FR2802413A1 - COSMETIC OR DERMOPHARMACEUTICAL COMPOSITIONS CONTAINING THE N-PALMYTOYL-GLY-HYS-LYS TRIPEPTIDE FOR ELIMINATING, REDUCING OR PREVENTING THE APPEARANCE OF WRINKLES, WHEREVER THE LOCATION AND THE CAUSE - Google Patents

Abstract

The patent describes the use of the tripeptide N-palmytoyl-Gly-Hys-Lys, in cosmetic or dermopharmaceutical compositions. This tripeptide can be obtained by synthesis, by biotechnology or by controlled hydrolysis of vegetable proteins. Used topically, these compositions are advantageously used to eliminate, reduce or prevent the appearance of wrinkles, whatever their location and cause, as well as to improve the shape of the mouth.

Description

Aging, especially that of the skin, causes significant

  intimate biochemical tissue disturbances manifested by gross changes, usually considered unsightly, which have continued

  worry both women and men: wrinkles.

  Other components of our current way of life, such as the physical and chemical aggressions of pollution; alcohol and tobacco consumption promote and worsen the aging process. It is the same for the perpetual quest for tanning by natural or artificial UV, through

  heliodermia (Dr C. Musy-Preault, (1994)).

  The cosmetic industry is constantly on the lookout for new ingredients capable of repairing the irreparable outrage for years, and therefore of minimizing the main stigmas of wrinkles, by delaying the appearance or by

decreasing importance.

  It is now certain that the decrease in the quantity and quality of substance in the extracellular matrix is the main cause of the appearance of wrinkles. The improvement or reconstruction of the extracellular matrix is essential because we now know that not only does this structure play "the role of a framework stabilizing the physical structure of the tissues" but that it also plays a role

  20 .... in regulating the behavior of cells which are in contact with it -

  influencing their development, their migration, their proliferation, their form and their functions (Molecular Biology of the Cell, 3rd ed. Medicine-Sciences,

Flammarion, Paris, 1997: 972).

  As collagen is the main constituent of the cell matrix, acting on the quantity and quality of tissue collagen is equivalent to acting in a beneficial way

on the latter.

  In the context of this patent, the effects of aging on collagens can be summarized by: - The decrease in its synthesis by fibroblasts, a decrease due to the conjunction of two causes: on the one hand, the rate of renewal of these producer cells decreases with age and, on the other hand, the quantity of molecules

  secreted by these cells also decreases.

  When we know that collagen represents approximately 80% of skin proteins, it is easy to understand that the slightest decrease in its tissue concentration can have significant consequences on

  mechanical and physiological properties of the skin.

  - The appearance of structural modifications of the neosynthesized molecules which

  lead to the crosslinking of the fibers and therefore to their stiffening.

  For collagen, variations in the aC chains modify the distribution of its different forms. For example, the proportion of collagen type mI increases in the epidermis when collagen type IV accumulates in the basement membrane. We also note the appearance of reactions, enzymatic or not (of the Maillard reaction type), which create so-called crossed bonds, either between two collagen fibers, or between the collagen itself and molecules of

  glucose, thus stiffening the collagen fiber networks.

  The skin therefore loses its substance by the decrease in the amount of its constituents, hardens by the loss of elasticity of the collagen fibers and by its dehydration and this contributes to giving the aged skin its characteristic aspects: absence of flexibility, finesse, fragility, and above all, wrinkles more or

  less numerous and more or less deep.

  Acting on the quantity and quality of skin collagen is therefore the best

  approach to fight wrinkles.

  It is only to be convinced that the growing number of Dermatologists who use the intradermal injection of collagen to fill the tissue voids responsible for wrinkles. This method however has certain more or less significant drawbacks. In addition to the cost of these procedures, the collagen injected can be recognized as part of the non-self and thus cause, at the very least, necessarily undesirable inflammatory reactions. In addition, this artificially added biological material will quickly undergo normal enzymatic degradation, which will induce a cycle of collagen injections according to

  frequencies closer and closer together.

  The invention which is the subject of this patent application resides in the fact that we have developed a product, the tripeptide, N-palmitoylGly-His-Lys, which makes it possible to restore the concentration of a better collagen, which, being newly formed by the body itself, will erase or reduce the importance of wrinkles while ensuring perfect tolerance and painless to the user. These effects have been studied and demonstrated, both in vitro and in vivo, by sophisticated scientific tests which will be explained in the examples given in this patent application. This Gly-His-Lys sequence, linked to a copper atom, was described in 1989 (WO 89/12441) in cosmetic compositions intended to thicken adipose tissue

  subcutaneous of warm-blooded animals and / or the skin and its subunits in general.

