Tyrosinase inhibitors

Abstract

Disclosed is a compound of the formula (I), (IIA) or (IIB) for use to treat hyperpigmentation of human skin by inhibiting the activity of human tyrosinase in an animate substrate which is human skin containing human tyrosinase. (I) (IIA) (IIB) where: X is oxygen, sulphur, selenium or tellurium; R is OH, alkyl, aryl or peptide group: R1 is OH, alkyl, aryl or peptide group; R2 is OH, alkyl, aryl or peptide group; n is an integer from 2 to 5; m is an integer from 2 to 4 and p is an integer from 2 to 4. ##STR00001##

Claims

1. A method for treating hyperpigmentation of human skin by inhibiting the activity of human tyrosinase in human skin, the method comprising contacting said human skin with a compound of formula (I), (IIA) or (IIB): ##STR00013## wherein: X is oxygen; R is OH, alkyl, aryl, or peptide group, or a group of formula (III): ##STR00014## R.sub.1 is OH, alkyl, aryl or peptide group; R.sub.2 is OH, alkyl, aryl or peptide group; n is an integer from 2 to 5; m is an integer from 2 to 4; and p is an integer from 2 to 4.

2. The method as claimed in claim 1 wherein in said formula (I), said R is OH.

3. The method as claimed in claim 1 wherein in said formula (I), said R is a group of formula (III): ##STR00015##

4. The method as claimed in claim 1 wherein in said formula (IIA) and (IIB), at least one of said R.sub.1 or R.sub.2 is OH.

5. The method as claimed in claim 1 wherein in said formula (IIA) and (IIB), at least one of said R.sub.1 or R.sub.2 is a group of formula (IV): ##STR00016##

6. The method as claimed in claim 1, wherein the compound of the formula (I), (IIA) or (IIB) is contained in a cosmetic composition.

7. A method for inhibiting the activity of mushroom tyrosinase in a food or beverage composition, the method comprising contacting said food or said beverage composition with a compound of formula (I), (IIA) or (IIB): ##STR00017## wherein: X is oxygen; R is OH, alkyl, aryl, or peptide group, or a group of formula (III): ##STR00018## R.sub.1 is OH, alkyl, aryl or peptide group; R.sub.2 is OH, alkyl, aryl or peptide group; n is an integer from 2 to 5; m is an integer from 2 to 4; and p is an integer from 2 to 4.

8. The method claimed in claim 1 comprising: (i) 0.01 to 10% by weight a compound of the formula (I), (IIA), or (IIB); and (ii) a carrier.

Description

DETAILED DESCRIPTION OF THE INVENTION

(1) Crown ethers comprise a ring of repeating ether groups and are considered to be oligomers of ethylene oxide [(CH.sub.2CH.sub.2O).sub.n]. They contain a central cavity, which can accommodate cations (the well-known case being potassium/sodium) in the ring interior, coordinating with e.g., oxygen atoms.

(2) Metal ions (inorganic cations) play crucial role in various biological processes. The formation of melanin pigment is via the activity of tyrosinase. Similar reactions play a part in plant derived materials, especially in the context of browning of food/beverage compositions. Without wishing to be bound by theory, it is believed that copper chelators can inhibit the activity of tyrosinase and therefore such compounds are suitable for use in skin lightening products and for food products where the activity of tyrosinase needs to be controlled.

(3) Melanin is produced by a complex set of reactions within the melanocyte involving, at a basic level, the enzyme tyrosinase and the aminoacid L-tyrosine. It is well known that tyrosinase is an essential component of melanin synthesis. Tyrosinase catalyzes the conversion of L-tyrosine to dopaquinone via L-DOPA (L-3,4-dihydroxyphenylalanine) as an intermediate. Dopaquinone undergoes further conversion to form melanin. As disclosed earlier, a need exists for novel methods and compositions to inhibit tyrosinase activity.

(4) Disclosed in accordance with the first aspect of the invention is a compound of the formula (I), (IIA) or (IIB) for use to treat hyperpigmentation of human skin by inhibiting the activity of human tyrosinase in an animate substrate which is human skin containing tyrosinase.

