KR20170055920A - Multifunctional skin penetration peptide with whitening, skin elasticity and wrinkle improvement ability - Google Patents

Abstract

The present invention relates to a multifunctional peptide with skin whitening, skin elasticity improvement, and wrinkle alleviation effects based on a skin penetration function and to a use thereof, and more specifically, to a method for utilizing a multifunctional peptide, which has a skin penetration function, a melanin generation inhibiting function, a collagen generation promoting function, and a corneum stem cell proliferation function, as a material for cosmetics and medicines. The multifunctional skin penetration peptide according to the present invention has a property of effectively penetrating the skin, an excellent melanin synthesis inhibiting function, and a promoting effect on collagen synthesis and corneum stem cell proliferation, thereby being usefully utilized as cosmetics or medicines for skin whitening, skin elasticity improvement, and wrinkle alleviation even when being used alone.

Description

[0002] Multifunctional skin penetration peptides with whitening, skin elasticity and wrinkle improvement ability,

The present invention relates to a multifunctional peptide having a whitening, elasticity and wrinkle-reducing effect based on a skin permeation function, and a use thereof. More specifically, the present invention relates to a multifunctional peptide having cell and skin permeation function, melanin synthesis inhibition, collagen production promotion, To a method for utilizing the multifunctional peptide as a raw material for functional cosmetics and pharmaceuticals.

Skin is the largest surface organ in the human body and is the pathway that can effectively deliver the drug when appropriate methods are used. However, due to the extrinsic skin permeability of foreign substances due to the stratum corneum composed of dead cells at the outermost part of the skin, it is difficult to transfer a substance having a high molecular weight and a hydrophilic property. In order to overcome the defense mechanism against external substances of skin barrier during drug delivery to such skin, various skin permeation methods have been tried, but it has been attempted to physically transfer the drug through the characteristics of the chemical without damaging or stimulating the skin It is considered to be almost impossible, and development is required continuously due to various advantages expected from the material overcome.

In accordance with this demand, a number of studies have been reported on skin permeation delivery materials such as liposomes or nanoparticles, peptide ligands and the like for effectively delivering drugs with a large molecular weight [Lademann et al., 2007 (Eur J Pharm Biopharm. 2007 May 2006 (J Pharmacol Exp Ther 2006 Nov; 319 (2): 765-75), Chen et al., 2006b (Nat Biotechnol. 2006 Apr ; 24 (4): 455-60.)], The efficiency and stability of the drug to be delivered can not be guaranteed even though the development cost of the practical step is increased, and the material having both the skin permeation function and the body efficacy is developed .

On the other hand, the skin plays an important role in maintaining the body's homeostasis by protecting the body from the external environment. However, unnecessary metabolites, which are caused by various external stimuli and metabolic processes in themselves, reduce skin function and promote aging.

Collagen in skin is a structural protein produced by dermal fibroblasts. It is known that the decrease of production by aging and the change of structure are related to the wrinkling of the skin. The melanin pigment present in the skin protects the skin from ultraviolet rays and the active oxygen in the body, but abnormal melanocyte activation causes hyperpigmentation such as spots, freckles, and black spots and causes cosmetic problems. The skin corneal stem cells present in the basal layer of the skin epidermis are known to play an important role in the regeneration of the skin by regulating the growth and differentiation of keratinocytes as well as to the fibroblasts of the dermal layer. Therefore, the intrinsic homeostasis related to collagen production, melanin pigment inhibition and keratinocyte cell growth is a closely related factor affecting the health and beauty of the skin, and researches for its function and effective material discovery are still actively carried out. However, The efficacy of the external volume application material requires much effort due to the technical difficulties of passing through the epidermis and dermis.

Under these circumstances, the present inventors have made intensive efforts to solve the conventional problems of the peptides which are restricted in their use due to the problems such as reduction in effectiveness and safety due to the modification of the above-mentioned effective substances, or cost increase. As a result, , Which has skin permeation and delivery functions of various active substances, inhibits melanin synthesis, promotes collagen synthesis and proliferation of keratinocyte stem cells, and has completed the present invention.

Accordingly, one object of the present invention is to provide a skin permeable multifunctional peptide or derivative thereof having a skin permeation function, melanin synthesis inhibitory ability, collagen synthesis, and proliferation promoting activity of dermal keratinocyte.

It is still another object of the present invention to provide a skin permeable composite in which the skin permeable multifunctional peptide or a derivative thereof is linked with a physiologically active substance.

Furthermore, another object of the present invention is to provide a cosmetic composition for skin whitening, skin elasticity enhancement or wrinkle improvement comprising the skin permeable multifunctional peptide or derivative thereof.

Another object of the present invention is to provide a pharmaceutical composition for skin whitening, skin elasticity enhancement or wrinkle improvement comprising the skin permeable multifunctional peptide or a derivative thereof.

Still another object of the present invention is to provide a composition for transdermal delivery of the above-mentioned skin permeable multifunctional peptide or derivative thereof, and a physiologically active substance containing the peptide and a physiologically active substance.

