Agents for restoring or maintaining skin homeostasis

Abstract

The present invention relates to the cosmetic field, in particular to cosmetic agents able to restore or maintain cutaneous homeostasis. More precisely, the invention relates to the cosmetic use of an agent able to increase synaptopodin expression in keratinocytes for improving, restoring or maintaining cutaneous homeostasis, preferably epidermal homeostasis, in a healthy skin. The invention also relates to cosmetic compositions comprising the same as well as cosmetic methods using the same.

Description

[0001] AGENTS FOR RESTORING OR MAINTAINING SKIN HOMEOSTASIS

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to the cosmetic field, in particular to cosmetic agents able to restore or maintain skin homeostasis.

[0004] BACKGROUND OF THE INVENTION

[0005] The skin is the first barrier protecting the body from outer aggressions. This organ is formed of several tissue layers, namely the epidermis, which is the outermost portion of the skin, the dermis, a connective tissue consisting of fibroblasts and an extracellular matrix (ECM), which ensures the functions of cohesion and nutrition of the skin, and the hypodermis consisting of adipocytes.

[0006] Skin ageing leads to histological changes in the skin. The epidermis becomes thinner and the dermal-epidermal junction (DEJ) flattens, while the density of the underlying dermis decreases. In addition, the expression of components in the ECM such as type I and III collagens and elastin decreases while the synthesis and the activity of matrix metalloproteinases (MMPs) increase (Varani et al., Am J Pathol, 2006; 168(6): 1861-8). These alterations result in an age- related reduction of skin elastic modulus: mechanical measurements made on the human skin at different ages showed a reduction in skin elastic modulus overtime from 10 kPa for a 30 year old skin to 5.3 kPa for a 80 year old skin. In vitro studies showed that keratinocytes respond differently according to the rigidity of the culture support : a hard support promotes proliferation and migration whereby re-epithelization is induced whereas cell differentiation is inhibited. In contrast, primary human keratinocytes respond to a soft substrate by a decrease in adhesion focal points and proliferation and a rapid commitment in terminal differentiation. The morphology of the cells is also changed, with a decrease in cell spread and focal adhesions (Pailler-Mattei et al., J Meeh Behav Biomed Mater. 2013;28:474-83) .

[0007] Of note, Ya etal. (Archives of Dermatological Research, 2019, 311 (10), pp.741-751) show that such a terminal differentiation of keratinocytes on a soft substrate is reversible suggesting that keratinocytes are able to adapt their behavior (proliferation, migration and differentiation) in response to mechanical changes in their microenvironment.

[0008] These studies strongly suggest that age-related changes in dermal elasticity is likely to result in keratinocyte phenotypic disturbance leading to an alteration of epidermal homeostasis characterized by a decrease in cell regeneration and an increase in cell differentiation, and as a result by an alteration of the epidermis quality, and thus an alteration of the skin aspect. Thus, there is a need for cosmetic agents able to counterbalance the effect of age-related decrease in dermal stiffness and / or rebalance keratinocyte response to dermal stiffness.

[0009] SUMMARY OF THE INVENTION

[0010] The invention relates to the cosmetic use of an agent able to increase synaptopodin expression in keratinocytes for improving, restoring or maintaining cutaneous homeostasis, preferably epidermal homeostasis in a healthy skin.

[0011] In some embodiments, the agent is able to increase the expression of synaptopodin in keratinocytes on a soft hydrogel as compared to keratinocytes cultured on a soft hydrogel but not contacted with the agent.

[0012] In an embodiment, the agent is selected by a method of selection as described herein.

[0013] In some embodiments, the agent is used for stimulating or maintaining the regeneration of the epidermis, preferably for promoting keratinocyte proliferation and / or for slowing-down keratinocyte differentiation in the skin.

[0014] In some embodiments, the agent is for improving the visual appearance of the skin, in particular for attenuating, decreasing or treating one or more non-pathological alterations of the skin appearance such as fine lines, wrinkles, a loss of skin density, a loss of skin firmness, a loss of skin elasticity, an increase in skin roughness, a loss of radiance or glow, dull or non- uniform complexation and combinations thereof.

[0015] In some embodiments, the agent of the Invention is for decreasing, slowing down, or delaying the onset of keratinocyte differentiation, and / or for decreasing the expression of a differentiation marker and / or for promoting the production of procollagen type I and / or for maintaining or restoring dermal extracellular matrix, in a skin, such as in a aged skin.

[0016] In some embodiments, the agent is applied on the skin of a subject having at least 40 years old, preferably a woman of at least 40 years old. In some embodiments, the subject is a peri- menopausal or menopausal woman.

[0017] In some embodiments, the agent is used to prevent, slow-down or delay the onset of a decrease in collagen production, a decrease in dermal extracellular matrix quality, a loss of skin density and / or a loss of skin firmness in a subject of at most 40 years old.

[0018] Preferably, the agent is present as an active cosmetic ingredient in a cosmetic composition which is applied on the skin. The cosmetic composition may be of any type. The composition can be selected among skin care products, skin hygiene products or make-up products such as a cream e.g. a cream, a balm, an ointment, a serum, a gel, a mask, and a foundation. In a particular embodiment, the agent is selected from peptides and plant extracts, preferably a Vitis vinifera grape extract.

[0019] In some embodiments, the plant extract is an aqueous extract from white Vitis vinifera grapes said grapes being harvested after being frozen on the vine. Preferably, the plant extract is prepared from pomace obtained from Vitis vinifera grapes harvested after being naturally frozen on the vine

[0020] In a particular embodiment, the plant extract is obtained from ice wine grape pomace of Vidal variety. Said plant extract can be obtained by a method comprising a step of extracting the pomace with hot water e.g. at a temperature of at least 60°C such as under reflux.

[0021] The Vitis vinifera grape extract as described herein can have a total polyphenol content lower than 3.0%, preferably lower than 2.5% such as lower than 2.0% of the dry matter of the extract, the percentage being expressed in gallic acid equivalent.

[0022] The invention also relates to a cosmetic composition for improving, restoring or maintaining cutaneous homeostasis in a healthy skin comprising (i) an aqueous extract from Vitis vinifera as described herein as an active cosmetic ingredient and (ii) at least one cosmetically acceptable excipient.

[0023] The invention further relates to a cosmetic method for improving, restoring or maintaining cutaneous homeostasis in a healthy skin, comprising topically applying an agent able to increase synaptopodin, preferably a plant extract described herein, on the skin of the subject. In another aspect, the invention relates to a method for selecting an agent able to improve, restore or maintain cutaneous, preferably epidermal, homeostasis in a skin, preferably in a healthy and aged skin, said method comprising contacting keratinocytes with a candidate agent, and determining whether the candidate agent increases the expression of synaptopodin in the keratinocytes, as compared to a reference expression level.

[0024] The method may comprise the steps of : a) contacting keratinocytes with an agent candidate, b) quantifying the expression of synaptopodin in said keratinocytes, c) comparing the expression of synaptopodin in said keratinocytes with that of reference keratinocytes not contacted with the agent candidate, and d) selecting the agent candidate if said agent candidate increases the expression of synaptopodin in said keratinocytes as compared to the reference keratinocytes, and wherein the keratinocytes are human primary keratinocytes seeded on a soft substrate having an elastic modulus lower than 10 kPa, preferably from 3.0 to 6.0 kPa. In some embodiments, the reference keratinocytes are primary keratinocytes seeded on a similar soft substrate but not contacted with the candidate agent.

[0025] In some embodiments, the Invention relates to an ex vivo method for selecting an agent for improving, restoring or maintaining cutaneous, preferably epidermal homeostasis in a skin, preferably in a healthy and aged skin, said method comprising : a) contacting a skin explant with an agent candidate, b) quantifying the expression of synaptopodin in said skin explant, c) comparing the expression of synaptopodin in said skin explant with that of a reference skin explant not contacted with the agent candidate, and d) selecting the agent candidate if said agent candidate increases the expression of synaptopodin in said skin explant.

[0026] In some embodiments, the Invention relates to a method for preparing a cosmetic active ingredient, e.g. suitable for a mature skin, said method comprising the steps of a) implementing the method for selecting an agent as described herein, b) optionally formulating the agent selected in step a) with one or more cosmetically acceptable excipients, e.g. a vehicle, a pH adjusting agent and / or a preservative, so as to obtain the cosmetic active ingredient.

[0027] In some other embodiments, the Invention relates to a method for preparing a cosmetic product, e.g. suitable for a mature skin, said method comprising the steps of a) implementing the method for selecting an agent as described herein, b) formulating the agent selecting in step a) with one or more pharmaceutically acceptable excipients, and optionally with one or more additional cosmetic active ingredients whereby the cosmetic product is obtained.

[0028] FIGURES

[0029] Figure 1 shows the relative Synaptopodin mRNA expression normalized to housekeeping gene RPL13A for keratinocytes on rigid support (plastic) and on a soft substrate (soft hydrogel).

[0030] Figure 2A shows the relative mRNA expression of each investigated marker (Ki67, K10 and Involucrin) normalized to the housekeeping gene RPL13 A at the different phases : proliferation phase (D-2), confluence (DO) and differentiation phase (DI, D4 and D6). Figure 2B shows the relative mRNA expression of synaptopodin normalized to the housekeeping gene RPL13 A at the different phases : proliferation phase (D-2), confluence (DO) and differentiation phase (DI, D4 and D6).

[0031] Figure 3 shows that synaptopodin was mostly expressed in the basal layer of the epidermis, i.e. in undifferentiated keratinocytes.

[0032] Figures 4A and 4B show the expression of synaptopodin evidenced by immunofluorescent staining in histological skin sections depending on the age of skin explant donors (in complete epidermis and basal layer of epidermis, respectively). Figure 4C shows the synaptopodin expression as evidenced by immunofluorescent staining in histological skin sections from donors aged under 30 years old and donors above 60 years old. The results are shown for both the whole epidermis and the basal layer of epidermis.

[0033] Figure 5 shows the distribution of synaptopodin and collagen XVII (COL17) in skin explants. Synaptopodin and COL 17 are co-located, in particular in the basal layer of epidermis.

[0034] Figure 6A shows the intensity of fluorescence reported per cell treatment with the active ingredient of the invention (at 0.1 or 0.2%) compared to the untreated control cells. The ice wine grape pomace active ingredient significantly increases synaptopodin expression.

[0035] Figure 6B shows the intensity of fluorescence reported per cell treatment with the ice wine grape pomace active ingredient of the invention (Ice Wine) or with a pomace extract from white wine (White wine) compared to the untreated control cells. The ice wine grape pomace active ingredient significantly increases synaptopodin expression. The comparative extract (White wine) has no significant effect on synaptopodin expression.

[0036] Figure 7 shows the intensity of fluorescence reported per cell and compared to the untreated control. In the skin explant, synaptopodin protein level is significantly increased after treatment with 0.2% ice wine grape pomace active ingredient as compared to the untreated condition.

[0037] Figure 8A and 8B show the effect of Ice wine grape pomace active ingredient on procollagen production in NHDF. Incubation of NHDF with Ice wine grape pomace active ingredient (IW) at different concentrations do not significantly increase procollagen I production as compared to untreated NHDF (Untreated) (Figure 8A). In contrast, procollagen I production is significantly increased in NHDF incubated with culture medium from keratinocytes incubated with Ice wine grape pomace active ingredient (IW) as compared to untreated NHDF (untreated) or NHDF incubated with culture medium (CTRL-) (Figure 8B). Positive control : NHDF incubated with ascorbic acid (AA).

[0038] Figure 9 shows the K10 mRNA expression in keratinocytes (NHDF) cultured on a rigid substrate (plastic) or on a soft substrate (hydrogel) in the presence of with Ice wine grape pomace active ingredient (Ice wine) at a concentration of 0.2% or in the absence of said active ingredient (untreated).

[0039] Figure 10A and 10B show the intensity of fluorescence reported per cell treatment with the retinoic acid (AR - 5 pM) or resveratrol (RSV - 10 and 50 pM) compared to the untreated control cells respectively. The cells are keratinocytes cultures on soft hydrogel. Contrary to Ice wine grape pomace active ingredient, resveratrol and retinoic acid did not promote the expression of synaptopodin.

[0040] DETAILED DESCRIPTION OF THE INVENTION

[0041] The keratinocyte homeostasis is modulated by mechanical changes in their microenvironment. It was shown that a support with high elastic modulus, which mimics young skin, promotes proliferation of human primary keratinocytes. In contrast, a support with low elastic modulus, which mimics aged skin, promotes keratinocyte terminal differentiation but without altering their ability to proliferate back onto a rigid substrate

[0042] Ya et al. (see supra) also showed that human keratinocytes cultured on a soft substrate undergo genome reprogramming with a significant loss of expression in differentiation marker genes and an increase in the expression of genes involved in the proliferation machinery, when put in contact with a stiffer environment.

