WO2008054144A1

WO2008054144A1 - Skin external composition containing cathepsin g inhibitors for preventing skin aging and the screening method for development of antiaging materials

Info

Publication number: WO2008054144A1
Application number: PCT/KR2007/005451
Authority: WO
Inventors: Eui Dong Son; Hyae Kyoung Kim; Hyun Jung Choi; So Hee Lee; Jin Young Lee; Jae Won Yoo; Jae Sung Hwang; Ih Seop Jang
Original assignee: Amorepacific Corp
Current assignee: Amorepacific Corp
Priority date: 2006-11-03
Filing date: 2007-10-31
Publication date: 2008-05-08
Anticipated expiration: 2009-05-03
Also published as: KR100937263B1; KR20080040612A

Abstract

Disclosed herein is a skin external composition for preventing skin aging, which contains a cathepsin G inhibitor as an active ingredient. Also disclosed is a method for screening anti-aging substances.

Description

[DESCRIPTION]

[invention Title]

Skin external composition containing cathepsin G inhibitors for preventing skin aging and the screening method for development of antiaging materials [Technical Field]

The present invention relates to a skin external composition for preventing skin aging, which contains a cathepsin G inhibitor as an active ingredient, and to a method for screening anti-aging substances, which are capable of inhibiting cathepsin G activity. [Background Art]

Skin aging may be divided into photoaging, resulting from external factors such as UV light, and intrinsic aging, resulting from a change in hormones and a change in internal factors such as stresses. Due to the combined action of such aging phenomena, an increase in skin wrinkles or a decrease in skin elasticity appears. As skin aging progresses for this reason, the content and arrangement of collagen, elastin, hyaluronic acid, proteoglycan, glycosaminoglycan, fibronectin and sugar protein in the skin are changed or decreased, and their ability to bind to moisture is reduced. As a result, the substrates of the skin are reduced, and thus the skin' s ability to maintain moisture, leading to conditions, such as loss of skin firmness, skin drying and skin sagging.

In order to develop cosmetic materials or drugs capable of preventing or treating such changes in the skin, various studies are in progress. In current studies related to substances for inhibiting aging, studies on the production of collagen and the suitable regulation of matrix metalloproteinase, an enzyme which degrades collagen by UV light, are being actively conducted. In order to develop active ingredients capable of preventing and reducing such skin aging phenomena, in vitro screening methods for developing anti-aging substances are currently mainly used. Such methods include a method comprising irradiating skin cells with UV light and then treating the cells with a candidate substance to reduce the expression of metalloproteinase, and a method comprising treating skin cells with a substance to increase collagen. Antiwrinkle substances developed through such methods can somewhat overcome the expression of metalloproteinase, which directly affects cells by UV light, but problems associated with the expression of metalloproteinase caused by indirect factors are difficult to solve. Also, other major methods include methods for developing collagen-like peptides, elastase inhibitors, antioxidant substances, etc. Fibronectin is an adhesion protein that connects cells with collagen in the dermal layer of the skin in a normal state and has been reported to be a mediator that is involved in the migration, proliferation or differentiation of cells and transmits intracellular and extracellular signals. Fibronectin is found in all vertebral animals and is a dimer consisting of two subunits. It is a glycoprptein of 440 kDa and is present in two forms: soluble plasma Fn (pFN) and cellular Fn (cFN) . pFN is mostly produced in the liver, cFn is produced in various cells, including fibroblasts, epidermal cells macrophage cells, and such two forms of Fn are found in normal skin

(Roberger DJ. Fibronectin. Int J Biochem Cell Biol 1997;

29:939-943) . It is known that fibronectin shows various biological functions, including the promotion of cell adhesion and elongation, the promotion of cell migration, the regulation of cell differentiation and the restoration of tissue, by binding either to an integrin receptor, a glycoprotein which is expressed on the cell surface and consists of two subunits, α and β, or to a non-integrin receptor (Couchman JR, et al., Fibronectin-cell interactions. J. I. D. 1990; 94: 7s-14s; Hynes RO, et al., Fibronectin; multifunctional modular glycoprotein. J. cell boil., 1982; 92; 369-377).

