Method for diagnosing skin exposed to ozone

Abstract

Method for diagnosing skin exposed to ozone. The present invention relates to a method for diagnosing skin exposure to ozone, comprising measuring the amount 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 deleterious effects of ozone on the skin. Figure for abstract: none

Description

Title of the invention: Method for diagnosing skin exposed to ozone

[0001] The present invention relates to a method for diagnosing skin exposure to ozone comprising measuring the amount of methionine sulfoxide, as well as a method for identifying an active compound and a method for evaluating the effectiveness of a compound, in order to prevent and / or treat the deleterious effects of ozone on skin.

[0002] Ozone, or trioxygen, has the chemical formula O3. It is an allotropic form of oxygen, much less stable than dioxygen (O2), into which it naturally tends to decompose. Ozone is naturally present in the Earth's atmosphere, forming an ozone layer in the stratosphere that intercepts the Sun's ultraviolet rays. However, in the lower layers of the atmosphere (the troposphere), it is one of the most dangerous air pollutants for health. This very powerful oxidant can be produced by forest fires and high temperatures; it is also produced in industrial areas or urban centers with strong sunlight. Thus, certain environments are regularly subjected to ozone peaks. Individuals in their daily environment, and particularly in urban areas, can be subjected to multiple forms of damage to keratinous materials, especially the skin, by this air pollutant.Indeed, ozone has harmful effects related to the oxidation of skin components and the modulation of biomarker expression that it induces.

[0003] From the foregoing, it is clear the importance of finding biomarkers to detect skin exposure to ozone, in order to treat the skin in a targeted manner. The present invention aims to meet this need.

[0004] The applicant has surprisingly discovered a new biomarker that is significantly modulated in skin exposed to ozone. This biomarker is an oxidized form of an amino acid found in the skin's epidermis, methionine sulfoxide.

[0005] The methods according to the invention are therefore based on the measurement of this biomarker modulated in a significant way, in reconstructed tissues exposed to ozone and / or on skin samples exposed to ozone.

[0006] Thus, according to a first of its aspects, the invention relates to a diagnostic method, preferably in vitro or ex vivo, of skin exposure to ozone in a subject, said method comprising measuring the quantity of methionine sulfoxide in a skin sample of said subject.

[0007] Optionally, this diagnostic method according to the invention further comprises the following step: based on the measured quantity of methionine sulfoxide, de- to conclude if the skin of said subject has been exposed to ozone.

[0008] The invention also relates to a method for identifying a compound for preventing and / or treating the harmful effects of ozone on the skin, said method comprising the following steps: a. measure the amount of methionine sulfoxide in a skin sample from a subject, said sample being treated with said compound; b. compare said quantity measured in step a) to the quantity of methionine sulfoxide in a skin sample not treated with said compound; c. identify said candidate compound as a compound for preventing and / or treating the harmful effects of ozone on the skin, when the amount of methionine sulfoxide measured in the skin sample that has been treated with said compound is less than the amount of methionine sulfoxide measured in the skin sample that has not been treated with said compound.

[0009] The invention also relates to a method for identifying a compound for preventing and / or treating the harmful effects of ozone on the skin, said method comprising the following steps: a. to perform a treatment on a skin sample chosen from keratinocytes, a reconstructed epidermis and reconstructed skin, by bringing the skin sample into contact with said compound; b. perform ozone exposure of said skin sample; c. measure the amount of methionine sulfoxide in the skin sample treated with said compound; d. compare 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. identify said candidate compound as a compound enabling the prevention and / or treatment of the deleterious effects of ozone on the skin when the amount of methionine sulfoxide measured in the skin sample that has been treated with said compound is less than the amount of methionine sulfoxide measured in the skin sample that has not been treated with said compound;

[0010] it being understood that step b can be carried out after step a and vice versa, and that steps a and b can be repeated at least 2 times.

[0011] The invention also relates to a method for evaluating the effectiveness of a compound for preventing and / or treating the harmful effects of ozone on a subject's skin, said method comprising the following steps: a. measure the amount of methionine sulfoxide in a skin sample of said subject before treatment with said compound. b. treat the skin of said subject with said compound, c. measure the amount of methionine sulfoxide, after step b, in a second skin sample of said subject; d. compare the said quantities measured in step a. and in step c.; e. evaluate the efficacy of said candidate compound in preventing and / or treating the harmful effects of ozone on the skin.