In one aspect, the present invention relates to a skin permeable multifunctional peptide having the amino acid sequence of SEQ ID NO: 1, or a derivative thereof, as described below.

[SEQ ID NO: 1] TGSTSAS

Specifically, the derivative means a peptide having a sequence in which at least one amino acid of the amino acid sequence of SEQ ID NO: 1 is substituted with alanine (Ala). Preferably a peptide having an amino acid sequence selected from SEQ ID NOS: 2-7.

The synthesis of the peptides of the present invention can be carried out, for example, using a device or using genetic engineering techniques. When synthesized using a device, the desired peptide is synthesized by the Fmoc solid-phase method in an automatic peptide synthesizer (PeptrEX-R48, Peptron, Daejeon, Korea). Synthetic peptides were separated from the resin and purified by reversed phase HPLC (reverse-phase HPLC, Prominence LC-20AB, Shimadzu, Japan) using a Shiseido capsel pack C18 analytical RP column (Shiseido capcell pak C18 analytical RP column) ≪ / RTI > The final peptide is identified using a mass spectrometer (HP 1100 Series LC / MSD, Hewlett-Packard, Roseville, USA). When a genetic engineering technique is used, the desired nucleotide sequence of the desired peptide is introduced into a protein expression vector, and then a bacterium in which protease deficient such as BL21 (λDE3) and BL21 (λDE3) pLys is deficient, Induced expression using IPTG and pure separation.

In one specific embodiment, the skin permeable multifunctional peptides according to the present invention were identified using phage display techniques and screened by validating the efficacy of the identified peptides. In the step of identifying the peptides permeating the skin, a random phage display library is applied to the pig skin, and the skin epidermis is removed after a certain time, and the phage remaining in the skin except for the epidermis is amplified to obtain information about the peptide sequence and frequency .

Furthermore, in order to verify the efficacy of the identified peptides, it was found that 1) the peptide having the skin permeation function, 2) the melanin synthesis inhibitory effect in mouse melanocytes, 3) the collagen synthesis effect in human fibroblasts, 4) Multifunctional peptides were selected through validation of the proliferation effect.

The peptides according to the present invention have cell and skin permeability and thereby can transport or transmit various active substances such as low molecular substances, and at the same time, they can be used as a multifunctional, specifically excellent skin whitening, skin elasticity enhancement, And is useful for drugs or cosmetics.

Furthermore, when a variety of physiologically active substances are transported or transferred, the physiologically active substance and the peptide according to the present invention may be bound to each other by covalent bonding or the like to be transported or delivered in the complex form of the skin permeable multi-functional peptide or its derivative- And it is also possible to easily transfer the desired active substance by simply mixing the physiologically active substance and the peptide according to the present invention. In particular, when the covalent bond such as a peptide is made, denaturation, Can be effectively avoided. At this time, the method of covalently bonding the active substance to the skin permeable multifunctional peptide according to the present invention can be suitably prepared by using known techniques.

Specifically, the physiologically active substance refers to any substance that promotes or suppresses the function of a living body when living organisms live, including drugs capable of exerting a desired effect in vivo. Examples of the physiologically active substance include, but are not limited to, peptides, polypeptides, polynucleotides, nanoparticles, DNA, RNA, and low molecular compounds. As a specific example, the present inventors confirmed transdermal delivery by the skin permeable multifunctional peptide according to the present invention using exendin-4, 10-mer and 20-mer random peptide and MTX as physiologically active substances. According to one embodiment of the present invention, the skin permeable multifunctional peptide of the present invention not only exhibits excellent skin permeability as compared with the transmittance of TAT protein known as PTD (Protein transduction domain), but also excellently Melanin biosynthesis inhibitory effect, collagen synthesis effect, and keratinocyte cell proliferation effect.

Furthermore, in another aspect, the present invention relates to a cosmetic composition for skin whitening, skin elasticity enhancement or wrinkle improvement comprising the above-mentioned skin permeable multi-functional peptide as an active ingredient.

In the present invention, the term "wrinkle " means a scarred skin caused by a genetic cause, a decrease in collagen and elastin present in the skin dermis, and an external environment. In the present invention, "wrinkle improvement" refers to inhibiting or inhibiting the generation of wrinkles on the skin, or alleviating already formed wrinkles.

The term "skin elasticity" as used herein refers to elastic fibers composed of elastin present in the dermal layer. The term "skin elasticity enhancement" means that elastic fibers composed of elastin and collagen, The skin elasticity is maintained.

The cosmetic composition according to the present invention can be used as a cosmetic composition in the form of a solution, an ointment for external use, a cream, a foam, a nutritional lotion, a softening water, a pack, a soft water, an emulsion, a makeup base, But are not limited to, emulsions, emulsions, pastes, gels, lotions, powders, soaps, surfactant-containing cleansing, oils, powder foundations, emulsion foundations, wax foundations, patches and sprays It is not. In addition, the cosmetic composition of the present invention may further comprise at least one cosmetically acceptable carrier incorporated in a cosmetic composition for general skin, and examples thereof include oil, water, a surfactant, a moisturizer, a lower alcohol, A thickening agent, a chelating agent, a coloring matter, an antiseptic, a perfume, and the like may be appropriately compounded, but the present invention is not limited thereto.

