Method for diagnosing a skin exposed to ozone

Abstract

The present invention relates to a method for diagnosing skin exposure to ozone comprising measuring the quantity of methionine sulfoxide, as well as a method for identifying an active compound and a method for evaluating the efficacy of a compound, in order to prevent and / or treat the harmful effects of ozone on skin.

Description

[0001] TITLE: Method for diagnosing a skin exposed to ozone

[0002] The present invention relates to a method for diagnosing skin exposure to ozone comprising measuring the quantity of methionine sulfoxide, as well as a method for identifying an active compound and a method for evaluating the efficacy of a compound, in order to prevent and / or treat the harmful effects of ozone on skin.

[0003] Ozone or trioxygen has the chemical formula O3, and is an allotropic variety of oxygen, much less stable than dioxygen O2, into which it tends to decompose naturally. Ozone is naturally present in the Earth's atmosphere, forming in the stratosphere an ozone layer which intercepts ultraviolet rays from the Sun. However, in the lower layers of the atmosphere (troposphere), it is one of the most hazardous air pollutants for health. This very powerful oxidant may be produced by forest fires, high temperatures, it is also produced in industrial areas or urban centers with strong sunshine. Thus some environments are regularly subjected to ozone peaks. The individual in their daily environment and particularly in an urban zone can be subjected to multiple aggressions on keratin materials, and in particular the skin, by this air pollutant. Indeed, ozone has harmful effects linked with oxidation of the constituents of the skin and modulation of the expression of the biomarkers that it induces.

[0004] From the above, the importance of finding biomarkers making it possible to detect skin exposure to ozone, in order to be able to treat the skin in a targeted way, is understood. The aim of the invention is to meet this need.

[0005] The applicant surprisingly discovered a novel biomarker that is significantly modulated in skins exposed to ozone. This biomarker is an oxidized form of an amino acid of the epidermis of the skin, methionine sulfoxide.

[0006] The methods according to the invention are therefore based on measuring this significantly modulated biomarker, in reconstructed tissues exposed to ozone and / or on skin samples exposed to ozone.

[0007] Thus, according to a first of its aspects, the invention relates to a method for diagnosing, preferably in vitro or ex vivo, skin exposure to ozone, in a subject, said method comprising measuring the quantity of methionine sulfoxide in a skin sample from said subject. Optionally, this diagnostic method according to the invention further comprises the following step: based on the quantity of methionine sulfoxide measured, determining whether said subject's skin has been exposed to ozone.

[0008] The invention also relates to a method for identifying a compound making it possible to prevent and / or treat the harmful effects of ozone on skin, said method comprising the following steps: a. measuring the quantity of methionine sulfoxide in a skin sample from a subject, said sample being treated with said compound; b. comparing said quantity measured in step a) to the quantity of methionine sulfoxide in a skin sample not treated with said compound; c. identifying said candidate compound as a compound making it possible to prevent and / or treat the harmful effects of ozone on skin, when the quantity of methionine sulfoxide measured in the skin sample which was treated with said compound is lower than the quantity of methionine sulfoxide measured in the skin sample which was not treated with said compound.

[0009] The invention also relates to a method for identifying a compound making it possible to prevent and / or treat the harmful effects of ozone on skin, said method comprising the following steps: a. performing a treatment of a skin sample chosen from among keratinocytes, a reconstructed epidermis and a reconstructed skin, by contacting the skin sample with said compound; b. performing an exposure of said skin sample to ozone; c. measuring the quantity of methionine sulfoxide in the skin sample treated with said compound; d. comparing said quantity measured in step c) to the quantity of methionine sulfoxide in a skin sample exposed to ozone and not treated with said compound; e. identifying said candidate compound as a compound making it possible to prevent and / or treat the harmful effects of ozone on skin, when the quantity of methionine sulfoxide measured in the skin sample which was treated with said compound is lower than the quantity of methionine sulfoxide measured in the skin sample which was not treated with said compound; it being understood that step b may be carried out after step a and vice versa, and that steps a and b may be repeated at least 2 times. The invention also relates to a method for evaluating the efficacy of a compound making it possible to prevent and / or treat the harmful effects of ozone on a subject's skin, said method comprising the following steps: a. measuring the quantity of methionine sulfoxide in a skin sample from said subject before the treatment with said compound. b. treating said subject's skin with said compound, c. measuring the quantity of methionine sulfoxide, after step b, in a second skin sample from said subject; d. comparing said quantities measured in step a. and in step c.; e. evaluating the efficacy of said candidate compound in terms of preventing and / or treating the harmful effects of ozone on skin.

