Patent Grant
8569243
This application claims priority to French Patent Application Serial No. FR 10 02698, filed Jun. 29, 2010 under the original title “Nouveaux peptides activateurs de la sirtuine 6 et composition cosmétique ou pharmaceutique les comprenant,” hereby incorporated by reference.
The present invention is situated in the cosmetic and pharmaceutical field, and more particularly in the dermatology field. The present invention relates to sirtuin 6 (SIRT6) activating peptides, derived from highly conserved regions of human SIRT proteins.
The present invention also relates to a cosmetic or pharmaceutical composition, comprising a SIRT6 activating peptide, used alone or in combination with at least one other active agent, in a physiologically acceptable medium. The invention also relates to the utilization of this novel peptide as an active agent in a cosmetic composition. The invention further relates to the utilization of a cosmetic composition to prevent and/or repair DNA degradation, improve telomere maintenance and reduce cellular senescence. Lastly, the invention applies to a cosmetic treatment process intended to prevent and/or treat the cutaneous signs of aging and photo aging, according to which an effective quantity of active agent, or a composition containing the active agent, is applied to the areas to be treated.
Aging corresponds to the set of physiological processes that modify the structure and functions of the organism according to the time and stresses undergone. Intrinsic aging due to genetic factors and biochemical modifications that take place during states of fatigue and stress and hormonal changes such as pregnancy, etc., may be distinguished from extrinsic aging due to environmental factors to which the organism is subjected throughout its life, such as pollution, sunlight, disease, lifestyle, etc. Aging is a slow and progressive process that affects all cells and organs. Thus this applies to the skin, which constitutes a barrier between the external environment and the inner medium and protects the organism against external stresses. During aging, the appearance of the skin changes and thus wrinkles and fine lines, hyper- or hypopigmentation spots, dryness and even dehydration of the skin, thinning of the epidermis, elastosis, etc., may appear.
Intrinsic aging is closely linked to the repeated divisions of cells. Thus, in human somatic cells, telomeres shorten the rhythm of cellular division, until dysfunctional telomeres appear that induce senescence or apoptosis, depending on the cellular type. This phenomenon constitutes the biological clock that explains the fact that human somatic cells are programmed for a limited number of divisions.
Cellular senescence phenomena are accelerated by oxidative damage, particularly in areas of the body where the skin is exposed to the sun; Photo aging is then superimposed on intrinsic aging. Oxidative damage is promoted by various agents, both endogenous (metabolism, inflammation, redox cycles) and exogenic, such as UV radiation and ionizing radiation, tobacco abuse and various molecules supplied by the diet (toxic metals, alcohol). Damage caused by oxidative stress also reaches the DNA and lipids and proteins. At the DNA level, oxidative stress causes many structural modifications (mutations, cleavage, covalent protein cross-links). 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 novel compounds capable of both combating localized damage caused to the DNA by oxidative stress and slowing down cellular senescence by promoting telomere stability.
Such being the case, the inventors have recently identified an interesting molecular target capable of fulfilling these various functions.
SIRT proteins are nuclear or mitochondrial proteins, bearing a NAD+dependent deacetylase function and belonging to the sirtuin family. The deacetylase or mono-ADP-ribosyltransferase activity of sirtuins enables them to modulate the acetylation level of some histones, which suggests their involvement, particularly with 1, 2 and 3 sirtuins, in the regulation of epigenetic phenomena.
The human sirtuin family comprises 7 proteins, very conserved throughout evolution, named SIRT1 to SIRT7.
SIRT6 is a nuclear sirtuin specifically associated with telomere chromatin and plays a role in the maintenance and stabilization of telomeric structures (Michishita et al. Nature. 2008 Mar. 27; 452(7186):492-6). Thus, in the mouse invalidated for the SIRT6 gene, premature aging and a short lifespan are observed, as well as an increase in the replicative senescence of keratinocytes (Kawahara T L et al. Cell. 2009 Jan. 9; 136(1):62-74).
Telomeres are structures that cover the ends of chromosomes and protect chromosomes against enzymatic degradation, recombination and interchromosomal fusion. In humans, these structures are constituted of a DNA sequence repeated thousands of times, associated with specific proteins, such as TRF1 and TRF2. Recent studies have shown that the TRF2 expression declines during cell aging (Amoyel et al., J. Invest. Dermatol. April 2009; 129 (Supplement 1s), s70).
