Method for diagnosing a skin exposed to ozone

Abstract

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 harmful effects linked with the oxidation of certain compounds of the skin and with the modulation of biomarker expression in response to ozone exposure.

Description

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

[0002] The present invention relates to a method for diagnosing skin exposure to ozone, as well as a method for identifying an active compound and a method for evaluating the efficacy of a compound, in order to prevent and / or treat the harmful effects of ozone on skin linked with the oxidation of certain constituents of the skin and with the modulation of biomarker expression in response to ozone exposure.

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

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

[0005] The applicant surprisingly discovered several biomarkers significantly modulated in skins exposed to ozone. These biomarkers are substances obtained from the oxidation of skin lipids, they comprise aldehydes and oxidized forms of cholesterol also known as oxysterols.

[0006] The list of biomarkers is as follows: tridecenal, pentadecanal, octadecanal, 5a-6a- epoxycholesterol, 5p-6p-epoxycholesterol and 7-ketocholesterol.

[0007] The methods according to the invention are therefore based on measuring significantly modulated biomarkers, in reconstructed tissues exposed to ozone and / or on skin samples exposed to ozone.

[0008] Numerous publications describe the ozone-induced degradation products of skin lipids. The reaction of ozone on unsaturated bonds induces aldehyde formation. The substances which can be formed, theoretically, from squalene have already been described. However, in view of the reactivity of ozone, the complexity of skin lipids and also the relative stability of the substances formed, it is not obvious to predict which of the aldehyde family might be the correct markers of ozone-induced stress.

[0009] As ozone-induced stress may have an impact over several hours, it is important to analyze markers accumulating on skin. Due to the poor retention of volatile molecules on skin, the search for biomarkers has concentrated on aldehydes and oxysterols.

[0010] T o the inventors' knowledge, among the many aldehydes found on skin, surprisingly, tridecenal, pentadecanal, octadecanal have never been disclosed in the literature as being significantly correlated with ozone-induced stress.

[0011] Furthermore, nonanal, decanal and / or dodecanal are also significantly correlated with ozone-induced stress.

[0012] Similarly, among the oxidized forms of cholesterol, 5a-6a-epoxycholesterol, 5p-6p- epoxycholesterol and 7-ketocholesterol have never been disclosed in the literature as being significantly correlated with ozone-induced stress.

[0013] As such, 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 the following step: a. measuring the quantity of at least one substance obtained from the oxidation of skin lipids chosen from among tridecenal, pentadecanal, octadecanal, 5a-6a- epoxycholesterol, 5p-6p-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: b. based on the quantity measured in step a., determining whether the skin of said subject has been exposed to ozone.

[0015] The invention also relates to a method for identifying a compound making it possible to prevent and / or treat the harmful effects of ozone on skin, said method comprising the following steps: a. measuring the quantity of at least one substance obtained from the oxidation of skin lipids chosen from among tridecenal, pentadecanal, octadecanal, 5a-6a- epoxycholesterol, 5p-6p-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. identifying said candidate compound as a compound making it possible to prevent and / or treat the harmful effects of ozone on skin when the quantity of aldehyde or oxysterol measured in the skin sample which was treated with said compound is lower than the quantity of aldehyde or oxysterol measured in the skin sample which was not treated with said compound.

[0016] The invention also relates to a method for identifying a compound making it possible to prevent and / or treat the harmful effects of ozone on skin, said method comprising the following steps: a. performing a treatment of a skin sample chosen from among keratinocytes, a reconstructed epidermis and a reconstructed skin, by contacting the skin sample with said compound; b. performing an exposure of the skin sample to ozone; c. measuring the quantity of at least one substance obtained from the oxidation of skin lipids chosen from among tridecenal, pentadecanal, octadecanal, 5a-6a- epoxycholesterol, 5p-6p-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. identifying said candidate compound as a compound making it possible to prevent and / or treat the harmful effects of ozone on skin when the quantity of aldehyde or oxysterol measured in the skin sample which was treated with said compound is lower than the quantity of aldehyde or oxysterol measured in the skin sample which was not treated with said compound; it being understood that step b may be carried out after step a and vice versa, and that steps a and b may be repeated at least 2 times.

[0017] The invention also relates to a method for evaluating the efficacy of a compound making it possible to prevent and / or treat the harmful effects of ozone on a subject's skin, said method comprising the following steps: a. measuring the quantity of at least one substance obtained from the oxidation of skin lipids chosen from among tridecenal, pentadecanal, octadecanal, 5a-6a- epoxycholesterol, 5p-6p-epoxycholesterol and / or 7-ketocholesterol, in a skin sample from said subject before the treatment with said compound. b. treating said subject's skin with said compound, c. after step b., measuring the quantity of the same substance(s) obtained from the oxidation of skin lipids in a second skin sample from said subject; d. comparing said quantities measured in step a. and in step c.; e. evaluating the efficacy of said candidate compound in terms of preventing and / or treating the harmful effects of ozone on skin.

[0018] The invention will become more apparent on reading the following description, given merely as a non-limiting example.

[0019] General definitions

[0020] "Subject" is understood to mean a human, preferably aged from 1 month to 90 years. Advantageously, the subject is likely to have been exposed to ozone, in particular to chronic or acute exposure.

[0021] "Exposure to ozone" here means chronic or acute exposure to ozone.

[0022] "Chronic exposure" here means regular, in particular daily, exposure during a given period, preferably a period of more than one year, in particular a period of twelve months optionally stopping on one or more occasions for short durations, typically durations of less than one month, preferably less than one week.

[0023] "Acute exposure" here means brief exposure, in particular from 12 hours to 5 days, to at least an ozone level greater than 0.05 ppm, preferably greater than 0.07 ppm, more preferably greater than 0.09 ppm, even more preferably greater than 0.1 ppm.

[0024] The stratum corneum is the outermost layer of the epidermis, and comprises the skin surface. It is essentially made up of dead cells.

[0025] "Stratum corneum sample" here means a sample taken from this layer. For example, such a sample may be taken by rubbing the skin surface or using an adhesive surface such as a D-squame® disc applied on skin.