[0010] The invention will become more apparent on reading the following description, given merely as a non-limiting example.

[0011] General definitions

[0012] "Subject" is understood to mean a human, preferably aged from 1 month to 90 years. Advantageously, the subject is likely to have been exposed to ozone, in particular to chronic or acute exposure.

[0013] The stratum corneum is the outermost layer of the epidermis, and comprises the skin surface. It is essentially made up of dead cells.

[0014] "Stratum corneum sample" here means a sample taken from this layer. For example, such a sample may be taken by rubbing the skin surface or using an adhesive surface such as a D-squame® disc applied on skin.

[0015] "Exposure to ozone" here means chronic or acute exposure to ozone.

[0016] "Chronic exposure" here means regular, in particular daily, exposure during a given period, preferably a period of more than one year, in particular a period of twelve months optionally stopping on one or more occasions for short durations, typically durations of less than one month, preferably less than one week.

[0017] "Acute exposure" here means brief exposure, in particular from 12 hours to 5 days, to at least an ozone level greater than 0.05 ppm, preferably greater than 0.07 ppm, more preferably greater than 0.09 ppm, even more preferably greater than 0.1 ppm. Methionine sulfoxide is an oxidized methionine derivative having the following formula

[0018] (I):

[0019] Formula (I)

[0020] The measurement of the quantity of methionine sulfoxide may be carried out with any technique known to a person skilled in the art and in particular with liquid chromatography coupled with a UV detector (LC-LIV) or with liquid chromatography coupled with mass spectrometry for example high-resolution mass spectrometry (LC-HRMS).

[0021] In a preferred embodiment, the measured quantity of methionine sulfoxide in the sample corresponds to the quantity of methionine sulfoxide without taking into account oxidized methionine residues in peptides or proteins. Hence, preferably the measurement of the quantity of methionine sulfoxide is carried out without performing hydrolysis beforehand on the sample.

[0022] "Harmful effects of ozone" mean irritations or discomfort reactions on skin (also including the skin of the scalp) which may result in particular in redness, itching, sensations of heat, stinging and tautness.

[0023] Method for diagnosing skin exposure to ozone

[0024] The present invention relates to a method for diagnosing skin exposure to ozone, in a subject, said method comprising measuring the quantity of methionine sulfoxide in a skin sample from said subject.

[0025] Preferably, this method of the invention is a method for diagnosing chronic or acute exposure to ozone.

[0026] The method of the invention is preferably implemented in vitro or ex vivo.

[0027] The skin sample of the subject used in the diagnostic method according to the invention, is a sample taken, preferably non-invasively, on the subject's skin, preferably on the subject's face, in particular on the subject's cheek. Preferably, the skin sample is a biopsy, a microbiopsy in particular carried out using a needle, or a stratum corneum sample.

[0028] Thus according to an embodiment, this method according to the invention comprises a step of taking the skin sample from the subject before measuring the quantity of methionine sulfoxide. This step is preferably performed non-invasively, and in particular does not require local anesthetic. According to a preferred embodiment, the step of taking the sample is performed by rubbing the skin surface or using an adhesive surface such as a D-squame® disc.

[0029] Optionally the diagnostic method according to the invention further comprises the following step: based on the quantity measured, determining whether said subject's skin has been exposed to ozone. In a particular embodiment, this step is carried out after comparing the quantity of methionine sulfoxide measured with a reference value. In an embodiment, the reference value is determined by the mean quantity of methionine sulfoxide measured in a defined population not subjected to exposure to ozone, in particular acute or chronic exposure to ozone.

[0030] In a particular embodiment, the reference value is determined by the mean quantity of methionine sulfoxide measured in humans aged from 1 month to 90 years. According to the invention, a reference value may be determined with a plurality of samples, preferably more than 20, 30, 50, 100, 200 or 500 samples.

