Method for identifying retinoid-mimetic anti-aging compounds

Abstract

Method for Identifying Retinoid-Mimetic Anti-Aging Compounds. The present invention relates to a method for identifying an anti-aging compound that mimics retinoic acid, retinol, or one of their derivatives. It also relates to the cosmetic use of at least one anti-aging compound that mimics retinoic acid, retinol, or one of their derivatives, identified by such a method, for preventing and / or treating skin aging. It further relates to a method for evaluating the efficacy of a cosmetic composition comprising, as an active ingredient, retinol, retinoic acid, one of their derivatives, or an anti-aging compound identified by such a method. Figure for the abstract: none

Description

Title of the invention: Method for identifying retinoid-mimetic anti-aging compounds

[0001] The present invention relates to a method for identifying an anti-aging compound having a mimetic effect of retinoic acid, retinol, or one of their derivatives. It also relates to the cosmetic use of at least one anti-aging compound having a mimetic effect of retinoic acid, retinol, or one of their derivatives, identified by such a method, for preventing and / or treating skin aging. It further relates to a method for evaluating the efficacy of a cosmetic composition comprising, as an active agent, retinol, retinoic acid, one of their derivatives, or an anti-aging compound identified by such a method.

[0002] Glycosaminoglycans (GAGs) are a major component of the extracellular matrix. They are large, linear polysaccharides composed of repeating disaccharide units. The six types of GAGs include chondroitin sulfate (CS), dermatan sulfate (DS), keratan sulfate (KS), heparin sulfate (HS), heparin (HP), and hyaluronic acid (HA). GAGs interact with a variety of molecules, including cell adhesion molecules and growth factors.

[0003] Several studies have shown various changes in GAGs, proteoglycans (PGs) and glycoproteins in aged skin (Donovan et al, N-glycans are stratum comeum biomarkers of aging skin, J Invest Dermatol 2022 Aug 31; S0022-202X(22)01877-2), suggesting that these molecules are important contributors to skin aging (Carrino et al., Age-related changes in the pro-teoglycans of human skin, Archives of Biochemistry and Biophysics, Volume 373, Issue 1, 1 January 2000, Pages 91-101).

[0004] There is therefore a need for methods of identifying effective active ingredients in the prevention and / or treatment of skin aging. There is also a need for cosmetic active ingredients capable of improving skin hydration.

[0005] The present invention meets this need.

[0006] Surprisingly, the Applicant has now discovered that applying retinol or retinoic acid to the skin decreases the expression of 14 glycoside hydrolase enzymes that cleave glycosidic bonds in glycosides. These enzymes are primarily involved in the deglycosylation of several types of N-glycans, gangliosides, HS, DS, CS, and KS. These results show that retinoids inhibit glycan degradation by decreasing the expression of specific glycoside hydrolases: this ensures protection of the oligosaccharide chains of GAGs against potential shortening.

[0007] The present invention therefore relates to a method for identifying an anti-aging compound having a mimetic action of retinoic acid, retinol or one of their derivatives, said method comprising the following steps: a. bring at least one candidate compound into contact with a skin sample from a subject, or with a skin model or cell model in which at least one glycoside hydrolase is expressed, and then measure the level of expression and / or enzymatic activity of that glycoside hydrolase in said sample or model; b. compare the expression measured in step a) to the expression of this glycoside hydrolase in a skin sample from the subject or in a skin model or cell model not exposed to said compound; and c. identify said candidate compound as having an anti-aging effect mimicking retinoic acid, retinol or one of their derivatives, when a decrease in the expression of this glycoside hydrolase in the subject's skin sample or skin model or cell model that has been exposed to said candidate compound is detected, compared to the expression of this glycoside hydrolase in the subject's skin sample or skin model or cell model that has not been exposed to the candidate compound.

[0008] It also relates to the cosmetic use of at least one anti-aging compound having a mimetic action of retinoic acid, retinol or one of their derivatives, identified by the process according to the invention, to prevent and / or treat skin aging.

[0009] It also relates to a method for evaluating the effectiveness of a cosmetic composition comprising, as an active agent, retinol, retinoic acid, one of their derivatives or an anti-aging compound identified by the process according to the invention, comprising the following steps: a. the measurement of the level of expression and / or enzymatic activity of at least one glycoside hydrolase in a skin sample from a subject after application of the cosmetic composition; then b. comparison of the measurement from step a) with a control sample.

[0010] All the methods described in the present invention are in vitro methods.

