Patent Application
20170216179
The invention relates to a cosmetic and/or dermatological composition to be used for colouring the skin.
The benefits of sunlight are well known: it boosts morale and promotes the synthesis of vitamin D. However, we also know that any unprotected sun exposure is a danger to the skin. That is why it has been ensured that it is no longer necessary to sunbathe or to conduct irradiation sessions with ultraviolet rays in appropriate cabins in order to obtain a tanned skin. Therefore, products have been proposed to preserve the “sun resistance” of the human body, i.e. the amount of radiation that it can withstand without irreversible damage.
To meet this demand of the cosmetics industry, many topical solutions of self-tanning products such as dihydroxyacetone (DHA) or erythrulose, have been proposed, which allow obtaining a tanned skin without exposure to harmful radiations. The coloration obtained results from the combination of the self-tanning product and the amino acids making up the proteins of the corneocytes of the stratum corneum of the skin according to the Maillard reaction (non-enzymatic reaction) inducing the formation of coloured products on the surface of the skin. These colorations appear after a few hours but disappear pretty quickly and are not generally evenly distributed on the skin.
Tanning by solar radiation generates, in the superficial layers of the skin, an increase of melanin, which is not the case with the current self-tanning products; the result is a significant difference of colorations obtained by the self-tanning products, even when homogeneous, from those obtained under the effect of sun radiation.
Moreover, it is known that tanning due to the increase of melanin in all the surface layers of the skin, reduces the impact of skin burns of subjects who have already suffered excessive exposure. It was determined that, under the effect of ultraviolet rays, keratinocytes secrete the αMSH hormone (melanocyte-stimulating hormone), which binds to melanocytes via receptors, and results in an increase in intracellular cyclic AMP and stimulates melanin production. Melanin is stored in vesicular structures called melanosomes, which mature in melanocytes and are transported in the multiple indentations of the melanocytes (see Nature Reviews, Molecular Cell Biology, Vol. 2, October 2001, p. 1-11). The melanosomes thus transported are transferred up to the skin keratinocytes, allowing for the migration of melanin in the successive layers of the skin up to the stratum corneum and therefore to skin tanning. Therefore, it appears that to increase skin tanning, it is best to stimulate the natural production of melanin and to act on the transfer of melanosomes from melanocytes to keratinocytes.
Regarding the first process mentioned above, it has already been proposed to apply, topically to the skin, a cosmetic milk rich in glycyrrhetinic acid. However, the effect of such a product is limited because melanogenesis stimulation is very poor, if any, in an in vitro model (Jung et al, Exp. Mol. Med. 2001, 33, 131). It was also proposed to increase the synthesis of melanin using a subcutaneous implant consisting of an analogue of the α-MSH hormone mentioned above, namely afamelanotide, but this implant implementation cannot be considered compatible with normal use in cosmetics: it is in fact a medical device, particularly useful for treating vitiligo.
Patent application WO 03/089449 describes novel derivatives of sterols; this patent application indicates that the new compounds it describes tend to redifferentiate tumour cells, which are dedifferentiated during their malignant transformation. Therefore, a person skilled in the art deduces that these compounds have an action that has no interest if one tries to act on a normal differentiated cell; therefore, he/she would not attempt to use them in cosmetics to obtain skin tanning with no malignant transformation, although it has been mentioned that some of these derivatives were efficient in stimulating melanin secretion in melanoma cells (de Medina et al. J. Med. Chem. 2009, 52, 7765). However, it has now been found, according to the invention, that if the compounds mentioned above were used in topically delivered compositions and despite the skin being a major obstacle to the penetration of active molecules, surprisingly a significant improvement of skin tanning would be obtained thanks to significant penetration of some molecules of patent application WO 03/089449, their stability after penetration and their photostability after sun exposure.
