ES2360388T3 - COSMETIC OR PHARMACEUTICAL COMPOSITION CONTAINING PEPTIDES WITH THE ARG-GLY-SER SEQUENCE. - Google Patents

Abstract

Cosmetic and / or dermatological and / or pharmaceutical composition characterized in that it contains in an acceptable medium, as active ingredient, at least one peptide of formula (I): (AA) n-Arg-Gly-Ser- (AA) n (I ) where (AA) is any amino acid or one of its derivatives, n is an integer between 0 and 3, the peptide being present in the composition at a concentration between about 0.005 and 500 ppm.

Description

The invention relates to the field of cosmetics and pharmaceuticals, particularly to the field of dermatology.

A subject of the present invention is a cosmetic and / or dermatological and / or pharmaceutical composition comprising as active ingredient at least one peptide of sequence (AA) n-Arg-Gly-Ser- (AA) n, where (AA) is any amino acid or one of its derivatives, being between 0 and 3.

The skin is a lining organ that covers the entire body surface. It is a vital organ that fulfills several functions such as sensory, protective functions against multiple external, immune, metabolic or thermoregulatory aggressions. These functions are possible thanks to a complex structure that associates varied tissue structures. The skin consists of three different superimposed compartments: the epidermis, the dermis and the hypodermis. The epidermis is a lining epithelium that constitutes the outer structure of the skin and fulfills the protective function. This function is carried out by cohesion of epithelial cells and by the production of a filamentous and resistant protein, keratin. The dermis is a connective tissue consisting of a fundamental substance in which fibroblasts, collagen fibers and elastin fibers, protein fibers synthesized by fibroblasts are immersed. Collagen fibers are responsible for a large part of the solidity of the dermis, contribute to elasticity and especially to the tonicity of the skin and / or mucous membranes. Under the dermis is a layer of adipose tissue: the hypodermis. The hypodermis is made up of a layer of reserve fat, or white adipose tissue, attached to the lower part of the dermis by expansions of collagen fibers and elastic fibers. It consists of large vacuolated cells, adipocytes, almost completely filled with triglycerides. These cells can change volume quickly, in the case of weight loss or weight gain and can measure from 40 to 120 µm in diameter, which is equivalent to a volume variation of 27 times. Adipose tissue also contains connective tissue in which there are, among other, particular fibroblasts and preadipocytes. This adipose tissue is capable of storing lipids in the form of triglycerides or releasing them in the form of fatty acids and glycerol.

Currently, health and cosmetic professionals use more and more means to discover new active ingredients capable of acting on the skin. This industry seeks assets capable of not only protecting and maintaining it, but also capable of improving its appearance and the well-being of the individuals who use them. These products are required to have several properties simultaneously and, consequently, to have an improved efficiency spectrum. These assets must therefore have a general mode of action on the skin, and therefore have to act at different levels.

The object of the present invention is to provide a new substance, usable in the field of cosmetics and pharmacy, which presents, in addition to the skin care properties, a preventive and curative action on the manifestations of skin aging, also a stimulating and revitalizing action. This asset may therefore, at the same time, combat the phenomena of skin aging, protect the skin in an effective manner and, for example, have a slimming action.

Adenosine 5’-triphosphate (ATP) is a molecule that plays a central role in many cellular mechanisms. It is, in effect, the main energy source of the cells: this molecule is able to reserve chemical energy and restore it very easily in the reactions that need energy to be produced. ATP plays a particularly important role at the skin level: it is a marker of the vitality and activity of cells. Because, in effect, there is a close correlation between cellular activity and, for example, an increase in the synthesis of essential molecules such as proteins or DNA.

Therefore, by increasing the amount of intracellular ATP, the cell is stimulated and provided with the energy necessary to synthesize the enzymes that will induce activation mechanisms and thus allow to increase cellular metabolism. In this way, by stimulating, for example, the synthesis of the molecules essential for the proper functioning of the skin, such as extracellular matrix proteins (collagen, elastin, fibronectin) or keratin, then it will be allowed to skin fight better against the phenomena of aging and promote its renewal (by increasing proliferation and cell differentiation). The skin may also develop its repair process better, or fight more effectively against UV damage.

The inventors managed to select particular substances that have remarkable properties when applied to the skin.

Unexpectedly, the inventors discovered that the peptides corresponding to the general formula (I):

(AA) n-Arg-Gly-Ser- (AA) n (I)

where (AA) is any amino acid or one of its derivatives, and n is an integer between 0 and 3, they have remarkable properties as a skin care agent.

The asset thus obtained has particularly notable effects at the skin level. In addition to its care properties, it exerts a true stimulating and revitalizing action on the skin and on the cells that compose it. This compound thus has slimming and anti-cellulite properties, protective properties, but also a very effective action in the fight against the manifestations of skin aging. Indeed, it was discovered that this peptide has an effect on the modulation of the concentration of ATP in the cell, on the intracellular concentration of calcium and on the production and activation of essential proteins for the skin.

According to a first aspect, the present invention has as its object a cosmetic and / or dermatological and / or pharmaceutical composition characterized in that it contains in an acceptable medium, as an active principle, at least one peptide of formula (I):

(AA) n-Arg-Gly-Ser- (AA) n (I)

where (AA) is any amino acid or one of its derivatives, n is an integer between 0 and 3, the peptide being present in the composition at a concentration between about 0.005 and 500 ppm.

The term "amino acid" refers herein to any natural or non-natural organic acid having the formula (II):

-NHR-CR-C (O) -O- (II)

where each -R is independently selected from a hydrogen and an alkyl group having between 1 and 12 carbon atoms. Preferably, at least one -R group of each amino acid is a hydrogen.

