KR20160006994A - Composition for wound-healing or inhibiting wrinkle formation on skin comprising peptide fragments derived form epidermal growth factor - Google Patents

Abstract

Provided in the present invention are a composition for treating a wound, comprising peptide fragments or small peptides derived from an epidermal growth factor (EGF); and a composition for inhibiting skin aging or skin wrinkle formation. The peptide activates an epidermal growth factor acceptor, and has an activity of growing dead cells and promoting angiogenesis of the skin. The peptide is used in wound treatment, and control of skin aging or skin wrinkle formation.

Description

Technical Field [0001] The present invention relates to a composition for inhibiting wound healing or skin wrinkle formation comprising a peptide fragment derived from epidermal growth factor,

The present invention relates to a composition for treating wounds comprising peptide fragments (small peptides) derived from an epidermal growth factor (EGF); And a composition for inhibiting skin aging or skin wrinkle formation. The peptide activates the epidermal growth factor receptor and also has an activity to promote dermal keratinocyte proliferation and angiogenesis.

Wounds are sometimes referred to as wounds, and the symptoms include pain, bleeding, and dysfunction. The wound creates a unique microenvironment consisting of growth factors, cytokines, neurohormones, and extracellular matrix, secreted from surrounding cells, blood-derived cells and sensory neurons. Some factors of the wound microenvironment lasted long enough to spread to the peripheral blood, which then affected the bone marrow stem cells, leading to migration of bone marrow cells to peripheral blood, supply to wound sites, and participation in wound healing .

In order to maintain the elasticity and strength of the skin without destroying the connective tissue of the skin, extracellular matrix such as collagen I, III (Type I, III collagens), elastin, fibronectin (ECM: Extracellular matrix) proteins are important. Chronic exposure to natural aging or ultraviolet light reduces ECM protein loss and elasticity and strength of skin tissue, and these phenomena are strongly associated with characteristic skin aging, such as wrinkling. The matrix metalloproteinases (MMPs) act as enzymes that are important in the degradation of dermal ECMs. Overexpression of MMPs in keratinocytes and dermal fibroblasts under stress conditions, such as ultraviolet irradiation, contribute to the degradation of the ECM protein, thereby creating wrinkles in the skin.

On the other hand, epidermal growth factor (EGFR) binds to epidermal growth factor receptor (EGFR) and is one of the growth factors that regulate the proliferation, differentiation and survival of various cells. In particular, it plays a major role in re-epithelization by promoting keratinocyte proliferation and cell migration, and is known to function excellently in wound healing. EGF is secreted by platelets, macrophages, and fibroblasts, and acts as a paracrine fashion in keratinocytes. EGF increases the expression of keratin K6 and K16 and activates cell proliferation signal transduction. Clinically, when external epidermal growth factor is applied to chronic wounds such as tissue grafts and diabetic foot stones, it is known that epithelization is accelerated and the wound healing rate is shortened (Barrientos S, Stojadinovic O, Golinko M, Brem H, Vinay Tomic-Canic M. 2008. Growth factors and cytokines in wound healing. Wound Rep Reg . 16: 585). When the epidermal growth factor receptor is activated, it induces the production of growth factors such as VEGF and HGF to promote the growth of keratinocytes and fibroblasts, and induces the production of extracellular matrix such as collagen in fibroblasts, Or improve skin wrinkles.

The present inventors have designed various small peptide fragments that activate the epidermal growth factor receptor expressed in the keratinocytes and fibroblasts of the skin. Surprisingly, certain peptide fragments (peptide fragments or small peptides) And promotes epidermal re-epithelization by promoting keratinocyte proliferation and angiogenesis.

Accordingly, it is an object of the present invention to provide a pharmaceutical composition for promoting wound healing or wound healing, which comprises a specific peptide fragment derived from epidermal growth factor as an effective ingredient.

It is another object of the present invention to provide a cosmetic composition for inhibiting skin aging or inhibiting the formation of wrinkles of skin comprising the above specific peptide fragment.

According to one aspect of the present invention, there is provided a pharmaceutical composition comprising a peptide selected from the group consisting of peptides of SEQ ID NOS: 1-15 as an active ingredient, and a pharmaceutically acceptable carrier, for promoting wound healing or wound healing. In one embodiment, a pharmaceutical composition comprising the peptide of SEQ ID NO: 9 is provided as an active ingredient.

According to another aspect of the present invention, there is provided a cosmetic composition for inhibiting skin aging or preventing skin wrinkle formation comprising, as an active ingredient, a peptide selected from the group consisting of peptides of SEQ ID NOS: 1 to 15. In one embodiment, a cosmetic composition comprising the peptide of SEQ ID NO: 9 is provided as an active ingredient.

