KR20150117185A - A Composition for Improving Lipid Metabolism and Inhibiting Fat Accumulation Containing Dipeptides of WE or WR - Google Patents

Abstract

The present invention relates to a pharmaceutical composition or a food containing dipeptide WE or WR as an active ingredient and, more specifically, to a pharmaceutical composition or a food containing dipeptide WE or WR as an active ingredient, capable of effectively reducing neutral lipid or cholesterol concentrations in blood or liver by activating peroxisome proliferator-activated protein α (PPAR α), particularly, which is closely related to life-habit disease and metabolic syndrome, and inhibiting fat accumulation, or treating or alleviating fatty liver by improving lipid metabolism inside a body.

Description

[0001] The present invention relates to a composition for improving lipid metabolism and inhibiting lipid accumulation, which comprises dipeptide WE or WR as an active ingredient,

TECHNICAL FIELD The present invention relates to a pharmaceutical composition or food containing the dipeptide WE or WR as an active ingredient and more particularly to a pharmaceutical composition or food containing the dipeptide WE or WR as an active ingredient by activating PPAR alpha (peroxisome proliferator-activated protein alpha), which is particularly closely related to lifestyle diseases and metabolic syndrome, The present invention relates to a pharmaceutical composition or food containing an effective amount of a dipeptide WE or WR, which can effectively reduce neutral lipid or cholesterol concentration in blood or liver and improve lipid metabolism in the body to inhibit fat accumulation or treat or improve fatty liver will be.

Metabolic syndrome refers to the combination of diseases such as impaired glucose tolerance, hypertension, hyperlipidemia, obesity, and cardiovascular atherosclerosis. According to various epidemiologic data to date, about 25% of the population of developed or developing countries have metabolic syndrome, and the prevalence of the disease continues to increase in Korea.

In recent years, as a predictor of metabolic syndrome, fat is accumulating in hepatocytes, irrespective of alcohol, and non-alcoholic fatty liver disease, which accounts for more than 5% of the liver weight, is increasing. In particular, nonalcoholic fatty liver has been associated with all the factors of metabolic syndrome such as obesity, diabetes and hyperlipidemia, and it has been reported that the amount of liver fat is four times higher in people with metabolic syndrome disease than those without metabolic syndrome, According to the survey, the prevalence of adult nonalcoholic fatty liver disease in Korea has increased from 11.5% in 2004 to 23.6% in 2010. If the nonalcoholic fatty liver is persistent, liver fat degeneration, inflammatory cell infiltration, hepatocellular necrosis, and the like result, resulting in fatty liver, which can lead to liver cirrhosis, liver failure and hepatocellular carcinoma.

Nonalcoholic fatty liver disease is a chronic disease that is usually caused by lifestyle habits, and it is known that changing this habit can treat some degree. However, most of the chronic illnesses are difficult to correct by lifestyle alone, and management including lipid-lowering drug treatment is necessary. Lipid-lowering drugs should be administered for a lifetime, and there is a risk of complications such as elevated liver function levels and muscular dysfunction. Especially, when the liver function level is increased three times or more, There is a need for studies on useful active ingredients that can treat non-alcoholic fatty liver

Several methods have been known for the prevention and treatment of major lifestyle / metabolic diseases. Recently, however, a mechanism for regulating intracellular nuclear receptors has been newly recognized for a decade. Specifically, when a ligand binds to a nuclear receptor present in the cytoplasm and is activated, it is transferred to the nucleus and then binds to a series of target gene promoters to act as a transcriptional regulator and regulate the expression of a gene involved in a similar metabolic process .

Of these nuclear receptors, there are PPARs (peroxisome proliferator-activated proteins), which are particularly closely related to lifestyle-related diseases and metabolic syndrome, and PPARs have three sub-types of PPAR-a, -γ, -β / δ. Among them, PPARα plays a major role in liver tissue, and it is known that PPARα is involved in lipid metabolism, lipoprotein metabolism, and cholesterol metabolism by regulating genes involved in fatty acid and neutral lipid synthesis and neutral lipolysis related gene expression. As new roles and roles of PPARα are known, they have been used in the development of new drugs. Fibrate has been developed as a neutral lipid lowering agent in the body, and has been widely marketed worldwide. However, fibrates have an excellent effect of lowering triglycerides in dyslipidemic patients, but have side effects that can cause digestive disorders and gallstones.

A dipeptide is an amino acid polymer in which two amino acids are linked by a peptide bond and is one of the products in which the dietary protein is degraded in the body. Since it can be absorbed through the small intestine faster than free amino acid and transported to the blood, , And is physiologically superior. In particular, the dipeptide has low bio-toxicity, is excellent in selectivity for disease target proteins, and can be chemically synthesized, making it widely used in various fields of research and application in various fields such as medicine, new materials, nanoscience, molecular imaging, drug delivery.

Under these circumstances, the present inventors have made intensive efforts to develop a component capable of effectively inhibiting lipid accumulation or treating or improving fatty liver by effectively lowering neutral lipid or cholesterol concentration in blood or liver and improving lipid metabolism in the body. As a result, diepeptide WR Or WE exhibit the desired efficacy, thereby completing the present invention.

It is an object of the present invention to provide a medicinal composition or food which is capable of effectively lowering neutral lipid or cholesterol concentration in blood or liver and improving lipid metabolism in the body to inhibit fat accumulation or to treat or ameliorate fatty liver.

