JPH03123717A - Melanocyte-stimulating hormone suppressant and skin external preparation containing the same suppressant - Google Patents

Abstract

PURPOSE:To obtain a skin external preparation preventing and improving symptom of stain, freckle, etc., following sthenia of melanocyte function, containing a peptide having a specific pentapeptide amino acid sequence in the molecule. CONSTITUTION:A skin external preparation containing a peptide having an amino acid sequence shown by formula I in the molecule, preferably a peptide shown by formula II [X is H, acyl, amino acid residue, acylated material thereof, peptide residue (preferably Ile-Leu or Leu-His-Ala-Leu-Gln-Leu-Leu-Leu-Ile- Leu) having 2-40 amino acid residues or acylated material thereof) Y is OH, NH2, amino acid residue, amidated material thereof, peptide residue (preferably Ile-Ser-Pro-Gly-Arg-Arg) having 2-40 amino acid residues or amidated material thereof)]. The peptide preferably has 532-10,000, especially 532-4,000 molecular weight and 6-81, especially 5-31 amino acid residues.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a melanocyte-stimulating hormone (also called melanotropin) that activates the function of melanocytes, which are melanin pigment-producing cells in living bodies.

Hereinafter abbreviated as MSH. ), and also relates to preparations such as external preparations for the skin that can prevent and improve symptoms such as spots and freckles caused by advanced melanocyte function such as excessive production of melanin pigments. .

[Conventional technology]

MSH is a polypeptide hormone that exists in vertebrates, and types such as α-1β-1γ- are known.

These are known to bind to receptors located on the surface of melanocytes and sequentially activate adenylate cyclase, tyrosinase, and melanin production.
MSH is said to have a strong effect as described above.

In the past, many proposals have been made as a method for suppressing melanocyte function, such as the use of inhibitory substances that have an action site on tyrosinase within melanocytes. Some examples have been put into practical use, such as kojic acid disclosed in No. 18739. However, conventional methods are not necessarily satisfactory in terms of effectiveness, safety, etc. Furthermore, at present, there have been almost no proposals regarding the use of substances that suppress the action of melanocyte-stimulating hormone itself.

[Problem to be solved by the invention]

Therefore, an object of the present invention is to provide an MSH inhibitor that is highly effective in suppressing melanocyte functions such as melanin production and is highly safe, as well as formulations containing the same, such as external preparations for skin use.

[Means to solve the problem]

As a result of synthesizing various peptides and examining the relationship between their structure and function, the present inventors found that peptides with a specific structure have an affinity for MSH itself, and as a result, MSH
The new findings suggest that melanin production in melanocytes can be specifically suppressed by inhibiting the action of H, and that the above problems can be effectively solved by incorporating the peptide into ordinary cosmetics and pharmaceutical base materials. This was done based on new findings.

That is, the present invention provides a melanocyte-stimulating hormone inhibitor comprising a peptide having an amino acid sequence represented by the following formula IF in its molecule.

-Leu-Ala-Cys-Ala-Arg -...(
IF Note that in the present invention, any compound can be used as long as it has an amino acid sequence represented by the formula ['IE] in its molecule; however, the peptide represented by the following general formula [■] is preferred.

X-Leu-Ala-Cys-Ala-Arg-Y ・
(:] I:1 ([■] In the formula, X is preferably hydrogen, an acyl group, -11e-Leu or -Leu -His
-Ala -Leu -Gin -Leu -Leu
-Leu -Ile -Peptide residues containing the amino acid sequence shown by -Leu and their acylated products.

On the right, the above acyl group, the acylated product has 1 to 1 carbon atoms.
2, preferably 1 to 6. Y represents a hydroxyl group, an amino group, an amino acid residue and its amidated product, or a peptide residue having 2 to 36 amino acid residues, preferably 2 to 16 amino acid residues, and its amidated product. Y is preferably a hydroxyl group, an amino group or I le -Set -P
These are peptide residues containing the amino acid sequence ro -Gly-Arg-Arg- and amidated products thereof. )
As amino acid residues and peptide residues of XSY, MSH
Various combinations can be used as long as they do not inhibit the inhibitory function, and the peptide residue is preferably a physiologically inert group.

In addition, a sugar chain may be attached to XSY.

