METHODS OF EVALUATING THE EFFECTS OF DEHYDRATION ON CHILDREN'S SKIN

专利摘要:
The present invention relates to biological markers of children's skin, and in particular that of infants, the expression of which is modified when the skin is dry. Such markers are particularly advantageous because they make it possible to follow the response of the skin to dehydration. The inventors have developed methods for evaluating the in vitro efficacy of formulations on the prevention of the effects of dehydration for the skin of children, using a skin model specifically capable of reproducing the characteristics of children's skin.
公开号:FR3045669A1
申请号:FR1562528
申请日:2015-12-16
公开日:2017-06-23
发明作者:Stephanie Bredif；Caroline Baudouin
申请人:Laboratoires Expanscience；
IPC主号:

专利说明:

INTRODUCTION
The skin is a set of cells and macromolecules grouped in the form of a resistant and flexible fabric, covering the entire body. It consists of two joined layers: the epidermis and the dermis to which the subcutaneous tissues can be associated. The epidermis whose main role is the protection of the body is the most superficial layer of the skin and ensures the impermeability of the skin and its resistance. Four distinct cell layers, a basal layer (stratum basalis), a spinous layer (stratum spinosum), a granular layer (stratum granulosum), and a stratum corneum (stratum corneum) can be identified in the latter. If different cell types coexist in the epidermis, keratinocytes are largely the majority (90%). Their characteristic activity is the synthesis of keratins, fibrous proteins and insoluble in water which represent 95% of the total proteins of the epidermis.
The main function of the skin is to establish a barrier against environmental damage while allowing certain exchanges between the indoor and outdoor environments. The barrier function is provided primarily by the stratum corneum, which makes the skin impermeable and hydrophobic, thus protecting the dermis from a massive influx of water. It is also the stratum corneum that resists chemical attack. It is composed of cells, called corneocytes, dead and coreless, but filled with keratins and other products such as lipids, fatty acids and ceramides. The corneocytes are connected by particular tight junctions, the corneodesmosomes, forming a compact layer whose cohesion is further reinforced by a lipid cement. Under the granular layer, the tight junctions in the granular layer also participate in the barrier function of the skin (see, for example, Hogan et al., J Allergy, 2012: 901940, 2012).
In everyday life, the skin has to face various attacks. It is exposed, for example, to chemical agents such as soap and physical stresses such as friction with clothing and exposure to the sun. The epidermis and epidermal appendages must therefore be constantly renewed to keep the skin in good condition. Stem cells make these maintenance and repair processes possible. More particularly, the regenerative capacity of the epidermis is conferred by adult stem cells which allow regular replacement of differentiated cells removed during keratinization. The epidermal stem cells thus give rise to keratinocytes, which will eventually differentiate into corneocytes, which are exfoliated during the desquamation process. This process is particularly crucial for the maturation and maintenance of the barrier function.
The barrier function is particularly important especially to limit the loss of water from the epidermis.
Indeed, whatever the type of skin, it can undergo a state of dehydration passenger that can be related to extrinsic factors (wind, cold, sun, detergents ...) and / or intrinsic (psoriasis, eczema, senescence ...). This lack of water causes a decrease in the effectiveness of the cutaneous barrier. A cascade of events, called "dry skin cycle", is then put in place. Firstly, the reduction of the barrier function leads to an amplification of the drop in the water content as well as the barrier fault. In return, the proliferation of keratinocytes increases (hyperkeratosis) and moderate inflammatory changes are induced, the skin trying to repair itself. This inflammatory hyperproliferative state is crucial in the dry skin cycle as it leads to aberrant differentiation and the production of poor quality materials and structures. Finally, the reduced activity of the desquamation enzymes leads to thickening and loss of hygroscopy of the stratum corneum.
Adaptation to ectopic life is a process that begins at birth and continues throughout the first year of life. The first months of postnatal life constitute a period of structural and functional reorganization of the skin that allows a physiological adaptation to the ectopic environment. For example, the immaturity of the newborn's skin is underlined by the difference in structure and molecular composition of the stratum corneum with respect to the adult. These are incomplete and thus continue to develop during the first 12 months at least after birth (Chiou et al., Skin Pharmacol Physiol, 17: 57-66, 2004, Nikolovski et al., J. Invest Dermatol, 128: 1728-1736, 2008; Stamatas et al., Pediatr Dermatol, 27: 125-131, 2010; Telofski et al., Dermatol Res Pract, 2012: 198789, 2012). Moreover, the results of two recent clinical studies (Fluhr et al., Br J Dermatol, 166 (3): 483-90, 2012 and Fluhr et al., Br.
Dermatol, 2014, doi: 10.1111 /bjd.12880) suggest that the skin of infants presents some immaturity on its ability to capture water and regulate the mechanisms associated with it. In addition, these studies have shown that the epidermal barrier is structurally organized from birth to 2 years of age and is therefore not completely competent during this period. This helps explain the fragility of the skin of infants and young children and the susceptibility of it to chemical, physical and microbial attacks.
In addition, an incomplete maturation of the skin can have important clinical consequences. It is therefore important to allow the skin to build and develop properly and harmoniously, otherwise its functional and structural organization could be overburdened. In this respect, it is crucial to preserve the barrier function and the renewal capacity of the epidermis.
Thus, the immaturity of the barrier and the mechanisms of regulation of hydration in the baby's skin could make it even more vulnerable to exogenous or endogenous factors involved in dry skin.
There is always a need, by integrating knowledge about the skin of the child and the role of dehydration to identify and characterize assets and improved formulations to better support the dry skin of children.
DESCRIPTION
The present inventors have shown that the skin of children, and in particular that of infants, is particularly sensitive to dehydration. In particular, they have been able to identify biological markers whose expression is modified in dry skin. Such markers are particularly advantageous because they make it possible to follow the response of the skin under desiccation conditions.
The inventors have developed methods for evaluating the in vitro efficacy of active ingredients and formulations on the prevention of the effects of dehydration for the skin of children, using a skin model specifically capable of reproducing the characteristics of children's skin. , and in particular that of very young children like infants. Prior art studies have relied on the use of adult populations to analyze the skin's response to dehydration (De Benedetto et al., J Allergy Clin Immunol., 127 (3): 773-786, 2011) or reconstructed skin models of adults (Yokota et al., Exp Dermatol., 23 Suppl 1: 27-31, 2014). However, the properties of the skin evolve during the first years of life (see for example Fluhr et al., Exp Dermatol., 19 (6): 483-492, 2010, Fluhr et al., Br J Dermatol, 166 ( 3): 483-90, 2012 and Fluhr et al., Br J Dermatol., 2014, doi: 10.1111 / bjd.12880) and it is unlikely that it is possible to accurately determine the effects of dehydration on the skin children from adult skin samples.
In contrast, the inventors have developed models of reconstructed skin from samples from children and have been able to test the effect of dehydration on these models. They were able to observe that the expression of certain biological markers was impaired when these models of reconstructed skin of children were cultivated under conditions of desiccation. Some markers, such as markers of inflammation were thus more strongly expressed, while the expression of others, such as markers of stem cells or those of the barrier function was decreased. On the other hand, the expression variations of these markers were reduced, or even faded, when the models were treated with active agents or formulations known to treat or prevent dry skin. This result highlights the physiological relevance of these markers. The importance of using reconstructed skin models of children and not adults to isolate such markers is reinforced.
By "dry skin" is meant here a skin that has suffered a loss of water. Dry skin is rough, rough, scaly and uncomfortable skin that is characterized by a deficit in water of the stratum corneum. Dry skin is also known as a xerotic skin.