However, to improve skin penetration of these sterol derivatives, it was also proposed according to the invention, the implementation of a catanionic combination, according to patent application WO 2011/039379, in which the sterol derivative mentioned above is combined with a surfactant, the combination between the two molecules being obtained by an acid-base reaction and the active ingredient being an amphiphilic counter-ion having therapeutic properties. The carrier/active ingredient combination is bound by electrostatic interactions, allowing for an easy release of the active ingredient in tissues and spreading of the cosmetic effect by a delay related to the separation of the active ingredient. In other words, the carrier may play a triple role: facilitating the passage of the active component, having its own cosmetic effect on skin cells and having a delayed action for the release of the active ingredient. The carrier/active ingredient assembly allows for the combination of a positive ionization group of the active ingredient with the acid group of the carrier. Therefore, the active ingredient can be combined with the carrier by the R3 substituent carried by carbon 6 of the cholesterol skeleton of the compound of formula (I) defined below.
Moreover, it should be highlighted that the compounds described in patent application WO 03/089449 have anticancer activity and that those of them which, according to the invention, may allow some tanning as indicated above, are present in skin areas subjected to sun exposure, and are, therefore, in place in irradiated sites where damage could occur, which would then allow them to play their anticancer role if such were the case.
Therefore, an object of the present invention relates to a topically applicable cosmetic composition to ensure skin tanning of a human subject, with or without irradiation under ultraviolet rays, characterized in that it contains, in a cosmetically acceptable vehicle, an aqueous solution of at least one compound of formula (I):
wherein
R1 designates a hydrogen atom or R10—CO, where R10 represents H, CH3 or C2H5,
R2 represents hydrogen or OH,
R3 represents a group X—(CH2)n—Y, where n is an integer from 1 to 4, X represents S, O or NH, and Y represents NH2, imidazol-5-yl, indol-3-yl, piperidin-2-yl, piperidin-3-yl, piperidyn-4-yl, piperazin-2-yl, piperazin-3-yl, pyridin-2-yl, pyridin-3-yl or pyridin-4-yl,
R4 represents hydrogen or OH in position 20, 22, 24, 25, 26 or 27 of the ring shown above to define formula (I), where OH is positioned so as to obtain an R or S asymmetric centre,
Z1 and Z2 represent the possibility of having single or double bonds between the C7 and C8 carbons, on the one hand, and the C22 and C23 carbons, on the other hand,
T1, T2, T3 are, independently from one another, H or CH3 in α or β, T4 is H, CH3 or C2H5 positioned to form an R or S asymmetric centre.
According to a first aspect of the invention, the composition contains a mixture of both compounds of formula (I) wherein R1=R4=T1=T2=T3=H, R2=OH, R3=NH—(CH2)2-imidazol-5-yl and T4=CH3 or C2H5.
According to another aspect of the invention, the composition contains from 0.01 to 500 g/L of compound(s) of formula (I).
According to another aspect of the invention, the composition contains at least one tanning compound other than the compound(s) of formula (I).
According to another aspect of the invention, the tanning compound(s) it contains, independently from the compound(s) of formula (I), is dihydroxyacetone and/or erythrulose and/or at least one water-soluble dye.
According to another aspect of the invention, the composition contains at least one melanogenesis activator compound other than the compound(s) of formula (I) selected from the group made up of substrates for melanin biosynthesis and melanogenesis biological activators able to act by stimulating the synthesis of melanin and/or the transfer of melanosomes from melanocytes to keratinocytes.
According to another aspect of the invention, when the composition contains one (or more) melanogenesis activator(s) independently of the compound(s) of formula (I), said activator(s) is (or are) chosen from the group made up of L-tyrosine or derivatives thereof such as N-acetyl-L-tyrosine, L-dihydrophenylalanine, aliphatic diols such as propylene glycol, cyclic diols, adenosine-1 receptor agonists, adenosine-2 receptor agonists, α-hydroxyacids and derivatives thereof, β-hydroxyacids and derivatives thereof, retinoids and derivatives thereof, pro-opiomelanocortic peptides, α-MSH or its analogs, MC1R receptor agonists, cAMP analogs, psoralens, PAR-2 receptor activity activators.
