Composition for treating wound or inhibiting skin wrinkle formation

Abstract

The present invention provides: a pharmaceutical composition for wound healing and a cosmetic composition for inhibiting skin wrinkle formation, both comprising a specific peptide. The peptide acts as an agonist for IGF-1R, thereby effectively inducing IGF-1R signal transduction, promoting cell proliferation, and inducing the synthesis of collagen, elastin, and HAS2. The peptide can be advantageously used for wound healing and the inhibition of skin wrinkle formation.

Description

Composition for wound healing or skin wrinkle prevention

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

[0002] IGF-1 / IGF-1R is a growth factor that promotes cell proliferation and inhibits apoptosis, playing a crucial role in cell growth and maintenance. IGF-1 / IGF-1R protects keratinocytes from external stress and helps repair DNA damage in cells, thereby inhibiting apoptosis. Furthermore, it promotes keratinocyte proliferation and collagen and elastin production, thereby regenerating damaged skin tissue and aiding wound healing. (Jennifer M Kavran, et al., Elife. 2014 Sep 25:3:e03772; Weikang Cai, et al., Nature communications, 2017, 8.1: 14892, etc.)

[0003] It is known that IGF-1 transmits signals into cells through various pathways such as phosphoinositide 3-kinase (PI3K)-Akt and Ca2+ when bound to IGF-1R of skin keratinocytes (Hwa Jin Jung, at al., Frontiers in genetics, 2015, 5: 472; Antero Salminen et al., Inflammation Research,2021, 7010-12: 1043-1061). When stimulated, IGF-1 receptors regenerate tissues and heal 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 remodeling of new tissues (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 that can activate IGF-1R can act not only as active substances for wound healing or promoting wound healing, but also as functional cosmetic substances for inhibiting skin aging (e.g., skin wrinkle formation).

[0004] 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).

[0005] The present inventors conducted various studies to develop an active substance based on small peptides as a substance capable of activating IGF-1R. As a result, we 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, and inducing cell proliferation and collagen, elastin, and HAS2 (Hyaluronan synthase 2) synthesis, and thus can be usefully applied to wound healing or suppressing skin wrinkle formation.

[0006] Accordingly, the present invention aims to provide a pharmaceutical composition for wound treatment or wound healing promotion 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 wrinkle formation, which comprises the specific peptide.

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

[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 the peptide of the above chemical formula 1 or a pharmaceutically acceptable salt thereof.

[0012] The present invention has revealed 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 collagen, elastin, and HAS2 (Hyaluronan synthase 2) synthesis. The present invention has also revealed that the peptide according to the present invention effectively induces collagen 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, including inhibiting skin wrinkle formation.

[0013] Figure 1 shows the results of measuring the signal reduction of the peptide (VE-Igrofin)-rhodamine of the present invention composed of Arg-Arg-Leu and IGF-1R-FITC by treatment with IGF-IR α / β siRNA.

[0014] Figure 2 shows the results of analyzing changes in GRB2 / SOS1 interaction due to peptide treatment of the present invention.

[0015] Figure 3 shows the results of analyzing changes in Akt phosphorylation by peptide treatment of the present invention.

[0016] Figure 4 shows the results of analyzing changes in Akt phosphorylation and IGF-1R expression by treatment with the peptide of the present invention and IGF-1R siRNA.

[0017] Figure 5 shows the results of analyzing the cell proliferation rate by peptide treatment of the present invention.

[0018] Figure 6 shows the results of analyzing changes in the expression levels of collagen and HAS2 by peptide treatment of the present invention.

[0019] Figure 7 shows the results of analyzing changes in elastin expression levels due to peptide treatment of the present invention.

[0020] Figure 8 shows the results of analyzing the proliferation of keratinocytes and fibroblasts and changes in the expression level of collagen proteins by peptide treatment of the present invention in a 3D human skin model.

[0021] The present invention provides a pharmaceutical composition for wound treatment or wound healing promotion, comprising a peptide of the following chemical formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[0022] <Chemical Formula 1>

[0023]

[0024] In addition, the present invention provides a cosmetic composition for inhibiting skin wrinkle formation, comprising the peptide of the above chemical formula 1 or a pharmaceutically acceptable salt thereof.

