METHOD FOR DIAGNOSING AND COMBAT THE SIGNS OF SKIN AGING

Abstract

The present invention relates to methods for determining skin age, and particularly the severity of skin aging, including wrinkles, as well as for identifying compounds with anti-aging properties. These methods are based on measuring the Corynebacterium durum content within the skin microbiome of subjects. The inventors have demonstrated a correlation between the abundance of Corynebacterium durum in the skin microbiome and the severity of skin signs of aging.

Description

Title of the invention: METHOD FOR DIAGNOSING AND COMBAT THE SIGNS OF SKIN AGING technical field

[0001] The present invention relates to the field of cosmetics, and particularly to a diagnostic method for determining skin age, and in particular the severity of signs of aging, especially wrinkles. The present invention also relates to methods for identifying new compounds with anti-aging activity to combat the signs of skin aging. Technological background

[0002] The skin is mainly made up of three layers, namely, starting from the most superficial, the epidermis, the dermis and the hypodermis.

[0003] The epidermis is primarily composed of keratinocytes, melanocytes (involved in skin pigmentation), and Langerhans cells. Its function is to protect the body from the external environment and ensure its integrity, in particular by inhibiting the penetration of microorganisms or chemical substances and preventing the evaporation of water contained in the skin.

[0004] The dermis is composed of fibroblasts, which produce an extracellular matrix containing collagen and elastin fibers providing elasticity and support to the skin.

[0005] With age, the proliferation and activity of skin cells, particularly fibroblasts, decrease. This leads to a reduction in the production of collagen and elastin, the direct consequence of which is the appearance of wrinkles and sagging skin.

[0006] While these phenomena appear naturally over time, certain external factors such as ultraviolet rays, tobacco, the environment and pollution can also induce oxidative stress damaging cellular constituents (DNA, proteins, lipids) and thus accelerating skin aging.

[0007] In order to prevent or correct these different phenomena, it is known to apply to the skin cosmetic compositions containing active ingredients acting on one or more biological targets involved in skin aging processes.

[0008] Many anti-aging active ingredients are currently available. However, these compounds have very diverse mechanisms of action and are not suitable for all biological processes and the different phases of skin aging. Compounds products designed to combat the appearance of the first wrinkles will, for example, not be suitable for so-called "mature" skin with deep-set wrinkles.

[0009] Skin aging is generally assessed clinically, by visual assessment, or using analysis tools based on high-resolution photographs of the face.

[0010] Unfortunately, these methods face many limitations and their results may vary depending on the individual responsible for the analysis.

[0011] There is therefore a need for new methods enabling a better understanding and determination of the factors involved in skin aging. It would be particularly advantageous to be able to identify specific markers correlated with skin aging, which could also represent promising targets for preventing or reducing the processes involved in the appearance of signs of aging.

[0012] The involvement of the skin microbiome in skin quality and function has now been demonstrated in various scientific studies (Smythe, P., Wilkinson, HN Int. J. Mol. Sci. 2023, 24, 3950). Resident bacteria, known as commensal bacteria, interact with the skin through the secretion of various metabolites involved in maintaining the skin barrier function, regulating pH, and protecting against pathogens.

[0013] Recently, cohort studies have demonstrated a correlation between loss of balance in the skin microbiome, or dysbiosis, and skin aging (Ratanapokasatit, Y., et al. Life 2022, 12, 936). However, the exact influence of the microbiome on the skin aging process remains unknown. One limitation to this understanding is that these studies describe the skin microbiome at the taxonomic level of the phylum or genus, and not at the species level. Not all species within the same genus have the same impact on the skin.

[0014] It would therefore be particularly advantageous to be able to precisely identify species of the human skin microbiome that are correlated with skin aging. Identifying such species would make it possible to identify new cosmetic ingredients capable of specifically targeting the activity of these bacteria for an anti-aging effect. Summary

[0015] Thanks to the identification and quantification of the abundance of bacterial species present on the face of each subject according to age and gender, as well as correlations with biophysical and clinical parameters, the present inventors were able to establish a link between the abundance of Corynebacterium durum and the signs of age, particularly in women.