Dry skin can be caused by prolonged exposure to low humidity so that the dehydration stress increases to such an extent that the normal hydration gradient of the stratum corneum is changed. In this regard, it has been shown that a moisture content in the atmosphere of less than 30% results in dry skin (Sunwoo et al., J Physiol Anthropol, 25: 7-14, 2006). External conditions such as, for example, intense and dry cold, physical or chemical irritations, or sun exposure, can trigger and / or maintain dehydration of the skin.
Dry skin can also develop as a result of physical or chemical changes in the skin, such as aging, leading to changes in the normal moisture gradient. These changes may in particular be caused by pathological situations, such as atopic dermatosis.
By "desiccation condition" is meant here any condition leading to a release of water from the skin. The desiccation conditions according to the invention are therefore conditions which lead a model of normally hydrated child's skin to become dry. The water outlet of said model may be caused by external conditions in which the humidity level in the atmosphere is lowered. It can also be caused by a direct alteration of the barrier function of the skin, for example by an alteration of the Natural Moisturizing Factors (NMF) or the lipid film of the stratum corneum. The inventors have shown that a child skin model cultivated under conditions in which the humidity level in the atmosphere is abruptly lowered has physiological and histological characteristics quite similar to those of dry skin of children. . According to a preferred embodiment, the desiccation conditions according to the invention are conditions in which the humidity level in the atmosphere is lowered compared to usual skin cell culture conditions. More preferably, the humidity level in the atmosphere is less than or equal to 50%. Even more preferably, it is less than or equal to 45%, 40%, 35%, 30% or 25%. According to a particularly preferred embodiment, it is less than or equal to 25%.
By "child" is meant according to the invention an individual whose age is less than 16 years. 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, and the children themselves, who are at least 2 years old. A "newborn", as we understand it here, may as well be born at term as it is premature.
For the avoidance of doubt, the term "child" used in this application without further specification must be understood in its broadest sense, that is, referring to a person under 16 years of age. An "adult" within the meaning of the present invention is a person who is not a child, that is, a person over 16 years of age.
Preferably, the method of the invention can be used regardless of the ethnic or geographical origin of the skin, or the phototype thereof. It can be of Caucasian, African, Asian, South American, Melanesian or other origin; it may also present a phototype I, II, III, IV, V or VI, without this affecting the invention. The aim is to identify biological markers that characterize any type of skin and depend only on the age of the donor.
The methods of the invention are therefore based on the use of a suitable skin model, reproducing the skin of a child, as well as on the use of biological markers, the expression of which is affected by dehydration in a particular way in the skin of children. The invention thus makes it possible to precisely determine which active agents have an advantageous effect on the prevention or treatment of the effects of dehydration of the skin. The methods of the invention are also suitable for evaluating the activity of formulations. The inventors have thus been able to show that certain formulations were more effective than others in preventing and / or limiting the effects of dry skin, thus demonstrating the utility of the approach taken.
According to a first aspect, the subject of the invention is a method for evaluating the in vitro efficacy of an active ingredient or a formulation for preventing or treating the effects of the dehydration of the skin of a child, said method comprising the determination of the level of expression and / or activation of at least one biological marker.
More specifically, the method of the invention preferably comprises the following steps: a) bringing said asset or said formulation into contact with a reconstructed skin model, said model being obtained from a skin sample from a child; b) cultivating the reconstructed skin model of step a) under desiccation conditions; c) measuring the level of expression of at least one biological marker in the skin model of step b); and d) evaluating the effectiveness of said asset or said formulation as a function of the level of step c).
In a first preferred embodiment, the reconstructed skin model is cultured under desiccation conditions in step b) in the presence of the active ingredient or formulation. According to another preferred embodiment, the active ingredient or formulation is removed prior to exposure of said reconstructed skin model to drying during said step b).
Moreover, the subject of the invention is also a method for evaluating the in vitro efficacy of an active ingredient or a formulation in reducing the effects of dehydration on the skin of a child, characterized in that the said The method comprises the following steps: a) culturing under a drying condition a reconstructed skin model, said model being obtained from a skin sample from a child; b) contacting said asset or said formulation with the reconstructed skin model of step a); c) measuring the level of expression of at least one biological marker in the skin model of step b); and d) evaluating the effectiveness of said asset or said formulation as a function of the level of step c). Those skilled in the art will understand without difficulty that steps a) and b) can be performed simultaneously or successively, according to their needs. In other words, the reconstructed skin model can be cultured under desiccation conditions in step b) in the presence of the active ingredient or formulation. Alternatively, the skin model can first be grown under desiccation conditions and then brought into contact with the asset or formulation.
By "the effectiveness of a formulation or an active ingredient in the prevention or reduction of the effects of dehydration of the skin of a child" is meant within the meaning of the present application the capacity of the formulation or the active to cancel or reduce said effects of dehydration of the skin of a child. In the present case, prevention refers to a treatment which is administered before the development of the effects of dehydration, whereas the reduction corresponds to a treatment which is administered once the effects of dehydration have appeared.
According to a more preferred embodiment, the donor of the sample is more particularly a donor having an age of between 0 and 1 month, between 1 month and 2 years or between 2 years and 16 years. In other words, according to this embodiment, the donor of the sample is selected from the group consisting of newborns, whose age is between 0 and 1 month, infants, whose age is between 1 month and 2 years, and children between 2 and 16 years old. More preferably, the donor of the sample is a newborn or an infant.
In a first embodiment, the level of expression of said biological marker of step a) is compared to a reference expression level.
It is important to check that the active ingredients and formulations of the invention are well tolerated. For example, some currently marketed products may cause irritation if used regularly. Such an effect can only worsen a state of dry skin in development or already present.
According to another aspect, the subject of the invention is therefore a method for evaluating the tolerance of an active ingredient or formulation when the child's skin is dried, said method comprising the following steps: a) bringing said active ingredient into contact with each other or said formulation with a reconstructed skin model, said model being obtained from a skin sample from a child; b) cultivating under desiccation conditions the reconstructed skin model of step a) in the presence of the active ingredient or formulation; c) measuring the level of expression of at least one biological marker in the skin model of step b); and d) determining whether said active agent, active ingredient or formulation is well tolerated by the skin of a child as a function of the level of step c).
The method of the invention may further include a comparison of cell viability in the reconstructed skin model treated with the asset, composition or formulation and in the reconstructed skin model, i.e. dehydrated skin but which has not been treated with the active ingredient or formulation. In this case, the active ingredient or the cosmetic formulation is well tolerated by the child's skin if the cell viability of the reconstructed skin model is not affected by the presence of the agent or formulation.
According to another preferred embodiment, the method of the invention therefore comprises an additional step of determining cell viability in the dehydrated reconstructed skin model treated with the active ingredient or the cosmetic formulation, for determining the cell viability of the cell. model of reconstructed skin witness and comparison between the two.
Many tests for determining cell viability are available to those skilled in the art and are commonly used in cosmetology. In particular, the MTT test, described for example in Mosman et al. (J Immunol Methods, 65 (1-2): 55-63, 1983).
In another aspect, the invention makes it possible to isolate formulations or active agents having an effect in preventing the effects of dehydration of the skin of a child. As shown by the experimental examples, the invention makes it possible in particular to distinguish the active agents or the formulations according to their activity in order to prevent the effects of the dehydration of the skin of a child. The invention is therefore particularly suitable for identifying formulations or active agents that are suitable for this very specific skin. The subject of the invention is therefore also a method for identifying an active ingredient or a formulation for preventing the effects of dehydration of the skin of a child, characterized in that said method comprises the following steps: contacting an asset or a candidate formulation with a reconstructed skin model, said model being obtained from a skin sample from a child; b) cultivating under desiccation conditions the skin model of step a); c) measuring the level of expression of at least one biological marker in the skin model of step b); and d) determining whether said active or said candidate formulation is a formulation or an active agent for preventing the effects of dehydration of the skin of children as a function of the level of step c).
