WO2012156419A2

WO2012156419A2 - Lmna, fnta and face-1 activators for preventing and/or attenuating skin ageing and/or hydrating skin

Info

Publication number: WO2012156419A2
Application number: PCT/EP2012/059060
Authority: WO
Inventors: Christelle Lasserre; Fedorova Elena
Original assignee: Chanel Parfums Beaute
Priority date: 2011-05-17
Filing date: 2012-05-15
Publication date: 2012-11-22
Also published as: WO2012156419A3

Abstract

The invention relates to the identification and the use of compounds which activate the expression of at least one gene selected from LMNA, FNTA and FACE-1, for preventing and/or attenuating ageing, and/or for hydrating skin. The invention thus relates to an in vitro method for screening for candidate compounds for preventing and/or attenuating ageing of the skin, and/or for hydrating the skin, comprising the following steps: a. bringing at least one test compound in contact with a sample of keratinocytes; b. measuring the expression of at least one gene selected from LMNA, FACE-1 and FNTA, in said keratinocytes; c. selecting the compounds for which an activation of at least 1.6 fold of the expression of at least one of said genes is measured in the keratinocytes treated in a. compared with the untreated keratinocytes.

Description

LMNA, FNTA AND FACE-1 ACTIVATORS FOR PREVENTING AND/OR

ATTENUATING SKIN AGEING AND/OR HYDRATTNG SKIN

The invention relates to the identification and the use of compounds which activate the expression of at least one gene selected from LMNA, FACE-1 (or ZMPSTE24) and FNTA genes, for preventing and/or attenuating ageing, particularly photo-ageing, and/or for hydrating skin.

Nuclear lamina is a meshwork of intermediate filaments localized under the inner nuclear membrane. A-type lamins are major proteins of the nuclear lamina that form a platform that acts as a scaffold for protein complexes that regulate nuclear structure and functions. A-type lamins are encoded by the LMNA gene. A-type lamins include major isoforms Lamins A and C, that are formed through alternative splicing of pre-mRNA encoded by LMNA gene. Precursor forms of lamin A undergo 4-step post-translational modifications involving farnesylation (thanks to a farnesyl-transferase encoded by the FNTA gene) and methylation of the terminal cysteine and double cleavage by ZMPSTE24 endoprotease (also called FACE-1 ) to generate mature Lamin A. A-type lamins are expressed in nearly all differentiated cell types including skin cells (Broers JL et al, Nuclear Lamins: laminpathies and their role in premature ageing. Physiol Rev 2006:86:967-1008; Andres V et al, Role of A-type lamins in signaling, transcription and chromatin organization. J Cell Biol 2009: 187:945-957; Hutchison CJ et al, Lamins: building blocks or regulators of gene expression? Nat Rev Mol Cell Biol 2002:3:848-858; Navarro CL et al, Molecular bases of progeroid syndromes, Hum Mol Genet 2006: 15 Spec No 2:R151 -161 ; De Vos WH et al, Increased plasticity of the nuclear enveloppe and hypermobility of telomeres due to the loss of A- type lamins, Biochim Biophys Acta: 1800:448-458).

A-type lamins contribute to the mechanical properties of the whole cell through their connection with cytoskeletal proteins. Lamins are also involved in mitosis, chromatin organization, DNA replication and repair, RNA transcription and splicing, and regulation of gene expression patterns through direct interactions with DNA, cell cycle progression, cell differentiation or cell migration (Olins AL et al, Cytoskeletal influences on nuclear shape in granulocytic HL-60 cells, BMC Cell Biol 2004:5:30; Verstraeten et al, The nuclear envelope, a key structure in cellular integrity and gene expression. Curr Med Chem 2007: 14: 1231-1248).

Mutations in LMNA or genes encoding proteins that affect its end products, like FACE-1 or FNTA, lead to diseases or laminopathies, like muscular dystrophies, lipid dystrophies, neuropathies, and the premature-ageing syndromes: restrictive dermopathy and Hutchinson-Gilford Progeria Syndrome (HGPS). The cause of premature-ageing syndromes is a defective maturation of prelamin A: activation of a cryptic splice site generates a truncated form of prelamin A (progerin), that lacks endoproteolytic cleavage site needed for correct lamin A maturation. Accumulation of progerin is toxic to cells (Navarro CL et al, Loss of ZMPSTE24 (FACE-1) causes autosomal recessive restrictive dermopathy and accumulation of Lamin A precursors, Hum Mol Genet 2005: 14: 1503-1513; Eriksson M et al, Recurrent de novo point mutations in lamin A cause Hutchinson-Gilford progeria syndrome, Nature 2003:423:293-298; Cao K et al, A lamin A protein isoform overexpressed in Hutchinson-Gilford progeria syndrome interferes with mitosis in progeria and normal cells, Proc Natl Acad Sci USA 2007: 104:4949- 4954).

