NEW PEPTIDES AND PHARMACEUTICAL AND COSMETIC COMPOSITIONS: CONTAINERS

Abstract

The present invention relates to novel peptides having the same LRIRX1TYX2 motif, where X1 is selected from amino acids A or F, and X2 is selected from amino acids G or K. The present invention also relates to their use in the treatment of skin lesions of various origins, as well as to pharmaceutical and cosmetic compositions comprising them and a cosmetic process. More particularly, the treatment of said lesions consists notably of strengthening the dermo-epidermal junction, cell-cell adhesion and / or cell-matrix adhesion, and cell migration at the level of the epidermis, and promoting skin surface repair.

Description

Title of the invention: NEW PEPTIDES AND PHARMACEUTICAL AND COSMETIC COMPOSITIONS: CONTAINERS FIELD OF INVENTION

[0001] The present invention relates to novel peptides having the same LRIRXiTYX2 motif, where X1 is chosen from amino acids A or F; and X2 is chosen from amino acids G or K. The present invention also relates to their use in the treatment of skin lesions of various origins, as well as to pharmaceutical and cosmetic compositions comprising them and a cosmetic process. More particularly, the treatment of said lesions consists in particular of strengthening the dermo-epidermal junction, cell-cell adhesion and / or cell-matrix adhesion and cell migration at the level of the epidermis and promoting the repair of the skin surface. STATE OF THE ART

[0002] The most common and natural skin alteration is simply that due to skin aging. Skin aging is a complex phenomenon caused by numerous intrinsic (genetic factors) and extrinsic (such as sun exposure, diet, and exposure to cigarette smoke) factors. Clinically, this manifests as the appearance of wrinkles and fine lines, a loss of skin elasticity, sagging of the cutaneous and subcutaneous tissues, and impaired wound healing.

[0003] Numerous research avenues have been proposed to combat skin aging, including protection against environmental factors (sun, pollution, etc.), activation of cell regeneration, and strengthening of the extracellular matrix (collagen and elastin). Recent studies have demonstrated the importance of cell adhesion, both between cells and between cells and the extracellular matrix, in the skin aging process, and therefore the need to strengthen these adhesions to prevent or even treat skin laxity.

[0004] Dermatological studies recently focused on the use of peptides in cutaneous biology (sequences derived from alpha-MSH, certain neuropeptides, RGD peptide...), are oriented towards the search for peptides having strong activity at the cutaneous level.

[0005] The cosmetics industry is also constantly awaiting new peptides capable of increasing cell adhesion to the extracellular matrix and / or cell adhesion to each other.

[0006] Interactions between cells and the extracellular matrix (ECM) play an important role in controlling cell behavior. This control occurs through specific interactions between matrix molecules and cells at transmembrane receptors, primarily of the integrin family, whose intracellular domain is linked to cytoskeletal components. This allows the matrix to transmit intracellular signals that modulate, depending on the case, the adhesion, migration, proliferation, differentiation, or apoptosis of epidermal cells. This control of cell behavior is crucial during development as well as during tissue remodeling.

[0007] Depending on their constituent molecules and the resulting organization, several types of ECMs can be distinguished, with basal laminae being arguably the most specialized. These are fine, continuous protein lattices upon which the various cell layers of the organism rest. They were long defined as discrete morphological structures whose function was limited to partitioning tissue compartments. It is only in the last 25 years that, despite their low representation and high insolubility, significant progress has been made in investigating their molecular composition and functions. It appears that these structures play a fundamental role in controlling cell behavior, both during embryonic development and in maintaining the integrity of differentiated cell phenotypes.

[0008] The structure of the skin is composed of:

[0009] • of a lining epithelium: the epidermis, • of connective tissue: the dermis (a thick layer that determines the morphology of the skin and contains blood and lymphatic vessels, and nerves), and • of adipose tissue: the hypodermis.

[0010] The dermis and epidermis are connected by a unique and complex structure, the dermo-epidermal junction (DEJ) or epidermal basal lamina. Anatomically, it corresponds to the area between the basal cells of the epidermis and the most superficial layers of the dermis. It is a zone of adhesion that creates a partition between the polarized epithelium and the underlying stroma, ensuring the maintenance and cohesion of adjacent cells. The DEJ, composed exclusively of extracellular matrix (ECM), performs two roles:

[0011] 1°) mechanical role, its unique molecular architecture gives it a great mechanical stability allowing it to ensure solid anchoring of the epidermis;

[0012] 2°) Biological role, the proteins of the DEJ maintain with the basal cells of the epidermis has close relationships and transmits important morphogenetic information to them via receptors of the integrin family.

[0013] The JDE is also a diffusion filter with respect to nutrients and meta- bolic. It therefore allows the transmission of biological information.

[0014] During skin aging, the DEJ flattens and gradually loses its characteristic undulations, which considerably reduces the epidermis-dermis interface. In addition, the molecular networks become disorganized, the protein framework weakens, and the adhesion of basal keratinocytes is diminished.

[0015] As a result, the mechanical function (support) and the biological function (exchange of information and molecules) of the JDE are altered.

[0016] When a skin injury occurs, the dermal-epidermal junction (DEJ) is damaged and its constituent molecules are degraded by specific enzymes. Under favorable conditions, epidermal re-epithelialization begins a few hours after the trauma. As soon as the epithelium has covered the wound bed, the DEJ proteins reappear sequentially and in an orderly fashion.

[0017] With skin aging, each of these stages occurs more slowly. In particular, a decrease in the expression of matrix adhesion proteins and membrane receptors has been observed, which could be the major cause of the delay observed in the DEJ regeneration process and the extreme fragility of scar tissue in elderly subjects.

