EP1996606A1 - Oligopeptide substitue par de la tropolone et de l'hydroxybenzol pour depigmentation - Google Patents

Oligopeptide substitue par de la tropolone et de l'hydroxybenzol pour depigmentation

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Publication number
EP1996606A1
EP1996606A1 EP07726729A EP07726729A EP1996606A1 EP 1996606 A1 EP1996606 A1 EP 1996606A1 EP 07726729 A EP07726729 A EP 07726729A EP 07726729 A EP07726729 A EP 07726729A EP 1996606 A1 EP1996606 A1 EP 1996606A1
Authority
EP
European Patent Office
Prior art keywords
peptide
acid
skin
oxo
compounds according
Prior art date
Legal status (The legal status 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 status listed.)
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Application number
EP07726729A
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German (de)
English (en)
Inventor
Klaus-Heinrich RÖHM
Catrin Scherner
Bernd Schwarz
Michael Wendt
Rainer Wolber
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Beiersdorf AG
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Beiersdorf AG
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Application filed by Beiersdorf AG filed Critical Beiersdorf AG
Publication of EP1996606A1 publication Critical patent/EP1996606A1/fr
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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/05—Dipeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/04—Antipruritics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/14—Drugs for dermatological disorders for baldness or alopecia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the pigmentation of the skin is essentially determined by the skin's own pigment melanin. This is formed by specialized cells of the epidermis, the melanocytes. In melanin synthesis, tyrosinase plays a crucial role as a pacemaker enzyme.
  • Lactic acid, glycolic acid, but also vitamin A acid used Lactic acid, glycolic acid, but also vitamin A acid used.
  • Skin-lightening treatments are also known in which antioxidants such as vitamin C and its derivatives are used, or those in which the intracellular stability of the pacemaker enzyme tyrosinase is reduced (for example by linolenic acid).
  • tyrosinase inhibitors are phenolic or catecholic structures.
  • Commonly known tyrosinase inhibitors such as koji acid, arbutin and hydroquinone are used.
  • Hydroquinone is often described in the literature as the "gold standard.” However, effective concentrations in vivo also often have significant side effects.
  • Tropolone and its derivatives are already described in the patent literature as tyrosinase inhibitors and skin lightening agents.
  • JP 63033313 describes the use of tropolone for treating skin spots and freckles.
  • JP 1979-82213 and US 4361581 describe the skin lightening effect of isopropyltropolone, a naturally occurring tropolone derivative also called thujaplicin.
  • EP 308919 describes the use of tropolone in combination with other whitening agents.
  • n can assume the values 1, 2, 3,
  • R 2 is a group chosen from p-hydroxyphenyl, 4-hydroxyhepta-1, 3,6-trien-5-one-1-yl or 2,4-dihydroxyphenyl, 4 [(hydroxyamino) carbonyl] phenyl, ( 5-hydroxy-4-oxo-4H-pyran-2-yl) -, (3 Hydroxy-4-oxopyridin-1 - (4H) -yl) -, (2-thioxo-1,3-oxazolidin-5-yl) -, 4-
  • R 3 is a group chosen from -OH, NH 2 , remedy the disadvantages of the prior art.
  • this relates to the reduction of unwanted pigmentation, which may naturally occur quite naturally in the respective skin type (whitening) as well as in the various forms of local hyperpigmentation (pigmentation disorders such as age spots, melasma, chlosma, freckles, liver spots, various forms of postinflammatory hyperpigmentation, such as after sunburn, ingrown (beard) hair, after removal of body hair, shaving and inflammatory lesions of the skin (for example, acne) etc).
  • n 1 or 2 in the compounds according to the invention.
  • R 1 is H, tert-butyloxycarbonyl or benzyloxycarbonyl.
  • R 1 H or benzyloxycarbonyl.
  • the compounds according to the invention are oligopeptides which are C-terminally terminal of tyrosine, its tropolone analog or 2,4-dihydroxytyrosine, which is peptidically linked to 2,4-diaminobutyric acid or orinithin.
  • the compounds according to the invention are tri-, preferably dipeptides.
  • the invention also encompasses the use of compounds according to the invention for inhibiting human tyrosinase and for reducing skin pigmentation at a use concentration of 0.0001 to 15% by weight, preferably 0.01 to 1% by weight.