The molecular weight of the compound of the formula [■] is composed of at least the above five amino acid residues, so the lower limit is 532,
The upper limit is preferably up to 10,000, but the molecular weight range is
Preferably 532-6000, particularly preferably 532-6000
It is 4000. In addition, the number of amino acid residues is 5 to 81.
The number is preferably 5 to 41, particularly preferably 5 to 31.

As a compound represented by the general formula [■] used in the present invention,
Specifically, the compounds shown in Table 1 below are exemplified.

In Table 1, the amino acid residues constituting the peptide are I
UPAC abbreviation was used.

Here, following the usual notation, the amine end of the peptide (
The N-terminus) is shown on the left, and the carboxyl terminus (C-terminus) is shown on the right. (All are the same in this specification.

) Note that when the N-terminus of X(7) is hydrogen and the C-terminus of Y is a hydroxyl group, the description of H- or -OH is omitted.

Further, Ac represents an acetyl group, and 8u represents a butyryl group.

(The same applies hereinafter.) The MSH inhibitor according to the present invention can be obtained using a liquid phase method or a solid phase method, which are common peptide synthesis methods.

In the case of a liquid phase method, the amine group of one amino acid is protected with a benzyloxycarbonyl group or t-butoxycarbonyl group, the carboxyl group of the other amino acid or peptide is protected with a benzyl ester, etc., and DCC (N, N
'-dicyclohexylcarbodiimide), etc. By repeating this operation, the protecting group can be removed and the compound of the present invention can be purified. In the case of the solid phase method, the C-terminal amino acid is first coupled to the crosslinked polystyrene resin, and then t-butoxycarbonyl amino acids are sequentially coupled one by one toward the N-terminus. After completion of the reaction, the compound of the present invention can be obtained by detachment from the resin, removal of the protecting group, and purification.

Furthermore, it is also possible to incorporate the DNA encoding the active ingredient of the present invention into microorganisms such as Escherichia coli for production.

The MSH inhibitor of the present invention is in the form of a white powder at room temperature.

The MSH inhibitor of the present invention can be used to treat symptoms associated with advanced skin melanocyte function due to various causes, such as age spots, which are epidermal or dermal pigment spots caused by overproduction of melanin pigment.
It is effective in the prevention and treatment of melanoma (melasma), freckles (chloasma), birthmarks, etc., as well as in suppressing the abnormal growth of melanoma, which is cancerous melanocytes, and its usage method and amount should be controlled. It is also possible to adjust skin color or hair color, and can be applied as a skin whitening agent. Furthermore, it has recently become known that MSH exhibits a number of physiological functions in the living body, such as adipogenesis, sebum production, pheromone production, steroid production, aldosterone biosynthesis, and acts as a neurohormone in the brain. In these cells and tissues that exhibit physiological effects, the active ingredient of the present invention can control various actions of MSH.

Therefore, the MSH inhibitor of the present invention can be used not only in humans but also in mammals such as dogs and cats, reptiles, both species, and fish.
Applicable to animals endogenously producing SH. Naturally, it can also be used as an in vitro diagnostic agent or biochemical reagent.

When the MSH inhibitor of the present invention is used in a living body, the animal species to which it is applied, the site of cells and tissues, the ease of reaching the target site,
Various dosage forms can be selected based on ease of use, etc. That is,
It can be blended into cosmetics or pharmaceutical base materials and used in any form such as external preparations for the skin, oral preparations, injections, etc., and the active ingredient of the present invention can be blended at any concentration. Depending on the purpose, form, and frequency of use, 0.0000
About 000001 to 1% by weight (hereinafter abbreviated as %),
Preferably, it is blended in an amount of 0.00000001 to 0.1%. In this case, it is preferable that the remainder be water as an inert carrier, and more preferably 1 to 99% water.

Among the preparations containing the MSH inhibitor of the present invention, the skin external preparation contains, in addition to the above-mentioned essential ingredients, raw materials normally used for external preparations, such as surfactants, oils, alcohols, humectants, and additives. Adhesive, preservative, antioxidant, chelating agent, pH
Conditioners, fragrances, pigments, ultraviolet absorbing/scattering agents, vitamins, amino acids, water, etc. can be added.

