HK1187063B - New dermatopontin-activating peptides and cosmetic compositions including same - Google Patents

Description

Novel dermatopontin-activating peptide and cosmetic composition comprising the same

Technical Field

The present application relates to the field of cosmetic and dermopharmaceutically active ingredients and compositions comprising them.

The present application aims to provide novel molecules useful for preventing or combating signs of aging (signs) of the skin and in particular wrinkles, sagging and loss of skin volume and elasticity.

More specifically, the invention relates to peptides activating the expression of dermopontines (dermatoponins), cosmetic or pharmaceutical compositions comprising such peptides, the use of such compositions for increasing the protein expression of the extracellular matrix and for preventing the UV degradation of collagen and elastic fibres, and finally to cosmetic care methods aimed at preventing and/or treating the signs of aging and photoaging of the skin.

Background

The skin is a covered organ composed of multiple layers (dermis, dermoepidermal junction, epidermis). The dermis is the tissue that supports the skin and is composed of water, elastic fibers and collagen fibers (70% of the dermal fibers) encapsulated in an interstitial matrix of proteoglycans. Fibroblasts are the main cellular component of the dermis and are the source of collagen and elastic fiber synthesis.

The outermost part is the epidermis, a multi-layered epithelium consisting essentially of keratinocytes closely connected to each other. The basal layer of the epidermis consists of a layer of proliferating cells (mainly keratinocytes and melanocytes) which are fixed to the dermal-epidermal junction (DEJ). DEJ is an extracellular network structure that makes up the gap between the dermis and epidermis.

Like all other organs, skin is subject to a complex physiological aging process. Intrinsic or chronological aging as a result of genetically programmed aging is distinct from biochemical changes due to endogenous factors. In the skin, it is characterized by a slow regeneration of cells and extracellular matrix, which leads to atrophy, dryness, reduction of elasticity and appearance of fine lines and wrinkles of the dermis and epidermis.

Extrinsic aging is due to environmental stresses experienced by the body during life, such as pollution, sunlight, disease, lifestyle habits, and the like. Their effects are combined with those of intrinsic aging in areas exposed to sunlight for long periods; this is called photoaging. The major changes associated with photoaging are located at the dermis and include: the appearance of pigmented spots, the reduction and rupture of collagen fibers that cause wrinkles, and the accumulation of dystrophic elastic fibers that constitute solar elastosis.

Various research approaches have been developed to identify active agents capable of combating skin aging, among which protection against environmental stress (sunlight, pollution, etc.), activation of cell regeneration and enhancement of extracellular collagen and elastomeric matrices. This research has led to the market release of a variety of more or less effective active agents. Therefore, it remains important to identify new compounds capable of preventing or combating skin aging. The problem more specifically addressed by the present invention is to combat the tissue destruction of the fibrillar structure of the extracellular matrix of the skin during aging or photoaging.

The inventors have recently identified beneficial molecular goals for achieving this function. This target is cutaneous desmin, a small acidic protein, rich in tyrosine, poor in the dermis and more particularly surrounding collagen fibers. It plays a key role in the structuring of collagen fibrils (jonathanet al, j. biol. chem.,1993, vol.268, 19826-. These properties confer an important role for cutaneous desmin healing (akamotoetal, Biochem,2010, vol.49, p. 147-.

These results were confirmed, and the absence of skin bridge in the genetically modified mice resulted in a reduction in dermal thickness and dermal collagen content, as well as a reduction in skin elasticity (takedaet al j. invest. dermatol.2002, vol.119, p 678-683).

Independent of its structural role, dermatopontin can bind decorin and transforming growth factor beta (TGF β) to form a trimeric complex and thus potentiate the effect of transforming growth factor β 1 (Okamotoetal, biochem J.1999Feb1;337(3): 537-41).

Document JP2008201777 discloses the use of peptides derived from dermatopontin as agents promoting cell adhesion and healing.

Document US20050065089 discloses that natural cutaneous desmin can be used to enhance the activation of transforming growth factor β in the context of biological treatment of disc herniation.

However, until now, no peptide according to the invention has been described for activating cutaneous desmin in skin cells and preventing or repairing the signs of skin aging and photoaging.

Disclosure of Invention

The inventors have verified peptides having the following general formula (I):

R₁-(AA)_n-X₁-Arg-X₂-Trp-X₃-X₄-X₅-X₆(R₃)-(AA)_p-R₂

is a good activator of dermatopontin. Therefore, these peptides are useful against skin aging and photo aging.

The peptides according to the invention are characterized by the fact that:

-activating dermatopontin expression;

-increasing the expression of collagen types I and III and fibronectin;

-preventing degradation of the fibrillar structure of collagen and elastic fibers in skin subjected to UV radiation.

The term "activating cutaneous desmins or peptides or active agents capable of activating cutaneous desmins" refers to any peptide having the general formula (I) which increases the amount of cutaneous desmins present in a cell either by increasing protein synthesis through direct or indirect regulation of gene expression or by other biological methods such as stabilization of proteins or stabilization of messenger RNA transcription.

The term "skin" means all the overlying tissues that form the skin, mucous membranes and skin appendages, including hair, eyelashes and eyebrows.