  Among other peptide sequences, N-acylated or not, in 1990, we had described (FR90 / 13349) Gly-His-Lys usable in cosmetic preparations,

  without however claiming any particular physiological activity.

  Since the filing of our patent, certain documents have claimed general cosmetic activity: - by excluding the possibility of N-acylation by a palmitic acid (FR-95 13543 dul S / 11/95), - by mentioning that a tripeptide, not N-acylated, must contain any possible association of amino acids found in thymulin or in thymopoietin AA (FR-95 13544) without the Gly-His-Lys sequence being mentioned, glycosylated and preferably linked to an atom metallic like zinc, - by only describing the use of 3-methyl-L-His, linked or not to another indeterminate molecule to facilitate transcutaneous passage, in topical use against collagen damage due to UV radiation ( WO

/ 06102),

  - by only mentioning an amino acid including histidine which can be grafted to a fatty acid chain of 6 to 24 carbons, and claiming emulsifying, antioxidant, bactericidal, chelating or sunscreen activities (JP

24655/91).

  Finally, which was not yet the case in 1990, it is now well known that there is no single kind of collagen and that the quality of skin tissue depends not only on the quantity of tissue collagen but also on the harmonious distribution of its different forms. Types I, II, II V and XI, fibrillar forms, fill in the empty spaces while type IV forms the lamellar network of the basal lamina and type VII constitutes the anchor fibrils of the stratified squamous epithelia (for example Albert B et al., Molecular Biology

  de la cellule, 3rd ed., Médecine Science, Flammarion ed., Paris, 1997: 979-980).

  Surprisingly, following extensive studies, we discovered that the tripeptide of sequence Gly-His-Lys, had a spectacular and unpredictable effectiveness in reducing, even eliminating, wrinkles but also in preventing of their appearance and this, whatever the cause: aging, prolonged and excessive exposure to natural or artificial UV,

  approximate lifestyle .......

  We have also found, as is sometimes the case (FR 90 13349), that acylating the N-terminal amine or esterifying the carboxyl group of this (these) peptide (s), increases the stability of the molecule and tissue availability by facilitating the passage of the skin lipid barrier of this type of structure

essentially hydrophilic.

  In this case, not only is the anti-wrinkle activity preserved, but it is even more spectacular. The tripeptides, which are the subject of this patent application, can be obtained either by conventional chemical synthesis (in heterogeneous phase or in homogeneous phase), or by enzymatic synthesis (Kullman et al., J. Biol. Chem. 1980, 255, 8234) from

  constituent amino acids or their derivatives.

  The small size of the tripeptide allows it to be synthesized industrially, at an advantageous cost. Its large demonstrated activity authorizes commercial use in a large number of cosmetic or dermopharmaceutical products.

financially acceptable.

  The tripeptide, can also be obtained by fermentation of a strain of bacteria, modified or not by genetic engineering, to produce the sequences

  sought or their different fragments.

  Finally, the tripeptide can be obtained by extraction of proteins of animal or vegetable origin, preferably vegetable, capable of containing these sequences within their structure, followed by a controlled hydrolysis, enzymatic or not, which releases the peptide fragment of sequence Gly-His-Lys in plants which are likely to contain these sequences within their structure. Hydrolysis

  made it possible to release these peptide fragments.

  To carry out the invention, it is possible, but not necessary, to extract either the proteins concerned first and then hydrolyze them, or to carry out the hydrolysis first on a crude extract and to purify the peptide fragments then. It is also possible to use the hydrolyzate without extracting the peptide fragments in question therefrom, making sure however to have stopped the enzymatic hydrolysis reaction in time and to measure the presence of the peptides in question by appropriate analytical means (tracing by radioactivity,

  immunofluorescence or immunoprecipitation with specific antibodies, etc.).

  Other simpler or more complex processes, leading to cheaper or purer products, are easily conceivable by those skilled in the art knowing the

  profession of extraction and purification of proteins and peptides.

