WO2005084612A1

WO2005084612A1 - The new usage of elastase inhibitors

Info

Publication number: WO2005084612A1
Application number: PCT/CN2005/000214
Authority: WO
Inventors: Fengming Liu
Original assignee: Fengming Liu
Priority date: 2004-02-24
Filing date: 2005-02-23
Publication date: 2005-09-15

Abstract

A new usage of elastase inhibitors, in particular in the field of manufacturing anti-wrinkle cosmetics. The elastase inhibitors which are creatively applied to cosmetic compositions accroding to the present invention include sivelestat and eglin. And the anti-wrinkle activity is believed to be the results of the effective elastase inhibition, reduced hydrolytic rate and the increased content of elastin in derma cortex by the said elastase inhibitors.

Description

New uses of elastase inhibitors TECHNICAL FIELD

The present invention relates to new uses of elastase inhibitors, and particularly to the use of elastase inhibitors in the preparation of anti-wrinkle beauty cosmetics. Background technique

The human skin structure is usually divided into three layers, from deep to shallow, subcutaneous tissue, dermis and epidermis. The epidermis is the most active layer in the skin. It consists of the basal layer, the granular layer, and the stratum corneum. It continuously protects the skin through keratinization. Subcutaneous tissue is composed of connective tissue, nerve fibers, lymphatic vessels, and small blood vessels. Its main function is to reduce impact, heat insulation, and store heat. There are few cells inside the dermis, mainly composed of fibrous connective tissue such as collagen fibers, elastic fibers, and reticular fibers. They have a very important relationship with the skin's elasticity, gloss, and tension. Happened in the dermis.

Sivelestat (sivelestat sodium hydrate, ONO-5046, Elaspol), is a highly specific elastase inhibitor.

Pharmaceutical) for the treatment of acute lung injury with systemic inflammatory response syndrome. Civilastadol has a molecular formula of i ^ ie ^ S. 4¾0 and a molecular weight of 528.51, and its structural formula is like formula I. It was licensed for production in Japan on April 11, 2002, and clinically conducted in the United States, Europe, and China. test. The mechanism of anti-lung injury induced by seleviride is to improve the respiratory function of patients with acute lung injury with systemic inflammatory response syndrome by inhibiting elastase released by neutrophils, and shorten the time for patients to use respirators Reduce the incidence of pressure injury and respiratory tract infection caused by respirator installation, improve systemic inflammatory response syndrome and idiopathic pulmonary fibrosis

(Formula I)

i Hirudin is an elastase inhibitor isolated by Jung et al. In Leech (The Journal of Biological Chemistry, 1995, 270: 13879-13884), which can efficiently and specifically inhibit elastase activity in the acid-base state. Both are very stable. Hirudin is a protein having the amino acid residue sequence of SEQ N24 in the Sequence Listing, and its coding gene is the nucleotide sequence of SEQ N21 in the Sequence Listing. At present, genetic engineering has been used to achieve high-efficiency expression of hirudin in vitro (Chinese invention patent application 02146788. 9).

Invention Disclosure

An object of the present invention is to provide a new use of an elastase inhibitor.

The inventors of the present invention have confirmed through experiments that the application of an elastase inhibitor to the skin surface of an animal can increase the elastin content in the skin tissue without significant adverse reactions, indicating that the elastase inhibitor can be used as an effective ingredient in anti-wrinkle cosmetic products. Can be used in cosmetics.

Among them, commonly used elastase inhibitors include selelestat and I or hirudin. The cosmetics prepared by using the elastase inhibitor as a raw material may be various dosage forms such as creams, ointments, liquids, powders, oils, and the like. The cosmetic preparations of the above various dosage forms may be prepared according to conventional methods in the field of cosmetic preparations.

Another object of the present invention is to provide an elastase inhibitor skin cream and a method for preparing the same.

The elastase inhibitor skin cream provided by the present invention comprises an elastase inhibitor and an auxiliary skin cream base, the auxiliary skin cream base contains the following parts by weight: cocoate 9-11, sixteen, stearyl alcohol and Hexadecyl octadecyl polyglucose 7-8, Azone 0.5-5-6, Isohexadecanol 4-6, Polydimethylsiloxane 14-16, 2-Isooctyl-2 Cyano-3,3-diphenylacrylate 4-6, Vitamin E 2-4, glyceryl stearate 1-3, deionized water 40-45, glycerol 4-6, hydrolyzed almond protein 2-4 5, sunscreen 0.5-1.5.