[0043] Such results suggest that the age-related behavior changes observed in keratinocytes are reversible.

[0044] In that context, the Applicants sought to identify a cosmetic target that could rebalance the keratinocyte behavior in aged skin, in particular able to promote a shift from differentiation- prone behavior to a proliferation-prone behavior.

[0045] As illustrated in the Example section, the Applicants identified that synaptopodin, an actin- associated protein, is a cosmetic target of interest for modulating epidermal homeostasis.

[0046] To the knowledge of the Applicants, there is no publication in the prior art suggesting a possible involvement of synaptopodin in epidermal homeostasis. As of today, most of the studies concerning synaptopodin focus on very different cell types such as neurons and renal podocytes, in which synaptopodin was shown to be associated with the cytoskeleton and involved in the regulation of intracellular calcium. It was also suggested that synaptopodin plays a role in modulating actin-based shape and motility of dendritic spines and renal podocyte foot processes. However, to the knowledge of the Applicants, there is no publication in the state of the art suggesting a possible involvement of synaptopodin in epidermal homeostasis. Surprisingly, the Applicants showed that the expression of SYNPO gene in keratinocytes is strongly modulated by the support stiffness. As illustrated in Example 1, synaptopodin mRNA was decreased by 80% in Normal Human Epidermal Keratinocytes (NHEK) cultured on a soft substrate as compared to that observed on a rigid substrate (Figure 1). SYNPO expression was also correlated with keratinocyte phenotype. The Applicants showed that in in vitro 2D differentiation model, synaptopodin is expressed at a higher level in proliferating cells than in differentiating ones. When the cells reach confluence, synaptopodin expression tends to decrease compared to proliferating state and this decrease is significant when the cells enter the differentiation stage (Example 2, Figure 2b).

[0047] The Applicants also showed that these in vitro data were correlated with the synaptopodin expression profile in human skin. Immunostaining performed on skin explant sections showed that synaptopodin is mostly expressed in the basal layer of the epidermis, namely in undifferentiated keratinocytes (Example 3, Figure 3). The Applicants further demonstrated a clear decrease in synaptopodin expression related to age. Immunostaining of skin explant section showed a significant negative correlation between the age of the explant donor and synaptopodin expression levels: synaptopodin expression decreases with age and this decrease is more pronounced in the basal layer of the epidermis. A clear decrease in synaptopodin expression was observed in the epidermis and the basal layers in skin biopsies from donors above 60 years old as compared to skin biopsies obtained from donors under 30 years old (Examples 4A and 4B, Figure 4A, Figure 4B, and Figure 4C).

[0048] The Applicants further show a co-location of synaptopodin and collagen XVII (COL 17) in skin explants, in particular in the basal layer of the epidermis (Example 5, Figure 5). COL17 is a transmembrane collagen expressed in epidermal basal keratinocytes. Its expression is found at the hemidesmosomes of the epidermal basement membrane where COL17 is involved in epidermal-dermal attachment. COL 17 is also found at the intercellular spaces between basal keratinocytes, where COL 17 is not co-localized with desmosomal proteins. It is believed that COL 17 may play a role in keratinocyte physiology and homeostasis It was shown that physiological ageing reduces non-hemidesmosomal location of COL 17 due to altered epidermal cell polarity. (Natsuga et al., Experimental Dermatology, 2019:281135-1141). Collagen XVII expression has been shown to decrease with age. ColXVII is important for cell renewal, with high expression in epidermal stem cells to promote symmetric cell division. Loss of COL17A1 limits stem cell division, leading to ageing. (Liu et al., Nature, 2019: 568, 344- 350). Without wishing to be bound by any theory, the Applicants believe that synaptopodin may play a role in COL 17 distribution and / or expression in keratinocytes and may contribute to restore cell polarity and epidermis attachment to dermis.

[0049] Altogether these results strongly support that synaptopodin is a key cosmetic target to modulate or maintain keratinocyte homeostasis.

[0050] In addition, the Applicants showed that a particular plant extract obtained from non-fermented ice wine pomace (namely from naturally frozen grapes of Vidal variety) significantly increases synaptopodin expression in keratinocytes cultured on a soft substrate mimicking old skin (Example 6, Figure 6A). This vine extract was also showed to significantly increase (+52%) the synaptopodin in skin explants from a woman of 52 years old (Example 6, Figure 7). The Applicants also showed that this particular vine extract was able to decrease the expression of a well-known marker of differentiation (K10) in keratinocytes cultured on a soft substrate and to mainly restrict the expression of involucrin, another marker of differentiation, in the uppermost layers of reconstructed epidermis. Such results strongly suggest that the extract of the invention, by inducing the expression of synaptopodin, can slow-down or reverse the differentiation phenotype in keratinocytes in mature skins.

[0051] It was also shown that the supernatant from keratinocytes treated with the extract of the invention significantly increases the procollagen production in fibroblasts. Such a result indicates that the increase in synaptopodin expression is likely to promote interactions between keratinocytes and fibroblasts, leading to an increase in pro collagen and thus in an improvement of the extracellular matrix. Thus, by inducing synaptopodin expression in keratinocytes, it can be possible to promote the secretion of particular factors by keratinocytes, able to stimulate the production of procollagen by fibroblasts and by the way, improve the dermal extracellular matrix in mature skin.

[0052] Of note, the Applicants also showed that resveratrol and retinoic acid, that would be described as promoting the expression of synaptopodin in podocytes or renal cortex cells in the prior art, are unable to promote the expression of synaptopodin in keratinocytes cultured on soft hydrogel mimicking aged skin.

[0053] Accordingly, the cosmetic effects and mechanism of action described herein with the Ice wine grape pomace active ingredient are unique and unexpected over the prior art.

[0054] The Invention relates to a non-therapeutic, cosmetic, use of an agent able to increase synaptopodin expression in keratinocytes for improving, restoring or maintaining epidermal homeostasis in a skin, preferably in a healthy skin. The Invention further relates to a cosmetic method for improving, restoring or maintaining epidermal homeostasis in a subject comprising topically applied an agent able to increase synaptopodin expression in keratinocytes.

[0055] The Invention also relates to a cosmetic composition for improving, restoring or maintaining epidermal homeostasis comprising (i) an agent able to increase synaptopodin expression in keratinocytes and (ii) at least one pharmaceutically acceptable excipient. Vitis Vinifera extracts able to increase synaptopodin expression in keratinocytes are also described herein.

[0056] In a further aspect, the Invention also relates to said Vitis Vinifera extracts and their non- therapeutic, cosmetic, use for improving, restoring or maintaining epidermal homeostasis in a in a skin, preferably in a healthy skin.

[0057] The Invention further relates to a method for selecting agents for improving, restoring or maintaining epidermal homeostasis.

[0058] Cosmetic uses accordins to the Invention

[0059] The Invention relates to the cosmetic use of an agent able to increase the expression of synaptopodin for improving, restoring or maintaining skin homeostasis, in particular epidermal homeostasis.

[0060] Skin homeostasis refers to the maintenance of the steady state of the skin, which includes among others the maintenance of skin barrier function and skin regeneration.

[0061] As used herein, “Epidermal homeostasis" refers to the maintenance of the epidermis, the outermost layer of the skin, through a balanced process of cell proliferation and differentiation. The epidermis is mainly composed of keratinocytes that are distributed in different layers reflecting the proliferation and / or the differentiation state of the cells. Keratinocytes undergo a tightly regulated process of proliferation from stem cells in the basal layer and terminal differentiation as they migrate outward, constantly replenishing the epidermis. During this process, keratinocytes profoundly change their morphology and start to produce a variety of keratin isoforms and different kinds of cell mediators modulating the activity of other cutaneous cells as well as immune cells in the skin. Keratinocytes divide in the basal to spinous layer (stratum basal and stratum spinosum. respectively). Mature, differentiated keratinocytes are pushed towards the surface of the skin by the proliferative action of immature keratinocytes in the underlying layers. While being moved through the stratum granulosum and into the stratum corneum, keratinocyte nuclei dissolve and bioactive lipids are squeezed into the extracellular space. The cells are fully differentiated at the stratum corneum, where they are eventually shed into the environment.

[0062] The balance between proliferation and differentiation in keratinocytes is critical for epidermal homeostasis (and more generally for skin homeostasis).

[0063] Indeed, homeostasis leads to the maintenance of the epidermal thickness , the mechanical and visual properties and the barrier function of the skin. Impairment in epidermal homeostasis leads to the loss of cellular turnover, a decrease in barrier function and skin thickness, and more generally an alteration in the quality of the skin in terms of mechanical and / or visual properties. As used herein, “epidermal homeostatic imbalance", “keratinocyte homeostatic imbalance". refers to changes in the phenotype and / or behavior of keratinocytes resulting in an imbalance between proliferation and differentiation leading to non-pathological alteration of the epidermis quality and by the way, by non-pathological alterations of the visual and / or mechanical properties of the skin.

[0064] The epidermal homeostasis can be affected by physiological factors such as age, menopause, puberty as well extrinsic factors (UV, pollution, climatological stresses (wind, cold, humidity), poor diet, tabaco, smoke etc).

[0065] For instance, a decrease in regenerative potential of epidermis is observed with age : the keratinocytes in the basal layer show a decrease in proliferation and a tendency through differentiation resulting in a slowdown or decrease in epidermis renewal. This phenomenon is even accentuated by the menopause due to hormonal changes.

[0066] Other changes are observed with physiological ageing such as alterations in keratinocyte polarity, in keratinocyte attachment to dermis, and in production or location of matrix proteins by keratinocytes.

[0067] In preferred embodiments, the agent able to increase the expression of synaptopodin is used for delaying the onset, preventing, decreasing or alleviating age-related imbalance of epidermal homeostasis.

[0068] An imbalance in epidermal homeostasis, in particular when associated with age, can result in an alteration of the skin barrier function as well as an alteration of the skin appearance and mechanical properties. Visual signs of epidermal homeostasis imbalance encompass, without being limited to, roughness, fine lines, wrinkles, dull complexion and dark circles. Mechanical signs of epidermal homeostasis imbalance encompass, without being limited to, loss in skin density, loss in skin firmness, a loss in skin density and loss in skin elasticity.

[0069] As used herein, “ synaptopodin" refers to the human protein encoded by SYNPO gene e.g. identified under NCBI Gene ID: 11346. Synaptopodin can be any one of isoforms encoded by SYNPO. In some embodiments, synaptopodin refers to an isoform SEQ ID NO: 1, SEQ ID NO:2 or SEQ ID NO:3. In preferred embodiments, synaptopodin is the isoform of SEQ ID NO:3, said isoform being the one mainly expressed in keratinocytes.

[0070] The agent of the invention can be of any type, for instance a small molecule, a peptide, e.g. a peptide having from 2 to 50 amino acids in length, preferably from 2 to 10 amino acids in length, a plant extract, an oligonucleotide, and combinations thereof, with proviso that it is able to increase the expression of synaptopodin and suitable for a use as a cosmetic agent on the skin when used in appropriate amounts. It goes without saying that the agent able to increase synaptopodin expression is adapted for a cosmetic use when used in appropriate amounts topically, namely exerts cosmetic effects without any significant toxicity or side-effects in particular for the skin.

[0071] The ability of an agent to increase synaptopodin expression can be assessed by any appropriate method of the prior art, for instance by quantification of the corresponding mRNA or by quantification of the protein per se. Preferably, the increase in synaptopodin expression is determined by detecting the synaptopodin per se, for instance by immunolabelling e.g. immunostaining of a cell culture or a skin histological section and by comparison with a reference value.

[0072] For instance, the ability of an agent to increase synaptopodin expression can be assessed in keratinocyte culture or in a skin explant.

[0073] In preferred embodiments, the agent of the invention is able to increase the expression of synaptopodin in human keratinocytes, e.g. in human keratinocytes cultured on a soft substrate and / or in keratinocytes present in a human skin explant, e.g. from a subject of at least 40 year old, preferably of at least 50 year old.

[0074] In some embodiments, the agent of the invention is able to increase the expression of synaptopodin in normal human epidermal keratinocytes (NHEK) cultured in vitro on a soft hydrogel. The increase in synaptopodin expression is preferably determined by quantification of synaptopodin protein through immunostaining by comparison of synaptopodin expression in reference cells, e.g. NHEK cultured on the same soft hydrogel but not contacted with the agent. Alternatively, the increase in synaptopodin expression can be quantified through the quantification of mRNA of SYNPO gene in NHEK cultured on the soft hydrogel and contacted with the agent as compared to reference NHEK cultured on the soft hydrogel but not contacted with the agent.