In a first preferred embodiment, the reconstructed skin model is cultured under desiccation conditions in step b) in the presence of the active ingredient or formulation. According to another preferred embodiment, the active ingredient or formulation is removed prior to exposure of said reconstructed skin model to drying during said step b).
Similarly, the method of the invention makes it possible to isolate active ingredients or formulations making it possible to reduce the effects of the dehydration of the skin of a child. According to this embodiment, this method comprises the following steps: a) cultivating under a desiccation condition a reconstructed skin model, said model being obtained from a cutaneous sample from a child; b) contacting a candidate asset or formulation with the skin model of step a); c) measuring the level of expression of at least one biological marker in the skin model of step b); and d) determining whether said active or said candidate formulation is a formulation or an asset for reducing the effects of dehydration of the skin of a child as a function of the level of step c). Those skilled in the art will understand without difficulty that steps a) and b) can be performed simultaneously or successively, according to their needs. In other words, the reconstructed skin model can be cultured under desiccation conditions in step b) in the presence of the active ingredient or formulation. Alternatively, the skin model can first be grown under desiccation conditions and then brought into contact with the active ingredient or formulation.
The candidate formulation is a formulation for the prevention or reduction of the effects of dehydration of the skin of a child, 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 according to the nature of the biological marker, to an increase or decrease in the expression of said marker. For example, it may be advantageous to isolate formulations that minimize the effects of dehydration on markers preferentially expressed in stem cells, these formulations making it possible to preserve the renewal capacity of the fragile epidermis of children. Similarly, it would be advantageous to identify formulations that minimize the effects of dehydration on barrier markers in children, in order to maintain the integrity of the skin barrier. Finally, it may be desirable to isolate formulations that do not induce markers of inflammation.
Similarly, the candidate active agent is an asset for the prevention or reduction of the effects of dehydration on the skin of a child, if said active candidate 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 according to the nature of the biological marker, to an increase or decrease in the expression of said marker.
Firstly, the formulation or the asset of interest is brought into contact with a reconstructed skin culture obtained from a sample from a child. This contact of the asset of interest with the skin model can be made directly. Alternatively, it may be advantageous to formulate the asset of interest, for example so as to obtain a liquid composition, to facilitate its contact with the skin model. Thus, according to one embodiment of the invention, the method further comprises a step of formulation of the active agent, in particular in the form of a liquid solution, in particular an aqueous solution, prior to the step of contacting said active ingredient. with a skin model.
The inventors have previously shown that the expression profiles of specific categories of genes (for example, genes for barrier, inflammation, defense, stem cells) change according to age (application WO 2014/009566 ). Those skilled in the art can thus easily characterize the skin at the molecular level from birth to adulthood. More particularly those skilled in the art will note that the cells of the child's skin have a specific expression profile of the genes involved in particular physiological processes, in particular cell metabolism, stress response, inflammation and immunity. , apoptosis, growth / proliferation and cell cycle, cell signaling, migration and differentiation, epidermal barrier, adhesion and pluripotent stem cells of the skin.
Within the meaning of the invention, the reconstructed skin model obtained from a cutaneous sample from a child may be any tissue model comprising skin cells, in particular keratinocytes, and in which said skin cells have been obtained. from a sample from a child.
For the purposes of the invention, the term "skin sample" is intended to mean any sample containing cells of the skin. The cutaneous samples according to the invention thus comprise both the fresh skin explants obtained directly from the patient, the cutaneous skin cell cultures in suspension, the monolayer skin cell cultures, the bilayer skin cell cultures and the tissue models. including reconstructed skin cultures and reconstructed mucosal cultures. Since it is often difficult to work on fresh explants, it is particularly advantageous in the context of the present invention to use skin cell cultures. Advantageously, the cutaneous cells according to the invention comprise normal, healthy or pathological cells, or cells derived from lines. For example, skin cells cultured may be cells obtained from skin tissue explant. By "explant" or "skin explant" is meant herein a sample of cells or skin tissue, which may be made for surgical purposes or for performing analyzes.
In particular, an explant can be obtained during a surgical excision. By "resection" is meant here a surgical procedure of cutting (excise) a more or less wide or deep part of the skin to treat an abnormality or growth. Excision is performed either to remove a cancerous tumor or suspected of being, or to treat a benign skin abnormality that is troublesome, whether for functional or aesthetic reasons. An excision within the meaning of the invention includes for example the skin samples obtained after plastic surgery (mammary plasty, abdominal, facelift, preputial sampling, otoplasty, that is to say, ear gluing, syndactyly or supernumerary finger, etc. .).
An explant can also be obtained by biopsy. By "biopsy" is meant here a sample of cells or skin tissue made for analysis. Several types of biopsy procedures are known and practiced in the field. The most common types include (1) incisional biopsy, in which only a sample of the tissue is removed; (2) excisional biopsy (or surgical biopsy), which consists of the total removal of a tumor mass, thus making a therapeutic and diagnostic gesture, and (3) the needle biopsy, in which a tissue sample is taken with a needle, it can be wide or thin. Other types of biopsies exist, such as smear or curettage, and are also encompassed by the present invention.
Alternatively, 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).
The cutaneous cells according to the invention, whether from a biopsy or obtained by differentiation of stem cells, comprise at least one type of cell usually present in the hypoderm, the dermis and / or the epidermis. . These cells thus comprise, inter alia, keratinocytes, melanocytes, fibroblasts, adipocytes, endothelial cells, mast cells, Langerhans cells and / or Merkel cells. Preferably, the cutaneous 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.
Many methods of culturing cutaneous cells are known to those skilled in the art. Any of these methods can be used to culture the skin cells of the invention. Advantageously, the cutaneous cells are cultured and / or preserved under conditions now, at least partially, a cellular metabolism and / or cellular functions. The skin cell culture according to the invention therefore comprises both cutaneous skin cell cultures in suspension, monolayer skin cell cultures or bilayer skin cell cultures, and tissue models, including reconstructed skin cultures and cultures. of reconstructed mucosa.
For example, suspension cell cultures have been routinely performed in a very large number of laboratories for several decades. Similarly, monolayer or bilayer skin cell cultures have been known and used for a very long time.
Furthermore, many tissue models, including reconstructed skin models and reconstructed mucosal models (Rosdy et al., In Vitro Toxicol., 10 (1): 39-47, 1997; Ponec et al., J. Invest Dermatol., 109 (3): 348-355, 1997, Ponec et al., Int J Pharm., 203 (1-2): 211-225, 2000, Schmalz et al., Eur J Oral Sci., 108 (5): 442-448, 2000. Black et al., Tissue Eng, 11 (5-6): 723-733, 2005, Dongari-Batgtzoglou and Kashleva, Nat Protoc, 1 (4): 2012-2018, 2006 Bechtoille et al., Tissue Eng, 13 (11): 2667-2679, 2007. Vrana et al., Invest
Ophthalmol VisSc, 49 (12): 5325-5331, 2008; Kinicoglu et al., Biomaterials, 30 (32): 6418-6425, 2009; Auxenfans et al., Eur J Dermatol, 19 (2): 107-113, 2009; Kinicoglu et al., Biomaterials, 32 (25): 5756-5764, 2011; Costin et al., Altern Lab Anim, 39 (4): 317-337, 2011; Auxenfans et al., Tissue Eng Regen Med, 6 (7): 512-518, 2012; Lequeux et al., Skin Pharmacol Physiol, 25 (1): 47-55, 2012; EP 29,678; EP 285,471; EP 789,074; EP 1 451 302 B1; EP 1 878 790 B1; EP 1 974 718; US 2007 / 0148,771; US 2010 / 0,099,576; WO 02/070729; WO 2006/063864; WO 2006 / 0.63865; WO 2007/064305) are at the disposal of those skilled in the art and are included in the scope of the invention.