  • the invention also encompasses cosmetic preparations containing a compound according to the invention.
  • the invention also encompasses the use of compounds according to the invention in preparations or the non-therapeutic use of preparations according to the invention to lighten the natural hair color, to reduce the post-reactions of the skin to UV light (sunburn), the after-reaction of the skin to the shave (razor burn). to reduce the skin's own protective and repair mechanisms (for example, for dysfunctional enzymes, DNA, lipids, proteins), as well as for the pre- and post-treatment in topical application of laser and Ableifnessen, z. B. the reduction of skin wrinkles and Scars are used to counteract the resulting skin irritation and to promote the regeneration processes in the injured skin.
  • the compounds according to the invention may be oligopeptides, in particular dipeptides in which a C-terminal tyrosine residue or its analogue, which is derived from the various residues R 2 according to FIG. 2, are orinithin or preferably 2,4-diaminobutyric acid or especially preferably linked to 2,3-diaminopropanoic peptidic act.
  • Figure 1 shows the general structure of these compounds.
  • the particular effectiveness consists in the combination of the peptide structure with a radical R 2 which acts in complex with tyrosinase as a ligand of the essential copper ions of the enzyme.
  • a sterically large, hydrophobic substituent R 1 can improve the inhibitory effect. • In addition to an unprotected N-terminal amino function, this can also be protected by various protective groups.
  • the N-terminal protecting group can be selected from hydrophobic aliphatic linear, aliphatic branched or cyclic and particularly preferably aromatic radicals.
  • C O-cyclohexyl, Coc, N- (4,4-dimethyl-1-oxopentyl), N- (1-oxo-4-pentenyl), N- (4,6-dimethyl-1-oxo-2, 4-dodecadienyl), N- (1-oxo-9, 12,15-octadecatrienyl), N- (4-methyl-1-oxopentyl).
  • N- (1-oxo-10-undecenyl N- (2-methyl-1-oxopropyl) and Fmoc, homo-Z, Moz,
  • the stereochemistry (L- or D-configuration of the two amino acids) has to be taken into account, which has a decisive influence on the effectiveness of the structures. In most cases, L configuration is preferred, but in case of ornithine it is D configuration.
  • preparations according to the present invention can advantageously also contain more than one peptide according to the invention.
  • the preparations according to the present invention may also advantageously contain self-tanning substances such as, for example, dihydroxyactone, melanin derivatives and erythrulose, these substances being used in concentrations of 0.1% by weight up to 8% by weight, preferably 0.4-2% by weight. % based on the total weight of the preparation.
  • the formulations according to the invention can also advantageously contain, in addition to dihydroxyacetone, also nu- tus extracts, as well as other substances which are intended to obtain or produce or additionally strengthen the tan.
  • Moisturizers are substances or mixtures of substances which give cosmetic or dermatological preparations the property of reducing the moisture release of the horny layer (also called transepidermal water loss (TEWL) after application or spreading on the skin surface) and / or hydrating the skin Horny layer to influence positively.
  • TEWL transepidermal water loss
  • the moisturizer (s) are selected from the group: glycerol, lactic acid and / or lactates, in particular sodium lactate, butylene glycol, propylene glycol, biosaccharide Gu ⁇ m-1, glycine soy, ethylhexyloxyglycerol, pyrrolidone carboxylic acid and urea.
  • polymeric moisturizers from the group of water-soluble, water-swellable or water-gellable polysaccharides.
  • glycerol as a moisturizer, preferably in a concentration of 0.05 to 30 wt .-%, particularly preferably 1 to 10 wt .-%, each based on the total weight of the preparation.
  • active ingredients to be used in the context of the present invention are those which have a positive effect on the condition of the skin, in particular active ingredients for positively influencing the aging skin, which reduce the formation of wrinkles or even existing wrinkles.
  • biochinones in particular ubiquinone (Q10), ubiquinol, folic acid and its derivatives (in particular tetrahydrofolic acid and dihydrofolic acid), niacin and its derivatives (especially niacinamide), creatine and creatinine, carnitine, biotin, isoflavone, cardiolipin, lipoic acid, anti-freezing proteins , Hops and hops malt extracts.