[0075] As used herein, a “soft substrate" refers to any substrate suitable for cell culture and having a stiffness (also called elastic modulus) of at most 10 kPa, preferably from 2 to 10 kPa, and more preferably from 3 to 6 kPa or from 3.5 to 5.5 kPa such as 4 or 5 kPa. A soft substrate with such an elastic modulus is able to mimic the mechanical environment of keratinocytes in an aged skin.

[0076] The soft substrate can be a hydrogel, for instance a polyacrylamide hydrogel.

[0077] The preparation of an appropriate hydrogel is described, for instance, in Ya et al (Supra). Briefly, the hydrogel can be prepared by mixing acrylamide / bisacrylamide solutions and initiating polymerization by adding ammonium persulfate and TEMED. The resulting polyacrylamide solution can be pipetted onto a chlorosilane slide and covered by a functionalized coverslip. After the completed polymerization, the top coverslip with the attached polyacrylamide gel is slowly peeled off and rinsed.

[0078] Other soft substrate can be used such as hydrogels made of PEG and poly(L-lysine) dendri grafts (DGL). Such hydrogels can be prepared by reacting PEG-bis(Nsuccinimidyl succinate) with DGL as described in Carranca et al. Journal of Biomedical Materials Research

[0079] Part A, 2020, 10.1002 / jbm.a.3708. Such type of hydrogel can be designated under “PEG-DGL hygrogel” or “PEG crosslinked with DGL”

[0080] To facilitate keratinocyte attachment, the gel can be functionalized e.g. with an extracellular matrix molecule such as collagen isoforms, e.g. collagen I or IV, or proteins from basal lamina such as laminin through covalent or non-covalent coating.

[0081] For instance, when a polyacrylamide gel is used, theextracellular matrix molecules are preferably covalently bound to the hydrogel, e.g. by means of a crosslinker such as Sulfo- SANPAH (sulfosuccinimidyl 6-(4'-azido-2'-nitrophenylamino) hexanoate).

[0082] As used herein, the term “ stiffness of the soft substrate" refers to the elastic modulus of the soft substrate. The elastic modulus can be determined by any methods known in the prior art, for instance by Dynamical Mechanic Analysis, e.g. as described in Ya et al, supra or by Indentation Type-AFM (IT-AFM) as described in Bouchonville et Nicolas in Santos and Carvalho, Atomic Force Microscopy: Methods and Protocols, Methods in Molecular Biology, vol. 1886, pages 281-290 or in Paiva et al. ACS Biomater. Sci. Eng. 2020, 6, 1, 340-351.

[0083] Preferably, the elastic modulus of the hydrogel is determined by AFM indentation.

[0084] Appropriate soft substrates are also commercially available (e.g. see those marketed by Cell&Soft and SoftWell)

[0085] In a particular embodiment, the agent able to increase the expression of synaptopodin is selected by the screening method (also called method of selection) as described herein.

[0086] In another embodiment, the agent able to increase the expression of synaptopodin is a plant extract, in particular an extract of Vitis vinifera grapes as described herein. As used herein, the term “skin” refers to any part of the skin of the human body, in particular the skin of the face, including the lips and eyelids, neck skin, hand skin and scalp.

[0087] The skin is healthy, which means that the skin does not show any wounds or skin disorders such as skin infection, eczema, or dermatitis including atopic dermatitis.

[0088] In some embodiments, the skin is a mature (also called aged) skin. Accordingly, the skin can show one or several signs of ageing such as wrinkles, fine lines, loss of elasticity and / or firmness and / or density, dull complexion, dark circles, and the likes.

[0089] In some embodiments, the agent able to increase the expression of synaptopodin is applied on an aged and healthy skin.

[0090] The subject may be any human of any gender. In some embodiments, the subject is an adult above 40 years old, preferably above 50 years old.

[0091] As used herein, “a mature skin” or “an aged skin” refers to the skin of a subject of at least 40 years old, preferably of at least 45 years old e.g., of at least 50 years old or of at least 55 years old. A mature skin (or an aged skin) refers to a skin showing one or more non-pathological alterations of the visual aspect and / or mechanical properties of the skin induced by ageing. In a particular embodiment, a mature skin refers to a skin that can show one or several non- pathological alterations such as a decrease in collagen production, loss of skin density, loss of firmness, slackening skin, dull complexion, fine lines, wrinkles, roughness, thin epidermis and / or combinations thereof.

[0092] In some embodiments, the subject can be a woman of at least 40 years old such as of at least 45 years old or 50 years old. The subject may be a pre-menopausal, peri -menopausal or menopausal woman. In a particular embodiment, the subject is a woman in peri-menopause or menopause.

[0093] As used herein, “perimenopause” means “around menopause” and to refers to the time during which the woman body makes the natural transition to menopause, marking the end of the reproductive years. Women can start perimenopause at different ages. Signs of progression toward menopause, such as menstrual irregularity (irregular periods in length and / or periodicity) and menopause-like symptoms such as hot flashes and sleep disorders, can be observed around 40 years old. Physiological estrogen decline observed in perimenopause and in menopause can impair the quality, the visual aspect and the comfort of the skin and can result in skin ageing. Perimenopause period is considered as achieved and thus menopause established when the woman does not experiment any periods during twelve consecutive months. In some embodiments, the agent able to increase the expression of synaptopodin is used to provide prophylaxis against unbalance in skin homeostasis, in particular in epidermis homeostasis. The agent can be used to maintain the expression of synaptopodin in the skin. For instance, the agent of the invention can be used to prevent, slow-down or delay the onset of one or several non-pathological alterations in the skin such as decrease in collagen production, loss of skin density, loss of firmness, slackening skin, dull complexion, fine lines, wrinkles, roughness, thin epidermis and / or combinations thereof. Preferably, the agent can be used to prevent, slow-down or delay the onset of a decrease in collagen production, a decrease in dermal extracellular matrix quality, a loss of skin density and / or a loss of skin firmness.

[0094] In some embodiments, the agent of the invention is applied on the skin of a subject of at most 40 years old, typically of 20 to 40 years old e.g. from 25 to 35 years old.

[0095] In some embodiments, the agent is used for prophylactic purposes on a young skin, namely on a skin which does not show, or solely exhibit very light signs of skin ageing.

[0096] In some embodiments, the agent of the invention is used in a subject of less than 40 years old that is exposed to a lifestyle that can induce a premature skin aging, such as unbalanced diet (e.g. high fat diet), consumption of tobacco and / or alcohol, staggered work rhythm (e.g. night work), get lag and the like.

[0097] In some embodiments, the agent of the invention is applied on the skin of a woman of at most 40 years old, e.g. from 20 to 40 years old.

[0098] The agent of the invention is to be used topically, namely applied on the skin. As used herein, “a cosmetic effec ' refers to any non-therapeutic effect aiming at improving and / or modifying the appearance of the skin, and / or the mechanical properties of the skin.

[0099] A “cosmetic effect” includes preventing, slowing-down, delaying the onset and / or alleviating any non-pathological alterations of the skin due to aging, lifestyle (e.g. jet lag, lack of sleep, night work, poor diet such as high fat diet, consumption of alcohol and / or tabaco), or exposure to external stress such as climatic conditions (wind, cold, heat, sun, dryness), pollution, smoke, chemicals and the like.

[0100] In the context of the invention, a cosmetic effect preferably refers to the prevention, the retardation, the alleviation or the improvement of one or several non-pathological alterations of the skin resulting from an age-related imbalance in epidermal homeostasis. Such non pathological alterations encompass, without being limited to, as fine lines, wrinkles, a loss of skin density, a loss of skin firmness, a loss of skin elasticity, increase in skin roughness, a loss of radiance or glow, a dull or non-uniform complexation, dark circles and combinations thereof As used herein, “a cosmetic agent” refers to an active ingredient able to promote a cosmetic effect, namely to improve and / or modify the appearance of the skin, and / or the mechanical properties of the skin.

[0101] In a particular embodiment, the agent able to increase the expression of synaptopodin in keratinocytes is used to modulate the phenotype of keratinocytes in an aged skin by rebalancing the equilibrium between proliferation / differentiation in keratinocytes.

[0102] In a particular aspect, the invention relates to the use of an agent able to increase the expression of synaptopodin in keratinocytes for promoting or increasing keratinocyte proliferation and / or for decreasing or delaying the onset of keratinocyte differentiation. In some embodiments, said agent is used to increase the proliferative phase in keratinocytes and delay the onset of keratinocyte differentiation in an aged skin.

[0103] In a particular aspect, the invention relates to the use of an agent able to increase the expression of synaptopodin in keratinocytes for decreasing, slowing down, or delaying the onset of cell differentiation in epidermis.

[0104] In a particular aspect, the invention relates to the use of an agent able to increase the expression of synaptopodin in keratinocytes for shifting keratinocyte phenotype from differentiation-prone phenotype to proliferation-prone phenotype.

[0105] In some embodiments, said agent is used to delay keratinocyte differentiation or to decrease differentiation rate of keratinocytes, preferably in an aged skin.

[0106] In some embodiments, said agent is used to decrease a differentiation marker such as involucrin or K10, preferably in an aged skin.

[0107] In a particular embodiment, the invention relates to the use of an agent able to increase the expression of synaptopodin to maintain, promote or increase epidermis renewal and / or to maintain, promote or increase epidermis thickness in a healthy skin, preferably in an aged and healthy skin.

[0108] In an embodiment, the invention relates to the use of an agent able to increase the expression of synaptopodin for maintaining, restoring or increasing the regeneration of the epidermis.

[0109] In an embodiment, the invention relates to the use of an agent able to increase the expression of synaptopodin for restoring or maintaining keratinocyte polarity.

[0110] In an embodiment, the invention relates to the use of an agent able to increase the expression of synaptopodin for maintaining or restoring collagen XVII distribution and / or expression in keratinocytes, preferably in a mature skin.

[0111] In some embodiments, the invention relates to the use of an agent able to increase the expression of synaptopodin for promoting the production of procollagen type I, preferably in an aged skin. In some embodiment, the invention relates to the use of an agent able to increase the expression of synaptopodin for maintaining or restoring dermal extracellular matrix, preferably in an aged skin.

[0112] In some embodiment, the invention relates to the use of an agent able to increase the expression of synaptopodin for maintaining or restoring skin density, preferably in an aged skin.

[0113] The agent of the invention can also be used to provide mature or elderly skin with a more youthful appearance, and more generally to make the skin look and / or feel healthier and / or younger.

[0114] In particular embodiments, the agent of the Invention is used as a cosmetic active agent to prevent (e.g. slow-down or delay), attenuate, decrease or treat one or more non-pathological alterations of the skin appearance or mechanical properties, in particular in aged skin. Non- pathological alterations of the skin appearance encompass, without being limited to: roughness, fine lines, wrinkles, loss of radiance or glow, dull or non-uniform complexation, loss of firmness, loss of density, loss of elasticity and combinations thereof.

[0115] In another embodiment, the agent of the Invention may be used towards at least one (e.g. 1, 2, 3 or more) of the following cosmetic purposes:

[0116] - Reduce signs of aging, including wrinkling and loss of tissue elasticity.

[0117] - Homogenize or unify the complexion of the face,

[0118] - Make the complexion of the face brighter and / or more radiant,

[0119] - Prevent or treat a dull complexion, a blurred complexion and / or drawn features,

[0120] - Make the skin, especially on the face, younger, and / or

[0121] - Soothe facial features,

[0122] In an embodiment, the agent of the Invention is used to maintain, restore or increase the barrier properties of the skin and / or maintain, restore or increase skin sensitivity, in particular towards external stimuli. In a particular embodiment, the agent of the Invention is used to decrease or prevent over-reactivity of the skin in particular towards external factors.

[0123] In the uses and methods as described herein, the agent of the invention (i.e. an agent able to increase the expression of synaptopodin) is administered topically, namely applied on the skin. Typically, the agent is applied by means of a cosmetic composition. In other words, the agent of the invention is present in a cosmetic composition which is applied on the skin of the subject. Cosmetic compositions of interest are described further below.

[0124] In certain embodiments, the agent of the Invention can be directly incorporated into a cosmetic composition. In other embodiments, the agent of the Invention is pre-formulated into a cosmetic active ingredient before being incorporated into a cosmetic composition.

[0125] The agent of the Invention, e.g. the Vitis vinifera extract as described herein, is typically present in an amount from 0.005% to 10.0 % by weight, preferably from 0.01% to 5.0% e.g. such as 0. 5 % to 2.0 % by weight in the cosmetic composition.