[0016] The present inventors have shown, in particular, that women, and especially Caucasian women, with a more pronounced skin aging score, as well as those showing more severe wrinkles relative to their age, have a significantly higher relative abundance of Corynebacterium durum. This is the only species that has been commonly found to be associated with these two specific parameters of skin aging. The direct correlation (i.e., not resulting from other external factors such as pollution, for example) between the abundance of this bacterial species and the signs of skin aging in women has never been described in the literature before. In light of these results, the present inventors have demonstrated that it is possible to use Corynebacterium durum as a model organism to identify new anti-aging biological targets.

[0017] The inventors also demonstrated a significant correlation between Corynebacterium durum and sebum production in women: women with a higher abundance of Corynebacterium durum have lower sebum production. This result is particularly interesting given that another study showed that sebum production decreases with age in women (Jdid et al., 2024). This suggests that the level of Corynebacterium durum abundance in women could be an indicator of skin age: not only through correlation with skin aging scores and wrinkle severity, but also with sebum production.

[0018] Also, according to a first aspect, the present invention relates to a method for determining the skin age of a subject, said method comprising a step of evaluating, in a skin microbiome sample obtained from said subject, the content of Corynebacterium durum. In the context of the present invention, this Corynebacterium durum content is correlated with skin aging.

[0019] In the context of the present invention, the subject is preferably a woman, and even more preferably a Caucasian woman.

[0020] The present invention also relates to an in vitro method for screening cosmetic compounds to prevent and / or treat the signs of skin aging, said method comprising the steps of: a. Evaluation, in a sample of skin microbiome, of the content of Corynebacterium durum; b. Contacting a candidate cosmetic compound with said skin microbiome sample; c. Evaluation, in said cutaneous microbiome sample, of the content of Corynebacterium durum; d. Selection of the candidate cosmetic compound when the Corynebacterium durum content measured in step c. is lower than that measured in step a. Brief description of the figures Fig. 1

[0021] [Fig.1]: Comparison of the skin microbiome of Caucasian women according to the severity of skin aging. Detailed description of the invention

[0022] As explained above, the present inventors have succeeded in demonstrating a correlation between the abundance of Corynebacterium durum within the skin microbiome and signs of aging. The results presented in this application demonstrate that:

[0023] - it is possible to use this correlation as a diagnostic tool allowing to to determine the severity of skin aging, particularly wrinkles, which is greater in women.

[0024] - it is possible to test and select compounds with anti-aging activity in view of their impact on a population of Corynebacterium durum, and

[0025] - the increase in the abundance of Corynebacterium durum is directly related to the severity of signs of aging, particularly wrinkles.

[0026] Also, according to a first aspect, the present invention relates to a method for determining the age of the skin, and in particular the severity of signs of aging such as wrinkles, in a subject, said method comprising an evaluation step, in a sample of cutaneous microbiome obtained from said subject, of the content of Corynebacterium durum.

[0027] “Corynebacterium durum” is a bacterial species belonging to the genus Corynebacterium. This genus comprises Gram-positive, non-motile, and often commensal bacteria, meaning they live in symbiosis with their host without causing harm. Originally identified in samples taken from the respiratory tracts of healthy individuals (Riegel, Philippe, et al. International Journal of Systematic and Evolutionary Microbiology 47.4 (1997): 1107-1111), Corynebacterium durum was subsequently identified as a resident bacterium of the human skin microbiome, playing a role in maintaining the balance of the skin's bacterial flora. Analysis of the 16S ribosomal RNA of Corynebacterium durum is reported in Barrett, Sara L. Rassoulian, et al. (Journal of Clinical Microbiology 39.3 (2001): 943-948).