  By way of example illustrating the invention, a few cosmetic formulas which are representative but not limiting of the invention are cited: Example No. 1: Carbopol 1342R gel 0.3 Propylene glycol 2.0 Glycerin 1.0 White petrolatum 1.5 Cylomethicone 6 .0 Sipol C16C18S3 0.5 LubrajelR MS 10 triethanolamine 0.3 N-Palmitoyl-Gly-His-Lys 0.0005

  Water, preservatives, perfume qs 100 g.

  Example 2: BrijR 721 cream 2.4 BrijR 72 2.6 ArlamolR E 8.0 Beeswax 0.5 AbilR ZP 2434 3.0 Propylene glycol 3.0 CarbopolR 941 0.25 Triethanolamine 0.25 N-Palmitoyl-Gly-His-Lys 0.005 Water, preservatives, perfumes qs 100 g activities described at the beginning of this request are illustrated by the examples

following.

  Example 3: Increase in collagen synthesis: in vitro The method chosen is a variant of that described by Augustin C. et al. (Skin Pharmacol. (1997); 10: 63-70) in that we used human skin explants instead of human pulmonary fibroblasts, in order to make our

  results directly usable in cosmetology.

  These explants, coming from mammary or abdominal plasty, are incubated, for 72 hours in the presence of 3H-proline, with the tripeptide in three concentrations

  final in the culture medium (1.1 04%, 3.1 0'4% and 1.10-3%; ie 1, 3 and 10 ppm).

  The explants are then washed, the dermis and epidermis of each explant are separated, homogenized and lysed. The measurement of the incorporation of 3H-proline is then

  performed in each lysate. The tests are carried out in triplicate.

  At the same time, negative controls are carried out under the same conditions but in the absence of the tripeptide. Positive controls, for their part, are carried out by replacing the tripeptide tested with vitamin C. In the presence of 1, 3 or 10 ppm of the tripeptide, the incorporation of 3H-proline which translates the synthesis of collagen, is increased 15.3 (+ 1)%, 34.7 (+ 4)% and 75.3 (+ 4)% respectively compared to what is found in the experiments

witnesses (without the tripeptide).

  Under the same conditions, the reference product, ascorbic acid, at the

  0.5mM concentration, increases the synthesis of collagen by 32.7 (+ 5)%.

  Additional studies allowed us to refine these raw results and to note that collagen type I, but also collagens of types IV and VII increased in such a way that collagen I neoformé could fill volumes whose edges consisted of type IV and VII collagens, also newly formed, thus providing the entire tissue with new

  homogeneity and a lasting effect on the reduction or elimination of wrinkles.

  Example No. 4: Skin Firming Activity: In Vitro For 24 hours, human fibroblates originating from the same cell culture are placed in the presence of supplemented standard culture medium, or not for

  controls, with different concentrations of tripeptide (1, 3 and 10 ppm).

  The stimulation of protein synthesis is evaluated by colorimetry (so-called reaction

Biuret).

  To standardize the results, the quantity of protein measured is expressed per 1000 cells present in the test. Compared to the control experiments, in the presence of 1, 3 and 10 ppm of tripeptide, the increase in the protein concentration is 15.3 (+ 1)%, 32.2 (+3)% and 53 respectively, 5 (+ 3)%. Thus, this in vitro test demonstrates the stimulating potential, dependent concentration, at the cutaneous level of the tripeptide, effect directly linked to a firming and a

  thickening of excessively thin skin.

  Example 5: Anti-wrinkle activity: in vivo 25 This example reports the anti-wrinkle effect obtained, in vivo, on a panel made up of 150 adult female volunteers, aged 35 to 63 years. The anti-wrinkle power of the cream of Example 2, containing N-Palmitoyl-Gly-His-Lys, at a concentration of 0.005% (i.e. 50 ppm), is compared to that of a placebo cream (same cream but without the asset). The creams are applied to precisely identified sites, located on the corner of the right or left eye, in a randomized distribution, twice a day, for 28 days. This experiment was carried out according to a so-called double-blind protocol since neither the volunteer nor the experimenters knew the nature of the product applied. It was only when all the results were analyzed that the codes were lifted. The parameter taken into account is the skin relief, at the level of the eye contour (wrinkles called crow's feet). The quantifications of the various relief variables are carried out by video-computer analysis of silicone impressions taken on the surface of the skin according to the protocols described by Corcuff et al. (1985, Int. J Cosm. Sci.7: 117-126) and Corcuff et al. (1995, in Handbook of non-invasive methods and the skin, Serup & Jemec

eds., CRC Press: 89-96).

