JP6656890B2 - Filaggrin production promoter - Google Patents

Description

  The present invention relates to a filaggrin production promoter that promotes production of filaggrin.

  Filaggrin is a type of basic protein produced in epidermal granule cells, and aggregates keratin filaments in the stratum corneum and plays an important role in the construction of the tissue structure of the stratum corneum. In addition, the production process of filaggrin has already been elucidated, and is considered as follows. First, profilaggrin, a precursor of filaggrin, is produced in the epidermal granular layer. The produced profilaggrin undergoes phosphorylation to form keratohyalin granules in granule cells. When the granule cells migrate into the stratum corneum following the turnover of the epidermis, profilaggrin is dephosphorylated and subjected to the action of a protease to produce filaggrin.

  In recent years, it has been reported that reduced function of filaggrin is one of the causes of atopic dermatitis and hay fever, and it has been clarified that filaggrin is involved in the development of various allergic diseases. Furthermore, it has been clarified that a decrease in the expression level of filaggrin is involved in the development of ichthyosis vulgaris.

  For the prevention or treatment of such filaggrin-related diseases, it is effective to promote the expression of filaggrin in vivo. Therefore, recently, various components for promoting the expression of filaggrin have been studied. For example, as a component for promoting the expression of filaggrin, an extract of a beautiful woman under the moon (Patent Literature 1), rosmarinic acid and / or eriodictyol 7-O-rutinoside (Patent Literature 2), Sansho extract (Patent Literature 3) Are found.

  However, development of new components that promote the expression of filaggrin has been desired in order to meet demands such as diversification of pharmaceutical formulations and further improvement of filaggrin production promoting action.

JP 2009-107965 A JP 2011-68594 A

  An object of the present invention is to provide a filaggrin production promoter that promotes the expression of filaggrin production.

  The present inventors have conducted intensive studies to solve the above-mentioned problems, and found that sulfated glucosaminoglycan has an action of increasing the expression level of profilaggrin mRNA in epidermal cells and promotes the expression of filaggrin production. I found what I could do. The present invention has been completed by further study based on such knowledge.

That is, the present invention provides the following aspects of the invention.
Item 1. A filaggrin production promoter comprising sulfated glucosaminoglycan.
Item 2. Item 2. The filaggrin production promoter according to Item 1, wherein the sulfated glucosaminoglycan is a heparin-like substance.
Item 3. Item 3. The filaggrin production promoter according to Item 1 or 2, which is a skin external preparation.
Item 4. Item 4. The filaggrin production promoter according to any one of Items 1 to 3, which is used for prevention or treatment of atopic dermatitis, hay fever, or ichthyosis vulgaris.

  According to the filaggrin production promoter of the present invention, since the expression of profilaggrin in epidermal cells can be promoted and the production of filaggrin production can be promoted, it is useful for the prevention or treatment of diseases and symptoms caused by a decrease in the function of filaggrin. It is valid.

FIG. 4 is a view showing the results of culturing normal epidermal model cells in the presence of a heparin-like substance and measuring the expression level of profilaggrin mRNA. It is a figure which shows the result of having culture | cultivated the epidermis upper layer model cell in presence of a heparin analogue, and measuring the expression level of profilagulin mRNA.

  The filaggrin production promoter of the present invention is characterized by containing sulfated glucosaminoglycan. Hereinafter, the filaggrin production promoter of the present invention will be described in detail.

Sulfated glucosaminoglycan Sulfated glucosaminoglycan is a mucopolysaccharide containing an amino sugar as one of its constituent units and having a sulfate group.

  The type of sulfated glucosaminoglycan used in the present invention is not particularly limited, as long as it is pharmaceutically acceptable, but, for example, heparin analog, heparan sulfate, chondroitin sulfate, keratan sulfate, heparin, etc. Is mentioned. The sulfated glucosaminoglycan may be in the form of a metal salt such as sodium, potassium, calcium, magnesium, iron and manganese; and a salt such as an ammonium salt.

  One of these sulfated glucosaminoglycans may be used alone, or two or more thereof may be used in combination. Among them, preferred is a heparin-like substance from the viewpoint of promoting filaggrin production more effectively.

  The origin of the sulfated glucosaminoglycan is not particularly limited, for example, those obtained by polysulfating mucopolysaccharides, tissues of edible animals (for example, in the case of a heparin-like substance, And lungs including tracheal cartilage of cattle and pigs).