(5) ##STR00002##
where:

(6) X is oxygen, sulphur, selenium or tellurium; R is OH, alkyl, aryl or peptide group: R.sub.1 is OH, alkyl, aryl or peptide group; R.sub.2 is OH, alkyl, aryl or peptide group; n is an integer from 2 to 5; m is an integer from 2 to 4 and p is an integer from 2 to 4.

(7) Compounds of the formulae (I) generally belong to the class of benzo crown ethers. On the other hand, the compounds of the formula (IIA) and (IIB) belong to the class of dibenzo crown ethers. (IIA) and (IIB) are positional isomers.

(8) The special technical feature common between formulae (I), (IIA) and (IIB) is the benzo crown ether part, which is believed to be responsible for inhibition of tyrosinase.

(9) Formulae (I), (IIA) and (IIB) may be considered alternatives having a common activity, which is the inhibition of the activity of tyrosinase, mushroom as well as human. The formulae (I), (IIA) and (IIB) also contain structural elements of the benzo crown ether nucleus. The compounds represented by (I), (IIA) and (IIB) share a common chemical structure which occupies a large portion of their structures, or, alternatively, it may also be said that the commonly shared structure constitutes a structurally distinctive portion.

(10) Although it is possible to have compounds which contain sulphur or tellurium, it is preferred that in each said formula, each said X is oxygen.

(11) It is preferred that in formula (I), the R is OH. In other words, the substituent is a carboxylic acid group.

(12) Alternatively, in said formula (I), said R is an amide group of the formula (III), where the carbonyl carbon in said formula (I) is bonded to the nitrogen atom of the formula (III) which is indicated by the arrow.

(13) ##STR00003##

(14) In the case of each of the compounds of the formulae (IIA) and (IIB), it is preferred that at least one of the R.sub.1 or R.sub.2 is OH. In other words, at least one substituent is a carboxylic group. Alternatively, there are two carboxylic groups, in which case, each one of R.sub.1 and R.sub.2 is OH group.

(15) Alternatively, in the case of the compound of formula (IIA) and (IIB), it is preferred that least one of the R.sub.1 or R.sub.2 is a amide group of the formula (IV), where the carbonyl carbon in said formula (IIA or IIB) is bonded to the nitrogen atom of the formula (IV) which is indicated by the arrow.

(16) ##STR00004##

(17) Table 1 has some exemplary compounds particularly preferred for use in accordance with the invention.

(18) TABLE-US-00001 TABLE 1 (a) (b) (c) (d)

(19) Compounds (a) and (b) are exemplary embodiments of the compound of the general formula (I).

(20) In the case of (a), each X is oxygen; R is OH and n is equal to 3.

(21) In the case of compound (b), each X is oxygen; n is equal to 3 and R is:

(22) ##STR00009##

(23) In the case of compound (c), each X is oxygen, R1 is OH; R2 is OH, m is equal to 3, p is equal to 3.

(24) Similarly, in the case of compound (d), each X is oxygen, m is equal to 3, p is equal to 3 and each one of R1 and R2 is:

(25) ##STR00010##
The Tyrosinase

(26) The tyrosinase is either human or mushroom tyrosinase. The animate substrate is human skin.

(27) Alternatively, the compound is used to inhibit the activity of tyrosinase in an inanimate substrate which is a food or beverage composition.

(28) In accordance with another aspect of the invention is disclosed the non-therapeutic use of a compound of the formula (I), (IIA) or (IIB) to inhibit the activity of tyrosinase in an animate or inanimate substrate.

(29) The use is for non-therapeutic applications to inhibit the activity of human tyrosinase in an animate substrate which is human skin containing said human tyrosinase, more preferably for cosmetic applications such as in cosmetic creams and lotions which are available in stores and supermarkets and which are used by many people who desire a beautiful, even-tone and blemish-free complexion. Persons skilled in the art know the differences between medical and cosmetic use. Cosmetic compositions are used to maintain the condition of skin or to beautify it, whereas medical compositions are useful to mitigate a medical condition.

