TW590771B - Serine protease and topical retinoid compositions useful for treatment of acne vulgaris and production of anti-aging effects - Google Patents

Abstract

This invention is related to methods for treating acne vulgaris and/or for producing anti-aging effects on the skin of a mammal, and compositions effective for the same. More specifically, the present invention is directed to the use of serine proteases, as the sole active in a composition effective for the treatment of acne vulgaris and/or for producing anti-aging effects on the skin of a mammal, or in combination with a retinoid compound in a composition effective for the same.

Description

590771 A7 B7 V. Description of the invention (i) Consumers' cooperation with the Central Bureau of Standards of the Ministry of Economic Affairs of the People's Republic of China ^ Two exemptions before and after the exemption of related applications 037,605, which claims the benefit of patent scope, is fully incorporated herein by reference. Field of the Invention The present invention relates to a method for treating acne vulgaris and / or anti-aging effect on mammalian skin, and an effective composition for the same. More specifically, the present invention is directed to the use of serine proteases, which can be used alone or in combination with retinoid compounds in pharmaceutical or cosmetic compositions. BACKGROUND OF THE INVENTION Acne vulgaris is a disorder of the hair follicle sebaceous gland unit that can affect to some extent all young people, as well as many adults. The initial damage of this disease is thought to be caused by the hyperkeratosis and hyperkeratinization of the infondibulum, a process that helps the sebaceous glands to transform into acne. When the funnel ruptures and sebum enters the dermis, these epithelium are damaged, causing inflammatory damage. Therefore, traditional treatments focus on three pathological processes that primarily cause acne vulgaris. These treatments, such as topical Thunder Nord, are used to combat sebaceous gland obstruction caused by abnormal peeling of the cystic epithelium. Hormone agents target increased sebum production caused by androgen stimulation. Finally, the role of antibiotics is to reduce and / or stop the proliferation of propionic acid bacteria in the sacs due to inflammation. Benzoyl peroxide, salicylic acid, and various cleaning agents are also used for similar purposes. Thunder Nodule for topical use is considered to be one of the most effective acne-resolving agents for the treatment of acne vulgaris. However, the clinical effectiveness of this product is 3 paper standards applicable to the Chinese National Standard (CNS) 8 7 ^ grid (210X297 mm). --fc I J .---- 衣-(Please read the precautions on the back before filling in this page)

、 1T 590771 A7 B7 V. Description of the invention (2) The work of the Central Standards Bureau of the Ministry of Economic Cooperation and Consumption Cooperative Seal-¾ Limited to their stimulating effects. Thundertop for topical use has also been applied to the surface of mammalian dermis to produce an anti-aging effect. Although they are familiar with this technique and one of the effective topical therapies available for Yuan, these compounds are limited by their stimulating effects. A method for treating acne vulgaris, which is as effective as traditional acne treatment, but without high irritation, would be desirable. It would be desirable to provide a method for producing an anti-aging effect on the skin surface of mammals, which is as effective as Thunder Nuode treatment but does not have the same irritant effect. Summary of invention! In accordance with the present invention, we have discovered a method for treating acne vulgaris and / or for producing anti-aging effects on mammalian skin, comprising 'substantially including, or including topical application to an effective content of mammalian skin A combination of the first protease-containing topical active agent and the second Thunder-Nord-containing topical active agent. In another specific embodiment of the present invention, we have discovered a pharmaceutical or cosmetic composition, which contains, basically includes or includes: a) the first topical active agent containing a protease; and b) the Two topical active agents containing Thunder Nord. The composition and method of the present invention provide a unique and convenient method for treating acne vulgaris and / or producing anti-aging effects on mammalian skin. I Icon Jane! 4 This paper size applies to China National Standard (CNS) A4 specification (210X297 male splash) (Please read the precautions on the back before filling this page)

590771 A7 B7 Central Bureau of Standards of the Ministry of Economic Affairs-Industrial and Industrial Co., Ltd .: V. Description of Invention (3) This patent file contains many color pictures. A copy of the patent containing the color picture can be requested from the Patent and Trademark Office and provided with payment of the required fee. With reference to the following detailed description of the invention and the accompanying pictures, the invention has the advantages of making it more obvious, and it is more fully understood. Figure 1 (a) illustrates that after one hour of treatment with fluorescently labeled trypsin, rhino-like (Rhino) An image of a cross-section of a mouse skin. Figure 丨 ⑼ illustrates a cross-section of the skin of rhinoceros-like mice four hours after treatment with fluorescently labeled catalase. Fig. 1 (c) illustrates an image of a cross-section of the skin of a rhino-like mouse, four hours after treatment with fluorescently labeled trypsin, followed by daily treatment with 1% (W / V) trypsin. Figures 2 (a) and 2 (b) illustrate that after the skin of rhinoceros-like mice was treated with h & e staining, the tadpoles were untreated, and (b) the daily content contained o.io/^w/v) Histological images of GDL 圹 somes treated with trypsin for up to five days. Figures 3 (a) and 3 (b) are color images illustrating cross-sections of rhino-like mouse skin after paraffin sections and elastin staining. Circle 3 (a) is treated with vehicle, and Figure 3 (b) is treated with trypsin. Figures 3 (c) and 3 (d) are color images illustrating a wide cross section of the skin of C57B1 / 6 mice treated with paraffin sections and elastin staining. Figure 3 (a) is treated with a vehicle, and Figure 3 (d) is treated with trypsin. Figure 4 (ac) shows that rhino-like mouse skin is treated with h & e staining, and ⑻ is treated with 0.005% (w / v) All-trans retinoic acid treatment, ⑼ is treated with 0.005% (w / v) riding protease, and (c) is treated with 0,005 / o (w / v) all-trans Histological images of flavonic acid and 0,005% (w / v) trypsin treatment. 5 This paper size applies to China National Standard (CNS) A4 (210X297). (Please read the precautions on the back before filling this page)

