WO2017082692A1

WO2017082692A1 - Multifunctional skin-permeating peptide having whitening, skin elasticity, wrinkle improvement, and wound healing activities

Info

Publication number: WO2017082692A1
Application number: PCT/KR2016/013033
Authority: WO
Inventors: 최호일; 김동석; 송상용; 양현; 이정옥; 고희주; 송강; 김태균; 김희경; 정다은
Original assignee: 주식회사 펩트론
Priority date: 2015-11-12
Filing date: 2016-11-11
Publication date: 2017-05-18

Abstract

The present invention relates to a multifunctional peptide and use of same, the multifunctional peptide having whitening, elasticity, and wrinkle improvement effects based on a skin-permeating function, and more specifically, to a method for utilizing, as a raw ingredient in functional cosmetics and medicine, a multifunctional peptide having the effects of skin permeation, melanin production inhibition, collagen production promotion, and keratinous stem cell proliferation promotion. The multifunctional skin-permeating peptide, according to the present invention, has the characteristic of easily permeating skin and is exceptionally effective in inhibiting melanin synthesis and promoting collagen synthesis and keratinous stem cell proliferation, thus rendering the peptide useful, even independently, as cosmetics or medicine for whitening skin, improving skin elasticity, improving wrinkles, or healing wounds.

Description

【Specification】

[Name of invention]

Multifunctional skin penetration peptide with whitening, skin elasticity and wrinkle improvement activity

The present invention relates to a multifunctional peptide having a skin permeation function-based whitening, elasticity and wrinkle improvement effect, and more particularly, to cells and skin permeation function, inhibiting melanin production, promoting collagen production, and proliferating skin keratin stem cells. The present invention relates to a method for utilizing a multifunctional peptide having a function as a raw material for functional cosmetics and pharmaceuticals.

Background Art

Skin is the organ with the largest surface area in the human body, and is a route for effective drug delivery when used properly. However, due to the stratum corneum, which is composed of dead cells on the outermost side of the skin, the skin permeability of the foreign material is extremely low, which makes it difficult to deliver substances with high molecular weight and hydrophilicity. The drug delivery to the skin protects against foreign substances in the skin barrier.

Although many researches have been attempted to overcome these problems, it is considered impossible to deliver drugs through the properties of chemicals without physically damaging or irritating the skin. There is a continuous need for development.

In response to these demands, a number of studies have been reported on skin permeation delivery materials such as liposomes or nanoparticles and peptide ligands for effectively delivering large molecular weight drugs [Lademann et al., 2007 (Eur J Pharm Biopharm. 2007 May). 66 (2): 159-64.), Chen et al, 2006a (J Pharmacol Exp Ther. 2006 Nov; 319 (2): 765-75.), Chen et al., 2006b (Nat Biotechnol. 2006 Apr; 24 (4): 455-60.)], While the development cost of the commercialization step increases, the efficiency and stability of the drug to be delivered and delivered cannot be guaranteed. It is required.

On the other hand, the skin protects the body from the external environment and plays an important role in maintaining homeostasis. However, skin function is deteriorated and aging phenomenon is caused by endless external stimuli and unnecessary metabolites (metabolites) generated by its own metabolic processes. Is promoted.

Collagen in the skin is a structural protein produced by skin fibroblasts, and it is known that the decrease in production due to aging and the change in its structure are related to wrinkle formation of the skin. Melanin in the skin acts to protect the skin from UV rays and free radicals in the body, but abnormal activation of melanocytes causes hyperpigmentation such as blemishes, freckles and blotch, resulting in aesthetic problems. Skin keratin stem cells present in the basal epidermal layer of the skin are known to play an important role in skin regeneration by controlling the growth and differentiation of keratinocytes as well as the fibroblasts of the dermis. Therefore, body homeostasis related to collagen production, melanin pigment suppression, and keratin stem cell growth are closely related factors to the health and beauty of skin. The effectiveness of external treatment materials applied requires a great deal of effort due to the technical difficulties of passing through the epidermis and dermis.

Under this background, the present inventors have limitations in utilization due to problems such as reduced efficacy and safety, or increased cost due to the modification of the above-mentioned efficacy substance.

As a result of intensive efforts to solve the conventional problems of peptides, not only have skin permeation function and skin permeation and delivery function of various active substances, but also inhibits the production of dalanine and promotes collagen synthesis and the proliferation of skin keratin stem cells. The present invention was completed by discovering a multifunctional peptide material.

[Detailed Description of the Invention]

[Technical problem]

Accordingly, one object of the present invention is to provide a skin permeable multifunctional peptide or derivative thereof having a function of inhibiting melanin synthesis and collagen synthesis, and promoting the proliferation of skin keratinocytes in addition to skin permeation function.

Another object of the present invention is to provide a skin permeation complex in which the skin permeation multifunctional peptide or a derivative thereof and a bioactive substance are connected.

