HK1176890B - Novel sirtuin 6 activating peptides and cosmetic or pharmaceutical composition containing them - Google Patents

Description

SIRTUIN 6 activating peptide and cosmetic or pharmaceutical composition containing the same

Technical Field

The present invention is in the field of cosmetics and pharmacy, more particularly in the field of dermatology. The invention relates to sirtuin 6(SIRT6) activating peptides derived from a highly conserved region of human SIRT protein.

The invention also relates to cosmetic or pharmaceutical compositions comprising a SIRT6 activating peptide in a physiologically acceptable medium, alone or in combination with at least one other active agent. The invention also relates to the use of this novel peptide as an active agent in cosmetic compounds. The invention also relates to the use of cosmetic compounds for preventing and/or repairing DNA degradation, improving telomere maintenance and reducing cellular senescence. Finally, the invention relates to a cosmetic treatment method for preventing and/or treating cutaneous signs of aging and photoaging, in which an effective amount of an active agent or of a composition comprising said active agent is applied to the area to be treated.

Background

Aging corresponds to a series of physiological processes that alter the structure and function of an organism as a function of time and stress experienced. Intrinsic aging, which is due to genetic factors as well as biochemical changes occurring during fatigue and stress states and hormonal changes such as pregnancy, can be distinguished from extrinsic aging, which is due to environmental factors experienced by the organism throughout life, such as pollution, sunlight, disease, lifestyle, etc. Aging is a slow and progressive process that affects all cells and organs. This is therefore applied to the skin, which constitutes a barrier between the external environment and the internal medium and protects the organism against external stresses. During aging, the appearance of the skin changes and, as a result, wrinkles and fine lines, pigmented spots or pigment-reducing spots, dry and even dehydrated skin, thinned epidermis, elastosis may occur.

Intrinsic aging is closely related to the repeated division of cells. Thus, in human cells, telomeres shorten the rhythm of cell division until dysfunctional telomeres appear, inducing senescence or apoptosis, depending on the cell type. This phenomenon constitutes a biological clock, which explains the programming of a limited number of divisions by human cells.

The phenomenon of cellular senescence is accelerated by oxidative damage, particularly in the body regions where the skin is exposed to the sun; then photo-aging is superimposed on the aging. Oxidative damage is promoted by a variety of substances, both endogenous (metabolism, inflammation, redox cycle) and exogenous, such as UV and ionizing radiation, tobacco abuse, and dietary supply of a variety of molecules (toxic metals, alcohols). Damage caused by oxidative stress also reaches DNA and lipids and proteins. At the DNA level, oxidative stress causes numerous structural modifications (mutations, cleavage, covalent protein cross-linking). Oxidized bases such as 8-oxo-guanine increase with age and may reach up to 10,000 bases per day and per cell.

To combat aging, it is therefore of interest to identify new compounds capable of combating local damage to DNA caused by oxidative stress and slowing cellular senescence by promoting telomere stability.

Thus, the present inventors have recently identified interesting molecular targets that are capable of performing these different functions.

SIRT proteins are nuclear or mitochondrial proteins that have NAD + -dependent deacetylase function and belong to the sirtuin family. sirtuin deacetylase or mono-ADP-ribosyltransferase activities make them capable of modulating the acetylation levels of some histones, suggesting that they are involved in the regulation of epigenetic phenomena, particularly 1, 2 and 3 sirtuins.

The human sirtuin family comprises 7 proteins, well conserved throughout evolution, designated SIRT1 through SIRT 7.

SIRT6 is a nuclear sirtuin that is particularly associated with telomeric chromatin and plays a role in the maintenance and stabilization of telomeric structure (Michishita et al. Nature.2008Mar 27;452(7186): 492-6). Thus, in mice in which the SIRT6 gene was null, premature aging and short lifespan were observed, as well as an increase in replicative senescence of keratinocytes (Kawahara TL et al. cell.2009Jan 9;136(1): 62-74).

Telomeres are structures that cover the ends of chromosomes and protect chromosomes from enzymatic degradation, recombination, and interchromosomal fusion. In humans, these structures consist of DNA sequences that are repeated thousands of times, associated with specific proteins such as TRF1 and TRF 2. Recent studies have demonstrated that TRF2 expression decreases during cell aging (Amoyel et al, J.invest.Dermatol.Apr 2009;129(Supplement 1s), s 70).

On the other hand, SIRT6 plays an important role in DNA repair by base excision, a DNA repair mechanism that cells utilize when DNA has been damaged by an oxidizing agent. These findings indicate that SIRT6 is essential for the regulation of genomic integrity and the phenomenon of aging and may be directly involved in the increase in cell life (Mostoslavsky et al, cell.2006Jan 27;124(2): 315-29).