Lamin mutations affect the physical integrity of nuclei and nucleo-cytoskeletal interactions, resulting in increased susceptibility to mechanical stress and altered gene expression (De Vos WH et al, Increased plasticity of the nuclear enveloppe and hypermobility of telomeres due to the loss of A-type lamins, Biochim Biophys Acta: 1800:448-458).

Lamin A plays important role in skin physiological ageing. Cell nuclei from old skin fibroblast cell lines acquire defects similar to those of HGPS patient cells, including changes in histone modifications and increased DNA damage (Scaffidi P et al, Lamin A-dependent nuclear defects in human ageing, Science 2006:312: 1059-1063).

Surprisingly, the applicant demonstrated that LMNA and FNTA expressions decrease in skin explants from donors of different age.

Normal aging has been shown to be associated with reduced expression and altered distribution of lamin A/C in nuclei of cardiomyocytes (Am J Physiol Heart Circ Physiol 293: H1451-H1456, 2007).

It is thus desirable and important to provide products or active agents which prevent, reduce or even inhibit the cellular senescence, particularly the keratinocyte senescence, more particularly UV-induced cell senescence.

The present invention thus provides a method for identifying such useful agents.

The present invention thus relates to an in vitro method for screening for candidate compounds for preventing and/or attenuating ageing of the skin, and/or for hydrating the skin, comprising the following steps: a. bringing at least one test compound in contact with a sample of keratinocytes ; b. measuring the expression of at least one gene selected from LMNA, ZMPSTE24 and FNTA, in said keratinocytes;

c. selecting the compounds for which an activation of at least 1.6 fold of the expression of at least one of said genes is measured in the keratinocytes treated in a. compared with the untreated keratinocytes.

According to a first embodiment, step b. is performed before and after step a. In this case, the expression of at least one gene selected from LMNA, ZMPSTE24 and FNTA, measured in the keratinocytes before step a. corresponds to the control value (i.e. untreated keratinocytes). Thus, step c. comprises the selection of the compounds for which an activation of at least 1.6 fold of the expression of at least one gene selected from LMNA, ZMPSTE24 and FNTA is measured in the keratinocytes treated in a. compared with the same keratinocytes before step a.

According to another embodiment, the method comprises a first step a', of preparing samples of keratinocytes. Thus, preferably, the present invention relates to an in vitro method for screening for candidate compounds for preventing and/or attenuating ageing of the skin, and/or for hydrating the skin, comprising the following steps:

a' . preparing at least two samples of keratinocytes ;

a. bringing one of the samples into contact with at least one test compound ; then b. measuring the expression of at least one gene selected from LMNA, ZMPSTE24 and FNTA, in said samples; and

c. selecting the compounds for which an activation of at least 1.6 fold of the expression of at least one of said genes is measured in the keratinocytes treated in a. as compared to the untreated keratinocytes.

In this second embodiment, the expression of at least one gene selected from LMNA, ZMPSTE24 and FNTA, measured in the sample of keratinocytes not submitted to step a. corresponds to the control value (i.e. untreated keratinocytes). By the expression "ageing of the skin" is intended any change in the external appearance of the skin due to ageing, preferably due to photo-induced ageing or photo-aging, such as, for example, wrinkles and fine lines, withered skin, flaccid skin, thinned skin, and skin lacking elasticity and/or tonus, and also any internal change in the skin which is not systematically reflected by a changed external appearance, such as, for example, any internal degradation of the skin, particularly of collagen, following exposure to ultraviolet radiation.

By "hydrating the skin", it is meant maintaining the natural humidity of the skin and preventing its drying, notably by improving skin cell mechanical properties and function, including improving barrier function.

The test compounds may be of any type. They may be of natural origin or may have been produced by chemical synthesis. This may involve a library of structurally defined chemical compounds, uncharacterized compounds or substances, or a mixture of compounds.

Natural compounds include compounds from animal or vegetal origin, like plants. Preferably, the test compounds are vegetal, preferably chosen from botanical extracts.

According to step a., the test compound is put into contact with a sample of keratinocytes.

According to step b., the expression of at least one gene selected from LMNA, ZMPSTE24 and FNTA is measured in said keratinocytes. Preferably, the expression of the LMNA gene is measured in step b.

The term "expression of at least one gene selected from LMNA, ZMPSTE24 and FNTA" is intended to mean the mRNA of the corresponding gene, or the corresponding protein. Said gene expression may thus be measured by quantifying the mRNA or the protein, for example by measuring the staining intensity and distribution. This is notably shown in examples 1, 2, 4 and 6.