[0031] The term "comparison" here refers to determining whether the quantity of methionine sulfoxide is essentially identical to a reference value or differs therefrom. Preferably, the quantity of methionine sulfoxide is considered to be different from a reference value if the observed difference is statistically significant. If the difference is not statistically significant, the quantity of methionine sulfoxide and the reference value are essentially identical.

[0032] On the basis of this comparison, it is possible to determine whether the skin has been exposed to ozone. Typically, the skin is considered to have been exposed to ozone when the quantity of methionine sulfoxide is significantly greater than the reference value, and the skin is not considered to have been exposed to ozone when the quantity of methionine sulfoxide is essentially identical or significantly lower than the reference value.

[0033] Preferably, the method according to the invention comprises a step of treating the sample so as to prepare the measurement of the quantity of methionine sulfoxide. For example, if the skin sample has been obtained with an adhesive surface such as a D- squame® disc, methanol may be added to the surface then the liquid obtained is resuspended in a tube.

[0034] Optionally, the diagnostic method according to the invention further comprises the following step: if said subject's skin is determined as having been exposed to ozone, treating said subject's skin with a cosmetic composition making it possible to prevent and / or treat the harmful effects of ozone.

[0035] "Cosmetic composition making it possible to prevent and / or treat the harmful effects of ozone" is understood to mean any cosmetic composition known to a person skilled in the art making it possible to reduce and / or slow down the appearance of the harmful effects of ozone such as irritations or discomfort reactions on skin, redness, itching, sensations of heat, stinging or tautness caused by ozone. In an embodiment of the invention, the cosmetic composition comprises a compound making it possible to prevent and / or treat the harmful effects of ozone on skin, preferably identified with the method of the invention.

[0036] Methods for identifying a compound making it possible to prevent and / or treat the harmful effects of ozone on skin

[0037] The methods for identifying a compound of the invention are preferably implemented in vitro or ex vivo.

[0038] The invention also relates to a method for identifying a compound making it possible to prevent and / or treat the harmful effects of ozone on skin, said method comprising the following steps: a. measuring the quantity of methionine sulfoxide in a skin sample from a subject, said sample being treated with said compound; b. comparing said quantity measured in step a) to the quantity of methionine sulfoxide in a skin sample not treated with said compound; c. identifying said candidate compound as a compound making it possible to prevent and / or treat the harmful effects of ozone on skin, when the quantity of methionine sulfoxide measured in the skin sample which was treated with said compound is lower than the quantity of methionine sulfoxide measured in the skin sample which was not treated with said compound.

[0039] According to a preferred embodiment, the skin samples from a subject used in the method for identifying a compound making it possible to prevent and / or treat the harmful effects of ozone on skin according to the invention are chosen from among a human skin explant, a skin biopsy, a microbiopsy in particular carried out using a needle and a stratum corneum sample.

[0040] The method according to the invention comprises at least one step of taking a skin sample from the subject. This step is preferably performed non-invasively, and in particular does not require local anesthetic. According to a preferred embodiment, the step of taking the sample is performed by rubbing the skin surface, using an adhesive surface such as a D-squame® disc.

[0041] Preferably, the method according to the invention comprises a step of treating the sample so as to prepare the measurement of the quantity of methionine sulfoxide. For example, if the skin sample has been obtained using an adhesive surface such as a D- squame® disc, methanol may be added to the adhesive surface then the liquid obtained resuspended in a tube.

[0042] In a particular embodiment, the method according to the invention hereinabove comprises an additional step of exposing the samples to ozone. This step is preferably carried out on both samples under the same conditions. It may be carried out before or after treating the skin sample with said compound.

[0043] The invention also relates to a method for identifying a compound making it possible to prevent and / or treat the harmful effects of ozone on skin, said method comprising the following steps: a. performing a treatment of a skin sample chosen from among keratinocytes, a reconstructed epidermis and a reconstructed skin, by contacting the skin sample with said compound; b. performing an exposure of said skin sample to ozone; c. measuring the quantity of methionine sulfoxide in the skin sample treated with said compound; d. comparing said quantity measured in step c) to the quantity of methionine sulfoxide in a skin sample exposed to ozone and not treated with said compound; e. identifying said candidate compound as a compound making it possible to prevent and / or treat the harmful effects of ozone on skin, when the quantity of methionine sulfoxide measured in the skin sample which was treated with said compound is lower than the quantity of methionine sulfoxide measured in the skin sample which was not treated with said compound; it being understood that step b may be carried out after step a and vice versa, and that steps a and b may be repeated at least 2 times. Preferably, this method according to the invention comprises a step of treating the samples so as to prepare the measurement of the quantity of methionine sulfoxide.

