WO2025110755A1 - Composition for wound healing or inhibiting skin wrinkle formation or moisturizing skin - Google Patents

Abstract

The present invention provides: a pharmaceutical composition for wound healing, comprising a specific peptide; and a cosmetic composition for inhibiting skin wrinkle formation or moisturizing skin. The peptide acts as an agonist for a GLP -2 receptor, thereby effectively inducing GLP-2 signaling and inducing cell proliferation, and promoting the expression of fatty acid synthase, ceramide synthase 3 (CERS3), filaggrin, involucrin, and loricrin, which are proteins related to the skin barrier. Therefore, the peptide can be effectively used for: wound healing; and inhibiting skin wrinkle formation or moisturizing skin.

Description

Composition for wound healing or skin wrinkle prevention or skin moisturizing

The present invention relates to a pharmaceutical composition for wound treatment comprising a specific peptide; and a composition for inhibiting skin wrinkle formation or moisturizing the skin. The peptide has an activity of activating a glucagon-like peptide 2 (GLP-2) receptor and promoting the expression of fatty acid synthase, ceramide synthase 3 (CERS3), filaggrin, involucrin, and loricrin, which are proteins related to the skin barrier.

GLP-2 is a peptide hormone consisting of 33 amino acids produced in the L cells of the small intestine, and plays a role in energy absorption and protection, and in activating intestinal cell function. (https:/www.proteinatlas.org/ENSG00000065325-GLP2R/tissue; Curr Protein Pept Sci.(2004) 5(1):51-65). In addition, GLP-2 is known to help skin regeneration by inducing skin cell proliferation (Daniel J Drucker, et al, GMol Endocrinol. 2003 Feb;17(2):161-171).

The skin barrier is a barrier formed in the outermost layer of the skin, the stratum corneum, that protects the skin, preventing moisture and electrolytes from escaping and bacteria and pathogens from penetrating the skin. Therefore, the role of the skin barrier is known to be very important in skin hydration and aging. The skin barrier is composed of keratinocytes containing natural moisturizing factors and intercellular lipids, that is, a wall structure of lipid components that connect keratinocytes and each other. The intercellular lipid layer has a lamellar structure in which several layers of membranes are arranged in a row, and is composed of lipid components such as ceramides and fatty acids. In addition, a structural support protein layer composed of proteins such as filaggrin, involucrin, and loricrin forms the cell shell of keratinocytes and plays a role in maintaining the skin barrier (Yeonjoon Kim, et al., Arch Pharm Res. 2021 Jan;44(1):36-48; Byung Eui Kim, et al., Allergy Asthma Immunol Res. 2018 May;10(3):207-215, etc.). Therefore, a substance that can activate the GLP-2 receptor can act not only as an active substance for wound healing or promoting wound healing, but also as a functional cosmetic substance for inhibiting skin aging (e.g., skin wrinkle formation) or moisturizing the skin.

The present inventors have disclosed that a peptide derived from fibroblast growth factor (FGF) activates the fibroblast growth factor receptor to induce collagen synthesis and also has the activity of promoting skin keratinocyte proliferation and angiogenesis (Korean Patent Registration No. 10-1772048).

The present inventors have conducted various studies to develop an active substance based on small peptides as a substance capable of activating the GLP-2 receptor. As a result, it was found that a specific peptide, that is, a 4-mer peptide composed of Leu-Ser-Ser-Phe, acts as a substance capable of activating the GLP-2 receptor [i.e., an agonist for the GLP-2 receptor], thereby effectively inducing GLP-2 signaling, and inducing cell proliferation and promoting the expression of fatty acid synthase, ceramide synthase 3 (CERS3), filaggrin, involucrin, and loricrin, which are proteins related to the skin barrier, and thus can be usefully applied to wound healing, inhibition of skin wrinkle formation, or skin moisturizing.

Accordingly, the present invention aims to provide a pharmaceutical composition for wound treatment or promoting wound treatment comprising the specific peptide as an active ingredient.

In addition, the present invention aims to provide a cosmetic composition for inhibiting skin aging or moisturizing skin, which contains the specific peptide.