[0028] The "skin microbiome" or "skin microbiota" is a concept well known to those skilled in the art. It refers to all the microorganisms, primarily of Bacteria, fungi, viruses, and mites reside on the surface of human skin. These microorganisms form a complex and dynamic ecosystem, interacting with each other and with their human host. The skin microbiome plays a crucial role in maintaining skin health, notably by contributing to the skin's barrier function, regulating skin pH, producing beneficial metabolites, and protecting against pathogens. The interactions between the microorganisms of the skin microbiome and skin cells are essential for maintaining skin balance. A disruption of this balance, called dysbiosis, can be associated with various skin conditions, such as acne, eczema, or psoriasis.

[0029] In the context of the present invention, a sample of the skin microbiome is obtained in order to assess the content of Corynebacterium durum within this microbiome. This step makes it possible to evaluate the abundance of bacterial species, including Corynebacterium durum, in the skin microbiome, that is to say, in relation to the other microorganisms present in the microbiome. It is on the basis of this content, and particularly this abundance, that the skin age of the subject is determined, and particularly the severity of the signs of skin aging.

[0030] The term "skin microbiome sample" refers to any sample taken to detect and study the microorganisms that make up the skin microbiome of a subject. This sample is generally taken using a sterile swab, which is rubbed on a specific area of ​​the skin to collect the microorganisms present. The sample can also be obtained using the "tape stripping" technique, a well-known method that involves applying an adhesive strip to the subject's skin. The microorganisms of the skin microbiome are thus fixed to the adhesive strip and can then be analyzed.

[0031] The process of collecting a skin microbiome sample using a swab generally involves several steps:

[0032] 1. Preparation of the sampling area: The area of ​​skin to be sampled is often The skin is cleaned in the days preceding the sample collection to remove external contaminants without disturbing resident microorganisms. However, the skin is not washed on the day of the sample collection itself.

[0033] 2. Sampling using a swab: A sterile swab impregnated in a The sampling swab is used to gently rub the skin surface, using back-and-forth movements, in order to collect microorganisms.

[0034] 3. Sample preservation: The swab is then placed in a tube containing the sampling buffer to stabilize the microorganisms. The sample is typically stored at -80°C until analysis.

[0035] 4. Sample analysis

[0036] A person skilled in the art is familiar with numerous techniques for detecting and analyzing the content of specific microorganisms within a sample. Typically, the nucleic acids (DNA / RNA) of the microorganisms present in the sample are extracted, purified, amplified, and sequenced. A commonly used method involves subjecting isolated RNA to reverse transcription (“RT”) coupled to polymerase chain reaction (“PCR”) using oligonucleotide primers specific to the genes of interest, which are specific to the target microorganism. In the experimental section below, the variable regions VI to V3 of 16S ribosomal T DNA were amplified. Quantification of mRNAs can generally be performed using one of two real-time quantification technologies called SYBR Green® or Quant-IT.The sequences obtained are then analyzed to identify the bacterial species and other microorganisms present, as well as to assess their relative abundance.

[0037] The method according to the present invention makes it possible to determine the "skin age" of a subject. "Skin age" is a measure of the skin's aging state, which may differ from a person's chronological age. It is assessed by taking into account various clinical and biophysical signs of the skin, such as wrinkles, texture, pigmentation, elasticity, and skin laxity. Skin age reflects the appearance and health of the skin, influenced by genetic, environmental, and lifestyle factors.

[0038] Skin age is generally classified into different grades determined on the basis of one or more given criteria. For example, skin age, as characterized by the Glogau scale, classifies skin age into four different types based on the severity of wrinkles and sun damage. Type I corresponds to an absence of wrinkles, youthful skin, generally observed in people aged 20 to 30; type II corresponds to the presence of dynamic wrinkles (appearing during facial movements), generally observed in people aged 30 to 40; type III corresponds to the presence of resting wrinkles (present even without facial movement), generally observed in people aged 40 to 50; and type IV corresponds to the presence of deep wrinkles and severely sun-damaged skin, generally observed in people over 50.Other methods for classifying skin age that can be mentioned include, for example, the Larnier scale or the Bazin scale. The inventors have also developed, validated and published photographic ordinal scales of skin aging (Jdid, R. et al, Skin Res Technol. 2018;24:196-202).

