Use of peptides for the treatment of the epidermis

Abstract

Peptides have the general formula X-(Xaa) n K * TFK * -(Xaa) m -Z. The peptide serves to maintain or improve the condition of the epidermis. * is selected from lysine (K), ornithine, diaminobutyric acid or diaminopropionic acid, or hydroxylated derivatives thereof. n and (Xaa) m corresponds independently to a sequence of n or m amino acids Xaa selected independently from Gly, Ala, Pro, Val, Leu, Ile and Phe, where n and m are integers between 0 and 5, which may be equal or different. The N-terminal X is H, -CO-R 1 , -SO2-R 1 or a biotinoyl group. Z at the C-terminus is selected from OH, OR 1 , NH2, NHR 1 or NR 1 R 2 Selected from: R 1 and R 2 are independently selected from alkyl, aryl, aralkyl, alkylaryl, alkoxy, sugar and aryloxy groups, which may be linear, branched, cyclic, polycyclic, unsaturated, hydroxylated, carbonylated, phosphorylated and / or sulfurized, said groups having from 1 to 24 carbon atoms and optionally containing O, S and / or N heteroatoms in their backbone. A preferred peptide is Pal-KTFK.

Description

[Technical Field]

[0001] The present invention relates to cosmetic or dermatological peptide-based treatments, to novel cosmetic or dermatological ingredients based on said peptides, and to compositions containing same. More particularly, the present invention relates to peptides intended for the treatment of the skin and its appendages in humans or mammals.

[0002] This is particularly relevant to the cosmetic, dermatological, hygiene and personal care industries. [Background technology]

[0003] Peptides have important signaling functions and regulate many biochemical processes. As a result, they have long been essential and promising active ingredients, especially in the cosmetics industry. New compounds are constantly being sought to beautify the skin and its appendages, i.e., to improve their normal condition.

[0004] Most of the peptides currently proposed act on the dermis through stimulation of extracellular matrix components, mainly collagen and elastin. Many peptides based on this principle have been proposed, particularly by the applicant. For example, Pal-KTTKS (SEQ ID NO 1), sold under the trade name MATRIXYL®, a mixture containing Pal-GHK and Pal-GQPR (SEQ ID NO 2), sold under the trade name MATRIXYL® 3000, Pal-KMO2K (MO2 corresponds to methionine dioxide), sold under the trade name MATRIXYL® Synthe' 6®, or more recently Pal-K(P)HG (with proline grafted to lysine), sold under the trade name MATRIXYL® Morphomics™, Pal-VGVAPG (SEQ ID NO 3), sold under the trade name Dermaxyl™ or Biopeptide EL™. 3), or N-acetyl-Tyr-Arg-O-hexadecyl, sold under the trade names Idealift™ or Calmosensine™.

[0005] The beauty and health of skin depend largely on the quality and thickness of the epidermis, particularly through optimal differentiation of keratinocytes and the ability of the epidermis to form its outermost layer, the stratum corneum, and through its periodic renewal by shedding. The epidermis, particularly the stratum corneum, actually forms a natural skin barrier essential for protection against external environmental molecules and attacks (photoirradiation, pollutants, etc.). For example, by properly protecting this skin barrier, the risk of epidermal microinflammation, which can cause premature skin aging, can be advantageously limited. Such protection is necessary for sensitive skin. It also limits the risk of moisture loss and helps maintain good epidermal hydration. Summary of the Invention

[0006] The present invention aims to provide peptides that have activity acting on the properties of the epidermis, particularly the stratum corneum, ie, peptides that can act on the first layer of the skin surface.

[0007] To this end, According to the first aspect, The present invention provides the use of a peptide having the following general formula 1 for a non-therapeutic cosmetic treatment to maintain or improve the condition of the epidermis:

[0008] [ka]

[0009] In the general formula 1: ·K * is selected from the group comprising lysine, ornithine, diaminobutyric acid or diaminopropionic acid or their hydroxylated derivatives, and two K * are the same or different; (Xaa) n and (Xaa) m corresponds independently to a sequence of n or m amino acids Xaa selected independently from Gly, Ala, Pro, Val, Leu, Ile and Phe, where n and m are integers between 0 and 5, which may be equal or different; N-terminal X is H, -CO-R 1 , -SO2-R 1 or a biotinoyl group; C-terminal Z is OH, OR 1 , NH2, NHR 1 or NR 1 R 2 Selected from; where R 1 and R 2are independently selected from alkyl, aryl, aralkyl, alkylaryl, alkoxy, sugar and aryloxy groups, which may be linear, branched, cyclic, polycyclic, unsaturated, hydroxylated, carbonylated, phosphorylated and / or sulfurized, and which have from 1 to 24 carbon atoms and may have one or more O, S and / or N heteroatoms in their backbone.

[0010] Therefore, the peptides used according to the invention contain the amino acid sequence K, which is physiologically active on the epidermis. * TFK * The active sequence K is characterized by containing at least * TFK * may be flanked on both sides by a sequence of 1 to 5 nonpolar amino acids selected from Gly, Ala, Pro, Val, Leu, Ile and Phe, preferably selected from Gly, Ala, Pro and Val, preferably selected from Gly and Ala.

[0011] Preferably, according to the present invention, K * is lysine or ornithine, more preferably lysine. The preferred hydroxylated derivative is hydroxylysine.

[0012] More preferably, according to the present invention, n and m are between 0 and 2, preferably equal to 0, and said peptide has the following general formula 2:

[0013] [ka]

[0014] Preferably, the peptide has the following general formula 3:

[0015] [ka]

[0016] K is lysine (Lys), and T is threonine (Thr), and F is phenylalanine (Phe), and X and Z are as defined above.

[0017] The results of in vitro tests (on keratinocyte cultures, skin explants or reconstructed skin) are described below and show that the peptides according to the invention allow for the maintenance / protection and improvement of the condition of the epidermis, in particular by strengthening the skin barrier function and harmonizing the natural maturation process of the epidermis.

[0018] These studies demonstrated the targeted action of the peptides according to the invention at several sites, in particular: · The peptide stimulates the production of several molecules that constitute the skin barrier or actively intervene in the differentiation of keratinocytes at the barrier's origin; Peptides increase hydration of the upper epidermis; · Peptides improve the regeneration of the stratum corneum due to shedding; · The peptide stimulates the production of alpha-crystallin; Peptides reduce the levels of many molecules involved in micro-inflammation of the skin caused by a variety of small everyday insults (light exposure, pollutants, etc.).

[0019] The results of the in vivo tests described below demonstrate the subsequent real cosmetic benefits associated with the peptides of the present invention: smoother skin, better moisturization (thus clearer, suppler and softer skin), and improved radiance and evenness of the complexion. Skin imperfections are avoided or reduced by the present invention.

[0020] Preferably, the peptides of the present invention are modified at the N-terminal or C-terminal position, except when X=H and Z=OH.

[0021] According to another preferred feature of the present invention: ·R 1 and / or R 2is an alkyl chain of 1 to 24 carbon atoms, preferably a lipophilic alkyl chain of 3 to 24 carbon atoms; and / or X is an acyl group CO-R 1 and preferably octanoyl (C8), decanoyl (C 10 ), Lauroyl (C 12 ), myristoyl (C 14 ), palmitoyl (C 16 ), stearoyl (C 18 ), biotinoyl, elaidoyl, oleoyl and lipoyl; more preferably lauroyl (C 12 ), myristoyl (C 14 ) and palmitoyl (C 16 ) selected from; and / or Z is selected from OH, OMe, OEt and NH2, preferably OH; and / or X is palmitoyl (C 16 ), myristoyl (C 14 ) and lauroyl (C 12 ), more preferably palmitoyl (C 16 ) and Z is OH.

[0022] Peptides containing specific acid derivatives at the N- or C-terminal position, such as ascorbic acid, retinoic acid, cinnamic acid, oleanolic acid, hyaluronic acid, nicotinic acid, lipoic acid, gallic acid, or pantothenic acid, are also encompassed by the present invention.

[0023] A preferred peptide of the present invention is Pal-KTFK-OH (also referred to as Pal-KTFK, SEQ ID NO: 5), which corresponds to the N-terminal palmitoyl chain substitution (X=Pal) and the C-terminal no substitution (Z=OH) in the following formula 4:

[0024] [ka]

[0025] The peptides of the present invention may be optically pure and consist of the L or D isomer, or a mixture thereof. The naturally occurring L isomer may be preferred. The peptides may also be in the form of salts, in particular hydrochlorides or acetates, where appropriate.

[0026] The present invention also encompasses peptide derivatives (with modified and / or added chemical functionality, but without altering the carbon backbone), peptide analogs (with modified and / or added chemical functionality, but with further alterations to the carbon backbone), and complexes with other species, such as metal ions (e.g., copper, zinc, manganese, magnesium, and the like).

[0027] For use according to the invention, the peptides can be solubilized in a physiologically acceptable lipophilic or hydrophilic matrix, if appropriate using solubilizing agents, depending on the form of galenic formulation envisaged.