According to one aspect of the present invention, a pharmaceutical composition for wound treatment or promoting wound treatment is provided, comprising a peptide of the following chemical formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

<Chemical Formula 1>

According to another aspect of the present invention, a cosmetic composition for inhibiting skin wrinkle formation or moisturizing skin is provided, comprising the peptide of the chemical formula 1 or a pharmaceutically acceptable salt thereof.

The present invention has revealed that a peptide according to the present invention (i.e., a peptide composed of Leu-Ser-Ser-Phe) binds to an intracellular GLP-2 receptor, induces GLP-2 signaling in a concentration-dependent manner, induces cell proliferation in a concentration-dependent manner, and promotes the expression of fatty acid synthase, ceramide synthase 3 (CERS3), filaggrin, involucrin, and loricrin, which are proteins related to the skin barrier. The present invention has also revealed that the peptide according to the present invention effectively induces filaggrin synthesis in a 3D human skin model. Therefore, the peptide according to the present invention can be usefully applied to a pharmaceutical composition for wound healing or promoting wound healing; and a cosmetic composition for inhibiting skin aging or moisturizing skin, including inhibition of skin wrinkle formation.

Figure 1 shows the results of measuring the signal reduction of the peptide (VE-Glytin)-AMC of the present invention composed of Leu-Ser-Ser-Phe by GLP-2 siRNA treatment.

Figure 2 shows the results of analyzing the change in β-catenin/TCF interaction by peptide treatment of the present invention.

Figure 3 shows the results of analyzing the change in β-catenin/TCF interaction by the peptide of the present invention and GLP2 siRNA treatment.

Figure 4 shows the results of analyzing the cell proliferation rate by peptide treatment of the present invention.

Figure 5 shows the results of analyzing the change in the expression level of skin barrier-related proteins by peptide treatment of the present invention.

Figure 6 shows the results of analyzing the change in the expression level of skin barrier-related proteins by peptide treatment of the present invention in a 3D human skin model.

The present invention provides a pharmaceutical composition for wound treatment or promoting wound treatment, comprising a peptide of the following chemical formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

<Chemical Formula 1>

In addition, the present invention provides a cosmetic composition for inhibiting skin wrinkle formation or moisturizing the skin, comprising the peptide of the chemical formula 1 or a pharmaceutically acceptable salt thereof.

In the pharmaceutical composition or cosmetic composition of the present invention, the peptide of Chemical Formula 1 may also be expressed as "Leu-Ser-Ser-Phe". The amino acids constituting the peptide of Chemical Formula 1 may independently be in the form of L-amino acid or D-amino acid. Pharmaceutically acceptable salts of the peptide derivative of Chemical Formula 1 include, but are not limited to, acid addition salts, for example.

The pharmaceutical composition of the present invention may contain excipients such as lactose and corn starch, lubricants such as magnesium stearate, emulsifiers, suspending agents, buffers, isotonic agents, etc. that are known and usable, and may be formulated as a parenteral administration form, preferably a parenteral administration form including a skin external preparation. In the case of intramuscular, intraperitoneal, subcutaneous, and intravenous administration forms, a sterile solution of the active ingredient is usually prepared, and a buffer capable of suitably adjusting the pH of the solution may be included, and in the case of intravenous administration, a isotonic agent may be included to impart isotonicity to the preparation. 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 intramuscular bloodstream of a patient in the form of a solution. In addition, it may be formulated as a transdermal administration formulation such as an external solution, emulsion, ointment, or patch according to a conventional pharmaceutical method. The pharmaceutical composition of the present invention can be administered to patients with various wounds at a dosage of about 1 to 50 mg/kg per day. The appropriate dosage can generally be changed depending on the patient's age, weight, and symptoms.

The cosmetic composition of the present invention may be in the form of a functional cosmetic composition containing the above-described peptide as an effective ingredient. The cosmetic composition may be produced in various forms according to a conventional cosmetic production method. For example, the cosmetic composition may be produced in the form of a cosmetic product, toner, cream, lotion, etc. containing the peptide, which may be diluted with a conventional cleansing solution, astringent, or moisturizing solution and used. In addition, the cosmetic composition may include conventional excipients such as a stabilizer, a solubilizer, a vitamin, a pigment, and a fragrance commonly used in the field of cosmetic compositions. In the cosmetic composition, the content of the peptide may be contained in an amount effective for inhibiting skin aging, particularly for inhibiting skin wrinkle formation, and/or for moisturizing the skin, for example, in an amount of 1 x 10 ^-5 to 1 x 10 ^-2 wt% based on the total weight of the composition, and preferably in an amount of about 1 x 10 ^-4 to 1 x 10 ^-3 wt%.