On the other hand, SIRT6 plays an important role in DNA repair by bases excision, a DNA repair mechanism utilized by the cell when the DNA has been damaged by oxidants. These discoveries suggest that SIRT6 is necessary for regulating genome integrity and aging phenomena and may be directly involved in the increase of cellular longevity (Mostoslaysky et al., Cell. 2006 Jan. 27; 124(2):315-29).
It is known that the utilization of SIRT1 protein activating peptides (FR 2883751, FR 2883752, FR 2883753, FR 2883754), enables cosmetic or pharmaceutical compositions useful for protecting the skin and combating aging to be prepared, or else that certain SIRT7 inducer pharmaceutical compounds are useful for treating age-related diseases (EP 1955715). However, to date, no peptide compound capable of activating the SIRT6 protein in skin cells has been described, while the need for this type of skin care exists.
In one aspect, a peptide derived from the peptide sequence of highly conserved regions of human SIRT proteins is disclosed herein. The peptide has the general formula (I):
R1-(AA)n-X1—X2—X3—X4—X5—X6-(AA)p-R2 (I)
in which,
The peptide may correspond to one of the following sequences:
In another aspect, compositions were prepared that include the peptide discussed above.
The inventors have demonstrated that peptides derived from highly conserved regions of human SIRT proteins of the following general formula (I):
R1—(AA)n—X1—X2—X3—X4—X5—X6-(AA)p—R2 (I)
were very good SIRT6 activating agents, and would enable DNA degradation caused by external stresses and particularly by UV radiation to be prevented and/or effectively repaired, telomere maintenance to be improved and cellular senescence to be reduced. Consequently, these peptides are suitable for combating aging and photo aging of the skin.
Peptides according to the invention are characterized by the fact that they: (1) activate SIRT6 expression in skin cells; (2) reduce DNA degradation of skin cells subjected to UVB radiation; (3) promote the protection of skin cells subjected to oxidative stress; (4) stimulate the expression of TRF2 protein, specifically associated with telomeres; (5) increase the expression of proteins from the extracellular matrix by fibroblasts; and (5) optimize the barrier function of the epidermis.
“Peptide or SIRT6 activating active agent or active agent capable of activating human SIRT6” is understood to refer to any peptide of general formula (I) capable of increasing the quantity of SIRT6 present in the cell, or by increasing protein synthesis by direct or indirect modulation of the gene expression, or by other biological processes such as protein stabilization or else messenger RNA transcript stabilization.
“Skin” is understood to refer to all of the covering tissues constituting the skin, mucous membranes and epithelial appendages.
Alignment of peptide sequences of 7 proteins from the SIRT family was carried out by using the ClustalW2 multiple peptide sequence alignment program from the European Bioinformatics Institute presented in
“Highly conserved region of human SIRT proteins” is understood to refer to peptide sequences comprising at least 2 absolutely identical consecutive amino acids in 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 the His-Gly peptide sequence.
Thus, the first object of the invention is a peptide of 6 to 10 amino acids, derived from the peptide sequence of a highly conserved region of human SIRT proteins that responds to general formula (I):
R1-(AA)n-X1—X2—X3—X4—X5—X6-(AA)p-R2 (I)
in which, X1 is glycine or threonine or histidine, X2 is alanine or glutamine or glycine, X3 is glycine or asparagine or serine, X4 is valine or isoleucine or leucine, X5 is serine or aspartic acid or phenylalanine, X6 is alanine or glutamic acid or lysine, and when X1 is glycine then X2 is alanine and X3 is glycine, when X1 is threonine then X3 is asparagine, or when X1 is histidine then X2 is glycine, and AA represents any amino acid, or one of its derivatives, and n and p are integers between 0 and 2, and R1 represents the primary amino function of the N-terminal amino acid, free or substituted by an acyl type group having either an alkyl chain from C1 to C30, saturated or unsaturated, that may be an acetyl group, or an aromatic group that may be chosen from among a benzoyl, tosyl or benzyloxycarbonyl type group, and R2 represents the hydroxyl group of the carboxyl function of the C-terminal amino acid, free or substituted by a group that may be chosen from among an alkyl chain from C1 to C30, or an NH2, NHY or NYY group with Y representing an alkyl chain from C1 to C4.
Said sequence of general formula (I) is constituted of 6 to 10 residues of amino acids.
According to a particularly preferred embodiment of the invention, the peptide has the sequence:
According to a particularly interesting embodiment, the peptide corresponds to the SEQ ID No. 4 or to the SEQ ID No. 5.