Among them, the elastase inhibitor may be cevilastel and / or hirudin, the concentration of velevilastin in the skin cream base of the adjuvant is 0.05 to 10 μπιοΙ / L, and hirudin in the adjuvant The skin cream base has a concentration of 10 ng / ml-10 mg / ml.

The preparation method of the elastase inhibitor skin cream includes the following steps:

1) Preparation of auxiliary skin cream base: Pour the oil phase into a container, stir and heat to 75-80 ° C, mix the water phase in another container, stir and heat to 75-80 ° C, add the aqueous phase with stirring The oil phase is mixed with heat preservation, cooled to 40-50 ° C, and then additives are added, and the mixture is stirred and cooled to room temperature to obtain the auxiliary skin cream base; the oil phase contains the following parts by weight: coconut oil ester 9-11 , Sixteen, Octadecyl alcohol and hexadecyl polyglucose 7-8, Azone 0.5-6.0, isohexadecanol 4-6, polydimethylsiloxane 14-16, 2-isooctyl ~ 2 cyanide -3,3-diphenylacrylate 4-6, vitamin E2-4, glyceryl ¾ fatty acid ester 1-3; the aqueous phase contains the following parts by weight: deionized water 40-45, glycerol 4-6; the additive contains the following parts by weight of the material: hydrolyzed almond protein 2-4, sunscreen 0.5-1.5, the pH was adjusted to 5.5 with citric acid;

2) Preparation of skin cream: Mix the elastase inhibitor with the auxiliary skin cream base and stir well to obtain the elastase inhibitor skin cream. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the inhibition rate of selelestat enzyme activity;

Figure 2 shows the effects of selelestat cream on skin elastin content in mice;

FIG. 3 is an electrophoresis map of hirudin by PCR;

Figure 4 is a map of the PPIC9K-GUM expression plasmid;

FIG. 5 is an electrophoresis diagram of a culture fluid before and after the induction of hirudin;

FIG. 6 is an enzyme activity inhibition rate of hirudin;

Figure 7 shows the effect of hirudin cream on mouse skin elastin content.

The best way to implement the invention

Example 1.Effect of selelestat on elastase activity

Take 4 test tubes, and add 40 μ1 0.2M Tris-HCl (pH 8.0), 20 1 0.37mg / ml porcine pancreatic elastase (SIGMA, E-1250), and then add 40 μΐ normal saline, 0.375 μM sive Lelistat (Shanghai Julong Drug Research and Development Co., Ltd.), 1.25 μM civillestat, 3.75 μM civillestat, mix well, and then add 400 μΐ 0.1 mM reaction substrate SucA3PNA (SIGMA), then the experimental group The final concentrations of selevirastam were 0.03 μM, 0.1 μM, and 0.3 μM, respectively. After reacting at room temperature for 10 minutes, the color was measured at 410 nm, and the percentage of enzyme activity inhibition was calculated using physiological saline as a comparison, and the calculation formula was 0D ₄₁ . Assay / 0D ₄₁ . Saline. The results are shown in Figure 1. The results show that selelestat has a good elastase inhibitory effect, which is a dose-dependent enzyme activity inhibitory effect. The selelestat 0.3 μM enzyme inhibition percentage is 72%.

Example 2. Effect of selelestat cream on elastin content in mouse skin tissue

1. Preparation of selelestat cream

Take 99 g of excipient skin cream base, add 1.0 μl of 30 μM selelestat, mix and stir Spread evenly, dispense, and set aside, that is, selelestat skin cream. Then take 99 g of supplementary skin cream base, add 1.0 ml of normal saline, mix and stir well, and dispense, which is a normal saline skin cream.

Skin cream base formula is (W / W,%): A, oil phase: coconut oil (caprylic / decanoate) ester 10. 00; cetyl, stearyl alcohol and cetyl, octadecyl polyglucose 7.50 Azone 3.5, isohexadecanol 5. 00; polydimethylsiloxane 350csks 15. 00; 2-isooctyl-2 cyano-3, 3-diphenylacrylate 5. 00; Vitamin E 3. 00; glyceryl stearate 2. 00. B water phase: deionized water 40.00; glycerin 5. 00.

C additive: hydrolyzed almond protein 3. 00; sunscreen 1. 00, adjust pH to 5.5 with 12.5% citric acid.

Skin cream base preparation method: Pour oil phase A into a container, stir to 75-80 ° C, mix water phase B in another container, and stir to 75-80 ° C. Add component B to component A with stirring, and keep mixing for 15 minutes. Cool to 50 ° C and add Additive C. Cool to room temperature with stirring.

