FR3099057A1 - Inhibitor of the LAT-1 / SLC7A5 transporter for its use for the regulation of skin pigmentation and a composition comprising said inhibitor - Google Patents

Abstract

Inhibitor of the LAT-1 / SLC7A5 transporter for its use in the regulation of skin pigmentation and a composition comprising said inhibitor The invention relates to an inhibitor of the LAT-1 / SLC7A5 membrane transporter for its use in the regulation of skin pigmentation. According to another aspect, the invention relates to a composition for its use for the regulation of skin pigmentation comprising an effective amount of an inhibitor of the membrane transporter LAT-1 / SLC7A5 or of a cosmetically or pharmaceutically acceptable salt thereof. It finds a particularly advantageous application in the field of skin care and more particularly for the regulation of skin pigmentation including skin depigmentation or skin bleaching.

Description

LAT-1/SLC7A5 transporter inhibitor for its use in regulating skin pigmentation and a composition comprising said inhibitor

The invention relates to the field of cosmetics and/or dermatology. It finds a particularly advantageous application in the field of skin care and more particularly for the regulation of skin pigmentation, including skin depigmentation or else skin whitening.

The regulation of cutaneous pigmentation, and in particular depigmentation of the skin or whitening, may be desired in various circumstances, either for an overall lightening of the latter or either to eliminate or attenuate spots caused by local disorders of pigmentation. in the context of cosmetics, or even to treat hyperpigmentations in the context of dermatology.

The skin has several integrated layers, ranging from the superficial layer, the epidermis (epithelial tissue), to the deeper layers, the dermis and the hypodermis (connective tissue), and each has specific properties allowing the whole to react and adapt to the conditions of its environment.

Skin pigmentation results from the presence of melanin in the epidermis and dermis. Melanin is produced by melanocytes located mainly in the basal layer.

There are pathologies associated with poor functioning of melanocytes causing hyperpigmentation such as age spots (actinic lentigo) or melasma. These pigment defects do not endanger the lives of those affected, but have a significant psychological impact.

Many products are currently available on the market to alleviate these hyperpigmentation problems. Most of these products include in their formulas one or more ingredients that are now controversial because they are responsible for undesirable effects. Indeed, these products contain ingredients whose safety is severely questioned by the health authorities of different countries or communities.

An object of the present invention is therefore to provide a composition for the regulation of skin pigmentation exhibiting improved safety.

The other objects, features and advantages of the present invention will become apparent from a review of the following description and the accompanying drawings. It is understood that other benefits may be incorporated.

SUMMARY

To achieve this objective, according to one embodiment the present invention provides an inhibitor of the LAT-1 / SLC7A5 transporter for its use in the regulation of skin pigmentation.

Surprisingly, the inventors noticed that the inhibition of the transporter LAT-1/SLC7A5 had an effect on melanogenesis and in particular a reduction in the production of melanin which makes it possible to regulate skin pigmentation.

The use of an inhibitor of this LAT-1/SLC7A5 transporter makes it possible, by limiting, or even blocking, the production of melanin to ensure skin whitening or skin depigmentation.

The LAT-1 /SLC7A5 transporter is known as a protein whose function is the cellular transport of large neutral amino acids, or also called L-type amino acids, such as phenylalanine, tyrosine, leucine, arginine, tryptophan. The LAT-1 transporter is a transmembrane transporter. The LAT-1 transporter is encoded by the SLC7A5 gene.

The LAT-1 transporter is expressed in a large number of cell types ranging from T cells to cancer cells or even in the endothelial cells of the brain.

The involvement of the LAT-1 transporter in melanogenesis has not been documented prior to the present application. Thus, the inhibition of this LAT-1 transporter to regulate skin pigmentation is surprising.

Advantageously, the inhibitor of the LAT-1/SLC7A5 transporter is BCH or 2-amino-2-norbornanecarboxylic acid.

