WO2021043906A1

WO2021043906A1 - Method for diagnosing old skin

Info

Publication number: WO2021043906A1
Application number: PCT/EP2020/074604
Authority: WO
Inventors: Mark Donovan; Dominique Bernard
Original assignee: L'oreal
Priority date: 2019-09-05
Filing date: 2020-09-03
Publication date: 2021-03-11
Also published as: FR3100452B1; FR3100452A1

Abstract

The present invention relates to a method for diagnosing an old skin in a subject, comprising a step (a) of measuring, in a skin sample of the subject, the level of at least one N-glycan chosen from the group consisting of glycan of the type rich in mannose M7, glycan of the type rich in mannose M8, glycan of the type rich in mannose M9, glycan of the complex type F(6)A2G(4)2S1 and glycan of the complex type A2G1GalNAc1.

Description

Method for diagnosing old skin

The present invention relates to a method for diagnosing old skin.

During aging the skin is on the overall subjected to much damage, reflections of functional and structural alterations and of degradations of all of its compartments, at the same time as a drop in the quality of the vascularisation. At the cellular level, many metabolic functions are affected. The epidermis becomes thinner with age, the keratinocytes lose their renewal and repair capacity, the synthesis of hyaluronic acid, of proteoglycans collapses with age, the quality and the cohesion of the dermis-epidermis junction drops making skin tissue thinner and more fragile, the thickness of the epidermis is thinner, the dermis-epidermis junction loses its invaginations major characteristics of a young skin.

The skin can also lose its shine, have alterations in its microrelief causing the appearance of a weathered texture, an appearance of wrinkles and lines, have an increase in the heterogeneity of the diameter of the pores, all signs that are perceived as "poor health" or fatigue and that modify the perception of the apparent age.

From the above, the importance of finding biomarkers that make it possible to detect the signs of aging of the skin is understood and in particular when the latter are not yet visible, so as to be able to reduce and/or slow down the progression of these signs of aging of the skin.

The aim of the invention is to respond to this need.

Glycosylation corresponds to the adding of an oligo- or polysaccharide chain to a protein, a lipid or any other organic molecule, and is a key post-translational modification in all biological functions. This is the most common post-translational modification and is found on more than 60% of the eucaryotic proteins. It can influence many aspects of the structure and the function of the protein: its biological activity, its immunogenicity, its solubility, its aggregation, its half-life. Glycoproteins play a key role in a multitude of biological functions in all tissues, such as cell signaling, recognition of receptors, inflammation, cell adhesion, migration and differentiation.

Insofar as the synthesis of glycans is not carried out by a model, contrary to the synthesis of DNA or protein, it was difficult to study in detail the changes in the structures of glycans in diseased or old tissues due to the lack of sufficiently sensitive molecular tools. However, the recent development of qualitative and quantitative glycomic tools based on the availability of specific glycosidases, and of sensitive chromatography and mass spectrometry methods, has allowed glycobiologists to characterize the glycome of tissues in detail.

The importance of glycosylation in hemostasis of the skin was thus underlined in congenital disorders of glycosylation. Neonatal patients suffering from these diseases have mutations in the genes encoding the enzymes involved in the glycosylation pathways, which results in several anomalies of the skin, such as a wrinkled and sagging skin, orange peel, dry skin and ichthyosis (Kouwenberg et al. (2014) Pediatr. Dermatol. 31 :e1-5).

Few glycomic studies have been conducted on the skin, but Uematsu et al. (2009) Mol. Cell. Proteomics 8 :232-244 showed that the N-glycomes in the human and mouse epidermis were very similar. The epidermis is characterized by an abundance of oligosaccharides rich in mannose with a lower presence of sialylated and fucosylated species. A reciprocal profile was identified in the dermis.

However, as far as the inventors know, no study has revealed old skin markers in the N-glycome of the skin.

This invention is the result of the unexpected discovery by the inventors that only 3 glycans of the type rich in mannose, glycans M7, M8 and M9, and 2 glycans of the complex type, glycans F(6)A2G(4)2S1 and A2G1GalNAc1 , among all the N-glycans present in the stratum corneum of the skin, were expressed differentially in an old skin with respect to a young skin. They indeed showed a decrease in the levels of the N-glycans M7, M8, M9 and F(6)A2G(4)2S1 , and an increase in the level of the N-glycan A2G1 GalNAd , in samples of old skin with respect to young skins.

