WO2019124603A1 - Composition for preventing or treating keloid, containing iwr-1 as active ingredient - Google Patents

Abstract

The present invention relates to a composition for preventing or treating keloid, containing IWR-1 as an active ingredient. The composition containing IWR-1 as an active ingredient may be provided as a pharmaceutical composition and a cosmetic composition for preventing or treating fibrosis diseases such as keloid, as it has been confirmed that said IWR-1 exhibits the effects of inhibiting the proliferation and migration of keloid fibroblasts without affecting the TGF-β signaling system, and inhibiting collagen synthesis while increasing the expression of MMP which is a collagen degrading factor.

Description

Composition for preventing or treating keloid containing IWR-1 as an active ingredient

And a composition for preventing or treating keloid containing IWR-1 as an active ingredient.

Keloid disease is a benign skin fibro-proliferative tumor characterized by excessive accumulation of extracellular matrix proteins leading to an excess of collagen formation. Abnormal skin scar formation can occur after injury in genetically sensitive individuals.

Currently, no effective single treatment regimen for the treatment of keloids or prevention of keloid growth after surgery has been established. Existing therapies include the following: closed dressings, compression therapy, intra-lesion steroid injections, cryosurgery, surgical resection, laser therapy, radiation therapy, canallog (triamcinolone), interferon therapy, bleomycin, 5- Verapamil, imiquimodem cream, and combinations thereof. Although closed dressings of both silicone and non-silicone based have been used extensively as clinical options for keloids over the past three decades, since all of these methods lead to very limited efficacy, new therapies for keloids It is widely recognized.

In addition, various forms of radiotherapy have been tried as monotherapy for keloids, but there remains considerable debate as a case report of cancer after treatment. Laser therapy with argon, CO2, and pulse dyes has been tried over the last 40 years, none of which has been proven efficacious.

A number of studies have shown that all three forms of laser therapy have a recurrence rate of up to 90%, with little or no effect. Cryotherapy has been used as a monotherapy. However, pain at the treatment site, and hyperpigmentation or hypopigmentation are included in the side effects associated with this approach. Although injection of triamcinolone acetone in a lesion, which is a kind of corticosteroid, is often used as the primary therapy for the treatment of keloids, the actual reported clinical efficacy is highly variable. In addition, as the side effects of injection pain, skin atrophy, telangiectasias, and pigmentation degeneration have been confirmed, research on safer and more effective treatment methods is needed.

In order to solve the above problems, the present invention provides a composition containing IWR-1 as an active ingredient as a pharmaceutical composition and a cosmetic composition for preventing or treating keloid.

The present invention can provide a pharmaceutical composition for preventing or treating keloid containing IWR-1 as an active ingredient.

Further, the present invention can provide a cosmetic composition for preventing or improving keloid containing IWR-1 as an active ingredient.

According to the present invention, IWR-1 inhibits proliferation and migration of keloid fibroblasts without the influence of the TGF-beta signaling system, increases the expression of collagen degrading factor MMP and inhibits collagen synthesis , The composition containing the IWR-1 as an active ingredient can be provided as a pharmaceutical composition and a cosmetic composition for preventing or treating fibrosing diseases such as keloid.

Fig. 1A shows the chemical structure of IWR-1. Fig. 1B shows the results of confirming the cytotoxicity of IWR-1 in fibroblasts. As a result, IWR-1 at a concentration of 0 to 50 μM was incubated with human normal fibroblasts (NF) (KF) for 24 hours, and LDH activity in the culture medium. FIG. 1C shows the effect of IWR-1 on endogenous Wnt / β-catenin signal. TOPflash or FOPflash reporter adenovirus was detected overnight (* P < 0.01) after transfection and treating IWR-1 at a concentration of 0 to 20 [mu] M for 24 hours and then confirming luciferase activity.

FIG. 2A shows the results of confirming the cell proliferation effect of IWR-1. As a result, human normal skin fibroblasts (NF) and keloid fibroblasts (KF) were treated with 0 to 20 μM of IWR- FIG. 2B shows the effect of IWR-1 on cell migration by confirming the level of confluence of NF and KF after the production of a scratch wound. FIG. 2B shows the result of confirming the effect of IWR- (* P <0.01, Scale bar = 50 μm).