In order to achieve the above object, the present invention provides a method of inhibiting lipid accumulation, comprising the steps of: administering a dipeptide tryptophan-glutamic acid (H-Trp-Glu-OH: WE) or tryptophan-arginine Treatment, blood triglyceride or cholesterol lowering, or hepatic triglyceride, or cholesterol lowering.

In addition, it has been reported that inhibition of fat accumulation, improvement of fatty liver, serum triglyceride (H-Trp-Glu-OH: WE) or tryptophan-arginine Or cholesterol lowering, or intrahepatic triglyceride, or cholesterol lowering.

In the present invention, since the dipeptide WE or WR is used as an active ingredient, it is possible to produce a safe drug having less adverse side effects than those of statins, which have been conventionally used, and to improve the fat metabolism or treat the related diseases It can be used for the prevention of fat accumulation, improvement of fatty liver, blood triglyceride or cholesterol lowering, or intrahepatic triglyceride or cholesterol lowering by preparing and taking a health functional food containing the dipeptide WE or WR, .

Figure 1 shows the structure, structure and molecular weight of the dipeptide WE (left) or WR (right).
FIG. 2 shows the results of the simultaneous determination of pSG5-PPARa and pCMV-3xPPRE-Luc vectors with the dipeptide WE (left: 10, 50, 100, 500 μM) or WR (right: 50, 100, 500, 1000 μM) composition and GW7647 (WE or WR) after the treatment with transfected CHO-K1 cells for 24 hours. The results are shown in the following table. ≪ tb >< TABLE > One-way ANOVA was used for the significance test between the two groups, and the error bars of each graph were expressed as mean ± SEM.
Figure 3 shows the results of a TR-FRET assay verifying whether the dipeptide WE (left) or WR (right) and GW7647 can act as direct ligands for PPARa (significance test: one-way ANOVA; Mean ± SEM).
Figure 4 demonstrates surface plasmon resonance (SPR) experiments using a Biacore 2000 instrument to verify whether the dipeptide WE (intermediate) or WR (bottom) binds directly to the PPAR alpha ligand binding domain (LBD) The results are shown. GW7647 (above) was used as a positive control.
FIG. 5 is a graph showing the results of qPCR of PPAR alpha and its target gene by treating the cells with the dipeptide WE (10, 50, 100, 500 μM) composition and GW7647 (1 μM) for 24 hours (significance test: one-way ANOVA ; Error bars: mean ± SEM). Abbreviations: PPARα, Peroxisome proliferator-activated receptors alpha; FATP4, fatty-acid transport protein 4; ACS, acyl-CoA synthetase; CPT1, carnitine palmitoyltransferase 1; ACOX, acyl-CoA oxidase.
FIG. 6 is a graph showing the results of qPCR of PPAR alpha and its target gene treated with the dipeptide WR (50, 100, 500, 1000 μM) composition and GW7647 (1 μM) for 24 hours (significance test: one-way ANOVA ; Error bars: mean ± SEM). Abbreviations: PPAR α, Peroxisome proliferator-activated receptors alpha; FATP4, fatty-acid transport protein 4; ACS, acyl-CoA synthetase; CPT1, carnitine palmitoyltransferase 1; ACOX, acyl-CoA oxidase.
FIG. 7 shows the results of treatment of GW7647 (1 [mu] M) with diepeptide WE (top, 10, 50, 100, 500 [mu] M) or WR (ANOVA: mean ± SEM) were used to determine the intracellular fatty acid uptake.
FIG. 8 shows the intracellular cholesterol content (left) and triglyceride content (right) after 24 hour treatment with the dipeptide WE (10, 50, 100, 500 μM) composition and hepatocyte H4IIE with lipid accumulation with GW7647 (COBAS C111). The results of the analysis are shown in Fig. The intracellular lipid components were normalized by measuring the intracellular total protein content. One-way ANOVA was used for the significance test between the two groups, and the error bars of each graph were expressed as mean ± SEM.
FIG. 9 is a graph showing the changes in intracellular cholesterol content (left) and triglyceride content (right) after 24 hour treatment with lipopolysaccharide hepatocyte H4IIE with a dipeptide WR (50, 100, 500, 1000 μM) composition and GW7647 (COBAS C111). The results of the analysis are shown in Fig. The intracellular lipid components were normalized by measuring the intracellular total protein content. One-way ANOVA was used for the significance test between the two groups, and the error bars of each graph were expressed as mean ± SEM.

The present invention relates to a method for inhibiting lipid accumulation, treatment of fatty liver, blood neutrality (blood circulation), and the like, which comprises dipeptide tryptophan-glutamic acid (H-Trp-Glu-OH: WE) or tryptophan-arginine Fat or cholesterol lowering, or intrahepatic triglyceride or cholesterol lowering.

The improvement of lipid metabolism caused by the inhibition of lipid accumulation, the treatment of fatty liver, the blood triglyceride or cholesterol lowering, or the intrahepatic triglyceride or cholesterol lowering according to the present invention is caused by the improvement of lipid metabolism, Which is induced by the accumulation of fat in the body by the factor, inhibiting the accumulation of body fat, inhibiting the anti-obesity and weight gain. In addition, it may mean inhibiting the concentration of serum LDL cholesterol, inhibiting elevation of serum triglyceride concentration, inhibiting hepatocyte cholesterol synthesis, and inhibiting ACAT activity. Through the improvement of lipid metabolism, diseases such as hypertension, hyperlipidemia, arteriosclerosis, myocardial infarction, cerebrovascular disorder, metabolic syndrome can be treated, prevented or improved.