[0026] "Substance obtained from the oxidation of skin lipids" here means oxidized lipids that may be used as biomarkers according to the invention, i.e.: nonanal, decanal, dodecanal, tridecenal, pentadecanal, octadecanal, 5a-6a-epoxycholesterol, 5p-6p-epoxycholesterol and 7-ketocholesterol. "Aldehyde" here means nonanal, decanal, dodecanal, tridecenal, pentadecanal, octadecanal.

[0027] "Oxysterol" here means 5a-6a-epoxycholesterol, 5p-6p-epoxycholesterol and 7- ketocholesterol.

[0028] Tridecenal is an aldehyde of formula C13H24O with an unsaturation. Preferably, the tridecenal is chosen from among 2 tridecenal, 4 tridecenal, 5 tridecenal, and mixtures thereof.

[0029] Pentadecanal is an aldehyde of formula CH3(CH2)i3CHO.

[0030] Octadecanal is an aldehyde also known as stearyl aldehyde of formula CH3(CH2)i6CHO.

[0031] Nonanal also known as nonanaldehyde, pelargonaldehyde or aldehyde C-9, is an aldehyde of formula CH3(CH2)7CHO.

[0032] Decanal is an aldehyde of formula CH3(CH2)8CHO.

[0033] Dodecanal also known as lauraldehyde or dodecyl aldehyde is an aldehyde of formula CH3(CH2)IOCHO.

[0034] 5a-6a-epoxycholesterol, and 5p-6p-epoxycholesterol are stereoisomers.

[0035] 5a-6a-epoxycholesterol has the following formula (I):

[0036] Formula (I)

[0037] 5p-6p-epoxycholesterol has the following formula (II): Formula (II)

[0038] 7-ketocholesterol has the following formula (III):

[0039] Formula (III)

[0040] "Corresponding quantity" means the quantity of the same aldehyde or the same oxysterol as that for which the quantity was measured in a preceding step.

[0041] "Harmful effects of ozone" mean irritations or discomfort reactions on skin (also including the skin of the scalp) which may result in particular in redness, itching, sensations of heat, stinging and tautness.

[0042] The measurement of the quantity of oxidized lipids may be carried out with any technique known to a person skilled in the art and in particular with liquid chromatography coupled with a UV detector (LC-LIV) or with liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS). For the aldehyde measurement, the measurements are made preferably after derivatization with 2,4-dinitrophenylhydrazine (DNPH).

[0043] Preferably, this measurement is made from a skin sample and not in the air in the vicinity of the skin.

[0044] Method for diagnosing skin exposure to ozone

[0045] The present invention relates to a method for diagnosing skin exposure to ozone, in a subject, said method comprising the following step: a) measuring the quantity of at least one substance obtained from the oxidation of skin lipids chosen from among tridecenal, pentadecanal, octadecanal, 5a-6a- epoxycholesterol, 5p-6p-epoxycholesterol and 7-ketocholesterol, in a skin sample from said subject.

[0046] Preferably, the at least one substance obtained from the oxidation of skin lipids is chosen from among 5a-6a-epoxycholesterol, tridecenal, pentadecanal and octadecanal.

[0047] Preferably, this method of the invention is a method for diagnosing chronic or acute exposure to ozone.

[0048] The method of the invention is preferably implemented in vitro or ex vivo.

[0049] Step a. of this method may further comprise measuring the quantity of nonanal, decanal and / or dodecanal in the skin sample from said subject.

[0050] The skin sample of the subject used in the diagnostic method according to the invention, is a sample taken, preferably non-invasively, on the subject's skin, preferably on the subject's face, in particular on the subject's cheek. Preferably, the skin sample is a biopsy, a microbiopsy in particular carried out using a needle, or a stratum corneum sample.

[0051] Thus according to an embodiment, this method according to the invention comprises a step of taking the skin sample from the subject. This step is preferably performed non- invasively, and in particular does not require local anesthetic. According to a preferred embodiment, the step of taking the sample is performed by rubbing the skin surface and / or using an adhesive surface such as a D-squame® disc and / or by wiping the skin with absorbent paper (such as filter paper).

[0052] In an embodiment of the invention, to measure the quantity of one or more aldehyde(s), the step of taking the sample is performed by rubbing the skin surface with absorbent paper, preferably soaked in a monoalcohol such as ethanol.

[0053] In an embodiment of the invention, to measure the quantity of one or more oxysterol(s), the step of taking the sample is performed using an adhesive surface such as a D-squame® disc.

[0054] Optionally the diagnostic method according to the invention further comprises the following step: b. based on the quantity measured in step a., determining whether the skin of said subject has been exposed to ozone.

[0055] In a particular embodiment, step b. is carried out after comparing the quantity of this aldehyde or oxysterol measured in step a. with a reference value. In an embodiment, the reference value is determined by the mean quantity of this aldehyde or oxysterol measured in a defined population not subjected to exposure to ozone, in particular acute or chronic exposure to ozone.

[0056] In a particular embodiment, the reference value is determined by the mean quantity of this aldehyde or oxysterol measured in humans aged from 1 month to 90 years. According to the invention, a reference value may be determined with a plurality of samples, preferably more than 20, 30, 50, 100, 200 or 500 samples.

[0057] The term "comparison" here refers to determining whether the quantity of this aldehyde is essentially identical to a reference value or differs therefrom. Preferably, the quantity of this 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 quantity of this aldehyde or oxysterol and the reference value are essentially identical.

[0058] On the basis of this comparison, it is possible to determine whether the skin has been exposed to ozone. Typically, the skin is considered to have been exposed to ozone when the quantity of 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 quantity of this aldehyde or oxysterol is essentially identical or significantly lower than the reference value.

[0059] Preferably, the method according to the invention comprises a step of treating the sample so as 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 for example soaked in ethanol), the sample may be evaporated then resuspended in an acetonitrile solution in order to carry out derivatization for example derivatization with DNPH.