[0012] The invention will become clearer upon reading the following description, given solely by way of non-limiting example.

[0013] General definitions

[0014] By "subject" we understand a human being, preferably aged from 1 month to 90 years.

[0015] Advantageously, the subject is likely to have been exposed to ozone, in particular to chronic or acute exposure.

[0016] The stratum corneum is the outermost layer of the epidermis and comprises the surface of the skin. It is composed mainly of dead cells.

[0017] The term "stratum corneum sample" herein means a sample taken from this layer. For example, such a sample can be taken by rubbing the surface of the skin or by using an adhesive surface such as a D-squame® disc applied to the skin.

[0018] “Ozone exposure” here means chronic or acute exposure to ozone.

[0019] By "chronic exposure" is meant here regular exposure, in particular daily exposure, over a given period, preferably a period of more than one year, in particular a period of twelve months possibly interrupted on one or more occasions for short periods, typically periods of less than one month, preferably less than one week.

[0020] By "acute exposure" is meant here a momentary exposure, in particular from 12 hours to 5 days, to at least an ozone level above 0.05 ppm, preferably above 0.07 ppm, more preferably above 0.09 ppm, even better above 0.1 ppm.

[0021] Methionine sulfoxide, or methionine sulfoxide, is an oxidized derivative of methionine having the following formula (I): OO 11 AM nh2

[0022] Formula (I)

[0023] The measurement of the quantity of methionine sulfoxide can be carried out by any technique known to those skilled in the art, and in particular by liquid chromatography coupled with a UV detector (LC-UV) or by liquid chromatography coupled with mass spectrometry, for example high-resolution mass spectrometry (LC-HRMS).

[0024] By "harmful effects of ozone" is meant irritations or reactions of discomfort on the skin (including also the skin of the scalp) which may result in particular in redness, itching, sensations of heat, tingling and pulling.

[0025] Method for diagnosing skin exposure to ozone

[0026] The present invention relates to a method for diagnosing skin ozone exposure in a subject, said method comprising measuring the amount of methionine sulfoxide in a skin sample of said subject.

[0027] Preferably, this method of the invention is a method for diagnosing chronic or acute exposure to ozone.

[0028] The method of the invention is preferably implemented in vitro or ex vivo.

[0029] The skin sample from the subject used in the diagnostic method according to the invention is a sample taken, preferably non-invasively, from the subject's skin, preferably from the subject's face, in particular from the subject's cheek. Preferably, the skin sample is a biopsy, a micro-biopsy, in particular performed using a needle, or a sample of stratum corneum.

[0030] Thus, according to one embodiment, this method according to the invention includes a step of collecting a skin sample from the subject before measuring the amount of methionine sulfoxide. This step is preferably performed non-invasively and, in particular, does not require local anesthesia. According to a preferred embodiment, the sample collection step is performed by rubbing the skin surface or using an adhesive surface such as a D-squame® disc.

[0031] Optionally, the diagnostic method according to the invention further comprises the following step: based on the measured quantity, determining whether the skin of said subject has been exposed to ozone. In a particular embodiment, this step is performed after comparing the measured quantity of methionine sulfoxide with a reference value. In one embodiment, the reference value is determined by the average quantity of methionine sulfoxide measured in a specified population that has not been subjected to ozone exposure, in particular acute or chronic ozone exposure.

[0032] In a particular embodiment, the reference value is determined by the average amount of methionine sulfoxide measured in humans aged from 1 month to 90 years. According to the invention, a reference value can be determined from a plurality of samples, preferably more than 20, 30, 50, 100, 200, or 500 samples.

[0033] The term "comparison" here refers to determining whether the amount of methionine sulfoxide is essentially the same as or different from a reference value. Preferably, the amount of methionine sulfoxide is considered different from a reference value if the observed difference is statistically significant. If the difference is not statistically significant, the amount of methionine sulfoxide and the reference value are essentially the same.

[0034] Based on 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 amount of methionine sulfoxide is significantly higher than the reference value, and the skin is not considered to have been exposed to ozone when the amount of methionine sulfoxide is essentially the same as or significantly lower than the reference value.

[0035] Preferably, the method according to the invention includes a sample treatment step to prepare the sample for measurement of the amount of methionine sulfoxide. For example, if the skin sample was obtained using an adhesive surface such as a D-squame® disc, methanol can be added to the surface and then the resulting liquid is collected in a tube.

