EP1874328A1

EP1874328A1 - Use of a peptide in the form of a slimming active principle

Info

Publication number: EP1874328A1
Application number: EP06743691A
Authority: EP
Inventors: Nouha Domloge; Claude Dal Farra
Original assignee: Societe dExtraction des Principes Actifs SA
Current assignee: Ashland Specialties France
Priority date: 2005-04-15
Filing date: 2006-04-14
Publication date: 2008-01-09
Also published as: FR2884418B1; WO2006108963A1; FR2887447A1; FR2884418A1; FR2887447B1

Abstract

The invention relates to the use of at least one peptide of a (AA)n-Pro-Gly-Pro-(AA)n sequence, wherein (AA) is any amino acid or one of the derivatives thereof and n is an integer ranging from 0 to 3, in the form of an active agent in or for preparing a cosmetic and/or dermatological and/or pharmaceutical composition. The use of said peptide for treating cellulite and/or the use thereof for reducing, eliminating or preventing subcutaneous fat overloads are also disclosed.

Description

USE OF A PEPTIDE AS A THINNING ACTDJ PRINCIPLE

The present invention is in the field of pharmaceuticals, and more particularly in the field of dermatology and cosmetology. The subject of the present invention is the use of at least one peptide of sequence (AA) n-Pro-Gly-Pro- (AA) n, in which (AA) is any amino acid or a derivative thereof, n being between 0 and 3, as an active agent, in or for the preparation of a cosmetic and / or dermatological and / or pharmaceutical composition. Said peptide being, in particular, for the treatment of cellulite and / or used to reduce, eliminate or prevent subcutaneous fat overload.

The skin is a covering organ covering the entire surface of the body. It is a vital organ providing multiple functions such as sensory functions, protective against multiple external attacks, immune, metabolic or thermoregulatory. These roles are made possible by a complex structure that combines various tissue structures.

The skin consists of three distinct compartments superimposed: the epidermis, the dermis and the hypodermis. The epidermis is a coating epithelium that constitutes the outer structure of the skin and provides its protective function. The dermis is a connective tissue that participates in the resistance, elasticity and especially the tonicity of the skin and / or mucous membranes. Below the dermis is a layer of adipose tissue: rhypoderm. The hypodermis consists of a layer of reserve fat, or white adipose tissue, attached to the lower dermis by expansions of collagen fibers and elastic fibers. It consists of large vacuolated cells, the adipocytes, almost entirely filled with triglycerides. These cells can change rapidly in volume, slimming or weight gain, and can measure 40 to 120 microns in diameter, which corresponds to a variation of 27 times in volume. Adipose tissue also contains connective tissue in which, inter alia, particular fibroblasts and preadipocytes are present. This adipose tissue is the largest energy reservoir in the body. It is capable of storing lipids as triglycerides or releasing them as fatty acids and glycerol. The lipid reserves of the body are constantly renewed and there is a balance, constantly adapted to the energy needs of the body, between the phenomenon of lipolysis, which releases fatty acids, and the phenomenon of lipogenesis which stores them. If an imbalance settles in the body between these two phenomena as, for example, if the quantities of stored fat become greater than the quantities of fats eliminated by lipolysis, the fat is stored in the adipocytes, whose volume and the number increases (We speak of hypertrophy and hyperplasia of adipocytes). The excessive development of fat mass can then result in changes in the appearance of the skin, or even progress to overweight the individual, or progress to a real obesity.

Cellulite is a particular configuration of adipose tissue, considered, today, as unsightly. It refers to a quilted and padded appearance of the skin that corresponds, schematically, to the accentuation of adipose tissue in certain areas of the body, in particular, in the woman, at the hips, thighs, buttocks, knees and forearms. At an advanced stage of cellulite formation, the skin spontaneously takes on the appearance of "orange peel" or capiton, which is characterized by a succession of small bumps and depressions due to traction of the epidermis towards the deep tissues. From a pathological point of view, two main phenomena contribute to the formation of cellulite tissue: hypertrophy of adipose cells by overloading triglycerides and hyperviscosity of the substance (by polymerization of polysaccharides). These modifications interfere with the cellular exchanges and the mobility of the connective fibers (collagen, elastin and reticulin), which results in a retention of water and a slowing down of the venous and lymphatic circulation.

