Title of Invention

SKIN LIGHTENING AGENTS, COMPOSITIONS AND METHODS

Abstract

Cosmetic compositions and methods are provided, particularly for skin lightening, using 7-hydroxy-2-phenyl coumarin and derivatives of general formula (I) as skin lightening agents alone or in combination with other skin benefit agents and together with a cosmetic vehicle: (I) wherein R1 and/or R2 may be positioned at the 1-, 2-, 3-, 5-, and/or 6- positions on the phenyl ring; each or both R1 and/or R2 represents a hydrogen atom, OH, C1-C4 acyl group, or C1-C4 alkyl group; and R represents a hydrogen atom, C1-C4 acyl group, or C1-C4 alkyl group.

Full Text

FORM - 2 THE PATENTS ACT, 1970 (39 of 1970) & The Patents Rules, 2003 COMPLETE SPECIFICATION (See Section 10 and Rule 13) SKIN LIGHTENING AGENTS, COMPOSITIONS AND METHODS HINDUSTAN UNILEVER LIMITED, a company incorporated under the Indian Companies Act, 1913 and having its registered office at Hindustan Lever House, 165/166, Backbay Reclamation, Mumbai -400 020, Maharashtra, India The following specification particularly describes the invention and the manner in which it is to be performed WO 2007/112853 - PCT/EP2007/002440 SKIN LIGHTENING AGENTS, COMPOSITIONS AND METHODS The invention relates to cosmetic compositions and methods using 7-hydroxy-3-phenyicoumarin and derivative compounds, and more specifically, using 7-hydroxy-3-phenylcoumarin and derivative compounds as skin lightening agents. Many people are concerned with the degree of pigmentation of their skin. For example, people with age spots or freckles may wish such pigmented spots to be less pronounced. Others may wish to reduce the skin darkening caused by exposure to sunlight or to lighten their natural skin color. To meet this need, many attempts have been made to develop products that reduce the pigment production in the melanocytes. However, the substances identified thus far tend to have either low efficacy or undesirable side effects, such as, for example, toxicity or skin irritation. Therefore, there is a continuing need for new cosmetic skin lightening agents, with improved overall effectiveness. For example, certain resorcinol derivatives, particularly 4-substituted resorcinol derivatives, are usefuf in cosmetic compositions for skin tightening benefits, as disclosed in Hu et al., U.S. Patent No. 6,132,740; Bradley, et al., U.S. Patent Nos. 6,504,037 and 6,861,564; Japanese published patent applications JP 2001-010925 and JP2000-327557; and Harichian et al., U.S. Patent No. 6,852,310. Aminophenol derivatives have been described as optical brighteners in, for example, Chevalier et al., U.S. Patent No. 6, 403,065 (for skin). Applicants have now discovered that 7-hydroxy-3-phenylcoumarin and derivative compounds deliver skin lightening benefits. The general chemical formulas and structures of these compounds are discussed in more detail hereinbelow. The 7-hydroxy-3-phenylcoumarin and derivative compounds have been found to be cosmetically effective and possibly less irritating to the skin. These compounds of the present invention have not been used in cosmetics, nor, specifically, for lightening skin. The 7-hydroxy-3-phenyicoumarin is available from Sigma-Aldrich as a fluorescent pH probe for the physiological pH range. Raue GB 933042 disloses 3-phenyl-7-hydroxycoumarin compounds as optical brightening agents for textiles. WO 2007/112853 PCT/EP2007/002440 -2- SUMMARY OF THE INVENTION The use of compounds of the general formula I, and compositions including same, delivers skin lightening benefits with potential reduced irritation. The present invention provides a cosmetic composition and method of skin lightening using a composition comprising in 5 addition to a cosmetically acceptable vehicle, about 0.000001 to about 50 % of a compound of general formula I: (!) wherein R1 and/or R2 may be positioned at the 1-, 2-, 3-, 5-, and/or 6- positions on the 10 phenyl ring; each or both R1 and/or R2 represents a hydrogen atom, OH, CrC4 acyl group, or C1-C4 alkyl group; and R represents a hydrogen atom, C1-C4 acyl group, or C1-C4 alkyl group. 