According to another particularly interesting embodiment, the peptide corresponds to the SEQ ID No. 6 or to the SEQ ID No. 7.
The amino acids, constituting the peptide according to the invention and designated by the terms AA or X, may be under isomeric configuration L- and D-. Preferentially, the amino acids are in L form.
The term “peptide” designates a linkage of two or more amino acids interlinked by peptide linkages or by modified peptide linkages.
“Peptide” is also understood to refer to the natural or synthetic peptide of the invention as described, or at least one of its fragments, whether obtained by proteolysis or synthetically, or else any natural or synthetic peptide whose sequence is partially or totally constituted by the sequence of the peptide previously described.
The peptide derivatives particularly relate to amino acids interconnected by a pseudo-peptide linkage. “Pseudo-peptide linkage” is understood to refer to all types of linkages capable of replacing “conventional” peptide linkages.
So as to improve resistance to degradation, it may be necessary to use a protected form of the peptide according to the invention. Preferably, to protect the primary amine function of the N-terminal amino acid, a substitution by an R1 group of the acyl type having an alkyl chain from C1 to C30, saturated or unsaturated, that may be chosen from among an acetyl group or an aromatic group, may be utilized. Preferably, to protect the carboxyl function of the C-terminal amino acid, a substitution by an R2 group of the C1 to C30 alkyl chain type, or an NH2, NHY or NYY group with Y representing an alkyl chain from C1 to C4 is utilized. The peptide according to the invention may be protected at the region of the N-terminal end, C-terminal end or at the region of the two ends.
Thus, the invention relates to a composition such as previously defined, characterized by the fact that the peptide of SEQ ID No. 1 to SEQ ID No. 8 is in protected or unprotected form.
The peptide of general formula (I) according to the invention may be obtained either by conventional chemical synthesis (in solid phase or in homogeneous liquid phase), or by enzymatic synthesis (Kullman et al., J. Biol. Chem. 1980, 225, 8234), from constituent amino acids.
The peptide according to the invention may be of natural or synthetic origin. Preferentially, 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 peptide according to the invention is advantageously solubilized in one or more physiologically suitable solvents, such as water, glycerol, ethanol, propanediol, butylene glycol, dipropylene glycol, ethoxylated diethylene glycol or propoxylated diethylene glycol, cyclic polyols or any mixture of these solvents. The diluted peptide is then sterilized by sterile filtration.
After this dilution step, the peptide may be encapsulated or included in a cosmetic or pharmaceutical carrier such as liposomes or any other microcapsule utilized in the cosmetic field or adsorbed on powdery organic polymers, mineral supports such as talcs and bentonites.
“Physiologically suitable” is understood to mean that the solvent chosen is suitable for entering in contact with the skin without causing toxicity or intolerance reactions.
The peptide according to the invention may be utilized as a medication.
The second object of the invention is a cosmetic or pharmaceutical, in particular a dermatological composition comprising, in a physiologically suitable medium, a peptide of general formula (I) as a human SIRT6 activating active agent.
According to an advantageous embodiment of the invention, the active agent according to the invention is present in the compositions of the invention at a concentration of between approximately 10−9 M and 10−3 M, and preferentially at a concentration of between 2×10−8 M and 10−5 M with relation to the total weight of the final composition.
This range of concentrations represents the effective quantity of active agent corresponding to the quantity necessary to obtain the desired result, that is, to activate the SIRT6, reduce DNA degradation and improve telomere maintenance.
In a preferred manner, the composition according to the invention is present in a form suitable for topical application comprising a medium that is physiologically suitable for the skin. “Physiologically suitable” is understood to refer to media that are suitable for a use in contact with the skin or with human epithelial appendages, without risk of toxicity, incompatibility, instability, allergic response or other secondary effects.
“Topical application” is understood to refer to the act of applying or spreading the active agent according to the invention, or a composition containing the agent, to or on the surface of the skin.
The compositions intended to be applied on the skin may be present in the form of an aqueous or hydroalcoholic solution, water in oil emulsion or oil in water emulsion, microemulsion, aqueous or anhydrous gel, serum, or else vesicle dispersion, patch, cream, spray, ointment, pomade, lotions, colloid, solution, suspension or other forms.
These compositions may particularly be present in the form of an aqueous solution, hydroalcoholic or oily solution; an oil in water emulsion, water in oil emulsion or multiple emulsions. They may also be present in the form of creams, suspensions or else powders, suitable for application on 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, paste or foam. They may also be present in solid form, such as a stick, or may be applied on the skin in aerosol form. They may be utilized as a care product and/or as a skin makeup product.
