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, 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 related to the oxidation of skin compounds and the modulation of biomarker expression in response to ozone exposure. 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 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 linked to the oxidation of certain constituents of the skin and to the modulation of the expression of biomarkers in response to exposure to ozone.

[0002] Ozone or trioxygen has the chemical formula O3, it is an allotropic variety 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 which 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, high temperatures, it is also produced in industrial areas or urban centers with strong sunlight. Thus certain environments are regularly subject to ozone peaks. The individual in his daily environment and particularly in urban areas, can be subjected to multiple attacks on keratinous materials, and in particular the skin, by this air pollutant.Indeed, ozone has deleterious effects linked to the oxidation of certain constituents of the skin and to the modulation of the expression of the biomarkers that it induces.

[0003] From the above, it is understood the importance of finding biomarkers that can detect ozone exposure of the skin, in order to be able to treat the skin in a targeted manner. The present invention aims to satisfy this need.

[0004] The applicant surprisingly discovered several biomarkers significantly modulated in skin exposed to ozone. These biomarkers are products resulting from the oxidation of skin lipids; they include aldehydes and oxidized forms of cholesterol, also called oxysterols.

[0005] The list of biomarkers is as follows: tridecenal, pentadecanal, octadecanal, 5a-6a-epoxycholesterol, 5[3-6[3-epoxycholesterol and 7-ketocholesterol.

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

[0007] Numerous publications describe the degradation products of skin lipids by ozone. The reaction of ozone on unsaturated bonds causes the formation of aldehydes. The products that can be formed, theoretically, at from squalene have already been described. However, given the reactivity of ozone, the complexity of skin lipids and also the relative stability of the products formed, it is not easy to predict which of the aldehyde family could be the right marker(s) of ozone-induced stress.

[0008] Since ozone-induced stress can have an impact over several hours, it is important to analyze markers that accumulate on the skin. Because of the low retention of volatile molecules on the skin, biomarker research has focused on aldehydes and oxysterols.

[0009] To the inventors' knowledge, among the numerous aldehydes found on the skin, surprisingly, tridecenal, pentadecanal, octadecanal have never been disclosed in the literature as being significantly correlated with ozone-induced stress.

[0010] Furthermore, nonanal, decanal and / or dodecanal are also significantly correlated with ozone-induced stress.

[0011] Similarly, among the oxidized forms of cholesterol, 5a-6a-epoxycholesterol, 5[3-6[3-epoxycholesterol and 7-ketocholesterol have never been disclosed in the literature as being significantly correlated with ozone-induced stress.

[0012] Thus, according to a first of its aspects, the invention relates to a method of diagnosis, preferably in vitro or ex vivo, of exposure to ozone of the skin, in a subject, said method comprising the following step:

[0013] a. measuring the quantity of at least one product resulting from the oxidation of skin lipids chosen from tridecenal, pentadecanal, octadecanal, 5a-6a-epoxycholesterol, 5[3-6[3-epoxycholesterol and 7-ketocholesterol, in a skin sample from said subject.

[0014] Optionally, this diagnostic method according to the invention further comprises the following step:

[0015] b.based on the quantity measured in step a., determining whether the skin of said subject has been exposed to ozone.

[0016] 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. measuring the quantity of at least one product resulting from the oxidation of skin lipids selected from tridecenal, pentadecanal, octadecanal, 5a-6a-epoxycholesterol, 5[3-6[3-epoxycholesterol and 7-ketocholesterol in a skin sample from a subject, said sample being treated with said compound; b. compare the said quantity measured in step a) with the corresponding quantity in a skin sample not treated with said compound; c. identify said candidate compound as a compound for preventing and / or treating the deleterious effects of ozone on the skin when the amount of aldehyde or oxysterol measured in the skin sample that has been treated with said compound is less than the amount of aldehyde or oxysterol measured in the skin sample that has not been treated with said compound.

[0017] 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. 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. perform ozone exposure of the skin sample; c. measure the quantity of at least one product resulting from the oxidation of lipids in the skin selected from tridecenal, pentadecanal, octadecanal, 5a-6a-epoxycholesterol, 5[3-6[3-epoxycholesterol and / or 7-ketocholesterol in the skin sample treated with said compound; d. comparing said quantity measured in step c) with the corresponding quantity 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 aldehyde or oxysterol measured in the skin sample that has been treated with said compound is less than the amount of aldehyde or oxysterol measured in the skin sample that has not been treated with said compound;

[0018] 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.

[0019] 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 the skin of a subject, said method comprising the following steps: a. measuring the amount of at least one product resulting from the oxidation of skin lipids selected from tridecenal, pentadecanal, octadecanal, 5a-6a-epoxycholesterol, 5[3-6[3-epoxycholesterol and / or 7-ketocholesterol in a skin sample of said subject before treatment with said compound. b. treating the skin of said subject with said compound, c. after step b., measure the quantity of the same product(s) from oxidation of skin lipids in a second skin sample from 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 deleterious effects of ozone on the skin.

[0020] The invention will appear more clearly on reading the description which follows, given solely by way of non-limiting example.

[0021] General definitions

[0022] By “subject” we mean a human being, preferably aged from 1 month to 90 years.

[0023] Advantageously, the subject is likely to have been exposed to ozone, in particular to chronic or acute exposure.

[0024] By "exposure to ozone" is meant herein chronic or acute exposure to ozone.

[0025] By "chronic exposure" is meant here regular exposure, in particular daily, 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.

[0026] By "acute exposure" is meant here a momentary exposure, in particular from 12 hours to 5 days, to at least one ozone level greater than 0.05 ppm, preferably greater than 0.07 ppm, more preferably greater than 0.09 ppm, even better greater than 0.1 ppm.

[0027] The stratum corneum is the outermost layer of the epidermis, and comprises the surface of the skin. It is composed mainly of dead cells.

[0028] By "stratum corneum sample" is meant here 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.