[0036] Optionally the diagnostic method according to the invention further includes the following step: if the skin of said subject is determined to have been exposed to ozone, treat the skin of said subject with a cosmetic composition enabling the prevention and / or treatment of the deleterious effects of ozone.

[0037] The term "cosmetic composition for preventing and / or treating the harmful effects of ozone" means any cosmetic composition known to those skilled in the art that reduces and / or slows the onset of the harmful effects of ozone, such as skin irritation or discomfort, redness, itching, and sensations of heat, tingling, and tightness caused by ozone. In one embodiment of the invention, the cosmetic composition comprises a compound for preventing and / or treating the harmful effects of ozone on the skin, preferably identified by the method of the invention.

[0038] Methods for identifying a compound for preventing and / or treating the harmful effects of ozone on the skin

[0039] The methods for identifying a compound of the invention are preferably implemented in vitro or ex vivo.

[0040] The invention also relates to a method for identifying a compound for preventing and / or treating the harmful effects of ozone on the skin, said method comprising the following steps: a. measure the amount of methionine sulfoxide in a skin sample from a subject, said sample being treated with said compound; b. compare said quantity measured in step a) to the quantity of methionine sulfoxide in a skin sample not treated with said compound; c. identify said candidate compound as a compound for preventing and / or treating the harmful effects of ozone on the skin, when the amount of methionine sulfoxide measured in the skin sample that has been treated with said compound is less than the amount of methionine sulfoxide measured in the skin sample that has not been treated with said compound.

[0041] According to a preferred embodiment, the skin samples from a subject used in the method for identifying a compound to prevent and / or treat the deleterious effects of ozone on the skin according to the invention are chosen from a human skin expiant, a skin biopsy, a microbiopsy in particular carried out using a needle and a sample of stratum corneum.

[0042] The method according to the invention includes at least one step of collecting a skin sample from the subject. This step is preferably performed non-invasively and, in particular, does not require local anesthesia. According to a preferred embodiment, the sample collection step is performed by rubbing the skin surface using an adhesive surface such as a D-squame® disc.

[0043] Preferably, the method according to the invention includes a sample treatment step to prepare the sample for measurement of the amount of methionine sulfoxide. For example, if the skin sample was obtained using an adhesive surface such as a D-squame® disc, methanol can be added to the adhesive surface and the resulting liquid collected in a tube.

[0044] In a particular embodiment, the method according to the above invention includes an additional step of exposing the samples to ozone. This step is preferably carried out on both samples under the same conditions. It can be carried out before or after treatment of the skin sample with said compound.

[0045] The invention also relates to a method for identifying a compound for preventing and / or treating the harmful effects of ozone on the skin, said method comprising the following steps: a. to perform a treatment on a skin sample chosen from keratinocytes, a reconstructed epidermis and reconstructed skin, by bringing the skin sample into contact with said compound; b. perform ozone exposure of said skin sample; c. measure the amount of methionine sulfoxide in the skin sample treated with said compound; d. compare 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. identify said candidate compound as a compound enabling the prevention and / or treatment of the deleterious effects of ozone on the skin when the amount of methionine sulfoxide measured in the skin sample that has been treated with said compound is less than the amount of methionine sulfoxide measured in the skin sample that has not been treated with said compound;

[0046] it being understood that step b can be carried out after step a and vice versa, and that steps a and b can be repeated at least 2 times.

[0047] Preferably, this method according to the invention includes a sample processing step to prepare for the measurement of the amount of methionine sulfoxide.

[0048] In a preferred embodiment, the method for identifying a compound to prevent and / or treat the harmful effects of ozone on the skin comprises the following steps: a. perform a first treatment of a skin sample chosen from keratinocytes, a reconstructed epidermis and reconstructed skin, by bringing the skin sample into contact with said compound; b. perform a first exposure to ozone of said skin sample; c. perform a second treatment of the skin sample by contacting of the skin sample with said compound; d. perform a second ozone exposure of said skin sample; e. perform a third treatment of the skin sample by placing in contact of the skin sample with said compound; f. perform a third ozone exposure of said skin sample; g. Measure the amount of methionine sulfoxide in the treated skin sample with the said compound; h. compare 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. identify said candidate compound as a compound enabling the prevention and / or treatment of the deleterious effects of ozone on the skin when the amount of methionine sulfoxide measured in the skin sample that has been treated with said compound is less than the amount of methionine sulfoxide measured in the skin sample that has not been treated with said compound.

