KR20040052389A - Novel peptide compound and uses thereof - Google Patents

Abstract

PURPOSE: Provided is a peptide compound and use thereof, which peptide compound acts an antagonist of alpha-MSH(alpha-melanocyte stimulating hormone), so that melanin formation within cells can be inhibited. CONSTITUTION: The peptide compound, 5-phenylvaleric acid-(D)His-Arg-Trp-(Lys)n, represented by formula (1) is provided, wherein n is an integer of 0 to 21; and R is OH or NH2. A composition for prevention and treatment of disease associated with alpha-MSH(alpha-melanocyte stimulating hormone) comprises the peptide compound of formula (1) as an efficient component. A composition for whitening the skin comprises 0.00001 to 10 wt.% of the peptide compound of formula (1) as an efficient component.

Description

Novel Peptide Compounds and Their Uses {NOVEL PEPTIDE COMPOUND AND USES THEREOF}

The present invention relates to novel peptide compounds and their use.

It is everyone's constant desire to have white, fair skin. Human skin color is determined by the concentration and distribution of melanin in the skin. In addition to genetic factors, it is also influenced by environmental or physiological conditions such as ultraviolet rays, fatigue and stress. Melanin is a type of amino acid tyrosine (tyrosine) enzyme called tyrosinase (tyrosinase) acts to convert to dopa (DOPA), dopaquinone (dopaquinone) is produced through a non-enzymatic oxidation reaction.

When such synthesis of melanin occurs excessively in the skin, it may darken the skin tone, and may cause spots and freckles. Therefore, by inhibiting the synthesis of melanin pigment in the skin, not only can brighten the skin tone to realize skin whitening, but also improve skin hyperpigmentation such as spots, freckles, etc. caused by ultraviolet rays, hormones and genetic causes. have.

Therefore, conventionally, a substance having an inhibitory activity against tyrosinase such as hydroquinone, ascorbic acid, kojic acid, glutathione, and the like is blended into a cosmetic such as skin ointment or essence. Thus, skin whitening was realized or skin hyperpigmentation such as blemishes and freckles was improved. However, these materials are limited in use due to problems such as severe skin irritation or poor product stability.

Therefore, the technical problem to be achieved by the present invention is to provide a novel peptide compound, and uses such as skin whitening of the compound.

Hereinafter, the peptide compound and its use according to the present invention will be described in detail.

Melanin is produced in specific cells called melanocytes (Melanocyte) in the basal layer of the skin, and the activity of these cells determines the amount of melanin produced. The most important factors affecting the activity of melanocytes are known to be alpha melanocyte stimulating hormone (α-MSH). α-MSH is a hormone that directly affects the growth of melanocytes and the biosynthetic activity of melanin, as indicated by its name, and has been known to affect the skin through blood vessels produced by the pituitary gland. It is also produced in (melanosite, keratinocytes) and has been shown to play an important role in skin blackening. In addition, studies have shown that skin color became dark when a large amount of α-MSH was administered to humans, and that melanin production increased with cell proliferation when α-MSH was administered to melanocytes cultured and isolated from humans of various races. It became.

As such, α-MSH plays a very important role in the pigmentation process by external stimulation such as ultraviolet rays as well as human skin color. The action of α-MSH on melanocytes is via the melanocortin-1 receptor (MC1R), known as the α-MSH receptor present on the melanocytes' cell membrane. When α-MSH binds to MC1R, an enzyme called adenylate cyclase linked to MC1R is activated to increase the concentration of cyclic adenosine monophosphate (cAMP) in the cell and increase the cAMP. It is known that a series of processes such as the proliferation of melanocytes, the synthesis of melanin, and the transfer of melanin to keratinocytes act as signaling agents.

Therefore, the present inventors synthesized a new material capable of antagonizing the action of α-MSH by focusing on the action of α-MSH, which plays a very important role in the process of pigmentation of the skin. 5-phenyl valerian acid- (D) histidyl alginyl tryptophanyl (lysine) _n , a peptide derivative represented by, has an excellent inhibitory effect on the proliferation and melanogenesis of melanocytes triggered by α-MSH. Revealed.

<Formula 1>

In Formula 1, n is an integer selected from 0 to 21, R is OH or NH ₂ .

Since the peptide compound of the present invention antagonizes the action of α-MSLAN (α-melanocyte stimulating hormone), for example, by selectively adding a pharmaceutically acceptable carrier, excipient, diluent and treating the disease related to α-MSH and Not only can be usefully used in prophylactic pharmaceutical compositions, but can also be added to skin whitening compositions such as skins, lotions, creams, foundations, essences, gels, packs, foam cleansing, cosmetics such as soaps, and pharmaceuticals such as external skin ointments. It can show strong skin whitening effect without any side effects. It is apparent that the peptide compound of the present invention can be easily synthesized with a conventional peptide synthesizer using 5-phenyl valeric acid and raw material amino acids of (D) histidine, arginine, tryptophan and lysine.