Specifically, the surfactants include lipophilic glycerin monostearate, self-emulsifying glycerin monostearate, polyglycerin monostearate, sorbitan monooleate, polyethylene glycol monostearate,
Nonionic surfactants such as polyoxyethylene sorbicun monooleate, polyoxyethylene cetylethenope polyoxyethylenated sterol, polyoxyethylene lanolin, polyoxyethylated beeswax, polyoxyethylene hydrogenated castor oil, sodium stearate, Potassium palmitate, sodium cetyl sulfate, sodium lauryl phosphate, triethanolamine valmitate,
Anionic surfactants such as polyoxyethylene sodium lauryl phosphate, sodium N-acylglutamate,
Examples include cationic surfactants such as stearyldimethylbenzylammonium chloride and stearyltrimethylammonium chloride, amphoteric surfactants such as alkylaminoethylglycine hydrochloride, and lecithin.

Oils include vegetable oils such as castor oil, olive oil, cacao butter, camellia oil, coconut oil, wood wax, jojoba oil, grapeseed oil, and avocado oil; animal oils and fats such as mink oil and egg yolk oil; beeswax and spermaceti , lanolin, carnauba wax, candelilla wax, etc.; hydrocarbons such as liquid paraffin, squalane, microcrystalline wax, ceresin wax, paraffin wax, petrolatum; lauric acid, myristic acid, stearic acid, oleic acid, isostearic acid, Natural and synthetic fatty acids such as behenic acid; Natural and synthetic higher alcohols such as cetanonopestearyl alcohol, hexyldecanonopeoctyldodecanol, lauryl alcohol: imbropinomyristate, improvinope balmitate, octyldodecyl myristate, Examples include esters such as octyldodecyl oleate and cholesterol oleate.

Humectants include glycerin, propylene glycol,
113 Polyhydric alcohols such as butylene glycol, sorbitonopeboliglycerin, polyethylene glycol, dipropylene glycol; amino acids, sodium lactate,
Examples include NMF components such as sodium pyrrolidone carboxylate; water-soluble polymer substances such as hyaluronic acid, collagen, mucopolysaccharide, and chondroitin sulfate.

Thickeners include natural polymers such as sodium alginate, xanthan gum, aluminum silicate, quince seed extract, tragacanth gum, and starch; semisynthetic polymers such as methylcellulose, hydroxyethylcellulose, carboxymethylcellulose, soluble starch, and cationized cellulose. Substances: Examples include synthetic polymeric substances such as carboxyvinyl polymers and polyvinyl alcohol.

Preservatives include benzoate, salicylate, sorbate, dehydroacetate, paraoxybenzoic acid estenope 2.4.4') dichloro-2'hydroxydiphenyl ether, 3.4.4' trichlorocarbanilide. , benzalkonium chloride, hinokithionoberezorcin, ethanol and the like.

Examples of antioxidants include dibutylhydroxytoluene, butylated hydroxyanisone, furovir gallate, ascorbic acid, etc., and chelating agents include disodium edetate, ethylenediaminetetraacetate, birophosphate, hexametaphosphate, citric acid, Tartaric acid, gluconic acid, etc.
Examples of the pH adjuster include sodium hydroxide, triethanolamine, citric acid, sodium citrate, boric acid, borax, and potassium hydrogen phosphate.

For ultraviolet absorbing and scattering agents, 2-hydroxy-4-
Examples include methoxybenzophenone, octyldimethylparaaminobenzoate, ethylhexylparamethoxycynamate, titanium oxide, kaolin, and talc.

Examples of vitamins include vitamin A1 vitamin B1 vitamin C1 vitamin D1 vitamin E1 vitamin F1 vitamin 1 vitamin P1 vitamin U1 carnitine, ferulic acid, γ-oryzanol, α-lipoic acid, orotic acid and derivatives thereof. .

Amino acids include glycine, alanine, valine, leucine, isoleucine, serine, threonine, phenylalanine, tyrosine, tryptophan, cystine, cysteine, methionine, proline, hydroxyproline,
Examples include aspartic acid, glutamic acid, arginine, histidine, lysine, and transparent conductors thereof.

