Method for diagnosing skin exposed to ozone

Abstract

The present invention relates to a method for diagnosing skin exposure to ozone, and to a method for identifying active compounds and a method for evaluating the effectiveness of compounds in order to prevent and / or treat adverse effects associated with the oxidation of specific compounds in the skin and the modulation of biomarker expression in response to ozone exposure.

Description

[Technical Field]

[0001] The present invention relates to a method for diagnosing skin exposure to ozone, and to a method for identifying active compounds and a method for evaluating the effectiveness of compounds in order to prevent and / or treat the harmful effects of ozone on the skin, which are related to the oxidation of specific components of the skin and the modulation of biomarker expression in response to ozone exposure. [Background technology]

[0002] Ozone, or trioxygen, has the chemical formula O3 and is a much less stable allotropic variant of oxygen than dioxygen (O2), tending to spontaneously decompose into dioxygen (O2). Ozone is naturally present in the Earth's atmosphere and forms the ozone layer in the stratosphere, blocking ultraviolet radiation from the sun. However, in the lower atmosphere (troposphere), ozone is one of the most harmful air pollutants to health. This extremely potent oxidizing agent can be generated by forest fires, high temperatures, and also in industrial areas or sunny urban centers. Thus, some environments are routinely exposed to ozone peaks. Individuals in their daily environments, especially in urban areas, may be subjected to multiple attacks on keratinous substances, particularly the skin, by this air pollutant. In fact, ozone has harmful effects associated with the oxidation of certain components of the skin and the modulation of ozone-induced biomarker expression. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] M. Picardo et al., Sebaceous gland lipids, Dermato-endocrinology 2009 [Non-Patent Document 2] Curpen et al., 2020(A novel method for evaluating the effect of pollution on the human skin under controlled conditions, Skin Res Technol.) [Overview of the project] [Problems that the invention aims to solve]

[0004] From the above, it is understood that it is important to discover biomarkers that enable the detection of skin exposure to ozone, allowing for targeted skin treatment. The objective of the present invention is to satisfy this need. [Means for solving the problem]

[0005] The applicant has surprisingly discovered several biomarkers that are significantly regulated in skin exposed to ozone. These biomarkers are substances derived from the oxidation of skin lipids, and they include oxidized forms of cholesterol, also known as aldehydes and oxysterols.

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

[0007] Therefore, the method according to the present invention is based on measuring significantly modified biomarkers in reconstructed tissue and / or skin samples exposed to ozone.

[0008] Numerous publications describe ozone-induced degradation products of skin lipids. The reaction of ozone with unsaturated bonds induces aldehyde formation. Theoretically, substances that can be formed from squalene have already been described. However, considering the reactivity of ozone, the complexity of skin lipids, and the relative stability of the formed substances, it is not clear which aldehyde family members can serve as precise markers of ozone-induced stress.

[0009] Because ozone-induced stress can have effects for several hours, it is important to analyze markers that accumulate in the skin. Due to the poor retention of volatile molecules in the skin, the search for biomarkers focused on aldehydes and oxysterols.

[0010] To the best of our knowledge, among the many aldehydes found in the skin, it has been surprisingly never disclosed in the literature that tridecenal, pentadecanal, and octadecanal are significantly correlated with ozone-induced stress.

[0011] Furthermore, nonanal, decanal, and / or dodecanal also show a significant correlation with ozone-induced stress.

[0012] Similarly, it has not been disclosed in the literature that among oxidized forms of cholesterol, 5α-6α-epoxycholesterol, 5β-6β-epoxycholesterol, and 7-ketocholesterol are significantly correlated with ozone-induced stress.

[0013] Therefore, according to a first aspect of the present invention, the present invention provides a method for diagnosing skin exposure to ozone in a subject, preferably in vitro or ex vivo, comprising the following steps: a. A step of measuring the amount of at least one substance obtained from the oxidation of skin lipids selected from tridecenal, pentadecanal, octadecanal, 5α-6α-epoxycholesterol, 5β-6β-epoxycholesterol, and 7-ketocholesterol in a skin sample derived from the subject. relates to a method, comprising:

[0014] Optionally, this diagnostic method according to the present invention comprises the following steps: b. A step of determining whether the skin of the subject is exposed to ozone based on the amount measured in step a. It further comprises:

[0015] The present invention also relates to a method for identifying a compound that makes it possible to prevent and / or treat the harmful effects of ozone on the skin, the method comprising the following steps: a. A step of measuring the amount of at least one substance obtained from the oxidation of skin lipids selected from tridecenal, pentadecanal, octadecanal, 5α-6α-epoxycholesterol, 5β-6β-epoxycholesterol, and 7-ketocholesterol in a skin sample derived from a subject, wherein the sample has been treated with the compound; b. A step of comparing the amount measured in step a) with the corresponding amount in a skin sample not treated with the compound; c. A step of identifying the candidate compound as a compound that makes it possible to prevent and / or treat the harmful effects of ozone on the skin, when the amount of aldehyde or oxysterol measured in the skin sample treated with the compound is less than the amount of aldehyde or oxysterol measured in the skin sample not treated with the compound; relates to a method, comprising:

[0016] The present invention also relates to a method for identifying a compound that makes it possible to prevent and / or treat the harmful effects of ozone on the skin, the method comprising the following steps: a. A step of performing treatment of a skin sample selected from keratinocytes, reconstructed epidermis, and reconstructed skin by bringing the skin sample into contact with the compound; b. A step of performing exposure of the skin sample to ozone; c. measuring the amount of at least one substance obtained from the oxidation of skin lipids selected from tridecenal, pentadecanal, octadecanal, 5α-6α-epoxycholesterol, 5β-6β-epoxycholesterol, and / or 7-ketocholesterol in a skin sample treated with the said compound; d. comparing the said amount measured in step c) with the corresponding amount in a skin sample that has been exposed to ozone and not treated with the said compound; e. identifying the candidate compound as a compound that makes it possible to prevent and / or treat the harmful effects of ozone on the skin, when the amount of aldehyde or oxysterol measured in the skin sample treated with the said compound is less than the amount of aldehyde or oxysterol measured in the skin sample not treated with the said compound comprising, It relates to a method, wherein it is understood that step b may be performed after step a or vice versa, and that steps a and b may be repeated at least twice.