Accordingly, a first object of the present invention is to provide peptides having the general formula (I)

R₁-(AA)_n-X₁-Arg-X₂-Trp-X₃-X₄-X₅-X₆(R₃)-(AA)_p-R₂

Wherein

X₁Means aspartic acid or an amino acid free,

X₂represents a compound of the formula glutamine or glutamic acid,

X₃denotes asparagine or lysine or glutamine or no amino acid,

X₄represents phenylalanine or tyrosine or no amino acid,

X₅represents tyrosine or alanine or no amino acid,

X₆denotes a cysteine or the absence of an amino acid,

AA represents any amino acid and n and p are integers between 0 and 2;

R₁representing substituted or unsubstituted by radicals of acyl type (R-CO-)Primary amine function of the N-terminal amino acid, in which the R group is a saturated or unsaturated C of the acetyl type₁-C₃₀Alkyl chains or aromatic groups of the benzoyl, tosyl or benzyloxycarbonyl type;

R₂represents having an unsubstituted hydroxyl group or having a substituent selected from C₁-C₃₀Radicals of alkyl chains or NH₂a-NHY or-NYY 'group (Y and Y' represent C₁-C₄Alkyl chain) a carboxyl function of the C-terminal amino acid of the substituted hydroxyl group;

R₃is shown at X₆A thiol functional group of a cysteine at a position, which thiol group is unsubstituted or substituted by a methyl or acetyl group, or covalently bound to another cysteine through a disulfide bond.

The sequence having the general formula (I) consists of 3 to 12 amino acid residues and may be in the form of a salt.

According to an advantageous embodiment of the invention, the peptides preferably correspond to the general formula (I)

R₁-(AA)_n-X₁-Arg-X₂-Trp-X₃-X₄-X₅-X₆(R₃)-(AA)_p-R₂

Wherein

X₁Means aspartic acid or an amino acid free,

X₂represents a compound of the formula glutamine or glutamic acid,

X₃denotes asparagine or lysine or glutamine or no amino acid,

X₄represents phenylalanine or tyrosine or no amino acid,

X₅represents tyrosine or alanine or no amino acid,

X₆denotes a cysteine or the absence of an amino acid,

AA represents any amino acid other than arginine, cysteine, leucine, glycine and glutamic acid; and n and p =0 or 1, wherein n is not equal to p.

R₁Represents a primary amine function (primaryamine function) with an N-terminal amino acid, unsubstituted or substituted by a radical (R-CO-) of the acyl type, in which the radical R is a saturated or unsaturated C of the acetyl type₁-C₃₀Alkyl chains or aromatic groups of the benzoyl, tosyl or benzyloxycarbonyl type;

R₂represents having an unsubstituted hydroxyl group or having a substituent selected from C₁-C₃₀Radicals of alkyl chains or NH₂a-NHY or-NYY 'group (Y and Y' represent C₁-C₄Alkyl chain) substituted hydroxyl group.

R₃Is shown at X₆A thiol functional group of a cysteine at a position, which thiol group is unsubstituted or substituted by a methyl or acetyl group, or covalently bound to another cysteine through a disulfide bond. The sequence having the general formula (I) consists of 3 to 7 amino acid residues and may be in the form of a salt.

According to a particularly preferred embodiment of the invention, the peptide has the following sequence:

(SEQIDNo1):Asp-Arg-Gln-Trp-NH₂

(SEQIDNo2):Asp-Arg-Glu-Trp-NH₂

(SEQIDNo3):Asp-Arg-Gln-Trp-Asn-Tyr-NH₂

(SEQIDNo4):Arg-Glu-Trp-Gln-Phe-Tyr-Cys-NH₂

(SEQIDNo5):Arg-Glu-Trp-Gln-Phe-Tyr-Cys(Cys)-(NH₂)

(SEQIDNo6):Asp-Arg-Glu-Trp-Gln-Phe-NH₂

(SEQIDNo7):Asp-Arg-Gln-Trp-Asn-Tyr-Ala-Cys-NH₂

(SEQIDNo8):Asp-Arg-Gln-Trp-Asn-Tyr-Ala-Cys(Cys)-(NH₂)

(SEQIDNo9):Asp-Arg-Glu-Trp-Gln-Phe-Tyr-Cys-NH₂

(SEQIDNo10):Asp-Arg-Glu-Trp-Gln-Phe-Tyr-Cys(Cys)-(NH₂)

(SEQIDNo11):Asp-Arg-Gln-Trp-Lys-Phe-NH2

(SEQIDNo12):Arg-Glu-Trp-Gln-Phe-Tyr-NH2

(SEQIDNo13):Arg-Glu-Trp-Gln-Phe-Tyr.

according to a particularly advantageous embodiment, the peptide corresponds to the sequence seq id no 5.

According to another particularly advantageous embodiment, the peptide corresponds to the sequence of seq id no9 or to the sequence of seq id no 10.

According to another even more advantageous embodiment, the peptide corresponds to the sequence seq id no12 or to the sequence seq id no 13.

The amino acids constituting the peptide according to the invention and indicated by the term AA may be in any isomeric L-and D-configuration. Preferably, the amino acid is in the L form.