It is known that the use of SIRT1 protein-activating peptides (FR 2883751, FR 2883752, FR 2883753, FR 2883754) enables the preparation of cosmetic or pharmaceutical compositions useful for protecting the skin and combating ageing, or certain SIRT7 inducer pharmaceutical compounds useful for the treatment of age-related diseases (EP 1955715). However, to date, peptide compounds capable of activating SIRT6 protein in skin cells have not been described, although there is a need for this type of skin care.

Disclosure of Invention

The present inventors have demonstrated that peptides derived from the highly conserved region of human SIRT protein having the following general formula (I) are very good SIRT6 activators and enable the prevention and/or effective repair of DNA degradation caused by external stress, in particular UV radiation, improved telomere maintenance and reduced cellular senescence:

R₁-(AA)_n-X₁-X_2--X₃-X₄-X₅-X₆-(AA)_p-R₂

therefore, these peptides are suitable for combating the aging and photoaging of the skin.

The peptides of the invention are characterized in that they:

activating SIRT6 expression in skin cells,

-reducing DNA degradation in skin cells subjected to UVB radiation,

-promoting the protection of skin cells undergoing oxidative stress

-stimulating the expression of TRF2 protein specifically associated with telomeres,

-increasing the expression of proteins from the extracellular matrix by fibroblasts,

-optimizing the barrier function of the epidermis.

"peptide capable of activating human SIRT6 or SIRT6 activating agent or agent" is understood to mean any peptide of the general formula (I) which is capable of increasing the amount of SIRT6 present in a cell, or increasing protein synthesis by directly or indirectly modulating gene expression or by other biological processes such as protein stabilization or messenger RNA transcript stabilization.

Skin is understood to mean all the overlying tissues that constitute the skin, mucous membranes and epithelial appendages.

Alignment of peptide sequences from 7 proteins of the SIRT family was performed using the ClustalW2 multiple peptide sequence alignment program from European Bioinformatics Institute, as shown in figure 1. The best alignment showed 3 highly conserved regions.

A "highly conserved region of human SIRT proteins" is understood to mean a peptide sequence comprising at least 2 contiguous amino acids of absolute identity among the 7 sirtuins of the family when the sequences have been aligned on the basis of the highest homology.

The first highly conserved region comprises the Gly-Ala-Gly peptide sequence.

The second highly conserved region comprises the Gln-Asn peptide sequence.

The third highly conserved region comprises a His-Gly peptide sequence.

Accordingly, a first object of the invention is a 6-10 amino acid peptide derived from a peptide sequence corresponding to a highly conserved region of a human SIRT protein of the following general formula (I):

R₁-(AA)_n-X₁-X₂-X₃-X₄-X₅-X₆-(AA)_p-R₂

wherein

X₁Glycine or threonine or histidine;

X₂is alanine or glutamine or glycine;

X₃glycine or asparagine or serine;

X₄is valine or isoleucine or leucine;

X₅is serine or aspartic acid or phenylalanine;

X₆is alanine or glutamic acid or lysine;

and is

When X is present₁In the case of glycine, X₂Is alanine and X₃Is glycine;

when X is present₁When threonine, X₃Is asparagine;

when X is present₁When it is histidine, X₂Is glycine;

AA represents any amino acid or one of its derivatives, and n and p are integers from 0 to 2;

R₁represents the primary amino function of the N-terminal amino acid, which is free or saturated or unsaturated by C₁-C₃₀An alkyl chain, which may be acetyl, or substituted with an acyl type group having an aromatic group, which may be selected from benzoyl, tosyl or benzyloxycarbonyl type groups;

R₂represents the hydroxyl group of the carboxyl function of the C-terminal amino acid, which is free or may be chosen from C₁-C₃₀Radical substitution of alkyl chains, or NH₂NHY or NYY radical, wherein Y represents C₁-C₄An alkyl chain.

The sequence of the general formula (I) consists of 6-10 amino acid residues.

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

(SEQ ID No.1)Glu-Ile-His-Gly-Ser-Leu-Phe-Lys-NH₂

(SEQ ID No.2)His-Gly-Ser-Leu-Phe-Lys-NH₂

(SEQ ID No.3)Leu-Val-Gly-Ala-Gly-Val-Ser-Ala-NH₂

(SEQ ID No.4)Gly-Ala-Gly-Val-Ser-Ala-Glu

(SEQ ID No.5)Gly-Ala-Gly-Val-Ser-Ala-Glu-NH₂

(SEQ ID No.6)Thr-Gln-Asn-Ile-Asp-Glu-Leu

(SEQ ID No.7)Thr-Gln-Asn-Ile-Asp-Glu-Leu-NH₂

(SEQ ID No.8)Val-Ile-Thr-Gln-Asn-Ile-Asp-Ala-NH₂

according to a particularly interesting embodiment, said peptide corresponds to the sequence of SEQ ID No.4 or to the sequence of SEQ ID No. 5.