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

Example 1. Synthesis of peptides

The peptide consisting of Leu-Ser-Ser-Phe was 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 (Prominence LC-20AB, Shimadzu, Japan) using a C18 analytical RP column (Shiseido capcell pak), and identified using mass spectrometry (HP 1100 Series LC/MSD, Hewlett-Packard, Roseville, USA).

Example 2. Preparation of a composition containing a peptide

The peptide (peptide composed of Leu-Ser-Ser-Phe) manufactured in Example 1 was dissolved in triple-distilled water to prepare a concentration of 1000 ppm. The obtained peptide solution was used in the following test examples.

Test Example 1: GLP-2 Receptor Binding Evaluation

In order to confirm whether the peptide of the present invention (a peptide composed of Leu-Ser-Ser-Phe) binds to the target protein, GLP-2 receptor, the peptide-AMC of the present invention labeled with a fluorescent substance, AMC (7-amino-4-methyl coumarine), was treated to cells and whether it binds to the GLP-2 receptor was confirmed through immunofluorescence.

(1) Test material

- Test substance preparation

The peptide of the present invention (peptide composed of Leu-Ser-Ser-Phe) was dissolved in triple-distilled water to prepare a concentration of 1000 ppm.

- Test system

1) Cell line: Human Keratinocyte (HaCaT, CLS)

2) Cell management: The cell line was frozen and thawed, inoculated into a 100 cm2 animal cell culture dish containing culture medium, and cultured in an incubator (5% _CO2 , 37°C), and subcultured with new culture medium every 2 to 3 days.

3) Medium: DMEM (Dulbecco's Modified Eagle Medium)

Composition: 10% fetal bovine serum, 1% antibiotic / Storage conditions: Refrigerated storage / Manufacturer: GIBCO

- Test materials

1) Peptide of the present invention -AMC (7-amino-4-methyl coumarine)

Storage conditions: -20℃ frozen storage / Manufacturer: Peptron

2) Phalloidin-Rhodamin

Storage conditions: -20℃ frozen storage / Manufacturer: : INVITROGEN, R415

3) siRNA transfection

3-1) GLP2R siRNA(h)

Storage Conditions: -20℃ frozen storage / Manufacturer: SANTA CRUZ BIOTECHNOLOGY, sc-60695

3-2) Lipofectamine™ RNAiMAX Transfection Reagent

Storage conditions: Refrigerated at 4℃ / Manufacturer: INVITROGEN, 13778075

3-3) Opti-MEM ^® Medium

Storage Conditions: Refrigerated at 4℃ / Manufacturer: GIBCO, 31985062

(2) Test method

- Composition of the test group

- Exam process

1) 2.5 x 10 ⁴ cells were seeded into each well of a 24-well culture plate with a 12 mm microscope circular cover glass. After culturing for 24 hours, the monolayer culture status of the cells was checked and the test was performed when the confluency of the cells was 50% or higher.

2) The medium was replaced with a dedicated medium (DMEM + 10% FBS) for siRNA treatment.

3) siRNA + Opti-MEM ^® medium and transfection reagent + Opti-MEM ^® medium were mixed in a 1:1 ratio, slowly mixed, and reacted at room temperature for 5 minutes.

4) 3) The solution was added to cultured cells and treated for 48 hours to conduct the test.

5) The peptide-AMC (1:100) of the present invention was reacted at 4°C for 16 hours and then washed three times with a washing solution (PBS).

6) To stain the cytoskeleton, phalloidin-rhodamine (1:2000) was reacted at room temperature for 40 minutes, and then washed three times with washing solution (PBS).

7) Mounting was performed using a mounting solution.

8) The fluorescence signal (AMC) detected in the cells was observed and photographed using a digital fluorescence imaging system (LOGOS BIOSYSTEMS, CS20002).

- Observe and judge the results

The luminescence level of the peptide-AMC of the present invention in the GLP-2R siRNA treatment group was compared and analyzed based on the control siRNA negative control group.