In another aspect, the present invention relates to a pharmaceutical composition for enhancing skin whitening, wrinkle or skin elasticity, which comprises the multifunctional skin permeation peptide. The compositions of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of pharmaceutical compositions. The composition of the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterilized injection solutions according to a conventional method Examples of carriers, excipients and diluents that can be contained in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. In the case of the composition according to the present invention, the multifunctional skin permeation peptide, which is an active ingredient, is preferably in the form of a transdermal preparation. The preferred dosage of the pharmaceutical composition of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the route of administration and the period of time, but can be appropriately selected by those skilled in the art. However, for the desired effect, the pharmaceutical composition of the present invention may be administered once a day or divided into several doses. Accordingly, the dosage is not limited in any way to the scope of the present invention.

The cosmetic or pharmaceutical composition according to the present invention may further comprise at least one substance having a function of skin whitening, skin wrinkle improvement or skin elasticity enhancement.

In another aspect, the present invention provides a skin permeable multifunctional peptide or derivative thereof, and a composition for transdermal delivery of a physiologically active substance containing the peptide and a physiologically active substance. Preferably, the skin permeable multifunctional peptide or derivative thereof and the physiologically active substance in the transdermal delivery composition may be mixed or may be in the form of a complex linked by covalent bonds.

The skin permeable multifunctional peptide or derivative thereof according to the present invention has a property of effectively permeating the skin and can effectively transfer the foreign substance into the skin when the foreign substance is bound, It exhibits excellent melanin synthesis inhibitory ability, collagen synthesis, and proliferation promoting effect of keratinocyte stem cells, and can be effectively used as cosmetics and medicines for skin whitening, skin elasticity enhancement or wrinkle improvement even with peptide alone.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a graph quantitatively showing the skin permeation effect when the exendin-4 complex is mixed with exendin-4 in combination with the skin permeable multifunctional peptide according to the present invention.
FIG. 2 is a graph quantitatively showing skin permeation effects when the skin permeable multifunctional peptide according to the present invention is mixed with 10-mer and 20-mer random peptides and when the random peptides are used alone.
FIGS. 3 to 7 are photographs showing improvement of psoriasis and therapeutic effect when treated with a skin-permeable multifunctional peptide according to the present invention and MTX-conjugated complex administered to psoriasis-induced mouse skin.
FIG. 8 is a graph quantitatively showing the degree of erythema of a complex of a skin permeable multifunctional peptide according to the present invention and MTX linked to psoriasis-induced mouse skin.
FIG. 9 is a graph quantitatively showing the degree of keratinization when treated with a complex of a skin permeable multi-functional peptide and MTX according to the present invention on mouse skin induced by psoriasis.
FIG. 10 is a graph quantitatively showing the thickness of skin when treated with mouse skin induced by psoriasis, in which a skin-permeable multifunctional peptide according to the present invention and MTX-linked conjugate are treated.
FIG. 11 is a photograph showing a cell permeation activity of a skin permeable multi-functional peptide according to the present invention by a confocal microscope.

Hereinafter, the present invention will be described in detail with reference to examples. These examples are for further illustrating the present invention, and the scope of the present invention is not limited by these examples.

< Example  1> Peptides  Synthesis of derivatives

In this example, the synthesis of various peptide derivatives was carried out according to a general peptide solid phase synthesis method (Wang C. Chan, Perter D. White, "Fmoc Solid Phase Peptide Synthesis", Oxford). More specifically, coupling was performed one by one from the 5 C-terminal using an automatic synthesizer (ASP48S, Peptron, Inc.) using Fmoc-SPPS (9-Fluorenyl methyl oxycarbonyl solid phase peptide synthesis) method. All monomeric materials used in the synthesis of peptide derivatives are protected with Fmoc at the N-terminus and amino acids protected with trityl (Trt), t-butyloxycarbonyl (Boc), t-butyl Respectively. As the coupling reagent, HBTU (2- (1H-Benzotirazloe-1-yl) -1,1,3,3-tetramethyluronium hexafluorophophate / HOBt (Hydroxybenzo triazloe) / NMM (N-methylmorpholine) was used.

(1) Protected amino acid (8 eq.) And coupling agent HBTU (8 eq.) / NMM (16 eq.) Were dissolved in DMF (dimethyl formamide) and reacted at room temperature for 2 hours.

(2) To remove Fmoc, 20% (v / v) piperidine / DMF was added and reacted at room temperature twice for 5 minutes.

After repeating the above reactions (1) and (2) to produce a peptide derivative, the fluorescent peptides for confirming the skin permeability include 6-amino hexanoic acid as a linker to the N- ) And fluorescein isothiocyanate (FITC).