In addition, any of the compositions disclosed herein may comprise any additive commonly utilized in the contemplated field of application as well as the adjuvants necessary for their formulation, such as co-solvents (ethanol, glycerol, benzyl alcohol, humectants, etc.), thickeners, diluents, emulsifiers, antioxidants, colorants, sunscreens, pigments, fillers, preservatives, fragrances, odor absorbers, essential oils, trace elements, essential fatty acids, surface active agents, film-forming polymers, chemical or mineral filters, moisturizing agents or thermal waters, etc. For example, one may include hydrosoluble polymers of the natural polymer type, such as polysaccharides or polypeptides, cellulosic derivatives of the methylcellulose type or hydroxypropylcellulose type, or else synthetic polymers, poloxamers, carbomers, siloxanes, PVA or PVP and particularly the polymers sold by the ISP company.
In all cases, the person skilled in the art will make sure that these adjuvants as well as their proportions are chosen so as to not harm the desired advantageous properties of the composition according to the invention. These adjuvants may, for example, be present at concentrations ranging from about 0.01 to about 20% of the total weight of the composition. When the composition of the invention is an emulsion, the fatty phase may represent from about 5 to about 80% by weight and preferably from about 5 to about 50% by weight with relation to the total weight of the composition. The emulsifiers and co-emulsifiers utilized in the composition will be chosen from among those conventionally utilized in the field under consideration. For example, they may be utilized in a proportion going from about 0.3 to about 30% by weight with relation to the total weight of the composition.
It is understood that the active agent according to the invention may be utilized alone or in combination with other active agents. Advantageously, the usable compositions according to the invention contain, also, at least one other active agent intended to promote the action of the active agent according to the invention and intended, in particular, for the prevention and/or treatment of age-related disorders. In a non-limiting manner, the following classes of ingredients may be cited: other peptide active agents, vegetable extracts, cicatrizant, anti-age, anti-wrinkle, smoothing, anti-radical, anti-UV agents, agents stimulating the synthesis of dermal macromolecules or energy metabolism, moisturizing, antibacterial, antifungal, anti-inflammatory, anesthetic agents, agents modulating cutaneous differentiation, pigmentation or depigmentation, agents stimulating nail or hair growth. In one embodiment, an anti-radical or antioxidant agent, or an agent stimulating the synthesis of dermal macromolecules, or else an agent stimulating energy metabolism will be utilized.
In another embodiment, the composition may comprise, in addition to the peptide disclosed herein, at least one cytochrome c activating compound, at least one moisturizing compound, such as an aquaporin activating compound, at least one sirtuin activating compound and in particular the peptides cited in patents FR 2 883754, US11/910,098, EP 1868631, incorporated by reference herein, at least one compound that increases cell adhesion, at least one compound that increases the production of matrix proteins such as collagen, fibronectin, laminin, mucopolysaccharide, at least one compound modulating proteasome activity, at least one compound modulating the circadian rhythm, at least one compound modulating HSP proteins, at least one compound that increases cell energy, at least one compound modulating skin pigmentation, at least one coenzyme Q10 activating compound, at least one compound improving the barrier function, such as transglutaminase activating compounds or HMG-CoA reductase activating compounds, at least one mitochondrial protector compound, at least one compound protecting or modulating the adult somatic cells of the epidermis or dermis, at least one compound protecting or repairing DNA degradation, and combinations thereof.
Said compounds above may be of natural origin, such as vegetable, animal or microorganism peptide hydrolysates, or else of synthetic origin, such as peptides.
Independently of their functions, the other active agents associated with the active agent in the composition may have very diverse chemical structures. In a non-limiting manner the other active agents may include other peptides, vitamin C and its derivatives, vitamins from group B, DHEA (dihydroepiandrosterone), phytosterols, salicylic acid and its derivatives, retinoids, flavonoids, sugar amines, azole compounds, metallic salts, peptide extracts of natural origin or else natural or synthetic polymers.
Another object of the invention is a pharmaceutical composition comprising, in a physiologically acceptable medium, the peptide according to the invention as a medication. The pharmaceutical composition according to the invention will improve dermatological symptoms connected with premature aging or photo aging, among which xerosis, depigmentation or conversely brown spots, keratosis, etc.