By the term "alkyl" is meant here a carbon chain that can be linear or branched, substituted (monoo poly-) or unsubstituted, saturated, monosaturated (a double or triple bond in the chain) or polyunsaturated (two or several double bonds , two or several triple links, one or several double links and one or several triple links in the chain).

According to the applicant's knowledge, the use of a sequence peptide (AA) n-Arg-Gly-Ser- (AA) n where (AA) is any amino acid, or one of its derivatives, was never described in the prior art, and n an integer between 0 and 3, the peptide being present in the composition at a concentration between about 0.005 and 500 ppm, in cosmetics and / or in dermatology and / or in pharmaceuticals.

The peptide used according to the invention may contain in particular 3 to 9 amino acid residues, and in particular 3, 4 or 5 amino acid residues.

The invention particularly relates to the use of peptides containing at least the Arginine-Glycine-Serine peptide sequence and the use of derivatives of these peptides. According to a presently preferred embodiment, the aforementioned peptide is preferably the Arg-Gly-Ser sequence peptide.

When a peptide containing the Arginine-Glycine-Serine tripeptide is used, it is understood that it is chosen so that the amino acids surrounding the Arginine-Glycine-Serine motif, both by its nature and by the secondary structure of the peptide that are going to induce, do not prevent it from exercising the activity for which it is used in the present invention.

Amino acid derivatives and peptide derivatives are, for example, those of which at least one functional group (in particular the amino and carboxylic groups) is protected by a protective group. Indeed, it is possible that for reasons due to resistance to degradation, it is necessary to use a protected form of the peptide according to the invention. The form of protection must obviously be a biologically compatible form and must be compatible with a use in the field of cosmetics or pharmacy.

Numerous biologically compatible forms of protection can be considered, which are well known to those skilled in the art, such as acylation or acetylation of the aminoterminal end, or amidation or esterification of the carboxyterminal end. Thus, the invention relates to a use as defined above characterized by the fact that the peptide is in protected form or not.

Preferably, a protection based on either acylation or acetylation of the aminoterminal end, or on amidation or esterification of the carboxyterminal end, or both is used.

Amino acid derivatives and peptide derivatives also refer to amino acids and peptides linked together by a pseudopeptide bond. "Pseudopeptide link" means all types of links that can replace "classic" peptide bonds.

In the field of amino acids, the geometry of the molecules is such that they can theoretically be presented in the form of different optical isomers. There is indeed a molecular configuration of amino acid (AA) such that it shifts the plane of polarization of light to the right (D or D-aa configuration), and a molecular configuration of amino acid (aa) such that it deflects to the left the plane of polarization of the light (configuration L or L-aa). Nature retained only the L configuration for natural amino acids. Consequently, a naturally occurring peptide consists only of L-aa type amino acids. However, chemical synthesis in the laboratory allows the preparation of amino acids with the two possible configurations. From this base material, it is thus possible to incorporate in the peptide synthesis both amino acids in the form of optical isomers of configuration D and of configuration L. Thus, the amino acids constituting the peptide according to the invention can be under the configuration L - or D-, preferably, the amino acids are in the form L. The peptide according to the invention can then be in the form L-, D- or DL-.

The peptides, objects of the present patent, can be obtained either by classical chemical synthesis (solid phase or liquid homogeneous phase), or by enzymatic synthesis (Kullman et al., J. Biol. Chem. 1980, 225, 8234) from constituent amino acids or their derivatives. The peptides according to the invention can also be obtained by fermentation of a strain of modified or non-bacteria, by genetic engineering to produce the previously indicated sequence peptides and their fragments, or by extraction of animal or plant proteins, preferably of origin vegetable, followed by controlled hydrolysis that releases peptide fragments of medium or small sizes of which it is a question, provided that the released elements contain at least the Arg-Gly-Ser sequence.

A large number of proteins found in plants are likely to contain these sequences in their structure. The hydrolysis carried out allows these peptide fragments to be released. It is possible, but not necessary to carry out the invention, either to extract the involved proteins first and then hydrolyze them, or to carry out the hydrolysis first in a crude extract and purify the peptide fragments later. The expert in the field of synthesis, extraction and purification of proteins and peptides may consider other simpler or more complex processes. Thus, the peptide according to the invention can be a peptide of natural or synthetic origin. Preferably according to the invention, the peptide is obtained by chemical synthesis.

According to an advantageous embodiment of the invention, the aforementioned peptide is previously solubilized in one or more cosmetically or pharmaceutically acceptable solvents, classically used by those skilled in the art, such as water, ethanol, propylene glycol, butylene glycol, dipropylene glycol, ethoxylated or propoxylated diglycols, cyclic polyols, petrolatum, a vegetable oil or any mixture of these solvents.

Even according to another advantageous embodiment of the invention, the aforementioned peptides are previously solubilized in a cosmetic or pharmaceutical vector such as liposomes or are absorbed in pulverulent organic polymers, mineral supports such as talc and bentonite, and more generally they are solubilized, or set, in any cosmetically or pharmaceutically acceptable vector.

It is of course obvious that the peptide according to the invention can be used alone or in association with at least one other active agent, in or for the preparation of a cosmetic and / or dermatological and / or pharmaceutical composition.

According to another aspect, the object of the invention is also a non-therapeutic cosmetic treatment method that consists in applying an effective amount of a composition according to the invention, for the care and treatment of the skin and / or plows that allow to activate the energy metabolism mobile; a non-therapeutic cosmetic treatment procedure consisting in applying an effective amount of a composition according to the invention, for the care and treatment of the skin and / or pimples that have cytostimulant properties, in particular as a care agent that favors the tissue regeneration; a non-therapeutic cosmetic procedure that consists in applying an effective amount of a composition according to the invention, to stimulate the synthesis of extracellular matrix proteins and / or stimulate the synthesis of keratins; a non-therapeutic cosmetic treatment method that consists in applying an effective amount of a composition according to the invention, to fight against the manifestations of skin aging and / or to prevent it; a non-therapeutic cosmetic procedure that consists in applying an effective amount of a composition according to the invention, to protect the skin and / or hair against all types of external aggression; and a non-therapeutic cosmetic treatment method consisting in applying an effective amount of a composition according to the invention, against cellulite and / or orange peel; and / or in order to reduce, eliminate or prevent subcutaneous fat overloads.