Those skilled in the art are familiar with the techniques for quantitatively or semi- quantitatively detecting the mRNA of the gene of interest, and thus, determining said gene expression. Techniques based on hybridization of the mRNA with specific nucleotide probes are the most common, like Northern blotting, RT-PCR (reverse transcriptase polymerase chain reaction), quantitative RT-PCR (qRT-PCR).

The expression of at least one gene selected from LMNA, ZMPSTE24 and FNTA after treatment with the test compound is then compared to a control value, i.e. a value obtained in the same keratinocytes before treatment, or a value obtained in another sample of keratinocytes which are untreated. According to step c, the useful compounds are those for which an activation of at least 1.6 fold of the expression of at least one of said genes is measured in the keratinocytes treated in a. as compared to the untreated keratinocytes. Preferably, the activation of the expression of at least one of said genes is of at least 2 fold.

The compounds selected by means of the screening methods defined herein can subsequently be tested on other in vitro models and/or in vivo models (in animals or humans) for their effects on skin ageing and/or skin hydration. The useful compounds according to the invention are activators of the targeted LMNA, ZMPSTE24 and/or FNTA genes.

A subject of the invention is also the cosmetic use of an activator of the expression of at least one gene selected from LMNA, ZMPSTE24 and FNTA, which can be obtained according to the above described method, for preventing and/or attenuating ageing of the skin and/or for hydrating the skin.

According to another aspect, an objet of the present invention is the use of at least one activator of the expression of at least one gene selected from LMNA, ZMPSTE24 and FNTA, which can be obtained according to the above described method, to make a therapeutic composition for preventing and/or attenuating ageing of the skin and/or for hydrating the skin. The present invention thus also relates to the use of at least one activator of the expression of at least one gene selected from LMNA, ZMPSTE24 and FNTA, which can be obtained according to the above described method, for preventing and/or attenuating ageing of the skin and/or for hydrating the skin.

The activator refers to a compound which substantially increases the expression of at least one gene selected from LMNA, ZMPSTE24 and FNTA, or the expression of at least one of the corresponding proteins. The term "substantially" means an increase of at least 1.6 fold, preferably of at least 2 fold.

The activator can be used in a proportion of from 0.001 to 10% by weight, preferably in a proportion of from 0.01 to 5% by weight of the composition.

The activator may be chosen from small organic molecules, but may also be a botanical extract. The activators identified thanks to the screening method described above can be formulated within a composition, in combination with a physiologically acceptable carrier, preferably a cosmetically acceptable medium, i.e. a medium that is suitable for use in contact with human skin without any risk of toxicity, incompatibility, instability or allergic response and especially that does not cause any sensations of discomfort (redness, tautness, stinging, etc.) that are unacceptable to the user. These compositions may be administered, for example, orally, or topically. Preferably, the composition is applied topically. By oral administration, the composition may be in the form of tablets, gel capsules, sugar-coated tablets, syrups, suspensions, solutions, powders, granules, emulsions, suspensions of microspheres or nanospheres or lipid or polymeric vesicles for controlled release. By topical administration, the composition is more particularly for use in treating the skin and the mucous membranes and may be in the form of salves, creams, milks, ointments, powders, impregnated pads, solutions, gels, sprays, lotions or suspensions. It may also be in the form of suspensions of microspheres or nanospheres or lipid or polymeric vesicles or polymeric patches or hydrogels for controlled release. This composition for topical application may be in anhydrous form, in aqueous form or in the form of an emulsion. The composition for topical application may be in the form of an oil- in-water, water-in-oil or multiple emulsion (W/O/W or 0/W/O), which may optionally be microemulsions or nanoemulsions, or in the form of an aqueous dispersion, a solution, an aqueous gel or a powder. In a preferred variant, the composition is in the form of a gel, a cream or a lotion.

The physiologically acceptable carrier of the composition generally comprises water and optionally other solvents such as ethanol. This composition is preferably used as a care and/or cleansing product for facial and/or bodily skin and it may especially be in the form of a fluid, a gel or a mousse, conditioned, for example, in a pump-dispenser bottle, an aerosol or a tube, or in the form of cream conditioned, for example, in a jar. As a variant, it may be in the form of a makeup product and in particular a foundation or a loose or compact powder.