However, the effects of degradation products of such adhesion proteins on the expression of metalloproteinase have been much studied in the cartilage cells of arthritis patients. That is, it was reported that the degradation products of fibronectin stimulated the expression of cytokines (interleukin-1 and-6, and TNF-I) mediating various inflammatory reactions (Arthritis & Rheumatism, 2002) and increased the expression of metalloproteinase (J. Cell Bio. (1983)), but the direct effect of degradation products of fibronectin on human skin cells is not yet reported. It was only reported in animal studies that, upon UV irradiation, changes in structural ECM proteins, such as a decrease in collagen and an increase in MMP-I, were observed, the activity of elastase was increased upon UV irradiation to stimulate the degradation of fibronectin, and such degradation products of fibronectin would have adverse effects on skin aging (Journal of photochemistry and photobiology, 2000) .

Also, although the role of cathepsin has not been much studied, many proteases, particularly serine proteases and metalloproteinases, are present in the skin. However, with respect to intracellular and extracellular pH effects, it is known that serine proteases, cathepsin B, D, H, L, K and S, are enzymes acting in cells having an acidic pH, and cathepsin G is an enzyme acting in cells having a neutral pH (Arthritis Research Campaign 1998) . Cathepsin G has a molecular of about 30 kDa, and as substrates of cathepsin G, collagen and fibronectin have been reported. In the year 2002, it was reported that UV light promoted the expression of cathepsin G in skin fibroblasts (Biochemistry 2002) . It is currently known that cathepsin G is predominantly expressed in lesion sites such as atopy, and it is not present in the epidermis and is expressed at high levels in the dermis and the like.

Meanwhile, typical in vitro screening methods for developing anti-aging substances, which are currently performed, include a method comprising irradiating skin cells with UV light and then treating the UV-irradiated skin with a candidate substance in order to inhibit the expression of metalloproteinase, and a method comprising treating normal skin fibroblasts with substances and then screening a substance increasing the production of collagen. However, in the human skin, the expression and activity of metalloproteinase are increased due to various factors such as inflammatory factors, in addition to direct factors such as UV light, and thus said methods are unsuitable for screening anti-aging substances against such various factors . [Disclosure] [Technical Problem] The present inventors have investigated a method of inhibiting rapid skin aging caused by UV light and of effectively controlling intrinsic aging and, as a result, have found that the degradation products of a cell adhesion protein (fibronectin) promotes the expression of metalloproteinase in skin cells and that cathepsin G is involved in the degradation of the adhesion protein. Also, the present inventors have found that a cathepsin G inhibitor, obtained through cathepsin G inhibitor screening, has the effect of inhibiting the activity of cathepsin G and the anti-aging effect of preventing the degradation of the adhesion protein to inhibit the production of metalloproteinase in skin cells. Accordingly, the present inventors have found that the use of a skin external composition containing a cathepsin G inhibitor can prevent the adhesion protein from being degraded due to intrinsic aging and photoaging, reduce the production of metalloproteinase and inhibit the activity of metalloproteinase, and thus anti-aging substance more potent than the existing anti-aging substances can be developed, thereby completing the present invention.

Therefore, it is an object of the present invention to provide a skin external composition for preventing skin aging, which contains a cathepsin G inhibitor as an active ingredient . Another object of the present invention is to identify the function of cathepsin G and to provide a screening method capable of developing anti-aging substances, which effectively inhibit the quantitative and qualitative increase of metalloproteinase, resulting from the degradation of an adhesion protein by cathepsin G in skin cells without direct UV irradiation. [Technical Solution]

On the basis of the fact that the degradation products of a cell adhesion protein (fibronectin) , which increase in aged skin, promote the expression of metalloproteinase, the present inventors have found that an enzyme degrading the adhesion protein is cathepsin G and that, when cells were treated with ISMD 38 as a cathepsin G inhibitor, the expression and activity of metalloproteinase in the cells were significantly decreased.