[0018] Laminin 5 (LN-5), or laminin 332, is a protein specifically expressed in the basal laminae of specialized squamous and transitional epithelia such as the dermal-epidermal junction (DEJ) of the skin. LN-5 results from the heterotrimeric assembly of the alpha 3, beta 3, and gamma 2 subunits and is synthesized exclusively by epithelial cells as a 460 kDa precursor. Under physiological conditions, each of the alpha 3 and gamma 2 subunits undergoes extracellular post-translational cleavage of its carboxy- and amino-terminal ends, respectively, resulting in the mature tissue form. Studies indicate that the entire gamma 2 precursor chain is required for the secretion and integration of LN-5 into the extracellular matrix (ECM). Other studies have shown that the gamma 2 precursor chain is involved in keratinocyte migration during the skin healing process.

[0019] The major role of LN-5 is highlighted by the existence of hereditary or acquired pathologies resulting from an abnormality in the synthesis and / or expression of one of its constituent subunits. These diseases, called junctional epidermolysis bullosa, lead in particular to fragility of the dermal-epidermal junction (DEJ) of the skin, characterized by the spontaneous formation of epidermal blisters. LN-5 thus plays a crucial and irreplaceable role in epithelial-mesenchymal cohesion. LN-5 carries key biological signals, as it enables the adhesion of adjacent epithelial cells via integrins and induces the assembly of stable adhesion structures, namely hemidesmosomes.

[0020] International patent application WO 00 / 66731 in the name of Biostatum describes the Production of whole LN-5 in recombinant form in eukaryotic cells (L5r) and documentation of its activity in improving wound healing, proliferation, and cell adhesion. Other peptides derived from Laminin-5 are described in US-B1-6294356 (Jones).

[0021] Patent EP1784423, filed by the Applicant, characterizes the efficacy of a peptide of sequence TALRIRATYGEY, a sequence present on the gamma 2 chain of LN-5, which specifically induces the adhesion of keratinocytes of the epidermis and other epithelial cells.

[0022] There remains a need for alternatives for the treatment of skin alterations of various origins, such as those described above, which also have increased efficacy.

[0023] Today, the Applicant has surprisingly and unexpectedly identified that an effective amount of a peptide comprising the motif LRIRXiTYX2, where XI is selected from amino acids A or F and X2 is selected from amino acids G or K, specifically induces the adhesion of epidermal keratinocytes and other epithelial cells. This peptide not only increases cell adhesion to the ECM but also enhances keratinocyte migration. Due to its small size and high stability, the peptide possesses all the necessary characteristics to effectively cross the epidermis and reach its target, the DEJ, interact with basal keratinocytes, and transmit adhesion induction signals. Its chemical synthesis process is simple and applicable on an industrial scale.It does not use animal cell cultures, nor growth factors and / or sera or serum derivatives. Being small in size, it will be subject to little or no specific and / or non-specific proteolytic degradation.

[0024] The general inventive concept according to the invention is the use of a peptide comprising the sequence: LRIRXiTYX2, where:

[0025] • Xiestchoisi entre les acids aminos A or F ; and • X2 is chosen from among the amino acids G or K.

[0026] It is this motif that specifically induces the effects mentioned below. The variants allowing cyclization or doubling of the presence of this peptide are merely format variants of this motif, which may slightly influence its effectiveness. Description of the invention

[0027] According to a first aspect, the invention relates to a peptide consisting of the sequence: LRIRXjTYX,, where:

[0028] • Xiestchoisi entre les acids aminos A or F ; and • X2 is chosen from among the amino acids G or K.

[0029] According to one embodiment, said peptide has the following sequence: LRIRFTYK (SEQ ID NO: 1)

[0030] According to one embodiment, said peptide has the following sequence: LRIRFTYG (SEQ ID NO: 5)

[0031] According to one embodiment, said peptide has the following sequence: LRIRATYK (SEQ ID NO: 9)

[0032] According to one embodiment, said peptide has the following sequence: LRIRATYG (SEQ ID NO: 13)

[0033] According to a second aspect, the invention relates to a peptide consisting of the sequence: CLRIRXiTYX2C, where:

[0034] • Xiestchoisi parmi les acids aminos A ou F ; et • X2 is chosen from among the amino acids G or K.

[0035] The addition of these two cysteines to the ends of the peptide allows its cyclization.

[0036] According to one embodiment, said peptide has the following sequence: CL-RIRFTYKC (SEQ ID NO: 2)

[0037] According to one embodiment, said peptide has the following sequence: CL-RIRFTYGC (SEQ ID NO: 6)

[0038] According to one embodiment, said peptide has the following sequence: CL-RIRATYKC (SEQ ID NO: 10)

[0039] According to one embodiment, said peptide has the following sequence: CL-RIRATYGC (SEQ ID NO: 14)

[0040] According to a third aspect, the invention relates to a peptide consisting of the sequence: CSGLRIRXjTYXzSGC, where:

[0041] • Xiestchoisi entre les acids aminos A or F ; and • X2 is chosen from among the amino acids G or K.

[0042] The addition of these two cysteines as well as Serine-Glycine spacers to the ends of the peptide allows its cyclization.

[0043] According to one embodiment, said peptide has the following sequence: CSGL-RIRFTYKSGC (SEQ ID NO: 3)

[0044] According to one embodiment, said peptide has the following sequence: CSGL-RIRFTYGSGC (SEQ ID NO: 7)

[0045] According to one embodiment, said peptide has the following sequence: CSGLRL RATYKSGC (SEQ ID NO: 11)

[0046] According to one embodiment, said peptide has the following sequence: CSGLRL RATYGSGC (SEQ ID NO: 15)

[0047] According to a fourth aspect, the invention relates to a peptide consisting of the sequence LRIRXjTYXzSGLRIRXjTYX,, where:

[0048] • Xiestchoisi entre les acids aminos A or F ; and • X2 is chosen from among the amino acids G or K.

[0049] The addition of these Serine-Glycine spacers makes it possible to space out this peptide which includes twice the motif of the peptide according to the invention.