  • agents that promote the restructuring of the connective tissue such as natural and synthetic isoflavo noids and isoflavonoid-containing plant extracts - such.
  • soy and clover extracts - can be used very well in the formulations of the invention.
  • the formulations are particularly suitable as active ingredients for supporting the skin functions in dry skin (such as, for example, vitamin C, biotin, carnitine, propionic acid, green tea extracts, eucalyptus oil, urea and mineral salts (such as, for example, NaCl, Marine minerals) as well as osmolytes (such as inositol, betaine, quaternary ammonium compounds)).
  • agents such as sericosides, various extracts of licorice, licochalcone, especially licochalcone A, silymarin, silyphos, dexpanthenol, inhibitors of prostaglandin metabolism, in particular cyclooxygenase and leukotriene metabolism, in particular the 5-lipoxygenase, but also the 5-lipoxvgenase inhibitor protein , FLAP.
  • active compounds are furthermore particularly suitable for applying or renewing an (energy) depot and for activating the repair of various cellular structures, in particular of the DNA.
  • rosin sulphate dioic acid (8-hexadecene-1, 16-dicarboxylic acid) as well as lipoic acid and liponamide, glabridin, various extracts of licorice, mulberry, beech, horse chestnut, witch hazel, as well as baikal helmet, pantethein sulphonates , Kojic acid, Hydroquinone, arbutin, alpha-arbutin, deoxyarbutin, fruit acids, especially alpha-hydroxy acids (AHAs), bearberry extracts (Bearberry or Uvae ursi), ursolic acid, rucinol, deoxyarbutin, Bio377, H-Tyr-Gly-Trp- OH, tropolone, thujaplicin ascorbic acid, green tea extracts, aminoguanidine, pyridoxamine, niacinamide and its derivatives, inhibitors of proteinase activated receptor 2 (PAR-2).
  • PAR-2 proteinase
  • formulations according to the invention which comprise further active ingredients which bring about increased or faster tanning of the skin, whether with or without the influence of UV light. This is extremely true for such formulations of the invention, which serve to reduce local hyperpigmentation (including age spots, freckles, melasma).
  • active ingredients for example, advanced glycation end products (AGE), lipofuscins, glycerrhetin and glycyrrhetinic acid, nucleic acid oligonucleotides, purines and pyrimidines, NO-releasing substances, tyrosine and its derivatives (especially N-acetyl-tyrosine), phenylalanine and its derivatives in particular N-acetyl-phenylalanine and activators of Proteinase Activated Receptor 2 (PAR-2) are used.
  • AGE advanced glycation end products
  • lipofuscins for example, lipofuscins, glycerrhetin and glycyrrhetinic acid, nucleic acid oligonucleotides, purines and pyrimidines, NO-releasing substances, tyrosine and its derivatives (especially N-acetyl-tyrosine), phenylalanine and its derivatives
  • compositions are also obtained when antioxidants are used as additives or active ingredients.
  • the preparations advantageously contain one or more antioxidants.
  • antioxidants As favorable, but nevertheless optional to be used antioxidants all suitable or common for cosmetic and dermatological applications or conventional antioxidants can be used.
  • water-soluble antioxidants can be used, such as vitamins, eg. As ascorbic acid and its derivatives - in particular ascorbyl palmitate, Na and Mg ascorbyl phosphate and ascorbyl acetate - and rutinic acid and derivatives thereof in particular alpha glucosylrutin, quercetin and isoquercetin.
  • vitamins eg. As ascorbic acid and its derivatives - in particular ascorbyl palmitate, Na and Mg ascorbyl phosphate and ascorbyl acetate - and rutinic acid and derivatives thereof in particular alpha glucosylrutin, quercetin and isoquercetin.
  • antioxidants are also vitamin E and its derivatives (in particular vitamin E acetate), vitamin A and its derivatives (in particular vitamin A palmitate) and carnosine, lipoic acid and its derivatives (in particular dihydrolipoic acid and liponamide), butylhydroxytoluene, butylated hydroxyanisole , beta-alanine and carotenoids (especially beta-carotene) and phytoene.
  • the amount of the antioxidants (one or more compounds) in the preparations is preferably 0.001 to 30 wt .-%, particularly preferably 0.05 to 20 wt .-%, in particular 0.1 to 10 wt .-%, based on the total weight the preparation. If vitamin E and / or its derivatives represent the antioxidant (s), it is advantageous to choose their respective concentrations from the range from 0.001 to 10% by weight, based on the total weight of the formulation.
