WO2016006733A1 - Composition containing fibroblast growth factor-derived peptide fragments for wound healing or inhibiting skin wrinkle formation - Google Patents

Abstract

Provided is a composition containing peptide fragments or small peptides derived from fibroblast growth factor (FGF) for wound healing or inhibiting aging of the skin or formation of skin wrinkles. The peptides activate FGF receptors to induce collagen synthesis and have activities promoting keratinocyte proliferation and angiogenesis in the skin. Accordingly, the peptides can be effectively utilized in wound healing and inhibiting aging of the skin or formation of skin wrinkles.

Description

Composition for inhibiting wound or skin wrinkle formation comprising peptide fragment derived from fibroblast growth factor

The present invention relates to a wound healing composition comprising peptide fragments (small peptides or small peptides) derived from fibroblast growth factor (FGF); And it relates to a composition for inhibiting skin aging or wrinkle formation. The peptide activates the fibroblast growth factor receptor to induce collagen synthesis, and also has activity to promote skin keratinocyte proliferation and angiogenesis.

Wounds are sometimes referred to as wounds, and their symptoms include pain, bleeding, and dysfunction. Wounds create a unique microenvironment consisting of growth factors, cytokines, neurohormones, and extracellular matrix secreted from surrounding cells, blood derived cells, and sensory neurons. Some factors in the wound microenvironment persist long enough to spread into peripheral blood and then affect stem cells in the bone marrow, causing the bone marrow cells to migrate into peripheral blood, supply to the wound site, and participate in wound healing. It is known.

Extracellular matrix such as collagen I, III (Type I, III collagens), elastin, and fibronectin present in the skin to maintain its elasticity and strength without breaking the connective tissue of the skin Extracellular matrix (ECM) proteins are important. Natural aging or chronic exposure to UV rays reduces the loss of ECM proteins and the elasticity and strength of skin tissues, which are strongly associated with characteristic skin aging such as wrinkles. Matrix metalloproteinases (MMPs) are important enzymes in the degradation of dermal ECM. Under stress conditions such as UV irradiation, overexpression of MMPs in keratinocytes and dermal fibroblasts contributes to the degradation of ECM proteins, thereby forming wrinkles on the skin.

Meanwhile, fibroblast growth factor is one of growth factors that control the proliferation, migration, differentiation and survival of various cells including fibroblasts, and is composed of a group of 23 proteins. FGF is produced in keratinocytes, fibroblasts, vascular endothelial cells, striated myocytes, chondrocytes and mast cells and exhibits the above-described function by activating four kinds of fibroblast growth factor receptors (FGF Receptors) expressed in various cells. In particular, FGF-2, FGF-7 and FGF-10 are known to play an important role in skin regeneration (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). FGF-2 increases the expression of acute wounds and functions as re-epithelialization of the epidermis, and acts on fibroblasts in the dermal layer to induce granulation tissue formation and tissue remodeling through the production of extracellular matrix such as collagen. It is also known to promote the expression of skin barrier components and hair growth. FGF-7 and FGF-10 are involved in the re-epithelial process by promoting the proliferation of keratinocytes, and also plays a role in maintaining keratinocytes through the detoxification of free radicals. FGF-7 also stimulates the growth of fibroblasts and keratinocytes by inducing the production of growth factors such as VEGF and HGF.

The present inventors have designed various small peptide fragments that activate fibroblast growth factor receptors expressed in keratinocytes and fibroblasts of the skin, and surprisingly, specific peptide fragments or small peptides derived from fibroblast growth factor are derived from fibroblasts. It has been found that activating growth factor receptors and promoting the proliferation and angiogenesis of keratinocytes can promote epithelial re-epithelialization. In addition, it has been found that the formation of extracellular matrix such as collagen can be inhibited by acting on fibroblasts of the dermal layer to inhibit skin wrinkle formation.

Accordingly, an object of the present invention is to provide a pharmaceutical composition for treating wounds or promoting wound healing comprising a specific peptide fragment derived from fibroblast growth factor as an active ingredient.

In addition, an object of the present invention is to provide a cosmetic composition for inhibiting skin aging or inhibiting skin wrinkle formation comprising the specific peptide fragment.