[0060] Optionally the diagnostic method according to the invention further comprises the following step: c. if said subject's skin is determined as having been exposed to ozone in step b., treating said subject's skin with a cosmetic composition making it possible to prevent and / or treat the harmful effects of ozone. "Cosmetic composition making it possible to prevent and / or treat the harmful effects of ozone" is understood to mean any cosmetic composition known to a person skilled in the art making it possible to reduce and / or slow down the appearance of the harmful effects of ozone such as irritations or discomfort reactions on skin, redness, itching, sensations of heat, stinging or tautness caused by ozone. In an embodiment of the invention, the cosmetic composition comprises a compound making it possible to prevent and / or treat the harmful effects of ozone on skin identified with the method of the invention.

[0061] Methods for identifying a compound making it possible to prevent and / or treat the harmful effects of ozone on skin

[0062] The invention also relates to a method for identifying a compound making it possible to prevent and / or treat the harmful effects of ozone on skin, said method comprising the following steps: a. measuring the quantity of at least one substance obtained from the oxidation of skin lipids chosen from among tridecenal, pentadecanal, octadecanal, 5a-6a- epoxycholesterol, 5p-6p-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. identifying said candidate compound as a compound making it possible to prevent and / or treat the harmful effects of ozone on skin when the quantity of aldehyde or oxysterol measured in the skin sample which was treated with said compound is lower than the quantity of aldehyde or oxysterol measured in the skin sample which was not treated with said compound.

[0063] Preferably, the at least one substance obtained from the oxidation of skin lipids is chosen from among 5a-6a-epoxycholesterol, tridecenal, pentadecanal and octadecanal.

[0064] The identification method according to the invention may also comprise measuring the quantity of nonanal and / or decanal and / or dodecanal in the skin sample from said subject in step a, this measurement may then be compared to the corresponding quantity in a skin sample not treated with said compound in step b, in order to identify said candidate compound as a compound making it possible to treat and / or prevent the harmful effects of ozone on skin. According to a preferred embodiment, the skin samples from a subject used in the method for identifying a compound making it possible to prevent and / or treat the harmful effects of ozone on skin hereinabove are chosen from among a human skin explant, a skin biopsy, a microbiopsy in particular carried out using a needle, skin and a stratum corneum sample.

[0065] The method according to the invention comprises a step of taking the skin sample from the subject. This step is preferably performed non-invasively, and in particular does not require local anesthetic. According to a preferred embodiment, the step of taking the sample is performed by rubbing the skin surface and / or using an adhesive surface such as a D-squame® disc, and / or by wiping the skin with absorbent paper (such as filter paper).

[0066] In an embodiment of the invention, to measure the quantity of one or more aldehyde(s), the step of taking the sample is performed by rubbing the skin surface with absorbent paper, preferably soaked in a monoalcohol such as ethanol.

[0067] In an embodiment of the invention, to measure the quantity of one or more oxysterol(s), the step of taking the sample is performed using an adhesive surface such as a D-squame® disc.

[0068] Preferably, the method according to the invention comprises a step of treating the samples so as 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 for example soaked in ethanol), the sample may be evaporated then resuspended in an acetonitrile solution and derivatization then carried out for example derivatization with 2,4-dinitrophenylhydrazine (DNPH).

[0069] 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 may be carried out before or after treating the skin sample with said compound.

[0070] The invention also relates to a method for identifying a compound making it possible to prevent and / or treat the harmful effects of ozone on skin, said method comprising the following steps: a. performing a treatment of a skin sample chosen from among keratinocytes, a reconstructed epidermis and a reconstructed skin, by contacting the skin sample with said compound; b. performing an exposure of the skin sample to ozone; c. measuring the quantity of at least one substance obtained from the oxidation of skin lipids chosen from among tridecenal, pentadecanal, octadecanal, 5a-6a- epoxycholesterol, 5p-6p-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. identifying said candidate compound as a compound making it possible to prevent and / or treat the harmful effects of ozone on skin when the quantity of aldehyde or oxysterol measured in the skin sample which was treated with said compound is lower than the quantity of aldehyde or oxysterol measured in the skin sample which was not treated with said compound; it being understood that step b may be carried out after step a and vice versa, and that steps a and b may be repeated at least 2 times.

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

[0072] Preferably, the method according to the invention comprises a step of treating the samples so as 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 for example soaked in ethanol), the sample may be evaporated then resuspended in an acetonitrile solution and derivatization then carried out for example derivatization with 2,4-dinitrophenylhydrazine (DNPH).

[0073] In a preferred embodiment, the method for identifying a compound making it possible to prevent and / or treat the harmful effects of ozone on skin comprises the following steps: a. performing a first treatment of a skin sample chosen from among keratinocytes, a reconstructed epidermis and a reconstructed skin, by contacting the skin sample with said compound; b. performing a first exposure of said skin sample to ozone; c. performing a second treatment of the skin sample by contacting the skin sample with said compound; d. performing a second exposure of said skin sample to ozone; e. performing a third treatment of the skin sample by contacting the skin sample with said compound; f. performing a third exposure of said skin sample to ozone; g. measuring the quantity of at least one substance obtained from the oxidation of skin lipids chosen from among 5a-6a-epoxycholesterol, 5p-6p-epoxycholesterol and 7-ketocholesterol, in the skin sample treated with said compound; h. comparing said quantity measured in step g) to the corresponding quantity in a skin sample exposed to ozone and not treated with said compound; i. identifying said candidate compound as a compound making it possible to prevent and / or treat the harmful effects of ozone on skin when the quantity of oxysterol measured in the skin sample which was treated with said compound is lower than the quantity of oxysterol measured in the skin sample which was not treated with said compound.