It may comprise various adjuvants, such as at least one compound chosen from:

- oils, which may be chosen especially from: linear or cyclic, volatile or nonvolatile silicone oils, such as polydimethylsiloxanes (dimethicones), polyalkylcyclosiloxanes (cyclomethicones) and polyalkylphenylsiloxanes (phenyl dimethicones); synthetic oils such as fluoro oils, alkylbenzoates and branched hydrocarbons such as polyisobutylene; plant oils and especially soybean oil or jojoba oil; and mineral oils such as liquid petroleum jelly;

- waxes such as ozokerite, polyethylene wax, beeswax or carnauba wax;

- silicone elastomers obtained especially by reaction, in the presence of a catalyst, of a polysiloxane containing at least one reactive group (especially hydrogen or vinyl) and bearing at least one alkyl group (especially methyl) or phenyl, in a terminal and/or side position, with an organosilicone such as an organohydrogenopolysiloxane;

- surfactants, preferably emulsifying surfactants, whether they are nonionic, anionic, cationic or amphoteric, and in particular fatty acid esters of polyols such as fatty acid esters of glycerol, fatty acid esters of sorbitan, fatty acid esters of polyethylene glycol and fatty acid esters of sucrose; fatty alkyl ethers of polyethylene glycol; alkylpolyglucosides; polysiloxane-modified polyethers; betaine and derivatives thereof; polyquaterniums; ethoxylated fatty alkyl sulfate salts; sulfosuccinates; sarcosinates; alkyl and dialkyl phosphates, and salts thereof; and fatty acid soaps;

- co- surfactants such as linear fatty alcohols and in particular cetyl alcohol and stearyl alcohol;

- thickeners and/or gelling agents, and in particular crosslinked or non-crosslinked, hydrophilic or amphiphilic homopolymers and copolymers, of acryloylmethylpropanesulfonic acid (AMPS) and/or of acrylamide and/or of acrylic acid and/or of acrylic acid salts or esters; xanthan gum or guar gum; cellulose derivatives; and silicone gums (dimethiconol);

- organic screening agents, such as dibenzoylmethane derivatives (including butyl- methoxydibenzoylmethane), cinnamic acid derivatives (including ethylhexyl methoxycinnamate), salicylates, para-aminobenzoic acids, β,β'-diphenyl acrylates, benzophenones, benzylidenecamphor derivatives, phenylbenzimidazoles, triazines, phenylbenzotriazoles and anthranilic derivatives;

- inorganic screening agents, based on mineral oxides in the form of coated or uncoated pigments or nanopigments, and in particular based on titanium dioxide or zinc oxide;

- dyes;

- preserving agents;

- sequestrants such as EDTA salts;

- fragrances; - and mixtures thereof, without this list being limiting.

Examples of such adjuvants are especially mentioned in the CTFA dictionary (International Cosmetic Ingredient Dictionary and Handbook published by The Cosmetic, Toiletry and Fragrance Association, 11th edition, 2006), which describes a wide variety, without limitation, of cosmetic and pharmaceutical ingredients usually used in the skincare industry, that are suitable for use as additional ingredients in the compositions according to the present invention.

The composition may also comprise at least one compound with an optical effect such as fillers, pigments, nacres, tensioning agents and matting polymers, and mixtures thereof.

The term "fillers" should be understood as meaning colorless or white, mineral or synthetic, lamellar or non-lamellar particles suitable for giving the composition body or rigidity and/or softness, a matt effect and uniformity immediately on application. Fillers that may especially be mentioned include talc, mica, silica, kaolin, Nylon® powders such as Nylon- 12 (Orgasol® sold by the company Atochem), polyethylene powders, polyurethane powders, polystyrene powders, polyester powders, optionally modified starch, silicone resin microbeads such as those sold by the company Toshiba under the name Tospearl®, hydroxyapatite, and hollow silica microspheres (Silica Beads® from the company Maprecos).

The term "pigments" should be understood as meaning white or colored, mineral or organic particles that are insoluble in the medium, which are intended to color and/or opacify the composition. They may be of standard or nanometric size. Among the mineral pigments that may be mentioned are titanium dioxide, zirconium dioxide and cerium dioxide, and also zinc oxide, iron oxide and chromium oxide.

The term "nacres" should be understood as meaning iridescent particles that reflect light. Among the nacres that may be envisaged, mention may be made of natural mother-of-pearl, mica coated with titanium oxide, with iron oxide, with natural pigment or with bismuth oxychoride, and also colored titanium mica.

The mass concentration in the aqueous phase of these fillers and/or pigments and/or nacres is generally from 0.1% to 20% and preferably from 0.2% to 7% by weight relative to the total weight of the composition. The term "tensioning agent" should be understood as meaning a compound suitable for making the skin taut and, by means of this tension effect, making the skin smooth and reducing or even immediately eliminating wrinkles and fine lines therefrom. Tensioning agents that may be mentioned include polymers of natural origin. The term "polymer of natural origin" means polymers of plant origin, polymers derived from integuments, egg proteins and latices of natural origin. These polymers are preferably hydrophilic. Polymers of plant origin that may especially be mentioned include proteins and protein hydrolyzates, and more particularly extracts of cereals, of legumes and of oil-yielding plants, such as extracts of corn, of rye, of wheat, of buckwheat, of sesame, of spelt, of pea, of bean, of lentil, of soybean and of lupin. The synthetic polymers are generally in the form of a latex or a pseudolatex and may be of polycondensate type or obtained by free-radical polymerization. Mention may be made especially of polyester/polyurethane and polyether/polyurethane dispersions. Preferably, the tensioning agent is a copolymer of PVP/dimethiconyl acrylate and of hydrophilic polyurethane (Aquamere S-2001 ® from the company Hydromer) .