[0050] According to one embodiment, said peptide has the following sequence: LRIRF-TYKSGLRIRFTYK (SEQ ID NO: 4)

[0051] According to one embodiment, said peptide has the following sequence: LRIRF-TYGSGLRIRFTYG (SEQ ID NO: 8)

[0052] According to one embodiment, said peptide has the following sequence: LRIRA-TYKSGLRIRATYK (SEQ ID NO: 12)

[0053] According to one embodiment, said peptide has the following sequence: LRIRA-TYGSGLRIRATYG (SEQ ID NO: 16)

[0054] The present invention also relates to other peptides resulting from one or more modifications of the above peptides, the deletion or substitution of one or more amino acids, preferably as indicated in Table 1, and / or from oligomerization, cyclization or folding of the above peptides, it being understood that these modifications do not in any way diminish the adhesive activity of the reference peptide, or even improve it.

[0055] Table 1: Possible amino acid substitutions of peptides according to the invention Position of amino acids SEQ Possible replacement amino acids 1 LL, M, I, V, F, E or R 2 RR, N, E, Q, H, K or Y 3 II, M, L, V, F, T or K 4 RR, N, E, Q, H, K or T 5 X (A or F) A, C, S, G, V or D 6 TT, S, P, G, N, D, E, Q, H, or K 7 YY, H, F, W, D, I or Q 8 X (G or K) G, S, T, A or N

[0056] * X can be any amino acid.

[0057] More particularly, the addition or removal of one or more amino acids can be carried out on the carboxy- and / or amino-terminal end of said peptide. The invention also relates to any analogue or derivative resulting from the grafting of a motif of interest (natural or synthetic molecules, proteins and / or sugars) onto said peptide. It also relates to any dermatologically active fragment of the peptide of the invention, modified or not.

[0058] Preferably, the peptides according to the invention are obtained by chemical synthesis.

[0059] The invention also relates to the use of said peptides according to the invention as a medicinal product.

[0060] According to one embodiment, said peptides according to the invention are used in the strengthening of the dermo-epidermal junction, cell-cell adhesion and / or cell-matrix adhesion and cell migration at the level of the epidermis.

[0061] As previously stated, the most commonly observed alteration of the epidermis and the dermal-epidermal junction (DEJ) is that resulting from skin aging. However, other skin alterations can occur independently of aging and can, for example, be induced by certain dermatological conditions. Examples include eczema, psoriasis, pruritus, irritant dermatitis, sun damage, keratoses, fungal infections, and ichthyosis. In addition to the symptoms specific to each of these conditions, the skin undergoes alterations that the present invention aims to address as a complementary therapy. It is clear that the present invention is not intended to treat such conditions but rather to restore the damaged DEJ and cell adhesion in these conditions and to regenerate the epidermis by promoting keratinocyte migration.

[0062] Thus the present invention allows the repair, regeneration and / or restructuring of the skin.

[0063] The skin can also be weakened by cosmetic or therapeutic skin treatments which, while treating the targeted condition, generate side effects on the skin that must be treated independently of the condition itself. This is particularly the case with acne treatments, PUVA therapy, surgical procedures (dermatological or otherwise), laser skin treatments, dermabrasion, peels, or cancer treatments by radiotherapy.

[0064] According to one embodiment, said peptides according to the invention are used in the treatment of skin alterations induced by dermatological pathologies.

[0065] According to one embodiment, said peptides according to the invention are used in the treatment of alterations to skin weakened by cosmetic or therapeutic treatments.

[0066] According to one embodiment, said peptides according to the invention are used in the curative or preventive treatment of skin aging such as wrinkles, sagging, loss of elasticity, poorer wound healing, keloid, hypertrophic or atrophic scars, senile xerosis, changes in the skin's pigmentary system, and depletion of the skin's vascular network. irregular skin texture, skin atrophy, and alteration of the hair and nails.

[0067] Indeed, the aforementioned pathologies, skin fragility, and various treatments generate alterations on the skin such as decreased adhesion of the epidermis and epidermal cohesion, or even less effective restructuring of the skin surface.

[0068] According to another aspect, the invention relates to a pharmaceutical composition comprising at least one peptide according to the invention.

[0069] Preferably, said pharmaceutical composition according to the invention comprises from 0.00002% to 5%, preferably from 0.00005% to 0.1% and more preferably from 0.0001% to 0.001% by weight of peptide according to the invention and at least one pharmaceutically acceptable excipient.

[0070] According to one embodiment, said composition further comprises at least one other dermatologically active principle.

[0071] More particularly, the composition according to the present invention further comprises at least one other dermatologically active principle acting either on the strengthening of the dermo-epidermal junction, the adhesion of skin cells to the matrix, and / or the cell-cell adhesion and / or the migration of keratinocytes or otherwise on the skin according to the nature of the agent(s) used such as a moisturizing agent, a re-lipidating agent, a superfatting agent, an exfoliating agent, a keratolytic agent, an antioxidant agent, a soothing agent, a softening agent, a sedative agent, a cleansing agent, a makeup-removing agent, a purifying agent, an antibacterial agent, an antiseptic agent, an antiseborrheic agent, a brightening agent, an anti-wrinkle agent, a decongestant agent, a revitalizing agent, a cell renewal activator or one or more sun filters.

[0072] The compositions of the invention must be in a dermatologically acceptable form, i.e. compatible with the skin, hair, mucous membranes and / or hair.

[0073] Thus, preferably, said pharmaceutical composition according to the invention is in the form of a cream, ointment, salves, mask, serum, milk, lotion, paste, foam, aerosol, stick, powder, solution, suspension, shampoo, conditioner, patches, aqueous hydroalcoholic or oily solution, oil-in-water or water-in-oil or multiple emulsions, aqueous or oily gel, suture threads, surgical glue, dressing, anhydrous liquid, paste or solid product, and / or an oil dispersion in an aqueous phase using spherules, these spherules being able to be polymeric nanoparticles such as nanospheres and nanocapsules or vesicles ionic and / or non-ionic lipids.

[0074] Preferably, this composition is intended for topical application.

[0075] Possible additional compounds, active or not, may be added to the composition of the invention and a person skilled in the art shall choose them so that their addition does not alter the advantageous properties of said composition.