Accordingly, the present invention provides a skin external composition for preventing skin aging, which contains a cathepsin G inhibitor as an active ingredient. Also, the present invention aims to identify the functions of cathepsin G and provides a screening method capable of developing anti-aging substances, which effectively inhibit the quantitative and qualitative increase of metalloproteinase, resulting from the degradation of an adhesion protein by cathepsin G in skin cells without direct UV irradiation. [Advantageous Effects]

In the inventive skin external composition for preventing skin aging, which contains a cathepsin G inhibitor as an active ingredient, the cathepsin G inhibitors prevents the degradation of fibronectin to inhibit the production of metalloproteinase in skin cells so as to the quantitative and qualitative changes of metalloproteinase, resulting from enzymes in the skin in addition to the direct effect of UV light, thus reducing wrinkles which are skin aging conditions. Particularly, because the cathepsin G inhibitor has the effect of inhibiting the activity of metalloproteinase, it can contribute to the reduction of skin wrinkles and the improvement of skin elasticity, and thus can be used as an effective substance in the future development of cosmetic materials for inhibiting skin aging.

Also, according to the inventive method for screening antiaging substances, the antiaging substances can be screened by adding an anti-aging candidate substance to a substrate in the presence of cathepsin G enzyme and measuring the inhibition of activity of the cathepsin G enzyme on the basis of the concentration of metalloproteinase produced. [Description of Drawings]

FIG. 1 is a flowchart showing a process in which wrinkles are increased due to the activity of cathepsin G in aged skin. FIG. 2 illustrates a graphic diagram showing an increase in the activity of cathepsin G in aged cells and UV-irradiated cells, measured in Test Example 1, and illustrates immunofluorescence photographs showing the comparison of cathepsin G between young skin and aged skin. FIG. 3 is a graphic diagram showing the expression levels of metalloproteinase (MMP-I) by the degradation products of fibronectin, measured in Test Example 2.

FIG. 4 shows the results of Western blot analysis conducted to identify a fibronectin-degrading enzyme in Test Example 3.

FIG. 5 shows the results of Western blot analysis conducted to examine the degradation of fibronectin in human fibroblast cells in Test Example 3. FIG. 6 shows the results of Western blot analysis conducted to examine the quantitative change of fibronectin in human fibroblast cells in Test Example 3.

FIG. 7 is a graphic diagram showing the inhibition of cathepsin G activity, measured in Test Example 4. FIG. 8 shows the results of Western blot analysis conducted to examine the inhibition of expression of MMP-I by ISMD 38 in Test Example 5.

FIG. 9 is a graphic diagram showing wrinkle measurement results, obtained in hairless mice as photoaging animal models in Test Example 6. In FIG. 9, Rl: deep wrinkles, R2 : thin wrinkles, R3 : middle between deep wrinkles and thin wrinkles, R4 : weak roughness of skin surface, and R5 : strong roughness of skin surface.

[Best Mode] Hereinafter, the present invention will be descried in further detail.

In the inventive skin external composition for preventing skin aging, the cathepsin G inhibitor is characterized in that it prevents the degradation of adhesion protein fibronectin to inhibit the production of metalloproteinase, which is the degradation product of fibronectin, a collagen-degrading enzyme, in skin cells. As shown in FIG. 1, when the activity of cathepsin G enzyme in aged skin is increased, a cell adhesion protein will be rapidly degraded so as to increase the expression and activity of metalloproteinase, which is the degradation product of the adhesion proteins, thus increasing wrinkles. However, when a cathepsin G inhibitor is applied to inhibit the activity of the cathepsin G enzyme, it will prevent the degradation of the adhesion protein to inhibit the production of metalloproteinase, a collagen-degrading enzyme, in skin cells, thus exhibiting the effects of preventing skin wrinkles and improving skin elasticity. The cathepsin G inhibitor according to the present invention is selected from among compounds represented by the following formulas 1 to 4 and is preferably contained in an amount of 0.01-10 wt% based on the total weight of the skin external composition: [Formula 1]

[Formula 2]

[Formula 3]

[Formula 4]

If the compounds represented by the formulas 1 to 4 are contained in an amount of less than 0.01 wt% based on the total weight of the skin external composition, the effect of inhibiting the cathepsin G enzyme cannot be obtained, and if it is contained in an amount of more than 10 wt%, the skin external composition can be deteriorated, it will be difficult to control the viscosity of the skin external composition, and the economic efficiency will be reduced.

The compounds of the formulas 1 to 4 are derivatives of 3 , 5-dihydroxybenzoic acid and are known not only to have the effect of inhibiting elastase, but also to effectively eliminate free radicals produced by UV light.

