Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
  • C12N5/0629—Keratinocytes; Whole skin
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0697—Artificial constructs associating cells of different lineages, e.g. tissue equivalents
  • C12N5/0698—Skin equivalents
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2500/00—Specific components of cell culture medium
  • C12N2500/30—Organic components
  • C12N2500/36—Lipids
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2500/00—Specific components of cell culture medium
  • C12N2500/70—Undefined extracts
  • C12N2500/72—Undefined extracts from bacteria
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2503/00—Use of cells in diagnostics
  • C12N2503/02—Drug screening
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2529/00—Culture process characterised by the use of electromagnetic stimulation
  • C12N2529/10—Stimulation by light

Definitions

• the field of the invention relates to a new skin model integrating a microbial component and a lipid component.
• This model is particularly suitable for studying and developing new active ingredients/products in the dermo-cosmetic field. Indeed, to this integrated model, a physical or chemical stress can be added to reproduce lesions or pathological situations, which can cause imbalances in the skin microbiota.
• the skin is much more than an outer envelope, it is indeed a real organ necessary for life. It constitutes a ground of exchange between our internal environment and the external environment and is therefore exposed to multiple attacks and variations to which it adapts permanently.
• the skin therefore ensures, among other things, a real barrier function, formerly attributed to its structure and composition alone.
• the skin is made up of cells grouped together to form a fabric that is both resistant and flexible. These cells are divided into three layers: the hypodermis, the dermis and the epidermis.
• the hypodermis is the deepest layer of the skin. It is made up of white adipose tissue which helps protect the body from shocks, builds up an energy reserve and thermoregulates the body.
• the dermis is the layer between the hypodermis and the epidermis. It is the thickest layer of the skin.
• the dermis houses blood and lymphatic vessels, nerves, sweat glands that produce sweat, and pilosebaceous follicles. It is mainly composed of macromolecules, fibroblast cells and immune system cells.
• the epidermis is the thinnest surface of the skin. It is made up of 90% keratinocytes which are used to synthesize keratin and, thanks to their differentiation, ensure impermeability and protection for the skin.
• the epidermis is made up of four main layers: the basal layer (basal stratum), made up of keratinocytes with large nuclei, which ensure the constant renewal of the epidermis; the spinous layer (stratum spinosuni), made up of voluminous keratinocytes which gradually flatten as they move towards the upper layers; the granular layer (stratum granulosuni), composed of flattened keratinocytes which allow the keratinocytes to be transformed into comeocytes (anucleate cells) and finally the horny layer (stratum corneum), the upper layer composed of comeocytes which will desquamate.
• the epidermis is not only composed of comeocytes but also of lipids between these different cells. Lipids are organized in the form of lipid bilayers. The main classes of these lipids are ceramides, cholesterol and free fatty acids. They partly ensure the barrier function of the skin.
• Sebum is a hydrolipidic film produced by the sebaceous glands. It is excreted on the surface of the skin where it will form a protective layer, but also serve as a substrate for the skin microbiota.
• the sebum is mainly lipophilic and anaerobic, and will therefore allow the preservation of lipophilic and anaerobic microbial species. It will also protect the skin by lubricating it and acidifying it to regulate the skin's pH.
• the skin microbiota also plays a key role in the protective functions of the skin. It also participates in the regulation of the pH of the skin.
• the skin microbiota also called skin flora, is located in the epidermis. It is made up of microorganisms such as bacteria, viruses, fungi and parasites.
• the skin microbiota evolves as you age, reaching an average of 1,000 billion bacteria, and 1,000 species of viruses, parasites and fungi.
• bacteria the most known and studied strains are Staphylococcus aureus, Staphylococcus epidermidis and Cutibacterium acnes. Some are aerobic and therefore live with the supply of oxygen and others are anaerobic and live deeper in the stratum corneum without the supply of oxygen. Some of these bacteria produce fatty acids, others antimicrobial peptides and in some cases even amino acids. Most of these bacteria use sebum lipids to grow and produce these compounds.
• the skin microbiota varies quantitatively and qualitatively from one person to another. Variables such as age, sex, immune system, pH, temperature or even humidity can modify the composition of the microbiota of the skin. For example, the use of cosmetic or pharmaceutical products create differences. However, two distinct categories can be identified within the skin microbiota: “resident” flora and “transient” flora.
• the resident skin flora lives in total symbiosis with the skin. These are called commensal organisms. It is important for the defense against pathogenic microorganisms, because the place it occupies saturates the space and prevents the attachment of undesirable organisms.
• the transient flora does not establish itself permanently on the surface of the skin, it varies during the day, depends on the activities carried out and variations in the surrounding conditions. It can be present for a few hours or a few days.
• the microorganisms that make it up are mostly harmless, called saprophytes.
• This flora can also consist of opportunistic pathogenic bacteria and lead to diseases, if the host defenses weaken.
• One of the most common transient species is S. aureus, implicated in atopic dermatitis (AD).
• the microorganisms present on the skin and making up the resident flora are not opportunistic, they perform very useful functions for the protection of the organism. Indeed, the skin microbiota plays different roles in maintaining the integrity of our skin. It constitutes a barrier by regulating the homeostasis of the skin, by supplying it with nutrients or even by competing with pathogens.
• S. epidermidis for example, secretes antimicrobial peptides against S. aureus.
• Corynebacterium striatum and C. acnes inhibit the expression of virulence genes of the latter (Sanford and Gallo, Seminars in Immunology, 25, pp370-7, 2013).
• dysbiosis The symbiosis between host and microorganisms is particularly essential for the good health of the skin. When this balance is broken, it is called dysbiosis. It has been shown in numerous studies that dysbioses are correlated with the appearance of certain inflammatory skin pathologies, such as acne, atopic dermatitis or eczema. Understanding the interactions between the cutaneous microbiota and the skin is therefore an issue in the development of new cosmetic and pharmaceutical treatments.
• lipids having a protective role for the skin and the microbiota and which are secreted by the sebaceous glands does not contain viable keratinocytes.
• viable keratinocytes does not contain viable keratinocytes.
• 3D models of reconstructed epidermis with a microbial compound have also been used to study the interactions between the microbiota and the skin (Rademacher et al., Experimental Dermatology, 27, pp489-494, 2018).
• most of these models include the colonization of a single species of microorganism, such as C. amycolatum, and therefore do not reflect the actual conditions of our skin. The presence of lipids in the models healthy skin is also not described.
• the inventors have developed a new skin model comprising a microbial component and a lipid component which advantageously makes it possible to approach real skin.
• the cost associated with such a model is notably reduced compared to explants.
• this model can be used with different stresses to mimic skin disorders. Indeed, the inventors observed in particular that the application of an exogenous stress on the skin model induced molecular and metabolic characteristics similar to those observed in vivo in subjects having skin exposed to the sun or to inflammatory conditions.
• the model can advantageously be used as a tool to screen active ingredients or even cosmetic or pharmacological formulations of interest.
• the first object of the invention therefore relates to a reconstituted skin model comprising:
• Another object of the invention relates to a method for obtaining a model of reconstituted skin comprising: a) Supply of a skin substitute, b) Application to said skin substitute, concomitantly or sequentially, of a component microbial and a lipid component.
• the invention also relates to a screening method for the in vitro identification of an active ingredient or of a cosmetic formulation for preventing and/or improving at least one skin disorder induced following at least one exogenous stress comprising the following steps: a ) Applying said active ingredient or said cosmetic formulation to the reconstituted skin model according to the invention; b) The realization of at least one exogenous stress on said model; c) At least one measurement of the level of expression of at least one biological marker; And d) Determining whether said active ingredient or said cosmetic formulation makes it possible to prevent and/or improve at least one skin disorder as a function of at least one level of expression measured in step c).
• the invention also relates to a method for evaluating the in vitro efficacy of an active ingredient or of a cosmetic formulation for preventing and/or treating at least one skin disorder induced following at least one exogenous stress comprising the following steps: a) Applying said active ingredient or said cosmetic formulation to the reconstituted skin model according to the invention; b) The realization of at least one exogenous stress on the said model; c) At least one measurement of the level of expression of at least one biological marker; and d) Evaluating the efficacy of said active ingredient or of said cosmetic formulation as a function of at least one level of expression measured in step c).
• the invention also relates to a method for in vitro evaluation of the tolerance of an active ingredient or of a cosmetic formulation, comprising the following steps: a) The application of the said active ingredient or of the said cosmetic formulation on the reconstituted skin model according to invention; b) At least one measurement of the level of expression of at least one biological marker in the skin model of step a); and c) Assessing whether said active ingredient or cosmetic formulation is well tolerated by the skin model.