It has been found that certain peptide fragments derived from epidermal growth factor according to the present invention activate epidermal growth factor receptors and promote epidermal re-epithelization by promoting keratinocyte proliferation and angiogenesis. Therefore, the peptide can be usefully applied to a pharmaceutical composition for promoting wound healing or wound healing, and a cosmetic composition for inhibiting skin aging or inhibiting skin wrinkle formation.

Figures 1 and 2 show the results of evaluating activation of epidermal growth factor receptor by peptides according to the present invention.
FIGS. 3 and 4 show results of evaluating phosphorylation of ERK and Akt by peptides according to the present invention in human keratinocyte HaCaT (FIG. 3) and mouse fibroblast NIH3T3 (FIG. 4), respectively.
FIG. 5 shows the results of evaluating the effect of peptides according to the present invention on proliferation of keratinocytes.
FIG. 6 shows the results of evaluating the activity of promoting angiogenesis by the peptides according to the present invention.

As used herein, the term "wound " refers to damage to epithelial and connective tissue caused by intrinsic and extrinsic factors, and may preferably be skin damage.

The term "skin aging" refers to aging of the skin caused by intrinsic and external factors, and may be skin photoaging accompanied by skin wrinkle formation, more preferably skin And skin photo-aging by ultraviolet stimulation accompanied by wrinkle formation.

Also, "skin wrinkle" refers to wrinkle formation on skin.

The present invention provides a pharmaceutical composition comprising a peptide selected from the group consisting of the peptides of SEQ ID NOS: 1 to 15 as an active ingredient and a pharmaceutically acceptable carrier for promoting wound healing or wound healing.

In addition, the present invention provides a cosmetic composition for inhibiting skin aging or inhibiting skin wrinkle formation, comprising a peptide selected from the group consisting of peptides of SEQ ID NOS: 1 to 15 as an active ingredient.

The inventors prepared fragments of various lengths from epidermal growth factor and searched for their activity. Surprisingly, as a peptide derived from epidermal growth factor, a specific peptide fragment having a short length composed of 3 or 4 amino acids activates epidermal growth factor receptor, promotes keratinocyte proliferation and angiogenesis, . Therefore, the peptide according to the present invention has excellent activity for wound healing and skin regeneration. In addition, it can be effectively applied to a cosmetic composition for improving skin aging and / or suppressing the formation of wrinkles of skin, which is accompanied by skin inflammation caused by external stimuli such as ultraviolet rays. The peptide fragments are shown in Table 1 below. In one embodiment, the peptide of the invention may be the peptide of SEQ ID NO: 9.

Peptide name SEQ ID NO: Amino acid sequence SE208A SEQ ID NO: 1 VCMY SE208 B SEQ ID NO: 2 YACN SE208 C SEQ ID NO: 3 ACNC SE208 D SEQ ID NO: 4 CNCV SE208 E SEQ ID NO: 5 NCVV SE208 F SEQ ID NO: 6 CVVG SE208 G SEQ ID NO: 7 VVGY SE208 H SEQ ID NO: 8 VCM SE208 I SEQ ID NO: 9 YAC SE208 J SEQ ID NO: 10 ACN SE208 K SEQ ID NO: 11 CNC SE208 L SEQ ID NO: 12 NCV SE208 M SEQ ID NO: 13 CVV SE208 N SEQ ID NO: 14 VVG SE208 O SEQ ID NO: 15 VGY

The pharmaceutical composition of the present invention may contain an excipient such as lactose, corn starch, a lubricant such as magnesium stearate, a known emulsifier, a suspending agent, a buffer, an isotonic agent, etc., And may be formulated into parenteral administration forms containing external preparations for skin. For intramuscular, intraperitoneal, subcutaneous, and intravenous dosage forms, it may usually comprise a buffer which is capable of producing a sterile solution of the active ingredient and suitably adjusting the pH of the solution, and may be isotonic Lt; / RTI > The pharmaceutical composition of the present invention may also be in the form of an aqueous solution comprising a pharmaceutically acceptable carrier such as a saline solution having a pH of 7.4 and can be introduced locally into the intramuscular blood stream of the patient in the form of a solution have. In addition, it can be formulated into a transdermal dosage form such as a solution for external use, an emulsion, an ointment, a patch or the like according to a conventional pharmaceutical method. The pharmaceutical composition of the present invention may be administered to a patient having various wounds at a dose of about 1 to 2000 mg / kg per day. Appropriate dosages can generally vary depending on the age, weight and symptoms of the patient.

