Compositions for wound-healing or inhibiting skin-wrinkle formation

Abstract

The present invention provides a pharmaceutical composition for wound healing comprising a specific peptide; and a cosmetic composition for inhibiting skin wrinkle formation. The peptide acts as an agonist for IGF-1R, thereby effectively inducing IGF-1R signaling, as well as inducing cell proliferation and the synthesis of collagen, elastin, and HAS2. The peptide can be usefully used for wound healing and for inhibiting skin wrinkle formation.

Description

Technology Field

[0001] The present invention relates to a pharmaceutical composition for wound healing comprising a specific peptide; and a composition for inhibiting skin wrinkle formation. The peptide has the activity of inducing collagen synthesis by activating the insulin-like growth factor-1 receptor (IGF-1R). Background Technology

[0002] IGF-1 / IGF-1R are growth factors that promote cell proliferation and inhibit apoptosis, playing a crucial role in cell growth and maintenance. IGF-1 / IGF-1R protects keratinocytes from external stress and inhibits apoptosis by helping to repair DNA damage. Furthermore, by promoting keratinocyte proliferation and the production of collagen and elastin, it regenerates damaged skin tissue and aids in wound healing. (Jennifer M Kavran, et al., Elife. 2014 Sep 25:3:e03772; Weikang Cai, et al., Nature communications, 2017, 8.1: 14892 et al.)

[0003] It is known that when IGF-1 binds to IGF-1R in skin keratinocytes, it transmits signals into the cell via various pathways, such as phosphoinositide 3-kinase (PI3K)-Akt and Ca2+ (Hwa Jin Jung, et al., Frontiers in genetics, 2015, 5: 472; Antero Salminen et al., Inflammation Research, 2021, 7010-12: 1043-1061). When stimulated, the IGF-1 receptor regenerates tissues and heals wounds through cell proliferation and differentiation (Andrea M Vincent et al., Growth hormone & IGF research, 2002, 124: 193-197; NL Ge, et al., Blood, Volume 96, Issue 8, 15 October 2000, Pages 2856-2861). IGF receptors play an important role in skin cell proliferation and are known to promote the secretion of collagen, elastin, and hyaluronic acid for the rearrangement of new tissue (Anne Chetty, et al., Pediatr Res. 2006 Oct;60(4): 389-94. doi: 10.1203; Nour Qa'aty, et al., J Dermatol Sci. 2015 Dec;80(3):175-85. doi: 10.1016). Therefore, substances capable of activating IGF-1R can not only act as active substances for wound healing or promoting wound healing, but also as functional cosmetic substances to inhibit skin aging (e.g., skin wrinkle formation).

[0004] The inventors have disclosed that a peptide derived from fibroblast growth factor (FGF) activates fibroblast growth factor receptors to induce collagen synthesis and also has the activity of promoting skin keratinocyte proliferation and angiogenesis (Korean Patent Registration No. 10-1772048). The problem to be solved

[0005] The inventors conducted various studies to develop an active substance based on small peptides as a substance capable of activating IGF-1R. As a result, they discovered that a specific peptide, namely a 3-mer peptide composed of Arg-Arg-Leu, acts as a substance capable of activating IGF-1R [i.e., an agonist for IGF-1R], thereby effectively inducing IGF-1R signaling, as well as inducing cell proliferation and the synthesis of collagen, elastin, and HAS2 (Hyaluronan synthase 2), which can be usefully applied for wound healing or inhibiting skin wrinkle formation.

[0006] Accordingly, the present invention aims to provide a pharmaceutical composition for wound healing or promoting wound healing comprising the specific peptide as an active ingredient.

[0007] In addition, the present invention aims to provide a cosmetic composition for inhibiting skin aging or inhibiting skin wrinkle formation comprising the specific peptide above. means of solving the problem

[0008] According to one aspect of the present invention, a pharmaceutical composition for wound healing or promoting wound healing is provided, comprising as an active ingredient a peptide of the following formula 1 or a pharmaceutically acceptable salt thereof.

[0009] <Chemical Formula 1>

[0010]

[0011] According to another aspect of the present invention, a cosmetic composition for inhibiting skin wrinkle formation is provided, comprising a peptide of Formula 1 or a pharmaceutically acceptable salt thereof. Effects of the invention

[0012] It has been revealed by the present invention that a peptide according to the present invention (i.e., a peptide composed of Arg-Arg-Leu) binds to intracellular IGF-1R, induces IGF-1R signaling in a concentration-dependent manner, induces cell proliferation in a concentration-dependent manner, and induces the synthesis of collagen, elastin, and HAS2 (Hyaluronan synthase 2). It has also been revealed by the present invention that a peptide according to the present invention effectively induces collagen synthesis in a 3D human skin model. Accordingly, the peptide according to the present invention can be usefully applied to pharmaceutical compositions for wound healing or promoting wound healing; and cosmetic compositions for inhibiting skin aging, including inhibiting skin wrinkle formation. Brief explanation of the drawing