1T 5. Invention description (4) A7 B7 Consumption of employees of the Central Bureau of Standards of the Ministry of Economic Affairs 1) ⑻, Figures 5 说明 and 5 (b) illustrate rhino-like mice treated with a carrier, which have broad skin when TUNEL stained Cross-section image. Figures 5 (c) and 5 (d) illustrate images of cross-sections of skin tissue of trypsin-treated rhinoceros-like mice when stained with TUNEL. FIG. 6 illustrates cross-sectional images of gene expression obtained from trypsin-treated rhino-dermis-like mouse skin cells under various concentrations of tenase by reverse transcription-polymerase chain reaction (" RT · PCR ") detection. Detailed description of the invention As used herein, "(w / v)" means the number of grams of the specified ingredient per 100 ml of the total composition. Suitable for use in the composition of the present invention as the first topical active agent containing protease, It contains, but is not limited to, serine proteases. Preferably, the first topical active agent is selected from trypsin, carboxyl protease-Y, protease VI, subtilysin, or a mixture thereof. The selected protease is trypsin. Preferably, the content of the protease is from about 0% (w / v) to about 5% (w / v), and more preferably from about 0% (w / v) to the total volume of the composition according to the present invention. 0.01% (w / v) to about 1% (w / v). Without intending to be bound by any theory, the first topical active agent of the present invention is believed to treat hyperkeratinization associated with acne vulgaris and / Or it can produce anti-aging effect on the skin.Although the first topical active agent is used for ordinary Acne treatment and / or anti-aging effect on the skin is the only active agent in the composition, but more comprehensively, in the treatment of acne vulgaris, a test of a local active agent and a second topical active agent are combined. Once again, I do not want to be bound by any theory, the second type of topical active test (please read the precautions on the back before filling this page) Order-V * 's Zhang scale towel making home; f 婢 (CNS) M specifications ( 2 Shu 297 Gong 590771 A7 V. Description of the invention (5) The agent 'preparation' is used to treat the hyperkeratinization and obstruction of sebaceous glands associated with acne vulgaris, but it also has an anti-aging effect on the skin, which can be active with the first topical application The reagent produced a match. Therefore, as demonstrated in Example 6 herein, the first characteristic of the first-type and second local activity test combination is that the treatment obtained is aimed at at least related to acne vulgaris. For the two pathological processes, ffij does not sacrifice the benefit of the first kind of local activity test to prevent aging. The second feature of the first combination with the second topical active agent is confirmed by Example 4 herein. display When the first topical active agent is combined with the second topical active agent, the stimulating effect related to the second topical active agent can be relieved. Therefore, the treatment of acne vulgaris and / or the skin has anti-aging effects. Compared with the treatment involving only the second topical active agent, the treatment of the present invention is almost the same, but the stimulating effect normally related to the second topical active agent is substantially reduced. Central Bureau of Standards, Bureau of Consumption, printed r-shirts— (Please read the precautions on the back before filling out this page) The third feature of the first and the second topical active test shot combination is as in the examples in this article. It has been confirmed that the combination of the first topical active agent and the second topical active agent can substantially reduce the time required to produce an effect when compared with the use of only the second topical active agent. Therefore, the therapeutic effect is still almost the same when compared with the treatment with only the second topical active agent, but the time required to refer to the results normally related to the use of the second topical active agent is essentially affected by the second «Combination of topical active agents with this first topical active agent is reduced. __ 7 Shi's Zhang scale suitable financial standard (CNS) domain (2ωχ297 public A7 --_ _B7 V. Description of the invention (6) The work and consumption of the Central Standards Bureau of the Ministry of Economic Affairs is suitable for use in the composition of the present invention as the first Two kinds of topical activity test! 'These include these finolides, which include but is determined by retinoic acid, vitamin A, alcohol, vitamins, ethyl acetate, brown retinal vinegar, and other = Biological, quasi-like or mixed compounds. The selected Tian Ling Nuode is all-trans retinoic acid. Preferably, the content presented by Ray Ling Nuode is an approximation of the total volume of the composition according to the present invention G _% (w / V) to about 0.5 / 〇 (y / v) 'more preferably from about 0% (she) to about 5% (w / v). ^ The first kind of "activity test _When the release parameter is required, the pharmaceutical and cosmetic composition of the present invention is preferably one that can further contain- a kind of medical or cosmetically acceptable, its wealth is used as a release secret, and it can be worn by live agents. Penetrates into human oval capsules. Although any commercially available first release active topical test fabric for the appropriate skin appendage (in this case the oval capsule) is suitable as a reliance It is acceptable on the carrier, but the fat body is better. It is more non-ionic and contains: (a) glycerol dilaurate or glyceryl distearate; ⑼ has alcohol as found in cholesterol Backbone compounds; and ⑷ has a purpose of from about 12 to about 18 carbon atoms, wherein the composition of the compound of composition is from about 53:10:22 to about 63: 2G: 32 in proportion, preferably each Do not go from about 55:12:24 to about 61.18.30. Liposomes composed of glycerol dilaurate / cholesterol / polyoxyethylene. Stearin ("GDL") is the best. Preferably, lipid The content exhibited by the body is from about 10 mg / mL to about 100 mg / mL, more preferably from about 25 mg / mL to about% Cock / mL, based on the total volume of the composition according to the present invention. The ratio of about 58:15:27 is the best. It is suitable for liposomes, and it is better to record. 2 Nakasaki mentioned above is prepared, but other ___ 8 This paper size is applicable to China A4 specifications (210χ ^^ (please first Read the precautions on the back and fill out this page} Φ, can 590771 A7 B7 M Central Ministry of Economics and Technology Bureau of the Central Bureau of Standards and Consumers of the United States and the United States of America and the United States and the United States, the technical description (7) The method is also acceptable. The above-mentioned liposomes can be used in combination with their required ingredients in an appropriate container and used under non-ionic conditions under any surrounding conditions, as known to those skilled in the art. Liposomal formulations are prepared by mixing with conventional shear mixing tools, such as those disclosed in Niemiec et al., "Nonionic liposome compositions are released into the hair follicle sebaceous glands alone 70 · in vivo in a topical peptide agent. Studies using hamster ear patterns "12 Drug Research (Pharm. Res.) 1184_88 (1995) (" Niemiec, "), which is fully incorporated herein by reference. In alternative specific embodiments, the pharmaceutical and cosmetic composition of the present invention may be combined with other content components, such as a moisture-increasing agent, a cosmetic adjuvant, an antioxidant, a surfactant, a foaming agent, a regulator, a fish moisturizer, as needed. Agents, fragrances, viscosifiers, buffers, sunscreens, colorants, preservatives, etc. without destroying the liposome structure (if present) to make cosmetic or medicinal products. When used in combination with each other, the first and second topical active agents of the present invention can be applied to mammalian skin simultaneously or at different times. For example, in the first example, if a daily combination of the first and second topical active agents is needed, the first topical active agent may be administered in the morning and the second topical agent in the afternoon. A topical active agent. In the second example, which is used as an example only, the second topical active agent can be administered in the morning and the first topical active agent can be administered in the afternoon. In the related example, this is only used as an example, and the first and the second topical active agents can be administered together. In the fourth example, it is still only used as

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V. Description of the invention (8) The employee of the Central Standards Bureau of the Ministry of Economic Affairs consumes Hezhu 590771 Α7 ___ B7 Example ‘The first and second types of topical active agents can be administered alternately every other day. In addition, in the fifth example, it is only used as an example. The first and second topical active agents administered are not one-to-one, so the first one can be administered in the first two days. For topical active agents, the first topical active agent is administered on the third day. Of course, in the fifth example, there are many variations. The previous five examples merely provide illustrations of some of the many different treatment regimens that are possible in the method of the invention. It will be appreciated that these examples are by no means limiting the method of treatment of the present invention, many other methods are also possible. An effective amount of a pharmaceutical and cosmetic composition is administered to treat acne vulgaris and / or produce anti-aging effects on mammalian skin. As used herein, "effective content" means a broad surface that is sufficient to cover the intended treatment of acne vulgaris and / or produce an anti-aging effect. Preferably, the composition applied to the skin surface on the skin surface to be treated for acne vulgaris and / or has an anti-aging effect is from about 2 microliters per square centimeter to About 8 μl / cm 2 of topical active agent. The invention disclosed herein by way of example may suitably be operated without any constituents, elements or steps not specifically disclosed herein. Many of the examples set forth below further illustrate the nature and operation of the invention. However, the invention should not be considered limited to its detailed description. Example Yixiong Example 1 and Shame V, Muntia • Rhinopithoid-like mice were used as experimental acne models to screen for topical active dissolving powder pupae and anti-keratinizing agents, such as Sandberg 10 papers Standards are applicable to China National Standard (CNS) Α4 specifications (210 × 297 mm) (Please read the precautions on the back before filling this page)