Another aspect of the present invention relates to a composition for its use for the regulation of skin pigmentation comprising an effective amount of an inhibitor of the LAT-1/SLC7A5 transporter or a cosmetically or pharmaceutically acceptable salt thereof.

Another aspect of the present invention relates to an inhibitor of the expression of the SLC7A5 gene for its use for the regulation of skin pigmentation. Advantageously, the inhibitor being an siRNA.

Another aspect of the present invention relates to a method for reducing or inhibiting expression of the SLC7A5 transporter comprising administering an inhibitor of SLC7A5.

The aims, objects, as well as the characteristics and advantages of the invention will emerge better from the detailed description of an embodiment of the latter which is illustrated by the following accompanying drawings in which:

Figures 1 and 2 represent the level of expression of SLC7A5 in B16F10 cells transfected with a control siRNA or two different siRNAs directed against SLC7A5 by quantitative PCR (figure 1), and by Western Blot (figure 2).

FIG. 3 represents the quantification of the level of melanin produced in the B16F10 cells after transfection with a control siRNA or two different siRNAs directed against SLC7A5 and stimulation of the pigmentation with Forskolin and IBMX, in a spectrophotometer at 405 nm.

Figure 4 shows the viability of B16F10 cells after 72 hours of treatment with different doses of BCH.

FIG. 5 represents the quantification of the level of melanin produced in the B16F10 cells after treatment with different doses of BCH and stimulation of the pigmentation with Forskolin and IBMX, in the spectrophotometer at 405 nm.

Figure 6 shows the viability of MNT 1 cells after 72 hours of treatment with different doses of BCH.

FIG. 7 represents the quantification of the level of melanin produced in the MNT1 cells after treatment with two doses of BCH for 4 to 7 days in a spectrophotometer at 405 nm.

FIG. 8 represents the quantification of the melanin level of the reconstructed human pigmented epidermis after treatment with the BCH inhibitor at 5 or 10 mM for 7 or 12 days.

The drawings are given by way of example and do not limit the invention.

Before starting a detailed review of embodiments of the invention, optional characteristics are set out below which may possibly be used in combination or alternatively:

Advantageously, the regulation of skin pigmentation includes depigmentation of the skin.

Advantageously, the regulation of skin pigmentation includes bleaching of the skin.

Advantageously, the composition comprises, as inhibitor of the LAT-1/SLC7A5 transporter, 2-amino-2-norbornanecarboxylic acid (BCH).

Advantageously, the composition is suitable for topical application.

A composition wherein the regulation of skin pigmentation comprises skin whitening and/or skin depigmentation.

• « application topique », le fait d'appliquer ou d'étaler les compositions selon l'invention à la surface de la peau,
• « cosmétiquement ou pharmaceutiquement acceptable », que la composition selon l'invention est appropriée pour entrer en contact avec le corps et plus particulièrement la peau sans provoquer de réactions de toxicité ou d'intolérance.
• « quantité efficace » et « quantité suffisante » les quantités nécessaires soit des constituants soit de la composition qu'il convient d'utiliser pour obtenir une efficacité de régulation de la pigmentation cutanée sur la peau sans engendrer d'effet indésirable.

In the present invention, the following terms mean:

• "topical application", the fact of applying or spreading the compositions according to the invention on the surface of the skin,
• "cosmetically or pharmaceutically acceptable", that the composition according to the invention is suitable for coming into contact with the body and more particularly the skin without causing toxicity or intolerance reactions.
• “effective amount” and “sufficient amount” the necessary amounts either of the constituents or of the composition that should be used to obtain effectiveness in regulating skin pigmentation on the skin without causing any undesirable effect.