According to another particularly interesting embodiment, said peptide corresponds to the sequence of SEQ ID No.6 or to the sequence of SEQ ID No. 7.

The amino acids which constitute the peptides of the invention and are designated by the terms AA or X may be in the isomeric configurations L-and D-. Preferably, the amino acid is in the L form.

The term "peptide" denotes a linkage of two or more amino acids linked to each other by peptide bonds or modified peptide bonds.

"peptide" is also understood to mean a natural or synthetic peptide according to the invention, or at least one of its fragments, whether obtained by proteolysis or synthetically, or any natural or synthetic peptide, the sequence of which is partially or totally constituted by the peptide sequence previously described.

Peptide derivatives particularly refer to amino acids linked to each other by pseudopeptide bonds. "pseudopeptide bond" is understood to mean all types of bond which can replace the "conventional" peptide bond.

In order to improve resistance to degradation, it may be necessary to use protected forms of the peptides of the invention. Preferably, for protection of the primary amine function of the N-terminal amino acid, a compound having C₁-C₃₀Acyl-type substituent R of saturated or unsaturated alkyl chain₁The substituents may be selected from acetyl or aromatic groups. Preferably, to protect the carboxyl function of the C-terminal amino acid, C is used₁-C₃₀Alkyl chain type substituent group R₂Or R is₂Is a NH2, NHY or NYY group, wherein Y represents C₁-C₄An alkyl chain.

The peptide of the present invention may be protected in the N-terminal, C-terminal region or both regions.

The invention therefore relates to a composition, as defined previously, characterized in that the peptides of SEQ ID No.1 to SEQ ID No.8 are in protected or unprotected form.

The peptides of general formula (I) of the invention can be obtained from the constituent amino acids by conventional chemical synthesis (in solid or homogeneous liquid phase) or by enzymatic synthesis (Kullman et al, J.biol.chem.1980,225, 8234).

The peptides of 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, a mixture of peptides or peptide derivatives.

The peptides of 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 diluted peptide was then sterilized by sterile filtration.

After this dilution step, the peptides can be encapsulated or contained in cosmetic or pharmaceutical carriers such as liposomes or any other microcapsules used in the cosmetic field, or adsorbed on powdered organic polymers, mineral supports such as talc and bentonite.

"physiologically suitable" is understood to mean that the solvent chosen is suitable for contact with the skin without causing toxic or intolerable reactions.

The peptides of the invention can be used as medicaments.

A second object of the invention is a cosmetic or pharmaceutical composition, in particular a dermatological composition, comprising a peptide of general formula (I) as human SIRT6 activator active agent in a physiologically suitable medium.

According to an advantageous embodiment of the invention, the active agent of the invention is present in a quantity of about 10 relative to the total weight of the final composition^-9M-10^-3The concentration of M is present in the compositions of the invention, and is preferably 2.10^-8M-10^-5The concentration of M.

This concentration range represents an effective amount of active agent corresponding to the amount necessary to achieve the desired result (i.e., activation of SIRT6, reduction of DNA degradation, and improvement of telomere maintenance).

In a preferred manner, the composition of the invention is in a form suitable for topical application, comprising a physiologically compatible medium. "physiologically suitable" is understood to mean a medium suitable for use in contact with the skin or human epithelial appendages without the risk of toxicity, incompatibility, instability, allergic response or other secondary effects.

"topical application" is understood to mean the action of applying or spreading the active agent of the invention or a composition containing said active agent on the skin surface.

The composition to be applied to the skin may be in the form of an aqueous or hydroalcoholic solution, a water-in-oil or oil-in-water emulsion, a microemulsion, an aqueous or anhydrous gel, a serum, or a vesicular dispersion, a patch, a cream, a spray, an ointment, a pomade, a lotion, a colloid, a solution, a suspension, or other forms.

These compositions may be in particular in aqueous, hydroalcoholic or oily solution; the oil-in-water emulsion, the water-in-oil emulsion or the composite emulsion exists; they may also be present in the form of creams, suspensions or powders suitable for application to the skin, mucous membranes, lips and/or epithelial appendages. These compositions may be more or less fluid and have the appearance of a cream, lotion, milk, serum, pomade, gel, patch or foam. They may also be present in solid form, such as a stick, or may be applied to the skin in aerosol form. They can be used as care products and/or skin make-up products.