Separation of peptides in Resin was performed using Trifluoroacetic acid / EDTA (1,2-ethanedithiol) / Thioanisole / TIS (Triisopropylsilane) / H2O (mixing ratio (weight basis) = 90 / 2.5 / 2.5 / 2.5 / 2.5) Respectively.

The thus obtained mixed solution is subjected to an excess treatment of refrigerated diethyl ether solvent to produce a precipitate. The resulting precipitate was centrifuged to complete precipitation, excess trifluoroacetic acid, thianyzole, and ethanedithiol were firstly removed, and then the same procedure was repeated twice to obtain a solidified precipitate.

The resulting precipitate was dissolved in a water-containing solution containing 0.1% (v / v) Trifluoroacetic acid using a high performance liquid chromatographic apparatus (Shimadzu Prominence HPLC, Japan) using a C18 column (250 mm × 22 mm, 10 μm, Vydac Everest, USA) acetonitrile liner gradient (Acetonitrile concentration: 10 ~ 75% (v / v)). The molecular weight of the purified peptide derivative was confirmed using LC / MS (Agilient HP 1100 series), and the pure purified fractions were lyophilized to obtain the following peptides as TFA (Trifluoroacetic acid) salt in the form of a white powder.

Specifically, the sequences and molecular weights of the synthesized peptide derivatives are shown below.

Heptapeptide 1: TGSTSAS (SEQ ID NO: 1) : Rt = 2.217 min (various concentration gradient from 5% (v / v) to 100% (v / v) acetonitrile / water containing 0.01% (v / v) TFA over 30 minutes); MS (ESI) 609.66 m / e, [M + H] &lt; ⁺ &gt; = 610

Heptapeptide 2: AGSTSAS (SEQ ID NO: 2) : Various concentration gradient from 5% (v / v) to 100% (v / v) acetonitrile / water containing 0.01% (v / v) TFA over 30 minutes) Rt = 3.892 min; MS (ESI) 579.25 m / e, [M + H] &lt; ⁺ &gt; = 580

Heptapeptide 3: TASTSAS (SEQ ID NO: 3): Rt = 4.117 bun (0.01% (v / v) 5% containing TFA (v / v) to 100% (v / v) acetonitrile / different gradient over 30 minutes from water of ); MS (ESI) 623.61 m / e, [M + H] &lt; ⁺ &gt; = 624

Heptapeptide 4: TGATSAS (SEQ ID NO: 4): Rt = 2.153 bun (0.01% (v / v) 5% containing TFA (v / v) to 100% (v / v) acetonitrile / different gradient over 30 minutes from water of ); MS (ESI) 593.58 m / e, [M + H] &lt; ⁺ &gt; = 594

Heptapeptide 5: TGSASAS (SEQ ID NO: 5): Rt = 3.892 bun (0.01% (v / v) 5% containing TFA (v / v) to 100% (v / v) acetonitrile / different gradient over 30 minutes from water of ); MS (ESI) 579.56 m / e, [M + H] &lt; ⁺ &gt; = 580

Heptapeptide 6: TGSTAAS (SEQ ID NO: 6): Rt = 3.125 bun (0.01% (v / v) 5% containing TFA (v / v) to 100% (v / v) acetonitrile / different gradient over 30 minutes from water of ); MS (ESI) 593.59 m / e, [M + H] &lt; ⁺ &gt; = 594

Heptapeptide 7: TGSTSAA (SEQ ID NO: 7): Rt = 3.267 bun (0.01% (v / v) 5% containing TFA (v / v) to 100% (v / v) acetonitrile / different gradient over 30 minutes from water of ); MS (ESI) 593.59 m / e, [M + H] &lt; ⁺ &gt; = 594

Fluorescent heptapeptide 1: FITC-linker-TGSTSAS : Rt = 5.900 min (from v / v) to 100% (v / v) acetonitrile / water containing 0.01% (v / &Lt; / RTI &gt; MS (ESI) 1111.81 m / e, [M + H] &lt; ⁺ &gt; = 1111

Fluorescent heptapeptide 2: FITC-linker-AGSTSAS (SEQ ID NO: 2) : Rt = 7.575 min (various concentration gradient from 5% (v / v) to 100% (v / v) acetonitrile / water containing 0.01% (v / v) TFA over 30 minutes); MS (ESI) 1081.80 m / e, [M + H] &lt; ⁺ &gt; = 1082

Fluorescent heptapeptide 3: FITC-linker- TASTSAS (SEQ ID NO: 3): Rt = 7.600 bun (0.01% (v / v) 5% containing TFA (v / v) to 100% (v / v) acetonitrile / different gradient over 30 minutes from water of ); MS (ESI) 1125.83 m / e, [M + H] &lt; ⁺ &gt; = 1126

Fluorescent heptapeptide 4: FITC-linker- TGATSAS (SEQ ID NO: 4): Rt = 5.792 bun (0.01% (v / v) 5% containing TFA (v / v) to 100% (v / v) acetonitrile / different gradient over 30 minutes from water of ); MS (ESI) 1095.82 m / e, [M + H] &lt; ⁺ &gt; = 1095