Fig. 3 shows the results of confirming the effect of IWR-1 on collagen production. Fig. 3A shows the results of treatment of human normal skin fibroblasts (NF) and keloid fibroblasts (KF) with 20 [mu] M IWR- FIG. 3B is a Western blot analysis showing the expression levels of collagen-1α1 and collagen-1α2 proteins. Collagen type I protein (collagen- 1α1 and collagen-1α2), and the right photograph shows the expression level of collagen type I protein at 0, 24 and 48 hours in 20 μM of IWR-1 treated cells, FIG. 3C is an ELISA analysis (* P < 0.01) showing the level of procollagen secreted in the culture medium after culturing the cells for 20 hours at a concentration of 20 μM for 48 hours, As a result of the shrinkage analysis, NF or KF The IWR-1 of 20 μM contained in the collagen gel is the result confirming the excellent shrinkage seven days after the treatment compared to the control as a result of confirming the degree of shrinkage Gel IWR-1 is treated group (* P <0.01).

FIG. 4A shows the effect of IWR-1 on TGF-β1 / Smad signal and myofibroblast differentiation. FIG. 4A shows the results of pretreatment of 20 μM of IWR-1 in NF and KF for 1 hour, Western blot analysis of Smad phosphorylation and α-SMA expression levels after stimulation with ng / ml TGF-β1 showed that IWR-1 did not affect the phosphorylation of Smad protein induced by TGF-β1 , FIG. 4B shows the result of treatment of 20 μM IWR-1 with NF and KF for 1 hour and stimulation with 5 ng / ml TGF-β1 for 48 hours, followed by rhodamine-conjugated paloidine staining (red) As a result, the nuclei were contrasted with DAPI, and the results showed that stress fiber formation was reduced by IWR-1 treatment (bar = 25 μm).

FIG. 5A shows the effect of MMP (matrix metalloproteinase) production on IGF-1 in normal human fibroblast (NF) and keloid fibroblast (KF) extracts treated with 20 μM IWR-1 for 48 hours. As a result of checking the level, the left photograph shows the result of Western blot analysis confirming the level of MMP in the cell extract, and the right photograph shows the concentration of secreted MMP by collecting the same amount of culture medium. As a result, Western blot analysis using a concentrated culture medium And the same amount of sample was loaded through Ponceau S staining. FIG. 5B shows the result of RT-PCR analysis of the mRNA levels of MMP-1, MMP-3 and MMP-13, wherein GAPDH was used as an internal control and the expression of MMPs was increased by IWR- 5C was the result of gelatinization analysis of electrophoresis after enriching the cell culture medium. According to the previous report [26], the band of about 90 kDa was predicted as MMP9, the band of about 70 kDa as MMP2, The low molecular weight band can be predicted by MMP13.

Hereinafter, the present invention will be described in more detail.

The present invention can provide a pharmaceutical composition for preventing or treating keloid containing IWR-1 as an active ingredient.

The IWR-1 can inhibit proliferation and migration of fibroblasts.

As the fibrosis diseases such as keloid are more proliferated and the migration of fibroblasts is increased, the effect of IWR-1 on the proliferation and migration of fibroblasts is examined. As a result, as shown in Fig. 2A, (NF) and keloid fibroblasts (KF). As shown in FIG. 2B, it was confirmed that NF and KF migration was significantly inhibited by IWR-1 in a dose-dependent manner.

The IWR-1 can inhibit collagen synthesis.

More specifically, the most important feature of fibrosis disease, including keloid, is the increase in collagen synthesis and accumulation. The effect of IWR-1 on collagen synthesis was confirmed by Western blot analysis. As a result, The collagen synthesis effect was significantly reduced in normal and keloid tissues. Also, referring to FIG. 3C, which confirms the amount of procollagen released from fibroblasts, it was confirmed that the release of procollagen was significantly reduced by IWR-1 treatment in both NF and KF, in accordance with the collagen synthesis effect as described above.

In addition, IWR-1 may be one that increases the production of matrix metalloproteinase (MMP), a collagen degrading factor.

5A, IWR-1 expresses MMP-1, MMP-3, and MMP-13, respectively, as shown in FIG. 5A. , And the MMP-1, MMP-3 and MMP-13 secretion levels were significantly increased in the cell culture medium by IWR-1 treatment.