The peptide according to the invention is advantageously used as a care and treatment agent for skin and / or skin. A care and treatment agent means, in the sense of the present invention, agents that have, in general, a reparative and revitalizing activity that allows, among other things, the skin and / or the legs to react better to aggressions. They can know.

This peptide exerts a favorable action at the level of the energy metabolism of skin cells, such as fibroblasts and adipocytes, and a cytostimulant activity. The peptide according to the invention is advantageously used as a care and treatment agent for the skin and / or skin that allows to activate the cellular energy metabolism. By agent that allows to activate the cellular energy metabolism is understood, for example, compounds capable of increasing the synthesis of intracellular ATP of skin cells or capable of increasing the concentration of intracellular calcium. Indeed, it was demonstrated that the peptide according to the invention allows to increase the synthesis of intracellular ATP, particularly, at the level of fibroblasts and adipocytes; but it is also capable of causing an increase in the concentration of intracellular calcium in skin cells. This stimulation makes it possible to activate various favorable mechanisms for the cell, particularly in order to help it fight against stress and aging. The peptide according to the invention can also exert an antioxidant action.

The cytostimulant and revitalizing activity of the peptide according to the invention is characterized by an increase in cell differentiation and regeneration. Indeed, the peptide according to the invention favors cell differentiation, particularly that of keratinocytes. The latter migrate more rapidly to the surface of the epidermis and guarantee better resistance of the corneal layer. When progressing to the upper layers, the keratinocytes flatten and pour into the extracellular space a cement consisting of lipids, cholesterol, saturated free fatty acids and ceramides that increase cohesion between the cells and thus contribute to the barrier function of the epidermis. The peptide then effectively increases the skin barrier function of the skin and thus promotes tissue regeneration.

The compound according to the present invention, by its cytostimulant action, will intervene effectively at the level of all the phenomena related to skin aging and cell stress. The energy reserve constituted by the peptide according to the invention improves the synthesis of the proteins at the skin cell levels and / or improves their stability, very particularly at the level of the epidermal cells.

It was shown that the peptides, according to the invention, have beneficial effects on the proteins of the extracellular matrix, particularly that they allow to increase and favor their synthesis. By "extracellular matrix protein" is meant, for example, proteins such as collagen, fibronectin or elastin. In the same way, it was shown that the peptide of formula (I) exerts an effective action at the level of keratinocytes. The invention then has for another object the use of at least one peptide as defined above, in or for the preparation of a composition, in order to stimulate the synthesis of keratins.

The peptide according to the invention is then particularly well adapted to a use in order to combat the phenomena of skin aging and / or prevent it. Skin modifications of aging means any modification of the external appearance of the skin due to aging, such as wrinkles and crow's feet, withered skin, soft skin, thinned skin, lack of elasticity and / or of skin tone, dull and dull skin but also any internal modification of the skin that is not systematically translated by a modified external appearance such as, for example, any internal degradation of the skin following an exposure to ultraviolet radiation.

Increasing the concentration of ATP will also allow the cell to be protected and better resist the stress caused by the environment. The cell will then be protected against all types of external aggression. The present invention then relates to the use of at least one peptide of formula (I) as defined above, in or for the preparation of a composition, in order to protect the skin and / or hair against all types of external aggression The term “external aggression” means the aggressions that the environment can produce. These aggressions can be of chemical, physical, biological or thermal origin. As an example, aggressions such as pollution, UV, rubbing, water of high calcareous concentration, temperature variations or irritating products such as surfactants, preservatives or perfumes can be cited.

On the other hand, due to its stimulating activity, the peptide has a particular and pronounced activity on lipolysis, which makes it particularly useful in preparations that have as a purpose the thinning. In fact, it was found that the peptide according to the invention, or the composition containing it, exerts an effective action at the level of adipocytes. This peptide can thus be used in or for the manufacture of a cosmetic and / or pharmaceutical composition, for topical use, intended for the treatment of cellulite and / or for the treatment of orange peel. It is used in a more general way to reduce, eliminate or prevent subcutaneous fat overloads. Cellulite is a particular configuration of adipose tissue. Designates a padded and padded appearance of the skin that corresponds, schematically, to the accentuation of adipose tissues in certain regions of the body due to an increase in the amount of fat, stored in the adipocytes, whose volume and number increases. In an advanced stage of cellulite formation, the skin spontaneously takes on the "orange peel" appearance.

The peptide according to the invention contributes to significantly decrease the amount of triglycerides contained in adipocyte vacuoles. This phenomenon is due to an increase in the phenomenon of lipolysis in adipocytes, a mechanism that goes through an increase in the amount of intracellular ATP followed by an increase in the amount of intracellular cAMP. This increase in the amount of cAMP results in an increase in the stimulation of the activity of Triglyceride lipase, an enzyme that allows the hydrolysis of triglycerides in fatty acids. Since lipolysis is the elimination reaction of triglycerides stored in adipocytes, an increase in this phenomenon will allow a greater elimination of triglycerides and an increase in the release of free fatty acids and glycerol in the extracellular environment. Thus, when the amount of triglycerides present in adipocyte vacuoles decreases, their volume decreases. The skin thus gradually returns to its "normal" appearance: the cellulite tissue decreases, the orange peel effect is attenuated, the ugly appearance of the body gradually diminishes. Thus, the degree of peptide lipolytic activity according to the invention and its ability to act on lipolysis will vary depending on the amount of cAMP. The peptide will then reduce, slow down or eliminate fatty deposits. The active according to the invention or the composition containing it will have a slimming activity and will improve the appearance of the skin and, in particular, attenuate the appearance of "orange peel".