The term "peptide" means a chain of two or more amino acids bound to each other by peptide bonds.

The term "peptide" also means at least part of a natural or synthetic peptide of the invention, as described above, or a fragment thereof, whether it is obtained by proteolysis or by synthesis, or any natural or synthetic peptide whose sequence constitutes, in whole or in part, the sequence of the aforementioned peptide.

In order to improve the resistance to degradation, it may be necessary to use protected forms of the peptides according to the invention. Preferably, to protect the primary amine function of the N-terminal amino acid, R of the acyl type (R-CO-) is used₁Substituted by radicals in which the R radical is a saturated or unsaturated C of the acetyl type₁-C₃₀Alkyl chains or aromatic groups of the benzoyl, tosyl or benzyloxycarbonyl type, even more preferably acetyl groups. Preferably, to protect the carboxyl function of the C-terminal amino acid, C is used₁-C₃₀R of alkyl chain type₂Radicals or NH₂a-NHY or-NYY 'group (Y and Y' represent C₁-C₄Alkyl chain), even more preferably NH₂And (4) substituting the group.

The peptide according to the invention may be protected at the N-or C-terminus or both.

To inhibit dimerization of the peptides according to the invention, the thiol function of the C-terminal cysteine may be substituted by a methyl or acetyl group or another cysteine. In the last such case, the substitution results in the formation of a disulfide bond between the two cysteine residues.

The present invention therefore relates to a composition as defined above, characterized in that: the peptides according to the invention and advantageously having the sequences SEQ ID no1 to SEQ ID no13 are or are not in a protected form, preferably in a protected form at the C-terminus.

The peptides of general formula (I) according to the invention can be obtained by classical chemical synthesis (in solid phase or liquid homogeneous phase) or by biochemical enzymatic synthesis from constitutive amino acids (kullmanet. j. biol. chem.,1980, vol.225, page 8234).

The peptides according to the invention may be of natural or synthetic origin. Preferably, according to the invention, the peptide is of synthetic origin obtained by chemical synthesis.

According to the invention, the active agent may be a single peptide or a mixture of peptides.

The peptides according to the invention are advantageously dissolved in one or more physiologically suitable solvents, such as water, glycerol, ethanol, propylene glycol, butylene glycol, dipropylene glycol, ethoxylated or propoxylated diethylene glycol, cyclic polyols or any mixtures of these solvents.

The term "physiologically suitable" means that the selected solvent is suitable for use in contact with the skin without causing toxic or intolerant reactions.

The diluted peptide was then sterilized by sterile filtration.

After this dilution step, the peptides may be encapsulated or contained in cosmetic or pharmaceutical carriers such as liposomes or any other microcapsules used in the cosmetic field, or absorbed onto powdered organic polymers, mineral carriers such as talc and bentonite, and more generally dissolved or bound to any physiologically suitable carrier.

According to a second subject of the invention, the peptides having general formula (I) are useful as medicaments.

Particularly advantageously, the medicaments of the general formula (I) can be used as healing agents.

It is another object of the present invention to provide a pharmaceutical healing composition comprising a peptide according to the present invention in a physiologically suitable medium.

According to a particular aspect of the invention, said peptides are useful for treating dermatological symptoms associated with aging or premature photoaging, in particular delayed healing, relaxation and skin atrophy, solar elastosis and reduced dermoepidermal adhesions.

Advantageously, according to this embodiment of the invention, the pharmaceutical composition will be suitable for topical use or may be in a suitable liquid form for injection under or in the skin, in particular for compound epidermal local meso-therapeutic (type) injection.

A third object of the present invention is to provide a cosmetic composition comprising a peptide of general formula (I) as a skin desmin activator in a physiologically suitable medium.

According to an advantageous embodiment of the invention, the active agent according to the invention is present in a quantity of about 10% relative to the total weight of the final composition^-9M and 10^-3Concentration between M, and preferablyAt 10^-8M and 10^-5Concentration between M, and even more preferably at 5.10^-5M and 5.10^-6Concentrations between M are present in the compositions of the present invention.

Concentrations within this range represent the necessary amount of active agent to obtain the desired molecular effect, i.e., to activate the expression of dermatopontin, type I and III collagen, and fibronectin.

Preferably, the composition according to the invention is in a form suitable for topical application, comprising a physiologically compatible medium for application to the skin. By "physiologically acceptable" is meant suitable for use in contact with the skin or human skin appendages without risk of toxicity, incompatibility, instability, allergic response, or other adverse effect.

By "topical application" is meant that the active agent according to the present invention or a composition comprising the same is applied or coated on the skin surface.

These compositions may in particular be in the form of: water, hydroalcoholic or oily solutions; an oil-in-water or water-in-oil emulsion or a multiple emulsion; water or non-hydrogel; and (3) colloid. These compositions may also be in the form of creams, suspensions, or powders suitable for application to the skin, mucous membranes, lips, and/or skin appendages. These compositions may be more or less fluid and have the appearance of a cream, lotion, milk, serum, wax, cream, paste or foam. They may also be in solid form, such as a stick, or they may be applied to the skin in aerosol form. They can also be used as care products and/or cosmetic products for the skin.