Advantageously, the tissue model includes reconstructed skin models and reconstructed mucosa models. Preferably, the reconstructed skin model is selected from the group comprising dermal models, containing mainly stromal cells, and more particularly fibroblasts, epidermal models consisting mainly of keratinocytes, hypodermic models, skin comprising a dermis and an epidermis, and skin models comprising a dermis, an epidermis and a hypodermis. Models comprising at least one dermis form connective tissues, while models comprising at least one epidermis form stratified epithelia comprising the characteristic layers of the tissue in question. For example, a basal layer (stratum basalis), a thorny layer (stratum spinosum), a granular layer (stratum granulosum), and a horny layer (stratum corneum) can be identified in epidermal models. On the other hand, preferably, the reconstructed mucosal model according to the invention is a mucosal model of the mouth, gum, vagina or cornea.
Advantageously, said model is a dermal matrix connective-type tissue model comprising a matrix support preferably chosen from: an inert support selected from the group consisting of a semi-permeable synthetic membrane, in particular a semi-permeable nitrocellulose membrane , a semi-permeable nylon membrane, a teflon membrane or sponge, a polycarbonate or polyethylene membrane, polypropylene, polyethylene terephthalate (PET) semi-permeable, an inorganic membrane Anopore semi-permeable, acetate or cellulose ester (HATF), semi-permeable Biopore-CM membrane, semi-permeable polyester membrane, membrane or polyglycolic acid film.
This group includes, for example, Skin2 ™ model ZK1100 and Dermagraft® and Transcyte® (Advanced Tissue Sciences) dermal models; treated cell culture plastic (dermal leaf formation: Michel et al., In Vitro Cell, Dev Biol.-Animal, 35: 318-326, 1999); a gel or a membrane based on hyaluronic acid (Hyalograft® 3D - Fidia Advanced Biopolymers) and / or collagen (such as for example an equivalent dermis or collagen lattices) and / or fibronectin and / or fibrin; in this group, for example, there is the Vitn'x® dermal model (Organogenesis); a porous matrix, surfaced or non-surface-treated (for example an equivalent dermis), made from collagen which may contain one or more glycosaminoglycans and / or possibly chitosan (EP0296078A1, WO 01/911821 and WO 01/92322).
In this group, for example, we find the Mimederm® (Coletica) dermal model.
These matrix supports comprise stromal cells, in particular fibroblasts.
Advantageously, said skin model is an epidermis model comprising a matrix support preferably chosen from: an inert support selected from the group consisting of a semi-permeable synthetic membrane, in particular a semi-permeable nitrocellulose membrane, a membrane semipermeable nylon, a teflon membrane or sponge, a polycarbonate or polyethylene, polypropylene, polyethylene terephthalate (PET) semi-permeable membrane, an inorganic Anopore semi-permeable membrane, acetate or cellulose (HATF), a semipermeable Biopore-CM membrane, a semi-permeable polyester membrane; in this group are the reconstructed Epidermis models (Skinethic®) as well as the EpiDerm® model (Mattek Corporation); a film or membrane based on hyaluronic acid and / or collagen and / or fibronectin and / or fibrin.
In this group we can particularly mention the models: Laserskin® (Fidia Advanced Biopolymers), Episkin® (L'Oreal).
These models can be seeded by fibroblasts in the dermal part.
These models, in which fibroblasts may or may not be integrated, serve as a support for the seeding of keratinocytes and the reconstitution of the epidermis. Advantageously, are introduced, in addition to keratinocytes, pigment cells, immunocompetent cells, nerve cells; preferably the immunocompetent cells are Langerhans cells.
Advantageously, said tissue model is a reconstructed skin or mucosal tissue model comprising a dermal or chorion matrix support preferably chosen from: an inert support selected from the group consisting of a semi-permeable synthetic membrane, in particular a membrane of semi-permeable nitrocellulose, a semi-permeable nylon membrane, a teflon membrane or sponge, a polycarbonate or polyethylene membrane, polypropylene, semi-permeable polyethylene terephthalate (PET), an inorganic membrane Anopore semi-permeable, d acetate or cellulose ester (HATF), a semipermeable Biopore-CM membrane, a semi-permeable polyester membrane, said inert support containing or not stromal cells, in particular fibroblasts, a collagen-based gel and / or hyaluronic acid and / or fibronectin, and / or fibrin comprising stromal cells, in particular fibroblasts, a pore matrix use, surfaced or non-surfaced, made from collagen which may contain one or more glycosaminoglycans and / or possibly chitosan, these porous matrices incorporating stromal cells, in particular fibroblasts, a desepidermized or dermal dermis, human or animal.
In this group, Mimeskin (Coletica), EpidermFT ™, EpiAirway ™, EpiOccular ™ EpiOral ™, EpiGingival ™, EpiVaginal ™ (MatTek Corporation), Human Corneal Epithelium (HCE), Human Oral Epithelium (HOE), Human Gingival Epithelium (HGE), Human Vaginal Epithelium (HVE) (Skinethic®), Phenion® Full Thickness Skin Model (Phenion) Apligraf® (Organogenesis), ATS-2000 (CellSystems® Biotechnology Vertrieb) and Skin 2TM (ZK1200-1300 2000 Advanced Science).
There are also models dedicated to tissue therapy that can also be used in the context of the present invention. Epidex (Modex Therapeutics), Epibase® (Juniper Lab), Epicell ™ (Genzyme), Autoderm ™ and Transderm ™ (Innogenetics) models can be cited.
The matrix support is then inoculated with keratinocytes to reconstruct the epidermis and finally obtain a reconstructed skin.
Advantageously, 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 hair, hair, sebaceous glands.
After exposing the reconstructed skin model of the invention to desiccation conditions, one skilled in the art can measure the level of expression of the biological markers of the invention.
For the purpose of this application, the term "biological marker" is intended to mean a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacological responses to a therapeutic intervention. A biological marker therefore refers to a variety of different substances and parameters. For example, a biological marker may be a substance whose detection indicates a particular disease state (for example the presence of activated protein C as a marker of infection), or on the contrary a substance whose detection indicates a physiological state specific. 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.
According to one embodiment of the present invention, the biological marker is a gene, the products of a gene such as transcripts or peptides, a lipid, a sugar or a metabolite whose expression changes, in particular level of expression, correlate with a physiological state of the child's skin. Those skilled in the art seeking to determine which class a gene or protein marker belongs to can easily consult the relevant scientific literature or refer to public databases such as, for example, those grouped on the website of the National Center for Biotechnology. Information (http://www.ncbi.nlm.nih.gov/guide/).
The inventors have particularly selected markers whose variation in the level of expression varies after dehydration surprisingly and unexpectedly in children. The markers selected are therefore of particular interest in the context of the method of the invention, insofar as their level of expression is measured on a skin model reproducing the characteristics of the skin of a child.
In particular, the inventors have shown that the desiccation conditions affect the amount of lipids and NMFs produced by a child skin model. In addition, the inventors have shown that at the same time, the expression of the markers of the barrier decreases, as well as those of the NMF synthesis. Finally, the markers of the stem cells are also affected and therefore the renewal capacity of the epidermis. On the other hand, bringing the skin into contact with an active formulation against the effects of dehydration of the skin makes it possible to prevent and correct the variations in the expression of said markers, which underlines the relevance of these.
The biological marker of the invention is therefore advantageously a marker selected from the group of lipids of the stratum corneum, in particular ceramides, NMFs, markers of inflammation, markers of the barrier function and markers preferentially expressed in cells. strains.