Advantageously, according to this form of the invention, the compositions will be suitable for oral administration for pharmaceutical use. Thus, the compositions may, in particular, be present in the form of tablets, capsules, gel capsules, chewable pastes, powders to consume as is or to be mixed immediately before use with a liquid, syrup, gel or any other form known to the person skilled in the art. They will contain suitable formulation excipients, such as colorants, sweeteners, flavorings, bulking agents, binders and preservatives.
The third object of the invention is a cosmetic composition comprising the peptide of general formula (I), as an active agent, to prevent and/or repair DNA degradation. “Active agent to prevent and/or repair DNA degradation” is understood to refer to a peptide capable of limiting DNA degradation or promoting the repair of damage due to photochemical reactions between DNA bases.
The fourth object of the invention is a cosmetic composition comprising the peptide of general formula (I), as an active agent, to improve telomere maintenance and reduce cellular senescence. “Active agent to improve telomere maintenance and reduce cellular senescence” is understood to refer to a peptide capable of increasing the synthesis of proteins specifically associated with telomeres and participating in their stability, such as TRF2 and SIRT6.
The fifth object of the invention is the utilization of a cosmetic composition comprising the peptide of general formula (I) as an active agent to increase the expression of keratinocyte differentiation markers and to promote the expression of extracellular matrix proteins by fibroblasts of the skin. These particular properties of the active agent according to the invention, improve the quality of the dermis and thus the firmness of the skin, and optimize the barrier function of the epidermis.
The sixth object of the invention is the utilization of a composition comprising the peptide of general formula (I), as an active agent, to protect the skin against all types of external stresses. The expression “external stresses” is understood to refer to stresses that the environment may produce. By way of example, such stresses may include pollution, UV radiation or else irritating products such as surface active agents, preservatives or fragrances, or mechanical stresses, such as abrasions, shaving or epilation. Pollution is understood to refer to both “external” pollution, due for example to diesel particles, ozone or heavy metals and to “internal” pollution, that may be particularly due to the emissions from paint, adhesive or wallpaper solvents (such as toluene, styrene, xylene or benzaldehyde), or else to cigarette smoke.
In particular, the object of the invention is the utilization of a cosmetic composition comprising an effective quantity of peptide according to the invention to prevent or treat damage caused to the skin by exposure to UV radiation and by oxidative stress.
The seventh object of the invention is a cosmetic treatment method characterized in that a composition comprising an effective quantity of active agent according to the invention is topically applied to the skin to be treated to prevent and/or treat cutaneous signs of aging and photo aging. “Cutaneous signs of aging” is understood to refer to any modifications in the external appearance of the skin and epithelial appendages due to aging such as, for example, superficial roughness of the horny layer of the epidermis, wrinkles and fine lines, but also any internal modification of the skin that is not systematically manifested in a modified external appearance such as, for example, thinning of the dermis or any other internal degradation of the skin following exposure to UV radiation. In particular, the invention relates to a cosmetic treatment method intended to protect the skin against stresses due to UV radiation.
Other advantages and characteristics of the invention will more clearly appear upon reading the examples given for illustrative and non-limiting purposes.
The object of this study is to determine the influence of peptides SEQ ID No. 5 and SEQ ID No. 7 on Sirtuin 6 expression in human skin. To do this, specific labeling by immunofluorescence was carried out on normal human keratinocytes (NHK) culture and on normal human fibroblast cultures.
Protocol: NHK or normal human fibroblasts are treated once per day with a solution at 10−6M or at 3×10−6M of peptide SEQ ID No. 5 or peptide SEQ ID No. 7.
Short-term treatment studies for 24, 48 and 72 hours were carried out.
Long-term treatment studies were also carried out between passage 5 and passage 17 (or 12 passages) for fibroblasts and between passage 3 and passage 5 (or 2 subcultures) for NHK.
For immunolabelling by anti SIRT6 antibodies, the cells are washed and fixed with paraformaldehyde at 3.7% for 10 minutes. The cells are then incubated in the presence of a specific anti SIRT6 antibody (Abeam, ref ab62738, polyclonal rabbit), and then a secondary suitable antibody, coupled with a fluorescent dye. After mounting in a particular medium, the slides are observed by epifluorescence microscope (Nikon Eclipse E 80i microscope). Fluorescence intensity is quantified by analyzing the image using the Image-Pro Analyser version 5 software.