[0044] In a preferred embodiment, the method for identifying a compound making it possible to prevent and / or treat the harmful effects of ozone on skin comprises the following steps: a. performing a first treatment of a skin sample chosen from among keratinocytes, a reconstructed epidermis and a reconstructed skin, by contacting the skin sample with said compound; b. performing a first exposure of said skin sample to ozone; c. performing a second treatment of the skin sample by contacting the skin sample with said compound; d. performing a second exposure of said skin sample to ozone; e. performing a third treatment of the skin sample by contacting the skin sample with said compound; f. performing a third exposure of said skin sample to ozone; g. measuring the quantity of methionine sulfoxide in the skin sample treated with said compound; h. comparing said quantity measured in step g) to the quantity of methionine sulfoxide in a skin sample exposed to ozone and not treated with said compound; i. identifying said candidate compound as a compound making it possible to prevent and / or treat the harmful effects of ozone on skin when the quantity of methionine sulfoxide measured in the skin sample which was treated with said compound is lower than the quantity of methionine sulfoxide measured in the skin sample which was not treated with said compound.

[0045] Preferably, this method according to the invention comprises a step of treating the samples so as to prepare the measurement of the expression level.

[0046] Preferably, in the methods for identifying a compound of the invention, the only difference between the skin sample treated with said compound and the skin sample not treated with said compound is said treatment(s) with the compound.

[0047] Typically, the treatment of the sample with said compound comprises contacting the skin sample with said compound for at least 5 minutes, at least 10 minutes or at least 20 minutes. Typically, the measurement of the quantity of methionine sulfoxide is carried out at least 5 minutes, at least 10 minutes or at least 20 minutes after the last treatment of the sample with said compound.

[0048] Steps b, d or h. of comparing the identification methods hereinabove refer to determining whether the quantity of methionine sulfoxide measured in step a. is essentially identical to the quantity of methionine sulfoxide measured in a skin sample not treated with said compound. Preferably, the quantity of methionine sulfoxide is considered to be different if the observed difference is statistically significant. If the difference is not statistically significant, the two quantities of methionine sulfoxide are essentially identical.

[0049] On the basis of this comparison, it is possible to determine whether the compound makes it possible to prevent and / or treat the harmful effects of ozone on skin. Typically, the compound is considered to make it possible to prevent and / or treat the harmful effects of ozone on skin, when the quantity of methionine sulfoxide is significantly lower in the skin sample treated with said compound with respect to the quantity of methionine sulfoxide measured in the sample which was not treated with said compound, alternatively the compound is not considered to make it possible to prevent and / or treat the harmful effects of ozone on skin, when the quantity of methionine sulfoxide is essentially identical or significantly greater in the skin sample treated with said compound with respect to the quantity of methionine sulfoxide measured in the sample which was not treated with said compound.

[0050] Preferably, the steps of exposure to ozone are carried out identically on the sample treated with said compound and the sample which was not treated with said compound.

[0051] Typically, the samples are incubated in an ozone-enriched atmosphere.

[0052] In an embodiment, the steps of exposure to ozone b, d or f last at least 5 hours, more particularly at least 10 hours and preferably at least 18 hours. These steps may also be interspersed with periods of incubation in a normal, ambient atmosphere. The samples are incubated in an ozone-enriched atmosphere for 18h followed by a normal atmosphere for the next 6 hours and once again in an ozone-enriched atmosphere for 18h.

[0053] The methods for identifying a compound according to the invention make it possible to identify compounds making it possible to prevent and / or treat the harmful effects of ozone on skin following chronic or acute exposure to ozone.