[0039] In the context of the present invention, the abundance of Corynebacterium durum is positively correlated with skin aging, and particularly with the severity of signs of aging, for example, wrinkles. Indeed, as explained above, the Inventors have demonstrated that the higher the abundance of Corynebacterium durum in the skin microbiome of women, the more advanced the skin age.

[0040] Within the scope of the present invention, the Corynebacterium durum content within the skin microbiome is thus determined. This content makes it possible to determine the relative abundance of Corynebacterium durum within the skin microbiome, and therefore the relative quantity, expressed as a percentage, of Corynebacterium durum compared to the total number of other species in the sample. The Corynebacterium durum content can be determined in absolute terms, or in relative terms, by comparison with the content of microorganisms other than Corynebacterium durum within the microbiome, or by comparison with a reference value.

[0041] The "subject" in the context of the present invention is a human, and preferably a woman, that is to say a subject of the female sex.

[0042] In one particular embodiment, the subject is of Caucasian type. "Caucasian type" here refers to a group of people with specific physical and genetic characteristics, often associated with populations originating from Europe, the Middle East, and parts of Central and South Asia. This term is used to describe specific skin traits and responses to treatments or environmental conditions.

[0043] Typical characteristics of Caucasian people include:

[0044] - skin color: the skin of Caucasian people varies from very light to dark. It is generally more susceptible to damage from UV rays, which can lead to premature aging and an increased risk of skin cancer.

[0045] - Hair color: Hair can be of different colors, including blond, brown, red and black.

[0046] - eye color: the eyes can be of different colors, including blue, green, grey, hazelnut and brown.

[0047] - skin texture: the skin of Caucasian people can vary in terms of texture, but it is often finer.

[0048] - response to the sun: Caucasian people, especially those with the most People with fair skin tend to burn more easily in the sun and tan less easily compared to other populations. This is often assessed using the Fitzpatrick scale, which classifies skin based on its reaction to sun exposure.

[0049] As explained herein, the concentration of Corynebacterium durum within the skin microbiome allows for the determination of a subject's skin age. This bacterium and its role in skin aging therefore represent an excellent model for studying a compound's ability to influence this skin age, and in particular its ability to prevent and / or combat skin aging.

[0050] The present invention also relates to an in vitro method for screening cosmetic compounds to prevent and / or treat the signs of skin aging, said method comprising the steps of: a. Evaluation, in a sample of skin microbiome, of the content of Corynebacterium durum; b. Contacting a candidate cosmetic compound with said skin microbiome sample; c. Evaluation, in said cutaneous microbiome sample, of the content of Corynebacterium durum; d. Selection of the candidate cosmetic compound when the Corynebacterium durum content measured in step c. is lower than that measured in step a.

[0051] The candidate cosmetic compound can be of any type. It can be of natural origin or produced by biotechnology. It can be a library of structurally defined chemical compounds, uncharacterized compounds or substances, or a mixture of compounds. Natural compounds include compounds of plant origin, such as plants.

[0052] Preferably, the candidate cosmetic compound is plant-based, preferably chosen from botanical extracts.

[0053] In the above-described screening process, the skin microbiome sample used can advantageously be selected according to the intended application. For example, the sample may be representative of young skin when the process aims to identify compounds that can prevent or reduce the appearance of the first signs of aging. Similarly, the sample may be representative of mature skin when the process aims to identify compounds that can reduce the signs of aging that are already present.