  • vitamin A or vitamin A derivatives, or carotenes or derivatives thereof are the antioxidant (s), it is advantageous if their respective concentrations are in the range from 0.001 to 10% by weight, based on the total weight of the formulation choose.
  • the cosmetic preparations according to the present invention contain cosmetic or dermatological active ingredients, where preferred active ingredients are antioxidants which can protect the skin against oxidative stress.
  • the preparations according to the invention preferably contain at least one further UV-A, UV-B and / or broadband filter substance.
  • the formulations may, although not necessary, optionally also contain one or more organic and / or inorganic pigments as UV filter substances, which may be present in the water and / or the oil phase.
  • Such UV filter substance may be: phenylene-1,4-bis (2-benzimidazyl) -3,3'-5,5'-tetrasulfonic acid salts; 2-phenylbenzimidazole-5-sulfonic acid salts; 1,4-di (2-oxo-10-sulfo-3-bomylidenemethyl) benzene and its salts; 4- (2-oxo-3-bomylidenemethyl) benzenesulfonic acid salts; 2-methyl-5- (2-oxo-3-bomylidenemethyl) sulfonic acid salts; 2,2'-methylene-bis (6- (2H-benzotriazol-2-yl) -4- (1,1,3,3-tetramethylbutyl) -phenol); 2- (2H-Benzotriazol-2-yl) -4-methyl-6- [2-methyl-3- [1,3,3,3-tetramethyl-1 - [(trimethylsilyl) oxy] disiloxanyl
  • Preferred inorganic pigments are metal oxides and / or other sparingly water-soluble or insoluble metal compounds, in particular oxides of titanium (TiO 2 ), zinc (ZnO), iron (eg Fe 2 O), zirconium (ZrO 2 ), silicon (SiO 2 ) 2 ), manganese (eg MnO), aluminum (Al 2 Os), cerium (eg Ce 2 O 3 ), mixed oxides of the corresponding metals as well as mixtures of such oxides and the barium sulfate (BaSO 4 ).
  • the pigments can also be used advantageously in the form of commercially available oily or aqueous predispersions.
  • dispersants and / or solubilizers can be added to these predispersions.
  • the pigments can advantageously be treated superficially, in which case, for example, a hydrophilic, amphiphilic or hydrophobic character is to be formed or retained.
  • This surface treatment may consist in providing the pigments with a thin hydrophilic and / or hydrophobic inorganic and / or organic layer by processes known per se.
  • the various surface coatings may also contain water for the purposes of the present invention.
  • Inorganic surface coatings in the context of the present invention may consist of aluminum oxide (Al 2 O 3 ), aluminum hydroxide Al (OH) 3 or aluminum oxide hydrate (also: alumina, CAS No .: 1333-84-2), sodium hexametaphosphate (NaPO 3 ) 6 , sodium metaphosphate (NaPOs) n , silica (SiO 2 ) (also: silica, CAS No .: 7631-86-9), or iron oxide (Fe 2 O 3 ).
  • Al 2 O 3 aluminum oxide
  • Al aluminum hydroxide Al
  • Al oxide hydrate also: alumina, CAS No .: 1333-84-2
  • sodium hexametaphosphate (NaPO 3 ) 6 sodium metaphosphate (NaPOs) n
  • silica (SiO 2 ) also: silica, CAS No .: 7631-86-9
  • iron oxide Fe 2 O 3
  • Organic surface coatings for the purposes of the present invention may consist of vegetable or animal aluminum stearate, vegetable or animal stearic acid, lauric acid, dimethylpolysiloxane (also: dimethicone), methylpolysiloxane (methicone), simethicone (a mixture of dimethylpolysiloxane with an average chain length of 200 to 350 Dimethylsiloxane units and silica gel) or alginic acid.
  • These organic surface coatings may be present alone, in combination and / or in combination with inorganic coating materials.
  • Zinc oxide particles which are suitable according to the invention and predispersions of zinc oxide particles are obtainable from the following companies under the following commercial names:
  • Suitable titanium dioxide particles and predispersions of titanium dioxide particles are available from the following companies under the following trade names:
  • Latex particles which are advantageous according to the invention are those described in the following documents: US Pat. No. 5,663,213 or EP 0 761 201. Particularly advantageous latex particles are those which are formed from water and styrene / acrylate copolymers and, for. B. under the trade name "AHiance SunSphere" at the company. Rohm & Haas are available.
  • the total amount of inorganic pigments, in particular hydrophobic inorganic micropigments, in the finished cosmetic or dermatological preparations is advantageously from the range from 0.1 to 30% by weight, preferably from 0.1 to 10.0, in particular from 0.5 to 6.0 % By weight, based on the total weight of the preparations.