[0054] In another embodiment, the steps of exposure to ozone b, d or f last from 10 to 120 minutes, preferably from 20 to 60 minutes. These steps may also be interspersed with periods of incubation in a normal, ambient atmosphere. "Atmosphere" wherein the skin sample is incubated means the gas or gas mixture wherein the sample is placed. Preferably, the sample is incubated in an atmosphere similar to ambient air.

[0055] "Ozone-enriched atmosphere" means an atmosphere similar to ambient air comprising at least 0.05 ppm (parts per million) of ozone, preferably at least 0.07 ppm of ozone, at least 0.1 ppm of ozone or at least 0.3 ppm of ozone.

[0056] Method for evaluating the efficacy of a compound making it possible to prevent and / or treat the harmful effects of ozone on a subject's skin

[0057] The invention also relates to a method for evaluating the efficacy of a compound making it possible to prevent and / or treat the harmful effects of ozone on a subject's skin, said method comprising the following steps: a. measuring the quantity of methionine sulfoxide in a skin sample from said subject before treatment with said compound. b. treating said subject's skin with said compound, c. measuring the quantity of methionine sulfoxide, after step b, in a second skin sample from said subject; d. comparing said quantities measured in step a. and in step c.; e. evaluating the efficacy of said candidate compound in terms of preventing and / or treating the harmful effects of ozone on skin.

[0058] The method of the invention is preferably implemented in vitro or ex vivo.

[0059] The skin samples of the subject used in the evaluation method according to the invention are preferably taken non-invasively, on the subject's skin, preferably on the subject's face, in particular on the subject's cheek.

[0060] Preferably, the skin sample is a biopsy, a microbiopsy in particular carried out using a needle, or a stratum corneum sample.

[0061] Thus according to an embodiment, this method according to the invention comprises several steps for taking skin samples from the subject. This step is preferably performed non-invasively, and in particular does not require local anesthetic. According to a preferred embodiment, the step of taking the sample is performed by rubbing the skin surface or using an adhesive surface such as a D-squame® disc. Preferably, the only difference between the skin sample of step a. and the skin sample of step c. is said treatment with the compound.

[0062] The skin sample of step c. is sampled after said subject's skin has been treated with said compound. In an embodiment, the skin sample of step c. is sampled after said subject's skin has been treated with said compound for at least 14 days, preferably at least 28 days. Typically, this sample is taken at least 5 minutes, at least 10 minutes or at least 20 minutes after the last treatment of the sample with said compound.

[0063] Preferably, the method according to the invention comprises a step of treating the sample so as to prepare the measurement of the quantity of methionine sulfoxide. For example, if the skin sample has been obtained using an adhesive surface such as a D- squame® disc, methanol may be added to the adhesive surface then the liquid obtained is resuspended in a tube.

[0064] Comparison step d. here refers to determining whether the quantity of methionine sulfoxide measured in step a. is essentially identical to the quantity of methionine sulfoxide measured in step c. Preferably, the quantity of methionine sulfoxide is considered to be different if the observed difference is statistically significant. If the difference is not statistically significant, the two quantities of methionine sulfoxide are essentially identical.

[0065] On the basis of this comparison, it is possible to evaluate the efficacy of a compound in preventing and / or treating the harmful effects of ozone on skin. Typically, the lower the quantity of methionine sulfoxide in the skin sample treated with said compound with respect to the quantity of methionine sulfoxide measured in the sample which was not treated with said compound, the more effective the compound is considered to be for preventing and / or treating the harmful effects of ozone on skin. Alternatively, the compound is not considered to be effective for preventing and / or treating the harmful effects of ozone on skin when the quantity of methionine sulfoxide measured is essentially identical or significantly greater in the skin sample treated with said compound with respect to the quantity of methionine sulfoxide measured in the sample which was not treated with said compound.

[0066] Example:

[0067] Example 1 :

[0068] Materials and methods:

[0069] 1- In vitro ozone exposure system 1.1- Device outside the incubator:

[0070] To imitate ozone pollution, the laboratory uses a MEGATEC 49i ozone analyzer / generator coupled with an external pump, and also connected to a culture incubator regulated at 37°C, 5% CO2 and saturated in humidity (approximately 95%).

[0071] The external pump makes it possible to extract ambient air and supply the MEGATEC ozone analyzer / generator. The air will then pass in front of the UV lamp of the apparatus (set to the wavelength of 185nm) which will convert some of the oxygen molecules (02) from the air into ozone molecules (03). The air is then ozone-enriched.