[0029] By "product resulting from the oxidation of skin lipids" is meant here the oxidized lipids which can be used as biomarkers according to the invention, that is to say: nonanal, decanal, dodecanal, tridecenal, pentadecanal, octadecanal, 5a-6a-epoxycholesterol, 5[3-6[3-epoxycholesterol and 7-ketocholesterol.

[0030] By "aldehyde" is meant here nonanal, decanal, dodecanal, tridecenal, pentadecanal, octadecanal.

[0031] By "oxysterol" is meant here 5a-6a-epoxycholesterol, 5[3-6[3-epoxycholesterol and 7-ketocholesterol.

[0032] Tridecenal is an aldehyde of formula C13H24O with unsaturation. Preferably, tridecenal is chosen from 2-tridecenal, 4-tridecenal, 5-tridecenal, and mixtures thereof.

[0033] Pentadecanal is an aldehyde of formula CH3(CH2)i3CHO.

[0034]

[0035]

[0036]

[0037]

[0038]

[0039] Octadecanal is an aldehyde also known as stearyl aldehyde with the formula CH3(CH2)i6CHO. Nonanal, also known as nonanaldehyde, pelargonaldehyde, or C-9 aldehyde, is an aldehyde with the formula CH3(CH2)7CHO. Decanal is an aldehyde with the formula CH3(CH2)8CHO. Dodecanal, also called lauraldehyde or dodecyl aldehyde, is an aldehyde with the formula CH3(CH2)10CHO. 5a-6a-epoxycholesterol, and 5[3-6[3-epoxycholesterol are stereoisomers. 5a-6a-epoxycholesterol has the following formula (I):

[0040]

[0041] Formula (I) 5[3-6[3-epoxycholesterol has the following formula (II):

[0042]

[0043] Formula (II) 7-ketocholesterol has the following formula (III):

[0044] Formula (III)

[0045] By "corresponding quantity" is meant the quantity of the same aldehyde or the same oxysterol as that whose quantity was measured in a previous step.

[0046] By "deleterious effects of ozone" is meant irritations or discomfort reactions at the level of the skin (also including the skin of the scalp) which can result in particular in redness, itching, sensations of heat, tingling and tightness.

[0047] The measurement of the quantity of oxidized lipids 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 high-resolution mass spectrometry (LC-HRMS). For the measurement of aldehydes, the measurements are preferably carried out after derivatization with 2,4-dinitrophenylhydrazine (DNPH).

[0048] Preferably, this measurement is carried out from a skin sample and not in the air near the skin.

[0049] Method for diagnosing ozone exposure of the skin

[0050] The present invention relates to a method for diagnosing ozone exposure of the skin in a subject, said method comprising the following step: a. measuring the quantity of at least one product resulting from the oxidation of skin lipids chosen from tridecenal, pentadecanal, octadecanal, 5a-6a-epoxycholesterol, 5[3-6[3-epoxycholesterol and 7-ketocholesterol, in a skin sample of said subject.

[0051] Preferably, the at least one product resulting from the oxidation of skin lipids is chosen from 5a-6a-epoxycholesterol, tridecenal, pentadecanal and octadecanal.

[0052] Preferably, this method of the invention is a method for diagnosing chronic or acute exposure to ozone.

[0053] The method of the invention is preferably carried out in vitro or ex vivo.

[0054] Step a. of this method may further comprise measuring the amount of nonanal, decanal and / or dodecanal in the skin sample of said subject.

[0055] The skin sample of the subject used in the diagnostic method according to the invention is a sample taken, preferably non-invasively, from the skin of the subject, preferably from the face of the subject, in particular from the cheek of the subject. Preferably the skin sample is a biopsy, a microbiopsy in particular carried out using a needle, or a sample of stratum corneum.

[0056] Thus, according to one embodiment, this method according to the invention comprises a step of taking the skin sample from the subject. This step is preferably carried out in a non-invasive manner, and in particular does not require local anesthesia. According to a preferred embodiment, the step of taking the sample is carried out by rubbing the surface of the skin and / or using a surface adhesive such as a D-squame® disc and / or by wiping the skin with absorbent paper (such as filter paper).

[0057] In one embodiment of the invention, to measure the quantity of one or more aldehyde(s), the sample collection step is carried out by rubbing the surface of the skin with absorbent paper, preferably soaked with a monoalcohol such as ethanol.

[0058] In one embodiment of the invention, to measure the amount of one or more oxysterol(s), the sample collection step is carried out using an adhesive surface such as a D-squame® disc.

[0059] Optionally, the diagnostic method according to the invention further comprises the following step:

[0060] b. based on the quantity measured in step a., determining whether the skin of said subject has been exposed to ozone.

[0061] In a particular embodiment, step b. is carried out after comparing the measured quantity of this aldehyde or oxysterol in step a. with a reference value. In one embodiment, the reference value is determined by the average quantity of this aldehyde or oxysterol measured in a given population, which has not been subjected to exposure to ozone, in particular acute or chronic exposure to ozone.

[0062] In a particular embodiment, the reference value is determined by the average quantity of this aldehyde or oxysterol measured in human beings aged from 1 month to 90 years. According to the invention, a reference value can be determined by a plurality of samples, preferably more than 20, 30, 50, 100, 200 or 500 samples.

[0063] The term "comparison" herein refers to determining whether the amount of such aldehyde is substantially the same as or differs from a reference value. Preferably, the amount of such aldehyde or oxysterol is considered to be different from a reference value if the observed difference is statistically significant. If the difference is not statistically significant, the amount of such aldehyde or oxysterol and the reference value are substantially the same.

[0064] Based on this comparison, it can be determined whether the skin has been exposed to ozone. Typically, the skin is considered to have been exposed to ozone when the amount of this aldehyde or oxysterol is significantly greater than the reference value and the skin is not considered to have been exposed to ozone when the amount of this aldehyde or oxysterol is essentially the same as or significantly less than the reference value.