• the invention finally relates to a kit comprising a microbial component and a lipid component intended to be applied to a skin substitute, the microbial component comprising at least four genera of microorganisms selected from the genera Cutibacterium, Staphylococcus, Streptococcus, Burkholderia, Finegoldia, Gemella, Veillonella, Kocuia, Corynebacterium, Methylobacterium, and Brevibacterium.
• the invention relates to a reconstituted skin model comprising:
• skin substitute is meant a culture of skin cells composed of at least one layer.
• the skin substitutes according to the invention include in particular cultures of skin cells in monolayer, cultures of skin cells in bilayer and tissue models, including cultures of reconstructed epidermis or of reconstructed skin. Indeed, as it is often difficult to work on fresh explants, it is particularly advantageous, in the context of the present invention, to use cutaneous cell cultures.
• reconstructed epidermis is meant an epidermis generated in vitro by conventional techniques well known to those skilled in the art (cf., for example, Limât and Hunziger.
• reconstructed skin is meant a culture containing at least two compartments: a dermal compartment, and an epidermal compartment.
• the epidermal component of the reconstructed skin contains keratinocytes.
• the dermal component contains fibroblasts.
• the skin cells comprise normal, healthy or pathological cells which can cause skin disorders, or cells resulting from lines.
• Cultured skin cells can in particular be obtained from skin tissue explant.
• explant or “skin explant” is meant here a removal of cells or of skin tissue, which can be carried out for a surgical purpose or to carry out analyses.
• an explant can be obtained during surgical excision.
• excision we mean here a surgical intervention consisting in cutting (excising) a more or less large or deep part of the skin to treat an anomaly or an outgrowth. Excision is performed either to remove a tumor that is cancerous or suspected of being cancerous, or to treat a benign skin abnormality that is bothersome, whether for functional or aesthetic reasons.
• An excision within the meaning of the invention includes, for example, skin samples obtained after plastic surgery (mammaplasty, abdominal surgery, facelift, preputial removal, otoplasty, that is to say ear reattachment, syndactyly or supernumerary finger, etc. .).
• An explant can also be obtained by biopsy.
• biopsy is meant here a sample of cells or skin tissue taken for analysis purposes. Several types of biopsy procedures are known and practiced in the field.
• incisional biopsy in which only a sample of the tissue is taken
• excisional biopsy or surgical biopsy
• needle biopsy in which a sample of tissue is taken with a needle, which can be large or fine.
• others types of biopsy exist, such as smear or curettage, for example, and are also included in the present invention.
• said skin cells can be obtained by differentiation of stem cells (Guenou et al., Lancet, 374(9703): 1745-1753, 2009; Nissan et al., Proc. Natl. Acad. Sci., 108(36) : 14861-14866, 2011; Kraehenbuehl et al., Nature Methods, 8: 731-736, 2011).
• Said stem cells are not human embryonic stem cells.
• the skin cells according to the invention comprise at least one type of cells usually present in the hypodermis, the dermis and/or the epidermis. . These cells thus include, inter alia, keratinocytes, melanocytes, fibroblasts, adipocytes, endothelial cells, mast cells, Langerhans cells and/or Merkel cells.
• the skin cells according to the invention comprise at least keratinocytes and/or fibroblasts. More preferably, the skin cells according to the invention comprise keratinocytes and/or fibroblasts.
• the skin substitute is cultured and/or stored under conditions maintaining, at least partially, cellular metabolism and/or cellular functions.
• the culture of the skin substitute therefore includes both cultures of skin cells in monolayer (eg of keratinocytes) or cultures of skin cells in bilayer and tissue models, including cultures of reconstructed skin.
• the reconstructed skin model is selected from the group comprising epidermis models consisting mainly of keratinocytes, skin models comprising a dermis and an epidermis, and skin models comprising a dermis, an epidermis and a hypodermis.
• the models comprising at least one dermis form tissues of the connective type, while the models comprising at least one epidermis form stratified epithelia comprising the characteristic layers of the tissue considered.
• stratified epithelia comprising the characteristic layers of the tissue considered.
• said skin model is an epidermis model comprising a matrix support preferably chosen from: an inert support chosen from the group consisting of a semi-permeable synthetic membrane, in particular a semi-permeable nitrocellulose membrane, a of semi-permeable nylon, a membrane or a sponge of Teflon, a membrane of polycarbonate or of polyethylene, polypropylene, of semi-permeable polyethylene terephthalate (PET), a semi-permeable inorganic Anopore membrane, of acetate or ester of cellulose (HATF), semi-permeable Biopore-CM membrane, semi-permeable polyester membrane; in this group we find the reconstructed Epidermis models (Skinethic®) as well as the EpiDerm® model, (Mattek Corporation); a film or a membrane based on hyaluronic acid and/or collagen and/or fibronectin and/or fibrin.
• an inert support chosen from the group consisting of a semi-per
• Laserskin® Fidia Advanced Biopolymers
• Episkin® L'Oréal
• These models can be seeded by fibroblasts in the dermal part.
• fibroblasts may or may not be integrated, serve as a support for the seeding of keratinocytes and the reconstitution of the epidermis.
• pigment cells, immunocompetent cells, nerve cells are introduced; preferably immunocompetent cells are Langerhans cells.
• the matrix support is then seeded with keratinocytes to reconstruct the epidermis and finally obtain reconstructed skin.
• the skin model used comprises a model in which at least one complementary cell type has been incorporated, such as endothelial cells (EC) and/or immune cells such as lymphocytes, macrophages, mast cells, dendritic cells and/or cells fat and/or skin appendages, such as body hair, hair, sebaceous glands.
• EC endothelial cells
• immune cells such as lymphocytes, macrophages, mast cells, dendritic cells and/or cells fat and/or skin appendages, such as body hair, hair, sebaceous glands.
• the reconstituted skin model of the invention is particularly simple to implement. In addition, it does not require the use of a particular commercial cell line, and is advantageously adaptable.
• microbial component is meant a set of microorganisms composed of at least four different genera of bacteria.
• the microbial component is a skin microbial component.
• cutaneous microbial component is meant a set of microorganisms composed of at least four different genera of bacteria, said at least four genera of bacteria being present on the skin of a subject, preferably of a human subject.
• the microbial component comprises at least four, five, six, seven, eight, nine, or ten genera of microorganisms selected from the genera Cutibacterium, Staphylococcus, Streptococcus, Burkholderia, Finegoldia, Gemella, Veillonella, Kocuia, Corynebacterium, Methylobacterium, and Brevibacterium, more preferably the set of genera described above.
• the at least four genera includes the following genera: Cutibacterium, Staphylococcus, Streptococcus, and Burkholderia.
• the microbial component may also include one or more genera or species of fungus and/or virus.
• the microbial component corresponds to the microbiota of the skin of at least one subject.
• microbiota is meant the set of microorganisms present in an environment.
• the skin microbiota therefore corresponds to the set of microorganisms present on the skin.
• the composition of the microbiota can vary depending on the site of the skin.
• the cutaneous microbial component corresponds to the microbiota of the skin face, preferably the forehead, of at least one subject.
• the cutaneous microbial component comes from a sample taken from at least one subject, more preferentially at the level of the face and even more preferentially at the level of the forehead.
• the sampling of the microbial component can be done on an area of skin affected by a skin disorder such as, for example, an inflammatory dermatosis.
• a skin disorder such as, for example, an inflammatory dermatosis.
• the microbial component has a viability of 5,000 RLU (2 x 10 5 CFU/ml) when it is added to the surface of the skin substitute. After 48 hours of incubation, the microbial component preferably has a viability of approximately 100,000 RLU/0.66 cm 2 of skin substitute. Viability is determined using the "BacTiter ATP Lite" kit from Promega.
• lipid component is meant a mixture of at least two, three or four components chosen from free fatty acids, triglycerides, squalene, wax and/or wax esters, and cholesterol and/or esters of cholesterol.
• the lipid component is a source of nutrients for at least one bacterial species present in the microbial component.
• the lipid component makes it possible to protect the skin substitute and/or at least one bacterial species from desiccation.
• the lipid component comprises a mixture of free fatty acids, triglycerides, squalene, wax and/or wax esters, and cholesterol and/or cholesterol esters.
• the free fatty acids include C16:0, C18:0, C16:1, C18:1, and/or C18:2.