[0017] The present invention also relates to a method for evaluating the effectiveness of a compound that makes it possible to prevent and / or treat the harmful effects of ozone on the skin of a subject, comprising the following steps: a. measuring the amount of at least one substance obtained from the oxidation of skin lipids selected from tridecenal, pentadecanal, octadecanal, 5α-6α-epoxycholesterol, 5β-6β-epoxycholesterol, and / or 7-ketocholesterol in a skin sample derived from the subject before treatment with the said compound; b. treating the skin of the said subject with the said compound; c. after step b., measuring the amount of the same substance obtained from the oxidation of skin lipids in a second skin sample derived from the said subject; d. comparing the said amounts measured in step a. and step c.; e. evaluating the effectiveness of the said candidate compound in relation to the prevention and / or treatment of the harmful effects of ozone on the skin This includes methods. [Modes for carrying out the invention]

[0018] The present invention will become clearer by reading the following description, which is merely presented as a non-limiting example.

[0019] General definition The term "subject" is understood to mean a human being, preferably between 1 month and 90 years of age.

[0020] Advantageously, the subjects are likely to have been exposed to ozone, particularly chronic or acute exposure.

[0021] Here, "exposure to ozone" refers to chronic or acute exposure to ozone.

[0022] Here, “chronic exposure” means routine, especially daily, exposure over a given period, preferably more than one year, particularly 12 months, with optional short periods, typically less than one month, preferably less than one week, with one or more interruptions.

[0023] Here, "acute exposure" means short-term exposure, particularly for 12 hours to 5 days, to ozone levels of at least 0.05 ppm, preferably 0.07 ppm, more preferably 0.09 ppm, and even more preferably 0.1 ppm.

[0024] The stratum corneum is the outermost layer of the epidermis and includes the skin surface. It is essentially composed of dead cells.

[0025] Here, "stratum corneum sample" refers to a sample taken from this layer. For example, such a sample can be obtained by rubbing the skin surface or by using an adhesive surface such as a D-squame® disc applied to the skin.

[0026] Here, "substances obtained from the oxidation of skin lipids" refers to oxidized lipids that can be used as biomarkers according to the present invention, namely nonanal, decanal, dodecanal, tridecenal, pentadecanal, octadecanal, 5α-6α-epoxycholesterol, 5β-6β-epoxycholesterol, and 7-ketocholesterol.

[0027] Here, "aldehyde" refers to nonanal, decanal, dodecanal, tridecenal, pentadecanal, and octadecanal.

[0028] Here, "oxysterol" refers to 5α-6α-epoxycholesterol, 5β-6β-epoxycholesterol, and 7-ketocholesterol.

[0029] Tridecenal is an unsaturated compound of C 13 H 24 It is an aldehyde of O. Preferably, the tridecenal is selected from 2-tridecenal, 4-tridecenal, 5-tridecenal, and mixtures thereof.

[0030] Pentadecanal is given by the formula CH3(CH2) 13 It is the aldehyde CHO.

[0031] Octadecanal is a compound with the formula CH3(CH2), also known as stearyl aldehyde. 16 It is the aldehyde CHO.

[0032] Nonanal, also known as nonanaldehyde, pelargonaldehyde, or aldehyde C-9, is an aldehyde with the formula CH3(CH2)7CHO.

[0033] Decanale is an aldehyde with the formula CH3(CH2)8CHO.

[0034] Dodecanal, also known as raulaldehyde or dodecylaldehyde, has the formula CH3(CH2) 10 It is the aldehyde CHO.

[0035] 5α-6α-epoxycholesterol and 5β-6β-epoxycholesterol are stereoisomers.

[0036] 5α-6α-epoxycholesterol is given by the following formula (I):

[0037] [ka]

[0038] It holds.

[0039] 5β-6β-epoxycholesterol is given by the following formula (II):

[0040] [ka]

[0041] It holds.

[0042] 7-Ketocholesterol is given by the following formula (III):

[0043] [ka]

[0044] It holds.

[0045] "Corresponding quantity" refers to the same amount of aldehyde or oxysterol as measured in the preceding process.

[0046] "The harmful effects of ozone" specifically refer to skin (including scalp) irritation or unpleasant reactions that may result in redness, itching, burning, stinging, and tightness.

[0047] The amount of oxidized lipids can be measured using any technique known to those skilled in the art, particularly liquid chromatography coupled with a UV detector (LC-UV) or liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS). Regarding aldehyde measurement, the measurement is preferably performed after derivatization with 2,4-dinitrophenylhydrazine (DNPH).

[0048] Preferably, this measurement is performed on a skin sample and not in the air surrounding the skin.

[0049] Methods for diagnosing skin exposure to ozone The present invention relates to a method for diagnosing skin exposure to ozone in a subject, comprising the following steps: a) A step of measuring the amount of at least one substance obtained from the oxidation of skin lipids selected from tridecenal, pentadecanal, octadecanal, 5α-6α-epoxycholesterol, 5β-6β-epoxycholesterol, and 7-ketocholesterol in a skin sample derived from the subject. This includes methods.

[0050] Preferably, at least one substance obtained from the oxidation of skin lipids is selected from 5α-6α-epoxycholesterol, tridecenal, pentadecanal, and octadecanal.

[0051] Preferably, the present invention is a method for diagnosing chronic or acute exposure to ozone.

[0052] The method of the present invention is preferably carried out in vitro or ex vivo.

[0053] Step a of this method may further include measuring the amounts of nonanal, decanal and / or dodecanal in the skin sample derived from the subject.

[0054] The skin sample used in the diagnostic method according to the present invention is a sample taken from the skin of the subject, preferably from the face of the subject, and more particularly from the cheek of the subject, preferably non-invasively. Preferably, the skin sample is a biopsy, particularly a microbiopsy performed using a needle, or a stratum corneum sample.

[0055] Accordingly, according to one embodiment, the method according to the present invention includes the step of taking a skin sample from a subject. This step is preferably carried out non-invasively and, in particular, does not require local anesthesia. According to a preferred embodiment, the step of taking a sample is carried out by rubbing the skin surface and / or using an adhesive surface such as a D-squame® disc and / or wiping the skin with absorbent paper (such as filter paper).

[0056] In one embodiment of the present invention, the step of taking a sample to measure the amount of one or more aldehydes is carried out by rubbing the skin surface with absorbent paper soaked in a monoalcohol such as ethanol.

[0057] In one embodiment of the present invention, the step of taking a sample to measure the amount of one or more oxysterols is carried out using an adhesive surface such as a D-squame® disc.

[0058] Optionally, the diagnostic method according to the present invention includes the following steps: b. A step of determining whether the skin in question has been exposed to ozone based on the amount measured in step a. It also includes.