[0065] Preferably, the method according to the invention comprises a step of treating the sample in order to prepare the measurement of the quantity of this aldehyde or oxysterol. For example, if the skin sample was obtained by wiping the skin with absorbent paper (such as filter paper soaked in ethanol), the sample can be evaporated and then taken up in an acetonitrile solution in order to carry out a derivatization, for example a DNPH derivatization.

[0066] Optionally, the diagnostic method according to the invention further comprises the following step:

[0067] c. if the skin of said subject is determined to have been exposed to ozone in step b., treating the skin of said subject with a cosmetic composition for preventing and / or treating the deleterious effects of ozone.

[0068] By "cosmetic composition for preventing and / or treating the harmful effects of ozone" is meant any cosmetic composition known to those skilled in the art for reducing and / or slowing down the appearance of the harmful effects of ozone such as irritations or discomfort reactions in the skin, redness, itching, 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 identified by the method of the invention.

[0069] Methods for identifying a compound for preventing and / or treating the deleterious effects of ozone on the skin

[0070] 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. measuring the quantity of at least one product resulting from the oxidation of skin lipids selected from tridecenal, pentadecanal, octadecanal, 5a-6a-epoxycholesterol, 5[3-6[3-epoxycholesterol and 7-ketocholesterol, in a skin sample from a subject, said sample being treated with said compound; b. comparing said quantity measured in step a) to the corresponding quantity in a skin sample not treated with said compound; c. identify said candidate compound as a compound for preventing and / or treating the deleterious effects of ozone on the skin when the amount of aldehyde or oxysterol measured in the skin sample that has been treated with said compound is less than the amount of aldehyde or oxysterol measured in the skin sample that has not been treated with said compound.

[0071] Preferably, the at least one product resulting from the oxidation of skin lipids is chosen from 5a-6a-epoxycholesterol, tridecenal, pentadecanal and octadecanal.

[0072] The identification method according to the invention may also comprise measuring the quantity of nonanal and / or decanal and / or dodecanal in the sample of skin of said subject in step a, this measurement can then be compared to the corresponding quantity in a sample of skin not treated with said compound in step b, in order to identify said candidate compound as a compound making it possible to prevent and / or treat the deleterious effects of ozone on the skin.

[0073] According to a preferred embodiment, the skin samples of a subject used in the method for identifying a compound for preventing and / or treating the deleterious effects of ozone on the skin above, are chosen from an explant of human skin, a skin biopsy, a microbiopsy, in particular carried out using a skin needle, and a sample of stratum corneum.

[0074] The method according to the invention comprises a step of taking the skin sample from the subject. This step is preferably carried out in a non-invasive manner, and in particular does not require local anesthesia. According to a preferred embodiment, the step of taking the sample is carried out by rubbing the surface of the skin and / or using an adhesive surface such as a D-squame® disc, and / or wiping the skin with absorbent paper (such as filter paper).

[0075] In one embodiment of the invention, to measure the quantity of one or more aldehyde(s), the sample collection step is carried out by rubbing the surface of the skin with absorbent paper, preferably soaked with a monoalcohol such as ethanol.

[0076] In one embodiment of the invention, to measure the amount of one or more oxysterol(s), the sample collection step is carried out using an adhesive surface such as a D-squame® disc.

[0077] Preferably, the method according to the invention comprises a step of treating the samples in order to prepare the measurement of the quantity of aldehyde or oxysterol. For example, if the skin sample was obtained by wiping the skin with absorbent paper (such as filter paper, for example, soaked in ethanol), the sample can be evaporated and then taken up in an acetonitrile solution and then a derivatization carried out, for example a derivatization with 2,4-dinitrophenylhydrazine (DNPH).

[0078] In a particular embodiment, the method according to the invention comprises 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.

[0079] 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. 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. perform ozone exposure of the skin sample; c. measure the quantity of at least one product resulting from the oxidation of lipids in the skin selected from tridecenal, pentadecanal, octadecanal, 5a-6a-epoxycholesterol, 5[3-6[3-epoxycholesterol and / or 7-ketocholesterol in the skin sample treated with said compound; d. comparing said quantity measured in step c) with the corresponding quantity 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 aldehyde or oxysterol measured in the skin sample that has been treated with said compound is less than the amount of aldehyde or oxysterol measured in the skin sample that has not been treated with said compound;

[0080] 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.

[0081] Preferably, the at least one product resulting from the oxidation of skin lipids is chosen from 5a-6a-epoxycholesterol, tridecenal, pentadecanal and octadecanal.

[0082] Preferably, the method according to the invention comprises a step of treating the samples in order to prepare the measurement of the quantity of aldehyde or oxysterol. For example, if the skin sample was obtained by wiping the skin with absorbent paper (such as filter paper, for example, soaked in ethanol), the sample can be evaporated and then taken up in an acetonitrile solution and then a derivatization carried out, for example a derivatization with 2,4-dinitrophenylhydrazine (DNPH).

[0083] In a preferred embodiment, the method for identifying a compound for preventing and / or treating the deleterious effects of ozone on the skin comprises the following steps: a. carrying out a first treatment of a skin sample chosen from keratinocytes, a reconstructed epidermis and a reconstructed skin, by bringing the skin sample into contact with said compound; b. carry out a first exposure to ozone of said skin sample; c. carry out a second treatment of the skin sample by contacting of the skin sample with said compound; d. carry out a second exposure to ozone of said skin sample; e. carry out a third treatment of the skin sample by putting in contact of the skin sample with said compound; f. carry out a third exposure to ozone of said skin sample; g. measure the quantity of at least one product resulting from the oxidation of lipids of the skin selected from 5a-6a-epoxycholesterol, 5[3-6[3-epoxycholesterol and 7-ketocholesterol in the skin sample treated with said compound; h. comparing said quantity measured in step g) with the corresponding quantity in a skin sample exposed to ozone and not treated with said compound i. identify said candidate compound as a compound for preventing and / or treating the deleterious effects of ozone on the skin when the amount of oxysterol measured in the skin sample that has been treated with said compound is lower than the amount of oxysterol measured in the skin sample that has not been treated with said compound.