(3) Test results

As a result of evaluating the degree of binding of the test substance to the GLP-2 receptor, in the control siRNA treatment group, the test substance bound to the GLP-2 receptor and an AMC (blue fluorescence) signal was observed, but in the GLP-2R siRNA treatment group in which the expression of the GLP-2 receptor was suppressed, the AMC signal was reduced (Fig. 1).

(4) Conclusion

Compared to the negative control group, it was observed that the peptide-AMC signal of the present invention was reduced in the GLP-2R siRNA treatment group. Therefore, it is judged that the peptide of the present invention (a peptide composed of Leu-Ser-Ser-Phe) specifically binds to the GLP-2 receptor.

Test Example 2: Evaluation of GLP-2 Receptor Signaling Induction Efficacy

In order to verify the potential of the peptide of the present invention as a GLP-2 receptor agonist, the change in the interaction of β-catenin/TCF, a downstream signaling pathway of the GLP-2 receptor, was observed through in situ PLA, and whether the peptide of the present invention transmits a signal through GLP-2R was confirmed by treating with GLP-2R siRNA.

(1) Test material

- Test substance preparation

The peptide of the present invention (peptide composed of Leu-Ser-Ser-Phe) was dissolved in triple-distilled water to prepare a concentration of 1000 ppm.

- Test system

1) Cell line: Human dermal fibroblast (HDF Passage 5-10, CEFObio)

2) Cell management: The cell line was frozen, thawed, and inoculated into a 100 cm2 animal cell culture dish containing culture medium. The cells were cultured in an incubator (5% _CO2 , 37°C), and subcultured with new culture medium every 2–3 days.

3) Badge: CEFOgro Human MSC Growth medium

Composition: 10% Fetal bovine serum, 1% antibiotics / Storage conditions: Refrigerated storage / Manufacturer: CEFObio

- Test materials

1) Anti-β-catenin (S33/S37/T41) antibodies

Storage Conditions: -20℃ frozen storage / Manufacturer: CELL SIGNALING, 4270

2) Anti-TCF-4 (D-4) antibody

Storage Conditions: -20℃ frozen storage / Manufacturer: SANTA CRUZ BIOTECHNOLOGY, SC-166699

3) siRNA transfection

3-1) GLP2R siRNA(h)

Storage Conditions: -20℃ frozen storage / Manufacturer: SANTA CRUZ BIOTECHNOLOGY, sc-60695

3-2) Lipofectamine™ RNAiMAX Transfection Reagent

Storage conditions: Refrigerated at 4℃ / Manufacturer: INVITROGEN, 13778075

3-3) Opti-MEM ^® Medium

Storage Conditions: Refrigerated at 4℃ / Manufacturer: GIBCO, 31985062

4) NaveniFlex 100RM

Storage Conditions: -20℃ frozen storage / Manufacturer: NaveniFlex, NV C-NF MR.100

5) Prolong™ diamond antifade mountant with DAPI

Storage conditions: -20℃ frozen storage / Manufacturer: INVITROGEN, P36962

6) Digital Fluorescence Imaging System

Manufacturer: LOGOS BIOSYSTEMS, CS20002

(2) Test method

(2-1) in situ PLA

- Composition of the test group

- Exam process

1) 2.5 x 10 ⁴ cells were seeded into each well of a 24-well culture plate with a 12 mm microscope circular cover glass. After culturing for 24 hours, the monolayer culture status of the cells was checked and the test was performed when the confluency of the cells was 50% or higher.

2) For the siRNA treatment group, the medium was replaced with a dedicated medium (DMEM + 10% FBS).

3) siRNA + Opti- ^MEM® medium and transfection reagent + Opti-MEM® medium were mixed slowly in a 1:1 ratio and reacted at room temperature for 5 minutes.

4) 3) The solution was added to cultured cells and treated for 48 hours to conduct the test.

5) The negative control and test substances were treated for 1 hour at the appropriate concentration for each treatment group.

6) After fixing the cells using 4% paraformaldehyde, pretreatment was performed to increase the permeability of cell antibodies by perforating the cells with 0.1% Triton X-100.

7) Subsequent tests were conducted using the In Situ PLA Kit (NaveniFlex 100RM) and the tests were conducted according to the manufacturer’s instructions.

8) After washing once with PBS, blocking was performed with blocking solution at 37°C for 30 minutes.