• the waxes and/or wax esters comprise C16:0/C16:0, C18:0/C18:0, C16:1/C16:1, and/or Cl 8:1/C18:1.
• the triglycerides include C16:0, C18:0, C16:1, and/or C 18:1.
• the lipid component comprises 30 to 50% w/w triglycerides.
• the lipid component comprises 15 to 30% w/w free fatty acids.
• the lipid component comprises 25 to 30% w/w waxes and/or wax esters.
• the lipid component comprises 12-20% w/w squalene.
• the lipid component comprises 4.5 to 8.5% w/w cholesterol and/or cholesterol esters.
• the lipid component is composed of 30 to 50% w/w of triglycerides, 15 to 30% w/w of free fatty acids, 25 to 30% w/w of waxes and/or wax esters, from 12 to 20% w/w squalene and from 4.5 to 8.5% w/w cholesterol and/or cholesterol esters.
• the lipid component comprises 3.0 to 6.0% w/w cholesterol and 1.5 to 2.5% w/w cholesterol esters.
• the lipid component is in hydrolipid form, ie.
• the lipid component is sebum.
• the lipid component comes from a sample taken from at least one subject, more preferably from the face and even more preferentially at the level of the forehead.
• the lipid component is added at a final concentration of 30 to 70 ug/cm 2 , more preferably at a final concentration of 40 ug/cm 2 of skin substitute.
• the skin substitute being composed of at least one layer of skin cells, the microbial component and the lipid component are present on the surface of said substitute.
• subject is meant here any human person, whether an adult or a child.
• child according to the invention, is meant an individual whose age is less than or equal to 16 years. Are thus included in the category of children according to the invention, newborns, whose age is between 0 and 1 month, infants, who are between 1 month and 2 years old, and children themselves, who are at least 2 years old. A “newborn”, as we understand it here, can be born at term as well as premature.
• An "adult” within the meaning of the present invention is a person who is not a child, in other words a person over the age of 16.
• Another object of the invention relates to a method for obtaining a model of reconstituted skin comprising: a) Supply of a skin substitute, b) Application to said skin substitute, concomitantly or sequentially, of a component microbial and a lipid component.
• the skin substitute is as described above.
• the lipid component can be applied before the microbial component, or vice versa.
• the microbial component and the lipid component are mixed together before application to the skin substitute.
• the microbial component and/or the lipid component is/are applied to the skin substitute using a pipette and a finger cot.
• the microbial component is applied fresh; in other words, it has not been frozen beforehand.
• the reconstituted skin is stressed.
• the method for obtaining further comprises a step of producing at least one exogenous stress.
• the method for obtaining a model of reconstituted skin is characterized in that the reconstituted skin is stressed and in that the method further comprises a step c) of carrying out at least one stress exogenous.
• Step c) can be carried out before or after step b) of the process for obtaining the model.
• step c) can be carried out on the microbial component and/or the component lipid before their application on the skin substitute.
• step c) is carried out after step b) of the process.
• exogenous stress is meant any external factor affecting the integrity of the skin and which may lead, where appropriate, to a progressive reduction in the effectiveness of its functions. Exogenous stress can be chemical and/or physical stress.
• Physical stresses include environmental stresses (eg temperature) and mechanical stresses (eg friction).
• the physical stress is chosen from ultraviolet radiation, sunlight, infrared, near infrared, heat, hertzian radiation including microwaves and waves from mobile phones, ionizing radiation including beta, gamma, X, non-ionizing radiation , the radiation of a magnetic field, ozone, a change in pressure, heat, cold, friction, stretching, particles.
• the physical stress is radiation mimicking solar radiation, more preferably radiation in the UV range including UVA and/or UVB; and/or visible light including visible and/or infrared high energy blue light.
• ultraviolet radiation we mean electromagnetic rays whose wavelength is between 153 nm and 400 nm.
• exposure to UV according to the invention comprises exposure to UVA and/or UVB.
• UVA is meant in the sense of the invention radiation whose wavelength is between 400 and 320 nm.
• UVB is meant in the sense of the invention radiation whose wavelength is between 320 and 290 nm.
• “Visible light” means radiation whose wavelength is between 400 and 700 nm.
• “Visible high-energy blue light” means visible light with a wavelength of 400 to 450 nm.
• infrared we mean radiation whose wavelength is between 700 nm and 2500 nm.
• the exposure to UV according to the invention comprises or consists of exposure to UVA.
• the exposure to UV according to the invention comprises or consists of exposure to UVB.
• the exposure to UV according to the invention comprises or consists of exposure to UVA and to UVB.
• the exposure to radiation according to the invention comprises or consists of exposure to UVA and/or UVB and/or visible and/or IR high-energy blue light.
• the wavelengths applied are between 290 and 450 nm or between 290 and 400nm.
• the skin model is exposed to a single acute UV dose of 16.5 J/cm 2 for approximately 45 minutes.
• the dose of UVB is between 80 and 150 mJ/cm 2 of UVB; preferably 120 mJ/cm 2 of UVB.
• the realization of an irradiation can be carried out under conditions well known to the person skilled in the art (cf. for example Lordanov et al, J. Biol. Chem. 1998).
• the chemical stress is at least one chemical substance whose topical application leads to an alteration of the structure and/or the function of the skin.
• the chemical stress is at least one allergen, a polluting agent, a surfactant, a solvent, or at least one pro-inflammatory stimulation reagent such as a cytokine; preferably a cocktail of specific cytokines capable of inducing the skin disorder studied.
• a pro-inflammatory stimulation reagent such as a cytokine; preferably a cocktail of specific cytokines capable of inducing the skin disorder studied.
• reagents are well known, and are described for example in application WO 2015/014949.
• the chemical stress comprises one or two components chosen from interleukin 1b, a ligand of the TLR-2 receptor, and a ligand of the TLR-3 receptor.
• the chemical stress comprises interleukin 1b and optionally the TLR ligand (Poly (1:C) and/or Pam3CSK4, more preferably all three components.
• Performing chemical stress comprising the application of at least interleukin IL-1 P on the reconstituted skin model advantageously makes it possible to mimic DA
• other chemical stresses can also make it possible to mimic DA, such as the combination of Poly I:C and TNF ⁇ ; the combination IL4 and IL13; the Poly LC, IL-4, IL13 combination; the IL-4, IL-13, IL-31 and optionally TNF ⁇ combination; the IL-4, IL-13, IL-22, TNF ⁇ combination.
• particles or molecules can in particular be brought into contact with the skin substitute.
• particles matter PM10
• particles matter 10 ⁇ L/cm 2 of solution of fine particles suspended in acetone can be deposited on the surface of the reconstructed epidermis before adding the microbiota and the sebum.
• benzo(a)pyrene B(a)P
• B(a)P is a good representative of outdoor pollutants because it leads to very toxic metabolites.
• TABPA Tetrabisphenol A
• cortisol is known as the stress hormone.
• Negative effects may include unwanted visible signs of aging such as fine lines and wrinkles, thinning of the skin, reduced elasticity and reduced functionality of the skin barrier.
• step (c) may correspond to bringing the skin substitute into contact with cortisol, for example at a concentration of 4 ⁇ M.
• the invention also relates to a model of reconstituted skin obtained by the method as described here.
• At least two exogenous stresses are carried out on the reconstituted skin model according to the invention.
• the invention makes it possible to identify active agents or formulations making it possible to prevent and/or improve at least one skin disorder or to evaluate the effectiveness of the active agents or formulations making it possible to prevent and/or improve at least a skin disorder.
• the invention makes it possible in particular to distinguish the active agents or the formulations according to their activity for preventing and/or improving at least one skin disorder.
• the invention therefore also relates to a screening method for the in vitro identification of an active ingredient or of a cosmetic formulation to prevent and/or improve at least one skin disorder induced following at least one exogenous stress comprising the steps following steps: a) Applying said active ingredient or said cosmetic formulation to the reconstituted skin model; b) The realization of at least one exogenous stress on the said model; c) At least one measurement of the level of expression of at least one biological marker; and d) Determining whether said active ingredient or said cosmetic formulation makes it possible to prevent and/or improve at least one skin disorder according to at least one level of expression measured in step c).
• the invention also relates to a method for evaluating the in vitro efficacy of an active or a cosmetic formulation for preventing and/or treating at least one skin disorder induced following at least one exogenous stress comprising the following steps: a) applying said active ingredient or said cosmetic formulation to the reconstituted skin model; b) The realization of at least one exogenous stress on said model; c) At least one measurement of the level of expression of at least one biological marker; and d) Evaluating the efficacy of said active ingredient or of said cosmetic formulation as a function of at least one level of expression measured in step c).