Furthermore, all these compositions comprise any additives commonly used in the envisaged field of use, as well as adjuvants necessary for their formulation, such as co-solvents (ethanol, glycerol, benzyl alcohol, humectants, etc.), thickeners, diluents, emulsifiers, antioxidants, colorants, opacifiers, pigments, fillers, preservatives, perfumes, odor absorbers, essential oils, trace elements, essential fatty acids, surfactants, film-forming polymers, chemical or mineral filtration media, hydrating or hot water, etc. For example, water-soluble polymers of the natural type, such as polysaccharides, or polypeptides, cellulose derivatives of the methylcellulose or hydroxypropylcellulose type, or synthetic polymers, poloxamers, carbomers, silicones, PVA or PVP and in particular the polymers sold by the ISP company may be cited.

In any case, the person skilled in the art will ensure that: these adjuvants and their proportions are chosen so as not to counteract the advantageous properties sought in the composition according to the invention. These adjuvants may be present, for example, in concentrations of 0.01 to 20% based on the total weight of the composition. When the composition of the invention is an emulsion, the fatty phase may represent 5 to 80% by weight and preferably 5 to 50% by weight, relative to the total weight of the composition. The emulsifiers and co-emulsifiers used in the composition will be chosen from those conventionally used in the field under consideration. For example, they may be used in a proportion of 0.3 to 30% by weight relative to the total weight of the composition.

Of course, the active agents according to the present invention may be used alone or in combination with other active agent compositions.

Advantageously, the compositions that can be used according to the invention also comprise at least one other active agent intended to enhance the action of the active agent according to the invention in the field of preventing and improving the signs of skin ageing, or another active agent that makes it possible to extend the range of properties of the compositions considered.

The following categories of ingredients may be cited in a non-limiting manner: rejuvenating agents, anti-ageing agents, anti-wrinkle agents, thickening agents, anti-radical agents, anti-glycation agents, hydrating agents, antibacterial agents, antifungal agents, keratolytic agents, muscle relaxants, exfoliating agents and toning agents, agents stimulating synthesis or energy metabolism of dermal macromolecules, agents regulating skin differentiation, pigmentation or depigmentation, agents stimulating nail or hair growth, agents stimulating microcirculation, sunscreens or metalloproteinase inhibitors.

In a particular embodiment of the invention, the composition according to the invention will comprise, in addition to the peptide according to the invention:

at least one cytochrome c activating compound, and/or

At least one hydration compound, such as an aquaporin activating compound, and/or

-at least one sirtuin activating compound, and/or

At least one compound that increases cell adhesion, and/or

At least one compound that increases the production of matrix proteins such as collagen, fibronectin, laminin, glycosaminoglycans, and/or

At least one proteasome activity regulating compound, and/or

At least one circadian rhythm regulator compound, and/or

At least one HSP protein-modulating compound, and/or

At least one compound which increases the energy of the cells, and/or

At least one skin pigmentation-regulating compound, and/or

At least one coenzyme Q10 activating compound, and/or

At least one compound improving barrier function, such as a transglutaminase or an HMG-CoA reductase activating compound, and/or

-at least one mitochondrial protecting compound.

The above-mentioned compounds may be peptide hydrolysates of natural origin, such as plants, or of synthetic origin, such as peptides.

Independent of their function, the other active agents combined with the active agent according to the invention may have very different chemical structures. Peptides, vitamin C and its derivatives, vitamins of group B, DHEA (dihydroepiandrosterone), phytosterols, salicylic acid and its derivatives, retinoids, flavonoids, sugar amines, azoles, metal salts, peptide extracts of plant origin or polymers may be cited in a non-limiting manner.

A fourth object of the present invention relates to the following uses of a cosmetic composition comprising as active agent a peptide having general formula (I): enhancing the dermoepidermal junction, stimulating expression of components of the dermoepidermal junction, increasing densification of the region supporting the dermoepidermal junction, or even improving anchoring of the basal keratinocytes and/or melanocytes to the dermoepidermal junction.

A fifth object of the invention relates to the use of a cosmetic composition comprising, as an active agent, a peptide having general formula (I), in order to increase the protein expression of the extracellular matrix by fibroblasts of the skin.

According to an advantageous aspect of the invention, the active agent makes it possible to increase the density and elasticity of the dermis and, therefore, the firmness of the skin, preventing or resisting sagging of the facial lines, loss of volume, thinning of the skin, loss of tension, fine lines, deep wrinkles and atrophy of the dermis.

A sixth object of the present invention relates to the use of a cosmetic composition comprising as active agent a peptide having general formula (I), in order to prevent the degradation of collagen and elastic fibers in the skin subjected to external pressure.

The term "external pressure" means the pressure that can be generated by the environment. For example, pressure such as contamination, UV radiation or stimulation may be cited. Contamination means both "external" contamination by, for example, diesel particles, ozone or heavy metals and "internal contamination" which can be caused in particular by emissions from coating solvents, glues or wallpaper (for example toluene, styrene, xylene or benzaldehyde) or cigarette smoke.