The inventive skin external composition for preventing skin aging is used to increase the adhesion of skin cells and to prevent the degradation of the adhesion protein, thus inhibiting an increase in skin wrinkles, increasing skin elasticity and preventing the skin from sagging, and there is no particular limitation on the formulation of the composition. For example, the skin external composition of the present invention can be formulated into a cosmetic formulation, such as skin lotion, astringent lotion, milk lotion, nourishing cream, essence, massage cream, eye cream, pack, body lotion, body cream, body oil or body essence, or a transdermal formulation, such as lotion, ointment, gel, cream, patch or spray.

Also, the inventive skin external composition for preventing skin aging may contain, in addition to the cathepsin G inhibitor, other components which can increase the main effect of the present invention, within a range not to impair the main effect of the present invention.

The method for screening the anti-aging substance cathepsin G inhibitor showing the effect of inhibiting the degradation of the adhesion protein, is performed by adding an anti-aging candidate substance to a substrate in the presence of cathepsin G enzyme and measuring the inhibition of activity of the cathepsin G enzyme, in which the inhibition of activity of the cathepsin G enzyme can be determined by measuring the concentration of metalloproteinase produced by the degradation of the adhesion protein.

[Mode for Invention]

Hereinafter, the construction, operation and effect of the present invention will be described in further detail with reference to the following examples and test examples. However, the scope of the present invention is not limited only to these examples.

Test Example 1: (a) change in activity of cathepsin G in aged skin cells and UV- irradiated skin cells and (b) change in activity of cathepsin G in young skin and aged skin

The activities of cathepsin G in young cells, a culture medium of fibroblasts intrinsically aged through passage cultures (38 passages) and a culture medium of fibroblasts irradiated three times with UV light were measured. The measurement of the activities was performed by reacting 80 βg of each of culture medium proteins with 2 mM cathepsin G substrate (Suc-AAPF-pNA) in buffer solution (HEPES; pH 7.5), and then measuring the absorbance at 405 nm showing yellow color development due to the release of p-nitroaniline (pNA) from the substrate by cathepsin G. Also, in order to examine the quantitative change of cathepsin G in human skin, the buttock skin of three young persons (20-years-old age group) and the buttock skin of three old persons (70-year-old age group) were biopsied, and then the quantitative change of cathepsin G in the skin was analyzed by immunofluorescence staining. The analysis results are shown in FIG. 2. As can be seen from the results in FIG. 2, the aged cells and the UV-irradiated cells showed an increase in the activity of cathepsin G. This suggests that a substance inhibiting the activity of cathepsin G has an anti-aging effect. Test Example 2j Expression of matrix metalloproteinase-1 in degradation products of adhesion protein in skin cells

The results of the comparison of cytotoxicity between four degradation products (commercially available from Sigma; Fn-fl20, Fn-f70, Fn-f45 and Fn-f30) of a cell adhesion protein (fibronectin) are shown in Table 1 below. As can be seen from cell viabilities in Table 1, the fibronectin degradation product 1 (Fn-f120; 120 kDa) had toxicity at 0.5 μM, but the other degradation products had no toxicity. For this reason, in subsequent tests, human skin fibroblasts were treated with the fibronectin degradation products 2, 3 and 4 (Fn-f70, Fn-f45 and Fn-f30; 70 kDa, 40 kDa and 30 kDa) having no toxicity, and the expression levels of metalloproteinase in the fibroblasts were measured with an ELISA kit. Specifically, human fibroblasts were placed in a 6-well microtiter plate containing 10% fetal bovine serum-containing DMEM media (fibroblast growth media), at a cell density of 10,000 cells/well. After cell adhesion, the media were replaced with fetal bovine serum-free media, and the cells were cultured to a confluency of about 70-80%. Then, the cells were treated with the fibronectin degradation products at the concentrations shown in Table 1 below, and after 2 days, the number of viable cells was measured to analyze the toxicities of the degradation products for skin cells and to compare the quantitative change of matrix metalloproteinase (hereinafter, referred to as "MMP-I"). Herein, a group untreated with the degradation products of cell adhesion protein fibronectin was used as a control group. The test results are shown in Table 1 and FIG. 3 as ratios relative to the control group taken as 100. [Table l]

As can be seen from Table 1 and FIG. 3, the fibronectin degradation products increased the amount of metalloproteinase in skin fibroblasts. As shown in FIG. 3, such results were increased by a maximum of 50% compared to the case irradiated with UVB. Currently, the degradation products of fibronectin are reported to increase the expression of metalloproteinase in joint cells, but were not reported in skin cells. Accordingly, it can be predicted through the results in FIG. 3 that the fibronectin degradation products are important aging factors that promote skin aging by increasing metalloproteinase.