Nowadays, many research efforts are made to find an effective way to fight against cellulite and fat overload in general. Many methods have been used, such as certain medical-surgical techniques such as lipoplasty, lymphatic drainage, mesotherapy, iontophoresis techniques ... However, these techniques, although effective, are cumbersome and restrictive. Biological pathways have been studied in order to act in a gentle and non-invasive way on the mechanisms of formation of subcutaneous fat overloads. Solutions have been proposed to intervene, especially on the metabolism of fatty acids. The cosmetic and pharmaceutical assets will therefore act at different levels so as to prevent the appearance of cellulite. For example, they will promote lipolysis or to inhibit lipogenesis, that is to say to reduce the formation of triglycerides. A particularly effective way to act on the metabolism of fatty acids and promote their elimination, is to act on the level of lipolysis. Lipolysis is regulated by the level of intracellular cAMP, which stimulates Triglyceride-lipase, enzyme allowing the hydrolysis of triglycerides. This enzyme cleaves triglycerides into free fatty acids and glycerol, which are released into the extracellular medium and eliminated in the bloodstream. CAMP is itself regulated by activation of adenylcyclase or inhibition of phosphodiesterase. Thus, one of the possibilities for increasing lipolysis in fat cells is to develop substances capable of stimulating the synthesis of FAMPc, by activating adenylcyclase, or substances capable of decreasing the destruction of cAMP, such as, for example by inhibiting phosphodiesterase.

Today, many advances have been made in the field of slimming actives and a number of substances, introduced in cosmetics or pharmaceuticals, have emerged. However, there is still room for improvement in order to have cosmetic or pharmaceutical products that can address these problems in a truly satisfactory way. There is therefore still a need for an asset with a truly effective action as a slimming agent.

The present invention aims to fill this gap and its main purpose is to provide a new slimming active ingredient. Indeed, the inventors have succeeded in selecting particular substances having remarkable properties when they are applied to the skin.

They thus discovered that a peptide corresponding to the general formula (I):

(AA) Pro-Gly-Pro - (AA) n (I) wherein (AA) is any amino acid or a derivative thereof, n is an integer of 0 to 3; has a localized thinning effect when applied to the skin; and, more particularly, that it is capable of reducing, eliminating or preventing subcutaneous fat overloads. This peptide can thus be used in or for the manufacture of a cosmetic and / or pharmaceutical composition, for topical use, for the treatment of cellulite and / or the treatment of orange peel. It is used in a more general way to reduce, eliminate or prevent subcutaneous fat overload. It has been found that the peptide according to the invention, or the composition containing it, has an effective action at the level of adipocytes. Indeed, it helps to significantly reduce the amount of triglyceride contained in the hypodermal adipocytes. This phenomenon is probably due to an increase in the phenomenon of lipolysis, which increases the amount of intracellular cAMP. This increase in the amount of cAMP results in increased stimulation of Triglyceride-lipase, an enzyme that allows the hydrolysis of triglycerides to fatty acids. Lipolysis, being 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 medium. Thus, when the amount of triglycerides present in the adipocyte vacuoles decreases, their volume decreases. The skin gradually resumes its "normal" appearance: the cellulite tissue is reduced, the orange peel effect is reduced. The unsightly appearance of the body fades away. Thus, the subject of the present invention is therefore the use of an effective amount of at least one peptide corresponding to the general formula (I):

(AA) _n - Pro-Gly-Pro - (AA) _n (I) wherein (AA) is any amino acid or a derivative thereof, n being between 0 and 3, as active ingredient in or for the preparation a cosmetic and / or dermatological and / or pharmaceutical composition.

According to a method of embodiment of the presently preferred invention, the peptide of formula (I) according to the invention will advantageously be used as a slimming active agent. According to another aspect, the peptide according to the invention can be used in or for the manufacture of a composition, for topical use, intended for the treatment of cellulite and / or the treatment of orange peel. The peptide or the composition containing it is advantageously used in order to reduce, eliminate or prevent subcutaneous fat overloads.