[0126] Cosmetic compositions of interest, encompass, without being limited to, skin care products, a skin hygiene products or a make-up products such as a cream e.g. a cream, a balm, an ointment, a serum, a gel, a mask, and a foundation. Examples of cosmetic compositions of interest are detailed further below.

[0127] An additional object of the present invention is a cosmetic method for providing one or more cosmetic effects as described herein, said method comprising applying an agent able to increase the expression of synaptopodin, or a cosmetic composition thereof, on the skin of the subject. The extract is preferably applied in the form of a cosmetic composition as described herein.

[0128] For instance, the cosmetic method may be for promoting or increasing keratinocyte proliferation and / or for decreasing or delaying the onset of keratinocyte differentiation and / or maintaining promoting or increasing epidermis renewal and / or maintaining, promoting or increasing epidermis thickness in a healthy skin, preferably in an aged and healthy skin.

[0129] As a further example, the cosmetic method of the invention may be for maintaining, restoring or increasing the regeneration of the epidermis.

[0130] In a particular aspect, the cosmetic method of the invention can achieve one or several of the following effects in the subject:

[0131] - Reduce signs of aging, including wrinkling and loss of tissue elasticity in the skin.

[0132] - Homogenize or unify the complexion of the face,

[0133] - Make the complexion of the face brighter and / or more radiant,

[0134] - Prevent or treat a dull complexion, a blurred complexion and / or drawn features,

[0135] - Make the skin, especially on the face, younger, and / or

[0136] - Soothe facial features,

[0137] As mentioned above, the subject is preferably an adult with a mature skin e.g. a menopausal or peri-menopausal woman.

[0138] In the cosmetic methods and uses as described herein, the dose to be administered and the frequency of administration of the agent according to the invention vary according to the cosmetic effect sought, the characteristics of the individual, in particular the sex, age, and skin type thereof, and the chemical nature of the agent to be administered. Typically, the agent of the invention, or the cosmetic composition comprising it, can be applied to a skin area to be treated once or twice daily, e.g. after skin cleaning, for several consecutive weeks or even months, for example for at least 3 months. For example, the subject can apply a dose of 1 g to 2 g of cosmetic composition on the face in the morning and / or in the evening.

[0139] - Method for selectins an agent of the invention

[0140] An agent able to increase the expression of synaptopodin in keratinocytes can be determined, for instance, in vitro or ex vivo, by the methods described e.g. in Example 6, items 3 or 4, preferably by the in vitro method described in example 6, item 3.

[0141] In an additional aspect, the invention relates to a method for selecting an agent for improving, restoring or maintaining cutaneous, preferably epidermal homeostasis in a skin, said method comprising the steps of a) contacting keratinocytes with a candidate agent, and b) determining whether the candidate agent increases the expression of synaptopodin in the keratinocytes, as compared to a reference expression level.

[0142] The reference expression level typically refers to the synaptopodin expression level in keratinocyte not contacted with the candidate agent. The reference expression level can refer to literature data. Preferably, the reference expression level is experimentally determined in keratinocytes similar to those used in the method, in the absence of the candidate agent, the other conditions being similar.

[0143] Keratinocytes are preferably human keratinocytes present in a skin sample (e.g. a skin explant) and / or in a cell culture.

[0144] In some embodiments, the method of the Invention is performed on a skin explant.

[0145] In an additional aspect, the Invention relates to an ex vivo method for selecting an agent for improving, restoring or maintaining cutaneous, preferably epidermal homeostasis in a skin, preferably in a healthy and aged skin, said method comprising : a) contacting a skin explant with an agent candidate, b) quantifying the expression of synaptopodin in said skin explant, c) comparing the expression of synaptopodin in said skin explant with that of a reference skin explant not contacted with the agent candidate, and d) selecting the agent candidate if said agent candidate increases the expression of synaptopodin in said skin explant.

[0146] The skin explant is from a human subject, preferably from an adult subject of at least 40 years old. In some preferred embodiments, the skin explant is from a woman of at least 50 years old. The skin explant can be typically any skin biopsies sampled e.g. during esthetical surgery such as abdominoplasty. In step a), typically, the skin explant is placed in an appropriate culture medium. Preferably, the dermis is fully submerged in the culture medium while the epidermis is left dry at the air-liquid interface. Then, the agent candidate to be tested is diluted in an appropriate medium and then applied on the epidermis of the skin explant. The skin explant is preferably incubated with the agent candidate during few hours, e.g. during 12h or 24h.

[0147] After step b, the expression of synaptopodin in the skin explant is quantified, preferably in the epidermis layer. The quantification of synaptopodin expression can be performed by any method known by the skilled artisan, through the quantification of the protein per se or that of the corresponding mRNA. In preferred embodiments, the synaptopodin expression is quantified through the quantification of the protein in the skin explant per se. Said quantification can be performed by immunostaining of histological section obtained from the skin explant. The histological section can be prepared by any method known by the skilled artisan, e.g. as described in Example 4, item 4. The quantification of the protein can be determined by immunofluorescence, by using anti-synaptopodin antibody (e.g. labelled with a fluorophore or revealed by a secondary antibody coupled to a fluorophore).

[0148] In step c), the level of synaptopodin expression in the skin explant treated with the candidate agent is compared with that quantified in a reference explant not treated with the candidate agent. Preferably, the reference explant corresponds to a skin explant from the same skin biopsy as, and subjected to the same treatment, as the explant treated with the agent candidate except that the reference explant is not treated with the agent candidate.

[0149] In step d), a candidate agent is selected if the synaptopodin expression in the treated skin explant is increased by at least 5%, preferably by at least 10%, 15%, 20%, 30% or 40% as compared to the reference explant.

[0150] For illustration of such a method, one can refer to Example 4, item 4, further below.

[0151] In some other embodiments, the method of the Invention is performed on a keratinocyte culture. In an additional aspect, the Invention also relates to an in vitro method for selecting an agent for improving, restoring or maintaining cutaneous, preferably epidermal, homeostasis in a skin, preferably in a healthy and aged skin, said method comprising the steps of : a) contacting keratinocytes with an agent candidate, b) quantifying the expression of synaptopodin in said keratinocytes, c) comparing the expression of synaptopodin in said keratinocytes with that of reference keratinocytes not contacted with the agent candidate, and d) selecting the agent candidate if said agent candidate increases the expression of synaptopodin in said keratinocytes.

[0152] In preferred embodiments, the method is performed on a primary cell culture of human keratinocytes. In some embodiments, the keratinocytes used in the primary culture are normal human epidermal keratinocytes (NHEK cells). NHEKs are preferably isolated from healthy skin biopsy from human adult, preferably from adult woman.

[0153] In step a), typically, keratinocytes are seeded on an appropriate substrate and cultured in an appropriate medium. The keratinocytes are then contacted with the candidate agent. The candidate agent is typically diluted in an appropriate medium and added in the culture medium. In some embodiments, the keratinocytes are incubated with the candidate agent, for instance during few hours e.g. during 12h or 24h.

[0154] After step b) the expression of synaptopodin in the cells is quantified, The quantification of synaptopodin expression can be determined by any method known by the skilled artisan, through the quantification of the protein per se or that of the corresponding mRNA. In preferred embodiments, the synaptopodin expression is quantified through the quantification of the protein per se. Said quantification can be performed by immunostaining by any method known by the skilled artisan, e.g. as described in Example 4, item 3. The quantification of the protein can be determined by immunofluorescence, by using anti-synaptopodin antibody (e.g. labelled with a fluorophore or revealed by a secondary antibody coupled to a fluorophore).

[0155] In step c), the level of synaptopodin expression in the cells treated with the candidate agent is compared with that quantified in reference cells not treated with the candidate agent. The reference cells preferably correspond to the same keratinocytes, and subjected to the same treatment, as the keratinocytes treated with the agent candidate except that the reference cells are not treated with the agent candidate.

[0156] A candidate agent is selected if the synaptopodin expression in the treated cells is increased by at least 5%, preferably by at least 10%, 15%, or 20%, as compared to the reference cells.

[0157] In preferred embodiments, the keratinocytes are cultured on a soft substrate. Using a soft substrate enables to mimic the mechanical environments of keratinocytes in aged skin. Indeed, with age, the dermis, on which the epidermis is based, loses its elasticity. In some embodiments, the keratinocytes are seeded (cultured) on a soft substrate having an elastic modulus of at most 10 kPa, preferably from 2 to 10 kPa, and more preferably from 3.0 to 6.0 kPa or from 3.5 to 5.5 kPa such as 4 or 5 kPa. A soft substrate with such an elastic modulus is able to mimic the mechanical environment of keratinocytes in an aged skin. The soft substrate can be any appropriate hydrogel suitable for cell culture. In some embodiments, the soft substrate is made of polyacrylamide hydrogel or a PEG-DGL hydrogel.

[0158] As described above, the hydrogel can be prepared by any appropriate methods described in the prior art. As described herein, the hydrogel can be functionalized (e.g. by covalent or non- covalent coating) with extracellular matrix molecules such as collagen to promote keratinocyte attachment.

[0159] For illustration of such a method, one can refer to Example 6, item 3, further below.

[0160] In an additional aspect, the invention relates to a method for preparing a cosmetic active ingredient which comprises the step a) of implementing the method for selecting an agent of the invention, as described herein.

[0161] In some embodiments, said method comprises the step of formulating the agent of step a) with one or more cosmetically acceptable excipients, e.g. a vehicle, a pH adjusting agent and / or a preservative, so as to obtain the cosmetic active ingredient for aged skin.

[0162] In some embodiments, the selected agent is formulated with a vehicle selected from triethyl citrate, a C2-C5 alkanediol such as propanediol, butylene glycol, pentylene glycol and isomers thereof, ketal esters such a methyl or ethyl levulinate, glycerin, water, maltodextrin and mixtures thereof.

[0163] The cosmetic active ingredient can be as described herein.

[0164] In an additional aspect, the invention relates to a method for preparing a cosmetic product comprising the steps of: a) implementing the method for selecting an agent, as described herein, b) formulating the agent selecting in step a) with one or more pharmaceutically acceptable excipients, and optionally with one or more additional cosmetic active ingredients whereby the cosmetic product is obtained.

[0165] The cosmetic product can be as described herein.

[0166] - Particular agents to implement the cosmetic methods and uses of the Invention.

[0167] As mentioned above, the agent to implement the cosmetic methods or uses of the invention can be of any type, with proviso that it increases the expression of synaptopodin in keratinocytes, preferably as evidenced by a method of testing (also called herein method for selecting) as described herein. The Applicants identified that certain Vitis vinifera extracts are able to significantly increase synaptopodin expression in skin explants or in keratinocytes cultured on soft substrates.

[0168] In a particular embodiment, the Invention relates to the non-therapeutic, cosmetic, use of an Vitis vinifera extract able to increase synaptopodin expression in keratinocytes for improving, restoring or maintaining epidermal homeostasis in a healthy skin, preferably in an aged healthy skin.

[0169] Vitis vinifera extract can be of any type with respect that it increases synaptopodin expression in keratinocytes, preferably as evidenced by a method for selecting as described herein.

[0170] In preferred embodiments, the Vitis vinifera extract is aqueous or hydroalcoholic extract, preferably an aqueous extract of Vitis vinifera grapes.

[0171] Any cultivars or varieties of Vitis vinifera are contemplated herein.

[0172] In some embodiments, the extract of the invention is obtained from white grapes.

[0173] In an embodiment, the extract is prepared from a Vitis vinifera variety suitable for the preparation of ice wine. Such varieties encompass, without being limited to, Riesling, Vidal, Sylvaner, Gewurztraminer, Pinot gris, Gruner Veltliner, and Chardonnayr

[0174] As used herein, icewine (or ice wine) refers to a wine produced from grapes that have been frozen while still on the vine. The sugars and other dissolved solids do not freeze, but the water does, allowing for a more concentrated grape juice to develop. The crushed, mashed grapes are then pressed from the frozen grapes, resulting in a smaller amount of more concentrated, very sweet juice. With icewines, the freezing happens before the fermentation, not afterwards. Ice wine grapes are preferably not be affected by Botrytis cinerea or noble rot.

[0175] The solids remaining after pressing the grapes refer to the pomace (also called marc) which is thus a by-product in wine industry.

[0176] In a particular embodiment, the extract of the invention is obtained from white grapes suitable for the preparation of ice wine, preferably from Vidal or Riesling varieties, and more preferably from Vidal variety.

[0177] In embodiment, the extract of the invention is obtained from grapes which have frozen on the vine before being harvested, said grapes being preferably of the Vidal variety.