(30) Alternatively, the use is for therapeutic applications as in treatment of any medical condition such as hyperpigmentation.

(31) In another aspect the invention provides a method of treat hyperpigmentation of human skin by inhibiting the activity of human tyrosinase in an animate substrate which is human skin, by contacting said substrate with a compound of the formula (I), (IIA) or (IIB). Further, preferably the method is non-therapeutic. Alternatively, it is of therapeutic nature as explained earlier.

(32) In accordance with yet another aspect is disclosed a composition comprising: (i) 0.01 to 10% by weight a compound of the formula (I), (IIA) or (IIB); and, (ii) a carrier,
for use to treat hyperpigmentation of skin by inhibiting the activity of tyrosinase in an animate substrate containing said human tyrosinase which is human skin.

(33) In accordance with yet another aspect is disclosed use of a compound of the formula (I), (IIA) or (IIB) to inhibit the activity of mushroom tyrosinase in a food or a beverage composition.

(34) In accordance with yet another aspect is disclosed the use of a compound of the formula (I), (IIA) or (IIB) in the preparation of a food or beverage composition to inhibit the activity of mushroom tyrosinase in said composition.

(35) Methods of inhibiting tyrosinase activity may involve application of the aforementioned composition. The composition is applied to a substrate in need of tyrosinase inhibition. Substrates in need of tyrosinase inhibition include any substrate containing tyrosinase which an individual selects for inhibition. In certain embodiments, the animate substrate is a human skin surface. A suitable skin surfaces include facial skin surfaces, hand and arm skin surfaces, foot and leg skin surfaces, and neck and chest skin surfaces. In certain embodiments, a particular area or areas of the skin surface may be selected for tyrosinase inhibition. In one embodiment, the area may be the facial skin surface including the forehead, perioral, chin, periorbital, nose, and/or cheeks.

(36) The area on the skin surface could be a hyperpigmented spot or other area with increased melanin production. Hyperpigmented spots may be identified by the user or a third party such as a dermatologist, cosmetician, or other caregiver. Identification may be by visual inspection of the skin for hyperpigmented spots in need of treatment based on size and/or colour. Commercially available imaging devices such SIAscope V may be used to identify hyperpigmented spots.

(37) The composition may be applied and left on the substrate for a sufficient time or may be repeatedly applied a sufficient number of times to inhibit the activity of tyrosinase. In certain embodiments, the contact time is greater than about 1 hour, 2 hours, 6 hours, 8 hours, 12 hours, or 24 hours. The contact time is time from application of the composition until the composition is removed. In certain embodiments, the composition may be removed by rinsing or washing the substrate. When human skin surface is the animate substrate, the composition may be removed by washing or rinsing the skin. The treatment period may involve a single application or multiple applications. The composition may be applied at least once daily. In other embodiments, the composition is applied at least twice daily. Multiple applications may occur over the course of at least about one week. Alternately, the treatment period may last more than about 4 weeks or more than about 8 weeks. In certain embodiments, the treatment period will extend over multiple months (i.e., 3 to 12 months) or multiple years. In the case of cosmetic composition, the composition may be applied daily for prolonged period.

(38) Where such compositions are cosmetic compositions, they are applied to maintain the tone, colour or texture of the skin. In such case, the use generally is prolonged involving daily application of the composition. The user may thereafter continue application.

(39) Other Ingredients:

(40) When the composition in accordance with the invention is cosmetic composition, it preferably comprises one or more of fragrance, skin lightening compound, surfactant, organic sunscreen, inorganic sunscreen, extender pigment and preservative.

(41) The skin lightening compound may be selected from the group consisting of niacinamide, resorcinols, koijic acid, retinol, retinyl esters including retinyl palmitate, 12-hydroxystearic acid, lactic acid, ascorbic acid, glabradin, dioic acids including octadecenedioic acid and azeleic acid, resveratrol, ascorbyl phosphate, ascorbyl palmitate, acetyl glucosamine, calcium pantothenate, alpha arbutin, climbazole, pitera extract, soybean extract, undecylenoyl phenylalanine, aqueous extract of bearberry and mixtures thereof.