Furthermore, all these compositions comprise any additives generally used in the fields of application considered and the adjuvants necessary for their formulation, such as cosolvents (ethanol, glycerol, benzyl alcohol, humectants, etc.), thickeners, diluents, emulsifiers, antioxidants, colorants, sunscreens, pigments, fillers, preservatives, fragrances, deodorants, essential oils, trace elements, essential fatty acids, surfactants, film-forming polymers, chemical or mineral filters, moisturizers or hot water, etc. For example, mention may be made of water-soluble polymers of the natural polymer type, such as polysaccharides or polypeptides, cellulose derivatives of the methylcellulose or hydroxypropyl cellulose type, or synthetic polymers, poloxamers, carbomers, silicones, PVA or PVP, in particular those sold by the ISP company.

In each case, the person skilled in the art will ensure that these additives and their proportions are selected in such a way as not to impair the advantageous properties desired for the compositions of the invention. These adjuvants may be present, for example, in a concentration of 0.01 to 20% by weight of the total composition. When the composition of the invention is an emulsion, the fatty phase may represent from 5 to 80% by weight, and preferably from 5 to 50% by weight, of the total weight of the composition. The emulsifiers and co-emulsifiers used in the composition are chosen from those commonly 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.

It is to be understood that the active agents of the present invention may be used alone or in combination with other active agents.

Advantageously, the compositions that can be used according to the invention comprise at least one further active agent intended to promote the action of the active agent of the invention, in particular to prevent and/or treat age-related disorders.

The following classes of ingredients may be mentioned in a non-limiting manner: other peptide active agents, plant extracts, cicatrizants, anti-ageing agents, anti-wrinkle agents, smoothing agents, anti-radical agents, anti-UV agents, substances stimulating the synthesis of skin macromolecules or the metabolism of energy, moisturizers, antibacterial agents, antifungal agents, anti-inflammatory agents, anesthetics, substances regulating the differentiation of the skin, the pigmentation of the skin or the depigmentation of the skin, and substances stimulating the growth of nails and hair.

Preferably, an anti-radical agent or an antioxidant is used, or a substance that stimulates the synthesis of skin macromolecules, or a substance that stimulates energy metabolism. In a particularly preferred embodiment, the composition of the invention may comprise, in addition to the peptide of the invention, a peptide of the invention

At least one cytochrome c activating compound, and/or

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

At least one sirtuin activating compound, in particular a peptide as mentioned in patents FR 2883754, US11/910,098, EP 1868631, 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, mucopolysaccharide, and/or

At least one compound which modulates proteasome activity, and/or

At least one compound regulating the circadian rhythm, and/or

At least one compound which modulates the activity of HSP's, and/or

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

At least one compound regulating skin pigmentation, and/or

At least one coenzyme Q10 activating compound, and/or

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

At least one mitochondrial protecting compound, and/or

At least one compound for protecting or modulating adult somatic cells of the epidermis or dermis, and/or

-at least one compound that protects or repairs DNA degradation.

The compounds may be of natural origin, such as plant, animal or microbial peptide hydrolysates, or of synthetic origin, such as peptides.

Regardless of their function, other active agents in the composition that are associated with the active agents of the present invention can have a very diverse chemical structure. Mention may be made, in a non-limiting manner, of peptides, vitamin C and its derivatives, vitamins from the B group, DHEA (dihydroepiandrosterone), phytosterols, salicylic acid and its derivatives, retinoids, flavonoids, sugar amines, pyrrole compounds, metal salts, peptide extracts of natural origin or natural or synthetic polymers.

Another object of the invention is a pharmaceutical composition comprising a peptide of the invention as a medicament in a physiologically acceptable medium. The pharmaceutical composition of the invention can improve the dermatological symptoms associated with premature aging or photoaging, among which mention may be made of xerosis, depigmentation or, conversely, tan, keratosis, etc.

Advantageously, according to this form of the invention, the composition is suitable for oral administration for pharmaceutical use. Thus, the composition may be presented in the form of tablets, capsules, gel capsules, chewable pastes, powders for direct consumption or for mixing with liquids immediately before use, syrups, gels or any other form known to the person skilled in the art. They will contain suitable formulating excipients such as coloring agents, sweetening agents, flavoring agents, fillers, binders and preservatives.

A third object of the present invention is a cosmetic composition comprising a peptide of general formula (I) as active agent to prevent and/or repair DNA degradation.