[0011] Method for identifying an anti-aging compound having a mimetic action of retinoic acid, retinol or one of their derivatives

[0012] The present invention relates to a method for identifying an anti-aging compound having a mimetic action of retinoic acid, retinol or one of their derivatives, said method comprising the following steps: has. bring at least one candidate compound into contact with a skin sample from a subject, or with a skin model or a cell model in which less a glycoside hydrolase is expressed, then measure the level of expression and / or enzymatic activity of that glycoside hydrolase in said sample or model; b. compare the expression measured in step a) to the expression of this glycoside hydrolase in a skin sample from the subject or in a skin model or cell model not exposed to said compound; and c. identify said candidate compound as having an anti-aging effect mimicking retinoic acid, retinol or one of their derivatives, when a decrease in the expression of this glycoside hydrolase in the subject's skin sample or skin model or cell model that has been exposed to said candidate compound is detected, compared to the expression of this glycoside hydrolase in the subject's skin sample or skin model or cell model that has not been exposed to the candidate compound.

[0013] The identification method of the invention is preferably carried out ex vivo.

[0014] The anti-aging compounds identified by the method according to the invention have a mimetic effect on retinoic acid, retinol, or one of their derivatives. They are retinoid mimetics, particularly on skin glycoside hydrolases.

[0015] A “derivative” of retinoic acid or retinol means a natural or synthetic derivative. In particular, such a natural derivative is a compound selected from the isomers of retinoic acid or retinol, vitamin A, 9-cis-retinoic acid, and retinal. Such a synthetic derivative is a compound selected from 13-cis-retinoic acid, acitretin, tazarotene, adapalene, and bexarotene.

[0016] The subject is preferably a human subject, preferably a woman aged at least 40 years, preferably at least 45 years, even better at least 50 years. Preferably, the subject is preferably a human subject, preferably a woman aged 40 to 80 years, preferably 45 to 75 years, preferably 50 to 70 years.

[0017] The subject sample is preferably a skin sample. Typically, the sample can be obtained by biopsy and / or D-squame.

[0018] The skin model or cell model in which at least one glycoside hydrolase is expressed is any in vitro model of reconstructed skin, or in vitro cell model, in which the level of expression and / or enzymatic activity of a glycoside hydrolase is detected. The cell model is, for example, composed of keratinocytes, preferably human keratinocytes.

[0019] By "glycoside hydrolase" is meant an enzyme capable of hydrolyzing a glycosidic bond. Preferably, the glycoside hydrolase is present in the skin.

[0020] Preferably, glycoside hydrolase is involved in the degradation of glycosaminoglycans (GAGs).

[0021] Preferably, glycoside hydrolase is involved in the degradation of at least a GAG chosen from chondroitin sulfate (CS), dermatan sulfate (DS), keratan sulfate (KS), heparin sulfate (HS), heparin (HP) and hyaluronic acid (HA).

[0022] Preferably, the glycoside hydrolase is selected from aspartylglucosaminidase, fucosidase alpha-L-1, glucosidase alpha, galactosamine-6-sulfate sulfatase, acid beta glucosidase, galactosidase beta 1, hexosaminidase A, hexosaminidase B, iduronate 2-sulfatase, mannosidase alpha class 2B member 2, N-acetylgalactosaminidase alpha, sialidase 2, N-sulfoglucosamine sulfohydrolase and glucosamine (N-acetyl)-6-sulfatase.

[0023] Aspartylglucosaminidase (AGA) is a lysosomal hydrolase that participates in one of the final steps in the degradation of N-glycosylated proteins. AGA cleaves the bond between N-acetylglucosamine and asparagine after the polypeptide backbone has been degraded. The human AGA protein is available in Uniprot under the number P20933.

[0024] Fucosidase alpha-L-1 (FUCA1) is a lysosomal enzyme involved in the degradation of fucose-containing glycoproteins and glycolipids. It involves the cleavage of the alpha 1,2-fucoside bond during N-glycan degradation. The human FUCA1 protein is available in Uniprot under the number P04066.

[0025] Glucosidase alpha (GAA) is a lysosomal carbohydrate hydrolase that hydrolyzes non-reducing alpha-glucose terminal residues to release a single alpha-glucose molecule. It is responsible for removing glucose molecules from N-glycans of endoplasmic reticulum glycoproteins. The human GAA protein is available in Uniprot under number P10253.

[0026] Galactosamine-6-sulfate sulfatase (GALNS) is a lysosomal enzyme that hydrolyzes the 6-sulfate groups of the N-acetyl-D-galactosamine 6-sulfate units of chondroitin sulfate and the D-galactose 6-sulfate units of keratan sulfate. The human GALNS protein is available in Uniprot under number P34059.

[0027] Beta-glucosidase (GBA) is a lysosomal enzyme that hydrolyzes the glycosidic bond at a terminal non-reducing beta-D-glucosyl residue, releasing glucose. The human GBA protein is available from Uniprot under number P04062-2.