Test Example 3 : Identification of fibronectin- degrading enzyme and increase in expression of metalloproteinase

In order to identify a fibronectin-degrading enzyme, 0.5 βg of a fibronectin protein was allowed to react with 1.5 mU of cathepsin G in an eppendorf tube at 37 ^°C for 24 hours, and then the degradation of fibrobectin was observed by Western blot analysis in comparison with the case in which cathepsin G was not added (FIG. 4) .

Also, in order to observe the degradation of fibronectin in human fibroblasts, fibroblasts were cultured in a 48 -well microtiter plate at a cell density of 10⁴ cells/well, while the media were replaced with media containing 1 mU and 5 mU of cathepsin G. After 24 hours of the culture, the supernatant was collected, and then the degradation of fibronectin in the supernatant was analyzed by Western blot. Then, the expression level of metalloproteinase was observed by Western blot analysis, and the observation results are shown in FIG. 5 in comparison with the case in which cathepsin was not added.

Meanwhile, while the skin cells were cultured, the cells were treated with cathepsin G at concentrations of 0.1 mU and 1 Mu, and the expression of metalloproteinase (MMP-I) in the cells was observed by Western blot analysis. The observation results are shown in FIG. 6.

As shown in FIG. 4, when fibronectin was treated with cathepsin G, various fibronectin degradation products appeared, suggesting that cathepsin G was an enzyme degrading fibronectin. Also, as shown in FIG. 5, when skin cells were treated with cathepsin G, the degradation of fibronectin was observed. From the results in FIG. 6, it could be observed that the amount of metalloproteinase, a fibronectin degradation product, was increased due to the change in the degradation of fibronectin. That is, as can be seen in FIG. 6, the case of treatment with low- concentration cathepsin G (0.1 mU) showed an increase in the amount of proMMP-1 compared to the control group, but in the case of treatment with 1 mU of cathepsin G, proMMP-1 was reduced, while active MMP-I was increased. Accordingly, from the results in FIGS. 4 to 6, it could be found that cathepsin G was an enzyme promoting the degradation of fibronectin. Also, a band having a size of 42 kDa, which is the size of active metalloproteinase, was observed (FIG. 6) , and thus the size of active metalloproteinase, which was not observed in cells irradiated with UV light, could be observed. In addition, it could be observed that aging was promoted by cathepsin G, the expression of which was promoted in aged skin without UV irradiation. Accordingly, it could be observed that the cathepsin G enzyme promoted the degradation of the cell adhesion protein in skin cells to increase the amount and activity of metalloproteinase.

Test Example 4 : Cathepsin G activity inhibitory effect and anti-aging substance screening

In a 96-well microtiter plate, a cathepsin G enzyme, a substrate (Sue-AAPF-pNA) , a candidate substance (ISMD 38) of the present invention or a positive control group (inhibitor; commercially available from Amersham) , shown in Table 2 below, and a buffer solution, were mixed with each other in the amounts shown in Table 3 below so as to make a total volume of 200 μlt . Then, the mixture was allowed to react at 37 ^°C for 3 hours, and then the absorbance at 504 nm was measured in order to examine whether the cathepsin G enzyme had activity. Herein, a group, containing only cathepsin G enzyme and substrate, was used as a control group. The test results are shown in FIG. 7 as ratios relative to the control group taken as 100. Also, in FIG. 7, if the cathepsin G enzyme had activity, the substrate would be degraded to show a yellow color. [Table 2]

[Table 3]

As can be seen in FIG. 7, the positive control group showed an IC₅₀ at 1 μM, and the ISMD 38 of the present invention showed a cathepsin G inhibitory effect of 40% at 10 mM.