[0178] In a particular embodiment, the extract of the Invention is obtained from wine grape pomace, preferably from grape pomace of white wine such as those made of Vidal or Riesling varieties. In a preferred embodiment, the extract of the Invention is obtained from pomace, preferably pomace of ice wine, e.g. from Vidal or Riesling grape varieties.

[0179] In a preferred embodiment, the extract of the Invention is an aqueous or hydroalcoholic extract of grape pomace of a white wine, preferably a white ice wine, of the Vidal variety. In other words, the extract of the invention may be prepared from ice wine pomace prepared from grapes, preferably of the Vidal variety. In other words, the extract of the invention may be prepared from a wine grape pomace obtained from white grapes which have been naturally frozen at the vine, preferably at a temperature below -8°C, before being harvested. Without wishing to be bound by any theory, the Applicant believes that the natural freezing of the grapes on the vines enables to activate some stress and / or defense mechanism in the grapes resulting in the production of secondary metabolites that may contribute to the biological activities of the extract, in particular its capacity to promote the expression of synaptopodin in keratinocytes, as described herein.

[0180] In some embodiments, the extract is prepared from grapes of vine, preferably of the Vidal variety, cultured in Canada or from any derivatives thereof such as grape pomace.

[0181] In some embodiments, the extract is non-fermented. In particular, the extract is obtained from non-fermented pomace e.g non-fermented pomace of ice wine of the Vidal variety.

[0182] As used herein, “an aqueous or hydroalcoholic extract' refers to an extract obtainable or prepared by extracting the patent material with a solvent selected from water, a lower alcohol including glycols, and combinations thereof.

[0183] Such an extract can be also obtainable by extraction with a natural deep eutectic solvent. Deep eutectic solvents (DES) are a subclass of ionic liquid (IL). A DES is a eutectic mixture of two or three constituent components, generally interacting through hydrogen bonding, that have a lower melting point than that of each component when combined at the proper molar ratio. A natural DES (NADES) refers to an eutectic mixture of naturally occurring molecules such as sugars, organic acids and bases, and amino acids. NADES can be grouped into: (1) ionic liquid NADES (made from an acid and a base), (2) neutral NADES (made from sugars only or sugars and polyalcohol), (3) neutral NADES with acids (made from sugar / polyalcohol and organic acids), (4) neutral NADES with bases (made from sugar / polyalcohol and organic bases), and (5) amino acids-based NADES (made from amino acids and organic acids / sugars). For review in that matter one can refer to Hikmawanti et al., Plants (Basel). 2021 Oct; 10(10): 2091.

[0184] As herein, a “lower alcohol” encompasses C2-C6 alkanols, C2-C6 alkanediols, C2-C6 polyols and combinations thereof. Appropriate lower alcohols, encompass, without being limited to, ethanol, propanol, isopropanol, butanol, ethylene glycol, propanediol, glycerol, butanediol, pentanediol, isomers thereof and combinations thereof. For instance, the lower alcohol can be selected from ethanol, isopropanol, glycerin and C2-C5 alkanediols of the glycol type such as 1,2-propylene glycol, 1,3-propane diol, butylene glycol, pentylene glycol and isomers thereof. In a preferred embodiments, the extract is obtainable by aqueous extraction.

[0185] In a particular embodiment, the extract is prepared with hot water e.g. at a temperature of at least 60°C, 70°C, 80°C or 90°C. In some embodiments, the extraction with hot water is performed under reflux.

[0186] Typically, the extract of the Invention can be prepared by subjecting vine grapes, preferably grape pomace as described above (e.g. ice wine grape pomace of the Vidal variety) to extraction with the solvent, preferably water. Before the extraction step, the grapes, preferably the wine pomace, may be subjected to one or several pre-extraction steps such as drying, milling, crushing and / or debacterization. Debacterization may be performed by any method known in the art such as flash pasteurization or debacterization by saturated steam under vacuum. In preferred embodiments, the extraction is performed on non-fermented pomace.

[0187] Preferably, the extraction is performed under reflux. The weight ratio “solvent / grape” is typically from 0.5 to 20, for instance from 5 to 15 such as about 10. The extraction can last few minutes to several hours, preferably about one hour.

[0188] At the end of the extraction step, the liquid phase is recovered by any appropriate methods such as centrifugation or filtration. If the liquid phase it too cloudy, it may be also subjected to a clarification step, e.g. by use of a pectinase, or by depth filtration or any other alternative method known by the skilled artisan. If needed, the liquid phase can be partially concentrated so as to obtain an extract of the invention having a dry matter content of at least 50% such as of at least 70% in weight.

[0189] More generally, the extract can be subjected to additional steps such as filtration, ultrafiltration, sterilization e.g. by nanofiltration and / or formulation steps (e.g. by mixing the liquid phase) with a cosmetically acceptable carrier.

[0190] The vine grape extract of the invention, preferably the aqueous extract from grape pomace as described above (e.g. ice wine grape pomace of the Vidal variety) is characterized by a low polyphenol amount.

[0191] In some embodiments, the total polyphenol content in the extract is lower than 3.0%, preferably lower than 2.5% such as lower than 2.0% of the dry matter of the extract, the percentage in total polyphenols being expressed in gallic acid equivalent and preferably determined by Folin- Ciocalteu method e.g. as described in Singleton et al., Am. J. Enol. Vitic. 1965, 16, 144-158.

[0192] The extract of the invention can be also characterized by:

[0193] - a proanthocyanin content of at most 0.5%, preferably of at most 0.2% relative to the dry matter of the extract, the proanthocyanin percentage being expressed in cyanidin chloride equivalent and preferably determined by Porter method e.g. as described in Porter et al., Phytochemistry 1986, 25, 223-230, and / or

[0194] - a flavonoid content of at most 0.5%, preferably of at most 0.2% relative to the dry matter of the extract, the flavonoid percentage being expressed in rutin equivalent and preferably determined by A1C13 method e.g. as described in Shraim et al., Food Science and Technology 2021, 150, 111932.

[0195] The vine grape extract of the invention, preferably the aqueous extract from grape pomace as described above (e.g. ice wine grape pomace of the Vidal variety) is also enriched in carbohydrates and organic acids. In some embodiments, the extract of the Invention comprises:

[0196] - at least 40%, preferably from 45% to 55%, of total carbohydrates relative to the dry matter of the extract, the percentage of carbohydrates being expressed in glucose equivalents and preferably determined by the phenol-sulfuric acid method e.g. as described in Dubois et al., Analytical Chemistry 1956, 28, 350-356, and / or

[0197] - at least 5%, preferably from 5% to 20% of organic acids such as tartaric acid, malic acid and succinic acid, relative to the dry matter of the extract, the organic acid percentage preferably being determined by UHPLC by using organic acid standards to identify the retention times.

[0198] The vine grape extract of the invention, preferably the aqueous extract from grape pomace as described above (e.g. ice wine grape pomace of the Vidal variety) may also comprise amino acids at an amount of at most 5%, preferably from 1% to 3% relative to its dry matter, the amino acid percentages being expressed in arginine equivalent and determined by ninhydrin spectro- colorimetric method.

[0199] In some embodiments, the extract of the invention can be diluted or dissolved in a liquid cosmetically acceptable carrier such as triethyl citrate, a C2-C5 alkanediol such as propanediol, butylene glycol, pentylene glycol and isomers thereof, ketal esters such a methyl or ethyl levulinate, glycerin, water, and mixtures thereof. Alternatively, the extract of the invention can be adsorbed on a solid carrier such as maltodextrin.

[0200] In some preferred embodiments, the extract of the invention is formulated into glycerin to give a cosmetic ingredient that can be directly applied on the skin or incorporated in a cosmetic product.

[0201] The cosmetic ingredient may comprise from 50% to 80% of glycerin, and from 20% to 50% of the extract of the Invention.

[0202] One or several cosmetically acceptable excipients such as antioxidant, preservative, or pH adjusting agents may be also added preferably at an amount at most 5%, preferably at most 1% by weight of the total weight of the cosmetic ingredient. In other embodiments, the extract of the Invention can be directly incorporated into a cosmetic composition.

[0203] - Cosmetic active ingredient and cosmetic composition according to the invention

[0204] In a particular aspect, the Invention relates to a cosmetic ingredient comprising (i) an agent of the Invention, preferably a Vitis vinifera extract as described herein, e.g. an aqueous extract from an ice wine grape pomace of the Vidal variety, and (ii) a cosmetically acceptable carrier as described above. For instance, said carrier can be selected from tri ethyl citrate, a C2-C5 alkanediol such as propanediol, butylene glycol, pentylene glycol and isomers thereof, ketal esters such a methyl or ethyl levulinate, glycerin, water, and mixtures thereof. A preferred carrier is glycerin or glycerin / water mixtures. The cosmetic ingredient may comprise from 50% to 80% of glycerin, and from 20% to 50% of the extract of the Invention.

[0205] Typically, the active ingredient may comprise from (i) 1% to 5% of dry matter (w / v) from the extract in a mixture glycerin / water with a volume ratio from 1 / 1 to 4 / 1.

[0206] In some embodiments, the cosmetic ingredient of the Invention further comprises one or more cosmetically acceptable excipients, preferably selected from antioxidants, preservatives, pH adjusting agents and combinations thereof. Said antioxidants, preservatives, and pH adjusting agents can be as described above. The one or more cosmetically acceptable excipients account for 0.01% to 5.00%, preferably for 0.05% to 1.00% by weight of the total weight of the cosmetic ingredient of the Invention.

[0207] The cosmetic ingredient of the Invention is typically incorporated in a cosmetic composition at an amount from 0.1% to 10%, for instance from 0.5% to 5 % by weight of the total weight of the cosmetic composition.

[0208] In an additional aspect, the Invention further relates to a cosmetic composition comprising an agent of the invention, as described herein.

[0209] Typically, the cosmetic composition of the Invention comprises (i) an agent of the Invention, in particular a Vitis vinifera extract as described herein, e.g. an aqueous extract from an ice wine grape pomace of the Vidal variety and (ii) one or more cosmetically acceptable excipients.

[0210] The cosmetically acceptable excipients may be selected from standard excipients depending on the cosmetic composition to formulate.

[0211] Cosmetically acceptable excipients encompass, without being limited to, diluents, dispersing agents, gelling agents, emollients, vectorizing agents (such as polycationic polymers, phospholipids, liposomes, lamellar systems, lipid or polymeric vesicles, nanospheres, micro or nano-particles of natural or non-natural polymers, hydrogels), gums, resins, solvents, in particular water, C2-C6 alcohols or polyols including ethanol, isopropanol, dipropylene glycol, butylene glycol, propanediol, glycerin, sorbitol, propylene glycol and combinations thereof, fillers such as modified and polymerized starches, preservatives, pearlescent agents, odor absorbers, pH regulators, lubricating agents, thickening agents, surfactants including anionic, cationic, amphoteric or nonionic surfactants, humectants, wetting agents, stabilizing agents, bulking agents, dispersants, perfumes, dyes, organic or mineral pigments such as iron oxides, oily agents such as plant or animal oils or fats such as triglycerides, fatty alcohols, fatty acids or salts thereof, synthetic oils such as paraffins or isoparaffins, silicone oils (cyclomethicone), fatty alcohol esters, fluorinated oils, waxes, and / or other substances commonly used in cosmetic formulations.

[0212] In a particular embodiment, the cosmetic composition comprises from 0.005% to 10.0 % by weight, preferably from 0.01% to 0.5% e.g. such as 0.05 % to 2.0 % by weight of the agent of the invention in particular Vitis vinifera extract as described herein, e.g. an aqueous extract from an ice wine grape pomace of the Vidal variety, the percentage referring to the total weight of the cosmetic composition.

[0213] More particularly, said cosmetic composition may comprise:

[0214] - from 0.005% to 10.0 % by weight of a Vitis vinifera extract as described herein, e.g. an aqueous extract from an ice wine grape pomace of the Vidal variety and

[0215] - from 70 % to 99.995 % by weight of one or more cosmetically acceptable excipients.

[0216] In some embodiments, the cosmetic composition further comprises one or more active agents with additional cosmetic effect(s). Said one or more active agents can account for up to 29.995% by weight of the total weight of the composition. When present, the one or more additional cosmetic active ingredients account for 0.0001% to 29.995% of the total weight of the cosmetic composition.

[0217] The term "active agent with cosmetic effect or cosmetic active agent or ingredient" refers to an agent able to exert at least one cosmetic effect on the skin or its appendages. "Cosmetic effect" means any non-therapeutic effect aiming at modifying and / or improving the visual appearance and mechanical properties of the skin or mucous membranes such as the lips, protecting them from external aggressions (sun, wind, moisture, dryness, chemicals), preventing and / or correcting phenomena related to their aging, or preventing or treating the effects caused by stress or fatigue on the skin.