[0049] Preferably, this method according to the invention includes a sample processing step to prepare for the measurement of the expression level.

[0050] 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 (or said) treatment(s) with the compound.

[0051] Typically, the treatment of the sample with said compound includes bringing the skin sample into contact with said compound for at least 5 minutes, at least 10 minutes or at least 20 minutes.

[0052] 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.

[0053] Steps b, d, or h of comparing the above identification methods refer to determining whether the amount of methionine sulfoxide measured in step a is essentially the same as the amount of methionine sulfoxide measured in a skin sample not treated with said compound. Preferably, the amount of methionine sulfoxide is considered different if the observed difference is statistically significant. If the difference is not statistically significant, the two amounts of methionine sulfoxide are essentially the same.

[0054] Based on this comparison, it can be determined whether the compound prevents and / or treats the harmful effects of ozone on the skin. Typically, the compound is considered to prevent and / or treat the harmful effects of ozone on the skin when the amount of methionine sulfoxide is significantly lower in the skin sample treated with said compound compared to the amount of methionine sulfoxide measured in the sample that was not treated with said compound. Alternatively, the compound is not considered to prevent and / or treat the harmful effects of ozone on the skin when the amount of methionine sulfoxide measured is essentially the same or significantly higher in the skin sample treated with said compound compared to the amount of methionine sulfoxide measured in the sample that was not treated with said compound.

[0055] Preferably, the ozone exposure steps are carried out identically on the sample treated with said compound and the sample not treated with said compound.

[0056] Typically the samples are incubated in an ozone-enriched atmosphere.

[0057] In one embodiment, the ozone exposure steps 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 incubation periods in a normal, ambient atmosphere. Typically, the samples are incubated in an ozone-enriched atmosphere for 18 hours, then in a normal atmosphere for the next 6 hours, and then again in an ozone-enriched atmosphere for 18 hours.

[0058] The methods for identifying a compound according to the invention make it possible to identify compounds that can prevent and / or treat the harmful effects of ozone on the skin following chronic or acute exposure to ozone.

[0059] In another embodiment, the ozone exposure steps b, d or f last from 10 to 120 minutes, preferably from 20 to 60 minutes. These steps may also be interspersed with incubation periods in a normal, ambient atmosphere.

[0060] By "atmosphere" in which the skin sample is incubated, we mean the gas or mixture of gases in which the sample is placed. Preferably, the sample is incubated in an atmosphere similar to ambient air.

[0061] By "ozone-enriched atmosphere" is meant 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.

[0062] Method for evaluating the effectiveness of a compound for preventing and / or treating the harmful effects of ozone on a subject's skin

[0063] The invention also relates to a method for evaluating the effectiveness of a compound for preventing and / or treating the harmful effects of ozone on a subject's skin, said method comprising the following steps: a. measure the amount of methionine sulfoxide in a skin sample of said subject before treatment with said compound. b. treat the skin of said subject with said compound, c. Measure the amount of methionine sulfoxide, after step b, in a second skin sample of said subject; d. compare the said quantities measured in step a. and in step c.; e. evaluate the efficacy of said candidate compound in preventing and / or treating the harmful effects of ozone on the skin.

[0064] The method of the invention is preferably implemented in vitro or ex vivo.

[0065] The skin samples of the subject used in the evaluation method according to the invention are preferably taken in a non-invasive manner from the skin of the subject, preferably from the face of the subject, in particular from the cheek of the subject.

[0066] Preferably the skin sample is a biopsy, a micro-biopsy in particular carried out using a needle, or a sample of stratum corneum.

[0067] Thus, according to one embodiment, this method according to the invention comprises several steps for collecting a skin sample from the subject. This step is preferably performed non-invasively and, in particular, does not require local anesthesia. According to a preferred embodiment, the sample collection step is performed by rubbing the skin surface or by using an adhesive surface such as a D-squame® disc.

[0068] Preferably, the only difference between the skin sample from step a. and the skin sample from step c. is said treatment with the compound.

[0069] The skin sample from step c is taken after the subject's skin has been treated with the compound. In one embodiment, the skin sample from step c is taken after the subject's skin has been treated with the 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 the compound.

[0070] Preferably, the method according to the invention includes a sample treatment step to prepare the sample for measurement of the amount of methionine sulfoxide. For example, if the skin sample was obtained using an adhesive surface such as a D-squame® disc, methanol can be added to the adhesive surface and then the resulting liquid is collected in a tube.