It has been verified that active agents enable to limit the degradation and tissue destruction of elastic fibers and to stimulate the reorganization (reorganization) of collagen fibers after UV radiation, and more particularly UVA radiation.

"shuffling of elastic fibers" means the accumulation of dystrophic elastic fibers typical of solar elastosis and all modifications due to repeated exposure to sunlight. These changes, which are difficult to reverse and partly responsible for the age-related sagging or sagging of the skin, are mainly caused by UVA radiation penetrating deeply through the dermis.

A particular embodiment of the invention relates to the following uses of a cosmetic composition comprising a peptide according to the invention: preventing or resisting non-aesthetic signs associated with solar elastosis and/or tissue disruption (disorganization) of the elastic fiber caused by exposure to UV radiation, and in particular UVA radiation.

A seventh object of the present invention relates to the use of a cosmetic composition comprising as active agent a peptide having general formula (I) for increasing the regeneration of the epidermis and dermis.

The phrase "regeneration of the epidermis and dermis" means: according to this advantageous aspect of the invention, the peptides of the invention cause a greater proliferation and migration of keratinocytes and fibroblasts, thus promoting an accelerated renewal of the epidermis and a more comprehensive and better regeneration of the skin.

An eighth object of the present invention relates to a method for cosmetic care intended to prevent and/or treat the signs of skin aging and photoaging, characterized in that: the composition according to the invention is topically applied to the skin to be treated.

The phrase "signs of aging of the skin" means all modifications of the external appearance of the skin and skin appendages caused by aging, such as skin thinning, sagging, loss of elasticity and tension, deep lines and wrinkles, loss of firmness and tone, dermal atrophy or any other internal degradation of the skin caused by exposure to UV radiation.

In particular, the invention relates to a method for cosmetic treatment intended to protect the skin from the pressure caused by UV radiation.

In one embodiment, the composition is applied prior to exposure to sunlight as a pre-solar care treatment to prevent tissue damage of the elastic fibers.

In a second embodiment of the invention, the composition is applied after sun exposure as a post-sun care treatment to repair damage to which collagen and elastic fibers are subjected.

Other advantages and features of the present invention will become clearer in view of the following examples, provided for illustrative and non-limiting purposes.

Example 1: verifying that the peptides of SEQ ID No5 and SEQ ID No9 express dermatopontin Activating effect

The objective of this study was to determine the effect of the peptide of seq id no5 and the peptide of seq id no9 on the expression of dermatopontin in fibroblasts. For this purpose, specific immunofluorescent labeling was performed on normal human fibroblast cultures and on skin preserved in vitro cultures.

The implementation scheme is as follows:culturing normal human fibroblasts for 24 or 48 hours and using the peptide SEQ ID NO5 or 10 of the peptide SEQ ID NO9^-6M solution was treated once daily. The cells were then washed and fixed with 3.7% formaldehyde for 10 minutes at ambient temperature.

Human skin samples were placed in culture at the air/liquid interface. Topical administration of peptide SEQ ID NO5 or 10 of peptide SEQ ID NO9^-6M solution, and then incubated with the sample for 24 hours or 48 hours. These skin samples were subsequently fixed with formaldehyde and then either embedded in paraffin or fixed with OCT by freezing at-20 ℃. Then, 3-4 μm sections (6 μm for cold sections) were prepared. Immunolabeling of the samples embedded in paraffin was performed after unmasked specific sites. Immunolabeling of samples embedded in OCT was performed after fixation at 37 ℃ and with cold acetone.

The fixed cells or sections are incubated in the presence of a specific rabbit polyclonal antibody to the skin desmin (Proteinterchgroup, Ref:10537-1-AP) and then replaced with a second antibody conjugated to a fluorescent label. After the cells were mounted in the adhoc medium, the cells were then examined with an Epi-fluoroscene microscope (nikon eclipse 600 microscope).

As a result:under all conditions tested, at a temperature of from 10^-6More intense fluorescence was observed in fibroblasts treated with the peptide of seq id no5 or seq id no9 under M than under control conditions.

Human skin showed more intense fluorescence at the dermis in samples treated with the peptide of seq id no5 or the peptide of seq id no 9.

And (4) conclusion:the peptide of seq id no5 and the peptide of seq id no9 stimulated very significantly the expression of dermatopontin by fibroblasts of the dermis.

Example 2: study of ultrastructure of skin cells treated with the peptide SEQ ID No5

The objective of this study was to observe the effects of the drug delivery system by measuring the flux at 10^-6Subcellular structures of keratinocytes and fibroblasts treated with the peptide of seq id no5 under M.