The present invention thus relates to a method for diagnosing an old skin in a subject, comprising a step (a) of measuring, in a skin sample of the subject, the level of at least one N-glycan chosen from the group consisting of glycan of the type rich in mannose M7, glycan of the type rich in mannose M8, glycan of the type rich in mannose M9, glycan of the complex type F(6)A2G(4)2S1 and glycan of the complex type A2G1 GalNAd .

The present invention also concerns a method for evaluating the efficacy of a cosmetic treatment against old skin in a subject, said method comprising, after a step (pre- a) of applying the cosmetic treatment against old skin, a step (a) of measuring the level of at least one N-glycan chosen in the group consisting of glycan of the type rich in mannose M7, glycan of the type rich in mannose M8, glycan of the type rich in mannose M9, glycan of the complex type F(6)A2G(4)2S1 and glycan of the complex type A2G1 GalNAd , in a skin sample of said subject. The present invention also concerns a method for screening active compounds to treat old skin, said method comprising the following steps:

(A) contacting the candidate active compound with a skin sample,

(B) measuring the level of at least one N-glycan chosen from the group consisting of glycan of the type rich in mannose M7, glycan of the type rich in mannose M8, glycan of the type rich in mannose M9 and glycan of the complex type F(6)A2G(4)2S1 and glycan of the complex type A2G1 GalNAd , in said skin sample,

(C) comparing the level of said at least one N-glycan measured in step (B) with a control, and

(D) selecting the candidate active compound as being an active compound for treating old skin when i. the level of the glycan of the type rich in mannose M7 measured in step (B) is higher than the level of the control, ii. the level of the glycan of the type rich in mannose M8 measured in step (B) is higher than the level of the control, iii. the level of the glycan of the type rich in mannose M9 measured in step (B) is higher than the level of the control, iv. the level of the glycan of the complex type F(6)A2G(4)2S1 measured in step (B) is higher than the level of the control and/or v. the level of the glycan of the complex type A2G1 GalNAd measured in step (B) is lower than the level of the control.

Detailed description of the invention

N-glycans

"N-glycan" here means a polymer of monosaccharides linked together by a glycosidic bond, and which is bonded to a protein, to form a glycoprotein, via an amide bond at an asparagine residue (Asn).

N-glycans typically contain a common pentasaccharidic core, known as being the trimannosyl core, which can be fucosylated on the first N-acetyl-glucosamine (core fucosylation).

The N-glycans are branched oligosaccharides of three types:

- glycans of the type rich in mannose, which consist of branched chains of mannose;

- glycans of the hybrid type, in which N-acetylglucosamine, galactose or fucose and/or sialic acid are added to a chain of the oligomannose structure; and - glycans of the complex type, in which terminal sialic acids are added to the structures of N-glycans of the hybrid type

(see Stanley et al. N-Gylcans; In: Varki et al. Ed. Essentials of Glycobiology. 2nd ed. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 2009. Chapter 8). These three types of N-glycans are diagramed in Figure 1 .

As is well known to those skilled in the art, several types of notation exist to designate N-glycans. The main ones are the Oxford Notation system and the Essentials notation system. In the framework of this invention, the N-glycans are designated using the Oxford Notation or using text format. The Oxford Notation system was designed by scientists of the Oxford Glycobiology

University in 2009, as follows. All N-glycans have two N-acetyl-glucosamines (GlcNAcs) cores; F at the beginning of the abbreviation indicates a fucose core; Mx: number (x) of mannoses on the GlcNAcs core; Ax: number of antennas (GlcNAc) on the trimannosyl core (for example A2 = two antennas with each one of the GlcNAcs bonded in a1-2); Gx: number of galactoses bonded on the antennas (for example [3]G1 = a galactose on the antenna of the mannose in a1-3, while [6]G1 = one galactose on the antenna of the mannose in a1-6); Sx: number of sialic acids (N-acetylneuraminic acids) bonded to the galactose.