[0013] Figure 1 shows the results of measuring the signal reduction of the peptide (VE-Igrofin)-rhodamine and IGF-1R-FITC of the present invention composed of Arg-Arg-Leu by IGF-IR α / β siRNA treatment. Figure 2 is the result of analyzing the change in GRB2 / SOS1 interaction by peptide treatment of the present invention. Figure 3 is the result of analyzing the change in Akt phosphorylation by peptide treatment of the present invention. Figure 4 shows the results of analyzing changes in Akt phosphorylation and IGF-1R expression by treatment with the peptide and IGF-1R siRNA of the present invention. Figure 5 is the result of analyzing the cell proliferation rate by peptide treatment of the present invention. Figure 6 is the result of analyzing the change in expression levels of collagen and HAS2 by peptide treatment of the present invention. Figure 7 is the result of analyzing the change in elastin expression amount by peptide treatment of the present invention. Figure 8 shows the results of analyzing the proliferation of keratinocytes and fibroblasts and the change in collagen protein expression levels caused by the peptide treatment of the present invention in a 3D human skin model. Specific details for implementing the invention

[0014] The present invention provides 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.

[0015] <Chemical Formula 1>

[0016]

[0017] In addition, the present invention provides a cosmetic composition for inhibiting skin wrinkle formation, comprising a peptide of Formula 1 or a pharmaceutically acceptable salt thereof.

[0018] In the pharmaceutical composition or cosmetic composition of the present invention, the peptide of Formula 1 may also be denoted as "Arg-Arg-Leu". The amino acids constituting the peptide of Formula 1 may independently be in the form of L-amino acids or D-amino acids. Pharmaceutically acceptable salts of the peptide derivative of Formula 1 include, for example, acid addition salts, but are not limited thereto.

[0019] The pharmaceutical composition of the present invention may include excipients such as lactose and corn starch, lubricants such as magnesium stearate, known and usable emulsifiers, suspending agents, buffers, isotonic agents, etc., and may be formulated in a parenteral administration form, preferably a parenteral administration form including a topical preparation for the skin. In the case of intramuscular, intraperitoneal, subcutaneous, and intravenous administration forms, a sterile solution of the active ingredient is typically prepared, and a buffer capable of appropriately adjusting the pH of the solution may be included; in the case of intravenous administration, an isotonic agent may be included to impart isotonicity to the preparation. Furthermore, the pharmaceutical composition of the present invention may be in the form of an aqueous solution containing a pharmaceutically acceptable carrier, such as saline solution with a pH of 7.4, and may be introduced topically into the intramuscular bloodstream of a patient in the form of a solution. Additionally, it may be formulated into transdermal administration formulations such as topical solutions, emulsions, ointments, and patches according to conventional pharmaceutical methods. The pharmaceutical composition of the present invention may be administered to patients with various wounds at a dose of about 1 to 10 mg / kg per day. The appropriate dosage may generally be changed depending on the patient's age, weight, and symptoms.

[0020] The cosmetic composition of the present invention may be in the form of a functional cosmetic composition comprising the above-described peptide as an active ingredient. The cosmetic composition may be prepared in various forms according to conventional cosmetic manufacturing methods. For example, the cosmetic composition may be prepared in the form of cosmetic products, toners, creams, lotions, etc., containing the above-described peptide, and may be used by diluting with conventional cleansing liquids, astringent liquids, and moisturizing liquids. In addition, the cosmetic composition may include conventional auxiliary agents such as stabilizers, solubilizers, vitamins, pigments, and fragrances commonly used in the field of cosmetic compositions. In the cosmetic composition, the content of the above-described peptide is an amount effective for achieving the effect of inhibiting skin aging, particularly inhibiting the formation of skin wrinkles, for example, 1 x 10⁻⁶ with respect to the total weight of the composition.-5 ~ 2 x 10 -3 It may be contained in a weight % content, preferably about 1 x 10 -4 ~ 1 x 10 -3 It may be contained in a weight % content.

[0021] The present invention will be explained in more detail below 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.

[0022] Example 1. Synthesis of Peptides

[0023] A peptide composed of Arg-Arg-Leu was synthesized using the FMOC solid-phase method with an automated synthesizer (PeptrEx-R48, Peptron, Daejeon, South Korea). The synthesized peptide was purified and analyzed using reverse-phase high-speed liquid chromatography (reverse-phase HPLC) with a C18 analysis RP column (Shiseido capcell pak) (Prominence LC-20AB, Shimadzu, Japan), and identified using a mass spectrometer (HP 1100 Series LC / MSD, Hewlett-Packard, Roseville, USA).

[0024] Example 2. Preparation of a composition containing a peptide

[0025] The peptide prepared in Example 1 (a peptide composed of Arg-Arg-Leu) was dissolved in triple-distilled water to a concentration of 1000 ppm. The obtained peptide solution was used in the following test examples.

[0026] Test Example 1: Evaluation of IGF receptor binding

[0027] IGF-1 (insulin-like growth factors)-1 is known to transmit signals into cells through IGF-1R. Immunofluorescence was used to confirm whether the peptide of the present invention (a peptide composed of Arg-Arg-Leu) binds to IGF-1R.

[0028] (1) Test materials

[0029] - Preparation of test substances

[0030] The peptide of the present invention (a peptide composed of Arg-Arg-Leu) was dissolved in triple distilled water to produce a concentration of 1000 ppm.