[0074] Preferably, the at least one substance obtained from the oxidation of skin lipids is 5a-6a-epoxycholesterol,

[0075] In another preferred embodiment, the method for identifying a compound making it possible to prevent and / or treat the harmful effects of ozone on skin comprises the following steps: a. performing a treatment of a skin sample chosen from among keratinocytes, a reconstructed epidermis and a reconstructed skin, by contacting the skin sample with said compound; b. performing an exposure of the skin sample to ozone; c. measuring the quantity of at least one substance obtained from the oxidation of skin lipids chosen from among tridecenal, pentadecanal and octadecanal in the skin sample treated with said compound; d. comparing said quantity measured in step c) to the corresponding quantity in the skin sample exposed to ozone and not treated with said compound; e. identifying said candidate compound as a compound making it possible to prevent and / or treat the harmful effects of ozone on skin when the quantity of aldehyde measured in the skin sample which was treated with said compound is lower than the quantity of aldehyde measured in the skin sample which was not treated with said compound; it being understood that step b. may be carried out after step a and vice versa.

[0076] Preferably, the method according to the invention comprises a step of treating the samples so as 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 for example soaked in ethanol), the sample may be evaporated then resuspended in an acetonitrile solution and derivatization then carried out for example derivatization with 2,4-dinitrophenylhydrazine (DNPH).

[0077] The methods for identifying a compound of the invention are preferably implemented in vitro or ex vivo.

[0078] Measuring steps a, c or g of these methods for identifying a compound according to the invention may further comprise measuring the quantity of nonanal, decanal and / or dodecanal in the skin sample. This measurement may then be compared to the corresponding quantity in a skin sample not treated with said compound, in order to identify said candidate compound as a compound making it possible to prevent and / or treat the harmful effects of ozone on skin.

[0079] 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.

[0080] In a particular embodiment, said reconstructed epidermis or reconstructed skin may be covered with squalene before the ozone exposure step(s), for example steps b, d or f, in particular in the case where aldehydes are measured in steps a, c or g.

[0081] Typically, the treatment of the sample with said compound comprises contacting the skin sample with said compound for at least 5 minutes, at least 10 minutes or at least 20 minutes.

[0082] 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.

[0083] Steps b, d or h of comparing the identification methods here refer to determining whether the quantity of aldehyde or oxysterol measured in measuring steps a, c or g is essentially identical to the quantity of the same aldehyde or oxysterol measured in a skin sample not treated with said compound. Preferably, the quantity of this aldehyde or oxysterol is considered to be different if the observed difference is statistically significant. If the difference is not statistically significant, the two quantities of this aldehyde or oxysterol are essentially identical. On the basis of this comparison, it is possible to determine whether the compound makes it possible to prevent and / or treat the harmful effects of ozone on skin. Typically, the compound is considered to make it possible to prevent and / or treat the harmful effects of ozone on skin, when the quantity of this aldehyde or oxysterol is significantly lower in the skin sample treated with said compound with respect to the quantity of this aldehyde or oxysterol measured in the sample which was not treated with said compound, alternatively the compound is not considered to make it possible to prevent and / or treat the harmful effects of ozone on skin when the quantity of this aldehyde or oxysterol is essentially identical or significantly greater in the skin sample treated with said compound with respect to the quantity of this aldehyde or oxysterol measured in the sample which was not treated with said compound.

[0084] In an embodiment, the steps of exposure to ozone b, d or f last at least 5 hours, more particularly at least 10 hours and preferably at least 18 hours. These steps may also be interspersed with periods of incubation in a normal, ambient atmosphere. The samples are incubated in an ozone-enriched atmosphere for 18h followed by a normal atmosphere for the next 6 hours and once again in an ozone-enriched atmosphere for 18h.

[0085] In another embodiment, the steps of exposure to ozone b, d and f last from 10 to 120 minutes, preferably from 20 to 60 minutes. These steps may also be interspersed with periods of incubation in a normal, ambient atmosphere.

[0086] "Atmosphere" wherein the skin sample is incubated means the gas or gas mixture wherein the sample is placed. Preferably, the sample is incubated in an atmosphere similar to ambient air.

[0087] "Ozone-enriched atmosphere" means an atmosphere similar to ambient air comprising at least 0.05 ppm (parts per million) of ozone, preferably at least 0.07 ppm of ozone, at least 0.1 ppm of ozone or at least 0.3 ppm of ozone.

[0088] Method for evaluating the efficacy of a compound making it possible to prevent and / or treat the harmful effects of ozone on skin

[0089] The invention also relates to a method for evaluating the efficacy of a compound making it possible to prevent and / or treat the harmful effects of ozone on a subject's skin, said method comprising the following steps: a. measuring the quantity of at least one substance obtained from the oxidation of skin lipids chosen from among tridecenal, pentadecanal, octadecanal, 5a-6a- epoxycholesterol, 5p-6p-epoxycholesterol and 7-ketocholesterol, in a skin sample from said subject before the treatment with said compound. b. treating said subject's skin with said compound, c. after step b., measuring the quantity of the same substance(s) obtained from the oxidation of skin lipids, in a second skin sample from said subject; d. comparing said quantities measured in step a. and in step c.; e. evaluating the efficacy of said candidate compound in terms of preventing and / or treating the harmful effects of ozone on skin.

[0090] The method of the invention is preferably implemented in vitro or ex vivo.

[0091] Preferably, the at least one substance obtained from the oxidation of skin lipids is chosen from among 5a-6a-epoxycholesterol, tridecenal, pentadecanal and octadecanal.

[0092] 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 from said subject. Comparing these measures makes it possible to evaluate the efficacy of said candidate compound in terms of preventing and / or treating the harmful effects of ozone on skin.

[0093] The skin samples of the subject used in the evaluation method according to the invention are preferably taken non-invasively, on the subject's skin, preferably on the subject's face, in particular on the subject's cheek.

[0094] Preferably, the skin sample is a biopsy, a microbiopsy in particular carried out using a needle, or a stratum corneum sample.

[0095] Thus according to an embodiment, this method according to the invention comprises several steps for taking skin samples from the subject. This step is preferably performed non-invasively, and in particular does not require local anesthetic. According to a preferred embodiment, the step of taking the sample is performed by rubbing the skin surface or using an adhesive surface such as a D-squame® disc, and / or by wiping the skin with absorbent paper (such as filter paper).

