EP1353644A1

EP1353644A1 - Use of an isothiocyanate, a thiocyanate or a mixture thereof as depigmenting agent

Info

Publication number: EP1353644A1
Application number: EP02700395A
Authority: EP
Inventors: Daniel Jean
Original assignee: LMD SAS
Current assignee: LMD SAS
Priority date: 2001-01-26
Filing date: 2002-01-24
Publication date: 2003-10-22
Also published as: PL364826A1; US20040077715A1; JP2004520371A; FR2820036A1; KR20030086256A; MXPA03006731A; CA2435942A1; WO2002058664A1; FR2820036B1; HUP0303018A2; SK9612003A3; CZ20032012A3

Abstract

The invention concerns the use if isothiocyanate of general formula (I): R1-N=C=S wherein: R1 represents an alkyl, alkenyl, alkynyl, aryl, acetyl, alkylcarbonyl, alkoxy, cycloalkyl, aryloxy, arylcarbonyl group, carboxylic acid or a -(CH2)nR3 group wherein n represents an integer ranging between 1 and 5 and R3 represents a polar functional group, advantageously a halogen atom, a sulphoxide, carbonyl, nitro, thioester, thioether, sulphonyl, sulphinyl, nitrile group, carboxylic acid, carboxylic ester, alkylthio or hydroxyl, a thiocyanate of general formula (II): R2-S=C=N wherein: R2 represents an alkyl, alkenyl, alkynyl, aryl, acetyl, alkylcarbonyl, alkoxy, cycloalkyl, aryloxy, arylcarbonyl group, carboxylic acid, carboxylic ester or a -(CH2)nR3 group, wherein n represents an integer ranging between 1 and 5 and R3 represents a polar functional group, advantageously a halogen atom, a sulphoxide, carbonyl, nitro, thioester, thioether, sulphonyl, sulphinyl, nitrile group, carboxylic acid, carboxylic ester, alkylthio or hydroxyl, or mixtures thereof for making a medicine or a cosmetic composition for inhibiting tyrosinase.

Description

Use of an isothiocyanate, a thiocyanate or a mixture thereof as a depigmenting agent

The present invention relates to depigmentants and in particular the use of isothiocyanates or thiocyanates as depigmentants.

Skin pigmentation in humans comes from a complex series of cellular processes that take place in a single population of cells called melanocytes. Melanocytes are located in the lower part of the epidermis, and their function is to synthesize a brown pigment, melanin, which protects the body from the damaging effects of ultraviolet radiation. Melanin is deposited in the melanosomes, vesicles present inside the melanocytes. Melanosomes are expelled from melanocytes and transported to the surface of the skin by keratinocytes, which assimilate the melanin contained in the melanosomes. The dark shade of the skin is proportional to the amount of melanin synthesized by the melanocytes and transferred to the keratinocytes. In some cases, it is better to reduce or inhibit melanogenesis, for example, to lighten the skin, to remove age spots or to reduce the hyperactivity of melanocytes.

For a long time, cosmetic compositions containing a peroxide such as hydrogen peroxide or zinc peroxide have been used for the purpose of removing spots, such as freckles, which appear on the skin. However, peroxides are extremely unstable and, therefore, their storage is problematic. In addition, the stable incorporation of these peroxides in cosmetic bases is difficult and the peroxides themselves do not have a sufficiently whitening effect.

On the other hand, cosmetic preparations comprising vitamin C, cysteine or colloidal sulfur have started to be used for the purpose of bleaching the skin. However, the effects of these substances are not satisfactory. For a long time, hydroquinone has been the reference depigmenting molecule and used in many dermo-cosmetic preparations. However, this product is not safe and has significant cytotoxicity for melanocytes which can cause irreversible depigmentations.

Recently, kojic acid has been used effectively as a substance that inhibits the formation of melanin in human skin. Consequently, various cosmetic preparations intended for depigmenting the skin and containing kojic acid (Japanese patent publication No. 56-18569) or an ester of kojic acid with an aromatic carboxylic acid such as cinnamic acid or l benzoic acid (Japanese Patent Publication No.

60/100005) or kojic acid diesters (Japanese patent publications Nos. 61-60801 and 60-17961) have been described. These kojic acids and esters of kojic acid are therefore known to be substances capable of inhibiting melanogenesis. However, kojic acid has variable efficacy depending on the individual and on average insufficient.

Consequently, the search for other depigmenting products is still current.

Surprisingly, the applicants have discovered that certain molecules belonging to the thiocyanate and isothiocyanate family have a very clear inhibitory effect on tyrosinase in vitro.