[0218] Examples of such agents are, among others, anti-wrinkle agents, anti-aging agents, antioxidant agents, moisturizing agents, soothing agents, anti-redness agents, exfoliating agents, seboregulating agents, lightening agents, concealers, anti-dark spot agents, anti-pollution agents, anti-stress agents, sunscreen agents, and combinations thereof.

[0219] In a particular embodiment, the cosmetic composition of the invention may comprise one or more additional cosmetic active agents selected from an anti-wrinkle agent, an anti-redness agent, an antioxidant agent, a moisturizing active, a soothing agent, a sebo-regulating agent, a lightening agent, an anti-stress agent, an anti-fatigue agent, an anti-pollution active, a concealer, a sunscreen agent, and combinations thereof.

[0220] Examples of anti-pollution agents include extracts of Chrysanthellum Indicum polysaccharides, especially marine from a fermentation medium of Alteromonas and salts of Nigari.

[0221] Examples of concealer agents include extracts of Chrysantellum Indicum or extracts and sulfated polysaccharides of algae, including Ascophyllum nodosum or Asparagopsis Armata. Examples of moisturizing agents include urea, pidolic acid (PCA) and its derivatives in particular its salts such as arginine PCA, chitosan PCA, its copper salts (Copper PCA), magnesium (magnesium PCA), sodium (sodium PCA) or zinc, ethylhexyl PCA, calcium gluconate, hyaluronic acid and its salts and other glycosaminoglycans, trehalose, polydextrose, sucrose (Sucrose), maltitol, mannitol, sorbitol, xylitol and other carbohydrates and derivatives, polyethylene glycols such as PEG-7, PEG-8, PEG-10, PEG-12 or PEG-14, glycerin, propylene glycol, butylene glycol, betaine, citrulline, collagen and its derivatives, histidine, silk, keratin or soy hydrolysates, plant extracts rich in polysaccharides and / or polyphenols, for example extracts of aloe, blueberry (Centaurea cyanus), extracts rich in polysaccharides, especially from fermentation media of marine microorganisms such as Alteromonas or Porphyridium and combinations thereof.

[0222] Examples of anti-aging agents include ascorbic acid and its derivatives such as magnesium ascorbyl phosphate, glycosaminoglycans and their derivatives such as hyaluronic acid, retinoids such as retinol, Cyathea polysaccharides, collagen, flaxseed extracts (Linum usitatissimum), peptides such as Caprooyl-Tetrapeptide-3 and trifluoroacetyl tripeptide-2, extracts of Polygonum aviculare, extracts of brown algae, in particular Ascophyllum nodosum, extracts of Cyathea cumingii, extract of Anigozanthos flavidus marketed under the tradename Skinectura™.

[0223] Examples of anti-stress agents include rose extracts such as Rosality™ which is a combination of rose water and rose essential oil and pink flowering cistus extract, for example marketed under the brand name IBR-Chill™.

[0224] Examples of anti-fatigue agents include liposoluble extract of Lavandula hybrid and liposoluble extract of Melaleuca alternifolia marketed under the tradename ImmunightTMand Regenight™. Examples of soothing agents include allantoin, extracts of aloe, birch (e.g. Betula alba , fireweed (e.g. Epilobium angustifolium), chestnut (e.g. Castenea sativa), blueberry (e.g. Centaurea cyanus), centella (e.g. Centella asialica), horsetail (e.g. Equisetum arvense), fennel (e.g. Foeniculum vulgare ), witch hazel (e.g. Hamamelis virginiana), ivy (e.g. Hedera helix), Hibiscus sabdariffa, lily (e.g. Lilium candidum), mallow (e.g. Malva sylvestris), lemon balm (e.g. Melissa officinalis), skullcap (e.g. Scutellaria baicalensis), mimosa (e.g Mimosa tenuiflora), cinquefoil (e.g. Potentilla erecta , an extract of oligosaccharides or an oligosaccharide, for example flax, peptides such as palmitoyl tripeptide-8, extracts of polysaccharides, especially extracts of exopolysaccharides from the fermentation medium of Alteromonas or Porphyridium.

[0225] Examples of antioxidant agents include HMR (hydroxy methyl resorcinol), ascorbic acid and its derivatives, vitamin B9, histidine hydrochloride, or fireweed extract (Epilobium augustifolium), tocopherol, carotenoids and extracts containing carotenoids (e.g. extracts of Dunaliella salina , and the likes.

[0226] Examples of sebo-regulating agents include flax lignans, rice powder, zinc gluconate, sarcosine, an extract of Cinnamomum zeylanicum bark, an extract of avocado, an extract of Backhousia citriodora and combinations thereof.

[0227] Examples of anti-redness agents encompass saponins, flavonoids, ruscogenins, esculosides, and extracts containing them, for example extracts of Ruscus, as well as certain essential oils, for example lavender or rosemary.

[0228] Examples of anti-spot agents include extracts such as licorice (Glycyrrhyza glabra , jackfruit extract (Artocarpus heterophyllus), Rumex extract (R occidental is), plant extracts belonging to the genus citrus, resveratrol, peptides such as oligopeptide-68, nonapeptide- 1, kojic acid, magnesium ascorbyl phosphate and combinations thereof.

[0229] Preferred additional cosmetic agents encompass pink flowering cistus extract, for example marketed under the brand name IBR-Chill™., liposoluble extract of Lavandula hybrid e.g. marketed under the tradename Immunight™ , liposoluble extract of Melaleuca alternifolia e.g. marketed under the tradename Regenight™, quinoa starch and derivatives thereof e.g. marketed under the tradename Pickmulse™ and combinations thereof.

[0230] The cosmetic composition of the invention is typically applied on the skin. The cosmetic composition may be in any form suitable for such a topical administration. In a particular embodiment, the cosmetic composition of the invention is selected from the group consisting of aqueous solutions, hydroalcoholic solutions, oil-in-water emulsions (O / W) or water-in-oil (W / O) or multiple), including nanoemulsions, aqueous gels (also called hydrogels), emulgels, suspensions, preferably in aqueous or hydroalcoholic medium, liposome suspensions, oily compositions, serums, powders, lotions, milks, creams, ointments, gels, foams, balms, foams, and aerosols.

[0231] In some embodiments, the cosmetic composition according to the invention is selected from a cream, a balm, a serum, or a lotion.

[0232] More generally, the composition according to the Invention can also take the form of any cosmetic or dermocosmetic product. It may be a skin care product, a make-up product or a body hygiene product, for example a lotion, a milk, a serum, an aqueous or oily gel, an emulsion, a cream, a gel-cream, an ointment, a balm, foundation, spray, mascara, stick, lipstick, gloss, deodorant, nourishing mask, shower gel, an aftershave, cleansing oil, soap, shampoo or conditioner, hair mask or oil including massage oil. In some preferred embodiments, the composition of the Invention is a skin care product e.g. a cream, a balm, a serum, a gel cream or an ointment.

[0233] Other aspects and advantages of the present invention will become apparent from the following examples, which are to be regarded as illustrative and in no way limiting.

[0234] EXAMPLE 1: SYNPO expression on soft vs. rigid support

[0235] Objective : Using an in vitro model that mimics the skin rigidity, the main aim of this experiment is to evaluate the synaptopodin mRNA expression in keratinocytes cultivated on different supports. Two types of supports were used, a rigid and a soft supports to mimic young skin and old skin, respectively.

[0236] Method : Normal Human Epidermal Keratinocytes (NHEK) cells were isolated from healthy human epidermis (female) undergoing plastic surgery and maintained in complete KGM2 medium containing 1 % of antibiotics (penicillin-streptomycin) at 37 °C under 5 % CO2 and 95 % humidity. NHEK were seeded at 105cells on the surface of hydrogels at 4 kPa (soft support) and 2.104cells on plastic plate (rigid support).

[0237] The initial cell density on the two supports were different due to the proliferation rate of keratinocytes that is affected by the support stiffness. While the cells proliferate “normally” on a rigid support, they stop their proliferation on a soft one. The initial cell density was optimized to achieve an equivalent cell number at 72 hours post-seeding..

[0238] Thus, after 72 hours, the cells were collected, washed with cold PBS IX and, then total RNA was extracted using the RNeasy mini kit (Qiagen). The amount and purity of RNA was determined spectrophotometrically using the NanoDrop ND-2000 (Thermo Scientific). By calculating the absorbance ratio at 260 / 280 nm, the purity of the mRNA and the absence of contaminating proteins were determined and with the absorbance ratio at 260 / 230 nm, allow to evaluate the contamination or not of organic molecules could be assessed. The mRNAs used had an A260 / A280 ratio > 1.8 and an A260 / A230 ratio > 1.6.

[0239] Reverse transcription (RT) of mRNA was performed by using PrimeScriptTM RT Reagent Kit (Perfect Real Time) (TaKaRa, Ozyme) according to the manufacturer's recommendations in order to obtain cDNA (complementary DNA).

[0240] Subsequently, qPCR was performed with 12.5 ng of previously prepared cDNA by using Faststart Universal SYBR® Green Master (ROX, Roche). The qPCR results were standardised against a housekeeping gene (RPL13A). Each sample was run in duplicate.

[0241] Results : the results are shown in Figure 1. Figure 1 shows the relative Synaptopodin mRNA expression normalized to the housekeeping gene RPL13A for keratinocytes on rigid support (plastic) and on a soft substrate (soft hydrogel). Synaptopodin mRNA expression was decreased by 80% in NHEK cultivated on soft support compared to rigid support. The expression of synaptopodin is affected by the rigidity of the support on which the keratinocytes are cultivated.

[0242] EXAMPLE 2: Decrease in synaptopodin expression during keratinocyte differentiation

[0243] - Validation of the differentiation model

[0244] Objective :To analyze the involvement of synaptopodin in the differentiation process of human keratinocytes, we used an early 2D differentiation model. This model consists of inducing the first stages of epidermal differentiation in keratinocytes cultured as monolayers. Markers from the different layers of the epidermis were used to validate the stages of differentiation.

[0245] Method: Human primary keratinocytes were seeded at a density of 23.103cells / cm2in 6-well microplates in a specific medium containing growth factors. When the cells reached confluence, the culture medium was replaced with growth factor-free medium to force the process of differentiation.

[0246] The cells were stopped at different times: during the proliferation phase (D-2), when the cells were confluent (DO) and during their differentiation phase (D2, D4 and D6), corresponding to days 1, 2, 4, 6 and 8 after seeding.

[0247] For all conditions, total RNA was extracted using the RNeasy mini kit (Qiagen) to assess the genetic expression of synaptopodin and differentiation markers (see below). The amount and purity of RNA was determined spectrophotometrically using the NanoDrop ND-2000 (Thermo Scientific). Reverse transcription of mRNA to obtain cDNA (complementary DNA) was performed by using the PrimeScript™ RT Reagent Kit (Perfect Real Time) (TaKaRa, Ozyme) according to the manufacturer's recommendations.

[0248] The qPCR was performed with 12.5 ng of previously prepared cDNA using the Fast Start universal SYBR Green Master Kit (ROX, Roche). The qPCR results were standardized against a housekeeping gene (RPL13A). Each sample was run in duplicate.

[0249] The specific differentiation markers examined were Ki67, a proliferation marker, cytokeratin 10, a trabecular meshwork marker (K10) and involucrin a tardive differentiation marker, and synaptopodin.

[0250] Results : The results are shown in Figure 2A. Figure 2A shows the relative mRNA expression of each investigated marker (Ki67, K10 and Involucrin) normalized to the housekeeping gene RPL13 A at the different phases : proliferation phase (D-2), confluence (DO) and differentiation phase (D2, D4 and D6). One-way ANOVA was used to determine the correlation between gene expression and keratinocytes differentiation state. The statistical significant values are p< 0.01 ** and p<0.001 ***.

[0251] As expected, the Ki67 marker was expressed at a higher level in proliferating cells than in differentiating cells, while the differentiation markers were expressed in differentiating cells only. In addition, the increase in differentiation marker expression was delayed in time, with an early expression on day 2 for the early differentiation marker (K10) and later expression on day 6 for the late differentiation marker (involucrin). These results confirm the in vitro model to mimic the different stages of keratinocytes.

[0252] Figure 2B shows the relative mRNA expression of synaptopodin normalized to the housekeeping gene RPL13A at the different stages: proliferation phase (D-2), confluence (DO) and differentiation phase (D2, D4 and D6).

[0253] When the cells reached confluence, synaptopodin expression tended to decrease (#, p-value= 0.052) compared to the proliferating stage, and this decrease was significant when the cells entered the differentiation stage. The decrease in synaptopodin RNA expression on differentiated cells was confirmed by immunolabelling of synaptopodin to quantify expression at the protein level on different cell phases (data not shown).