[0031] - Test

[0032] 1) Cell line: Human dermal fibroblast (HDF Passage 5-10, CEFObio)

[0033] 2) Cell management: The cell line was thawed from cryopreservation and inoculated into a 100 cm² animal cell culture dish containing culture medium, cultured in an incubator (5% CO2, 37℃), and subcultured with fresh culture medium every 2 to 3 days.

[0034] 3) Medium: CEFOgro Human MSC Growth Medium

[0035] Composition: 10% Fetal Bovine Serum, 1% Antibiotic / Storage: Refrigerated / Manufacturer: CEFObio

[0036] - Test materials

[0037] 1) The peptide of the present invention labeled with Rhodamine

[0038] Storage Conditions: Store frozen at -20℃ / Manufacturer: Peptron

[0039] 2) Anti-IGF-IR (7G11) antibody

[0040] Storage conditions: Store frozen at -20℃ / Manufacturer: SANTA CRUZ BIOTECHNOLOGY, sc-37193

[0041] 3) FITC-labeled anti-mouse IgG

[0042] Storage conditions: Keep refrigerated at 4℃ / Manufacturer: INVITROGEN, A11001

[0043] 4) siRNA transfection

[0044] 4-1) IGF-IR α / β siRNA

[0045] Storage conditions: Store frozen at -20℃ / Manufacturer: SANTA CRUZ BIOTECHNOLOGY, sc-29358

[0046] 4-2) Lipofectamine™ RNAiMAX Transfection Reagent

[0047] Storage conditions: Keep refrigerated at 4℃ / Manufacturer: INVITROGEN, 13778075

[0048] 4-3) Opti-MEM ® Medium

[0049] Storage conditions: Keep refrigerated at 4℃ / Manufacturer: GIBCO, 31985062

[0050] (2) Test method

[0051] - Composition of the test group

[0052] Sample throughput n per test group Control siRNA 1 mL 5 IGF-IR α / β siRNA 10 nM 1 mL 5 IGF-IR α / β siRNA 50 nM 1 mL 5

[0053] - Exam process

[0054] 1) Place a 12 mm circular microscope cover glass in a 24-well culture plate and 2.5 x 10 4 After dispensing cells into each well, the monolayer culture status of the cells was checked after 24 hours of culture, and the test was conducted when the cell confluence was 50% or higher.

[0055] 2) For siRNA treatment, the medium was replaced with a dedicated medium (DMEM + 10% FBS).

[0056] 3) siRNA + Opti-MEM ® Media and transfection reagent + Opti-MEM ® The culture medium was prepared in a 1:1 ratio, slowly mixed, and then reacted at room temperature for 5 minutes.

[0057] 4) The solution from 3) was added to cultured cells and treated for 48 hours to conduct the test.

[0058] 5) The peptide of the present invention (1:100) labeled with Rhodamine was reacted at 4°C for 16 hours, and then washed three times with a washing solution (PBS).

[0059] 6) The IGF-1R antibody (1:100) was treated and reacted at room temperature for 40 minutes, then washed three times with washing solution (PBS).

[0060] 7) The secondary antibody (1:2000) was treated and reacted at room temperature for 40 minutes, then washed three times with a washing solution (PBS).

[0061] 8) Mounting was performed using a mounting solution containing DAPI (nuclear staining).

[0062] 9) Fluorescence signals detected in cells were observed and captured using a Digital Fluorescence Imaging System (LOGOS BIOSYSTEMS, CS20002).

[0063] - Observation and judgment of results

[0064] Based on a control siRNA negative control, the luminescence levels of the peptide of the present invention (labeled with rhodamine) and IGF-1R-FITC in the IGF-IR α / β siRNA treatment group were compared and analyzed.

[0065] (3) Test results

[0066] Compared to the negative control group, it was observed that the signals of the peptide-rhodamine and IGF-1R-FITC of the present invention were reduced in the IGF-IR α / β siRNA-treated group. In the negative control group, the test substance bound to IGF-1R and showed a rhodamine (red fluorescence) signal, and the IGF-1R antibody showed a FITC (green fluorescence) signal; when the expression of IGF-1R was inhibited by treatment with IGF-1R siRNA, the rhodamine signal and the FITC signal were reduced (Fig. 2).

[0067] (4) Conclusion

[0068] As a result of evaluating the degree of IGF-1R binding of the test substance, the signal expression sites of the peptide-rhodamine of the present invention and IGF-1R-FITC coincided. Therefore, it is determined that the peptide of the present invention (a peptide composed of Arg-Arg-Leu) specifically binds to IGF-1R.

[0069] Test Example 2. Evaluation of IGF signaling induction efficacy

[0070] When IGF-1 binds to IGF-1R on skin keratinocytes, phosphoinositide 3-kinase (PI3K)-Akt, Ca2 + It is known that signals are transmitted into cells through various pathways. To verify the potential of the peptide of the present invention as an IGF-1 receptor agonist, changes in Akt phosphorylation and the interaction of GRB2 / SOS1, which are downstream signaling pathways of the IGF-1 receptor, were observed through Western blot analysis and in situ PLA, respectively, and it was confirmed whether the peptide of the present invention transmits signals through IGF-1R by treating with IGF-1R siRNA.

[0071] (1) Test materials

[0072] - Preparation of test substances

[0073] The peptide of the present invention (a peptide composed of Arg-Arg-Leu) was dissolved in triple distilled water to produce a concentration of 1000 ppm.