[0028] "Physiologically acceptable medium" according to the present invention means, but is not limited to, an aqueous or aqueous-alcoholic solution, a water-in-oil emulsion, an oil-in-water emulsion, a microemulsion, an aqueous gel, an anhydrous gel, a serum, a vesicle dispersion, or a powder.

[0029] "Physiologically acceptable" means that ingredients and compositions containing the peptides of the present invention can be ingested or injected into the skin without risk of toxicity, incompatibility, instability, allergic reaction, etc., and are suitable for topical or transdermal use in contact with the mucous membranes, nails, scalp, hair, body hair and skin of mammals, more particularly humans. This "physiologically acceptable medium" generally forms what is called the excipient of the composition.

[0030] The peptides according to the invention may also be used in vectorized form by being bound, entrapped or absorbed onto / into macro-, micro- or nanoparticles such as capsules, spheres, liposomes, oleosomes, chylomicrons, sponges, in the form of micro- or nanoemulsions, or by being adsorbed onto, for example, organic polymer powders, talc, bentonite, spores or exines, and other inorganic or organic supports.

[0031] Preferably, the peptides of the present invention are used in a specific form "entrapped" in an oil-wax surfactant microemulsion and water to slow their penetration and make them more physiologically available at the surface of the skin, i.e., at the epidermal site.

[0032] This particular form substantially prevents the peptide from entering the dermis, allowing it to exert its physiological action slowly and uniformly from the stratum corneum and in all parts of the epidermis.

[0033] According to a second aspect of the present invention, Cosmetic or dermatological ingredients are provided that include a peptide having the following general formula 1:

[0034] [ka]

[0035] In general formula 1: ·K * is selected from lysine, ornithine, diaminobutyric acid or diaminopropionic acid, or hydroxylated derivatives thereof, and two K * are the same or different; (Xaa) n and (Xaa) m corresponds independently to a sequence of n or m amino acids Xaa selected independently from Gly, Ala, Pro, Val, Leu, Ile and Phe, where n and m are integers between 0 and 5, which may be equal or different; N-terminal X is H, -CO-R 1 , -SO2-R 1 or a biotinoyl group; C-terminal Z is OH, OR 1 , NH2, NHR 1 or NR 1 R 2 Selected from; where R 1 and R 2 are independently selected from alkyl, aryl, aralkyl, alkylaryl, alkoxy, sugar and aryloxy groups, which may be linear, branched, cyclic, polycyclic, unsaturated, hydroxylated, carbonylated, phosphorylated and / or sulfurized, said groups having from 1 to 24 carbon atoms and optionally containing one or more O, S and / or N heteroatoms in their backbone; The physiologically acceptable medium allows the peptide to penetrate the skin slowly and act on the epidermis.

[0036] According to a first aspect of the invention, the peptide is more precisely as defined above.

[0037] Preferably, the vehicle or vector is an oil-wax surfactant microemulsion and water in which the peptide is solubilized, more preferably comprising a wax phase containing lecithin.

[0038] In the microemulsion formed, the peptide finds itself "trapped" by solvation, establishing weak ionic, hydrogen, and van der Waals bonds between the peptide and the wax particles via the surfactant.

[0039] The forces and energy induced during topical application then break these weak bonds, allowing the release of the peptides within the stratum corneum by slow and gradual desolvation.

[0040] According to a third aspect of the present invention,There is provided a cosmetic or dermatological composition comprising an ingredient according to the second aspect of the present invention.

[0041] The composition comprising the peptide according to the present invention, particularly the peptide according to the third aspect of the present invention, is ultimately applied by the user.

[0042] The composition may be provided in any galenical form (examples of which are given in the description below) and may be delivered via a textile support of natural fibers, synthetic fibers, wool, or any material adapted to contact the skin, or applied to clothing such as daytime or nighttime underwear, handkerchiefs, or tissues, so as to exert a cosmetic or dermatological effect upon contact with the skin / textile and allow continuous topical delivery.

[0043] In a particularly advantageous form, according to the invention, the peptide may be combined with at least one additional active agent adapted to act as a reinforcer of the activity and / or to act complementary to one or more other activities.

[0044] A variety of additional active agents for this purpose are described in the detailed description below.

[0045] According to the fourth form, The present invention provides a method for improving the aesthetic appearance of the skin, which method comprises topically applying to the skin an effective amount of a cosmetic or dermatological composition according to the third aspect of the invention described above.

[0046] According to the fifth form, The present invention proposes a kit for carrying out a self-tanning cosmetic treatment, comprising in separate compartments: a) A composition according to the invention as defined above or a composition comprising, in a physiologically acceptable medium, a peptide according to the invention as defined above according to the first aspect. b) A composition comprising a self-tanning active agent in a physiologically acceptable medium.

[0047] Particularly advantageously, the peptides according to the invention can be used to achieve a natural, soft smoothing before the application of a self-tanning active agent, in particular when the peptides are applied at least 3 days, preferably 6 days, more preferably 8 days before the application of the self-tanning active agent.

[0048] "Topical treatment" or "topical use" means, according to the invention, an application intended to act where it is applied, ie on the skin, mucous membranes and appendages.

[0049] The peptide or a composition of the present invention comprising the peptide can be applied topically to the target area.

[0050] An "effective" amount depends on various factors, such as the age, condition of the patient, the severity of the disorder or pathology, the mode of administration, etc. An effective amount means a non-toxic amount sufficient to achieve the desired effect.

[0051] In the cosmetic compositions according to the invention, the peptides are present in an effective amount, generally in proportions ranging from 0.000001% to 5%, preferably from 0.00001% to 0.1%, more preferably from about 0.0005% to 0.005% relative to the total weight of the composition, depending on the purpose of the composition and the desired effect, which may be more or less pronounced.

[0052] All percentages and ratios used herein are by weight of the total composition and all measurements are made at 25°C unless otherwise specified.

[0053] For example, for facial cosmetic procedures, the European Cosmetics Directive recommends a limit of 2.72 mg / cm per person. 2 The standard application amount of cream per day is 0.5mg / cm 2 A standard application amount of body lotion per day has been set.

[0054] According to another feature, the cosmetic treatment method of the present invention may be combined with one or more other treatment methods that target the skin, such as, for example, light therapy, thermotherapy, vibration therapy, electrotherapy, microneedle patches or aromatherapy treatments.

[0055] According to the present invention, it is possible to provide a device or kit with several compartments intended for carrying out the methods described above, which may contain, for example but not limited to, a composition comprising the active ingredient according to the invention in a first compartment and additional excipients and / or active agents in a second compartment, wherein the compositions contained in the first and second compartments are considered in particular as a combined composition for simultaneous, separate or stepwise use in one of the above-mentioned treatment methods.

[0056] The compositions according to the invention are suitable for therapeutic treatment, in particular for the treatment of skin, in particular for the treatment of skin with a diseased epidermis.

[0057] According to a sixth aspect, there is provided a peptide for the cosmetic uses, methods, compositions and kits according to the invention, said peptide having the following general formula 1:

[0058] [ka]

[0059] In the general formula 1: ·K * is selected from the group comprising lysine (K), ornithine, diaminobutyric acid or diaminopropionic acid, or hydroxylated derivatives thereof, and two K * are the same or different; (Xaa) n and (Xaa) mcorresponds independently to a sequence of n or m amino acids Xaa selected independently from Gly, Ala, Pro, Val, Leu, Ile and Phe, where n and m are integers between 0 and 5, which may be equal or different; N-terminal X is H, -CO-R 1 , -SO2-R 1 or a biotinoyl group; C-terminal Z is OH, OR 1 , NH2, NHR 1 or NR 1 R 2 Selected from; where R 1 and R 2 are independently selected from alkyl, aryl, aralkyl, alkylaryl, alkoxy, sugar and aryloxy groups, which may be linear, branched, cyclic, polycyclic, unsaturated, hydroxylated, carbonylated, phosphorylated and / or sulfurized, said groups having from 1 to 24 carbon atoms and optionally containing one or more O, S and / or N heteroatoms in their backbone; The peptides Pal-KTFK-OH and Ela-KTFK-OH are excluded.

[0060] According to the preferred characteristics of the peptide ·K * is lysine (K) or ornithine; and / or n and m are independently any one of 0, 1, or 2; and / or the peptide is modified at the N-terminal or C-terminal position, except when X=H and Z=OH; and / or ·R 1 and / or R 2 is an alkyl chain of 1 to 24 carbon atoms; and / or X is an acyl group CO-R 1 and Z is selected from OH, OMe, OEt and NH2; and / or The vehicle or vector in which the peptide is solubilized is preferably an oil-wax surfactant microemulsion and water, more preferably a wax phase containing lecithin.

[0061] The present invention will be better understood with reference to the following description of embodiments and in vitro and in vivo tests.