[0076] According to another aspect, the invention relates to a non-therapeutic cosmetic composition comprising at least one peptide according to the invention.

[0077] Preferably, said cosmetic composition according to the invention comprises from 0.00002% to 5%, preferably from 0.00005% to 0.1% and more preferably from 0.0001% to 0.001% by weight of peptide and at least one cosmetically acceptable excipient.

[0078] According to one embodiment, said composition further comprises at least one other cosmetically active ingredient.

[0079] The composition of the invention may also contain the usual adjuvants used in the cosmetic and dermatological fields, such as hydrophilic or lipophilic gelling agents, hydrophilic or lipophilic active ingredients, preservatives, antioxidants, solvents, perfumes, fillers, filters, pigments, odor absorbers, and colorants. The quantities of these various adjuvants are those typically used in the fields concerned, and for example, from 0.01 to 20% of the total weight of the composition. These adjuvants, depending on their nature, may be introduced into the oil phase, the aqueous phase, lipid vesicles, and / or nanoparticles.

[0080] Preferably, said composition is in the form of a cream, ointment, salves, mask, serum, milk, lotion, paste, foam, aerosol, stick, powder, solution, suspension, shampoo, conditioner, patches, aqueous hydroalcoholic or oily solution, oil-in-water or water-in-oil or multiple emulsion, aqueous or oily gel, anhydrous liquid, paste or solid product, and / or an oil dispersion in an aqueous phase using spherules, these spherules being polymeric nanoparticles such as nanospheres and nanocapsules or ionic and / or non-ionic lipid vesicles.

[0081] According to one embodiment, said composition is a cosmetic composition for the care of skin aging such as wrinkles, sagging, and loss of elasticity.

[0082] According to one embodiment, said composition is a cosmetic composition for skin regeneration or repair care.

[0083] According to one embodiment, said composition is a cosmetic composition for scalp care such as an anti-hair loss treatment.

[0084] According to one embodiment, said composition is a cosmetic composition for the care of the skin's pigmentary system, irregular skin texture, and / or alteration of the hair and nails.

[0085] According to another aspect, the invention relates to the non-therapeutic use of at least one peptide according to the invention in a cosmetic composition.

[0086] According to a final aspect, the invention relates to a cosmetic skin care process characterized in that it includes a step of applying a cosmetic composition according to the invention to the skin.

[0087] Preferably, an amount of 0.2 to 3 mg / cm2 of cosmetic composition is applied to the skin area, preferably 2 mg / cm2.

[0088] Preferably, the cosmetic composition according to the invention is applied once or twice a day, preferably twice a day, for at least 7 days, preferably at least 15 days, even more preferably for at least one month, and particularly preferably for at least two months. Most preferably, the cosmetic composition is applied in the process according to the invention twice a day for at least two months. DESCRIPTION OF THE FIGURES

[0089] Other features, purposes and advantages of the invention will become apparent from the following description, which is purely illustrative and not limiting, and which should be read in conjunction with the accompanying drawings on which:

[0090] [Fig.1] Fig.1 illustrates the results of the cell adhesion test for the peptide TALRIRATYGEY (SEQ ID NO: 17) as a positive control and the peptides LRIRATYG (SEQ ID NO: 13), LRIRATYGLRIRATYG (SEQ ID NO: 18) and LRL RATYGSGLRIRATYG (SEQ ID NO: 16).

[0091] [Fig.2] Fig.2 illustrates the results of the cell adhesion assay for the peptide TALRIRATYGEY (SEQ ID NO: 17) as a positive control and the peptides LRIRATYG (SEQ ID NO: 13), LRIRATYGLRIRATYG (SEQ ID NO: 18), CSGL-RIRATYGSGC (SEQ ID NO: 15) and LRIRATYGSGLRIRATYG (SEQ ID NO: 16).

[0092] [Fig. 3] Figure 3 illustrates the results of the wound closure test of a confluent mat of primary keratinocytes for the peptide TALRIRATYGEY (SEQ ID NO: 17), the peptides LRIRATYG (SEQ ID NO: 13), LRIRATYGLRIRATYG (SEQ ID NO: 18), the cyclic peptide CSGLRIRATYGSGC (SEQ ID NO: 15), a positive control (EGF), and a negative control (culture medium alone). (The asterisks * correspond to: * p < 0.05; ** p < 0.005; **** p < 0.0001).

[0093] [Fig. 4] [Fig. 4] illustrates the results of a second wound closure test of a confluent mat of primary keratinocytes for the TALRIRATYGEY peptide (SEQ ID NO: 17), the LRIRATYG peptide (SEQ ID NO: 13), the cyclic CSGLRIRATYGSGC peptide (SEQ ID NO: 15), a positive control (EGF), and a negative control (culture medium alone). (The * denote: * p<0.05; **p<0.005; ****p<0.0001).

[0094] [Fig. 5] Figure 5 illustrates the results of the cell adhesion test for the peptide TALRIRATYGEY (SEQ ID NO: 17) as a positive control and peptides LRIRATYG (SEQ ID NO: 13), LRIRATYK (SEQ ID NO: 9), and LRIRKTYG (SEQ ID NO: 19).

[0095] [Fig. 6] Figure 6 illustrates the results of the cell adhesion test for the peptide TALRIRATYGEY (SEQ ID NO: 17) and the peptides LRIRATYG (SEQ ID NO: 13), LRIRFTYK (SEQ ID NO: 1) and LRIRFTYG (SEQ ID NO: 5).

[0096] [Fig.7] Figure [Fig.7] illustrates the results of the wound closure test of a mat confluent of primary keratinocytes for the TALRIRATYGEY peptide (SEQ ID NO: 17) and the LRIRATYG (SEQ ID NO: 13), LRIRFTYG (SEQ ID NO: 5) and LRIRFTYK (SEQ ID NO: 1) peptides, a positive control (EGF) and a negative control (culture medium alone). (The * correspond to: * p<0.05; **p<0.005; *** p<0.0005; **** p<0.0001).