[0059] In certain embodiments, step b is performed after comparing the amount of this aldehyde or oxysterol measured in step a with a reference value. In some embodiments, the reference value is determined by the average amount of this aldehyde or oxysterol measured in a defined population that has not been exposed to ozone, particularly acute or chronic exposure to ozone.

[0060] In certain embodiments, the reference value is determined by the average amount of this aldehyde or oxysterol measured in humans aged 1 month to 90 years. According to the present invention, the reference value can be determined by a plurality of samples, preferably 20, 30, 50, 100, 200, or more than 500 samples.

[0061] Here, the term "comparison" refers to determining whether the amount of this aldehyde is essentially the same as or different from the reference value. Preferably, if the observed difference is statistically significant, the amount of this aldehyde or oxysterol is considered different from the reference value. If the difference is not statistically significant, the amount of this aldehyde or oxysterol and the reference value are essentially the same.

[0062] Based on this comparison, it is possible to determine whether the skin is exposed to ozone. Typically, if the amount of this aldehyde or oxysterol is significantly higher than the baseline, the skin is considered to be exposed to ozone; if the amount of this aldehyde or oxysterol is essentially the same as the baseline or significantly lower than the baseline, the skin is considered not to be exposed to ozone.

[0063] Preferably, the method according to the present invention includes a step of preparing the sample to measure the amount of the aldehyde or oxysterol. For example, if a skin sample is obtained by wiping the skin with absorbent paper (e.g., filter paper soaked in ethanol), the sample can be evaporated and then resuspended in an acetonitrile solution to carry out derivatization, for example, with DNPH.

[0064] Optionally, the diagnostic method according to the present invention includes the following steps: c. If it is determined that the skin in question has been exposed to ozone in step b, the step of treating the skin in question with a cosmetic composition that can prevent and / or treat the harmful effects of ozone. It also includes.

[0065] "A cosmetic composition that enables prevention and / or treatment of the harmful effects of ozone" is understood to mean any cosmetic composition known to those skilled in the art that enables the occurrence of harmful effects of ozone, such as irritation or unpleasant reactions to the skin caused by ozone, such as redness, itching, burning, stinging, or tightness. In one embodiment of the present invention, the cosmetic composition comprises a compound that enables prevention and / or treatment of the harmful effects of ozone to the skin, as specified by the method of the present invention.

[0066] Method for identifying compounds that can prevent and / or treat the harmful effects of ozone on the skin. The present invention also relates to a method for identifying compounds that enable the prevention and / or treatment of the harmful effects of ozone on the skin, comprising the following steps: a. A step of measuring the amount of at least one substance obtained from the oxidation of skin lipids selected from tridecenal, pentadecanal, octadecanal, 5α-6α-epoxycholesterol, 5β-6β-epoxycholesterol, and 7-ketocholesterol in a skin sample derived from a subject, wherein the sample is treated with the compound; b. A step of comparing the amount measured in step a) with the corresponding amount in a skin sample that has not been treated with the compound; c. If the amount of aldehyde or oxysterol measured in a skin sample treated with the compound is less than the amount of aldehyde or oxysterol measured in a skin sample not treated with the compound, the candidate compound is identified as a compound that can prevent and / or treat the harmful effects of ozone on the skin. This includes methods.

[0067] Preferably, at least one substance obtained from the oxidation of skin lipids is selected from 5α-6α-epoxycholesterol, tridecenal, pentadecanal, and octadecanal.

[0068] The identification method according to the present invention may also include, in step a, measuring the amount of nonanal and / or decanal and / or dodecanal in a skin sample derived from the subject, and then, in step b, comparing this measurement to the corresponding amount in a skin sample not treated with the compound, to identify the candidate compound as a compound that can treat and / or prevent the harmful effects of ozone on the skin.

[0069] In a preferred embodiment, the skin sample derived from a subject used in the method for identifying compounds that enable the prevention and / or treatment of the harmful effects of ozone on the skin described herein is selected from human skin explants, skin biopsies, particularly microbiopsies performed using a needle, skin, and stratum corneum samples.

[0070] The method according to the present invention includes the step of taking a skin sample from a subject. This step is preferably carried out non-invasively and, in particular, does not require local anesthesia. According to a preferred embodiment, the step of taking a sample is carried out by rubbing the skin surface and / or using an adhesive surface such as a D-squame® disc and / or wiping the skin with absorbent paper (such as filter paper).

[0071] In one embodiment of the present invention, the step of taking a sample to measure the amount of one or more aldehydes is carried out by rubbing the skin surface with absorbent paper soaked in a monoalcohol such as ethanol.

[0072] In one embodiment of the present invention, the step of taking a sample to measure the amount of one or more oxysterols is carried out using an adhesive surface such as a D-squame® disc.

[0073] Preferably, the method according to the present invention includes a step of preparing the sample to measure the amount of the aldehyde or oxysterol. For example, if a skin sample is obtained by wiping the skin with absorbent paper (e.g., filter paper soaked in ethanol), the sample can be evaporated, then resuspended in an acetonitrile solution, and then derivatized with, for example, 2,4-dinitrophenylhydrazine (DNPH).

[0074] In certain embodiments, the method according to the present invention includes an additional step of exposing the sample to ozone. This step is preferably carried out for both samples under the same conditions. This step may be carried out before or after treating the skin sample with the compound.

[0075] The present invention also relates to a method for identifying compounds that enable the prevention and / or treatment of the harmful effects of ozone on the skin, comprising the following steps: a. A step of treating a skin sample selected from keratinocytes, reconstructed epidermis, and reconstructed skin by contacting the skin sample with the compound; b. A step of exposing a skin sample to ozone; c. A step of measuring the amount of at least one substance obtained from the oxidation of skin lipids selected from tridecenal, pentadecanal, octadecanal, 5α-6α-epoxycholesterol, 5β-6β-epoxycholesterol, and / or 7-ketocholesterol in a skin sample treated with the compound; d. A step of comparing the amount measured in step c) with the corresponding amount in a skin sample that has been exposed to ozone and not treated with the compound; e. If the amount of aldehyde or oxysterol measured in a skin sample treated with the compound is less than the amount of aldehyde or oxysterol measured in a skin sample not treated with the compound, the candidate compound is identified as a compound that can prevent and / or treat the harmful effects of ozone on the skin. Includes, The present invention relates to a method in which it is understood that step b may be performed after step a, or vice versa, and that steps a and b may be repeated at least twice.

