine, an appetite suppressant, was originally used as an antidepressant and suppresses the reabsorption of serotonin at synapses. However, the drug has been reported to have a number of side effects, including cardiovascular, central, hepatic and renal failure. Orlistat inhibits the function of lipase, a digestive enzyme that helps absorb the body by breaking down fat, and releases fat out of the body. However, it has been pointed out as a side effect as a side effect. There is a drug whose effect is unclear.

Adipocytes are made by stem cells, which differentiate into adipocytes, and then, progenitor cells, which differentiate into adipocytes. Fat cells play a key role in maintaining blood glucose homeostasis by decomposing the stored triglycerides when triglycerides are stored in the form of triglycerides and decomposing stored triglycerides when the body is depleted for exercise or fasting. It is a cell. On the contrary, the proliferation and differentiation of adipocytes increases excess fat intake and increases the amount of fat stored in the body, which is a direct cause of obesity. The 3T3-L1 adipocyte cell line is an in vitro model widely used for the study of the proliferation and differentiation of adipocytes. The treatment of hormonal stimulants to these cells undergoes a mitotic clonal expansion (MCE) step in which cell division resumes. Differentiation progresses with expression of genes involved in the development of fat cells such as EBPα, PPARγ, adipocyte protein-2 / fatty acid-binding protein (Spiegelman et al., 1996; Fajas et al., 1998) ).

Cyclic AMP response element binding protein (CREB) has been reported to be a key transcription factor in cells that are expressed continuously during the differentiation of adipocytes and adipocytes before adipocyte differentiation begins (Klemm et al., 1998; Reusch et al., 2000). CREB is activated by insulin, dexamethasone and 3-isobutyl-1-methyxanthine (IBMX), which are differentiation inducing substances of adipocytes. CREB regulates transcription of these genes by binding to promoter sites of genes specific for adipocytes (Reusch et al., 2000; Spiegelman et al., 1980). Therefore, activation of CREB is known to be essential for adipocyte differentiation. In cells, protein kinase A (PKA) phosphorylates the Ser133 position of the target protein, CREB, and activates CREB by promoting its migration to the nucleus (Student et al., 1980; Gonzalez et al., 1989). Activated CREB promotes C / EBPβ expression in the early stages of differentiation of adipocytes. As in the case of other proteins, C / EBPβ is produced through protein synthesis from C / EBPβ mRNA. During this process, C / EBPβ-liver-enriched activator protein (C / EBPβ-LAP) and C / EBPβ-LIP (liver) -enriched inhibitory protein) has a unique biosynthetic pathway in which four proteins are produced (Ossipow et al., 1993; Timchenko et al., 1999). Subsequently, expression of genes such as C / EBPa and PPARγ specific for adipocytes is successively promoted, resulting in traits of mature adipocytes (Zhang et al., 2004).

Therefore, the development of drugs with inhibitory effect on C / EBPβ-LAP expression, which acts as a key factor in the differentiation of adipocytes, and the inhibition of C / EBPα and PPARγ activity in differentiated adipocytes It is expected to be a new drug therapy for the treatment of obesity.

On the other hand, metadoxin (pyridoxol L-2-pyrrolidone-5-carboxylate) is a complex compound of pyridoxine (pyridoxine) and pyrrolidone carboxylate having a structure represented by the following formula (1).

Metadoxine has been studied as a therapeutic for alcoholic fatty liver (Ki et al., 2007; Caballeria et al., 1998; Gutierrez et al., 2003). In animal models, metadoxin has been reported to increase the release of alcohol and acetaldehyde and to reduce the damage caused by free radicals to restore intracellular ATP and GSH to normal levels (Caballeria et al., 1998). Metadoxine is also known to be effective against alcoholic fatty liver as a drug currently being used clinically (Gutierrez et al., 2003). Although metadoxin has been shown to inhibit the accumulation of fat in the liver, the effects on the proliferation and differentiation of fat cells have not been studied.

The present invention is to solve the problem to find a method for preventing, improving or treating obesity and the complications and the composition therefor.

The present inventors have announced through the previous study that the alcoholic fatty liver disease is effectively improved by the combined administration of metadoxin and garlic oil (Korean Patent Application No. 10-2006-0119935). Based on the results of these previous studies, studies on the relationship between metadoxin and obesity have shown that metadoxin inhibits PKA-CREB signaling, which affects the expression of key transcription factors that regulate the differentiation of adipocytes. By suppressing the differentiation process of fat cells thereby revealing an excellent effect on the prevention or treatment of obesity and its complications, the present invention has been completed based on this.

The composition containing the metadoxin of the present invention as an active ingredient inhibits the differentiation of fat cells through the inhibition of PKA-CREB signal by metadoxine, as well as inhibits the accumulation of fat in the cells, thereby causing obesity and its complications with little side effects. It can be usefully used as a prophylactic or therapeutic use of.

The present invention provides a composition for preventing or treating obesity containing metadoxin as an active ingredient and a pharmaceutically acceptable carrier, and a method for preventing or treating obesity using the same.

The present invention also provides a food composition for the prevention or improvement of obesity containing metadoxin as an active ingredient.

The present invention also provides a composition for the prevention, improvement or treatment of obesity and related complications, including metadoxin and other obesity treatment substances (eg, sibutramine, orlistat) or the prevention, improvement or Provide treatment.