Fluorescent heptapeptide 5: FITC-linker- TGSASAS (SEQ ID NO: 5): Rt = 7.500 bun (0.01% (v / v) 5% containing TFA (v / v) to 100% (v / v) acetonitrile / different gradient over 30 minutes from water of ); MS (ESI) 1081.80 m / e, [M + H] &lt; ⁺ &gt; = 1082

Fluorescent heptapeptide 6: FITC-linker- TGSTAAS (SEQ ID NO: 6): Rt = 6.608 bun (0.01% (v / v) 5% containing TFA (v / v) to 100% (v / v) acetonitrile / different gradient over 30 minutes from water of ); MS (ESI) 1095.82 m / e, [M + H] &lt; ⁺ &gt; = 1096

Fluorescent heptapeptide 7: FITC-linker- TGSTSAA (SEQ ID NO: 7): Rt = 6.750 bun (0.01% (v / v) 5% containing TFA (v / v) to 100% (v / v) acetonitrile / different gradient over 30 minutes from water of ); MS (ESI) 1095.82 m / e, [M + H] &lt; ⁺ &gt; = 1096

< Example  2> Skin Permeability  Measure

The extracted pig skin (1.77 cm ² ) was fixed between donor chamber and receiver chamber of a Franz diffusion cell (FCDV-15, Labfine, Korea) and phosphate buffer (pH 7.4) Respectively. The temperature of the receiver chamber was maintained at 37 ± 0.5 ° C, and stirring was performed at a speed of 600 rpm using a magnetic bar. The conditions were maintained until the end of the experiment. Heptapeptide labeled with fluorescence (FITC) and derivatives thereof and TAT labeled with positive control fluorescence are applied to the surface of pig skin contacted with the donor chamber. After 20 hours, peptides and their derivatives migrated into the receiver chamber are measured. Peptide quantification was performed using a spectrophotometer (Nanodrop 2000; Thermoscientific, USA).

Table 1 shows the results of analyzing the permeability of the heptapeptide. Table 2 shows the results of field scanning of the heptapeptide for validation of the heptapeptide.

Group (s) Skin penetration rate
[占 퐂 / cm2 / hr] Negative control group 0.16 ± 0.003 Positive control group 1.89 + 0.095 Fluorescent heptapeptide 1 5.32 + 0.169

Group (s) Skin penetration rate (%)
(vs. FITC-linker- TGSTSAS) Fluorescent heptapeptide 2 0.47 + 0.04 Fluorescent heptapeptide 3 0.79 + 0.03 Fluorescent heptapeptide 4 0.39 0.25 Fluorescent heptapeptide 5 0.84 0.34 Fluorescent heptapeptide 6 0.30 ± 0.00 Fluorescent heptapeptide 7 0.89 ± 0.16

As shown in Table 1, it was confirmed that the fluorescence-labeled heptapeptide 1 (TGSTSAS) exhibited a skin permeability of 5.32 ㎍ / cm ² / hr and an excellent skin permeability of about 2.8 times that of the positive control TAT Respectively. Further, the skin permeability (%) between the derivatives of the heptapeptide 1 (TGSTSAS) in Table 2 was 0.47 times higher than that of the fluorescence heptapeptide 2 and 0.79 times higher than that of the fluorescence heptapeptide 1 (FITC-linker-TGSTSAS) Fold, fluorescence heptapeptide 4, fluorescence heptapeptide 5 and fluorescein heptapeptide 4 were 0.39-fold, 0.84-fold, 0.30-fold and 0.89-fold, respectively.

< Example  3> Measurement of Melanin Biosynthesis Inhibitory Effect

In addition to the anti-tyrosinase inhibiting substance as a whitening cosmetic material, a substance inducing inhibition of melanin synthesis in skin cells has sufficient potential for skin whitening. Therefore, whitening can be expected by the melanin biosynthesis inhibiting effect of melanoma cells.

B16F10 melanoma cells (ATCC) were inoculated into a 6-well plate containing DMEM (Dulbecco's modified eagle medium, Gibco) medium containing 10% FBS (1 × 10 ⁵ cells / well) in the CO ₂ incubator and cultured for 24 hours on 37 ℃. After 24 hours, the medium was removed from each well, and the medium containing arbutin (Arbutin, Sigma Aldrich) as a positive control, the medium containing 20 ppm heptapeptide as a test group, the normal medium as a negative control, and re-cultured for 48 hours . After 48 hours, the cells were washed with phosphate buffered saline (PBS), and the cells were recovered by trypsin-EDTA treatment. The recovered cells were centrifuged at 13,000 rpm for 3 minutes, and the supernatant was removed to obtain a precipitate. The obtained precipitate was washed once with PBS, and the supernatant was removed. Then 200 ul of 1N NaOH containing 10% DMSO was added and the cells were allowed to stand at 60 ° C for 1 hour to dissolve melanin in the cells. The solution was transferred to a microplate and the absorbance at 405 nm was measured to determine the amount of melanin. The experimental results are shown in Table 4.