The experimental scheme is as follows:culturing NHEK or fibroblasts in dishes or in the Transwell system, or 10 using the peptide of SEQ ID No5^-6M solution treatment of in vitro cultured skin for 48 hours (medium change every 24 hours). Skin cells or samples were washed with PBS and then fixed by a Karnosky high tension fixation device (4% paraformaldehyde, 5% glutaraldehyde in 0.08M phosphate buffer) at ambient temperature for 1 hour, then at 4 ℃ for 24 hours. The cells were separated from the substrate by scraping by centrifugation at 1000rpm for 5 minutes at 4 ℃. The supernatant was removed and 0.1M sodium cacodylate buffer was deposited on round particles (pellet). The cells were mixed with 2% agar and then fixed thereafter for 1 hour by osmium tetroxide. The sample was subsequently dehydrated by successive passes (50-100%) through the alcohol train. Then coating the skin cells with a resinOr a sample. The polymerization was carried out at 60 ℃ for about 12 hours. A0.5 μm semi-thin section was taken with a microtome. The sections were deposited on a bottom (sub) slide under heat and then stained with toluidine blue. The slide is then dehydrated again and mounted in a suitable medium. After selecting the best study area, the block size is adjusted to the desired size and ultra-fine slices are prepared. Only sections with "silver-gray" color and appropriate size were mounted in an electron microscope grid double labeled with uranyl acetate and lead citrate and examined with a transmission microscope at 60 or 80 KV.

As a result:ultrastructural studies showed an enrichment of organelles, and in particular mitochondria, in keratinocytes treated with the peptide of seq id no 5. In thatIn cultures in the system, intercellular contact between keratinocytes appears more adhesive and refers to appears to stain more strongly than untreated controls. Similarly, in vitro skin cultures, a narrower cell contact between the cells of the basal layer was observed.

In fibroblasts in culture treated with the peptide of seq id no5, significant staining of the body of the fossa (caveolar), rough endoplasmic reticulum and golgi apparatus was observed more than in control cells. A stronger secretion of the components of the extracellular matrix was also observed compared to the untreated control.

And (4) conclusion:in keratinocytes in culture, at 10^-6The peptide of seq id no5 under M causes a general stimulation of cellular activity, in particular with an increase in mitochondrial density.

In fibroblasts, the peptide of seq id no5 induces an increase in the synthesis of components of the extracellular matrix.

In vitro skin cultures, the peptide of seq id no5 increased intercellular contact of basal keratinocytes.

Example 3: verification of the activating Effect of the peptide of SEQ ID No5 on fibronectin expression

The objective of this study was to determine the effect of the peptide of seq id no5 on the expression of fibronectin, an extracellular matrix protein synthesized by fibroblasts. For this purpose, specific immunofluorescent labeling was performed on fibroblast cultures and on in vitro skin cultures.

The implementation scheme is as follows:10 with the peptide of SEQ ID No5^-6The M solution treats human dermal fibroblasts in culture once a day (replacement of medium containing active agent every 24 hours). The cells were then washed and fixed with 3.7% formaldehyde for 10 minutes at ambient temperature.

Human skin samples were placed in culture at the air/liquid interface. Topical application 10^-6M solution of the peptide SEQ ID No5 or SEQ ID No9, and then incubated for 24 hours or 48 hours. These skin samples were subsequently fixed with formaldehyde and then either embedded in paraffin or fixed with OCT by freezing at-20 ℃. Sections of 3-4 μm (6 μm for cold sections) were then prepared for immunolabeling of samples embedded in paraffin after unmasked specific locations. Immunolabeling of samples embedded in OCT was performed after fixation at 37 ℃ and with cold acetone.

The fixed cells or sections were incubated in the presence of a specific rabbit fibronectin polyclonal antibody (Sigma, Ref: F-3648) and then in the presence of a second antibody linked to a fluorescent marker. The cells were then examined with an Epi-fluoroscene microscope (Nikon eclipse 600 microscope).

As a result:is used in 10^-6More intense cytoplasmic fluorescence was observed in the peptide seq id no5 treated fibroblasts under M than in the control cells.

In extracorporeal skin, fluorescence is localized primarily in the upper part of the papillary dermis, just below the dermoepidermal junction.

And (4) conclusion:the peptide of seq id no5 stimulated very significantly the expression of fibronectin in fibroblasts.

In human skin, the peptide of seq id no5 stimulates densification of the region supporting the dermoepidermal junction.

Example 4: verification of the Components of the collagen I and III dermal extracellular matrices by the peptide SEQ ID No5 Activation effect of seed expression

The aim of this study was to determine the effect of the peptide of seq id no5 on the molecular expression of the dermal extracellular matrix. For this purpose, the expression of collagen I and III in human fibroblasts obtained from the dermis was investigated.

The implementation scheme is as follows:culturing normal human fibroblasts for 24 or 48 hours and using the peptide of SEQ ID NO5 or 10 of SEQ ID NO9^-6M solution was treated once daily. The cells were then washed and fixed with 3.7% formaldehyde for 10 minutes at ambient temperature.

Human skin samples were placed in culture at the air/liquid interface. Topical administration of the peptide of SEQ ID No5 or 10 of the peptide of SEQ ID No9^-6M solution, and then incubated with the sample for 24 hours or 48 hours. These skin samples were subsequently fixed with formaldehyde and then either embedded in paraffin or fixed with OCT by freezing at-20 ℃. Then, 3-4 μm sections (6 μm for cold sections) were prepared. Immunolabeling of the samples embedded in paraffin was performed after unmasked specific sites. Immunolabeling of samples embedded in OCT was performed after fixation at 37 ℃ and with cold acetone.