More generally, the composition according to the invention contains the active compound as defined above. Thus, the peptide present in the composition is chosen from those of which at least one functional group is protected by a protective group, this protecting group being either an acylation or an acetylation of the aminoterminal end or an amidation or an esterification of the end. carboxy terminal, or both.

It is understood that the peptide according to the invention can be used alone or in association with at least one other active agent.

In the composition according to the invention, the peptide may be a mixture of peptide derivatives and / or be constituted by amino acid derivatives.

The composition containing the peptide according to the invention can be a cosmetic or dermatological or pharmaceutical composition. Preferably according to the invention, the composition is a cosmetic composition, since it is intended to improve the appearance and general skin behaviors of the individual using it. The composition according to the invention is preferably a cosmetic and / or dermatological composition adapted to administration by the skin topical route comprising a cosmetically or pharmaceutically acceptable medium.

It is clear that the invention is intended for mammals in general and more particularly for humans.

The effective amount of active ingredient corresponds to the amount necessary to obtain the desired result. According to an advantageous embodiment of the invention, the aforementioned peptide is present in the compositions of the invention at a concentration between about 0.005 and 500 ppm (parts per million), and preferably at a concentration between 0.1 and 50 ppm approximately with respect to the total weight of the final composition.

Whatever the form of the invention, the composition according to the invention can be ingested, injected or applied to the skin (on any skin area of the body), hair, nails or mucous membranes. According to the mode of administration, the composition according to the invention can be presented in all the galenic forms normally used. Preferably, the compositions according to the present invention will be presented in a galenic form adapted to administration by the topical cutaneous route, and will cover all cosmetic or dermatological forms. These compositions must then contain an acceptable cosmetic medium, that is, compatible with the skin, mucous membranes, hairs or hair. These compositions may in particular be presented in the form of an aqueous, hydroalcolic or oily solution; of an oil-in-water emulsion, water in oil or multiple emulsions; They can also come in the form of creams, suspensions or powders, adapted to an application on the skin, mucous membranes, lips and / or hair. These compositions can be more or less fluid and have the appearance of a cream, a lotion, a milk, a serum, an ointment, a gel, a paste or a foam. They can also be presented in solid form, such as a bar or applied to the skin in aerosol form. They can be used as a care product and / or as a skin makeup product.

These compositions also comprise any additive usually used in the field of application considered here and the additives necessary for its formulation, such as solvents, thickeners, diluents, antioxidants, dyes, sunscreens, self tanning agents, pigments, fillers, preservatives, perfumes , odor absorbers, cosmetic or pharmaceutical assets, essential oils, vitamins, essential fatty acids, surfactants, film-forming polymers, etc. In all cases, the person skilled in the art will ensure that these additives and their proportions are chosen in such a way that they do not harm the desired advantageous properties of the composition according to the invention. These additives may, for example, be from 0.01 to 20% of the total weight of the composition. When the composition of the invention is an emulsion, the fatty phase may represent 5 to 80% by weight and preferably 5 to 50% by weight in relation to the total weight of the composition. The emulsifiers and co-emulsifiers used in the composition will be chosen from those used classically in the field considered. For example, they can be used in a proportion ranging from 0.3 to 30% by weight, in relation to the total weight of the composition.

Of course, the person skilled in the art will make sure to choose the possible complementary compounds, active or non-active, and / or their amounts, so that the advantageous properties of the mixture are not, or are not noticeably altered, by the addition considered.

The compositions according to the invention will particularly find an application as cosmetic or pharmaceutical compositions for the skin, mucous membranes and / or semi-mucous membranes, but also as cosmetic or pharmaceutical compositions for the faneras and / or the hair. They find very particularly an application as a protection and / or skin care product, or as an anti-wrinkle and / or anti-aging composition, or as a slimming and / or firming composition. The slimming and / or firming composition may be applied locally on the areas of the face or body to be refined, in particular on the hips, buttocks, thighs, belly, face. It can also be considered an application in the field of makeup compositions of the skin of the face and body, such as lipsticks, bases, base creams, concealers,

or antisolar or artificial tanning compositions.

The compositions, object of the invention, find their application in numerous particularly cosmetic or dermatological treatments, and may constitute a cosmetic composition, particularly for the treatment, protection, care, make-up and / or cleaning of the skin, of the lips and / or hair, and / or for the makeup of the skin, lips, eyelashes and / or body. The composition according to the invention may also consist of solid preparations which also comprise soaps or cleaning tablets. The composition may also be presented in the form of an aerosol composition which also comprises a propellant under pressure. The composition can also have oral use, for example a toothpaste. The composition of the invention may also be a cosmetic composition intended for oral administration. For oral administration, the composition according to the invention can be presented in any adapted form, particularly in the form of a drinkable solution, a syrup, a tablet, a dragee, a capsule,

or of a food or nutritional supplement. According to the invention, other active agents may be added to the composition of the invention intended, inter alia, for the prevention and / or treatment of skin manifestations of aging, or intended for the protection of the skin against external aggressions, or assets for the treatment of the skin against external aggressions, or assets for the treatment of cellulite and the phenomenon of "orange peel".