Indeed, the peptide has interesting effects in cosmetics and dermatology, mainly because of its particular and pronounced activity on lipolysis, which makes it particularly useful in slimming preparations. It will increase the elimination of triglycerides contained in the adipocyte vacuoles. This compound will help reduce, slow down or resorb fat deposits. The peptide 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, to reduce the appearance "orange peel".

The peptide used according to the invention may contain from 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 Proline-Glycine-Proline peptide sequence as well as the use of derivatives of these peptides. According to a presently preferred embodiment, the aforementioned peptide is preferably the tripeptide of Pro-Gly-Pro sequence. When a peptide containing the Proline-Glycine-Proline tripeptide is used, it is understood that it is chosen such that the amino acids surrounding the Proline-Glycine-Proline motif, both by their nature and by their structure. secondary of the peptide that they will induce, do not prevent it from carrying out the activity for which it is used in the present invention.

The invention also relates to variant forms of these peptides. The term "variant" herein refers to a polypeptide or peptide that differs from, for example, the sequence of a reference peptide while retaining its essential properties. Generally, the differences are limited so that the sequences of the reference peptide and those of the variant are quite similar and, in some regions, identical. Preferentially, the variant forms are those which vary from the reference sequences, by the substitution of chemically equivalent amino acids (or homologues), that is to say by the substitution of one residue for another having the same characteristics. Generally, the differences are limited so that the sequences of the reference peptide and those of the variant are quite similar and, in some regions, identical. A variant peptide and a reference peptide may thus differ from the amino acid sequence by one or more substitutions, additions, deletions in all combinations.

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

-NHR-CHR-C (O) -O- (H) where each -R is independently selected from hydrogen and an alkyl group having from 1 to 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 which may be linear or branched, substituted (mono- or poly-) or unsubstituted; saturated, mono-saturated (a double or triple bond in the chain) or polyunsaturated (two or more double bonds, two or more triple bonds, one or more double bonds and one or more triple bonds in the chain). The invention furthermore relates to the peptide derivatives of the peptide as defined above as well as peptides consisting of amino acid derivatives. The amino acid derivatives and peptide derivatives are, for example, those in which at least one functional group (in particular amino and carboxyl groups) is protected with a protective group. Indeed, it may be that for resistance to degradation, it is necessary to use, according to the invention, a protected form of the peptide. The form of protection must obviously be a biologically compatible form and must be compatible with use in the field of cosmetics or pharmacy. Many biologically compatible forms of protection can be envisaged, they are well known to those skilled in the art, such as, for example, the acylation or acetylation of the amino-terminal end, or the amidation or esterification of the carboxy terminus. Thus, the invention relates to a use as defined above characterized in that the peptide is in protected form or not. Preferably, protection based on either acylation or acetylation of the amino-terminus, or amidation or esterification of the carboxy terminus, or both is used.

Amino acid derivatives and peptide derivatives also relate to amino acids and peptides linked together by a pseudo-peptide bond. The term "pseudo-peptide bond" means all types of bonds likely to replace the "conventional" peptide bonds. In the field of amino acids, the geometry of the molecules is such that they can theoretically be in the form of different optical isomers. There is indeed a molecular conformation of the amino acid (AA) as it deviates to the right the plane of polarization of light (dextrorotatory conformation or D-aa), and a molecular conformation of the amino acid (aa) as it deviates on the left the plane of polarization of the light (conformation levogyre or L-aa). Nature has retained for natural amino acids only the levorotatory conformation. Consequently, a peptide of natural origin will consist only of amino acids of the L-aa type. However, Chemical synthesis in the laboratory makes it possible to prepare amino acids having both possible conformations. From this basic material, it is thus possible to incorporate during the synthesis of peptide both amino acids in the form of optical isomers dextrorotatory or levorotatory. Thus, the amino acids constituting the peptide according to the invention can be in L- and D- configuration. Preferably, the amino acids are in L- form. The peptide according to the invention can therefore be in L-, D- or DL- form.