[0062] A) Preparation of Pal-KTFK peptide (SEQ ID NO 5) according to the present invention The Pal-KTFK peptide is prepared by peptide synthesis. Lysine is coupled to the resin via its terminal acid functional group (using a coupling agent, e.g., DCC (dicyclohexylcarbodiimide) / NHS (N-hydroxysuccinimide) or HBTU (2-(1H-benzotriazol-1-yl)-1,3,3-tetramethyluronium hexafluorophosphate) / HOBT (1-hydroxybenzotriazole)). The protected lysine is then reacted with a threonine derivative in the presence of a coupling agent, followed by the addition of phenylalanine and a second lysine in the same manner. The latter is then acylated with its amine functional group using an activated palmitic acid derivative (e.g., palmitoyl chloride) in the presence of base. After cleavage of the peptide chain from the resin in an acidic medium, precipitation, washing, and drying, the palmitoyl-lysyl-threonyl-phenylalanyl-lysine product is obtained in solid form.

[0063] B) Preparation of a cosmetic active ingredient according to the invention containing Pal-KTFK (SEQ ID NO. 5) The Pal-KTFK peptide is amphiphilic: the Pal chain is hydrophobic and the phenylalanyl moiety is hydrophilic. The peptide is solubilized in a water / glycol matrix using an appropriate surfactant.

[0064] C) Preparation examples of cosmetic active ingredients according to the present invention having vectorized peptides procedure:

[0065] Step 1: Preparation of the "excipient" phase Aqueous phase: water + surfactant + Wax phase: Wax + lecithin surfactant +Oil phase: oil + surfactant

[0066] An oil-in-water emulsion is formed by vigorous stirring, which results in the formation of a heterogeneous phase containing a liquid portion and a solid portion containing wax particles.

[0067] Step 2: Preparation of the phase containing the active peptide of the present invention Aqueous phase: Water + Pal-KTFK peptide (200 ppm)

[0068] Step 3: Add the "active" phase to the "excipient" phase under moderate agitation.

[0069] Step 4: Optionally, add one or more phases containing preservatives into the final emulsion.

[0070] Step 5: Optionally, add a pH adjuster to the final emulsion (desired pH is about 4.5).

[0071] The wax may be composed of multiple waxes, similar to the oil.

[0072] Resulting product: In the emulsion formed, the peptide establishes weak ionic, hydrogen, and van der Waals bonds between the peptide and the wax particles via lecithin, finding itself "trapped" by solvation in the previously formed oil-in-water emulsion.

[0073] Because the Pal-KTFK peptide is amphiphilic, it diffuses and acts uniformly after desolvation and has high affinity for binding between keratinocytes.

[0074] D) In ​​vitro efficacy testing Tests were performed on Pal-KTFK, a preferred peptide according to the invention, in its unsolvated form.

[0075] The following in vitro tests were carried out on epidermal cells: normal human keratinocytes (NHK), skin explants, or reconstructed skin. The peptides according to the invention were tested in solution in an inert solvent (ethanol) at the concentrations recommended for use in the skin.

[0076] 1 / Improvement of epidermal barrier and harmonization of epidermal maturation The stratum corneum or stratum corneum is a highly complex assembly that combines, on the one hand, flat, anuclear cells that are tightly bound to one another, and, on the other hand, lipids and proteins, the composition and assembly of which ensure the unique properties of this structure, which make it highly resistant to the physical, chemical and biological attacks of the environment.

[0077] The following studies show that the peptides according to the invention induce epidermal homeostasis and strengthening of the skin barrier by analyzing various markers involved in epidermal differentiation and barrier formation in three different biological systems (human keratinocyte thin layer cultures or cultures of human skin explants or reconstructed skin).

[0078] Keratinocytes gradually mature due to the formation of a strong outer shell made up of interconnected proteins called involucrin and loricrin, mediated by the calcium-sensitive enzyme transglutaminase. Other proteins involved in stratum corneum maturation and homeostasis, such as SPRRs (small proline-rich proteins), strengthen this protein shell by forming flexible and strong crosslinks between proteins through the activity of transglutaminase. NICE-1 is a protein whose gene is colocalized with many layer proteins. Its composition makes it an ideal substrate for transglutaminase and is similar to that of loricrin. LEC (late cornified membrane protein) is also one of the last components to be crosslinked during maturation. Furthermore, keratin 10, keratin 1, and ceramides play crucial roles in the formation and quality of the stratum corneum and its proteolipid matrix, and cosmetic active agents that stimulate their synthesis are also important.

[0079] A good barrier function also relies heavily on filaggrin and filaggrin-2, produced by keratinocytes, which undergo significant metabolism. They stabilize the corneocytes by binding to keratin and are eventually broken down into amino acids, which generate essential components of the natural moisturizing factors (NMFs) present in the stratum corneum. Hyaluronic acid, which surrounds keratinocytes, also keeps the epidermis hydrated, acting as a natural sponge that can attract and retain up to 1,000 times its own weight in water.

[0080] Finally, α-crystallin is a small protein related to the HSP family (or heat shock proteins). It is present in the epidermis, where it protects epithelial cells, restores mitochondrial function, and increases resistance to oxidative stress. This protein has the property of being present both inside and in its immediate vicinity within the cell. Its presence protects the skin from UV rays, inflammation, and more generally, oxidative attacks.

[0081] 1.1 / DNA-array principle: The peptides of the present invention were contacted with confluent KHN (n=3) for 6 or 24 hours. The KHN mats were then washed, the cells were disrupted, and their mRNA was extracted. These mRNAs were deposited on a DNA chip and amplified using a method similar to qRT-PCR (real-time quantitative reverse transcription polymerase chain reaction), then converted into small DNA sequences for analysis. The changes in mRNA induced by the peptides were compared with those observed in a control sample (peptide solvent).

[0082] result: Table 1 : Changes in the regulation of expression of genes encoding epidermal differentiation proteins. Effect of 4 ppm of the peptide according to the present invention.

[0083] [Table 1] No toxic effects were observed. *6 hours of contact; **24 hours of contact

[0084] These studies demonstrated that several physiological targets were induced early (6 to 24 hours) in NHKs exposed to the peptides of the present invention. The genes for loricrin, filaggrin, and filaggrin 2, three proteins involved in keratinocyte assembly or stratum corneum moisturization, were statistically significantly more expressed than the solvent control (p<0.01). Further qRT-PCR analysis of filaggrin gene expression revealed that doses equivalent to 4 ppm and 6 ppm of the peptide of the present invention increased expression by 2.87-fold and 3.66-fold, respectively, compared to the control (p<0.01).

[0085] 1.2 / LC-MS / MS (liquid chromatography coupled with mass spectrometry) study principle: The same incubation procedure as for DNA arrays (1.1 / above) was used, but the incubation period (7 days) was longer, resulting in longer detection of protein production with this LC-MS / MS method. The NHK medium (n=3) was replaced every 3 days. At the end of this incubation period, the cells were lysed and proteins were extracted. The analysis was performed in the form of lysates using a method that combined the action of proteases on the lysates, separation of the fragments by liquid chromatography combined with mass spectrometry, and analysis of the identity and concentration of the existing proteins according to the nature and quantity of the fragments obtained (LC-MS / MS). The protein concentration of the lysates was measured for LC-MS / MS analysis of equivalent amounts of protein.

[0086] Analysis of variance and Student's t-test were performed to assess the significance of the results.

[0087] result: Table 2 : Changes in the regulation of proteins involved in homeostatic maturation of the epidermis. Effect of 4 ppm of the peptide according to the invention.

[0088] [Table 2] No toxic effects were observed.

[0089] These results confirm that the peptides of the present invention positively induce the production of differentiation-related proteins: filaggrin, NICE-1, and SPRR1A, all three of which increased. At the same time, keratin-10, which is also known to be produced during epidermal differentiation, increased. Keratin is used to strengthen the epidermis via desmosomes (cell-to-cell junctions), positively influencing epidermal homeostasis.

[0090] Interestingly, and unexpectedly, α-crystallin protein expression appeared to be more influential than in control cases, leading to complementary exploration.

[0091] 1.3 / α-crystallin in reconstructed skin principle: Normal human fibroblasts were seeded into the matrix. After a few days, keratinocytes were deposited on top of this dermal equivalent, forming a uniform layer of cells. At this stage, the whole was transferred to an air-liquid interface (immersion), allowing the formation of a multilayered epidermis. After (approximately) 21 days, mature skin (with an epidermis containing a stratum corneum) was obtained.

[0092] The night before the immersion step, the peptides of the present invention were applied to a confluent layer of human keratinocytes from these skins for 24 hours and then for 18 days (the period during which the epidermis forms). Cryosections (7 μm) were cut and α-crystallin was labeled with a fluorescent antibody (immunohistology). Fluorescence was analyzed photographically, with equivalent areas analyzed for all photographs (n=51 to 59). Student's t-test was performed.

[0093] result: Table 3 : Expression of α-crystallin protein in reconstructed skin epidermis. Effect of 4 ppm and 6 ppm of the peptide of the present invention.