According to another aspect, the present invention relates to a non-therapeutic cosmetic treatment method intended to treat aged skin and / or to combat and / or prevent the phenomena of skin aging consisting of applying, on the surface of the skin, an amount effective of a composition as defined above. That is, a composition containing the peptide corresponding to the general formula (I), in order to obtain the desired action. The present invention relates, in the same way, to a non-therapeutic cosmetic treatment procedure in order to protect the skin and / or hair against all types of external aggression. According to another aspect of the invention, the present invention relates to a non-therapeutic cosmetic treatment method in order to favor cell differentiation and / or to promote tissue regeneration and / or to strengthen the skin's skin barrier consisting of to apply, on the surface of the skin, an effective amount of a composition as defined above. The present invention also relates to a non-therapeutic cosmetic care procedure intended for the purpose of obtaining a slimming action, and non-therapeutic cosmetic care procedure intended to reduce, eliminate and / or prevent subcutaneous fat overloads, and / or intended to fight against cellulite and / or fight against the phenomenon of orange peel, which consists of applying, topically, on the affected areas of the skin, an effective amount of a composition as defined above. Particular embodiments of this cosmetic treatment procedure also result from the above description.

The cosmetic treatment method of the invention can be implemented particularly by applying cosmetic compositions as defined above, according to the usual technique of use of these compositions, for example: application of creams, gels, serums, lotions, milks , of shampoos or antisolar compositions, on the skin or hair, or application of toothpaste on the gums.

Other advantages and features of the invention will appear more clearly when reading the examples given by way of illustration and not limitation.

Example 1 - Evidence of the effect of the peptide on the amount of intracellular ATP.

The purpose of this study is to determine the influence of the peptide according to the invention on the production of ATP by different skin cells. This study is carried out with a Kit, "ATP Bioluminescence Assay Kit HS II", which allows to determine the concentration of intracellular ATP. The study is carried out in fibroblasts and in a differentiated 3T3-L1 preadipocyte cell line.

The test was performed, for one part, on fibroblasts. These fibroblasts were cultured and then put in culture in 12-well plates (Labteks). When the cells reached 80% confluence, a solution containing the Arg-Gly-Ser sequence peptide at a concentration of 10-6 M was applied to the cells for periods of 5, 15, 30 and 60 minutes Control tests were performed applying in the cells solutions that did not contain active.

In the same way, the test was performed on 3T3-L1 fibroblasts in culture. These cells have the distinction of differentiating themselves, under the action of a hormonal cocktail, in preadipocytes and then in adipocytes loaded with triglycerides. The preadipocyte cells were seeded in Labteks and kept up to 100% confluence in a DMEM 10% and SVF culture medium. When they reach confluence, the cells are grown in a medium of classical culture, which contains differentiation inducers (IBMX, dexamethasone and insulin), to pass the terminal differentiation phases and thus obtain mature adipocytes. The adipocytes are then treated with the Arg-Gly-Ser sequence peptide, representative of the peptide family according to the invention, dissolved at 1% of a solution at 50 ppm, or with a solution that does not contain the active. The adipocytes are incubated for different periods: 5 minutes, 15 minutes and 3 hours.

At the end of the incubation time, the different plates, containing the fibroblasts or adipocytes, are emptied, and then rinsed with 2 ml of cold PBS and then 250 µl of a lysis buffer, supplied by the kit, is added. The cells are then collected separately in 14 ml tubes and each well is rinsed with 2x500 µl of cold PBS. Then, each tube is passed to the polytron for 10 seconds at 18000 rev / min. A dilution of 1/1200 is made for each condition, with cold PBS, before each reading.

The ATP determination is performed on these samples: 50 µl of this dilution is deposited in a Lumacuvette cuvette and 50 µl of luminol are added. After 10 seconds, the luminescence reading begins. The measurements are made with one device: the Bioconteur M2010A LUMAC® / 3M.

The results obtained express the percentage increase in luminescence. This luminescence was measured after different incubation periods, in the cells treated with the active ones with respect to the untreated cells, in the fibroblasts and in the adipocytes. This increase in luminescence expresses the increase in the amount of intracellular ATP, the luminescence being proportional to the amount of ATP present in the cells.

Weather

t = 5 min t = 15 min t = 30 min t = 3 hours

In active treated fibroblasts

+ 60% + 75% + 45% + 0.2%

In adipocytes treated with active

+ 1% + 30% - + 16%

5

10

fifteen

twenty

25

30

35

40

Four. Five

These results demonstrate that in the presence of the peptide the amount of intracellular ATP increases significantly with respect to cells not treated with the active. This increase in luminescence is observed in both fibroblasts and adipocytes, which means that the increase in ATP is carried out in both types of cells.

However, at the level of the fibroblasts the increase begins at 5 minutes and reaches its maximum 15 minutes after the administration of the asset according to the invention, to return approximately to its baseline state after 2 hours. While at the level of adipocytes, the maximum is reached 15 minutes after the application of the asset and the concentration of ATP is maintained for at least 3 hours.

Example 2 - Evidence of the effect of the peptide on the expression of extracellular matrix proteins.

The object of the study is to determine the influence of the peptide according to the invention on the synthesis of fibronectin, on the synthesis of type I and type III collagen, by the fibroblasts, by means of the immunofluorescence technique.

Immunofluorescence is a semi-quantitative technique that allows you to determine the concentration of each of the proteins present in the cell cytoplasm.

Human fibroblasts are seeded in Labteks and then grown overnight. When the cells reach approximately 40 to 70% of confluence, after an HBS buffer rinse, a composition containing a concentration of 10-6 M of the Arg-Gly-Ser sequence peptide, representative of the family, is added of peptides according to the invention, or a control composition that does not contain peptide. The cells are then incubated for 24 hours. After removing the supernatants and rinsing the cultures, the cells are fixed with polyacetal for 30 minutes at 4 ° C and then rinsed with PBS buffer. 200 µl of anti-fibronectin antibodies and / or anti-collagen I antibodies and / or anti-collagen III antibodies are then added. The incubation lasts 30 minutes at room temperature. Supernatants are removed and cells are rinsed with PBS. Then 200 µl of secondary antibody coupled to a fluorescent marker (fluorescein) is added. After 30 minutes of incubation at room temperature, the supernatants are removed and the cells are rinsed with PBS. The sheets are then mounted and examined under an inverted fluorescence microscope. The amounts of fibronectin and / or collagen type I and / or type III, synthesized by the cells, are proportional to the intensity of flowering.