[0025] In the pharmaceutical composition or cosmetic composition of the present invention, the peptide of Chemical Formula 1 may also be expressed as "Arg-Arg-Leu". The amino acids constituting the peptide of Chemical Formula 1 may independently be in the form of L-amino acids or D-amino acids. Pharmaceutically acceptable salts of the peptide derivative of Chemical Formula 1 include, but are not limited to, acid addition salts, for example.

[0026] The pharmaceutical composition of the present invention may include excipients such as lactose and corn starch, lubricants such as magnesium stearate, and known and usable emulsifiers, suspending agents, buffers, isotonic agents, etc., and may be formulated as a parenteral dosage form, preferably a parenteral dosage form including a topical skin 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 dosage form such as a topical 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 10 mg / kg per day. The appropriate dosage may generally vary depending on the patient's age, weight, and symptoms.

[0027] The cosmetic composition of the present invention may be in the form of a functional cosmetic composition containing the above-described peptide as an active ingredient. The cosmetic composition may be manufactured in various forms according to a conventional cosmetic manufacturing method. For example, the cosmetic composition may be manufactured 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 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 peptide is an amount effective for achieving an effect of inhibiting skin aging, particularly, inhibiting skin wrinkle formation, for example, 1 x 10 based on the total weight of the composition.-5 ~ 2 x 10 -3 It may be contained in a content of weight %, preferably about 1 x 10 -4 ~ 1 x 10 -3 It can be contained in a content of weight%.

[0028] 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.

[0029] Example 1. Synthesis of peptides

[0030] A peptide consisting of Arg-Arg-Leu 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).

[0031] Example 2. Preparation of a composition containing a peptide

[0032] The peptide (peptide composed of Arg-Arg-Leu) prepared 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.

[0033] Test Example 1: IGF Receptor Binding Evaluation

[0034] IGF-1 (insulin-like growth factors) is known to transmit signals into cells through IGF-1R. Whether the peptide of the present invention (a peptide composed of Arg-Arg-Leu) binds to IGF-1R was confirmed through immunofluorescence.

[0035] (1) Test materials

[0036] - Preparation of test substances

[0037] The peptide of the present invention (a peptide composed of Arg-Arg-Leu) was dissolved in triple-distilled water to prepare a concentration of 1000 ppm.

[0038] - Test system

[0039] 1) Cell line: Human dermal fibroblast (HDF Passage 5-10, CEFObio)

[0040] 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.

[0041] 3) Medium: CEFOgro Human MSC Growth medium

[0042] Composition: 10% fetal bovine serum, 1% antibiotic / Storage conditions: Refrigerated / Manufacturer: CEFObio

[0043] - Test materials

[0044] 1) Peptide of the present invention labeled with Rhodamine

[0045] Storage conditions: -20℃ frozen storage / Manufacturer: Peptron

[0046] 2) Anti-IGF-IR (7G11) antibody

[0047] Storage conditions: -20℃ frozen storage / Manufacturer: SANTA CRUZ BIOTECHNOLOGY, sc-37193

[0048] 3) FITC-labeled anti-mouse IgG

[0049] Storage conditions: Refrigerated at 4℃ / Manufacturer: INVITROGEN, A11001

[0050] 4) siRNA transfection

[0051] 4-1) IGF-IR α / β siRNA

[0052] Storage conditions: -20℃ frozen storage / Manufacturer: SANTA CRUZ BIOTECHNOLOGY, sc-29358

[0053] 4-2) Lipofectamine™ RNAiMAX Transfection Reagent

[0054] Storage conditions: Refrigerated at 4℃ / Manufacturer: INVITROGEN, 13778075

[0055] 4-3) Opti-MEM ® Medium

[0056] Storage conditions: Refrigerated at 4℃ / Manufacturer: GIBCO, 31985062

[0057] (2) Test method

[0058] - Composition of the test group

[0059]

[0060] - Exam process

[0061] 1) Place 12 mm microscope round cover glasses in a 24-well culture plate and add 2.5X10 4 After dispensing the 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 confluency of the cells was 50% or higher.