590771 A7 B7 Consumer cooperation of the Central Standards Bureau of the Ministry of Economic Affairs. 5. Description of invention (9 (Sundberg) J.P. et al. '"Hairless and rhino-like mice, chromosome 14," with abnormal skin and hair. It is described in the Mouse Mutation Handbook 291-312 (1994), which is fully incorporated herein by reference. A recessive mutation in chromosome 14 will cause mice aged 25 days to have wrinkled skin and no body hair. There At the end of the hair cycle, the cystic nipples cannot recover the hair follicles later, but are isolated in the dermis. The nipples are no longer associated with the cystic epithelium to stimulate a new hair follicle cycle. The upper remnants of the hair follicles are full of sores, keratinocytes' And on their bases, oval sacs containing sebaceous glands are formed, similar to a kind of open acne. The surface of rhino-like skin expands, which causes the underdeveloped hair follicles to fill with horny residues, and progressively become loose, forming continuous folds and bulges. The ellipsoid sac progressively grows to form a hair cyst (pseudoacne), which is caused by a flared cystic funnel full of horny residues. It will be obtained from Jackson Labs (Barr (Bar) Port, Maine, RHJ / LH hairless ("rhino-like"), male mice aged 5-7 weeks, treated with "Mezick" and others for "local and systemic use" The role of Ray Lingnuo in rhino-like mice filled with keratin sugar bursa: Quantitative Thunder Nuode's "anti-keratinizing" mode of action ,, 83. Journal of Dermatology 110 (J. Invest. Dermatol.) 110-113 (1984) ("Mezick"), the article is fully incorporated herein as a reference. 磉 Example 2: The living ministry uses living gold to release the remaining y 逄 from Sieg Sigma-Aldrich (St. Louis, Missouri) lyophilized trypsin in sufficient quantity, mixed under the trade name "Hepes", obtained from Life Technology (Life Techn〇l〇gies) -L .-------- Cloth ------ Order ----- (Please read the precautions on the back before filling this page) 11 590771 A7 B7 Central Ministry of Economic Affairs Standards Bureau Shellfish Consumption Printed by Bamboo Fir 5. Description of Invention (10) Company (Gaithersburg, Maryland) 〇05 Μ Ν-2 · hydroxyethylhexahydro Burning acid buffered water In the solution, the pH value of the obtained solution was about 7.4, and trypsin in the solution was about 2% (w / v). Then, one volume of the obtained trypsin solution was mixed with one volume of water (50 / 〇). Glycerol dilaurate / cholesterol / polyethylene-10-stearyl ether liposomes are prepared by the method described by Niemiec to produce 1% (w / v) of the obtained topical active product Trypsin concentration. Glyceryl dilaurate is marketed under the trade name "EmulsyntGDL" and is available from Van Dyke (Belleville, New Jersey), an international specialty product. Cholesterol is available under the trade name "cholesterol VSP / NF" from Croda Corporation (Parsippany, New Jersey). Polyethylene · 10-stearyl ether is available under the trade name "Brij 76" from ICI Surfactants Americas (Wilmington, Delaware). The volume-to-volume ratio of each trypsin to GDL liposome can be adjusted to produce various concentrations of trypsin liposome composition. The retinoic acid composition includes an ethanol / 1,2-propanediol carrier containing 70% (w / v) obtained from Quantum Chemicals (Tuscola, Illinois). Ethanol (ethyl alcohol, 200 standard alcohol) with 30% (w / v) 1,2-propanediol obtained from Fisher Scientific (Pittsburgh, Pennsylvania). All-trans retinoic acid used in retinoic acid composition was obtained from BASF Thieves Company (Ludwigshafen, Germany). All-trans retinoic acid to ethanol / 1,2-propanediol carrier volume-to-volume ratio, 12 This paper size applies to Chinese national standards (CNS> A4 size (210X297 mm) (Please read the precautions on the back before filling (This page). ^. Order 590771 A7 ____ ^ B7 V. Description of the invention (11) Consumption of Hezhu by employees of the Central Bureau of the Ministry of Economic Affairs, printed, which can be adjusted to produce various concentrations of retinoic acid composition. Example 3 : Guangdan Club Luhao Chufeng applied about 100 microliters of topical active trypsin composition in Example 2 to the back of each rhinoceros-like mouse. According to the molecular probe (M-Probe) The prodrug white ft light labeling kit is attached to the procedure and will be used for trypsin fluorescent labeling in this composition (1996). One and four hours after treatment with fluorescent trypsin, cut out from each mouse with scissors 1 cm X 2 cm skin samples, fixed at 25t, with a buffered formaldehyde solution of 10% pH from 6.9-7.1 by Stephens Scientific, and then formed into paraffin according to well-known methods Block and verify it with a fluorescence microscope using well-known methods As shown in Figure 1 (a), almost all fluorescent markers were found in the oval capsule and the sebaceous glands. At 1 hour intervals (Figure 1 (a)) and 4 hours interval (Figure 1 (b)), Identified mice showed the same histological staining pattern, but at a later time point 'no additional skin penetration. This observation suggests that the possibility of digestion by non-specific extracellular matrix through proteases is likely to occur at a later time point' It is shown that the fluorescent dye can penetrate deeper into the stratum corneum. Except for the treatment with fluorescent trypsin, the 1% (w / v) protease composition in Example 2 was not as small as this daily. Five days after the rat treatment, this example was repeated with a rhino-like mouse similar to that in Example 1. Four hours after the fifth day of fluorescent trypsin treatment, the skin of these mice was subjected to a similar fluorescent microscope method. Analysis. As shown in Figure 1 (c), no significant changes in trypsin '· into the oval sac and sebaceous glands of the treated skin were observed. However, there were some changes in the outer layer of the stratum corneum of the skin after trypsin treatment. 13 This paper size applies to Chinese national standards CNS) Α4 Specification (21〇χ297 mm) (Please read the back of the precautions to fill out this page)