[0028] Galactosidase beta 1 (GLB1) is a lysosomal hydrolase that catalyzes the hydrolysis of galactosides into monosaccharides by breaking a glycosidic bond. Galactosides include galactose-containing carbohydrates where the glycosidic bond is located above the galactose molecule. Substrates of various galactosidases include GM1 ganglioside, lactosylceramides, lactose, and various glycoproteins. The human GLB1 protein is available in Uniprot under the number P16278-3.

[0029] Hexosaminidase A (HEXA) and hexosaminidase B (HEXB) are two subunits of hexomidase, a lysosomal enzyme involved in the hydrolysis of N- residues Acetyl-D-hexosamine terminals are converted to N-acetyl-D-hexosaminides. This enzyme, in association with the activating cofactor protein GM2, catalyzes the degradation of ganglioside GM2. Human HEXA protein is available in Uniprot under the number H3BP20, and human HEXB protein is available in Uniprot under the number P07686.

[0030] Iduronate 2-sulfatase (IDS) is involved in the lysosomal degradation of heparan sulfate and dermatan sulfate. The human IDS protein is available in Uniprot under number P22304.

[0031] Mannosidase alpha class 2B member 2 (MAN2B2) is required for the catabolism of N-linked carbohydrates released during glycoprotein turnover. It cleaves all known types of alpha-mannosidic bonds. The human MAN2B2 protein is available in Uniprot under the number Q9Y2E5.

[0032] N-acetylgalactosaminidase alpha (NAGA) is a lysosomal enzyme that cleaves alpha-N-acetylgalactosaminyl groups from glycoconjugates. The human NAGA protein is available in Uniprot under number P17050.

[0033] Sialidase 2 (NEU2) is a lysosomal sialidase that catalyzes the removal of terminal sialic acids from sialyloconjugates. The human NEU2 protein is available in Uniprot under the number Q9Y3R4.

[0034] N-sulfoglucosamine sulfohydrolase (SGSH) is a sulfamidase, one of several enzymes involved in the lysosomal degradation of heparan sulfate. The human SGSH protein is available in Uniprot under number P51688.

[0035] Glucosamine (N-acetyl)-6-sulfatase (GNS) is a lysosomal enzyme present in all cells. It is involved in the catabolism of heparin, heparan sulfate, and keratan sulfate. The human GNS protein is available in Uniprot under number P15586.

[0036] Preferably, the glycoside hydrolase is chosen from glucosidase alpha, ga-lactosamine-6-sulfate sulfatase, mannosidase alpha class 2B member 2 and N-acetylgalactosaminidase alpha.

[0037] By "glycoside hydrolase expression level" is meant the amount of mRNA produced, or the amount of protein produced, by the expression of the gene encoding the glycoside hydrolase in question. This amount may be expressed as such, or as a concentration or ratio.

[0038] The expression level of glycoside hydrolase can be measured by any suitable technique known in the prior art. To measure the amount of mRNA, RNA can be extracted from skin by any RNA extraction method and then processed for specific mRNA quantification by any mRNA quantification method, for example, by quantitative PCR. To measure the amount of protein, protein can be extracted using a suitable buffer for protein extraction from skin, for example, RIPA Buffer or a urea buffer, then be processed by any specific protein assessment method, for example Western blot or ELISA.

[0039] To measure the enzymatic activity of a glycoside hydrolase, one can evaluate the quantity of GAGs degraded.

[0040] In particular, the level of expression and / or enzymatic activity of glycoside hydrolase is measured by quantitative PCR, NGS sequencing, mass spectrometry, Western blot or ELISA.

[0041] Preferably, the identification method according to the invention comprises the following steps: a. Contact at least one candidate compound with a skin sample from a subject, or with a skin model or cell model in which at least (i) glucosidase alpha, (ii) galactosamine-6-sulfate sulfatase, (iii) mannosidase alpha class 2B member 2 and (iv) N-acetylgalactosaminidase alpha are expressed, and then measure the level of expression and / or enzymatic activity of each of the glycoside hydrolases (i) to (iv) in said sample or model; b. compare the expressions measured in step a) to the expressions of these glycoside hydrolases (i) to (iv) in a skin sample from the subject or in a skin model or cell model not exposed to said compound; and c. Identify said candidate compound as having an anti-aging effect mimicking retinoic acid, retinol, or one of their derivatives, when the expression of each of the glycoside hydrolases (i) to (iv) in the subject's skin sample or skin or cell model that has been exposed to said candidate compound is decreased, compared to the expression of these glycoside hydrolases in the subject's skin sample or skin or cell model that has not been exposed to the candidate compound. Preferably, the expression of glycoside hydrolases (i) to (iv) in the subject's skin sample or skin model that has been exposed to said candidate compound is at least 1.2 times lower than the expression of these glycoside hydrolases in the subject's skin sample or skin or cell model that has not been exposed to the candidate compound.

