Method for diagnosing and combatting signs of skin aging

Abstract

The present invention relates to methods for determining the age of the skin, and particularly the severity of skin aging, in particular wrinkles, and for identifying compounds having anti-aging properties. These methods are based on measuring the amount of Corynebacterium durum in the skin microbiome of subjects. The present inventors have demonstrated a correlation between high levels of Corynebacterium durum in the skin microbiome and the severity of signs of skin aging.

Description

Description Title: 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 (responsible for skin pigmentation), and Langerhans cells. Its function is to protect the body from the external environment and ensure its integrity, specifically by inhibiting the penetration of microorganisms or chemical substances and preventing the evaporation of water from the skin.

[0004] The dermis is composed of fibroblasts, which produce an extracellular matrix containing collagen and elastin fibers that provide 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 cosmetic compositions to the skin containing active ingredients that act on one or more biological targets involved in skin aging processes.

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

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

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

[0011] Therefore, there is a need for new methods to better understand and identify 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 is now 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's barrier function, regulating pH, and protecting against pathogens.

[0013] Recently, cohort studies have demonstrated a correlation between imbalances in the skin microbiome, or dysbiosis, and skin aging (Ratanapokasatit, Y., et al. Life 2022, 12, 936). However, the precise 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, rather than 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 allow for the development of 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 signs of aging, particularly in women.

[0016] The inventors have shown, in particular, that women, and especially Caucasian women, with a more pronounced skin aging score (this score taking into account multiple clinical signs of aging, including wrinkles, sagging skin, and the presence of age spots), as well as those showing more severe wrinkles relative to their age, had 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 inventors have demonstrated that it was It is possible to use Corynebacterium durum as a model study to identify new anti-aging biological targets.

[0017] The inventors also highlighted a significant correlation between Corynebacterium durum and sebum production in women: women with higher Corynebacterium durum abundance had lower sebum production. This finding 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 its correlation with skin aging scores and wrinkle severity, but also with sebum production.

[0018] According to a first aspect, the present invention relates to a method for determining skin age in a subject, said method comprising a step of evaluating the content of Corynebacterium durum in a skin microbiome sample obtained from said subject. Within the framework 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 skin microbiome sample, of the Corynebacterium durum content; b. Contacting a candidate cosmetic compound with said skin microbiome sample; c. Evaluation, in said skin microbiome sample, of the Corynebacterium durum content; 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 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 based on their impact on a population of Corynebacterium durum; and

[0025] - The increase in the abundance of Corynebacterium durum is directly linked 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 skin 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 well-known concept among those in the field. It refers to all the microorganisms, primarily bacteria, fungi, viruses, and mites, that 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 evaluate the content of Corynebacterium durum within this microbiome. This step makes it possible to assess the abundance of bacterial species, including Corynebacterium durum, in the skin microbiome, that is to say, in relation to 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] A "skin microbiome sample" refers to any sample taken to detect and study the microorganisms that make up a subject's skin microbiome. This sample is usually collected 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 then fixed to the adhesive strip and can subsequently be analyzed.

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

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

[0033] 2. Sampling using a swab: A sterile swab impregnated with a sampling buffer is used to gently rub the surface of the skin, making 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 with 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 V1 to V3 of 16S ribosomal 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, and 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 based on one or more given criteria. For example, the Glogau skin age scale classifies skin age into four different types according to the severity of wrinkles and sun damage. Type I corresponds to an absence of wrinkles, youthful skin, generally observed in people between 20 and 30 years old; type II corresponds to the presence of dynamic wrinkles (appearing during facial movements), generally observed in people between 30 and 40 years old; type III corresponds to the presence of resting wrinkles (present even without facial movement), generally observed in people between 40 and 50 years old; and type IV corresponds to the presence of deep wrinkles and severely sun-damaged skin, generally observed in people over 50 years old.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, such as 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 concentration of Corynebacterium durum within the skin microbiome is determined. This concentration allows for the determination of the relative abundance of Corynebacterium durum within the skin microbiome, and therefore the relative percentage of Corynebacterium durum compared to the total number of other species in the sample. The concentration of Corynebacterium durum can be determined in absolute terms, or relatively, by comparison to the concentration of microorganisms other than Corynebacterium durum within the microbiome, or by comparison to 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: Caucasian people's skin 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 various colors, including blonde, brown, red, and black.

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

[0047] - Skin texture: Caucasian people's skin can vary in texture but is often thinner.

[0048] - Response to the sun: Caucasian people, especially those with lighter 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 according to its reaction to sun exposure.

[0049] As explained here, the concentration of Corynebacterium durum in the skin microbiome allows us to determine an individual's skin age. This bacterium and its role in skin aging therefore represent an excellent model for studying a compound's ability to influence 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 skin microbiome sample, of the Corynebacterium durum content; b. Contacting a candidate cosmetic compound with said skin microbiome sample; c. Evaluation, in said skin microbiome sample, of the Corynebacterium durum content; 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 those of plant origin, such as plants.