15 Preferably, R represents a hydrogen atom, as represented by compound of general formula II: (H) In a more preferred embodiment, R represents a hydrogen atom, and both R1 and R2 represent hydrogen, resulting in the 7-hydroxy-3-phenyicoumarin of formula III (also 20 called 3-phenylumbellifeone): (III) WO 2007/112853 PCT/EP2007/002440 -3- Further skin benefit agents may be included in the inventive cosmetic compositions. Organic and inorganic sunscreens may also be included. The inventive compounds and compositions may be used for reducing overall skin 5 pigmentation and the reduction of discrete hyperpigmentation, such as blemishes and freckles, as well as for reducing the irritation associated with irritating skin benefit agents, such as retinol. DETAILED DESCRIPTION OF THE INVENTION 10 As used herein, the term "cosmetic composition" is intended to describe compositions for topical application to human skin. The term "skin" as used herein includes the skin on the face, neck, chest, back, arms, axilla, hands, legs, and scalp. 15 Except in the examples, or where otherwise explicitly indicated, all numbers in this description indicating amounts of material or conditions of reaction, physical properties of materials and/or use are to be understood as modified by the word "about". All amounts are by weight of the composition, unless otherwise specified. 20 It should be noted that in specifying any range of concentration, any particular upper concentration can be associated with any particular lower concentration. The term "comprising" is used herein in its ordinary meaning and means including, made 25 up of, composed of, consisting and/or consisting essentially of. In other words, the term is defined as not being exhaustive of the steps, components, ingredients, or features to which it refers. SKIN LIGHTENING AGENTS 30 The invention is concerned with the use of compounds of general formula I, shown below, and compositions including same, as skin cosmetic agents, particularly as skin lightening agents. A particular advantage of the inventive compositions and methods is that compounds of general formula I can be less irritating to the skin than known skin lightening compounds, 35 WO 2007/112853 PCT/EP2007/002440 (I) wherein R, and/or R2 may be positioned at the 1-, 2-, 3-, 5-, and/or 6- positions on the phenyl ring; 5 each or both R1and/or R2 represents a hydrogen atom, C1-C4 acyl group, or C1-C4 alkyl group;and R represents a hydrogen atom, C1-C4 acyl group, or C1-C4 alkyl group. Preferably R represents a hydrogen atom, as represented by compound of general 10 formula II: (II) In a more preferred embodiment, R represents a hydrogen atom, and both R1 and R2 15 represent hydrogen, resulting in the 7-hydroxy-3-phenylcoumarin of formula 111 (also called 3-phenylumbelliferone): (III) WO 2007/112853 PCT/EP2007/002440 - 0- Further skin benefit agents may be included in the inventive cosmetic compositions. Organic and inorganic sunscreens may also be included. The inventive cosmetic compositions and methods have effective skin lightening 5 properties and may be less irritating to the skin. The compositions generally contain about 0.000001 to about 50 % of compounds of general formula I. Compounds ol formula II and/or III are preferred. The amount of the compound of general formula I or formula II or formula III is preferably in the range of 10 about 0.00001 % to about 10 %, more preferably about 0.001 to about 7 %, most preferably from 0.01 to about 5 %, of the total amount of a cosmetic composition. OPTIONAL SKIN BENEFIT AGENTS Preferred cosmetic compositions are those suitable for the application to human skin 15 according to the method of the present invention, which optionally, but preferably, include a further skin benefit agent. Suitable additional skin benefit agents include anti-aging, wrinkle-reducing, skin whitening, anti-acne, and sebum reduction agents. Examples of these include alpha-20 hydroxy acids, beta-hydroxy acids, polyhydroxy acids, hyaluronic acid, hydroquinone, t-butyl hydroquinone, vitamin B derivatives, vitamin C derivatives, allantoin (a placenta extract), dioic acids, retinoids and resorcinol derivatives. COSMETICALLY ACCEPTABLE CARRIER 25 The cosmetically acceptable vehicle may act as a dilutant, dispersant or carrier for the skin benefit ingredients in the composition, so as to facilitate their distribution when the composition is applied to the skin. The vehicle may be aqueous, anhydrous or an emulsion. Preferably, the compositions are 30 aqueous or an emulsion, especially water-in-oil or oil-in-water emulsion, preferentially oil in water emulsion. Water when present wili