The term "matting polymers" means herein any polymer in solution, in dispersion or in the form of particles, which reduces the sheen of the skin and which unifies the complexion. Examples that may be mentioned include silicone elastomers; resin particles; and mixtures thereof. Examples of silicone elastomers that may be mentioned include the products sold under the name KSG® by the company Shin-Etsu, under the name Trefil®, BY29® or EPSX® by the company Dow Corning or under the name Gransil® by the company Grant Industries.

The composition used according to the invention may also comprise active agents other than the activator, and in particular at least one active agent chosen from: agents that stimulate the production of growth factors; anti-glycation or deglycating agents; agents that increase collagen synthesis or that prevent its degradation (anti-collagenase agents and especially matrix metalloprotease inhibitors); agents that increase elastin synthesis or prevent its degradation (anti- elastase agents); agents that stimulate the synthesis of integrin or of focal adhesion constituents such as tensin; agents that increase the synthesis of glycosaminoglycans or proteoglycans or that prevent their degradation (anti-pro teoglycanase agents); agents that increase fibroblast proliferation; depigmenting or anti-pigmenting agents; antioxidants or free-radical scavengers or anti-pollution agents; and mixtures thereof, without this list being limiting. Examples of such agents are especially: plant extracts and in particular extracts of Chondrus crispus, of Thermus thermophilus, of Pisum sativum (Proteasyl® TP LS), of Centella asiatica, of Scenedesmus, of Moringa pterygosperma, of witch hazel, of Castanea sativa, of Hibiscus sabdriffa, of Polianthes tuberosa, of Argania spinosa, of Aloe vera, of Narcissus tarzetta, or of liquorice; an essential oil of Citrus aurantium (Neroli); cc-hydroxy acids such as glycolic acid, lactic acid and citric acid, and esters thereof; β-hydroxy acids such as salicylic acid and derivatives thereof; plant protein hydrolyzates (especially of soybean or of hazelnut); acylated oligopeptides (sold especially by the company Sederma under the trade names Maxilip®, Matrixyl® 3000, Biopeptide® CL or Biopeptide® EL); yeast extracts and in particular of Saccharomyces cerevisiae; algal extracts and in particular of laminairia; vitamins and derivatives thereof such as retinyl palmitate, ascorbic acid, ascorbyl glucoside, magnesium or sodium ascorbyl phosphate, ascorbyl palmitate, ascorbyl tetraisopalmitate, ascorbyl sorbate, tocopherol, tocopheryl acetate and tocopheryl sorbate; arbutin; kojic acid; ellagic acid; and mixtures thereof. As a variant or in addition, the composition used according to the invention may comprise at least one elastase inhibitor (anti-elastase), such as an extract of Pisum sativum seeds that is sold especially by the company Laboratoires Serobiologiques/Cognis France under the trade name Proteasyl TP LS®. The composition may also contain inert additives or combinations of these additives, such as wetting agents, stabilizers, moisture regulators, pH regulators, osmotic pressure modifiers, or UV-A and UV-B screens.

The following examples illustrate the invention without limiting the scope thereof.

EXAMPLES

Example 1: Test of the expression of LMNA in human keratinocytes

Protocol: The expression of the messenger RNA (mRNA) of LMNA was evaluated in cultured human keratinocytes by PCR and the expression of the LMNA protein was evaluated in keratinocytes in human skin samples and reconstructed human skin equivalents (SE). Keratinocytes derived from neonatal foreskins (Cascade Biologies/ Invitrogen, Portland, OR, USA) were placed in 6-well plates and cultured in keratinocyte growth culture medium with supplement (EpiLife, Invitrogen). After culturing for 24 hours in an incubator at 37°C, the medium was changed to basal keratinocyte growth culture medium, and cells were cultured for additional 24 hours. The 70 % confluent cells were washed with PBS buffer (Invitrogen) and then used for mRNA extraction.

The mRNA was isolated using the Qiagen RNeasy kit (Qiagen, CA) and quantified using the Quantlt kit (Invitrogen, CA). To quantify the expression level of the mRNA of LMNA in cultured keratinocytes, real-time reverse transcription polymerase chain reaction (RT-PCR) was used. The results were normalized relative to the expression of housekeeping genes of these samples. The results were expressed in terms of the number of times of change of expression level of the target gene LMNA.