The present inventors have here shown that 3 N-glycans of the type rich in mannose and 2 N-glycans of the complex type made it possible to diagnose old skin

These 3 N-glycans of the type rich in mannose are:

- N-glycan M7, also designated according to the following formula (I):

Manct- Mana 3 6

Manp 4GlcNAcp 4GlcNAC

3

Mana-2Mana'

(I)

N-glycan M8, also designated according to the following formula (II):

Manou,

Mana-2

^Mana

Mana- ^Manp-4GlcNAcp-4GlcNAC

Mana-2 Mana-2Mana-

(II)

N-glycan M9, also designated according to the following formula (III): Mana

Mana Mana-2 Mana-2Mana '

The N-glycans of the complex type used as a marker in the framework of the present invention are :

- N-glycan F(6)A2G(4)2S1 , also designated according to the following formula (IV):

Fuca

GaIp-4GIcNAcp-2IVIana_v 6

6

Manp-4GlcNAcp-4GlcNAC

3

Neu5Aca2,3Galp-4GlcNAcp-2Mana '

(iv),

N-glycan A2G1 GalNAd , also designated according to the following formula (V):

(V).

Method for diagnosing

The method for diagnosing according to the invention is a method for diagnosing old skin in a subject. "Old skin" here means a general state of the skin resulting from chronological aging and/ or photo-induced aging. An old skin typically has skin signs of aging.

"Skin signs of aging" means all the modifications of the exterior aspect of the skin due to aging whether of chronological and/or photo-induced origin. As examples of these modifications in the exterior aspect of the skin due to aging whether chronological and/or photo-induced, mention can be made of wrinkles and lines, an alteration in the microrelief, loose skin, flabby skin, lack of elasticity and/or tonus of the skin, thinning of the dermis and/or degradation of the collagen fibers, which results in the appearance of soft and wrinkled skin.

The skin is more particularly the skin of the face, in particular the skin of the cheeks and/or of the forehead, the skin of the neckline, the skin of the neck, the skin of the arms and forearms, the skin of the feet. More preferably, the skin is the skin of the face, in particular the skin of the cheeks and/or of the forehead or the skin, more particularly the skin of the crow's feet wrinkle zones.

The method for diagnosing according to the invention comprises a step (a) of measuring, in a skin sample of the subject, the level of at least one N-glycan chosen from the group consisting of glycan of the type rich in mannose M7, glycan of the type rich in mannose M8, glycan of the type rich in mannose M9, glycan of the complex type F(6)A2G(4)2S1 and glycan of the complex type A2G1GalNAc1 , as defined in the section "N-glycans" hereinabove.

The level of said N-glycans can be measured by any suitable technique. In particular, the level of said N-glycans can be measured by high-throughput glycomic tools.

Thus, in a particular embodiment, the level of said N-glycans can be measured by hydrophilic interaction liquid chromatography (HILIC), HPLC/UPLC or mass spectrometry, and optionally digestions by specific exoglycosidases.

Typically, the method for measuring implemented in step (a) can include:

• a step of releasing N-glycans from the skin sample, in particular from tissues contained in the skin sample, by using for example a peptide enzyme N glycanase F (PNGase F) which cleaves any type of N-glycan from its core protein;

• a step of labelling isolated glycans with a fluorescent molecule such as 2 aminobenzamide;

• a step of analyzing by hydrophilic interaction liquid chromatography (HILIC), HPLC/UPLC or mass spectrometry; and

• a step of analyzing in detail the glycan chain by digestions by specific exoglycosidases and determining glycan structures from chromatograms obtained on the basis of a relational database, such as the Glycobase 3.1 database which consists of nearly 1 ,000 glycan structures defined to date.

This measuring technique is typically described in Knezevic etal. (2010) Glycobiology 20 :959-969.

In a particular embodiment, the method for diagnosing according to the invention further comprises the steps consisting of:

(b) comparing the level of said at least one N-glycan measured in step (a) with a control, and

(c) based on the comparison in step (b), determining if the skin of said subject is an old skin. In a particular embodiment, the control is a reference value.

In a particular embodiment, the reference value is determined by the mean value of the level of said N-glycan in a determined population, for example a population in a defined age-group, and/or having a defined skin type.

In a particular embodiment, the control is the mean value of the level of said N- glycan in a population of subjects, in particular subjects as defined hereinbelow, having a young skin.