• Step a) can be carried out before or after step b) of the screening method for the in vitro identification of an active ingredient or of a cosmetic formulation or in the method for evaluating the in vitro efficacy an active ingredient or a cosmetic formulation.
• carrying out step a) before step b) can in particular make it possible to evaluate the ability of an active ingredient or cosmetic formulation to prevent at least one skin disorder
• carrying out step a) after step b) can in particular make it possible to evaluate the ability of an active ingredient or cosmetic formulation to improve at least one skin disorder.
• the application of the active ingredient of interest to the skin model according to step a) can be done directly.
• the method further comprises a step of formulating the active ingredient, in particular in the form of a liquid solution, in particular aqueous, prior to step a) of applying said active with a skin model.
• the candidate active ingredient is an active ingredient for the prevention and/or improvement of at least one skin disorder if said candidate active ingredient makes it possible to modulate the expression of at least one biological marker of the invention.
• This modulation may correspond, depending on the case, and in particular depending on the nature of the biological marker, to an increase or a decrease in the expression of said marker.
• the candidate formulation is a formulation for preventing and/or improving at least one skin disorder, if said candidate formulation makes it possible to modulate the expression of at least one biological marker of the invention. This modulation may correspond, depending on the case, and in particular depending on the nature of the biological marker, to an increase or a decrease in the expression of said marker.
• the in vitro efficacy of an active ingredient or of a cosmetic formulation for preventing and/or treating at least one skin disorder is meant within the meaning of the present application the ability of the formulation or of the active ingredient to cancel or decrease the effects related to at least one skin disorder.
• Prevention in the present case means treatment that is administered before the development of the skin disorder (i.e. before the realization of an exogenous stress in step b)), while reduction corresponds to a treatment that is administered once the effects of the skin disorder have appeared (i.e. after the achievement of an exogenous stress in step b)).
• creased means a larger amount, e.g., an amount slightly greater than the original amount, or e.g., an amount in great excess of the original amount. , including all quantities in between.
• crease may refer to an amount or activity that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% more than the quantity or activity for which the increased quantity or activity is compared , or at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 800%, 900%, 950% , 1000%, 1100%, 1200%, 1300%
• decreased means a lesser amount, for example, a slightly lower amount than the original amount, or for example a greatly reduced amount compared to the original amount, including all quantities in between.
• decrease may refer to an amount or activity that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% less than the quantity or activity for which the decreased quantity or activity is compared , or at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 800%, 900%, 950% , 1000%, 1100%, 1200%, 1300%, 1400%, 1500%
• the level of expression of said marker can be compared with a reference level of expression.
• step d) making it possible to determine whether the active ingredient or the cosmetic formulation prevents and/or improves at least one skin disorder, can comprise a comparison of the level of expression of the biological marker of step c) with a reference level of expression.
• a reference level of expression of a biological marker is meant within the meaning of the present application any level of expression of said marker used as a reference.
• a baseline expression level can be obtained by measuring the expression level of the marker of interest in a normal skin model.
• a normal skin model can, for example, correspond to a reconstituted skin model obtained from skin cells of a healthy subject or without apparent pathology. It is also possible to use, as a model of normal skin, a model of reconstituted skin without realization of the exogenous stress.
• the expression of the biological marker can be measured before step b); the level thus determined is then the reference expression level of said biological marker.
• the reference level of expression of a biological marker corresponds to the level of expression of said marker in the skin model in the absence or in the presence of a particular treatment.
• the reference level of expression of a biological marker is obtained by measuring the expression of said marker in the skin model which has not been brought into contact with an active ingredient or a formulation and/or which has not been subjected to exogenous stress.
• the expression of said marker is measured in the skin model treated with an active ingredient or a formulation known to be effective against a skin disorder.
• step d) is preferably carried out between measurements of expression levels obtained for skin models obtained from skin substitutes of similar, or even identical, histological structures.
• similar histological structures is meant within the meaning of the present application that the relative proportions of the cell types included in the compared skin models are similar.
• the relative proportions of the cell types and/or types of microorganisms comprised in the skin model of step a) do not differ by more than 5% from the relative proportions of the cell types and/or of types of microorganisms included in the skin model used to obtain the reference expression level of step d).
• relative proportion of a cell type is meant within the meaning of the present application the ratio of the number of cells corresponding to this cell type to the number of total cells included in the skin model.
• the proportion of keratinocytes on the number of total cells in the skin model of step a) does not differ by more than 5% from the proportion of keratinocytes on the number of total cells in the skin model used to obtain the reference expression level of step d).
• relative proportion of a type of microorganism is meant within the meaning of the present application the ratio of the abundance of a genus or species of microorganism to the number of total microorganisms included in the model of skin.
• the relative proportion of a genus or species of bacteria is the ratio of the abundance of said genus or species to the number of total bacteria.
• the relative proportions of the cell types included in the compared skin models are identical.
• the relative proportions of the cell types included in the skin model of step a) are identical to the relative proportions of the cell types included in the skin model used to obtain the level of expression of reference from step d) when they do not differ by more than 0.1%.
• the proportion of keratinocytes to the number of total cells in the skin model of step a) does not differ by more than 0.1% from the proportion of keratinocytes to the number of total cells in the skin model used to obtain the reference expression level of step d).
• step d) is preferably carried out between measurements of expression levels obtained for skin models which are of similar size, volume or weight, or even identical.
• the size, volume, or weight of the skin model of step a) does not differ by more than 5% from the size, volume, or weight of the skin model used for the obtaining the reference expression level of step d).
• the size, and the volume and the weight of the skin model of step a) do not differ by more than 5% from the size, the volume and the weight of the skin model used to obtain the level step reference expression d).
• the size, and the volume and the weight of the skin model of step a) do not differ by more than 0.1% from the size, the volume and the weight of the skin model used for the obtaining the reference expression level of step d).
• the person skilled in the art can standardize the level obtained in step c) and the reference level in step d ) using a normalization factor.
• This normalization factor could for example be a directly accessible physical marker such as the mass of cells in the sample, or the mass of a cellular constituent, such as the mass of cellular DNA or the mass of cellular proteins.
• the housekeeping genes according to the invention include for example the RPS28, GAPDH, B2M, TFRC, YWHAZ, RPLO, 18S, GUSB, UBC, TBP, GAPDH, PPIA, POLR2A, ACTB, PGK1, HPRT1, IPO8 and HMBS genes.
• Step c) is carried out after steps a) and b).
• the determination of step d) will advantageously be done by comparing the level of expression of step c) with a reference level of expression (see above).
• the skin model which has not been treated with the active ingredient or the formulation can be used as a control.
• the level of expression of the biological marker of the invention is measured in said skin model before and after application of the active ingredient or of the cosmetic formulation.
• the level of expression of the biological marker of the invention is measured in a skin model without application of the active ingredient or of the cosmetic formulation and in a skin model with active ingredient or of the cosmetic formulation .
• the level of expression of the biological marker of the invention can also be compared with that measured in vivo in normal skin or in vivo in skin having undergone exogenous stress.
• normal skin is meant here skin originating from a healthy subject or one with no apparent skin pathology.
• the reference expression level is preferably the expression level of said biological marker in a skin model which has not been in contact with the active ingredient or the formulation, which makes it possible to carry out a significant comparison between the expression level of step c) and said reference level.
• a skin model that has not been treated with the active ingredient or the formulation i.e. without performing step a) or before performing step a)
• the level of expression of the biological marker of the invention is measured in said skin model with and without application of the active agent or the cosmetic formulation (or before and after performing step a)).
• skin disorders it is here understood all the abnormal reactions which can occur on the skin of an individual. These conditions affect both the skin itself (i.e. the epidermis, dermis and/or hypodermis), as well as the pores of the skin, the sweat and sebaceous glands attached to it, and/or the microbiota.
• the skin disorders according to the invention result more particularly in lesions, which corresponds to skin that is damaged or in poor condition.
• Damaged skin includes for example reactive sensitive skin, dry skin (xerosis), skin damaged by the sun, by radiation, by cold, by stress or by pollution, by an allergy, by hives, by eczema and other forms of dermatitis such as atopic dermatitis, impetigo, irritative dermatitis, in particular irritative diaper dermatitis or diaper rash, contact dermatitis, seborrheic dermatitis of the skin and scalp ( cradle cap), psoriasis, Lainer-Moussous disease, or wounds or burns.