[0254] Thus, synaptopodin expression decreases in differentiated keratinocytes. This result is in agreement with the immunofluorescence result showing a high expression of synaptopodin in the basal layer representing the proliferative stage of the cells (see Example 3 below).

[0255] EXAMPLE 3: synaptopodin location in the skin Objective : The main purpose of this experiment is to identify the presence and location of synaptopodin in the skin using skin explants.

[0256] Biological materials : Healthy biopsy samples of skin obtained from abdominoplasty reductions of Caucasian women aged 20 to 60 years were collected by Alphenyx company.

[0257] Method : Upon receipt of human explants, the skin biopsies were cut into small pieces (0.6 cm2), then skin samples were frozen before to be embedded in optimal cutting temperature (OCT), and sectioned at 5pm by using a cryostat (Epredia™). Sections were mounted on glass slides and stored at -20°C until use. Synaptopodin was specifically labelled with a synaptopodin antibody (abeam) diluted at 1 / 250 in PBS - 0.5% BSA overnight at 4°C. After three PBS IX washes, Alexa 546 secondary antibody 1 / 1000 was added for one hour at room temperature in the dark to detect synaptopodin labelling. Microscopic observations and slides were imaged using a lame scanner Zeiss Axioscan 7 to confirm the synaptopodin location.

[0258] Results : Figure 3 is a picture of an histological section stained with anti-synaptopodin antibody firoma skin explant of a Female of 33 years old. This picture is representative of the all explants observed. Figure 3 shows that synaptopodin was mostly present in the basal layer of the epidermis, i.e. undifferentiated keratinocytes. Synaptopodin is not detected in the layers corresponding to the most differentiated keratinocytes such as in stratum corneum. Some expression of synaptopodin is detected in the dermis but it mainly corresponds to blood vessels, sebaceous glands and hair follicles.

[0259] Thus, synaptopodin is mainly associated with undifferentiated keratinocytes in the skin.

[0260] EXAMPLE 4: Age-dependent decrease in synaptopodin expression

[0261] • EXAMPLE 4A

[0262] Objective : Using frozen skin explants, the aim of this experiment was to analyse the expression of synaptopodin in relation to age.

[0263] Method: Frozen skin biopsies of approximatelt 8mm diameter from abdominoplasty or breast procedures of Caucasian women aged 19 to 71 years were obtained from the company Alphenyx or DermoBiotec center. Each frozen skin samples were embedded in OCT and sectioned at 5pm thichness using a cryostat cryotome (Epredia™). Sections were mounted on glass slides and stored at -20°C until use. Prior to immunostaining, the OCT component was removed from the skin sections and rehydrated in PBS IX. Slides were then labelled with synaptopodin (abeam) and cytokeratin 14 (Invitrogen) antibodies overnight at 4°C. After three washes in PBS IX, the primary stainings were revelated with Alexa 488 and Alexa 546 secondary antibodies (Bio-rad), respectively, added for 1 hour at room temperature in the dark. The nuclei were counterstained with DAPI (Sigma).

[0264] Results: Slides were imaged using a lame scanner Zeiss Axioscan 7. Synaptopodin staining was quantified in the basal layer of the epidermis by the QuPath software with a region of interest determined by the cytokeratin 14 labelling.

[0265] The correlation coefficient was calculated to highlight the relationship between the level of synaptopodin expression and the age of the corresponding human explant. To do that, the Pearson r was used to determine the correlation between synaptopodin expression and explant age. The statistical significant value is p< 0.05 *.

[0266] The results are shown in Figure 4A and 4B. Figures 4A and 4B show the expression of synaptopodin evidenced by immunofluorescent staining in histoological skin sections depending on the age of skin explant donors. In the whole epidermis and basal layer, a significant negative correlation is observed between explant age and synaptopodin protein expression levels. These results show that synaptopodin expression decreases with age and this decrease is more pronounced in the basal layer of the epidermis.

[0267] • EXAMPLE 4B

[0268] The same assay as in EXAMPLE 4A was performed on skin biopsies from abdominoplasty surgeries in Caucasian women aged under 30 or over 60.

[0269] The results are shown in Figure 4C. This experiment confirms a clear decrease in synaptopodin expression in epidermis with age: synaptopodin expression was significantly lower in explants from donors over 60 years old than in explants from donors under 30 years old, in both the whole epidermis and the basal layer.

[0270] EXAMPLE 5: Colocation of synaptopodin and COLXVII in the skin

[0271] Objective: The main purpose of this experiment is to study the distribution of synaptopodin and collagen XVII in the skin using skin explants.

[0272] Biological materials: Healthy biopsy samples of skin obtained from abdominoplasty reductions of Caucasian women aged 20 to 60 years were collected by Alphenyx company.

[0273] Method: Upon receipt of human explants, the skin biopsies were cut into small pieces (0.6 cm2), then skin samples were frozen before to be embedded in optimal cutting temperature (OCT), and sectioned at 5pm by using a cryostat (Epredia™). Sections were mounted on glass slides and stored at -20°C until use. Collagen XVII was pecifically labelled with a synaptopodin antibody (abeam) diluted at 1 / 100 in PBS - 0.5% BSA overnight at 4°C. After three PBS IX washes, Alexa 488 secondary antibody 1 / 1000 (Bio-rad) was added for one hour at room temperature in the dark to detect synaptopodin labelling. Then, synaptopodin was specifically labelled with a synaptopodin antibody (abeam) diluted at 1 / 250 in PBS - 0.5% BSA overnight at 4°C. After three PBS IX washes, Alexa 546 secondary antibody 1 / 1000 (Bio-rad) was added for one hour at room temperature in the dark to detect synaptopodin labelling.

[0274] Results: Microscopic observations and slides were imaged using a fluorescent microscopy (EVOS-FL, ThermoFisher, Scientific) and showed that synaptopodin and collagen XVII are collocated, in particular in the basal layer of epidermis (Figure 5).

[0275] EXAMPLE 6: Ice wine grape pomace extract increases synaptopodin expression

[0276] 1. Preparation of the ice wine grape pomace extract

[0277] Ice wine pomace of Vidal variety was collected in a Canadian vineyard. The grapes were harvested after being naturally frozen on the vine. The pomace (i.e. by-product derived from wine manufacture) was recovered before fermentation. The pomace was dried in an oven (50°C), milled and subjected to debacterization by saturated steam under vacuum

[0278] The resulting material was extracted with water at reflux (1 kg of pomace for about 10 L of water) during about one hour. The liquid fraction was recovered by filtration and clarified thanks to pectinase addition during one hour at 50°C. After pectinase degradation (10’ at 80°C) and filtration, the obtained extract was partially concentrated and diluted to glycerin to obtain a dry matter of about 2.5% (w / v) in glycerin / water (about 7 / 3) (v / v) so as to active ingredient of the Invention (called hereunder ice wine grape pomace active ingredient).

[0279] 2. Chemical characterization o f the Extract

[0280] Table 1 hereunder shows the chemical characterization of the ice wine grape pomace extract. The analyses were performed before dilution in glycerin. The percentages are relative to the dry matter of the extract.

[0281] 3. Biological characterization : Ice wine grape pomace active ingredient increases synaptopodin expression in keratinocytes on a soft stand (soft hydrogels 5kPa)

[0282] Skin ageing is associated with a number of histological changes. In fact, the epidermis becomes thinner and the dermal-epidermal junction (DEJ) flattens, while the density of the underlying dermis decreases. In addition, the extracellular matrix (ECM) is affected by a reduction of several component expression such as type I and III collagens and elastin, while the activity of MMPs is increased, resulting in a significant loss of skin elasticity. These changes have a direct impact on the mechanical properties of the skin, with the overall elastic modulus decreasing progressively with age.

[0283] The mechanical properties of the skin can be measured and it has been shown that an elastic modulus of 10 kPa corresponds to a 30-year-old skin, 7.5 kPa to a 60-year-old skin and 5.3 kPa to an 80-y ear-old skin. These variations in stiffness affect the properties of keratinocytes which must adapt to changes in their microenvironment variation. The ability of keratinocytes to sense and adjust their behaviour (proliferation, migration and differentiation) in response to mechanical changes may be a crucial factor in the skin's response to these changes.

[0284] In order to mimic skin ageing as closely as possible, a 5 kPa support may be a support of choice for several reasons. This level of stiffness is close to that measured in the skin of an elderly person, making it a relevant approximation of the skin stiffness associated with ageing. The choice of a 5 kPa support allows the mechanical conditions associated with skin ageing, and to mimicked and the cellular mechanisms involved to be studied.

[0285] Methods: 48-wells plates containing 5 kPa hydrogels (indentation measurement, Cell&Soft) were rehydrated with sterile PBS IX for 24 hours. Then, hydrogels were equilibrated with cold medium (to avoid cracking) for 1 hour at 37 °C, 5 % CO2 and 95 % humidity. At the end of the incubation period, NHEKs were seeded at 23.103cells / cm2in complete medium. After 72 hours, the cells were treated with different concentrations of active ingredient of the invention (0.2% and 0.1%) for an additional 24h period. At the end of the incubation period, the cells were fixed and stained to assess the expression of synaptopodin. The cell nuclei were then counterstained with DAPI (Sigma).

[0286] Synaptopodin images were acquired using the imaging section of the BioTek Cytation 1 using Gen5 software.

[0287] Results: Figure 6 shows the intensity of fluorescence reported per cell treatment with the extract of the invention (at 0.1 or 0.2%) compared to the untreated control cells. The Ice wine grape pomace active ingredient significantly increases synaptopodin expression. On a soft support, mimicking old skin, the treatment of keratinocytes with 0.2% or 0.1% ice wine pomace active ingredient significantly increases synaptopodin expression.

[0288] 4. Biological characterization : Ice wine grape pomace active ingredient increases synaptopodin expression in skin explant

[0289] Objective : In order to mimic the physiology of human skin as closely as possible, the aim of this experiment was to evaluate the effect of topical treatment with active ingredient of the Invention on ex vivo human skin explants.

[0290] Biological materials : Frozen skin biopsies from Caucasian women aged 52 who had undergone abdominoplasty were collected by the company Alphenyx.

[0291] Method : Upon receipt, the skin biopsies were cut into small pieces (1 cm2). The skin samples were then placed in culture inserts with 8 pm pore diameter (Thincert™ transparent membrane, Greiner Bio-One), epidermis side up. Specific culture medium (DMEM + 1% Pen / Strep + 1% non-essential amino acids) was added to fully submerge the dermis, leaving the epidermis dry at the air-liquid interface. The biopsies were then treated or not with 0.2% of the active ingredient topically for 24 hours at 37°C and 5% CO2. At the end of the incubation period, each biopsy was wrapped in aluminum foil and stored at -80°C. Each frozen skin samples were then embedded in OCT and sectioned in 5pm thichness using a cryostat cryotome. Sections were mounted on glass slides and stored at -20°C until use. Prior to immunostaining, the OCT component was removed from the skin sections and rehydrated in PBS IX. Slides were labelled with synaptopodin antibody (abeam) over night at 4°C. After three washes in PBS IX, the primary staining were revelated with Alexa 488 secondary antibody, added for 1 hour at room temperature in the dark. The nuclei were counterstained with DAPI (Sigma).

[0292] Results: Figure 7A shows the fluorescence intensity reported per cell and compared to the untreated control. In the skin explant, synaptopodin protein levels increased significantly by 49% after treatment with 0.2% Ice wine grape pomace active ingredient compared to the untreated condition. Treatment with 0.2% ice wine active ingredient significantly increases synaptopodin expression in skin explant.

[0293] 5. Biological characterization: Ice wine grape pomace active ingredient versus standard extract from white wine pomace (without any natural freeze of the grapes)

[0294] White wine pomace of Vidal variety was collected in a Canadian vineyard. The grapes were harvested before any natural freezing of the grapes on vines. The pomace was recovered before fermentation and subjected to an extraction process similar to that used to obtain the Ice wine grape pomace active ingredient of the Invention (see above).

[0295] Objective: to demonstrate the beneficial effect of the ingredient of the invention versus extract obtained from pomace of standard white wine on synaptopodin expression

[0296] Method: Human primary keratinocytes were seeded at a density of 7.103cells / well in 96-well plates in specific medium containing growth factors. 24 hours later, the cells were treatment with Ice wine grape pomace active ingredient (Ice wine) or standard pomace extract (white wine) at different concentrations for an additional 24 hours. At the end of incubation, the cells were fixed and stained to assess the expression of synaptopodin. The cell nuclei were then counterstained with DAPI. Synaptopodin images were acquired using the imaging section of the BioTek Cytation 1 using Gen5 software

[0297] Result: Figure 6B shows the intensity of fluorescence reported per cell treatment with the extract of wine versus extract of white wine (at 0.1 or 0.2%) compared to the untreated control cells. The active ingredient of the invention significantly increases synaptopodin expression whereas the standard pomace extract has not impact on synaptopodin expression versus untreated control cells.