[0062] 2) The medium was replaced with a dedicated medium (DMEM + 10% FBS) for siRNA treatment.

[0063] 3) siRNA + Opti-MEM ® Media and transfection reagent + Opti-MEM ® The mixture was mixed in a 1:1 ratio and slowly mixed, then left to react at room temperature for 5 minutes.

[0064] 4) The solution of 3) was added to cultured cells and treated for 48 hours to conduct the test.

[0065] 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).

[0066] 6) After treating with IGF-1R antibody (1:100), the cells were incubated at room temperature for 40 minutes and washed three times with washing solution (PBS).

[0067] 7) After treating with secondary antibody (1:2000), reacting at room temperature for 40 minutes, the cells were washed three times with washing solution (PBS).

[0068] 8) Mounting was performed using a mounting solution containing DAPI (nuclear staining).

[0069] 9) The fluorescent signals detected in the cells were observed and photographed using a digital fluorescence imaging system (LOGOS BIOSYSTEMS, CS20002).

[0070] - Observe and judge results

[0071] 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 using the control siRNA negative control group as a standard.

[0072] (3) Test results

[0073] 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 treatment group. In the negative control group, the test substance bound to IGF-1R and exhibited a rhodamine (red fluorescence) signal, and the IGF-1R antibody exhibited a FITC (green fluorescence) signal. When the expression of IGF-1R was suppressed by treating with IGF-1R siRNA, the rhodamine and FITC signals were reduced (Fig. 2).

[0074] (4) Conclusion

[0075] As a result of evaluating the degree of IGF-1R binding of the test substance, the signal expression locations of the peptide-rhodamine of the present invention and IGF-1R-FITC were identical. Therefore, it is determined that the peptide of the present invention (a peptide composed of Arg-Arg-Leu) specifically binds to IGF-1R.

[0076] Test Example 2. Evaluation of IGF signaling induction efficacy

[0077] When IGF-1 binds to IGF-1R of skin keratinocytes, it activates phosphoinositide 3-kinase (PI3K)-Akt, Ca2 + It is known that signals are transmitted into cells through various pathways such as the backbone. In order to verify the possibility of the peptide of the present invention as an IGF-1 receptor agonist, changes in Akt phosphorylation and GRB2 / SOS1 interaction, which are downstream signaling pathways of the IGF-1 receptor, were observed through Western blot analysis and in situ PLA, respectively, and whether the peptide of the present invention transmits signals through IGF-1R was confirmed by treating with IGF-1R siRNA.

[0078] (1) Test materials

[0079] - Preparation of test substances

[0080] The peptide of the present invention (a peptide composed of Arg-Arg-Leu) was dissolved in triple-distilled water to prepare a concentration of 1000 ppm.

[0081] - Test system

[0082] 1) in situ PLA

[0083] 1-1) Cell line: Human Keratinocyte (HaCaT, CLS)

[0084] 1-2) 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 2-3 days.

[0085] 1-3) Medium: DMEM (Dulbecco's Modified Eagle Medium)

[0086] Composition: 10% fetal bovine serum, 1% antibiotic / Storage conditions: Refrigerated storage / Manufacturer: GIBCO

[0087] 2) Western blot analysis (1), (2)

[0088] 2-1) Cell line: Human keratinocyte (HaCaT, CLS)

[0089] 2-2) 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 2-3 days.