In one aspect, the dipeptide WE or WR may be characterized by increasing the activity of PPAR alpha (Peroxisome proliferator-activated receptor alpha). As a result of luciferase assay, 53% and 62% PPARα activities were increased at 100 μM and 500 μM, respectively, and 98%, respectively, when treated with WE dipeptide, compared with the negative control group . In addition, it was confirmed that PPARα activity was significantly increased by 51% at 1000 μM when treated with WR dipeptide. This means that PPARa activity is increased when the WE or WR dipeptide is treated on liver tissue cells.

Both the positive control group GW7647 and the test substances WE and WR were shown to act as agonists for increasing the activity of PPARα and the EC ₅₀ value corresponding to 50% of the maximum activity having the active effect was GW7647 of 18.6 nM , WE was 83.4 μM, and WR was 437 μM. WE and WR were compared with EC ₅₀ values.

The positive control group, GW7647, binds to PARα-ligand binding domain in the range of 100 μM to 25 μM, dipeptide WE to 5 mM to 1 mM, and WR to 10 mM to 2.5 mM. Kinetic dissociation constants (KD) were calculated for GW7647 at about 96.4 nM, WE at about 120 μM, and WR at about 604 μM. Thus, both types of dipeptides are capable of binding PPARα-LBD, and the binding efficiency of the dipeptide WE is superior to that of WR in the same manner as the TR-FRET test results.

QRT-PCR analysis of the expression of PPARα and dipeptide-related genes regulated by PPARα and PPARα on the mRNA level of dipeptide WE and WR in the H4IIE cell line, a rat hepatocyte model, revealed that 100 μM , And 500 μM, respectively. The CPT1 and ACOX genes were significantly increased (500 μM) when WE was treated. FATP4 and ACS are genes whose expression is regulated by PPARα and produce proteins that mediate fatty acid uptake in liver tissue cells. The CPT1 and ACOX genes are genes whose expression is regulated by PPARα and produce fatty acid beta oxidation enzymes. In conclusion, gene expression results show that the two dipeptides bind to PPARα and are then activated to increase the expression of the target gene, thereby promoting the oxidative degradation of the fatty acid by absorbing the fatty acid into the liver, thereby inhibiting the blood lipid lowering and the lipid accumulation It can be seen that

In the case of dipeptide WR, PPARα activity was significantly increased at high concentration (1000 μM). In addition, ACOX gene significantly increased gene expression at both 500 μM and 1000 μM concentrations, and FATP4, ACS, and CPT1 genes were expressed at high concentrations (1000 μM) when treated with WR. In conclusion, when dipeptides WE and WR were treated, the expression of PPARα gene and PPARα-related sub-gene related to lipid metabolism was increased, and the activity of these genes showed that the intracellular lipid content could be reduced do.

Based on the results of the qRT-PCR, it was confirmed that the expression of FATP4 and ACS, which are related to fatty acid uptake, was high in the dipeptide WE or WR, and the fatty acid absorption by the two types of dipeptide WE or WR Change was measured. When 100 .mu.M and 500 .mu.M of WE were treated, the absorption of fluorescently labeled fatty acid was significantly increased by 20% as compared to the negative control. In the case of WR, fatty acid uptake was significantly increased by 20% compared to negative control when treated at high concentration (1000 μM). These results suggest that PPARα activity of the two dipeptides activates the fatty acid uptake mechanism in hepatocytes. It is known that when PPARα is activated, the fatty acids derived from the neutral lipids in the blood are absorbed into the liver tissue cells and decomposed through the fatty acid beta oxidation process, so that the neutral lipids in the blood are lowered, and both of the dipeptides WE and WR have this action .

Finally, the intracellular lipid lowering effect was evaluated when the dipeptide (WE, WR) was treated with hepatocyte cultured cells in which lipid was sufficiently accumulated. The intracellular cholesterol content was significantly decreased when treated with 100 μM and 500 μM of dipeptide WE, and the intracellular triglyceride content was significantly decreased when treated with 50 μM, 100 μM and 500 μM concentrations. This means that both types of dipeptides can improve non-alcoholic fatty liver.

In conclusion, lipid lowering and lipid accumulation inhibitory effects of such a dipeptide WE or WR in liver cells can be interpreted as a result of the activity of PPARα. In the present invention, the two types of dipeptide compositions act as activators for increasing the activity of PPARα, which promotes the absorption of fatty acids derived from neutral lipids in blood and oxidizes the absorbed fatty acids by PPARα activity to induce degradation. Therefore, the two types of dipeptides, WE or WR, are expected to promote the oxidation and absorption of neutral lipids in the liver, thereby improving liver lipid metabolism and having a non-alcoholic fatty liver therapeutic effect.

The pharmaceutical compositions of the present invention are for parenteral, topical, subcutaneous, intramuscular, intravenous, oral, intranasal, intraperitoneal, or topical administration (e.g., in the form of a cream) for oral use, prophylactic and / or therapeutic use. Preferably, the compositions of the invention are administered parenterally, intramuscularly or intranasally. The WE or WR compositions herein have the advantage of providing the desired effect without toxicity at very low doses.

