JP2019210239A - Pharmacological use of 8-hydroxyeicosapentaenoic acid - Google Patents

Abstract

To provide a novel pharmacological use of 8-hydroxyeicosapentaenoic acid.SOLUTION: The present invention provides an ABCA1 gene expression promoter and an IL-1β gene expression inhibitor containing 8-hydroxyeicosapentaenoic acid as an active ingredient. 8-hydroxyeicosapentaenoic acid exhibits, based on these actions, preventive and therapeutic effects on angiopathy, diabetes or the like.SELECTED DRAWING: Figure 1

Description

  The present invention relates to the pharmacological use of 8-hydroxyeicosapentaenoic acid.

  8-Hydroxyeicosapentaenoic acid (8-HEPE) is a metabolite of eicosapentaenoic acid (EPA), which is one of ω3 fatty acids, and its existence has been known for a long time. Because it was not possible, little was known about the utility value. However, in recent years, our research group has found that 8-HEPE can be isolated and obtained from the marine organism Euphausiacea and that 8-HEPE can activate fatty acid metabolism and promote body fat burning. PPAR (Peroxisome Proliferator-Activated Receptor: peroxisome proliferator activity known to bring about effects such as inhibition of obesity, inhibition of fatty liver, prevention and improvement of diabetes, prevention and improvement of insulin resistance, and improvement of endurance (Patent Document 1), and attention has been focused on its pharmacological action. However, the full pharmacological action of 8-HEPE has not yet been elucidated.

JP2015-163607A

  Therefore, an object of the present invention is to provide a novel pharmacological application of 8-HEPE.

  As a result of intensive investigations in view of the above points, the present inventors have found that 8-HEPE promotes ABCA1 (ATP binding cassette transporter A1) gene expression and suppresses IL (Interleukin) -1β gene expression in foamed macrophages. It was found to have an action.

As described in claim 1, the ABCA1 gene expression promoter of the present invention made based on the above findings comprises 8-HEPE or an alkyl ester thereof or a pharmaceutically acceptable salt thereof as an active ingredient.
Moreover, the IL-1β gene expression inhibitor of the present invention comprises, as described in claim 2, 8-HEPE or an alkyl ester thereof or a pharmaceutically acceptable salt thereof as an active ingredient.
Moreover, the preventive / therapeutic agent for vascular disorders of the present invention comprises, as described in claim 3, 8-HEPE or an alkyl ester thereof or a pharmaceutically acceptable salt thereof as an active ingredient.
Moreover, the preventive / therapeutic agent for diabetes of the present invention comprises, as described in claim 4, 8-HEPE or an alkyl ester thereof or a pharmaceutically acceptable salt thereof as an active ingredient.

  According to the present invention, ABCA1 gene expression promoter and IL-1β gene expression inhibitor can be provided as novel pharmacological uses of 8-HEPE.

It is a graph which shows the (A) ABCA1 gene expression promotion effect and (B) IL-1β gene expression suppression effect of 8-HEPE in Experiment 1. It is (A) photograph and (B) graph which show the anti-arteriosclerosis effect | action of 8-HEPE in Experiment 2. FIG. 10 is a graph showing the blood glucose lowering effect of 8-HEPE in Experiment 3.

  As 8-HEPE which is an active ingredient of ABCA1 gene expression promoter and IL-1β gene expression inhibitor provided by the present invention, for example, 8-hydroxy-5Z, 9E, 11Z, 14Z, 17Z having the following chemical structure: -Eicosapentaenoic acid. Since 8-HEPE has an asymmetric carbon, it has optical isomers, and since it has a plurality of double bonds, various cis-trans isomers exist. It is included.

  In the present invention, 8-HEPE as an active ingredient is isolated from a natural product, synthesized by an organic synthetic chemistry method, EPA-oxidized with lipoxygenase or the like non-enzymatically. It may be an oxidized one (for example, commercially available from Cyman Chemical). Isolation acquisition from the natural product of 8-HEPE can be performed by extraction operation from krill as described in Patent Document 1, for example. The krill used as the raw material is not particularly limited, and it may be Euphausia superba, in addition to Euphausia pacifica, which is called Isada in the Sanriku region and is used for food. 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 an organic solvent and / or water soluble in water such as alcohol (methanol, ethanol, isopropanol, etc.), acetone, acetonitrile or the like. In order to improve the purity of isolated 8-HEPE, use ion exchange resin, non-ionic adsorption resin, gel filtration chromatography, chromatography with activated carbon, alumina, silica gel and other adsorbents, and high performance liquid chromatography. In addition to the separation operation, various operations such as a crystallization operation, a vacuum concentration operation, and a freeze-drying operation may be performed alone or in appropriate combination.