[0054] By "preventing and / or treating the signs of skin aging," we mean preventing, delaying the appearance, or even reducing the visible and measurable manifestations of the changes that occur in the skin with age. The main signs of skin aging include wrinkles and fine lines, loss of skin elasticity and firmness, age-related dryness, age spots, a dull and uneven complexion, thinning of the skin, and loss of volume due to the loss of dermal tissue components. In one particular embodiment, the signs of aging targeted by the present invention are wrinkles and fine lines.

[0055] The present description also relates to a method for treating the signs of aging, said method comprising a step in which skin identified as aged according to the methods disclosed above is treated with an effective amount of an anti-aging compound.

[0056] The invention will now be illustrated by means of the following examples. Examples

[0057] MATERIALS AND METHODS

[0058] 1 / Cohort description

[0059] A study was conducted between October 3, 2022 and February 21, 2023 on 198 Caucasian individuals, 99 women and 99 men, aged between 20 and 70 years, in order to study the link between skin condition and the skin microbiome.

[0060] 2 / Evaluation of clinical signs

[0061] High-resolution photographs of each individual's face were taken using a camera in a dark room. The camera used was a Nikon D5600 with an f / 6.3 lens. It was mounted on a monopod and attached to a chair to ensure that the face was positioned identically relative to the camera lens. Two lamps reproducing the continuous spectrum of daylight were positioned symmetrically at 45° to each face profile to provide standardized lighting for each photograph.

[0062] Six digital photographs of 13 MB (4000 x 6000 pixels) were taken for each subject, one front view and two profile views (right / left), with eyes open and eyes closed each time.

[0063] After image acquisition, a deep-learning algorithm trained to reproduce clinical ratings performed by a dermatologist was applied to the photographs. This algorithm was trained on a large database of photographs from different populations (N = 5646 photographs of phototypes I to VI) and rated by dermatologists using validated ordinal photographic scales (Jdid et al., 2018). The performance of this algorithm was validated using the Mean Absolute Error (MAE) on the photographs not used to develop the algorithm (test sample).

[0064] Thus, the severity of clinical signs is assessed and 2 scores are calculated:

[0065] - a wrinkle score

[0066] - a skin age score summarizing overall aging of the skin.

[0067] The higher these scores are, the more significant the skin aging.

[0068] These scores were constructed on the basis of the photographs which made it possible to develop the Deep-Learning algorithm using a partial least squares (PLS) structural equation model with second-order constructs.

[0069] 3 / Measurement of biophysical parameters

[0070] Biophysical parameters were measured after a 30-minute acclimatization period in a controlled atmosphere.

[0071] Sebum was measured on the foreheads of individuals using the Courage and Khazaka SM810 Sebumeter.

[0072] 4 / Sampling and sequencing of the skin microbiome

[0073] The skin microbiome was collected from the cheek of each individual using a swab impregnated in a sampling buffer, stored at -80° before being analyzed according to the DNA extraction, purification, amplification, sequencing and taxonomic assignment protocols summarized below (in English)

[0074] - DNA extraction and purification:

[0075] DNA extraction and purification were performed with the MagMAX™ Microbiome Ultra Nucleic Acid Isolation kit (ThermoFisher Scientific Cat # A42358).

[0076] - Ribosomal DNA amplification and sequencing

[0077] The variable regions VI to V3 of 16S ribosomal DNA were amplified using the primers "27F" and "534R" described in the Human Microbiome Project Consortium sequencing protocol (available at hmpdacc.org, via a link hmpdacc.org / hmp / doc / 16S_Sequencing_SOP_4.2.2).

[0078] The amplicon sequencing was carried out with the Illumina MiSeq device.

[0079] - Sequence processing and taxonomic assignment:

[0080] The Fastq files from the sequencing were processed to filter, denoise and merge sequences, remove chimeras and assign taxonomy using Qihne2 software.

[0081] For each OTU, its abundance corresponds to the number of associated reads. The abundance of taxa was estimated by summing the abundances of the OTUs associated with each taxon.