、 1T 590771 Α7 ___ Β7 V. Description of the invention (12) Printed by Hezhu Co., Ltd., employee of Central Standard Bureau of Jingyi Department Minimal staining, showing that the integrity of the barrier is partially defective. The barrier defect can thus reflect the value of < TEWL ,, > as described in Example 4 and Table 10 herein. This example shows that the broad surface of rhino-like mouse skin is applied topically. When a trypsin-containing composition is used, the short-term and long-term use of the trypsin-containing composition will lead to the release of the proteases into the oval capsule and the sebaceous glands. Paper ~ 'Protein method to reduce the size of the work bag 4, without reading the dermal stimulation of the rhino-dermal-like mice of Example 1, the trypsin composition of Example 2 (0.001% (w / v)-1% (w / v)) local treatment once for five days. On day 8, the animal was sacrificed and the reduction in sac sac size was quantified using image analysis. For image analysis, the entire mounting epithelium must be treated and according to Mezick and Bererad et al., "Rhino-skin-like mouse model topical application of all-trans retinoic acid and CD271 to the epithelium Microstructure and the role of pseudoacne oval capsule wall, 283 (2) Pi Guangxue (Arch · Dermatol. Res ·) 100-107 (1991), and Bouclier et al., "Yu In a rhino-like mouse model, using standardized image analysis techniques to quantify the histological changes in epithelium induced by local application of Thunder Nord and CD271, "4 (2) SkinPharmacol 65-73 (1991) The methods described above are used for microscopic measurements, and each of them is fully incorporated herein as a reference. On a Gateway 2000 P5-100 computer, the Empire Imagins database version 1.1 was used to capture the images .Using Image Pro Plus version 1.3 as the measurement, and using Microsoft Excel version 5.0 for data processing. (Please read the precautions on the back before filling out this page} Standards apply to Chinese national standards (CNS) A4 size (210 × 29 * 7 male ί 590771 A7 B7) Consumption of Hezhu and printing by employees of the Central Standards Bureau of the Ministry of Lithology 5. Description of the invention (13 pairs of treatment groups (3 rhino-like mice), use Five random areas, two measurements per animal per area to calculate average ellipsoid capsule diameter (μ) and average sebaceous gland size (μ2). According to Finney, DJ · "Parallel line analysis, statistics of biological analysis Method, "the method described in Charles & Griffen Co., Ltd. 69-104 (1978), to calculate the percentage reduction in the diameter of the oval capsule, which is fully incorporated herein as a reference. As shown in Table 1 The trypsin-induced dose was dependent on the size of the ellipsoid capsule, and reached ~ 0.1% (w / v) trypsin to make it reach the plateau of the curve. Compared to the liposome control, further increase of trypsin concentration did not lead to rounding There was a reduction of more than 55% in the size of the capsule. A small decrease in the diameter of the oval capsule was observed with the liposome carrier only. When analyzed after seven days (not shown), a single trypsin (1% (W / V)) Treatment does not affect the size of the oval capsule Table \ 'Teng protein_ induction dose dependent reduction of the size of the oval capsule (please read the precautions on the back before filling in this page) Staple trypsin 0.001% (w / v) Trypsin 0.005% (w / v) Trypsin 0.01 % (w / v) Trypsin 0.05% (w / v) Trypsin 0.1% (w / v) Trypsin 0.5% (w / v) L0% (w / v) Reduction of oval capsule size (%) For liposomes Control: 26.85 + 4.38 19.58 ± 3.06 33.08 ± 2 · 15 43.84 ± 0.62 50 · 67 ± 0 · 83 54.31 ± 1.33 54.85 ± 1.02 TEWL (g / m2) 26.40 ± 1 77 28.53 ± 2.18 36.57 ± 2. 7 42 · 80 ± 4.33 36 · 23 ± 1.24 42 · 00 ± ι · 14 15 This paper size is applicable to China National Standard (CNS) A # specification (21〇297297 mm 590771 A7 ___ B7 Employee consumption cooperation of the Central Standards Bureau of the Ministry of Economic Affairs And imprint five, invention description (! 4) _g purpose plastid carrier 13 · 2 ± 1 · 82 * 19 · 8 ± 1 · 14 ^^ ga ^ aasggaaa = g = s =: s · ι caasso; ··· ι ··· ρι ··· ~ κιγγ ass ^ sassss ^ B ^ saK ^ gassssfsfsssBss ^ sax ^^^! imm The percentage reduction in the oval sac size during plastid-loaded Zhao treatment is calculated relative to the untreated control to further characterize the trypsin on rhinoceros The effect of mouse-like skin, we measured transdermal dehydration using an evaporator, which is available from Servomed AB. First, the evaporator was standardized by ambient humidity. Then, place the probe on the skin of the back of the test object, and obtain a TEWL reading at that point. As shown in Table 1, TEWL increases in a dose-dependent manner, and is ~ 0.05% (w / v ) When trypsin is used, a stable curve can be reached. This is about the same concentration as when the diameter of the elliptical capsule is maximally reduced. There is no correlation between the increase in TEWL and the visible stimulus. A small number of descaling and erythema observed throughout the experiment were not doses Dependent, even under as low as 1% (W / V) trypsin. In addition, TEWL in mice treated with trypsin is low Thunderwort alone was administered to TEWL in mice. Histological analysis of the skin of untreated, liposome-controlled, and trypsin-treated rhinoceros-like mice showed significant changes in the skin treated with trypsin. H & E staining and group Weaving analysis was performed using standard methods as described by jgheehand and Harpchak, 1980. As shown in Figure 2 (b), when compared with untreated epithelium as shown in Figure 2 (a) In comparison, treatment of epithelium with trypsin will proliferate as the number of cystic epithelium and epithelial cell layers increases. The major changes observed in the granular and stratum corneum are due to the restoration of peeling and improvement of the epidermal structure. These epithelium Changed to use for living 16 This paper size applies Chinese National Standard (CNS) A4 specification (210X29 * 7mm) (Please read the precautions on the back before filling this page) · «Cloths" Order 590771 A7 B7 Central Standard of the Ministry of Economic Affairs Consumption cooperation with staff of the Bureau, ^ V. Description of the invention (15) The complete characteristics of in vivo Thunder Novo active markers are related to its effective clinical efficacy. To further assert that trypsin is not related to dermal stimulation, Figure 2 (b) shows It shows no inflammatory cells, which will normally appear under irritated conditions. As shown in this example, trypsin caused a decrease in the size and dose of the oval capsule. The reduction in oval capsule size is related to the effective clinical efficacy of the composition for treating acne vulgaris. Related. Therefore, this example further shows that trypsin is effective in the treatment of acne vulgaris. This example also shows that topical trypsin treatment will not induce skin irritation. Feng Example 5: 腐 蛋 # 薜 液 will be an example Rhinoceros-like mice of Example 1 were treated with the proteinase composition of Example 2 and showed a significant effect on skin elasticity. To quantify this effect, a cutometer was used for analysis. The dermatometers we used were obtained from Academy (Menlo Park, California), and using Couturaud et al., "Pearl Biomechanics Nature · Evaluation of the effects of non-destructive methods in vivo on age and body parts 1 Skin Res. And Technol. 63-73 (1995) and Eisner et al., "Humans Mechanical properties of forearm and vulvar skin, "122 The method described in Br. J. Dermatol 607_ 614 (1990) was performed 'These two lines are fully incorporated herein as a reference. Subject to negative pressure After release, suction was used to pass through the 2 mm aperture and the corresponding skin movement and recovery were measured. In human studies, the deformation parameters Ua / Uf (skin wide fatigue, or total recovery under self-load), Ur / uf (bioelasticity) , Or the elastic recovery after negative load) to Ur / Ue (hardness, or to improve the resistance to deformation of the skin) ratio, showing the better skin tension and elasticity. Deformation 17 This paper size applies to China National Standard (CNS) A4 size (210x297 public holiday) (Please read the precautions on the back before filling this page)