[0042] Preferably, the identification method according to the invention further comprises the following steps:

[0043] d. compare the expression measured in step a) to the expression of this glycoside hydrolase in a skin sample from the subject or in a skin model or cell model, said sample or model being exposed to retinoic acid, retinol, or one of their derivatives; and

[0044] e. identify said candidate compound as having an anti-aging mimic effect of retinoic acid, retinol or one of their derivatives, when the expression of this glycoside hydrolase in the subject's skin sample or skin or cell model that has been exposed to said candidate compound is not significantly different from the expression of this glycoside hydrolase in the subject's skin sample or model exposed to retinoic acid, retinol or one of their derivatives.

[0045] By "compound having an anti-aging effect mimicking retinoic acid, retinol or one of their derivatives", we mean a compound which decreases the level of expression and / or the enzymatic activity of at least one glycoside hydrolase. Preferably, the glycoside hydrolase is chosen from aspartylglucosaminidase, fucosidase alpha-L1, glucosidase alpha, galactosamine-6-sulfate sulfatase, acid beta glucosidase, galactosidase beta 1, hexosaminidase A, hexosaminidase B, iduronate 2-sulfatase, mannosidase alpha class 2B member 2, N-acetylgalactosaminidase alpha, sialidase 2, N-sulfoglucosamine sulfohydrolase and glucosamine (N-acetyl)-6-sulfatase.

[0046] Preferably, the compound having an anti-aging effect mimicking retinoic acid, retinol or one of their derivatives decreases the level of expression and / or enzymatic activity by at least 2, preferably by at least 3, preferably by at least 4, preferably by at least 5, preferably by at least 6, preferably by at least 7, preferably by at least 8, preferably by at least 9, preferably by at least 10, preferably by at least 11, preferably by at least 12, preferably by at least 13, preferably of all glycoside hydrolases, the glycoside hydrolases being chosen from the aforementioned list.

[0047] In step e), the candidate compound is identified as having an anti-aging effect mimicking retinoic acid, retinol, or one of their derivatives, when the expression of glycoside hydrolase in the subject's skin sample or the skin or cell model that was exposed to said candidate compound is not significantly different from the expression of this glycoside hydrolase in the subject's skin sample or the model exposed to retinoic acid, retinol, or one of their derivatives. In other words, under the process conditions, the candidate compound has effects similar to retinoic acid, retinol, or one of their derivatives.

[0048] By "is not significantly different", it is meant that the expression of glycoside hydrolase in the sample or model exposed to said candidate compound is identical or similar to the expression of this glycoside hydrolase in the sample or model exposed to retinoic acid, retinol or one of their derivatives.

[0049] Preferably, an anti-aging compound identified by the process according to the invention is adenosine.

[0050] Cosmetic use

[0051] The present invention also relates to the cosmetic use of at least one an anti-aging compound having a mimetic action of retinoic acid, retinol or one of their derivatives, identified by the process according to the invention, to prevent and / or treat skin aging.

[0052] The anti-aging compound identified by the process according to the invention is a mimetic of retinoic acid, retinol or one of their derivatives.

[0053] Preferably, such an anti-aging compound is adenosine.

[0054] The anti-aging compound identified by the process according to the invention is preferably applied topically, i.e. on the skin and / or mucous membranes.

[0055] Such an anti-aging compound identified by the process according to the invention can be formulated in a cosmetic composition. The cosmetic composition preferably comprises a physiologically acceptable medium, i.e., a medium compatible with the skin and / or mucous membranes. Preferably, the cosmetic composition is an anti-aging composition. It can be applied by any suitable route of administration, in particular topically. It can be in the form of, for example, a cream, lotion, gel, or suspension.

[0056] Method for evaluating the effectiveness of a cosmetic composition

[0057] The invention also relates to a method for evaluating the effectiveness of a cosmetic composition comprising, as an active agent, retinol, retinoic acid, one of their derivatives or an anti-aging compound identified by the process according to the invention, comprising the following steps: a. the measurement of the level of expression and / or enzymatic activity of at least one glycoside hydrolase in a skin sample from a subject after application of the cosmetic composition; then b. comparison of the measurement from step a) with a control sample.

[0058] Preferably, the composition is effective when the level of expression and / or enzymatic activity of glycoside hydrolase measured in step a) in the subject's skin sample is lower, in particular significantly lower, than a control.