• skin disorder we thus means disorders as diverse as scabs, angiomas (including tuberous, subcutaneous or flat), hemangiomas, infant acne, adolescent acne, ichthyosis (eg vulgaris, congenital, harlequin...), rosacea, pruritus, etc.
• a skin disorder can also be caused or exacerbated by an external infection, for example of parasitic, viral, bacterial or fungal origin.
• parasitic, viral, bacterial or fungal origin are thus included in particular in skin disorders warts, prurigo strophulus, scabies, pediculosis of the scalp, or even mycoses.
• the latter are parasitoses caused by the proliferation of parasitic microscopic fungi in the body.
• the skin disorder is chosen from disorders linked to ageing, pollution, radiation; inflammatory dermatoses, preferably atopic dermatitis, acne, psoriasis; rosacea; pruritus; impaired barrier function; reactive skin; xeroses.
• biological marker is meant within the meaning of the present application a characteristic which is objectively measured and evaluated as an indicator of normal biological processes, of pathogenic processes, or of pharmacological responses to a therapeutic intervention.
• a biological marker therefore designates a whole range of substances and various parameters.
• a biological marker can be a substance whose detection indicates a particular pathological state (for example the presence of C-reactive protein as a marker of an infection), or on the contrary a substance whose detection indicates a physiological state. specific.
• said biological marker is a biological marker of a skin disorder if its level of expression is different in healthy skin and in skin exhibiting the clinical characteristics of said skin disorder.
• said marker characterizes a skin disorder if said marker is differentially expressed in a skin model in which an exogenous stress has been carried out and the skin model not having undergone exogenous stress. This results in a ratio in step d) that is different from 1 when the expression level measurement is compared between these two conditions.
• the biological marker according to the invention is preferably a gene, the products of a gene such as its transcripts and the peptides derived from its transcripts, a lipid, a sugar or a metabolite.
• a gene such as its transcripts and the peptides derived from its transcripts, a lipid, a sugar or a metabolite.
• biological markers according to the invention are in particular provided in Table 1, as well as below.
• many methods are available to the person skilled in the art for measuring the expression of said biological marker.
• at least two, three, four, five, six, seven, eight, nine, or ten biomarkers are measured. Said biomarkers are discriminating, thus making it possible to screen cosmetic actives or formulations for preventing or treating skin disorders or to evaluate the effectiveness of cosmetic actives or formulations for preventing or treating skin disorders.
• the biological marker of the invention is advantageously a marker selected from markers of inflammation, oxidative stress, barrier function, and/or microbial component.
• the biomarker can be measured in the skin substitute and/or in the microbial component/lipid component which are present on the surface of said model skin substitute.
• the biomarker is measured at least in the microbial component, preferably in the microbial component.
• the biological marker is measured at least in the skin substitute, preferably in the skin substitute.
• the "markers of inflammation” include markers which are modulated during an inflammatory response in the skin. They include cytokines, such as IL-8 or TSLP, as well as anti-microbial peptides, such as psoriasin, encoded by the S100A7 gene, and beta-defensin 2, encoded by the DEFB4A gene.
• cytokines such as IL-8 or TSLP
• anti-microbial peptides such as psoriasin, encoded by the S100A7 gene, and beta-defensin 2, encoded by the DEFB4A gene.
• the IL-8 protein is a cytokine which is induced early in cutaneous inflammation. It is in particular produced by de novo synthesis in keratinocytes stimulated by ILla and/or TNF. It is therefore a predictive marker of a biological response, even in the absence of visible signs of skin irritation.
• the protein sequence of human interleukin IL-8 corresponds to the NCBI reference sequence: NP 000575. This protein is encoded by the human IL8 gene (NCBI reference: Gene ID: 3576). The sequence of this one is accessible under the NCBI reference: NM_000584.
• the TSLP protein is an inflammatory cytokine characteristic of AD. Its secretion is clearly stimulated in the case of atopic dermatitis and promotes the initiation of an inflammatory response by Th2. Therefore, it is a good marker of AD. Other markers, such as RANTES/CCL5, MCP-3/CCL7 and/or IL-6 are also indicative of AD and can be measured within the scope of the present invention.
• the "markers of oxidative stress" according to the invention include markers which are expressed in the skin and/or in the microbial component in the event of oxidative stress.
• the oxidative stress markers are metabolites.
• at least one metabolite of xanthine, uric acid, and glutathione is measured.
• the “barrier function markers” according to the invention include markers which are specifically expressed in the outermost layers of the epidermis and which participate in the barrier function.
• the barrier function markers according to the invention are markers expressed in the stratum corneum or markers expressed in the tight junctions of the stratum granulosum.
• markers include in particular claudins, including claudin-1 (CLDN1), transglutaminases, such as for example transglutaminase 1 (TGM1), keratins, in particular keratin 1 (KRT1) and keratin 10 (KRT10), peptidyl arginine human deiminase type 1 (PAD1), caspase 14 (CASP14), aquaporin 3 (AQP3), loricin (LOR), sciellin (SCEL), BARX Homeobox protein 2 (BARX2), desmoglein-1 (DSG1 ), filaggrin (FLG), involucrin (IVL), sphingomyelinase or sphingomyelin diesterase (SMPD), comeodesmosine (CSDN), etc.
• CLDN1 claudin-1
• transglutaminases such as for example transglutaminase 1 (TGM1)
• keratins in particular keratin 1 (
• the barrier function markers comprise one or more barrier function discriminating metabolites.
• at least one metabolite of octanoic acid, serine, glutamate, phosphorylcholine, histidine, L-glutamine, L-alanine, methionine, and xanthine is measured.
• the "microbial component markers” include markers which are modulated in the microbial component in response to an exogenous stress and/or an active ingredient or cosmetic formulation. They include in particular metabolites, such as formate, acetate, glucose and maltose, glycerol, betaine, octanoate, tyrosine, panthenol, phenylalanine.
• metabolites such as formate, acetate, glucose and maltose, glycerol, betaine, octanoate, tyrosine, panthenol, phenylalanine.
• the expression of a gene can be measured for example at the nucleotide level, by measuring the amount of transcripts of said gene, and can also be measured for example at the peptide level, by measuring for example the amount of proteins from said transcripts.
• the level of expression of a gene is meant within the meaning of the invention the measurement of the quantity or of the cellular concentration of product of the gene in its peptide form or in its nucleotide form.
• the expression of at least one gene e.g. 16S
• the expression of at least one gene can be measured in order to determine the presence or absence of at least one microorganism in the model microbiota.
• the expression of the biological marker according to the invention will be detected in vitro from the reconstituted skin model.
• the method of the invention may comprise one or more intermediate steps between obtaining the reconstituted skin model and measuring the expression of the biological marker, said steps corresponding to the extraction from said reconstructed skin model of a lipid sample, an NMF sample, an mRNA sample (or the corresponding cDNA) or a protein sample. This can then be directly used to measure the expression of the marker.
• the preparation or extraction of mRNA (as well as the reverse transcription thereof into cDNA), proteins, lipids or NMFs from a sample of skin cells are only well-known routine procedures of the person in the trade.
• the expression of the marker can be measured.
• the method used to do this then depends on the type of transformation (mRNA, cDNA or protein) and the type of sample available.
• transcriptome analysis When the expression of the marker is measured at the mRNA (or corresponding cDNA) level, any technology usually used by the person skilled in the art can be implemented.
• gene expression level analysis technologies include well-known methods such as PCR (Polymerase Chain Reaction, if starting from DNA), RT-PCR (Reverse Transcription-PCR, if starting from RNA) or quantitative RT-PCR or even nucleic acid chips (including DNA chips and oligonucleotide chips) for higher throughput.
• nucleic acid chips we mean here several different nucleic acid probes which are attached to a substrate, which can be a microchip, a glass slide, or a bead the size of a microsphere.
• the microchip can be made of polymers, plastics, resins, polysaccharides, silica or silica-based material, carbon, metals, inorganic glass, or nitrocellulose.
• the probes can be nucleic acids such as cDNAs (“cDNA chip”), mRNAs (“mRNA chip”) or oligonucleotides (“oligonucleotide chip”), said oligonucleotides typically being able to have a length of between about 25 and 60 nucleotides.
• cDNA chip cDNA chip
• mRNA chip mRNA chip
• oligonucleotide chip oligonucleotides
• nucleic acid corresponding to all or part of said gene is labeled, then brought into contact with the chip under hybridization conditions, leading to the formation of complexes between said nucleic acid labeled target and probes attached to the chip surface that are complementary to that nucleic acid. The presence of labeled hybridized complexes is then detected.
