JPH03123716A - Melanocyte-stimulating hormone antagonist and skin external preparation containing the same antagonist - 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 tetrapeptide amino acid sequence in the molecule. CONSTITUTION:A skin external preparation containing a peptide having an amino acid sequence shown by formula I or formula II in the molecule, preferably a peptide shown by formula III or formula IV (X and P are H, acyl, amino acid residue, acylated material thereof, peptide residue having 2-40 amino acid residues or acylated material thereof; Y and Q are OH, NH2, amino acid residue, amidated material thereof, peptide residue having 2-40 amino acid residues or amidated material thereof). The peptide preferably has 584-10,000, especially 600-3,000 molecular weight and 4-84, especially 4-24 amino acid residues. When the compounds shown by formula III and formula IV have D-form or nonnatural type amino acid residue, the compounds have improved stability to peptidase.

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 formulations such as external preparations for 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 peptide hormone that is present in many vertebrates, and types such as α-1β-1T- 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.

Conventionally, various 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 in melanocytes, and Japanese Patent Application Laid-Open Nos. 53-3538, 53-6432 and 53-18. Some examples have been put into practical use, such as kojic acid disclosed in '739. 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 antagonist that is highly effective in suppressing melanocyte functions such as melanin production and is highly safe, and formulations such as external skin preparations containing the MSH antagonist.

[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 affinity for melanocyte receptors, and that MSH
New knowledge that the peptide specifically suppresses melanin production in melanocytes by competing with the peptide, and a new finding that the above problems can be effectively solved by incorporating the peptide into ordinary cosmetics and pharmaceutical base materials. This was done based on knowledge.

That is, the present invention provides a melanocyte-stimulating hormone antagonist comprising a peptide having an amino acid sequence represented by the following formula 1''I] or [II] in its molecule.

-Hrs-Ser-Arg-Trp-...CI)-
Trp-Arg-Ser-His - (II) (
[I] or in formula C11, His, Ser.

Argz Trp represents a histidine, serine, arginine, or tryptophan residue, respectively. ) In the present invention, any compound can be used as long as it has an amino acid sequence represented by the formula [I] or [■] in its molecule, but compounds with the following general formula CI [I
) or [■] is preferably used.

X-His-3X-Arg-Ser-Ar...(
III) P-Trp-Arg-Ser-His-Q
...(IV) ([■], in the CIVE formula, His
XSerSArg.

Trp is [”I), same as CIII formula. HisSArg
.

Trp is preferably an L-integral residue. X1P is hydrogen, an acyl group, an amino acid residue and its acylated product, or 2 to 40 amino acid residues. Preferably 2 to 20 peptide residues and their acylated products are shown. XSP is preferably an acyl group, an acylated amino acid residue, or an acylated peptide residue. The above-mentioned acyl group and acylated product include those having 1 to 12 carbon atoms, preferably 1 to 6 carbon atoms. Y and Q are hydroxyl groups, amine groups, amino acid residues and amidated products thereof, or 2 to 40 amino acid residues,
Preferably 2 to 20 peptide residues and amidated products thereof are shown.

YSQ is preferably an amino group, an amidated product of an amino acid residue, or an amidated product of a peptide residue. ) As the amino acid residues and peptide residues of X, YSP, and Q, various combinations can be used as long as they do not inhibit the MSH antagonistic function, and the peptide residues are preferably physiologically inactive groups. In addition, individual amino acid residues can be used either as L- or D-units in steric structure, and may be derived from unnatural amino acids such as norleucine and norvaline.

In addition, a sugar chain may be attached to X, Y, and PSQ.

By containing a D-unit amino acid residue or a non-natural amino acid residue as XSY or PSQ, the stability of the compound of the present invention against various peptide degrading enzymes is improved, and the compound exhibits a sustained MSH antagonistic effect. can be done.

Moreover, X, Y, and PSQ may further contain various substituents, such as hydroxyl group, halogen, and the like.

In addition, as X, Y, P, Q, -Ser-Tyr -5
It is more preferable to include an amino acid sequence consisting of er- because it has a high affinity with the ligand. It is also possible to improve the stability and physiological activity by binding biotin or the like to the site consisting of the above three amino acid residues.