Results: Under all the conditions tested, more intense fluorescence was observed in cultures treated by the peptide SEQ ID No. 5 and by the peptide SEQ ID No. 7 at 10−6M or at 3×10−6 M than under the control conditions. In the fibroblasts, a maximum increase of 47% of the fluorescence is observed in the cells treated for 24 hours by 10−6 M of peptide SEQ ID No. 5, with relation to the control cells (
Conclusions: Peptides SEQ ID No. 5 and SEQ ID No. 7 increase sirtuin 6 expression very significantly in normal human fibroblasts and NHK in short-term cultures. In addition, the sirtuin 6 expression stimulation effect is maintained for the long term.
The goal of this study is to determine the influence of peptide SEQ ID No. 4 on TRF2 protein expression in human skin, a protein specifically associated with telomeres and involved in their maintenance. To do this, specific labeling by immunofluorescence was carried out on normal human keratinocytes (NHK) culture and on normal human fibroblast cultures on a long-term basis.
Protocol: NHK or normal human fibroblasts in culture are treated once per day with a solution at 10−6M or at 3×10−6M of peptide SEQ ID No. 4, between passage 5 and passage 17 (or 12 passages) for fibroblasts and between passage 1 and passage 2 (or 1 passage and 10 days of treatment) for NHK.
For immunolabelling by anti TRF2 antibodies, the cells are washed and fixed with paraformaldehyde at 3.7% for 10 minutes. The cells are then incubated in the presence of a specific anti TRF2 antibody (Abeam, ref ab13579, polyclonal mouse), and then a secondary suitable antibody, coupled with a fluorescent dye. After mounting in a particular medium, the slides are observed by epifluorescence microscope (Nikon Eclipse E 80i microscope). Fluorescence intensity is quantified by analyzing the image using the Image-Pro Analyser version 5 software.
Results: Under all the conditions tested, more intense fluorescence was observed in cultures treated by the peptide SEQ ID No. 4 at 10−6 M or at 3×10−6 M than under the control conditions. In the fibroblasts, a maximum increase of 63% of the fluorescence is observed in the cells treated for 12 subcultures by 10−6 M of peptide SEQ ID No. 4, with relation to the control cells. The fluorescence increase is dose-dependent. In the NHK, a maximum increase of 39% of the fluorescence is observed in cells treated for 10 days by 3×10−6 M of peptide SEQ ID No. 4, with relation to the control cells. The fluorescence increase is dose-dependent.
Conclusions: Peptide SEQ ID No. 4 increases TRF2 protein expression very significantly in normal human fibroblasts and NHK, in a dose-dependent manner, in long-term cultures.
The goal of this study is to determine the influence of peptide SEQ ID No. 5 on epidermal differentiation. To do this, the expression of the main epidermal differentiation markers, specifically expressed in the keratinocytes of suprabasal layers cultivated on a long-term basis, was studied. The markers tested are transglutaminase 1 and involucrin.
Protocol: NHK in culture are treated once per day with a solution at 10−6M or at 3×10−6M of peptide SEQ ID No. 5, between passage 1 and passage 3 (or 2 passages and 11 days of treatment). The cells are then washed and fixed. After unmasking the specific sites, the cells are incubated in the presence of a specific antibody directed against TG1 (TEBU, ref sc-25786, polyclonal rabbit), a specific antibody directed against involucrin (Novocastra NCL-INV, mouse monoclonal, clone SYS), and then incubated in the presence of a suitable secondary antibody, coupled with a fluorescent dye. For greater ease of observation, the cell nuclei may be counterstained by DAPI (4′,6′ Di Amidino-2-Phenylindole), a fluorescent blue molecule capable of strongly bonding to DNA). After mounting in a particular medium, the slides are observed by epifluorescence microscope (Nikon Eclipse E 80i microscope).
Results: More intense fluorescence is observed in cultures and on the sections of skin treated by the peptide SEQ ID No. 5 at 10−6 M or at 3×10−6 M than under the control conditions.
Conclusions: Peptide SEQ ID No. 5 at 10−6 M or at 3×10−6 M improves NHK differentiation and this optimizes the barrier function of the epidermis.
The goal of this study is to determine the influence of peptide SEQ ID No. 5 on the expression of dermal extracellular matrix molecules. To do this, the expression of collagens I and III in normal human fibroblasts cultivated on a long-term basis was studied.
Protocol: Normal human fibroblasts are treated once per day with a solution at 10−6 M or at 3×10−6 M of peptide SEQ ID No. 5, between passage 5 and passage 17 (or 12 passages). The cells are then washed and fixed with cold methanol for 5 minutes. After unmasking specific sites, the cells are incubated in the presence of a specific antibody directed against collagen I (TEBU, ref 600-401-103, polyclonal rabbit) or against collagen III (TEBU, ref 600-401-105, polyclonal rabbit), and then incubated in the presence of a suitable secondary antibody, coupled with a fluorescent dye. After mounting in a particular medium, the slides are observed by epifluorescence microscope (Nikon Eclipse E 80i microscope).