[0074] - Test

[0075] 1) in situ PLA

[0076] 1-1) Cell line: Human keratinocyte (HaCaT, CLS)

[0077] 1-2) Cell management: The cell line was thawed from cryopreservation and inoculated into a 100 cm² animal cell culture dish containing culture medium, cultured in an incubator (5% CO2, 37℃), and subcultured with fresh culture medium every 2 to 3 days.

[0078] 1-3) Medium: DMEM (Dulbecco's Modified Eagle Medium)

[0079] Composition: 10% Fetal Bovine Serum, 1% Antibiotic / Storage: Refrigerated / Manufacturer: GIBCO

[0080] 2) Western blot analysis (1), (2)

[0081] 2-1) Cell line: Human keratinocyte (HaCaT, CLS)

[0082] 2-2) Cell management: The cell line was thawed from cryopreservation and inoculated into a 100 cm² animal cell culture dish containing culture medium, then cultured in an incubator (5% CO2, 37℃) and subcultured with fresh culture medium every 2 to 3 days.

[0083] 2-3) Medium: DMEM (Dulbecco's Modified Eagle Medium)

[0084] Composition: 10% Fetal Bovine Serum, 1% Antibiotic / Storage: Refrigerated / Manufacturer: GIBCO

[0085] - Test materials

[0086] 1) in situ PLA

[0087] 1-1) Anti-GRB2 Antibody

[0088] Storage conditions: Keep refrigerated at 4℃ / Manufacturer: Invitrogen, PA1-10033

[0089] 1-2) Anti-SOS1 (C-23) Antibody

[0090] Storage conditions: Keep refrigerated at 4℃ / Manufacturer: SANTA CRUZ BIOTECHNOLOGY, sc-256

[0091] 1-3) NaveniFlex 100RM

[0092] Storage Conditions: Store frozen at -20℃ / Manufacturer: NaveniFlex, NV C-NF MR.100

[0093] 1-4) Prolong™ diamond antifade mountant with DAPI

[0094] Storage conditions: Store frozen at -20℃ / Manufacturer: INVITROGEN, P36962

[0095] 1-5) Digital Fluorescence Imaging System

[0096] Manufacturer: LOGOS BIOSYSTEMS, CS20002

[0097] 2) Western blot analysis

[0098] 2-1) Anti-Akt1 / 2 / 3-phospho(S473)-R antibody

[0099] Storage Conditions: Keep refrigerated at 4℃ / Manufacturer: SANTA CRUZ BIOTECHNOLOGY, sc-7985-R

[0100] 2-2) Hang-Akt-1 (F-8)

[0101] Storage conditions: Keep refrigerated at 4℃ / Manufacturer: SANTA CRUZ BIOTECHNOLOGY, sc-271149

[0102] 2-3) Goat Anti-Rabbit IgG Fc-HRP

[0103] Storage Conditions: Keep refrigerated at 4℃ / Manufacturer: ABFRONTEIR

[0104] 2-4) Goat Anti-Mouse IgG Fc-HRP

[0105] Storage Conditions: Keep refrigerated at 4℃ / Manufacturer: ABFRONTEIR

[0106] 2-5) NP40 Cell Lysis Buffer

[0107] Storage conditions: Store frozen at -20℃ / Manufacturer: INVITROGEN, FNN0021

[0108] 2-6) Bovine serum albumin (BSA)

[0109] Storage Conditions: Keep refrigerated at 4℃ / Manufacturer: CELLCONIC, FNN0021

[0110] 2-7) Protein assay dye reagent concentrate

[0111] Storage conditions: Keep refrigerated at 4℃ / Manufacturer: BIO_RAD, #5000006

[0112] 2-8) Immuno-bolt for Protein Blotting ® PVDF membrane

[0113] Storage conditions: Store at room temperature / Manufacturer: BIO-RAD, #1620177

[0114] 2-9) WEST SAVE GOLD,

[0115] Storage Conditions: Keep refrigerated at 4℃ / Manufacturer: AB FRONTIER, LF-QC0103

[0116] 2-10) Da Vinci Western Imaging System

[0117] Manufacturer: DAVINCH-K, CAS-400SM

[0118] 3) siRNA transfection

[0119] 3-1) IGF-IR α / β siRNA

[0120] Storage conditions: Store frozen at -20℃ / Manufacturer: SANTA CRUZ BIOTECHNOLOGY, sc-29358

[0121] 3-2) Lipofectamine™ RNAiMAX Transfection Reagent

[0122] Storage conditions: Keep refrigerated at 4℃ / Manufacturer: INVITROGEN, 13778075

[0123] 3-3) Opti-MEM ® Medium

[0124] Storage conditions: Keep refrigerated at 4℃ / Manufacturer: GIBCO, 31985062

[0125] (2) Test method

[0126] (2-1) in situ PLA

[0127] - Composition of the test group

[0128] Sample throughput n per test group Control 1 mL 5 1 ppm of the peptide of the present invention 1 mL 5 5 ppm of the peptide of the present invention 1 mL 5 10 ppm of the peptide of the present invention 1 mL 5

[0129] - Exam process

[0130] 1) Place a 12 mm circular microscope cover glass in a 24-well culture plate and 2.5 x 10 4 After dispensing cells into each well, the monolayer culture status of the cells was checked after 24 hours of culture, and the test was conducted when the cell confluence was 50% or higher.