9) Two antibodies for confirmation were diluted to 10 μg/mL in antibody diluent, reacted at 4°C for 16 hours, and washed three times with TTBS (0.01 M Tris, 0.15 M NaCl, 0.05% Tween 20, pH 7.4).

10) The PLA probe was added and reacted at 37°C for 1 hour, then washed three times with TTBS.

11) Reactions A, B, and C were sequentially treated and reacted at 37°C for 1 hour, 30 minutes, and 90 minutes, respectively. Finally, the samples were washed twice with TBS (0.01 M Tris, 0.15 M NaCl) and mounted using a mounting solution containing DAPI (nuclear stain).

12) The PLA signal detected in the cells was observed and photographed using a digital fluorescence imaging system (LOGOS BIOSYSTEMS, CS20002).

- Observe and judge the results

To compare and analyze the luminescence signal due to the interaction of β-catenin/TCF in the test substance treatment group, the PLA fluorescence signal was quantitatively analyzed using NIS-Elements BR3.1 with the negative control group as the standard.

(3) Test results

Compared to the negative control group, it was observed that the interaction of β-catenin/TCF increased in a concentration-dependent manner in the test substance treatment group (Fig. 2), and when the expression of GLP-2R was suppressed by treating with GLP-2R siRNA, the signal was reduced (Fig. 3).

(4) Conclusion

It was confirmed that the test substance activates the downstream signaling pathway through the GLP-2 receptor. Therefore, it is judged that the peptide of the present invention (a peptide composed of Leu-Ser-Ser-Phe) can act as a GLP-2 receptor agonist.

Test Example 3: Evaluation of cell proliferation induction efficacy

GLP-2 is known to help skin regeneration by inducing proliferation of skin cells. Using a proliferation assay kit (CCK-8), it was confirmed whether the peptide of the present invention (a peptide composed of Leu-Ser-Ser-Phe) induces proliferation of skin keratinocytes like GLP-2.

(1) Test material

- Test substance preparation

The peptide of the present invention (peptide composed of Leu-Ser-Ser-Phe) was dissolved in triple-distilled water to prepare a concentration of 1000 ppm.

- Test system

1) Cell line: Human dermal fibroblast (HDF Passage 5-10, CEFObio)

2) Cell management: The cell line was frozen, thawed, and inoculated into a 100 cm2 animal cell culture dish containing culture medium. The cells were cultured in an incubator (5% _CO2 , 37°C), and subcultured with new culture medium every 2–3 days.

3) Badge: CEFOgro Human MSC Growth medium

Composition: 10% Fetal bovine serum, 1% antibiotics / Storage conditions: Refrigerated storage / Manufacturer: CEFObio

- Test materials

1) Cell viability assay kit

Storage Conditions: Refrigerated at 4℃ / Manufacturer: BYLABS, BYVA0500

2) Microplate reader

Manufacturer: BIO-TEK, EL808

(2) Test method

- Composition of the test group

- Exam process

1) ^5X103 cells were dispensed into each well of a 96-well culture plate at 0.1 mL.

2) The negative control and test substances were treated for 24, 48, and 72 hours at the concentrations of each treatment group.

3) Each well was treated with 10 ㎕ of cell viability analysis solution and reacted at 37°C for 2 hours.

4) The absorbance (450 nm) of each test group was measured using a microplate reader.

- Observe and judge the results

The degree of cell proliferation was confirmed by measuring absorbance (450 nm) using a microplate reader, and the degree of cell proliferation in the test substance treatment group was compared and analyzed based on the negative control group.

(3) Test results

Compared to the negative control group, it was observed that cell proliferation increased in a concentration-dependent manner in the test substance treatment group (Fig. 4).

(4) Conclusion

As a result of evaluating the skin cell proliferation inducing efficacy of the test substance, it was confirmed that cell proliferation induction was promoted in a concentration-dependent manner by treatment with the test substance. Therefore, it is determined that the peptide of the present invention (peptide composed of Leu-Ser-Ser-Phe) has skin cell proliferation inducing efficacy.

Test Example 4: Evaluation of the efficacy of inducing skin barrier-related protein synthesis

It was confirmed through Western blot analysis whether the peptide of the present invention has the effect of maintaining and strengthening the skin barrier by inducing the synthesis of lipid components and proteins related to the skin barrier.