The composition of the present invention is remarkable for the prevention and treatment of obesity by inhibiting the differentiation of fat cells through the inhibition of PKA-CREB signaling process in which the treatment of metadoxin affects the expression of key transcription factors that regulate adipocyte differentiation. It shows an excellent effect.

Since obesity is a problem not only in humans but also in many other animals, the present invention also provides methods and compositions for treating or preventing obesity in other animals than humans.

The inventors of the present invention have found that metadoxin inhibits the differentiation of 3T3-L1 adipocytes into adipocytes by identifying triglycerides in the cytoplasm (FIG. 1B). In addition, the expression of PPARγ, C / EBPa, ACC, and sterol regulatory element binding protein (SREBP), which are indicator proteins of adipose differentiation, was also reduced by metadoxin, and confirmed by immunochemical analysis (FIG. 1C). Treatment of adipose progenitor cells with MDI (3-isobutyl-1-methylxanthine, dexamethasone and insulin) caused mitotic clonal expansion, leading to a sharp increase in cell numbers, whereas metadoxin treatment did not significantly inhibit this fat cell proliferation (FIG. 2A). ). Significant inhibitory effects were not observed by metadoxin on the expression of p27, p21, a protein that inhibits cyclin-dependent kinase (CDK) activity, and cyclin A, which indicates the progression of S phase of the cell cycle. 2B, it is interpreted that the effect of inhibiting the differentiation of adipocytes by metadoxin is not a phenomenon caused by disturbing the cell cycle.

The inventors of the present invention have observed different effects of metadoxin-induced differentiation of hormone-induced adipocytes at different stages of the differentiation process. Results: When metadoxin was treated for 8 days of differentiation induction, triglyceride accumulation was strongly inhibited (Fig. 3A, Experiment # 3), and the drug was induced between 4 and 8 days later in the process of inducing differentiation of adipocytes. Triglyceride accumulation was effectively inhibited when treatment was performed (Fig. 3A, Experiments # 3, # 4, # 5), but when the drug was treated until the first 4 days of adipocyte differentiation induction process, fat accumulation progressed to a similar level as the control group. It confirmed that it became. These results indicate that metadoxin acts during the differentiation of adipocytes, not the early stages of differentiation. Inhibition of the accumulation of neutral lipids by metadoxin was also confirmed through expression changes of proteins of FAS and ACC, which are marker proteins of adifferentiation (FIG. 3B). These results suggest that the inhibition of adipocyte differentiation by metadoxin is not due to the blocking of mitotic clonal expansion, which is a preliminary stage of lipodifferentiation, but by the inhibition of the latter stage.

Transcriptional stimulation of luciferase gene in 3T3-L1 adipocytes cultured and differentiated by MDI for 2 days was reduced by metadoxin treatment (FIG. 4A), while PPARγ and RXRα dependent reporter activity in HEK293 cells was not affected by metadoxin treatment. Not changed (Fig. 4B). Therefore, the change in PPRE luciferase activity in the adipocyte differentiation program is thought to be due to inhibition of cellular differentiation, but not inhibition of PPARγ dependent gene transcription by metadoxin.

The inventors of the present invention, using an antibody that specifically binds to the Ser133 position of CREB phosphorylated by PKA, the degree of phosphorylation of CREB induced by MDI at the initial time after metadoxin treatment (FIG. 5A) and expression of total CREB It was observed that the amount decreased. In addition, the degree of binding of activated CREB to the CREB consensus sequence site was observed by gel delay analysis. Was observed (FIG. 5B). The suppression of the differentiation of adipocytes by metadoxin is interpreted as being disrupted by the process of phosphorylation and activation of CREB by MDI treatment.

We also demonstrated that metadoxin has an inhibitory effect on the differentiation of adipocytes by reducing phosphorylation of CREB by disrupting the PKA-adenylate cyclase signaling system (FIGS. 6A, B, C).

Hormone stimulation induced the differentiation of adipocytes, and the expression of C / EBPβ-LAP and C / EBPβ-LIP was observed. The hormonal stimulation induced C / EBPβ activity decreased 24 hours after metadoxin treatment. (FIG. 6), it was observed that the ratio of LAP / LIP in the metadoxin treatment group decreased from 12 hours to 48 hours compared to the control group (FIG. 7). This means that metadoxin formed a transcription factor complex in the direction of inhibiting adipocyte differentiation.

Based on the above experimental results, the present invention provides a pharmaceutical composition for the prevention or treatment of obesity or its complications, including metadoxin, a pharmaceutically acceptable salt thereof, or a solvate or hydrate thereof as an active ingredient, or It is used to provide a method of preventing or treating obesity or the complications thereby. The pharmaceutical compositions of the present invention are used to treat or prevent obesity and the complications of humans as well as other animals.

The present invention also provides a food composition for preventing or ameliorating obesity or its complications, including metadoxin, a food acceptable salt thereof, or a solvate or hydrate thereof as an active ingredient, or using the composition or obesity or It provides a method for preventing or ameliorating complications. The food composition of the present invention is used to prevent or ameliorate the obesity and the complications of humans as well as other animals.