The results showed that heptapeptide 1 inhibited melanin biosynthesis in melanoma cells as shown in Table 3.

Group (s) Melanin inhibition [%] Negative control group 100 ± 0.6 Positive control group 73.5 ± 2.7 Heptapeptide 1
(TGSTSAS) 71.1 ± 1.6

< Example  4> Measurement of collagen synthesis of human dermal fibroblast

The amount of collagen synthesis in fibroblasts is an indicator of wrinkle formation in the skin. Wrinkles are known to occur as the synthesis of collagen is reduced and collagen is broken down. As a result, promotion of collagen synthesis of fibroblasts can lead to suppression of wrinkle formation and improvement of existing wrinkles.

Fibroblast (human dermal fibroblast, ATCC), the FBM (fibroblast basal medium; PCS- 201-020; ATCC) inoculated in 96-well plates containing a medium and ⁽³ X 10 3 cells / well) in a 5% CO ₂ concentration Lt; RTI ID = 0.0 &gt; 37 C &lt; / RTI &gt; for 24 hours. After 24 hours, the medium was removed from each well, treated with vitamin C containing medium as a positive control, medium containing 20 ppm heptapeptide as an experimental group, serum-free medium as a negative control, and re-cultured for 48 hours. After 48 hours, the cell culture medium was collected to prepare a sample. The amount of collagen in the sample was determined by measuring the amount of procollagen type I C-peptide (PICP) using a collagen measurement kit (Procollagen Type I C-peptide EIA kit; MK101; Takara, Kyoto, Japan) Respectively. Experiments were performed according to the methods described in the manual. The experimental results are shown in Table 4.

As can be seen from the results in Table 4, heptapeptide 1 increased the collagen synthesis of fibroblasts to an extent comparable to that of the positive control group.

Group (s) Collagen synthesis [%] Negative control group 100 ± 6.3 Positive control group 124.1 ± 0.22 Heptapeptide 1
(TGSTSAS) 121.1 ± 3.8

< Example  5> epidermal keratinocytes keratinocyte  progenitor cell) Proliferation promoting activity measurement

The persistent division of keratinocyte stem cells in the epidermal basal layer decreases the proliferative capacity with age, leading to the aging phenomenon such as a decrease in elasticity due to a decline in skin regeneration ability. The skin elasticity can be improved by promoting the growth of keratinocyte cells associated with skin regeneration and elasticity due to aging.

Human dermal keratinocytes were purchased from CellnTec (USA) and cultured according to the Cell &amp; The cell culture medium was prepared by adding cnt-57 to 500 ml of cnt-BM medium according to the method of Cell & Tech. Human dermal keratinocytes were inoculated into 96-well plates (5 x 10 ³ cells / well) and cultured in a 5% CO ₂ incubator at 37 ° C for 24 hours. After 24 hours, the medium was removed from each well and treated with the medium containing rutin (Rutin, Sigma Aldrich) as a positive control, the medium containing 20 ppm heptapeptide as a test group, the normal medium as a negative control, and re-cultured for 72 hours. The degree of cell proliferation was confirmed by CCK-8 kit (CK04; Dojindo), and the experimental procedure was performed according to the kit instructions. Table 5 shows the results of the above experiment.

From the results shown in Table 5, it is possible to confirm the effect of heptapeptide 1 on keratinocyte proliferation at a concentration of 20 ppm.

Group (s) Cell proliferation [%] Negative control group 100 ± 5.4 Positive control group 130.0 + - 14.0 Heptapeptide 1
(TGSTSAS) 123. 9 ± 12.1

< Example  6> cells Survival  Measure

Human skin keratinocyte stem cells were purchased from CellnTec (USA) and grown in 500 ml Cnt-BM medium with growth factor Cnt-57. Human dermal keratinocytes were inoculated into 96-well plates (5 x 10 ³ cells / well) and cultured in a 5% CO ₂ incubator at 37 ° C for 24 hours. After 24 hours, the cells were treated at 100, 200 and 400 ppm concentrations to confirm the cytotoxicity of the heptapeptide. After 48 hours, the degree of cytotoxicity was confirmed by the CCK-8 kit (CK04; Dojindo). As a positive control, 20 ppm of doxorubicin hydrochloride (Sigma) was treated at the same time.

From the results of Table 6, it was confirmed that heptapeptide 1 was not cytotoxic at 100 to 200 ppm.

Group (s) Cell viability [%] Negative control group 100 ± 13.4 Positive control group 10.3 ± 0.7 Heptapeptide 1
(TGSTSAS) 100 ppm 115.4 ± 4.7 200 ppm 118.4 ± 10.6 400 ppm 78.4 ± 6.5

< Example  7 > Peptides and Exendin -4 skin by simple mixing Permeability  Measure

Exendin-4 (exendin-4) was used as a target substance for skin permeation in order to confirm the effect of the heptapeptide according to the present invention on improving the skin permeability of various substances including drugs. Specifically, exendin-4, a skin permeation target substance, was introduced from Chengdu Kaijie Biopharm Co., Ltd. The mixture was mixed with heptapeptide 1: exendin-4 = 1: 5 in a weight ratio of 45: 1 with 45% EtOH / PBS (v / v) buffer Respectively.