The composition of the present invention containing WE or WR can be formulated in such a way that it is absorbed into the bloodstream. The composition of the present invention is a therapeutic agent for improving lipid metabolism and inhibiting fat accumulation, which induces a change in an intracellular amount so as to cause a subsequent change that is no longer dependent on the presence of the composition in an intracellular process. It has been observed that the effect of the peptide lasts for a long period of time, say weeks to months, despite the peptide being somewhat faster, e.g. within 5 minutes.

While WE or WR of the present invention are water soluble at the generally used lower concentrations, they are preferably administered in the form of acids or in pharmaceutically acceptable preparations such as acetic acid, citric acid, maleic acid, Is used in the form of an alkali salt formed with potassium, ammonium or zinc. The salts in which the WE or WR of the present invention are freely dissolved can also be converted to a slightly water-soluble pharmaceutically acceptable salt such as, for example, tannic acid or palmoic acid, or can be covalently coupled to a larger carrier, May be converted into a salt having low solubility in body fluids, such as contained in a formulation or contained in a capsule that is liberated over time.

The WE or WR pharmaceutical preparations of the present invention may be used in the form of free peptides or water-soluble pharmaceutically acceptable salts such as sodium, potassium, ammonium or zinc salts. It will be appreciated that the dipeptides of the present invention may be administered with other active ingredients that impart activity independently to the composition. Pharmaceutically acceptable salts can conveniently be prepared from WE or WR (or an agonist thereof) by conventional methods. Thus, such salts may be prepared, for example, by treating WE or WR with an aqueous solution of a preferred pharmaceutically acceptable metal hydroxide or other metal base and evaporating the resulting solution, preferably under reduced pressure, under a nitrogen atmosphere. Alternatively, a solution of WE or WR may be mixed with the alkoxide of the desired metal and subsequently the solution may be evaporated to dryness. Pharmaceutically acceptable hydroxides, bases and alkoxides include those having cations for this purpose, including, but not limited to, potassium, sodium, ammonium, calcium and magnesium. Other exemplary pharmaceutically acceptable salts include the hydrochloride, hydrobromide, sulfate, bisulfate, acetate, oxalate, ballalate, oleate, laurate, borate, benzoate, lactate, phosphate, Malate, fumarate, succinate, tartrate and the like.

In the case of parenteral administration, the present invention encompasses pharmaceutical preparations comprising a solution of WE or WR dissolved in a pharmaceutically acceptable carrier, preferably an aqueous carrier, or a polymeric, multimeric or cyclic form thereof, or a derivative thereof, Lt; / RTI > Various aqueous carriers such as water, buffers, 0.4% saline, 0.3% glycine and the like may be used to increase the stability, such as albumin, lipoproteins, globulins and the like and / or proteins and / or glycoproteins. These compositions can be sterilized by conventional well-known sterilization methods. The resulting aqueous solution may be packaged for use or filtered under aseptic conditions and lyophilized, and the lyophilized preparation is mixed with the sterile aqueous solution prior to administration. Such compositions may include, for example, pharmaceutically acceptable auxiliary substances necessary for approximating physiological conditions such as pH control and buffering agents, tonicity adjusting agents and the like, for example sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride and the like have. It may be desirable to stabilize WE or WR, analogs, derivatives, agonists, etc. to increase the shelf life and weak dynamic half life. Storage stability can be determined by a) a hydrophobizing agent (e.g., glycerol); b) sugars (such as sucrose, mannose, sorbitol, rhamnose, xylose); c) complex carbohydrates (such as lactose); And / or d) an excipient such as a bacteriostatic agent. The weak dynamic half-life of the peptide may be determined by coupling to the carrier peptide, polypeptide and carbohydrate by chemical derivatization (e. G., By coupling to the side chain or N- or C- terminus), or by coupling the amino acid with another amino acid ). ≪ / RTI > The weak dynamic half-life and pharmacodynamics of the peptide may also be a) encapsulated (e. G., In liposomes); b) controlling the degree of hydration (e. g., controlling the degree and type of glycosylation of the peptide); c) modulating the electrostatic charge and hydrophobicity of the peptide.

The WE or WR-containing composition according to the present invention may be administered as a conventional pharmaceutical agent suitable for parenteral administration (for example, intravenous, subcutaneous, intramuscular administration). The formulations may be conventionally treated with pharmaceuticals, such as sterilization, and may contain adjuvants such as preservatives, stabilizers, wetting agents, and the like.

The WE or WR composition is typically biologically active at doses of from about 0.5 μg / kg to about mg / kg, preferably from about 1 to about 50 μg / kg. The concentration of WE or WR in the pharmaceutical composition may vary, i.e. from about 0.001% to 15% or 20% by weight, depending on the particular need for treatment and the mode of administration of the patient, Will be selected. When used intramuscularly, it is used as an injectable solution having an effective amount for inhibiting angiogenesis, for example, an active ingredient of about 0.001 to 0.01% by weight of WE or WR. When prepared in the form of tablets, capsules or suppositories, it is preferred that the active substance is present in an amount of about 0.1 mg of WE or WR per tablet, suppository or capsule. A pharmaceutically acceptable vehicle for this injection form may be any pharmaceutically acceptable solvent, such as 0.9% aqueous sodium chloride, distilled water, Novocaine solution, Ringer solution, glucose solution and the like . In this form, the capsules, suppositories or tablets may also contain other conventional excipients such as fillers, starches, glucose and the like and vehicles. In the case of topical preparations, WE or WR is generally contained in a concentration of about 0.1 to 10,000 ppm, preferably about 1 to 1000 ppm, and most preferably about 10 to 100 ppm for urea-based softener, petroleum-based ointment and the like. Actual methods of preparing parenteral, oral, and topical dosage forms are well known or will be readily apparent to those skilled in the art and are described, for example, in Remington's Pharmaceutical Science, 17th Ed., Mack Publishig Company, Easton, PA (1985)]