  Further, for example, when a heparin-like substance is used as the sulfated glucosaminoglycan, a heparin-like substance listed in the Japanese Pharmacopoeia Non-Pharmaceutical Standards is preferable.

The blending amount of the sulfated glucosaminoglycan in the filaggrin production promoter of the present invention may be appropriately set according to the formulation of the filaggrin production promoter, for example, 0.01 to 5% by weight, preferably, 0.05 to 3% by weight, more preferably 0.1 to 1% by weight.
Other Ingredients The filaggrin production promoter of the present invention may contain other pharmacological components, if necessary, in addition to the sulfated glucosaminoglycan. Such pharmacological components include, for example, antihistamines (dipotassium glycyrrhizinate, glycyrrhizic acid, diphenhydramine, diphenhydramine hydrochloride, etc.), local anesthetics (procaine, tetracaine, bupivacaine, mepipacaine, chloroprocaine, proparacaine, meprilcaine or salts thereof. , Orthocaine, oxesazein, oxypolyentoxydecane, funnel extract, percaminepase, tesit decithin, etc., anti-inflammatory agents (allantoin, indomethacin, felbinac, diclofenac sodium, loxoprofen sodium, etc.), skin protective agents (collodion, castor oil, etc.), blood circulation promotion Ingredients (nonylate wanilylamide, nicotinic acid benzyl ester, capsaicin, pepper extract, etc.), freshener (menthol, can Le etc.), vitamins (vitamin A, etc.), mucopolysaccharides (sodium chondroitin sulfate, glucosamine, etc.) and the like.

  In addition, the filaggrin production promoter of the present invention may contain a base and additives as necessary in order to obtain a desired formulation. Such bases and additives are not particularly limited as long as they are pharmaceutically acceptable. For example, water, lower alcohols (such as ethanol and isopropanol), and polyhydric alcohols (glycerin, propylene glycol, dipropylene) Aqueous bases such as glycols, 1,3-butylene glycol, etc .; oils (olive oil, safflower oil, soybean oil, camellia oil, corn oil, rapeseed oil, sunflower oil, cottonseed oil, peanut oil, lard, squalane, fish oil, etc. ), Mineral oils (liquid paraffin, paraffin, gelled hydrocarbons, petrolatum, etc.), waxes and waxes (beeswax, carnauba wax, candelilla wax, ceresin, rice wax, microcrystalline wax, etc.), ester oils (isopropyl myristate) , Isopropyl adipate, sebacic acid Ethyl, isopropyl sebacate, isopropyl palmitate, cetyl palmitate, ethyl oleate, etc.), fatty acid alkyl esters, fatty acids (stearic acid, oleic acid, palmitic acid, behenic acid, linoleic acid, lanolin, etc.), fatty acid esters (palmitic acid) Cetyl, isopropyl palmitate, ethyl linoleate, etc.), higher alcohols (stearyl alcohol, cetanol, behenyl alcohol, myristyl alcohol, oleyl alcohol, hexadecyl alcohol, lanolin alcohol, etc.), cholesterol, glyceryl tri-2-ethylhexanoate, 2-ethyl Oily bases such as cetyl hexanoate, silicone oil (dimethylpolysiloxane, cyclic silicone, etc.); POE (10 to 50 mol) phytosterol ether, POE ( 0-50 mol) dihydrocholesterol ether, POE (10-50 mol) 2-octyldodecyl ether, POE (10-50 mol) decyltetradecyl ether, POE (10-50 mol) oleyl ether, POE (2-50 mol) ) Cetyl ether, POE (5-50 mol) behenyl ether, POE (5-30 mol) polyoxypropylene (5-30 mol) 2-decyltetradecyl ether, POE (10-50 mol) polyoxypropylene (2 30 mol) polyoxyethylene alkyl ethers such as cetyl ether, their phosphoric acid / phosphates (eg, POE cetyl ether sodium phosphate), POE (20 to 60 mol) sorbitan monooleate, POE (10 to 60 mol) sorbitan Monoisostearate, POE (10- 80 mol) glyceryl monoisostearate, POE (10-30 mol) glyceryl monostearate, POE (20-100 mol) polyoxypropylene-modified silicone, POE alkyl-modified silicone, polyethylene glycol monolaurate, polyethylene monopalmitate Glycol, polyethylene glycol monostearate, polyethylene glycol dilaurate, polyethylene glycol dipalmitate, polyethylene glycol distearate, polyethylene glycol dioleate, polyethylene glycol diricinoleate, polyoxyethylene hydrogenated castor oil (5-100), polysorbate (20 -85), glycerin fatty acid esters (glyceryl monostearate, etc.), hydrogenated soybean phospholipids, hydrogenated lanoli Surfactants such as alcohol; fresheners (menthol, camphor, borneol, peppermint water, peppermint oil, etc.), preservatives (methylparaben, propylparaben, benzoic acid, sodium benzoate, sorbic acid, etc.), flavoring agents (citral) , 1,8-sionone, citronellal, farnesol, etc.), coloring agents (tar dyes (brown 201, blue 201, yellow 4, yellow 403, etc.), cocoa dyes, chlorophyll, aluminum oxide, etc.), thickeners (Carboxyvinyl polymer, hypromellose, polyvinylpyrrolidone, sodium alginate, ethylcellulose, sodium carboxymethylcellulose, xanthan gum, carrageenan, etc.), pH adjusters (phosphoric acid, hydrochloric acid, citric acid, sodium citrate, succinic acid, tartaric acid, sodium hydroxide, Hydroxy acid Potassium, triethanolamine, triisopropanolamine, etc.), wetting agents (sodium dl-pyrrolidonecarboxylate solution, D-sorbitol solution, macrogol, etc.), stabilizers (dibutylhydroxytoluene, butylhydroxyanisole, sodium edetate, methaline) Sodium acid, L-arginine, L-aspartic acid, DL-alanine, glycine, sodium erysorbate, propyl gallate, sodium sulfite, sulfur dioxide, chlorogenic acid, catechin, rosemary extract, etc.), antioxidant, ultraviolet absorption And chelating agents, adhesives, buffers, solubilizers, solubilizers, preservatives and other additives.