[0076] Preferably, at least one substance obtained from the oxidation of skin lipids is selected from 5α-6α-epoxycholesterol, tridecenal, pentadecanal, and octadecanal.

[0077] Preferably, the method according to the present invention includes a step of preparing the sample to measure the amount of the aldehyde or oxysterol. For example, if a skin sample is obtained by wiping the skin with absorbent paper (e.g., filter paper soaked in ethanol), the sample can be evaporated, then resuspended in an acetonitrile solution, and then derivatized with, for example, 2,4-dinitrophenylhydrazine (DNPH).

[0078] In a preferred embodiment, a method for identifying compounds that can prevent and / or treat the harmful effects of ozone on the skin includes the following steps: a. A step of performing a first treatment on a skin sample selected from keratinocytes, reconstructed epidermis, and reconstructed skin by contacting the skin sample with the compound; b. A step of performing a first exposure of the skin sample to ozone; c. A step of performing a second treatment on the skin sample by bringing the skin sample into contact with the compound; d. A step of performing a second exposure of the skin sample to ozone; e. A step of performing a third treatment on the skin sample by bringing the skin sample into contact with the compound; f. A step of performing a third exposure of the skin sample to ozone; g. A step of measuring the amount of at least one substance obtained from the oxidation of skin lipids selected from 5α-6α-epoxycholesterol, 5β-6β-epoxycholesterol, and 7-ketocholesterol in a skin sample treated with the compound; h. A step of comparing the amount measured in step g) with the corresponding amount in a skin sample that has been exposed to ozone and not treated with the compound; i. If the amount of oxysterol measured in a skin sample treated with the compound is less than the amount of oxysterol measured in a skin sample not treated with the compound, the candidate compound is identified as a compound that can prevent and / or treat the harmful effects of ozone on the skin. Includes.

[0079] Preferably, at least one substance obtained from the oxidation of skin lipids is 5α-6α-epoxycholesterol.

[0080] In another preferred embodiment, a method for identifying compounds that can prevent and / or treat the harmful effects of ozone on the skin includes the following steps: a. A step of treating a skin sample selected from keratinocytes, reconstructed epidermis, and reconstructed skin by contacting the skin sample with the compound; b. A step of exposing a skin sample to ozone; c. A step of measuring the amount of at least one substance obtained from the oxidation of skin lipids selected from tridecenal, pentadecanal, and octadecanal in a skin sample treated with the compound; d. A step of comparing the amount measured in step c) with the corresponding amount in a skin sample that has been exposed to ozone and not treated with the compound; e. If the amount of aldehyde measured in a skin sample treated with the compound is less than the amount of aldehyde measured in a skin sample not treated with the compound, the candidate compound is identified as a compound that can prevent and / or treat the harmful effects of ozone on the skin. Includes, It is understood that step b may be performed after step a, or vice versa.

[0081] Preferably, the method according to the present invention includes a step of preparing the sample to measure the amount of the aldehyde or oxysterol. For example, if a skin sample is obtained by wiping the skin with absorbent paper (e.g., filter paper soaked in ethanol), the sample can be evaporated, then resuspended in an acetonitrile solution, and then derivatized with, for example, 2,4-dinitrophenylhydrazine (DNPH).

[0082] The method for identifying the compounds of the present invention is preferably performed in vitro or ex vivo.

[0083] Steps a, c, or g of these methods for identifying compounds according to the present invention may further include measuring the amounts of nonanal, decanal, and / or dodecanal in a skin sample. The measured amounts can then be compared with the corresponding amounts in a skin sample not treated with the compound to identify the candidate compound as one that can prevent and / or treat the harmful effects of ozone on the skin.

[0084] Preferably, in a method for identifying the compound of the present invention, the only difference between a skin sample treated with the compound and a skin sample not treated with the compound is the treatment with the compound.

[0085] In certain embodiments, the reconstructed epidermis or reconstructed skin may be coated with squalene before the ozone exposure step, for example, steps b, d, or f, particularly if aldehydes are measured in step a, c, or g.

[0086] Typically, the treatment of a sample with the compound involves contacting the skin sample with the compound for at least 5 minutes, at least 10 minutes, or at least 20 minutes.

[0087] Typically, the amount of aldehyde or oxysterol is measured at least 5 minutes, at least 10 minutes, or at least 20 minutes after the final treatment of the sample with the compound.

[0088] Here, step b, d, or h, which compares the identification methods, refers to determining whether the amount of aldehyde or oxysterol measured in step a, c, or g is essentially the same as the amount of the same aldehyde or oxysterol measured in a skin sample not treated with the compound. Preferably, if the observed difference is statistically significant, the amounts of this aldehyde or oxysterol are considered different. If the difference is not statistically significant, the two amounts of this aldehyde or oxysterol are considered essentially the same.

[0089] Based on this comparison, it is possible to determine whether the compound can prevent and / or treat the harmful effects of ozone on the skin. Typically, if the amount of the aldehyde or oxysterol in the skin sample treated with the compound is significantly less than the amount of the aldehyde or oxysterol measured in the sample not treated with the compound, the compound is considered to be able to prevent and / or treat the harmful effects of ozone on the skin. Alternatively, if the amount of the aldehyde or oxysterol in the skin sample treated with the compound is essentially the same as or significantly more than the amount of the aldehyde or oxysterol measured in the sample not treated with the compound, the compound is considered not to be able to prevent and / or treat the harmful effects of ozone on the skin.

[0090] In one embodiment, steps b, d, or f of exposure to ozone last for at least 5 hours, more specifically at least 10 hours, preferably at least 18 hours. These steps may also be interrupted by incubation periods in a normal ambient atmosphere. The sample is incubated in a rich ozone atmosphere for 18 hours, followed by 6 hours in a normal atmosphere, and then again in a rich ozone atmosphere for 18 hours.

[0091] In another embodiment, steps b, d, and f of exposure to ozone last for 10 to 120 minutes, preferably 20 to 60 minutes. These steps may also be interrupted by an incubation period in a normal ambient atmosphere.

[0092] The “atmosphere” in which the skin sample is incubated refers to the gas or gas mixture in which the sample is placed. Preferably, the sample is incubated in an atmosphere similar to ambient air.

[0093] "Ozone-rich atmosphere" means an atmosphere similar to ambient air containing at least 0.05 ppm (parts per million) of ozone, preferably at least 0.07 ppm, at least 0.1 ppm, or at least 0.3 ppm of ozone.