Intramuscular and intranasal routes are preferred for administration of the compositions of the present invention. One preferred dose of the composition of the invention for intramuscular administration is about 50 to 100 [mu] g WE or WR (in the case of a total treatment of 300-1000 [mu] g) per single administration for an adult; About 10 μg per dose for infants under one year of age; About 10 to 20 [mu] g per single dose for infants 1 to 3 years of age; About 20 to 30 μg per single dose for infants aged 4 to 6 years; For children between 7 and 14 years old, about 50 μg per single dose. All of the above-mentioned dosages are useful for 3 to 10 days of treatment and may be varied according to the needs of the patient. Treatment may be repeated within 1 to 6 months, as needed. In another preferred embodiment, the dosage of the WE or WR pharmaceutical agent is from about 10 μg / kg to about 1 mg / kg for a period of from about 6 days to about 10 days, optionally, for a period of up to about 30 days, ≪ / RTI > In one preferred course of treatment, the WE or WR regimen is intramuscularly administered at a dose of 1 to 100 μg / kg for 5 to 7 days and then stopped for 1 to 6 months before the same injection regimen is repeated.

The WE or WR composition may be administered alone or in combination with a pharmaceutically acceptable carrier in a single dose or in multiple doses. Suitable pharmaceutical carriers include inert solid diluents or fillers, sterile aqueous solutions and various non-toxic organic solvents. WE or WR is mixed with a pharmaceutically acceptable carrier and a selective antibiotic to produce a pharmaceutical composition. The combined therapeutic agents of the invention are then conveniently administered in a variety of dosage forms such as tablets, lozenges, syrups, injection solutions, and the like. The combination therapy may also contain WE or WR in the same unit dosage form. Optionally, the pharmaceutical carrier may contain additional ingredients such as flavoring agents, binders, excipients, and the like.

Thus, in the case of oral administration, starch, preferably potato or tapioca starch, various disintegrants such as alginic acid and specific complex silicates, and binders such as polyvinylpyrrolidone, sucrose, gelatin and acacia, Tablets containing various excipients such as calcium carbonate and calcium phosphate may be used. Lubricants such as magnesium stearate, sodium lauryl sulfate and talc are also often useful for purification purposes. Solid compositions of a similar type may also be used as fillers in salt and hard-filled gelatin capsules. Preferred materials for this purpose include lactose or milk sugars and high molecular weight polyethylene glycols. When an aqueous suspension of elixir is desired for oral administration, the essential active WE or WR components therein may be formulated with various sweetening, flavoring, coloring matter or flavoring agents, such as water, ethanol, propylene glycol, glycerin and mixtures thereof, Dyes, and, if desired, emulsifying or suspending agents.

In the case of parenteral administration, solutions of WE or WR in a sterile aqueous broth solution of homoe oil or groundnut oil or aqueous polypropylene glycol, and the corresponding water-soluble pharmaceutically acceptable metal salts mentioned above, can be used. This aqueous solution should be suitably buffered as required and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal injection. The sterile aqueous media employed may all be readily obtained by standard techniques known to those skilled in the art. It is also possible to administer the above-described compounds locally (for example, via a catheter placed on the spot) using a solution suitable for this purpose.

An amount sufficient for more than 50% of the treated individuals to be treated is defined as "therapeutically effective dose ". Treatment of an acute disease will generally take about 3 to 10 days. The treatment or prophylactic treatment of a chronic disease has the same process, but can be repeated after a long period of time of about 1 to 6 months or more. In some cases, it is preferable to administer the composition intermittently daily for a period of from about 2 to about 20 days, preferably from about 3 to about 14 days, more preferably from about 4 to about 10 days, , Preferably about 20 days or more, or about 1 to 6 months or more.

The delivery route of the WE or WR composition is determined by the disease and site requiring treatment. In the case of topical application, it is possible to apply the WE or WR composition to a localized site (e.g., by placing the needle in the tissue at that site), or to place the invading bandage on the tumor site, e.g., after surgical removal; For other diseases, it may be desirable to administer the WE or WR composition systemically. For other applications, the WE or WR composition may be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intranasally and intradermally, and by inhalation (e.g., using a sprayer), transdermal delivery (using a liposoluble carrier of the patch), or by intragastric delivery (e.g., using a capsule or tablet).

Generally, the acid addition salt of the WE or WR composition of the present invention, e.g., a pharmaceutically acceptable acid, of the L-Trp-L-Glu composition will be biologically equivalent to the WE or WR composition of the present invention itself.