The results obtained demonstrate that the addition of peptides in the culture medium of the fibroblasts has the effect of increasing the synthesis of fibronectin and / or the synthesis of type I and type III collagen by the cells. This stimulation was observed considerably.

Indeed, when the fibroblasts are incubated in the presence of the composition containing the peptide, after 24 hours an increase in the intensity of the fluorescence is observed, with respect to the untreated cells, thus translating a stimulation of the synthesis of fibronectin and / or a stimulation of the synthesis of type I collagen and / or a stimulation of the synthesis of type III collagen by fibroblasts.

Example 3 - Evidence by immunofluorescence of the effect of the peptide on the expression of keratins.

The object of the study is to determine the influence of the peptide according to the invention on the synthesis of keratins, by keratinocytes, by the immunofluorescence technique.

A study was carried out using the immunofluorescence technique, identical to that of example 2, in human HaCat keratinocytes, with Pan cK anti-keratin antibodies.

The results obtained demonstrate that the addition of the Arg-Gly-Ser sequence peptide, representative of the peptide family according to the invention, in the keratinocyte culture medium has the effect of increasing the synthesis of keratin by the cells. This stimulation was observed considerably. Indeed, when the keratinocytes are incubated in the presence of the composition containing the active ingredients, after 24 hours, an increase in the intensity of the fluorescence is observed, thus translating a stimulation of the keratin synthesis by the keratinocytes.

Example 4 - Evidence of the effect of the peptide on cell differentiation.

The object of the study is to determine the influence of the peptide according to the invention on cell differentiation, by means of the immunofluorescence technique. The principle of this technique lies in the detection of markers of cell differentiation such as, for example, implicacrine and the ERG molecule.

A study was carried out using the immunofluorescence technique, identical to that of Examples 2 and 3, in human HaCat keratinocytes, with anti-ERG 1-2 antibodies (the ERG 1-2 molecule being a very early cell differentiation marker).

The results obtained demonstrate that the addition of the Arg-Gly-Ser sequence peptide, representative of the peptide family according to the invention, in the keratinocyte culture medium has the effect of increasing the amount of ERG molecules present in the cells . This stimulation was observed considerably. Indeed, when the keratinocytes are incubated in the presence of the composition containing the peptide, an increase in fluorescence intensity is observed after 24 hours, thus translating a stimulation of the synthesis of ERG molecules by keratinocytes, that is an increase due to the degree of cell differentiation.

Example 5 - Evidence of peptide activity at the level of adipocytes.

1) Principle of the in vitro test:

The effect of the peptide was evaluated by microscopic observation of the number and size of the lipid vacuoles present in the adipocytes after coloring, incubating or not adipocytes in the presence of the substance to be tested.

2) Experimental model:

Evidence of the biological activity of the active substance was carried out in the preadipocyte cell line 3T3-L1, maintained in a suitable medium, these preadipocytes being able to enter, under certain conditions, in the phase of terminal differentiation.

The cells are seeded in Labteks and up to 100% confluence is maintained in a DMEM 10% and SVF culture medium. When they reach confluence, the cells are cultured in a classic culture medium, which contains differentiation inducers (IBMX, dexamethasone and insulin) to pass the terminal differentiation phases and thus obtain mature adipocytes. The adipocytes are then treated with the Arg-Gly-Ser sequence peptide, representative of the peptide family according to the invention, dissolved at 1% of a solution at 50 ppm. The adipocytes are incubated for different periods: 30 minutes, 3 hours and 6 hours. After the different incubation periods, in the presence, or not, of the active agent to be tested, the adipocytes are colored with the "Oil Red" solution (Sigma, O-0625). This solution is prepared by adding 0.5 g of product in 100 ml of isopropanol and diluting to 4/10 in distilled water, and then filtering. The adipocytes are fixed for 10 minutes in a solution of 4% formalin and NaCl, the Red Oil solution is applied for 15 minutes. A countercolouration with Hematoxylin, for 30 seconds, is possible. The cells are then rinsed with warm water and mounted on sheets, in hydrophilic medium (Aquatex). The observation is carried out under an optical microscope.

3) Results:

The results of the observation of the cells demonstrate that, contrary to the mature mature adipocytes (in which the active one was not applied), that they have a spherical bulky form and a considerable accumulation of intracytoplasmic lipid vesicles; mature adipocytes, treated with a solution containing the active according to the invention, have a less rounded morphology and a clearly reduced intraadipocyte lipid vesicles content.

The solution containing the peptide according to the invention then appears naturally effective in the process of limiting adipocyte hypertrophy by probably increasing the phenomenon of lipolysis. It allows then a elimination of triglycerides contained in intraadipocyte vesicles.

These results are confirmed by the determination of the concentration of glycerol released by adipocytes in the supernatant medium, according to the HPLC analysis technique and by an enzymatic determination technique.

Example 6 - Evidence of peptide activity on the amount of intracellular cAMP.

1) Principle of the test:

The objective of this test is to measure the increase in intracellular cAMP concentration in adipocytes, in order to deduce the activation of the lipolysis phenomenon.

Lipolysis is a mechanism that releases triglycerides contained in adipocyte vacuoles. This reaction is carried out thanks to an enzyme, Triglyceride lipase, which cuts triglycerides into free fatty acids and glycerol. The latter are released in the extracellular environment and eliminated in the bloodstream. Triglyceride lipase is

5

10

fifteen

twenty

25

30

35

regulates by the concentration of intracellular cAMP. Thus, by increasing the concentration of intracellular cAMP, the activity of the enzyme is increased allowing the hydrolysis of triglycerides and thus the phenomenon of lipolysis is stimulated.