[0082] RESULTS

[0083] 1 / Correlation between the abundance of the species Corynebacterium durum and the skin aging score in women

[0084] To study the link between the skin microbiome and skin condition, the microbiome was compared between subjects with less severe skin aging and subjects with more severe skin aging relative to their chronological age using the LEfSe (Linear Discriminant Analysis Effect Size) method. Correlations between biophysical parameters and relative abundances were also studied.

[0085] The study highlighted a link between the relative abundance of Corynebacterium durum and skin condition in Caucasian women. Those with a higher relative abundance of Corynebacterium durum had higher overall skin aging scores for their age ([Fig. 1]).

[0086] Furthermore, a Wilcoxon rank correlation test confirmed, in a target population, a correlation between the relative abundance of Corynebacterium durum and skin condition, but also demonstrated that, in this same population, there is no correlation between skin aging and other bacteria previously described as being correlated with certain pathological conditions affecting the skin. In particular, the inventors demonstrated that there was no correlation between skin aging and species such as Haemophilus parainfluenzae, Micrococcus luteus, Streptococcus mitis, Streptococcus oralis, Neisseria sp., or Staphylococcus epidermidis.

[0087] 2 / Correlation between the abundance of the species Corynebacterium durum and a high wrinkle score in women

[0088] The study revealed a significant correlation between the abundance of Corynebacterium durum and the severity of wrinkles, particularly in Caucasian women. Those with a higher relative abundance of Corynebacterium durum had higher facial wrinkle scores for their age.

[0089] Previous results indicate that the abundance of Corynebacterium durum in women is specifically correlated with a greater severity of skin age score and wrinkles relative to their chronological age.

[0090] 3 / Correlation between the abundance of the species Corynebacterium durum and the rate sebum in women

[0091] The correlation of the skin microbiome and biophysical parameters also made it possible to highlight that Caucasian women with a higher relative abundance of Corynebacterium durum have a lower sebum level compared to their chronological age.

[0092] The correlation is equal to r = -0.22, p = 0.0354

[0093] With age, the sebum level in women decreases and is associated with a decrease The abundance of the most dominant commensal bacterial species on the face, Cutibacterium acnes, is a factor. A decrease in this species would explain the increase in microbiome diversity in women with age, as described in several studies (Townsend and Kalan, 2023). This correlation between sebum levels and the abundance of Cutibacterium acnes could serve as an indicator of the skin aging process in women.

[0094] The study showed that Corynebacterium durum is more abundant in women with more pronounced visible signs of skin aging. Given the decrease in sebum levels in women with age, the correlation between the abundance of Corynebacterium durum and the low measured sebum levels is significant. would confirm the involvement of this species in the signs of skin aging in women.

Claims

Demands

1. A method for determining the age of the skin in a subject, said method comprising a step of evaluating, in a skin microbiome sample obtained from said subject, the content of Corynebacterium durum.

2. A method according to claim 1 wherein the content of Corynebacterium durum is correlated with skin aging.

3. A method according to claim 1 or 2, wherein said skin microbiome sample was taken using a swab brought into contact with the skin surface of said subject.

4. A method according to any one of claims 1 to 3, wherein the subject is a woman.

5. A method according to any one of claims 1 to 4, wherein said subject is of Caucasian type.

6. An in vitro process for screening cosmetic compounds to prevent and / or treat the signs of skin aging, said process comprising the steps of: a. Evaluating the Corynebacterium durum content in a skin microbiome sample; b. Contacting a candidate cosmetic compound with said skin microbiome sample; c. Evaluating the Corynebacterium durum content in said skin microbiome sample; d. Selecting the candidate cosmetic compound when the Corynebacterium durum content measured in step c is lower than that measured in step a.

7. A method for screening cosmetic compounds according to claim 6, wherein said candidate cosmetic compound is a plant extract.

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