In particular, the inventors have shown that the amount of lipid in the skin decreases when the child skin model is cultured under desiccation conditions. In the horny layer, the lipids are arranged in lamellar planes in the spaces between the corneocytes, thus forming a cement which contributes to the protection of the skin against external aggressions and to the maintenance of a good level of intraepidermal water. These lipids are phospholipids, cholesterol and glucosylceramides, which are modified in the intercorneocyte spaces, by specialized enzymes, ceramides, cholesterol, cholesterol sulfate and free fatty acids. (Jungerstend et al., Contact Dermitis, 58 (5): 255-262, 2008). Ceramides, sterols and free fatty acids are also found in sebum, among other components. Finally, the surface of the skin is covered with a protective film that allows the skin to maintain its hydration and protect itself against external aggressions. This skin surface film includes, inter alia, a hydrolipidic film, which consists essentially of sweat, water, sebum and other lipids. These include ceramides, triglycerides and fatty acids in approximately equal proportions.
According to a particular embodiment, the biological marker according to the invention is therefore a lipid marker. By "lipids" is meant any natural liposoluble (i.e., lipophilic) molecule. Lipids are a heterogeneous group of compounds with many essential biological functions. The lipids may be defined more particularly as small hydrophobic or amphiphilic molecules, which originate wholly or in part from ketoacyl or isoprene groups. For an overview of all lipid classes, refer to "Lipid Metabolites and Pathways Strategy (LIPID MAPS) Classification System" (National Institute of General Medical Sciences, Bethesda, MD). In particular, the lipid marker according to the invention is chosen from ceramides, phospholipids, glucosylceramides, sterols, triglycerides and free fatty acids. Even more preferably, the lipid marker according to the invention is chosen from ceramides.
A "ceramide" according to the invention is a lipid resulting from the amidification reaction of sphingosine and a fatty acid. A ceramide thus consists of a sphingosine or phytoshingosine base linked by an amide bond to α-hydroxy, ω-hydroxy or non-hydroxy acids having variable hydrophobic chain lengths. In the human stratum corneum, 9 classes of ceramides, called CERs 1 to 9, have been identified (see for example Dayan, Cosm & Toil, 121 (1): 37-44, 2006; Jungerstend et al., Contact Dermitis, 58 (5): 255-262, 2008, Farwick et al., Cosm et al., 124 (2): 63-72, Masukawa et al., J Lipid Res., 50 (8): 1708-1719, 2009) . The ceramide according to the invention is more preferably chosen from the group consisting of the ceramides CER1 to 9. On the other hand, the present inventors have shown that the NMFs are affected by the desiccant conditions. The natural moisturizing factor, also called NMF (Natural Moisturizing Factor) is derived from the proteolysis of filaggrin according to a cascade of reactions involving enzymes including caspase 14 and peptidylarginine deiminase (PAD1). NMF is a mixture of hygroscopic substances having the property of retaining water (Fluhr et al., Exp DermatoL, 19 (6): 483-492, 2010). Among them, the sodium salt of pyrrolidone carboxylic acid or PCA Na (from the cyclization of glutamic acid released by decomposition of profilaggrin) and lactates are the most hygroscopic substances. NMF also includes free amino acids (serine, citrulline ...), citrates and formates, urea, ions, nitrogen, uric acid, glycosamine, creatinine , phosphates, as well as compounds not yet identified. The amount of NMF can be measured by any method known to those skilled in the art, in particular by Raman microspectroscopy. According to another preferred embodiment, the biological marker according to the invention is therefore the NMF.
The inventors have thus shown that markers of inflammation are particularly expressed after the skin of a child has been incubated in a dry atmosphere. Inflammation is a normal defense reaction of the body, but it can contribute to the decrease of the integrity of the skin. In addition, the inventors have shown that at the same time, dehydration induces in a child skin model a reduction in the expression of the markers of the barrier function and the markers preferentially expressed in the stem cells.
For the purposes of the invention, the term "markers of cutaneous inflammation" is intended to mean markers whose expression variation correlates with cutaneous inflammation.
The term "inflammation" according to the invention means all the reaction mechanisms of defense by which the body recognizes, destroys and eliminates all substances that are foreign to it. "Inflammation of the skin" corresponds more particularly to a reaction of the immune system in response to an attack on the skin, such as an attack on the environment, causing or not a wound, or even vascular damage if any. Skin inflammation may manifest as erythema, characterized by redness associated with local vasodilatation, edema, characterized by swelling, and feeling of warmth. In addition, cutaneous inflammation is accompanied by a variation in the level of expression or concentration of gene or protein markers well known to those skilled in the art, which may refer for example to Vahlquist (Acta Derm Venereol; : 161, 2000).
The onset and continuation of inflammation, its diffusion from the initial focus, involve factors that are synthesized locally or are inactive as precursors in the circulation. Depending on the type of mediators synthesized, one can differentiate particular processes in the inflammation reaction. Thus, the skin inflammation according to the invention groups together at least three distinct processes, protein inflammation, lipid inflammation and neurogenic (or neurogenic) inflammation.
By "protein inflammation" or "nonspecific inflammation" is meant here the production in response to external aggression of inflammatory protein mediators, such as cytokines IL-1, IL-2, IL-6, IL8 and TNFα, the complement, or the proteins involved in coagulation, if any. By "lipidic inflammation" is meant here the production of said external aggression of lipid mediators, in particular prostaglandins and leukotrienes which are both synthesized from arachidonic acid, as well as the activation of the enzymes responsible for this production ( Shimizu, Annu Rev Pharmacol Toxico, 49: 123-150, 2009). The protein and lipid mediators thus produced will induce a cascade of reactions within the skin involving other inflammatory cells, in particular, immune and vascular cells. The clinical result is reflected in particular redness or edema.
In response to external aggression, the neurosensory system may be stimulated and associated with the inflammatory response then involving other cellular actors such as nerves (or nerve endings) and cells such as mast cells. By "neurogenic inflammation" is meant here the release by the nerve terminals, in response to external aggression, of specific mediators, including neuropeptides (including tachykinins including substance P, and Calcitonin Gene-Related Peptide or CGRP); also involved in the neurogenic inflammation according to the invention, the activity of particular receptors such as the substance P receptor or the TRPV1 receptor. Neurogenic inflammation most often results in a sensation of pain and / or discomfort and / or itching sensation (pruritus).
The marker of cutaneous inflammation according to the invention is preferably chosen from markers of protein inflammation, markers of lipid inflammation and markers of neurogenic inflammation.
Preferably, the protein inflammation marker is selected from the group consisting of interleukins, preferably IL1a and IL8. Human IL-1 interleukin has a protein sequence represented by the NCBI reference sequence: NP_000566. This protein is encoded by the human IL1A gene (reference NCBI: Gene ID: 3552), whose sequence corresponds to the reference NCBI: NM_000575. The protein sequence of human IL-8 interleukin 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 is accessible under the reference NCBI: NM_000584.
The NGFR nerve growth factor receptor has a protein sequence represented by the reference sequence NP_002498. This protein is encoded by the NGFR gene (NCBI reference: Gene ID: 4804). The sequence of this is accessible under the reference NCBI: NM_002507.
The OSMR receptor ("Oncostatin-M specifies receptor subunit beta" or "oncostatin M receptor") is a type I cytokine receptor, whose ligand is oncostatin. The OSMR protein corresponds to the sequence represented by the accession number Genbank NP_001161827, and is encoded by the OSMR gene (reference NCBI: Gene ID: 9180), of sequence NM_001168355.