[0059] In a particular embodiment, the control is a reference value.

[0060] Preferably, the control is a reference value determined by the value average level of expression and / or enzymatic activity of glycoside hydrolase, measured in a given population, for example a population aged 40 to 80 years.

[0061] The measurement in step a) is preferably carried out as described in the section "Method for identifying an anti-aging compound having a mimetic action of retinoic acid, retinol or one of their derivatives" above.

[0062] The glycoside hydrolase, the skin sample of said subject and the subject may be as described in the section “Method for identifying an anti-aging compound having a mimetic action of retinoic acid, retinol or one of their derivatives” below. above.

[0063] The cosmetic composition is as described above. It can be applied by any suitable route of administration, in particular topically. It may be in the form of a cream, lotion, gel or suspension, for example.

[0064] Preferably, the cosmetic composition is evaluated as effective when the level of expression and / or enzymatic activity of a glycoside hydrolase measured in step a) in the subject's skin sample is at least 1.2 times lower, preferably significantly lower, than a control.

[0065] In a particular embodiment, the reference value is determined by an ROC study (“The ROC (receiver operating characteristic) curve: principles and main applications in clinical biology”, H. Delacour et al., Ann Biol Clin 2005; 63 (2): 145-54).

[0066] By "significantly lower" for the purposes of the invention, means a statistically significant variation in the level of expression and / or enzymatic activity of a glycoside hydrolase.

[0067] The present invention will be described in more detail by the examples below. Examples

[0068] Example 1: Clinical study with retinol and retinoic acid Materials & Methods Clinical study

[0069] The clinical results obtained from the one-year clinical study have been partially published (Tancrede-Bohin et al., In vivo multiphoton imaging for non-invasive time course assessment of retinoids effects on human skin, Skin Res Technol 2020 Nov;26(6):794-803). The clinical study was conducted in Paris, France, between February 2011 and April 2012, and the experimental protocol was approved by the ethics committee of Saint Louis Hospital (reference EC 2010 / 58) in accordance with the Declaration of Helsinki.

[0070] This study involved 30 female volunteers (aged 50-65) with a constitutive skin color determined by an ITA value between 10° and 41° (ITA group III / IV). Fifteen volunteers applied 0.3% retinol (RE) (L'Oréal Group formula) to one dorsal forearm versus a control product (white paraffin-containing excipient, DIPROBASE® Bayer) to the other forearm, and fifteen volunteers applied 0.025% all-trans retinoic acid (RA) (0.025% formula, RETACNYL® Galderma) versus a control product (white paraffin-containing excipient, DIPROBASE® Bayer) to the other forearm. The product (250 mg) was applied once daily for 1 year. Non-invasive stripping (DSquames™) was performed on the area at months M00 (March), M03 (June), M06 (September), M12 (March + 1 year) (Tancrède-Bohin et al., In vivo multiphoton imaging for non-invasive time course assessment of retinoids effects on human skin, Skin Res Technol 2020 Nov;26(6):794-803).

[0071] Protein extraction, iTRAO labeling and spectrometric analysis of mass

[0072] Protein extractions were performed from each individual DSquame as follows:

[0073] 550 μl of DOC buffer (0.5% sodium deoxycholate, 50 mM bicarbonate ammonium, 50 mM DTT, pepstatin 1 pM and EDTA-free inhibitor cocktail) were added / DSquame.

[0074] Mechanical extraction using a tissue lyser (Retsch MM300) and stainless steel beads was performed with two 2-minute cycles at 30 Hz. The supernatant was filtered through 0.45 µm PVDF membranes by centrifugation (Millipore). The proteins were precipitated overnight in 4 volumes of ice-cold acetone at -20°C followed by centrifugation at 16,000 g for 20 minutes at 4°C. The protein pellet was resuspended in 50 µl of TEAB buffer.

[0075] (0.5 M) in the presence of 0.5% sodium deoxycholate. The samples have The samples were then sonicated in water for 5 min to resolubilize the proteins. The total protein concentration was measured using the Bradford assay (Pierce BCA). For the analysis, 35 pg of protein was reduced in TCEP (5 mM) for 60 min at 60°C, then alkylated by the addition of 6 mM MMTS for 10 min at room temperature (RT) in the dark. 0.25% sodium deoxycholate was then added to each sample before trypsin digestion by adding 1.5 pg of modified porcine trypsin (Promega) and incubating for 18 h at 37°C.

[0076] After digestion, the samples are acidified to pH 2 with 5% formic acid and incubated for 10 min at room temperature. Centrifugation at 16,000 g for 5 min removes sodium deoxycholate. The peptides are then processed through a desalting column (Oasis HLV, 1cc / 10mg, Waters). After elution and lyophilization, the samples are resuspended in 30 µl of TEAB 500 mM. Next, 200 µl of isopropanol are added to a vial of iTRAQ 4-plex reagent (AB SCIEX, Toronto, Canada) and centrifuged twice.