The pharmaceutical composition may contain 0.001 to 50 parts by weight of IWR-1, based on 100 parts by weight of the total amount of the pharmaceutical composition.

In one embodiment of the present invention, the pharmaceutical composition for preventing or treating keloid containing the IWR-1 as an active ingredient can be administered orally or parenterally in the form of injections, granules, powders, tablets, pills, capsules, suppositories, , Emulsions, drops, and liquid preparations can be used.

In another embodiment of the present invention, the pharmaceutical composition for the prevention or treatment of keloids containing IWR-1 as an active ingredient may be formulated with a suitable carrier, excipient, disintegrant, sweetener, coating agent, swelling agent, lubricant , At least one additive selected from the group consisting of lubricants, flavors, antioxidants, buffers, bacteriostats, diluents, dispersants, surfactants, binders and lubricants.

Specific examples of carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. Solid formulations for oral administration may be in the form of tablets, pills, powders, granules, capsules These solid preparations can be prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc., into the composition. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, syrups and the like, and various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin which are commonly used simple diluents. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories, and the like. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As the suppository base, witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like can be used.

According to one embodiment of the present invention, the pharmaceutical composition may be administered orally, intraarterally, intraperitoneally, intramuscularly, intraarterally, intraperitoneally, intrasternally, transdermally, nasally, inhaled, topically, rectally, &Lt; / RTI &gt; can be administered to the subject in a conventional manner.

The preferred dosage of the above IWR-1 may vary depending on the condition and body weight of the subject, the type and degree of disease, the drug form, the administration route and the period, and may be appropriately selected by those skilled in the art. According to one embodiment of the present invention, the daily dose may be 0.01 to 200 mg / kg, specifically 0.1 to 200 mg / kg, more specifically 0.1 to 100 mg / kg, though it is not limited thereto. The administration may be performed once a day or divided into several times, and thus the scope of the present invention is not limited thereto.

In the present invention, the 'subject' may be a mammal including a human, but is not limited thereto.

Further, the present invention can provide a cosmetic composition for preventing or improving keloid containing IWR-1 as an active ingredient.

The cosmetic composition may contain, in addition to the active ingredient IWR-1, conventional additives such as stabilizers, solubilizers, vitamins, pigments and flavors, and carriers.

The cosmetic composition may be prepared in any form conventionally produced in the art and may be in the form of a solution, suspension, emulsion, paste, gel, cream, lotion, powder, oil, powder foundation, emulsion foundation, Wax foundation, spray, and the like. However, the present invention is not limited thereto. More specifically, it can be manufactured in the form of a sun cream, a flexible lotion, a convergent lotion, a nutritional lotion, a nutritional cream, a massage cream, an essence, an eye cream, a pack, a spray or a powder.

When the formulation is a paste, cream or gel, an animal oil, a vegetable oil, a wax, a paraffin, a starch, a tracer, a cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as a carrier component .

When the formulation is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component. In the case of a spray, in particular, chlorofluorohydrocarbons, propane / Or propellants such as dimethyl ether.

When the formulation is a solution or an emulsion, a solvent, a solubilizing agent or an emulsifying agent is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, - butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or fatty acid esters of sorbitan.

When the formulation is a suspension, a carrier such as water, a liquid diluent such as ethanol or propylene glycol, a suspension such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, a microcrystalline cellulose , Aluminum metahydroxide, bentonite, agar or tracant, etc. may be used.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended to illustrate the contents of the present invention, but the scope of the present invention is not limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

<References> Reagents and Antibodies

IWR-1 was purchased from Selleckchem (Houston, Tex.). Collagen-1 alpha 1, collagen-1 alpha 2, MMP-1, MMP-3 and alpha-SMA (Santa Cruz Biotechnology, Santa Cruz, Calif.) As primary antibodies; MMP-13 (Abcam, Cambridge, Mass.); (Rhodamine phalloidin) was transformed into Invitrogen (Eugene, St. Louis, Mo.), p-Smad2, Smad2, p-Smad3, Smad3 (Cell Signaling Technology, Beverly, MA) OR).

&Lt; Experimental Example 1 > Cell culture

Normal human skin and keloid tissue were obtained from several donors with written consent. This study was conducted with approval of the Ethics Committee of the Clinical Trial Committee of Chungnam National University Medical School.