Furthermore, another object is to provide a cosmetic composition for skin whitening, skin elasticity enhancement or wrinkle improvement comprising the skin permeable multifunctional peptide or derivatives thereof. Another object is to provide a pharmaceutical composition for skin whitening, skin elasticity enhancement or wrinkle improvement comprising the skin permeation multifunctional temperide or derivatives thereof.

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

Furthermore, another object of the present invention is to provide a method for skin whitening, skin elasticity improvement or wrinkle improvement, which comprises using the skin permeable multifunctional peptide or derivatives thereof.

In addition, another object of the present invention is to provide a method for transdermal delivery of a physiologically active substance, using the skin permeable multifunctional peptide or derivatives thereof.

【Effects of the Invention】

The skin permeable multifunctional temptide or derivative thereof according to the present invention has a property of effectively penetrating the skin, and when foreign substances are combined, it can be effectively moved into the skin, and thus can be used as a tool for mass transfer. ， Excellent melanin synthesis inhibitory ability and collagen synthesis, showing the effect of promoting the proliferation of skin keratin stem cells, the peptide alone may be useful as a cosmetic, medicine for skin whitening, skin elasticity or wrinkle improvement.

[Brief Description of Drawings]

1 is a graph quantitatively illustrating the skin permeation effect of the complex of the skin permeable multifunctional peptide and the accendin-4 according to the present invention and the use of the accendin-4 alone.

Figure 2 is a graph quantitatively illustrating the skin permeation effect when the skin permeable multifunctional peptide according to the present invention is mixed with a random peptide of lOmer and 20mer and when the random peptides are used alone.

3 to 7 are photographs confirming the improvement and treatment effect of psoriasis when the complex of the skin permeable multifunctional peptide and MTX is connected to psoriasis induced mouse skin. 8 is a graph quantitatively showing the degree of erythema when the complex of the skin-permeable multifunctional peptide and MTX linked to the psoriasis-induced mouse skin according to the present invention.

Figure 9 shows the extent of keratinization when the skin-permeable multifunctional peptide and MTX-linked complexes were treated to psoriasis-induced mouse skin.

It is a quantitative graph.

Figure 10 is a graph quantitatively showing the skin thickness when treated with a skin-permeable multifunctional peptide and MTX-linked conjugate to mouse skin induced psoriasis.

Figure 11 is a photograph confirming the cell permeation activity of the skin permeable multifunctional temperate according to the present invention by confocal microscopy.

[Best form for implementation of the invention]

As one embodiment for solving the above problems, the present invention relates to a skin permeable multifunctional peptide having the amino acid sequence of SEQ ID NO: 1 or derivatives thereof.

[SEQ ID NO 1] TGSTSAS

The derivative specifically refers to a peptide having a sequence in which one or more amino acids of the amino acid sequence of SEQ ID NO: 1 are substituted with alanine (Ala).

Preferably it means a peptide having an amino acid sequence selected from SEQ ID NOs: 2 to 7.

Synthesis of the peptides of the invention can be carried out, for example, using a device or using genetic engineering techniques. Automated Peptides When Synthesizing Using an Instrument

Fmoc to the desired peptide in a synthesizer (PeptrEX-R48, Peptron, Daejeon, Korea)

It is synthesized by the Fmoc solid-phase method. After separating the synthetic peptide from the resin, it was performed by reverse-phase HPLC (Reverse-phase HPLC, Prominence LC-20AB, Shimadzu, Japan) using Shiseido capcell pak Cl 8 analytical RP column. Peptides are purified and analyzed. Final peptides are identified using a mass spectrometer (HP 1100 Series LC / MSD, Hewlett-Packard, Roseville, USA). When using genetic engineering techniques, the corresponding nucleotide sequence of the desired peptide After introduction into the protein expression vector, expression is induced and purified by IPTG in bacteria lacking protease such as BL21 (DE3) and BL21 (^ DE3) pLys.

In one specific embodiment, the skin permeable multifunctional peptide according to the present invention was identified using phage display technique and selected by verifying the efficacy of the identified peptide. In the step of identifying peptides that penetrate the skin, after applying a random phage display library to the pig skin, the skin epidermis is removed after a certain time and information about peptide sequence and appearance frequency is amplified by remaining phages remaining inside the skin except the epidermis. Can be secured.

Furthermore, in order to verify the efficacy of the identified peptide, 1) the peptide having a skin permeation function 2) melanin synthesis inhibitory effect in mouse melanocytes, 3) human

Multifunctional peptides were selected through validation of collagen synthesis in fibroblasts, 4) proliferation effects in human skin keratinocytes.