The desired therapeutic composition, inoculum, route and dosage will vary depending on the clinical application as well. In the case of intramuscular injection, the inoculum is typically prepared from a dried peptide (or peptide conjugate) by suspending the peptide in a physiologically acceptable diluent such as water, saline or phosphate buffered saline. Depending on the condition of the patient being treated, some change in dosage is required and in any case the physician will determine the appropriate dosage for the individual patient. The effective amount of the peptide per unit dose depends on body weight, physiological status and selected inoculation therapy among others. The unit dose of the peptide means the weight of the peptide (if carrier is used), excluding the weight of the carrier. Effective treatment will be achieved when the concentration of WE or WR, for example L-Trp-L-Glu, at the tissue site in the microenvironment of the cell approaches 10 ^-5 to 10 ^-9 M. Skilled doctors can use clinical and laboratory indicators (as described above) to observe the patient's response to the treatment of the present invention and adjust the dosage accordingly.

Since the pharmacokinetics and pharmacodynamics of WE or WR, agonists, antagonists, etc., will vary from patient to patient, the most preferred method for obtaining therapeutic concentrations in the tissues is to gradually increase the dosage and to monitor clinical and laboratory indicators ). In the case of therapeutic regimens for increasing such doses, the initial dosage will depend on the route of administration. When WE or WR with an approximate molecular weight of 200 to 400 Daltons is administered intravenously, an initial dose of about 0.5 [mu] g / kg body weight is administered and the dosage increases by 10-fold for each interval of the dose-increasing therapy .

When provided in the form of tablets, capsules or suppositories, it is preferred that the active substance is present in an amount of about 0.1 mg per tablet, suppository or capsule. When provided in this form, the capsules, suppositories or tablets may also contain other conventional excipients and vehicles such as fillers, starches, glucose, and the like.

In another aspect, the present invention relates to a food for inhibiting fat accumulation, improving liver fat, serum triglyceride or cholesterol, or intracranial triglyceride or cholesterol lowering which contains WE or WR as an active ingredient.

The food of the present invention can be variously used for medicines for preventing oxidation, foods and beverages. The functional food of the present invention can be used in the form of powder, granule, tablet, capsule or beverage, for example, various foodstuffs, candy, chocolate, beverage, gum, tea, vitamin complex and health supplement.

The food of the present invention may be in the form of a food or beverage. The amount of the active ingredient in the food or beverage may be generally 0.01 to 50% by weight, preferably 0.1 to 20% by weight of the total food, and the health beverage composition may contain 0.02 to 10 g, And preferably 0.3 to 1 g.

The health beverage composition is not particularly limited to liquid ingredients other than those containing WE or WR as essential ingredients in the indicated ratios, and may contain various flavors or natural carbohydrates such as ordinary beverages as an additional ingredient. Examples of the above-mentioned natural carbohydrates include monosaccharides such as disaccharides such as glucose and fructose, polysaccharides such as maltose, sucrose and the like, such as dextrin, cyclodextrin and the like And sugar alcohols such as xylitol, sorbitol and erythritol. Natural flavors (tau martin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavors other than those described above The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 mL of the composition of the present invention.

In addition to the above, flavorings such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, colorants and heavies (cheese, chocolate etc.), pectic acid and its salts, alginic acid and its salts, Protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like. In addition, the compositions of the present invention may contain natural fruit juice and pulp for the production of fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The proportion of such additives is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the food.

Hereinafter, the present invention will be described in more detail with reference to Examples. These embodiments are only for illustrating the present invention, and thus the scope of the present invention is not construed as being limited by these embodiments.

Example 1. Assay of PPAR? Activity of dipeptide WE or WR

In order to verify the effect of the dipeptides WE and WR used in the examples, the cells were cultured in a rat hepatocyte model H4IIE (Rat reuber hepatoma, Korea Cell Line Bank (KCLB)) cell line and a hamster ovary CHO-K1 (Chinese hamster ovary, KCLB) cell line was used. For each culture, 10% inactivated fetal bovine serum (Logan, UT, USA) and 1% penicillin-streptomycin antibiotic solution were added to MEM / EBSS, DMEM / F-12 And 5% CO2.

The characteristics of the dipeptides WE (H-Trp-OH) and WR (H-Trp-Arg-OH) used in this experiment are shown in FIG. A reporter gene assay was performed to verify the PPARα activity of the dipeptide WE or WR. Luciferase, an enzyme that catalyzes the action of light emission by oxidizing a specific substrate, was used as a reporter gene, and PPARE of the promoter region of the vector inserted by transfection into the cultured cells was added to PPARα / RXR When the dimer is bound, luciferase is designed to express its activity. Based on this principle, the degree of binding of PPRE and PPARα was measured by PPARα activity following treatment with dipeptide by concentration. More specifically, CHO-K1 (Chinese hamster ovary) cells were cultured in a 24-well plate at a concentration of 2 × 10 ⁵ per well, cultured for 24 hours, and cultured in Hilymax (Dojindo, MD, USA) The transfection reagent was used to transfect pCMV-3xPPARE-Luc vector and pSG5-PPARα (Addgene, MA, USA) vector for 24 hours at the same time. Two types of dipeptides WE (10-500 μM) or WR (50-1000 μM) were treated with PPARα synthetic antagonist GW7647 (1 μM) for 24 hours and then the Firefly luciferase assay kit (Biotium, Hayward, CA, USA) Was used to measure luciferase activity.

