JP6315536B2 - PPAR activator - Google Patents

Description

  The present invention relates to a PPAR activator.

  PPAR (Peroxisome Proliferator-Activated Receptor: Peroxisome proliferator-activated receptor) is a protein that functions as a nuclear receptor type transcriptional regulator. PPAR is an α-type that has the effect of leading to a decrease in blood triglyceride concentration through peroxisome proliferation, is involved in adipocyte differentiation by being distributed in adipose tissue, γ-type that is a target molecule for improving insulin resistance, skeleton Three types of subtypes of δ type (β type) are known which have an action of suppressing fat accumulation in peripheral tissues by increasing muscle energy consumption. Therefore, activation of PPAR has effects such as activation of fatty acid metabolism, promotion of body fat burning, suppression of obesity, suppression of fatty liver, prevention and improvement of diabetes, prevention and improvement of insulin resistance, and improvement of endurance. Therefore, since it is effective against lifestyle-related diseases and visceral fat syndrome (metabolic syndrome) in the modern society, for example, fibrates are already on the market as drugs that activate PPARα. However, the search of the component which activates the more superior PPAR is still energetically performed (for example, patent document 1).

JP 2011-63557 A

  Then, an object of this invention is to provide the novel component which activates PPAR.

  The present inventors have been researching the pharmacological action of components contained in the marine organism krill (Euphausiacea), and as a result, the krill water-soluble extract has the effect of inhibiting fat accumulation in fat cells. And has an inhibitory effect on weight gain (JP 2012-153646 A). Therefore, the present inventors have examined the components contained in krill in more detail, and found that specific hydroxyeicosapentaenoic acid (HEPE) isolated and obtained from krill has an excellent PPAR activation effect.

As described in claim 1, the PPARδ activator of the present invention based on the above findings is effective in HEPE having a hydroxyl bonding position at the 8-position or an alkyl ester thereof, or a pharmaceutically acceptable salt thereof. Ingredients .
Also, process for preparing a PPARδ activator of the present invention, as claimed in claim 2, wherein, due to HEPE isolate acquires bonding position of the hydroxyl group is 8-position by extraction from krill.
Further, preparation method of claim 3, wherein, in the preparation process of claim 2 wherein the extraction operation using an organic solvent and / or water-soluble in water.

  ADVANTAGE OF THE INVENTION According to this invention, the novel PPAR activator which uses as an active ingredient HEPE which the coupling | bonding position of a hydroxyl group is any one of 8th-position can be provided.

It is a graph which shows the evaluation result of each PPAR activation effect | action of 5-HEPE, 8-HEPE, 9-HEPE, 12-HEPE, and 18-HEPE isolated and acquired from krill in Example 1. It is a graph which shows the density | concentration dependence of each PPAR activation action of 8-HEPE, 9-HEPE, and 18-HEPE. It is a graph which shows the evaluation result of the PPAR activation action in each low concentration of various HEPE. It is a chart which shows the result of isolation acquisition of various HEPE from krill by HPLC in Reference Example 1. It is a chart which shows the result of recycle HPLC.

  As HEPE in which the bonding position of the hydroxyl group, which is an active ingredient of the PPAR activator provided by the present invention, is any of the 8th to 12th positions, for example, 8-HEPE (8-hydroxy-5Z) having the following chemical structure: , 9E, 11Z, 14Z, 17Z-eicosapentaenoic acid), 9-HEPE (9-hydroxy-5Z, 7E, 11Z, 14Z, 17Z-eicosapentaenoic acid), 11-HEPE (11-hydroxy-5Z, EZ12E) , 14Z, 17Z-eicosapentaenoic acid), 12-HEPE (12-hydroxy-5Z, 8Z, 10E, 14Z, 17Z-eicosapentaenoic acid). HEPE having a hydroxyl bonding position in any of the 8th to 12th positions has an asymmetric carbon, so there are optical isomers, and since it has multiple double bonds, there are various cis-trans isomers. However, the present invention includes both of them within the scope of the right.

  In the present invention, HEPE in which the hydroxyl bonding position is any of the 8th to 12th positions may be isolated and obtained from natural products, synthesized by an organic synthetic chemical method, or eicosapentaene. An acid (EPA) that is artificially oxidized (this is commercially available from, for example, Cyman Chemical) may be used. According to the study by the present inventors, by extraction from krill, HEPE having a hydroxyl bonding position in any of positions 8 to 12, especially 8-HEPE, 9-HEPE, 12-HEPE is isolated. Can be acquired. The krill used as a raw material is not particularly limited, and may be euphorius pacifica, which is called Isada in the Sanriku region, and may be euphausia superba. The krill may be raw or frozen. Moreover, you may use the dried material and salted material of krill. The extraction operation can be performed using, for example, an organic solvent soluble in water such as alcohol (methanol, ethanol, isopropanol, etc.), acetone, acetonitrile, and / or water. Separation using high-performance liquid chromatography and chromatography using adsorbents such as activated carbon, alumina, and silica gel, and ion-exchange resins, nonionic adsorption resins, gel filtration chromatography, and high-performance liquid chromatography In addition to the operations, various operations such as a crystallization operation, a vacuum concentration operation, and a freeze-drying operation may be performed alone or in appropriate combination.