Results: More intense fluorescence is observed in cultures and on the sections of skin treated by the peptide SEQ ID No. 5 at 10−6M or at 3×10−6M than under the control conditions.
Conclusions: Peptide SEQ ID No. 5 at 10−6M or at 3×10−6M applied on a long-term basis increases the expression of collagen I and collagen III, two essential proteins from the dermal extracellular matrix.
The goal of this study is to determine the protective effect of peptide SEQ ID No. 5 on damage caused to the DNA by UV radiation. To do this, a comet assay, that enables damage caused to the DNA at the cellular level to be quantified, was performed.
Protocol: Normal human fibroblasts are cultured for 24 hours with the peptide of sequence SEQ ID No. 5 at a concentration of 10−6M or 3×10−6M, and then irradiated with UVB radiation at a rate of 60 mJ/cm2, and then treated again for 24 hours by the peptide at a concentration of 10−6M or 3×10−6M. A control condition is carried out in the absence of treatment. The cells are then detached from their support by the trypsin, then centrifuged at 1200 rotations/min for 10 minutes in order to concentrate and count them.
A defined number of cells (25,000 cells) is then included in a Low Melting agarose gel at 0.75%, and then deposited on a glass slide previously covered with agarose at 1%. The slides are then immersed in a lysis solution for 1½ hours at 4° C., and then in an alkaline solution for 20 min at 4° C. The cells are thus lyzed and the DNA is denatured. The slides are immersed in an electrophoresis solution before applying an electrical field (20 V-250 mA). The DNA thus denatured is subjected to migration within the agarose gel at 4° C., for 30 min. The application of a DNA fluorescent dye, propidium iodide at 2 μg/ml, on the slides for 20 minutes enables the DNA, in the shape of comet tails if it has been damaged, to be observed with a microscope.
Quantification software enables the mean “Tail Moment” (or length of the comet tail) applied to each condition tested to be determined This parameter provides information on the level of DNA damage: the higher this parameter, the greater the DNA degradation.
Results: The results show a reduction of 24.8% of the Tail Moment when the cells are treated by the peptide of SEQ ID No. 5 at 3×10−6 M, compared to the control conditions.
Conclusion: The DNA of the cells treated and then subjected to UVB radiation has undergone less damage than the DNA of the control cells. These results confirm the preventive protector and curative effect of the peptide of sequence SEQ ID No. 5 in relation to UVB radiation.
The goal of this study is to determine the protective effect of peptide SEQ ID No. 5 on keratinocytes during oxidative stress. To do this, the expression of Sirtuin 6 was qualitatively and quantitatively evaluated by specific immunolabelling after oxidative stress by H2O2.
Protocol: The NHK in culture are treated for 24 hours with a solution at 10−6M or 3×10−6M of peptide SEQ ID No. 5. The cells are then incubated in the presence of H2O2 at 2 mM, rinsed and then treated for another 24 hours with a solution at 10−6M or 3×10−6M of peptide SEQ ID No. 5. A control that was not treated and not subjected to the H2O2 stress (control 0), as well as a control that was not treated but was subjected to the H2O2 stress (control 1) were carried out.
For immunolabelling by anti SIRT6 antibodies, the cells are washed and fixed with paraformaldehyde at 3.7% for 10 minutes. The cells are then incubated in the presence of a specific anti SIRT6 antibody (Abcam, ref ab62738, polyclonal mouse), and then a secondary suitable antibody, coupled with a fluorescent dye. After mounting in a particular medium, the slides are observed by epifluorescence microscope (Nikon Eclipse E 80i microscope). Fluorescence intensity is quantified by analyzing the image using the Image-Pro Analyser version 5 software.
Results: Quantitative analysis shows an increase of respectively 16% and 20% in SIRT6 expression when the NHK are treated by peptide SEQ ID No. 5 at 10−6M or 3×10−6 M, and subjected to a H2O2 stress, with relation to control 1.
Conclusions: The cells treated by peptide SEQ ID No. 5, preventively and subsequent to oxidative stress, have an SIRT6 protein content greater than that of the control cells. These results confirm that the peptide of sequence SEQ ID No. 5 promotes NHK protection during oxidative stress.