By peptide is meant the natural or synthetic peptide of the invention as described above or at least one of these fragments, whether obtained by proteolysis or synthetically or any natural or synthetic peptide which the sequence is totally or partially constituted by the sequence of the peptide previously described. The peptides, subject of the present patent, can be obtained either by conventional chemical synthesis (in solid phase or in homogeneous liquid phase) or by enzymatic synthesis (Kullman et al., J. Biol Chem 1980, 225, 8234) to from constituent amino acids or their derivatives. The peptides according to the invention can also be obtained by fermentation of a strain of bacteria modified or not, by genetic engineering to produce the peptides of sequence indicated above and their fragments, or by extraction of proteins of animal or vegetable origin, preferably of vegetable origin, followed by controlled hydrolysis which releases the peptide fragments of medium sizes and small sizes in question, with the proviso that the released elements must contain at least the Pro-Gly-Pro sequence. Many proteins found in plants are likely to contain these sequences within their structure.

According to an advantageous embodiment of the invention, the aforementioned peptide is extracted from seaweeds of the genus Palmaria or Rhodymenia (sea red algae of the family Palmariaceae or Rhodymeniaceae). Extraction can be performed from fresh, frozen or dehydrated thalli.

Preferably the algae are desalinated beforehand by immersion in fresh water. This operation is performed at a temperature below 10 ⁰ C by simple soaking or under a stream of running water. The algae are then drained. Under these conditions, about 40% of the dry mass is discarded (salts and other solutes). In order to maximize the nitrogen content (proteins), an extraction of the porysaccharides, in particular, xylanes which represent 20% of the total weight can be produced. For this, the thalli are finely ground in an acid solution (pH between 0 and 3) and stirred for a few hours, cold. Under these conditions the polysaccharides are solubilized. The insoluble fraction is recovered by centrifugation or filtration. However, this operation, like the previous one, is optional because for certain cosmetic applications, it may be wise to keep the polysaccharide fraction.

The insoluble fraction is resuspended in an aqueous solution and is then subjected to a series of gentle hydrolysis to clear the biologically active peptide fragments. The hydrolysis can be carried out chemically or enzymatically using one or more proteases. The hydrolyzate can then be purified by fractionation, in particular by a method of the chromatographic type or purified by a dialysis process, making it possible to eliminate the high molecular weight protein compounds. Any of the more or less purified forms of the hydrolyzate is ultimately sterilized by ultrafiltration.

The peptide extract of alga obtained according to the invention is quantitatively analyzed for its content of proteinaceous compounds and more particularly for its biologically active peptide content, of formula (AA) _n -Pro-Gly-Pro- (AA) _n . These are demonstrated by chromatographic analysis, SDS gel electrophoresis or other conventional techniques well known to those skilled in the art, and / or by measuring their biological activity. By peptide extract is meant an aqueous solution of fragments of proteins, peptides and free amino acids.

Other simpler or more complex processes can be envisaged by those skilled in the art of synthesis, extraction and purification of proteins and peptides. Thus the peptide according to the invention may 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 first solubilized in one or more cosmetically or pharmaceutically acceptable solvents, conventionally used by those skilled in the art, such as water, ethanol, glycerol (glycerine) , xylitol, sorbitol, propylene glycol, butylene glycol, dipropylene glycol, ethoxylated or propoxylated diglycols, cyclic polyols, petroleum jelly, a vegetable oil or any mixture of these solvents.

According to yet another advantageous embodiment of the invention, the aforementioned peptide is first solubilized in a cosmetic or pharmaceutical vector such as liposomes or adsorbed on powdery organic polymers, mineral supports such as talcs and bentonites, and more generally solubilized in or attached to any cosmetically or pharmaceutically acceptable carrier.

The peptides according to the invention can 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 the treatment of orange peel. They are used in a more general way to reduce, eliminate or prevent subcutaneous fat overload.

Thus, according to another aspect, the invention relates to a cosmetic and / or dermatological and / or pharmaceutical composition containing, in an acceptable medium, as active ingredient, a cosmetically effective amount of at least one peptide corresponding to the general formula (I ). According to a particularly advantageous embodiment of the invention, the composition contains the Pro-Gly-Pro sequence peptide. In a more general manner, the composition according to the invention contains the active compound as defined above. In the composition according to the invention, the peptide may be a mixture of peptide derivatives and / or consisting of amino acid derivatives. It is understood that the peptide according to the invention may be used alone or in combination with at least one other active agent.