[0090] 2-3) Medium: DMEM (Dulbecco's Modified Eagle Medium)

[0091] Composition: 10% fetal bovine serum, 1% antibiotic / Storage conditions: Refrigerated storage / Manufacturer: GIBCO

[0092] - Test materials

[0093] 1) in situ PLA

[0094] 1-1) Anti-GRB2 antibody

[0095] Storage conditions: Refrigerated at 4℃ / Manufacturer: Invitrogen, PA1-10033

[0096] 1-2) Anti-SOS1 (C-23) antibody

[0097] Storage conditions: Refrigerated at 4℃ / Manufacturer: SANTA CRUZ BIOTECHNOLOGY, sc-256

[0098] 1-3) NaveniFlex 100RM

[0099] Storage conditions: -20℃ frozen storage / Manufacturer: NaveniFlex, NV C-NF MR.100

[0100] 1-4) Prolong™ diamond antifade mountant with DAPI

[0101] Storage conditions: -20℃ frozen storage / Manufacturer: INVITROGEN, P36962

[0102] 1-5) Digital Fluorescence Imaging System

[0103] Manufacturer: LOGOS BIOSYSTEMS, CS20002

[0104] 2) Western blot analysis

[0105] 2-1) Anti-Akt1 / 2 / 3-phospho (S473)-R antibody

[0106] Storage conditions: Refrigerated at 4℃ / Manufacturer: SANTA CRUZ BIOTECHNOLOGY, sc-7985-R

[0107] 2-2) Anti-Akt-1 (F-8)

[0108] Storage conditions: Refrigerated at 4℃ / Manufacturer: SANTA CRUZ BIOTECHNOLOGY, sc-271149

[0109] 2-3) Goat anti-rabbit IgG Fc-HRP

[0110] Storage conditions: Refrigerated at 4℃ / Manufacturer: ABFRONTEIR

[0111] 2-4) Goat anti-mouse IgG Fc-HRP

[0112] Storage conditions: Refrigerated at 4℃ / Manufacturer: ABFRONTEIR

[0113] 2-5) NP40 cell lysis buffer

[0114] Storage conditions: -20℃ frozen storage / Manufacturer: INVITROGEN, FNN0021

[0115] 2-6) Bovine serum albumin (BSA)

[0116] Storage conditions: Refrigerated at 4℃ / Manufacturer: CELLCONIC, FNN0021

[0117] 2-7) Protein assay dye reagent concentrate

[0118] Storage conditions: Refrigerated at 4℃ / Manufacturer: BIO_RAD, #5000006

[0119] 2-8) Immuno-bolt for protein blotting ® PVDF membrane

[0120] Storage conditions: Store at room temperature / Manufacturer: BIO-RAD, #1620177

[0121] 2-9) WEST SAVE GOLD,

[0122] Storage conditions: Refrigerated at 4℃ / Manufacturer: AB FRONTIER, LF-QC0103

[0123] 2-10) DaVinci Western Imaging System

[0124] Manufacturer: DAVINCH-K, CAS-400SM

[0125] 3) siRNA transfection

[0126] 3-1) IGF-IR α / β siRNA

[0127] Storage conditions: -20℃ frozen storage / Manufacturer: SANTA CRUZ BIOTECHNOLOGY, sc-29358

[0128] 3-2) Lipofectamine™ RNAiMAX Transfection Reagent

[0129] Storage conditions: Refrigerated at 4℃ / Manufacturer: INVITROGEN, 13778075

[0130] 3-3) Opti-MEM ® Medium

[0131] Storage conditions: Refrigerated at 4℃ / Manufacturer: GIBCO, 31985062

[0132] (2) Test method

[0133] (2-1) in situ PLA

[0134] - Composition of the test group

[0135]

[0136] - Exam process

[0137] 1) Place 12 mm microscope round cover glasses in a 24-well culture plate and add 2.5X10 4 After dispensing the 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 confluency of the cells was 50% or higher.

[0138] 2) The negative control and test substances were treated for 15 minutes at the concentrations of each treatment group.

[0139] 3) After fixing the cells using 4% paraformaldehyde, pretreatment was performed to increase the permeability of the cell antibody by perforating the cells with 0.1% Triton X-100.

[0140] 4) Subsequent tests were conducted using the In situ PLA Kit (NaveniFlex 100RM) and the tests were conducted according to the manufacturer's instructions.

[0141] 5) After washing once with PBS, blocking was performed with blocking solution at 37°C for 30 minutes.

[0142] 6) Two antibodies for confirmation were diluted to 10 μg / mL in antibody dilution solution, 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).

[0143] 7) After adding the PLA probe and reacting at 37°C for 1 hour, it was washed three times with TTBS.