Isothiocyanates can be extracted from various cruciferous plants, including broccoli, Lepidium dabra and radishes, such as sulforaphane and sulforaphene.

Sulforaphane and some of its synthetic analogues are known to protect against the mutagenic effect of chemicals such as those found in tobacco smoke, for example. This effect goes through the induction of enzyme systems involved in the evacuation of mutagenic molecules out of the body. It would also seem that these molecules also act directly on the mechanism of mutagenesis (WO 94/19948, Carcinogenesis, 8, 12, 1987, pages 1971-1973; Cancer Research, 51, 13, 1991, pages 2063-2068).

However, the action of these substances as a depigmenting agent has never been described.

The present invention therefore relates to the use of an isothiocyanate of general formula I below: Rι-N = C = SI in which Ri represents an alkyl, alkenyl, alkynyl, aryl, acetyl, alkylcarbonyl, alkoxy, cycloalkyl, aryloxy, arylcarbonyl, carboxylic acid, carboxylic ester group or a - (CH ₂ ) _n R group in which n represents an integer ranging from 1 to 5 and R ₃ represents a polar functional group, advantageously a halogen atom, a sulfoxide, carbonyl, nitro, thioester, thioether, sulfonyl, sulfinyl, nitrile, carboxylic acid, carboxylic ester group, alkylthio or hydroxyl, of a thiocyanate of general formula II below:

R ₂ -S = C = N π in which R ₂ represents an alkyl, alkenyl, alkynyl, aryl, acetyl, alkylcarbonyl, alkoxy, cycloalkyl, aryloxy, arylcarbonyl, carboxylic acid, carboxylic ester or a group - (CH) _n R ₃ in which n represents an integer ranging from 1 to 5 and R ₃ represents a polar functional group, advantageously a halogen atom, a sulfoxide, carbonyl, nitro, thioester, thioether, sulfonyl, sulfinyl, nitrile, carboxylic acid group, carboxylic, alkylthio or hydroxyl ester, or mixtures thereof for the manufacture of a medicament or a cosmetic composition intended to inhibit tyrosinase.

By the term "alkyl group" is meant in the sense of the present invention any alkyl group of 1 to 10 carbon atoms, linear or branched substituted or not, in particular the group CH. By the term "alkenyl group" is meant in the sense of the present invention any alkenyl group of 2 to 10 carbon atoms, linear or branched, substituted or not, in particular the vinyl group.

By the term "alkynyl group" is meant within the meaning of the present invention any alkynyl group of 2 to 10 carbon atoms, linear or branched, substituted or not, in particular the ethynyl group. By the term "alkylcarbonyl group" is meant in the sense of the present invention any alkyl group as defined above linked via a carbonyl group. An example of an alkylcarbonyl group is the acetyl group.

By the term "alkoxy group" is meant within the meaning of the present invention any alkoxy group of 1 to 10 carbon atoms, linear or branched substituted or not, in particular the OCH group.

By the term "cycloalkyl group" is meant in the sense of the present invention any cycle composed of alkyl group of 1 to 10 carbon atoms, substituted or not, in particular, the cyclohexyl group. By the term "aryl group" is meant within the meaning of the present invention one or more aromatic rings having 5 to 8 carbon atoms, which can be joined or fused, substituted or not. In particular, the aryl groups can be phenyl or naphthyl groups and the substituents of the halogen atoms, alkoxy groups as defined above, alkyl groups as defined above or the nitro group.

By the term "aryloxy group" is meant in the sense of the present invention any aryl group as defined above, linked via an alkoxy group as defined above.

By the term "aralkyl group" is meant in the sense of the present invention any aryl group as defined above, linked via an alkyl group as defined above. In particular an aralkyl group is a benzyl group.

By the term "arylcarbonyl group" is meant in the sense of the present invention any aryl group as defined above, linked via a carbonyl group. An example of an arylcarbonyl group is the benzoyl group.

By the term "carboxylic acid" is meant in the sense of the present invention any alkyl group as defined above to which is linked a carboxy group (-COOH).

By the term "sulfonyl group" is meant in the sense of the present invention any alkyl, cycloalkyl or aryl group as defined above, linked via an SO ₂ group. By the term "sulfinyl group" is meant within the meaning of the present invention any alkyl, cycloalkyl or aryl group as defined above, linked via an SO group.

By the term "alkylthio group" is meant in the sense of the present invention any alkyl group as defined above linked via a sulfur atom.