According to another aspect of the invention, the composition is applied to the skin in an amount between 0.0001 and 100 mg/cm2/day of compound(s) of formula (I).
As previously stated, the invention also provides improvement of the transport of the compound(s) of formula (I) to the skin keratinocytes by combining this (these) compound(s) with at least one acidic amphiphilic carrier (T), to form catanionic mixture complexes by electrostatic interactions.
According to another aspect of the invention, the composition, therefore, contains an ionic solution of catanionic mixture complexes, each mixture is formed by a molecule of formula (I) and one or two molecules of an amphiphilic acid carrier (T).
According to another aspect of the invention, a catanionic mixture is obtained by at least one acid-base reaction carried out between the acid group of a carrier (T) and an amino group of substituent R3 of carbon in position 6 of the compound of formula (I).
According to another aspect of the invention, the carrier (T) is a bolaform surfactant derived from sugar comprising an acid function within its structure.
According to another aspect of the invention, the carrier (T) acid function is selected from the group made up of carboxylic acid COOH, sulfuric acid O—SO3H, sulfonic acid SO3H, phosphoric acid O—P(O) (R5O)OH, wherein R5 is a C1-C6 alkyl group, phosphonic acid O—P(O)R6OH and phosphinic acid P(O)R6OH, wherein R6 is H or a C1-C6 alkyl group.
According to another aspect of the invention, the sugar derivative is a monosaccharide or polysaccharide derivative.
According to another aspect of the invention, the sugar derivative contained in the composition is a glucose or lactose derivative.
According to another aspect of the invention, the sugar acid derivative comprises a sugar structure on which a linear or branched aliphatic chain (CH2)p is bonded by a NH group of which the end opposite to the sugar structure carries an acid group, p being an integer having a value from 4 to 10.
According to another aspect of the invention, the ratio of the weight amounts A of compound(s) of formula (I) and B of carrier(s) (T) is between 0.001 and 1000.
According to another aspect of the invention, the composition contains from 0.01 to 150 g of the totality of components A and B per litre of composition.
According to another aspect of the invention, the composition comprises at least one adjuvant selected from the group made up of at least one thickener and/or water-soluble dye and/or fragrance and/or alcohol and/or vitamin and/or emulsifier, used alone or in combination with optionally a co-emulsifier and/or oil or another fat and/or preservative and/or antioxidant and/or bactericide.
According to another aspect of the invention, the composition contains at least one chemical or physical sun filter, or a combination of such filters.
According to another aspect of the invention, the composition is packaged as nanocapsules or nanosized catanionic vesicles suspended in a liquid medium.
According to another aspect of the invention, the composition is packaged in the form of hydrogels, creams, lotions, emulsions or aerosols.
To better understand the aim of the invention, several embodiments will be described now as purely illustrative and non-limitative examples. The results of the examples detailed below have been shown in the drawings.
In the drawings:
The process described below is extracted from the published patent application WO03/089449. The compound (hereinafter referred to as DDA) corresponds to the systematic name: 5α-hydroxy-6β-[2-(1H-imidazol-4-yl)ethylamino]cholestan-3β-ol.
Meta-chloro-peroxybenzoic acid (0.73 g, 4.25 mmol, purity of 70-75% by weight) was dissolved in methylene chloride (10 ml) and added dropwise, under stirring, to a mixture of cholesterol (1 g, 2.5 mmol) dissolved in methylene chloride (25 ml). Stirring was maintained overnight. The reaction mixture was washed with aqueous sodium sulfite solution (10% by weight) and sodium hydrogen carbonate (5% by weight) and a saturated solution of a mixture of sodium chloride and potassium chloride. The organic phase was dried over anhydrous magnesium sulfate. Vacuum evaporation of the organic solvent gave 0.7 g of white needles (70% yield).