The present invention also provides a composition for the prevention or treatment of obesity or its complications, including metadoxin, a pharmaceutically acceptable salt thereof, or a solvate or hydrate thereof and other obesity treatment substances as an active ingredient, or the composition To provide a method of preventing or treating obesity or the complications thereof. Other obesity therapeutic agents include, but are not limited to, for example, sibutramine, orlistat, and the like. The pharmaceutical compositions of the present invention are used to prevent or treat obesity and the complications of humans as well as other animals. The metadoxin and the anti-obesity substance are preferably contained in a weight ratio of 25-1: 1-25, more preferably 10-1: 1-10.

The composition of the present invention can also be used for the prevention, improvement or treatment of obesity or abdominal obesity of each part of the body, the target abdominal obesity, living for a long time sitting in a chair, lack of exercise, alcohol (alcohol) intake, stress, etc. But not limited to, but not limited to.

In the present invention, the complications include, but are not limited to, diseases caused by elevated blood concentrations of triglycerides, cholesterol, and low density lipids. In addition, complications due to obesity, which can be prevented or treated by the composition of the present invention, for example, metabolic syndrome (intestinal fat syndrome, metabolic syndrome, etc.), hypertriglyceridemia, hypoHDLemia, angina pectoris, myocardial infarction , Sleep apnea, premenstrual syndrome, urinary incontinence including stress urinary incontinence, hyperactivity disorder, chronic fatigue syndrome, osteoarthritis, cancer associated with weight gain, orthostatic hypotension, pulmonary hypertension, menstrual disorders, diabetes, hypertension, impaired internal Glucose, coronary thrombosis, stroke, depression, anxiety, psychosis, tardive dyskinesia, drug addiction, drug abuse, cognitive impairment, Alzheimer's disease, cerebral ischemia, obsessive compulsive behavior, panic attack, social phobia, macrophage, atherosclerosis, cholelithiasis Reproductive disorders such as gallbladder disease, anorexia, polycystic ovary diseases, infections, skin diseases such as varicose veins, epidermal growth and eczema, insulin Resistance, chronic arterial occlusion, orthopedic injury, thromboembolism, heart disease, urinary disease, lipid syndrome, hyperglycemia, stress, and the like.

In the composition of the present invention, as an active ingredient, a pharmaceutically or food acceptable salt of metadoxin may be used. Examples of such salts include acid addition salts and quaternary ammonium salts. Examples of acid addition salts include inorganic acid salts such as hydrochloride, hydrobromide, hydroiodide, sulfate, and phosphate, oxalate, maleic acid salt, fumaric acid salt, lactic acid salt, malic acid salt, succinic acid salt, tartaric acid salt, and ancin. Organic acid salts, such as a salt of a salt and a methanesulfonic acid salt, are mentioned. As the quaternary ammonium salts, for example, lower alkylhalogenides such as methyl iodide, methyl bromide, ethyl iodide, ethyl bromide and the like; Lower alkylsulfonates such as methylmethanesulfonate and ethylmethanesulfonate; And quaternary ammonium salts such as lower alkylarylsulfonate such as methyl-p-toluenesulfonate.

In addition, since metadoxin or a pharmaceutically or food-acceptable salt thereof may exist as a solvate or hydrate, solvates or hydrates thereof may be used as an active ingredient of the therapeutic agent of the present invention.

The present invention also provides a pharmaceutical composition wherein the pharmaceutical composition is formulated as an oral preparation and an injection. The preparation for oral administration is preferably selected from capsules, tablets, suspensions, syrups or powders.

Metadoxin, an active ingredient of the composition of the present invention, a pharmaceutically or food acceptable salt thereof, or a solvate or hydrate thereof may be administered to the patient itself, but in general, one or two of these active ingredients. Compositions containing more than one species may be prepared or administered, or may be administered in combination with other useful anti-obesity drugs in a formulated form. In practice, it is formulated and administered in a unit dosage form suitable for oral administration according to conventional methods used in the art. Oral dosage forms suitable for this purpose include hard and soft capsules, tablets, powders, suspensions, syrups and the like. Such oral preparations include, in addition to two or more active ingredients, one or more inert conventional carriers such as excipients such as starch, lactose, carboxymethylcellulose, kaolin, water, gelatin, alcohols, glucose, gum arabic And additional additive components such as binders such as tragacanta rubber, disintegrants such as starch, dextrin, sodium alginate, and suspending agents such as talc, stearic acid, magnesium stearate, liquid paraffin, and the like. In the present invention, a dissolution aid for dissolution may also be added.

The daily dose of the drug according to the present invention depends on various factors such as the degree of obesity progression, onset time, age, health condition, complications, etc. of the subject to be administered, but generally based on the adult weight ratio mentioned above 50 mg to 2 g, preferably 200 to 750 mg of the combined composition may be administered once or twice a day. However, patients with severe obesity or complications can be administered by increasing the drug of the present invention to a large amount outside the above range in order to improve treatment efficiency. Most preferably, 1-2 unit doses containing 200-500 mg of metadoxin are orally administered once or twice daily. When the composition of the present invention is a food composition, the mixed amount of the active ingredient can be suitably determined according to the purpose of use (prevention, health or therapeutic treatment). In general, metadoxin may be added in amounts of 0.0001 to 10% by weight, preferably 0.1 to 5% by weight, based on the raw materials in the manufacture of foods or beverages and the like. However, in the case of long-term intake for health and hygiene or health control, the amount may be below the above range, but the active ingredient may be used in an amount above the above range because there is no problem in terms of safety. Self-explanatory Foods using the food composition of the present invention include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, dairy products including other noodles, gum, ice cream, various soups, drinks, tea, drinks, Alcoholic beverages and vitamin complexes, and the like, but are not limited thereto.