"agent preventing and/or repairing DNA degradation" is understood to mean a peptide capable of limiting DNA degradation or of promoting repair of damage due to photochemical reactions between DNA bases.

A fourth object of the present invention is a cosmetic composition comprising a peptide of general formula (I) as an active agent to improve telomere maintenance and reduce cellular senescence.

An "agent that improves telomere maintenance and reduces cellular senescence" is understood to mean a peptide capable of increasing the synthesis of proteins specifically associated with telomeres and involved in their stability (such as TRF2 and SIRT 6).

A fifth object of the present invention is the use of a cosmetic composition comprising as active agent a peptide of general formula (I) for increasing the expression of keratinocyte differentiation markers and for promoting the expression of extracellular matrix proteins by fibroblasts of the skin.

These unique properties of the active agents of the present invention improve the quality of the dermis and thus the firmness of the skin, and optimize the barrier function of the epidermis.

A sixth object of the invention is the use of a composition comprising as active agent a peptide of general formula (I) for protecting the skin against all types of external stress.

The expression "external stress" is understood to mean the stress that the environment can produce. By way of example, mention may be made of stresses such as soiling, UV radiation or irritating products such as surfactants, preservatives or fragrances, or mechanical stresses such as scratching, shaving or depilation. Contamination is understood to mean "external" contamination, for example due to diesel particles, ozone or heavy metals, and "internal" contamination, which may be due in particular to the release of paint, adhesives or wallpaper solvents, such as toluene, styrene, xylene or benzaldehyde, or to cigarette smoke.

In particular, the object of the present invention is the use of a cosmetic composition comprising an effective amount of a peptide according to the invention for preventing or treating skin damage caused by exposure to UV radiation and oxidative stress.

A seventh object of the present invention is a cosmetic treatment method characterized in that a composition comprising an effective amount of the active agent of the invention is topically applied to the skin to be treated in order to prevent and/or treat the cutaneous signs of aging and photoaging.

Signs of aging skin are understood to mean any change in the appearance of the skin and epithelial appendages due to aging, such as surface roughness, wrinkles and fine lines of the stratum corneum of the epidermis, and also any internal change of the skin that is not systematically manifested in the changed appearance, such as thinning of the dermis or any other internal degradation of the skin after exposure to UV radiation.

In particular, the invention relates to a cosmetic treatment method aimed at protecting the skin against stress due to UV radiation.

Other advantages and characteristics of the invention will appear more clearly on reading the examples given for the purpose of illustration and not of limitation.

Drawings

FIG. 1: alignment of human SIRT protein peptide sequences (alignment by using ClustalW2 multiple peptide sequence alignment programs from European bioinformatics institute)

FIG. 2: quantification of sirtuin 6(SIRT6) immunolabeling in normal human fibroblasts treated for 24 hours with the peptide SEQ ID No. 5.

Examples

Example 1: demonstration of the activating Effect of the peptides SEQ ID No.5 and SEQ ID No.7 on sirtuin 6 expression

The objective of this study was to determine the effect of peptides SEQ ID No.5 and SEQ ID No.7 on Sirtuin 6 expression in human skin. For this purpose, cultures of Normal Human Keratinocytes (NHK) and normal human fibroblasts are labeled specifically for immunofluorescence.

Scheme(s): NHK or Normal human fibroblasts were treated with 10^-6M or 3.10^-6A solution of the peptide SEQ ID No.5 or the peptide SEQ ID No.7 of M is treated once daily.

Short-term treatment studies of 24, 48 and 72 hours were performed.

Long-term treatment studies were also performed between passage 5 and passage 17 (or passage 12) of fibroblasts and between passage 3 and passage 5 (or 2 passages) of NHK.

For immunolabeling by the anti-SIRT 6 antibody, cells were washed and fixed with 3.7% paraformaldehyde for 10 minutes.

The cells were then incubated in the presence of specific anti-SIRT 6 antibody (Abcam, ref. ab62738, polyclonal rabbit) and then with a suitable secondary antibody conjugated to a fluorescent dye. After mounting in a specific medium, the slides were observed by an epifluorescence microscope (Nikon Eclipse E80 i microscope).

Fluorescence intensity was quantified by analyzing the images using Image-Pro analyzer version 5 software.

As a result: at passage 10, compared to the control conditions, under all conditions tested^-6M or 3.10^-6Stronger fluorescence was observed in peptide SEQ ID No.5 and peptide SEQ ID No.7 treated cultures of M.

In fibroblasts, in the passage 10^-6A maximum increase in fluorescence of 47% relative to control cells was observed in cells treated for 24 hours with the peptide SEQ ID No.5 of M (FIG. 2).