The fixed cells or sections were incubated in the presence of a specific rabbit collagen I polyclonal antibody (Rockland, Ref:600-401-103) or a specific rabbit collagen III polyclonal antibody (Rockland, Ref:600-401-105) and then replaced with a second antibody coupled to a fluorescent label. After the cells were mounted in adhoc medium, the cells were then examined with an Epi-fluoroscope microscope (nikon eclipse 600 microscope).

As a result:is used in 10^-6More intense fluorescence was observed in the M and skin sections of the culture and skin treated with the peptide seq id no5 than under the control conditions.

And (4) conclusion:at 10^-6The peptide seq id no5 under M increased the expression of collagen I and collagen III (two basic fibrillar proteins of the dermal extracellular matrix).

Example 5: verification of the protective Effect of the peptide SEQIDNo5 on elastic fibers subjected to UV radiation Fruit

The aim of this study was to determine the effect of the peptide of seq id no5 on the elastic fibers present in the dermis. To accomplish this, specific staining of elastic fibers was performed on skin samples cultured in vitro.

The implementation scheme is as follows:human skin samples were placed in culture at the air/liquid interface. Topical administration of peptide SEQ ID No 510^-6M solution, then subjecting the sample to 5J/cm²Before UVA irradiation, the samples are incubated for 24 hours or 48 hours. Untreated irradiated controls were also prepared.

These skin samples were subsequently fixed with formaldehyde and then embedded in paraffin. Subsequently, 4 μm sections were prepared. These skin portions were removed from paraffin one after the other and then dehydrated in different xylene and alcohol baths. The elastic fibers were dyed using the ElastikavanGiesen dyeing kit (VWR, Ref: 1.15974). The stained skin sections were dehydrated and then mounted in Eukitt mounting medium and observed under a microscope.

As a result:observation under microscope was irradiated and passed at 10^-6Less degradation of the elastic fibers and better protection of the elastic fiber tissue was shown in the sample treated with the peptide seq id no5 under M than under control conditions.

And (4) conclusion:at 10^-6The peptide seq id no5 under M protects elastic fibers that are altered by UV radiation.

Example 6: verifying the regeneration effect of the peptide SEQIDNo5

The aim of this study was to determine the regenerative effect of the peptide of seq id no5 on dermal fibroblasts and normal human keratinocytes (NHEK). To accomplish this, the Ibidi in vitro healing model (integrated biodiagnostics, Munich, Germany) was used.

The implementation scheme is as follows:this test involves maintaining a cell-free area at the middle of the cell layer and evaluating the time required for cells disposed on either side of the "scar" to migrate and proliferate and fill the gap. Self-adhesive silicone culture inserts were placed at the vessel base of the culture plate. These inserts have particular features: consisting of two distinct chambers separated by an impermeable membrane having a thickness of 500 μm. Each of the two chambers was seeded with cells of the same cell type and cultured until confluent. The insert was then removed with sterile forceps, creating a 400 μm wide cell-free region between the two cell layers. Then 10 of the peptide of SEQ ID No5^-6M or 3.10^-6M solution was added to the culture medium to treat the cells, and the peptides were refreshed every 24 hours. Phase contrast microscopy observations (OlympusCK 40 microscope X5 coupled to an OlympusE-510 camera) were performed at different times (0, 6, 24, 30 and 48 hours) until the proliferation and migration processes resulted in filling of the gaps.

As a result:microscopic observation showed that the composition was used at 10 hours after 30 hours^-6The gaps of human keratinocytes treated with the peptide of seq id no5 under M were completely filled. At the same time, the cell gap was not filled under the control conditions.

For use in 10^-6Human fibroblasts treated with the peptide SEQ ID No5 under MThe gap was completely filled after 48 hours. At the same time, the cell gap was not filled under the control conditions.

The effect of the peptide is dose dependent, as it is used at 10^-6Gap filling was faster when cells were treated with the peptide seq id no5 under M.

And (4) conclusion:fibroblasts and normal human keratinocytes (NHEK) were cultured in the presence of the peptide of seq id no5 and they filled the gap faster compared to control conditions.

The peptide of seq id no5 stimulates the proliferation and migration of fibroblasts and NHEK, thereby promoting skin regeneration.

Example 7: preparation of the composition

1-sunshine protective cream

The components of phase A and phase B are heated between 70 ℃ and 75 ℃ respectively. Emulsify phase B in phase a with stirring. Phase C was added at 45 ℃ with increasing stirring. Subsequently, when the temperature is below 40 ℃, phase D is added. Cooling was continued until 25 ℃ with active stirring.

2-face-beautifying cream

Phase A was prepared and melted at 65-70 ℃. Heat phase C to 65-70 ℃. Phase B is added to phase a immediately after a is emulsified in phase B. The carbomer was neutralized by the addition of phase D at about 45 ℃. Phase E was then added with gentle stirring and cooling continued until 25 ℃. Phase F is then added if necessary.

3-day protective cream

Phase a was prepared with stirring and heated to 75 ℃. Phase B was prepared by dispersing carbopol and then xanthan gum under stirring. And (5) standing. Heating to 75 ℃. A was emulsified in B under rotor-stator stirring at temperature. Neutralize phase C under rapid stirring. After cooling at 40 ℃, phase D is added followed by phase E. Continue cooling with gentle stirring and add phase F.