2.Effect of selelestat cream on elastin content in mouse skin tissue

The content of elastin in the dermis is an important parameter to characterize the quality of the skin. Increasing the content of elastin in the dermis can play an anti-wrinkle and cosmetic effect. In the present invention, the elastin content of mouse skin tissue is used as a standard for evaluating the cosmetic effect of selelestat cream.

Twenty Kunming mice purchased from the Animal Center of the Academy of Military Medical Sciences, half male and half female, weight

18-22 grams, randomly divided into two groups, namely the civilistel group and the normal saline group, hair removal on the back, respectively, sievelestine skin cream and normal saline control skin cream, three times a day, 30 consecutive applications day. Mice were sacrificed the next day after deactivation, skin peeled, fat removed, weighed, and the elastin content in skin tissues was determined by Sandberg et al (Connective Tissue Research. 25; 139-48, 1990). The results are shown in Figure 2. As can be seen from the figure, selelestat skin cream can significantly increase the elastin content in mouse skin tissues, and has good anti-wrinkle and cosmetic effects.

Example 3. Preparation of hirudin

I. Synthesis of hirudin expression gene

1. Small fragment artificial synthesis: The sequence 1 hirudin gene is divided into two segments, the first segment is the positive cDNA strand, and the second segment is the cDNA complementary strand. At the 3 ′ end of the first paragraph, add 8 bases complementary to the second paragraph 3, with a total length of 105 bases, which is sequence 2 in the sequence listing. At the 3, end of the second paragraph, add 8 and the first The 3 'end of the segment is complementary bases, with a total length of 111 bases, and is Sequence 3 in the sequence listing. Ndel and Notl restriction sites were added to the ends of the two fragments 5, respectively, and synthesized with a DNA synthesizer.

The fragment synthesized by the DNA synthesizer was connected to the full-sequence hirudin gene of synthetic sequence 1 by a single-cycle PCR reaction, as shown in FIG. 3, which proves that the obtained sequence is correct. In vitro expression of hirudin

1. Cut the PCR fragment and load it into the pGEM-T-EASY plasmid to construct the pGEM-GUM clone vector.

2.Construction of PPIC9K- GUM expression plasmid

The high-efficiency expression plasmid pPIC9K purchased from Invitrogen, USA was used to digest the pGEM-GUM plasmid and pPIC9K plasmid with Ndel and Notl, respectively. The target fragments were collected by gel electrophoresis and ligated with T 4 ligase at 16 ° C overnight. Transformation, picking bacteria, and amplification to obtain expression plasmids by conventional methods

The structure of PPIC9-GUM is shown in Figure 4. The enzyme digestion and identification proved that the structure of the expression plasmid was correct.

3. The plasmid PPIC9K- GUM was linearized with Sacl. Using the Zhejiang Xinzhi Electric Gene Introduction Instrument, the linearized pPIC9K- GUM was introduced into Pichia pastoris GS115 (purchased from Invitrogen) and cultured, selected, and screened. In the process, a monoclonal bacterium resistant to G418 mg / ml was obtained.

4. Select the monoclonal bacteria, inoculate them in 5ml medium, incubate at 30 ° C overnight, then transfer to 250ml medium, and continue to culture to 0D ₆ . . = 10-20, collect bacterial cells, and dilute to 0D ₆ with carbon-free medium. . = 50, continue to culture for 24 hours, and add methanol to a final concentration of 2% to induce expression, and add methanol once every 24 hours to 96 hours. The supernatant was centrifuged to perform gel electrophoresis analysis and functional determination. 10 ml of the supernatant was taken, electrophoresed by 12% SDS-PAGE, and stained by Coomassie brilliant blue. The results showed that there was induced protein expression at the 6KD position, and the expression was estimated to be 103 mg / L based on the BSA control.

In this example, it is significant to use methanol to induce monoclonal bacteria when culturing monoclonal bacteria. Before the induction, the content of the target protein in the culture solution is very low, as shown in FIG. 5, where lane 1 is a molecular weight marker, 2 It is the supernatant before methanol induction, and 3 is the supernatant after methanol induction. There was a significant target protein band in the culture medium after induction, the same size as the protein of sequence 4, but there was no significant target protein band in the culture medium before induction.