(42) The resorcinol may be selected from the group consisting of 4-ethyl resorcinol, 4-hexyl resorcinol, 4-isopropyl resorcinol and phenylethyl resorcinol. It is particularly preferred that the skin-lightening compound is a non-tyrosinase inhibiting skin-lightening compound. The composition could comprise 0.0001 to 10, preferably 0.01 to 2, most preferably 0.1 to 1% by weight of the skin lightening compound. Sunscreens include those materials which block harmful ultraviolet light. Preferred suncreens are the derivatives of p-aminobenzoic acid (PABA), cinnamate and salicylate. For example, avobenzophenone (Parsol 1789), octyl methoxycinnamate and 2-hydroxy-4-methoxy benzophenone (also known as oxybenzone) can be used. Octyl methoxycinnamate and 2-hydroxy-4-methoxy benzophenone are commercially available under the trade marks, Parsol MCX and Benzophenone-3, respectively. Ecamsule, a benzylidene camphor derivative, and drometrizole trisiloxane, a benzotriazole, may also be used. Further examples include Octocrylene, phenylbenzimidazole sulfonic acid (also known as Ensulizole), ethylhexyl salicylate, diethylhexyl naphthylate, bimotrizinole (trade marked as Tinosorb S) and bisoctrizole (Tinosorb M). Inorganic sunscreens include oxides like titanium dioxide and zinc oxide which reflect or scatter the sunrays. The quantity of sunscreens present in the compositions could vary depending upon the degree of protection desired from UV radiation. Preferably, the compositions comprise 0.01 to 15% by weight, more preferably 0.1 to 10 and most preferably 0.5 to 7.5% by weight sunscreen.

(43) Illustrative examples of the types of fragrances that may be used include those comprising terpenes and terpene derivatives like those described in Bauer, K., et al., Common Fragrance and Flavor Materials, VCH Publishers (1990). Further examples include myrcene, dihydromyrenol, citral, tagetone, cis-geranic acid, citronellic acid, mixtures thereof.

(44) The carrier acts as diluent or dispersant for the ingredients of the compositions. The carrier may be aqueous-based, anhydrous or an emulsion, whereby a water-in-oil or oil-in-water emulsion is generally preferred. If the use of water is desired, water typically makes up the balance of the composition, which most preferably is from 40 to 80% by weight of the composition.

(45) In addition to water, organic solvents may optionally be included as carrier to assist any other carrier in the compositions of the present invention. Examples include alkanols like ethyl and isopropyl alcohol.

(46) Other suitable organic solvents include ester oils like isopropyl myristate, cetyl myristate, 2-octyldodecyl myristate, avocado oil, almond oil, olive oil and neopentylglycol dicaprate. Typically, such ester oils assist in emulsifying the compositions, and an effective amount is often used to yield a stable, and most preferably, water-in-oil emulsion.

(47) Emollients may also be used, if desired, as a carrier. Alcohols like 1-hexadecanol (i.e. cetyl alcohol) are preferred. Other emollients include silicone oils and synthetic esters. Silicone oils suitable for use include cyclic or linear polydimethylsiloxanes containing from 3 to 9, preferably from 4 to 5 silicon atoms. Non-volatile silicone oils useful as emollients include polyalkyl siloxanes, polyalkylaryl siloxanes and polyether siloxane copolymers. The non-volatile polyalkyl siloxanes useful polydimethylsiloxanes. Silicone elastomers may also be used. The ester emollients that may optionally be used are: (i) alkenyl or alkyl esters of fatty acids having 10 to 20 carbon atoms. Examples thereof include isoarachidyl neopentanoate, isononyl isonanonoate, oleyl myristate, oleyl stearate, and oleyl oleate; (ii) ether-esters such as fatty acid esters of ethoxylated fatty alcohols; (iii) polyhydric alcohol esters. Ethylene glycol mono and di-fatty acid esters, diethylene 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 di-fatty acid esters, polyglycerol poly-fatty esters, ethoxylated glyceryl mono-stearate, 1,3-butylene glycol monostearate, 1,3-butylene glycol distearate, polyoxyethylene polyol fatty acid ester, sorbitan fatty acid esters, and polyoxyethylene sorbitan fatty acid esters are satisfactory polyhydric alcohol esters; (iv) wax esters such as beeswax, spermaceti, stearyl stearate and arachidyl behenate; and, (v) sterols esters, of which cholesterol fatty acid esters are examples.