In the preparation of the composition for skin whitening according to the present invention, the content of the peptide compound is preferably 0.00001 to 10% by weight based on the total weight of the composition, more preferably 0.001 to 1% by weight, based on the skin whitening effect and economic efficiency. to be.

Hereinafter, the present invention will be described in detail with reference to Examples. However, embodiments according to the present invention can be modified in many different forms, the scope of the present invention should not be construed as limited to the embodiments described below. Embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art.

Examples 1-12

The peptide compounds of Examples 1 to 12 were synthesized using a peptide synthesizer (PEPTIDE SYNTHESIZER, ABI433A from APPLIED BIOSYSTEMS). Where (D) H is D-Histidine, R is Arginine, W is Tryptophan, (K) _n is Lysine and its number, NH ₂ is peptide C-end Indicates short-term amidation.

Example 1 5-phenylvaleric acid- (D) HR-WNH ₂

Example 2 5-phenylvaleric acid- (D) HRW- (K) ₃ NH ₂

Example 3: 5-phenylvaleric acid- (D) HRW- (K) ₆ NH ₂

Example 4: 5-phenylvaleric acid- (D) HRW- (K) ₉ NH ₂

Example 5: 5-phenylvaleric acid- (D) HRW- (K) ₁₂ NH ₂

Example 6: 5-phenylvaleric acid- (D) HRW- (K) ₁₅ NH ₂

Example 7: 5-phenylvaleric acid- (D) H-R-W

Example 8 5-phenylvaleric acid- (D) HRW- (K) ₃

Example 9: 5-phenylvaleric acid- (D) HRW- (K) ₆

Example 10 5-phenylvaleric acid- (D) HRW- (K) ₉

Example 11 5-phenylvaleric acid- (D) HRW- (K) ₁₂

Example 12 5-phenylvaleric acid- (D) HRW- (K) ₁₅

Experimental Example 1

5-phenyl valerian acid- (D) histidyl alginyl tryptophanyl (lysine) _n of Examples 1 to 12 were isolated from human normal melanocytes and added to the cultured medium, followed by α-MSH. The inhibitory effect on the promotion of melanocyte cell division was examined. Human normal melanocytes were inoculated in a 24 well culture vessel (2 × 10 ⁴ cells / well), and then cultured for 3 days, and then exchanged with a medium without α-MSH for 2 days. Subsequently, the 5-phenyl valeric acid- (D) heat of Examples 1 to 12 was added to a negative control group without α-MSH, a positive control group with 1 nM α-MSH and a medium with 1 nM α-MSH. Tidyl alginyl tryptophanyl (lysine) _n was added to the final concentration of 10μM (test group) were each divided for 2 more days incubation. At this time, radioisotope ³ H-thymidine was added to each well 1 μCi to measure the proliferation of the cells. After 2 days, the medium was removed, washed with PBS, and the amount of isotope (CPM) inserted into the cells was measured by using a radiometer (β-counter), and the inhibition rate was calculated according to the following equation. Indicated.

% Inhibition = (amount of isotopes in positive control group-isotope amount in test group) / (amount of isotopes in positive control group-amount of isotopes in negative control group) X 100

MC1R 10μM CPM ± Std ¹ % Inhibition α-MSH free negative control group 6262 ± 854 100 1 nM α-MSH positive control group 11146 ± 364 - Example 1 5-phenylvaleric acid- (D) HR-WNH ₂ 8558 ± 153 53 Example 2 5-phenylvaleric acid- (D) HRW- (K) ₃ NH ₂ 8063 ± 321 63 Example 3 5-phenylvaleric acid- (D) HRW- (K) ₆ NH ₂ 6512 ± 183 95 Example 4 5-phenylvaleric acid- (D) HRW- (K) ₉ NH ₂ 6015 ± 231 105 Example 5 5-phenylvaleric acid- (D) HRW- (K) ₁₂ NH ₂ 6123 ± 260 103 Example 6 5-phenylvaleric acid- (D) HRW- (K) ₁₅ NH ₂ 6090 ± 266 103 Example 7 5-phenylvaleric acid- (D) H-R-W 8589 ± 195 52 Example 8 5-phenylvaleric acid- (D) HRW- (K) ₃ 7965 ± 337 65 Example 9 5-phenylvaleric acid- (D) HRW- (K) ₆ 6620 ± 222 93 Example 10 5-phenylvaleric acid- (D) HRW- (K) ₉ 6099 ± 285 103 Example 11 5-phenylvaleric acid- (D) HRW- (K) ₁₂ 6285 ± 293 100 Example 12 5-phenylvaleric acid- (D) HRW- (K) ₁₅ 6196 ± 331 101