• any current or future technology that can determine gene expression based on the amount of mRNA in the sample can be used.
• the person skilled in the art can measure the expression of a gene by hybridization with a labeled nucleic acid probe, such as for example by Northern blot (for mRNA) or by Southern blot (for cDNA), but also by techniques such as the Serial Gene Expression Analysis (SAGE) method and its derivatives, such as LongSAGE, SuperSAGE, DeepSAGE, etc.
• SAGE Serial Gene Expression Analysis
• tissue microarrays also known as TMAs
• Typical tests employed with tissue microarrays include immunohistochemistry and fluorescent in situ hybridization.
• tissue microarrays can be coupled with fluorescent in situ hybridization.
• RNA-Seq or “Whole Transcriptome Shotgun Sequencing”.
• massive parallel sequencing methods are available. Such methods are described in, for example, US 4,882,127; US 4,849,077; US 7,556,922; US 6,723,513; WO 03/066896; WO 2007/111924; US 2008/0020392; WO 2006/084132; US 2009/0186349; US 2009/0181860; US 2009/0181385; US 2006/0275782; EP-B1-1141399; Shendure & Ji, Nat Biotechnol., 26(10):1135-45, 2008; Pihlak et al., Nat Biotechnol., 26(6):676-684, 2008; Fuller et al., Nature Biotechnol., 27(11):1013-1023, 2009; Tuesdays, Genome Med., 1(4): 40,
• marker expression When marker expression is measured at the protein level, specific antibodies can be employed, particularly in well-known technologies such as immunoprecipitation, immunohistology, western blot, dot blot, ELISA or ELISPOT, protein chips, antibody chips, or tissue chips coupled with immunohistochemistry.
• FRET or BRET techniques flow cytometry, microscopy or histochemistry methods, including in particular confocal microscopy, fluorescence microscopy, electron microscopy, atomic force microscopy, methods based on the use of one or more excitation wavelengths and a suitable optical method, such as an electrochemical method (voltammetry and amperometry techniques), and methods of radiofrequency, such as multipolar, confocal and non-confocal resonance spectroscopy, such as the detection of fluorescence, luminescence, chemiluminescence, absorbance, reflectance, transmittance, birefringence or index of refraction (for example, by surface plasmon resonance, or "surface plasmon resonance” in English, by ellipsometry, by resonant mirror method, etc.), radioisotopic or magnetic resonance imaging, analysis by polyacrylamide gel electrophoresis (SDS-PAGE); by two-dimensional electrophoresis, by spectrophotometry, by
• the biological marker is a lipid, in particular a ceramide or a fatty acid, or a metabolite
• the person skilled in the art can use all the methods at his disposal to measure the lipid content in a sample of skin cells. These methods include, among others, liquid chromatography (HPLC, see for example Sullivan et al., Arch Ophthalmol., 120(12): 1689-99, 2002), liquid chromatography coupled with an evaporative light scattering detector (HPLC-ESD, see Nordbâck et al., J. High Résolut. Chromatogr., 22: 483-486, 1999; Torres et al., J. Chromatogr.
• HPLC liquid chromatography
• HPLC-ESD evaporative light scattering detector
• the NMF assay is a procedure well known to those skilled in the art. In particular, it is possible to measure NMF using in vivo confocal Raman microspectroscopy. This is a commonly used procedure in the field for at least 15 years.
• the evaluated asset is at least one sunscreen.
• sunscreen is meant any substance capable of filtering solar radiation to protect a surface, typically the skin or the hair, against the harmful effects of this radiation.
• the sunscreen is chosen from those conventionally known including organic sunscreens such as triazine derivatives, benzotriazole and phenyl benzotriazole derivatives, dibenzoylmethane derivatives, amino-substituted benzophenone and 2-hydroxybenzophenone derivatives, merocyanine derivatives including methoxypropylamino cyclohexenylidene ethoxyethylcyanoacetate, para-aminobenzoic acid derivatives, salicylic derivatives, cinnamic derivatives, P,P'-diphenylacrylate derivatives, benzylidenecamphor derivatives, phenylbenzimidazole, anthranilic derivatives, imidazoline derivatives, and benzalmalonate derivatives.
• organic sunscreens such as triazine derivatives, benzotriazole and phenyl benzotriazole derivatives, dibenzoylmethane derivatives, amino-substituted benzophenone and 2-
• the active evaluated in is at least one sunscreen.
• the method for evaluating the in vitro efficacy of at least one sunscreen or of a cosmetic formulation comprising it or them for preventing and/or treating at least one skin disorder induced following at least one exogenous stress consisting of UV, infrared, or blue light irradiation.
• Method for in vitro evaluation of the tolerance of an active ingredient or of a cosmetic formulation comprising the following steps: a) The application of the said active ingredient or of the said cosmetic formulation on the reconstituted skin model; b) At least one measurement of the level of expression of at least one biological marker in the skin model of step a); and c) Assessing whether said active ingredient or cosmetic formulation is well tolerated by the skin model.
• the active ingredient is an active ingredient which is well tolerated by the skin if said active ingredient does not significantly modulate the expression of the biological marker.
• the cosmetic formulation is well tolerated if the expression of the biological marker is not significantly modulated by its addition to the reconstituted skin model.
• a statistical test such as a univariate Wilcoxon test, and having a p value less than 0.05, preferably a p value less than 0.01, more preferably a p-value of less than 0.001.
• Said modulation may correspond, depending on the case, and in particular depending on the nature of the biological marker, to an increase or a decrease in the expression of said marker.
• the expression of inflammatory markers is known to be increased when the skin is attacked, for example by exogenous stress.
• the expression of these markers of inflammation is not affected by well-tolerated active ingredients or formulations.
• the biological marker of step b) is an inflammation marker, for example among those described here.
• the tolerance consists in maintaining the balance of the microbial component.
• maintaining the balance is meant that at least one microorganism, preferably at least one bacterial genus or species, is maintained in the microbial component of the model following application of the cosmetic agent or formulation to the step a).
• the abundance of said at least one microorganism is not significantly modified following the application of the cosmetic agent or formulation in step a).
• the presence of the microorganism, or its level of abundance is compared to a condition or reference level, for example in a model in which the agent was not applied or in normal skin in vivo.
• kits comprising a microbial component and a lipid component intended to be applied to a skin substitute, the microbial component comprising at least four genera of microorganisms selected from the genera Cutibacterium, Staphylococcus, Streptococcus, Burkholderia, Finegoldia, Gemella, Veillonella, Kocuia, Corynebacterium, Methylobacterium, and Brevibacterium.
• the microbial component comprises at least five, six, seven, eight, nine, or ten genera of microorganisms selected from the genera Cutibacterium, Staphylococcus, Streptococcus, Burkholderia, Finegoldia, Gemella, Veillonella, Kocuia, Corynebacterium, Methylobacterium, and Brevibacterium, more preferably the set of genera described above.
• the at least four genera includes the following genera: Cutibacterium, Staphylococcus, Streptococcus, and Burkholderia.
• the microbial component may also include one or more genera or species of fungus and/or virus.
• the microbial component corresponds to the microbiota of the skin of at least one subject.
• microbiota we mean the set of microorganisms present in an environment.
• the skin microbiota therefore corresponds to the set of microorganisms present on the skin.
• the composition of the microbiota can vary depending on the site of the skin.
• the cutaneous microbial component corresponds to the microbiota of the skin of the face, preferably of the forehead, of at least one subject.
• the cutaneous microbial component comes from a sample taken from at least one subject, more preferentially at the level of the face and even more preferentially at the level of the forehead.
• the microbial component has a viability of 5000 RLU.
• the lipid component is as described above.
• the kit further comprises an instruction manual.
• the kit further comprises at least one chemical component as described herein.
• the kit comprises an allergen, a polluting agent, a surfactant, a solvent, or at least one pro-inflammatory stimulation reagent such as a cytokine, or cortisol. Said kit advantageously makes it possible to obtain the reconstituted skin model.
• the sebum is sampled from the forehead of 10 volunteers with no apparent skin pathology with a standard sampling kit. Each collection tube is then washed 3 times with hexane to extract the sebum. The extracts are then combined and then the solvent is evaporated under vacuum. The mass of sebum collected is checked (eg 1100 mg for a preparation) then the sebum is taken up in 20 ml of hexane. 100 vials of sebum are produced with 9 mg of substance per vial and packaged under argon.
• the vials are then taken up with 250 ⁇ l of absolute ethanol (Fischer Scientific, Illkirch, France) then diluted to 1/20 th in physiological saline.