(1) Test material

- Test substance preparation

The peptide of the present invention (peptide composed of Leu-Ser-Ser-Phe) was dissolved in triple-distilled water to prepare a concentration of 1000 ppm.

- Test system

1) Cell line: Human Keratinocyte (HaCaT, CLS)

2) Cell management: The cell line was frozen, thawed, and inoculated into a 100 cm2 animal cell culture dish containing culture medium. The cells were cultured in an incubator (5% _CO2 , 37°C), and subcultured with new culture medium every 2–3 days.

3) Medium: DMEM (Dulbecco's Modified Eagle Medium)

Composition: 10% fetal bovine serum, 1% antibiotic / Storage conditions: Refrigerated storage / Manufacturer: GIBCO

- Test materials

1) Anti-Fatty acid synthase antibodies

Storage Conditions: Refrigerated at 4℃ / Manufacturer: SANTA CRUZ BIOTECHNOLOGY, sc-20140

2) Anti-LASS3 (CerS3) antibody

Storage Conditions: Refrigerated at 4℃ / Manufacturer: ABCAM, ab272552

3) Anti-Filaggrin (AKH1) antibody

Storage Conditions: Refrigerated at 4℃ / Manufacturer: SANTA CRUZ BIOTECHNOLOGY, sc-66192

4) Anti-Involucrin (SY5) antibody

Storage Conditions: Refrigerated at 4℃ / Manufacturer: SANTA CRUZ BIOTECHNOLOGY, sc-21748

5) Anti-Loricrin (W-22) antibody

Storage Conditions: Refrigerated at 4℃ / Manufacturer: SANTA CRUZ BIOTECHNOLOGY, sc-133757

6) Goat anti-mouse IgG Fc-HRP

Storage conditions: Refrigerated at 4℃ / Manufacturer: ABFRONTEIR

7) NP40 cell lysis buffer

Storage conditions: -20℃ frozen storage / Manufacturer: INVITROGEN, FNN0021

8) Bovine serum albumin (BSA)

Storage Conditions: Refrigerated at 4℃ / Manufacturer: CELLCONIC, FNN0021

9) Protein assay dye reagent concentrate

Storage Conditions: Refrigerated at 4℃ / Manufacturer: BIO_RAD, #5000006

10) Immuno-bolt ^® PVDF membrane for protein blotting

Storage Conditions: Store at room temperature / Manufacturer: BIO-RAD, #1620177

11) WEST SAVE GOLD,

Storage Conditions: Refrigerated at 4℃ / Manufacturer: AB FRONTIER, LF-QC0103

12) DaVinci Western Imaging System

Manufacturer: DAVINCH-K, CAS-400SM

(2) Test method

(2-1) Immunofluorescence

- Composition of the test group

- Exam process

1) ^5X106 cells were seeded into each well of a 6-well culture plate. After 24 hours of culture, the monolayer culture status of the cells was checked, and the test was performed when the cell confluency was 50% or higher.

2) The negative control and test substances were treated for 24 hours at a concentration appropriate for each treatment group.

3) Cells were lysed using NP40 cell lysis buffer, and cell extracts for electrophoresis were prepared through quantification using the Bradford assay method.

4) Electrophoresis was performed by loading 20 ㎍ of cell extracts quantified on a sodium dodecyl sulfate-polyacrylamide gel into each well.

5) The proteins developed in SDS-PAGE were transferred to a PVDF membrane.

6) The PVDF membrane was treated with a blocking solution (3% BSA, 0.05% Tween 20, TBS) and reacted at room temperature for 1 hour.

7) The primary antibody was reacted at room temperature for 2 hours, and washed three times with washing solution (0.05% Tween 20, TBS).

8) The secondary antibody was reacted at room temperature for 1 hour and washed 5 times with washing solution.

9) After photosensitization using an antibody detection kit, the result was confirmed using a Western blot imaging system.

- Observe and judge the results

The expression levels of each protein treated with the test substance were evaluated by quantitative analysis using ImageJ based on the expression levels of β-Actin, which was used as a loading control, by photographing using a Western blot imaging system. The changes in the expression levels of fatty acid synthase, CERS3 (ceramide synthase 3), Filaggrin, Involucrin, and Loricrin in the test substance treatment group were compared and analyzed based on the negative control group.