2) Experimental model:

The test was performed on 3T3-L1 fibroblasts in culture. These cells have the distinction of differentiating themselves, under the action of a hormonal cocktail, in preadipocytes and then in adipocytes loaded with triglycerides.

3T3-L1 cells are plated in 24-well plates and maintained up to 100% confluence in a DMEM 10% and SVF culture medium. When they reach confluence, the cells are grown in a medium of classical culture, which contains differentiation inducers (IBMX, dexamethasone and insulin), to pass the terminal differentiation phases and thus obtain mature adipocytes. The adipocytes are then treated with the Arg-Gly-Ser sequence peptide, representative of the peptide family according to the invention, dissolved at 1% of a solution at 50 ppm, or with a solution that does not contain the active. A negative control test is performed in the presence of a solution that does not contain active. The adipocytes are incubated in the presence, or not, of the active to be tested. The amount of intracellular cAMP is measured in different periods (15 minutes, 30 minutes and 2 hours).

After the different incubation periods, in the presence, or not, of the asset to be tested, the amount of cAMP contained in the adipocytes is measured with the Amersham Biosciences Kit "ciot Biotrak® EIA System". At the end of the incubation time, the plates are emptied, and then rinsed with cold PBS. The cAMP determination protocol is carried out according to the instructions provided by the kit. The cAMP is determined by reading the OD at 450 nm (the amount of cAMP being proportional to the measured OD).

3) Results:

The results express the intracellular concentration of cAMP, in nmol / ml, read after different incubation times, and the percentage increase in the concentration in the cells treated with the active ones with respect to the untreated cells.

Weather

t = 0 t = 15 min t = 30 min t = 2 h

Concentration in nmol / ml

226  246 304  287

% increase

- + 8.6% + 33% + 28%

These results demonstrate that in the presence of the asset, the amount of intracellular cAMP increases significantly with respect to the cells not treated with the assets. This increase begins at approximately 5 minutes, and reaches its maximum 30 minutes after administration of the peptide according to the invention. No increase in concentration is observed in cells not treated with the active.

This increase in the amount of cAMP in the adipocytes, combined with the results obtained in example 5, makes it possible to conclude that the peptide according to the invention acts, at the level of the adipocytes, on the mechanism of lipolysis. Indeed, by increasing the amount of intracellular cAMP in adipocytes, the activity of Triglyceride lipase is increased, and thus lipolysis is favored. The peptide according to the invention then favors the elimination of triglycerides in intraadipocyte vesicles.

Example 7 - Preparation of compositions

These compositions are obtained by simply mixing the different compounds. The amounts indicated are given in percentage by weight.

1. Oil in water emulsion

Tradenames

INCI names % of dough

OIL PHASE

Montanov 68

Cetearyl Alcohol (and) Cetearyl Glucoside 5.00

Jojoba oil

Simmondsia Chimensis Seed Oil 5.00

Vaseline oil

Paraffinum Liquidum (Mineral oil) 5.00

Isopropyl Palmitate

Isopropyl Palmitate 7.00

AQUEOUS PHASE

Glycerin

 Glycerin 5.00

Allantoin

 Allantoin 0.10

Arg-Gly-Ser Peptide

1.5 ppm

Sepigel 305

Polyacrylamide (and) C13-14 Isoparaffin (and) Laureth-7 0.30

Conservative

0.50

Fragrance

 Parfum (Fragrance) 0.50

Demineralized water

Aqua (Water) Qsp

2. Lotion

Tradenames

INCI names % of dough

Monopropylene Glycol

Propylene Glycol 1.00

Allantoin

 Allantoin 0.30

Glycerin

 Glycerin 1.00

Cetiol HE

PEG-7 Glyceryl Cocoate 1.00

Arg-Gly-Ser Peptide

0.1 ppm

Conservative

0.20

Fragrance

 Parfum (Fragrance) 0.50

Demineralized water

Aqua (Water) qsp

3. Gel 4. Slimming cream

Tradenames

INCI names % of dough

Carbopol Ultrez 10 (2%)

Carbomer 25.00

Triethanolamine

 triethanolamine 0.50

Arg-Gly-Ser Peptide

1 ppm

Conservative

0.20

EDTA

 Tetrasodium EDTA 0.10

Fragrance

 Parfum (Fragrance) 0.50

Water soluble dye

Qsp

Demineralized water

Aqua (Water) Qsp

Tradenames

INCI names % of dough

PHASE A

Montanov 68

Cetearyl Alcohol (and) Cetearyl Glucoside 5.00

Squalane

 Squalane 2.50

IPP DUB

Isopropyl Palmitate 3.50

Eutanol G

Octyldodecanol 1.50

Phenonip

 Phenoxyethanol (and) Methylparaben (and) Ethylparaben (and) Butylparaben and) Propylparaben (and) Isobutylparaben 0.50

PHASE B

Demineralized water

Aqua (Water) Qsp

Glycerin

 Glycerin 3.00

Butylene Glycol

Butylene glycol 3.00

PHASE C

Simulgel EG

Sodium Acrylate / Acryloyldimethyl Taurate Copolymer (and) Isohexadecane (and) Polysorbate 80 0.60

PHASE D

Arg-Gly-Ser Peptide

1.25 ppm

Fragrance

 Parfum (Fragrance) Qsp

Colorant

Qsp

The components of phase A melt at 75 ° C and the components of phase B are heated to 75 ° C. Phase A is emulsified with B, and then the mixture is cooled to a temperature below 40 ° C. Then phases C and D are added constantly stirring.