Claims (17)

1. Peptides having the general formula (I)

R₁-(AA)_n-X₁-Arg-X₂-Trp-X₃-X₄-X₅-X₆(R₃)-(AA)_p-R₂

Wherein

X₁Is aspartic acid or is free of amino acids,

X₂is a mixture of glutamine or glutamic acid,

X₃asparagine or lysine or glutamine or no amino acid,

X₄is phenylalanine or tyrosine or has no amino acid,

X₅is tyrosine or alanine or is free of amino acids,

X₆is a cysteine or a non-amino acid,

AA represents any amino acid other than arginine, cysteine, leucine, glycine and glutamic acid, and n and p are 0 or 1, where n is not equal to p.

R₁Is a primary amine function with an N-terminal amino acid, unsubstituted or substituted by a radical of the acyl type (R-CO-), in which the radical R is a saturated or unsaturated C of the acetyl type₁-C₃₀An alkyl chain, or an aromatic group of the benzoyl, tosyl or benzyloxycarbonyl type;

R₂is unsubstituted hydroxyl group or has a substituent selected from C₁-C₃₀Radicals of alkyl chains or NH₂A carboxyl function of the C-terminal amino acid of a hydroxyl group substituted by a-NHY or-NYY 'group, wherein Y and Y' represent C₁-C₄An alkyl chain;

R₃is at X₆A thiol functional group of cysteine at a position, which thiol functional group is unsubstituted or substituted by a methyl or acetyl group, or which thiol functional group is covalently bound to another cysteine by a disulfide bond,

characterized in that the peptide corresponds to one of the following sequences:

(SEQIDNo5):Arg-Glu-Trp-Gln-Phe-Tyr-Cys(Cys)-(NH₂) (ii) a And

(SEQIDNo9):Asp-Arg-Glu-Trp-Gln-Phe-Tyr-Cys-NH₂。

2. the peptide according to claim 1, characterized in that it is dissolved in one or more physiologically suitable solvents selected from the group consisting of: water, glycerol, ethanol, propylene glycol, butylene glycol, dipropylene glycol, ethoxylated or propoxylated diethylene glycol, cyclic polyols or any mixtures of these solvents.

3. Use of a peptide according to claim 1 or 2 for the manufacture of a medicament.

4. The use as claimed in claim 3, as a healing agent.

5. Cosmetic composition comprising, in a physiologically suitable medium, as activator of dermatopontin, at least one peptide according to claim 1 or 2.

6. Cosmetic composition according to claim 5, characterized in that said peptide is present in a quantity of 10^-8M and 10^-5Concentrations between M are present.

7. Cosmetic composition according to claim 5 or 6, characterized in that it is intended for topical application.

8. Cosmetic composition according to any one of claims 5 to 7, characterized in that it further comprises at least one other active agent.

9. Cosmetic composition according to claim 8, characterized in that the other active agents are selected from: rejuvenating agents, anti-ageing agents, anti-wrinkle agents, thickening agents, anti-radical agents, anti-glycation agents, hydrating agents, antibacterial agents, antifungal agents, keratolytic agents, muscle relaxants, exfoliating agents and toning agents, agents stimulating synthesis or energy metabolism of dermal macromolecules, agents regulating skin differentiation, pigmentation or depigmentation, agents stimulating nail or hair growth, agents stimulating microcirculation, sunscreens or metalloproteinase inhibitors.

10. Use of a peptide according to claim 1 or 2 as active agent in a physiologically suitable medium for the preparation of a cosmetic composition for increasing the protein expression of the extracellular matrix by fibroblasts of the skin.

11. Use of the peptide according to claim 1 or 2 as an active agent in a physiologically suitable medium for the preparation of a cosmetic composition for increasing the density of the dermis and the elasticity of the skin, preventing or counteracting sagging or loss of volume of the facial lines, thinning of the skin, loss of tension, fine lines, deep wrinkles and atrophy of the dermis.

12. Use of a peptide according to claim 1 or 2 as active agent in a physiologically suitable medium for the preparation of a cosmetic composition for preventing or resisting the degradation of collagen and elastic fibres in the skin subjected to UV radiation.

13. Use of a peptide according to claim 1 or 2 for the preparation of a cosmetic composition for preventing or combating non-aesthetic signs related to solar elastosis and/or tissue damage of elastic fibres caused by exposure to UV radiation.

14. Use according to claim 13, wherein the UV radiation is UVA radiation.

15. Use of a peptide according to claim 1 or 2 as an active agent in a physiologically suitable medium for the preparation of a cosmetic composition for increasing the regeneration of the epidermis and the dermis.

16. Use of a peptide according to claim 1 or 2 as active agent in the preparation of a cosmetic composition for preventing and/or treating the signs of skin aging and photoaging, wherein a composition according to one of claims 5 to 9 is topically applied to the skin to be treated.

17. Use of a peptide according to claim 16, characterized in that: the composition is applied before exposure to sunlight to prevent tissue damage of the elastic fibers, or after exposure to sunlight to repair damage sustained by the collagen and elastic fibers.