[0072] This ozone-enriched air can then be transferred into the incubator containing culture plates with the T-Skin™ reconstructed skin model.

[0073] The MEGATEC analyzer / generator makes it possible to set and monitor:

[0074] The ozone-enriched air flow rate (supplying the incubator) by means of a relief valve placed upstream from the circuit in the MEGATEC analyzer / generator.

[0075] The ozone concentration generated by setting the power of the UV lamp of the MEGATEC analyzer / generator.

[0076] Moreover, the MEGATEC analyzer / generator has a second pump and an internal analysis system which makes it possible to measure the actual ozone concentration: either at the apparatus outlet (generated concentration, upstream from the exposure in the incubator), or in the incubator (residual concentration, downstream from the exposure).

[0077] 1.2- Device inside the incubator:

[0078] Inside the incubator, a fluidic system developed in the laboratory makes it possible to dispense the same quantity of ozone on each reconstructed skin insert. This system is semiopen to enable at the same time: a homogeneous distribution of ozone on the top of the plates, and satisfactory regulation of the culture conditions of the reconstructed skins.

[0079] Moreover, the lid of each culture plate (generally made of polystyrene, ozone-sensitive material) is replaced by a specific lid for exposure: it consists of a PTFE or PEEK lid perforated above each reconstructed skin insert, and thus allowing direct exposure of the surface of the surface of the reconstructed skins to oxygen-enriched air. Furthermore, this lid makes it possible to protect the culture medium to prevent its oxidation with ozone, so as only carry out exposure of the skin surface. 2- In vitro exposure of reconstructed skins to ozone

[0080] 2.1- Receipt and culture of the T-Skin™ reconstructed skin model.

[0081] The reconstructed skin model used is the T-Skin™ reconstructed skin model produced and supplied by EPISKIN SA (FR-69007, Lyon).

[0082] It consists of a reconstructed skin model composed of:

[0083] - A reconstructed dermis consisting of a collagen 1 lattice, containing fibroblasts of human origin extracted from mammoplasties from healthy adult donors (surgical waste obtained with the patients' consent following cosmetic breast reduction procedures).

[0084] - A reconstructed epidermis produced from keratinocytes of human origin extracted from mammoplasties from healthy adult donors (surgical waste obtained with the patients' consent following cosmetic breast reduction procedures).

[0085] This model is cultured in a 4.5cm2culture insert which makes it possible to suspend it above the culture medium and culture it at the air-liquid interface.

[0086] In this insert, the collagen lattice, or dermal equivalent, covers a surface area which can vary from 2 to 2.5 cm2at the center of the insert. The epidermis generally covers the whole 4.5cm2of the insert.

[0087] The T-Skin™ model is received at the laboratory after 12 days of culture of the dermis (D12). The epidermis is at 7 days of culture and it is relatively immature (with little differentiation). During transport, these inserts are placed on a semi-solid nutrient culture medium suitable for transport.

[0088] On receipt, the inserts are transferred without delay into new so-called "deep well" 6-well culture plates making it possible to place 8.5ml of liquid culture medium under each insert (culture at the air-liquid interface). These plates may be Corning brand reference 355467 plates. The culture medium used is that marketed by EPISKIN for T-Skin™ kits: medium known as "Culture medium for T-Skin".

[0089] The reconstructed skins are then placed in a culture incubator regulated at 37°C, 5% CO2 and with a saturated humidity (approximately 95% humidity).

[0090] The culture medium is then replaced in the same way on D13 and D15 (dermis).

[0091] Finally on D18, the inserts are placed in standard 6-well culture plates in SBS format (COSTAR brand reference 3506 plate type) with 2.5ml of culture medium for TSKIN under each insert.

[0092] These plates are subsequently replaced with culture medium at least every day.

[0093] 2.2- Repeated exposures of T-Skin™ model to 0.1 ppm ozone: For this type of exposure, the ozone generator is set to low values:

[0094] Ozone generated at apparatus outlet: approximately 0.130 ppm for a concentration measured inside the incubator lower than 0.1 ppm

[0095] The ozone-enriched air outlet flow rate above each reconstructed skin is approximately 20 ml / min (note that the ozone outlets are more than 10cm from the reconstructed skins inside the incubator to prevent direct outlet flow stress).