[0071] The comparison step d here refers to determining whether the amount of methionine sulfoxide measured in step a is essentially the same as the amount of methionine sulfoxide measured in step c. Preferably, the amount of methionine sulfoxide is considered different if the observed difference is statistically significant. If the difference is not statistically significant, the two amounts of methionine sulfoxide are essentially the same.

[0072] Based on this comparison, the effectiveness of a compound in preventing and / or treating the harmful effects of ozone on the skin can be evaluated. Typically, the lower the amount of methionine sulfoxide in the skin sample treated with said compound compared to the amount of methionine sulfoxide measured in the sample that was not treated with said compound, the more effective the compound is considered to be in preventing and / or treating the harmful effects of ozone on the skin. Alternatively, the compound is not considered effective in preventing and / or treating the harmful effects of ozone on the skin when the amount of methionine sulfoxide measured is essentially the same as, or significantly higher than, the amount of methionine sulfoxide measured in the skin sample treated with said compound compared to the amount of methionine sulfoxide measured in the sample that was not treated with said compound. Example: Example 1: Materials and methods: 1- In vitro ozone exposure system 1.1-Device external to the incubator:

[0073] To mimic ozone pollution, the laboratory uses a MEGATEC 49i ozone analyzer / generator coupled to an external pump, and also connected to a culture incubator regulated at 37°C, 5% CO2 and saturated with humidity (approximately 95%).

[0074] The external pump draws in ambient air and supplies it to the MEGATEC ozone analyzer / generator. The air then passes in front of the device's UV lamp (set to a wavelength of 185 nm), which transforms some of the oxygen molecules (O2) in the air into ozone molecules (O3). The air is thus enriched with ozone.

[0075] This ozone-enriched air can then be transferred into the incubator containing the culture plates with the T-Skin™ reconstructed skin model.

[0076] The MEGATEC analyzer / generator allows for the adjustment and control of: - The flow of ozone-enriched air (supplying the incubator) by means of a pressure regulator placed upstream of the circuit in the MEGATEC analyzer / generator. - The concentration of ozone generated by adjusting the power of the UV lamp of the MEGATEC analyzer / generator.

[0077] Furthermore, the MEGATEC analyzer / generator has a second pump and an internal analysis system that allow the actual ozone concentration to be measured: either at the device outlet (generated concentration, upstream of exposure in the incubator), or in the incubator (residual concentration, downstream of exposure). 1.2-Internal device in the incubator:

[0078] Within the incubator, a fluidic system developed in the laboratory distributes the same amount of ozone onto each reconstructed skin insert. This system is semi-open to allow both: homogeneous distribution of ozone over the surface of the plates, and precise regulation of the culture conditions of the reconstructed skin.

[0079] Furthermore, the lid of each culture plate (generally made of polystyrene, a material sensitive to ozone) is replaced by a specific lid for exposure: this is a PTFE or PEEK lid with a hole above each reconstructed skin insert, thus allowing direct exposure of the surface of the reconstructed skin to ozone-enriched air. In addition, this lid protects the culture medium from oxidation by ozone, ensuring that only the surface of the skin is exposed.

[0080] 2-Exposure to ozone of in vitro reconstructed skin

[0081] 2.1- Reception and culture of the T-Skin1^ reconstructed skin model.

[0082] The reconstructed skin model used is the T-Skin™ reconstructed skin model, produced and supplied by the company EPISKIN SA (FR-69007, Lyon).

[0083] This is a reconstructed skin model composed of: - A reconstructed dermis consisting of a collagen 1 lattice, containing human fibroblasts extracted from breast augmentations using tissue from healthy adult donors (surgical waste obtained with the consent of patients during cosmetic breast reduction surgery procedures). mammary glands). - From a reconstructed epidermis made from human keratinocytes extracted from breast augmentations from healthy adult donors (surgical waste obtained with the consent of patients during cosmetic breast reduction surgery procedures).

[0084] This model is cultivated in a 4.5cm2 culture insert which allows it to be suspended above the culture medium and cultivated at the air-liquid interface.

[0085] In this insert, the collagen lattice, or dermal equivalent, covers an area that can vary from 2 to 2.5 cm² at the center of the insert. The epidermis generally covers the entire 4.5 cm² of the insert.

[0086] The T-Skin™ model is received at the laboratory after 12 days of dermal culture (Day 12). The epidermis is 7 days into culture and is relatively immature (poorly differentiated). During transport, these inserts are placed on a semi-solid nutrient culture medium suitable for transport.