[0131] 2) The negative control and test substance were each treated for 15 minutes according to the concentration of each treatment group.

[0132] 3) After fixing the cells using 4% paraformaldehyde, the cells were perforated with 0.1% Triton X-100 to perform a pretreatment that increases the permeability of cell antibodies.

[0133] 4) Subsequent tests were conducted using an In situ PLA Kit (NaveniFlex 100RM) and were performed according to the manufacturer's instructions.

[0134] 5) After washing once with PBS, the blocking solution was blocked at 37°C for 30 minutes.

[0135] 6) 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).

[0136] 7) After adding the PLA probe and reacting at 37°C for 1 hour, the mixture was washed 3 times with TTBS.

[0137] 8) Reactions A, B, and C were treated sequentially 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 staining).

[0138] 9) The PLA signal detected in the cells was observed and captured using a digital fluorescence imaging system (LOGOS BIOSYSTEMS, CS20002).

[0139] - Observation and judgment of results

[0140] The PLA fluorescence signal was evaluated by quantitative analysis using NIS-Elements BR3.1. The luminescence signal due to the interaction of β-catenin / TCF in the test substance treatment group was compared and analyzed against a negative control.

[0141] (2-2) Western Blot Analysis

[0142] - Composition of the test group

[0143] Sample throughput n per test group Control 1 mL 3 1 ppm of the peptide of the present invention 1 mL 3 5 ppm of the peptide of the present invention 1 mL 3 10 ppm of the peptide of the present invention 1 mL 3

[0144] Sample throughput n per test group Control 1 mL 3 10 ppm peptide of the present invention + Control siRNA 1 mL 3 Peptide 10 ppm of the present invention + IGF-IR α / β siRNA 10 uM 1 mL 3

[0145] - Exam process

[0146] 1) 5 x 10⁶ in a 6-well culture plate 6 Cells were dispensed into each well. After 24 hours of culture, the monolayer culture status of the cells was checked, and the test was conducted when the cell confluence was 80% or higher.

[0147] 1-1) In the case of the siRNA-treated group, the medium was replaced with dedicated medium (DMEM + 10% FBS) when the cell confluence was 50%.

[0148] 1-2) siRNA + Opti-MEM ® Media and transfection reagent + Opti-MEM ® The culture medium was prepared in a 1:1 ratio, slowly mixed, and then reacted at room temperature for 5 minutes.

[0149] 1-3) The solution from 1-2) was added to cultured cells and treated for 6 hours, then the culture medium was replaced with fresh medium and cultured for 24 hours to conduct the test.

[0150] 2) The negative control and test substances were treated for 15 minutes according to the concentration of each treatment group.

[0151] 3) Cells were lysed using NP40 cell lysis buffer, and a cell extract for electrophoresis was prepared through quantification using the Bradford assay method.

[0152] 4) 25 μg of the quantified cell extract was loaded into each well of a sodium dodecyl sulfate-polyacrylamide gel and electrophoresis was performed.

[0153] 5) The proteins developed on SDS-PAGE were transferred to a PVDF membrane.

[0154] 6) A blocking solution (3% BSA, 0.05% Tween 20, TBS) was applied to a PVDF membrane and reacted at room temperature for 1 hour.

[0155] 7) The primary antibody was reacted at room temperature for 2 hours, and washed 3 times with a washing solution (0.05% Tween 20, TBS).

[0156] 8) The secondary antibody was reacted at room temperature for 1 hour and washed 5 times with the washing solution.

[0157] 9) After exposure using an antibody detection kit, the results were confirmed through a Western blot imaging system.

[0158] - Observation and judgment of results

[0159] The expression levels of each protein treated with the test substance were evaluated by quantitative analysis using ImageJ, based on the expression level of β-Actin used as a loading control, which was captured using a Western blot imaging system. Changes in Akt phosphorylation and IGF1R in the test substance treatment group were observed relative to the negative control.

[0160] (3) Test results

[0161] (3-1) in situ PLA

[0162] Compared to the negative control group, the interaction between GRB2 and SOS1 was observed to increase in a concentration-dependent manner in the test substance treatment group (Fig. 2).

[0163] (3-2) Western Blot Analysis

[0164] Compared to the negative control group, it was confirmed that Akt phosphorylation increased in a concentration-dependent manner in the test substance treatment group (Fig. 3), and when IGF1R expression was inhibited by treatment with IGF1R siRNA, Akt phosphorylation and a decrease in IGF1R expression were observed (Fig. 4).

[0165] (4) Conclusion

[0166] It was confirmed that the test substance activates the downstream signaling pathway through the IGF-1 receptor. Therefore, the peptide of the present invention (a peptide composed of Arg-Arg-Leu) is considered to be capable of acting as an IGF-1 receptor agonist.

[0167] Test Example 3: Evaluation of Cell Proliferation Induction Efficacy

[0168] It is known that when stimulated, the IGF-1 receptor regenerates tissues through cell proliferation and differentiation and heals wounds. We observed whether the peptide of the present invention (a peptide composed of Arg-Arg-Leu) induces cell proliferation using a proliferation assay kit (CCK-8).