Also, according to the above-described process, an anti-aging substance can be screened by adding an anti- aging candidate substance to a substrate and measuring the inhibition of activity of cathepsin G enzyme. Test Example 5: Analysis of inhibition of MMP-I expression by ISMD 38

In order to examine whether ISMD 38 having a cathepsin G activity inhibitory effect acts even in the cell level, skin fibroblasts were treated with ISMD 38, and the expression of metalloproteinase in the cells was measured by Western blot analysis. Specifically, human fibroblasts were placed in a 6-well microtiter plate containing 10% fetal bovine serum-containing DMEM media (fibroblast growth media), at a cell density of 10,000 cells/well. After cell adhesion, the media were replaced with fetal bovine serum-free DMEM media, and then the cells were cultured to a confluency of about 70-80%. Then, the cells were treated with each of cathepsin G (1 mU) and ISMD 38 (0, 0.1, 1 and 10 μM) , and after 2 days, the supernatants were collected, and changes in the amounts of metalloproteinase in the supernatants were observed by Western blot analysis. The observation results are shown in FIG. 8. Herein, a group untreated with cathepsin G or ISMD 38 was used as a control group.

As can be seen from the results in FIG. 8, the group treated only with cathepsin G showed increases in the expression and active form of metalloproteinase compared to the group untreated with cathepsin G. This is because cathepsin G degraded the cell adhesion protein to increase the degradation product metalloproteinase.

However, the group treated with ISMD 38 showed decreases in the expression and active form of metalloproteinase compared to the group treated only with cathepsin G, even though it was treated with cathepsin G. Test Example 6 : Examination of wrinkle reduction in hairless mice as photoaging animal models

In order to examine the wrinkle-reducing effect of the inventive skin external composition, an application test was performed on about 7-week-old SKH-I female hairless mice. For this purpose, skin external formulations were prepared as shown in Table 4 below and were applied to the back portion of hairless mice for 12 weeks together with UV irradiation. The UV irradiation was carried out every other day, and the application of the formulations was carried out every day. Then, the reduction of wrinkles in the mice was examined by replica analysis, and the examination results are shown in Table 9. [Table 4]

In the results in FIG. 9, Rl indicates deep wrinkles; R2, thin wrinkles; R3, the middle between deep wrinkles and thin wrinkles; R4 and R5; roughness of skin surface; R4, weak roughness; and R5, strong roughness. As can be seen from the results in FIG. 9, when ISMD 38 was used alone, it showed the effect of reducing wrinkles (Rl, R2 and R3) .

Claims

[CLAIMS]

[Claim l]

A skin external composition for preventing skin aging, which contains a cathepsin G inhibitor as an active ingredient . [Claim 2]

The skin external composition of Claim 1, wherein the cathepsin G inhibitor is one or more selected from the group consisting of compounds represented by the following formulas 1 to 4 : [Formula 1]

[Formula 2]

[Claim 3 ]

The skin external composition of Claim 2, wherein the compounds represented by the formulas 1 to 4 are contained in an amount of 0.01-10 wt% based on the total weight of the skin external composition. [Claim 4]

The skin external composition of Claim 1, which has a formulation selected from among skin lotion, astringent lotion, milk lotion, nourishing cream, essence, massage cream, eye cream, pack, body lotion, body cream, body oil and body essence. [Claim 5]

The skin external composition of Claim 1, which has a transdermal formulation selected from among lotion, ointment, gel, cream, patch and spray. [Claim 6]

A method for screening anti-aging substances, the method comprising adding an anti-aging candidate substance to a substrate in the presence of cathepsin G enzyme and measuring the inhibition of activity of the cathepsin G enzyme . [Claim 7]

The method of Claim 6, wherein the inhibition of activity of the cathepsin G enzyme is determined by measuring the concentration of metalloproteinase produced. [Claim 8]

A use of a cathepsin G inhibitor for anti-aging, in which the cathepsin G inhibitor prevents the degradation of fibronectin to inhibit the production of metalloproteinase. [Claim 9]

The use of Claim 8, wherein the cathepsin G inhibitor is one or more selected from the group consisting of compounds represented by the following formulas 1 to 4 : [Formula 1]

[Formula 2]