[0144] 8) Reactions A, B, and C were processed in sequence and reacted at 37°C for 1 hour, 30 minutes, and 90 minutes, respectively. Finally, the cells were washed twice with TBS (0.01 M Tris, 0.15 M NaCl) and mounted using a mounting solution containing DAPI (nuclear staining).

[0145] 9) The PLA signal detected in the cells was observed and photographed using a digital fluorescence imaging system (LOGOS BIOSYSTEMS, CS20002).

[0146] - Observe and judge results

[0147] The PLA fluorescence signal was quantitatively analyzed and evaluated using NIS-Elements BR3.1. The luminescence signal due to the interaction of β-catenin / TCF in the test substance-treated group was compared and analyzed with the negative control group as the standard.

[0148] (2-2) Western blot analysis

[0149] - Composition of the test group

[0150]

[0151]

[0152] - Exam process

[0153] 1) 5X10 in a 6-well culture plate 6 Cells were seeded into each well. After 24 hours of culture, the monolayer culture status of the cells was confirmed, and testing was performed when the cell confluency was 80% or higher.

[0154] 1-1) For the siRNA treatment group, the medium was replaced with a dedicated medium (DMEM + 10% FBS) when the cell confluency reached 50%.

[0155] 1-2) siRNA + Opti-MEM ® Media and transfection reagent + Opti-MEM ® The mixture was mixed in a 1:1 ratio and slowly stirred, then allowed to react at room temperature for 5 minutes.

[0156] 1-3) The solution of 1-2) was added to the cultured cells and treated for 6 hours. The culture solution was replaced and cultured for 24 hours to conduct the test.

[0157] 2) The negative control and test substances were treated for 15 minutes according to the concentration of each treatment group.

[0158] 3) Cells were lysed using NP40 cell lysis buffer, and cell extracts for electrophoresis were prepared through quantification using the Bradford assay method.

[0159] 4) Electrophoresis was performed by loading 25 μg of cell extracts quantified on a sodium dodecyl sulfate-polyacrylamide gel into each well.

[0160] 5) The proteins developed on SDS-PAGE were transferred to a PVDF membrane.

[0161] 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.

[0162] 7) The primary antibody was reacted at room temperature for 2 hours, and washed three times with washing solution (0.05% Tween 20, TBS).

[0163] 8) The secondary antibody was reacted at room temperature for 1 hour and washed 5 times with washing solution.

[0164] 9) After exposure using an antibody detection kit, it was confirmed using a Western blot imaging system.

[0165] - Observe and judge results

[0166] The expression levels of each protein after treatment 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 phosphorylation of Akt and changes in IGF1R in the test substance-treated group were observed based on the negative control group.

[0167] (3) Test results

[0168] (3-1) in situ PLA

[0169] Compared to the negative control group, it was observed that the interaction of GRB2 / SOS1 increased in a concentration-dependent manner in the test substance treatment group (Fig. 2).

[0170] (3-2) Western blot analysis

[0171] 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 siRNA was treated to suppress IGF1R expression, a decrease in Akt phosphorylation and IGF1R expression was observed (Fig. 4).

[0172] (4) Conclusion

[0173] The test substance was confirmed to activate a downstream signaling pathway via the IGF-1 receptor. Therefore, the peptide of the present invention (a peptide composed of Arg-Arg-Leu) is believed to be capable of acting as an IGF-1 receptor agonist.

[0174] Test Example 3: Evaluation of cell proliferation induction efficacy

[0175] The IGF-1 receptor is known to regenerate tissues and heal wounds through cell proliferation and differentiation when stimulated. The peptide of the present invention (a peptide composed of Arg-Arg-Leu) was observed to induce cell proliferation using a proliferation assay kit (CCK-8).

[0176] (1) Test materials

[0177] - Preparation of test substances

[0178] The peptide of the present invention (a peptide composed of Arg-Arg-Leu) was dissolved in triple-distilled water to prepare a concentration of 1000 ppm.