Since the molecular weight of the compound of the formula 1"III], [■] is composed of at least the above-mentioned four amino acid residues, the lower limit is 584 and the upper limit is preferably up to toooo, but the molecular weight range is preferably 600 to 6000, particularly preferably 600 to 3000. The number of amino acid residues is preferably 4 to 84, preferably 4 to 44, particularly preferably 4 to 24.

Specific examples of the compounds represented by the general formulas [■] and [■] used in the present invention include the compounds shown in Table 1 below.

In Table 1, the amino acid residues constituting the peptide are expressed using IUPAC abbreviations; L- is omitted in the case of L-units, and D-3et is written only in the case of D-units. The residues of norleucine and norvaline, which are non-natural amino acids, are each Nle.

It was expressed as NVa. Here, according to the usual notation, the amine terminus (N-terminus) of the peptide is shown on the left, and the carboxyl terminus (C-terminus) is shown on the right.

(All are the same in this specification.) Also, Ac represents an acetyl group, and Bu represents a butyryl group. (The same applies hereinafter.) The MSH antagonist according to the present invention can be obtained using a liquid phase method or a surface phase method, which are common peptide synthesis methods.

In the case of the liquid phase method, the amino 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 phase 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.

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

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

The MSH antagonist 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 antagonist 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 antagonist 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, o, ooo may be present in various compositions.
It is advisable to mix it in an amount of about oooooooi to 1% by weight (hereinafter abbreviated as %), preferably 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 antagonist of the present invention, the skin external preparation contains, in addition to the above-mentioned essential ingredients, raw materials normally used in 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 cetyl ether, polyoxyethylene sterol, polyoxyethylene lanolin, polyoxyethylene beeswax, polyoxyethylene hydrogenated castor oil, sodium stearate, valmitin potassium acid, sodium cetyl sulfate, sodium lauryl phosphate, triethanolamine palmitate,
Anionic surfactants such as polyoxyethylene sodium lauryl phosphate, sodium N-acylglutamate,
Examples include cationic surfactant adjuvants such as stearyldimethylbenzylammonium chloride and stearyltrimethylammonium chloride, amphoteric surfactants such as alkylaminoethylglycine hydrochloride solution, 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, and other waxes; liquid paraffin, squalane, microcrystalline wax, ceresin wax, paraffin wax, petrolatum, and other hydrocarbons; 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 cetanol, stearyl alcohol, hexyldecanonopeoctyldodecanonoperauryl alcohol; isopropyl myristate, imbropinope valmitate, octyldodecinoveolein myristate; Examples include esters such as octyldodecyl acid and cholesterol oleate.

Moisturizers include polyhydric alcohols such as glycerin, propylene glycol 1.3 butylene glycol, sorbitol, polyglycerin, and polyethylene glyconopedipropylene 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; and 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, hinokithionoperesorcin, ethanol, and the like.

Antioxidants include dibutylhydroxytoluene, butylhydroxyanisole, gallic acid provinopea corbic acid, etc., and chelating agents include edetate disodium, ethylenediaminetetraacetate, pyrophosphate, 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 F, vitamin P1 vitamin U1 carnitine, ferulic acid, γ-oryzanone α-lipoic acid, orotic acid and its derivatives. .

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 derivatives 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 serums, packs, under makeup, foundations, jelly preparations, and ointments.

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.
Composition containing 70% surfactant, 2-7% surfactant, 1-10% humectant, purified water balance, trace amount of preservative, trace amount of fragrance, as emulsion: Essential ingredients 0.00000001-0.1%, oil content 10~
40%, alcohol 0-15%, surfactant 1-5%
, moisturizer 1-10%, thickener 0-2%, purified water balance, trace amount of preservative, composition containing trace amount of fragrance, lotion, beauty serum: Essential ingredients 0.00000001-0.1% , 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, Composition containing a trace amount of fragrance, as a pack agent: Essential ingredients: 0. OOO00001-0.1%, alcohol 2-10%, humectant 2-10%, inorganic powder 0-20
%, a film-forming agent of 10 to 20%, a purified water balance, a trace amount of a preservative, and a composition containing a trace amount of a fragrance.

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

Oral preparations containing the MSH antagonist 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 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. o o oμg/kg.