The cosmetic composition of the present invention may be in the form of a functional cosmetic composition containing the peptide as an active ingredient. The cosmetic composition may be manufactured in various forms according to a conventional cosmetic preparation method. For example, the cosmetic composition may be prepared in the form of a cosmetic product containing the peptide, a lotion, a cream, a lotion or the like, which may be diluted with a conventional cleansing solution, a converging solution and a moisturizing solution. The cosmetic composition may also contain conventional adjuvants such as stabilizers, solubilizers, vitamins, pigments, and flavorings commonly used in the cosmetic composition arts. In the cosmetic composition, the content of the peptide may be an amount effective to achieve skin aging inhibition, particularly skin wrinkle formation inhibiting effect, for example, in an amount of 0.001 to 10% by weight based on the total weight of the composition, By weight may be contained in an amount of about 0.01 to 1% by weight.

Hereinafter, the present invention will be described in more detail through examples and test examples. However, these examples and test examples are for illustrating the present invention, and the present invention is not limited to these examples and test examples.

Example  One. Of peptide  synthesis

Peptides of SEQ ID NOS: 1 to 15 were synthesized by the FMOC solid-phase method using an automated synthesizer (PeptrEx-R48, Peptron, Daejeon, Korea). The synthesized peptides were purified and analyzed by reversed-phase high-performance liquid chromatography (RP-20AB, Prominence LC-20AB, Shimadzu, Japan) using a C18 analysis RP column (Shiseido capcell pak) LC / MSD, Hewlett-Packard, Roseville, USA).

Example  2. Peptides  Preparation of a Composition Containing

Peptides of SEQ ID NOS: 1 to 15 were each dissolved in PBS to prepare a 1 M concentration. The resulting protein solution was used in the following test examples.

Test Example  1. Evaluation of epidermal growth factor receptor activation

In situ proximity ligation assay was used to confirm that the peptide of the present invention activates epidermal growth factor receptor. The test was performed by purchasing a Duolink In Situ reagent from Olink Bioscience, Sweden.

1 mL of DMEM medium was added to a 24-well plate on which 12 mm glass was placed, 4.5 × 10 ⁴ human keratinocyte HaCaT cells (DKFZ, Heidelberg, Germany) were plated and stabilized for 24 hours. A solution obtained by dissolving the peptides of SEQ ID NOS: 1 to 15 in serum-free DMEM medium was added to 10 [mu] M and fixed with 4% formaldehyde after 15 minutes. Control group means untreated group. One drop of blocking solution was added to the cells of each well and incubated at 37 占 폚 for 30 minutes. (Gab1 Antibody, Cell Signaling Technology, INC., USA) and anti-Shp2 mouse monoclonal antibody (SH-1) were used to measure the degree of interaction between Gab1 and Shp2, PTP2 (B-1): sc-7384 mouse mAb, Santa Cruz Biotechnology, INC.) Was treated at 37 ° C. After 30 minutes, the cells were treated with PLA probe solution, incubated at 37 ° C for 1 hour, treated with ligation solution, and incubated for 30 minutes. amplification solution and incubated for 100 minutes. The cells treated as described above were washed with washing buffer, and the number of red dots was measured using confocal microscopy. The results are shown in FIGS. 1 and 2. As can be seen from Fig. 1, when the peptides of SEQ ID NOS: 1 to 7 having four amino acids were treated, the interaction between Gab1 and Shp2 was increased to about 50 to 100% as compared with the control. As can be seen from Fig. 2, when the peptide of SEQ ID NOS: 8 to 15 having three amino acids was treated, the interaction between Gab1 and Shp2 was increased to about 30 to 170% as compared with the control. This indicates that the peptide according to the present invention activates epidermal growth factor receptor which plays a major role in skin regeneration.

Test Example  2. ERK Wow Akt  Activation evaluation

In order to evaluate activation of the epidermal growth factor receptor signaling pathway in keratinocytes and fibroblasts by the treatment of the peptides of the present invention, peptides of SEQ ID NO: 9 were added to serum-free DMEM medium at 0.05, 0.1, 0.5, 1, 5, (DKFZ, Heidelberg, Germany) and mouse fibroblast NIH3T3 (ATCC CRL-1658, USA) were treated with human keratinocyte HaCaT (DKFZ, Heidelberg, Germany) with a solution (1 ml) After 15 minutes, the cell extracts were subjected to Western blotting analysis to determine changes in phosphorylation of ERK and Akt. The results are shown in FIGS. 3 and 4. FIG. As can be seen in Figs. 3 and 4, the degree of phosphorylation of ERK and Akt was increased in a concentration-dependent manner by the peptide of SEQ ID NO: 9. Thus, it can be confirmed that the peptide according to the present invention activates epidermal growth factor receptor expressed in keratinocytes and fibroblasts.