• the sebum was characterized by GC/MS ( Figure 1).
• microbiota is sampled from 18 volunteers with no apparent skin pathology using a swab soaked in physiological saline (Fischer Scientific, Illkirch, France) containing 0.1% by volume of Triton X-100 (Fischer Scientific , Illkirch, France).
• the swab is rubbed energetically on the forehead of the donor for 4 seconds.
• Each swab is then placed in a tube to be centrifuged. After centrifugation, a colorless liquid is recovered (approximately 60 pL) and its volume is adjusted up to 120 pL with saline.
• Each microbiota is then mixed with the sebum in order to be able to make the deposit.
• 22 pL of the sebum/microbiota mixture is deposited on the surface of a reconstructed human epidermis and spread using a finger cot.
• a given sebum/microbiota mixture can be applied to up to 6 reconstructed epidermis.
• the reconstructed human epidermis are incubated at 32° C., 5% CO2, 60% humidity.
• the incubation time is set at 48 h which allows to have a sufficient quantity of bacteria without damaging the reconstructed human epidermis.
• the inventors deposited on the reconstructed epidermis of 0.6 cm 2 , 24 pg of sebum which corresponds to a concentration of 40 pg/cm 2 .
• Example 2 Establishment of the reconstructed skin model with or without the addition of a stress Methods
• the epidermis was reconstructed from skin resections from plastic surgery according to the method described by Frankart et al. (Frankart et al., Exp. Dermatol. 2012, 21(11), 871-875).
• the cells were isolated from skin resections, then cultured before being seeded on culture inserts immersed in culture medium then the culture inserts are placed at the air/liquid interface in an incubator at 37°C in a humidified atmosphere with 5% CO2, to form the stratum corneum.
• the reconstructed epidermis are placed in a culture medium without antibiotics.
• the reconstructed epidermis are placed in 12-well plates with maintenance medium without antibiotics (see Frankart et al., 2012) and with Phenol Red.
• a sebum/microbiota mixture obtained as described in Example 1, is deposited on the surface of each reconstructed human epidermis and spread using a finger cot.
• the reconstructed human epidermis are seeded with an inoculum of microbiota with a viability of approximately 5000 RLU.
• the survival of the epidermis is carried out under sterile conditions.
• Each insert containing an epidermis is placed in a well of a new 12-well plate containing the new maintenance medium alone or supplemented in order to induce stress.
• the plate is incubated at 32°C under 5% CO2 and 60% relative humidity.
• the culture medium is renewed every 24 hours up to 48 hours of incubation.
• the medium used is maintenance medium (1 mL/well) supplemented with interleukin 10 at 0.06 ug/mL, TLR ligand (Poly (1:C) at lOpg/ ml, and Pam3CSK4 at 5pg/ml).
• the viability of the microbiota is measured using the BacTiter ATP Lite kit, Promega following the supplier's protocol.
• the viability of the microbiota after culture is evaluated using this same kit.
• the viability measurement is a bioluminescent test involving luciferase.
• the amount of light emitted is proportional to the amount of ATP present in the sample and therefore to the number of living cells.
• a viability reading is carried out in duplicate on the microbiota sampled from each epidermis (BacTiter-Glo Microbial Viability Cell Assay kit, Promega).
• the ATP mix and the microbiota are placed in each well in a 1:1 ratio.
• the plate Low Binding Greiner 655209
• the reading is then carried out using a Clariostar plate reader.
• the DNA of microorganisms before and/or after growth on reconstructed epidermis is extracted using a Qiacube following the QIAamp DNA Investigator Kit protocol from Qiagen.
• Droplet Digital PCR is a digital PCR technique based on the generation of droplets using a water/oil emulsion. From a sample, it makes it possible to generate more than 20,000 droplets within which the PCR amplification is carried out. After amplification, each droplet is analyzed to quantify the number of positive droplets in the sample. This technique has the particular advantage of allowing the amplification of DNA present in small quantities in a sample.
• the ddPCR is performed in a 96-well plate: 5 pL of sample are added to 15 pL of ddPCR mix as explained in the Biorad protocol.
• the probes used correspond to the species sought, e.g. S. epidermidis, S. aureus, C. acnes.
• Droplets are generated using Biorad's QX200 Droplets Generator device, then the plate is placed in the thermal cycler for amplification. The plate is then read using Biorad's QX200 Droplet Reader device and analyzed using QuantaSoft software.
• the sequencing was carried out on a MiSeq sequencer (Illumina) with the primers adapted to each of the targets.
• the pre-processing of the sequences is carried out by a computer pipeline developed by INRAE running under Mothur (version 1.44.0).
• the pre-processing of the sequences is carried out by a computer pipeline developed by INRAE running under Mothur (version 1.44.0). Barcodes and primers as well as chimeras were removed from sequence files during filtering steps. The sequences presenting 100% homology between them were grouped into unique sequences, then into OTU (operational taxonomy unit: threshold of 97%) which will be identified subsequently.
• the bioinformatics analysis of the sequencing data makes it possible to identify the microorganisms present at different taxonomic levels (phylum, genus (e.g. for Staphylococcus), and species (e.g. for C. acnes)).
• the analysis allowing the phylogenetic affiliation down to the genus level was carried out using bioinformatics tools for processing large amounts of sequence data (Mothur). Identification was performed based on the Greengenes taxonomy for bacteria.
• the bioinformatics analysis of the sequencing data makes it possible to identify the Staphylococcus present at different taxonomic levels (genus and species are targeted).
• the analysis allowing the phylogenetic affiliation down to the species level was carried out using bioinformatics tools for processing large amounts of sequence data (Mothur). The identification was carried out on the list of Staphylococcus.
• barrier function proteins Claudin 1, Filaggrin and TGM1
• human antimicrobial peptides DEFB4A and S100A7
• the reconstructed epidermis halves are stored in 300 ⁇ l of a 1% ⁇ -mercaptoethanol RLT (Qiagen) solution and frozen at ⁇ 80° C. while awaiting extraction.
• RLT 1% ⁇ -mercaptoethanol
• the RHEs are separated from their membrane using forceps.
• a 5 mm Stainless Steel Beads ball Qiagen
• the Tissue Lyser Qiagen
• the RNA is extracted using Qiagen's RNA Fibrous Tissue Mini Kit extraction protocol. The extractions are carried out via Qiacube (Qiagen).
• the quantity of RNA extracted is measured with the Nanodrop.
• RNA is then adjusted so as to reverse-transcribe 1 pg of RNA per sample.
• the cDNAs obtained are then diluted to one-twentieth and placed in a 384-well plate (triplicates) using Qiagen's EpMotion robot. 5.5 pL of reaction mix (5 pL of TaqMan Fast Advanced Master Mix + 0.5 pL of probes per reaction) are then added to each well. GAPDH, YWHAZ and UBC are the reference genes chosen for qPCR analysis.
• IL-8 is assayed in the culture supernatants (24H, 48H and 72H) using the Bioplex ELISA IL-8 kit from R&D Bio-Techne.
• the supernatants are assayed in triplicates. Plate reading (Costar 3590 Corning) is performed on the Clariostar plate reader.
• the TSLP is assayed in the supernatants using the Bioplex TSLP kit from R&D Bio-Techne.
• the monitoring of viability during the time of implementation of this model accounts for the growth of the microbiota on the epidermis.
• microbiota growth results on healthy or inflammatory epidermis are presented in Table 2 below.
• microbiota The growth of microbiota on complete epidermis is 1 to 2 log in 48 hours. Significantly greater growth is observed on inflammatory epidermis than on healthy epidermis. Without being bound by theory, one can think that the barrier is altered in the inflamed epidermis, which allows microorganisms to access additional nutrients and therefore to grow more.
• Cutibacterium especially C. acnes
• Staphylococcus Streptococcus
• Burkholderia Finegoldia
• Gemella Veillonella
• Kocuia Corynebacterium, Methylobacterium and Brevibacterium.
• Staphylococcus species have been determined more precisely. Staphylococcus species identified in the microbiota are: S. epidermidis, S. capitis, S. warneri, S. hominis and S. aureus.
• microbiota/sebum on healthy epidermis leads to the production of IL-8, so there is a reaction of the epidermis to the presence of microorganisms.
• TSLP TSLP is an inflammatory cytokine characteristic of atopic dermatitis. Its secretion is clearly stimulated in the case of atopic dermatitis and promotes the initiation of an inflammatory response by Th2. It is measured in the supernatant after 24 hours of culture.