  The tables below indicate the difference, in percentage, of the average values obtained between T +28 or T + 60 days and TO for the average depths of the main wrinkle (column A) or for all the folds (col. B ); for the density of the main folds (col. C) as well as for the measurement of roughness

(col. D).

  * After 28 Days of treatment ABC b Placebo 0.2 + 0.2 - 0.7 + 1.0 Tripeptide - 15.7 - 17.8 - 29.7 - 27.3 After 60 Days of treatment = BC b Placebo 0.3 + 0.2 - 0.9 + 1.2 Tripeptide - 25.3 - 28.9 - 41.3 - 34.4 It should be noted that, under the same experimental conditions, the placebo cream does not present absolutely no effect if N-Palmitoyl-Gly-His-Lys has not been incorporated into it, which clearly shows that it is only NPalmitoyl-Gly-His-Lys

  that we can attribute the observed beneficial effect.

  The cream containing the N-Palmitoyl-Gly-His-Lys previously described therefore clearly demonstrates a powerful anti-wrinkle effect since there is a significant progressive difference between the start and the end of the study in vivo and this, on

  all of the four parameters conventionally used in this indication.

  The most remarkable result we saw at the end of this study carried out on 150 volunteers was the spontaneous personal appreciation of the volunteers. Indeed, at the time of each measurement (T + 28 and T + 60 days), among other questions on the tolerance and cosmetic comfort of the creams tested, each volunteer had to indicate which side seemed to him the best. The final analysis of the results demonstrated that at T + 28 days and T +60 days, respectively 78% and 82% of the volunteers had designated the side receiving the

cream with tripeptide.

  The double-blind protocol further demonstrates the dramatic effect of the

N-Palmitoyl-Gly-His-Lys tripeptide.

  Example 6: Activity on the curve of the mouth: in vivo The study evaluated in 10 female volunteers (aged 18 and 45) with thin and dry lips, the effect of a labial "stick" on hydration

  superficial, smoothing and volume of the lips.

  This stick was of classic composition if it is not the addition of our NPalmitoyl-Gly-

  His-Lys at a concentration of 10 ppm.

  The level of surface hydration was rated by the dermatologist in charge of the study; the volume measured by Toposkin (from two photos taken simultaneously from two different angles, suitable software allows you to restore

  the three-dimensional image and make volumetric measurements).

  In addition, the volunteers had to fill out questionnaires based on subjective criteria. The following table shows the means (arbitrary units) and the standard deviations obtained at TO and T +30 days on the hydration evaluated at the level of the lip.

  upper and lower lip and on the flobal labial volume.

  Lip hydration Lower upper volume

T 0 2.0 + 07 1.7 _ 0.7 3.7 _ + 1.0

  T +30 3.2 _ + 0.6 2, + + 0.5 4.9 + 1.5

  The self-assessment showed that an obvious improvement in the state of the lips (in% of volunteers), satisfaction with skin comfort (70%), a feeling of protection (70%), an improvement in roughness ( 70%) and an improvement in

  flaking judged as moderate (40%) to large (20%).

  This study clearly demonstrates the beneficial effect of the lip stick containing our N-Palmitoyl-Gly-His-Lys, at a concentration as low as 10 ppm), on subjective and objective parameters qualifying the appearance and physiological state of the lips. . By grafting a fatty acid chain, from 2 to 22 carbons, hydroxylated or not, saturated or not, linear or branched, sulfated or not, on the N-terminal amine of the tripeptide Gly-His-Lys to obtain the N-Palmitoyl-Gly-His-Lys, it is advantageous to confer on these peptides, hydrophilic by nature, lipophilic properties to increase their cutaneous passage and therefore their availability and to facilitate their

  incorporation in cosmetic and dermopharmaceutical compositions.

  The tripeptide of this patent, acylated or not, can be obtained by chemical synthesis, by enzymatic route, by fermentation, by extraction of proteins of vegetable origin, by conventional peptide synthesis in homogeneous or heterogeneous phase or

  by enzymatic synthesis from the constituent amino acids.

  The tripeptide of this patent, acylated or not, subject of this patent, can be obtained by extraction of plant proteins, followed by hydrolysis, enzymatic or non-enzymatic, so as to generate peptide fragments of average size between 300 and 1500 daltons, part of the fragments released in front

contain the Gly-His-Lys sequence.

  The tripeptide of this patent, acylated or not, can be used at concentrations varying between 0.1 and 1000 ppm (w / w), preferably between 1 and 200 ppm (w / w)

  in the finished cosmetic or dermopharmaceutical product.