  • the cosmetic and / or dermatological sunscreen formulations may be composed as usual and serve for cosmetic and / or dermatological sunscreen, furthermore for the treatment, care and cleansing of the skin and / or the hair and as a make-up product in decorative cosmetics.
  • the cosmetic and dermatological preparations are applied according to the invention in the usual manner for cosmetics on the skin and / or hair in sufficient quantity.
  • cosmetic and dermatological preparations which are in the form of a sunscreen.
  • these may additionally comprise at least one further UVA filter and / or at least one further UVB filter and / or at least one inorganic pigment, preferably an inorganic micropigment.
  • Very particularly preferred organic UV filters are ethylhexyl methoxycinnamate, ethylhexyl salicylate, butylmethoxydibenzoylmethane, phenylbenzimidazole sulfonic acid, phenyldibenzimidazole tetrasulfonic acid, benzophenone-3 (oxybenzone) and 4 (2-hydroxy-4-methoxybenzophenone-5-sulfonic acid), terephthalalidenedicamphor sulfonic acid, ethylhexyltriazone, bis (benzyl) Ethylhexyloxyphenolmethoxyphenyltriazine, 3- (4-methylbenzylidene) camphor, 2-ethylhexyl-2-cyano-3,3-diphenylacrylate, homomenthylsalicylate.
  • metal oxides and / or other water-insoluble or insoluble metal compounds in particular oxides of titanium (TiOa), zinc (ZnO), iron (eg Fe 2 O 3), zirconium (ZrOa), silicon ( SiO 2), manganese (for example MnO), aluminum (Al 2 O 3 ), cerium (for example Ce 2 O 3 ), mixed oxides of the corresponding metals and mixtures of such oxides and the barium sulfate (BaSO 4 ) ,
  • the pigments can advantageously be treated superficially, in which case, for example, a hydrophilic, amphiphilic or hydrophobic character is to be formed or retained.
  • This surface treatment can consist in providing the pigments with a thin hydrophilic and / or hydrophobic inorganic and / or organic layer by processes known per se.
  • the various surface coatings may also contain water for the purposes of the present invention.
  • Inorganic surface coatings for the purposes of the present invention may consist of aluminum oxide (Al 2 O 3 ), aluminum hydroxide Al (OH) 3 , or aluminum oxide hydrate (also: alumina, CAS No .: 1333-84-2), sodium hexametaphosphate (NaPO 3 ).
  • sodium metaphosphate (NaPOs) n silicon dioxide (SiO 2 ) (also: silica, CAS No .: 7631-86-9), or iron oxide (Fe 2 Os).
  • SiO 2 silicon dioxide
  • Fe 2 Os iron oxide
  • Organic surface coatings for the purposes of the present invention may consist of vegetable or animal aluminum stearate, vegetable or animal stearic acid, lauric acid, dimethylpolysiloxane (also: dimethicone), methylpolysiloxane (methicone), simethicone (a mixture of dimethylpolysiloxane with an average chain length of 200 to 350 Dimethylsiloxane units and silica gel) or alginic acid.
  • These organic surface coatings may be present alone, in combination and / or in combination with inorganic coating materials.
  • icylate, homosalate ( 3,3,5-trimethylcyclohexyl-2-hydroxybenzoate)
  • the cosmetic and dermatological preparations according to the invention may contain cosmetic adjuvants, such as are commonly used in such preparations, for.
  • cosmetic adjuvants such as are commonly used in such preparations, for.
  • Preservatives, bactericides, perfumes, foaming inhibitors, dyes, pigments having a coloring effect, thickeners, moisturizing and / or moisturizing substances, fats, oils, waxes or other common ingredients of a cosmetic or dermatological formulation such as alcohols, Polyols, polymers, foam stabilizers, electrolyte ⁇ , organic solvents or silicone derivatives.
  • the K values shown in Table 1 are based on a novel tyrosinase assay for which previously recombinant human tyrosinase is recovered in a eukaryotic expression system (see Appendix 1).
  • this assay the principle of which is published, the tyrosinase L-DOPA oxidase activity is exploited, converting L-DOPA to dopaquinone.
  • the formed dopachinone is reacted with MBTH (Besthorn 's Hydrazone).
  • the pink reaction product can be measured photometrically.
  • the main advantage of this assay for drug development is that it allows the inhibitor properties of drug candidates to be screened for the first time on human tyrosinase, which has been shown to differ structurally from any currently available fungal tyrosinase.
  • Table 1 shows some compounds that have been found to be particularly effective (+++).