The present invention also relates to the use of an isothiocyanate of general formula I, of a thiocyanate of general formula II or of their mixtures for the manufacture of a medicament or of a cosmetic composition intended to lighten or depigment the epidermis. or to remove age spots.

Advantageously, the thiocyanate is a thiocyanate of general formula II in which R ₂ represents the aralkyl group, even more advantageously, it is benzylthiocyanate. Advantageously, the thiocyanate of general formula II is in the form of a salt, even more advantageously of sodium or potassium.

Thiocyanates can be obtained in parallel with isothiocyanates during the breakdown of cruciferous glucosinolates by myrosinase

(Pharmacognosy, Phytochemistry, Medicinal Plants, Bruneton, ed. Lavoisier, Paris, 1993, p. 177). Some are synthetic and commercially available, such as benzylthiocyanate, from the company Fluka (ref. 13929).

In a particular embodiment, the isothiocyanate of general formula I is a synthetic isothiocyanate, in particular wherein R _i represents a group, aryl, acetyl, alkylcarbonyl, cycloalkyl, arylalkyl or arylcarbonyl. Advantageously, the isothiocyanate is chosen from the group consisting of cyclohexylisothiocyanate, benzylisothiocyanate, acetylisothiocyanate and benzoylisothiocyanate.

Synthetic isothiocyanates are commercially available. Thus cyclohexylisothiocyanate, benzylisothiocyanate and benzoylisothiocyanate are available from the company Aldrich (ref. Cl 0-540-6; 25,249-2 and 26,165-

3 respectively) and acetylisothiocyanate from the company Fluka (ref. 01230). The other isothiocyanates can be synthesized according to the method and the examples indicated in US Pat. No. 5,411,986.

In another particular embodiment, the isothiocyanate of general formula I is obtained by extraction of a cruciferous plant, advantageously chosen from the group consisting of broccoli, Lepidium dabra and radish. Even more advantageously, it is chosen from the group consisting of sulforaphane or sulforaphene.

In particular, the cruciferous extraction process comprises the following stages: treatment of the cruciferous plant, advantageously lyophilized, with a water-miscible solvent or a water-solvent mixture, advantageously acetone,

- Concentration of the solution obtained, advantageously under reduced pressure, - Filtration of the product obtained,

- Treatment with silver nitrate at 0 ° C,

- Filtration of the complex silver precipitate thus formed,

- Displacement of this complex by sodium thiosulfate,

- Extraction of the suspension obtained with an organic solvent immiscible with water, advantageously chosen from the group consisting of chloroform, ether, ethyl acetate or their mixture, even more advantageously an ethyl ether mixture -chloroform,

- Drying of the organic phase,

- Possibly purification of the product obtained, in particular by plate chromatography.

The following examples of the preparation of sulphoraphane and sulphoraphene by cruciferous extraction are given as a non-limiting indication.

Example 1. Preparation of sulforaphane:

90 g of freeze-dried broccoli (Brassica oleracea italica) are treated with three reflux decoctions in 75% acetone. The extracting solutions are combined and concentrated under reduced pressure up to 100 g. We filter on paper. The filtrate is placed at 0 ° C. and 100 ml of a 60% aqueous solution of silver nitrate are added. The precipitate is filtered on sintered glass and rinsed with three times 100 ml of distilled water. The precipitate is then treated with 100 ml of a 60% aqueous solution of sodium thiosulfate and the mixture is left to act at 0 ° C. with stirring for two hours.

The suspension obtained is then extracted into a separatory funnel with six times 50 ml of an ethyl ether-chloroform mixture (8/2 v / v). The organic phase is dried over sodium sulfate and then evaporated under reduced pressure. 32 mg of crude sulforaphane are obtained. The residue is deposited on a preparative chromatography plate of silica gel and eluted with a mixture of isopropanol and methanol (7/3 v / v).

The plaque is revealed by ammoniacal silver nitrate on a small part in order to determine the migration zone of the sulforaphane. This area is scratched and the sulforaphane is extracted from the silica with chloroform. The chloroform is evaporated and 9 mg of sulforaphane are obtained. Sulforaphane is identified by gas chromatography coupled to a mass spectrometer.

Example 2: preparation of sulforaphene: The procedure is the same as on broccoli but using radish seeds (raphanus sativus).

7 mg of sulforaphene are obtained after purification by plate chromatography.