[0094] [Table 3] No toxic effects were observed.

[0095] These results show that α-crystallin is very significantly stimulated in the skin by the peptide according to the invention (+61% and +164% compared to the control, p<0.01). This protein has been shown to exert a protective effect on keratinocytes, their proteins and their mitochondria present in the epidermis.

[0096] 1.4 / Regulation of epidermal differentiation and moisturizing components principle: Confluent NHKs (n=3) were cultured for 7 days as before in the presence or absence (control) of the peptides according to the present invention, and then immunocytochemical marking was carried out using antibodies against proteins characteristic of epidermal maturation: involucrin, loricrin, and filaggrin, or proteins characteristic of moisturization: filaggrin and ceramide (structuring elements of the stratum corneum).

[0097] The moisturizing properties were evaluated by analyzing the hyaluronic acid produced by keratinocytes using the ELISA method.

[0098] Finally, the effect of the peptides of the present invention on transglutaminase activity and transglutaminase-5 gene expression was evaluated.

[0099] For this purpose, confluent NHKs were contacted with the peptides of the present invention for up to 21 days. Transglutaminase activity was assessed using a procedure that utilizes fluorescent dansylcadaverine, which binds to stratum corneum proteins via these enzymes. Fluorescence analysis was performed and the results were compared with those of a solvent control.

[0100] For transglutaminase-5, qRT-PCR was performed after 72 hours of contact between the peptides of the present invention and KHN.

[0101] For the four studies described in this part, quantification of cell numbers was performed in parallel to reduce the results to cell numbers.

[0102] result: Table 4 Induction of involucrin, loricrin, filaggrin, and ceramide formation in NHKs (immunofluorescence). Effect of 2 ppm of the peptide according to the invention.

[0103] [Table 4] No toxic effects were observed.

[0104] These results demonstrate that the peptide of the present invention effectively promotes NHK maturation, with the effect being evident at 2 ppm.

[0105] Table 5 : Induction of hyaluronic acid secretion in NHK. Effect of 4 ppm and 6 ppm of the peptide according to the present invention.

[0106] [Table 5] No toxic effects were observed.

[0107] These results indicate that the peptide of the present invention is involved in moisturizing the epidermis.

[0108] Table 6 : Changes in transglutaminase activity and transglutaminase-5 gene expression in KHN. Effect of 4 ppm and 6 ppm of the peptide according to the present invention.

[0109] [Table 6]

[0110] These results demonstrate the effect of the peptides of the present invention on the expression of the transglutaminase-5 gene, which is known to bind in particular to the proteins loricrin and involucrin, thereby contributing to the formation of the stratum corneum. At the same time, the activity of the transglutaminase protein in keratinocytes is actually increased.

[0111] All these results, obtained by complementary methods, show that contact of NHK with the peptides according to the invention leads to a stimulation of the synthesis of key cellular components that result in a strengthening of the skin barrier against possible environmental attacks and promotes moisturization of the superficial layers of the epidermis.

[0112] 1.5 / Mechanism of stimulation of maturation: TRPC6 The TRPC6 receptor (Transient Receptor Potential Canonical 6) is involved in calcium-induced keratinocyte differentiation. Its simple stimulation is sufficient to induce the construction of structures necessary for epidermal differentiation and subsequent formation of the stratum corneum. TRPC6 is not present in basal layer keratinocytes. TRPC6 is specific to the mature part of the epidermis.

[0113] DNA arrays (see 1.1 above) revealed an increase in the production of mRNA encoding TRPC6, a protein that provides intracellular calcium flux, in addition to stimulating genes encoding the formation of the stratum corneum. This highly significant increase associated with the peptide of the invention (4 ppm) was 6.05-fold compared to the solvent control at 6 hours.

[0114] principle: Confluent keratinocytes were loaded with a specific calcium probe. This molecule becomes highly fluorescent in the presence of intracellular free calcium. The appearance of fluorescence was monitored under a microscope. Once loaded, the cells were washed and then treated with 6 ppm or 15 ppm of the peptides of the present invention or a positive control (bradykinin).

[0115] Analysis of variance and Student's t-test were performed to assess the significance of the results (n=3 for each condition).

[0116] result: The visual results show that the peptides according to the invention indeed induce intracellular calcium mobilization in confluent keratinocytes (visualized under a fluorescent microscope after approximately 30 minutes of contact between the cells and the peptides). This calcium release mechanism is well known to promote keratinocyte differentiation by allowing the coordinated formation of elements of the stratum corneum.

[0117] 1.6 / Epigenetic regulation of maturation:ANCR ANCR (anti-differentiation non-coding RNA) is an 855-nucleotide lnc-RNA (long non-coding RNA). ANCR is essential for maintaining epidermal homeostasis. ANCR is overexpressed in the basal epidermis and conversely, is highly underexpressed in the differentiating and maturing epidermis. ANCR is part of a complex network of mechanisms involved in epidermal renewal and epidermal barrier formation. Experimental depletion of ANCR in keratinocytes promotes late keratinocyte differentiation, accompanied by the expression of early and late markers of epidermal maturation: filaggrin, loricrin, and involucrin. This is an example of epigenetic regulation applied to skin barrier formation.

[0118] principle: In this test, confluent keratinocytes were contacted with the peptides of the present invention for 24 hours, after which the cells were washed, disrupted, and their RNA extracted, converted to DNA and then analyzed by qRT-PCR.

[0119] Analysis of variance and Student's t-test were performed to assess the significance of the results (n=4 for each condition).

[0120] result: Table 7 : Changes in expression of lnc-RNA ANCR over 24 hours; effect of the peptide of the present invention (results are expressed as a ratio to the control).

[0121] [Table 7]

[0122] These results show that the peptides of the present invention are capable of significantly, but moderately, reducing this lnc-RNA, an epigenetic regulator of epidermal differentiation, in a dose-dependent manner.

[0123] 1.7 / Desmosome Desmosomes form plaques that connect keratinocytes together. These plaques are highly resistant. These plaques are formed, in particular, by desmogleins. Desmosomes allow the intracellular anchoring of keratin-10 fibers and are involved in the cell binding of the epidermis.

[0124] principle: Desmoglein was detected on sections of abdominal skin from a female, Caucasian, 54 years old, to which a cream containing 6 ppm of the peptide according to the invention or a placebo cream had been applied. After 6 days (daily application of the cream), the skin was frozen and sectioned (fluorescence immunohistochemistry) to detect desmoglein using an antibody against this protein. Photographs were taken using a fluorescence microscope, and the images were analyzed using appropriate software. The results were compared with those of the placebo cream.

[0125] result: Table 8 : Changes in desmoglein production on day 6 in an abdominal skin explant model; effect of the peptide according to the present invention (results are expressed as a ratio to the control).

[0126] [Table 8]

[0127] These results indicate that the peptides of the present invention favor the formation of desmosomes, since they significantly increase the expression of desmoglein proteins, which are essential components of desmosomes.

[0128] 1.8 / Effects on dropouts KLK5 (kallikrein 5) is an enzyme involved in the regeneration of the stratum corneum, allowing it to naturally fall off and providing a soft, natural polishing effect ("soft polish").

[0129] principle: Using the same sections and the same method (fluorescent immunohistology) as used for desmoglein, the effect of the peptides according to the invention on this shedding marker was analyzed and quantified.

[0130] result: Table 9 : Changes in KLK5 production on day 6 in an abdominal skin explant model; effect of the peptide according to the present invention (results are expressed as a ratio to the control).

[0131] [Table 9]

[0132] Skin homeostasis requires the formation of an effective skin barrier as well as the possibility of ensuring the renewal of keratinocytes through shedding. A significant increase in KLK5 was observed when using the peptides of the present invention, which is known to ensure a soft and natural smoothing phenomenon.

[0133] 2 / Reducing the production of inflammatory markers Skin is exposed to constant stressors (such as exposure to UV rays, smoke, and pollutants), some of which can trigger direct or indirect inflammatory responses. Uncontrolled or constant inflammatory responses, although low in intensity, induce the production of cytokines such as IL-1α, IL-1β, IL-6, and TNFα, as well as lipids such as PGE2, which attract or stimulate other cells, triggering a cascade reaction. The pro-inflammatory microenvironment thus formed alters skin homeostasis, gradually altering or even destroying biomolecules in cells and tissues. It also induces disruption of the skin barrier integrity. Thus, the inflammatory mediators IL-6 and PGE-2 are known to cause premature aging through microinflammation. Furthermore, sensitive, inflammatory skin is characterized by abnormally high secretion of cytokines, pro-inflammatory peptides (e.g., IL-1, IL-6), and pro-inflammatory lipids (e.g., PGE-2).

[0134] To test its activity, keratinocytes were cultured under mild stress conditions (application of UVB irradiation) to mimic experimental skin microinflammation. In this context, a significant reduction in the secretome of inflammatory mediators is interpreted as a skin repair and protective effect.