The peptide according to the present invention has a cell and skin permeability, thereby transporting or delivering various active substances such as low molecular weight materials, and at the same time, by itself, have a multifunctional, particularly excellent skin whitening, skin elasticity and wrinkle improvement effect. It is characterized by showing, the usefulness regarding a drug or cosmetics is large. Furthermore, when transporting or delivering various bioactive substances,

The bioactive substance and the peptide according to the present invention may be combined or covalently linked to each other to transport or deliver a complex of a skin permeable multifunctional peptide or a derivative-physiologically active substance thereof. By combining, the desired active substance can be easily delivered, and in particular, it is possible to effectively avoid problems such as denaturation and difficulty in synthesis and purification, which may occur when covalent bonds such as peptides are used. At this time, the method of covalently binding the active substance and the skin permeable multifunctional peptide according to the present invention can be appropriately prepared using known techniques.

Specifically, the physiologically active substance is a substance that enhances or inhibits the function of a living body in living life, and means any substance including a drug that can exert a desired effect in a living body. The bioactive substance is for example Examples include, but are not limited to, peptides, polypeptides, polynucleotides, nanoparticles, DNA, RNA, small molecule compounds, and the like. As a specific example, the present inventors confirmed transdermal delivery by the skin-permeable multifunctional peptide according to the present invention using random peptides of exendin-4, 10mer and 20mer and MTX as bioactive substances. According to one embodiment of the present invention, the skin permeable multifunctional peptide of the present invention not only shows excellent skin permeability compared to the transmittance of the TAT protein known as PTD (Protein transduction domain), but also excellent by using only itself Melanin biosynthesis inhibitory effect, collagen synthesis effect and keratin stem cell proliferation effect are shown.

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

In the present invention, the term "wrinkle" refers to the lines produced by the decay of the skin, can be caused by the cause of the gene, the reduction of collagen and elastin in the skin dermis, external environment and the like. In the present invention, "wrinkle improvement" refers to inhibiting or inhibiting wrinkles from being generated on the skin, or alleviating wrinkles already generated.

In the present invention, the term "skin elasticity" is represented by elastic fibers composed of elastin present in the dermal layer, and "enhancement of skin elasticity" refers to skin in a state in which elastic fibers composed of elastin and collagen, which are collagen fibers, are present in layers. It is said that the elasticity is maintained.

The cosmetic composition according to the present invention includes a solution, an external ointment, a cream, a product, a nourishing cosmetic, a flexible cosmetic, a pack, a flexible water, an emulsion, a makeup base, an essence, a soap, a liquid detergent, a bath, a sunscreen cream, sun oil, a suspension, 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 include one or more cosmetically acceptable carriers formulated in general skin cosmetics, and as conventional components, for example, oil, water, surfactant, moisturizer, lower alcohol , Thickening crabs, chelating agents, pigments, preservatives, fragrances and the like can be blended properly It is not limited.

As another aspect, the present invention relates to a pharmaceutical composition for skin whitening, wrinkle improvement or skin elasticity enhancement, comprising the multifunctional skin permeation temptide. The composition of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions. The compositions of the present invention may be used in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, oral dosage forms, external preparations, suppositories, and sterile injectable solutions, respectively, according to conventional methods. Carriers, excipients and diluents that may be included in the composition include lactose, dextrose, sucrose, sorbbi, manny, xylly, erythris, malty, starch, acacia rubber, alginate, gelatin, calcium Phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyridone, water, methylhydroxy benzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, diluents or excipients such as commonly used layering agents, extenders, binders, wetting agents, disintegrating agents and surfactants are used. In the case of the composition according to the present invention, the multifunctional skin permeable 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 depends on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art. But

For the desired effect, the pharmaceutical composition of the present invention may be administered once a day or divided several times. Therefore, the above dosage does not limit the scope of the present invention in any aspect.

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

As yet another aspect, the present invention is to provide a composition for transdermal delivery of the skin-permeable multifunctional peptide or derivatives thereof and the bioactive material containing the peptide and the bioactive material. Preferably, the skin permeable multifunctional peptide or derivatives thereof and the bioactive material in the transdermal delivery composition may be in a mixed form or in a complex form covalently linked to each other.

As another aspect, the present invention is characterized in that using the skin permeable multifunctional peptide and a cosmetic or pharmaceutical composition comprising the same as an active ingredient Skin whitening, skin elasticity enhancement or wrinkle improvement method, and the skin permeable multifunctional peptide or derivatives thereof, the skin permeable multifunctional peptide or derivative-bioactive substance complex or composition comprising them as an active ingredient The present invention relates to a method for transdermal delivery of a bioactive substance, wherein the amount of the skin-penetrating multifunctional peptide used is varied depending on the patient's condition, weight, degree of skin condition, form of cosmetic or pharmaceutical composition, administration and application route and duration. However, it may be appropriately selected by those skilled in the art.

[Form for implementation of invention]

Hereinafter, the present invention will be described in detail through examples. These examples are intended to illustrate the present invention in more detail, and the scope of the present invention is not limited by these examples.