The proportion of α and β epoxide isomers was determined by proton NMR: 78% of α-epoxide, 22% of β-epoxide were found.
Characterization of the product: 1H NMR (200 MHz, MeOD) δ ppm 2.89 (d, 1H, J=4.37 Hz, H-6); 3.04 (d, J=2.43 Hz, H-6) 3.91 (m, 1H, H-3); MS DCI/NH3MH+ 403] DCI/MNH4+, m/z: 403 [MNH4]+. The α and β isomers were separated by liquid chromatography on silica (toluene/ethyl ether 85/15). The α isomer had a melting point Mp=141-142° C.; the β-isomer had a melting point Mp=131-132° C.
To complete characterization, thin layer chromatography was performed (ethyl acetate); we obtained: Rf=0.69 (brown colour after developing with a mixture of sulfuric acid-methanol).
b) Synthesis of the amino sterol from the α-epoxy-sterol obtained in step a)
Lithium perchlorate (0.75 mmol) and an amine in its basic form (1 mmol), namely, 2-(1H-imidazol-4-yl)-ethylamine, were dissolved in anhydrous ethanol (1 ml). This solution was added under an argon stream to an ethanol solution (3 ml) of the α-epoxy-sterol obtained according to a) above (100 mg, 0.25 mmol). The reaction mixture was stirred and refluxed for 6 days. Progress of the reaction was monitored by thin layer chromatography (TLC). The solvent was removed by evaporation and the residue was washed with ethyl ether (5×3 ml) and hexane (5×20 ml). The residue was dissolved in water and acidified with 2M HCl (2 ml).
The solution was pre-purified on a grafted silica cartridge (“sep-pack cartridge C18 RP”, 500 mg, Waters); excess polyamine was removed by flushing water on the cartridge (5 ml); the product was eluted with 1/1 CH3CN/H2O (5 ml). The product was purified by reverse phase HPLC using a linear gradient of a starting mixture of 95/5/0.1 H2O/CH3CN/TFA to 95/5/0.1 CH3CN/H2O/TFA achieved over 60 minutes (flow rate=1 ml/min; X=210 nm). The fraction of interest was purified again under isocratic conditions using a mobile phase consisting of a mixture of 44% of (95/5/0.1 CH3CN/H2O/TFA) 56% of (95/5/0.1 H2O/CH3CN/TFA) (flow rate=1 ml/min; λ=210 nm).
The product obtained was first characterized by TLC (methanol). Then high performance chromatography (HPLC) was performed on a “Perkin-Elmer LC200 Series” device equipped with “Ultrasep ES100RP10” column (6 μm particles), 250 mm length and 8 mm diameter, manufactured by “Bischoff”.
HPLC profile: 44% B #=220 nm rt=44-50 min
Characterization of the product: ESI-MS, m/z 514.5 [M+H]+.
A mixture of sitosterol and campesterol (70/30 by weight) was used as sterol, in step a) of Example 1. For the reaction on the obtained α-epoxysterol, the same amine as in Example 1 step b) was used.
A mixture of the 2 following compounds was obtained:
5α-hydroxy-6β-[2-(1H-imidazol-4-yl)ethylamino]sitostan-3β-ol and
5α-hydroxy-6β-[2-(1H-imidazol-4-yl)ethylamino]campestan-3β-ol.
The product obtained (hereinafter referred to as “AF130”) was characterized by TLC (ethyl acetate): Rf=0.69
Characterization of the product:
ESI-HRMS m/z: 528.8323 [M+H]+(Δm=0.3 mDa) (campesterol derivative) and 524.8590 [M+H]+(Δm=0.4 mDa) (sitosterol derivative)
a) Preparation of the amphiphilic carrier used to prepare the catanionic mixture complex implemented in the subsequent examples. Lactobionic acid was directly reacted with 8-aminooctanoic acid (see Patent Publication WO 2011/039379).