[0084] Preferably, the at least one product resulting from the oxidation of skin lipids is 5a-6a-epoxycholesterol,

[0085] In another preferred embodiment, the method for identifying a compound for preventing and / or treating the deleterious effects of ozone on the skin, comprises 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. perform ozone exposure of the skin sample; c. measure the quantity of at least one product resulting from the oxidation of lipids in the skin selected from tridecenal, pentadecanal and octadecanal in the skin sample treated with said compound; d. comparing said quantity measured in step c) with the corresponding quantity in the 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 aldehyde measured in the skin sample that has been treated with said compound is less than the amount of aldehyde measured in the skin sample that has not been treated with said compound;

[0086] it being understood that step b can be carried out after step a and vice versa.

[0087] Preferably, the method according to the invention comprises a step of treating the samples in order to prepare the measurement of the quantity of aldehyde or oxysterol. For example, if the skin sample was obtained by wiping the skin with absorbent paper (such as filter paper, for example, soaked in ethanol), the sample can be evaporated and then taken up in an acetonitrile solution and then a derivatization carried out, for example a derivatization with 2,4-dinitrophenylhydrazine (DNPH).

[0088] The methods for identifying a compound of the invention are preferably carried out in vitro or ex vivo.

[0089] The measuring steps a, c or g of these methods for identifying a compound according to The invention may further comprise measuring the amount of nonanal, decanal and / or dodecanal in the skin sample. This measurement may then be compared to the corresponding amount in a skin sample not treated with said compound, in order to identify said candidate compound as a compound for preventing and / or treating the deleterious effects of ozone on the skin.

[0090] 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.

[0091] In a particular embodiment, said reconstructed epidermis or reconstructed skin may be covered with squalene before the step(s) of exposure to ozone, for example steps b, d or f, in particular in the case where the aldehydes are measured in steps a, c or g.

[0092] Typically, treating 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.

[0093] Typically, the measurement of the quantity of aldehyde or oxysterol 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.

[0094] Steps b, d or h of comparing the identification methods herein refer to determining whether the amount of aldehyde or oxysterol measured in measuring steps a, c or g is essentially identical to the amount of that same aldehyde or oxysterol measured in a skin sample not treated with said compound. Preferably, the amount of that aldehyde or oxysterol is considered different if the observed difference is statistically significant. If the difference is not statistically significant, the two amounts of that aldehyde or oxysterol are essentially identical.

[0095] Based on this comparison, it can be determined whether the compound is capable of preventing and / or treating the deleterious effects of ozone on the skin. Typically, the compound is considered to be capable of preventing and / or treating the deleterious effects of ozone on the skin when the amount of this aldehyde or oxysterol is significantly lower in the skin sample treated with said compound compared to the amount of this aldehyde or oxysterol measured in the sample that has not been treated with said compound, alternatively the compound is not considered to be capable of preventing and / or treating the deleterious effects of ozone on the skin when the amount of this aldehyde or oxysterol measured is essentially the same or significantly higher in the skin sample treated with said compound compared to the amount of this aldehyde or oxysterol measured in the sample that has not been treated with said compound.

[0096] 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 a normal atmosphere for the following 6 hours, and then again in an ozone-enriched atmosphere for 18 hours.

[0097] In another embodiment, the ozone exposure steps b, d and 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.

[0098] By "atmosphere" in which the skin sample is incubated is meant the gas or mixture of gases in which the sample is placed. Preferably the sample is incubated in an atmosphere similar to ambient air.

[0099] 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.

[0100] Method for evaluating the effectiveness of a compound for preventing and / or treating the harmful effects of ozone on the skin

[0101] The invention also relates to a method for evaluating the effectiveness of a compound for preventing and / or treating the deleterious effects of ozone on the skin of a subject, said method comprising the following steps: a. measuring the amount of at least one product resulting from the oxidation of skin lipids selected from tridecenal, pentadecanal, octadecanal, 5a-6a-epoxycholesterol, 5[3-6[3-epoxycholesterol and 7-ketocholesterol, in a skin sample of said subject before treatment with said compound. b. treating the skin of said subject with said compound, c. after step b., measuring the quantity of the same product(s) resulting from the oxidation of skin lipids, in a second sample of skin 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 deleterious effects of ozone on the skin.

[0102] The method of the invention is preferably carried out in vitro or ex vivo.

[0103] Preferably, the at least one product resulting from the oxidation of skin lipids is selected from 5a-6a-epoxycholesterol, tridecenal, pentadecanal and octadecanal.

[0104] Steps a. and c. of this method may further comprise measuring the quantity of nonanal, and / or decanal and / or dodecanal in the skin samples of said subject. The comparison of these measurements making it possible to evaluate the effectiveness of said candidate compound in preventing and / or treating the deleterious effects of ozone on the skin.

[0105] The skin samples of the subject used in the evaluation method according to the invention are preferably taken non-invasively, from the skin of the subject, preferably from the face of the subject, in particular from the cheek of the subject.

[0106] Preferably the skin sample is a biopsy, a microbiopsy in particular carried out using a needle, or a sample of stratum corneum.

[0107] Thus, according to one embodiment, this method according to the invention comprises several steps of taking a skin sample from the subject. This step is preferably carried out in a non-invasive manner, and in particular does not require local anesthesia. According to a preferred embodiment, the step of taking the sample is carried out by rubbing the surface of the skin or by using an adhesive surface such as a D-squame® disc, and / or by wiping the skin with absorbent paper (such as filter paper).

[0108] In one embodiment of the invention, to measure the amount of one or more aldehyde(s), the sample collection steps are carried out by rubbing the surface of the skin with absorbent paper, preferably soaked with a monoalcohol such as ethanol.