The constituents of phase A and phase B are heated separately between 70° C. and 75° C. Phase B is emulsified in phase A under stirring. Phase C is added at 45° C., by increasing the stirring. Phase D is then added when the temperature is below 40° C. The cooling is continued until 25° C. under intensive stirring.
Prepare and melt phase A at 65-70° C. Heat phase C to 65-70° C. Phase B is added to phase A just before emulsifying A into B. At approximately 45° C., the carbomer is neutralized by adding phase D. Phase E is then added under mild stirring and cooling is continued until 25° C. Phase F is then added if desired.
Prepare phase A and heat to 75° C. under stirring. Prepare phase B by dispersing the carbopol and then the xanthan gum under stirring. Let rest. Heat to 75° C. and then emulsify A into B under rotor stator stirring while maintaining the 75° C. Neutralize with phase C under rapid stirring. After cooling to 40° C., add phase D, and then phase E. Cooling is continued under mild stirring and phase F is added.
Applicants incorporate by reference the material contained in the accompanying computer readable Sequence Listing entitled “Bv_10_142_SEQII_ST25.txt”, which was created on Sep. 18, 2013, and is 27,581 bytes in size, and hereby confirm that the information recorded in the computer readable form is identical to the written sequence listing.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10 02698 | Jun 2010 | FR | national |
| Number | Name | Date | Kind |
|---|---|---|---|
| 5516507 | N'Guyen et al. | May 1996 | A |
| 20030166057 | Hildebrand et al. | Sep 2003 | A1 |
| Number | Date | Country |
|---|---|---|
| 1955715 | Aug 2008 | EP |
| 1868631 | Jul 2010 | EP |
| 2883751 | Oct 2006 | FR |
| 2883752 | Oct 2006 | FR |
| 2883753 | Oct 2006 | FR |
| 2883754 | Oct 2006 | FR |
| WO9012879 | Nov 1990 | WO |
| WO 2005066337 | Jul 2005 | WO |
| WO 2007104062 | Sep 2007 | WO |
| Entry |
|---|
| Rudinger, Peptide Hormones, JA Parsons, Ed., 1976, pp. 1-7. |
| SIGMA, 2004, pp. 1-2. |
| Berendsen, A Glimpae of the Holy Grail?, Science, 1998, 282, pp. 642-643. |
| Voet et al, Biochemistry, John Wiley & Sons Inc., 1995, pp. 235-241. |
| Ngo et al, Computational Complexity, Protein Structure Protection, and the Levinthal Paradox, 1994, pp. 491-497. |
| Bradley et al., Limits of Cooperativity in a Structurally Modular Protein: Response of the Notch Ankyrin Domain to Analogous Alanine Substitut-ons in Each Repeat, J. Mol. BIoL (2002) 324, 373-386. |
| Physical Changes with Aging, from Merck Manual, Jun. 2009, pp. 1-4, accessed Oct. 15, 2012. |
| Siegel, Are Telomeres the Key to Aging and Cancer? from http://learn.genetics.utah.edu/content/begin/traits/telomeres/, pp. 1-3, accessed Oct. 15, 2012. |
| Aubert et al, Telomeres and Aging, Physiol. Rev., 2008, 88, pp. 557-579. |
| Callaway, Telomerase reverses ageing process: Nature News, 2010, pp. 1-13. |
| Merck Manual Home Edition, Effects of Aging on the Skin, Oct. 2006, p. 1, accessed Apr. 9, 2012. |
| Chronic Effects of Sunlight, from Merck Manual, Aug. 2007, pp. 1-2, accessed Aug. 23, 2012. |
| Sequence listing of WO 2005/066337 A2, pp. 1-4, Jul. 2005. |
| Michishita et al., Nature, vol. 452, pp. 492-496 (Mar. 27, 2008). |
| Kawahara, T.L.A. et al., “SIRT6 Links Histone H3 Lysine 9 Deacetylation to NF-κB-Dependent Gene Expression and Organismal Life Span,” Cell, 136, pp. 62-74 (Jan. 9, 2009). |
| Amoyel et al., Journal of Investigative Dermatology, vol. 129 (Supplement 1s), p. S70 (2009). |
| Mostoslaysky, R. et al., Cell, 124, pp. 315-329 (Jan. 27, 2006). |
| Kullman et al., J. Biol. Chem., vol. 255, No. 17, pp. 8234-8238 (1980). |
| Number | Date | Country | |
|---|---|---|---|
| 20110318284 A1 | Dec 2011 | US |