[0096] The exposures are carried out three times successively: 18h between D18 and D19, 18h between D19 and D20, and 18h between D20 and D21 . Between each exposure, the model is in the incubator without ozone for slightly less than 6h.

[0097] Once the skins are ready to be exposed, the standard polystyrene lids are replaced by perforated PEEK or PTFE lids.

[0098] Some of the samples are treated in ozone, and others, the controls, are prepared in exactly the same way, but incubated in a conventional incubator without ozone.

[0099] On D22, the reconstructed skin samples were removed, the epidermis was separated from the dermis, cut in two and frozen at -80°C for the analyses.

[0100] 2.3- Repeated exposures of T-Skin™ model to 0.4 ppm ozone:

[0101] For this type of exposure, the ozone generator is set to moderate ozone values, but slightly higher than realistic ozone peaks:

[0102] Ozone generated at apparatus outlet: approximately 0.4ppm for a concentration measured inside the incubator lower than 0.3ppm

[0103] The ozone-enriched air outlet flow rate above each reconstructed skin is approximately 20 ml / min (note that the ozone outlets are more than 10cm from the reconstructed skins inside the incubator to prevent direct outlet flow stress).

[0104] The exposures are carried out twice successively: 18h between D18 and D19, and 18h between D20 and D21. Between D19 and D20 and between D21 and D22, the reconstructed skins are placed in an incubator without ozone.

[0105] Once the skins are ready to be exposed, the culture plates and media are once again replaced (to prevent the risk of having raw material solution passing into the culture medium under the insert during application), and the standard polystyrene lids are replaced by perforated PEEK or PTFE lids.

[0106] Some of the samples are treated in ozone, and others, the controls, are prepared in exactly the same way, but incubated in a conventional incubator without ozone.

[0107] On D22, the reconstructed skin samples were removed, the epidermis was separated from the dermis, cut in two and frozen at -80°C for the analyses. 3- In vivo exposure of reconstructed skins to ozone

[0108] The clinical study included 10 volunteers (8 female and 3 male), having a mean age of 36.2 years, of phototype lll-IV and a quantity of sebum measured on the forehead greater than 140 pg / cm2(quantity of sebum of 146 to 299 pg / cm2).

[0109] The exposures in mini-zones were carried out at Cl DP Mauritius using bell jars connected to a generator of ozone, medical air or to ambient air, placed on the skin of the volunteers' backs (Controlled Pollution Exposure System (CPES)). The ozone generation system and the bell jars used are described in the publication Curpen et al., 2020 - A novel method for evaluating the effect of pollution on the human skin under controlled conditions, Skin Res Technol.). The exposures to ozone and to medical air were carried out randomly on the leftright on each volunteer. 3 conditions were carried out:

[0110] - 6 exposures to 0.3 ppm (gas flow rate: 0.2-0.3 l / min), three times a week for 2 weeks. Each exposure lasted 2 hours.

[0111] - 6 exposures to medical air (gas flow rate: 0.2-0.3 l / min), according to the same protocol.

[0112] D-squame samples were taken before and 1 h after each exposure on D1 (1 exposure), D5 (3 exposures) and D12 (6 exposures).

[0113] 4- Methionine sulfoxide analysis on reconstructed skin samples in vitro or D- squame samples in vivo

[0114] 4.1- Extraction of methionine sulfoxide for analysis

[0115] To extract methionine sulfoxide, methanol is added to each D-squame sample in a small vial (1 mL). It is stirred for 30 minutes at 37 °C, to be subsequently resuspended in a hemolysis tube and evaporated using a vacuum concentrator. The evaporated extract is resuspended with 200 pL of a 2:1 methanokisopropanol mixture. The samples are placed in an ultrasonic bath for 5 min and filtered at 0.2 pm.