1) Synthesis of sodium 8-lactobionamidooctanoate
To a solution of sodium hydroxide (300 mg, 5.59 mmol) in 50 mL of methanol were successively added 8-aminooctanoic acid (888 mg, 5.59 mmol) and lactobionic acid (2.00 g, 5.59 mmol). The mixture was stirred at 50° C. for 24 h; the solvent was then evaporated under reduced pressure; the residue obtained was purified by chromatography column on silica gel (eluent: CHCl3/CH3OH/H2O: 2.5/7/0.5, Rf=0.8). A white solid was collected at a yield of 56%.
Characterization of the product:
IR(neat) cm−1: 1643 (vc=o (amide)st);
1547 (vc=o (carboxylate) asymmetric);
1405 (vc=o (carboxylate) symmetric)
ESI-MS, m/z: 498 [M+Na]+.
The product obtained has the following formula:
2) Synthesis of 8-lactobionamidooctanoic acid
To a solution of sodium 8-lactobionamidooctanoate (1.00 g, 2 mmol) in 30 mL of H2O was added approximately 2 g of proton exchange resin (Dowex 50WX8). The reaction mixture was stirred for 2 h at room temperature, then filtered and concentrated. The residue obtained was purified by column chromatography on silica gel (eluent: acetone/H2O: 9/1; Rf: 0.5). A white solid was collected at a yield of 57%.
Characterization of the product:
1H NMR (300 MHz, MeOD) δ ppm: 1.05-2.15 (m, 10H, CH2β, CH2γ, CH2δ, CH2ε, CH2ζ); 2.17 (t, 2H, CH2α); 3.12 (m, 2H, CH2η); 3.46-4.12 (m, 10H, CH and CH2 of the sugar unit); 4.28 (m, 1H, H) 4.30 (m, 1H, H alpha of CONH); 4.54 (d, J=9 Hz, 1′H, anomeric H)
IR(neat) cm−1: 1645 (vc=o (amide) st);
1726 (vc=o (acid) st)
ESI-HRMS, m/z: 522.2162 [M+Na]+ (Δm=0.1 mDa).
The product obtained is called L7 and is represented by the following formula:
To a solution of 8-lactobionamidooctanoic acid (330 mg 0.66 mmol) in 30 mL of deionized H2O was added 339 mg of the product of Example 1 (0.66 mmol). The reaction mixture was then stirred at room temperature for 24 h. This gave a homogeneous solution which, after concentration, quantitatively gave the product represented by the below formula:
The resulting product is a pale yellow powder; it is hereinafter referred to as L7DDA as it consists of a L7 molecule combined with a DDA molecule.
Characterization of the product obtained:
1H NMR (300 MHz, MeOD) 5 ppm: 0.75 (s, 3H, CH3(19)); 0.89 (m, 3H, CH3); 0.91 (m, 3H, CH3); 1.05-2.15 (m, 45H, CH2β, CH2γ, CH2δ, CH2ε, CH2ζ, 35H for CH and CH2 of DDA); 2.21 (t, 2H, CH2α); 3.14 (m, 2H, CH2η); 3.42-4.10 (m, 12H, CH and CH2 of the sugar unit, +CH3 and CH6); 4.21 (m, 1H, H); 4.34 (m, 1H, H alpha to CONH); 4.50 (d, J=9 Hz, 1H, anomeric H); 6.92 (s, 1H, C=CH(NH); 7.80 (s, 1H, HN—CH=N).
The NMR spectrum is provided in
IR(neat) cm−1: 1643 (vc=o (amide) st); 1549 (vc=o (asymmetric carboxylate)); 1403 (vc=o (symmetric carboxylate)).
The IR spectrum, provided in
CAC (Critical Aggregation Concentration) (aqueous solution, 25° C.): 2.6×10−5M.