Formulation form The filaggrin production promoter of the present invention may be in any form such as an external preparation for the skin or an internal preparation, but from the viewpoint of promoting the production of filaggrin more effectively, an external preparation for the skin is preferably used. Is mentioned.

  When the filaggrin production promoter of the present invention is used as an external preparation for skin, its shape is not particularly limited as long as it can be applied transdermally, but, for example, liquid, solid, semi-solid (gel, ointment, Paste).

  In addition, when the filaggrin production promoter of the present invention is used as an external preparation for skin, its formulation is not particularly limited as long as it can be applied transdermally. And skin cleansing agents. When the filaggrin production promoter of the present invention is used as an external preparation for the skin, specifically, a cream, lotion, gel, emulsion, liquid, patch, aerosol, ointment, pack, etc. Dermatological products for skin; creams, lotions, gels, emulsions, liquids, patches, aerosols, ointments, packs, etc .; quasi-drugs for skin; creams, lotions, gels, emulsions And cosmetics such as liquids, ointments, and packs; and skin cleansers such as body shampoos, hair shampoos, and rinses. Among these formulation forms, preferably, a skin external medicine, more preferably, a cream, a lotion, a gel, an emulsion, a pack is used.

Use / Dose The filaggrin production promoter of the present invention promotes filaggrin production by increasing the expression level of profilaggrin mRNA which is a precursor of filaggrin in epidermal cells (particularly, cells present in the upper layer of the epidermis). be able to. Therefore, the filaggrin production promoter of the present invention is effective for the prevention or treatment of diseases and symptoms in which the decrease in filaggrin function is one of the onset factors. Diseases and symptoms in which decreased function of filaggrin are one of the causes are, for example, allergic diseases such as atopic dermatitis, asthma, rhinitis, hay fever, metal allergy, ichthyosis vulgaris, virus And skin infections caused by bacteria.

In addition, the dose of the filaggrin production promoter of the present invention may be appropriately set according to the dosage form, preparation form, degree of symptoms to be applied, and the like. For example, when the filaggrin production enhancer of the present invention is used as an external preparation for skin, as an example of the dose, the amount of sulfated glucosaminoglycan is about 0.1 to 3 mg per 1 cm ^{2 of} skin. The amount is about 1 to several times a day.

  Hereinafter, the present invention will be described more specifically with reference to Examples, but the present invention is not limited thereto.

Test Example 1: Effect of sulfated glucosaminoglycan on filaggrin production The following experiment was performed to evaluate the effect of sulfated glucosaminoglycan on filaggrin production of epidermal cells.