  8-HEPE 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 8-HEPE takes the form of a salt, pharmaceutically acceptable salts thereof include inorganic salts with sodium, potassium, magnesium, calcium, aluminum, etc., organic salts with lower alkylamines, lower alcoholamines, etc. In addition to basic amino acid salts with lysine, arginine, ornithine, ammonium salts and the like can be mentioned.

  In the present invention, the administration method when 8-HEPE as an active ingredient is administered to humans and animals as a pharmaceutical agent may be an oral administration method or a parenteral administration 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, 8-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 You may coat with a polymer etc. and may coat with two or more layers. Further, it may be encapsulated using ethyl cellulose or gelatin.

  8-HEPE as an active ingredient in the present invention has an ABCA1 gene expression promoting action and an IL-1β gene expression suppressing action. ABCA1 is one of the ABC proteins and is an essential membrane protein for the production of high density lipoprotein (HDL) called good cholesterol. IL-1β is an inflammatory cytokine produced primarily from monocytes / macrophages. Therefore, 8-HEPE having ABCA1 gene expression promoting action and IL-1β gene expression suppressing action corrects lipid abnormalities and suppresses inflammation by these actions, thereby preventing or treating vascular disorders such as arteriosclerosis. It can be used as an active ingredient of an agent. In addition, 8-HEPE brings about effects such as control of blood glucose and improvement of β-cell function based on the IL-1β gene expression inhibitory action. Use as an active ingredient of an inflammatory agent can also be expected. 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.

  Further, 8-HEPE may be eaten by adding an effective amount sufficient to exert ABCA1 gene expression promoting action and IL-1β gene expression suppressing action to foods of various forms (including supplements) (weight 1 kg). Ingestion of 0.1 mg to 100 mg per standard is standard).

  8-HEPE may be used in combination with EPA or docosahexaenoic acid (DHA), which is known to have an action of correcting lipid abnormalities.

  8-HEPE can also be used as a research reagent having an ABCA1 gene expression promoting action, a research reagent having an IL-1β gene expression suppressing action, and the like.

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

Experiment 1: Effect of 8-HEPE on expression of ABCA1 gene and IL-1β gene (experimental method)
J774.1 cells (monocyte / macrophage-like cell line producing IL-1) derived from a mouse obtained from the JCRB cell bank were seeded at 1 × 10 ⁶ cells / well in a 6-well plate, and RPMI1640 containing 10% FBS. After culturing at 37 ° C. for 24 hours in a 5% CO ₂ incubator using a medium, 8-hydroxy-5Z, 9E, 11Z, 14Z, 17Z-eicosapentaenoic acid (in Sanriku region according to the method described in Patent Document 1) The one obtained after isolation from the harvested Isada, the same below) was added to a final concentration of 50 μM. Subsequently, after culturing at 37 ° C. for 24 hours in a 5% CO ₂ incubator, cells were foamed by adding acetyl LDL cholesterol (AcLDL) to a final concentration of 50 μg / mL, and further cultured for 18 hours. did. Then, after rinsing the cells with PBS (−), total RNA was extracted using Sepazol RNA super G (Nacalai Tesque), RT-PCR was performed, and 8 for the expression of various genes in foamed macrophages. -The action of HEPE was investigated.

(Experimental result)
The effects of 8-HEPE on ABCA1 gene expression and IL-1β gene expression are shown in FIGS. 1A and 1B (H in the figure). FIG. 1 also shows the results of adding EPA as a control sample to a final concentration of 50 μM and the results of adding the same volume of PBS (−) as a control (the former is The latter is E in the figure and Vehi in the figure). As is apparent from FIG. 1, 8-HEPE exhibited an excellent ABCA1 gene expression promoting action and IL-1β gene expression suppressing action in foamed macrophages by acetyl LDL cholesterol loading. On the other hand, EPA did not show such effects.