"Young skin" here means a skin that has little or no skin signs of aging, typically a skin of a person 20 to 30 years of age.

In a particular embodiment, the skin of said subject is diagnosed as being an old skin when: i. the level of glycan of the type rich in mannose M7 measured in step (a) in the skin sample of the subject is lower than, in particular statistically lower than, a control level, ii. the level of glycan of the type rich in mannose M8 measured in step (a) in the skin sample of the subject is lower than, in particular statistically lower than, a control level, iii. the level of glycan of the type rich in mannose M9 measured in step (a) in the skin sample of the subject is less than, in particular statistically less than, a control level, iv. the level of glycan of the complex type F(6)A2G(4)2S1 measured in step (a) in the skin sample of the subject is lower than, in particular statistically lower than, a control level and/or v. the level of glycan of the complex type A2G1 GalNAd measured in step (a) in the skin sample of the subject is higher than, in particular statistically higher than, a control level, the control level being typically the mean level of said N-glycan in skin samples of a population of subjects, in particular of subjects as defined hereinbelow, having a young skin, and the level of said N-glycans being typically measured by hydrophilic interaction liquid chromatography (HILIC), HPLC/UPLC or mass spectrometry, and optionally digestions by specific exoglycosidases.

"Significantly lower" means in terms of the invention a statistically significant decrease in the level of N-glycan with respect to the control level. "Significantly higher" means in terms of the invention a statistically significant increase in the level of N-glycan with respect to the control level.

According to a preferred embodiment, the skin sample of the subject used in the method for diagnosing according to the invention, is a sample taken, preferably non- invasively, on the subject's skin, preferentially on the subject's face, in particularly on the subject's cheek and/or forehead or on the foot of the subject. Preferably the skin sample comes from the stratum corneum, in particular from the surface of the stratum corneum.

The stratum corneum is the outermost layer of the epidermis, and comprises the skin surface. It is essentially made up of dead cells.

According to an embodiment, the method for diagnosing according to the invention comprises a step of sampling the sample of the skin of the subject, in particular of the surface of the skin of the subject. This step is preferably performed non-invasively, and in particular does not require local anesthetic. According to one preferred embodiment, the step for taking the sample is performed by rubbing the skin surface, in particular with a grater such as a foot grater or a D-squame® disc.

In a particular embodiment, the skin sample is taken by rubbing the surface of the skin.

"Subject" here means a human being, preferably aged 20 to 70 years. Preferably, the subject is female.

The present invention also relates to a method for cosmetic treatment of an old skin, in a subject, said method comprising the following steps:

1) diagnosing the skin of the subject as being an old skin by implementing the method for diagnosing according to the invention,

2) if the skin of said subject is diagnosed as being an old skin, treating the skin of said subject with a cosmetic composition that makes it possible to reduce or abolish the signs of an old skin.

Cosmetic compositions that make it possible to reduce or abolish the signs of an old skin are well known to those skilled in the art and are for example described in French applications FR2825277, FR2994528, FR2811862, FR2946347, FR2864785, FR3027797, FR2991167 and FR3007650. Method for evaluating the efficacy of a cosmetic treatment

The present invention also relates to a method for evaluating the efficacy of a cosmetic treatment against old skin as defined in the section "Method for diagnosing" hereinabove, in a subject as defined in the section "Method for diagnosing " hereinabove, said method comprising, after a step (pre-a) of applying the cosmetic treatment against old skin, a step (a) of measuring the level of at least one N-glycan chosen in the group consisting of glycan of the type rich in mannose M7, glycan of the type rich in mannose M8, glycan of the type rich in mannose M9, glycan of the complex type F(6)A2G(4)2S1 and glycan of the complex type A2G1GalNAc1 , as defined in the section "N-glycans" hereinabove, in a skin sample of said subject.

The method for evaluating the efficacy is preferably implemented in vitro or ex vivo.

The subject is preferably a subject that has an old skin.

"Cosmetic treatment against old skin" means here any type of cosmetic treatment (topical or oral for example) that is supposed to make it possible to reduce or abolish the signs of an old skin.

Said cosmetic treatment can be applied in step (pre-a) by any suitable administration route, in particular topically or orally.