[0094] A method for evaluating the effectiveness of compounds that enable the prevention and / or treatment of the harmful effects of ozone on the skin. The present invention also provides a method for evaluating the effectiveness of a compound that enables the prevention and / or treatment of the harmful effects of ozone on the skin of a target, comprising the following steps: a. A step of measuring the amount of at least one substance obtained from the oxidation of skin lipids selected from tridecenal, pentadecanal, octadecanal, 5α-6α-epoxycholesterol, 5β-6β-epoxycholesterol, and 7-ketocholesterol in a skin sample derived from the subject before treatment with the compound; b. A step of treating the target skin with the compound; c. After step b, a step of measuring the amount of the same substance obtained from the oxidation of skin lipids in a second skin sample derived from the subject; d. A step of comparing the quantities measured in steps a. and c.; e. A step of evaluating the effectiveness of the candidate compound in preventing and / or treating the harmful effects of ozone on the skin. This includes methods.

[0095] The method of the present invention is preferably carried out in vitro or ex vivo.

[0096] Preferably, at least one substance obtained from the oxidation of skin lipids is selected from 5α-6α-epoxycholesterol, tridecenal, pentadecanal, and octadecanal.

[0097] Steps a. and c. of this method may further include measuring the amounts of nonanal and / or decanal and / or dodecanal in a skin sample derived from the subject. By comparing these measurements, it becomes possible to evaluate the effectiveness of the candidate compounds in preventing and / or treating the harmful effects of ozone on the skin.

[0098] The skin sample used in the evaluation method according to the present invention is preferably taken non-invasively from the skin of the subject, preferably from the face of the subject, and particularly from the cheek of the subject.

[0099] Preferably, the skin sample is a biopsy, particularly a microbiopsy performed using a needle, or a stratum corneum sample.

[0100] Accordingly, according to one embodiment, the method according to the present invention includes several steps of taking a skin sample from a subject. This step is preferably carried out non-invasively and, in particular, does not require local anesthesia. According to a preferred embodiment, the step of taking a sample is carried out by rubbing the skin surface, using an adhesive surface such as a D-squame® disc, and / or wiping the skin with absorbent paper (such as filter paper).

[0101] In one embodiment of the present invention, the step of taking a sample to measure the amount of one or more aldehydes is carried out by rubbing the skin surface with absorbent paper soaked in a monoalcohol such as ethanol.

[0102] In one embodiment of the present invention, the step of taking a sample to measure the amount of one or more oxysterols is carried out using an adhesive surface such as a D-squame® disc.

[0103] Preferably, the only difference between the skin sample from step a. and the skin sample from step c. is the treatment with the compound.

[0104] The skin sample in step c. is taken after the skin of the subject has been treated with the compound. In one embodiment, the skin sample in step c. is taken after the skin of the subject has been treated with the compound for at least 14 days, preferably at least 28 days. Typically, the sample is taken at least 5 minutes, at least 10 minutes, or at least 20 minutes after the last treatment of the sample with the compound.

[0105] Preferably, the method according to the present invention includes a step of preparing the sample to be used for measuring the amount of oxidized lipids. For example, if a skin sample is obtained by wiping the skin with absorbent paper (e.g., filter paper soaked in ethanol), the sample can be evaporated, then resuspended in an acetonitrile solution, and then derivatized with, for example, DNPH.

[0106] Here, comparison step d refers to determining whether the amount of aldehyde or oxysterol measured in step a is essentially identical to the amount of the same aldehyde or oxysterol measured in step c. Preferably, if the observed difference is statistically significant, the amounts of aldehyde or oxysterol are considered different. If the difference is not statistically significant, the two amounts of aldehyde or oxysterol are essentially identical.

[0107] Based on this comparison, it is possible to evaluate the effectiveness of a compound in preventing and / or treating the harmful effects of ozone on the skin. Typically, the less the amount of this aldehyde or oxysterol in a skin sample treated with the compound compared to the amount measured in a sample not treated with the compound, the more effective the compound is considered to be in preventing and / or treating the harmful effects of ozone on the skin. Conversely, if the amount of this aldehyde or oxysterol in a skin sample treated with the compound is essentially the same as or significantly higher than the amount measured in a sample not treated with the compound, the compound is considered to be less effective in preventing and / or treating the harmful effects of ozone on the skin. [Examples]

[0108] (Example 1) Aldehyde research and analysis Materials and methods: 1. In vitro ozone exposure system 1.1- Devices outside the incubator: To simulate ozone pollution, the laboratory uses a MEGATEC 49i ozone analyzer / generator connected to an external pump, as well as a culture incubator regulated to 37°C, 5% CO2, and saturated humidity (approximately 95%).

[0109] An external pump allows ambient air to be extracted and supplied to the MEGATEC ozone analyzer / generator. The air then passes in front of the device's UV lamp (set to a wavelength of 185 nm), where some of the oxygen molecules (O2) in the air are converted into ozone molecules (O3). The air is then enriched with ozone.

[0110] Next, this ozone-rich air can be transferred to an incubator containing culture plates of the T-Skin® reconstructed skin model.

[0111] The MEGATEC analyzer / generator allows you to configure and monitor the following: - A safety valve located upstream of the MEGATEC analyzer / generator circuit ensures a high ozone airflow rate (supplied to the incubator). - Ozone concentration generated by setting the output of the UV lamp in the MEGATEC analyzer / generator.

[0112] Furthermore, 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 device outlet (generated concentration, upstream of exposure in the incubator) or in the incubator (residual concentration, downstream of exposure).

[0113] 1.2- Devices inside the incubator: Inside the incubator, a fluid system developed in the laboratory allows for the dispensing of the same amount of ozone to each reconstructed skin implant. This system is semi-open, allowing for simultaneous homogeneous distribution of ozone on the top of the plate and precise control of the culture conditions for the reconstructed skin.

[0114] Furthermore, the lids of each culture plate (generally made of polystyrene, an ozone-sensitive material) are replaced with specific lids for exposure: these lids consist of PTFE or PEEK lids that are perforated over each reconstructed skin insert, thus allowing direct exposure of the surface of the reconstructed skin to oxygen-rich air. In addition, these lids protect the culture medium and prevent its oxidation by ozone, ensuring that only the skin surface is exposed.

[0115] 2. Ozone exposure of reconstituted skin in vitro for aldehyde analysis 2.1- Receipt and culture of T-Skin® reconstructed skin model The reconstructed skin model used is the T-Skin® reconstructed skin model, manufactured and supplied by EPISKIN SA (FR-69007, Lyon).