[0169] (1) Test materials

[0170] - Preparation of test substances

[0171] The peptide of the present invention (a peptide composed of Arg-Arg-Leu) was dissolved in triple distilled water to produce a concentration of 1000 ppm.

[0172] - Test

[0173] 1) Cell line: Human dermal fibroblast (HDF Passage 5-10, CEFObio)

[0174] 2) Cell management: The cell line was thawed from cryopreservation and inoculated into a 100 cm² animal cell culture dish containing culture medium, cultured in an incubator (5% CO2, 37°C), and subcultured with fresh culture medium every 2 to 3 days.

[0175] 3) Medium: CEFOgro Human MSC Growth Medium

[0176] Composition: 10% Fetal Bovine Serum, 1% Antibiotic / Storage: Refrigerated / Manufacturer: CEFObio

[0177] - Test materials

[0178] 1) Cell viability assay kit

[0179] Storage Conditions: Keep refrigerated at 4℃ / Manufacturer: BYLABS, BYVA0500

[0180] 2) Microplate reader

[0181] Manufacturer: BIO-TEK, EL808

[0182] (2) Test method

[0183] - Composition of the test group

[0184] Sample throughput n per test group Control 1 mL 3 1 ppm of the peptide of the present invention 1 mL 3 5 ppm of the peptide of the present invention 1 mL 3 10 ppm of the peptide of the present invention 1 mL 3

[0185] - Exam process

[0186] 1) 5 x 10⁶ in a 96-well culture plate 3 0.1 mL of cells were dispensed into each well.

[0187] 2) The negative control and test substances were administered for 24, 48, and 72 hours according to the concentrations of each treatment group.

[0188] 3) 10 µl of cell viability assay solution was added to each well and reacted at 37°C for 2 hours.

[0189] 4) The absorbance (450 nm) of each test group was measured using a microplate reader.

[0190] 5) The test was conducted according to the instructions in the ELISA kit manual.

[0191] - Observation and judgment of results

[0192] 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-treated group was compared and analyzed against a negative control group.

[0193] (3) Test results

[0194] Compared to the negative control group, it was observed that the degree of cell proliferation increased in a concentration-dependent manner in the test substance treatment group (Fig. 5).

[0195] (4) Conclusion

[0196] It was confirmed that cell proliferation is promoted in a concentration-dependent manner by treatment with the test substance. Therefore, the peptide of the present invention (a peptide composed of Arg-Arg-Leu) is judged to have skin cell proliferation-inducing efficacy.

[0197] Test Example 4: Evaluation of efficacy in inducing collagen, elastin, and HAS2 synthesis

[0198] IGF receptors play a crucial role in skin cell proliferation and are known to promote the secretion of collagen, elastin, and hyaluronic acid for the rearrangement of newly formed tissue. Immunofluorescence and Western blot analysis were used to observe whether the test substance demonstrates efficacy in activating IGF receptors to promote the secretion of collagen, elastin, and hyaluronic acid.

[0199] (1) Test materials

[0200] - Preparation of test substances

[0201] The peptide of the present invention (a peptide composed of Arg-Arg-Leu) was dissolved in triple distilled water to produce a concentration of 1000 ppm.

[0202] - Test

[0203] 1) Cell line: Human dermal fibroblast (HDF Passage 5-10, CEFObio)

[0204] 2) Cell management: The cell line was thawed from cryopreservation and inoculated into a 100 cm² animal cell culture dish containing culture medium, cultured in an incubator (5% CO2, 37℃), and subcultured with fresh culture medium every 2 to 3 days.

[0205] 3) Medium: CEFOgro Human MSC Growth Medium

[0206] Composition: 10% Fetal Bovine Serum, 1% Antibiotic / Storage: Refrigerated / Manufacturer: CEFObio

[0207] - Test materials

[0208] 1) Immunofluorescence

[0209] 1-1) Anti-COL1A2 (C-19) Antibody

[0210] Storage Conditions: Keep refrigerated at 4℃ / Manufacturer: SANTA CRUZ BIOTECHNOLOGY, SC-8786

[0211] 1-2) Anti-HAS2 (Y-14) Antibody

[0212] Storage Conditions: Keep refrigerated at 4℃ / Manufacturer: SANTA CRUZ BIOTECHNOLOGY, SC-34068

[0213] 1-3) Rabbit Anti-Goat IgG-FITC (Fluorescein isothiocyanate) Antibody

[0214] Storage conditions: Keep refrigerated at 4℃ / Manufacturer: INVITROGEN, A11078

[0215] 1-4) Bovine serum albumin (BSA)

[0216] Storage Conditions: Keep refrigerated at 4℃ / Manufacturer: CELLCONIC, FNN0021

[0217] 1-5) Phalloidin-Rhodamine

[0218] Storage Conditions: Store frozen at -20℃ / Manufacturer: INVITROGEN, R415

[0219] 1-6) Prolong™ diamond antifade mountant with DAPI

[0220] Storage conditions: Store frozen at -20℃ / Manufacturer: INVITROGEN, P36962

[0221] 1-7) Bovine serum albumin (BSA)

[0222] Storage Conditions: Keep refrigerated at 4℃ / Manufacturer: CELLCONIC, FNN0021