5.

 Spray firming - slimming

The components of phase A and phase B are heated separately at 65 ° C; It is incorporated by stirring phase B to phase A. The temperature of the mixture is increased to 83 ° C and then cooled to a phase inversion temperature. Then phase C is added. The asset is incorporated when the temperature reaches less than 40 ° C. Then you can add perfumes and / or dyes.

6.

Firming gel_ slimming - anti-cellulite

Tradenames

INCI names % of dough

PHASE A

Emulgade SEV

Glyceryl Stearate (and) Ceteareth-20 (and) Ceteareth-12 (and) Cetearyl Alcohol 4.60

Eumulgin B2

Ceteareth-20 1.40

Cetiol OE

Dicaprylyl Ether 3.00

DUB B1215

C12-C15 Alkyl Benzoate 5.00

Phenonip

 Phenoxyethanol (and) Methylparaben (and) Ethylparaben (and) Butylparaben and) Propylparaben (and) Isobutylparaben 0.50

DUB ININ

Isononyl Isononanoate 5.00

Phenonip

 Phenoxyethanol (and) Methylparaben (and) Ethylparaben (and) Butylparaben and) Propylparaben (and) Isobutylparaben 0.50

PHASE B

Demineralized water

Aqua (Water) 15.00

Glycerin

 Glycerin 3.00

PHASE C

Demineralized water

Aqua (Water) Qsp

PHASE D

Arg-Gly-Ser Peptide

1.50 ppm

Fragrance

 Parfum (Fragrance) qsp

Colorant

qsp

Tradenames

INCI names % of dough

Carbopol Ultrez 10 (2%)

Carbomer 25.00

Demineralized water

Aqua (Water) Qsp

DUB DIOL

Methyl Propanediol 3.00

EDTA

 Tetrasodium EDTA 0.10

Glydant Plus Liquid

DMDM Hydantoïn (and) Iodopropynyl butylcarbamate 0.20

Arg-Gly-Ser Peptide

1.25 ppm

TORCH

Triethanolamine 0.50

Fragrance

 Parfum (Fragrance) Qsp

Water soluble dye

Qsp

Carbopol gel was prepared at 2%. The ingredients are added by stirring in the order indicated above. The mixture is then neutralized with ASD. If necessary, perfume and dyes are added.

Claims (14)

1. Cosmetic and / or dermatological and / or pharmaceutical composition characterized in that it contains in an acceptable medium, as active ingredient, at least one peptide of formula (I): (AA) n-Arg-Gly-Ser- (AA) n (I) where (AA) is any amino acid or one of its derivatives, n is an integer between 0 and 3, the peptide being present in the composition at a concentration between about 0.005 and 500 ppm.

2.

 Composition according to claim 1 characterized in that the peptide is the Arg-Gly-Ser sequence.

3.

Composition according to any one of claims 1 or 2, characterized in that the peptide is chosen from among those of which at least one functional group is protected by a protective group, this protecting group being either an acylation or an acetylation of the aminoterminal end or a amidation or an esterification of the carboxy terminal terminal, or both.

Four.

Composition according to any of the preceding claims, characterized in that the peptide is present in the composition at a concentration of between 0.1 and 50 ppm approximately with respect to the total weight of the final composition.

5.

Composition according to any of the preceding claims, characterized in that it is presented in the form of a cosmetic and / or dermatological composition adapted to administration by the skin topical route comprising a cosmetically or pharmaceutically acceptable medium.

6.

Composition according to any of the preceding claims, characterized in that the peptide is previously solubilized in one or several cosmetically or pharmaceutically acceptable solvents, such as water, ethanol, propylene glycol, butylene glycol, dipropylene glycol, ethoxylated or propoxylated diglycols, cyclic polyols, petrolatum, a vegetable oil or any mixture of these solvents.

7.

Composition according to any of the preceding claims, characterized in that the peptide is previously solubilized in a cosmetic or pharmaceutical vector such as liposomes or absorbed in pulverulent organic polymers, mineral supports such as talc and bentonites, and more generally they are solubilized, or fixed in any cosmetically or pharmaceutically acceptable vector.

8.

Composition according to any of the preceding claims, characterized in that it is presented in the form of an aqueous, hydroalcolic or oily solution or in the form of an oil-in-water emulsion, water in oil or multiple emulsions or in the form of cream, suspensions or powders; These compositions may be more or less fluid or solid and have the appearance of a cream, a lotion of a milk, a serum, an ointment, a gel, a paste, a foam or a bar.

9.

Non-therapeutic cosmetic treatment method that consists in applying an effective amount of a composition as defined according to any one of claims 1 to 8, for the care and treatment of the skin and / or of the legs that allows to activate the energy metabolism mobile.

10.

Non-therapeutic cosmetic treatment method that consists in applying an effective amount of a composition as defined according to any of claims 1 to 8, for the care and treatment of the skin and / or of the legs that has cytostimulant properties, in particularly as a care agent that favors tissue regeneration.

eleven.

Non-therapeutic cosmetic treatment method consisting of applying an effective amount of a composition as defined according to any one of claims 1 to 8, to stimulate the synthesis of extracellular matrix proteins and / or stimulate keratin synthesis.

12.

Non-therapeutic cosmetic treatment method that consists in applying an effective amount of a composition as defined according to any of claims 1 to 8, to fight against the phenomena of skin aging and / or prevent it.

13.

Non-therapeutic cosmetic treatment method consisting of applying an effective amount of a composition as defined according to any of claims 1 to 8, to protect the skin and / or hair against all types of external aggression.

14.

Non-therapeutic cosmetic treatment method consisting in applying an effective amount of a composition as defined according to any of claims 1 to 8, against cellulite and / or orange peel; and / or to reduce, eliminate or prevent subcutaneous fat overloads.