In NHK, at a passage of 3.10^-6A maximum increase in fluorescence of 35% relative to control cells was observed in cells treated for 72 hours with the peptide SEQ ID No.5 of M. The increase in fluorescence was initially 72The hour is dose dependent.

On the other hand, the increase in SIRT6 expression was maintained during long-term treatment of both types of cells tested.

Conclusion: peptides SEQ ID No.5 and SEQ ID No.7 increased sirtuin 6 expression in normal human fibroblasts and NHK very significantly in short term culture. In addition, sirtuin 6 expression stimulatory effects are maintained over long periods.

Example 2: demonstration of the activating Effect of the peptide SEQ ID No.4 on the expression of TRF2 protein

The aim of this study was to determine the effect of the peptide SEQ ID No.4 on the expression of TRF2 protein, TRF2 being a protein specifically associated with telomeres and involved in their maintenance. For this purpose, cultures of Normal Human Keratinocytes (NHK) and normal human fibroblasts are labeled specifically with immunofluorescence on a long-term basis.

Scheme(s): for fibroblasts, between passage 5 and passage 17 (or passage 12), and for NHK, between passage 1 and passage 2 (or passage 1 and day 10 treatment), NHK or normal human fibroblasts in culture were used 10^-6M or 3.10^-6A solution of the peptide SEQ ID No.4 of M was treated once daily.

For immunolabeling by anti-TRF 2 antibody, cells were washed and fixed with 3.7% paraformaldehyde for 10 min.

The cells were then incubated in the presence of specific anti-TRF 2 antibodies (Abcam, ref. ab13579, polyclonal mice) and then with a suitable secondary antibody conjugated to a fluorescent dye. After mounting in a specific medium, the slides were observed by an epifluorescence microscope (Nikon Eclipse E80 i microscope).

Fluorescence intensity was quantified by analyzing the images using Image-Pro analyzer version 5 software.

Results: under all conditions tested, withComparison under control conditions at passage 10^-6M or 3.10^-6Stronger fluorescence was observed in the peptide of M, SEQ ID No.4 treated cultures.

In fibroblasts, in the passage 10^-6The peptide of M SEQ ID No.4 treated 12 secondary cells a maximal increase of 63% of fluorescence relative to control cells was observed. The increase in fluorescence is dose-dependent.

In NHK, at a passage of 3.10^-6A maximum increase in fluorescence of 39% relative to control cells was observed in cells treated for 10 days with the peptide SEQ ID No.4 of M. The increase in fluorescence is dose-dependent.

Conclusion: the peptide SEQ ID No.4 increased TRF2 protein expression in normal human fibroblasts and NHK very significantly in a dose-dependent manner in long-term culture.

Example 3: demonstration of the Effect of the peptide SEQ ID No.5 on epidermal differentiation and activation of the epidermal barrier function

The aim of this study was to determine the effect of the peptide SEQ ID No.5 on epidermal differentiation. For this reason, the expression of major epidermal differentiation markers, particularly in keratinocytes of the basal layer (suprabasal layer) cultured on a long-term basis, was investigated. The markers tested were transglutaminase 1 and endoglin.

Scheme(s): NHK in culture was used 10 between passage 1 and passage 3 (or treatment for 2 and 11 days)^-6M or 3.10^-6A solution of the peptide SEQ ID No.5 of M was treated once daily.

The cells were then washed and fixed. After exposure of the specific sites, the cells were incubated in the presence of specific antibodies against TG1 (TEBU, ref. sc-25786, polyclonal rabbit), specific antibodies against the endo-coat protein (Novocastara NCL-INV, mouse monoclonal, clone SY5) and then in the presence of a suitable secondary antibody coupled to a fluorescent dye. For easier visualization, nuclei can be counterstained by DAPI (4',6' diamidino-2-phenylindole, a fluorescent blue molecule capable of binding strongly to DNA). After mounting in a specific medium, the slides were observed by an epifluorescence microscope (Nikon Eclipse E80 i microscope).

Results: at passage 10 compared to control conditions^-6M or 3.10^-6Stronger fluorescence was observed in peptide SEQ ID No.5 treated cultures and on skin sections of M.

Conclusion：10^-6M or 3.10^-6The peptide SEQ ID No.5 of M increases NHK differentiation and this optimizes the barrier function of the epidermis.

Example 4: demonstration of the activating Effect of the peptide SEQ ID No.5 on the expression of skin extracellular matrix molecules

The objective of this study was to determine the effect of the peptide SEQ ID No.5 on the expression of skin extracellular matrix molecules. To this end, the expression of collagen I and III in normal human fibroblasts cultured on a long-term basis was investigated.