(3) Test results

Compared to the negative control group, it was confirmed that the expression levels of lipid component synthesis enzyme (Fatty acid synthase, CERS3) and structural support proteins (Filaggrin, Involucrin, Loricrin) of the skin barrier increased in a concentration-dependent manner in the test substance treatment group (Fig. 5).

(4) Conclusion

It was confirmed that the test substance promoted the expression of lipid component synthesis enzyme (Fatty acid synthase, CERS3) and structural support proteins (Filaggrin, Involucrin, Loricrin) of the skin barrier in a concentration-dependent manner. Therefore, it is judged that the peptide of the present invention (peptide composed of Leu-Ser-Ser-Phe) is effective in maintaining and strengthening the skin barrier.

Test Example 5: Evaluation of the efficacy of inducing filaggrin synthesis in a 3D human skin model

Through the above-described test, it was confirmed that the test substance strengthened the skin barrier by activating the GLP-2 receptor at the cellular level. It was confirmed through immunofluorescence that the test substance also showed the efficacy of activating the GLP-2 receptor and promoting the synthesis of filaggrin, a representative skin barrier protein, in a 3D human skin model (Neoderm-ED) similar to human skin.

(1) Test material

- Test substance preparation

The peptide of the present invention (peptide composed of Leu-Ser-Ser-Phe) was dissolved in triple-distilled water to prepare a concentration of 1000 ppm.

- Test system

1) 3D human skin model: Neoderm-ED

2) Management: Cultured in an incubator (5% _CO2 , 37℃) and tested within 3 days of receipt.

3) Badge: Maintenance medium

Composition: 10% Fetal bovine serum / Storage conditions: Refrigerated storage / Manufacturer: TEGO SCIENCE

- Test materials

1) Anti-Filaggrin (AKH1) antibody

Storage Conditions: Refrigerated at 4℃ / Manufacturer: SANTA CRUZ BIOTECHNOLOGY, sc-66192

2) Prolong™ diamond antifade mountant with DAPI

Storage conditions: -20℃ frozen storage / Manufacturer: INVITROGEN, P36962

3) Digital Fluorescence Imaging System

Manufacturer: LOGOS BIOSYSTEMS, CS20002

(2) Test method

- Composition of the test group

- Exam process

1) After receiving Neoderm-ED, a dedicated medium was added and cultured for 24 hours.

2) The negative control and test substances were treated for 48 hours at the appropriate concentration for each treatment group.

3) Neoderm-ED was separated from the insert well using a blade to produce a paraffin block.

4) Slides were prepared by cutting sections at 4 ㎛ thickness.

5) Paraffin washing and water dehydration process using xylene (Et-OH 100% >95%>90%>80%>70%) was performed.

6) To expose the antigen, citrate buffer was added, heated for 14 minutes, and then washed with PBS.

7) Pretreatment was performed to increase antibody permeability by treating with 0.1% Triton X-100 for 10 minutes.

8) The primary antibody was reacted at 4°C for 16 hours and washed three times with washing solution (PBS).

9) The secondary antibody was reacted at room temperature for 1 hour and washed 5 times with washing solution (PBS).

10) Mounting was performed using a mounting solution containing DAPI (nuclear stain).

11) The fluorescence signals detected in the tissue were observed and photographed using a digital fluorescence imaging system (LOGOS BIOSYSTEMS, CS20002).

- Observe and judge the results

The level of filaggrin expression in the test substance treatment group was compared and analyzed by comparing the amount of fluorescence signal based on the negative control group.

(3) Test results

Compared to the negative control group, an increase in the expression of Filaggrin (green fluorescence) was observed in the test substance treatment group (Fig. 6).

6-5. Conclusion

In a 3D human skin model, it was confirmed that the test substance promoted the expression of filaggrin, a representative structural support protein of the skin barrier. Therefore, it was confirmed that the peptide of the present invention (a peptide composed of Leu-Ser-Ser-Phe) maintains and strengthens the skin barrier not only at the cell level but also in the skin model.

Claims (2)

A pharmaceutical composition for wound healing or promoting wound healing, comprising a peptide of the following chemical formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient. <Chemical Formula 1>

A cosmetic composition for inhibiting skin wrinkle formation or moisturizing the skin, comprising a peptide of the following chemical formula 1 or a pharmaceutically acceptable salt thereof. <Chemical Formula 1>

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