[0097] [Fig.8] Figure 8 illustrates the results of the cell adhesion test for the peptide LRIRFTYK (SEQ ID NO: 1), and the cyclic peptides CLRIRFTYKC (SEQ ID NO: 2) and CSGLRIRFTYKSGC (SEQ ID NO: 3).

[0098] [Fig.9] Figure [Fig.9] illustrates the results of the cell viability test for peptides according to the invention TALRIRATYGEY (SEQ ID NO: 17), LRIRATYG (SEQ ID NO: 13), LRIRFTYK (SEQ ID NO: 1), as well as for the cyclic peptide CLRIRFTYKC (SEQ ID NO: 2).

[0099] [Fig. 10] [Fig. 10] illustrates the results of the wound closure test of a confluent mat of primary keratinocytes for the cyclic peptides according to the invention CLRIRFTYKC (SEQ ID NO: 2), CSGLRIRFTYKSGC (SEQ ID NO: 3) and for the peptides LRIRFTYK (SEQ ID NO: 1), LRIRATYG (SEQ ID NO: 13), TALRIRATYGEY (SEQ ID NO: 17), as well as for EGF, and the negative control. (The * correspond to: * p<0.05; ***p<0.0005; ****p<0.0001) EXAMPLES Example 1: Materials and methods The cells used for the study:

[0100] • The HT1080 cell line: Initially, cells from epithelial cell lines were used, tools commonly used for cell adhesion studies. The HT1080 cell line (fibrosarcoma, Human), American Type Culture Collection CCL-121. • Primary human keratinocytes: For the cell viability and migration test, freshly isolated normal human keratinocytes were used. The human keratinocytes were obtained from biopsy of Foreskin (surgical waste, Tissue and Cell Bank, Edouard Herriot Hospital). The culture medium used during the experiments was the KGM-Gold defined medium for keratinocyte culture, marketed by Lonza Bioscience, containing 0.15 mM CaCl₂, pH 7.2 to 7.4. Keratinocytes were obtained using the Boyce and Ham technique (Cultivation, frozen storage, and clonal growth of normal human epidermal keratinocytes in serum free-media, Tiss Cuit. Meth. 1985, 9:83-93). The skin fragments, after rinsing in PB S buffer containing antibiotics, were defatted and cut into 3 mm² pieces, then placed in a sterile 0.25% trypsin solution in PBS for 16 hours at 4°C. The dermis / epidermis separation was performed in a Petri dish containing culture medium to stop the action of trypsin. The epidermal fragments were aspirated and repulsed several times to detach the free basal cells.After centrifugation, 3104 live cells per cm² were seeded onto tissue culture plates (Corning, Polylabo, France). Keratinocytes were cultured at 37°C in a CO2 incubator (5% CO2, 95% air, and 98% humidity). Subculture occurred when the cells reached subconfluence. The cell mat was then rinsed with PBS and covered with a trypsin-EDTA solution (0.05–0.02%). After a short incubation at 37°C, the cells detached, were counted, and re-seeded. Cell viability test:

[0101] The effect of peptides on cell viability was analyzed using a colorimetric test (Cell Proliferation Kit XTT, Roche Diagnostics, Meylan, France) on normal human keratinocytes from young subjects.

[0102] The chemical reaction of the test is based on the production of NADPH by live cells, which reduces yellow XTT tretazolium salts to orange formazan salts. Absorbance is measured at 490 nm using an ELISA-Reader plate reader. Cells were seeded in 96-well plates at a density of 10⁴ cells per well (3 / condition) in KBM-Gold culture medium. After 24 h of culture at 37 °C in the presence of 5% CO₂, the culture media were removed and replaced with KBM-Gold medium containing the peptide quantities shown in the figures. Two days later, the media were removed and replaced with the test reagent. The plates were then placed in an incubator at 37 °C, and the absorbance reading was taken at 5 h. Controls without peptide were prepared on the same plate.The results are presented as the percentage of cell viability in cells exposed to the peptide compared to controls without the peptide. Cell viability was calculated using the formula: .

[0103] %viability = (Abs. cells with peptide / Abs. control cells) X 100.

[0104] Quantitative analysis of the cell adhesion properties of peptides of interest by a colorimetric test:

[0105] - Preparation of adhesion substrates

[0106] A decreasing concentration range of peptides was prepared by successive dilution in PBS (Phosphate Buffer Saline, KH2PO4 1.54 mM; Na2HPO4 1.42 mM; NaCl 131 mM), starting from a 1 mg / ml base solution. These solutions were immediately dispensed onto 96-well culture plates (plates selected according to immobilization) at a rate of 100 µL per well. The plates were then incubated at +4°C for 16 to 18 h. The solutions were then removed, and each well was saturated with a 1% PBS-SAB solution (bovine serum albumin). Three additional substrate-free wells underwent the same treatment and served as blanks.

[0107] - Cell adhesion test

[0108] HT1080 cells were detached from the culture dishes by a trypsin / EDTA solution (0.05-0.02%), then suspended in DMEM medium without additives. The number of cells seeded was 100,000 cells per well.

[0109] - Evaluation of the cell adhesion test

[0110] After inoculation of the cells, the plates were placed in an incubator at 37°C under a 5% CO2 atmosphere. After incubation for 30 to 60 minutes, the adhesion medium was removed and each well was washed with sterile PBS to remove any unadhered cells. The remaining cells, adhering to the substrate, were fixed with a 1% glutaraldehyde solution in PBS for 15 minutes. The glutaraldehyde solution was removed, and the cells were stained with a 1% crystal violet solution in distilled water for 30 minutes. After thorough rinsing, the cells were permeabilized with a 0.02% Triton solution for 15 minutes to solubilize the crystal violet. Absorbance was read at 570 nm using a plate reader (Victor2 V Spectrophotometer, Perkin Elmer). Each experimental point was performed in triplicate.The blank value is the average absorbance of the 3 control wells (SAB). This was subtracted from each of the absorbance values ​​obtained for the experimental points. The average absorbance values ​​for each condition were calculated and presented as a curve, with the average absorbance values ​​on the y-axis and the different quantities of substrate immobilized in the wells (in pg or mole) on the x-axis. [YES] Optimization and verification of peptide immobilization in plates

[0112] Since the different peptides tested do not necessarily have the same charges, they immobilize differently on the plates, which can lead to erroneous results. Therefore, it is necessary to test different types of plates and different buffers. immobilization to ensure proper immobilization of peptides, especially in comparative studies.