[0223] 1-8) Digital Fluorescence Imaging System

[0224] Manufacturer: LOGOS BIOSYSTEMS, CS20002

[0225] 2) Western blot analysis

[0226] 2-1) Anti-elastin (E-11) Antibody

[0227] Storage Conditions: Keep refrigerated at 4℃ / Manufacturer: SANTA CRUZ BIOTECHNOLOGY, SC-166543

[0228] 2-2) Goat anti-mouse e IgG Fc-HRP (secondary antibody)

[0229] Storage Conditions: Keep refrigerated at 4℃ / Manufacturer: ABFRONTEIR

[0230] 2-3) NP40 Cell Lysis Buffer

[0231] Storage conditions: Store frozen at -20℃ / Manufacturer: INVITROGEN, FNN0021

[0232] 2-4) Bovine serum albumin (BSA)

[0233] Storage Conditions: Keep refrigerated at 4℃ / Manufacturer: CELLCONIC, FNN0021

[0234] 2-5) Protein assay dye reagent concentrate

[0235] Storage conditions: Keep refrigerated at 4℃ / Manufacturer: BIO_RAD, #5000006

[0236] 2-6) Immuno-bolt for Protein Blotting® PVDF membrane

[0237] Storage conditions: Store at room temperature / Manufacturer: BIO-RAD, #1620177

[0238] 2-7) WEST SAVE GOLD,

[0239] Storage Conditions: Keep refrigerated at 4℃ / Manufacturer: AB FRONTIER, LF-QC0103

[0240] 2-8) Da Vinci Western Imaging System

[0241] Manufacturer: DAVINCH-K, CAS-400SM

[0242] (2) Test method

[0243] (2-1) Immunofluorescence

[0244] - Composition of the test group

[0245] Sample throughput n per test group Control 1 mL 5 1 ppm of the peptide of the present invention 1 mL 5 5 ppm of the peptide of the present invention 1 mL 5 10 ppm of the peptide of the present invention 1 mL 5

[0246] - Exam process

[0247] 1) Place a 12 mm circular microscope cover glass in a 24-well culture plate and 2.5 x 10 4 After dispensing cells into each well, the monolayer culture status of the cells was checked after 24 hours of culture, and the test was conducted when the cell confluence was 50% or higher.

[0248] 2) The negative control and test substances were treated at concentrations of 1, 5, and 10 ppm, respectively, for 24 hours.

[0249] 3) After fixing the cells using 4% paraformaldehyde, the cells were perforated with 0.1% Triton X-100 to perform a pretreatment that increases the permeability of cell antibodies.

[0250] 4) After washing once with PBS, block with a blocking solution (5% BSA in PBS) at 37°C for 10 minutes.

[0251] 5) To stain the cytoskeleton, Phalloidin-Rhodamine (1:500) was reacted at room temperature for 40 minutes, and then washed three times with washing solution (PBS).

[0252] 6) The antibody for confirmation was diluted 1:100, reacted at 4°C for 16 hours, and washed 3 times with PBS.

[0253] 7) The FITC-conjugated secondary antibody was diluted 1:500 and reacted at 37°C for 40 minutes, then washed 5 times with PBS.

[0254] 8) Mounting was performed using a mounting solution.

[0255] 9) Fluorescence signals detected in cells were observed and captured using a digital fluorescence imaging system (LOGOS BIOSYSTEMS, CS20002).

[0256] - Observation and judgment of results

[0257] The luminescence levels of collagen-FITC and HAS2-FITC in the test substance treatment group were compared and analyzed based on a negative control group.

[0258] (2-2) Western Blot Analysis

[0259] - Composition of the test group

[0260] Sample throughput n per test group Control 1 mL 3 1 ppm of the peptide of the present invention 1 mL 3 5 ppm of the peptide of the present invention 1 mL 3 10 ppm of the peptide of the present invention 1 mL 3

[0261] - Exam process

[0262] 1) 5 x 10⁶ in a 6-well culture plate 6 Cells were dispensed into each well. After 24 hours of culture, the monolayer culture status of the cells was checked, and the test was conducted when the cell confluence was 80% or higher.

[0263] 2) The negative control and test substances were treated at concentrations of 1, 5, and 10 ppm, respectively, for 15 minutes.

[0264] 3) Cells were lysed using NP40 cell lysis buffer, and a cell extract for electrophoresis was prepared through quantification using the Bradford assay method.

[0265] 4) 25 μg of the quantified cell extract was loaded into each well of a sodium dodecyl sulfate-polyacrylamide gel and electrophoresis was performed.

[0266] 5) The proteins developed on SDS-PAGE were transferred to a PVDF membrane.

[0267] 6) A blocking solution (3% BSA, 0.05% Tween 20, TBS) was applied to a PVDF membrane and reacted at room temperature for 1 hour.

[0268] 7) The primary antibody was reacted at room temperature for 2 hours, and washed 3 times with a washing solution (0.05% Tween 20, TBS).

[0269] 8) The secondary antibody was reacted at room temperature for 1 hour and washed 5 times with the washing solution.

[0270] 9) After exposure using an antibody detection kit, the results were confirmed through a Western blot imaging system.