The epilated dorsal skin (1.77 cm ² ) of the extracted Balb / c mouse was fixed between donor chamber and receiver chamber of a Franz diffusion cell (FCDV-15, Labfine, Korea) (pH 7.4) in the same manner. The temperature of the receiver chamber was maintained at 37 ± 0.5 ° C, and stirring was performed at 600 rpm using a magnetic bar. The conditions were maintained until the end of the experiment. Exendin-4 mixture with heptapeptide and fluorescence (FITC) labeled and exendin-4 as negative control was applied to the epidermal skin surface of Balb / c mouse abutted to the donor chamber. After 20 hours, The transferred fluorescently labeled exendin-4 was measured. Peptide quantification was performed using a spectrophotometer (Nanodrop 2000; Thermoscientific, USA). The results are shown in Fig.

As can be seen from Fig. 1, when the fluorescence-labeled heptapeptide 1 according to the present invention (SEQ ID NO: 1: TGSTSAS) and exendin-4 were simply mixed and permeated, And 54%, respectively, which was superior to that of the control.

< Example  In the present invention Peptides and  random Peptides  Skin by simple mixing Permeability  Measure

In order to confirm the effect of the heptapeptide according to the present invention on improving the skin permeability of various substances including drugs, experiments were conducted using fluorescently labeled 10-mer and 20-mer random peptides as target substances for skin permeation. Specifically, a 10-mer random peptide and a 20-mer random peptide were prepared by the same method as in Example 1 and fluorescently labeled with FITC as a substance to be permeated through the skin.

5% (v / v) to 100% (v / v) acetonitrile / acetonitrile containing 0.01% (v / v) TFA: FITC-linker- MRRACWMRER: Rt = 6.558 min. Various gradient of concentration over 30 minutes from water); MS (ESI) 1841.16 m / e, [M + H] &lt; + &gt; = 1841

5% (v / v) to 100% (v / v) acetonitrile / acetonitrile containing 0.01% (v / v) TFA: FITC-linker-DLDLEALAPYIPADDDFQLR: Rt = 5.650 min. Various gradient of concentration over 30 minutes from water); MS (ESI) 2793.08 m / e, [M + H] &lt; + &gt; = 1397 (M +

They were simply mixed with the heptapeptide 1 (SEQ ID NO: 1) according to the present invention, wherein the mixing ratio was 1: 5 heptapeptide 1: random peptide.

As a control, the mixture and the 10-mer peptide and 20-mer peptide alone were dispersed in a PBS buffer in a 45% EtOH / PBS (v / v) buffer and then applied to the dorsal skin of the hair of Balb / c mouse . Subsequently, a quantitative analysis of the peptides was performed using a spectrophotometer (Nanodrop 2000; Thermoscientific, USA). The results are shown in Fig.

As can be seen from FIG. 2, when the fluorescence-labeled heptapeptide 1 (SEQ ID NO: 1: TGSTSAS) according to the present invention and 10-mer and 20-mer random peptide were simply mixed and permeated, And 158% and 170%, respectively, as compared with the case where only the skin permeation was observed.

< Example  In the present invention Peptides -MTX due to skin permeation psoriasis  Check the improvement

In order to confirm the pharmacological effect of the transdermal drug covalently bound to the heptapeptide according to the present invention, methotrexate (hereinafter, referred to as MTX), which is known as a therapeutic agent for psoriasis, is used as a permeation target drug to improve the skin permeability of various substances including drugs In order to confirm this, it was tested using psoriasis as a skin permeation target material.

Specifically, MTX-covalently bonded material was prepared by the same method as described in Example 1 above as a material to be permeated through the skin.

(V / v) to 100% (v / v) of acetonitrile / water containing 0.01% (v / v) TFA over a period of 30 minutes. Concentration gradient); MS (ESI) 1044 m / e, [M + H] &lt; + &gt;

The hair of the back part of the Balb / c mice was epilated, and 80% of 5% imiquimod (IMQ, imiquimod) was applied to the epilated skin of the remaining groups except for the normal group for 6 days once a day for 6 days, Were prepared. In the control group, 0.1% (w / w) MTX and 0.1% (w / w) and 1% (w / w) of the vehicle were applied to the normal and negative control groups, respectively. Of the heptapeptide-MTX conjugate was mixed with 100 mg of the vehicle ointment type and applied. Due to the difference in molecular weight between MTX and heptapeptide-MTX conjugates, the 0.1% heptapeptide-MTX conjugate will contain a 2.3-fold lower content of MTX compared to 0.1% MTX at the same concentration. The degree of improvement of psoriasis after the treatment was confirmed, and the results are shown in Figs. The clinical PASI score indicating the degree of psoriasis was visually observed, and the erythema, the degree of keratinization and the skin thickness were scored (0, 1, 2, 3, 4 very severe) 10 &lt; / RTI &gt;

As can be seen in Figures 8-10, the heptapeptide-MTX conjugate in which the heptapeptide according to the present invention and the MTX are covalently linked has a lower content of MTX than the MTX of the same concentration And it was confirmed that it has excellent psoriasis improving effect.