According to one aspect of the invention, there is provided a pharmaceutical composition for the treatment of wounds or promoting wound healing comprising a peptide selected from the group consisting of the peptides of SEQ ID NOs: 1 to 13 as an active ingredient, and comprising a pharmaceutically acceptable carrier. In one embodiment, provided is a pharmaceutical composition comprising the peptide of SEQ ID NO: 1 as an active ingredient.

According to another aspect of the present invention, there is provided a cosmetic composition for inhibiting skin aging or skin wrinkle formation, comprising a peptide selected from the group consisting of the peptides of SEQ ID NOs: 1 to 13 as an active ingredient. In one embodiment, a cosmetic composition comprising a peptide of SEQ ID NO: 1 as an active ingredient is provided.

In accordance with the present invention it has been found that certain peptide fragments derived from fibroblast growth factor promote the re-epithelialization of the epidermis by activating the fibroblast growth factor receptor and promoting the proliferation and angiogenesis of keratinocytes. In addition, it has been found that the formation of extracellular matrix such as collagen can be suppressed by acting on fibroblasts of the dermal layer to inhibit skin wrinkle formation. Therefore, the peptides may be usefully applied to pharmaceutical compositions for treating wounds or promoting wound healing, and cosmetic compositions for inhibiting skin aging or inhibiting skin wrinkle formation.

1 and 2 show the results of evaluation of phosphorylation of ERK and Akt by the peptide according to the present invention in human keratinocytes HaCaT.

Figure 3 shows the results of the evaluation of phosphorylation of ERK and Akt by the peptide according to the invention in mouse fibroblast NIH3T3.

Figure 4 shows the results of evaluating the effect of the peptides according to the invention on the proliferation of keratinocytes.

Figure 5 shows the results of evaluating the effect of the peptides according to the invention on collagen synthesis in fibroblasts.

Figure 6 shows the results of evaluating the angiogenic activity by the peptides according to the present invention.

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

In addition, "skin aging" refers to aging of the skin caused by intrinsic and external factors, preferably skin photoaging accompanied with the formation of skin wrinkles, more preferably skin Skin photoaging due to ultraviolet irritation accompanied by wrinkle formation.

In addition, the term "skin wrinkle" refers to wrinkle formation on skin.

The present invention provides a pharmaceutical composition for promoting wound healing or wound treatment comprising a peptide selected from the group consisting of the peptides of SEQ ID NOs: 1 to 13 as an active ingredient, and comprising a pharmaceutically acceptable carrier.

The present invention also provides a cosmetic composition for inhibiting skin aging or inhibiting skin wrinkles, comprising a peptide selected from the group consisting of the peptides of SEQ ID NOs: 1 to 13 as an active ingredient.

We prepared fragments of various lengths from fibroblast growth factor and searched for their activity. Surprisingly, as a peptide derived from fibroblast growth factor, specific peptide fragments of short length consisting of 3 to 5 amino acids activate the fibroblast growth factor receptor, promote the growth of keratinocytes and promote angiogenesis. It has been found to promote epithelialization. In addition, it has been found that the formation of extracellular matrix such as collagen can be inhibited by acting on fibroblasts of the dermal layer to inhibit skin wrinkle formation. Thus, the peptides according to the invention have excellent activity on wound healing and skin regeneration. In addition, the present invention may be usefully applied to cosmetic compositions for improving skin aging and / or suppressing skin wrinkle formation accompanying skin irritation caused by external stimuli such as ultraviolet rays. The peptide fragments are shown in Table 1 below. In one embodiment, the peptide of the present invention may be a peptide of SEQ ID NO: 1.