[0135] principle: Cultured and confluent NHKs were exposed to the peptides of the present invention (or their solvent) for 24 hours in a medium that allowed their survival. The mats were then irradiated with UVB in a physiological buffer and returned to the test article for 24 hours. At the end of this incubation period, the culture medium was analyzed by ELISA to determine the amount of proinflammatory mediators produced by the cells in response to irradiation. The results were compared with solvent controls with and without irradiation. Cellular respiration assays were performed to assess cell number and normalize the results.

[0136] Analysis of variance and Student's t-test were performed to assess the significance of the results.

[0137] result: Table 10 : Alterations in the secretome of pro-inflammatory mediators by UVB-exposed NHK. Effect of the peptides of the present invention.

[0138] [Table 10]

[0139] These results demonstrate a very interesting effect of the peptides of the present invention in alleviating the secretion of pro-inflammatory mediators by NHKs exposed to UVB radiation, which strengthens the skin barrier and delays skin aging induced by radiation and exposure to various pollutants that cause microinflammation, especially for sensitive and inflamed skin.

[0140] E) Preparation of Galenic Formulations / Compositions of the Invention The peptides according to the invention may be formulated, in the form of an ingredient, or optionally with additional cosmetic active ingredients that support and / or complement their activity during the preparation of the final cosmetic composition for the consumer, which may be applied to the face, body, neck, scalp, hair, eyelashes or body hair, and may be delivered alone or as a premix, or in any form or medium known to those skilled in the art, in particular in the form of a solution, dispersion, emulsion, paste or powder.

[0141] In cosmetics, they may be proposed for use in particular in the skin care range for the face, body, hair, and body hair, and in the make-up care range.

[0142] These ingredients may be of any category depending on their function, the area to which they are applied (body, face, neck, bust, hands, hair, eyelashes, eyebrows, body hair, etc.), the desired end effect and the target consumer, such as antioxidant, anti-wrinkle, moisturizing, nourishing, protecting, smoothing, remodeling, volumizing (lipofilling), brightening the complexion, reducing under-eye bags and dark circles, anti-aging, anti-wrinkle, slimming, soothing, muscle relaxant, anti-redness, anti-stretch mark, sun protection, etc.

[0143] The International Cosmetic Ingredient Dictionary & Handbook (18th Edition, 2018), published by the CTFA (Cosmetic and Toiletries Manufacturers Association of America, Washington, D.C.), describes a wide variety of non-limiting cosmetic ingredients commonly used in the skin care and scalp care industries that are suitable for use as additional ingredients in the compositions of the present invention.

[0144] The active ingredients include at least one compound selected from vitamin B3 compounds, compounds such as niacinamide or tocopherol, retinoid compounds such as retinol, hexamidine, α-lipoic acid, resveratrol or DHEA, hyaluronic acid, and peptides, which are widely used in cosmetic or dermatological compositions.The peptides are particularly N-acetyl-Tyr-Arg-O-hexadecyl, Pal-VGVAPG (SEQ ID NO: 3), Pal-KTTKS (SEQ ID NO: 1), Pal-GHK, Pal-KMO2K, Pal-GQPR (SEQ ID NO: 2), and Pal-K(P)HG.

[0145] Other particularly useful additional skin care actives are listed in Sederma's trade literature and at www.sederma.com or www.crodarom.fr.

[0146] In enhancing the activity on the properties of the epidermis and / or stratum corneum, additional active agents may be selected from the group including: phospholipids, various ceramides, sphingosine, phytosphingosine, glycosphingolipids, cholesterol and its derivatives, sterols (especially those of canola and soy), fatty acids (especially linoleic acid, palmitic acid, lipoic acid, and thioctic acid), squalane (especially of olive), triglycerides (especially of coconut oil), lanolin, lanolin alcohol, lanosterol, vitamin D3, tocopherol nicotinate, various oils (especially argan oil, , rose, and baobab), ascorbic acid, N-acetylcysteine ​​and N-acetyl-L-serine, vitamin B3 compounds (niacinamide, nicotinic acid, etc.), panthenol, pseudofilaggrin, arginine, serine, PCA (pyrrolidone carboxylic acid) salts, Centella asiatica leaf extract (titrated with madecassoside and asiaticoside), some plant extracts (wild yam root, chestnut, cedar bud, and nightshade), plankton, and yeast.Mention may also be made of the following active ingredients sold by Sederma: Venuceane™ (Thermus thermophilus fermentation medium extract), Moist 24™ (Cogon grass root glycolic acid extract), Dermaxyl™ (Ceramide 2 conjugate with Pal-VGVAPG peptide), Senestem™ (Extract of an in vitro plant cell culture of Plantago lanceolata), Ceramide 2™ (Ceramide), Ceramide HO3™ (Hydroxyceramide), OptimHyal™ (Venuceane™) Hyal™ (somewhat acetylated oligosaccharides of glucuronic acid), Meiritage™ (conjugate of Bupleurum, Astragalus membranaceus and Astragalus maculatus root extracts), Revidrat™ (myristyl phosphomalate), Pacifeel™ (extract of Mirabilis herb), Hydrononesis™ (fermentation product of Salinococcus hispanicus), unsaponifiables of Shea™ and Citystem™ (extract of horehound in vitro plant cell culture).

[0147] More generally, the following commercially available activators may also be mentioned as examples: For example, betaine, glycerol, ActimoistBio2™ (Active organics), AquaCacteen™ (Mibelle AG Cosmetics), Aquaphyline™ (Silab), AquaregulK™ (Solabia), Carciline™ (Greentech), Codiavelane™ (Biotech Marine), Dermaflux™ (Arch Chemicals, Inc.), Hydra' Flow™ (Sochibo), Hydromoist L™ (Symrise), RenovHyal™ (Soliance), Seamoss™ (Biotech Marine). Marine), Argireline™ (a trademark of Lipotec's acetyl hexapeptide-3), spilanthol, or GatulineExtract of Boswellia serrata known under the trade name Boswellin™, Deepaline PVB™ (Seppic), Syn-AKE™ (Pentapharm), Ameliox™, Bioxilift™ (Silab), PhytoCellTec™ Argan (Mibelle), Papilactyl D™ (Silab), Preventhelia™ (Lipotec), or one or more of the following active ingredients sold by Sederma: Subliskin™, Venuceane™, Moist 24™ 24™, Vegesome Moist 24™, Essenskin™, Juvinity™, Revidrat™, Resistem™, Chronodyn™, Kombucha™, Chromocare™, Calmosensine™, Glycokin factor S™, Biobustyl™, Idealift™, Ceramide 2™, Ceramide A2™, Ceramide HO3HO3™, Legance™, Intenslim™, Prodizia™, Beautifeye™, Pacifeel™, Zingerslim™, Meiritage™, Senestem™, Sebuless™, Majestem™, Apiscalp™, Rubistem™, Citystem™, Neonyca™, NG Unsaponifiables of Shea Butter Karite™, Majestem™, Hydroonesis™, and Poretect™, or mixtures thereof.

[0148] Among the plant extracts (in the form of conventional extracts or prepared in vitro) which can be combined with the peptides according to the invention, there are listed: ivy, in particular English ivy, manchuria, bupleurum, arnica, rosemary, marigold), sage, ginseng, ginkgo, St. John's wort, thuja, meadowsweet, big-flowered Java tea, artichoke, algae, birch, green tea, kola nut, horse chestnut, bamboo, Centella asiatica, heather, fucus, willow, taraxacum, escin, kandu, Chrysanthelme indicum, plants of the genus Armeniacea, Atractylodis platycodon, Sinomenium, Farbitizis, Flemingia, Coleus, e.g. Coleus forskohlii, Coleus burmei. , extracts of Coleus esquiroli, Coleus scutellaroides, Coleus xanthos and Coleus barbatos, e.g. extract of Coleus barbatos root, Barotte, Guioa, Davaria), Terminalia, Barringtonia, Trema, Antilobia, Cecropia, Argan, Dioscorea, e.g. extract of Discolea opposita or Mexicana, extracts of Ammibisnaga, Mentha, particularly Mentha serrata, extracts of plants from the Ericaceae family, particularly extracts of cowberry or bearberry, Aloe vera, plants containing sterols (i.e. phytosterols), Manjistha (extract of plants from the genus Rubia, particularly extract of Rubia cordifolia), and Guggul (extract of plants from the genus Commiphora, particularly extract of Guggul), extract of kola, chamomile, extract of red clover, extract of kava (Kava kavaKava™, Sederma), Bacopa Monnieri Extract (Bacocalmine™, Sederma) and Whip Goat Extract, Glycyrrhiza Glabra (Licorice), Mulberry, Melaleuca Alternifolia (Tea Tree), Larrea Divaricata, Rabdosia Rubescens, Euglena Gracilis, Fibraurea Recisa Hildinea, Shallot Sorghum Extract, Sunflower Extract, Enantia Chlorantha, Mitracarpus Scaper, Butuber Lomus, Henna L, Capillus Capillifera L, Celandine, Helianthus Annuus Mention may in particular be made of extracts of chima, Japanese mandarin, tea plant, Imperata cylindrica, horn poppy, cypress, Solomon's seal, lovely hemlock, elderberry, lima bean, Centaurea japonica, Duckweed, Turnella diffusa, Anemone julibrissin, Portulaca dahurica, hops, coffee plant, Yerba mate, Globularia cordifolia, Albizia julibrissin, sourwood, ginger, Astragalus membranaceus, Plantain lanceolata, snowdrop, Mirabilis mirabilis, horehound, celery or orchid.