  • the structures mentioned in Table 1 also take into account the stereochemistry, which has a decisive influence on the effectiveness of the structures.
  • tyrosinase 70% confluent, adherent Sf9 cells were used at a MOI (multiplicity of infection, number of infectious particles per cell) of 10-15. The highest dopaoxidase activity was detectable three days after infection. At this time also the cell harvest took place. The cells were detached, washed twice with PBS (4 ° C) and the pellets stored at -80 ° C until further use. To prepare the cell extracts, the pellets are thawed on ice and resuspended in Lysis buffer (50 mM HEPES, 200 mM NaCl, 1% TritonX-100 or Chaps) and disrupted by ultrasound.
  • Lysis buffer 50 mM HEPES, 200 mM NaCl, 1% TritonX-100 or Chaps
  • the lysate is centrifuged for 15 minutes at 15,000 xg to remove debris and dialyzed (or rebuffered via Sephadex 25) to PBS (20 mM KP 1 , 150 mM NaCl, pH 7.3-7.4) 0.5% Triton X-100) Lysate can be stored for up to 2 weeks at 4 ° C and used for activity measurements become. Due to the observed stability of the enzyme, the addition of protease inhibitors was dispensed with. The recombinant enzyme was glycosylated and showed the kinetic properties and inhibition described in the literature by classical tyrosinase inhibitors such as kojic acid and mimosine.
  • the human tyrosinase was expressed by infecting the insect cells (Sf9) with the recombinant virus at a MOI (multiplicity of infection) of 10-15.
  • both adherently growing Sf9 cells and Sf9 suspension cultures can be used.
  • the infection took place in adherently growing cells at 70% confluence and in cells growing in suspension cultures at a cell density of 2 ⁇ 10 6 cells / ml.
  • the highest dopa oxidase activity was detectable three days after infection, at which time the cell harvest was also performed.
  • the cells were detached or harvested by centrifugation, washed twice with PBS and the pellets stored at -80 ° C until further use.
  • the cells were taken up in 25 mM Tris pH 8.0 and disrupted by pressing three times through a 27 ⁇ l gauge cannula.
  • the cell fragments were separated by centrifugation at 18,000 rpm (Beckman, Ja-20 rotor) for 15 minutes, and the supernatant containing the soluble proteins was discarded.
  • the pellet was taken up in PBS 2% chaps, suspended by means of a Potter homogenizer and incubated with stirring for one hour. Subsequently, the cell debris was separated by another 15-minute centrifugation at 18,000 rpm in the Ja-20 rotor. The supernatant, after reprecipitation (Amersham, G-25) on PBS 0.1% CHAPS, could be used directly for activity measurements or subjected to prepurification by ion exchange chromatography.
  • the extract was rebuffered to 25 mM Tris pH 8.0, 0.1% CHAPS and bound to Q-Sepharose (Amersham) equilibrated in the same buffer and eluted with 25 mM Tris pH8.0, 300 mM NaCl, 0.1% CHAPS , The eluate could be used after transient buffering (G-25) on PBS 0.1% CHAPS for activity measurements.
  • the step of anion exchange chromatography serves to enrich the tyrosinase in the protein extract.
  • the dopa oxidase assay used is based essentially on the protocol described by Winder and Harris [Winder AJ, Harris H (1991) "New assays for the tyrosine hydroxylase and dopa oxidase activities of tyrosinase" Eur J Biochem 198 (2),
  • the reactions were carried out in 50 mM potassium phosphate buffer pH 7.0, [pH 6.9 in Winder and Harris] 12% DMSO [2% DMF], 6 mM MBTH (Besthorn's hydrazone) and various concentrations of the substrate L-Dopa (0 - 7.5 mM) at 37 ° C in 96-well plates.
  • the volume per reaction mixture was 250 ⁇ l and depending on the activity of the respective preparation, 10 to 30 ⁇ l of the tyrosine extract from infected insect cells were used.
  • Tyrosinase catalyzes the oxidation of L-dopa to L-dopaquinone, which reacts in a further reaction with the MBTH used to form a stable, pink dye which can be measured photometrically at a wavelength of 505 nm (microplate photometer: 490 nm filter) ,
  • the standard use of 12% DMSO in the reaction mixtures ensures the solubility of the MBTH used even at elevated salt concentrations and significantly improves the solubility of many inhibitors tested with the assay.
  • Glycyrrhiza Glabra 0.05 0.05 0.05 0.05 0.05 0.05
  • Phenylbenzimidazole sulfonic acid 0.5 0.5 0.5 0.5 0.5 0.5
  • Diazolidinyl urea 0.2 0.2 0.2 0.2 0.2
  • Diazolidinyl urea 0, 25 0, 25 0, 25 0.25
  • Trisodium EDTA 0.5 0, 5 0, 5 0, 5
  • Diazolidinyl urea 0, 2 0, 2 0, 2 0, 2