* Repeat count = 3

Referring to Table 1, the 5-phenyl valeric acid- (D) histidyl alginyl tryptophanyl (lysine) _n of Examples 1 to 12 were melanocytes by α-MSH up to 52% to 105% at a concentration of 10 μM. It inhibited the growth of, and showed no toxicity. From these results, it can be seen that 5-phenyl valeric acid- (D) histidyl alginyl tryptophanyl (lysine) _n is a potent antagonist that inhibits the action of α-MSH, and as the number of C-terminal lysine increases It can be seen that the antagonism becomes strong. However, when the number of lysine was greater than 9, there was no increase in specific activity, and the presence or absence of the C-terminal NH ₂ group was not significantly affected.

Experimental Example 2

5-phenyl valeric acid- (D) histidyl alginyl tryptophanyl (lysine) _n of the above Examples 1 to 12 was isolated from human normal melanocytes and added to the culture medium to inhibit melanin synthesis. The effect was tested. Human normal melanocytes were inoculated in a 24well culture vessel (2 × 10 ⁴ cells / well) and then incubated for 3 days, followed by 5-phenyl valeric acid- (D) histidyl alginyl tryptophanyl (Examples 1 to 12). Lysine) _n was added to a final concentration of 1 μM and incubated for 2 more days. At this time, in order to measure the degree of synthesis of melanin, radioactive isotope ¹⁴ C-Tyrosine was added to each well 0.2μCi. After 2 days, the medium was removed, washed with PBS, and the amount of isotope (CPM) inserted into the cells was measured by using a radiometer (β-counter), and the inhibition rate was calculated according to the following equation. Indicated.

% Inhibition = (amount of control isotopes-amount of experimental isotopes) / amount of control isotopes X 100

MC1R 1μM CPM ± Std ¹ % Inhibition Control No addition 66034 ± 1972 - Example 1 5-phenylvaleric acid- (D) HR-WNH ₂ 38960 ± 2355 41 Example 2 5-phenylvaleric acid- (D) HRW- (K) ₃ NH ₂ 36318 ± 3213 45 Example 3 5-phenylvaleric acid- (D) HRW- (K) ₆ NH ₂ 23566 ± 6961 64 Example 4 5-phenylvaleric acid- (D) HRW- (K) ₉ NH ₂ 19188 ± 5114 71 Example 5 5-phenylvaleric acid- (D) HRW- (K) ₁₂ NH ₂ 18952 ± 2608 71.3 Example 6 5-phenylvaleric acid- (D) HRW- (K) ₁₅ NH ₂ 19003 ± 2966 71.2 Example 7 5-phenylvaleric acid- (D) H-R-W 40062 ± 2683 39.4 Example 8 5-phenylvaleric acid- (D) HRW- (K) ₃ 37562 ± 4337 43 Example 9 5-phenylvaleric acid- (D) HRW- (K) ₆ 24689 ± 2222 63 Example 10 5-phenylvaleric acid- (D) HRW- (K) ₉ 20325 ± 1285 70 Example 11 5-phenylvaleric acid- (D) HRW- (K) ₁₂ 19026 ± 6293 71.1 Example 12 5-phenylvaleric acid- (D) HRW- (K) ₁₅ 21008 ± 2331 68.2

* Repeat count = 3

Referring to Table 2, the 5-phenyl valerian acid- (D) histidyl alginyl tryptophanyl (lysine) _n of Examples 1 to 12 was characterized by melanin synthesis of melanocytes from about 40% to 70% at 1 μM concentration. Inhibited. From these results, 5-phenyl valeric acid- (D) histidyl alginyl tryptophanyl (lysine) _n is not only a potent antagonist that inhibits the action of α-MSH, but also has a strong melanin inhibitory effect. It can be seen that as the number of lysine C-terminal increases the antagonism becomes stronger. However, when the number of lysine was greater than 9, there was no increase in specific activity, and the presence or absence of the C-terminal NH ₂ group was not significantly affected.

In the following, 5-phenyl valeric acid- (D) histidyl alginyl tryptophanyl (lysine) _n is added to the skin ointment, cream, softening lotion, essence, pack and nutrient cosmetics to prepare a composition for skin whitening In addition, this study examines the effects of pigmentation inhibition by prescribing them in subjects.