• TSLP TSLP
• the DEEB4A gene encodes P-defensin 2, a skin antimicrobial peptide secreted by keratinocytes. The production of anti-microbial peptides is stimulated by the presence of microorganisms.
• S100A7 codes for the protein known as psoriasin with antimicrobial properties.
• RQ “Relative quantification”, RQ > 2: overexpression, 0.5 ⁇ RQ: underexpression
• RQ “Relative quantification”
• RQ > 2 overexpression
• RQ underexpression
• the presence of microbiota/sebum on healthy epidermis leads to a slight increase in the expression of the antimicrobial peptides DEFB4A and S100A7 by the epidermis.
• the presence of microbiota/sebum on healthy epidermis stimulates an “innate” immune response.
• the inventors quantified by RT-qPCR the expression of the gene coding for Claudin-1, a protein involved in tight junctions and the expression of the gene encoding Filaggrin.
• the TGM1 gene codes for transglutaminase-1, an enzyme involved in stratum corneum formation and barrier function.
• the presence of microbiota/sebum on healthy epidermis does not cause any significant modification in the expression of the genes coding for the proteins involved in the barrier function, Filaggrin and Claudine 1 (results not shown).
• the expression of the gene coding for the TGM1 enzyme is increased.
• IL-8 The production of IL-8 is multiplied by 1.7 to 2.2 times.
• TSLP TSLP by inflammatory epidermis
• TSLP is produced under inflammatory conditions compared to the "healthy" model (without addition of inflammatory stress), but the presence of the microbiota/sebum does not significantly modify the production of TSLP.
• microbiota/sebum on inflammatory epidermis leads to a significant increase in the expression of the antimicrobial peptides DEFB4A and S100A7 by the epidermis. As expected, an exacerbated sensitivity of the inflammatory epidermis is observed in the presence of microbiota/sebum.
• TGM1 The effects of microbiota/sebum on the expression of TGM1 are shown in Table 10. Expression of TGM1 by inflammatory epidermis with and without microbiota/sebum.
• the inventors clearly demonstrate the advantage of the reconstituted skin model comprising a skin substitute as well as a microbial component and a lipid component.
• a microbial component and a lipid component induces the production of certain inflammatory markers such as, for example, IL-8, and a slight increase in the expression of antimicrobial peptides thus stimulating innate immunity.
• the microbiota induces a basal level of inflammation under these conditions.
• the microbiota induces an exacerbated inflammatory response, which leads to an impaired barrier function.
• the model advantageously reproduces an inflammation of the atopic dermatitis type.
• This model is therefore quite suitable for evaluating new active ingredients that would be potentially useful in the prevention or treatment of atopic dermatitis.
• This model is also suitable for evaluating new active ingredients that would be potentially useful in the prevention or treatment of inflammatory dermatoses, such as psoriasis, acne, seborrheic dermatitis, chronic urticaria, eczema, rosacea.
• inflammatory dermatoses such as psoriasis, acne, seborrheic dermatitis, chronic urticaria, eczema, rosacea.
• this integrated model is particularly interesting for testing new active ingredients with a putative activity in the protection, or even the reinforcement of the barrier function.
• Example 3 Evaluation of the effect of a stress imitating sun exposure on the reconstructed skin model as well as the effect of a sunscreen
• the reconstructed epidermis are irradiated by simulated solar radiation using a Suntest CPS+ ATLAS chamber, Material Testing Technology BV, Moussy le Neuf, France) equipped with an NXE 1500 xenon lamp, and fitted with a UV filter to eliminate lengths waves below 290 nm.
• the irradiance in the UV spectra is about 70 W/m2 from 290 to 400 nm.
• the skin models are exposed to a single acute UV dose of 16.5 J/cm2 (approximately 45 min) and the irradiation chamber is maintained at 37°C using ice water and a air flow.
• Formulation A is applied at a dose of 2 mg/cm 2 on the surface of the skin model reconstituted 2 hours before exposure to radiation. After irradiation with the total solar spectrum (up to 790 nm), the model is maintained in culture for 24 hours at 32° C. and 60% humidity. At the end of the experiment, the surface of the reconstructed epidermis is washed twice with 100 ⁇ l of physiological serum, and then dried with a cotton swab. The purpose of this step is to recover the cutaneous microbiota as well as the lipids (here the sebum) for analysis separately from the skin cells.
• the reconstructed epidermis are separated from the insert and are weighed in order to standardize the results obtained with the metabolomic approach.
• the reconstructed epidermis are crushed and 108 samples are extracted. Briefly, the samples are ground in 2 ⁇ ImL of acetonitrile/water 1/9 (v:v) with a fastprep using Lysing Matrix M tubes, 6 cycles of 20 seconds at force 4, with 2 min on ice between each cycle. An 800pL volume of each sample was evaporated to dryness with a SpeedVac, then normalized in 220pL D2O. The samples were analyzed by NMR and then mass spectrometry.
• 1H NMR spectra are obtained at 300 K on a Bruker Avance III HD 600 MHz NMR spectrometer (Bruker Biospin, Rheinstetten, Germany), operating at 600.13 MHz for the 1H resonance frequency, using a 1H-13C-15N cryoprobe -31P 5 mm reverse sensing attached to a Cryoplatform (the preamp unit).
• 1H NMR spectra are acquired using the NOESY 1D experiment with pre-saturation for water removal (noesyprld), with a mixing time of 100 ms.
• the samples are transferred from the NMR tubes to the UHPLC vials.
• the samples are centrifuged at 9000 g for 5 min.
• a volume of 10 pL is then injected into the UHPLC ACQUITY system from Waters (Manchester, UK), using water/methanol/acetic acid 95/5/0.1 (v:v:v) as mobile phase A and 100/0.1 (v:v) methanol/acetic acid as mobile phase B, at a flow rate of 0.3 mL/min.
• the following gradient is used: 0-30 min: 0% to 100% B, 30-34 min: 100% B.
• the separation is carried out at 30°C with a Hypersil Gold C18 column (100 x 2.1 mm , 1.9 ⁇ m) from Thermo Scientific (Les Ulis, France).
• Thermo Scientific Les Ulis, France.
• the following electrospray parameters are applied: capillary voltage of 0.5 kV, sample cone voltage of 30 V, source temperature of 120°C, desolvation temperature of 350°C, cone gas flow of 50 L/h and 600 L/h desolvation gas flow in positive mode; capillary voltage of 0.5 kV, sample cone voltage of 30 V, source temperature of 120°C, desolvation temperature of 550°C, cone gas flow of 30 L/h and gas flow of desolvation of 600 L/h in mode negative.
• High-resolution mass spectra are acquired with a Synapt G2-Si mass spectrometer from Waters (Manchester, UK), between m/z 50 and 800 in sensitivity and centroid modes. The samples are analyzed randomly, and a QC sample corresponding to a pool of all samples, is analyzed 11 times along the sequence.
• Structural identifications of discriminating metabolites are carried out on an LTQ Orbitrap XL mass spectrometer (Thermo Scientific, Les Ulis, France) coupled to a U3000 liquid chromatography system (Thermo Scientific, Les Ulis, France).
• the data is normalized to allow quantification of signals detected in multiple samples.
• Principal component analysis is first applied to check the validity of the acquisition, to detect potential outliers and internal clusters.
• Multivariate analyzes are performed using SIM CA vl5 software (Umetrics, Umeâ, Sweden).
• the mixOmics package (Rohart et al., 2017) is used to perform multilevel analyses.
• the analytics pipeline includes the mapping metabolomic data to an organism's specific metabolic network, then applying graph-based methods and advanced visualization tools to enhance data analysis.
• MI Non-irradiated
• IR Irradiated
• CTRL Control
• FA Formulation A
• ns not significant
• results show a significant protective effect of a formulation comprising phenylene bis-diphenyltriazine (Formulation A) on oxidative stress due to sun exposure.
• the model also makes it possible to identify or evaluate the in vitro efficacy of an active ingredient in preventing and/or treating at least one skin disorder induced following at least one exogenous stress such as UV irradiation.
• MI Non-irradiated
• IR Irradiated
• CTRL Control
• FA Formulation A
• ns not significant
• C. acnes is a very abundant bacterial species in the model's microbiota.
• the inventors have demonstrated a significantly increased abundance under the effect of irradiation (see Table 15).
• MI Non-irradiated
• IR Irradiated
• CTRL Control
• FA Formulation A
• ns not significant
• This integrated model is particularly interesting because it makes it possible to approach the conditions observed in vivo.

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