Keloid tissue was collected from patients between 20 and 65 years of age (mean 32.0 ± 15.1 years). Normal fibroblasts (NF) and keloid-induced dermal fibroblasts (KF) were cultured by modification of the previously reported method (Peihua Zhang, et al., 2014).

Briefly, skin tissue was washed with phosphate-buffered saline (PBS) containing 1% penicillin-streptomycin, cut into small pieces and placed in a culture dish.

Fibroblasts were obtained from tissue cultured for one week. The cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal calf serum (FBS) and 1% penicillin-streptomycin (Life Technologies Corporation, Grand Island, NY) ; Welgene, Daegu, Korea).

Cells between 3 and 7 cells were used in all experiments and fibroblasts were incubated overnight in serum-free medium before IWR-1 treatment.

<Experimental Example 2> Confirmation of cytotoxicity and cell proliferation

Lactate dehydrogenase (LDH) analysis was performed to confirm cytotoxicity. Cells were seeded at a density of 2 × 10 ⁴ / well on a 24-well culture plate and treated with various concentrations of IWR-1. After 24 hours, the culture medium was collected and LDH activity was determined by a cytotoxicity detection kit (Roche, Indianapolis, IN) .

Cell proliferation was measured through cell counting.

NF or KF was dispensed into 60 mm dishes at the same cell number and treated with various concentrations of IWR-1 after 24 hours. The cells were separated from the culture dish at fixed intervals and the number of cells was counted using a hemocytometer.

<Experimental Example 3> TOPflash analysis

The TOPflash reporter adenovirus contains three copies of the motif with optimal TCF binding leading to luciferase expression.

Cells were plated in 24-well plates and cultured until confluence of 60%, then TOPflash reporter adenovirus was transformed with multiplicity of infection (MOI).

After overnight incubation, fresh medium containing IWR-1 was added to the cells, followed by further incubation for 24 hours. The intracellular extract was prepared using a cell lysis buffer, and then cultured in a Luciferase reporter assay system (Promega, Madison, Wis.) Luciferase activity was measured, and for control experiments, FOPflash reporter adenovirus containing TCF binding motif was used.

<Experimental Example 4> Migration Analysis

Cells were grown in 6-well plates to form confluent monolayers.

5 μg / ml Mitomycin C was pretreated with cells for 1 hour and replaced with fresh medium.

IWR-1 was treated with various concentrations of IWR-1 for 24 hours, scratched with a microwave pet tip to create artificial wounds, and the gap closure was monitored before and after treatment under a phase contrast microscope to determine the percentage of gap closure Respectively.

[(initial area - initial area) / 100

&Lt; Experimental Example 5 > Western blot analysis

Cells were lysed with Proprep solution (Intron, Daejeon, Korea) and cell protein concentration was quantified using BCA Protein Assay Reagent (Pierce, Rockford, Ill.).

A sample containing the same amount of protein was loaded on SDS-polyacrylamide gel and transferred to a nitrocellulose membrane. After blocking with 5% skim milk, the cells were incubated with the appropriate primary antibody and incubated with secondary antibody conjugated with the corresponding horseradish peroxidase.

Signals were visualized using a chemiluminescent substrate (Intron, Daejeon, Korea). For the detection of secreted proteins, the culture medium was concentrated using a protein concentration kit (Elpis Biotech, Daejeon, Korea).

<Experimental Example 6> ELISA (Enzyme-linked immunosorbent assay) analysis

Cells were seeded in a 24-well plate at a density of 1 × 10 ⁵ / well and treated with 20 μM IWR-1 for 48 hours. The culture medium was collected and centrifuged. The ELISA kit (TaKaRa, Shiga, Japan) Was used to confirm the release of procollagen.

<Experimental Example 7> Collagen gel contraction assay

Cell contraction assay kit (Cell Biolabs Inc., San Diego, Calif.) Was used to perform gel shrinkage analysis according to the manufacturer's instructions.

Briefly, 5 × 10 ⁵ cells were mixed with collagen gel and polymerized with Transwell permeable supports (3.0 μm polycarbonate membrane) (Corning Incorporated, Corning, NY).

After collagen polymerization, 5 ml of medium was added to each collagen gel lattice.