The protein F2RL1 (coagulation factor II (thrombin) receptor-like 1), which is also called PAR2 (Protease activated receptor 2) or GPR11 (G-protein coupled receptor 11), is a receptor that is encoded by the F2RL1 gene (reference NCBI : Gene ID: 2150) in humans. This receptor, which is part of the family of receptors with 7 transmembrane domains or GPCR, plays a role in the modulation of inflammatory responses. The sequence of this protein is accessible under the reference NCBI: NP_005233, while the sequence of the F2RL1 gene can be obtained with the accession number Genbank NM_005242.
The marker of lipid inflammation is advantageously chosen from prostaglandins, in particular prostaglandin E2, and the enzymes for the synthesis thereof from arachidonic acid, in particular PTGS2.
Prostaglandin E2 (PGE2) is a well-known derivative of arachidonic acid obtained by the action of cyclooxygenase. There are two isoforms of cyclooxygenases (COX): cyclooxygenase 1 which is constitutive in tissues and cyclooxygenase 2 which is induced by inflammatory phenomena. Proinflammatory stimulation (trauma, cytokines ...) leads to the synthesis of PGE 2 which is responsible for vasodilation (generating redness and edema), nociceptor sensitization to bradykinin and histamine (responsible for pain) and fever (with IL1 and IL6 cytokines). The enzyme cyclooxygenase 2, also called prostaglandin-endoperoxide synthase (PTGS), is encoded by the human PTGS2 gene (reference NCBI: Gene ID: 5743). The sequence of this gene is available under the reference NCBI: NM_000963 and the sequence of the protein under the reference NCBI: NP_000954.
Preferably, the marker of neurogenic inflammation is selected from neuropeptides and neuropeptide receptors, in particular TRPV1 and SRP receptors. TRPV1 receptor (Transient Receptor Potential Vanilloid 1) is a cationic channel type protein of the TRP family and NCBI reference sequence: NP_061197. In the skin, TRPV1 is expressed by keratinocytes, mast cells and nerve fibers. In response to an attacker, the activation of TRPV1 leads to the production of cytokines and neuropeptides and is an actor of neurogenic inflammation. The gene coding for the TRPV1 receptor is the TRPV1 gene (reference NCBI: Gene ID: 7442), the sequence of which is for NCBI reference: NM_018727. The TRPV3 gene, (NCBI reference: Gene ID: 162514), whose sequence has for reference NCBI: NM_001258205, encodes a receptor of the same family as TRPV1. The sequence of this TRPV3 receiver corresponds to the reference sequence NP_001245134.
Protachykinin-1 is a protein that is encoded in humans by the TAC1 gene (NCBI reference: Gene ID: 6863), the sequence of which is NCBI: NM_003182. The sequence of this protein is accessible under the reference NCBI: NP_003173. The SPR receptor (substance P receptor, also known as neurokinin 1 receptor, NK1R, or tachykinin receptor 1, TACR1) is a G protein-coupled receptor (GPCR) that transmits the signal of substance P (SP) and other tachykinins. . It is encoded by the human TACR1 gene whose sequence is for reference NCBI: NM_001058. Its peptide sequence is the NCBI reference sequence: NP_001049.
The MRGPRD receptor is a receptor of the transmembrane domain receptor family, also called GPCRs. The MRGD receptor (Mas-related G-protein coupled receptor member D) is more particularly expressed in a subpopulation of sensory neurons that participate in the detection of pain. This receptor is encoded by the gene encoded by the human MRGPRD gene (NCBI reference: Gene ID: 116512). The sequence of this gene is available under the reference NCBI: NM_198923 and the sequence of the protein under the reference NCBI: NP_944605.
Phospholipases 2A are an enzyme family that release proinflammatory mediators (lysophospholipids, oxidized fatty acids) by hydrolysis of short-chain oxidized phospholipids present on oxidized low-density lipoproteins (LDL). Of these, the Phospholipase A2 Group II F is encoded by the human PLA2G2F gene (NCBI reference: Gene ID: 64600). The sequence of this gene is available under the reference NCBI: NM_022819 and the sequence of the protein under the reference NCBI: NP_073730.3
According to the invention, the skin inflammation marker is therefore preferably selected from the group consisting of IL1A, IL8, PTGS2, NGFR, TAC1, TAC1R, TRPV1, TRPV3, MRGPRD, OSMR, PLA2G2F and F2RL1.
The present inventors have furthermore shown that dehydration induces a reduction in the expression of the markers of the barrier function and the markers preferentially expressed in the stem cells.
The "barrier markers" according to the invention include markers that are specifically expressed in the outermost epidermis layers and that participate in the barrier function.
As is well known to those skilled in the art, the main function of the skin is to establish a protective barrier against environmental damage while allowing certain exchanges between the internal environment and the external environment. This barrier function is mainly carried by the stratum corneum of the epidermis.
The intercellular lipids and corneodesmosomes as well as the horny corneocyte envelope are the key components.
However, under the stratum corneum, tight junctions constitute a second line of barrier function. These junctions constitute at the level of the stratum granulosum a barrier of selective paracellular diffusion preventing the penetration of deleterious molecules. The tight junctions are composed of different transmembrane proteins such as claudines, occludin and Z01.
The barrier functions carried by the stratum corneum and the tight junctions are closely related. Indeed, the alteration of one or the other can influence the formation of the other.
Preferably, the markers of the barrier function according to the invention are markers expressed in the stratum corneum or markers expressed in the tight junctions of stratum granulosum. In a more preferred embodiment, said epidermal barrier marker is selected from the group consisting of desmoglein 1 (DSG), sciellin (SCEL), peptidyl arginine deiminase 1 (PADI1), caspase 14 (CASP14), lon'crine (LOR), transglutaminase 1 (TGM1) and Claudine 1 (CLDN1).
The corneodesmosome is the only junctional structure of the stratum corneum, which emphasizes the importance of this structure for maintaining the integrity of the stratum corneum. Desmoglein 1 is a protein constituting corneodesmosomes whose sequence is available under the reference NP_001933. The DSG1 gene (NCBI reference: Gene ID: 1828) encodes desmoglein-1 and has the NM_001942 reference sequence. Sciellin, of sequence NP_001154178, and encoded by the SC gene (reference NCBI: Gene ID: 8796) which itself has the sequence NM_001160706 is a precursor of the horny envelope. Loricrin is a major protein component of the horny envelope, of which it accounts for about 70% by weight. This protein has the sequence NP_000418 and is encoded by the LOR gene (reference NCBI: Gene ID: 4014) of sequence NM_000427. Formation of the bonds between the protein components of the envelope of the horny envelope is provided by the enzyme transglutaminase 1. The sequence of this enzyme corresponds to that which can be found under the reference NP_000350. The TGM1 gene (NCBI reference: Gene ID: 7051) encodes desmoglein-1 and has the NM_000359 reference sequence.
The tight junctions represent one of the modes of cellular adhesion, in the epithelial tissues, They block the circulation of fluids between the cells and thus ensure the seal between two tissue compartments. They are localized at the apex of the epithelial cells where they form a continuous band all around which ensures the sealing. The CLDN1 gene (NCBI reference: Gene ID: 9076) encodes Claudine protein 1 which is one of the most important components of tight junctions. This protein has a sequence corresponding to that whose reference NCBI is NP_066924. The sequence of the CLDN1 gene is accessible under the reference NM_021101.
Barrier markers also include markers of NMF metabolism and intercorneocyte lipids. These compounds retain water in the epidermis during its ascent to the stratum corneum. Peptidyl arginine Deiminase 1 (PADI1) and caspase 14 (CASP14) are two enzymes involved in the maturation of filaggrin which makes it possible to obtain NMF. Peptidyl-arginine deiminase 1, of sequence NP_037490, is an enzyme which removes an imine function on filaggrin and keratin K1, which maintains the hydration of the stratum corneum, and consequently the skin barrier function. This enzyme is encoded by the PADI1 gene whose sequence (reference NCBI: Gene ID: 29943) can be found under the reference NM_013358. Caspase-14 is a member of the caspase family which is necessary for the degradation of filaggrin to NMF (Hoste et al., J. Invest Dermatol 131 (11): 2233-2241, 2011). This protein, of sequence NP_036246, is encoded by the CASP14 gene (reference NCBI: Gene ID: 23581) whose sequence is under the reference NM_012114.