Table 1

Peptide Name	SEQ ID NO:	Amino acid sequence
SE204 A	SEQ ID NO: 1	FLE
SE204 B	SEQ ID NO: 2	FRE
SE204 C	SEQ ID NO: 3	FAE
SE204 D	SEQ ID NO: 4	FEE
SE204 E	SEQ ID NO: 5	FTE
SE204 F	SEQ ID NO: 6	FKE
SE204 G	SEQ ID NO: 7	FFE
SE204 H	SEQ ID NO: 8	FGE
SE204 I	SEQ ID NO: 9	YNVYQ
SE204 J	SEQ ID NO: 10	YNVY
SE204 K	SEQ ID NO: 11	YNTY
SE204 L	SEQ ID NO: 12	YVIY
SE204 M	SEQ ID NO: 13	YTAL

The pharmaceutical composition of the present invention may include excipients such as lactose, corn starch, lubricants such as magnesium stearate, emulsifiers, suspending agents, buffers, tonicity agents, and the like which are well known, and may be used in parenteral dosage forms, preferably It may be formulated in parenteral dosage forms including external dermal preparations. For intramuscular, intraperitoneal, subcutaneous and intravenous dosage forms, a sterile solution of the active ingredient is usually prepared and may comprise a buffer which can suitably adjust the pH of the solution, and for intravenous administration isotonic in the formulation. May be included to impart this. In addition, the pharmaceutical composition of the present invention may be in the form of an aqueous solution containing a pharmaceutically acceptable carrier such as saline having a pH of 7.4, and may be locally introduced into the patient's intramuscular blood flow in the form of a solution. have. In addition, it may be formulated into a transdermal dosage form such as an external preparation, emulsion, ointment, patch, etc. according to conventional pharmaceutical methods. The pharmaceutical composition of the present invention can be administered to patients with various wounds at a dose of about 1 to 2000 mg / kg per day. Appropriate dosages will 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 comprising the peptide as an active ingredient. The cosmetic composition may be prepared 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, lotion, cream, lotion, etc., which may be diluted with a conventional cleansing liquid, astringent liquid and moisturizing liquid. In addition, the cosmetic composition may include conventional adjuvants such as stabilizers, solubilizers, vitamins, pigments, and flavorings commonly used in the field of cosmetic compositions. In the cosmetic composition, the content of the peptide may be contained in an amount effective to achieve an effect of inhibiting skin aging, in particular, skin wrinkle formation, for example, in an amount of 0.001 to 10% by weight based on the total weight of the composition. It 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 1 Synthesis of Peptides

Peptides of SEQ ID NOS: 1 to 13 were synthesized by the FMOC solid-phase method using an automated synthesizer (PeptrEx-R48, Peptron, Daejeon, Korea). The synthesized peptide was purified and analyzed by reverse-phase HPLC using a C18 analytical RP column (Shiseido capcell pak) (Prominence LC-20AB, Shimadzu, Japan), and mass spectrometer (HP 1100 Series). LC / MSD, Hewlett-Packard, Roseville, USA).

Example 2. Preparation of a Composition Comprising a Peptide

Peptides of SEQ ID NO: 1 to 13 were dissolved in PBS, respectively, to prepare a concentration of 1 M. The obtained protein solution was used in the following test example.

Test Example 1 Evaluation of Activation of Fibroblast Growth Factor Receptor in Keratinocytes

Human keratinocytes HaCaT (DKFZ, Heidelberg, Germany) were prepared using the peptides of SEQ ID NOs. The solutions obtained by dissolving in each were treated to 10 µM. Control means untreated group. After 15 minutes, Western blotting analysis was performed on the cell extracts to measure the change in phosphorylation of ERK and Akt, and the results are shown in FIGS. 1 and 2. As can be seen in Figures 1 and 2, the degree of phosphorylation of ERK and Akt was significantly increased by the peptides of SEQ ID NOS: 1 to 13. Therefore, it can be seen that the peptide according to the present invention activates the fibroblast growth factor receptor expressed in keratinocytes.

Test Example 2 ERK and Akt Activation Test in Fibroblasts

In order to evaluate whether the fibroblast growth factor receptor signaling pathway is activated in fibroblasts by treatment of the peptide of the present invention, the peptide of SEQ ID NO: 1 was prepared in serum-free DMEM medium of 0.05, 0.1, 0.5, 1, 5, 10 μM. Mouse fibroblast NIH3T3 (ATCC CRL-1658, USA) was treated with a solution (1 ml) obtained by lysis at a concentration. After 15 minutes, Western blotting analysis was performed on the cell extracts to measure the change in phosphorylation of ERK and Akt, and the results are shown in FIG. 3. As can be seen in Figure 3, the phosphorylation of ERK and Akt was increased in a concentration-dependent manner by the peptide of SEQ ID NO: 1. Therefore, it can be seen that the peptide according to the present invention activates the fibroblast growth factor receptor expressed in fibroblasts.