[0298] EXAMPLE 7: Impact of Ice wine grape pomace active ingredient on the crosstalk between keratinocytes and fibroblasts

[0299] Objectif: By using primary keratinocytes, the aim of this experiment was to evaluate the indirect effect of Ice wine grape pomace active ingredient on fibroblasts and the extracellular matrix. To this end, normal human dermal fibroblasts (NHDF) were cultured with conditioned media from treated normal human epidermal keratinocytes (NHEK), and the pro collagen I production was assessed. The Ice wine grape pomace active ingredient corresponds to the active ingredient described in Example 6 sections 1 and 2 above.

[0300] Method: Keratinocytes were treated with 0.2 % of ice wine grape pomace active ingredient for 24 hours, after which their culture medium was transferred onto fibroblasts that had been serum- starved for 24 hours. In parallel, fibroblasts that had been serum-starved for 24 hours were treated directly with Ice wine grape pomace active ingredient at different concentrations (0.2%, 0.1% and 0.05%).

[0301] Following incubation, the fibroblast supernatant was collected and procollagen type I quantification was performed using the Duoset ELISA kit (R&D Systems #DY6220-05), following the manufacturer’s instructions. The absorbance was read at 450 nm with a correction at 570 nm using a BioTek Cytation 1 plate reader. Ascorbic acid at a concentration of 25 pg / mL was used as a positive control for type I procollagen production (AA), while keratinocyte- specific medium that had not been in contact with cells served as a negative control.

[0302] Result: Of note, the direct incubation of NHDF with ice wine grape pomace active ingredient at different concentrations has no significant effects on type I procollagen levels in the supernatant, as compared to the control cells (untreated NHDF) (Figure 8A).

[0303] In contrast, NHDF treated with the culture medium of NHEK exposed to the ice wine grape pomace active ingredient (0.2%) show a significant increase in type I procollagen production as compared to control cells (untreated NHDF). Such results show that ice wine grape pomace active ingredient modulates type I procollagen secretion by fibroblasts through an indirect effect mediated by keratinocytes. In other words, these results illustrate a clear keratinocyte-fibroblast communication promoted by Ice wine grape pomace active ingredient.

[0304] EXAMPLE 8: Impact of Ice wine grape pomace active ingredient on keratinocyte differentiation

[0305] Objectif: The aim of this study was to evaluate the effects of Ice wine grape pomace active ingredient as described in Example 6 on K10 expression in human keratinocytes cultured on plastic and soft support (4 kPa hydrogel) by RT-qPCR. K10 is a marker of cell differentiation in keratinocytes.

[0306] Method: NHEK were seeded at 23.103 cells / cm2density in 6 well-microplates containing hydrogels at 4 kPa or plastic in complete medium. 72h hours later, the cells were treated with 0.2% of ice wine for additional 24h. At the end of incubation period, the cells were collected, then total RNA was extracted with RNeasy mini kit. Reverse transcription (RT) of mRNA was performed by using PrimeScriptTM RT Reagent Kit (Perfect Real Time) according to the manufacturer's recommendations in order to obtain cDNA (complementary DNA).

[0307] Then, qPCR was performed with 50 ng of previously prepared cDNA by using Faststart Universal SYBR® Green Master. The qPCR results were standardized against a housekeeping gene (RPL13A). Each sample was run in duplicate. Result: NHEK cultured on soft hydrogel are characterized by a decrease in synaptopodin expression and an increase in K10 expression, consistent with a differentiation phenotype. Treatment of NHEK on soft gel with Ice wine grape pomace active ingredient increases synaptopodin expression and leads to a decrease in K10 gene expression. These results suggest that Ice wine grape pomace active ingredient, by increasing synaptopodin expression, can limit or delay keratinocyte differentiation on a soft substrate and thus in mature skin (Figure 9).

[0308] EXAMPLE 9: Effect of Ice wine grape pomace active ingredient on reconstructed human epidermis (RHE)

[0309] Objectif: The aim of this study was to evaluate the effects of Ice wine grape pomace active ingredient as described in Example 6 on 3D keratinocytes differentiation.

[0310] Method: The 3D reconstructed epidermis model mimics natural skin development by allowing keratinocytes to stratify and differentiate at the air-liquid interface. To build the model, fibroblasts were first seeded on the underside of porous membrane inserts and given time to adhere before the inserts were flipped. Keratinocytes were then added to the upper surface and cultured for three days. 0,2% of Ice wine grape pomace active ingredient was applied for 24 hours before air exposure and was then continuously maintained in the culture medium throughout the differentiation process. Media were renewed daily to keep the epidermal surface dry. After 16 days of culture at the air-liquid interface, the tissues were fixed overnight in 4% PFA at 4 °C. Inserts were rinsed, processed, and embedded in paraffin. Sections were cut at 5 pm and stained using an antibody against involucrin to assess epidermal differentiation.

[0311] Result: The expression of Involucrin, a marker corresponding to tardive differentiation was analyzed. In 3D reconstructed epidermis model treated with Ice wine grape pomace active ingredient, Involucrin was mainly present in the uppermost layers of the epidermis and less detectable in the lower layers whereas in control condition (3D reconstructed epidermis model not treated with the active ingredient), involucrin expression extended into the spinous layer.

[0312] In conclusion, Ice wine grape pomace active ingredient, by increasing synaptopodin expression delays, epidermal differentiation, as evidenced by the more superficial localization of involucrin.

[0313] EXAMPLE 10: Effects of resveratrol and retinoic acid on synaptopodin expression in keratinocytes cultured on soft substrate (Comparative data). Objectif : The aim of this study was to evaluate the effects of retinoic acid (AR) and resveratrol (RSV) on synaptopodin expression in human keratinocytes cultured on hydrogel (4 kPa) by immunostaining.

[0314] Method: NHEK were seeded at 23.103cells / cm2density in 96 or 48 well-microplates in complete medium. 24h hours later, the cells were treated with different concentrations of retinoic acid (1 pM and 5 pM) and resveratrol (10 pM and 50 pM) were added for additional 24h. At the end of the incubation period, the cells were fixed and stained to perform immunostaining.

[0315] Synaptopodin immunolabelling: Synaptopodin was specifically labeled with a Synaptopodin antibody (abeam) diluted at 1 / 250 in PBS - 0.5% BSA over night at 4°C. After three washed Alexa 488 secondary antibody was added at 1 / 1000 for one hour at room temperature in the dark in order to reveal synaptopodin labelling. The nuclei were counter-stained using DAPI (Sigma).

[0316] Result: Neither retinoic acid nor resveratrol was able to significantly promote synaptopodin expression in keratinocytes cultured on hydrogel (Figures lOA and 10B).

[0317] EXAMPLE 11 : A cosmetic composition comprising the Ice wine grape pomace active ingredient (Illustrative example)

[0318] Day cream List of sequences

Claims

Claims1. Cosmetic use of an agent able to increase synaptopodin expression in keratinocytes for improving, restoring or maintaining cutaneous homeostasis, preferably epidermal homeostasis in the skin.

2. The cosmetic use of claim 1, wherein the agent is able to increase the expression of synaptopodin in keratinocytes on a soft hydrogel as compared to keratinocytes cultured on a soft hydrogel but not contacted with the agent.

3. The cosmetic use of claim 1, wherein the agent is selected by the method as described in any one of claims 17 to 19.

4. The cosmetic use of any one of claims 1 to 3, wherein the agent is used:• for decreasing, slowing down, or delaying the onset of keratinocyte differentiation, and / or• for decreasing the expression a differentiation marker in the skin and / or• for promoting the production of procollagen type I in the skin and / or• for maintaining or restoring dermal extracellular matrix, in a skin.

5. The cosmetic use of any one of Claims 1 to 4, wherein the agent is for improving the visual appearance of the skin, in particular for attenuating, decreasing or treating one or more non-pathological alterations of the skin appearance such as fine lines, wrinkles, a loss of skin density, a loss of skin firmness, a loss of skin elasticity, an increase in skin roughness, a loss of radiance or glow, dull or non-uniform complexation and combinations thereof.

6. The cosmetic use of any one of Claims 1 to 5, wherein the agent is topically applied on the skin of a subject having at least 40 years old, preferably a woman of at least 40 years old.

7. The cosmetic use of any one of Claims 1 to 4, wherein the agent is used to prevent, slow-down or delay the onset of a decrease in collagen production, a decrease in dermalextracellular matrix quality, a loss of skin density and / or a loss of skin firmness in a subject of less than 40 years old.

8. The cosmetic use of any one of Claims 1 to 7 wherein the agent is present as an active cosmetic ingredient in a cosmetic composition which is applied on the skin.

9. The cosmetic use of Claim 8, wherein the cosmetic composition is selected from a skin care product, a skin hygiene product or a make-up product, such as a cream, a balm, an ointment, a serum, a gel, a mask, and a foundation.

10. The cosmetic use of any one of claims 1 to 9, wherein the agent is selected from peptides and plant extracts.

11. The cosmetic use of claim 10 wherein the plant extract is an aqueous extract from white Vitis vinifera grapes said grapes being harvested after being frozen on the vine.

12. The cosmetic use of claim 10 or 11, wherein the plant extract is an aqueous extract obtained from ice wine grape pomace of Vidal variety.

13. The cosmetic use of claim 12 wherein the extract is obtained by a method of extraction comprising a step of extracting ice wine grape pomace of Vidal variety with hot water at a temperature of at least 60°C, preferably under reflux.

14. The cosmetic use according to any one of claims 11 to 13 wherein the Vitis vinifera grape extract has a total polyphenol content lower than 3.0%, preferably lower than 2.5% such as lower than 2.0% of the dry matter of the extract, the percentage being expressed in gallic acid equivalent.

15. A cosmetic composition for improving, restoring or maintaining cutaneous homeostasis in a healthy skin comprising (i) an aqueous extract from Vitis vinifera as defined in any one of Claims 11-14 as an active cosmetic ingredient and (ii) at least one cosmetically acceptable excipient.

16. A cosmetic method for improving, restoring or maintaining cutaneous homeostasis in a healthy skin, comprising topically applying an agent able to increase synaptopodin, preferably a plant extract as defined in any one of claims 11 to 14, on the skin of the subject.

17. A method for selecting an agent able to improve, restore or maintain cutaneous, preferably epidermal, homeostasis in a skin, preferably in a healthy and aged skin, said method comprising contacting keratinocytes with a candidate agent, and determining whether the candidate agent increases the expression of synaptopodin in the keratinocytes, as compared to a reference expression level.

18. The method of claim 17 which comprises the steps of : a) contacting keratinocytes with an agent candidate, b) quantifying the expression of synaptopodin in said keratinocytes, c) comparing the expression of synaptopodin in said keratinocytes with that of reference keratinocytes not contacted with the agent candidate, and d) selecting the agent candidate if said agent candidate increases the expression of synaptopodin in said keratinocytes as compared to the reference keratinocytes, and wherein the keratinocytes are human primary keratinocytes seeded on a soft substrate having an elastic modulus lower than 10 kPa, preferably from 3.0 to 6.0 kPa.

19. An ex vivo method for selecting an agent for improving, restoring or maintaining cutaneous, preferably epidermal homeostasis in a skin, preferably in a healthy and aged skin, said method comprising : a) contacting a skin explant with an agent candidate, b) quantifying the expression of synaptopodin in said skin explant, c) comparing the expression of synaptopodin in said skin explant with that of a reference skin explant not contacted with the agent candidate, and d) selecting the agent candidate if said agent candidate increases the expression of synaptopodin in said skin explant.

20. A method for preparing a cosmetic active ingredient, e.g. for improving, restoring or maintaining epidermal homeostasis in an aged skin, said method comprising the steps of a) implementing the method for selecting an agent as defined in any one of claims 17 to 19,b) optionally formulating the agent selected in step a) with one or more cosmetically acceptable excipients, e.g. a vehicle, a pH adjusting agent and / or a preservative, so as to obtain the cosmetic active ingredient.

21. A method for preparing a cosmetic product, e.g. for improving, restoring or maintaining epidermal homeostasis in an aged skin, said method comprising the steps of a) implementing the method for selecting an agent, as defined in any one of claims 17 to 19, b) formulating the agent selecting in step a) with one or more pharmaceutically acceptable excipients, and optionally with one or more additional cosmetic active ingredients whereby the cosmetic product is obtained.

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