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

[0053] Within the framework of the screening process described above, the skin microbiome sample used can advantageously be selected according to the intended application. For example, The sample can 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 can be representative of mature skin when the process aims to identify compounds that can reduce the signs of aging that are already present.

[0054] The term "preventing and / or treating the signs of skin aging" refers to preventing, delaying the onset, or even reducing the visible and measurable manifestations of age-related changes in the skin. The main signs of skin aging include wrinkles and fine lines, loss of skin elasticity and firmness, age-related dryness, pigmentation 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] This 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, whose distribution allows good representation over the whole of this age group, in order to study the link between skin condition and the skin microbiome.

[0060] 21 Evaluation of clinical signs

[0061] High-resolution photographs of each individual's face were taken in a darkened room using a Nikon D5600 camera with an f / 6.3 aperture. The camera was mounted on a monopod attached to a chair to ensure identical facial positioning relative to the camera lens. Two lamps reproducing the continuous spectrum of daylight were positioned symmetrically at 45° angles to each facial 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 from the front and two from the profiles (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 photos. 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 scales. validated ordinal photographs (Jdid et al., 2018). The performance of this algorithm was validated using the MAE (Mean Absolute Error) on the basis of 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 the overall aging of the skin.

[0067] The higher these scores, the more significant the skin aging. These scores allow for a comprehensive and standardized assessment of skin aging, based on a set of clinical signs (presence and severity of wrinkles, intensity of skin laxity, presence of pigment spots), and not just a specific clinical sign (for example, focused on the presence of a specific type of wrinkle) as was done previously.

[0068] These scores were constructed on the basis of the photographs which enabled the development of 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 using the MagMAX™ Microbiome Ultra Nucleic Acid Isolation kit (ThermoFisher Scientific Cat # A42358).

[0076] - Ribosomal DNA amplification and sequencing

[0077] The variable regions V1 to V3 of 16S ribosomal DNA were amplified using the primers "27F" and "534R" described in the Human Microbiome Project Consortium sequencing protocol (available on the hmpdacc.org website, via a link hmpdacc.org / hmp / doc / 16S_Sequencing_SOP_4.2.2).

[0078] The amplicon sequencing was performed using 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 Qiime2 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 investigate the link between the skin microbiome and skin condition, the microbiome was compared between subjects with less severe skin aging and those with more severe skin aging relative to their chronological age using the Linear Discriminant Analysis Effect Size (LEfSe) method. Correlations between biophysical parameters and relative abundances were also studied.

[0085] The study revealed 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 (Figure 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] 21 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 sebum levels in women

[0091] The correlation of the skin microbiome and biophysical parameters also revealed 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, sebum production in women decreases and is associated with a reduction in the abundance of the most dominant commensal bacterial species on the face: Cutibacterium acnes. The decrease in this species would explain the increase in microbiome diversity in women with age, as described by several studies (Townsend and Kalan, 2023). This correlation between sebum production and Cutibacterium acnes abundance 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 sebum levels measured would confirm the involvement of this species in the signs of skin aging in women.

[0095] 4 / Correlation between skin aging and the abundance of different species of the genus Corynebacterium in women

[0096] Several additional analyses were carried out in order to compare the results obtained with certain studies mentioning the possible impact of other species of the genus Corynebacterium, including Corynebacterium kroppenstedtii, Corynebacterium striatum, Corynebacterium minutissim and Corynebacterium xerosis, on skin aging.

[0097] Corynebacterium kroppenstedtii was not detected in the target cohort and Corynebacterium striatum was identified in only one individual in the cohort.

[0098] For Corynebacterium minutissim and Corynebacterium xerosis, the prevalence was also low in the population (i.e., found in less than 10% of individuals in the cohort): Corynebacterium minutissim (6.3%) and Corynebacterium xerosis (4.2%). The potential link between these bacteria and skin aging was nevertheless assessed using a Wilcoxon rank correlation coefficient.

[0099] The results showed no correlation between the severity of signs of age and the presence of Corynebacterium minutissim (p-value = 0.8) or Corynebacterium xerosis (p-value = 0.9).

[0100] 5 / Conclusion

[0101] The results of this study, which weighed a series of standardized clinical signs of wrinkles, skin laxity, and pigmentation spots, revealed a strong correlation between the abundance of Corynebacterium durum in the skin microbiome and the severity of signs of skin aging, particularly in women. The direct correlation (i.e., not resulting from other external factors such as pollution) between The abundance of this bacterial species in women with signs of skin aging has never been described in the literature before. These results demonstrate that Corynebacterium durum can be used as a model organism, particularly for identifying new anti-aging biological targets.

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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