The present invention is explained in more detail by the following experimental examples and examples, but the present invention is not limited thereto.

Experimental Example

Materials and Methods

Metadoxine was provided by Pharmaking. IBMX, dexamethasone (dexamethasone) and insulin (insulin) were used as products of Sigma-Aldrich (St. Louis, MO). Antibodies to PPARγ, C / EBPa, C / EBB, SREBP1, p21, p27 and cyclinA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). CREB, p-CREB (Ser133), acetyl coenzyme A carboxylase (ACC), ERK, p-ERK (Thr202 / Tyr204), JNK, p-JNK (Thr183 / Tyr185), p38 kinase and p-p38 kinase (Thr180 / Tyr182) was purchased from Cell Signaling Technology (Beverly, MA), anti-FAS antibody from BD Biosciences Pharmingen (San Jose, Calif.), And anti β-actin antibody from Sigma-Aldrich.

Cell culture Culture )

3T3-L1 adipocyte cell lines were obtained from ATCC (Rockville, MD). The cells were grown in medium containing Dulbecco's modified Eagle's medium (DMEM), 10% fetal bovine serum, 10 mM 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid, 50 units / ml penicillin and 50 μg / ml streptomycin. It was used at 37 ^o C, 5% CO _{2 in} a humidified atmosphere.

Differentiation of fat cells Adipocyte Differentiation )

To induce differentiation of 3T3-L1 adipocytes, change medium and incubate for 2 more days, then 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 1 mM dexamethasone, 1 mg / ml Replaced with DMEM medium containing insulin (MDI). After 2 days, the culture was replaced with DMEM medium containing 1 mg / ml insulin and then cultured for about 8-10 days with the same medium every 2 days. On day 8, cells were collected for Oil Red O staining or immunochemical analysis. If necessary, the cell count was counted with a hemocytometer under an optical microscope.

Oil Red Blot Dyeing ( Oil Red  O Staining )

To determine the status of adipocyte differentiation, the cells were washed with PBS, fixed with 10% formalin for 1 hour, and then stained with an optical microscope using Oil Red O staining.

Luciferase reporter gene analysis ( Luciferase Reporter Gene Assay )

Promega's dual-luciferase reporter assay system was used. 3T3-L1 cells (7 × 10 ⁵ ) were cultured in 6 well plates and serum depleted for 3 hours before transfection of the promoter-containing fluorescent reporter gene. Cells were lysed 48 hours after metadoxin treatment 12 hours later and activity was measured using lumino scan.

Immunoblot Analysis

The cells were buffered with 10 mM Tris-HCl, pH 7.1, 100 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% Triton X-100, 0.5% Nonidet P-40, 1 mM dithiothreitol and 0.5 mM phenylmethylsulfonyl fluoride Cell lysates were prepared by dissolving in a solution supplemented with a protase inhibitory cocktail (Calbiochem, La Jolla, CA).

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analysis were performed as previously described. Immune response proteins were stained with ECL chemiluminescence detection kit (Amersham Biosciences, Buckinghamshire, UK) or SuperSignal West Pico chemiluminescent substrate kit (Pierce, Rockford, IL). Whether the same amount of protein was loaded was confirmed by β-actin immunochemical analysis. At least three independent experiments with different samples were performed to confirm changes in protein levels.

Preparation of Nuclear Extracts

Cells were washed with cold PBS, detached from the surface and transferred to micro tubes. 50 μl of lysis buffer [10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.5% Nonidet-P40, 1 mM dithiothreitol and 0.5 mM phenylmethylsulfonylfluoride] was added to the cells and vortexed to destroy the cell membrane. Then, after standing in ice for 10 minutes, centrifuged for 5 minutes at 4 ℃, the pellet containing the nucleus 20 mM HEPES (pH 7.9), 400 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol and 1 mM phenylmethylsulfonylfluoride Resuspended in 25 μl of the extract buffer containing, left on ice for 30 minutes, and centrifuged at 10,000g for 10 minutes to obtain a supernatant containing nuclear extracts (nuclear extracts).

Gel Delay Assay (Gel Shift Assay )

Label the end with [γ-32P] ATP and T4 polynucleotide kinase on the double-stranded DNA probe (under 5'-AGAGATTGCC TGACGTCA GAGAGCTAG-3 'is the key nucleotide sequence) for the consensus sequence of CREB for gel shift analysis Was used. The reaction mixture is 5 × binding buffer 2 containing 20% glycerol, 5 mM MgCl ₂ , 250 mM NaCl, 2.5 mM EDTA, 2.5 mM dithiothreitol, 0.25 mg / ml poly dI-dC, 50 mM Tris-HCl, pH 7.5. The total amount was 10 μl, including μl, 10 μg nuclear extract and sterile distilled water, and the reaction was initiated by adding 1 μl (10 ⁶ cpm) probe, followed by 10 minutes of preculture and then 20 minutes of room temperature. Incubation at continued. The specificity of protein binding to DNA was confirmed by adding a 20-fold excess of unlabeled oligonucleotides to each reaction mixture prior to the addition of the radioisotope labeled probes for a competitive reaction. In some experiments, specificity of CREB binding to DNA consensus sequences was confirmed by supershift analysis using 2 μg of each specific antibody. Samples were loaded on 4% polyacrylamide gels and developed at 140 V, then gels were separated, dried and measured using radioisotopes.