Order 590771 A7 B7 V. Description of the invention (16) The consumption of employees of the Central Standards Bureau of the Ministry of Economic Affairs, the parameters of Ue, Uf, Ua and Ur are dependent on the thickness of the skin. Therefore, as described by Barel et al., "Suction method / dermatometer for measuring mechanical properties of skin, non-destructive method and skin handbook 335-340 (1995), use this ratio for evaluation, This article is fully incorporated herein as a reference. As shown in Table 2, trypsin treatment resulted in an increase in all of these parameters, which reflects an improved skin elasticity. Although the differences between animals are significant, they are derived on a per capita basis. The increase in properties is consistent, and increases with time and treatment length. Mechanical properties of rhino-dermal skin treated by trypsin. 7th, 12th, and 16th parameters. Protein untreated protein_Manzhao enzyme treatment control enzyme treatment treatment enzyme treatment treatment enzyme treatment Ua / Uf 0.541 ± 0.040 0.593 ± 0.009 0.656 ± 0.008 0.66310.010 0.429 ± 0.009 0.675 ± 0.003 Ur / Lte 0 · 408 ± 0 · 080 0.557 ± 0.021 0 · 242 ± 0.006 0.666 ± 0.024 0.243 ± 0 · 006 0.733 ± 0.018 Ur / Uf 0J0010.019 0,359 ± 0,022 0370 ± 0,005 0.548 ± 0.011 0.204 + 0.031 0.404 ± 0.008 Trypsin of Example 2 Extensive skin sections of rhino-like mice obtained from Example 1 treated with the composition, according to Kligman, LH, "Luna's" technology, a beautiful stain for elastin 3, (2) The method stated in The American Journal of Dermatology (The Amer J. Dermatopathol.) 199-200 (1981) 'stains elastin on its paraffin sections, which is fully incorporated into this 18 This paper size applies the Chinese National Standard (CNS) A4 specification (210 × 297 mm) (please read the precautions on the back before filling this page), 1T 590771 A7 B7 5. The description of the invention (17) is used as a reference. As shown in Figure 3 (b), when compared with the untreated mice shown in Figure 3 厚度, the thickness and density of elastin fibers (dyed in purple) near the sacs and sebaceous glands of rhino-like mice treated with trypsin. increase. The same experiment was performed with C57B1 / 6 mice obtained from Charles River Laboratory (Kingston, New York), and the results were similar. Figures 3 (c) and (d) show the results of the inelastic protein staining of the untreated and Jingteng-treated skin ', respectively. Table 3 below shows the increase in skin mechanical parameters after membrane protease treatment. Table 3.Mechanical properties of C57B1 / 6 skin treated with trypsin. Biophysical parameters. 16th: Untreated control Tuna protease treatment Ua / Uf 0.429 ± 0.014 675 ± 0.〇18 Ur / Ue 0.243 ± 0.021 〇. 7335 ± 0.〇2 Ur / Uf 0.204 ± 0.026 0.404 ± 0 · 01 (Please read the precautions on the back before filling out this page) Ordering and printing by the staff of the Central Bureau of Standards of the Department of Dispensing and Printing This example is based on the protease When topically treated, C57B1 / 6 and rhino-like mice showed increased skin elasticity. Skin elasticity is a property related to preventing aging. Therefore, this example further shows the effect of trypsin on the surface of the skin to prevent aging. Yichatu 6 •• The mechanism of tryptone is different from that of fuming_ Trypsin may affect the sebum composition of acne. It is evaluated using a hamster ear pattern system. Young 19 Standard (CNS) A4 Specification (210X297 mm) 590771 A7 B7 purchased from Charles River Laboratory (Wilmington, Massachusetts) 590771 A7 B7 V. Invention Description (18) Golden yellow Syrian hamster (Syrian hamster), 45_55 grams upon arrival. The ventral surface of the right ears of these hamsters were treated daily with 10 microliters of trypsin composition of Example 2 to three weeks in length every Friday and day, and the left ear was treated as an untreated control. As shown in Table 3 'In this system, trypsin has no effect on the size of the sebaceous glands. Table • Trypsin on sebaceous glands in hamster ears-_ size of fat-producing glands (μ2) percentage less (%) untreated 99112.4 ± 2904.0 liposome carrier 94698 · 9 ± 4997 · 1 4 · 45 (for untreated) trypsin 0.5% (w / v) 95043.0 ± 4269.1 _0.36 (for liposome carrier) (Please read the precautions on the back before filling this page) It was shown that the proteases had no effect on sebaceous gland size in the hamster ear model system. As is well known in this model, the reduction in the size of the sebaceous glands in hamster ears caused by Thunder Nord is dose-dependent. Therefore, this embodiment further mentions that the operation of trypsin is different from the mechanism of action of Thunder Nordic compounds. Example 7: Guangxiang Lingyou Dianrong ♦ Thin and Shame Saint ★ Ice bath fights the hemp The rhinoceros-like mice of the first group in Example 1 were treated with the suboptimal dose of the trypsin composition in Example 2. As used herein, "suboptimal," is defined as the concentration of the tenanase is lower than the optimal content for reducing the size of the oval capsule as described in Example 4. The second group of rhinoceros in Example 1 is small The rats were treated with the suboptimal dose of the trypsin composition in Example 2 and the all-trans retinoic acid composition in Example 2. In the third group of rhino-dermal mice, trypsin and all-trans retinoic acid were used. Each day at different times, the order is given d 20 This paper size is applicable to China National Standards (CNS) A4 specifications (21〇χ 297 mm 590771 A7 B7 V. Description of the invention (l9) Therapy (ie, trypsin in the morning And all-trans retinoic acid in the afternoon). Mice were sacrificed and their skins were subjected to histological examination using the procedure described in Example 3. As shown in Fig. 4 (c), although it was shown that individual treatment was given Compared with untreated skin (Figure 2 (a)), rhino-like mice treated with both trypsin and all-trans retinoic acid composition showed significant improvement when treated with trypsin and all-trans Comparison of retinoic acid treatments still shows considerable improvement in peeling effect (Figure 4 (a & b)). In addition, histological analysis revealed that after treatment, the open oval capsule on the skin surface is far less than that given to a single treatment alone. This example shows that trypsin and all-trans When combined with retinoic acid, it has an additive effect on skin characteristics such as the number of open oval capsules, which means that these compositions are effective for the treatment of acne vulgaris.