Example 1 Synthesis of Peptide Derivatives

In this embodiment, in order to synthesize various peptide derivatives, it proceeded according to a general tide solid phase synthesis method (Wang C. Chan, Perter D. White, "Fmoc Solid phase peptide synthesis", Oxford). More specifically, using an autosynthesizer (ASP48S, Peptron, Inc.) using the Fmoc-SPPS (9-Fluorenyl methyl oxycarbonyl solid phase peptide synthesis) method was coupled (coupling) one by one from the 5 C- terminal. All monomer raw materials used to synthesize peptide derivatives have amino acids protected with N-terminal Fmoc and residues protected with trityl (Trt), t-butyloxycarbonyl (Boc), t-butyl (t-Bu), etc. Used.

As a coupling reagent, HBTU (2- (lH-Benzotirazloe-l-yI) -l, 1,3,3-tetramethyluronium hexafluorophophate I HOBt (Hydroxybenzo triazloe) I NMM (N-methylmorpholine) was "tried". .

(1) Protected amino acid (8 equivalents) and coupling agent HBTU (8 equivalents) / NMM (16 equivalents)

After dissolving in DMF (Dimethyl formamide) and adding, the mixture was reacted at room temperature for 2 hours.

(2) Fmoc removal was performed by adding 20% (v / v) piperidine / DMF and reacting twice at room temperature for 5 minutes.

After repeating the reactions of (1) and (2) to make peptide derivatives, fluorescent peptides for confirming skin permeability are used as linkers at the N-terminal portion of the peptide. 6-amino hexanoic acid and fluorescein isothiocyanate (FITC) were combined. ^■

Peptide separation from Resin was performed using Trifluoroacetic acid (TFA) / EDT (l, 2-ethanedithiol) / Thioanisole I TIS (Triisopropylsilane) I H20 (mixture ratio (by weight) = 90 I 2.5 1 2.5 1 2.5 1 2.5) Separated.

The mixed solution thus obtained forms a precipitate by excessively treating the refrigerated diethyl ether solvent. The precipitate obtained was centrifuged to completely settle, and excess trifluoroacetic acid, thianizol and ethanedithi were firstly removed, and then the same procedure was repeated twice to obtain a precipitate which solidified.

The precipitate obtained was subjected to water containing 0.1% (v / v) Trifluoroacetic acid using a high performance liquid chromatography instrument (Shimadzu Prominence HPLC, Japan) using a C18 column (250mm x 22 mm, ΙΟμηι, Vydac Everest, USA). acetonitrile liner

Gradient (Acetonitrile concentration: 10 ~ 75% (v / v)) was separated by the method. The molecular weight of the purified peptide derivative was confirmed using LC / MS (Agilient HP 1 100 series), and pure purified fractions were lyophilized to obtain the following peptide as a TFA (Trifluoroacetic acid) salt in the form of a white powder.

Specifically, the sequence and molecular weight of the synthesized peptide derivatives are shown below. Heptapeptide 1: TGSTSAS (SEQ ID NO: 1): Rt = 2.217 min (() .01% (v / v) 5% (v / v) to 100% (v / v) acetonitrile / water containing TFA) Concentration gradient over 30 minutes); MS (ESI) 609.66 m / e, [M + H] ⁺ = 610

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

Heptapeptide 3: TASTSAS (SEQ ID NO: 3): Rt = 4. 1 17 min (0.01% (v / v) 5% (v / v) to 100% (v / v) acetonitrile / water containing TFA) Concentration gradient over 30 minutes); MS (ESI) 623.61 m / e, [M + H] ⁺ = 624

Heptapeptide 4: TGATSAS (SEQ ID NO: 4): Rt = 2.153 min (30 min from 5% (v / v) to 100% (v / v) acetonitrile / water containing 0.01% (v / v) TFA Varying concentration gradients); MS (ESI) 593.58 m / e, [M + H] ⁺ = 594 Heptapeptide 5: TGS AS AS (SEQ ID NO: 5): Rt = 3.892 min (0.01% (v / v) 5% (v / v) to 100% (v / v) acetonitrile / containing TFA Various concentration gradients over 30 minutes from water); MS (ESI) 579.56 m / e, [M + H] ⁺ = 580

Heptapeptide 6: TGSTAAS (SEQ ID NO: 6): Rt = 3.125 min (30 min from 5% (v / v) to 100% (v / v) acetonitrile / water containing 0.01% (v / v) TFA Varying concentration gradients); MS (ESI) 593.59 m / e, [M + H] ⁺ = 594

Hepta peptide 7: TGSTSAA (SEQ ID NO: 7): Rt = 3. from acetonitrile / water for ²⁶⁷ minutes (0.01% (v / v) 5% containing TFA (v / v) to 100% (v / v) Various concentration gradients over 30 minutes); MS (ESI) 593.59 m / e, [Μ + Η] ⁺ = 594