be in amounts which may range from 5 to 99%, preferably from 20 to 70%, optimally between 40 and 70% by weight. Besides water, relatively volatile solvents may also serve as carriers within compositions of 35 the present invention. Most preferred are monohydnc C1-C3 alkanols. These include ethyl WO 2007/112853 ■ PCT/EP2007/002440 - o- alcohol, methyl alcohol and isopropyl alcohol. The amount of monohydric alkanol may range from 1 to 70%, preferably from 10 to 50%, optimally between 15 to 40% by weight. Emollient materials may also serve as cosmetically acceptable carriers. These may be in 5 the form of silicone oils and synthetic esters. Amounts of the emollients may range anywhere from 0.1 to 50%, preferably between 1 and 20% by weight. Silicone oils may be divided into the volatile and non-voJatiie variety. The term "volatile" as used herein refers to those materials which have a measurable vapor pressure at ambient temperature. Volatile silicone oils are preferably chosen from cyclic or linear polydimethylsiloxanes containing from 3 to 9, preferably from 4 to 5, silicon atoms. Linear volatile silicone materials generally have viscosities less than about 5 centistokes at 25°C while cyclic materials typically have viscosities of less than about 10 centistokes. Non¬volatile silicone oils useful as an emollient material include polyaikyl siloxanes, polyalkylaryl siloxanes and polyether siloxane copolymers. The essentially non-volatile polyaikyl siloxanes useful herein include, for example, polydimethyl siloxanes with viscosities of from about 5 to about 25 million centistokes at 25°C. Among the preferred non-volatile emollients useful in the present compositions are the polydimethyl siloxanes having viscosities from about 10 to about 400 centistokes at 25°C. Among the ester emollients are: (1) Alkenyl or alkyl esters of fatty acids having 10 to 20 carbon atoms. Examples thereof include isoarachidyl neopentanoate, isononyl isonanonoate, oleyl myristate, oleyl 25 stearate, and oleyf oleate. (2) Ether-esters such as fatty acid esters of ethoxylated fatty alcohols. (3) Polyhydric alcohol esters. Ethylene glycol mono- and di-fatty acid esters, diethylene 30 glycol mono- and di-fatty acid esters, polyethylene glycol (200-6000) mono- and di-fatty acid esters, propylene glycol mono- and di-fatty acid esters, polypropylene glycol 2000 monooleate, polypropylene glycol 2000 monostearate, ethoxylated propylene glycol monostearate, glyceryl mono- and dnfatty acid esters, polyglycerol poly-fatty esters, ethoxylated glyceryl monostearate, 1,3-butylene glycol monostearate, 35 1,3-butylene glycol distearate, polyoxyethylene polyol fatty acid ester, sorbitan fatty WO 2007/112853 ' PCT/EP2007/002440 - (- acid esters, and poiyoxyethylene sorbitan fatty acid esters are satisfactory polyhydric alcohol esters. (4) Wax esters such as beeswax, spermaceti, myrtstyl myristate, stearyl stearate and 5 arachidyl behenate. (5) Sterols esters, of which cholesterol fatty acid esters are examples. Fatty acids having from 10 to 30 carbon atoms may also be included as cosmetically 10 acceptable carriers for compositions of this invention. IJIustrative of this category are pelargonic, lauric, myristic, palmitic, stearic, isostearic, hydroxystearic, oleic, linoleic, ricinoleic, arachidic, behenic and erucic acids. Humectants of the polyhydric alcohol-type may also be employed as cosmetically acceptable 15 carriers in compositions of this invention. The humectant aids in increasing the effectiveness of the emollient, reduces scaling, stimulates removal of built-up scale and improves skin feel. Typical polyhydric alcohols include glycerol, polyalkylene glycols and more preferably alkylene polyols and their derivatives, including propylene glycol, diprapylene glycol, polypropylene glycol, polyethylene glycol and derivatives thereof, sorbitol, hydroxypropyl 20 sorbitol, hexytene gfycof, 1,3-butylene glycol, 1,2,6-hexanethol, ethoxylated glycerol, propoxylated glycerol and mixtures thereof. For best results the humectant is preferably propylene glycol or sodium hyaluronate. The amount of humectant may range anywhere from 0.5 to 30%, preferably between 1 and 15% by weight of the composition. 