As shown in Fig. 2, the treatment with WE dipeptide resulted in an increase of 53% and 62% PPARα activity at 100 μM and 500 μM, respectively, in the positive control group treated with 1 μM, and a 98% increase in the positive control group treated with 1 μM. In addition, PPARα activity was significantly increased by 51% at 1000 μM when treated with WR dipeptide and increased by 59% when treated with positive control GW7647 (1 μM). As a result, the PPARα activity of the dipeptide WE or WR was confirmed to be increased when the dipeptide was treated at a high concentration.

Example 2. Analysis of PPAR? Ligand efficacy of dipeptides

A TR-FRET test was conducted to evaluate the activity of dipeptide as an agonist or an antagonist using a coactivator in order to evaluate whether it could act as a direct ligand to PPAR ?. When a ligand is bound to PPARα, the steric structure of PPARα is changed to facilitate the binding of coactivator, which induces transcription by PPARα, which is labeled with terbium through GST antibody When the ligand binds to PPAR alpha, the peptide bond of the fluorescently labeled coactivator is made in PPAR alpha. Terbium is excited at the wavelength of 340 nm, and this energy is transferred to the fluorescence signal of the coactivator peptide, which is measured at 520 nm. Thus, as the ligand binding to PPARα increases, the TR-FRET signal increases. In the present invention, the fluorescence-labeled PGC1? Peptide was used as a coactivator for PPAR ?, and the fluorescence wavelength was measured using a Spectra Max instrument (excitation 340 nm, emission 520 nm). Graphs and EC ₅₀ values were analyzed using the GraphPad ™ Prism 5.0 program and the dipeptide (WE or WR) efficacy was compared and analyzed using GW7647 developed as a PPARα agonist as a positive control.

As a result, as shown in FIG. 3, both the positive control group GW7647 and the test substance WE act as antagonists of PPARα, and the EC ₅₀ value corresponding to 50% of the maximum activity having the active activity was GW7647 18.6 nM, and WE was 83.4 μM. The dipeptide WR was also proved to act as an antagonist of PPARa and the EC ₅₀ value was calculated as 437 μM. As a result of TR-FRET, both types of dipeptides showed antagonistic tendency to increase PPARα activity, and WE was found to have higher activity when WE and WR were compared with EC ₅₀ values.

Example 3. Evaluation of binding affinity of dipeptide with PPARa

Surface plasmon resonance (SPR) experiments were performed using a Biacore 2000 instrument (GE healthcare product, Sweden) to confirm that the dipeptides WE and WR directly bind to the PPARα ligand binding domain (LBD). Specifically, the human-PPARα ligand binding domain (LBD) protein was immobilized on a CM5 sensor chip (GE healthcare, Sweden) and the dipeptide (WR or WE) was injected at a concentration of 5 mM each in the order of 0.5 mM. 25 < 0 > C. In SPR experiments, when a substance binds to human-PPARα LBD after injection of a dipeptide (WR or WE), the SPR signal, RU (response unit), increases and is expressed as a graph of signal intensity (RU) versus time. After a certain period of time, only the buffer solution (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.005% [v / v] surfactant P20, 1% DMSO, pH 7.4) The bond between the substances is released, and the RU value decreases. That is, the binding constant (KD) value was calculated using the binding ability (Ka value) and the releasing ability (Kd value) in the reaction. GW7647, a PPARα antagonist, was used as a positive control.

As shown in FIG. 4, the positive control GW7647 binds to the PARα-ligand binding domain in the range of 100 μM to 25 μM, the dipeptide WE to 5 mM to 1 mM, and the WR to 10 mM to 2.5 mM. Kinetic dissociation constants (KD) were calculated for GW7647 at about 96.4 nM, WE at about 120 μM, and WR at about 604 μM. As a result of the SPR test, both types of dipeptides were able to bind to PPARα-LBD, and the binding efficiency of dipeptide WE was superior to WR in the same manner as the TR-FRET test results.

Example 4. Regulation of PPAR alpha and related genes by treatment with dipeptide WE or WR

QRT-PCR (quantitative real-time PCR) was performed for analysis of PPARα and lipid metabolism-related target genes by dipeptides WE and WR. The present invention focuses on the improvement of liver lipid metabolism through PPARα activity. It has already been revealed that the PPARα target gene is related to fatty acid transport protein 4 (FATP4), acsyl-CoA synthetase (ACS) CPT1 (carnitine palmitoyltransferase 1) and ACOX (acyl-CoA oxidase) were selected. More specifically, H4IIE (Rat reuber hepatoma) cells were cultured in a 6-well plate for 24 hours. Then, PPAR [alpha] agonist GW7647 (1 [mu] M) and dipeptide WE (10, 50, 100, 500 μM) WR was treated with (50, 100, 500, 1000 μM) for 24 hours. Then, cDNA was synthesized (Mbiotech) using 2 μg of extracted RNA by adding 1 mL of RNAiso Plus reagent to each well. Then, 1 μL of the synthesized cDNA was added to a reaction mixture containing 10 μL of SYBR Green PCR Master Mix, 1 μL of a 10 pmol forward primer, 1 μL of a 10 pmol reverse primer, and 8 μL of distilled water and subjected to qRT-PCR PCR) was performed. Expression of each gene was quantified by standardizing glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a reference gene.

As shown in FIG. 5, when the negative control and the WE treatment groups were compared, the expression levels of the PPARα gene and PPARα-related lipid metabolism-related genes FATP4 and ACS were significantly increased at two concentrations of 100 μM and 500 μM, Expression of CPT1 and ACOX genes was significantly increased when WE was treated at high concentration (500 μM).