[0149] The compositions of the present invention may include one or more additional peptides, including, but not limited to, di-, tri-, tetra-, penta-, and hexapeptides and derivatives thereof. According to certain embodiments, the concentration of the additional peptide in the composition is greater than or equal to 1×10 -7 % to 20% by weight, and 1×10 -6 The range of 1×10 wt.% to 10 wt.% is preferred. -5 The range of 10% to 5% by weight is preferred. As used herein, the term "peptide" refers to peptides containing 10 or fewer amino acids, their derivatives, isotopes, and complexes with other species, such as metal ions (copper, zinc, manganese, magnesium, etc.). The term "peptides" refers to both natural and synthetic peptides. "Peptides" also refers to compositions containing peptides found in nature and / or commercially available.

[0150] Dipeptides suitable for use in the present invention include, but are not limited to, carnosine (βAH), YR, VW, NF, DF, KT, KC, CK, KP, KK, TT, PA, PM, or PP.

[0151] Tripeptides suitable for use in the present invention include, but are not limited to, RKR, HGG, GKH, GHK, GGH, GHG, KFK, KAvaK, KβAK, KAbuK, KAcaK, KPK, KMOK, KMO2K (MO2 is sulfoxide methionine dioxide), KVK, PPL, PPR, SPR, QPA, LPA, or SPA.

[0152] Tetrapeptides suitable for use as additional peptides in the present invention include, but are not limited to, RSRK (SEQ ID NO:6), GQPR (SEQ ID NO:7), KTAK (SEQ ID NO:8), KAYK (SEQ ID NO:9) or KFYK (SEQ ID NO:10).

[0153] Suitable pentapeptides include, but are not limited to, KTTKS (SEQ ID NO:11).

[0154] Suitable hexapeptides include, but are not limited to, GKTKS (SEQ ID NO: 12), VGVAPG (SEQ ID NO: 13).

[0155] Other peptides suitable for use in the present invention can be selected from the following, but this list is not limiting: lipophilic derivatives of peptides, preferably palmitoyl (Pal) or myristoyl (Myr) derivatives, and metal complexes thereof (e.g., copper complexes of the tripeptide HGG). Preferred dipeptides include, for example, N-palmitoyl-beta-Ala-His, N-acetyl-Tyr-Arg-hexadecyl ester (Calmosensine™, Idealift™, Sederma), Pal-RT or Pal-KT (Sederma). Preferred tripeptide derivatives include, for example, Pal-GKH and Pal-GHK (Sederma), the copper derivative of HGG (Lamin™, Sigma), lipospondin (N-Elideyl-KFK) and its conservatively substituted analogs, N-acetyl-RKR-NH2 (peptide CK+), N-Biot-GHK (Sederma), Pal-KAvaK, Pal-KβAlaK, Pal-KAbuK, Pal-KAcaK, or Pal-KMO2K (trademark Matrixyl® Synthe' 6®, Sederma), Pal-KVK (Syn-Coll™, DSM), and derivatives thereof.

[0156] Here, the general formula X-Pro * -Pro * Anti-aging tripeptides of -Xaa-Y may also be mentioned, where Xaa is selected from Leu, Arg, Lys, Ala, Ser, and Asp at the N-terminus, and X is H, -CO-R at the C-terminus. 1 and -SO2-R 1 Y is selected from OH, OR 1 , NH2, NHR 1 or NR 1 R 2 Selected from: R 1 and R 2are each independently selected from linear, branched, cyclic, polycyclic, unsaturated, hydroxylated, carbonylated, phosphorylated and / or sulfurized alkyl, aryl, aralkyl, alkylaryl, alkoxy and aryloxy groups, which may optionally contain heteroatoms in their backbone, in particular O, S and / or N. * corresponds to proline, its analogs or derivatives; for example, the general formula X-Pro * -Pro * -Xaa-Y includes Myr-PPL-OH and Myr-PPR-OH.

[0157] Further, the general formula X-(Xaa1)n-Pro disclosed in WO2014 / 080376 is * Reference can be made to pigmentation-promoting and / or ECM-promoting dipeptides and tripeptides of -Xaa2-Y. When n=0, 1, or 2, Xaa1 is a hydrophobic amino acid selected from Ala, Val, Met, Leu, Iso, Phe, and Pro, and analogs and derivatives thereof, or a polar amino acid selected from Ser, Thr, Tyr, Asp, and Glu, and analogs and derivatives thereof; when n=2, the two amino acids Xaa1 are the same or different; Xaa2 is a hydrophobic amino acid selected from Ala, Val, Met, Leu, Iso, and Phe, and analogs and derivatives thereof, or a basic amino acid selected from Arg, Lys, and His, and analogs and derivatives thereof; and at the N-terminus, X is H, -CO-R 1 and -SO2-R 1 at the C-terminus, Y is selected from OH, OR 1 , NH2, NHR 1 or NR 1 R 2 Selected from; R 1 and R 2are independently selected from alkyl, aryl, aralkyl, alkylaryl, alkoxy and aryloxy groups, which may be linear, branched, cyclic, polycyclic, saturated, unsaturated, hydroxylated, carbonylated, phosphorylated and / or sulfurized, said groups having or not O, S and / or N heteroatoms in the backbone, and Pro * corresponds to proline, its analogues or derivatives; for example, the general formula X-(Xaa1)n-Pro * -Xaa2-Y includes the following peptides Pal-SPR-OH, Pal-PPR-OH, Pal-QPA-OH, Pal-LPAOH, Myr-SPA-OH, Pal-PM-OH, Pal-PA-OH and Pal-PP-OH.

[0158] Tetrapeptide derivatives suitable for use as additional peptides in the present invention include, but are not limited to, Pal-GQPR (SEQ ID NO:2) (Sederma), Ela-KTAK (SEQ ID NO:14), Ela-KAYK (SEQ ID NO:15), or Ela-KFYK (SEQ ID NO:16). Pentapeptide derivatives suitable for use as additional peptides herein include, but are not limited to, Pal-KTTKS (SEQ ID NO:1) (available from Sederma as Matrixyl®), Pal-YGGFXaa (SEQ ID NO:17) where Xaa is Leu or Pro, or mixtures thereof.

[0159] Hexapeptide derivatives suitable for use herein include, but are not limited to, Pal-VGVAPG (SEQ ID NO:3), Pal-GKTTKS (SEQ ID NO:18), Pal-HLDIIXaa where Xaa is Trp, Phe, Tyr, Tic, 7-hydroxy-Tic, or Tpi (7-hydroxy-Tic ou Tpi) (SEQ ID NO:19), and derivatives thereof. Also included are mixtures of Pal-GHK and Pal-GQPR (SEQ ID NO:2) (Matrixyl® 3000, Sederma).

[0160] Preferred commercially available compositions containing tripeptides or derivatives include Sederma's Biopeptide-CL™, Maxilip™, Biobustyl™, Procapil™, and Matrixyl® Synthe' 6®. Preferred commercially available source compositions of tetrapeptides include Rigin™, Eyelis™, Matrixyl® Reloaded, and Matrixyl 3000®, containing between 50 and 500 ppm Pal-GQPR (SEQ ID NO:2) and excipients offered by Sederma.