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Abstract

L'invention concerne des composés de formule R<SUP>1</SUP>-NH-C*H((CH<SUB>2</SUB>)<SUB>n</SUB>-NH<SUB>3</SUB> <SUP>+</SUP>)
EP07726729A 2006-03-09 2007-03-09 Oligopeptide substitue par de la tropolone et de l'hydroxybenzol pour depigmentation Withdrawn EP1996606A1 (fr)

Applications Claiming Priority (2)

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DE102006011883A DE102006011883A1 (de) 2006-03-09 2006-03-09 Tropolon- und Hydroxybenzol-substituierte Oligopeptide zur Depigmentierung
PCT/EP2007/052206 WO2007101882A1 (fr) 2006-03-09 2007-03-09 Oligopeptide substitue par de la tropolone et de l'hydroxybenzol pour depigmentation

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EP1996606A1 true EP1996606A1 (fr) 2008-12-03

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CN (1) CN101400691A (fr)
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CN107699532A (zh) * 2017-08-21 2018-02-16 上海交通大学 3,7‑二羟基卓酚酮高产菌株及其发酵培养方法

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JP2010006732A (ja) * 2008-06-26 2010-01-14 Riron Soyaku Kenkyusho:Kk チロシナーゼ活性阻害剤および美白外用剤
US10729628B2 (en) 2015-12-24 2020-08-04 Conopco, Inc. Tyrosinase inhibitors

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JP2565513B2 (ja) * 1987-09-25 1996-12-18 三省製薬株式会社 メラニン生成抑制外用薬剤
US6602856B1 (en) * 1995-01-17 2003-08-05 J. Mark Quillan Antagonists of alpha-melanocyte stimulating hormone and methods based thereon
DE10306452A1 (de) * 2003-02-17 2004-09-02 Beiersdorf Ag Neue Aminosäurederivate
FR2870244B1 (fr) * 2004-05-11 2011-01-07 Centre Nat Rech Scient Conjugues dipeptidiques antagonistes de l'alpha-msh

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107699532A (zh) * 2017-08-21 2018-02-16 上海交通大学 3,7‑二羟基卓酚酮高产菌株及其发酵培养方法
CN107699532B (zh) * 2017-08-21 2020-12-29 上海交通大学 3,7-二羟基卓酚酮高产菌株及其发酵培养方法

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JP2009529513A (ja) 2009-08-20
CN101400691A (zh) 2009-04-01
DE102006011883A1 (de) 2007-09-13

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