Experimental Example 3. Proliferation promoting test of human keratinocyte line HaCaT

In each well of a 96-well microplate, 5 × 10 ³ HaCaT cells (DKFZ, Heidelberg, Germany) were dispensed in 100 μL of DMEM medium and incubated in 37 ° C., 5% CO ₂ incubator for 24 hours, and then the medium was removed, A solution obtained by dissolving the peptide of SEQ ID NO: 1 in a serum-free DMEM medium at a concentration of 0.01, 0.1, or 1 μM (100 μL) was added. The control group added only 100 μL of serum free medium. Then incubate for 24, 48 and 72 hours, treat for 2 hours with 10 μL of CCK-8 (Dojindo Laboratories, Japan) per well and absorbance at 450 nm with Spectramax plus 190 plate reader (Molecular Devices, USA). Measured. The result is shown in FIG. 4. As can be seen in Figure 4, when the peptide of SEQ ID NO: 1, the proliferation of HaCaT cells increased concentration-dependently depending on the concentration of the peptide. This indicates that the peptide according to the present invention promotes the proliferation of keratinocytes.

Test Example 4 Evaluation of Procollagen Type-1 Expression in Human Dermal Fibroblasts

The promotion of the synthesis of glue fibers is a major means to suppress skin aging caused by ultraviolet stimulation. It was evaluated using human dermal fibroblasts Detroit 551 (ATCC CCL-110, USA) to promote collagen synthesis in fibroblasts by treatment of the peptides of the present invention. Human dermal fibroblasts were treated with a solution (1 ml) obtained by dissolving the peptide of SEQ ID NO: 1 in serum-free DMEM medium at concentrations of 0.05, 0.1, 0.5, and 1 μM. After 48 hours, Western blotting analysis was performed on the cell extracts to measure the change in the expression level of procollagen type 1, and the results are shown in FIG. 5. As can be seen in Figure 5, the expression level of procollagen type 1 was increased in a concentration-dependent manner by the peptide of SEQ ID NO: 1. Therefore, it can be seen that the peptide according to the present invention can suppress aging of the skin due to collagen loss, in particular, skin wrinkles.

Test Example 5 In vitro Angiogenesis Promoting Test

The effect of the peptides according to the invention on angiogenesis was evaluated as follows. In general, the interaction of the vascular endothelial components with vascular endothelial cells is an important factor in the formation and maintenance of neovascularization. When the matrigel, which is a complex of the bottom membrane components, is put into a 96-well plate, a polymerization reaction occurs and a plug ). Human umbilical vein endothelial cells (HUVECs) were inoculated in a 96 well cell culture plate coated with Matrigel at a density of 1.5 x 10 ⁴ cells / well, and the peptide of SEQ ID NO: 1 was serum-free M199 medium. A solution obtained by dissolving at a concentration of 1, 10, or 100 μM ((100 μL) was added and cultured. As a control, Vascular Endothelial Growth Factor (VEGF) was added to a cell culture plate without adding a peptide solution. , 10 ng / ml) was added and not added, after 24 hours, magnified at x50 magnification under an inverted microscope to observe the formation of neovascularization and quantified the results are shown in Fig. 6. As can be seen, when treated with a solution containing the peptide of the present invention, it can be seen that angiogenesis is significantly promoted.

Claims (4)

1. A pharmaceutical composition for treating wounds or promoting wound care comprising a peptide selected from the group consisting of the peptides of SEQ ID NOs: 1 to 13 as an active ingredient, and comprising a pharmaceutically acceptable carrier.
2. The pharmaceutical composition according to claim 1, comprising the peptide of SEQ ID NO: 1 as an active ingredient.
3. A cosmetic composition for inhibiting skin aging or inhibiting skin wrinkles, comprising a peptide selected from the group consisting of the peptides of SEQ ID NOs: 1 to 13 as an active ingredient.
4. According to claim 3, Cosmetic composition characterized in that it comprises a peptide of SEQ ID NO: 1 as an active ingredient.

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