The LMNA PCR primers were obtained from Invitrogen (Invitrogen, CA).

Housekeeping gene was RPLPO. The RPLPO PCR primers were obtained from Invitrogen (Invitrogen, CA).

Reverse transcription was performed using the gene Amp RNA PCR kit (Invitrogen) according to the manufacturer's recommendations.

The real-time PCR measurement was performed using the iCYCLER IQ machine (Bio-rad, CA) with Taqman probes.

In all the tests, the cDNA was amplified using a standardized program. Each sample was charged with Taqman master-mix, Taqman primers, probes, and water. The final amount of cDNA per reaction corresponded to 75 ng of total RNA used for the reverse transcription. The relative quantification of the expression of the target gene was performed using the Pfaffl mathematical model (Pfaffl, MW, Nucleic Acids Res. 29(9), p. E45, 2001). The results were confirmed using cells from two different donors.

The expression of the LMNA protein was evaluated in reconstructed human SE.

The SEs were prepared as follows: a collagen solution containing type I collagen from rat tail (BD, CA), 10X DMEM medium (Invitrogen, CA), sodium bicarbonate (Invitrogen) and fibroblasts was poured into 24mm cell culture inserts (Falcon, Becton Dickinson, Schwechat, Austria), which were placed in six- well plates (Falcon). After two hours at 37 °C, the gels were equilibrated in keratinocyte growth culture medium with supplement (EpiLife, Invitrogen) at 37°C in an environment containing 5% C0₂/95 air, in a humidified incubator. After two hours, keratinocyte growth culture medium with supplement (EpiLife, Invitrogen), containing keratinocytes was added to the gel. After immersing the culture overnight, the medium was replaced with serum-free keratinocyte medium (SKDM, which is a medium rich in Ca²⁺ consisting of keratinocyte growth culture medium (EpiLife, Invitrogen) without bovine pituitary extract, with addition of transferrin (Sigma), BSA (Sigma) and L-ascorbic acid (Sigma) outside the insert, and the keratinocytes were maintained at the air-liquid interface. The culture medium of the reconstructed skins was replaced every two days with preheated fresh SKDM, and culturing was continued for up to seven days.

The expression of the LMNA protein was evaluated on frozen and paraffin-embedded skin samples from different donors (ZenBio inc. NC, USA; ProSci Inc., CA, USA; and Cybrdi, MD, USA) as well as on frozen and paraffin-embedded SE samples by immunohistochemistry (IHC),. Staining was performed on 6 μιη sections with anti-human LMNA rabbit antibodies (Sigma, MO, USA) and secondary goat anti-rabbit antibodies (Thermo Scientific, CA, USA). The staining was visualized using the AEC system (Thermo Scientific). Staining was assessed on 2-6 sections each from 3-7 donors.

Results: Evaluation of expression of the LMNA mRNA in keratinocytes from a single donor show that LMNA is expressed in normal human cultured keratinocytes. The data were confirmed in two donors of keratinocytes. Evaluation of LMNA IHC staining in human skin showed intense nuclear staining in the keratinocytes of the skin epidermis and SE.

It emerges from this test that LMNA is expressed in normal human epidermal keratinocytes.

Example 2: Assessment of the expression of LMNA protein with age

Protocol: The variation in expression of the LMNA protein was evaluated by IHC, on paraffin-embedded skin samples from donors of various age groups (ZenBio, NC, USA). Staining was performed on 6 μιη sections from donors in 3 age groups (21-30, 31-50, and 51-70 years old), with anti-LMNA antibodies (Sigma, MO, USA) and secondary antibodies (Thermo Scientific, CA, USA). The staining was visualized using the AEC system (Thermo Scientific). Staining was assessed on 2-6 images each from 3 donors in each age group by doing a blind visual assessment of the intensity of staining using a scale from 1 to 5 (1 = least intense, 5 = most intense). Mean values of ratings were compared for significance using the unpaired t-test.

Results:

Evaluation of LMNA staining in young adult skin (21-30 yrs old) showed intense nuclear staining in the epidermis which translated to a rating of 4.6- (±0.48). The intensity of staining then visibly decreased in sections of elder donor skin (31-50) and especially at 51-70 years of age, where staining intensity was rated 3.5 (±0.05) and 1.7 (±0.06), respectively. It emerges from this test that expression of LMNA protein is significantly diminished in epidermal keratinocytes of skin samples with increasing age. Example 3: Test of stimulation of the expression of the mRNA of LMNA in normal human keratinocytes with an active agent

Protocol:

The effect of Lipoic acid on the expression of the mRNA of LMNA was evaluated in cultured human keratinocytes by PCR.