The skin sample of said subject can be as described in the section “ Method for diagnosing " hereinabove. The level of said at least one N-glycan is preferably measured as described in the section " Method for diagnosing" hereinabove.

In a particular embodiment, the method for evaluating according to the invention further comprises the steps consisting of:

(c) based on the comparison in step (b), determining if said cosmetic treatment against old skin is effective in said subject.

In a particular embodiment, the control is the level of said at least N-glycan in a skin sample of said subject before application of said cosmetic treatment. Preferably, said cosmetic treatment against old skin is evaluated as being effective in said subject if: i. the level of glycan of the type rich in mannose M7 measured in step (a) in the skin sample of the subject is higher than, in particular statistically higher than, the control level, ii. the level of glycan of the type rich in mannose M8 measured in step (a) in the skin sample of the subject is higher than, in particular statistically higher than, the control level, iii. the level of glycan of the type rich in mannose M9 measured in step (a) in the skin sample of the subject is higher than, in particular statistically higher than, the control level, iv. the level of glycan of the complex type F(6)A2G(4)2S1 measured in step (a) in the skin sample of the subject is higher than, in particular statistically higher than, the control level, and/or v. the level of glycan of the complex type A2G1 GalNAd measured in step (a) in the skin sample of the subject is lower than, in particular statistically lower than, the control level, the control being the level of said at least N-glycan in a skin sample of said subject before application of said cosmetic treatment, and the level of said N-glycans being typically measured by hydrophilic interaction liquid chromatography (HILIC), HPLC/UPLC or mass spectrometry, and optionally digestions by specific exoglycosidases.

"Significantly lower" means in terms of the invention a statistically significant decrease in the level of N-glycan with respect to the control level.

"Significantly higher" means in terms of the invention a statistically significant increase in the level of N-glycan with respect to the control level.

Method for screening

The present invention also relates to a method for screening active compounds to treat old skin, said method comprising the following steps:

(A) contacting the candidate active compound with a skin sample,

(B) measuring the level of at least one N-glycan chosen from the group consisting of glycan of the type rich in mannose M7, glycan of the type rich in mannose M8, glycan of the type rich in mannose M9, glycan of the complex type F(6)A2G(4)2S1 and glycan of the complex type A2G1 GalNAd , as defined in the section "N-glycans" hereinabove, in said skin sample,

(D) selecting the candidate active compound as being an active compound for treating old skin when i. the level of the glycan of the type rich in mannose M7 measured in step (B) is higher, preferably statistically higher, than the control level, ii. the level of the glycan of the type rich in mannose M8 measured in step (B) is higher, preferably statistically higher, than the control level, iii. the level of the glycan of the type rich in mannose M9 measured in step (B) is higher, preferably statistically higher, than the control level, iv. the level of glycan of the complex type F(6)A2G(4)2S1 measured in step (B) is higher than, preferably statistically higher than, the control level and/or v. the level of the glycan of the complex type A2G1 GalNAd measured in step (B) is lower than, preferably statistically lower than, the control level, the control level being preferably the level of said at least N-glycan in the skin sample before contacting with the candidate active compound, and the level of said N-glycans being typically measured by hydrophilic interaction liquid chromatography (HILIC), HPLC/UPLC or mass spectrometry, and optionally digestions by specific exoglycosidases.

The method for screening is preferably implemented ex vivo.

According to a preferred embodiment, the skin sample used in the method for screening according to the invention, is a sample taken, invasively or non-invasively, on the skin of a subject having an old skin, for example a sample of a skin biopsy or a skin sample obtained by rubbing the surface of the skin, in particular with a grater such as a foot grater or a D-squame® disc.

Preferably the skin sample comes from the stratum corneum, in particular from the surface of the stratum corneum.

The level of said at least one N-glycan is preferably measured as described in the section " Method for diagnosing" hereinabove.

"Significantly higher" means in terms of the invention a statistically significant increase in the level of N-glycan with respect to the control level. The present invention shall be described in more detail by the figure and examples hereinbelow.

Brief description of the figures

Figure 1 shows the three types of N-glycans: (1) type rich in mannose, (2) complex type and (3) hybrid type.