The same process as for step b) of Example 3 can be used to form a combination complex between an amphiphilic acid carrier according to Example 3a2) and any compounds of formula (I) as all these compounds comprise at least one NH group allowing bonding with the carrier acid function. In particular, a complex mixture was obtained by combining the product of Example 2 with that of Example 3a2): this mixture was hereinafter named L7AF130.
The carrier/active component assembly allows to combine a positive ionization group of the active component with the acid group of the carrier. Therefore, the active component can be combined to the carrier by the R3 substituent carried by carbon 6 of the compound of formula (I). In the case of AF130 or DDA active components, we can combine two L7 carrier molecules on two amino functions of the R3 substituent. From the process for obtaining Example 3b, mix in water at room temperature, two equivalents of L7 molecule for one equivalent of active component, as described for example 3b. Once again, the mixture was initially a suspension but became a clear solution, indicating that the resulting complex, hereinafter named (L7)2(AF130) or (L7)2(DDA) depending on the active component, was water soluble.
Quantification of synthesized melanin by healthy murine melanocytes was performed according to the protocol described by Ando et al. (J. Lipid Res., 1999, 40, 1312).
Cells were seeded and treated for 24 h with the test product or the used vehicle (ethanol) and with, as a positive control, exposure to a dose of 20 mJ/cm2 of ultraviolet radiation.
5 Million cells were isolated after centrifugation at 1500 rpm for 5 min at 4° C. The pellet was washed twice with PBS and then transferred into an Eppendorf tube and centrifuged at 5000 g for 5 min. The supernatant was removed. 200 μL of water and 1 mL of EtOH/ether (1/1) were added (melanin was insoluble in this mixture). After 15 min at room temperature, the totality was again centrifuged at 5000 g for 5 min; the supernatant was removed. The pellet was then taken up with 300 μL of a solution of NaOH(1M) in H2O/DMSO 90/10 and placed at 80° C. for one hour. Absorbance was measured at 470 nm. The amount of synthesized melanin was expressed based on the control (=100%).
The results are shown in
The study was conducted on healthy murine melanocytes. The results were compared with those obtained with a positive control subjected to a dose of 20 mJ/cm2 of ultraviolet radiation. The cells were treated for 24 h with either the vehicle used (ethanol) or with the test product. The tyrosinase activity was quantified according to the protocol described by Ando et al. (J. Lipid Res. 1999, 40, 1312) as explained below.
5 Million cells were isolated after centrifugation at 1500 rpm for 5 min at 4° C. The pellet was washed twice with PBS and then transferred into an Eppendorf tube and centrifuged at 5000 g for 5 min. The supernatant was removed. 1 mL of 0.5% by mass sodium deoxycholate solution was added (cell lysis). After 15 min at 0° C., 3 mL of a solution of 0.1% by mass of L-DOPA in 0.1M phosphate buffer (pH 6.8) were added. The whole was placed at 37° C. for 10 min. Tyrosinase activity was spectrophotometrically analysed by following the oxidation of L-DOPA to DOPAchrome. Absorbance was measured at 475 nm. The tyrosinase activity was expressed based on the control (=100%), which determines the cell basal tyrosinase activity.
The results are illustrated in
Healthy murine melanocytes were treated as described in Example 6 by using untreated cells or cells treated with 1 μM of AF130 or 1 μM of (L7)2AF130. The melanin synthesis was visualized by staining according to the DAPI technique: the nuclei of cells fluoresce in blue and, by contrast, melanosomes are highlighted, which are visualized in black.
The DAPI technique is performed as follows: cells are seeded on previously sterilized slides by washing with ethanol; they are treated with the studied active molecule or with a vehicle (ethanol).
After 24 hours of treatment, cells were fixed using a formaldehyde solution diluted to one tenth (3.7% by weight) in PBS for 15 min at room temperature. Formaldehyde was then removed and the cells were washed with PBS. 10 μl of a DAPI solution to 1/500th in Mowiol (mounting solution) were deposited on a slide. The cover slip was placed on the drop, cells being present between the slide and the cover slip. The slides were placed at 4° C. overnight before being observed. For each sample, an image in “DAPI” mode was performed, only allowing visualization of nuclei as well as the same image in “visible” mode. The two images were then superimposed.