Preparation Example 1

5-phenyl valeric acid- (D) histidyl alginyl tryptophanyl (lysine) _n was added as an active ingredient, and a skin external ointment was prepared using the ingredients and contents shown in Table 3 below.

Comparative Example 1

A skin external ointment was prepared using the ingredients and contents shown in Table 3 below without adding 5-phenyl valeric acid- (D) histidyl alginyl tryptophanyl (lysine) _n .

Composition Preparation Example 1 (% by weight) Comparative Example 1 (% by weight) Peptide Compounds of Example 2 0.02 - Diethyl sebacate 8 8 Prepayment 5 5 Polyoxyethylene oleyl ether phosphate 6 6 Sodium benzoate Quantity Quantity vaseline to 100 to 100

Preparation Example 2

5-phenyl valeric acid- (D) histidyl alginyl tryptophanyl (lysine) _n was added as an active ingredient, and a cream was prepared with the ingredients and contents shown in Table 4 below.

Comparative Example 2

A cream was prepared with the ingredients and contents shown in Table 4 below without the addition of 5-phenyl valeric acid- (D) histidyl alginyl tryptophanyl (lysine) _n .

Composition Preparation Example 2 (% by weight) Comparative Example 2 (wt%) Peptide Compounds of Example 3 0.01 - Stearic acid 1.0 1.0 Cetanol 2.0 2.0 Fiji-20 sorbitan monostearate 1.0 1.0 Sorbitan monostearate 1.0 1.0 Mineral oil 10.0 10.0 Trioctanoate 5.0 5.0 Triethanolamine 0.5 0.5 Carbomer 0.2 0.2 glycerin 5.0 5.0 Propylene glycol 3.0 3.0 antiseptic Quantity Quantity incense Quantity Quantity Purified water to 100 to 100

Preparation Example 3

5-phenyl valeric acid- (D) histidyl alginyl tryptophanyl (lysine) _n was added as an active ingredient, and a softening softener was prepared using the ingredients and contents shown in Table 5 below.

Comparative Example 3

Softening water was prepared with the ingredients and contents shown in Table 5 below without adding 5-phenyl valeric acid- (D) histidyl alginyl tryptophanyl (lysine) _n .

Composition Preparation Example 3 (wt%) Comparative Example 3 (wt%) Peptide Compounds of Example 4 0.02 - ethanol 10.0 10.0 Polyoxyethylene Castor Oil 1.0 1.0 Methyl paraoxybenzoate 0.2 0.2 glycerin 5.0 5.0 1,3-butylene glycol 6.0 6.0 incense Quantity Quantity Pigment Quantity Quantity Purified water to 100 to 100

Preparation Example 4

5-phenyl valeric acid- (D) histidyl alginyl tryptophanyl (lysine) _n was added as an active ingredient, and an essence was prepared with the ingredients and contents shown in Table 6 below.

Comparative Example 4

Essence was prepared with the ingredients and contents shown in Table 6 below without the addition of 5-phenyl valeric acid- (D) histidyl alginyl tryptophanyl (lysine) _n .

Composition Preparation Example 4 (wt%) Comparative Example 4 (wt%) Peptide Compounds of Example 5 0.05 - Propylene glycol 10.0 10.0 glycerin 10.0 10.0 Sodium hyaluronate aqueous solution (1%) 5.0 5.0 ethanol 5.0 5.0 Polyoxyethylene Cured Castor Oil 1.0 1.0 Methyl paraoxybenzoate 0.1 0.1 Carbomer 0.3 0.3 Triethanolamine 0.4 0.4 incense Quantity Quantity Purified water to 100 to 100

Preparation Example 5

5-phenyl valeric acid- (D) histidyl alginyl tryptophanyl (lysine) _n was added as an active ingredient, and a pack was prepared with the ingredients and contents shown in Table 7 below.

Comparative Example 5

A pack was prepared with the ingredients and contents shown in Table 7 below without the addition of 5-phenyl valeric acid- (D) histidyl alginyl tryptophanyl (lysine) _n .

Composition Preparation Example 5 (% by weight) Comparative Example 5 (wt%) Peptide Compounds of Example 9 0.01 - glycerin 5.0 5.0 Propylene glycol 4.0 4.0 Polyvinyl alcohol 15.0 15.0 ethanol 8.0 8.0 Polyoxyethylene Cured Castor Oil 1.0 1.0 Polyoxyethylene oleylethyl 1.0 1.0 Methyl paraoxybenzoate 0.2 0.2 incense Quantity Quantity Pigment Quantity Quantity Purified water to 100 to 100

Preparation Example 6

5-phenyl valeric acid- (D) histidyl alginyl tryptophanyl (lysine) _n was added as an active ingredient, and nutritional longevity was prepared with the ingredients and contents shown in Table 8 below.