[0087] Upon receipt, the inserts are quickly transferred to new 6-well culture plates, also known as "deep well" plates, allowing 8.5 ml of liquid culture medium to be placed under each insert (air-liquid interface culture). These plates can be Corning brand plates, reference 355467. The culture medium used is that marketed by EpiSkin for T-Skin™ kits: a medium called "Culture medium for T-Skin".

[0088] The reconstructed skins are then placed in a culture incubator regulated at 37°C, 5% CO2 and with saturated humidity (approximately 95% humidity).

[0089] The culture medium is then replaced in the same way at J13 and J15 (dermis).

[0090] Finally on J18, the inserts are placed in standard 6-well culture plates in SBS format (COSTAR brand plate type reference 3506) with 2.5ml of culture medium for TSKIN under each insert.

[0091] Subsequently these plates are replaced with the culture medium at least every day.

[0092] 2.2- Repeated exposures of the T-Skin model to 0.1 ppm ozone:

[0093] For this type of exposure, the ozone generator is set to low values: - Ozone generated at the device outlet: approximately 0.130 ppm for a concentration measured inside the incubator of less than 0.1 ppm - The output flow rate of ozone-enriched air 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 avoid direct stress from the output flow).

[0094] The exposures are carried out three times successively: 18 hours between days 18 and 19, 18 hours between days 19 and 20, and 18 hours between days 20 and 21. Between each exposure, the model is at the incubator without ozone for a little less than 6 hours.

[0095] Once the skins are ready to be exposed, the standard polystyrene lids are replaced by perforated PEEK or PTFE lids.

[0096] Part of the samples are treated under ozone, and another part, the controls, are prepared in exactly the same way, but incubated in a conventional incubator without ozone.

[0097] On day 22, samples of reconstructed skin were taken, the epidermis was separated from the dermis, cut in half and frozen at -80°C for analysis.

[0098] 2.3 - Repeated exposures of the T-Skin model to 0.4 ppm ozone:

[0099] For this type of exposure, the ozone generator is set to moderate values ​​in ozone, but slightly higher than realistic ozone peaks: - Ozone generated at the device outlet: approximately 0.4 ppm for a concentration measured inside the incubator of less than 0.3 ppm - The output flow rate of ozone-enriched air 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 avoid direct stress from the output flow).

[0100] The exposures are carried out twice successively: 18 hours between J18 and J19, and 18 hours between J20 and J21. Between J19 and J20 and between J21 and J22 the reconstructed skins are placed in an ozone-free incubator.

[0101] Once the skins are ready to be exposed, the plates and culture media are replaced again (to avoid the risk of having raw material solution that has passed into the culture medium under the insert during application), and the standard polystyrene lids are replaced with perforated PEEK or PTFE lids.

[0102] Part of the samples are treated under ozone, and another part, the controls, are prepared exactly in the same way, but incubated in a conventional incubator without ozone.

[0103] At J22, samples of reconstructed skin were taken, the epidermis was separated from the dermis, cut in half and frozen at -80°C for analysis.

[0104] 3-Ozone exposure of in vivo reconstructed skin

[0105] The clinical study included 10 volunteers (8 women and 3 men), with a mean age of 36.2 years, phototype III-IV and a quantity of sebum measured on the forehead greater than 140 pg / cm2 (quantity of sebum from 146 to 299 pg / cm2).

[0106] Mini-zone exposures were conducted at the CIDP in Mauritius using bells connected to an ozone generator, medical air, or ambient air, placed on the skin of the volunteers' backs (Controlled Pollution Exposure System (CPES)). The ozone generation system and the bells 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.). Exposure to ozone and medical air was performed randomly, left-right, on each volunteer. Three conditions were tested: - 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. - 6 exposures to medical air (gas flow rate: 0.2-0.3 l / min), according to the same protocol.

[0107] D-scales were collected before and after each exposure at J1 (1 exposure), J5 (3 exposures) and J12 (6 exposures).