The composition containing the peptide according to the invention may be a cosmetic or dermatological or pharmaceutical composition. Preferably, according to the invention, the composition is a cosmetic composition, because it is intended to improve the appearance and general cutaneous performance of the individual who makes use of it. The composition according to the invention is preferably a cosmetic and / or dermatological composition suitable for topical administration by the cutaneous route comprising a cosmetically or pharmaceutically acceptable medium. It is obvious that the invention is directed to mammals in general and more particularly to humans. This composition is particularly intended to obtain a slimming action, and / or intended to reduce, eliminate or prevent subcutaneous fat overload. The composition according to the invention is also advantageously adapted to to fight against cellulite and / or to reduce or disappear the phenomenon of orange peel.

The effective amount of active ingredient is the amount needed to achieve the desired result. According to an advantageous embodiment of the invention, the abovementioned peptide is present in the compositions of the invention at a concentration of between about 0.005 and 500 ppm (parts per million), and preferably at a concentration of between 0, 1 and 50 ppm by weight relative to the total weight of the final composition.

By way of example, the composition according to the invention may be applied locally to the areas of the face or body to be refined, in particular on the hips, buttocks, thighs, belly or face. One of the many advantages of the present invention is to have an effective topical treatment against adiposity while employing "soft" methods.

Whatever the form of the invention, the composition according to the invention can be injected or applied to the skin (on any cutaneous area of the body), hair, nails or mucous membranes. Depending on the mode of administration, the composition according to the invention may be in any of the galenical forms normally used. Preferably, the compositions according to the present invention will be in a dosage form suitable for topical administration to the skin, and cover all cosmetic or dermatological forms. These compositions must therefore contain a cosmetically acceptable medium, that is to say, compatible with the skin, mucous membranes and integuments.

These compositions may especially be in the form of an aqueous solution, hydroalcoholic or oily; an oil-in-water, water-in-oil emulsion or multiple emulsions; they too can be in the form of creams, suspensions or powders, suitable for application to the skin, mucous membranes, lips and / or integuments. These compositions may be more or less fluid and have the appearance of a cream, lotion, milk, serum, ointment, gel, paste or paste. a foam. They can also be in solid form, as a stick or be applied to the skin in aerosol form. They can be used as a care product and / or as a make-up product for the skin. These compositions additionally comprise any additive usually used in the field of application envisaged as well as the adjuvants necessary for their formulation, such as solvents, thickeners, diluents, antioxidants, dyes, sunscreens, self-tanning agents, pigments, fillers, preservatives, perfumes, odor absorbers, cosmetic or pharmaceutical active ingredients, essential oils, vitamins, essential fatty acids, surfactants, film-forming polymers, etc. In all cases, those skilled in the art ensure that these adjuvants and their proportions are chosen in such a way as not to adversely affect the desirable properties of the composition according to the invention. These adjuvants may, for example, correspond to 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 from 5 to 80% by weight and preferably from 5 to 50% by weight relative to the total weight of the composition. The emulsifiers and co-emulsifiers used in the composition will be chosen from those conventionally used in the field under consideration. For example, they can be used in a proportion ranging from 0.3 to 30% by weight, relative to the total weight of the composition. Of course, those skilled in the art will take care to choose any additional compounds, active or non-active, and / or their amounts, so that the advantageous properties of the mixture are not, or not substantially, altered by the addition contemplated.

According to the invention, it is possible to add to the composition of the invention other active agents intended, inter alia, for the treatment of cellulite and the "orange peel" phenomenon. Preferentially, the slimming composition according to the invention will contain, for example, advantageously, in addition to the active as defined above, an active promoting lipolysis or inhibiting lipogenesis.

The compositions according to the invention find an application in particular as cosmetic or pharmaceutical compositions for the skin, mucous membranes and / or semi-mucous membranes. They find application as a protective and / or skin care product, but above all they find a particular application as a slimming composition and / or firming. The slimming and / or firming composition may be applied locally to the areas of the face or body to be refined, in particular on the hips, buttocks, thighs, stomach or face. It is also possible to envisage other applications in the field of compositions in combination, for example with other active agents. The present invention also relates to a cosmetic care method for obtaining a slimming action, and a cosmetic care method for reducing, eliminating and / or preventing subcutaneous fat overload. This process may also be intended to fight against cellulite and / or to fight against the phenomenon of orange peel. This method consists in applying, topically, to the relevant areas of the skin of the face and / or the body, an effective amount of the peptides, according to the invention, as defined previously or of a composition, as defined above, the container.