[0116] This consists of a reconstructed skin model comprising the following: - Reconstructed dermis consisting of a type I collagen lattice containing human-derived fibroblasts (surgical waste obtained with the patient's consent after cosmetic breast reduction surgery) extracted from breast augmentation surgery from a healthy adult donor. - Reconstructed epidermis generated from human-derived keratinocytes (surgical waste obtained with the patient's consent after cosmetic breast reduction surgery) extracted from breast augmentation surgery on healthy adult donors.

[0117] This model allows for suspension on a culture medium and cultivation at the gas-liquid interface, with a 4.5 cm diameter. 2 The culture is performed in the culture insert.

[0118] In this implant, the collagen lattice or dermal equivalent is 2-2.5 cm in the center of the implant. 2 It covers a surface area that can vary. The epidermis is generally 4.5 cm 2 It covers the entire insert.

[0119] T-Skin® models are received in the laboratory 12 days after dermal culture (D12). The epidermis is at 7 days of culture and is relatively immature (slightly differentiated). During transport, these inserts are placed in a suitable semi-solid nutrient culture medium.

[0120] For each insert, transfer them without delay to a so-called "deep well" 6-well culture plate that allows for the placement of 8.5 ml of liquid culture medium beneath each insert (cultivation at the gas-liquid interface). These plates may be Corning brand plates with reference number 355467. The culture medium used is the one supplied by EPISKIN for the T-Skin® kit: known as "T-Skin Culture Medium".

[0121] Next, the reconstructed skin is placed in a culture incubator controlled at 37°C, 5% CO2, and saturated humidity (approximately 95% humidity).

[0122] Next, the culture medium is similarly replaced on D13 and D15 (dermis).

[0123] Finally, on D18, the inserts are placed in standard 6-well culture plates of SBS format (COSTAR brand, reference number 3506 plate type) with 2.5 ml of culture medium for T-Skin™ under each insert.

[0124] Subsequently, these plates are also replaced with fresh culture medium before exposure on D19.

[0125] 2.2- Preparation of squalene solution for topical application to reconstructed skin models Squalene is a specific lipid compound of sebum that is present in relatively large amounts in sebum (approximately 12 to 20% according to M. Picardo et al., Sebaceous gland lipids, Dermato-endocrinology 2009).

[0126] Squalene is an unsaturated lipid with six double bonds.

[0127]

Chemical Formula

[0128] The commercial reference number S3626 supplied by SIGMA-ALDRICH is used.

[0129] This squalene is made into a solution in absolute ethanol at a concentration of 0.378 mg / ml.

[0130] When spreading onto T-Skin™, 30 μL of this solution is applied per insert, and this solution is dispensed only to the zone of the model where the collagen lattice is located. This surface area averages 2.27 cm 2 is estimated. As a result, approximately 5 μg of sebum / cm 2 is obtained.

[0131] 2.3- Protocol for topical application of squalene to T-Skin (trademark): The squalene solution is applied to D19 before ozone exposure according to the following protocol: - Place a precise volume of 30 μL of solution in the center of the model. - Take a glass rod with a rounded tip that has been pre-moistened with a small volume of squalene solution. - Place the liquid droplet, located in the center of the skin model, into contact with the glass rod and gradually move the droplet in a circular motion from the center of the skin towards the edges. The contact between the glass rod and the skin should be as light as possible to avoid stressing the model.

[0132] 2.4- Ozone exposure protocol for the T-Skin™ model: For this type of exposure, set the ozone generator to a low setting: - Ozone generated at the device outlet: Approximately 0.18 ppm when the measured concentration inside the incubator is less than 0.1 ppm. - The ozone-rich air outflow rate from the top of each reconstructed skin is approximately 30 ml / min (note that the ozone outlet is located more than 10 cm from the reconstructed skin inside the incubator to prevent direct outflow stress).

[0133] In this protocol, the inventors administer a single, very short exposure of 20 or 60 minutes on the T-Skin® D19 stage.

[0134] To model the presence of very simple sebum, several samples were treated with 5 μg / cm³ according to the protocol described above. 2 Squalene solution at a rate of approximately 2.27 cm² (corresponding to the surface area of ​​a complete skin model) 2 Apply 30 μL of 0.378 mg / ml squalene solution spread over the area.

[0135] When ready to expose skin, replace the standard polystyrene lid with a perforated PEEK or PTFE lid.

[0136] Some samples were treated in ozone, while others were prepared in exactly the same way as controls, except that they were incubated in a conventional incubator without ozone.

[0137] Once exposure is complete, reconstructed skin is sampled from D19 by separating the epidermis and dermis to analyze various aldehydes in the epidermis.

[0138] 3. Micro-regional exposure of healthy volunteers to ozone in vivo Clinical studies measured 140 μg / cm³ in the forehead for phototype III-IV patients. 2 Super (146~299μg / cm 2 The study included 10 volunteers (8 women and 3 men) with an average age of 36.2 years and sebum levels (at any given time).

[0139] Micro-area exposure was performed using ozone generators, medical air generators, or glass bells connected to ambient air (controlled pollutant exposure systems (CPES)) placed on the skin of volunteers' backs. The ozone generators and glass bells used are described in the publication Curpen et al., 2020 (A novel method for evaluating the effect of pollution on the human skin under controlled conditions, Skin Res Technol.). Exposure to ozone and medical air was performed randomly on each volunteer, either left or right. Three conditions were implemented: - Six exposures to 0.3 ppm (gas flow rate: 0.2-0.3 l / min), three times a week for two weeks. Each exposure lasted two hours. - Six exposures to medical air using the same protocol (gas flow rate: 0.2-0.3 l / min). - Six exposures to ambient air using the same protocol (glass bell without airflow).

[0140] Sebum samples were taken from filter paper before and one hour after exposures D1 (1 exposure), D5 (3 exposures), and D12 (6 exposures). The sebum was absorbed into a Millipore 0.45 μm filter that had been moistened with ethanol and wiped 10 times under pressure.

[0141] 4. Analysis of aldehydes in in vitro and in vivo samples 4.1- Extraction of in vitro epidermal samples from T-skin reconstructed skin Add 400 μl of acetonitrile (ACN) to Eppendorf containing epidermis. Stir for 5 cycles using a vibrating mill (30Hz, 2 minutes). Centrifug at 10,000 rpm for 5 minutes. Collect 200 μl of the supernatant into an amber glass vial. Add 20 μl of 2,4-dinitrophenylhydrazine (DNPH) solution (500 μg / ml ACN containing 1 M HCl). Wait for 1 hour DNPH can be used to prepare aldehydes for analysis.