As shown in FIG. 6, the expression of PPARα gene when treated with dipeptide WR significantly increased PPARα activity when treated at high concentration (1000 μM) as compared with negative control. In addition, ACOX gene significantly increased gene expression at both 500 μM and 1000 μM concentrations, and FATP4, ACS, and CPT1 genes were highly expressed when treated with WR at high concentration (1000 μM). As a result, RT-PCR was performed to confirm the effect of the dipeptide on the expression of the lipid metabolism-related gene. As a result, the expression of the PPARα gene and the sub-gene related to lipid metabolism by PPARα activity upon treatment with the dipeptide WE or WR And it was confirmed that the activity of the genes decreased the lipid content in the cells.

Example 5. Fatty acid uptake by two types of dipeptide WE or WR treatment

Among the PPARα-related genes identified in the qRT-PCR experiment, when the WE or WR dipeptide was treated, it was confirmed that the expression of FATP4 and ACS, which are related to fatty acid uptake, was high. In fact, two kinds of dipeptides WE Changes in fatty acid uptake by WR treatment were measured. For the present invention, a fatty acid and a flow cytometer (FACS Calibur; BD Biosciences, San Jose, CA, USA) labeled with fluorescence functionalized probe BODIPY (boron-dipyrromethene) was used. For the present invention, H4IIE (Rat reuber hepatoma) cells were plated on a 6-well plate at 1 × 10 ⁵ cells / well. As a positive control, the PPARα antagonist GW7647 (1 μM) and dipeptide WE (4 concentration: 10, 50, 100, 500 μM) or WR (4 concentration: 50, 100, 500, 1000 μM) for 24 hours. Then, the fatty acid with BODIPY was treated at room temperature for 1 minute, and then the reaction was terminated by adding a cold buffer solution (Hanks' buffered salt solution) containing 0.1% inactivated fetal bovine serum, (FACS Calibur; BD Biosciences, San Jose, Calif., USA).

As a result, as shown in FIG. 7, when 100 μM and 500 μM of WE were treated with H4IIE (Rat reuber hepatoma) cells, the fluorescence-labeled fatty acid absorption was significantly increased by 20% as compared to the negative control, 1 [mu] M) positive control group. In the case of WR, fatty acid uptake was significantly increased by 20% compared to the negative control when treated at high concentration (1000 μM). As a result, it was confirmed that the lipid acid uptake was increased by treatment with dipeptide WE or WR in the liver cultured cells. Taken together with the result of the qRT-PCR experiment performed earlier, the PPARα activity of the two kinds of dipeptides resulted in the fatty acid absorption mechanism Was evaluated as being activated.

Example 6. Assessment of hepatic cell lipid accumulation lowering efficacy

In order to investigate whether the dipeptides WR and WE decrease the hepatic lipid content, cultured cells were artificially accumulated lipid, and lipid was extracted from the cells treated with the dipeptide to measure the triglyceride and cholesterol concentration. More specifically, for the present invention, H4IIE (Rat reuber hepatoma) cells were seeded in a 6-well plate at a concentration of ⁵ × 10 ⁵ cells / well and cultured for 24 hours. Then, palmitate and oleic acid (4 concentrations: 50, 100, 500 μM) and GW7647 (1 μM) and dipeptide WE (4 concentrations: 10, 50, 100, and 500 μM) as the positive control groups after the oleate was added for 24 hours. , 500, 1000 [mu] M) for 24 hours. To the treated cells, 1 mL of hexane / isopropanol was added at a ratio of 2: 1 per well to each well, and the mixture was allowed to stand at room temperature for 30 minutes to extract lipids from the cells. After evaporation, the cells were diluted with 95% ethanol, and the residual cholesterol and triglyceride contents in the cells were analyzed using a liposome automation analyzer (COBAS C111). The intracellular lipid components were normalized by measuring the intracellular total protein content.

As a result, as shown in FIG. 8, the intracellular cholesterol content was significantly decreased at the level of 100 μM and 500 μM of the dipeptide WE at the level of GW7647 treated with heparinized cells, and 50 μM, 100 μM, The concentration of intracellular triglyceride was significantly decreased when the concentration was 500μM. In addition, as shown in FIG. 9, when the WR was treated at a high concentration (1000 μM), it was confirmed that the intracellular triglyceride content was significantly decreased.

In the statistical analysis of the above embodiments, a one-way ANOVA was used for the significance test between the two groups, and the error bars of each graph were expressed as mean ± SEM.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments. It will be obvious. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (4)

Inhibiting lipid accumulation, containing the dipeptide tryptophan-glutamic acid (H-Trp-Glu-OH: WE) or tryptophan-arginine (H-Trp-Arg-OH: WR) as an active ingredient, Depressed, or intrahepatic triglyceride, or cholesterol lowering.
The pharmaceutical composition according to claim 1, wherein the dipeptide WE or WR increases the activity of PPAR alpha (Peroxisome proliferator-activated receptor alpha).
Inhibition of fat accumulation, inhibition of fatty liver, serum triglyceride, or cholesterol (triglyceride), which contains dipeptide tryptophan-glutamic acid (H-Trp-Glu-OH: WE) or tryptophan-arginine Drop, or food in the liver for triglycerides, or cholesterol lowering.
4. The food according to claim 3, wherein the dipeptide WE or WR increases the activity of PPARa.

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