  HEPE in which the hydroxyl bonding position is any one of the 8th to 12th positions may be in the form of an alkyl ester. Examples of the alkyl ester include lower alkyl esters having 1 to 6 carbon atoms such as methyl ester, ethyl ester, butyl ester, and hexyl ester.

  When HEPE having a hydroxyl bonding position in any of positions 8 to 12 takes a salt form, the pharmaceutically acceptable salt includes inorganic salts with sodium, potassium, magnesium, calcium, aluminum, and the like. Examples include organic salts with lower alkyl amines, lower alcohol amines, etc., basic amino acid salts with lysine, arginine, ornithine, and ammonium salts.

  The administration method in the case of administering HEPE having a hydroxyl group binding position in any of positions 8 to 12 to humans or animals as a pharmaceutical may be an oral administration method or a parenteral administration. It may be a method. Examples of parenteral administration methods include intravenous injection, intramuscular injection, subcutaneous injection, intraperitoneal injection, transdermal administration, pulmonary administration, nasal administration, enteral administration, buccal administration, and transmucosal administration. In this case, HEPE is formulated and administered in a manner known per se into a form suitable for these administration methods. Examples of the dosage form include injections, suppositories, aerosols, transdermal absorption tapes, eye drops, nasal drops and the like. When preparing an injection, a pH adjuster, a buffer, a stabilizer, a solubilizing agent and the like are appropriately added to form an injection. Examples of oral preparations include tablets (including sugar-coated tablets, coated tablets, and buccal tablets), powders, capsules (including soft capsules), granules (including those coated), pills, troches, liquids, and the like. And a pharmaceutically acceptable sustained release preparation. Liquids include suspensions, emulsions, syrups (including dry syrups), elixirs and the like. For example, a tablet can be prepared with a pharmaceutically acceptable carrier, excipient, binder, disintegrant, lubricant, colorant and the like according to known pharmaceutical manufacturing methods. In this case, as the carrier or excipient, for example, lactose, glucose, sucrose, mannitol, potato starch, corn starch, calcium carbonate, calcium phosphate, calcium sulfate, crystalline cellulose, licorice powder, gentian powder and the like can be used. As the binder, for example, starch, tragacanth gum, gelatin, syrup, polyvinyl alcohol, polyvinyl ether, polyvinyl pyrrolidone, hydroxypropyl cellulose, methyl cellulose, ethyl cellulose, carboxymethyl cellulose and the like can be used. As the disintegrant, for example, starch, agar, gelatin powder, sodium carboxymethyl cellulose, carboxymethyl cellulose calcium, crystalline cellulose, calcium carbonate, sodium hydrogen carbonate, sodium alginate and the like can be used. As the lubricant, for example, magnesium stearate, talc, hydrogenated vegetable oil, macrogol and the like can be used. As the colorant, those allowed to be added to pharmaceuticals can be used. Tablets and granules are sucrose, gelatin, hydroxypropylcellulose, purified shellac, glycerin, sorbitol, ethylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, cellulose phthalate acetate, hydroxypropylmethylcellulose phthalate, methyl methacrylate, methacrylic acid as required It may be coated with a polymer or the like, or may be coated with two or more layers. Further, it may be encapsulated using ethyl cellulose or gelatin.

  HEPE in which the hydroxyl bonding position is any of positions 8 to 12 has at least one excellent activation action of PPARα, PPARγ, and PPARδ. Therefore, activation of fatty acid metabolism, promotion of body fat burning, obesity It has effects such as suppression, suppression of fatty liver, prevention and improvement of diabetes, prevention and improvement of insulin resistance, and improvement of endurance. Therefore, it is effective for lifestyle-related diseases and visceral fat syndrome in modern society. The dose may be appropriately set according to conditions such as the age and weight of the administration subject, the degree of symptoms, and the health condition, but typically, about 10 mg to about 10 g per day for an adult is orally or parenterally. In general, it may be administered once to several times a day.

  In addition, HEPE having a hydroxyl bonding position in any of positions 8 to 12 is added to various forms of foods (including supplements) by adding an effective amount sufficient to exert the PPAR activation effect. (0.1 mg to 100 mg intake per kg body weight is standard).