Example 3: synthesis of (D, L) -sulforaphane:

40 g of 4-chlorobutyronitrile (ref. Aldrich C 3,000-0) are dissolved in 800 ml of absolute ethyl alcohol previously distilled over sodium. 27 g of methane thioate (ref. Fluka 71742) are then added and the mixture is left stirring at 25 ° C. for 15 hours. The suspension is filtered on paper and evaporated under reduced pressure. The residue is taken up in 400 ml of ethyl ether. We filter again on paper. An ethereal solution is obtained containing 32 g of crude 4-methylthiobutyronitrile.

A suspension of 25 g of lithium aluminum hydride in 400 ml of ethyl ether is prepared. The solution of 4-methylthiobutyronitrile is gradually added to the suspension of lithium aluminum hydride, then the mixture is brought to reflux for 2 h 30 min.

The suspension is then neutralized by slowly adding 80 ml of distilled water under reflux. When boiling ceases, 120 ml of distilled water are then added to complete the neutralization of the remaining hydride. Filter on sintered glass. The insoluble material is washed on the filter with 200 ml of ethyl ether. The ethereal fractions are combined and evaporated to dryness. 26.9 g of methylthiobutylamine are obtained. The product obtained is taken up in 80 ml of acetone to which 23 ml of 35% hydrogen peroxide is added little by little. One night is placed in a water bath at 50 ° C.

Then a little activated carbon is added, filtered and 200 ml of chloroform containing 20 ml of thiophosgene are added slowly, followed by 300 ml of a 5% aqueous sodium hydroxide solution. Leave to act for 30 min. The mixture is then extracted against the current with 8 times 200 ml of dichloromethane. The organic phase is collected, dried over sodium sulfate and evaporated.

The residue is then rectified at 135 ° C. under 7.10 ^"2 Torr. 12.5 g of D, L-sulforaphane are obtained, the identity of which is verified by mass spectrometry.

The following examples of measuring the inhibitory power of tyrosinase are given by way of nonlimiting indication.

Measuring the tyrosinase inhibitory power:

The following reaction is used: L Dopa (L-3,4-dihydroxyphenylalanine, obtained from the company Sigma (ref D-9628)) colorless is oxidized to colored dopachrome absorbing at 475 nm. This reaction is catalyzed by tyrosinase fungal (EC 1.14.18.1, obtained from the company Sigma (ref T-7755)). The kinetics of the reaction are recorded by measuring the optical density (OD) as a function of time at 30 ° C. The compositions of the different solutions used are the following:

PH 6.5 buffer:

Monopotassium phosphate 0.70 g

Disodium phosphate 0.69 g

Distilled water q. s. 100 ml

Substrate solution:

L Dopa 0.35% (w / v) in pH 6.5 buffer solution

Inhibitor solutions: The inhibitor molecules are dissolved directly in the pH 6.5 buffer, in 50% methanol (methanol-distilled water) or in pure methanol depending on their solubility.

The weight-by-volume concentrations of the various inhibitor solutions are: 0.2%, 0.1%, 0.05%, 0.025%, 0.0125%, 0.00625% and 0.00312%.

Enzyme solution:

0.010% (w / v) Tyrosinase in pH 6.5 buffer solution.

The amounts of these different solutions used during the tests are presented in Tables 1 and 2 for each reaction studied: Table 1. Enzyme-substrate reaction

Table 2: Enzyme-substrate-inhibitor reaction

The action of tyrosinase is evaluated by the initial speed of the reaction measured on the D.O.

The initial rates of the reactions without inhibitors (concentration 0) and the rates at the various concentrations tested are plotted on a curve.

The inhibitory power of a molecule is defined as the concentration which reduces the action of tyrosinase by 50%.

The molecules tested as an inhibitor were acquired from the companies Aldrich or Fluka depending on the products, with the exception of sulforaphane and sulforaphene prepared as indicated in examples 1 and 2. The results obtained on the molecules tested are collated in the following table:

Sulforaphane inhibits tyrosinase about 1.5 times more than hydroquinone. It is therefore found that all the isothiocyanates used in the table are superior to hydroquinone and that the most active of them, the benzoylisothiocyanate is approximately 24 times more active than hydroquinone. Thiocyanates have an activity similar to that of hydroquinone.

Test of the depigmenting power of benzoylisothiocyanate and sulforaphane compared to kojic acid and hydroquinone in the pigmented guinea pig.

Piggy-skinned guinea pigs previously mowed, received twice a day, 5 days a week, applications of a glycerin-based cream containing 5% acid kojique (ref. Aldrich 22,046-9) or 5% hydroquinone (ref. Aldrich H 1,790-2) or 5% benzoylisothiocyanate (ref. Aldrich 30,818-8) or 5% synthetic sulforaphane prepared according to Example 3 within 1 to 2 months of treatment. These applications were carried out on circular areas of 2 cm in diameter identified by indelible marking in yellow ink.