Test Example  3. People Keratinocyte HaCaT Proliferation test

5 × 10 ³ HaCaT cells (DKFZ, Heidelberg, Germany) were dispensed into each well of a 96-well microplate in 100 μL of DMEM medium and incubated in a 5% CO ₂ incubator at 37 ° C. for 24 hours. Was dissolved in a serum-free DMEM medium at a concentration of 0.01, 0.1, or 1 μM (100 μL). Control group means untreated group. After incubation for 24, 48, and 72 hours, the cells were treated with 10 μL of CCK-8 (Dojindo Laboratories, Japan) for 2 hours per well and the absorbance at 450 nm was measured with Spectramax plus 190 plate reader (Molecular Devices, USA) Respectively. The results are shown in Fig. As can be seen from Fig. 5, when the peptide of SEQ ID NO: 9 was treated, the proliferation of HaCaT cells was increased in a concentration-dependent manner depending on the treatment concentration of the peptide. This indicates that the peptide according to the present invention promotes the proliferation of keratinocytes.

Test Example  4. In vitro in vitro ) Angiogenesis Promotion Test

The effects of the peptides according to the present invention on angiogenesis were evaluated as follows. In general, the interaction between the blood vessel component and the endothelial cells is an important factor for the formation and maintenance of the neovascularization. When Matrigel, a complex of the components of the basal membrane, is placed in a 24-well plate, ). Human umbilical vein endothelial cells (HUVECs) were inoculated into a 24-well cell culture plate coated with Matrigel at a density of 8 x 10 ⁴ cells / well, and the peptide of SEQ ID NO: 9 was cultured in serum-free M199 medium (1 mL) obtained by dissolving VEGF in a concentration of 1, 10, or 100 μM was added and cultured. As a control group, a cell culture plate to which no peptide solution was added was added with Vascular Endothelial Growth Factor (VEGF , 10 ng / ml) was added to each sample. After 24 hours, the cells were dilated with an x50 magnification under an inverted microscope to observe and quantitate the formation of neovascularization, and the results are shown in Fig. As can be seen, when treated with a solution containing the peptide of the present invention, angiogenesis is markedly promoted.

<110> KNU-INDUSTRY COOPERATION FOUNDATION <120> COMPOSITION FOR WOUND-HEALING OR INHIBITING WRINKLE FORMATION ON          SKIN COMPRISING PEPTIDE FRAGMENTS DERIVED FORM EPIDERMAL GROWTH          FACTOR <130> PN0697 <160> 15 <170> Kopatentin 2.0 <210> 1 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Peptide fragment <400> 1 Val Cys Met Tyr   One <210> 2 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Peptide fragment <400> 2 Tyr Ala Cys Asn   One <210> 3 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Peptide fragment <400> 3 Ala Cys Asn Cys   One <210> 4 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Peptide fragment <400> 4 Cys Asn Cys Val   One <210> 5 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Peptide fragment <400> 5 Asn Cys Val   One <210> 6 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Peptide fragment <400> 6 Cys Val Val Gly   One <210> 7 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Peptide fragment <400> 7 Val Val Gly Tyr   One <210> 8 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Peptide fragment <400> 8 Val Cys Met   One <210> 9 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Peptide fragment <400> 9 Tyr Ala Cys   One <210> 10 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Peptide fragment <400> 10 Ala Cys Asn   One <210> 11 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Peptide fragment <400> 11 Cys Asn Cys   One <210> 12 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Peptide fragment <400> 12 Asn Cys Val   One <210> 13 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Peptide fragment <400> 13 Cys Val Val   One <210> 14 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Peptide fragment <400> 14 Val Val Gly   One <210> 15 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Peptide fragment <400> 15 Val Gly Tyr   One

Claims (4)

A pharmaceutical composition comprising a peptide selected from the group consisting of peptides of SEQ ID NOS: 1 to 15 as an active ingredient and comprising a pharmaceutically acceptable carrier. The pharmaceutical composition according to claim 1, which comprises the peptide of SEQ ID NO: 9 as an active ingredient. A cosmetic composition for inhibiting skin aging or preventing skin wrinkle formation, comprising a peptide selected from the group consisting of peptides of SEQ ID NOS: 1 to 15 as an active ingredient. The cosmetic composition according to claim 3, which comprises the peptide of SEQ ID NO: 9 as an active ingredient.

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