[0142] 4.2- Extraction of in vivo sebum samples after ozone exposure The sample was taken immediately after ozone exposure.

[0143] Sample collection method: Wipe the sample with filter paper (Durapore HVLP04700) soaked in 150 μl of ethanol. Place the sample in a scintillation vial and store at -20°C until the analysis day. Extraction of filter paper by adding 4 ml of MeOH. Evaporate to dryness, then resuspend in 200 μl of ACN. Derivatization with DNPH is performed (as in 4.1), followed by analysis by LC-UV and MS.

[0144] 4.3- Analysis and quantification of aldehydes by liquid chromatography and LC-UV linked to a UV detector LC conditions: - ACENTIS RP amide expression column (2.1 × 150 mm, 2.7 μm Supelco 53914-u) - Eluent (0.5ml / min)

[0145] [Table 1]

[0146] - Inject 3 μl, UV detection at 360 nm

[0147] 4.4 Analysis and quantification of aldehydes by liquid chromatography (LC-HRMS) coupled with high-resolution mass spectrometry. LC conditions similar to those in Section 4.3 The Orbitrap Exploris 120 HRMS detector enables the identification of the morphology of DNPH derivatives.

[0148] result Aldehydes in vitro Table 2 shows that the amounts of the five aldehydes indicated (nonanal C9:0, pentadecanal C15:0, octadecanal C18:0, decanal C10:0, and dodecanal C12:0) increased after reconstructed skin was exposed to ozone at 0.2 ppm for 20 or 60 minutes in the presence of squalene, compared to a control that was not exposed to ozone (0 minutes).

[0149] [Table 2]

[0150] Aldehydes in vivo Tables 3 to 5 show that the amounts of the indicated aldehydes (nonanal C9:0, tridecenal, pentadecanal C15:0, octadecanal C18:0, decanal C10:0, and dodecanal C12:0) increased in samples taken on filter paper from the skin of volunteers exposed to ozone compared to those not exposed or exposed to medical air. The results were similar for D1, D5, and D12. The variability between subjects is substantial, representing the absolute value of the mean deviation. However, statistical analysis using the paired Wilcoxon test shows a significant difference between medical air and ozone conditions.

[0151] [Table 3]

[0152] [Table 4]

[0153] [Table 5]

[0154] Comparative test (not the present invention)

[0155] [Table 6]

[0156] [Table 7]

[0157] [Table 8]

[0158] Tables 3' to 5' (Tables 6 to 8) show that there was no significant difference in the amounts of aldehyde hexanal C6:0, octanal, and C8:0 present in samples taken on filter paper from the skin of ozone-exposed volunteers compared to those not exposed or exposed to medical air.

[0159] (Example 2) Research and analysis of oxysterols Materials and methods: 1. In vitro ozone exposure system and skin model The devices outside the incubator and the devices inside the incubator are the same as those described in sections 1.1 and 1.2 of Example 1. The reconstructed skin model used was the T-Skin® reconstructed skin model manufactured and supplied by EPISKIN SA (FR-69007, Lyon), prepared as described in Section 2.1 of the previous examples. Starting from D18, replace the plate medium with culture medium daily.

[0160] 2. Repeated exposure of the T-Skin™ model to 0.1 ppm ozone: For this type of exposure, set the ozone generator to a low setting: - Ozone generated at the device outlet: Approximately 0.130 ppm when the measured concentration inside the incubator is less than 0.1 ppm. - The ozone-rich air outflow rate from the top of each reconstructed skin is approximately 20 ml / min (note that the ozone outlet is located more than 10 cm from the reconstructed skin inside the incubator to prevent direct outflow stress).

[0161] The exposure will be carried out in three consecutive sessions: 18 hours between D18 and D19, 18 hours between D19 and D20, and 18 hours between D20 and D21. Between each exposure, the model will be placed in an incubator for just under 6 hours without ozone.

[0162] When ready to expose skin, replace the standard polystyrene lid with a perforated PEEK or PTFE lid.

[0163] Some samples were treated with ozone, while others were prepared in exactly the same way as controls, except that they were incubated in a conventional incubator without ozone.

[0164] On D22, reconstructed skin samples were taken, the epidermis was separated from the dermis, cut into two halves, and frozen at -80°C for analysis.

[0165] 3- Repeated exposure of the T-Skin™ model to 0.4 ppm ozone: For this type of exposure, the ozone generator should be set to a moderate ozone level, but slightly higher than the actual ozone peak. - Ozone generated at the device outlet: Approximately 0.4 ppm when the measured concentration inside the incubator is less than 0.3 ppm. - The ozone-rich air outflow rate from the top of each reconstructed skin is approximately 20 ml / min (note that the ozone outlet is located more than 10 cm from the reconstructed skin inside the incubator to prevent direct outflow stress).

[0166] The exposure will be carried out in two consecutive sessions: 18 hours between D18 and D19, and 18 hours between D20 and D21. Between D19 and D20, and between D21 and D22, the reconstructed skin will be placed in an incubator without ozone.

[0167] When ready to expose the skin, rearrange the culture plate and culture medium again (to prevent the risk of raw material solution seeping into the culture medium beneath the inserts during application), and replace the standard polystyrene lid with a PEEK or PTFE lid.

[0168] Some samples were treated with ozone, while others were prepared in exactly the same way as controls, except that they were incubated in a conventional incubator without ozone.

[0169] On D22, reconstructed skin samples were taken, the epidermis was separated from the dermis, cut into two halves, and frozen at -80°C for analysis.

[0170] 4. Micro-regional exposure of healthy volunteers to ozone in vivo Clinical studies measured 140 μg / cm³ in the forehead for phototype III-IV patients. 2 Super (146~299μg / cm 2 The study included 10 volunteers (8 women and 3 men) with an average age of 36.2 years and sebum levels (at any given time).

[0171] Micro-area exposure was performed using ozone generators, medical air generators, or glass bells (controlled pollutant exposure systems (CPES)) placed on the skin of volunteers' backs. The ozone generators and glass bells used are described in the publication Curpen et al., 2020 (A novel method for evaluating the effect of pollution on the human skin under controlled conditions, Skin Res Technol.). Exposure to ozone and medical air was performed randomly on each volunteer, either left or right. Three conditions were implemented: - Six exposures to 0.3 ppm (gas flow rate: 0.2-0.3 l / min), three times a week for two weeks. Each exposure lasted two hours. - Six exposures to medical air using the same protocol (gas flow rate: 0.2-0.3 l / min). - Six exposures to ambient air using the same protocol (glass bell without airflow).