This example shows in healthy murine melanocytes, that treatment with compositions according to the invention allows to increase the expression of genes corresponding to tyrosinase and TRP-1 and TRP-2 enzymes (TRP=Tyrosinase Related Protein), which are involved in melanogenesis.
Cells were seeded and treated for 24 h with the molecules the action of which we want to study, that is to say here, with DDA (1 μM), on the one hand, and with L7DDA (0.1 or 1 or 2.5 or 5 μM) on the other. The results were compared with irradiated ultraviolet cells as in Example 6.
To quantify the expression of these genes, RNA extraction from cells was performed. Melanocytes were treated for 24 h with the active molecule or the control agent. 1 mL of Trizol® was added; after 5 min at room temperature, the totality was placed in an Eppendorf tube. 200 μL of CHCl3 were added at 4° C. The Eppendorf tubes were stirred manually, by turning them during 15 sec, left for 5 min at room temperature and then centrifuged at 16000 g at 4° C. for 10 min. The colourless aqueous phase was isolated. 500 μL of isopropanol (−20° C.) were added. The Eppendorf tubes were stirred manually, by turning them during 15 sec, left for 15 min at room temperature and then centrifuged at 16200 g at 4° C. for 10 min. The supernatant was removed. The pellet was washed with 1 mL of 75% ethanol (−20° C.). After centrifugation at 16000 g at 4° C. for 5 min, the supernatant was removed. 10 min after drying, the pellet was resuspended with 30 μL of RNAase-free water. The quantity and purity of RNA were estimated by spectrophotometry (230, 260 and 280 nm).
A RT-qPCR was performed; in an Eppendorf tube, were added: 1 μL of reverse transcriptase, 4 μL of 5× reaction mixture “iScript®5×”, the amount of RNAase-free water needed to reach a total volume of 20 μL and the corresponding volume for 1 μg of RNA. The Eppendorf tubes were then vortexed, centrifuged for a few seconds and then placed in the thermocycler (program: 5 min at 25° C., then 30 min at 42° C., then 5 minutes at 85° C. and then back to 4° C.)
An amplification was then performed. The reference genes were: glyceraldehyde 3-phosphate dehydrogenase and Cyclophilin A1. Genes of interest were: Tyrosinase, TRP-1 and TRP-2. For each gene (reference or of interest), a mixture was prepared with RNAase-free water, Sybr Green® and the corresponding primer.
The resulting cDNAs were taken up with 180 μL of RNAase-free water. 5 μL of cDNA were added at the bottom of each well. 20 μL of mixture were added on the edge of each well. The plate was then covered with an adhesive plastic and centrifuged at 4000 rpm until no more bubbles were observed.
The plate was then placed in the thermal cycler: 95° C. for 3 min, followed by 50 amplification cycles of 15 sec at 95° C. and one minute at 60° C. At the end of these 50 cycles, the melting curves were generated at 95° C. for 1 min and 95° C. for 10 sec (80 cycles). One can thus refer, in
It was found that the treatment of cells with DDA and L7DDA products provided results showing that the enzymes involved in melanogenesis are stimulated as much as with UV irradiation.
In this example, the test treatment was carried out on healthy murine melanocytes. Treatments were performed with 1 μM of the DDA compound and also with 1 μM of L7DDA and (L7)2DDA and (L7)2AF130 compounds. The positive control was carried out with cells that had received a dose of 20 mJ/cm2 of UV radiation. The reference protein was actin (43KDa). After 24 hours of treatment, cells were scraped into cold PBS (4° C.) and then centrifuged at 1500 rpm at 4° C. for 5 min. The supernatant was removed.