5. Protein purification: An phenyl sepharose chromatography column containing 50 mM Tris-HCl (pH 8. 0) and 1M (N) ₂ S0 _{4 was} used for the experiment. The culture supernatant was applied at 1 ml / min. After washing, it was reused. 50mM Tris-HCl (pH 8. 0) was used for elution. The eluate was collected to obtain hirudin with a purity of about 95%. The protein content was determined by the Lowry method.

Example 4 Functional determination of hirudin

Take 3 test tubes and add 40 μ 1 0.2M Tris-HCl (pH 8. 0), 20 1 0.37 mg / ml porcine pancreatic elastase (SIGMA, E-1250), and then add 40 μ 1 saline (Supernatant before induction, purified solution of the supernatant after induction (α μπιοΙ / L)), mix well, and then add 400 1 0.1 μm reaction substrate SucA3PNA (SIGMA), after 10 minutes reaction at room temperature, colorimetrically at 410nm, Using physiological saline as a comparison, the percentage of inhibition of enzyme activity was calculated, and the calculation formula was 0D ₄₁ . Assay / 0D ₄₁ . Physiological salt Water. The results are shown in Figure 6. There was no significant difference between the supernatant before induction and the normal saline group, and the percentage inhibition of the enzyme activity of the supernatant after induction was 98%, which had a good elastase inhibitory effect.

Example 5. Effect of hirudin cream on the elastin content of mouse skin tissue I. Preparation of hirudin cream

Take 99 g of the auxiliary skin cream base, add 1.0 ml of the hirudin purified solution (3 mg / ml) obtained in Example 3, mix and stir well, dispense, and set aside, which is the hirudin skin cream. Then take 99 g of supplementary skin cream base, add 1.0 ml of normal saline, mix and stir evenly, and divide into packages, which is a normal saline skin cream.

Skin cream base formula is (W /,%): A, oil phase: coconut oil (caprylic / capric) ester 9. 00; cetyl, stearyl alcohol and cetyl, octadecyl polyglucose 7.50; Azone 3.5; Isohexadecyl alcohol 6. 00; Polydimethylsiloxane 350csks 14. 00; 2-Isooctyl-2 cyano-3, 3-diphenylacrylate 6. 00; Vitamins E 3. 00; glyceryl stearate 2. 00. B water phase: deionized water 40. 00; glycerol 5. 00.

C additive: hydrolyzed almond protein 3. 00; sunscreen 1. 00, with 12.5% citric acid to adjust _PH to 5.5.

Skin cream base preparation method: Pour the oil phase A component into a container, heat it to 75-8 ° C, mix the water phase B component in another container, and heat it to 75-80 ° C with stirring. Add group B while stirring. Divide into component A, mix and hold for 15 minutes, cool to 50Ό, add component C, and stir to cool to room temperature.

Effect of Hirudin Cream on Elastin Content of Mouse Skin Tissue

Twenty Kunming mice purchased from the Animal Center of the Academy of Military Medical Sciences, male and female, weighing 18-22 grams, were randomly divided into two groups, namely the hirudin group and the normal saline group, and their hairs were removed on the back and coated. The hirudin skin cream and saline control skin cream were applied three times a day for 30 consecutive days. Mice were sacrificed the next day after disabling, the skin was stripped, fat removed, and weighed. The Sandberg et al (Connective Tissue Research. 25; 139-48, 1990) method was used to determine the elastin content in skin tissues. The results are shown in Figure 7, It can be seen from the figure that the hirudin skin cream can obviously increase the elastin content in the skin tissue of mice, and has good anti-wrinkle and cosmetic effects.

Example 6.Sivelestat, Hirudin

I. Preparation of celeclastast and hirudin prostatin mixed cream

Take 98 g of the auxiliary skin cream base, and add the hirudin purified solution (3 mg / ml) obtained in Example 3, 1.0 ml, 30 μM cevillesstat 1.0 ml, mix and stir well, dispense For spare, it is sevirestat, hirudin mixed skin cream. Then take 99g of auxiliary skin cream base, add 2.0 ml of normal saline, mix and stir, and then distribute, which is the normal saline control skin cream. Skin cream base formula is (W / W,%): A, oil phase: coconut oil (caprylic / capric) ester 9. 00; cetyl, stearyl alcohol and cetyl, octadecyl polyglucose 7.50 ; Azone 3. 5; Isohexadecanol 6. 00; Polydimethylsiloxane 350csks l 4. 00; 2-Isooctyl-2 cyano-3, 3-diphenyl acrylate 6. 00; Vitamin E 3. 00; glyceryl stearate 2. 00. B water phase: deionized water 40.00; glycerin 5. 00.