[0096] In an embodiment of the invention, to measure the quantity of one or more aldehyde(s), the steps of taking the sample are performed by rubbing the skin surface with absorbent paper, preferably soaked in a monoalcohol such as ethanol. In an embodiment of the invention, to measure the quantity of one or more oxysterol(s), the steps of taking the sample are performed using an adhesive surface such as a D-squame® disc.

[0097] Preferably, the only difference between the skin sample of step a. and the skin sample of step c. is said treatment with the compound.

[0098] The skin sample of step c. is sampled after said subject's skin has been treated with said compound. In an embodiment, the skin sample of step c. is sampled after said subject's skin has been treated with said compound for at least 14 days, preferably at least 28 days. Typically, this sample is taken at least 5 minutes, at least 10 minutes or at least 20 minutes after the last treatment of the sample with said compound.

[0099] Preferably, the method according to the invention comprises a step of treating the samples so as 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 may be evaporated then resuspended in an acetonitrile solution and derivatization then carried out for example derivatization with DNPH.

[0100] Comparison step d. here refers to determining whether the quantity of aldehyde or oxysterol measured in step a. is essentially identical to the quantity of the same aldehyde or oxysterol measured in step c. Preferably, the quantity of this aldehyde or oxysterol is considered to be different if the observed difference is statistically significant. If the difference is not statistically significant, the two quantities of this aldehyde or oxysterol are essentially identical.

[0101] On the basis of this comparison, it is possible to evaluate the efficacy of a compound in preventing and / or treating the harmful effects of ozone on skin. Typically, the lower the quantity of this aldehyde or oxysterol in the skin sample treated with said compound with respect to the quantity of this aldehyde or oxysterol measured in the sample which was not treated with said compound, the more effective the compound is considered to be for preventing and / or treating the harmful effects of ozone on skin. Alternatively, the compound is not considered to be effective for preventing and / or treating the harmful effects of ozone on skin when the quantity of this aldehyde or oxysterol measured is essentially identical or significantly greater in the skin sample treated with said compound with respect to the quantity of this aldehyde or oxysterol measured in the sample which was not treated with said compound. Example:

[0102] Materials and methods:

[0103] 1- In vitro ozone exposure system

[0104] 1.1- Device outside the incubator:

[0105] To imitate ozone pollution, the laboratory uses a MEGATEC 49i ozone analyzer / generator coupled with an external pump, and also connected to a culture incubator regulated at 37°C, 5% CO2 and saturated in humidity (approximately 95%).

[0106] The external pump makes it possible to extract ambient air and supply the MEGATEC ozone analyzer / generator. The air will then pass in front of the UV lamp of the apparatus (set to the wavelength of 185nm) which will convert some of the oxygen molecules (O2) from the air into ozone molecules (O3). The air is then ozone-enriched.

[0107] This ozone-enriched air can then be transferred into the incubator containing culture plates with the T-Skin™ reconstructed skin model.

[0108] The MEGATEC analyzer / generator makes it possible to set and monitor:

[0109] The ozone-enriched air flow rate (supplying the incubator) by means of a relief valve placed upstream from the circuit in the MEGATEC analyzer / generator.

[0110] The ozone concentration generated by setting the power of the UV lamp of the MEGATEC analyzer / generator.

[0111] Moreover, the MEGATEC analyzer / generator has a second pump and an internal analysis system which makes it possible to measure the actual ozone concentration: either at the apparatus outlet (generated concentration, upstream from the exposure in the incubator), or in the incubator (residual concentration, downstream from the exposure).

[0112] 1.2- Device inside the incubator:

[0113] Inside the incubator, a fluidic system developed in the laboratory makes it possible to dispense the same quantity of ozone on each reconstructed skin insert. This system is semiopen to enable at the same time: a homogeneous distribution of ozone on the top of the plates, and satisfactory regulation of the culture conditions of the reconstructed skins. Moreover, the lid of each culture plate (generally made of polystyrene, ozone-sensitive material) is replaced by a specific lid for exposure: it consists of a PTFE or PEEK lid perforated above each reconstructed skin insert, and thus allowing direct exposure of the surface of the surface of the reconstructed skins to oxygen-enriched air. Furthermore, this lid makes it possible to protect the culture medium to prevent its oxidation with ozone, so as only carry out exposure of the skin surface.

[0114] 2- Ozone exposure of reconstructed skins in vitro for aldehyde analyses

[0115] 2.1- Receipt and culture of the T-Skin™ reconstructed skin model

[0116] The reconstructed skin model used is the T-Skin™ reconstructed skin model produced and supplied by EPISKIN SA (FR-69007, Lyon).

[0117] It consists of a reconstructed skin model composed of:

[0118] - A reconstructed dermis consisting of a collagen 1 lattice, containing fibroblasts of human origin extracted from mammoplasties from healthy adult donors (surgical waste obtained with the patients' consent following cosmetic breast reduction procedures).

[0119] - A reconstructed epidermis produced from keratinocytes of human origin extracted from mammoplasties from healthy adult donors (surgical waste obtained with the patients' consent following cosmetic breast reduction procedures).

[0120] This model is cultured in a 4.5cm2culture insert which makes it possible to suspend it above the culture medium and culture it at the air-liquid interface.

[0121] In this insert, the collagen lattice, or dermal equivalent, covers a surface area which can vary from 2 to 2.5 cm2at the center of the insert. The epidermis generally covers the whole 4.5cm2of the insert.

[0122] The T-Skin™ model is received at the laboratory after 12 days of culture of the dermis (D12). The epidermis is at 7 days of culture and it is relatively immature (with little differentiation). During transport, these inserts are placed on a semi-solid nutrient culture medium suitable for transport.

[0123] On receipt, the inserts are transferred without delay into new so-called "deep well" 6-well culture plates making it possible to place 8.5ml of liquid culture medium under each insert (culture at the air-liquid interface). These plates may be Corning brand reference 355467 plates. The culture medium used is that supplied by EPISKIN for T-Skin™ kits: medium known as "Culture medium for T-Skin".

[0124] The reconstructed skins are then placed in a culture incubator regulated at 37°C, 5% CO2 and with a saturated humidity (approximately 95% humidity). The culture medium is then replaced in the same way on D13 and D15 (dermis).