Scheme(s): the normal human fibroblasts were used 10 between passage 5 and passage 17 (or passage 12)^-6M or 3.10^-6A solution of the peptide SEQ ID No.5 of M was treated once daily.

The cells were then washed and fixed with cold methanol for 5 minutes. After exposure of the specific sites, the cells were incubated in the presence of specific antibodies against collagen I (TEBU, ref.600-401-103, polyclonal rabbit) or against collagen III (TEBU, ref.600-401-105, polyclonal rabbit) and then in the presence of a suitable secondary antibody coupled to a fluorescent dye. After mounting in a specific medium, the slides were observed by an epifluorescence microscope (Nikon Eclipse E80 i microscope).

Results: at passage 10 compared to control conditions^-6M or 3.10^-6Stronger fluorescence was observed in peptide SEQ ID No.5 treated cultures and on skin sections of M.

Conclusion: administered on a long term basis 10^-6M or 3.10^-6The peptide SEQ ID No.5 of M increases the expression of two essential proteins collagen I and collagen III from the extracellular matrix of the skin.

Example 5: demonstration of the effect of the peptide SEQ ID No.5 on DNA damage caused by UV radiation.

The aim of this study was to determine the protective effect of the peptide SEQ ID No.5 on DNA damage caused by UV radiation. For this purpose, a comet (comet) assay was performed, which enables quantification of DNA damage caused at the cellular level.

Scheme(s): normal human fibroblasts were treated at a concentration of 10^-6M or 3.10^-6M peptide of sequence SEQ ID No.5, then 60mJ/cm²Is irradiated with UVB radiation and then passed through a concentration of 10^-6M or 3.10^-6The peptide of M was treated again for 24 hours.

Control conditions were performed in the absence of treatment.

The cells were then detached from their support by trypsin and then centrifuged at 1200 rpm for 10 minutes to concentrate them and count them.

A certain number of cells (25,000 cells) were then contained in a 0.75% low-melting agarose gel and then deposited on a slide previously covered with 1% agarose. The slides were then immersed in lysis buffer at 4 ℃ 1¹/₂For hours, then in alkaline solution at 4 ℃ for 20 min. Thus the cells are lysed and the DNA is denatured. The slide was immersed in the electrophoretic solution, and then an electric field (20V-250 mA) was applied. The thus denatured DNA was allowed to migrate in an agarose gel at 4 ℃ for 30 min. The application of propidium iodide, a 2 μ g/ml DNA fluorescent dye, on a glass slide for 20 minutes enables the microscopic observation of DNA that is comet tail shaped if damaged.

The quantitative software enables the determination of the average "Tail Moment" (or the length of the comet Tail) for each test condition. This parameter provides information on the level of DNA damage: the higher this parameter, the greater the DNA degradation.

Results: the results show that when compared to the control conditions, a pass of 3.10^-6M peptide of SEQ ID No.5 reduced the tail moment by 24.8% when the cells were treated.

Conclusion: the DNA of the cells treated and then subjected to UVB irradiation experienced less damage than the DNA of the control cells. These results confirm the preventive protective and therapeutic effect of the peptide of sequence SEQ ID No.5 with respect to UVB radiation.

Example 6: demonstration of the protective Effect of SEQ ID No.5 during oxidative stress

The aim of this study was to determine the protective effect of the peptide SEQ ID No.5 on keratinocytes during oxidative stress. For this purpose, in the passage of H₂O₂After oxidative stress, Sirtuin 6 expression was assessed qualitatively and quantitatively by specific immunolabeling.

Scheme(s): NHK in culture was used 10^-6M or 3.10^-6M peptide SEQ ID No.5 for 24 hours. The cells were then incubated at 2mM H₂O₂Incubated in the presence of (2), rinsed and then incubated with 10^-6M or 3.10^-6The solution of the peptide SEQ ID No.5 of M is treated for an additional 24 hours. Untreated and not receiving H₂O₂Stressed control (control 0) and untreated but H-receiving₂O₂Stress control (control 1).

For immunolabeling by the anti-SIRT 6 antibody, cells were washed and fixed with 3.7% paraformaldehyde for 10 minutes.

The cells were then incubated in the presence of specific anti-SIRT 6 antibody (Abcam, ref. ab62738, polyclonal mice) and then with a suitable secondary antibody conjugated to a fluorescent dye. After mounting in a specific medium, the slides were observed by an epifluorescence microscope (Nikon Eclipse E80 i microscope).