(48) Emollients, when present, typically make up from 0.1 to 50% by weight of the composition, including all ranges subsumed therein.

(49) Fatty acids having from 10 to 30 carbon atoms may also be included as carriers. Examples of such fatty acids include pelargonic, lauric, myristic, palmitic, stearic, isostearic, oleic, linoleic, arachidic, behenic or erucic acid and mixtures thereof.

(50) Humectants of the polyhydric alcohol type may also be employed in the compositions. The humectant often aids in increasing the effectiveness of the emollient, reduces scaling at the skin surface, 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, dipropylene glycol, polypropylene glycol, polyethylene glycol and derivatives thereof, sorbitol, hydroxypropyl sorbitol, hexylene glycol, 1,3-butylene glycol, 1,2,6-hexanetriol, ethoxylated glycerol, propoxylated glycerol and mixtures thereof. For best results the humectant is preferably propylene glycol or sodium hyaluronate. Other humectants which may be used include hydroxyethyl urea. The amount of humectant may be 0.2 to 25% by weight and preferably from 0.5 to 15% by weight of the composition.

(51) Moisturisation may be improved through use of petrolatum or paraffin. Thickeners may also be utilized as a portion of the carrier in the compositions. Typical thickeners include cross-linked acrylates (e.g. Carbopol 982), hydrophobically-modified acrylates (e.g. Carbopol 1382), cellulosic derivatives and natural gums. Among useful cellulosic derivatives are sodium carboxymethylcellulose, hydroxypropyl methylcellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, ethyl cellulose and hydroxymethyl 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 0.001 to 5, optimally from 0.01 to 0.5% by weight of the composition.

(52) Surfactants may also be present. When present, the total amount of surfactants is 2 to 40% by weight, and preferably from 4 to 20% by weight, optimally from 5 to 12% by weight of the composition. The surfactant is selected from the group consisting of anionic, nonionic, cationic and amphoteric actives. Particularly preferred nonionic surfactants are those with a C.sub.10-20 fatty alcohol or acid hydrophobe condensed with from 2 to 100 moles of ethylene oxide or propylene oxide per mole of hydrophobe; 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 sorbitan as well as combinations thereof. Alkyl polyglycosides and saccharide fatty amides (e.g. methyl gluconamides) are also suitable nonionic surfactants.

(53) Preferred anionic surfactants include soap, alkyl ether sulfate and sulfonates, alkyl sulfates and sulfonates, alkylbenzene sulfonates, alkyl and dialkyl sulfosuccinates, C.sub.8 to 20 acyl isethionates, acyl glutamates, C.sub.8 to 20 alkyl ether phosphates and combinations thereof.

(54) Various other ingredients may also be used in compositions. Actives are defined as skin benefit agents other than emollients and other than ingredients that merely improve the physical characteristics of the composition. Although not limited to this category, general examples include extender pigments such as talcs and silicas, as well as alpha-hydroxy acids, beta-hydroxy acids and zinc salts.

(55) Beta-hydroxy acids include salicylic acid. Zinc oxide and zinc pyrithione are examples of useful zinc salts.