The "preferentially expressed markers in stem cells" according to the invention include markers, and more specifically genes and proteins, which are specifically present in epidermal stem cells.
For the purposes of the present invention, the term "epidermal stem cell" or "epidermal stem cell" is intended to mean a cell of the epidermis capable of long-term renewal. The epidermal stem cells of the invention include, inter alia, follicular stem cells, sebaceous stem cells and basal stem cells, the latter also known as interfollicular epidermal stem cells. The term "follicular stem cells", "sebaceous stem cells" and "basal stem cells" within the meaning of the invention means stem cells situated respectively in the bulge / bulge region of the hair follicle, in the sebaceous glands and in the basal layer of the epidermis. In a preferred embodiment of the invention, the epidermal stem cells of the invention are basal stem cells.
More specifically, the term epidermal stem cell in the sense of the present invention, a cell with a high potential for long-term renewal. By "renewal potential" is meant the ability to undergo at least one cell division cycle. A "high potential for long-term renewal" therefore represents the ability of a cell to enter successive cycles of cell division. It is well known that differentiated cells of the skin are not able to perform several successive divisions (Fortunel and Martin, J Soc Biol, 202 (1): 55-65, 2008). It is understood here that "successive" does not mean "consecutive" and that there may be periods during which a stem cell according to the invention remains quiescent without losing its high potential for long-term renewal.
The preservation of a high long-term renewal potential is expressed by an asymmetric division producing two different cells. The first daughter cell is a stem cell identical to the parent stem cell, while the second is a transient amplification cell that divides in a limited way over a short period of time and then enters the differentiation process. Advantageously, the epidermal stem cells of the invention are therefore also capable of generating at least one type of epidermal cell by differentiation. In other words, the transient amplification cell is capable of giving at least one type of epidermal cell by differentiation. Preferably, said epidermal cell is a keratinocyte. More preferably, the transient amplification cell is capable of giving all types of epidermal cells by differentiation.
Preferably, the markers expressed in the stem cells are markers that participate in the functions and protection of the stem cells. For example, the markers ΔΝρ63, BIRC5 (survivin), FN1 (fibronectin 1), MCSP (Melanoma-associated Chondroitin Sulfate Proteoglycan), LRIG1 (leucine-rich repeats and immunoglobulin-like domains protein 1), GJA1 (connexin 43), NID1 (nidogen 1), KRT15 (keratin 15), KRT19 (keratin 19), EGFR (epidermal growth factor receptor), CD71 (transferrin receptor), DSG3 (desmoglein 3), ITGB1BP1 (integrin betal binding protein), ITGA6 (integrin alpha 6), ITGB1 (integrin betal) and ITGB4 (integrin beta 4) or markers involved in the signaling and regulation of stem cell activity such as Wnt / beta catenin, hedgehog sonata (SHH), NOTCH1 (Notch homolog 1 translocation-associated). The markers ΔΝρ63 and survivin are markers of resistance to apoptosis, thus having a role in the survival of stem cells. Cytokeratin 15 and 19 are positive markers of stem cells, cytokeratin 15 being a marker of survival of these. MCSP colocalizes with integrins in cells that do not divide, while integrin betal (extracellular matrix adhesion marker of the basement membrane) and integrin alpha 6 (constituting hemidesmosomes binding marker of keratinocytes with each other) are surface proteins that participate in intercellular communication, regulate differentiation / proliferation processes, as well as interaction with the niche. The CD71 transferin receptor is a known surface marker of stem cells, which is used to isolate, in a population of integrin-alpha positive cells, high clonogenic potency cells. Finally, Lrigl is an antagonist of the Epidermal Growth Factor (EGFR) receptor, thus maintaining the quiescent stem cells, whereas the EGFR receptor, which is a marker whose absence characterizes the stem cells, on the contrary leads the cells the path of proliferation.
Preferably, the marker preferentially expressed in the stem cells is chosen from the group consisting of the markers ΔΝρ63, KRT15 (keratin 15), KRT19 (keratin 19), BIRC5 (survivin), and N0TCH1 (Notch Homolog 1). These markers are well known to those skilled in the art. The genes KRT15 (reference NCBI: Gene ID: 3866), KRT19 (reference NCBI: Gene ID: 3880), BIRC5 (reference NCBI: Gene ID: 332) and N0TCH1 (reference NCBI: Gene ID: 4851) thus correspond to the sequences represented. by the following Genbank accession numbers: NM_002275, NM_002276, NM_001012270, and NM_017617, respectively. The keratin 15, keratin 19, survivin and homolog 1 Notch proteins correspond to the sequences represented by the following Genbank accession numbers: NP_002266, NP_002267, NP_001012270 and NP_060087, respectively. The ΔΝρ63 protein is an isoform of the TP63 protein (Tumor protein 63) obtained by transcription from an internal promoter located in an intron. This protein corresponds to the sequence represented by the Genbank accession number: NP_001108452, and is encoded by the TP63 gene (NCBI reference: Gene ID: 8626), the sequence of which is available under the following Genbank accession number: NM_001114980.
It will also be obvious to those skilled in the art that the method of the invention will allow an evaluation of the effectiveness of the formulation or the more complete asset that a large number of markers of different types will be used. .
According to a preferred embodiment, the method of the invention comprises a step c) of measuring the level of expression of a combination of biological markers. Said combination according to the invention comprises at least two markers, said markers being chosen from at least two different categories of markers described above: NMF, lipids, including ceramides, inflammation markers, barrier markers and markers of stem cells. According to a preferred embodiment, said combination comprises more than two markers. According to a more preferred embodiment, each of the markers belongs to a distinct category of markers described above. It is also possible to use marker combinations as defined above, in which certain classes of markers are represented by more than one marker. The use of marker combinations comprising at least one marker of each of the various types indicated above is particularly advantageous.
For each of these markers, the term "level of expression" refers to the cell concentration of said marker. Thus, the expression level of the ceramides corresponds to the concentration of said lipids in the cell. If the marker is a gene, the "level of expression" within the meaning of the invention corresponds to the cellular concentration of at least one of the gene products of said marker. More specifically, the level of expression of said biological marker corresponds to the amount or the cell concentration of the transcript of said gene or of the protein derived from said transcript. According to a preferred embodiment, the level of expression of said biological marker corresponds to the quantity or the cellular concentration of the transcript of said gene. According to another embodiment, the level of expression of said biological marker corresponds to the quantity or the cellular concentration of the protein resulting from said transcript.
By "measuring the level of expression of a combination of biological markers" is meant within the meaning of the present application the measurement of the level of expression of each of the markers of the combination. 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, for example by measuring the quantity of proteins derived from said transcripts. Thus, by "measuring the level of expression of said gene" is meant in the sense of the invention the measurement of the amount of product of the gene in its peptide form or in its nucleotide form.
In general, the expression of the biological marker according to the invention will be detected in vitro from the reconstructed skin model.
In a particular embodiment, the method of the invention may comprise one or more intermediate steps between obtaining the reconstructed 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, NMF sample, mRNA sample (or corresponding cDNA) or protein sample. This can then be directly used to measure the expression of the marker. Preparation or extraction of mRNA (as well as retrotranscription thereof into cDNA), proteins, lipids or NMF from a skin cell sample are only routine procedures well known in the art. the skilled person.