Fluorescence intensity was quantified by analyzing the images using Image-Pro analyzer version 5 software.

Results: quantitative analysis showed that when NHK was used 10, relative to control 1^-6M or 3.10^-6Peptide SEQ ID No.5 of M and received H₂O₂When stressed, SIRT6 expression increased 16% and 20%, respectively.

Conclusion: cells that subsequently received oxidative stress treated prophylactically by the peptide SEQ ID No.5 had a greater SIRT6 protein content than control cells. These results demonstrate that the peptide of sequence SEQ ID No.5 promotes NHK protection during oxidative stress.

Example 7: preparation of the composition:

1, sunscreen cream:

the components of phase A and phase B are heated separately at 70 ℃ to 75 ℃. Emulsify phase B in phase a with stirring. Phase C was added by increasing stirring at 45 ℃. Phase D is then added at a temperature below 40 ℃. Cooling was continued up to 25 ℃ with vigorous stirring.

2, anti-aging cream:

phase A was prepared and melted at 65-70 ℃. Heat phase C to 65-70 ℃. Phase B was added to phase a just prior to emulsifying a into 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 desired.

3, protective day cream:

phase a was prepared and heated to 75 ℃ with stirring. Phase B was prepared by dispersing kappa (carbopol) and then xanthan with stirring. It was allowed to stand. Heat to 75 ℃.

Emulsifying a into B under rotor stator agitation at temperature. Neutralize phase C under rapid stirring. After cooling to 40 ℃, phase D and then phase E are added. Continue cooling and add phase F with gentle stirring.

Claims (12)

1. A peptide derived from a peptide sequence of a highly conserved region of a human SIRT protein having the general formula (I):

R₁-(AA)_n-X₁-X₂-X₃-X₄-X₅-X₆-(AA)_p-R₂

wherein

X₁Glycine or threonine or histidine;

X₂is alanine or glutamine or glycine;

X₃is glycineAcid or asparagine or serine;

X₄is valine or isoleucine or leucine;

X₅is serine or aspartic acid or phenylalanine;

X₆is alanine or glutamic acid or lysine;

and is

When X is present₁In the case of glycine, X₂Is alanine and X₃Is glycine;

when X is present₁When threonine, X₃Is asparagine;

when X is present₁When it is histidine, X₂Is glycine;

AA represents any amino acid, and n and p are integers from 0 to 2;

R₁represents the primary amino function of the N-terminal amino acid, which is free or saturated or unsaturated by C₁-C₃₀An alkyl chain substituted with an acyl type group, which is an acetyl group, or with an acyl type group having an aromatic group, which is selected from a benzoyl, tosyl or benzyloxycarbonyl type group;

R₂represents the hydroxyl group of the carboxyl function of the C-terminal amino acid, free or selected from C₁-C₃₀Radical substitution of alkyl chains, or NH₂NHY or NYY radical, wherein Y represents C₁-C₄An alkyl chain is arranged on the base,

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

(SEQ ID No.3)Leu-Val-Gly-Ala-Gly-Val-Ser-Ala-NH₂

(SEQ ID No.4)Gly-Ala-Gly-Val-Ser-Ala-Glu

(SEQ ID No.5)Gly-Ala-Gly-Val-Ser-Ala-Glu-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, white petrolatum, vegetable oils or any mixture of these solvents.

3. A peptide according to claim 1 or 2, characterised in that the peptide is for use as a medicament.

4. A cosmetic composition, characterized in that it comprises, in a physiologically suitable medium, at least one peptide as defined in claim 1 or 2 as SIRT6 activator, alone or in combination with at least one other active agent.

5. Composition according to claim 4, characterized in that the peptide is present in an amount of 10 relative to the total weight of the final composition^-9M-10^-3The concentration of M is present.

6. Composition according to claim 5, characterized in that the peptide is present in a quantity of 2 x 10 relative to the total weight of the final composition^-8M-10^-5The concentration of M is present.

7. Composition according to one of claims 4 to 6, characterized in that it is intended for topical administration.

8. Composition according to any one of claims 4 to 6, characterized in that it further comprises at least one other active agent which promotes the action of the peptide.

9. Use of a peptide as defined in claim 1 or 2, together with a physiologically suitable medium, for the preparation of a composition for protecting the skin against any type of external stress.

10. Use according to claim 9, characterized in that the external stress is caused by UV radiation.

11. Use according to claim 9, characterized in that the external stress is caused by oxidative stress.

12. Use of a peptide as defined in claim 1 or 2, for the preparation of a composition for preventing and/or treating cutaneous signs of aging, wherein said composition is topically applied to the skin to be treated.