Note that the optional components are not limited to these. By appropriately blending the above essential components and optional components, for example, the essential components may be 0.0000000001 to 1%, the optional components may be 0 to 80% oil, and 0 to 12% surfactant (preferably 0.05 to 8%). ), a skin external preparation containing 1 to 15% of a humectant, a balance of purified water, and a trace amount of a preservative can be provided. Specifically, creams, emulsions, lotions,
It can be used in a variety of product forms such as beauty serum, backing agent, under makeup, foundation, jelly, and ointment.

To give a specific example of use as a skin cosmetic, as a skin cream: essential ingredients 0.00000001-0.1%, oil content 20-0.
70% surfactant, 2-7% surfactant, 1-10% humectant, purified water balance, trace amount of preservative, trace amount of each ingredient, 7 Emulsion: Essential ingredients 0.00000001-0.1% , oil content 10~
40%, alcohol 0-15%, surfactant 1-5%
, a composition containing 1 to 10% moisturizer, 0 to 2% thickener, purified water balance, a trace amount of preservative, and a trace amount of fragrance, as a lotion or beauty serum: Essential ingredients 0.00000001 to 0.1% , alcohol 5-20%, surfactant 0-2%, humectant 2-8%,
Thickener 0-2%, antioxidant 0-0.5%, chelating agent 0-0.1%, pH adjuster 0-0.2%, purified water balance, trace amount of preservative, 0-trace amount of pigment, A composition containing a trace amount of fragrance, as a pack agent: essential ingredients 0.00000001-0.1%, alcohols 2-10%, humectants 2-10%, inorganic powders 0-20%
, film forming agent 10-20%, purified water balance, trace amount of preservative,
compositions containing trace amounts of fragrances.

As an ointment: Essential ingredients: 0.00000001-0.1%, oil content: 40-0.
Composition containing 60%, surfactant 1-12%, humectant 8-15%, purified water balance, trace amount of preservative (parent tree ointment), essential ingredients 0.00000001-0.1%, oil content 95% ~
Compositions (oil-based ointments) containing 99% purified water balance.

Oral preparations containing the MSH inhibitor of the present invention include:
Examples include solid preparations such as tablets in which excipients such as starch, glucose, and lactose are added to the essential ingredients of the present invention, and liquid preparations in which sugars, water, ethanol, and the like are added. In the case of oral administration, the dosage of the essential components of the present invention is 10 μg/kg to 10.0 μg/kg.
000μg/kg.

Injections include those in which the essential components of the present invention are dissolved in a solvent such as water or physiological saline, and can be used by injecting subcutaneously, intramuscularly, or intravenously. In the case of injections, the dosage of the essential ingredients of the present invention is 1 μg/kg.
~1. OOOμg/kg.

The details of the mechanism by which the active ingredient of the present invention exerts an excellent suppressive effect on MSH are still under investigation, but will be explained as follows.

That is, the active ingredient of the present invention has an amino acid sequence consisting of 5 residues shown by formula 13 in the molecule, but this part is M
It has a complementary relationship to the amino acid sequence that shows SH activity, and has structural affinity with MSH, and as a result, the binding between MSH and the receptor is blocked, and it exhibits an excellent melanin production suppressing effect. It is assumed that

Furthermore, it was confirmed that it has good stability in preparations such as external preparations for the skin, and is highly practical.

The above-mentioned active ingredients of the present invention have no practical problems in terms of safety such as skin irritation.

〔Effect of the invention〕

According to the present invention, it is possible to suppress the function of melanocytes through the MSH inhibitory effect, essentially solving symptoms associated with the enhancement of melanocyte function such as the appearance of age spots and freckles, and also to have excellent effects as a skin whitening agent. Provided are external preparations for skin use, oral preparations, injection preparations, etc., which have the following properties and are stable in terms of formulation.

Next, the present invention will be explained by examples.

Example 1 Compounds shown in Table 1, Nα13 and 32, which are active ingredients of the present invention, were synthesized by a phase phase method and purified.

First, Arg, which is an amino acid at the C-terminus, was coupled to a cross-linked polystyrene resin, and then amino acids whose amino groups were protected with t-butoxycarbonyl groups were coupled head-to-head one by one toward the N-terminus. . After all amino acids were coupled to the resin, the protected peptide was detached from the resin and the protecting groups were removed.