25 Thickeners may also be utilized as part of the cosmetically acceptable earner of compositions according to the present invention. Typical thickeners include crosslinked acrylates (e.g. Carbopol 932), hydrophobicatiy-modified acryiates (e.g. Carbopol 1382), cellulosic derivatives and natural gums. Among useful cellulosic derivatives are sodium carboxymeihylcellulose, hydroxypropyl methylcellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, ethyl 30 cellulose and hydroxymethyi cellulose. Natural gums suitable for the present invention include guar, xanthan, sclerotium, carrageenan, pectin and combinations of these gums. Amounts of the thickener may range from 0.0001 to 5%, usually from 0.001 to 1%, optimally from 0.01 to 0.5% by weight. WO 2007/112853 '______ PCT/EP2007/002440 Collectively the water, solvents, silicones, esters, fatty acids, humectants and/or thickeners will constitute the cosmetically acceptable carrier in amounts from 1 to 99.9%, preferably from 80 to 99% by weight. 5 An oil or oily material may be present, together with an emulsifier to provide either a water-in-oil emulsion or an oil-in-water emulsion, depending largely on the average hydrophilic-lipophilic balance (HLB) of the emulsifier employed. Surfactants may also be present in cosmetic compositions of the present invention. Total 10 concentration of the surfactant will range from 0.1 to 40%, preferably from 1 to 20%, optimally from 1 to 5% by weight of the composition. The surfactant may be selected from the group consisting of anionic, nonionic, cationic and amphoteric actives. Particularly preferred nonionic surfactants are those with a C10-C2o fatty alcohol or acid hydrophobe condensed with from 2 to 100 moles of ethylene oxide or propylene oxide per mole of hydrophobe; C2-Ct0 15 alkyl phenols condensed with from 2 to 20 moles of alkylene oxide; mono- and di- fatty acid esters of ethylene glycol; fatty acid monoglyceride; sorbitan, mono- and di- C8-C20 fatty acids; block copolymers (ethylene oxide/propylene oxide); and polyoxyethylene soroitan as well as combinations thereof. Alkyl polyglycosides and saccharide fatty amides (e.g. methyl gluconamides) are also suitable nonionic surfactants. 20 Preferred anionic surfactants include soap, alkyl ether sulfate and sulfonates, alkyl sulfates and sulfonates, alkylbenzene sulfonates, alkyl and dialkyl sulfosuccinates, C8-C20 acyl isethionates, acyl gfutamates, C8-C2O alkyl ether phosphates and combinations thereof. 25 OPTIONAL COMPONENTS Other adjunct minor components may also be incorporated into the cosmetic compositions. These ingredients may include coloring agents and/or pigments, opacifiers, perfumes, other thickeners, plasticizers, calamine, antioxidants, chelating agents, as welt as additional sunscreens, such as organic sunscreens. Amounts of these 30 other adjunct minor components may range anywhere from 0.001% up to 20% by weight of the composition. For use as sunscreen, metal oxides may be used alone or in mixture and/or in combination with organic sunscreens. Examples of organic sunscreens include but are 35 not limited those set forth in the table below: WO 2007/112853 PCT/EP2007/002440 -9- TABLE 1 CTFA Name Trade Name Supplier 5 Benzophenone-1 Benzophenone-2 Benzophenone-3 Benzophenone-4 Benzophenone-8 10 Methoxycinnamate Ethyl dihydroxypropyl-PABA Glyceryl PABA Homosaiate Methyl anthranilate 15 Octocrylene Octyl dimethyl PABA Octyl methoxycinnamate Octyl salicylate p-Amino benzoic acid (PABA) 20 2-Phenylbenzimidazole-5-sulphonic Triethanolamine (TEA) salicylate 3-(4-methylbenzylidene)-camphor Benzophenone-6 Benzophenone-12 25 4-lsopropyl dibenzoylmethane Butyl methoxydibenzoylmethane Etocrylene UVINUL 400 UVINUL D-50 UVINUL M-40 UVINUL MS-40 SPECRA-SORB UV-24 BERNEL HYDRO AMERSCREEN P NIPAG.M.P.A. KEMESTER HMS SUNAROME UVA UVINUL N-539 AMERSCOL PARSOL MCX SUNAROME WMO PABA acid EUSOLEX 232 SUNAROME W EUSOLEX 6300 UVINUL D-49 UVINUL 408 EUSOLEX 8020 PARSOL 1789 UVINUL N-35 BASF Chemical Co. BASF Chemical Co. BASF Chemical Co. BASF Chemical Co. American Cyanamide Bernel Chemical Amerchol Corp. Nipa Labs. Hunko Chemical Felton Worldwide BASF Chemical Co. Amerchol Corp. Bernel Chemical Felton Worldwide National Starch EM Industries Felton Worldwide EM Industries BASF Chemical Co. BASF Chemical Co. EM Industries Givaudan Corp. BASF Chemical Co. 30 The amount of the organic sunscreens in the cosmetic composition is preferably in the range of about 0.1 wt % to about 10 wt %, more preferably about 1 wt % to 5 wt %. Preferred organic sunscreens are Parsol MCX and Parsol 1789, due to their effectiveness 35 and commercial availability. USE OF THE COMPOSITION The method according to the invention is intended primarily as using a personal care product for topical application to human skin, for cosmetic benefits induding but not 40 limited to skin lightening. WO 2007/112853 [ _j PCT/EP2007/002440 -10 * The inventive compounds and compositions may be used for reducing overall skin pigmentation and the reduction of discrete hyperpigmentation, such as blemishes and freckles, as well as for reducing the irritation associated with irritating skin benefit agents, such as retinol. 