  The tripeptide of this patent, acylated or not, can be used in the form of a solution,

  dispersion, emulsion, or encapsulated in vectors such as macro-, micro-

  or nanocapsules, liposomes or chylomicrons, or included in macro-, micro- or nanoparticles, or in micro-sponges, or adsorbed on polymers

  powdery organic, talcs, bentonites and other mineral supports.

  The tripeptide of this patent, acylated or not, can be used in any galenical form: O / W and W / O emulsions, milks, lotions, gelling and viscous polymers, surfactants and emulsifiers, ointments, hair lotions, shampoos, soaps,

  powders, sticks and pencils, sprays, body oils.

  The tripeptide of this patent, acylated or not, can be used with any other ingredient usually used: extraction and / or synthetic lipids, gelling and viscosifying polymers, surfactants and emulsifiers, hydrosoluble or active ingredients,

  plant extracts, tissue extracts, marine extracts, sun filters, antioxidants.

  The tripeptide of this patent, acylated or not, can be used in cosmetic applications to eliminate, reduce or prevent the appearance of wrinkles, whether they

be the location and the cause.

  The tripeptide of this patent, acylated or not, can be used for the preparation of a medicament for eliminating, reducing or preventing the appearance of wrinkles, whether they are

be the location and the cause.

Claims (10)

Claims   1. Use of the tripeptide Gly-His-Lys in cosmetic or dermopharmaceutical compositions to eliminate, reduce or prevent the appearance of wrinkles, whatever their location and cause as well as to improve the curve from the mouth.   2. Use of the tripeptide in cosmetic or dermopharmaceutical compositions according to 1, characterized in that its lipophilicity is increased by grafting a fatty acid chain, from 2 to 22 carbons, hydroxylated or not, saturated or not, linear or branched, sulfated or not, on   the N-terminal amine and / or by the esterification of its carboxyl group.   3. Cosmetic compositions characterized in that they contain at least one tripeptides according to 1 to 2.   4. Cosmetic or dermopharmaceutical compositions according to 3, characterized in that the tripeptide (s) is / are obtained by chemical synthesis, by enzymatic route, by fermentation or by extraction of proteins of vegetable origin. 5. Cosmetic or dermopharmaceutical compositions according to 3 to 4, characterized in that the tripeptide (s) is / are obtained by conventional peptide synthesis in homogeneous or heterogeneous phase or by enzymatic synthesis with   from the constituent amino acids.   6. Cosmetic or dermopharmaceutical compositions according to 3 to 5, characterized in that the tripeptide (s) is / are obtained by extraction of plant proteins, followed by hydrolysis, enzymatic or non-enzymatic, so as to generate peptide fragments of average size between 300 and   1500 daltons, part of the fragments released must contain the sequence Gly-   His-Lys. 7. Cosmetic or dermopharmaceutical compositions according to 3 to 6 characterized in that the tripeptide (s) is / are used at concentrations varying between 0.1 and 1000 ppm (w / w), preferably between 1 and 200 ppm (w / w) in the final product.   8. Cosmetic or dermopharmaceutical compositions according to 3 to 7, characterized in that the tripeptide (s) is / are used in the form of a solution, dispersion, emulsion, or encapsulated in vectors such as macro-, micro- or nanocapsules, liposomes or chylomicrons, or included in macro-, micro- or nanoparticles, or in micro-sponges, or adsorbed on powdery organic polymers, talcs, bentonites and other mineral supports. 9. Cosmetic or dermopharmaceutical compositions according to 3 to 8, characterized in that the tripeptide (s) is / are used in any galenical form: O / W and W / O emulsions, milks, lotions, gelling and viscosifying polymers , surfactants and emulsifiers, ointments, hair lotions, shampoos,   soaps, powders, sticks and pencils, sprays, body oils.   10. Cosmetic or dermopharmaceutical compositions according to 3 to 9, characterized in that the tripeptide is used with any other ingredient usually used: extraction and / or synthetic lipids, gelling and viscosifying polymers, surfactants and emulsifiers, water-soluble or liposoluble active principles , plant extracts, tissue extracts, marine extracts, sun filters, antioxidants. 1 1. Cosmetic or dermopharmaceutical compositions according to 3 to 10 used for the preparation of a medicament for eliminating, reducing or preventing   the appearance of wrinkles, whatever their location and cause.