Keratinocytes derived from neonatal foreskins (Invitrogen, CA, USA) were cultured in 6-well plates in keratinocyte culture medium with supplement (EpiLife, Invitrogen). After culturing for 24 hours at 37°C, the 40-50% confluent cells were washed with PBS buffer (Invitrogen, CA) and incubated with basic keratinocytes culture medium (EpiLife, Invitrogen) containing the active agent to be tested, for 24 hours, at increasing concentrations. The cytotoxicity of the active agent was evaluated in human cultured keratinocytes before testing the activity.

A test similar to that of Example 1 was performed.

Results:

Results are confirmed in two donors of keratinocytes. The representative results from one donor are given in Table 1 below:

Table 1

0.0005% 2.15 0.07

0.00025% 1.66 0.03

⁽ the concentrations of the active agent is expressed as the weight of active agent per weight of preparation It emerges from this test that the active agent tested makes it possible to significantly stimulate the expression of mRNA of LMNA in normal human keratinocytes.

Example 4: Test of stimulation of the expression of LMNA protein in normal human keratinocytes with Lipoic acid

Protocol:

The effect of the Lipoic acid on the expression of the LMNA protein was evaluated in cultured human keratinocytes.

Keratinocytes derived from neonatal foreskin (Invitrogen, CA, USA) were cultivated in 6-well plates containing cover glasses coated with poly-L-ornithine (Sigma, MO) in keratinocyte culture medium with supplement (EpiLife, Invitrogen). After culturing for 24 hours at 37°C, the 40-50% confluent cells were washed with PBS buffer (Invitrogen, CA) and incubated with basal keratinocytes culture medium (EpiLife, Invitrogen) containing the extract to be tested, for 48 hours. The cytotoxicity of the extract was evaluated in human cultured keratinocytes before testing the activity. To quantify the expression of the LMNA protein in a treated sample relative to an untreated sample, immunocytochemical staining (ICC) of cover glasses with cultured keratinocytes was used. Staining was performed in triplicates, with primary anti-human LMNA rabbit antibodies (Sigma, MO, USA) and secondary goat anti-rabbit antibodies R-phycoerythrin conjugated (Invitrogen, OR). The extent of staining was assessed on thirty random images for each experimental condition, and a visual assessment of the staining was made using a scale from 1 to 5, with 1 being the least intense and 5 being the most intense. The significance of the difference between mean values was assessed using unpaired t test.

Results:

Evaluation of LMNA staining in keratinocytes treated with 0.001% Lpoic acid was 3.00 (±0.53), in keratinocytes treated with 0.0005% Lipoic acid it was 3.93 (±0.78), in keratinocytes treated with 0.00025% Lipoic acid it was 3.5 (±0.51) and in non-treated control keratinocytes it was 2.93 (±0.37). The difference of LMNA staining in untreated and in keratinocytes treated with 0.005% Lipoic acid was significant (p<0.0001). This demonstrates that the expression of LMNA protein is significantly increased with 0.005% Lipoic acid treatments. The data presented are from one donor of keratinocytes. Results are confirmed in two donors.

The results are given in Table 2 below: Table 2

⁽ the concentration of the extract is expressed as the weight of crude extract per weight of preparation.

It emerges from this test that Lipoic acid make it possible to stimulate the expression of LMNA protein in normal human keratinocytes. Example 5: Test of the expression of FNTA in human keratinocytes

Protocol: The expression of the mRNA of FNTA was evaluated in cultured human keratinocytes by PCR and the expression of the FNTA protein was evaluated in keratinocytes in human skin samples and reconstructed human SE.

Keratinocytes derived from neonatal foreskins (Cascade Biologies/ Invitrogen, Portland, OR, USA) were placed in 6- well plates and cultured in keratinocyte growth culture medium with supplement (EpiLife, Invitrogen). After culturing for 24 hours in an incubator at 37°C, the medium was changed to basal keratinocyte growth culture medium, and cells were cultured for additional 24 hours. The 70 % confluent cells were washed with PBS buffer (Invitrogen) and then used for mRNA extraction.

The test similar to that in the Example 1 was used.

The FNTA PCR primers were obtained from Invitrogen (Invitrogen, CA). Housekeeping gene was RPLPO. The RPLPO PCR primers were obtained from Invitrogen (Invitrogen, CA).

The expression of the FNTA protein was evaluated in reconstructed human SE. The SE were prepared similar to that in Example 1. The expression of the FNTA protein was evaluated by IHC, on frozen skin samples from different donors (ZenBio inc. NC, USA) as well as on frozen SE samples. Staining was performed on 6 μιη sections with anti-human FNTA rabbit antibodies (Sigma, MO, USA) and secondary goat anti-rabbit antibodies (Thermo Scientific, CA, USA). The staining was visualized using the AEC system (Thermo Scientific). Staining was assessed on 2-6 sections each from 3-5 donors.