8: PCD The kudus-like mouse in Example 1 was treated with the 0.1% (w / v) trypsin composition in Example 2 for five days, and sacrificed on the eighth day. Through the method stated in Example 3, 1 cm x 2 cm untreated, carrier-treated, and trypsin-treated skin samples were obtained through the method described in Example 3. Gavrieli et al. Analyzed the staining step of TdT-mediated dUTP-biotin nick end labeling (" TUNEL ") revealed by" identification of programmed cell death in situ by nuclear DNA fragment specific labeling. "During this step, Using the "ApopTag ™" additional in situ programmed cell death detection kit obtained from Oncor Corporation " ApopTag ™, press OnCor Corporation (February, 1955) KnApOpTagTM , '21 This paper size applies to China National Standards (CNS) A4 specifications (210X297 mm) 7 07 9 A7 ___B7 V. Description of the invention (20) Approved by the staff of the Central Standards Bureau of the Ministry of Foreign Affairs-TK. Printed in situ The programmed cell death detection kit process description is based on staining the prepared skin section according to the fragment DNA terminal label as described in Gavrieli. Figures 5 (a-d) show a tissue analysis where the stain has a peroxidase end point (blue) versus methyl green contrast staining. The resulting image is provided in Figure 5 (a & b), which was treated with a carrier, and 5 (c & d) was treated with trypsin. As shown in Fig. 5 (ad), TUNEL stained samples are based on morphology (concentrated or fragmented nucleus and cytoplasm or apoptotic body) and their stained color (in the concentrated nucleus, fragment DNA is stained brown) Define apoptotic cells. As shown in Figure 5 (a & b), TUNEL staining revealed unusually high levels of apoptotic bodies in cystic epithelium. As a result of trypsin treatment, all apoptotic bodies in the cystic epithelium disappeared, and in the granular layer, programmed cell death (PCD) would be restored as epithelial differentiation resumed (Figure 5 (c < fed)). This example shows that trypsin can restore the balance between cystic epithelium and intraepithelial cell death and proliferation. One of the pathological processes provided by acne vulgaris is hyperkeratinization, which is attributed to this equilibrium shift. Therefore, this example further shows that trypsin has the ability to restore the normal balance between epithelial cell death and proliferation, which may be a factor in its ability to treat acne vulgaris. Example 9: The name of the broad-boiled eggplant, Lianning Saining, Shame__ The rhinoceros-like mouse in Example 1 was prepared daily with the proteinase composition (0% (w / v)) as prepared in Example 2. , 0.0001% (w / v), 0. · 001 ° / 〇 (\ ν / ν and 0.01% (w / v)) were treated for five days, and they were sacrificed on the eighth day, as in Example 3. Obtained 22 mice treated with trypsin with carrier. The paper size is applicable to Chinese National Standard (CNS) A4 (210X297 mm) (Please read the precautions on the back before filling this page)

590771 A7 B7 V. Description of the invention (21) Enzyme treatment of mouse skin, and then use the "RNA Stat-60" reagent obtained from Tel_Test " B "company, according to Chomczymski" A single-step method for RNA isolation using acid guanidinium thiocyanate-phenol aerosol extraction, "162 Anal Biochem. 156-59 (1987), which extracts all of their RNA, and this article is fully incorporated herein as references. Then, using the method stated in Promega's (May, 1995) publication in the "RNase-free DNase" process, a sufficient amount of RNase-free DNase from Promega, trademark Named "RQ1 RNase-free DNase", it was added to the RNA extracted from each mouse so that each product contained 200 nanograms of DNase-activated RNA. The resulting 200 nanograms of DNase-treated RNA was published in "Superscript II Reverse Transcriptase" using Gibco-BRL (now Life Technologies) (April, 1992). The method stated in the scheme uses random hexamers as random primers, which are commercially available from Life Technologies, and perform reverse transcription (" RT "). Then, via polymerase chain reaction (& quot PCR "), using 0.5 units (per Γϋ (Γ microliter reaction) of thermostable DNA polymerase, which is commercially available from Perkin-Elmer-Cetus, Trade name "Taq Polymerase" and about 0.1 μmol / reactive mouse glyceraldehyde I phosphate dehydrogenase (G3PDH) primer, which was obtained from clone technology (ciomech) laboratory company ("cloning technology" ) Or the primers described in Table 4 (using the conditions in Table 4, or according to the procedure stated in the procedure attached to the clones (Clontech) primers) amplified RT products. 23 This paper-scale towel Guanjia County ( CNS) A4 specification (2iGX297 mm) 590771 A7 B7 Central Standard of the Ministry of Economic Affairs It ’s printed by local office workers. It ’s printed. 5. Description of invention (22) Table 4 shows some used DNA primers, MgCl2 content required for PCR reaction, and length of PCR cycle. Involucrin primer system For example, Marthinuss, et al. "Programmable cell death in Pam212, an epithelial keratinocyte cell line · The role of bcl-2 in epithelial differentiation." 6 Cell Growth Diff. 239-250 (1995), and this article is incorporated herein by reference in its entirety. Table DNA primer DNA primer MgCI2 loop sequence ID used in AIRT-PCR analysis (see attached sequence table) (mM) (分) @ ° c number number transglutaminase intentional stock 2.5 1 @ 94; 35 1 5f AACCCCAAGT TCCTGAAG 2 @ 55; 3 @ 72 transglutaminase antisense stock 2.5 1 @ 94; 35 2 5, TTTGTGCTGG GCCACTTC 2 @ 45; 3 @ 72 Elastin interested stock 5 1 @ 94; 35 3 5 »TAAGGCAGCC AAATATGGTG 2 @ 45; 3 @ 72 Elastin anti-interest stock 5 1 @ 94; 35 4 5 * ACCTGGATAA ATGGGAGAAA G 2 @ 55; 3 @ 72 For better and better observation, the PCR products of the results are well known Step, precipitate it with ethanol. When used for G3PDH primers, only 24 paper sizes are applicable to the Chinese National Standard (CNS) A4 specification (210X297 mm) (Please read the precautions on the back before filling this page) # 衣 · Order 590771 A7 B7 V. Description of the invention (23) The 10% PCR reaction product of the shellfish consumer cooperation of the Central Standards Bureau of the Ministry of Economic Affairs is used. The PCR product was then analyzed on a 2% agarose / ethidium bromide gel according to methods well known to those skilled in the art, in order to compare the exudate-treated and non-trypsin-treated mouse skin cells. , The degree of specific gene expression. RNA samples obtained from the skin of rhino-like mice without reverse transcription were used as a negative control group for each PCR amplification. When the positive control group is not commercially available, an RNA sample obtained from the skin of a 6-month-old rhinoceros-like mouse skin can be used as the positive control group. Gel analysis results showed that the RT_pCR product moved on the gel, which was always the same size as the positive control group and the reported amplification primers. Then, the relative quality of each RT-PCR reaction product was compared by analyzing the mRNA content of G3PDH in each product and the "housekeeper" gene. As shown in Figure 6, the G3PDH gene expression is similar in all tested samples. Therefore, it can analyze the relative gene expression of the desired gene. Transglutaminase, an enzyme involved in the cross-linking and formation of apoptotic bodies, shows high mRNA content in control animals and can be reduced below the detectable level as the trypsin concentration increases. This shows that trypsin restores the homeostasis of the oval capsule and eliminates abnormally high levels of programmed cell death in the cystic epithelium. With increasing trypsin concentration, elastin mRNA increased. Therefore, neo-elastin appeared after trypsin treatment, and as a result, the skin elasticity increased as described in Example 5. • Nepetin_white, a marker of epithelial differentiation, increases in content after trypsin-dependent treatment. This display shows normal epithelial conversion and analysis. This paper size is in accordance with Chinese National Standard (CNS) A4 (210X297 mm) (Please read the precautions on the back before filling this page)