[0179] - Test system

[0180] 1) Cell line: Human dermal fibroblast (HDF Passage 5-10, CEFObio)

[0181] 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 2-3 days.

[0182] 3) Medium: CEFOgro Human MSC Growth medium

[0183] Composition: 10% fetal bovine serum, 1% antibiotic / Storage conditions: Refrigerated / Manufacturer: CEFObio

[0184] - Test materials

[0185] 1) Cell viability assay kit

[0186] Storage conditions: Refrigerated at 4℃ / Manufacturer: BYLABS, BYVA0500

[0187] 2) Microplate reader

[0188] Manufacturer: BIO-TEK, EL808

[0189] (2) Test method

[0190] - Composition of the test group

[0191]

[0192] - Exam process

[0193] 1) 5X10 in a 96-well culture plate 3 0.1 mL of cells were dispensed into each well.

[0194] 2) The negative control and test substances were treated for 24, 48, and 72 hours according to the concentration of each treatment group.

[0195] 3) 10 ㎕ of cell viability assay solution was added to each well and reacted at 37°C for 2 hours.

[0196] 4) The absorbance (450 nm) of each test group was measured using a microplate reader.

[0197] 5) The test was conducted according to the instructions in the ELISA kit manual.

[0198] - Observe and judge results

[0199] 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.

[0200] (3) Test results

[0201] 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).

[0202] (4) Conclusion

[0203] It was confirmed that cell proliferation was 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 believed to have skin cell proliferation-inducing efficacy.

[0204] Test Example 4: Evaluation of the efficacy of inducing collagen, elastin, and HAS2 synthesis.

[0205] 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 remodeling of new tissue. The efficacy of the test substance in activating IGF receptors and promoting the secretion of collagen, elastin, and hyaluronic acid was observed using immunofluorescence and Western blot analysis.

[0206] (1) Test materials

[0207] - Preparation of test substances

[0208] The peptide of the present invention (peptide composed of Arg-Arg-Leu) was dissolved in triple-distilled water to prepare a concentration of 1000 ppm.

[0209] - Test system

[0210] 1) Cell line: Human dermal fibroblast (HDF Passage 5-10, CEFObio)

[0211] 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.

[0212] 3) Medium: CEFOgro Human MSC Growth medium

[0213] Composition: 10% fetal bovine serum, 1% antibiotic / Storage conditions: Refrigerated / Manufacturer: CEFObio

[0214] - Test materials

[0215] 1) Immunofluorescence

[0216] 1-1) Anti-COL1A2 (C-19) antibody

[0217] Storage conditions: Refrigerated at 4℃ / Manufacturer: SANTA CRUZ BIOTECHNOLOGY, SC-8786

[0218] 1-2) Anti-HAS2 (Y-14) antibody

[0219] Storage conditions: Refrigerated at 4℃ / Manufacturer: SANTA CRUZ BIOTECHNOLOGY, SC-34068

[0220] 1-3) Rabbit anti-goat IgG-FITC (Fluorescein isothiocyanate) antibody

[0221] Storage conditions: Refrigerated at 4℃ / Manufacturer: INVITROGEN, A11078

[0222] 1-4) Bovine serum albumin (BSA)

[0223] Storage conditions: Refrigerated at 4℃ / Manufacturer: CELLCONIC, FNN0021

[0224] 1-5) Phalloidin-Rhodamine

[0225] Storage conditions: -20℃ frozen storage / Manufacturer: INVITROGEN, R415

[0226] 1-6) Prolong™ diamond antifade mountant with DAPI

[0227] Storage conditions: -20℃ frozen storage / Manufacturer: INVITROGEN, P36962

[0228] 1-7) Bovine serum albumin (BSA)

[0229] Storage conditions: Refrigerated at 4℃ / Manufacturer: CELLCONIC, FNN0021

[0230] 1-8) Digital Fluorescence Imaging System

[0231] Manufacturer: LOGOS BIOSYSTEMS, CS20002

[0232] 2) Western blot analysis

[0233] 2-1) Anti-elastin (E-11) antibody

[0234] Storage conditions: Refrigerated at 4℃ / Manufacturer: SANTA CRUZ BIOTECHNOLOGY, SC-166543