The LAT-1/SLC7A5 transporter inhibitor, the SLC7A5 gene expression inhibitor and the composition according to the invention are intended to be used for the regulation of skin pigmentation. The regulation of cutaneous pigmentation is understood as the control of the pigmentation of the skin, including the scalp, and of the hair. The control of pigmentation includes the depigmentation of the skin, in particular of hyper-pigmented areas and/or the bleaching of the skin, in particular to obtain a homogeneous pigmentation of the skin and/or a uniformity of the complexion and/or an attenuation of the spots and/or skin lightening.

A role of LAT-1 and therefore of SLC7A5 in melanogenesis was identified by the inventors by studying the effect of the extinction or reduction of the expression of SLC7A5, according to example 1.

The reduction, advantageously the inhibition of the expression of the SLC7A5 gene is advantageously obtained according to the invention by a technique of small interfering RNA: siRNA silencing. Advantageously, the siRNA is directed against the SLC7A5 gene.

The inhibition of the LAT-1/SLC7A5 transporter is carried out by an inhibitor of said transporter. Preferably, the chosen inhibitor is BCH.

BCH is a molecule with the formula 2-Aminobicyclo[2.2.1]heptane-2-carboxylic acid or 2-amino-2-norbornanecarboxylic acid. BCH is represented below by the formula Chem. 1 below. The CAS number being 20448-79-7.

According to another aspect, the invention relates to a composition for its use for the regulation of skin pigmentation which comprises an inhibitor as described above. Advantageously, the composition comprises an inhibitor as described above or a cosmetically or pharmaceutically, in particular dermatologically, acceptable salt.

Preferably, the composition comprises at least one inhibitor of the LAT-1/SLC7A5 transporter. For example, the composition includes an effective amount of BCH. As a preferred example, the in vitro effective amount of BCH is advantageously less than or equal to 20 mM. The composition according to the invention for its use for regulating skin pigmentation advantageously comprises, as main active principle, an inhibitor of the transporter LAT-1/SLC7A5. Preferably, the main active ingredient is BCH.

According to one possibility, the composition comprises from 0.5% to 20% by weight of inhibitor of the LAT-1/SLC7A5 transporter relative to the total weight of the composition.

According to one possibility, the composition comprises in addition to an inhibitor of the LAT-1/SLC7A5 transporter, such as BCH, a synergistic agent. By depigmenting synergistic agents, mention may be made of hydroquinone, kojic acid, azelaic acid, phytic acid, mandelic acid, vitamin C and its derivatives, vitamin B and its derivatives, vitamin A and its derivatives, extracts plants such as Glycyrrhiza glabra, Morus Alba, resveratrol, undecylenoyl phenylalanine, peptides such as Acetyl Glycyl β-Alanine, Resorcinol derivatives, Hexyl resorcinol, Butyl Resorcinol, Phenylethyl Resorcinol.

Preferably, the composition according to the invention is a cosmetic or dermatological composition. Advantageously, the composition is in a form suitable for topical application.

The composition according to the invention may also contain other ingredients usual in cosmetology or dermatology, such as humectants, preservatives, colorants, perfumes, penetrating agents such as diethylene glycol monoethyl ether, etc.

The composition according to the invention advantageously comprises a suitable vehicle or suitable excipients for external topical administration, in particular excipients that are dermatologically and cosmetologically acceptable. These suitable excipients for the formulation are well known to those skilled in the art and include in particular penetrating agents such as ethoxydiphenol, phytantriol, octyl dodecanol and escin; thickeners such as natural gums and synthetic polymers; emollients and surfactants such as cetearyl octanoate, isopropyl myristate, cetearyl isononanoate, dimethicone, cyclomethicone, polyglyceryl 3-diisostearate, hydrogenated polyisobutene, cetyl alcohol, cetyl palmitate , cetyl phosphate, triglycerides of capric and caprylic acid; emulsifiers such as sorbitan esters, stearic or palmitic acid derivatives, sucro-esters or glucolipids of the myristyl glucoside or stearyl glucoside type; preservatives such as phenoxy-ethanol, methyl parahydroxybenzoate (methylparaben), ethyl parahydroxybenzoate (ethylparaben), propyl parahydroxybenzoate (propylparaben) and Phenonip® combining phenoxyethanol and methyl, ethyl, butyl and isobutyl; a combination with an antimicrobial effect of a lipoamino acid, a hydroxylated fatty acid with 8 to 12 carbon atoms and a glycerine alkyl ether; dyes; the perfumes ; etc