[0125] Finally on D18, the inserts are placed in standard 6-well culture plates in SBS format (COSTAR brand reference 3506 plate type) with 2.5ml of culture medium for T-Skin™ under each insert.

[0126] These plates are subsequently replaced with culture medium also on D19 before exposure.

[0127] 2.2- Preparation of squalene solution for topical application on the reconstructed skin model

[0128] Squalene is a specific lipid compound of sebum present in a relatively substantial quantity in sebum (approximately 12-20% as per M. Picardo et al., Sebaceous gland lipids, Dermatoendocrinology 2009)

[0129] Squalene is an unsaturated lipid which has 6 double bonds.

[0130] Formula (IV)

[0131] Commercial reference S3626 supplied by SIGMA-ALDRICH is used.

[0132] This squalene is placed in solution in absolute ethanol at a concentration of 0.378 mg / ml. When spreading on T-Skin™, 30pL of this solution is applied per insert, and this solution is only dispensed on the zone of the model where the collagen lattice is located. This surface area is estimated at 2.27cm2on average. Consequently, approximately 5pg of sebum / cm2is obtained.

[0133] 2.3- Topical application protocol of squalene on T-SkinTM:

[0134] The squalene solution is applied on D19 before ozone exposure according to the following protocol:

[0135] - An exact volume of 30pL of solution is deposited at the center of the model,

[0136] - A glass rod with a rounded end, pre-moistened with a small volume of squalene solution, is taken up,

[0137] Contact is made with the drop of solution placed at the center of the skin model, and the drop is carried in a circular motion gradually moving away from the center toward the edge of the skin. The contact between the glass rod and the skin must be as light as possible so as not to generate stress on the model.

[0138] 2.4- Ozone exposure protocol of T-Skin™ model:

[0139] For this type of exposure, the ozone generator is set to low values: Ozone generated at apparatus outlet: approximately 0.18ppm for a concentration measured inside the incubator lower than 0.1 ppm

[0140] The ozone-enriched air outlet flow rate above each reconstructed skin is approximately 30 ml / min (note that the ozone outlets are more than 10cm from the reconstructed skins inside the incubator to prevent direct outlet flow stress).

[0141] In this protocol, we carry out a single very short exposure of 20 or 60 min at the T-Skin™ D19 stage.

[0142] To model the presence of a very simplex sebum, some samples undergo an application of the squalene solution at a rate of 5pg / cm2(30pL of a 0.378mg / ml squalene solution spread over approximately 2.27cm2corresponding to the surface area of the complete skin model) prepared according to the protocol described above.

[0143] Once the skins are ready to be exposed, the standard polystyrene lids are replaced by perforated PEEK or PTFE lids.

[0144] Some of the samples are treated in ozone, and others, the controls, are prepared in exactly the same way, but incubated in a conventional incubator without ozone.

[0145] Once exposure is complete, the reconstructed skins are sampled from D19 by separating the epidermis and dermis in order to analyze the different aldehydes on the epidermises.

[0146] 3- Mini-zone exposure of healthy volunteers to ozone in vivo

[0147] The clinical study included 10 volunteers (8 female and 3 male), having a mean age of 36.2 years, of phototype lll-IV and a quantity of sebum measured on the forehead greater than 140 pg / cm2(casual level of 146 to 299 pg / cm2).

[0148] The exposures in mini-zones were carried out using bell jars connected to a generator of ozone, medical air or to ambient air, placed on the skin of the volunteers' backs (Controlled Pollution Exposure System (CPES)). The ozone generation system and the bell jars used are described in the publication Curpen et al., 2020 (A novel method for evaluating the effect of pollution on the human skin under controlled conditions, Skin Res Technol.). The exposures to ozone and to medical air were carried out randomly on the left-right on each volunteer. 3 conditions were carried out:

[0149] - 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.

[0150] - 6 exposures to medical air (gas flow rate: 0.2-0.3 l / min), according to the same protocol.

[0151] - 6 exposures to ambient air (bell jar without gas flow), according to the same protocol. Sebum samples on filter paper were taken before and 1 h after each exposure on D1 (1 exposure), D5 (3 exposures) and D12 (6 exposures). The sebum is absorbed on a Millipore 0.45 pm filter moistened with ethanol and wiped on the skin 10 times with application of pressure.

[0152] 4- Analysis of aldehydes on in vitro and in vivo samples

[0153] 4.1- Extraction of in vitro epidermis samples from T-skin reconstructed skins

[0154] Add 400pl acetonitrile (ACN) in the Eppendorf containing the epidermis

[0155] Stir for 5 cycles with a vibrating mill (30Hz, 2min)

[0156] Centrifuge for 5min at 10000 rpm

[0157] Retrieve 200pl of supernatant in an amber glass vial

[0158] Add 20pl of a 2,4-dinitrophenylhydrazine (DNPH) solution (500pg / ml ACN with 1M HCI) Wait 1 h

[0159] The aldehydes derivatized with DNPH will be ready to be analyzed

[0160] 4.2- Extraction of in vivo sebum samples after ozone exposure

[0161] The samples are taken immediately after the ozone exposures

[0162] 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 the analysis

[0163] Extraction of filter papers by adding 4ml of MeOH.

[0164] Evaporate to dryness then resuspend in 200pl ACN

[0165] Carry out derivatization with DNPH (as in 4.1) then analyze by LC-LIV and MS

[0166] 4.3- Analysis and quantification of aldehydes by liquid chromatography coupled with a UV detector. LC-UV

[0167] LC conditions:

[0168] -ACENTIS RP Amide Express column (2.1x150mm, 2.7pm Supelco 53914-u) -Eluent (0.5ml / min)

[0169] [Table 1] Table 1 :

[0170] -Injection 3pl, UV detection 360nm

[0171] 4.4- Analysis and quantification of aldehydes by liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS)

[0172] Similar LC conditions to section 4.3

[0173] Orbitrap Exploris 120 HRMS detector, enabling identification in the form of DNPH derivatives.