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

That is, it has been revealed by M.E. Hadley et al. that the minimum structure of MSH required to activate melanocytes is Ac-His-Phe-Arg-Trp-NH2 (Pigment Cell Research).
h Supplement l:69-78 (19
88)).

On the other hand, the MSH antagonist of the present invention has a structure similar to the amino acid sequence of the above-mentioned minimum structure in its molecule, and therefore shows strong affinity with the MSH receptor, and as a result, the binding between MSH and the receptor is inhibited. It is presumed that it exhibits an excellent melanin production inhibiting effect.

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.

[Effects of the Invention] According to the present invention, it is possible to suppress the function of melanocytes by MSH antagonism, and essentially solve symptoms associated with advanced melanocyte function such as the appearance of age spots and freckles. Provided are external preparations for skin use, oral preparations, injection preparations, etc., which have excellent effects as pharmaceuticals and are stable in terms of formulation.

Next, the present invention will be explained by examples.

Example 1 Compounds of Nα40 and 85 in Table 1, which are active ingredients of the present invention, were synthesized by a phase phase method and purified.

First, the C-terminal amino acid Val or Pr.

was coupled to a crosslinked polystyrene resin, and then amino acids each having an amine group protected with a t-butoxycarbonyl group were sequentially coupled one by one toward the N-terminus. After all the amino acids were coupled to the resin, the amine terminals were acetylated, and the protected peptide was detached from the resin to remove the protecting groups.

The thus obtained unpurified compound was purified by liquid chromatography to obtain the active ingredients of the present invention (compounds of Nα40 and 85 in Table 1).

Example 2 Compound of the present invention obtained in Example 1 (Table 1, Xα40
and compound No. 85) and the compound of the present invention synthesized in the same manner as in Example 1! (Table - Compounds of LNα14 and 74) were used to investigate their MSH antagonism in the following manner.

That is, a 7.5-day old yellow mouse (C57BL/6
Skin explant containing hair root melanocytes (J-Ay)
Approximately 1 mm x 1 mm) in Ham's F-12 medium.
The cells were cultured at ℃ for 2 days. At this time, (i) α-MSH
(11) A system containing α-M S H and the compound of the present invention, i.e., the compound of the present invention and a skin piece are pre-incubated in a medium, and then the skin piece is incubated in a new medium. and (iii) a system in which α-MSH is brought into contact with
A system without these additives (control) was 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 antagonist of α-MSH 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 11 shown in Table 3, the solution of components 1 to 7 was added thereto while stirring the solution of components 8 to 11, and emulsified. Thereafter, while cooling, component 12 was added 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).

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, pigment spots were clearly improved in the areas to which the cream containing the active ingredient of the present invention was applied, 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.

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-INα37.38.39.41,46.4 Compounds of Table-1 and Nα40 blended into the cream of Example 3
Creams were prepared by substituting compounds 7.82.83.84.85 or 86.

Table 1 above: Cream containing a compound of Nα85 (product of the present invention) and cream not containing a compound of Nα85 (comparative example)
When evaluated in the same manner as in Example 3, a clear improvement effect on pigment spots was observed.

Example 7 A milky lotion was prepared by replacing the compound No. 26 of Table 1 blended in the milky lotion of Example 4 with the compound No. 26 of Table IXα20.23.29.77 or 80.

Example 8 The compound of Table-1, Nα14 blended in the serum of Example 5 was replaced with the compound of Table-1, Nα8.11.15.17.74 or 7.
A beauty serum was prepared in place of the compound in Example 5.

Claims (3)

[Claims] (1) A melanocyte-stimulating hormone antagonist consisting of a peptide having an amino acid sequence represented by the following formula [I] or [II] in its molecule. -His-Ser-Arg-Trp-...[I]-
Trp-Arg-Ser-His-... [II] ([
I], [II], His, Ser, Arg, T
rp represents an L- or D-residue of histidine, serine, arginine, or tryptophan, respectively. ) (2) The melanocyte-stimulating hormone antagonist according to claim (1), wherein the peptide has a molecular weight of 584 to 10,000. (3) A skin external preparation containing the melanocyte-stimulating hormone antagonist according to claim (1) or (2).