Fluorescent heptapeptide 1: FITC-linker-TGSTSAS: Rt = 5.900 min (30 min from 5% (v / v) to 100% (v / v) acetonitrile / water containing 0.01% (v / v) TFA Varying concentration gradients); MS (ESI) 1 1 1 1.81 m / e, [M + H] ⁺ = 1 1 1 1

Fluorescent heptapeptide 2: FITC-linker- AGSTSAS: Rt = 7.575 min (30 min from 5% (v / v) to 100% (v / v) acetonitrile / water containing 0.01% (v / v) TFA) Varying concentration gradients); MS (ESI) 1081.80 m / e, [M + H] ⁺ = 1082

Fluorescent heptapeptide 3: FITC-linker-TASTSAS: Rt = 7.600 min (30 min from 5% (v / v) to 100% (v / v) acetonitrile / water containing 0.01% (v / v) TFA Varying concentration gradients); MS (ESI) 1 125.83 m / e, [M + H] ⁺ = 1 126

Fluorescent heptapeptide 4: FITC-linker-TGATSAS: Rt = 5.792 min (30 min from 5% (v / v) to 100% (v / v) acetonitrile / water with 0.01% (v / v) TFA) Varying concentration gradients); MS (ESI) 1095.82 m / e, [M + H] ⁺ = 1095

Fluorescent heptapeptide 5: FITC-linker-TGGSAS: Rt = 7.500 min (30 min from 5% (v / v) to 100% (v / v) acetonitrile / water containing 0.01% (v / v) TFA Varying concentration gradients); MS (ESI) 1081.80 m / e, [M + H] ⁺ = 1082

Fluorescent heptapeptide 6: FITC-linker-TGSTAAS: Rt = 6.608 min (30 min from 5% (v / v) to 100% (v / v) acetonitrile / water containing 0.01% (v / v) TFA Varying concentration gradients); MS (ESI) 1095.82 m / e, [M + H] ⁺ = 1096

Fluorescent heptapeptide 7: FITC-linker- TGSTSAA: Rt = 6.750 min (30 min from 5% (v / v) to 100% (v / v) acetonitrile / water containing 0.01% (v / v) TFA Various concentrations throughout gradient); MS (ESI) 1095.82 m / e, [M + H] ⁺ = 1096

Example 2 Measurement of Skin Permeability

Extracted porcine skin (1.77 cm ² ) was transferred to Franz diffusion cells (FCDV-15, Labfine,

^Λ } °] ° 1] of donor chamber and receiver chamber of Korea), and complete isotonic phosphate complete solution (pH 7.4) is added to each receiver chamber. The temperature of the receiver chamber is maintained ^at 37 ± 0.5 ^° C, and the magnetic bar is stirred at a speed of 600 rpm and the conditions are maintained until the end of the experiment. Fluorescent (FITC) -labeled hepta peptides and derivatives thereof and positive control fluorescence-labeled TAT are applied to the pig skin surface in contact with the donor chamber and peptides and derivatives thereof are transferred to the receiver chamber after 20 hours. Peptide quantification was _{spectrophotometer;:} was used (Nanodrop2000 Thermoscientific USA).

Table 1 shows the results of analyzing the permeability of heptapeptide, and Table 2 shows the results of performing alanine scanning as a method for verifying the effective sequence of heptapeptide.

Table 2

Fluorescent Heptapeptide 5 0.84 ± 0.34

Fluorescent Heptapeptide 6 0.30 ± 0.00

Fluorescent Heptapeptide 7 0.89 ± 0.16 As can be seen from Table 1, the skin permeability of fluorescence-labeled heptapeptide 1 was found to be 5.32 zg / cm ² / hr. It was confirmed that exhibited excellent skin permeability more than 2.8 times. Furthermore, in Table 2, the skin transmittance (%) between the derivatives of heptapeptide l (TGSTSAS) was 0.47-fold for fluorescent heptapeptide 2 (fluorine heptapeptide 3) compared to fluorescent heptapeptide l (FITC-linker-TGSTSAS), respectively. 79 times, fluorescent heptapeptide 4 was 39 times, fluorescent heptapeptide 5 was 0.84 times, fluorescent heptapeptide 6 was 0.30 times, and fluorescent heptapeptide 7 was 0.89 times.

Example 3 Measurement of Melanin Biosynthesis Inhibitory Effect

In addition to tyrosinase inhibitory substances, which are cosmetic materials for whitening, substances that induce melanin synthesis inhibition in skin cells also have a great potential for skin whitening. Thus, whitening can be expected as an inhibitory effect on melanin biosynthesis of melanoma cells.