Skin cream base preparation method: Pour oil phase A component into a container, stir to 75-80 ° C, mix water phase B component in another container, and stir to 75-80 ° C. Add component B to component A with stirring, and keep mixing for 15 minutes. Cool to 50 ° F, add Additive C, and cool to room temperature with stirring.

2. The effect of selelestat and hirudin mixed cream on the elastin content of mouse skin tissue

Twenty Kunming mice, male and female, weighing 18-22 grams, were purchased from the Animal Center of the Academy of Military Medical Sciences, randomly divided into two groups, namely celeclastine, hirudin mixed cream group, and normal saline. In the group, the back was depilated, and sevelestat, a hirudin mixed cream skin cream and a saline control skin cream were applied three times a day for 30 consecutive days. Mice were sacrificed the next day after disabling, the skin was stripped, fat removed, weighed, and the elastin content in the skin tissue was determined by Sandberg et aKConnective Tissue Research. 25; 139-48, 1990) method. Results The mixed skin cream of cevilastel and hirudin also significantly increased the elastin content in the skin tissue of mice. Compared with the saline control group, the mixed skin of velvestat and hirudin made the skin Tissue elastin content increased by 76%. Industrial applications

The invention creatively uses elastase inhibitors such as celeclastam and hirudin in cosmetic preparations, and through its effective inhibition of elastase, it reduces the rate of hydrolysis of elastin and improves the elasticity in the dermis layer. The content of protein can have anti-wrinkle and beauty effects, and has good application prospects.

Claims

Rights request

1. Application of elastase inhibitor in cosmetics.

2. The use according to claim 1, characterized in that: the elastase inhibitor is cevirestat and I or hirudin.

3. The application according to claim 1 or 2, characterized in that the cosmetic preparation is a cream, a paste, a water, a powder or an oil.

4. An elastase inhibitor skin cream comprising an elastase inhibitor and an auxiliary skin cream base, said auxiliary skin cream base contains the following parts by weight: cocoate 9-11, sixteen, stearyl alcohol and ten Hexadecyl polyglucose 7-8, Azone 0.5-6.0, Isohexadecanol 4-6, Polydimethylsiloxane 14-16, 2-Isooctyl-2 cyanide -3,3-diphenylacrylate 4-6, vitamin E 2-4, glyceryl stearate 1-3, deionized water 40-45, glycerol 4-6, 7 4. Sunscreen agent 0.5-1.5.

5. The elastase inhibitor skin cream according to claim 4, characterized in that the elastase inhibitor is selelestat and / or hirudin.

6. The elastase inhibitor skin cream according to claim 5, characterized in that: the concentration of the sevelestat in the excipient skin cream base is 0.05 to 10 μmol / L, and I or The concentration of the hirudin in the excipient skin cream base is 10 ng / ml-10 mg / ml ₀

7. The method for preparing an elastase inhibitor skin cream according to claim 4, comprising the following steps: 1) preparing an auxiliary skin cream base: pouring the oil phase into a container, stirring and heating to 75-80 ° C, and mixing the water in another container Phase, stir and heat to 75-8 ° C, add the water phase to the oil phase with stirring, keep it mixed, cool to 40-50 ° C, then add additives, stir and cool to room temperature to obtain the auxiliary skin cream base; The oil phase contains the following parts by weight: Cocoate 9-11, Cetyl, Octadecyl Alcohol and Cetyl Octadecyl Polyglucose 7-8, Azone 0.5-5. 0, Isodecyl Hexanol 4-6, polydimethylsiloxane 14-16, 2-isooctyl-2 cyano-3, 3-diphenylacrylate 4-6, vitamin E 2-4, glyceryl stearin Ester 1 to 3; the water phase contains the following parts by weight: deionized water 40-45, gan • oil 4-6; the additive contains the following parts by weight: hydrolyzed almond protein 2-4, 5-1. 5 sunscreen, adjust the pH to 5.5 with citric acid;

2) Preparation of skin cream: The elastase inhibitor skin cream is mixed with an auxiliary skin cream base, and the mixture is stirred to form the skin cream.

8. The method according to claim 7, wherein: the elastase inhibitor The agent is selelestat and / or hirudin.

9. The preparation method according to claim 7, characterized in that: the concentration of the sevelestat in the auxiliary skin cream base is 0.05-10 μmol / L, and I or the hirudin The concentration of the excipient skin cream base is 10 ng / ml-10 mg / mlo