[0077] Then 100 pl of iTRAQ labeling reagent were added to the samples and mixed.

[0078] The mixture was incubated for 2 hours at room temperature in the dark. The labeled peptides were then processed again in a desalting column (Oasis HLV, 1cc / 10mg, Waters), and then lyophilized in preparation for the next step. The peptides were then separated by isofocalization in a pH gradient gel ranging from 3 to 10. For this purpose, the peptides were resuspended in 320 µl of HPLC water containing 3 µl of ampholites (Biolyte 3-10, BioRad). This solution was used to re- hydrate a gel at pH immobilized by 18cm.

[0079] The migration of labeled peptides through the immobilized pH gel is as follows: passive rehydration (5 h at 20°C); 250 V for 15 min, 10,000 V for 3 h with a slow voltage increase, 10,000 V up to 60,000 V*h, and finally 500 V for 20 h. After peptide focusing, the pH gel is cut into 36 fragments of 5 mm. The peptides are eluted using 1 µL of formic acid / 2% acetonitrile solution and then with 1 µL of formic acid / 50% acetonitrile solution.

[0080] For MS analysis, the 36 fractions are grouped 2 at a time in order to finally obtain 18 fractions to be injected into the 5600 Triple-TOF mass spectrometer (ABSciex).

[0081] Mass spectrometry (MS) analyses were performed on an ABSciex 5600 Triple-TOF instrument coupled to an Agilent 1200 HPLC. The experimental setup allowed for three independent LC-MS / MS analyses. Briefly, 500 ng of samples were injected onto a trapping column for desalting followed by chromatographic separation on a C18 column, 75 µm x 15 cm. The samples were analyzed using a 90-minute gradient from 10 to 35% of solvent B (solvent A being a 0.1% formic acid solution and solvent B 0.1% formic acid in acetonitrile) at a flow rate of 300 nl / min. Data were acquired using positive ESI mode with a positive voltage of 2.4 kV. Complete scans were obtained in the mass range of 400-1250 in 250 ms. Data-dependent acquisition (DDA) was used for data collection for high-intensity ions in the m / z range of 100-1600.

[0082] Protein identification

[0083] The data files were submitted for simultaneous searches using Protein Pilot software version 4.0 (ABSciex) and the Mascot search engine (Matrix Science) with the following elements:

[0084] Criteria: Tryptic digestion, fixed modification on the cysteine ​​of methylthio, variable iTRAQ modification (4-plex iTRAQ(K), iTRAQ(N-term), iTRAQ(Y)) and methionine oxidation. Peptide charge was limited to +2, +3 and +4. Peptide mass tolerance (+ / -0.1) and fragment mass tolerance + / -0.1.

[0085] Proteins were defined as differentially modulated if they meet the following criteria: at least two peptides with a high confidence interval (95%) for identification (p-value<0.05) and with a protein variation factor greater than or equal to 11.51. Data analysis

[0086] Differential protein expression was calculated using the DESEQ2 tool (https: / / genomebiology.biomedcentral.com / articles / 10.1186 / sl3059-014-0550-8).

[0087] The differentially expressed proteins of the retinoid-treated subject and the subject placebos were defined by a Fold Change 11.51 in 2 or 3 replicates. The biological interpretation of these protein lists was performed by enrichment calculations using the R package ClusterProfiler (https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC3339379 / ) and using the "Gene Ontology" database (AmiGO: Carbon S, Ireland A, Mungall CJ, Shu S, Marshall B, Lewis S, AmiGO Hub, Web Working Group. AmiGO: Online access to ontology and annotation data. Bioinformatics, Jan. 2009; 25(2): 288-289) and the "KEGG" database (Kanehisa, M. and Goto, S.; KEGG: Kyoto Encyclopedia of Genes and Genomes. Nucleic Acids Res. 28, 27-30, 2000). A term from Gene Ontology or a pathway from the KEGG database was considered significantly impacted when it had a corrected p-value <0.05 with the given list of proteins as input. Results

[0088] The results are as follows:

[0089] [Tables 1] Accessio n Number Gene Protein Retinol Treatment Retinoic Acid Treatment FC* 3 M FC* 3 M P20933 AGA aspartylglucosaminidase -1.5 -2.2 P04066 FUCA1 fucosidase alpha-L-1 -1.8 -1.6 P10253 GAA glucosidase alpha -1.6 P34059 GALNS galactosamine-6-sulfate sulfatase -1.7 P04062-2 GBA glucosidase beta acid -1.6 P16278-3 GLB1 galactosidase beta 1 -1.9 -1.5 H3BP20 HEXA hexosaminidase A -1.6 -1.9 P07686 HEXB hexosaminidase B. -1,9 -2,2 P22304 IDS iduronate 2-sulfatase -1,7 -1,6 Q9Y2E5 MAN2B2 mannosidase alpha class 2B member 2 -1,6 P17050 NAGA N-acetylgalactosaminidase alpha -1,5 Q9Y3R4 NEU2 sialidase 2 -1,6 -1,5 P51688 SGSH N-sulfoglucosamine sulfohydrolase -1.7 -1.8 P15586 GNS glucosamine (N-acetyl)-6-sulfatase -1.4 -2.0

[0090] *Fold Change : FC

[0091] A significant impact of retinol and retinoic acid on glycan metabolism was observed, with 14 glycosidases involved in the deglycosylation of several glycan types being strongly downregulated, suggesting the shortening of GAGs, proteoglycans, and glycoproteins by both retinoids. In particular, GLB1 and Alpha-L-Fucosidase 1 (FUCA1) are both consistently downregulated by retinoids. These results demonstrate a significant impact of retinoids on modulating the glycan degradation pathway. Example 2: In vitro study with adenosine

[0092] Cultured human skin keratinocytes were treated with 10 pM adenosine for 24 hours versus control keratinocytes treated with DMSO for 24 hours. Each condition was performed in 3 replicates. RNA extraction

[0093] The extraction protocol was performed using the RNeasy micro-kit (Qiagen, Germantown, MD, USA) with DNase 1 treatment. The purified RNAs were eluted with 30–40 µl of RNase-free water and quantified in duplicate by spectrophotometry using the NanoDrop ND-1000 (NanoDrop Technologies, Wilmington, DE, USA). A 5 ng sample of total RNA was used for total RNA integrity verification using the Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA) with the RNA 6000 Pico Kit (Agilent). The integrity value (RIN), an indicator of RNA quality, ranged from 8.5 to 10 out of 10. Library preparation and sequencing

[0094] The NEBNext Ultra II directional RNA library prep kit for Illumina (New Englands Biolabs Inc., Ipswich, MA, USA) was used for the preparation of the RNA libraries, according to the manufacturer's instructions. The quality of the final libraries was verified on the TapeStation 2200 (Agilent Technologies, Santa Clara, CA, USA), and quantification was performed on the QBit 3.0 fluorometer (ThermoFisher Scientific, Canada). Subsequently, the mRNA-seq libraries, each with two unique indexes, were pooled in an equimolar ratio into seven pools. Each pool was loaded onto a high-performance S4 flowcell line and sequenced on an Illumina NovaSeq 6000 system (Illumina Inc., San Diego, CA, USA). A 100 bp paired sequencing was performed at the Next Generation Sequencing Platform of the CHU de Québec-Université Laval Research Centre, Quebec City, Canada. The average coverage per sample obtained was approximately 29 million paired reads. Bioinformatics analyses

[0095] The Reads were clipped using fastp v0.22.0 (Chen, S., Zhou, Y., Chen, Y., & Gu, J. (2018). fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics, 34(17), 1884-1890). Quality control was performed on raw materials and clipped data to ensure the quality of the Reads using FastQC v0.11.9 (Andrews, Simon. "FastQC: a quality control tool for high throughput sequence data." (2010)) and MultiQC v1.11 (Ewels, Philip, Mans Magnusson, Sverker Lundin, and Max Käller. "MultiQC: summarize analysis results for multiple tools and samples in a single report." Bioinformatics 32, no. 19 (2016): 3047-3048. J). Quantification was performed with Kallisto vO.46.1 (Bray, Nicolas L., Harold Pimentel, Pâli Melsted, and Lior Pachter. "Near-optimal probabilistic RNA-seq quantification." Nature bio-technology 34, no. 5 (2016): 525) against transcripts encoding proteins from the transcriptome of Homo sapiens (GRCh38 downloaded from Ensembl version 104).

[0096] Differential expression analysis was also performed using DESeq2 v 1.34.0 (Love, Michael L, Wolfgang Huber, and Simon Anders. "Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2." Genome biology 15, no. 12 (2014): 550). All R analyses were performed in R v4.1.2 (R: Team, R. Core. "R: A language and environment for statistical computing." (2013): 201).

[0097] The signature was obtained after differential expression analysis of the conditions treated with Adenosine versus the control conditions by fixing the parameters corrected qvalue <= 0.05 and the Fold-change >=11.21.