B16F10 melanoma cells (ATCC) containing 10% FBS

6-well plates containing DMEM (Dulbecco's modified eagle medium, Gibco) medium were inoculated (IX 10 ⁵ cells / well) and incubated at 37 ^° C for 24 hours in a C0 ₂ incubator at 5% concentration. After 24 hours, the medium was removed from each well, and the medium containing arbutin (Sigma Aldrich) as a positive control group, the medium containing 20 ppm heptapeptide as an experimental group,

Normal medium was treated as a negative control and incubated again for 48 hours. After 48 hours, the cells from which the medium was removed were washed with PBS (phosphate buffer saline) and treated with Trypsin-EDTA to recover the cells. The recovered cells were centrifuged at 13,000 rpm for 3 minutes, and then 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 lN NaOH containing 10% DMSO was added and allowed to stand at 60 ^° C for 1 hour to dissolve intracellular melanin. The solution was transferred to a microplate to measure melanin by measuring absorbance at 405 nm. The experimental results are shown in Table 4. The result is heptapeptide 0 as shown in Table 3.

It has been shown to inhibit melanin biosynthesis.

Table 3

Example 4 Measurement of Collagen Synthesis of Human Dermal Fibroblasts The amount of collagen synthesis of fibroblasts is an indicator of wrinkle formation in the skin. Wrinkles are known to occur due to decreased collagen synthesis and degradation of existing collagen. As a result, promoting collagen synthesis of fibroblasts can bring about the effect of inhibiting wrinkle formation and improving existing wrinkles.

Fibroblasts (human dermal fibroblasts, ATCCs) were introduced into the fibroblast basal medium (FBM);

201-020; ATCC) were inoculated into 96-well plates containing medium (3 × 10 ³ cells / well) and incubated at 37 ^° C. for 24 hours in a CO ₂ incubator at 5% concentration. After 24 hours, the medium was removed from each well, and the medium containing vitamin C as a positive control group and 20 ppm as an experimental group.

Heptapeptide containing medium, serum-free medium as negative control, and incubated for 48 hours. After 48 hours, cell culture medium was collected to prepare a sample. Sample ^ collagen amount was measured using a collagen measurement kit (Procollagen Type I C-peptide EIA kit; MK101; Takara, Kyoto, Japan) to measure the amount of Procollagen Type I C-peptide (PICP). It was. The experiment was performed according to the method described in the manual. The experimental results are shown in Table 4.

As can be seen from the results of Table 4, heptapeptide 1 increased the collagen synthesis of fibroblasts to the same degree as that of the positive control group.

Table 4

Positive control 124.1 ± 0.22

Heptapeptide 1 121.1 ± 3.8

(TGSTSAS)

Example 5 Measurement of Proliferation Promoting Activity of Epidermal Keratinocyte Progenitor Cells

Sustained division of keratin stem cells in the basal layer of the epidermis decreases with age, which leads to aging, such as decreased elasticity due to a decrease in skin regeneration. Continuous skin elasticity may be improved by promoting the growth of keratin stem cells associated with skin regeneration and elasticity with aging. Human skin keratinocytes were purchased from CellnTec (USA) and cultured according to Cell & Tech guidelines. As the cell medium, growth factor cnt-57 was used in 500 m cnt-BM medium according to Cell &Tech's method. Human skin keratinocytes were seeded in 96-well plates (5 × 10 ³ cells / well) and incubated at 37 ^° C. for 24 hours in a CO ₂ incubator at 5% concentration. After 24 hours, the medium was removed from each well, followed by the routine containing Rutin (Sigma Aldrich) as a positive control, the 20 ppm heptapeptide containing medium as the experimental group,

Treated with normal medium as negative control and incubated again for 72 hours. The degree of cell proliferation was confirmed using CCK-8 kit (CK04; Dojindo).

It was performed according to the instructions. Table 5 shows the results of the experiment.

From the results in Table 5, it can be confirmed that the keratin enjoyment cell proliferation effect of heptapeptide 1 at a concentration of 20 ppm.

Table 5

Example 6 Measurement of Cell Viability Human skin keratinocytes were purchased from Cell & Tek (CellnTec; USA) and used for culturing by adding growth factor Cnt-57 to 500 Cnt-BM medium according to the instructions. Human skin keratinocytes were seeded in 96-well plates (5 × 10 ³ cells / well) and incubated at 37 ^° C. for 24 hours in a CO ₂ incubator at 5% concentration. After 24 hours, cells were treated at 100, 200, 400 ppm concentrations to confirm the cytotoxicity of the heptapeptide, and after 48 hours, the degree of cytotoxicity was confirmed by CCK-8 kit (CK04; Dojindo). As a positive control, 20 ppm of doxorubicin hydrochloride (sigma) was treated under the same time condition. From the results of Table 6, heptapeptide 1 was found to be cytotoxic at 100 to 200 ppm.