[0161] Additional active ingredients may also include the following commercially available peptides: Vialox™ (INCI name = Pentapeptide-3 (synthetic peptide containing alanine, arginine, isoleucine, glycine, and proline)), Syn-Aake™ (β-Ala-Pro-Dab-NH-Bzl) or Syn-Coll™ (Pal-Lys-Val-Lys-OH), sold by Pentapharm; Argireline™ (Ac-Glu-Glu-Met-Gln-Arg-Arg-NH2 (INCI name = acetyl hexapeptide-3)) (SEQ ID NO: 20), Leuphasyl™ (Tyr-D-Ala-Gly-Phe-Leu) (SEQ ID NO: 21), Aldenine™ (Gly-His-Lys), Trylagen™ (INCI name = Pseudoalteromonas fermentation extract, wheat protein hydrolysate, soy protein hydrolysate, tripeptide-10 citrulline (reaction product of citrulline and tripeptide-10 (a synthetic peptide consisting of aspartic acid, isoleucine, and lysine)), tripeptide-1), and Eyeseryl™ (Ac-β-Ala-His-Ser-His) (SEQ ID NO: 22), all sold by Lipotec. ID NO: 22), Serilesine™ (Ser-Ile-Lys-Val-Ala-Val) (SEQ ID NO 23) or Decorinyl™ (INCI name: Tripeptide-10 Citrulline = reaction product of citrulline with tripeptide-10 (a synthetic peptide consisting of aspartic acid, isoleucine, and lysine); Collaxyl™ (Gly-Pro-Gln-Gly-Pro-Gln (SEQ ID NO: 24)) or Quintescine™ (Cys-Gly), available from Vincience; Cytokinol™ LS (casein hydrolysate), sold by Les Laboratoires Serobiologiques / Cognis; Kollaren™ (Gly-His-Lys), IP2000™ (Pal-Val-Tyr-Val) or Meliprene™ (INCI name = Monofluoroheptapeptide-1: reaction product of acetic acid with a synthetic peptide containing arginine, glycine, glutamic acid, histidine, norleucine, p-fluorophenylalanine and tryptophan), sold by the European Biological Institute (L'Institut Europeen de Biologie Cellulaire); Neutrazen™ (Pal-His-D-Phe-Arg-NH2), available from Introvations; BONT-L-Peptide™ (INCI name = Palmitoyl Hexapeptide-19: reaction product of palmitic acid with hexapeptide-19, a synthetic peptide consisting of asparagine, aspartic acid, lysine, and methionine), Timp-Peptide™ (INCI name = Acetyl Hexapeptide-20: reaction product obtained by acetylation of hexapeptide-20, a synthetic peptide consisting of alanine, glycine, lysine, valine, and proline), or ECM Moduline™ (INCI name = Palmitoyl Tripeptide-28: reaction product of palmitic acid with tripeptide-28, a synthetic peptide consisting of arginine, lysine, and phenylalanine), all sold by Infinitec Activos.

[0162] Various compositions / formulations according to the present invention are described below with examples of additional active ingredients.

[0163] The active ingredients according to the invention are as described in section C / , ie comprise vectorized peptides.

[0164] This component is generally present at between 1% and 5%, preferably 3%.

[0165] 1) Cream form , e.g., facial anti-aging day cream

[0166] [Table 10B]

[0167] Examples of additional active ingredients: 1. Moisturizing / smoothing ingredients such as: OPTIM HYAL™, sold by Sederma, contains oligosaccharides of acetylated glucuronic acid with a structure similar to fragments of hyaluronic acid. 2. Sebum-regulating ingredients such as: SEBULESS™: Sold by Sederma, a sebum-regulating agent containing lilac extract obtained through in vitro cell culture, cleanses, reduces shine, provides a cooling sensation, and reduces the appearance of fine lines. PORETECT™: Marketed by Sederma, this formulation of flaxseed and celery extracts titrated with cylolinopeptides and senkyunolides provides firming, tone, and density to the skin, strengthening the pore-holding structure that breaks down with age. 3. Ingredients that act on the elastic properties of the skin / skin barrier, such as: IDEALIFT™: Marketed by Sederma, it contains the lipodipeptide N-acetyl-tyrosyl-arginyl-O-hexadecyl ester, which combats facial sagging and improves resistance to gravity by specifically stimulating elastin. 4. DERMAXYL™: Sold by Sederma, this product combines ceramide 2, a stratum corneum binding agent, with palmitoyl-formulated matrikine, Pal-Val-Gly-Val-Ala-Pro-Gly, which smooths wrinkles and repairs the skin barrier.

[0168] 1) A mild, water-based serum

[0169] [Table 10C]

[0170] Examples of additional active ingredients : 1. Anti-aging ingredients such as: SENESTEM™, marketed by Sederma, contains plant cells obtained by in vitro cell culture of Plantago lanceolata, and is particularly effective in improving the viscoelastic properties of the skin and lightening age spots. 2. Antioxidants such as: MAJESTEM™, marketed by Sederma and based on plant cells of the snowdrop plant obtained in vitro in a cell culture, titrated with leontopodic acid; neutralizes oxidative stress (pollution, UV rays) and restores skin firmness.

[0171] 2) Gel form

[0172] [Table 10D]

[0173] Examples of additional active ingredients: 1. Anti-pollution ingredients such as: Citystem™, marketed by Sederma and based on plant cells obtained in vitro from horehounds with a high concentration of Forsythoside B; used against pollution attacks. Skin becomes softer and smoother, the texture is refined, the visibility of acne is reduced, and the skin is brightened and purified. 2. Soothing ingredients for sensitive skin such as: Pacifeel™, sold by Sederma, contains extract of Mirabilis mirabilis. 3. Moisturizing ingredients such as: AQUALANCE™, sold by Sederma, is an osmoprotective moisturizing active ingredient consisting of homarine and erythritol.

[0174] 3) Gel form for forming spray mask

[0175] [Table 10E]

[0176] Examples of additional active ingredients: 1. Brightening ingredients such as: EVERMAT™: Sold by Sederma, contains a combination of protoberberine-rich Enantia chlorantha extract and oleic acid; reduces pore size and shine; and refines the texture of acne-prone skin. 2. Ingredients with activating properties such as: Fruitliquid™ Kumquat™, sold by Crodarom.

[0177] 4) Cream form , for makeup base

[0178] [Table 10F]

[0179] Examples of additional active ingredients: 1. Ingredients that work on dark circles / eye contours, such as: HALOXYL™, sold by Sederma, combines two matrikines, Pal-GHK and Pal-GQPR, with N-hydroxysuccinimide and the flavonoid chrysin. EYELISS™, sold by Sederma, contains three ingredients: hesperidin methyl chalcone, the dipeptide valyltryptophan (VW), and the lipopeptide Pal-GQPR. PRODIZIA™, from Sederma, contains extract of the silk tree, which repairs and protects the skin from the damage caused by glycation, thereby reducing the visible signs of fatigue, such as dark circles, bags under the eyes, dullness, paleness, and a tense complexion. 2. Peptide-based anti-wrinkle / anti-aging ingredients such as: MATRIXYL®, MATRIXYL 3000™, MATRIXYL Synthe' 6™ and / or MATRIXYL Morphomics™, available from Sederma.

[0180] F) In vivo efficacy testing Test item :Cream 1) of item E) of the Galenic preparations above

[0181] principle : The efficacy of the peptides of the present invention was evaluated in three complementary studies on a set number of volunteers:

[0182] 1. First placebo-controlled study uses several complementary techniques to measure the improvement of defective skin and tests the following different parameters: · Defect assessment carried out against standard photographs; Imaging assessment of hydration performed using the Epsilon™ device; Assessment of skin smoothing, performed using a color camera; and -Evaluation of keratinocyte size (stratum corneum condition) by image analysis of "peeling" (peeling of the cell layer on the adhesive support).

[0183] 2. Second Examis a whole-face test that quantifies the perceived effect on complexion using the CLCT clinical assessment method (color, brightness, clarity, and transparency).

[0184] 3. Third placebo-controlled study aims to demonstrate the soft "polish" effect of peptides by analyzing the application uniformity and disappearance of a self-tanning product.

[0185] 1 / First Exam procedure Specific inclusion criteria The study was conducted on a panel of 29 women with a median age of 41 (30-50 years old). Women with visibly imperfect skin, i.e., dull / tired / stained complexion and / or irregular texture and roughness, were included. However, imperfections such as rosacea, breakouts, and redness were excluded.

[0186] Volunteers observed a one-month washout period using only one moisturizer and had to avoid scrubs or masks and strong sunlight.

[0187] Test, duration, and type of application The study was conducted in a single-blind manner on the face and both forearms. For 6 weeks, volunteers applied the cream of the present invention to one side in the morning and evening, and a placebo cream to the other side.

[0188] The outline of the test can be summarized as follows:

[0189] [Table 10G]

[0190] statistics Statistical tests were performed using the Student t-test or, where appropriate, the nonparametric Wilcoxon test. Two-tailed tests were performed on matched series. Chi-square tests were used for expert assessment.

[0191] Methods and Results Defect evaluation Standardized photographs were taken at T0 and T6 weeks using a photographic stage with a high-resolution digital camera, specific lighting, and a volunteer restraint system. Facial position, photograph, and lighting settings were standardized and controlled to ensure reproducibility over time.

[0192] The resulting photographs were evaluated by a panel of five expert judges. For each volunteer, the experts visualized the before- and after-treatment photographs and commented on the statement "improved complexion," which means a more even complexion, smoother skin, and fewer imperfections.

[0193] The results are shown below.

[0194] [Table 10H] **Significant difference compared to negative feedback: p<0.01 / $Significant difference from placebo: p<0.05.

[0195] Application of the cream according to the invention results in a significant visible improvement in complexion, with 54% favorable and 3% unfavorable, whereas use of placebo resulted in only 36% favorable and 8% unfavorable, a clear and significant difference (p<0.05).