The thus obtained unpurified compound was purified by liquid chromatography to obtain the active ingredients of the present invention (Table 1, compounds m of Nα13 and 32).

Example 2 Compounds of the present invention obtained in Example 1 (Table 1, No. 1
Using the compounds of the present invention (compounds No. 3 and 32) and the compounds of the present invention synthesized in the same manner as in Example 1 (Table 1, compounds No. 16 and 35), their MSH inhibitory effects were investigated in the following manner.

That is, a 7.5-day old yellow mouse (C57BL/6
J-A') skin explants (approximately 1 mm x 1 mm) containing hair root melanocytes were grown in Ham's F-12 medium at 37°C.
, and cultured for 2 days. At this time, (i) a system in which α-MSH is added to the medium, and (ii) a system containing α-MSH and the compound of the present invention, that is, a system in which α-MSH and the compound of the present invention are mixed in advance in the medium and α-MSH is added to the medium. -A system in which skin pieces were added after contacting MSH with the compound of the present invention, and (iii) a system in which these were not added (control) were set up. After the culture was completed, it was fixed with 10% formalin solution and a specimen was prepared according to a conventional method. The state of eumelanin (black melanin) production in the hair bulb was then observed under a microscope. The results are shown in Table-2.

From the results in Table 2, in the case of the control (no additives), the production of eumelanin was weak, which is a condition peculiar to yellow mice, whereas in the system to which α-MSH was added, strong eumelanin production was observed. Ta. However, α-MSH
In the system to which the compound of the present invention was added, the production of eumelanin was weak, as in the control case.

The above results indicate that the compound of the present invention acts as an α-MSH inhibitor and strongly inhibits the promotion of melanin production by α-MSH.

Example 3 After separately mixing and dissolving components 1 to 7 and 8 to 12 shown in Table 3, the solution of components 1 to 7 was added thereto while stirring the solution of components 8 to 12, and emulsified. Thereafter, component 13 was added during cooling, and the mixture was cooled to room temperature to prepare the cream shown in Table 3.

In addition, in the table, the compounding amount is shown in weight%. (same as below).

Table 4 The cream thus prepared was applied to the pigmented areas of eight men and women with pigmented spots (spots and freckles) twice a day in the morning and evening for two months. As a result, the areas to which the cream containing the active ingredient Nα13 or 32 of the present invention was applied showed a noticeable improvement in pigment spots compared to the areas to which the comparative example was applied. In addition, no abnormality was observed in the condition of the skin during and after using the above cream for two months.

Example 4 Components 1 to 7 shown in Table 4 were heated and dissolved at 70°C. On the other hand, components 8 to 13 were heated and dissolved at 70°C, the above-mentioned oil and fat solution (components 1 to 7) was added, and after emulsification, component 14 was added midway through cooling, and the mixture was cooled to room temperature. The emulsion shown in -4 was prepared.

When the emulsion with the composition shown in Table 4 (product of the present invention) and the emulsion containing no compound No. 16 (comparative example) were evaluated in the same manner as in Example 3, a clear improvement effect on pigment spots was observed. It was done.

Example 5 After separately dissolving components 1 to 4 and components 5 to 9 shown in Table-5,
A beauty serum was prepared by mixing.

Table 5 Example 6 A milky lotion was prepared by replacing the compound of Table 1, Nα16 blended in the milky lotion of Example 4 with the compound of Table INα14 or 15.

Example 7 A serum was prepared by replacing the compound No. 35 in Table 1 blended in the serum of Example 5 with the compound No. 33 or 34 in Table INα.

Example 8 The compounds of Table-1, No. 13 or 32 blended in the cream of Example 3 were added to Table-1, Nα2.7.20.24.
Creams were prepared by substituting compounds 28.37 or 39.

Claims (1)

[Scope of Claims] (1) A melanocyte-stimulating hormone inhibitor comprising a peptide having an amino acid sequence represented by the following formula [I] in its molecule. -Leu-Ala-Cys-Ala-Arg-... [
I](2) The melanocyte-stimulating hormone inhibitor according to claim (1), wherein the peptide has a molecular weight of 532 to 10,000. (3) A skin external preparation containing the melanocyte-stimulating hormone inhibitor according to claim (1) or (2).