The composition according to the invention is present in all the pharmaceutical forms normally used in the cosmetic and dermatological fields suitable for topical application, for example: serum lotion, emulsion of more or less fluid consistency, white or colored, obtained by dispersion of a fatty phase in an aqueous phase, oil-in-water emulsion (O/W) or conversely water-in-oil emulsion (W/O) of the milk, cream, gel, gel-emulsion type, of a mask or an anhydrous product balm oil, powder. They can also be in the form of a stick.

Among these preparations, creams, fluid or thick gels, or vaporizers are preferred, the application of which is simple and easy on all parts of the body.

The compositions according to the invention are applied topically in a sufficient quantity in humans, that is to say in a quantity corresponding to the usual application doses for the type of composition considered (gel, cream, lotion, etc. ). For example, in the case of a cream for the face, one applies from 0.5 to 3 g, and in particular from 1 to 2 g of cream per day in one or more exposures. For example, in the case of a body cream, 5 to 12 g, and in particular 7 to 10 g of cream, are applied per day in one or more exposures.

Example 1

The effect of the extinction of the expression of SLC7A5 was studied by an RNA silencing approach in a cell culture model of mouse melanoma (B16F10 cells), known for their response to chemical activators of pigmentation (Forskolin and IBMX: cAMP activators). For this, the cells received two different siRNAs directed against SLC7A5 by transfection with the HiPerfect reagent (si#1: Flexitube Qiagen, SI01424885, si#2: ON-TARGET plus SMARTpool L-041166-01). After 24 hours, the pigmentation process was induced by adding the cAMP activators mentioned above. After 48 hours, the cells were observed by microscopy in order to assess their degree of pigmentation. The silencing of the SLC7A5 gene leads to a very strong decrease in pigmentation in these cells, with the two siRNAs used.

Example 2

The expression level of SLC7A5 in response to the two siRNAs was then verified at the RNA level by quantitative PCR (Figure 1) and at the protein level by Western blot (Figure 2).

The two siRNAs used appear effective in decreasing the expression of SLC7A5 both at the RNA (figure 1) and protein (figure 2) levels, confirming that the effect observed on the pigmentation of the cells in example 1 is indeed due to the silencing of SLC7A5.

Example 3

The level of melanin production was then quantified in response to the extinction of SLC7A5 expression (Figure 3). For this, the B16F10 cells were transfected with a control siRNA or two different siRNAs directed against SLC7A5, and received pigmentation activators (Forskolin and IBMX) 24 hours later for 48 hours. The cells were then taken up in a 0.5M NaOH solution to quantify their melanin content by spectrophotometry at 405 nm. For each condition, the amount of melanin was related to the amount of protein present in the cells. It is observed in FIG. 3 that the silencing of SLC7A5 leads to a significant reduction in the production of melanin in the B16F10 cells, and this in a similar manner with the two siRNAs used.

These experiments have therefore made it possible to highlight a role of SLC7A5 in the process of cellular pigmentation, which had not been demonstrated until now.

Example 4

Study of the effect of a specific commercial inhibitor of the LAT-1 / SLC7A5 transporter, named BCH.

First, the effect of this inhibitor was analyzed on the viability of B16F10 cells after treatment with different doses for 72 hours. The viability was measured by spectrophotometry at 490 nm after addition of the Cell Titer reagent (Promega) (FIG. 4). From 20mM, the cell viability decreases to 60% compared to the control. It therefore appears necessary to work at concentrations below 20 mM on this cellular model.