Results: Evaluation of expression of the FNTA mRNA in keratinocytes from a single donor show that FNTA is expressed in normal human cultured keratinocytes. The data were confirmed in two donors of keratinocytes. Evaluation of FNTA staining in human skin and SE showed nuclear and cytoplasmic staining in the epidermal keratinocytes.

Example 6: Assessment of the expression of FNTA protein with age Protocol:

The variation in expression of the FNTA protein with age was evaluated on frozen skin samples from donors of various age groups (ZenBio, NC, USA) by IHC. Staining was performed on 6 μιη sections from donors in 3 age groups (21-30, 31-50, and 51-70 years old), with anti-FNTA antibodies (Sigma, MO, USA) and secondary antibodies (Thermo Scientific, CA, USA). The staining was visualized using the AEC system (Thermo Scientific). Staining was assessed on 2-6 images each from 3 donors in each age group by doing a blind visual assessment of the intensity of staining using a scale from 1 to 5 (1 = least intense, 5 = most intense). Mean values of ratings were compared for significance using the unpaired t-test.

Results:

Evaluation of FNTA staining in young adult skin (21-30 yrs old) showed intense cytoplasmic and nuclear staining in the epidermis which translated to a rating of 4.75 (±0.43). The intensity of staining then visibly decreased in sections of elder donor skin (31-50) and especially at 51-70 years of age, where staining intensity was rated 3.4 (±0.91) and 0.95 (±0.74), respectively.

It emerges from this test that expression of FNTA protein is significantly diminished with increasing age. Example 7: Test of the expression of FACE-1 in human keratinocytes

Protocol: The expression of the mRNA of FACE-1 was evaluated in cultured human keratinocytes by PCR.

Keratinocytes derived from neonatal foreskins (Cascade Biologies/ Invitrogen, Portland, OR, USA) were placed in 6-well plates and cultured in keratinocyte growth culture medium with supplement (EpiLife, Invitrogen). After culturing for 24 hours in an incubator at 37°C, the medium was changed to basal keratinocyte growth culture medium, and cells were cultured for additional 24 hours. The 70 % confluent cells were washed with PBS buffer (Invitrogen) and then used for mRNA extraction.

The test similar to that in the Example 1 was used.

The FACE-1 PCR primers were obtained from Invitrogen (Invitrogen, CA). Housekeeping gene was RPLPO. The RPLPO PCR primers were obtained from Invitrogen (Invitrogen, CA).

Results:

Evaluation of expression of the FACE-1 mRNA in keratinocytes from a single donor show that FACE-1 is expressed in normal human cultured keratinocytes. The data were confirmed in two donors of keratinocytes.

It emerges from this test that FACE-1 is expressed in normal human epidermal keratinocytes.

Example 8 : Cosmetic composition (O/W serum)

The following composition may be prepared in a classical manner for the man skilled in the art.

Claims

1. In vitro method for screening for candidate compounds for preventing and/or attenuating ageing of the skin, and/or for hydrating the skin, comprising the following steps:

a. bringing at least one test compound in contact with a sample of keratinocytes ; b. measuring the expression of at least one gene selected from LMNA, ZMPSTE24 and FNTA, in said keratinocytes;

2. Method according to Claim 1, characterized in that step b. is performed before and after step a.

3. Method according to Claim 1, characterized it comprises the following steps:

a' . preparing at least two samples of keratinocytes ;

4. Method according to any one of Claims 1 to 3, characterized in that the expression of at least one gene selected from LMNA, ZMPSTE24 and FNTA is measured by quantifying the mRNA of the corresponding gene.

5. Method according to any one of Claims 1 to 3, characterized in that the expression of at least one gene selected from LMNA, ZMPSTE24 and FNTA is measured by quantifying the activity of the corresponding protein.

6. Method according to any one of Claims 1 to 5, characterized in that the expression of the LMNA gene is measured in step b.

7. Method according to any one of Claims 1 to 6, characterized in that the test compounds are chosen from botanical extracts.

8. Method according to any one of Claims 1 to 7, characterized in that the activation of the expression of at least one of said genes measured in step c. is of at least 1.6 fold as compared to untreated keratinocytes.

9. Method according to any one of Claims 1 to 8, for screening for candidate compounds for preventing and/or attenuating photo-ageing.

10. Cosmetic use of an activator of the expression of at least one gene selected from LMNA, ZMPSTE24 and FNTA, which can be obtained according to the method of any one of Claims 1 to 9, for preventing and/or attenuating ageing of the skin and/or for hydrating the skin.

11. Activator of the expression of at least one gene selected from LMNA, ZMPSTE24 and FNTA, which can be obtained according to the method of any one of Claims 1 to 9, for preventing and/or attenuating ageing of the skin and/or for hydrating the skin.