Data Analysis

Scanning densitometry was performed using a Scan & Analysis System (Alpha-Innotech Corporation, San Leandro, Calif.). The area of each lane was calculated using AlphaEaseTM version 5.5 software.

Experimental Example 1 <Inhibition of Differentiation of Adipose Progenitor Cells into Adipocytes by Metadoxin>

In the present invention, as part of a study for discovering a drug having a prophylactic and therapeutic effect on obesity using the 3T3-L1 progenitor cell line model, it was observed whether metadoxin affects the differentiation of fat cells. 3T3-L1 adipocytes were treated with hormone-stimulated MDI and 0.1-1 mM of metadoxin at the same time, or treated with MDI alone without metadoxin and incubated in growth medium containing 10% FBS for 8 days. When metadoxin was treated, it was confirmed by staining triglycerides in the cytoplasm with Oil Red O staining that concentration-dependent inhibition of differentiation compared to the control group (FIG. 1B). From the accumulation of triglycerides stained with Oil Red O in the cytoplasm, it could be observed that hormonal treatment allows the fat precursor cells to differentiate well. On the other hand, treatment with metadoxine significantly inhibited differentiation into adipocytes in a dose-dependent manner.

Biochemical changes in adipose differentiation were confirmed by observing the expression levels of adipose differentiation marker proteins PPARγ, C / EBPa, ACC, and sterol regulatory element binding protein (SREBP). As expected, each lipo differentiation marker protein was confirmed by immunochemical assay that dose-dependently decreased by metadoxin (FIG. 1C).

Experimental Example 2 < Metadoxin  Effect on fat cell proliferation>

When hormonal stimulation is applied to 3T3-L1 progenitor cells that have ceased dividing, the cells undergo a mitotic clonal expansion step, which resumes dividing and occurs about 2-3 times of cell division, which is essential for the differentiation of fat cells. Known (Otto et al., 2005; Sherr et al., 1999; Morrison et al., 1999, Zhang et al., 2004). Therefore, we examined whether metadoxin inhibits the differentiation of adipocytes by disrupting the mitotic clonal expansion process. When MDI was treated with suspended progenitor cells, the number of cells in the control group increased rapidly as a function of time for about three days during the process of mitotic clonal expansion. As a result of measuring the relative number of cells in the metadoxin treated group and the control group, there was no significant change between the two groups (FIG. 2A).

Proteins that inhibit the activity of Cyclin-dependent kinase (CDK) also play an important role in regulating the cell cycle associated with the differentiation of adipocytes. Therefore, the expression levels of p27, p21, a CDK activity inhibitory protein, and cyclin A (Sherr et al., 1999), which indicates the progression of S phase of the cell cycle, were confirmed. p21, p27, cyclin A protein expression level was not significantly changed when compared with the metadoxin treatment group and the control group (Fig. 2B). The above results are interpreted that the effect of inhibiting the differentiation of fat cells by metadoxin is not a phenomenon caused by disturbing the cell cycle.

Experimental Example 3 < Metadoxin  Effect on adipocyte differentiation

The purpose of this study was to investigate the effects of metadoxin-induced differentiation of hormone-induced adipocytes. In this experiment, the treatment period of metadoxin or the timing of metadoxin treatment were differently observed. When metadoxin was treated for 8 days of differentiation induction process, triglyceride accumulation was strongly inhibited (FIG. 3A, Experiment # 3). It was confirmed that the accumulation of triglycerides was effectively inhibited when the drug was treated between 4 and 8 days, which is a late process of adipocyte differentiation (FIG. 3A, Experiment # 3, # 4, # 5). On the other hand, when the drug was treated until the first 4 days of adipocyte differentiation induction process, it was confirmed that fat accumulation proceeded to a similar degree as the control group. In addition, it was observed that accumulation of triglycerides was significantly inhibited when 3T3-L1 adipocytes were treated with the drug from 4 to 8 days (Fig. 3A, Experiment # 4, # 5). These results indicate that metadoxin acts during the differentiation process of adipocytes, not the early stages of differentiation.

Inhibition of accumulation of neutral lipids by metadoxin in each treatment group was also confirmed through the expression changes of proteins of FAS and ACC, which are marker proteins of adipose differentiation (FIG. 3B). These results suggest that the inhibition of adipocyte differentiation by metadoxin is not due to the blocking of mitotic clonal expansion, which is a preliminary stage of lipodifferentiation, but by the inhibition of the latter stage.