Experiment 2: Effect of 8-HEPE on arteriosclerosis (experimental method)
An apoE gene-deficient mouse, which is an arteriosclerosis-inducing model mouse, was added to a normal diet, Western feed, 8-hydroxy-5Z, 9E, 11Z, 14Z, 17Z-eicosapentaenoic acid-containing Western feed (8-HEPE per kg of feed: 50 mg). , EPA: 110 mg, DHA: 80 mg) for 3 months, the aorta (from the aortic arch to the common iliac bifurcation) was removed, the lumen was expanded, and then the lesion site ( The atherosclerotic site was dyed red. The stained aorta was photographed with a digital camera (Leica MC120HD) attached to a stereomicroscope, and the ratio of the lesioned part to the entire aorta was measured with image analysis software (Adobe photoshop C3 or photoshop CC 2017).

(Experimental result)
The results of staining of the aorta with Sudan IV and the quantitative results of the ratio of the lesioned part are shown in FIGS. 2A and 2B (WD + 8-HEPE in the figure), respectively. Moreover, in FIG. 2, the result at the time of feeding a normal diet and the result at the time of feeding a Western feed are shown together (the former is SD in the figure and the latter is WD in the figure). As apparent from FIG. 2, when the 8-HEPE-containing material-added western feed was ingested, arteriosclerosis caused by ingesting the western feed was suppressed. Western feed containing 8-HEPE-containing material contains EPA and DHA, which are known to have anti-arteriosclerotic action, but according to previous reports, each content has anti-arteriosclerotic action. Since it is not sufficient to show, the expression of the anti-arteriosclerosis effect by feeding the Western feed containing 8-HEPE-containing material together with the results of Experiment 1, 8-HEPE promotes the expression of ABCA1 gene. It was thought that this was due to the suppression of cholesterol deposition in peripheral tissues such as blood vessel walls and the exertion of anti-inflammatory action through suppression of IL-1β gene expression.

Experiment 3: Effect of 8-HEPE on blood glucose (experimental method)
In Experiment 2, blood was collected from the heart of an apoE gene-deficient mouse ingested for 3 months with a normal diet, a Western diet, or an 8-HEPE-containing material-added Western diet, and a glucose concentration measuring device for laboratory animals (Lab Gluco: Philgen) ) Was used to measure the blood glucose level.

(Experimental result)
As shown in FIG. Moreover, in FIG. 3, the result at the time of feeding a normal diet and the result at the time of feeding a Western feed are shown together (the former is SD in the figure and the latter is WD in the figure). As apparent from FIG. 3, when the 8-HEPE-containing material-added western feed was ingested, an increase in blood sugar caused by ingesting the western feed was suppressed. Western feed containing 8-HEPE-containing material contains EPA and DHA, which are known to have a hypoglycemic effect, but according to previous reports, each content shows a hypoglycemic effect. Since it is not sufficient, the expression of the hypoglycemic effect by feeding the Western feed containing 8-HEPE-containing material is considered together with the result of Experiment 1, 8-HEPE is mediated through the suppression of IL-1β gene expression. This was thought to be due to the effect of controlling blood glucose and improving β-cell function.

Formulation Example 1: Capsules 8-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.

  The present invention has industrial applicability in that it can provide an ABCA1 gene expression promoter and an IL-1β gene expression inhibitor as a novel pharmacological use of 8-HEPE.

Claims (4)

  An ABCA1 gene expression promoter comprising 8-hydroxyeicosapentaenoic acid or an alkyl ester thereof or a pharmaceutically acceptable salt thereof as an active ingredient.   An inhibitor of IL-1β gene expression comprising 8-hydroxyeicosapentaenoic acid or an alkyl ester thereof or a pharmaceutically acceptable salt thereof as an active ingredient.   A prophylactic / therapeutic agent for vascular disorders comprising 8-hydroxyeicosapentaenoic acid or an alkyl ester thereof or a pharmaceutically acceptable salt thereof as an active ingredient.   A prophylactic / therapeutic agent for diabetes comprising 8-hydroxyeicosapentaenoic acid or an alkyl ester thereof or a pharmaceutically acceptable salt thereof as an active ingredient.