  In addition, HEPE having a hydroxyl bonding position in any of the 8th to 12th positions can also be used as a research reagent having a PPAR activation effect.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is limited to the following description and is not interpreted.

Example 1: Evaluation of PPAR activation action of various HEPEs (experimental method)
PPAR ligand activity was measured by using Promega's Luciferaza reporter system for nuclear receptor analysis. Specifically, the base sequences of the ligand binding domains of each of PPARα, PPARγ and PPARδ (α: amino acids at positions 201 to 468, γ : Amino acids at positions 238 to 506 and δ corresponding to amino acids at positions 171 to 441), and a fusion protein expression vector of yeast-derived GAL4 protein DNA binding domain and PPAR ligand binding domain (hereinafter referred to as GAL4-PPAR expression vector) ) Was produced. Next, using Lipofectamine 2000 (Invitrogen), GAL4-PPAR expression vector and pGL4.35 were introduced into NIH3T3 cells at a ratio of 1: 1. 36 to 40 hours after gene introduction, the medium was replaced with a medium in which HEPE was added at various concentrations to a DMEM medium containing 10% FBS (Invitrogen) and an antibiotic-antifungal solution (Sigma-Aldrich). After a time, a cell lysate was prepared using Passive Lysis Buffer (Promega), and luciferase activity was measured. The luciferase activity was measured using a Promega Dual-Luciferase Reporter Assay System.

(Experiment result 1)
5-HEPE (5-hydroxy-6E, 8Z, 11Z, 14Z, 17Z-eicosapentaenoic acid), 8-HEPE, 9-HEPE, 12-HEPE, 18-HEPE (18) isolated and obtained from krill by the method described below. -Hydroxy-5Z, 8Z, 11Z, 14Z, 16E-eicosapentaenoic acid) at a concentration of 128 μM (test substances are dissolved in methanol) and cultured in PPARα, PPARγ, PPARδ FIG. 1 shows the respective activating effects (relative values where the luciferase activity is 1 when the cells are cultured in a medium containing only the same volume of methanol as that used when adding the test substance as a control). . FIG. 1 also shows the actions of EPA (Cyman Chemical Co., hereinafter the same) and ethyl ester (EPA-Et) (WAKO Co.). As is clear from FIG. 1, 8-HEPE and 9-HEPE had all excellent activation effects of PPARα, PPARγ, and PPARδ. 12-HEPE had a PPARδ activation effect. 5-HEPE and 18-HEPE did not have any activation effects of PPARα, PPARγ, and PPARδ, or very little if any.

(Experiment result 2)
When cells are cultured in a medium (test substance dissolved in methanol) containing 8-HEPE, 9-HEPE, and 18-HEPE isolated and obtained from krill by the method described below at various concentrations. , PPARα, PPARγ, and PPARδ are shown in FIG. 2 (the luciferase activity when cells were cultured in a medium containing only the same volume of methanol as that used when adding the test substance as a control). 1 relative value). FIG. 2 also shows the action of EPA. As is clear from FIG. 2, 8-HEPE and 9-HEPE had all the excellent activation effects of PPARα, PPARγ, and PPARδ in a concentration-dependent manner, but 18-HEPE has PPARα, PPARγ, It did not have any activating action of PPARδ or very little if any.

(Experiment result 3)
5-HEPE, 8-HEPE, 9-HEPE, 11-HEPE (11-hydroxy-5Z, 8Z, 12E, 14Z, 17Z-eicospentaenotic acid), 12-HEPE, 15-HEPE (15-) purchased from Cyman Chemical. hydroxy-5Z, 8Z, 11Z, 13E, 17Z-eicosapentaenoic acid) and 18-HEPE at a concentration of 5 μM, respectively, when the cells were cultured in PPARα, The activation effects of PPARγ and PPARδ are shown in FIG. 3 (the luciferase activity when the cells were cultured in a medium to which only the same volume of ethanol as the control was added as a control was defined as 1. Relative value) . FIG. 3 also shows the actions of palmitic acid (PA) (WAKO), arachidonic acid (AA) (WAKO), EPA, and various hydroxyeicosatetraenoic acids (HETE) (Cyman Chemical). . As is clear from FIG. 3, 11-HEPE had all excellent activation effects of PPARα, PPARγ, and PPARδ at low concentrations.

  From the above experimental results, HEPE having an excellent activating action of PPAR is HEPE near the center of the carbon chain where the hydroxyl bonding position is 8th to 12th, and the hydroxyl bonding position is 5th, 15th, It was found that HEPE near the end of the carbon chain, such as position 18, does not have or does not have the PPAR activation effect.