Experimental Example 4 < Metadoxin  by PPAR Effect on γ Gene Transcription>

Among PPARs, PPARγ is specific for adipocytes and is most highly expressed in adipose tissue and adipocyte lines. To determine whether the reduction of fat accumulation by metadoxin obtained from Oil Red O staining results from the change of PPAR binding to the target promoter, in 3T3-L1 adipocytes cultured and differentiated by MDI for 2 days The transcriptional promotion of the luciferase gene was monitored. When 3T3-L1 adipocytes were treated with metadoxin, PPRE luciferase activity was decreased in a concentration dependent manner (FIG. 4A). In order to confirm whether the decrease in PPRE luciferase activity was caused by direct PPARγ antagonism, PPRE luciferase activity was confirmed after transfection of PPARγ and RXRα into HEK293 cells. Metadoxin treatment did not alter PPRE luciferase activity increased by PPARγ and RXRα (FIG. 4B). Thus, changes in PPRE luciferase activity in adipocyte differentiation programs are thought to result from inhibition of cellular differentiation, but not inhibition of PPARγ dependent gene transcription by metadoxin.

Experimental Example 5 < Metadoxin  by CREB  Reduction of Activation>

CREB is a transcription factor that is continuously expressed during the differentiation of adipocytes and hormone-induced adipocytes and is known to regulate the proliferation and differentiation of adipocytes (Klemm et al., 1998; Reusch et al., 2000). The activated form of CREB overexpression is sufficient to allow the proliferation and differentiation of adipocytes. This was confirmed by the accumulation of triglycerides in the cytoplasm, changes in cell morphology, and an increase in marker proteins of fat differentiation. On the other hand, overexpression of inactivated forms of CREB inhibited adipocyte differentiation (Reusch et al., 2000). Therefore, we tried to determine whether the effect of inhibiting the differentiation of adipocytes by metadoxin is a phenomenon caused by disturbing the process of phosphorylation and activation of CREB by MDI treatment. The antibody that binds to phosphorylated CREB used in this experiment is an antibody specific for the Ser133 position of CREB phosphorylated by PKA. Immunohistochemical analysis confirmed that the phosphorylation of CREB induced by MDI was reduced at the initial time after metadoxin treatment (FIG. 5A). In addition, the expression of total CREB induced by MDI was also reduced by metadoxin treatment. This is because the expression of CREB is regulated by CRE (Meyer et al., 1993). In addition, the degree of binding of activated CREB to the CREB consensus sequence site was observed using a gel delay assay. As expected, the extent of binding of the protein of the nuclear fraction to the consensus sequence site of CREB was reduced after 15-30 min by treatment with metadoxin (FIG. 5B). These results were in agreement with those obtained by immunochemical analysis.

Experimental Example 6 < PKA  Dependent CREB  For phosphorylation Metadox  Impact>

To determine whether metadoxine directly reduced phosphorylation and activity of CREB, metadoxin was simultaneously treated with substances that increased cAMP by activating PKA in adipocytes. When 3T3-L1 adipocytes were treated with 8-Br-cAMP or forskolin, the expression level of phosphorylated CREB was increased. The expression of phosphorylated CREB increased by these substances was continuously decreased from the initial time by metadoxin treatment (Figures 6A, B). In contrast, pretreatment with H89, a chemical PKA inhibitor, reduced phosphorylation of CREB induced by MDI (FIG. 6C). These results demonstrate that metadoxin has an inhibitory effect on the differentiation of adipocytes by reducing the phosphorylation of CREB by disrupting the PKA-adenylate cyclase signaling system.

Example 7 <reduction of C / EBP β expression by single meta>

C / EBPβ is a key regulator early in the differentiation process of adipocytes (Yeh et al., 1995). C / EBPβ is a target gene of CREB and plays an important role in adipocyte differentiation. C / EBPβ activates the transcription of C / EBPa and PPARγ, and is known to be an important regulator of mitotic clonal expansion and late stage differentiation (Lane et al., 2005). Therefore, hormonal stimulation induced the differentiation of adipocytes and observed the expression of C / EBPβ-LAP and C / EBPβ-LIP. The hormonal stimulation-induced activity of C / EBβ decreased its expression after 24 hours of metadoxin treatment (FIG. 7). In addition, as a result of quantifying the intensity of the band region excluding the background, the ratio of LAP / LIP of the metadoxin treatment group decreased from 12 hours to 48 hours compared to the control group (FIG. 7). These results indicate that metadoxin formed a transcription factor complex in the direction of inhibiting adipocyte differentiation.

Various forms of preparations containing metadoxin as active ingredients were prepared as follows.

Formulation Example 1 Preparation of Tablet

Metadoxin 100 mg

Lactose 50 mg

Starch 10 mg

Magnesium stearate proper amount

The tablets were prepared by mixing the above components and tableting according to a conventional method for preparing tablets.

Formulation Example 2: Preparation of Powder

Metadoxin 250 mg

Lactose 30 mg

Starch 20 mg

Magnesium stearate proper amount

After the above ingredients were mixed intimately, a powder was prepared by filling and sealing a polyethylene-coated cloth.

Formulation Example 3: Preparation of Capsule

Metadoxin 500 mg

Lactose 30 mg

Starch 28 mg

Talc 2 mg

Magnesium stearate proper amount

The above ingredients were mixed and filled into gelatin light capsules according to a conventional method for preparing capsules to prepare capsules.

Formulation Example 4: Preparation of Suspending Agent

Metadoxin 50 mg

10 g of isomerized sugar

30 mg of sugar

Sodium Carboxymethylcellulose 100 mg

Lemon flavor

Add 100 ml of purified water

The above components were mixed to prepare a suspension according to a conventional method for preparing a suspension, and then filled into a 100 ml brown bottle and sterilized to prepare a suspension.