The pellet was taken up with 100 μL of lysis buffer (0.1M of TRIS-HCl, pH=7.4, 1% Igepal, 0.01% SDS, to which mixture were added protease inhibitors (1% by volume of the mixture)) and vortexed for several seconds. After 30 min at 4° C., the mixture was vortexed again and subjected to ultrasound for 5 sec. The Eppendorf tubes were finally centrifuged at 10000 rpm at 4° C. for 10 min. The supernatant (=protein extract) was transferred to a new Eppendorf tube. A Bradford assay was performed to quantify the proteins.
Based on the results of this assay, samples were prepared for deposit on SDS-PAGE gel (samples prepared at 1 μg/μL): 10 μL, i.e. 10 μg, of proteins were deposited for each sample.
After gel transfer in liquid phase of the proteins on a polyvinylidene fluoride membrane, membranes were incubated with the primary antibodies of interest (Tyrosinase: 1/2000th, Actin: 1/10000th, TRP-2: 1/2000th) and then with the secondary antibody directed against the gene of interest (in all cases, at 1/1000th), coupled with HRP (horseradish peroxidase). Finally, after an incubation of 5 min with 1 ml of ECL (enhanced chemiluminescence)/membrane, the membranes were developed in a darkroom. This information was also used to perform the dosages as described in Example 11 below (results in
The result of the Western Blot thus preformed is shown in
In this example, test treatment was carried out on “RHE/MEL light tanned” reconstituted skin epidermis marketed by the “StratiCELL®” company. The medium of the epidermis was changed every day before the test treatment. The treatment consists of adding 1 μM of the treatment product in the medium, twice a day for five days. The epidermi were isolated and placed in an Eppendorf tube with 400 μL of “Solvable” reagent (supplied by Perkin-Elmer) and then heated at 100° C. for 1 h. Absorbance was measured at 490 nm. The amount of melanin synthesized was expressed based on the control (=100%). The results are shown in
“RHE/MEL light tanned” reconstituted skin samples marketed by the “StratiCELL®” company were used. The sample was treated according to the procedure of Example 9 with the same UV positive control and the same control with actin. The sample was treated twice a day by action of 1 μM of DDA, L7DDA, (L7)2DDA and (L7)2AF130 products.
Details of the method used have already been provided in Example 9 (GP100: 1/500th, TRP-1 1/1000th, Rab27a: 1/500th).
The results are shown in
Examples 12-22 describe compositions according to the invention used for application on human reconstituted skin such as those used in Examples 10 and 11. The compounds are, depending on the case, described with their chemical names or INCI names (International Nomenclature of Cosmetic Ingredients) and the quantities indicated are percentages by weight (percentage of active material for the DDA and catanionic mixtures). Models of reconstituted skin were treated twice a day for three days with 2 mg/cm2 of the formulations described below (Examples 12-22). The results are illustrated in
Preparation of aqueous phase A:
Preparation of oil phase B:
Phase B was heated at 80° C. and phase A at the same temperature. Phase B was slowly introduced while under fast stirring into phase A. The mixture was homogenized and cooled under slow stirring: phase C was added when the temperature was below 40° C.
Preparation of aqueous phase A:
Preparation of oil phase B:
Phase B was heated at 70° C. and phase A at the same temperature. Phase B was slowly introduced while under fast stirring into phase A. The mixture was homogenized and cooled under slow stirring. Phase C was added when the temperature was below 40° C.
Preparation of phase A:
Passiflora incarnata (sold under the name “Cegesoft
Preparation of phase B:
Phase B was introduced into phase A under stirring and homogenized.
In all cases corresponding to Examples 12-22, the skin samples show a tanned, uniform and durable natural coloration. The self-tanning effect intensities are obtained by the Fontana-Masson coloration of histological sections and are summarized in the table below.
| Number | Date | Country | Kind |
|---|---|---|---|
| 14 00746 | Mar 2014 | FR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/FR2015/000065 | 3/24/2015 | WO | 00 |