, \ 呑 丁 590771 A7 __—____ B7 V. Description of the invention (24) The restoration has been resumed. Therefore, trypsin restores the balance of epithelial differentiation as described in Example 8_. The effect of trypsin on acne vulgaris and its ability to prevent aging shown in this example can be understood by testing a series of gene expression forms under a wide range of trypsin concentrations. Changes in mRNA content induced by trypsin have been clearly demonstrated to show that trypsin plays a regulatory role in pCD, apoptotic cell death, elastin expression, and epithelial differentiation. Glyceryl dilaurate / cholesterol / polyoxyethylene-10-stearyl ether lipid system is prepared according to the steps described by Niemiec, wherein the composition compounds of the liposomes are each about 58:15:27. proportion. Before mixing the liquid with the aqueous phase to form Niemiec liposomes, add 0.1% (w / v) ascorbic acid to the aqueous phase and add the ingredients listed in Table 5 to the liquid phase of the composition . The final pH of this composition is adjusted to a range of 4 to 7, preferably 4.5 to 5.5, with a buffer solution. Table 1 Ingredients% (w / v) added to the liquid phase Tretinoin 0.01 Methyl paraben 0.10 Propyl paraben 0.02 Second third- Butyl p-cresol 0.05 The second composition, which is produced at the central standard of the dripping department and is consumed by bamboo workers, is printed at pH 7.4 (sufficient to 100 ml), and contains 1.0 g dissolved in 0.05M Trypsin in Hepes buffer, according to 26 paper standards, applicable to Chinese National Standard (CNS) A4 (210X297 mm) 590771 A7 B7 V. Description of the invention (25) Every 8 parts of liposome composition, about 1 part of The ratio of the two components is added to the liposome composition. And this final composition is suitable for immediate topical application. (Please read the precautions on the back before filling out this page.) Consumer cooperation with the Central Bureau of Standards of the Ministry of Economic Affairs Π Yin Fan This paper size applies to China National Standard (CNS) A4 (210X297 mm)

Claims (1)