The cosmetic treatment method of the invention may be implemented in particular by applying the cosmetic compositions as defined above, according to the usual technique of use of these compositions, for example: application of creams, gels, serums , lotions, milks, or shampoos on the skin. Particular embodiments of this cosmetic treatment method also result from the foregoing description. Other advantages and characteristics of the invention will appear better on reading the examples given by way of illustration and not limitation.

Example 1 Preparation of peptides by extraction from plants of the species Palmaria palmata The red alga Palmaria palmata (or dulse) is rich in proteins (approximately 25%), vitamins (B6, B12), iron and fluorine in particular.

100 grams of dehydrated thalli of Palmaria palmata (Dulce or Dulse) are desalted in fresh water at 4 ° C by four times 2 liters of water. The desalted thalli are recovered by filtration. The desalted thalli are suspended in 1.5 liters of acidic aqueous solution at pH 1.5 and then ground with ultra-turrax. This mixture is stirred for two hours, cold, to allow the solubilization of the polysaccharide fractions. The insoluble residue is decanted by centrifugation and then hydrolyzed using an enzymatic cocktail. After hydrolysis, the medium is filtered to retain only the soluble fractions. Polypeptides and other high molecular weight molecules are removed by membrane dialysis. The dialysate is then sterilized by ultrafiltration on a 0.2 μm filter. An aqueous solution of brown color is then obtained. The peptide extract thus obtained is analyzed by HPLC (high performance liquid chromatography) and by SDS gel electrophoresis for its peptide content (AA) _n -Pro-Gly-Pro- (AA) _n . The peptide extract according to the invention contains about 50 milligrams per liter of biologically active peptides.

Example 2 Demonstration of the Activity of the Peptides According to the Invention on Adipocytes

1) Experimental protocol. 3T3-L1 preadipocyte cell line cells, undifferentiated, are inoculated in 8-well LabTek (for OII Red staining), in 24-well plates (for the assay of ¹ cAMP) and in 6-well plates ( for the determination of glycerol) as well as with DMEM culture medium (4.5 g / l) supplemented with antibiotics. These 3T3-L1 preadipocytes are able to return, under certain conditions, in terminal differentiation phase.

Once 3T3-L1 cells have reached 100% confluence, these cells are differentiated by culture in different media over a period of 6 to 8 days. During the first 2 days, the cells are cultured in the presence of IBMX, dexamethazone and insulin, diluted in 4.5 g / l DMEM culture medium. Then they are cultured for two days in the presence, only insulin diluted in DMEM 4.5 g / l culture medium and finally, the cells are cultured for 2 to 3 days in culture medium containing exclusively DMEM 4 , 5 g / 1. The differentiation of cells into mature adipocytes is visualized by the appearance of lipid droplets in the cytoplasm. The effect of the peptide according to the invention as well as its impact on the differentiated or differentiating 3T3-L1 cells has been evaluated through various techniques: the "OiI Red" staining, the dosage of the amount of the MPA. intracellular cyclic, - the measurement of glycerol released by the cells. 2) The "Oil Red" coloring:

The principle of this test is based on a microscopic observation of the number and size of the lipid vacuoles of the adipocytes after staining. The adipocytes being incubated or not, in the presence of the substance to be tested. The 3T3-L1 cells, seeded in 8-well LabTeks, are treated with a Pro-Gly-Pro sequence peptide, representative of the family of peptides according to the invention, dissolved in 1% of a 50 ppm solution. . The active ingredient was added at different stages of culture: 3 days during adipocyte differentiation and once the cells differentiated into adipocytes, the cells being treated with the active agent for 16 or 24 hours.