[0172] D-squame samples were taken before and one hour after each exposure: D1 (1 exposure), D5 (3 exposures), and D12 (6 exposures).

[0173] 5. Analysis of oxysterols in reconstituted skin samples in vitro or D-squame samples in vivo 5.1- Extraction of oxysterols for analysis To extract oxysterols, methanol is added to each tissue or D-squame sample in a small vial (1 mL). This is stirred at 37°C for 30 minutes, then resuspended in a hemolysis test tube and evaporated using a vacuum concentrator. The evaporated extract is resuspended in 200 μL of a 2:1 methanol:isopropanol mixture. The sample is placed in an ultrasonic bath for 5 minutes and filtered through a 0.2 μm filter.

[0174] 5.2- Chromatographic method for oxysterol analysis: ULC Vanquish (Thermofisher)

[0175] [Table 9]

[0176] 5.3 - Thermofisher Orbitrap Tribrid IDX Mass Spectrometer Setup Various analytes are detected with a resolution of 60,000 using an Orbitrap detector.

[0177] result Oxidized forms of cholesterol in vitro Table 7 (Table 10) shows that the amounts of the three oxidized forms of cholesterol (5α-6α-epoxycholesterol; 5β-6β-epoxycholesterol; and 7-ketocholesterol) increase in reconstructed skin exposed to 0.1 or 0.4 ppm of ozone.

[0178] [Table 10]

[0179] This test was repeated twice to compare the oxysterols according to the present invention, namely 7-ketocholesterol, 5α-6α-epoxycholesterol, and 5β-6β-epoxycholesterol, with 27-hydroxycholesterol (not included in the present invention). The obtained values ​​are shown in Table 7' (Table 11).

[0180] [Table 11]

[0181] The number of samples for 7-ketocholesterol, 5α-6α-epoxycholesterol, and 5β-6β-epoxycholesterol is n=7. The number of samples for 27-hydroxycholesterol is n=2.

[0182] Table 7' (Table 11) shows that the amounts of the three oxidized forms of cholesterol according to the present invention (5α-6α-epoxycholesterol; 5β-6β-epoxycholesterol; and 7-ketocholesterol) increase in reconstructed skin exposed to 0.4 ppm ozone, in contrast to 27-hydroxycholesterol (not according to the present invention).

[0183] Oxidized forms of cholesterol in vivo Table 8 (Table 12) shows that the amount of 5α-6α-epoxycholesterol increased in D-squame samples taken from volunteers exposed to ozone compared to those exposed to medical air, in 7 out of 10 volunteers, at D12.

[0184] [Table 12]

Claims

1. A method for diagnosing skin exposure to ozone in a subject, comprising the following steps: a. A step of measuring the amount of at least one substance obtained from the oxidation of skin lipids selected from tridecenal, pentadecanal, octadecanal, 5α-6α-epoxycholesterol, 5β-6β-epoxycholesterol, and 7-ketocholesterol in a skin sample derived from the subject. Methods that include...

2. The method according to claim 1, wherein step a further comprises measuring the amounts of nonanal, decanal, and / or dodecanal in a skin sample derived from the subject.

3. A method for identifying compounds that can prevent and / or treat the harmful effects of ozone on the skin, comprising the following steps: a. A step of measuring the amount of at least one substance obtained from the oxidation of skin lipids selected from tridecenal, pentadecanal, octadecanal, 5α-6α-epoxycholesterol, 5β-6β-epoxycholesterol, and 7-ketocholesterol in a skin sample derived from a subject, wherein the skin sample is treated with the compound; b. A step of comparing the amount measured in step a) with the corresponding amount in a skin sample that has not been treated with the compound; c. If the amount of aldehyde or oxysterol measured in a skin sample treated with the compound is less than the amount of aldehyde or oxysterol measured in a skin sample not treated with the compound, the candidate compound is identified as a compound that can prevent and / or treat the harmful effects of ozone on the skin. Methods that include...

4. The method according to claim 3, wherein step a further comprises measuring the amounts of nonanal, decanal, and / or dodecanal in a skin sample derived from the subject.

5. The method according to claim 3 or 4, wherein the skin sample is selected from human skin explants, skin biopsies, microbiopsies, and stratum corneum samples.

6. A method for identifying compounds that can prevent and / or treat the harmful effects of ozone on the skin, comprising the following steps: a. A step of treating a skin sample selected from keratinocytes, reconstructed epidermis, and reconstructed skin by contacting the skin sample with the compound; b. A step of exposing a skin sample to ozone; c. A step of measuring the amount of at least one substance obtained from the oxidation of skin lipids selected from tridecenal, pentadecanal, octadecanal, 5α-6α-epoxycholesterol, 5β-6β-epoxycholesterol, and / or 7-ketocholesterol in a skin sample treated with the compound; d. A step of comparing the amount measured in step c) with the corresponding amount in a skin sample that has been exposed to ozone and not treated with the compound; e. If the amount of aldehyde or oxysterol measured in a skin sample treated with the compound is less than the amount of aldehyde or oxysterol measured in a skin sample not treated with the compound, the candidate compound is identified as a compound that can prevent and / or treat the harmful effects of ozone on the skin. Includes, A method in which it is understood that step b may be performed after step a, or vice versa, and that steps a and b may be repeated at least twice.

7. A method for evaluating the effectiveness of a compound that enables the prevention and / or treatment of the harmful effects of ozone on the target skin, comprising the following steps: a. A step of measuring the amount of at least one substance obtained from the oxidation of skin lipids selected from tridecenal, pentadecanal, octadecanal, 5α-6α-epoxycholesterol, 5β-6β-epoxycholesterol, and 7-ketocholesterol in a skin sample derived from the subject before treatment with the compound; b. A step of treating the target skin with the compound; c. After step b, a step of measuring the amount of the same substance obtained from the oxidation of skin lipids in a second skin sample derived from the subject; d. A step of comparing the quantities measured in steps a. and c.; e. A step of evaluating the effectiveness of the candidate compound in preventing and / or treating the harmful effects of ozone on the skin. Methods that include...

8. The method according to claim 7, wherein steps a. and c. further include measuring the amount of nonanal and / or decanal and / or dodecanal in a skin sample derived from the subject.

9. The method according to any one of claims 1 to 8, wherein a skin sample is taken using a D-squame® disc.

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