[0174] Results

[0175] Aldehydes in vitro

[0176] Table 2 shows that the quantity of the 5 aldehydes shown (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, with respect to the control not exposed to ozone (0 min).

[0177] [Table 2] Table 2:

[0178] 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 not of squalene in topical application at 5000 ng / cm2. without; +: with; AMD Absolute Mean Deviation

[0179] Aldehydes in vivo

[0180] Tables 3-5 show that the quantity of aldehydes shown (nonanal C9:0, tridecenal, pentadecanal C15:0, octadecanal C18:0, decanal C10:0 and dodecanal C12:0) increases in samples taken on filter paper on the skin of volunteers exposed to ozone with respect to those not exposed or exposed to medical air. The results are similar on D1 , D5 and D12. The between-subject variability shows substantial absolute mean deviations. However, a statistical analysis using a paired Wilcoxon text shows a significance between the Medical air and ozone conditions.

[0181] [Table 3] Table 3:

[0182] [Table 4] Table 4:

[0183] [Table 5] Table 5:

[0184] Tables 3-5: Aldehydes quantified in arbitrary units on Day 1 , Day 5 or Day 12 in vivo on healthy volunteers exposed to ozone. AMD = Absolute Mean Deviation. The statistical analysis was carried using a paired Wilcoxon text between the Medical air and Ozone conditions.

[0185] Comparative tests (not in the invention)

[0186] Table 3’: Table 4’:

[0187] Table 5’:

[0188] Table 3' to 5': Aldehydes quantified in arbitrary units on Day 1 , Day 5 or Day 12 in vivo on healthy volunteers exposed to ozone. AMD = Absolute Mean Deviation. The statistical analysis was carried using a paired Wilcoxon text between the Medical air and Ozone conditions.

[0189] Tables 3'-5' show that there was no significant difference between the quantity of aldehydes hexanal C6:0, octanal, C8:0 present in samples taken on filter paper on the skin of volunteers exposed to ozone with respect to those not exposed or exposed to medical air.

[0190] Example 2: Study and analysis of oxysterols

[0191] Materials and methods:

[0192] 1- In vitro ozone exposure system and skin model

[0193] The device outside and the device inside the incubator are the same as those described in example 1 section 1.1 and 1.2.

[0194] The reconstructed skin model used is the T-Skin™ reconstructed skin model produced and supplied by EPISKIN SA (FR-69007, Lyon) prepared as described in section 2.1 of the preceding example.

[0195] From D18, the plate medium is replaced with culture medium every day.

[0196] 2- Repeated exposures of T-Skin™ model to 0.1 ppm ozone:

[0197] For this type of exposure, the ozone generator is set to low values: Ozone generated at apparatus outlet: approximately 0.130 ppm for a concentration measured inside the incubator lower than 0.1 ppm

[0198] The ozone-enriched air outlet flow rate above each reconstructed skin is approximately 20 ml / min (note that the ozone outlets are more than 10cm from the reconstructed skins inside the incubator to prevent direct outlet flow stress).

[0199] The exposures are carried out three times successively: 18h between D18 and D19, 18h between D19 and D20, and 18h between D20 and D21. Between each exposure, the model is in the incubator without ozone for slightly less than 6h.

[0200] Once the skins are ready to be exposed, the standard polystyrene lids are replaced by perforated PEEK or PTFE lids.

[0201] Some of the samples are treated in ozone, and others, the controls, are prepared in exactly the same way, but incubated in a conventional incubator without ozone.

[0202] On D22, the reconstructed skin samples were removed, the epidermis was separated from the dermis, cut in two and frozen at -80°C for the analyses.

[0203] 3- Repeated exposures of T-Skin™ model to 0.4 ppm ozone:

[0204] For this type of exposure, the ozone generator is set to moderate ozone values, but slightly higher than realistic ozone peaks:

[0205] Ozone generated at apparatus outlet: approximately 0.4ppm for a concentration measured inside the incubator lower than 0.3ppm

[0206] The ozone-enriched air outlet flow rate above each reconstructed skin is approximately 20 ml / min (note that the ozone outlets are more than 10cm from the reconstructed skins inside the incubator to prevent direct outlet flow stress).

[0207] The exposures are carried out twice successively: 18h between D18 and D19, and 18h between D20 and D21. Between D19 and D20 and between D21 and D22, the reconstructed skins are placed in an incubator without ozone.

[0208] Once the skins are ready to be exposed, the culture plates and media are once again replaced (to prevent the risk of having raw material solution passing into the culture medium under the insert during application), and the standard polystyrene lids are replaced by perforated PEEK or PTFE lids.

[0209] Some of the samples are treated in ozone, and others, the controls, are prepared in exactly the same way, but incubated in a conventional incubator without ozone.

[0210] On D22, the reconstructed skin samples were removed, the epidermis was separated from the dermis, cut in two and frozen at -80°C for the analyses.

[0211] 4- Mini-zone exposure of healthy volunteers to ozone in vivo The clinical study included 10 volunteers (8 female and 3 male), having a mean age of 36.2 years, of phototype lll-IV and a quantity of sebum measured on the forehead greater than 140 pg / cm2(casual level of 146 to 299 pg / cm2).

[0212] The exposures in mini-zones were carried out using bell jars connected to a generator of ozone, medical air or to ambient air, placed on the skin of the volunteers' backs (Controlled Pollution Exposure System (CPES)). The ozone generation system and the bell jars used are described in the publication Curpen et al., 2020 - A novel method for evaluating the effect of pollution on the human skin under controlled conditions, Skin Res Technol.). The exposures to ozone and to medical air were carried out randomly on the left-right on each volunteer. 3 conditions were carried out:

[0213] - 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.

[0214] - 6 exposures to medical air (gas flow rate: 0.2-0.3 l / min), according to the same protocol.

[0215] - 6 exposures to ambient air (bell jar without gas flow), according to the same protocol.

[0216] D-squame samples were taken before and 1h after each exposure on D1 (1 exposure), D5 (3 exposures) and D12 (6 exposures).