[0235] 2-2) Goat anti-mouse IgG Fc-HRP (secondary antibody)

[0236] Storage conditions: Refrigerated at 4℃ / Manufacturer: ABFRONTEIR

[0237] 2-3) NP40 cell lysis buffer

[0238] Storage conditions: -20℃ frozen storage / Manufacturer: INVITROGEN, FNN0021

[0239] 2-4) Bovine serum albumin (BSA)

[0240] Storage conditions: Refrigerated at 4℃ / Manufacturer: CELLCONIC, FNN0021

[0241] 2-5) Protein assay dye reagent concentrate

[0242] Storage conditions: Refrigerated at 4℃ / Manufacturer: BIO_RAD, #5000006

[0243] 2-6) Immuno-bolt for protein blotting ® PVDF membrane

[0244] Storage conditions: Store at room temperature / Manufacturer: BIO-RAD, #1620177

[0245] 2-7) WEST SAVE GOLD,

[0246] Storage conditions: Refrigerated at 4℃ / Manufacturer: AB FRONTIER, LF-QC0103

[0247] 2-8) DaVinci Western Imaging System

[0248] Manufacturer: DAVINCH-K, CAS-400SM

[0249] (2) Test method

[0250] (2-1) Immunofluorescence

[0251] - Composition of the test group

[0252]

[0253] - Exam process

[0254] 1) Place 12 mm microscope round cover glasses in a 24-well culture plate and add 2.5X10 4 After dispensing the 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 confluency of the cells was 50% or higher.

[0255] 2) The negative control and test substances were treated at concentrations of 1, 5, and 10 ppm, respectively, for 24 hours.

[0256] 3) After fixing the cells using 4% paraformaldehyde, pretreatment was performed to increase the permeability of the cell antibody by perforating the cells with 0.1% Triton X-100.

[0257] 4) After washing once with PBS, the cells were blocked with blocking solution (5% BSA in PBS) at 37°C for 10 minutes.

[0258] 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).

[0259] 6) The antibody for confirmation was diluted 1:100, reacted at 4°C for 16 hours, and washed three times with PBS.

[0260] 7) The FITC-conjugated secondary antibody was diluted 1:500 and incubated at 37°C for 40 minutes, then washed 5 times with PBS.

[0261] 8) Mounting was performed using a mounting solution.

[0262] 9) The fluorescent signals detected in the cells were observed and photographed using a digital fluorescence imaging system (LOGOS BIOSYSTEMS, CS20002).

[0263] - Observe and judge results

[0264] The luminescence levels of collagen-FITC and HAS2-FITC in the test substance treatment groups were compared and analyzed based on the control negative control group.

[0265] (2-2) Western blot analysis

[0266] - Composition of the test group

[0267]

[0268] -Testing process

[0269] 1) 5X10 in a 6-well culture plate 6 Cells were seeded into each well. After 24 hours of culture, the monolayer culture status of the cells was confirmed, and testing was performed when the cell confluency was 80% or higher.

[0270] 2) The negative control and test substances were treated at concentrations of 1, 5, and 10 ppm, respectively, for 15 minutes.

[0271] 3) Cells were lysed using NP40 cell lysis buffer, and cell extracts for electrophoresis were prepared through quantification using the Bradford assay method.

[0272] 4) Electrophoresis was performed by loading 25 μg of cell extracts quantified on a sodium dodecyl sulfate-polyacrylamide gel into each well.

[0273] 5) The proteins developed on SDS-PAGE were transferred to a PVDF membrane.

[0274] 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.

[0275] 7) The primary antibody was reacted at room temperature for 2 hours, and washed three times with washing solution (0.05% Tween 20, TBS).

[0276] 8) The secondary antibody was reacted at room temperature for 1 hour and washed 5 times with washing solution.

[0277] 9) After exposure using an antibody detection kit, it was confirmed using a Western blot imaging system.