5 In use, a small quantity of the composition, for example from 1 to 5 ml, is applied to areas of the skin, from a suitable container or applicator and, if necessary, it is then spread over and/or rubbed into the skin using the hand or fingers or a suitable device. 10 PRODUCT FORM AND PACKAGING The cosmetic composition useful for the method of the invention can be formulated as a lotion having a viscosity of from 4,000 to 10,000 mPas, a fluid cream having a viscosity of from 10,000 to 20,000 mPas or a cream having a viscosity of from 20,000 to 100,000 mPas or above. The composition can be packaged in a suitable container to suit its 15 viscosity and intended use by the consumer. For example a lotion or fluid cream can be packaged in a bottle or a roll-ball applicator or a propellant-driven aerosol device or a container fitted with a pump suitable for finger operation. When the composition is a cream, it can simply be stored in a non-deformable bottle or squeeze container, such as a tube or a lidded jar. When the composition is a solid or semi-solid stick, it may be 20 packaged in a suitable container for manually or mechanically pushing out or extruding the composition. The invention accordingly also provides a closed container containing a cosmetically acceptable composition as herein defined. 25 The following examples are by way of example, not by way of limitation, of the principles of the present invention, to illustrate the best mode of carrying out the invention. EXAMPLE 1 30 Cosmetic compositions within the scope of the invention were prepared. The 7-hydroxy-3-phenylcou ma rin was obtained from Sigrna-Aldrich. A base formulation shown in the table below was made by heating phase A ingredients to 70 to 85° C with stirring. Phase B ingredients were heated in a separate container to 70 35 to 85° C with stirring. Then phase A was added into phase B while both phases were WO 2007/11285* PCT/EP200 7/002440 - "ll - kept at 70 to 85° C. The mixture was stirred for at least 15 minutes at 70 to 85° C. then cooled. A base formulation is shown in the table below. TABLE 2 2a 2b %wt. %wt. Phase 6.00 6.00 A 3.00 3.00 A 2.00 2.00 A 1-50 1.50 A 1.50 1.50 A 3.00 4.00 A 1.20 1.20 B 1-00 1.00 A 1.00 1.00 A 0.60 0.60 B 0.10 0.10 A 0.50 0.50 A 0.01 0.01 B 0.20 0.20 B 0.50 0.50 B 0.10 0.10 B 0.05 0.05 B 0.05 0.05 B 0.05 2.00 B 1.00 1.00 B 2.50 5.00 B 0.15 0.15 B BAL" 100.00 BAL" 100.00 B Ingredients Isostearyl palmitate C12-C15 alky! ocianoate PEG-100stearate Glyceryl hydroxystearate Stearyl alcohol Stearic acid TEA, 99% Dimethicone Sorbitan monostearate Magnesium aluminum silicate Vitamin E acetate Cholesterol Simethicone Xanthan gum Hydroxyethylcellulose Propylparaben Disodium EDTA Butylated hydroxytolene 7-hydroxy-3-phenylcoumarin Niacinamide Metal oxide Methylparaben Water Tola! *BAL means Balance. 10 EXAMPLE 2 Additional cosmetic compositions within the scope of the invention were prepared. WO 2007/112853 - PCTYEP2007/002440 TABLE 3 Wt% Phase Water, Dl BALANCE A Disodium EDTA 0.05 A Magnesium aluminum silicate 0.6 A Methyl paraben 0.15 A Simethicone 0.01 A Butylene glycol 1,3 3.0 A Hydroxyethylcellulose 0.5 A Glycerine, USP 2.0 A Xanthan gum 0.2 A Triethanolamine 1.2 B Stearic acid 3.0 B Propyl paraben NF 0.1 B Glyceryl hydroxystearate 1.5 B Stearyl alcohol 1.5 B isostearyl palmitate 6.0 B C12-15 alcohols octanoate 3.0 B Dimethicone 1.0 B Cholesterol NF 0.5 B Sorbitan stearate 1.0 B Micronized titanium dioxide 5.0 C Tocopheryl acetate 0.1 B PEG-100 stearate 2.0 B Sodium stearoyl lactylate 0.5 B Hydroxycaprylic acid 0.1 C 7-Hydroxy-3-phenylcoumarin 10.0 C Parsol MCX 2.4 C Alpha-bisabolol 0.2 [C The composition of this example was prepared as follows: 5 1. Heat phase A to 80°C _ WO_2007/J12853 _ PCT/EP200 7/002440 - 13- 2. Heat phase B to 75°C in a separate container 3. Add phase B to phase A and mix with heat off for 30 min. 4. At 50°C add phase C and mix for 10 min. 5 EXAMPLES 3-10 A set of additional compositions useful in the methods of the present invention were prepared within the scope of the present invention and are listed in the table below. TABLE 4 Ingredients Phase Examples (weight %) 3 acid soap base 4 5 6 7 8 9 10 Stearic acid A 17.9 17.9 17.9 17.9 17.9 17.9 17.9 17.9 Sodium cetearyl sulfate (emuisifier) A 2.2 1 1.5 2 3 2 Myrj 59 (emuisifier) A 2 2 2 2 2 1 Span 60 (emulsifiers) A 2 2 2 2 2 1 7-hydroxy-3-phenylcoumarin B 0.05 0.05 2.0 2.0 3.5 3.5 5.0 10.0 Micronized zinc oxide B 2.50 5.00 5.00 2.50 2.50 5.00 2.50 5.00 KOH, 22% (form in situ soap with stearic acid) 2.20 Octy) methoxycinnamate 2.50 2.50 2.50 2.50 Water B BAL BAL BAL BAL BAL BAL BAL BAL Glycerin B 1 1 1 . 1 1 1 1 1 EXAMPLE 11 This example shows the skin lightening effect of using 7-hydroxy-3-phenylcoumarin as skin lightening agent in accordance with the inventive method. This experiment was carried out 15 using MatTek Corporation MeianoDerm cultures. Luminescence was measured using a chromameter to assay the degree of melanization of a 3-D skin model. Method for MeianoDerm Cultures MeianoDerm cultures were obtained from MatTek Corporation, Ashland, Massachusetts, 20 The MeianoDerm was maintained according to the manufacturer's instructions. The basal media used for the maintenance of the MeianoDerm cultures was Dulbeccco's Modified Eagle Media (DMEM) supplemented with unspecified quantities of epidermal WO 2007/112853 PCT/EP2007/002440 -14- growth factor, insulin, hydrocortisone, and proprietary epidermal differentiation compounds, in addition to anti-fungal agents and antibiotics. For the long term maintenance of the MelanoDerms, the basal media was supplemented 5 with both basic fibroblast growth factor (bFGF) and a-melanocyte-stimulating hormone (a- MSH), compounds which are stimulators of melanocyte growth and melanogenesis. The cultures were fed every other day for a total of two weeks. Fresh active preparations, prepared in dimethyl sutphoxide (DMSO) or culture media, were also applied to the MelanoDerms when feeding was performed. Each treatment condition was done in 10 duplicate and digital photographs were taken of the MelanoDerm cultures to assess overall pigmentation. Microscopic images of the MelanoDerms were taken to assess the cell viability of the keratinocytes and melanocytes. For further evidence that the treatments were/not cytotoxic, an lactate dehydrogenase (LDH) assay (Promega, Madison, Wt) was performed on the supematants from 24 hour post-treatment cultures. 15 Solvable Melanin Assay To prepare tissues for assay: After treatment tissues are usually frozen until completion of the experiment. Thaw tissues, a few at a time and place in Dulbecco's phosphated buffer solution (D-PBS) to 20 remove excess phenol red from the culture medium and residual test article. Remove a single tissue from the insert. Blot dry and place in 1.7 ml. microfuge tube. Repeat for all samples. Add 250 ul Solvable™ (Tissue and Gel Solubil'rzer 0.5 M—Packard BioScience Co. Catalogue No. 6NE9100 (NEF910)). Close the tube and make sure that the tissue is completely submerged. Incubate at 60° C overnight along with standards. In 25 the morning, vortex the samples. Sometimes thick tissues will require additional time to complete the solution process. To prepare standard: Dissolve melanin (Sigma cat. M 6631) in Solvable™ at 1mg/ml. The solution may be 30 warmed gently for 15 minutes at 37° C. Store solution in dark. To prepare standard curve: Prepare dilutions from the standard containing Oug to 250 ug of melanin in a total of 250 ul Solvable™. Incubate dilutions along with samples. 35 _ WO 2007/112853 __ PCT/EP20Q 7/002440 To read assay: Cool samples and standards. Centrifuge at 13,000 rpm for 5 minutes to pellet. Fill microwell plate (C-96) with 200 ul each of samples and standards. There is some foaming of samples when pipetted. Blow gentfy across the samples to break bubbles 5 prior to reading the plate. Read plate at 490nm. The results are shown in the table below. TABLE 5 mg/ml.mefantn standard -.■ .■■,,.'■.-.