Example 5

Study of the effect of the BCH inhibitor on melanin production in B16F10 cells after 72 hours of treatment for doses up to 10mM. For this, the cells received BCH at 1, 5 or 10 mM for 24 hours, and 24 hours later, cAMP activators (Forskolin and IBMX) were added for 48 hours as before. By observing the cells under the microscope, a visible decrease in pigmentation can be noted depending on the dose of BCH received, from 5 mM, and even more marked with 10 mM. The amount of melanin produced by B16F10 cells in response to cAMP activators is significantly reduced after treatment with 5 mM and 10 mM BCH (figure 5).

The BCH inhibitor, specific for the amino acid transporter SLC7A5, therefore has a significant effect on melanogenesis in B16F10 murine melanoma cells.

Example 6

In order to confirm these results, another model, namely MNT1 human melanoma cells, was studied. These cells have the particularity of being basally very pigmented and constitute a model of choice for studying the effect of depigmenting compounds.

The cell viability of the MNT1 line was first tested in response to treatment with increasing doses of BCH, and this for 72 hours (FIG. 6).

The cells appear resistant to BCH, with a slight decrease in viability from 20 mM, as previously observed with murine B16F10 cells. The doses of 5 mM and 10 mM were therefore used for the remainder of the experiments.

The melanin produced by MNT1 cells in response to BCH administered at 5 or 10 mM for 4 or 7 days of continuous treatment (FIG. 7) was analyzed. These two concentrations make it possible to reduce the production of melanin, in a dose-dependent and time-dependent manner. It is conceivable that longer treatment will have an even stronger effect on blocking melanogenesis.

Example 7

The effect of BCH was then tested on reconstructed human pigmented epidermis of phototype VI. For this, two groups of epidermis were used. In the first group, the epidermis received daily BCH at 5 or 10 mM for 7 days. In the second group, the melanocytes were first pretreated for 5 days with BCH at 5 or 10 mM before tissue reconstruction, and the epidermis thus reconstructed were then treated daily with BCH at 5 or 10 mM for 7 days (i.e. a total of 12 days of treatment). A histological analysis of these tissues makes it possible to appreciate a reduction in melanin by a Fontana Masson marking, showing the grains of melanin in brown, in the two groups of epidermis. This decrease was also observed by quantification of the level of melanin in the tissues at the end of the two treatment periods (FIG. 8).

exe simple 8

In the group where the melanocytes were pretreated with BCH for 5 days, we were able to observe from the first day of treatment of the reconstructed epidermis a decrease in pigmentation with BCH, whether the dose was 5 or 10 mM. This result remains visible after 7 days of treatment of the epidermis with BCH at 5 or 10 mM, the difference in pigmentation being even more marked at this precise time compared to the control. In the other group, the decrease in pigmentation appeared over the days, and seems to be very similar for the two doses of BCH administered.

The invention is not limited to the embodiments described above and extends to all the embodiments covered by the claims.

Claims (8)

LAT-1/SLC7A5 membrane transporter inhibitor for use in regulating skin pigmentation. Inhibitor for its use according to the preceding claim of formula 2-amino-2-norbornanecarboxylic acid (BCH). An inhibitor for its use according to any preceding claim wherein the regulation of skin pigmentation comprises depigmentation of the skin. An inhibitor for use according to any preceding claim wherein regulating skin pigmentation comprises bleaching the skin. A composition for its use in regulating skin pigmentation comprising an effective amount of an inhibitor of the LAT-1/SLC7A5 transporter or a cosmetically or pharmaceutically acceptable salt thereof. Composition for its use according to the preceding claim, in which the inhibitor of the SLC7A5 transporter is 2-amino-2-norbornanecarboxylic acid (BCH). Composition for its use according to any one of the two preceding claims, suitable for topical application. A composition according to any one of the three preceding claims wherein regulating skin pigmentation comprises skin whitening and/or skin depigmentation.