WO2005084612A1 - Utilisation des inhibiteurs de l'elastase - Google Patents

Utilisation des inhibiteurs de l'elastase Download PDF

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Publication number
WO2005084612A1
WO2005084612A1 PCT/CN2005/000214 CN2005000214W WO2005084612A1 WO 2005084612 A1 WO2005084612 A1 WO 2005084612A1 CN 2005000214 W CN2005000214 W CN 2005000214W WO 2005084612 A1 WO2005084612 A1 WO 2005084612A1
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WO
WIPO (PCT)
Prior art keywords
skin cream
elastase inhibitor
hirudin
cream base
skin
Prior art date
Application number
PCT/CN2005/000214
Other languages
English (en)
Chinese (zh)
Inventor
Fengming Liu
Original Assignee
Fengming Liu
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from CNB2004100047095A external-priority patent/CN100534410C/zh
Application filed by Fengming Liu filed Critical Fengming Liu
Publication of WO2005084612A1 publication Critical patent/WO2005084612A1/fr

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/22—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin
    • A61K31/222—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin with compounds having aromatic groups, e.g. dipivefrine, ibopamine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/55—Protease inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/55—Protease inhibitors
    • A61K38/57—Protease inhibitors from animals; from humans
    • A61K38/58—Protease inhibitors from animals; from humans from leeches, e.g. hirudin, eglin

Definitions

  • 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.
  • 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).
  • 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.
  • 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.
  • 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 ⁇ / L
  • 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:
  • auxiliary skin cream base pours 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 3 ⁇ 4 fatty acid ester 1-3;
  • the aqueous phase contains the following parts by weight: deionized water 40-45, g
  • 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.
  • 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.
  • polydimethylsiloxane 350csks 15.
  • 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.
  • 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.
  • the elastin content of mouse skin tissue is used as a standard for evaluating the cosmetic effect of selelestat cream.
  • mice purchased from the Animal Center of the Academy of Military Medical Sciences, half male and half female, weight
  • 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.
  • 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.
  • hirudin In vitro expression of hirudin
  • 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 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.
  • methanol it is significant to use methanol to induce monoclonal bacteria when culturing monoclonal bacteria.
  • 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.
  • lane 1 is a molecular weight marker
  • 2 It is the supernatant before methanol induction
  • 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.
  • 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 5 Effect of hirudin cream on the elastin content of mouse skin tissue I. Preparation of hirudin cream
  • auxiliary skin cream base 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.
  • 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 50 ⁇ , add component C, and stir to cool to room temperature.
  • 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
  • the Sandberg et al Connective Tissue Research. 25; 139-48, 1990
  • auxiliary skin cream base 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 ⁇ M 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.
  • 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 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.
  • 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.
  • 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.
  • elastase inhibitors such as celeclastam and hirudin
  • the content of protein can have anti-wrinkle and beauty effects, and has good application prospects.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Emergency Medicine (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Zoology (AREA)
  • Cosmetics (AREA)

Abstract

L'invention concerne une utilisation des inhibiteurs de l'élastase, notamment dans le domaine de la fabrication de produits cosmétiques antirides. Les inhibiteurs de l'élastase, appliqués de manière créative à des compositions cosmétiques selon l'invention, comprennent du sivelestat et de l'égline. L'inhibition efficace de l'élastase, le taux hydrolytique réduit et la teneur accrue en élastine dans la couche du derme obtenus grâce aux inhibiteurs de l'élastase créent l'activité antirides.
PCT/CN2005/000214 2004-02-24 2005-02-23 Utilisation des inhibiteurs de l'elastase WO2005084612A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
CNB2004100047095A CN100534410C (zh) 2004-02-24 2004-02-24 水蛭蛋白酶抑素在化妆品中的应用
CN200410004709.5 2004-02-24
CN200410006575.0 2004-03-10
CN200410006575 2004-03-10

Publications (1)

Publication Number Publication Date
WO2005084612A1 true WO2005084612A1 (fr) 2005-09-15

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WO (1) WO2005084612A1 (fr)

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6294181B1 (en) * 1998-11-05 2001-09-25 Protease Sciences, Inc Cosmetic compositions containing serine protease inhibitors
CN1345224A (zh) * 1999-03-31 2002-04-17 彭特法姆股份公司 美容的和皮肤病学的皮肤护理组合物
CN1365293A (zh) * 2000-03-27 2002-08-21 株式会社资生堂 皮肤基底模形成促进剂、人工皮肤形成促进剂和人工皮肤的制造方法
JP2003176221A (ja) * 2001-09-28 2003-06-24 Lion Corp 皮膚外用剤
JP2003252777A (ja) * 2002-02-27 2003-09-10 Showa Sangyo Co Ltd エラスターゼ阻害剤及び該剤を含有する化粧品、飲食品又は医薬品
JP2003300893A (ja) * 2002-04-09 2003-10-21 Maruzen Pharmaceut Co Ltd 皮膚化粧料及び美容用飲食品
JP2003342123A (ja) * 2002-05-31 2003-12-03 Ichimaru Pharcos Co Ltd コラゲナーゼ活性阻害剤、エラスターゼ活性阻害剤及び化粧料組成物

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6294181B1 (en) * 1998-11-05 2001-09-25 Protease Sciences, Inc Cosmetic compositions containing serine protease inhibitors
CN1345224A (zh) * 1999-03-31 2002-04-17 彭特法姆股份公司 美容的和皮肤病学的皮肤护理组合物
CN1365293A (zh) * 2000-03-27 2002-08-21 株式会社资生堂 皮肤基底模形成促进剂、人工皮肤形成促进剂和人工皮肤的制造方法
JP2003176221A (ja) * 2001-09-28 2003-06-24 Lion Corp 皮膚外用剤
JP2003252777A (ja) * 2002-02-27 2003-09-10 Showa Sangyo Co Ltd エラスターゼ阻害剤及び該剤を含有する化粧品、飲食品又は医薬品
JP2003300893A (ja) * 2002-04-09 2003-10-21 Maruzen Pharmaceut Co Ltd 皮膚化粧料及び美容用飲食品
JP2003342123A (ja) * 2002-05-31 2003-12-03 Ichimaru Pharcos Co Ltd コラゲナーゼ活性阻害剤、エラスターゼ活性阻害剤及び化粧料組成物

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
AKINOTO A. ET AL: "The effect of sivelestat sodium hydrate (ONO-5046-Na) on neutrophil phylaxis function in vitro.", JAPANESE PHARMACOLOGY & THERAPEUTICS-, vol. 31, no. 1, 2003, pages 85 - 91 *
LEE J.Y. ET AL: "Isolation and characterization of epidermal mucus from hirudo nipponia.", J.BIOCHEM.BIOL.MOL.BIOL., vol. 29, no. 3, 1996, pages 248 - 252 *
NISHIDA T. ET AL: "Effects of neutrophil elastase inhibitor(ONO-5046) on lung injury after intestinal ischemia-reperfusion.", JOURNAL OF APPLIED PHYSIOLOGY., vol. 91, no. 4, 2001, pages 1800 - 1807 *

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