[0098] Sequencing results show an underregulation of 4 glycoside hydrolase enzymes, which is consistent with the expected effect: Accession Number Gene Protein Adenosine Treatment Fold change Q9Y2E5 MAN2B 2 mannosidase alpha class 2B member 2 -1,2 P34059 GALNS galactosamine-6-sulfate sulfatase -1,3 P10253 GAA glucosidase alpha -1,4 P17050 NAGA N-acetylgalactosaminidase alpha -1,2

[0100] The under-regulation of these 4 glycoside hydrolases after treatment with adenosine, a compound known for its anti-aging effect, validates the invention and suggests an anti-aging effect mimicking retinol and / or retinoic acid.

Claims

Demands

1. A method for identifying an anti-aging compound having a mimetic action of retinoic acid, retinol, or one of their derivatives, said method comprising the following steps: a. bring at least one candidate compound into contact with a skin sample from a subject, or with a skin model or cell model in which at least one glycoside hydrolase is expressed, and then measure the level of expression and / or enzymatic activity of that glycoside hydrolase in said sample or model; b. compare the expression and / or activity measured in step a) to the expression and / or activity of this glycoside hydrolase in a skin sample from the subject or in a skin model or cell model, not exposed to said compound; c. identify said candidate compound as having an anti-aging effect mimicking retinoic acid, retinol or one of their derivatives, when a decrease in the expression and / or activity of this glycoside hydrolase in the subject's skin sample or skin model or cell model that has been exposed to said candidate compound is detected, compared to the expression and / or activity of this glycoside hydrolase in the subject's skin sample or skin model or cell model that has not been exposed to the candidate compound; d. compare the expression and / or activity measured in step a) to the expression and / or activity of this glycoside hydrolase in a skin sample from the subject or in a skin model or cell model, said sample or model being exposed to retinoic acid, retinol, or one of their derivatives; and e. identify said candidate compound as having an anti-aging effect mimicking retinoic acid, retinol or one of their derivatives, when the expression and / or activity of this glycoside hydrolase in the subject's skin sample or skin model or cell model that has been exposed to said candidate compound is not significantly different from the expression of this glycoside hydrolase in the subject's skin sample or model exposed to retinoic acid, to retinol or one of their derivatives.

2. A method according to claim 1, wherein the skin sample of a subject is a biopsy, preferably the subject is a human subject, preferably a woman aged at least 40 years, preferably at least 45 years, even better at least 50 years, preferably a woman aged 40 to 80 years, preferably 45 to 75 years, preferably 50 to 70 years.

3. A method according to any one of the preceding claims, wherein glycoside hydrolase is involved in the degradation of glycosaminoglycans (GAGs).

4. A method according to any one of the preceding claims, wherein the glycoside hydrolase is selected from aspartylglucosaminidase, fu-cosidase alpha-L-1, glucosidase alpha, galactosamine-6-sulfate sulfatase, acid beta glucosidase, galactosidase beta 1, hexosaminidase A, hexosaminidase B, iduronate 2-sulfatase, mannosidase alpha class 2B member 2, N-acetylgalactosaminidase alpha, sialidase 2, N-sulfoglucosamine sulfohydrolase and glucosamine (N-acetyl)-6-sulfatase; Preferably the glycoside hydrolase is chosen from glucosidase alpha, galactosamine-6-sulfate sulfatase, mannosidase alpha class 2B member 2 and N-acetylgalactosaminidase alpha.

5. A method according to any one of the preceding claims, wherein the level of expression is either the amount of mRNA produced, or the amount of protein produced, by the expression of the gene encoding glycoside hydrolase; and / or the enzymatic activity of glycoside hydrolase is the amount of GAGs degraded.

6. A method according to any one of the preceding claims, wherein the level of expression and / or enzymatic activity of glycoside hydrolase is measured by quantitative PCR, NGS sequencing, mass spectrometry, Western blot or ELISA.

7. A method for evaluating the efficacy of a cosmetic composition comprising, as an active agent, retinol, retinoic acid, one of their derivatives, or an anti-aging compound identified by the process according to any one of claims 1 to 6, comprising the following steps: a. measuring the level of expression and / or zymatic activity of at least one glycoside hydrolase in a skin sample from a subject after application of the com- cosmetic position; then b. comparison of the measurement from step a) with a control sample.

8. Method according to claim 7, wherein the composition is effective when the level of expression and / or enzymatic activity of glycoside hydrolase measured in step a) in the subject's skin sample is lower, in particular significantly lower, than a control.

9. Method according to claim 7 or 8, wherein the control is a reference value determined by the average value of the expression level and / or enzymatic activity of glycoside hydrolase, measured in a specified population, for example a population aged 40 to 80 years.