[0109] In one embodiment of the invention, to measure the amount of one or more oxysterol(s), the sample collection steps are performed using an adhesive surface such as a D-squame® disc.

[0110] Preferably, the only difference between the skin sample of step a. and the skin sample of step c. is said treatment with the compound.

[0111] The skin sample of step c. is taken after the skin of said subject has been treated with said compound. In one embodiment, the skin sample of step c. is taken after the skin of said subject 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.

[0112] Preferably, the method according to the invention comprises a step of treating the samples in order to prepare the measurement of the quantity of oxidized lipid measured. For example, if the skin sample was obtained by wiping the skin with absorbent paper (such as filter paper, for example, soaked in ethanol), the sample can be evaporated and then taken up in an acetonitrile solution and then a derivatization carried out, for example a derivatization with DNPH.

[0113] Comparison step d. here refers to determining whether the quantity of aldehyde or oxysterol measured in step a. is essentially identical to the amount of that same aldehyde or oxysterol measured in step c. Preferably, the amount of that aldehyde or oxysterol is considered different if the observed difference is statistically significant. If the difference is not statistically significant, the two amounts of that aldehyde or oxysterol are essentially identical.

[0114] Based on this comparison, the effectiveness of a compound in preventing and / or treating the deleterious effects of ozone on the skin can be evaluated. Typically, the lower the amount of this aldehyde or oxysterol in the skin sample treated with said compound compared to the amount of this aldehyde or oxysterol measured in the sample that was not treated with said compound, the more the compound is considered effective in preventing and / or treating the deleterious effects of ozone on the skin. Alternatively, the compound is not considered effective in preventing and / or treating the deleterious effects of ozone on the skin when the amount of this aldehyde or oxysterol measured is essentially the same or significantly higher in the skin sample treated with said compound compared to the amount of this aldehyde or oxysterol measured in the sample that was not treated with said compound. Example: Example 1: Study and analysis of aldehydes Materials and methods: 1-In vitro ozone exposure system 1.1- Device external to the incubator:

[0115] To mimic 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 with humidity (approximately 95%).

[0116] The external pump is used to take ambient air and supply the MEGATEC ozone analyzer / generator. The air will then pass in front of the device's UV lamp (set to a wavelength of 185nm) which will transform some of the oxygen molecules (O2) in the air into ozone molecules (O3). The air is then enriched with ozone.

[0117] This ozone-enriched air can then be transferred into the incubator containing the culture plates with the reconstructed T-Skin™ skin model.

[0118] The MEGATEC analyzer / generator allows you to adjust and control: - 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 ozone concentration generated by adjusting the power of the UV lamp of the MEGATEC analyzer / generator.

[0119] Furthermore, the MEGATEC analyzer / generator has a second pump and an internal analysis system which make it possible to measure the actual ozone concentration: either at the outlet of the device (generated concentration, upstream of exposure in the incubator), or in the incubator (residual concentration, downstream of exposure). 1.2- Internal device of the incubator:

[0120] Within the incubator, a fluidic system developed in the laboratory allows the same quantity of ozone to be distributed to each reconstructed skin insert. This system is semi-open to allow both: a homogeneous distribution of ozone on the top of the plates, and good regulation of the culture conditions of the reconstructed skins.

[0121] Furthermore, the cover of each culture plate (generally made of polystyrene, a material sensitive to ozone) is replaced by a specific cover for exposure: this is a PTFE or PEEK cover pierced above each reconstructed skin insert, and thus allowing direct exposure of the surface of the reconstructed skins to ozone-enriched air. In addition, this cover protects the culture medium to prevent its oxidation by ozone, in order to only expose the surface of the skins.

[0122] 2-Exposure to ozone of in vitro reconstructed skins for aldehyde analyses

[0123] 2.1- Reception and culture of the reconstructed skin model T-Skin—

[0124] The reconstructed skin model used is the T-Skin™ reconstructed skin model produced and supplied by the company EPISKIN SA (FR-69007, Lyon).

[0125] This is a reconstructed skin model composed of: - A reconstructed dermis made up of a collagen 1 lattice, containing human-derived fibroblasts extracted from breast plastic surgeries from healthy adult donors (surgical waste obtained with the consent of patients during cosmetic breast reduction surgery). - A reconstructed epidermis made from human-origin keratinocytes extracted from breast plastic surgeries from healthy adult donors (surgical waste obtained with the consent of patients during cosmetic breast reduction surgery).

[0126] This model is grown in a 4.5cm2 culture insert which allows it to be suspended above the culture medium and grown at the air-liquid interface.

[0127] In this insert the collagen lattice, or dermal equivalent, covers a surface which can vary from 2 to 2.5 cm2 in the center of the insert. The epidermis generally covers the entire 4.5 cm2 of the insert.

[0128] The T-Skin™ model is received at the laboratory after 12 days of dermis culture (D12). 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.

[0129] Upon receipt, the inserts are quickly transferred into new 6-well culture plates called “deep well” allowing 8.5 ml of liquid culture medium to be placed under each insert (culture at the air-liquid interface). These plates can be Corning brand plates reference 355467. The culture medium used is that supplied by the company EPISKIN for the T-Skin™ kits: medium called “Culture medium for T-Skin”.

[0130] The reconstructed skins are then placed in a culture incubator regulated at 37°C, 5% CO2 and with saturated humidity (approximately 95% humidity).

[0131] The culture medium is then replaced in the same way on D13 and D15 (dermis).

[0132] Finally, on D18, the inserts are placed in standard 6-well culture plates in SBS format (COSTAR brand plate type reference 3506) with 2.5 ml of culture medium for T-Skin™ under each insert.

[0133] Subsequently, these plates are replaced with the culture medium also on D19 before exposure.