[0113] Thus the peptide series were immobilized under the conditions described in the adhesion test, and after immobilization, the amount of immobilized peptide was measured by a colorimetric assay method.

[0114] This assay is performed using the micro-BCA colorimetric assay kit (PIERCE UPTIMA distributed by Interchim), in which the color intensity is directly proportional to the amount of peptide bonds that have reduced Cu2+ ions to Cu+ ions, in a concentration-dependent reaction. Bicinchonic acid is a copper(I)-specific chromogenic reagent that forms a purple complex with copper(I) having a maximum absorbance at 562 nm, which is directly proportional to the protein concentration. The proteins are assayed in each well using the BCA assay technique: 25 pL of PBS are added to the wells and incubated for 30 minutes at 37°C in the presence of 200 pL of BCA reagent prepared extemporaneously according to the protocol provided in the kit.The OD is then read at 562 nm (Victor2 V Spectrophotometer) and the protein level is determined relative to a standard curve made from a commercial SAB protein standard at 2mg / mL.

[0115] Wound closure test of a confluent mat of primary keratinocytes

[0116] Six-well plates (Costar) are coated with collagen I for 16 h and then rinsed with PBS. Three sterile Ibidi two-well inserts (Ibidi, part number 80209) are placed in each well. 2 x 10⁴ keratinocytes are placed in each insert well in KBM-Gold. After two hours of incubation in humid air at 37°C, composed of 5% CO₂ and 95% atmospheric air, the culture medium is aspirated and the inserts removed. KBM-Gold without supplementation is added to each well of the plate. The cells were prepared in the following ways: untreated (medium alone), treated with KBM-Gold without supplementation containing the peptides of interest (25 pg / ml), or treated with KBM-Gold without supplementation containing EGF at 1 ng / ml (Peprotech). Cultures were stopped at 12 h and 24 h, the media were aspirated, and the cells were fixed with 2% PB S-Paraformaldehyde for 20 minutes.After aspirating the fixative, the cells are stained with a 0.1% Crystal Violet solution in ultrapure water for 30 minutes. The Crystal Violet is removed and the wells are rinsed with water. Images are acquired and lesion quantification is performed using Adobe Photoshop CS3 software.

[0117] Example 2: Adhesion test - Comparison between the peptide TALRIRATYGEY and the peptides LRIRATYG, LRIRATYGLRIRATYG and LRIRATYGSGLRIRATYG

[0118] The adhesion test was performed according to the protocol described above using HT 1080 cells. The TALRIRATYGEY peptide (SEQ ID NO: 17) was used as a control peptide for the peptide test:

[0119] • LRIRATYG (SEQ ID NO: 13) • LRIRATYGLRIRATYG (SEQ ID NO: 18) • LRIRATYGSGLRIRATYG (SEQ ID NO: 16)

[0120] A protein assay was performed to determine the amount of peptide immobilized in each well in order to adjust it if necessary.

[0121] The results are presented in [Fig.1].

[0122] These results show that duplication of the LRIRATYG motif (SEQ ID NO: 13) leads to a doubling of adhesion induction activity at low concentration.

[0123] Example 3: Adhesion test - Comparison between the peptide TALRIRATYGEY, the peptides LRIRATYG, LRIRATYGLRIRATYG, LRIRATYGSGLRIRATYG and the cyclic peptide CSGLRIRATYGSGC.

[0124] The adhesion test was performed according to the protocol described above using HT 1080 cells. The TALRIRATYGEY peptide (SEQ ID NO: 17) was used as a control peptide for the peptide test:

[0125] • LRIRATYG (SEQ ID NO: 13) • LRIRATYGLRIRATYG (SEQ ID NO: 18) • CSGLRIRATYGSGC (SEQ ID NO: 15) • LRIRATYGSGLRIRATYG (SEQ ID NO: 16)

[0126] A protein assay was performed to determine the amount of peptide immobilized in each well in order to adjust it if necessary.

[0127] The results are presented in [Fig.2].

[0128] These results show in particular that the cyclization of the LRIRATYG motif (SEQ ID NO: 13), giving the peptide CSGLRIRATYGSGC (SEQ ID NO: 15) results in the activity being doubled at low concentration.

[0129] Example 4: Wound closure test of a confluent mat of freshly isolated primary keratinocytes, with the peptides TALRIRATYGEY, LRIRATYG, LRIRATYGLRIRATYG, and the cyclic peptide CSGLRIRATYGSGC.

[0130] The wound closure test was performed according to the protocol described above (Example 1) with freshly isolated primary human keratinocytes. Recombinant human Epidermal Growth Factor (EGF) (Peprotech, France) was used as a positive control and the culture medium alone as a negative control.

[0131] The peptides tested are:

[0132] • TALRIRATYGEY (SEQ ID NO: 17) • LRIRATYG (SEQ ID NO: 13) • LRIRATYGLRIRATYG (SEQ ID NO: 18) • CSGLRIRATYGSGC (SEQ ID NO: 15)

[0133] The results are presented in [Fig.3].

[0134] These results show that all the peptides studied significantly promote the Wound closure at 12h and / or 24h post-injury compared to the negative control. The cyclic peptide CSGLRIRATYGSGC (SEQ ID NO: 15) has significantly greater activity than the other peptides and very significantly greater activity than the negative control at both time points tested.