1. Experimental material (cell)
In this test example, the following two types of cells were used.
(A) Normal epidermal model cells (HaCaT cells)
HaCaT cells (human epidermal keratinocytes (Deutsches Krebsforschungsgentrum, Stiftung deoffentrichen Rechts (DKFZ), Heidelberg, Germany)) were used as normal epidermis model cells.
(B) Upper epidermis model cells (Syntaxin 4 high expressing HaCaT cells)
A cell model of the upper epidermis partly differentiated was prepared and used for the test. (I) epidermal cells move from the basal layer to the spinous layer / granular layer when keratinized; (ii) extrataxin 4 is secreted extracellularly as they move to the upper layer; and (iii) HaCaT cells It is known that keratinization proceeds when syntaxin 4 is overexpressed (Kadono N. et al., Mol Med 2015). Therefore, HaCaT cells overexpressing syntaxin 4 were produced as upper epidermis model cells and used in this test example. The fabrication method is as follows.
First, a gene encoding a mouse-derived IL-2 signal peptide consisting of 21 amino acid residues was added to the N-terminus of the mouse syntaxin 4 gene in order to forcibly display it outside the cell, and this was added to the pQCXIN retrovirus vector multimeric. It was inserted into the cloning site. Next, this was introduced into packaging cells PT67 using a transfection reagent Lipofectamine PLUS (manufactured by Thermo Fisher Scientific Co., Ltd.). A retrovirus was obtained from the culture supernatant of the obtained cells. By infecting HaCaT cells with the obtained virus, HaCaT cells (upper epidermis model cells) in which syntaxin 4 was forcibly expressed outside the cells were produced.

(Culture medium)
D-MEM / Ham's F-12 (Wako Pure Chemical Industries, Ltd.) / 10% FCS added

(Test substance)
Heparin analogue

2. Experimental method (1) Expression amount of profilaggrin mRNA in normal epidermis model cells Model cells were seeded at a concentration of 1 × 10 ⁵ cells / well in each well of a 12-well plate, and were incubated at 37 ° C. and 5% CO ₂ . Cultured. The next day, a heparin-like substance was added to each well at a concentration of 0.5 mg / mL, and the cells were cultured at 37 ° C. under 5% CO ₂ . The next day, the model cells were collected and total RNA was extracted using RNeasy mini kit (manufactured by Qiagen). Thereafter, reverse transcription was performed using ReverseTra Ace (manufactured by Toyobo Co., Ltd.) to synthesize cDNA. Next, the profilagulin gene was amplified by qRT-PCR using Fast Start Essential DNA Green Master on LightCycler Nano system (manufactured by Roche Diagnostics Co., Ltd.). β-actin was used as an internal standard to standardize the expression level of profilaggrin mRNA. The mRNA expression level was divided by the mRNA expression level (control) confirmed in the same manner as described above except that the heparin analog was not added.

(2) Expression level of profilaggrin mRNA in upper epidermal model cells The same procedure as in (1) above was carried out except that the upper epidermal model cells were used instead of the normal epidermal model cells, and the expression level of profilaggrin mRNA was confirmed. , Control (the mRNA expression level when no heparin analog was added).

  The results obtained are shown in FIGS. As can be seen from FIGS. 1 and 2, both the normal epidermal model cells and the upper epidermal model cells showed an increase in the expression level of profilaggrin mRNA, which is a precursor of filaggrin, under conditions to which a heparin-like substance was added. In the upper epidermis model cells, the expression level was significantly increased. That is, the present results indicate that sulfated glucosaminoglycan such as a heparin-like substance can express profilaggrin mRNA in epidermal cells, particularly cells in the upper layer of the epidermis (cells to which the administered sulfated glucosaminoglycan is directly affected). It has been found that it has the effect of increasing filaggrin and can promote the production of filaggrin. In addition, there was no significant difference in the expression level of mRNA between the control in the normal epidermis model cell and the control in the upper epidermis model cell.

Formulation Examples Creams (Formulation Examples 1 to 8) having compositions shown in Tables 1 and 2, lotions (Formulation Examples 9 to 14) shown in Table 3, gels (Formulation Examples 15 to 19) shown in Table 4, and tables Emulsions (Formulation Examples 20 to 29) shown in 5 to 6 were prepared. All of these preparations are expected to have an effect of increasing the expression level of profilaggrin mRNA in epidermal cells, as in the case of Test Example 1, and are effective in promoting filaggrin production.

Claims (3)

A filaggrin production comprising sulfated glucosaminoglycan, which is used for the prevention or treatment of hay fever (excluding those associated with atopic dermatitis and / or atopic disease). Accelerator.   The filaggrin production promoter according to claim 1, wherein the sulfated glucosaminoglycan is a heparin-like substance.   The filaggrin production promoter according to claim 1 or 2, which is a skin external preparation.