In addition, it was confirmed that the transdermal delivery of 1% heptapeptide-MTX conjugate showed an increased psoriasis improving effect compared to 0.1% heptapeptide-MTX conjugate.

In summary, the use of the heptapeptide-MTX conjugate shows excellent improvement effects even when transdermal MTX administered by oral administration or injection is conventionally administered. As a result, problems caused by conventional oral administration or injection administration (for example, The risk of adverse effects due to absorption, the risk of hepatotoxicity, and the risk of resistance to long-term use at high concentrations) can be directly applied to the skin requiring treatment, Respectively.

< Example  10> cells Permeability  Measure

In order to confirm the cytotoxic activity of the heptapeptide according to the present invention, MIA-PACA2 cells, one of the pancreatic cancer cell lines, were used to confirm the cytotoxic activity. Specifically, MIA-PACA2 cells were purchased from ATCC. Heptapeptide labeled with fluorescence (FITC) and TAT as a positive control were treated with MIA-PACA2 cells. After replacing with a normal culture medium, the degree of heptapeptide penetration into the cells was confirmed using a confocal microscope. 11. As can be seen from FIG. 11, the heptapeptide according to the present invention has excellent intracellular permeability.

<110> Peptron, Inc. <120> Multifunctional skin penetration peptide with whitening, skin          elasticity and wrinkle improvement ability <130> DPP20165175 <160> 9 <170> Kopatentin 2.0 <210> 1 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> 4ME skin penetration peptide <400> 1 Thr Gly Ser Thr Ser Ala Ser   1 5 <210> 2 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> 4ME skin penetration peptide derivative <400> 2 Ala Gly Ser Thr Ser Ala Ser   1 5 <210> 3 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> 4ME skin penetration peptide derivative <400> 3 Thr Ala Ser Thr Ser Ala Ser   1 5 <210> 4 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> 4ME skin penetration peptide derivative <400> 4 Thr Gly Ala Thr Ser Ala Ser   1 5 <210> 5 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> 4ME skin penetration peptide derivative <400> 5 Thr Gly Ser Ala Ser Ala Ser   1 5 <210> 6 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> 4ME skin penetration peptide derivative <400> 6 Thr Gly Ser Thr Ala Ala Ser   1 5 <210> 7 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> 4ME skin penetration peptide derivative <400> 7 Thr Gly Ser Thr Ser Ala Ala   1 5 <210> 8 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> 10mer random peptide <400> 8 Met Arg Arg Ala Cys Trp Met Arg Glu Arg   1 5 10 <210> 9 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> 20mer random peptide <400> 9 Asp Leu Asp Leu Glu Ala Leu Ala Pro Tyr Ile Pro Ala Asp Asp Asp   1 5 10 15 Phe Gln Leu Arg              20

Claims (10)

A skin-permeable multifunctional peptide or derivative thereof having the amino acid sequence of SEQ ID NO: 1. The skin permeable multifunctional peptide or derivative thereof according to claim 1, wherein the derivative is one in which at least one of the amino acid sequences of the skin-permeable multifunctional peptide is replaced with alanine. 3. The skin permeable multifunctional peptide or derivative thereof according to claim 2, wherein the derivative has an amino acid sequence selected from SEQ ID NOS: 2-7. A composition for transdermal delivery of a physiologically active substance, which comprises a skin permeable multi-functional peptide or a derivative thereof according to any one of claims 1 to 3 and a physiologically active substance. The composition for transdermal delivery of a physiologically active substance according to claim 4, wherein the skin-permeable multifunctional peptide or derivative thereof and the physiologically active substance are covalently bonded to each other. The composition for transdermal delivery of a physiologically active substance according to claim 4, wherein the skin-permeable multifunctional peptide or a derivative thereof and a physiologically active substance are mixed. A cosmetic composition for skin whitening, skin elasticity enhancement or wrinkle improvement comprising the multifunctional peptide of any one of claims 1 to 3 or a derivative thereof. 8. The cosmetic composition of claim 7, further comprising a cosmetically acceptable carrier. The composition according to claim 7, wherein the composition is selected from the group consisting of a solution, an external ointment, a cream, a foam, a nutritional lotion, a softening water, a pack, Wherein the composition has a formulation selected from the group consisting of suspensions, emulsions, pastes, gels, lotions, powders, soaps, surfactant-containing cleansing, oils, powder foundations, emulsion foundations, wax foundations, patches and sprays. Composition. A pharmaceutical composition for improving skin whiteness, skin elasticity or wrinkles, comprising the multifunctional peptide of any one of claims 1 to 3 or a derivative thereof.

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