[0135] Example 5: Wound closure test of a confluent mat of freshly isolated primary keratinocytes with the peptides TALRIRATYGEY, LRIRATYG, and the cyclic peptide CSGLRIRATYGSGC.

[0136] The wound closure test was performed according to the protocol described above (Example 1) with freshly isolated primary human keratinocytes. Recombinant human Epidermal Growth Factor (EGF) (Peprotech, France) was used as a positive control and the culture medium alone as a negative control.

[0137] The peptides tested are:

[0138] • TALRIRATYGEY (SEQ ID NO: 17) • LRIRATYG (SEQ ID NO: 13) • CSGLRIRATYGSGC (SEQ ID NO: 15)

[0139] The results are presented in [Fig.4].

[0140] These results show that the peptides studied, in particular the cyclic peptide CSGLRIRATYGSGC, have significantly greater activity at 12 and 24 hours compared to that of the peptides TALRIRATYGEY (SEQ ID NO: 17) and LRIRATYG (SEQ ID NO: 13). This test also shows that the peptides LRIRATYG and TALRIRATYGEY have equivalent activity at 12 and 24 hours, and that the 24-hour activity is significantly greater than that of the negative control. The activity of the cyclic peptide CSGLRIRATYGSGC is significantly greater than that of the negative control.

[0141] Example 6: Adhesion test - Comparison between the peptide TALRIRATYGEY and the peptides LRIRATYG, LRIRATYK, and LRIRKTYG

[0142] The adhesion test was performed according to the protocol described above using HT 1080 cells. The TALRIRATYGEY peptide (SEQ ID NO: 17) was used as a control peptide for the peptide test:

[0143] • LRIRATYG (SEQ ID NO: 13) • LRIRATYK (SEQ ID NO: 9) • LRIRKTYG (SEQ ID NO: 19)

[0144] A protein assay was performed to determine the amount of peptide immobilized in each well in order to adjust it if necessary.

[0145] The results are presented in [Fig.5].

[0146] These results show that the response dependent on the amount of peptide immobilized in the wells decreases when the K is positioned at the end of the sequence (LRIRATYK SEQ ID NO: 9) and disappears when the K is positioned in the middle of sequence (LRIRKTYG SEQ ID NO: 19)).

[0147] Example 7: Adhesion test - Comparison between the peptide TALRIRATYGEY and the peptides LRIRATYG, LRIRFTYK, and LRIRFTYG

[0148] The adhesion test was carried out according to the protocol described above with HT 1080 cells.

[0149] The peptide TALRIRATYGEY (SEQ ID NO: 17) was used as a control peptide for the peptide test:

[0150] • LRIRATYG (SEQ ID NO: 13) • LRIRFTYK (SEQ ID NO: 1) • LRIRFTYG (SEQ ID NO: 5)

[0151] A protein assay was performed to determine the amount of peptide immobilized in each well in order to adjust it if necessary.

[0152] The results are presented in [Fig.6].

[0153] The response dependent on the amount of peptide immobilized in the wells shows that for the peptides LRIRFTYK (SEQ ID NO: 1) and LRIRFTYG (SEQ ID NO: 5) the peptide adhesion capabilities are similar to the TALRIRATYGEY control.

[0154] Example 8: Wound closure test of a confluent mat of freshly isolated primary keratinocytes, with the peptides TALRIRATYGEY, LRIRATYG, LRIRFTYG and LRIRFTYK

[0155] The wound closure test was performed according to the protocol described above (Example 1) with freshly isolated primary human keratinocytes. The TALRIRATYGEY peptide (SEQ ID NO: 17) was used as the control peptide, along with recombinant human Epidermal Growth Factor (EGF) (Peprotech, France) as a positive control and culture medium alone as a negative control.

[0156] The peptides tested are:

[0157] • LRIRATYG (SEQ ID NO: 13) • LRIRFTYK (SEQ ID NO: 1) • LRIRFTYG (SEQ ID NO: 5)

[0158] The results are presented in [Fig.7].

[0159] These results show that the peptides studied have significantly greater activity at 12 and 24 hours, and very significantly greater activity for the LRIRFTYK peptide (SEQ ID NO: 1), compared to the negative control. The LRIRFTYK peptide (SEQ ID NO: 1) has significantly greater activity than the TALRIRATYGEY peptide (SEQ ID NO: 17) at 12 and 24 hours post-injury.

[0160] Example 9: Adhesion test for cyclized peptides fCLRIRFTYKC and CSGL-RIRFTYKSGC compared to LRIRFTYK.

[0161] The peptides CLRIRFTYKC (SEQ ID NO: 2) and CSGLRIRFTYKSGC (SEQ ID NO: 3) are cyclized versions of the peptide LRIRFTYK (SEQ ID NO: 1). The test Adhesion testing was carried out according to the protocol described above (example 1) with HT1080 cells.

[0162] The three peptides were immobilized. Assays were performed in the wells to determine the amount immobilized and to ensure that the same amount of peptide was present under the different conditions, and to adjust it if necessary.

[0163] The results are presented in [Fig.8].

[0164] The response dependent on the amount of peptide immobilized in the wells shows that the three peptides have equivalent adhesion activity. Thus, the cyclic peptide without spacers (without SG) is as effective as the cyclic peptide with spacers (SG).

[0165] Example 10: Cell viability assay on freshly isolated primary keratinocytes for the peptides TALRIRATYGEY, LRIRATYG, LRIRFTYK and CL-RIRFTYKC:

[0166] The cell viability test was performed according to the protocol described above (Example 1) with freshly isolated primary keratinocytes. The TALRIRATYGEY peptide (SEQ ID NO: 17) was used as the control peptide.

[0167] The peptides tested are:

[0168] • LRIRATYG (SEQ ID NO: 13) • LRIRFTYK (SEQ ID NO: 1) • CLRIRFTYKC (SEQ ID NO: 2)

[0169] The results are presented in [Fig.9].