After these different incubation periods, in the presence or absence of the active agent to be tested, the 3T3-L1 are stained by the "OiI Red" technique. The "OiI Red" solution (Sigma, O-0625) is prepared by adding 0.5 g of product in 100 ml of isopropanol and by dilution to 4/10 in distilled water, followed by filtration. The cells are fixed for 10 minutes in a 4% formalin solution and NaCl, the Red OiI solution is applied for 15 minutes. Hematoxylin counterstaining for 30 seconds is possible. The cells are then rinsed with warm water and mounted on slides in a hydrophilic medium (Aquatex). The observation is carried out under an optical microscope so as to distinguish the fatty vacuoles colored in red. The results of the observation of the cells demonstrate that, unlike the control adipocytes (that is to say on which the active has not been applied), which have a large spherical shape and a large accumulation of intra lipid vesicles -cytoplasmic; the adipocytes, treated with a solution containing the active agent according to the invention, have a less rounded morphology and a content in lipid vesicles intra-adipocyte significantly decreased. The solution containing the peptide according to the invention is therefore particularly effective in the process of limiting adipocyte hypertrophy by increasing, presumably, the phenomenon of lipolysis, thus allowing removal of triglycerides contained in the intra-adipocyte vesicles.

3) Determination of Intracellular Cyclic AMP: The objective of this test is to measure the variation of intracellular cAMP concentration in adipocytes. The test was performed on differentiated 3T3-L1 cells. These adipocytes are treated with the tripeptide of Pro-Gly-Pro sequence, representative of the peptide family according to the invention, dissolved in 0.5% of a 50 ppm solution for periods of 3 or 5 hours. . After the different incubation periods, in the presence of the active substance to be tested or in its absence, the quantity of cAMP contained in the adipocytes is measured by means of the "cAMP Biotrak® EIA System" kit from Amersham Biosciences as well as on a colorimetric principle of the ELISA type. The kit allows lysis of the cells with a solution thus allowing the hydrolysis of the cell membranes and the release of intracellular ¹ cAMP in the medium. Then binding to the capture antibody of the intracellular cAMP, extracted from the samples, is put into competition with cAMP coupled to an enzyme, peroxidase. Thus, after a revelation with the TMB substrate, the more cAMP will be in the samples to be assayed, the weaker the signal will be.

The results, presented in the table below, express the concentration of cAMP present in the adipocytes according to the different study conditions.

These results demonstrate that in the presence of the active, there is an increase in the amount of intracellular cAMP in the adipocytes, compared to the control condition, that is to say compared to the untreated treated cells.

4) Determination of glycerol:

The purpose of this study is to determine the influence of the peptide according to the invention on the release of glycerol by the adipocytes.

The 3T3-L1 cells, differentiated or undifferentiated, are treated at a dose of 1% of a 50 ppm solution containing the tripeptide of Pro-Gly-Pro sequence, representative of the peptide family according to the invention, during periods of 4 hours and 5 hours.

The determination of glycerol is carried out by means of a cascade enzymatic reaction leading to the formation of NADH whose quantity produced is evaluated by spectrophometric reading at 340 nm, the formation of NADH being proportional to the quantity of glycerol released into the culture medium. An internal control of glycerol at ImM was also used. The values are standardized with respect to the amount of protein for each sample.

The results obtained are shown in the table below. They allow us to observe that at the different treatment times studied, the release of glycerol increases significantly when the mature adipocytes are treated with the peptide according to the invention.

5) Conclusions:

These results thus tend to demonstrate that the peptides according to the invention have a really effective action on the level of adipocytes and that they prove to be particularly effective in the process of increasing lipolysis.

Indeed, the results of the "OiI Red" staining test allow us to conclude that the active agent according to the invention has a particularly effective effect in reducing the amount of intra-adipocyte lipid droplets. In addition, the peptide according to the invention induces an increase in the amount of intracellular cAMP. However, the increase in the amount of intracellular cAMP is an indicator of the activation of the lipolysis pathway: the level of intracellular cAMP regulating the activity of Triglyceride-lipase, the enzyme allowing the hydrolysis of triglycerides. Thus, an increase in the amounts of intracellular cAMP suggests that the active agent according to the invention acts by increasing the phenomenon of lipolysis. These results are, moreover, confirmed by the increase of glycerol release in the extracellular medium, glycerol being a product of lipolysis. In conclusion, the active agent according to the invention has a stimulating activity of the lipolysis mechanism in adipocytes. This increase in the phenomenon of lipolysis results in a decrease in the amount of triglycerides contained in the adipocyte vesicles of the adipocytes. This decrease in the content of Lipid vacuoles thus allow, on a larger scale, a reduction in fat mass.