[0217] 5- Analysis of oxysterols on reconstructed skin samples in vitro or D-squame samples in vivo

[0218] 5.1- Extraction of oxysterols for analysis

[0219] To extract the oxysterols, methanol is added to each tissue or D-squame sample in a small vial (1 mL). It is stirred for 30 minutes at 37 °C, to be subsequently resuspended in a hemolysis tube and evaporated using a vacuum concentrator. The evaporated extract is resuspended with 200 pL of a 2:1 methanokisopropanol mixture. The samples are placed in an ultrasonic bath for 5 min and filtered at 0.2 pm.

[0220] 5.2- Chromatographic method for oxysterol analysis: UPLC Vanquish

[0221] [Table 6] Table 6:

[0222] % B TO: percentage of solvent B at TO = 0-3 min

[0223] %B at Tx: percentage of solvent B at the different times indicated in the table

[0224] 5.3- Thermofisher Orbitrap Tribrid IPX mass spectrometer settings The various analytes are detected using the Orbitrap detector, with a resolution of

[0225] 60,000.

[0226] RESULTS Oxidized forms of cholesterol in vitro

[0227] Table 7 shows that the quantity of the three oxidized forms of cholesterol shown (5a-6a- epoxycholesterol; 5p-6p-epoxycholesterol; and 7-ketocholesterol) increases in reconstructed skins exposed to 0.1 or 0.4 ppm of ozone.

[0228] [Table 7] Table 7: Oxidized forms of cholesterol quantified in arbitrary units in vitro on reconstructed skins after exposure to ozone at 0.1 or 0.4 ppm. This test was repeated a second time in order to compare the oxysterols according to the invention, i.e. 7-ketocholesterol, 5a-6a-epoxycholesterol and 5p-6p-epoxycholesterol, with 27- hydroxy cholesterol (not included in the invention). The values obtained are shown in Table 7’.

[0229] Table 7’:

[0230] The number of samples is n=7 for 7-ketocholesterol, 5a-6a-epoxycholesterol, and 5p-6p- epoxycholesterol. The number of samples is n=2 for 27-hydroxycholesterol.

[0231] Table 7’ shows that the amount of the three oxidized forms of cholesterol according to the invention (5a-6a-epoxycholesterol; 5p-6p-epoxycholesterol; and 7-ketocholesterol) increases in reconstructed skin exposed to 0.4 ppm ozone in contrast to 27-hydroxy cholesterol (not in the invention).

[0232] Oxidized forms of cholesterol in vivo

[0233] Table 8 shows that the quantity of 5a-6a-epoxycholesterol increases in D-squame samples taken from volunteers exposed to ozone with respect to those exposed to medical air on D12 in 7 out of 10 volunteers.

[0234] [Table 8] Table 8: 5a-6a-epoxycholesterol quantified in arbitrary units in vivo on Day 12 after 6 exposures to ozone.

Claims

CLAIMS1. Method for diagnosing skin exposure to ozone, in a subject, said method comprising the following step: a. measuring the quantity of at least one substance obtained from the oxidation of skin lipids chosen from among tridecenal, pentadecanal, octadecanal, 5a-6a- epoxycholesterol, 5p-6p-epoxycholesterol and 7-ketocholesterol, in a skin sample from said subject.

2. Method according to claim 1 , wherein step a. of this method further comprises measuring the quantity of nonanal, decanal and / or dodecanal in the skin sample from said subject.

3. Method for identifying a compound making it possible to prevent and / or treat the harmful effects of ozone on skin, said method comprising the following steps: a. measuring the quantity of at least one substance obtained from the oxidation of skin lipids chosen from among tridecenal, pentadecanal, octadecanal, 5a-6a- epoxycholesterol, 5p-6p-epoxycholesterol and 7-ketocholesterol, in a skin sample from a subject, said skin 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. identifying said candidate compound as a compound making it possible to prevent and / or treat the harmful effects of ozone on skin when the quantity of aldehyde or oxysterol measured in the skin sample which was treated with said compound is lower than the quantity of aldehyde or oxysterol measured in the skin sample which was not treated with said compound.

4. Method according to claim 3, wherein step a. further comprises measuring the quantity of nonanal, decanal and / or dodecanal in the skin sample from said subject.

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

6. Method for identifying a compound making it possible to prevent and / or treat the harmful effects of ozone on skin, said method comprising the following steps:a. performing a treatment of a skin sample chosen from among keratinocytes, a reconstructed epidermis and a reconstructed skin, by contacting the skin sample with said compound; b. performing an exposure of the skin sample to ozone; c. measuring the quantity of at least one substance obtained from the oxidation of skin lipids chosen from among tridecenal, pentadecanal, octadecanal, 5a-6a- epoxycholesterol, 5p-6p-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. identifying said candidate compound as a compound making it possible to prevent and / or treat the harmful effects of ozone on skin when the quantity of aldehyde or oxysterol measured in the skin sample which was treated with said compound is lower than the quantity of aldehyde or oxysterol measured in the skin sample which was not treated with said compound; it being understood that step b may be carried out after step a and vice versa, and that steps a and b may be repeated at least 2 times.

7. Method for evaluating the efficacy of a compound making it possible to prevent and / or treat the harmful effects of ozone on a subject's skin, said method comprising the following steps: a. measuring the quantity of at least one substance obtained from the oxidation of skin lipids chosen from among tridecenal, pentadecanal, octadecanal, 5a-6a- epoxycholesterol, 5p-6p-epoxycholesterol and 7-ketocholesterol, in a skin sample from said subject before the treatment with said compound. b. treating said subject's skin with said compound, c. after step b., measuring the quantity of the same substance(s) obtained from the oxidation of skin lipids in a second skin sample from said subject; d. comparing said quantity measured in step a. and in step c.; e. evaluating the efficacy of said candidate compound in terms of preventing and / or treating the harmful effects of ozone on skin.

8. Method according to claim 7, wherein steps a. and c. of this method further comprise measuring the quantity of nonanal and / or decanal and / or dodecanal in the skin samples from said subject.

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

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