[0134] 2.2- Preparation of the squalene solution for topical application on the model of reconstructed skins

[0135] Squalene is a lipid compound specific to sebum present in relatively large quantities in sebum (approximately 12-20% according to M. Picardo et al., Sebaceous gland lipids, Dermato-endocrinology 2009)

[0136] Squalene is an unsaturated lipid that has 6 double bonds. ......... -, .. 1 - 4.

[0137] Formula (IV)

[0138] The commercial reference S3626 provided by SIGMA-ALDRICH is used.

[0139] This squalene is dissolved in absolute ethanol at a concentration of 0.378 mg / ml.

[0140] When spreading on T-Skin™, 30pL of this solution is applied per insert, and this solution is distributed only on the area of ​​the model where the collagen lattice is located. This surface area is estimated at 2.27cm2 on average. We therefore obtain approximately 5pg of sebum / cm2.

[0141] 2.3- Protocol for topical application of squalene on T-Skin—:

[0142] The squalene solution is applied on D19 before exposure to ozone according to the following protocol: - A precise volume of 30pL of solution is placed in the center of the model, - We take a glass rod with a round end that we pre-wet with a small volume of squalene solution, - We come into contact with the drop of solution placed in the center of the skin model, and we draw the drop in a circular movement which gradually moves away from the center towards the edge of the skin. The contact between the glass rod and the skin model must be as weak as possible so as not to cause stress on the model.

[0143] 2.4- Ozone exposure protocol of the T-Skin1^ model:

[0144] For this type of exposure the ozone generator is set to low values: - Ozone generated at the device outlet: approximately 0.18 ppm for a concentration measured inside the incubator less than 0.1 ppm - The output flow rate of ozone-enriched air above each reconstructed skin is approximately 30 ml / min (note that the ozone outlets are more than 10 cm from the reconstructed skins inside the incubator to avoid direct stress from the output flow).

[0145] In this protocol we perform a single very short exposure of 20 or 60 min at the T-Skin™ J19 stage.

[0146] To model the presence of a very simplex sebum, certain samples undergo an application of the squalene solution at a rate of 5 pg / cm2 (30 pL of a 0.378 mg / ml squalene solution spread over approximately 2.27 cm2 corresponding to the surface of the complete skin model) prepared according to the protocol described above.

[0147] Once the skins are ready for display, the standard polystyrene covers are replaced with perforated PEEK or PTFE covers.

[0148] A 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.

[0149] Once the exposure is finished, the reconstructed skins are taken from D19 by separating the epidermis and dermis in order to analyze the different aldehydes on the epidermis.

[0150] 3-Exposure of healthy volunteers to ozone in a mini-zone in vivo

[0151] The clinical study included 10 volunteers (8 women and 3 men), with an average age of 36.2 years, phototype III-IV and a quantity of sebum measured on the forehead greater than 140 pg / cm2 (casual level of 146 to 299 pg / cm2).

[0152] The mini-zone exposures were carried out 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.). Exposures to ozone and medical air were carried out randomly left-right on each volunteer. 3 conditions were

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[0173] carried out: - 6 exposures at 0.3 ppm (gas flow rate: 0.2-0.3 1 / min), three times a week for 2 weeks. Each exposure lasted 2 hours. - 6 exposures to medical air (gas flow: 0.2-0.3 1 / min), according to the same protocol. - 6 exposures to ambient air (bell without gas flow), according to the same protocol. Sebum samples on filter paper were collected before and 1 h after each exposure on D1 (1 exposure), D5 (3 exposures) and D12 (6 exposures). Sebum was absorbed onto a 0.45 µm Millipore filter moistened with ethanol and wiped onto the skin 10 times with pressure. 4-Analysis of aldehydes on in vitro and in vivo samples 4.1- Extraction of in vitro epidermis samples from T-skin reconstructed skin Add 400pl of acetonitrile (ACN) in the Eppendorf which contains the epidermis Shake 5 cycles in the vibro-grinder (30Hz, 2min) Centrifuge for 5 min at 10,000 rpm Collect 200pl of supernatant in an amber glass bottle Add 20 μl of 2,4-dinitrophenylhydrazine (DNPH) solution (at 500 μg / ml ACN with IM HCl) Wait Ih The aldehydes derived from DNPH will be ready for analysis 4.2- Extraction of sebum samples in vivo after exposure to ozone Samples are taken immediately after exposure to ozone. Sampling method: by wiping with filter paper (Durapore HVLP04700) soaked in 150pl Ethanol. Place in a scintillation vial and store at -20°C until the day of analysis. Extract the filter papers by adding 4ml of MeOH. Evaporate to dryness then resume in 200pl ACN Carry out the derivatization with DNPH (as in 4.1) then analyze by LC-UV and MS 4.3- Analysis and quantification of aldehydes by liquid chromatography coupled with a UV detector. LC-UV LC Conditions: -ACENTIS RP Amide Express column (2.1x150mm, 2.7pm Supelco 53914-u) -Eluent (0.5ml / min) [Table 1] Table 1: Time (min) H2O / ACN (50 / 50) Initial ACN 60 40 0.2 60 40 10 0 100 14 0 100 19 60 40

[0174] - 3pl injection, 360nm UV detection

[0175] 4.4- Analysis and quantification of aldehydes by liquid chromatography coupled with high resolution mass spectrometry (fLC-HRMS)

[0176] LC conditions similar to paragraph 4.3

[0177] HRMS Orbitrap detector model Exploris 120, allowing identification in the form of DNPH derivatives. Results Aldehydes in vitro

[0178] Table 2 shows that the quantity of the 5 aldehydes presented (nonanal C9:0, pentadecanal C15:0, octadecanal C18:0, decanal C10:0 and dodecanal C12:0) increases following exposure of the reconstructed skins to ozone for 20 or 60 minutes at 0.2 ppm, in the presence of squalene, compared to the control not exposed to ozone (0 min).