< Experimental Example  8> Reverse transcription  Reverse transcription-polymerase chain reaction (RT-PCR)

Total RNA was extracted from fibroblasts using Easy-blue RNA extraction kit (Intron, Daejeon, Korea) and 2 μg of total RNA was extracted with Molecular Rule Leukemia Virus (M-MLV) reverse transcriptase (Elpis Biotech, Daejeon , Korea).

The same amount of cDNA was used as a template for amplification, and primers of the following sequences were used: MMP-1, 5'-GATGGGAGGCAAGTTGAAAA and 5'-CCAGGTCCATCAAAAGGAGA; MMP-3, 5'-TGCTTTGTCCTTTGATGCTG and 5'-ATCGATTTTCCTCACGGTTG; MMP-13, 5'-TAAGGAGCATGGCGACTTCT and 5'-GTCTGGCGTTTTTGGATGTT; GAPDH, 5'-GTCAGTGGTGGACCTGACCT and 5'-AGGGGTCTACATGGCAACTG.

< Experimental Example  9> Gelatin Zymography (Gelatin zymography )

IWR-1 at a concentration of 20 μM in serum-free medium was treated with fibroblasts for 48 hours. The supernatant was concentrated and gelatinization was performed with the reported method (Substrate-zymography: A still worthwhile method for gelatinases analysis in biological samples).

Samples of the same amount were electrophoresed through 10% polyacrylamide gel (Sigma, St. Louis, MO) with gelatin (1 mg / ml) impregnated without boiling or deformation.

The gel was washed twice with 2.5% tryptone X-100 solution at room temperature for 30 minutes and then washed in a buffer containing 0.05 M Tris-HCl (pH 7.5), 0.15 M NaCl and 0.01 M CaCl ₂ at 37 ° C for 24 hours Lt; / RTI &gt;

The gel was then stained with 30% methanol, 10% acetic acid and 0.1% (w / v) Coomassie Brilliant Blue R250 and stained with 30% methanol and 10% acetic acid to clarify the band in the background gel, Activity was visualized.

<Example 1> Confirmation of cell proliferation and migration effect of IWR-1

In fibroblast activity, cytotoxicity of IWR-1 was confirmed in normal skin fibroblasts (NF) and keloid fibroblasts (KF) in order to confirm the effect of IWR-1, the compound of FIG. 1A.

As a result, no significant increase in LDH release was observed when the concentration of IWR-1 was increased to 20 μM as shown in FIG. 1B, but the LDH release was significantly increased in NF and KF cells when treated at a concentration of 50 μM.

From the above results, IWR-1 may cause some damage to the plasma membrane. Therefore, IWR-1 was further treated to 20 μM in fibroblasts.

IWR-1 was originally developed as a low-molecular inhibitor of Wnt / β-catenin signaling, confirming that IWR-1 indeed blocks intracellular Wnt / β-catenin signaling in fibroblasts. For this, TOPflash reporter, the most well-known analytical tool for Wnt / β-catenin signal validation, was used.

First, the basal activity of the Wnt / beta -catenin signal was confirmed after transfection of TOPflash reporter into NF and KF cells.

As expected, the basal TOPflash activity was found to be a major cause of keloid, with KF being higher than NF.

Next, TOPflash reporter was transfected into fibroblasts and then treated with IWR-1.

As a result, as shown in FIG. 1C, IWR-1 inhibited TOPflash activity in a dose-dependent manner in both NF and KF. From the above results, a potential effect on fibroblast Wnt /? - catenin signal was confirmed.

Fibrosis diseases such as keloids increase the proliferation and migration of fibroblasts, thus confirming the effect of IWR-1 on fibroblast proliferation and migration.

As a result, significant cell proliferation attenuation was observed in NF and KF fibroblasts treated with IWR-1 as shown in FIG. 2A, and NF and KF migration was significantly Respectively.

<Example 2> Confirmation of collagen synthesis inhibitory effect of IWR-1

The most important feature of fibrosis disease, including keloid, is increased collagen synthesis and accumulation, and the effect of IWR-1 on collagen synthesis was confirmed by western blot analysis.

As a result, as shown in Fig. 3A, all the normal and keloid tissues treated with IWR-1 exhibited greatly reduced collagen synthesis effect.

In addition, the inhibitory effect of IWR-1 on collagen synthesis was confirmed in a dose-dependent and time-dependent manner as shown in FIG. 3B, but unexpectedly, collagen mRNA levels did not significantly decrease with IWR-1 treatment.