Comparative Example 6

No nutrients were prepared with the ingredients and contents shown in Table 8 below without the addition of 5-phenyl valeric acid- (D) histidyl alginyl tryptophanyl (lysine) _n .

Composition Preparation Example 6 (wt%) Comparative Example 6 (wt%) Peptide Compounds of Example 10 0.01 - Polyoxyethylene Cured Castor Oil 1.0 1.0 Methyl paraoxybenzoate Quantity Quantity glycerin 6.0 6.0 1,3-butylene glycol 5.0 5.0 Carbomer 0.2 0.2 Triethanolamine 0.3 0.3 Propylene glycol 5.0 5.0 ethanol 3.2 3.2 Carboxy Vinyl Polymer 18.0 18.0 Pigment Quantity Quantity incense Quantity Quantity Purified water to 100 to 100

Experimental Example 3

The method used to verify the pigmentation inhibitory effect of the skin external ointment, cream, supple cosmetics, nutrient cosmetics, pack, essence prepared as described above is as follows.

First, 40 healthy men and women were selected and divided into two groups. Then, the upper arm of one arm was covered with three pieces of aluminum foil having two rows of 1.5 cm × 1.5 cm in diameter, and the positions of the holes were marked. Samples of Preparation Examples 1 to 3 were applied to the first group of test subjects in three rows of one row of foils, and samples of Comparative Examples 1 to 3 were applied to the other three rows of holes. In addition, to the second group test subjects, the samples of Preparation Examples 4 to 6 were applied to three rows of one row of foils, and the samples of Comparative Examples 4 to 6 were applied to three other rows of holes.

After two weeks, the light quantity of 120mJ / cm <2> was irradiated using the ORIEL solar simulaltor 1000W from the arm 50cm away. The irradiation site was washed well with 70% ethanol aqueous solution before irradiation. The same sample was applied twice a day for 4 weeks at the same location after UV irradiation. After 4 weeks, the pigmentation inhibition effect of the sample according to the Example for the control group (Comparative Example) was visually evaluated in three stages of distinct inhibitory effect, inhibitory effect, and no inhibitory effect, and also investigated the occurrence of skin side effects. The results are shown in Table 9 below.

Experimental substance Clear inhibitory effects (persons) Inhibitory effect (persons) No inhibitory effect (persons) Side effects (persons) Preparation Example 1 7 10 3 0 Preparation Example 2 8 10 2 0 Preparation Example 3 9 9 2 0 Preparation Example 4 11 6 3 0 Preparation Example 5 9 7 4 0 Preparation Example 6 7 9 4 0

As shown in Table 9, the composition for skin whitening of Preparation Examples 1 to 6 containing 5-phenyl valeric acid- (D) histidyl alginyl tryptophanyl (lysine) _n was at least 16 in 20 subjects. It showed a significant skin whitening effect on the above, and did not show any side effects in the skin.

As such, 5-phenyl valeric acid- (D) histidyl alginyl tryptophanyl (lysine) _n is a novel peptide compound that exhibits potent antagonism against α-MSH, and thus is a therapeutic agent for diseases related to α-MSH. In addition to being useful, skin, lotions, creams, foundations, essences, gels, packs, foam cleansing, cosmetics such as soaps, and cosmetics such as skin ointments can be added to the skin whitening composition without any adverse effects It can have a whitening effect.

Claims (5)

5-phenyl valeric acid- (D) histidyl alginyl tryptophanyl (lysine) _n {5-Phenylvaleric acid- (D) His-Arg-Trp- (Lys) _n } peptide compound represented by the following general formula (1) . <Formula 1> In Formula 1, n is an integer selected from 0 to 21, R is OH or NH ₂ . A pharmaceutical composition for preventing and treating diseases related to α-MSLAN (α-melanocyte stimulating hormone), comprising the peptide compound of claim 1 as an active ingredient. A skin whitening composition comprising the peptide compound of claim 1 as an active ingredient. According to claim 3, wherein the content of the peptide compound is a skin whitening composition, characterized in that 0.00001 to 10% by weight based on the total weight of the composition. The skin whitening composition of claim 3, wherein the skin whitening composition comprises any one of a formulation selected from the group consisting of skin, lotion, cream, foundation, essence, gel, pack, foam cleansing, soap, and external skin ointment. Composition.