[0179] [Table 2] Table 2: - Ozone + Squalene + Ozone + Squalene + Ozone + Squalene Ozone exposure time 0 min 20 min 60 min Nonanal C9:0 Median 3334433 2075700 8632513 DMA 229002 187521 892111 Decanal C10:0 Median 246962 266033 936877 DMA 90450 19494 37774 Dodecanal C12:0 Median 159394 290253 696688 DMA 14793 52725 105947 Pentadecanal C15:0 Median 2251134 2760404 6739628 DMA 474091 67636 269975 Octadecanal C18:0 Median 11310100 15271789 40903397 DMA 900111 479028 4997848

[0180] Table 2: Aldehydes quantified in arbitrary units in vitro on the T-skin model exposed to ozone (20 or 60 minutes at 0.2 ppm), in the presence or absence of squalene in ap topical application at 5000 ng / cm2. -: without; +: with; DMA Mean Absolute Deviation Aldehydes in vivo

[0181] Tables 3-5 show that the amount of the aldehydes presented (nonanal C9:0, tridecenal, pentadecanal C15:0, octadecanal C18:0, decanal C10:0 and dodecanal C12:0) increases in samples taken by filter paper on the skin of volunteers exposed to ozone compared to those not exposed or exposed to medical air. The results are similar on D1, D5 and D12. The inter-individual variability shows significant absolute mean deviations. However, a statistical analysis using a Wilcoxon test in paired conditions shows a significance between the Medical Air and Ozone conditions.

[0182] [Table 3] Table 3: Day 1 Medical Air Unexposed Ozone Medical Air vs Ozone Aldehyde Median DMA Median DMA Median DMA p-value Nonanal C9:0 1221.4 283.0 1144.0 208.1 1359.5 485.2 0.0153 Decanal C10:0 1155.2 215.5 1081.6 323.1 3228.1 2523.5 0.000036 Dodecanal C12:0 2111.7 785.9 2417.5 840.1 3530.0 2787.4 0.000105 Tridecenal 1.0 0.0 1.0 0.0 1553.3 1472.6 0.000254 Pentadecanal C15 0 2644.3 768.5 2581.0 753.5 5040.6 3514.3 0.0000095 Octadecanal C18:0 3043.0 1284.4 3113.8 708.8 5280.8 3261.5 0.0000057

[0183] [Table 4] Table 4: Day 5 Medical Air Unexposed Ozone Medical Air vs. Ozone Aldehyde Median DMA Median p-value Median DMA Nonanal C9:0 1271.9 209.7 1109.7 199.2 1507.8 280.9 0.0266 Decanal C10:0 1103.0 289.7 969.2 256.8 1724.2 908.7 0.0136 Dodecanal C12:0 2254.6 1016.3 2368.7 747.6 3829.9 1624.4 0.0023 Tridecenal C13:1 1.0 0.0 1.0 0.0 810.8 723.5 0.01018 Pentadecanal C15:0 2503.0 1176.0 2456.9 687.3 6060.9 3219.3 0.003654 Octadecanal C18:0 2801.0 1567.1 2845.4 872.0 6526.0 3173.5 0.002325

[0184] [Table 5] Table 5: Day 12 Medical Air Unexposed Ozone Medical Air vs Ozone Aldehyde Median DMA Median p-value Median DMA Nonanal C9:0 1208.7 173.9 1187.7 356.7 1431.4 406.3 0.0023 Dodecanal C12:0 1989.6 563.4 2459.8 957.0 3437.6 1772.4 0.00032 Tridecenal C13:1 1.0 0.0 1.0 0.0 614.2 909.1 0.00166 Pentadecanal C15:0 2322.3 632.3 2794.8 999.0 4386.7 2878.5 0.000168 Octadecanal C18:0 2805.6 751.2 3203.7 1092.2 4759.5 2847.8 0.000105

[0185] Tables 3-5: Aldehydes quantified in arbitrary units at Day 1, Day 5 or Day 12 in vivo on healthy volunteers exposed to ozone. DMA = Mean Absolute Deviation. Statistical analysis was performed using a Wilcoxon test in paired conditions between the Medical Air and Ozone conditions. Example 2: Study and analysis of oxysterols Material and method:

[0186] 1-In vitro ozone exposure system and skin model

[0187] The external device and the internal device of the incubator are the same as those described in example 1 part 1.1 and 1.2.

[0188] The reconstructed skin model used is the T-Skin™ reconstructed skin model produced and supplied by the company EPISKIN SA (FR-69007, Lyon) prepared as described in point 2.1 of the previous example.

[0189] From D18 the medium of the plates is replaced with the culture medium every day.

[0190] 2-Repeated exposures of the T-Skin model to 0.1 ppm ozone:

[0191] 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 10 cm from the reconstructed skins inside the incubator to avoid direct stress from the output flow).

[0192] The exposures are carried out three times successively: 18 hours between D18 and D19, 18 hours between D19 and D20, and 18 hours between D20 and D21. Between each exposure the model is in the incubator without ozone for a little less than 6 hours.

[0193] Once the skins are ready for display, the standard polystyrene lids are replaced with perforated PEEK or PTFE lids.

[0194] A 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.

[0195] At D22, the reconstructed skin samples were taken, the epidermis was separated from the dermis, cut in two and frozen at -80°C for analysis.

[0196] 3- Repeated exposures of the T-Skin model to 0.4 ppm ozone:

[0197] 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 10 cm from the reconstructed skins inside the incubator to avoid direct stress from the output flow).

[0198] The exposures are carried out twice successively: 18 hours between D18 and D19, and 18 hours between D20 and D21. Between D19 and D20 and between D21 and D22 the reconstructed skins are placed in an incubator without ozone.

[0199] Once the skins are ready for exposure, the plates and culture media are replaced again (to avoid the risk of having raw material solution which passed into the culture medium under the insert during application), and the standard polystyrene lids are replaced by perforated PEEK or PTFE lids.

[0200] A 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.

[0201] At D22, the reconstructed skin samples were taken, the epidermis was separated from the dermis, cut in two and frozen at -80°C for analysis.