Next, the amount of procollagen released from the fibroblasts was determined.

As a result, referring to FIG. 3C, it was confirmed that the procollagen release was significantly reduced by IWR-1 treatment in both NF and KF in accordance with the collagen synthesis effect confirmed above.

Finally, collagen gel shrinkage analysis was performed using a well established model for wound contraction [24].

As a result, significant gel shrinkage was observed in the IWR-1-treated NF and KF experimental groups, while in the control group, significant gel shrinkage occurred in a time-dependent manner, as shown in FIG. 3D.

From the above results, it has been confirmed that IWR-1 effectively inhibits collagen production of fibroblasts, thus, in the treatment of fibrosing disease, IWR-1 can be proposed as a potential therapeutic agent.

< Example  3> TGF -β / Smad  Signal and Muscle fiber  For differentiation IWR Influence of -1

The TGF-β / Smad signal plays an important role in keloid development by promoting fibroblast proliferation and collagen synthesis. Thus, we examined the effect of IWR-1 on TGF-β / Smad signal in NF and KF cells.

Western blot analysis showed that phosphorylation of Smad 2 and Smad 3 was significantly increased at 1 and 24 hours after TGF-β treatment as shown in FIG. 4A.

In addition, pretreatment with IWR-1 did not affect TGF-? -Induced phosphorylation of Smad 2 and Smad 3.

From the above results, it was confirmed that the effect of IWR-1 on collagen synthesis and cell proliferation of fibroblasts is not related to the TGF-β / Smad signal.

On the other hand, it has been confirmed that IWR-1 significantly inhibits collagen gel shrinkage, thereby confirming whether IWR-1 can affect the shrinking protein α-SMA, which plays an important role in muscle differentiation.

As a result, it was confirmed that α-SMA expression was increased in KF than in NF as shown in FIG. 4A, and the result implies the state of KF for myofibroblast differentiation. In addition, as a result of treatment with TGF-β for 24 hours, α-SMA increase was observed in NF, but not in KF.

Unexpectedly, IWR-1 treatment did not affect α-SMA expression regardless of TGF-β stimulation.

However, as a result of confirming stress fiber formation through paloidin staining, it was confirmed that IWR-1 suppresses stress fiber formation as compared with the control group. In the cells stimulated with TGF-β, On the other hand, it was confirmed that IWR-1 effectively blocked TGF-β induced stress fiber formation.

From the above results, it was confirmed that IWR-1 inhibits myoblast differentiation in stress fiber formation without affecting Smad signal.

< Example  4> MMP (matrix metalloproteinase ) On the production of IWR Check the effect of -1

Since MMP plays a role in degrading collagen, MMP production of IWR-1 was confirmed in NF and KF.

As a result, as shown in FIG. 5A, IWR-1 increased intracellular protein levels of MMP-1, MMP-3 and MMP-13 and similar results showed that MMP-1, MMP-3 and MMP- -13 secretion was significantly increased.

In addition, RT-PCR was performed to confirm whether IWR-1 can increase the expression of MMP gene.

As a result, as shown in FIG. 5B, IWR-1 increased mRNA levels of MMP-1, MMP-3 and MMP-13.

In addition, referring to FIG. 5C, it was confirmed that the result of the gelatin immunoassay was in agreement with the results of MMP expression confirmed above, and in particular, the MMP activity was increased at low molecular weight.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Claims (6)

A pharmaceutical composition for preventing or treating keloid containing IWR-1 as an active ingredient. [Claim 2] The pharmaceutical composition for preventing or treating keloid according to claim 1, wherein the IWR-1 inhibits proliferation and migration of fibroblasts. The pharmaceutical composition for preventing or treating keloid according to claim 1, wherein the IWR-1 inhibits collagen synthesis. The pharmaceutical composition for preventing or treating keloid according to claim 1, wherein the IWR-1 increases the production of matrix metalloproteinase (MMP), which is a collagen degrading factor. The pharmaceutical composition for preventing or treating keloid according to claim 1, wherein the pharmaceutical composition comprises 0.001 to 50 parts by weight of IWR-1, based on 100 parts by weight of the total amount of the pharmaceutical composition. A cosmetic composition for preventing or improving keloid containing IWR-1 as an active ingredient.

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