[0108] 4-Analysis of methionine sulfoxide on samples of in vitro reconstructed skin or in vivo D-squames

[0109] 4.1- Extraction for the analysis of methionine sulfoxide

[0110] To extract methionine sulfoxide, methanol is added to each D-squame in a small vial (1 mL). It is shaken for 30 minutes at 37 °C, then transferred to a hemolysis tube and evaporated using a vacuum concentrator. The evaporated extract is reconstituted with 200 µL of a 2:1 methanokisopropanol mixture. The samples are ultrasonically bathed for 5 minutes and filtered through a 0.2 µm filter. [YES] 4.2 - Chromatographic method for the analysis of methionine sulfoxide: UPLC Vanquish (for Thermofischer)

[0112] [Tables 1] Column Oven °C Eluents Flow Rate mL / min %B T0 %B at T x Duration Acquity CSH Fluoro-Phenyl - 2.1 x 150 xl,7pm -Waters 50 A: Water +0.1% formic acid B: Methanol 0.2 5% 0-0.8 min 0.7-2 min ramp to 99% 2-3 min 99% 3.1-7 min 5% 7 min

[0113] % B T0: percentage of solvent B at T0 = 0-0.8 min

[0114] %B at Tx: percentage of solvent B at the different times indicated in the table

[0115] 4.3 - Parameters of the Thermofisher Orbitrap Tribrid IPX Mass Spectrometer

[0116] The compound is detected using the Orbitrap detector, with the following parameters: Fashion : Scan: Full MS ESI Positive Scan range: 65 - 400 Resolution: 70,000 RESULTS Methionine sulfoxide in vitro

[0117] Table 2 shows that the amount of methionine sulfoxide increases in reconstructed skin exposed to 0.1 or 0.4 ppm of ozone.

[0118] [Table 2] Table 2: Methionine sulfoxide quantified in arbitrary units in vitro on reconstructed skin after exposure to ozone at 0.1 or 0.4 ppm. Metabolite Not exposed Standard deviation Ozone 0.4 ppm Standard deviation Ozone 0.1 ppm Standard deviation Methionine sulfoxide 1 0.148 23.118 0.606 10.226 0.775 Methionine sulfoxide in vivo

[0119] Table 3 shows that the amount of methionine sulfoxide increases in D-squames taken from volunteers exposed to ozone compared to those exposed to medical air at day 12 in 8 out of 10 volunteers.

[0120] [Table 3] Table 3: Methionine sulfoxide quantified in arbitrary units in vivo on Day 12 after 6 ozone exposures. Methionine sulfoxide Medical air Ozone Volunteer 1 2.514 7.019 Volunteer 2 1.352 1.625 Volunteer 3 4.370 6.325 Volunteer 4 3.012 3.734 Volunteer 5 3.098 NA Volunteer 6 2.488 2.962 Volunteer 7 4.358 2.388 Volunteer 8 1.670 2.141 Volunteer 9 7.027 21.035 Volunteer 11 2.330 2.601

Claims

Claims

1. A method of diagnosing ozone exposure of the skin of a subject, said method comprising measuring the amount of methionine sulfoxide in a skin sample of said subject.

2. A diagnostic method according to claim 1, comprising the following step: based on the measured amount, determining whether the skin of said subject has been exposed to ozone after comparing the measured amount of methionine sulfoxide with a reference value.

3. A method for identifying a compound for preventing and / or treating the deleterious effects of ozone on the skin, said method comprising the following steps: a. measuring the amount of methionine sulfoxide in a skin sample of a subject, said sample being treated with said compound; b. comparing said amount measured in step a) with the amount of methionine sulfoxide in a skin sample not treated with said compound; c. identifying said candidate compound as a compound for preventing and / or treating the deleterious effects of ozone on the skin, when the amount of methionine sulfoxide measured in the skin sample that has been treated with said compound is less than the amount of methionine sulfoxide measured in the skin sample that has not been treated with said compound.

4. The method of claim 3, wherein said skin samples are selected from: a human skin explant, a skin biopsy, a microbiopsy and a stratum corneum sample.

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

6. A method for identifying a compound for preventing and / or treating the deleterious effects of ozone on the skin, said method comprising the following steps: a. carrying out a treatment of a skin sample selected from keratinocytes, a reconstructed epidermis and a reconstructed skin, by bringing the skin sample into contact with said compound; b. carry out an ozone exposure of said skin sample; c. measuring the amount 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. identify said candidate compound as a compound for preventing and / or treating the deleterious effects of ozone on the skin when the amount of methionine sulfoxide measured in the skin sample that has been treated with said compound is less than the amount of methionine sulfoxide measured in the skin sample that has not been treated with said compound; it being understood that step b can be carried out after step a and vice versa, and that steps a and b can be repeated at least twice.