Example

Example 1

This example shows that the N-glycans M7, M8, M9, F(6)A2G(4)2S1 and A2G1 GalNAc1 make it possible to diagnose an old skin.

Materials and methods Samples of stratum corneum

The initial glycomic analysis was conducted on a stratum corneum (SC) of the foot obtained from foot gratings, D-squame or varnish strippings.

The SC samples studied were taken from the inside or outside leg, in a cohort of women in good health consisting of an old group (n = 10; 59-63 years) and of a younger group (n = 10; 20-27 years).

Analysis of the N-glycans

Preparation of the labelled N-glycans

The glycoproteins were extracted from SC samples in a multi-well plate in a buffer 25mM NaHC03 pH 8.5 / 50 mM DTT / 0.1% SDS, for 10 min at 70°C. The glycoproteins were then alkylated in the presence of 50 mM of isoacetamide for 30 min at room temperature. The samples were then clarified by successive filtrations through 1-pm and 0.22-pm membranes. The supernatants were then concentrated on an ultrafiltration cartridge (threshold of 10 kDa). The concentrate was then incubated with PNGase F for 1 h at 40°C to specifically release the N-glycans, and the latter were recovered by centrifugation by using 0.22-pm filtration cartridges (threshold of 10 kDa). 10 mI of N-glycans released and purified were then labelled by fluorescence with aminoquinoline N- hydroxysuccinimidylcarbamate (AQC) or with 2-aminobenzamide (2AB) for 5 min at room temperature. The free markers were eliminated by solid phase extraction.

UPLC analysis

The released N-glycans were separated by UPLC/HILIC chromatography by using a linear gradient of 12-88% MeCN on an ethylene bridged hybrid (BEH) glycan column 150 x 2.1 , i.d., 1 .7 mM. 40-min runs were conducted and the glycan peaks eluting were detected by using a fluorescence detector at A_ex of 245 nm and A_em of 395 nm respectively. The system was calibrated by using an external standard of oligomers of glucose hydrolyzed and labelled with 2-AB or with AQC, from which the retention times for the individual glycans were converted into glucose units (GU).

Digestions by exoglycosidase, and low anion exchange analyses - HPLC and LC- MS/MS were conducted to determine the structure of each glycan. The structural annotation of the resulting chromatographic peaks was carried out by comparing the retention time data expressed in glucose units (GU) with a glycan database established at NIBRT, Glycobase 3.1. The nomenclature of the N-glycans of Oxford and the symbolic representation were used.

Statistical analysis

The area of each peak integrated into the chromatogram was calculated and the quantities of N-glycans present in each area were expressed as a relative percentage of the total integrated chromatogram (total quantity of N-glycans in the sample). The peak areas for the duplicated samples were compared (i.e. standard deviation and coefficient of variance %CV) and the means areas were used for the statistical analysis.

The data was exported from Empower to Excel for an analysis by using the R statistic software package. A script developed at NIBRT (based on the Tukey test) was used to analyze the data. The script reports the p-values and generates the box diagrams for each peak in the glycan profile.

Results

The compared analyses of N-glycans present in the SC of women with old skin with respect to women with young skin showed that there was a significant decrease in N- glycans of the type rich in mannose M7, M8 and M9 and of glycan of the complex type F(6)A2G(4)2S1 and an increase in the N-glycan of the complex type A2G1GalNAc1 in the old skins compared to the young skins.

The results are presented in table 1 defined hereinbelow.

Table 1 : N-glycans of SC samples of old or young skin.

The means relative % peak area data was analyzed in R by using the Tukey test showing significant decreases for the N-glycans M7, M8, M9 and F(6)A2G(4)2S1 , and a significant increase for the N-glycan A2G1 GalNAd .

The inventors did not identify any difference for the other N-glycans detected in the SC samples between the young skins and the old skins.

Example 2

The method described in example 1 was applied to evaluate the efficacy of a cream against old skin.

Materials and methods The method described in example 1 was applied to evaluate the efficacy of a cream against old skin of the brand Yves Saint Laurent marketed since 2018 under the name L’Or Rouge creme fine, in a clinical trial on the full face in which 25 female volunteers in good health aged from 50 to 60 years applied the product twice a day for a period of 2 months. After the treatment, the glycomic analysis of D-squames^® samples obtained at the beginning and at the end of the study from a zone of the cheek, was conducted.