*$£ ;Op=490..nmi 800 2.727 400 1.321 200 0.573 100 0.321 50 0.184 25 0.065 controls ;OD=490„hrri DMSO 1.292 Untreated 1.259 7-hydroxy-3^phenylcbumarih (DM) "' OD=490:hni 25 1.674 50 1.339 75 0.849 100 0.293 From the results tabulated above it appears that 7-hydroxy-3-phenyicoumarin compounds of the present invention reduce melanin synthesis. 15 It should be understood that the specific forms of the invention herein illustrated and described are intended to be representative only. Changes, including but not limited to those suggested in this specification, may be made in the illustrated embodiments without departing from the clear teachings of the disclosure. Accordingly, reference should be made to the following appended claims in determining the full scope of the invention. WO 2007/112853 , PCT/EP2007/002440 16 — __. CLAIMS 1. A cosmetic method of skin lightening comprising applying to the skin a composition comprising; 5 a. 0.000001 to 50 % of a compound of general formula I: (0 wherein R1 and/or R2 may be positioned at the 1 -, 2-, 3-, 5-, and/or 6- positions 10 on the phenyl ring; each or both Ri and/or R2 represents a hydrogen atom, OH, C1-C4 acyl group, or C1-C4 alkyl group; and R represents a hydrogen atom, C1-C4 acyl group, or C1-C4 alkyl group; and b. a cosmetically acceptable carrier. 15 2. The cosmetic method of claim 1, wherein said compound has general formula II: 00 3. The cosmetic method of claim 1, wherein R1 and R2 represent hydrogen. 20 4. The cosmetic method of claim 1, wherein said compound is 7-hydroxy-3- phenylcoumarin of formula III: (III) * EP2007002440 -17- 5. The cosmetic method of claim 1, wherein said composition farther comprises a sunscreen. 6. The cosmetic method of claim 5, wherein said sunscreen is a micronized metal 5 oxide. 7. The cosmetic method according to claim 1, wherein said composition further comprises a skin benefit agent selected from the group consisting of alpha- hydroxy acids, beta-hydroxy acids, polyhydroxy acids, hydroquinone, t-butyl 10 hydroquinone, vitamin C derivatives, dioic acids, retinoids, resorcinol derivatives and mixtures thereof. 8. The cosmetic method of claim 1, wherein said composition further comprises an organic sunscreen selected from the group consisting of benzophenone-3, 15 benzophenone-4, behzophenone-8, DEA methoxycinnamaie, ethyl dihydroxypropyl-PABA, glyceryl PABA, homosafate, methyl anthranilate, octocrylene, octyl dimethyl PABA, octyl methoxycinnamate (Parsol MCX), octy) salicylate, PABA, 2-phenytbenzimidazo!e-5-sijlphonic acid, TEA salicylate, 3-(4- methylbenzylidenej-camphor, benzophenone-1, benzophenone-2, 20 benzophenone-6, benzophenone-12, 4~isopropyl dibenzoylmethane, butyl methoxydibenzoylmethane (Parsol 1789), etocrylene and mixtures thereof. 9. A cosmetic composition comprising: a. 0.000001 to 50 % of a compound of general formula I: 25 (I) wherein R, and/or R2may be positioned at the 1-, 2-, 3-, 5-, and/or 6- positions on the phenyl ring; each or both Ri arid/or R2 represents a hydrogen atom, OH, C1-C4 acyl group, 30 or C1-C4 alkyl group; "rMncncucFT iilisl -18-' EP2007002440 R represents a hydrogen atom, C1-C4 acyl group, or C1-C4 afkyf group; b. an organic and/or inorganic sunscreen; and c. a cosmetically acceptable carrier. 5 10. The cosmetic composition of claim 9, wherein said compound is a compound of general formula II: (II) 11. The cosmetic composition of claim 9, wherein said compound is a compound of 10 . formula III: (HI) 20 12. The cosmetic composition of claim 9, wherein R1 and R2 both represent hydrogen. 25 13. The cosmetic composition of claim 9, wherein said compound comprises 0.00001 % to 10 % of said composition. 14. The cosmetic composition of claim 9t"wherein said compound comprises 0.001 % to 7 % of said composition. 30 15. The cosmetic composition of claim 9, wherein said compound comprises 0.01 % to 5 % of said composition. 16. A cosmetic composition for skin lightening, comprising: 35 a. 0.000001 to 50 % of a compound of general formula I: AMENDED SHEET K3r I^T1 16th January 2008) EP2007002-: -19- (0 .5 10 wherein Rt and/or R2 may be positioned at the 1-, 2-, 3-, 5-, and/or 6- positions on the phenyl ring; each or both R, and/or R2 represents a hydrogen atom, OH, C1-C4 acyl group, or C1-C4 alkyl group; R represents a hydrogen atom, C1-C4 acyl group, or C1-C4 alkyl group; b. an organic and/or inorganic sunscreen; and c. a cosmetically acceptable carrier. Dated this 25th day of September 2008 AMENDED SHEET ' S#S