[0202] 4-Exposure of healthy volunteers to ozone in mini-zone / n vivo

[0203] The clinical study included 10 volunteers (8 women and 3 men), with an average age of 36.2 years, phototype III-IV and a quantity of sebum measured on the forehead greater than 140 pg / cm2 (casual level of 146 to 299 pg / cm2).

[0204] The mini-zone exposures were carried out 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.). Exposures to ozone and medical air were carried out randomly left-right on each volunteer. 3 conditions were carried out: - 6 exposures at 0.3 ppm (gas flow rate: 0.2-0.3 1 / min), three times a week for 2 weeks. Each exposure lasted 2 hours. - 6 exposures to medical air (gas flow: 0.2-0.3 1 / min), according to the same protocol. - 6 exposures to ambient air (bell without gas flow), according to the same protocol.

[0205] D-scales were collected before and 1 hour after each exposure on D1 (1 exposure), D5 (3 exposures) and D12 (6 exposures).

[0206] 5-Analysis of oxysterols on samples of in vitro reconstructed skin or in vivo D-squames 5.1- Extraction for the analysis of oxysterols

[0207] To extract the oxysterols, methanol is added to each tissue or D-squame in a small vial (1 mL). It is stirred for 30 minutes at 37 °C, then taken up in a hemolysis tube and evaporated using a vacuum concentrator. The evaporated extract is taken up with 200 pL of a 2:1 methanokisopropanol mixture. The samples are put in an ultrasonic bath for 5 min and filtered at 0.2 pm.

[0208] 5.2- Chromatographic method for the analysis of oxysterols: UPLC Vanquish (Thermofischer) [Table 6] Table 6: Column Oven °C Eluents Flow rate mL / min %B T0 % B at T x Time Acquity BEH C8 2.1 mm X 100 mm x 1.7 pm Waters 50 A: Water / MeOH 30 / 70 B: Iso-propanol+ 0.1% formic acid 0.2 0% 0-3 min 3-18 min rise from 0 to 35% 18 to 25 min rise to 100% 25-30 min 0% 30 min

[0210] % B T0: percentage of solvent B at T0 = 0-3 min

[0211] %B at Tx: percentage of solvent B at the different times indicated in the table

[0212] 5.3- Parameters of the Orbitrap Tribrid IPX Thermofisher Mass Spectrometer

[0213] The different analytes are detected using the Orbitrap detector, with a resolution of 60,000. RESULTS Oxidized forms of cholesterol in vitro

[0214] Table 7 shows that the amount of the three oxidized forms of cholesterol presented (5a-6a-epoxycholesterol; 5[3-6[3-epoxycholesterol; and 7-ketocholesterol) increases in reconstructed skin exposed to 0.1 or 0.4 ppm of ozone.

[0215] [Table 7] Table 7: Oxidized forms of cholesterol 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 7-ketocholesterol 1 0.091 1.706 0.075 1.368 0.294 5a-6a-epoxycholesterol 1 0.069 1.175 0.07 4 0.34 5[3-6[3-epoxycholesterol 1 0.0003 4.174 0.008 4.123 0.344 Oxidized forms of cholesterol in vivo

[0216] Table 8 shows that the amount of 5a-6a-epoxycholesterol increases in D-scales taken from volunteers exposed to ozone compared to those exposed to medical air on D12 in 7 out of 10 volunteers.

[0217] [Table 8] Table 8: 5a-6a-epoxycholesterol quantified in arbitrary units in vivo at Day 12 after 6 exposures to ozone. 5a-6a-epoxycholesterol Medical air Ozone Volunteer 1 0.797 3.679 Volunteer 2 1.946 1.357 Volunteer 3 0.582 1.010 Volunteer 4 0.953 1.625 Volunteer 5 1.851 1.427 Volunteer 6 0.558 0.542 Volunteer 7 2.629 3.254 Volunteer 8 0.502 0.686 Volunteer 9 4.890 15.886 Volunteer 10 0.631 1.007

Claims

Claims

1. A method for diagnosing ozone exposure of the skin in a subject, said method comprising the following step: a. measuring the amount of at least one product resulting from the oxidation of skin lipids chosen from tridecenal, pentadecanal, octadecanal, 5a-6a-epoxycholesterol, 5[3-6[3-epoxycholesterol and 7-ketocholesterol, in a skin sample of said subject.

2. The method of claim 1, wherein step a. further comprises measuring the amount of nonanal, decanal and / or dodecanal in the skin sample of said subject.

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 at least one product resulting from the oxidation of skin lipids selected from tridecenal, pentadecanal, octadecanal, 5a-6a-epoxycholesterol, 5[3-6[3-epoxycholesterol and 7-ketocholesterol in a skin sample from a subject, said skin sample being treated with said compound; b. comparing said amount measured in step a) with the corresponding amount in a skin sample not treated with said compound; c.identify said candidate compound as a compound for preventing and / or treating the deleterious effects of ozone on the skin when the amount of aldehyde or oxysterol measured in the skin sample that has been treated with said compound is less than the amount of aldehyde or oxysterol measured in the skin sample that has not been treated with said compound.

4. The method of claim 3 wherein step a. further comprises measuring the amount of nonanal, decanal and / or dodecanal in the skin sample of said subject.

5. A method according to claim 3 or 4, wherein said samples

6. skin samples are chosen from: a human skin explant, a skin biopsy, a microbiopsy and a sample of stratum corneum. 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. 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. perform ozone exposure of the skin sample; c. measure the quantity of at least one product resulting from oxidation skin lipids selected from tridecenal, pentadecanal, octadecanal, 5a-6a-epoxycholesterol, 5[3-6[3-epoxycholesterol and / or 7-ketocholesterol in the skin sample treated with said compound; d. comparing said quantity measured in step c) to the corresponding quantity 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 aldehyde or oxysterol measured in the skin sample that has been treated with said compound is less than the amount of aldehyde or oxysterol 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.