Results

The glycomic analysis shows a significant increase in N-glycans M8 and M9 in the stratum corneum of the skin of the face indicating that the formula promotes the profile of N-glycans observed in a young skin with respect to an old skin.

The results are detailed in table 2 defined hereinbelow.

Table 2: Significant increases in the relative quantities of N-glycans M8 and M9 in SC samples of volunteer females in good health (n = 25) aged 50 to 60 years after 2 months of topical treatment with the cosmetic treatment hereinabove. This example therefore confirms that the N-glycans M8 and M9 make it possible to determine the efficacy of a cosmetic treatment against old skin.

Claims

1. Method for diagnosing an old skin in a subject, comprising a step (a) of measuring, in a skin sample of the subject, the level of at least one N-glycan chosen from the group consisting of glycan of the type rich in mannose M7, glycan of the type rich in mannose M8, glycan of the type rich in mannose M9, glycan of the complex type F(6)A2G(4)2S1 and glycan of the complex type A2G1 GalNAd .

2. Method for diagnosing according to claim 1 , wherein the level of said at least one N-glycan is measured by hydrophilic interaction liquid chromatography (HILIC), HPLC/UPLC or mass spectrometry, and optionally digestions by specific exoglycosidases.

3. Method for diagnosing according to claim 1 or 2, the method further comprising the steps consisting of:

(c) based on the comparison in step (b), determining if the skin of said subject is an old skin.

4. Method for diagnosing according to claim 3, wherein said control is the mean value of the level of said N-glycan in a population of subjects having a young skin.

5. Method for diagnosing according to claim 4, wherein the skin of said subject is diagnosed as being an old skin when: i. the level of glycan of the type rich in mannose M7 measured in step (a) in the skin sample of the subject is lower than the level of the control, ii. the level of glycan of the type rich in mannose M8 measured in step (a) in the skin sample of the subject is lower than the level of the control, iii. the level of glycan of the type rich in mannose M9 measured in step (a) in the skin sample of the subject is lower than the level of the control, iv. the level of glycan of the complex type F(6)A2G(4)2S1 measured in step (a) in the skin sample of the subject is lower than the level of the control, and/or v. the level of glycan of the complex type A2G1 GalNAd measured in step (a) in the skin sample of the subject is higher than the level of the control.

6. Method for diagnosing according to any one of claims 1 to 5, wherein the skin sample is taken by rubbing the surface of the skin.

7. Method for evaluating the efficacy of a cosmetic treatment against old skin in a subject, said method comprising, after a step (pre-a) of applying the cosmetic treatment against old skin, a step (a) of measuring the level of at least one N-glycan chosen in the group consisting of glycan of the type rich in mannose M7, glycan of the type rich in mannose M8, glycan of the type rich in mannose M9, glycan of the complex type F(6)A2G(4)2S1 and glycan of the complex type A2G1GalNAc1 , in a skin sample of said subject.

8. Method for evaluating according to claim 7, further comprising the steps consisting of:

9. Method for evaluating according to claim 8, wherein the control is the level of said at least N-glycan in a skin sample of said subject before application of said cosmetic treatment.

10. Method for screening active compounds to treat old skin, said method comprising the following steps:

(A) contacting the candidate active compound with a skin sample,

(B) measuring the level of at least one N-glycan chosen from the group consisting of glycan of the type rich in mannose M7, glycan of the type rich in mannose M8, glycan of the type rich in mannose M9, glycan of the complex type F(6)A2G(4)2S1 and glycan of the complex type A2G1 GalNAd , in said skin sample,

(D) selecting the candidate active compound as being an active compound for treating old skin when i. the level of the glycan of the type rich in mannose M7 measured in step (B) is higher than the control level, ii. the level of the glycan of the type rich in mannose M8 measured in step (B) is higher than the control level, iii. the level of the glycan of the type rich in mannose M9 measured in step (B) is higher than the control level, iv. the level of the glycan of the complex type F(6)A2G(4)2S1 measured in step (B) is higher than the control level and/or v. the level of the glycan of the complex type A2G1 GalNAd measured in step (B) is lower than the control level.