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2050-MUMNP-2008-CORRESPONDENCE(24-1-2013).pdf

2050-MUMNP-2008-CORRESPONDENCE(29-12-2009).pdf

2050-MUMNP-2008-CORRESPONDENCE(IPO)-(7-3-2014).pdf

2050-mumnp-2008-correspondence.pdf

2050-mumnp-2008-description(complete).doc

2050-mumnp-2008-description(complete).pdf

2050-MUMNP-2008-DESCRIPTION(GRANTED)-(7-3-2014).pdf

2050-mumnp-2008-form 1.pdf

2050-MUMNP-2008-FORM 13(7-2-2012).pdf

2050-MUMNP-2008-FORM 18(29-12-2009).pdf

2050-MUMNP-2008-FORM 2(GRANTED)-(7-3-2014).pdf

2050-MUMNP-2008-FORM 2(TITLE PAGE)-(GRANTED)-(7-3-2014).pdf

2050-mumnp-2008-form 2(title page).pdf

2050-mumnp-2008-form 2.doc

2050-mumnp-2008-form 2.pdf

2050-MUMNP-2008-FORM 3(10-12-2013).pdf

2050-MUMNP-2008-FORM 3(12-8-2011).pdf

2050-MUMNP-2008-FORM 3(15-2-2011).pdf

2050-MUMNP-2008-FORM 3(15-2-2012).pdf

2050-MUMNP-2008-FORM 3(22-6-2013).pdf

2050-MUMNP-2008-FORM 3(23-1-2013).pdf

2050-MUMNP-2008-FORM 3(24-2-2010).pdf

2050-MUMNP-2008-FORM 3(4-8-2010).pdf

2050-MUMNP-2008-FORM 3(8-8-2012).pdf

2050-mumnp-2008-form 3.pdf

2050-mumnp-2008-form 5.pdf

2050-MUMNP-2008-GENERAL POWER OF ATTORNEY(23-7-2012).pdf

2050-mumnp-2008-other.pdf

2050-mumnp-2008-pct-ib-311.pdf

2050-mumnp-2008-pct-ipea-409.pdf

2050-mumnp-2008-pct-ipea-416.pdf

2050-mumnp-2008-pct-isa-210.pdf

2050-MUMNP-2008-REPLY TO EXAMINATION REPORT(23-7-2012).pdf

2050-MUMNP-2008-US DOCUMENT(23-7-2012).pdf

2050-mumnp-2008-wo international publication report a1.pdf

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