Formulation Example 1: Capsule 9-HEPE 200 mg, refined soybean oil 80 mg, beeswax 15 mg, and vitamin E 5 mg were thoroughly mixed while heating to 40 ° C. to obtain a homogeneous liquid. This was supplied to a capsule filling machine to produce a gelatin-coated capsule having an inner volume of 300 mg.

Reference Example 1: Isolation and acquisition of HEPE from krill (methanol extraction)
Isada (Tsunona krill) caught in the Sanriku region was dried with hot air at 100 ° C. for about 1 hour and then pulverized. A 10-fold amount (w / v) of methanol was added to the obtained powder and extracted for 2 hours or longer, and then filtered through a paper filter to obtain a methanol extract.

(Fractionation with HP-20)
This was performed using 30 g of synthetic resin adsorbent Diaion HP-20 (Mitsubishi Chemical Corporation) with respect to 200 mL of methanol extract. Specifically, first, 30 g of HP-20 was filled in a chromatographic tube, washed with 200 mL of 100% methanol, and then equilibrated with 200 mL of 30% aqueous methanol. Next, 200 mL of methanol extract was diluted with 800 mL of distilled water, applied, washed with 200 mL of 30% aqueous methanol, and then eluted with 200 mL of 80% aqueous methanol to obtain 200 mL of HP-20 eluate.

(Fractionation by InertSep C18)
One column (10 g) per 200 mL of HP-20 eluate was used. Specifically, the column was first washed with 100% methanol + 0.1% acetic acid solution (40 mL) and then equilibrated with 30% methanol aqueous solution + 0.1% acetic acid solution (40 mL). Next, 200 mL of the HP-20 eluate was diluted with 600 mL of distilled water, applied, washed with 40 mL of 30% aqueous methanol + 0.1% acetic acid, and then eluted with 40 mL of 100% methanol + 0.1% acetic acid. The eluate obtained was evaporated to give a yellow oil.

(Purification by HPLC)
A yellow oily substance was dissolved in methanol at 100 mg / mL, and then the reaction was performed under the following conditions.
Column: Inert Sustain-C18 (GL Science) φ20.0 mm × 250 mm (5 μm)
Sample: 10 mg / mL (100 mg / mL stock diluted 10 times with distilled water and filtered)
Injection: 1mL (10mg)
Mobile layer: 15 mL / min, A: distilled water + 0.2% formic acid solution, B: AcN, (A: B) 45: 55 → 45 min, 45:55 to 10: 90 → 20 min, 10: 90 → 10 min, 45:55 → 10min
Detector: UV235nm
Column oven: 40 ° C
The results are shown in FIG. Among the five peaks shown in FIG. 4, the components of the peaks 1, 2, and 5 were isolated and obtained, and the components of the peaks 3 and 4 were purified by recycle HPLC.

(Purification by recycle HPLC)
Column: Inert Sustain-C18 (GL Science) φ20.0 mm × 250 mm (5 μm)
Sample: 1 mg / mL (10% aqueous methanol solution) (10 mg / mL stock prepared by dissolving fractions of 3 and 4 peak components in methanol was diluted 10-fold with distilled water)
Injection: 500 μL (500 μg)
Moving bed: 60% AcN + 0.1% formic acid solution (Isocratic) 15 mL / min
Detector: UV235nm
Column oven: 40 ° C
Recycling: 19min ON, 71min OFF
Sorting: 85-94 min Signal 250 or more sorting result: FIG.

  As a result of collating the physicochemical characteristics of the five components isolated and obtained from Isada by the above process with the physicochemical characteristics of various HEPEs (standard substances) purchased from Cyman Chemical, the five components were 5-HEPE. (Yield from dry powder of Isada: 0.003%, the same applies hereinafter), 8-HEPE (yield: 0.006%), 9-HEPE (yield: 0.002%), 12-HEPE (yield) Rate: 0.003%) and 18-HEPE (yield: 0.008%). The identification results of the five components are shown in Table 1. Table 2 shows the retention times of the standard substances.

  INDUSTRIAL APPLICABILITY The present invention has industrial applicability in that it can provide a novel PPAR activator containing, as an active ingredient, HEPE whose hydroxyl bonding position is any of the 8th to 12th positions.

Claims (3)

A PPARδ activator comprising, as an active ingredient, hydroxyeicosapentaenoic acid having a hydroxyl group bonding position of 8 or an alkyl ester thereof, or a pharmaceutically acceptable salt thereof . A method for preparing a PPARδ activator by isolating and obtaining hydroxyeicosapentaenoic acid having a hydroxyl group at the 8- position by extraction from krill. The preparation method according to claim 2 , wherein the extraction operation is carried out using an organic solvent soluble in water and / or water.