After 4 weeks of treatment and after a period of peeling of the skin at the hydroquinone and benzoylisothiocyanate spots, there is a significant whitening of the skin for all the products except for kojic acid which has no effect.

Testing the inhibition power of melanin synthesis on melanocytes in culture by sulforaphane compared to kojic acid

Hydroquinone cannot be used as a reference product in this test because of its excessive cytotoxicity. These tests were carried out on batches of different cells during independent and repeated experiments (8 times).

The synthesis of melanin in kinetics or after 6 days of treatment (once a day) was tested; the viability of the cells is checked by MTT and / or by staining with crystal violet.

The results are expressed in mg / ml of melanin insofar as the cells are seeded at the same concentration.

At a concentration of 0.00035%, hydroquinone and sulforaphane inhibit the synthesis of melanin while kojic acid and benzoylisothiocyanate no longer have an effect. Out of all the tests (8 independent tests), we obtained a concentration of 0.00035% for all the products: 26.22% inhibition for sulforaphane, 1% for kojic acid, 9.5 % for benzoylisothiocyanate and 42% for hydroquinone. Test of the inhibition power of melanin synthesis on pigmented human skin reconstituted in culture:

Type 6 pigmented epidemics (corresponding to black skin), three samples per test were treated daily for 5 days with a control cream with the following composition (in% by weight):

Kojic acid at a concentration of 3.5.10 ^"5 (w / w) and sulforaphane at a concentration of 3.5.10 ^{" 6} (w / w) in the same cream serving as an excipient. At the end of the treatment, the skins are left in culture for a further two days and the melanin is extracted from the treated skins and the control skins with a mixture of solvents and sodium hydroxide (Solvable® from Packard Bioscience BV) and quantify the melanin extracted by colorimetry according to a method described above (Chemical Characterization of Hoir Melanins in Various Coat-Color Mutants of Mice; Hiroyuki Ozeki et al, J. Invest. Dermatol. 105, 3, 1995, 361 -366).

It is found that kojic acid inhibits the synthesis of melatonin by 8% and sulforaphane by 30% although its concentration is ten times lower than that of kojic acid.

Claims

1. Use of an isothiocyanate of general formula I below:

Rι-N = C = SI in which R ₁ represents an alkyl, alkenyl, alkynyl, aryl, acetyl, alkylcarbonyl, alkoxy, cycloalkyl, aryloxy, arylcarbonyl, carboxylic acid, carboxylic ester or a group - (CH ₂ ) _n R ₃ in which n represents an integer ranging from 1 to 5 and R represents a polar functional group, advantageously a halogen atom, a sulfoxide, carbonyl, nitro, thioester, thioether, sulfonyl, sulfinyl, nitrile, nitrile, carboxylic acid, carboxylic ester group , alkylthio or hydroxyl, of a thiocyanate of general formula LT as follows:

R ₂ -S = C = N π in which R ₂ represents an alkyl, alkenyl, alkynyl, aryl, acetyl, alkylcarbonyl, alkoxy, cycloalkyl, aryloxy, arylcarbonyl, carboxylic acid, carboxylic ester or a group - (CH) _n R in which n represents an integer ranging from 1 to 5 and R ₃ represents a polar functional group, advantageously a halogen atom, a sulfoxide group, carbonyl, nitro, thioester, thioether, sulfonyl, sulfinyl, nitrile, carboxylic acid, ester carboxylic, alkylthio or hydroxyl, or mixtures thereof for the manufacture of a medicament or a cosmetic composition intended to inhibit tyrosinase

2. Use according to claim 1, characterized in that the thiocyanate of general formula II is in the form of a salt, advantageously sodium or potassium.

3. Use according to claim 1, characterized in that the isothiocyanate of general formula I is obtained by extraction of a crucifer, advantageously chosen from the group consisting of broccoli, Lepidium dabra and radish.

4. Use according to claim 3, characterized in that the isothiocyanate is chosen from the group consisting of sulforaphane or sulforaphene.

5. Use according to claim 1 characterized in that the isothiocyanate of general formula I is a synthetic isothiocyanate selected from the group consisting of cyclohexylisothiocyanate, benzylisothiocyanate, acetylisothiocyanate and benzoylisothiocyanate.

6. Use according to one of the preceding claims for the manufacture of a medicament or a cosmetic composition intended to lighten or depigment the epidermis or to remove age spots.