Table 6

Example 7 Measurement of Skin Permeability by Simple Mixing of Peptides and Exendin-4 in the Present Invention

In order to confirm the effect of improving the skin permeability of various substances including drugs by the heptapeptide according to the present invention, experiments were performed using accendin-4 (exendin-4) as a target material for skin penetration. Specifically, exendin-4, a substance for skin permeation, was purchased from Chengdu aijie Biopharm Co., Ltd. It was simply mixed with heptapeptide 1 (SEQ ID NO: 1) according to the present invention, wherein the mixing ratio is by weight,

Heptapeptide 1: exendin-4 = 1: 5 was used and 45% EtOH / PBS (v / v) buffer was used. The removed dorsal dorsal skin (1.77 cm ² ) of the Balb / c mouse was fixed between the donor chamber of the Franz diffusion cell (FCDV-15, Labfine, Korea) and the receiver chamber and isotonic phosphate in the receiver chamber. The layer solution (pH 7.4) was provided in equal amounts to 13 mL.

The temperature of the receiver chamber is maintained ^at 37 ± 0.5 ^° C, 600 using a magnetic bar After stirring at the speed of rpm, the conditions were maintained until the end of the experiment.

Hextapeptide and fluorescence (FITC) -labeled accendin-4 complex and their negative control exendin-4 alone were applied to the epidermal skin surface of Balb / c mouse in contact with the donor chamber and 20 hours later Moved to the labeled axendin-4

Measured. Peptide quantitative analysis was performed using a spectrophotometer (Nanodrop2000; Thermoscientific, USA). The results are shown in FIG.

As can be seen in Figure 1, according to the present invention labeled fluorescence

When the heptapeptide 1 (SEQ ID NO: TGSTSAS) and exendin-4 were simply mixed and permeated, the skin permeability increased by 54% compared to when only the exendin-4 was permeated, indicating excellent skin permeability.

Example 8 Measurement of Skin Permeability by Simple Mixing of Peptides and Random Peptides According to the Present Invention

In order to confirm the skin permeability improvement effect of various substances including drugs by the heptapeptide according to the present invention, experiments were performed using fluorescently labeled lOmer and 20mer random peptides as target substances for skin penetration. Specifically, random peptides of 20mer and 20mer were prepared by the same method as Example 1 and fluorescently labeled with FITC as a substance for skin permeation.

lOmer random peptide (SEQ ID NO: 8): FITC-Unker-MRRACWMRER: Rt = 6.558 min (0.01% (v / v) 5% (v / v) to 100% (v / v) acetonitrile / containing TFA) Various concentration gradients over 30 minutes from water); MS (ESI) 1841.16 m / e, [M + H] < + > = 1841

20mer random peptide (SEQ ID NO: 9): FITC-linker-DLDLEALAPYIPADDDFQLR: Rt = 5.650 min (5% (v / v) to 100% (v / v) containing 0.01% (v / v) TFA

Various concentration gradients over 30 minutes from acetonitrile / water); MS (ESI) 2793.08 m / e, [^^ + H] + = 1397 (M + l) / 2

Each of them was simply mixed with heptapeptide 1 (SEQ ID NO: 1) according to the present invention, and the mixing ratio was weight ratio, and heptapeptide 1: random peptide = 1: 5.

As a control, the complex, lOmer peptide and 20mer peptide alone were dispersed in PBS buffer in 45% EtOH / PBS (v / v) buffer, and then Balb / c was dispersed. The hair of the mouse was applied to each of the dorsal skin of the hair. Subsequently, the peptide quantitative analysis was performed using a spectrophotometer (Nanodrop2000; Thermoscientific, USA). The results are shown in FIG.

As can be seen in Figure 2, according to the present invention labeled fluorescence

Excellent skin permeability increased by 158% and 170% skin permeability when heptapeptide 1 (SEQ ID NO: TGSTSAS) and lOmer and 20mer random peptides were permeated by simple mixing. It confirmed that it represents. Example 9 Confirmation of Psoriasis Improvement Effect Due to Penetration of Peptide-MTX in the Present Invention

In order to confirm the pharmacological effect of the drug transdermally covalently coupled to heptapeptide according to the present invention, methotrexate (hereinafter referred to as MTX), which is known as a therapeutic agent for psoriasis, improves the skin permeability of various substances including drugs as a penetrating drug. In order to confirm, it was tested using a psoriasis effect skin permeation target material. Specifically, a substance covalently bonded to MTX as a material for skin permeation was prepared by the same method as in Example 1.