[0170] The results, presented in [Fig.9], show similar positive results for the peptides LRIRATYG (SEQ ID NO: 13), LRIRFTYK (SEQ ID NO: 1)) and CLRIRFTYKC (SEQ ID NO: 2) to those of the peptide TALRIRATYGEY (SEQ ID NO: 17)

[0171] Example 11: Wound closure test of a confluent mat of freshly isolated primary keratinocytes for the peptides TALRIRATYGEY, LRIRATYG, LRIRFTYK, CSGLRIRFTYKSGC, and CLRIRFTYKC

[0172] The wound closure test was performed according to the protocol described above (Example 1) with freshly isolated primary keratinocytes. The TALRIRATYGEY peptide (SEQ ID NO: 17) was used as the control peptide, recombinant human Epidermal Growth Factor (EGF) (Peprotech, France) as the positive control, and culture medium alone as the negative control.

[0173] The peptides tested are:

[0174] • LRIRATYG (SEQ ID NO: 13) • LRIRFTYK (SEQ ID NO: 1) • CLRIRFTYKC (SEQ ID NO: 2) • CSGLRIRFTYKSGC (SEQ ID NO: 3)

[0175] The results are presented in [Fig.10].

[0176] The results show significantly higher activity than TAL- peptides RIRATYGEY (SEQ ID NO: 17) and LRIRATYG (SEQ ID NO: 13) for all peptides studied at 12 and 24h. The peptides with the highest activities are, in increasing order, LRIRFTYK (SEQ ID NO: 1), CSGL-RIRFTYKSGC (SEQ ID NO: 3) and CLRIRFTYKC (SEQ ID NO: 2).

[0177] Example 8: Examples of compositions comprising the peptides according to the invention,

[0178] Composition No. 1: W / O (water in oil) emulsion Ingredients % AQUA (WATER) QS 100 HEXYL LAURATE 37,000 GLYCERIN 5,000 METHYL GLUCOSE ISOSTEARATE 3,500 DIMETHICONE 3,000 DISTEARDIMONIUM HECTORITE 3,000 EUPHORBIA CERIFERA (CANDELILLA) WAX 1,500 PEG-45 / DODECYL GLYCOL COPOLYMER 1.000 SODIUM SALICYLATE 0.580 SODIUM BENZOATE 0.480 MAGNESIUM SULFATE 0.100 DISODIUM EDTA 0.100 LRIRFTYK PEPTIDE (SEQ ID N: 1) 0.0001

[0179] Composition No. 2: O / W (oil-in-water) emulsion Ingrédients % AQUA (WATER) QS 100 CYCLOMETHICONE 4.000 GLYCERIN 3.000 HYDROGENATED POLYISOBUTENE 3.000 PPG-15 STEARYL ETHER 2.000 ETHYLHEXYL PALMITATE 2.000 STEARETH-2 2.000 STEARETH-21 2.000 STEARIC ACID 1.750 CETYL ALCOHOL 1.500 SODIUM SALICYLATE 0.600 POLYACRLAMIDE, C13-14 ISO- PARAFFIN, LAURETH-7 0.400 XANTHAN GUM 0.300 SODIUM BENZOATE 0.200 PEPTIDE CLRIRFTYKC (SEQ ID N : 2) 0.001

[0180] Composition N°3 : lotion Ingrédients % AQUA (WATER) QS 100 GLYCERIN 5.000 SODIUM BENZOATE 0.700 SODIUM SALICYLATE 0.550 BIS-PEG-18 METHYL ETHER DIMETHYL SILANE 0.500 TETRASODIUM EDTA 0.200 PEPTIDE CSGLRIRFTYKSGC (SEQ ID N: 3) 0.003

[0181] Composition N°4 : Gel Ingrédients % AQUA (WATER) QS 100 CYCLOMETHICONE 4.000 GLYCERIN 3.000 SODIUM SALICYLATE 0.600 POLYACRLAMIDE, C13-14 ISO- PARAFFIN, LAURETH-7 0.500 CARBOMER 0.500 SODIUM BENZOATE 0.200 PEPTIDE LRIRFTYKSGLRIRFTYK (SEQ ID N : 4) 0.003

Claims

Claims

1. Peptide consisting of the sequence: LRIRXiTYX2, where: • X1 is chosen from amino acids A or F; and • X2 is chosen from amino acids G or K.

2. Peptide according to the preceding claim, characterized in that it has the following sequence: LRIRFTYK (SEQ ID NO: 1)

3. Peptide consisting of the sequence: CLRIRXiTYX2C, where: • X1 is chosen from amino acids A or F; and • X2 is chosen from amino acids G or K.

4. Peptide according to the preceding claim, characterized in that it has the following sequence: CLRIRFTYKC (SEQ ID NO: 2)

5. Peptide consisting of the sequence: CSGLRIRXiTYX2SGC, where: • X1 is chosen from amino acids A or F; and • X2 is chosen from amino acids G or K.

6. Peptide according to the preceding claim, characterized in that it has the following sequence: CSGLRIRFTYKSGC (SEQ ID NO: 3).

7. A peptide according to any one of claims 1 to 6 for use as a medicament.

8. Peptide according to the preceding claim, for use as a medicament in: • strengthening the dermo-epidermal junction, cell-cell adhesion and / or cell-matrix adhesion at the level of the epidermis; and / or • treating alterations of the skin induced by dermatological pathologies; and / or • treating alterations of the skin weakened by cosmetic or therapeutic treatments; and / or • curative or preventive treatment of skin aging such as wrinkles, sagging, loss of elasticity, poorer healing, senile xerosis, changes in the pigmentary system of the skin, depletion of the vascular network of the skin, irregularity of the skin texture, skin atrophy and alteration of the appendages.

9. Non-therapeutic cosmetic composition characterized in that it comprises at least one peptide according to any one of claims 1 to 6.

10. Cosmetic composition according to the preceding claim, characterized in that it comprises from 0.00002% to 5%, preferably from 0.00005% to 0.1% and more preferably still from 0.0001% to 0.001% by weight of peptide and at least one cosmetically acceptable excipient.

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