Formulation Example 5 Preparation of Soft Capsule (Content in 1 Tablet of Soft Capsule)

Metadoxin 500 mg

Polyethylene glycol 400 400 mg

Concentrated glycerin 55 mg

Purified water 35 mg

After mixing polyethylene glycol and concentrated glycerin, flavone was added while maintaining the mixture to which purified water was added at about 60 ° C., followed by uniform mixing while stirring at about 1,500 rpm with a stirrer. The mixture was cooled slowly to room temperature with gentle stirring, and the contents of the soft capsule were prepared by removing bubbles with a vacuum pump.

The soft capsule film is gelatin known in the art, soft formulation of plasticizer, gelatin 132 mg per 1 capsule, concentrated glycerin 52 mg, 70% dissolbitol solution 6 mg and the amount of ethyl vanillin as a flavoring agent, carnauba wax as coating agent It was prepared by the conventional manufacturing method using.

Formulation Example 6 Preparation of Injection

Metadoxin 200 mg

Mannitol 180 mg

Sterile distilled water for injection 2974 mg

Na ₂ HPO ₄ 12H ₂ O 26 mg

According to the conventional method for preparing an injection, the amount of the above ingredient is prepared per ampoule (2 ml).

Formulation Example 7 Preparation of Beverage

Metadoxin 0.01g

8.5 g citric acid

10g per bag

2.5 g of glucose

DL-apple acid 0.3 g

Purified water

A proper amount of purified water was added to the above-mentioned ingredient content to make a total of 100 mL, followed by stirring.

references:

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Abbreviations: Each abbreviation described in the present specification means the following.

ACC: acetyl CoA carboxylase; AMPK: AMP-activated protein kinase;

CDK: cyclin depend kinase; C / EBP: CCAAT / enhancer binding protein; CRE: cyclic AMP response element; CREB: cyclic AMP response element binding protein; FAS: fatty acid synthase; IBMX: 3-isobutyl-1-methylxanthine; MAPK: mitogen activated protein kinase; MDI: 3-isobutyl-1-methylxanthine, dexamethasone and insulin; PKA: protein kinase; PPAR: peroxisome proliferator activated receptor, SREBP: sterol regulatory element binding protein

1 is a result of observing the effect of inhibiting the differentiation of 3T3-L1 preadipocytes (fat progenitor cells) to adipocytes by metadoxin. Figure 1A is the chemical structure of metadoxin, Figure 1B is the result of Oil Red O staining triglyceride in the cytoplasm. FIG. 1C shows the expression level of PPARγ, C / EBPa, ACC, and SREBP, which are indicator proteins of adipose differentiation, by immunochemical analysis on cell lysates. The same amount of protein was loaded by β-actin. The results were confirmed by three independent experiments, and FIG. 1C is representative of them.

2 is a result of observing the effect of metadoxin on adipocyte proliferation and cell cycle control protein. The value in (A) is the mean ± S.E obtained from three separate experiments (* and ** mean p <0.05 and p <0.01, respectively, compared to the control).

(B) Immunohistochemical analysis of p27, p21 and cyclin A in cell lysates confirmed that β-actin was loaded with the same amount of protein, and the results were confirmed by three independent experiments.

Figure 3 is a result of observing the effect on the differentiation of metadoxin adipocytes with the treatment time of metadoxin. (A) Oil Red O staining of cytoplasmic triglycerides. 3T3-L1 adipocytes were treated with 1 mM metadoxin (indicated in Meta in FIG. 3) during the periods shown in bold during the MDI-induced differentiation program. The micrograph shows the result of Oil Red O staining with cells fixed at 10% formalin on day 8. (B) Cell lysates were analyzed by immunochemical analysis for FAS and ACC. Loading the same amount of protein was confirmed by β-actin, the results were confirmed by three independent experiments.

4 shows the effect of metadoxin on PPARγ reporter activity. (A) Relative activity of PPRE reporter in 3T3-L1 adipocytes. The MDI-induced differentiation program was applied to 3T3-L1 progenitor cells for 48 hours and the effect of metadoxin on PPRE luciferase activity was measured. Values are the mean ± S.E of results from at least three independent experiments. (** is statistically significant at p <0.01 compared to no treatment group; # and ## respectively are statistically significant at p <0.05, P <0.01 compared to MDI only treatment) (B) Relative PPRE luciferase activity in HEK293 cells was measured after treatment of HEK293 cells with 1 mM metadoxin for 48 hours. Values are the mean ± S.E of results from at least three independent experiments. (* Indicates p <0.05 statistically significant compared to mock transfection).

FIG. 5 shows the effects of CREB activation induced during the differentiation of adipocytes by metadoxin (Meta), showing the inhibitory effect of CREB phosphorylation by metadoxin (Meta) during MDI-induced differentiation program. . (A) Immunohistochemical analysis for CREB. Phosphorylated CREB (Ser133) and total CREB were measured in 3T3-L1 adipocytes treated with vehicle or 1 mM metadoxin for the time indicated in the figure. Loading the same amount of protein was confirmed by β-actin. (B) Gel Delay Assay. CREB-DNA binding activity was measured in 3T3-L1 adipocytes treated with vehicle or 1 mM metadoxin. The nuclear extract was reacted with CREB-binding oligonucleotides and binding specificity was confirmed by excess probe competition or supershift analysis. The results were confirmed by three independent experiments.