77 90 8 8 8 8 AB c D VI. Application for Patent Scope I Patent Application No. 87101981 J ROC Patent Appln. No. 87101981 Amended Claims Application for Spring Benefits Chinese Text-Attachment (I) Amended Claims in Chinese -Enel. (I) 3 (Submitted on November \ ^, 2003) (Submitted on November \ ^, 2003) Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs 1. A medicine used to produce anti-aging effects on the skin A composition containing a serine protease as an active ingredient. 2. The pharmaceutical composition according to item 1 of the application, wherein the active ingredient is selected from the group consisting of trypsin, carboxyl protease-Y, protease VI, subtilysin, or a mixture thereof. 3. The pharmaceutical composition according to item 2 of the patent application scope, wherein the active ingredient is trypsin. 4. The pharmaceutical composition according to item 3 of the scope of patent application, wherein the content of the active ingredient is from 0.01% (w / v) to 5% (w / v) of the total volume of the composition. The pharmaceutical composition under the scope of patent application No. 4 wherein the content of the active ingredient is from 0.01% (w / v) to 1% (w / v) based on the total volume of the composition. 6. The pharmaceutical composition according to item 1 of the patent application scope, wherein the composition further comprises a pharmaceutically acceptable carrier. _ 7. The pharmaceutical composition according to item 6 of the scope of patent application, wherein the pharmaceutically acceptable carrier is a liposome or a mixture thereof. 8. The pharmaceutical composition according to item 7 of the application, wherein the liposome is non-ionic. 9. The pharmaceutical composition according to item 8 of the scope of patent application, wherein the lipid -28-this paper size is applicable to China National Standard (CNS) A4 (210x297 mm) 87053B- 77 907 A8 B8 C8 _D8_ 6. The scope of the patent application system includes: (a) glyceryl dilaurate or glyceryl distearate; (b) cholesterol, or a compound with a steroid backbone as found in cholesterol, or Mixtures; and, <% (c) fatty acid ethers or mixtures thereof having from 12 to 18 carbon atoms. 10. The pharmaceutical composition according to item 9 of the scope of the patent application, wherein the lipid system comprises: (a) glycerol dilaurate; (b) cholesterol; (c) polyethylene oxide-10-stearin. 11. The pharmaceutical composition according to item 9 of the scope of patent application, wherein the composition of the liposome is from 53:10:22 to 63:20:32, respectively. 12. The pharmaceutical composition according to item 6 of the application, wherein the content of the pharmaceutically acceptable carrier is from 10 mg / ml to 100 mg / ml based on the total volume of the composition. Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs 13. The pharmaceutical composition according to item 1 of the scope of patent application, wherein the composition further includes a moisture-increasing agent, a cosmetic adjuvant, an antioxidant, a surfactant, a foaming agent, Regulators, humectants, fragrances > f, tackifiers, buffers, mothproofing agents, colorants, preservatives, etc. 14. A pharmaceutical composition for treating acne vulgaris and / or for producing an anti-aging effect on the skin, comprising a serine protease and a retinoid compound as active ingredients. -29-This paper size is in accordance with China National Standard (CNS) A4 (210x297 mm) 590771 A8 B8 C8 Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs —— —_D8 ________ Scope of patent application 15 · According to the scope of patent application The pharmaceutical composition according to item 14, wherein the first active ingredient is selected from the group consisting of trypsin, carboxyl protease-Y, protease VI, subtilysin, or a mixture thereof. 16. The pharmaceutical composition according to item 15 in the scope of the patent application, wherein the first active ingredient is trypsin 'Π · The pharmaceutical composition according to item π in the scope of the patent application, wherein the content of the first active ingredient, Based on the total volume of the composition from 0.01% (w / v) jl 5% (w / v) 〇18. The pharmaceutical composition according to item I of the patent application scope, wherein the content of the first active ingredient presents Is from 0.01% (w / v) to 1% (w / v) of the total volume of the composition. 19. The pharmaceutical composition according to item 14 of the application, wherein the second active ingredient is selected from the group consisting of retinoic acid, vitamin A alcohol, vitamin A aldehyde, ethyl retinyl acetate, retinyl palmitate, or Other derivatives, analogs or mixtures thereof. 20. The pharmaceutical composition according to item 19 of the application, wherein the second active ingredient is all-trans retinoic acid. 21. The pharmaceutical composition according to item 19 of the scope of patent application, wherein the content of the first active ingredient is from 0.0001% (w / magic to 0.5% (w / v)) 22. The pharmaceutical composition with scope of patent application f 21, wherein the content of the second active ingredient is from 0.001% (w / v) to 0.025% (w / v) ° 23. According to the scope of patent application 帛21 items of medicinal composition, of which this group -30-This paper size is applicable to Chinese National Standard (CNS) A4 specification (210x297) 590771. A8 B8 C8 D8 VI. Patent application scope The product further contains a medically acceptable Carrier. 24. The pharmaceutical composition according to item 23 of the patent application, wherein the pharmaceutically acceptable carrier is a liposome or a mixture thereof. 25. The pharmaceutical composition according to item 24 of the patent application, wherein the liposome is non- Ionicity. 26. The pharmaceutical composition according to item 25 of the application, wherein the lipid system comprises: a) glyceryl dilaurate or glyceryl distearate; b) having steroids as found in cholesterol Backbone compounds And c) a fatty acid or a mixture thereof having from 12 to 18 carbon atoms. 27. The pharmaceutical composition according to item 26 of the application, wherein the lipid system comprises: a) glyceryl dilaurate; b) cholesterol; c) polyethylene oxide-10-stearyl ether. 28. The pharmaceutical composition according to item 26 of the patent application, wherein the composition of the liposome is from 53:10:22 to 63:20:32, respectively. Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 29. The pharmaceutical composition according to item 23 of the scope of patent application, wherein the content of the pharmaceutically acceptable carrier is based on the composition > To 100 mg / ml. 30. The pharmaceutical composition according to item 14 of the scope of the patent application, wherein the composition further comprises a moisture-increasing agent, a cosmetic adjuvant, an antioxidant, a surfactant, a foaming agent, a regulator, a fisher agent, a perfume,- 31-This paper size is in accordance with Chinese National Standard (CNS) A4 (210x297 mm) 7 07 9 L A8 B8 C8 D8 VI. Patent application scope Tackifier, buffer, sunscreen, colorant, preservative, etc. 31. A cosmetic composition for producing an anti-aging effect on the skin, comprising a serine protease as an active ingredient. 32. The cosmetic composition according to item 31 of the scope of application, wherein the 谲 active ingredient is selected from the group consisting of trypsin, glutathione-Y, protease VI, subtilysin, or a mixture thereof. 33. The cosmetic composition according to item 32 of the application, wherein the active ingredient is a membrane protease. 34. The cosmetic composition according to item 33 of the scope of the patent application, wherein the content of the active ingredient is from 0.01% (w / v) to 5% (w / v) of the total volume of the composition. 35. According to The beauty composition of item 34 of the scope of patent application, wherein the content of the active ingredient is from 0.01% (w / v) to 1% (w / v) of the total volume of the composition. 36. According to the scope of patent application The cosmetic composition according to item 31, wherein the composition further comprises a pharmaceutically or cosmetically acceptable carrier. 37. The cosmetic composition according to claim 36, wherein the cosmetically acceptable carrier is a liposome or a mixture thereof. 38. The cosmetic composition according to item 37 of the application, wherein the liposome is nonionic. ~ 39. The cosmetic composition according to item 38 of the scope of the patent application, wherein the lipid system comprises: (a) glyceryl dilaurate or glyceryl distearate; (b) cholesterol, or having as found in cholesterol Steroid Backbone 32-This paper size applies to Chinese National Standard (CNS) A4 (210x297 mm) Printed by the Consumers' Cooperative of the Intellectual Property Bureau of the Ministry of Economic Planning and Economics 1 77 907 A8 B8 C8 D8 Mixtures; and (c) fatty acid ethers or mixtures thereof having from 12 to 18 carbon atoms. 40. A cosmetic product according to the scope of the patent application No. 39, wherein the lipid system comprises: (a) glycerol dilaurate; (b) cholesterol; ⑷polyethylene oxide-10-stearyl ether. 41. According to The beauty composition in the scope of patent application 39, wherein the composition of the liposomes is proportional from 53:10:22 to 63:20:32, respectively. 42. The beauty composition in accordance with the scope of patent application 36, wherein The content of the cosmetically acceptable carrier is from 10 mg / ml to 100 mg / ml according to the total volume of the composition. 43. The beauty composition according to item 31 of the patent application scope, wherein the composition further comprises an increase Moisturizers, cosmetic adjuvants, antioxidants, surfactants, foaming agents, conditioners, humectants, fragrances, thickeners, buffers, sunscreens, colorants, preservatives, etc. 44. A type used in treatment Acne vulgaris and / or a cosmetic composition for producing anti-aging effect on the skin, which contains ~ serine protease and retinoid compounds as active ingredients. 45. Beauty according to item 44 of the scope of patent application Content composition, wherein the first active ingredient is selected from the group consisting of trypsin, carboxyl protease-Y, protease VI, subtilysin, or a mixture thereof. 33 This paper is sized to the Chinese National Standard (CNS) A4 size (210x297 mm) Printed by the Consumers' Cooperative of the Intellectual Property Bureau of the Ministry of Economic Planning and Economics 590771 8 8 Op 8 / ABcfD / VI. Application for patent scope 46 · According to the scope of patent application for 帛 45 beauty composition, the first The active ingredient is trypsin. 47. The cosmetic composition according to the envious item of the patent application scope, wherein the content of the first active ingredient is from 0.01% (w / v) to 5 in the total volume of the composition in winter. % (w / v). ^ v 48. The beauty composition according to item 47 of the scope of patent application, wherein the content of the first active ingredient is from 0.01% (w / v) of the total volume of the composition To 1% (w / v). 49. The cosmetic composition according to item 44 of the patent application scope, wherein the second active ingredient is selected from the group consisting of retinoic acid, vitamin A alcohol, vitamin A aldehyde, and ethyl retinyl acetate. , Retinyl palmitate, or other Biological, analogues or mixtures thereof. 50. The cosmetic composition according to item 49 of the scope of the patent application, wherein the second active ingredient is all-trans retinoic acid. 51. According to the scope of the patent application, 49 beauty composition, Wherein, the content of the first active ingredient is from 0.0001% (w / v) to 0.5% (w / v). 52. The beauty composition according to the scope of application for item 51, wherein the first The content of an active ingredient printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs is from 0.001% (w / force to 0.025% (w / v)). 53. According to the towel, please specialize in the beauty of item 51. A composition, wherein the composition further comprises a cosmetically acceptable carrier. 54. The beauty composition according to item 53 of the patent application, wherein the acceptable carrier on the cereal is a liposome or a mixture thereof. -34-This paper size is in accordance with Chinese National Standard (CNS) A4 (210x297 mm) 7 07 9 L A8 B8 C8 D8 VI. Scope of patent application Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 55. According to the scope of patent application The cosmetic composition according to item 54, wherein the liposome is nonionic. 56. The cosmetic composition according to item 55 of the patent application scope, wherein the lipid system comprises: (a) glyceryl dilaurate or glycerol distearate; (b) having steroids as found in cholesterol Backbone compounds; and (c) fatty acid esters or mixtures thereof having from 12 to 18 carbon atoms. 57. The cosmetic composition according to item 56 of the application, wherein the lipid system comprises: (a) glycerol dilaurate; (b) cholesterol; (c) polyethylene oxide-10-stearyl ether. 58. The cosmetic composition according to item 56 of the scope of the patent application, wherein the composition of the liposomes is respectively from 53:10:22 to 63:20:32 in proportion. 59. The beauty composition according to item 53 of the application, wherein the content of the cosmetically acceptable carrier is from 10 mg / ml to 100 mg / ml based on the total volume of the composition. 60. The beauty composition according to item 44 of the scope of the patent application / wherein the composition further comprises a moisture-increasing agent, a cosmetic adjuvant, an antioxidant, a surfactant, a foaming agent, a conditioner, a wetting agent, a fragrance, and a thickener Agents, buffers, sunscreens, colorants, preservatives, etc. -35-This paper size applies to China National Standard (CNS) A4 (210 X 297 mm)