4ME-MTX: Methotrexacte-TGSTSAS: Rt = 6.742 minutes (various over 30 minutes from 5% (v / v) to 100% (v / v) acetonitrile / water containing 0.01% (v / v) TFA Concentration gradient); MS (ESI) 1044 m / e, [M + H] < + > = 1044

Psoriasis-induced mice were subjected to hair removal on the back of Balb / c mice by applying 5 mg of imiquimod (IMQ, 80%) once a day for 6 days to the epilated skin of the remaining groups except the normal group. Was prepared. 4 days after

The negative control group was applied with vehicle ointment type LOO mg, and the positive control group

0.1% (w / w) MTX, drug groups were applied with 0.1% (w / w) and l% (w / w) of heptapeptide-MTX conjugates in combination with 100 mg of vehicle ointment formulation, respectively. MTX

Due to the molecular weight difference of the heptapeptide-MTX conjugate, the 0.1% heptapeptide-MTX conjugate contains 2.3 times less MTX than the same concentration of 0.1% MTX. After the treatment, the degree of improvement of psoriasis was confirmed, and the results are shown in FIGS. 3 to 7. Indicated. In addition, by visually observing the clinical PASI score indicating the degree of psoriasis, scores of fungi, keratinogenesis and skin thickness (0 none, 1 slight, 2 moderate, 3 severe, 4 very severe), and the results are shown in FIG. 8. To 10. As can be seen in Figures 8 to 10, heptapeptide according to the present invention and

Heptapeptide-MTX conjugate covalently linked to MTX was found to have an excellent psoriasis improvement effect when treated with the same concentration of MTX, even though it contained a lower amount of MTX.

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

Overall, the use of the heptapeptide MTX conjugate shows an excellent improvement in the transdermal delivery of conventionally administered orally or MTX administered in the form of injection. Problems caused by conventional oral or injection (for example, systemic absorption The risk of side effects, the risk of hepatotoxicity, and the risk of resistance to long-term use at high concentrations).

Direct and low risk of side effects and toxicity.

Confirmed.

Example 10 Measurement of Cell Permeability

In order to confirm the cell permeation activity of the heptapeptide according to the present invention, cell permeation activity was confirmed using MIA-PACA2 cells, one of the pancreatic cancer cell lines. Specifically, MIA-PACA2 cells were purchased from ATCC. Fluorescent (FITC) -labeled hepta peptides and TAT as positive controls were treated with MIA-PACA2 cells, and after replacement with normal culture media, the degree of heptapeptide penetration into the cells was confirmed by confocal microscopy. As shown in FIG. 11, it can be seen that the heptapeptide according to the present invention has excellent intracellular permeation activity by itself.

Claims

[Range of request]

[Claim 1]

A skin permeable multifunctional peptide having the amino acid sequence of SEQ ID NO: 1 or a derivative thereof.

[Claim 2]

The method of claim 1, wherein the derivative is one or more of the amino acid sequence of the skin permeable multifunctional peptide is substituted with alanine, the skin permeable multifunctional peptide or derivatives thereof.

[Claim 3]

According to claim 2, wherein the derivative is a skin permeable multifunctional peptide or derivative thereof having an amino acid sequence selected from SEQ ID NO: 2 to 7.

[Claim 4]

Any one of claims 1 to skin permeation according to any one of claim 3, wherein the functional peptide or derivatives thereof and a physiologically active ^"material composition for transdermal delivery of a physiologically active substance comprising a.

[Claim 5]

According to claim 4, The skin permeable multifunctional peptide or derivatives thereof, and the bioactive material is in the form of covalently bonded to each other, the composition for transdermal delivery of the bioactive material.

[Claim 6]

The composition for transdermal delivery of a bioactive substance according to claim 4, wherein the skin permeable multifunctional peptide or a derivative thereof, and the bioactive substance are in a mixed form.

[Claim 7]

Claims 1 to 3, wherein the multifunctional peptide or derivative thereof, comprising a cosmetic composition for skin whitening, skin elasticity or wrinkle improvement.

[Claim 8]

8. The cosmetic composition of claim 7, further comprising a cosmetically acceptable carrier.

[Claim 9]

According to claim 7, wherein the composition is a solution, external ointment, cream, foam, nutrient cosmetics, Softener, Pack, Softener, Latex, Makeup Base, Essence, Soap, Liquid Cleanser, Bath, Sunscreen Cream, Sun Oil, Suspension, Emulsion, Paste, Gel, Lotion, Powder, Soap, Surfactant-containing Cleansing And a formulation selected from the group consisting of oils, powder foundations, emulsion foundations, wax foundations, patches and sprays.

[Claim 10]

Claims 1 to 3, wherein the multifunctional peptide or derivative thereof, comprising a pharmaceutical composition for skin whitening, skin elasticity or wrinkle improvement.

[Claim 1 11]

A method for skin whitening, skin elasticity improvement or wrinkle improvement, comprising using the multifunctional peptide of any one of claims U to 3 or a derivative thereof. [Claim 12]

A method for transdermal delivery of an active substance, characterized by using the multifunctional peptide of any one of claims 1 to 3 or a derivative thereof.