[0278] - Observe and judge results

[0279] The expression level of elastin in response to test substance treatment was evaluated by quantitative analysis using ImageJ based on the expression level of β-Actin, which was used as a loading control, by photographing using a Western blot imaging system. The expression level of elastin in the test substance treatment group was observed based on the negative control group.

[0280] (3) Test results

[0281] (3-1) Immunofluorescence

[0282] 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 the negative control group, a concentration-dependent increase was observed in the test substance treatment group (Fig. 6).

[0283] (3-2) Western blot analysis

[0284] As a result of observing the change in elastin expression level in the test substance treatment group based on the negative control group using a Western blot imaging system, it was observed that the expression level increased in a concentration-dependent manner in the negative control group and the test substance treatment group (Fig. 7).

[0285] (4) Conclusion

[0286] The test substance was confirmed to promote 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 believed to be effective in maintaining and strengthening skin tissue.

[0287] Test Example 5: Evaluation of collagen synthesis induction efficacy in a 3D human skin model.

[0288] The above test confirmed that the test substance activates IGF receptors at the cellular level. Using Verhoeff Van Gieson staining, we observed whether the test substance also activated IGF receptors and promoted collagen synthesis in a 3D human skin model (Neoderm-ED), which resembles human skin.

[0289] (1) Test materials

[0290] - Preparation of test substances

[0291] The peptide of the present invention (peptide composed of Arg-Arg-Leu) was dissolved in DMSO to prepare a concentration of 1000 ppm.

[0292] - Test system

[0293] 1) 3D human skin model: Neoderm-ED

[0294] 2) Management: Cultured in an incubator (5% CO2, 37℃) and tested within 3 days of receipt.

[0295] 3) Badge: Maintenance medium

[0296] Composition: 10% fetal bovine serum / Storage conditions: Refrigerated storage / Manufacturer: TEGO SCIENCE

[0297] - Test materials

[0298] 1) Elastic Stain Kit (Verhoeff Van Gieson EVG Stain)

[0299] Storage conditions: Room temperature / Manufacturer: abcam, ab150667

[0300] (2) Test method

[0301] - Composition of the test group

[0302]

[0303] - Exam process

[0304] 1) After receiving Neoderm-ED, a dedicated medium was added and cultured for 24 hours.

[0305] 2) The negative control and test substances were treated at a concentration of 5 ppm for 48 hours.

[0306] 3) Neoderm-ED was separated from the insert well using a blade to create a paraffin block.

[0307] 4) Slides were prepared by cutting sections at 4 ㎛ thickness.

[0308] 5) Paraffin washing and water dehydration process using xylene (Et-OH 100% >95%>90%>80%>70%) was performed.

[0309] 6) After rinsing in DW, the Elastic Stain Kit (Verhoeff Van Gieson EVG Stain) (abcam) was used, and the test was performed according to the manufacturer's instructions.

[0310] 7) Mix hematoxyline solution (5%), ferric chloride solution (10%), and Lugol's iodine solution to make an Elastic Stain Solution, apply it to the slide for 15 minutes, and wash the slide in running water.

[0311] 8) Rinse the slide 20 times in ferric chloride (2%) differentiating solution and then wash it again in running water.

[0312] 9) After treating with sodium thiosulfate solution for 1 minute, the slides were washed in running water.

[0313] 10) After treating with Van Gieson's solution for 2 minutes, it was washed with 95%>100% EtOH.

[0314] 11) Mounting was performed using a mounting solution.

[0315] 12) The stained tissue was observed and photographed using a microscope (OLYMPUS, BX53F2).

[0316] - Observe and judge results

[0317] The level of collagen expression in the test substance treatment group was compared and analyzed in the form of tissue stained red, based on the negative control group.

[0318] (3) Test results

[0319] 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).

[0320] (4) Conclusion

[0321] The test substance was confirmed to induce proliferation of keratinocytes in the epidermis, promote proliferation of fibroblasts in the dermal tissue, and promote expression of collagen, a skin structural protein. Therefore, the peptide of the present invention (a peptide composed of Arg-Arg-Leu) was confirmed to be effective in maintaining and strengthening skin structure not only at the cellular level but also in a skin model.

Claims

1. 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> 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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