[0196] Moisture rating The Epsilon™ E100 device (Biox) was used. This device provides not only single values ​​but also averages over several thousand points and images of moisture content ("capacitive imaging"). Its capacitive sensor with a spatial resolution of 50 μm yields an image area of ​​12.8 × 15 mm, equivalent to 76,800 pixels. Each pixel provides an ε value, the dielectric constant of skin, between 0 and 85, with air = 1 and water = 80 as benchmarks. The measurement depth reaches approximately 50 microns. In addition to its iconographic interest, this 2D measurement also provides information about skin topography and, therefore, skin homogeneity and potentially skin suppleness, since moisture is known to improve this parameter.

[0197] Table 11 : Changes in forearm moisture (n=3 measurements, 25 volunteers)

[0198] [Table 11] *Vs placebo: nonparametric test, one-sided; two-sided test, p<0.1. NSD: not significant

[0199] After 6 weeks of application of the cream according to the invention, the moisturizing value increased by +25.8%, while application of the placebo resulted in a small increase of +11.3% (it is normal for the placebo to have an effect, since usually no cream is applied to both arms).

[0200] Images obtained with Epsilon™ were also evaluated by a five-person expert review panel.

[0201] For each volunteer, an expert visualized the photograph after treatment with the cream according to the invention and the photograph after treatment with the placebo cream and selected which image was the most homogeneous.

[0202] Table 12: Percentage of positive or negative responses regarding skin evenness (n=25 volunteers, 5 judges)

[0203] [Table 12] **For "Skin becomes more uniform with placebo cream," a significant change was observed at p<0.01.

[0204] On the image, each black area conveys no information, but each pixel does. The distribution of moisture changes between the two images: the image corresponding to the areas where the cream according to the invention was applied has "darker" pixels than the image corresponding to the areas where the placebo was applied. This indicates that in addition to better moisture retention, the skin has become smoother and potentially more supple.

[0205] Smoothing Evaluation A color camera was used for this evaluation. It uses diffused, constant LED lighting with polarized light to avoid glare. It has an image correction algorithm that allows instant color calibration and ensures image uniformity (eliminating edge shadows). The measurement field of view is 16 x 12 mm, with a resolution of 10 megapixels. It also has a 50x zoom, allowing structures smaller than 20 μm in size to be seen. Furthermore, the system, based on stereophotometry technology, records multiple images under different lighting conditions and reconstructs them in 3D, allowing the analysis of skin topography.

[0206] This last function was used for testing the cream according to the invention to evaluate two parameters of the topography: complexity and depth of the microreliefs.

[0207] Table 13 : Changes in the topography of microrelief on the forearm (smoothing effect) (average of two locations, 28 volunteers)

[0208] [Table 13] * vs. placebo: parametric one-sided test; p<0.08 and two-sided test; p<0.05

[0209] First, there is a smoothing effect of the placebo (-8.5% and -14.3% for each parameter), which, as before, can be explained by the abnormal uptake of the cream in this location.

[0210] However, the effect of the cream according to the invention is notable and significantly higher, either in terms of the depth of the microrelief (-12.8%, p<0.04 vs. placebo) or its complexity (-20.9%, p<0.05 vs. placebo).

[0211] Assessment of keratinocyte size Several studies have shown that under conditions of microstress, keratinocyte size decreases, which correlates with an increased proportion of the epidermis that undergoes keratinocyte immaturity.

[0212] At weeks T0 and T6, keratinocytes were collected from the skin of volunteers using a standardized adhesive method, labeled with a fluorescent reagent, and visualized and quantified under a microscope. Approximately 300 keratinocytes were quantified per volunteer and case.

[0213] Table 14 : Change in facial keratinocyte size (n=300 keratinocytes, average of 29 volunteers)

[0214] [Table 14]

[0215] These results show that the size of the corneocytes did not change significantly between weeks T0 and T6 on the side treated with the cream according to the invention. A decrease in the size of the corneocytes was observed on the placebo side (-2.4%, p<0.01).

[0216] The test was carried out in autumn, a season of frequent weather and temperature changes. The skin of the volunteers may have "registered" these changes as stress, leading to a slight decrease in the size of the keratinocytes. The use of the cream according to the invention was able to maintain the homeostasis of the upper epidermis, in particular due to its properties of reducing pro-inflammatory mediators and strengthening the skin barrier, thereby calming the upper epidermis.

[0217] 2 / Second Exam procedure Specific inclusion criteria The study was conducted on a panel of 26 women (aged 22-44 years) with a mid-36 year old complexion (based on the Spincontrol Institute CLCT scale (Color, Lightness, Clarity, Transparency)) and phototype I to phototype III skin.

[0218] Test, duration, and type of application The test was carried out on the face: volunteers applied the cream according to the invention morning and night for 6 weeks.

[0219] statistics For the CLCT index, a one-tailed Student's t-test was performed against the theoretical mean values, and for the self-assessment by volunteers, a chi-square test was performed.

[0220] Methods and Results 1 / Evaluation of complexion using CLCT To assess complexion, the CLCT method is based on the visual analysis of seven descriptors characteristic of complexion by trained judges. · Skin color is defined by four shades that characterize Caucasian skin: pink-red, olive, beige, soft pink; · Lightness is defined by the intensity of "light spots" in prominent areas of the face; Clarity is the combination of the evenness of skin color and the regularity (homogeneity) of texture; and Transparency is characterized by the skin being visible through the skin, reflecting the fineness of the skin's texture.

[0221] The assessee sits between two "daytime" lamps and wears a cloak and black charlotte to protect from external influences. The judges rate the four colors on a color chart, and the other three descriptors on an analog scale from 0 to 100.

[0222] The "i" index is calculated from the seven descriptors, and the delta i(T6)-i(T0) is calculated to obtain the CLCT index "I". If this index has a significance probability of 5 or more, it can be said that the product is effective in improving the radiance of the complexion.

[0223] Table 15 : Changes in facial color perception (n=3 judges, 26 volunteers)

[0224] [Table 15]

[0225] Six weeks of application of the cream according to the invention changed the CLCT index by +8.4 units (33.5% vs. TO, p<0.01), making it an effective product for complexion radiance. The most significantly improved descriptors were olive (25% decrease), beige (4% decrease), and soft pink (16% improvement) (all p<0.01 vs. TO).

[0226] Self-assessment of complexion At the end of the six week period of applying the cream according to the invention, the volunteers commented on several points.

[0227] The results show that the volunteers gave very positive feedback, finding it comfortable (68%), quickly absorbed (71%), and not interfering with makeup (84%). The effects on the face were interesting, with skin feeling more hydrated and less rough (68% and 69%), and especially less dull (59%). All of this contributes to having fairer skin (66%) and a better appearance (62%).

[0228] 3 / Third Exam procedure Specific inclusion criteria The study was conducted on a panel of 20 women aged 37 years (20-49 years) with phototype I to phototype III skin.

[0229] Test, duration, and type of application The study was conducted in a single-blind manner in both arms: for 6 weeks, volunteers applied the cream according to the invention in the morning and evening and a placebo cream in the other arm.

[0230] Statistics information Statistical studies were performed using the chi-square test.

[0231] Methods and Results Evaluation of the effects of "Soft Polish" by monitoring DHA staining (docosahexaenoic acid) This test is based on the fact that before applying a self-tanner, a very soft abrasion or exfoliation of the skin should be performed to make the skin smoother and therefore ensure a more even and long-lasting application of the self-tanner on the skin.

[0232] To demonstrate the "soft polish" effect of the cream of the present invention, the cream was applied for 6 weeks, then the area was given DHA (a traditional self-tanner) and the color development and subsequent fading was captured with a C-Cube™ camera.

[0233] To assess the impact on the intensity and homogeneity of fading between days T2 and T7, the images acquired with the C-Cube™ camera were evaluated by an expert panel. For each volunteer, the experts had to prove which image pair (cream according to the invention or placebo cream) showed the most acceptable change in terms of fading (less discoloration) and / or homogeneity (less brown spots).

[0234] The results show a very clear preference for the areas where the cream of the present invention was applied, with 63% of votes compared to 25% who chose placebo. This result is statistically significant at p<0.01. Thus, skin preparation with the cream of the present invention resulted in a softening and natural smoothing of the stratum corneum, comparable to physical peeling.

Claims

1. A cosmetic or dermatological ingredient for non-therapeutic cosmetic treatment to maintain or improve the condition of the epidermis, comprising the peptide Pal-KTFK (SEQ ID NO5) and a physiologically acceptable vehicle, The physiologically acceptable medium comprises an oil, a wax, a surfactant, and water, and is an oil-in-water emulsion of the wax dispersed in an aqueous phase, the peptide is bound to the wax particles in the emulsion via the surfactant; The cosmetic treatment is for smoothing the skin, improving skin moisturization and / or complexion radiance, and for evenly toning the complexion. component.

2. A cosmetic or dermatological composition comprising the ingredients of claim 1.

3. In separate compartments, a) the composition of claim 2, and b) a composition comprising a self-tanning active agent in a physiologically acceptable medium; 1. A kit for performing a self-tanning cosmetic treatment comprising:

Images