(56) Compositions, especially those containing water, need to be protected against harmful microorganisms. Anti-microbial compounds, such as triclosan, and preservatives may become necessary. Suitable preservatives include alkyl esters of p-hydroxybenzoic acid, hydantoin derivatives, propionate salts, and a variety of quaternary ammonium compounds. Particularly preferred preservatives are methyl paraben, propyl paraben, phenoxyethanol and benzyl alcohol. Preservatives are from 0.1 to 2% by weight of the composition.

(57) The packaging could be a patch, bottle, tube, roll-ball applicator, propellant driven aerosol device, squeeze container or lidded jar.

(58) The invention will be explained in details with the help of non-limiting examples.

EXAMPLES

Example 1: Tyrosinase Assay by Oxidation of DOPA

(59) The (tyrosinase) inhibitory activity of a, b, c and d was measured by oxidation of DOPA.

(60) ##STR00011## ##STR00012##

(61) Each crown ether was tested against human Tyrosinase (in the form of melanocytic extract) and commercial (Worthington, USA) mushroom tyrosinase (MT), using the DOPA oxidation assay. Details of the assay mix are included in table 2.

(62) TABLE-US-00002 TABLE 2 0.5M Potassium Phosphate buffer (pH 6.8) 0.01 ml 3 mM aq. L-DOPA (SIGMA Cat.# D9628) stock 0.03 ml 2.5 mg/ml human melanocytic lysate OR 0.002 ml 1 mg/ml aqueous Mushroom Tyrosinase 10 mM test compound stock in 100% DMSO 0.01 ml Water 0.048 ml

(63) Assay examined the change (Optical Density) OD.sub.475 nm at 37 C., after 45 minutes reaction in a standard 96-well plate. All ON.sub.475 nm were corrected by subtracting the background OD.sub.475 nm value of the negative control sample lacking enzyme in the assay mix. The extent of inhibition was calculated using the formula:
% Inhibition=100*(a/b),
where a=(OD.sub.UOD.sub.T), b=(OD.sub.UOD.sub.N), U is the uninhibited enzyme alone sample, T is the test compound and N is the no enzyme control.

(64) The observations are summarised in Table 3. The background effect of 10% DMSO on tyrosinase activity was also examined for calculation purposes (i.e. the U sample).

(65) TABLE-US-00003 TABLE 3 % % Serial Concentration of OD.sub.475 nm Inhibition OD.sub.475 nm Inhibition No. Test Compound (HME) (HME) (MT) (MT) 1 Uninhibited 0.141 0 0.17 0 enzyme positive control 2 0.5 mM Kojic 0.076 46 0.084 51 acid 3 0.25 mM Kojic 0.100 29 0.111 35 acid 4 0.1 mM Kojic 0.124 12 0.131 23 acid 5 1 mM 0.029 79 0.068 60 Compound a 6 0.1 mM 0.126 11 0.128 25 Compound a 7 1 mM 0.162 No 0.122 28 Compound b Inhibition 8 0.01 mM 0.125 12 0.160 6 Compound b 9 1 mM 0.027 81 0.087 49 Compound c 10 0.1 mM 0.094 33 0.162 5 Compound c 11 0.01 mM 0.092 35 0.156 9 Compound c 12 1 mM 0.110 23 0.053 69 Compound d 13 0.1 mM 0.134 5 0.071 58 Compound d 14 0.01 mM 0.144 No 0.137 19 Compound d Inhibition Note: MT = mushroom tyrosinase, MHE = human tyrosinase

(66) The data in Table 3 indicates that compound a, b, c and d showed at least 10% inhibition of melanin compared to a vehicle control. It corresponds to inhibition of the activity of tyrosinase (at least one of human or mushroom tyrosinase).

(67) Kojic acid (0.5 mM) inhibits to the extent of 46%.

(68) A compound that inhibits colour by more than 10% of the control value is a tyrosinase inhibitor. The data indicates that compounds a, b, c and d do inhibit the activity of at least one of human or mushroom tyrosinase, in accordance with the assay.

(69) The illustrated examples indicate that exemplified compounds of the formula (I), (IIA) or (IIB) inhibit the activity of tyrosinase in at least one of an animate or inanimate substrate containing tyrosinase.