[0116] 4.2- Chromatographic method for methionine sulfoxide analysis: UPLC Vanquish (Thermofisher)

[0117] [Table 1]

[0118] % B TO: percentage of solvent B at TO = 0-0.8 min

[0119] %B at Tx: percentage of solvent B at the different times indicated in the table

[0120] 4.3- Thermofisher Orbitrap Tribrid IPX mass spectrometer settings

[0121] 5

[0122] Compound detection is carried out using the Orbitrap detector, with the following settings:

[0123] Mode: Full MS ESI

[0124] Scan: Positive

[0125] Scan range: 65 - 400

[0126] Resolution: 70,000

[0127] RESULTS

[0128] 10

[0129] In vitro methionine sulfoxide

[0130] Table 2 shows that the quantity of methionine sulfoxide (according to the invention) increases in reconstructed skins exposed to 0.1 or 0.4 ppm of ozone unlike cystine (not in the invention) - the oxidized form of cysteine - which is only present in trace amounts under 15 unexposed conditions and under 0.4 ppm ozone.

[0131] Thus, methionine sulfoxide represents a good marker of ozone exposure among oxidized forms of amino acids, unlike cystine.

[0132] [Table 2] Table 2: Methionine sulfoxide quantified in arbitrary units in vitro on reconstructed skins after exposure to ozone at 0.1 or 0.4 ppm.

[0133] *Not detected

[0134] In vivo methionine sulfoxide

[0135] Table 3 shows that the quantity of methionine sulfoxide increases in D-squame samples taken from volunteers exposed to ozone with respect to those exposed to medical air on

[0136] 5 D12 in 8 out of 10 volunteers.

[0137] [Table 3] Table 3: Methionine sulfoxide quantified in arbitrary units in vivo on Day 12 after 6 exposures to ozone.

Claims

CLAIMS1. Method for diagnosing skin exposure to ozone, in a subject, said method comprising measuring the quantity of methionine sulfoxide in a skin sample from said subject.

2. Diagnostic method according to claim 1 , comprising the following step: based on the quantity measured, determining whether said subject's skin has been exposed to ozone.

3. Method for identifying a compound making it possible to prevent and / or treat the harmful effects of ozone on skin, said method comprising the following steps: a. measuring the quantity of methionine sulfoxide in a skin sample from a subject, said sample being treated with said compound; b. comparing said quantity measured in step a) to the quantity of methionine sulfoxide in a skin sample not treated with said compound; c. identifying said candidate compound as a compound making it possible to prevent and / or treat the harmful effects of ozone on skin, when the quantity of methionine sulfoxide measured in the skin sample which was treated with said compound is lower than the quantity of methionine sulfoxide measured in the skin sample which was not treated with said compound.

4. Method according to claim 3, wherein said skin samples are chosen from among: a human skin explant, a skin biopsy, a microbiopsy and a stratum corneum sample.

5. Method according to claim 3 or 4 comprising an additional step of exposing said samples to ozone.

6. Method for identifying a compound making it possible to prevent and / or treat the harmful effects of ozone on skin, said method comprising the following steps: a. performing a treatment of a skin sample chosen from among keratinocytes, a reconstructed epidermis and a reconstructed skin, by contacting the skin sample with said compound; b. performing an exposure of said skin sample to ozone; c. measuring the quantity of methionine sulfoxide in the skin sample treated with said compound;d. comparing said quantity measured in step c) to the quantity of methionine sulfoxide in a skin sample exposed to ozone and not treated with said compound; e. identifying said candidate compound as a compound making it possible to prevent and / or treat the harmful effects of ozone on skin, when the quantity of methionine sulfoxide measured in the skin sample which was treated with said compound is lower than the quantity of methionine sulfoxide measured in the skin sample which was not treated with said compound; it being understood that step b may be carried out after step a and vice versa, and that steps a and b may be repeated at least 2 times.

7. Method for evaluating the efficacy of a compound making it possible to prevent and / or treat the harmful effects of ozone on a subject's skin, said method comprising the following steps: a. measuring the quantity of methionine sulfoxide in a skin sample from said subject before the treatment with said compound. b. treating said subject's skin with said compound, c. measuring the quantity of methionine sulfoxide, after step b, in a second skin sample from said subject; d. comparing said quantities measured in step a. and in step c.; e. evaluating the efficacy of said candidate compound in terms of preventing and / or treating the harmful effects of ozone on skin.

8. Method according to one of claims 1 to 7, wherein the skin sample(s) are taken using a D-squame® disc.

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