Example 3 - Preparation of compositions

1- Slimming cream

The components of phase A are melted at 75 ° C and the components of phase B heated to 75 ° C. Phase A is emulsified at B, then the mixture is cooled below 40 ° C. Phases C and D are then added with constant stirring. 2 - Firming Spray - Slimming

The components of phase A and phase B are heated separately at 65 ° C; phase B is incorporated in phase A with stirring. The temperature of the mixture is raised to 83 ° C and then cooled to a phase inversion temperature. Phase C is then added. The asset is incorporated when the temperature reaches less than 40 ° C. It is then possible to add perfumes and / or dyes. 3 - Firming Gel - Slimming - anti-cellulite

Carbopol gel is prepared at 2%. The ingredients are added in the order listed above with stirring. The mixture is then neutralized with the TEA. The perfume and the dyes are added if necessary.

Claims

Use of an effective amount of at least one peptide corresponding to the general formula (I): (AA) n-Pro-Gly-Pro- (AA) n (I) wherein (AA) is any acid amine or one of its derivatives, n is an integer from 0 to 3; as an active agent, in or for the preparation of a cosmetic and / or dermatological and / or pharmaceutical composition.

2. Use of an effective amount of at least one peptide according to claim 1 as a slimming active agent.

3. Use of an effective amount of at least one peptide according to claim 1 or 2, in or for the manufacture of a cosmetic and / or pharmaceutical composition for topical use; the peptide or the composition containing it being intended for the treatment of cellulite and / or orange peel; and / or to reduce, eliminate or prevent subcutaneous fat overloads.

4. Use according to any one of the preceding claims characterized in that the peptide has the Pro-Gly-Pro sequence.

5. Use according to any one of the preceding claims, characterized in that the peptide is chosen from peptides 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 amino end. or on an amidation or esterification of the carboxy terminus, or both.

6. Use according to any one of the preceding claims characterized in that the peptide is first solubilized in one or more cosmetically or pharmaceutically acceptable solvents such as water, ethanol, propylene glycol, butylene glycol, dipropylene glycol, diglycols ethoxylated or propoxylated, cyclic polyols, petroleum jelly, a vegetable oil or any mixture of these solvents.

7. Use according to any one of the preceding claims characterized in that the peptide is previously solubilized in a cosmetic or pharmaceutical vector such as liposomes or adsorbed on powdery organic polymers, inorganic supports such as talcs and bentonites, and more generally solubilized in, or attached to, any cosmetically or pharmaceutically acceptable carrier.

8. Cosmetic and / or dermatological and / or pharmaceutical composition, characterized in that it contains, in an acceptable medium, as active ingredient, at least one peptide corresponding to the general formula (I):

(AA) n-Pro-Gly-Pro- (AA) n (I) wherein (AA) is any amino acid or a derivative thereof, n is an integer of 0 to 3.

9. Composition according to claim 8, characterized in that the peptide has the Pro-Gly-Pro sequence.

10. Composition according to claim 8, characterized in that the peptide is derived from a seaweed extract of the genus rhodymenia.

11. Composition according to claim 10, characterized in that said alga is an algae of the species Palmaria palmata.

12. Composition according to any one of claims 8 to 11, characterized in that the peptide is present at a concentration of between 0.005 and 500 ppm, and preferably at a concentration between 0.1 and 50 ppm.

13. Composition according to any one of claims 8 to 12, characterized in that it is in the form of a cosmetic composition and / or dermatological method adapted for topical administration to the skin comprising a cosmetically or pharmaceutically acceptable medium.

14. Composition according to any one of claims 8 to 13, characterized in that it is in the form of an aqueous solution, hydroalcoholic or oily or in the form of an oil-in-water emulsion, water-in oil or multiple emulsions or in the form of creams, suspensions, or powders; these compositions may be more or less fluid or solid and have the appearance of a cream, a lotion, a milk, a serum, an ointment, a gel, a paste, a foam or a stick.

15. Cosmetic treatment method for reducing, eliminating and / or preventing subcutaneous fat overloads, and / or intended to fight against cellulite, and / or to fight against the phenomenon of orange peel, characterized in that an effective amount of a composition as defined in any one of claims 8 to 14 is applied to the surface of the skin.