Figure 6 shows the effect of metadoxin on PKA dependent CREB activation, showing the results for inhibition of PKA dependent CREB phosphorylation by metadoxin. (A) Effect of metadoxin (Meta) on CREB phosphorylation induced by 8-Br-cAMP. Phosphorylation of CREB at Ser133 was measured when 3T3-L1 adipocytes were treated with 100 μM 8-Br-cAMP and 1 mM metadoxin. (B) Effect of metadoxin on Forskolin induced CREB phosphorylation (Ser133). 10 μM forskolin was treated with 3T3-L1 adipocytes with or without metadoxine for 0.25-1 hour. (C) Inhibitory effect of 10 μM H89 (PKA inhibitor) on CREB phosphorylation in 3T3-L1 adipocytes exposed to MDI for 0.25-1 h. The results were confirmed by three independent experiments and showed representative blot results.

7 is a result of expression of C / EBβ gene, which is a target gene of CREB and a key transcription factor during differentiation in 3T3-L1 adipocytes treated with metadoxin. 3T3-L1 adipocytes were cultured for 1-48 hours in medium containing MDI with or without metadoxin and immunoblotting C / EBβ-LAP and C / EBβ-LIP. The results were confirmed by three independent experiments. Relative intensities of LAP and LIP were measured by densitometry. The lower right panel shows the ratio of LAP to LIP. The figure is the mean ± S.E of the results from three independent experiments. (* Indicates statistically significant at p <0.05 compared to MDI only treatment). (B) Effect of metadoxin on MAPK phosphorylation. Immunohistochemical analysis of phospho-ERK (Thr202 / Tyr204) and ERK, phospho-JNK (Thr183 / Tyr185) and JNK, phospho-p38 (Thr180 / Tyr182) and p38 in cell lysates (20 μg each) was performed. The results were confirmed by three independent experiments and showed representative blot results.

Claims (12)

Metadoxine (pyridoxol L-2-pyrrolidone-5-carboxylate, metadoxine) or a pharmaceutical composition for the prevention or treatment of obesity comprising a pharmaceutically acceptable salt thereof as an active ingredient. The pharmaceutical composition according to claim 1, wherein the obesity is abdominal obesity due to alcohol intake. A disease caused by elevated blood levels of triglycerides, cholesterol, high density lipids or low density lipids, including metadoxin (pyridoxol L-2-pyrrolidone-5-carboxylate, metadoxine) or a pharmaceutically acceptable salt thereof as an active ingredient. Pharmaceutical composition for the prevention or treatment of complications due to obesity selected from the group consisting of. Visceral fat syndrome, metabolic syndrome, hypertriglyceridemia, hypoHDLemia, angina, including metadoxin (pyridoxol L-2-pyrrolidone-5-carboxylate, metadoxine) or a pharmaceutically acceptable salt thereof as an active ingredient Myocardial infarction, osteoarthritis, cancer associated with weight gain, orthostatic hypotension, pulmonary hypertension, diabetes, hypertension, impaired glucose tolerance, coronary thrombosis, atherosclerosis, gallbladder diseases such as gallstones, insulin resistance, chronic arterial obstruction, thromboembolism, Pharmaceutical composition for the prevention or treatment of complications due to obesity selected from the group consisting of heart disease, lipid syndrome and hyperglycemia. The pharmaceutical composition according to any one of claims 1 to 4, wherein the prevention and treatment of obesity or the complications thereof are due to the inhibition of Protein Kinase A-Cyclic AMP response element binding protein (PKA-CREB). . The method according to any one of claims 1 to 4, wherein the prevention and treatment of obesity or the complications thereof are accompanied by the inhibition of expression of C / ATBP-LAP (CCAAT-enhancer-binding protein beta-Liver-enriched Activator Protein). A pharmaceutical composition, characterized in that it is due to the fat accumulation inhibitory effect of fat cells due to the inhibition of C / EBPα (CCAAT-enhancer-binding protein alpha) and PPARγ (peroxisome proliferator-activated receptor gamma) gene expression during cell differentiation. delete delete The pharmaceutical composition according to any one of claims 1 to 4, formulated as a preparation or injection for oral administration. The pharmaceutical composition according to claim 9, wherein the formulation for oral administration is a formulation selected from capsules, tablets, suspensions, syrups or powders. Diseases caused by elevated blood levels of obesity or neutral lipids, including metadoxine (pyridoxol L-2-pyrrolidone-5-carboxylate, metadoxine) or pharmaceutically acceptable salts thereof, as a result of elevated blood levels of cholesterol Diseases, diseases caused by elevated blood levels of high density lipids or low density lipids, visceral fat syndrome, metabolic syndrome, hypertriglyceridemia, hypoHDLemia, angina pectoris, myocardial infarction, osteoarthritis, cancer associated with weight gain, orthostatic hypotension, Obesity selected from the group consisting of pulmonary hypertension, diabetes, hypertension, impaired glucose tolerance, coronary thrombosis, atherosclerosis, gallbladder diseases such as cholelithiasis, insulin resistance, chronic arterial obstruction, thromboembolism, heart disease, lipid syndrome and hyperglycemia Food composition for the prevention or improvement of the complications caused. The food composition according to claim 11, wherein the obesity is abdominal obesity due to alcohol intake.

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