[0271] - Observation and judgment of results

[0272] The expression level of elastin resulting from treatment with the test substance was evaluated by quantitative analysis using ImageJ based on the expression level of β-Actin used as a loading control, which was captured using a Western blot imaging system. The expression level of elastin in the test substance treatment group was observed based on the negative control.

[0273] (3) Test results

[0274] (3-1) Immunofluorescence

[0275] When the changes in collagen and HAS2 expression levels measured in each test group using a digital fluorescence imaging system (LOGOS BIOSYSTEMS, CS20002) were compared with a negative control group, it was observed that the levels increased in a concentration-dependent manner in the test substance treatment group (Fig. 6).

[0276] (3-2) Western Blot Analysis

[0277] Changes in elastin expression levels in the test substance treatment group were observed using a Western blot imaging system relative to a negative control, and it was observed that there was a concentration-dependent increase in both the negative control and the test substance treatment group (Fig. 7).

[0278] (4) Conclusion

[0279] It was confirmed that the test substance promotes the expression of collagen, HAS2, and elastin in a concentration-dependent manner. Therefore, the peptide of the present invention (a peptide composed of Arg-Arg-Leu) is considered to be effective in maintaining and strengthening skin tissue.

[0280] Test Example 5: Evaluation of the efficacy of inducing collagen synthesis in a 3D human skin model

[0281] Through the above test, it was confirmed that the test substance activates IGF receptors at the cellular level. Verhoeff Van Gieson staining was used to observe whether the test substance activates IGF receptors and promotes collagen synthesis in a 3D human skin model (Neoderm-ED) similar to human skin.

[0282] (1) Test materials

[0283] - Preparation of test substances

[0284] The peptide of the present invention (a peptide composed of Arg-Arg-Leu) was dissolved in DMSO to prepare a concentration of 1000 ppm.

[0285] - Test

[0286] 1) 3D Human Skin Model: Neoderm-ED

[0287] 2) Management: Incubated in an incubator (5% CO2, 37℃) and tested within 3 days of receipt.

[0288] 3) Medium: Maintenance medium

[0289] Composition: 10% Fetal Bovine Serum / Storage: Refrigerated / Manufacturer: TEGO SCIENCE

[0290] - Test materials

[0291] 1) Elastic Stain Kit (Verhoeff Van Gieson EVG Stain)

[0292] Storage conditions: Room temperature / Manufacturer: abcam, ab150667

[0293] (2) Test method

[0294] - Composition of the test group

[0295] Sample throughput n per test group Control 1 mL 5 5 ppm of the peptide of the present invention 1 mL 5

[0296] - Exam process

[0297] 1) After receiving Neoderm-ED, dedicated medium was added and cultured for 24 hours.

[0298] 2) The negative control and test substances were treated at a concentration of 5 ppm for 48 hours.

[0299] 3) Neoderm-ED was separated from the insert well using a blade to produce a paraffin block.

[0300] 4) Slides were prepared by sectioning them to a thickness of 4 µm.

[0301] 5) Paraffin washing and hydration processes using xylene (Et-OH 100% >95%>90%>80%>70%) were carried out.

[0302] 6) After rinsing with DW, the test was performed using the Elastic Stain Kit (Verhoeff Van Gieson EVG Stain) (abcam), and the test was conducted according to the manufacturer's instructions.

[0303] 7) Hematoxyline solution (5%), ferric chloride solution (10%), and Lugol's iodine solution were mixed to make an Elastic Stain Solution, treated on a slide for 15 minutes, and then washed under running water.

[0304] 8) Rinse the slide 20 times in a Ferric Chloride (2%) Differentiating Solution and wash it again under running water.

[0305] 9) After treating with sodium thiosulfate solution for 1 minute, the slide was washed under running water.

[0306] 10) After treatment with Van Gieson's Solution for 2 minutes, wash with EtOH 95%>100%.

[0307] 11) Mounting was performed using a mounting solution.

[0308] 12) The stained tissue was observed and photographed using a microscope (OLYMPUS, BX53F2).

[0309] - Observation and judgment of results

[0310] The degree of collagen expression in the test substance-treated group was compared and analyzed based on the shape of the tissue stained red, relative to the negative control group.

[0311] (3) Test results

[0312] Compared to the negative control group, it was observed that the proliferation of keratinocytes and fibroblasts and the expression of collagen increased in the test substance treatment group (Fig. 8).

[0313] (4) Conclusion

[0314] It was confirmed that the test substance induces the proliferation of keratinocytes in the epidermis and promotes the proliferation of fibroblasts and the expression of collagen, a skin structural protein, in the dermal tissue. Therefore, it was confirmed that the peptide of the present invention (a peptide composed of Arg-Arg-Leu) is effective in maintaining and strengthening skin structure not only at the cellular level but also in a skin model.

Claims

Claim 1 A pharmaceutical composition for wound healing or promoting wound healing, comprising as an active ingredient a peptide of Chemical Formula 1 below or a pharmaceutically acceptable salt thereof, wherein the peptide induces collagen synthesis by activating IGF-1R (insulin-like growth factor-1 receptor). <Chemical Formula 1> Claim 2 A cosmetic composition for inhibiting skin wrinkle formation, comprising a peptide of the following Chemical Formula 1 or a pharmaceutically acceptable salt thereof. <Chemical Formula 1>

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