Once a sample of mRNA (or corresponding cDNA) or protein is obtained, the expression of the marker at either the mRNA level (i.e., all mRNAs or cDNAs) present in the sample), or proteins (i.e., all the proteins present in the sample), can be measured. The method used to do this depends on the type of transformation (mRNA, cDNA or protein) and the type of sample available.
When the expression of the marker is measured at the level of the mRNA (or corresponding cDNA), any technology usually used by those skilled in the art can be implemented. These technologies for analyzing the level of gene expression, such as, for example, transcriptome analysis, include well-known methods such as PCR (Polymerase Chain Reaction, based on DNA) and RT-PCR (reverse transcription). Transcription-PCR, starting from RNA) or quantitative RT-PCR or nucleic acid chips (including DNA chips and oligonucleotide chips) for higher throughput.
By "nucleic acid chips" is meant herein several different nucleic acid probes that are attached to a substrate, which may be a microchip, a glass slide, or a microsphere-sized bead. The microchip may consist of polymers, plastics, resins, polysaccharides, silica or a material based on silica, carbon, metals, inorganic glass, or nitrocellulose.
The probes can be nucleic acids such as cDNAs ("cDNA chips"), mRNAs ("mRNA chips") or oligonucleotides ("oligonucleotide chips"), said oligonucleotides typically having a length of between about 25 and 60 nucleotides.
To determine the expression profile of a particular gene, a nucleic acid corresponding to all or part of said gene is labeled and 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 surface of the chip that are complementary to this nucleic acid. The presence of labeled hybridized complexes is then detected.
These technologies make it possible to monitor the level of expression of a particular gene or of several genes or even of all genome genes (full genome or full transcriptome) in a biological sample (cells, tissues, etc.). These technologies are used routinely by those skilled in the art and therefore there is no need to detail them here. Examples of embodiments of the invention based on gene expression analysis (cDNA microarrays) and on quantitative PCR are described in the experimental section.
Alternatively, it is possible to use any current or future technology to determine gene expression based on the amount of mRNA in the sample. For example, one skilled in the art can measure the expression of a gene by hybridization with a labeled nucleic acid probe, for example by Northern blot (for mRNA) or by Southern blot (for cDNA) but also by techniques such as the Serial Analysis Method of Gene Expression (SAGE) and its derivatives, such as LongSAGE, SuperSAGE, DeepSAGE, etc. It is also possible to use fabric chips (also known as TMAs: "tissue microarrays"). The tests usually used with tissue chips include immunohistochemistry and fluorescent in situ hybridization. For mRNA analysis, the tissue chips can be coupled with fluorescent in situ hybridization. Finally, it is possible to use bulk sequencing in parallel to determine the amount of mRNA in the sample (RNA-Seq or Whole Transcn'ptome Shotgun Sequencing). For this purpose, several methods of mass sequencing in parallel are available. Such methods are described in, for example, US 4,882,127, U.S. 4,849,077; U.S. 7,556,922; U.S. 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 And Ji, Nat Biotechnol., 26 (10): 1135-45. 2008; Pihlak et al. Nat Biotechnology, 26 (6): 676-684, 2008; Fuller et al., Nature Biotechnol., 27 (11): 1013-1023, 2009; Tuesdays, Genome Med., 1 (4): 40, 2009; Metzker, Nature Rev. Genet., 11 (1): 31-46, 2010.
When the expression of the marker is measured at the protein level, it is possible to use specific antibodies, in particular in well-known technologies such as immunoprecipitation, immunohistology, western blot, dot blot, ELISA or the ELISPOT, the protein chips, the antibody chips, or the tissue chips coupled to immunohistochemistry. Other techniques that can be used include FRET or BRET techniques, microscopy or histochemistry methods, including confocal microscopy and electron microscopy, methods based on the use of one or more several excitation wavelengths and a suitable optical method, such as an electrochemical method (voltammetry and amperometry techniques), the atomic force microscope, and radiofrequency methods, such as multipole resonance spectroscopy, confocal and non-confocal, fluorescence detection, luminescence, chemiluminescence, absorbance, reflectance, transmittance, and birefringence or refractive index (for example, by surface plasmon resonance, or "plasmon resonance surface" in English, by ellipsometry, by resonant mirror, tec.), flow cytometry, radioisotopic or magnetic resonance imaging, ana polyacrylamide gel electrophoresis lysis (SDS-PAGE); by HPLC-Mass spectrophotometry, by liquid chromatography / mass spectrophotometry / mass spectrometry (LC-MS / MS). All these techniques are well known to those skilled in the art and it is not necessary to detail them here.
If the biological marker is a lipid, especially a ceramide, the skilled person 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 gas chromatography (HPLC, see, for example, Sullivan et al., Arch OphthalmoL, 120 (12): 1689-99, 2002), for example coupled to an evaporative diffraction detector of the invention. light (HPLC-ESD, see Nordbass et al., J. High Resolut Chromatogr., 22: 483-486, 1999, Torres et al., J. Chromatogr A, 1078: 28-34, 2005); thin layer chromatography (TLC, for example, Downing et al., J. Invest Dermatol., 77 (4): 358-360, 1981; Nordstrom et al., J. Invest Dermatol., 87 (2): 260-263, 1986); nuclear magnetic resonance (NMR, see for example Robosky et al., J Lipid Res., 49 (3): 686-692, 2008); in vivo confocal microspectroscopy of Raman; mass spectrometry, gas chromatography coupled with mass spectrometry (GC-MS, see O'Neill et al., J. Chromatogr Sci., 14 (1): 28-36, 1976); liquid chromatography coupled with mass spectrometry (see for example van Smeden et al., J Lipid Res, 52 (6): 1211-1221, 2011); high performance liquid chromatography (UPLC, see Rainville et al., J. Proteome Res., 6 (2): 552-558, 2007; Castro-Perez et al., J. Proteome Res., 10 (9): 4281-4290, 2011). We can also analyze the organization of these lipids in the skin and more particularly in the stratum corneum (or stratum corneum), lamellar or lateral organization, by techniques such as X-ray diffraction (Bouwstra et al., J Invest Dermatol 97 (6): 1005-1012, 1991, van Smeden et al., J Lipid Res., 52 (6): 1211-1221, 1991) or by Fourier transform infrared spectroscopy (Gorcea et al. , Int J Pharm, Nov. 10, 2011.) or by morphometric analysis by electron microscopy (Daehnhardt-Pfeiffer et al., Skin Pharmacol Physiol., 25 (3): 155-161, 2012) or by electron microscopic analysis vitreous skin section combined with molecular analysis (Iwai et al., J Invest Dermatol., Apr. 26, 2012).
The NMF assay is a procedure well known to those skilled in the art. In particular, it is possible to assay the NMF by the use of in vivo confocal microspectroscopy of Raman. This is a procedure commonly used in the field for at least 15 years. For example, the publications of Caspers et al. (J Invest Dermatol., 116 (3): 434-442, 2001), Vyumvuhore et al. (J Biomed
Opt., 19 (11): 111603, 2014), or Falcone et al. (Skin Pharmacol Physiol, 28: 307-317, 2015). It is also possible to determine the NMF by liquid chromatography coupled with mass spectrometry. For example, reference may be made to Piraud et al. (Rapid Commun Mass Spectrom, 19 (12): 1587-602, 2005), Petritis et al. (Journal of Chromatography A, 833 (2): 147-155, 1999), Henriksen et al. (J Am Soc Mass Spectrom, 16 (4): 446-455, 2005) or Yang (Application of Biophysics and Bioengineering to the assessment of skin barrier function.Thesis (Doctor of Philosophy (PhD)) University of Bath, UK , 2011).
By "a level of reference expression of a biological marker" is meant within the meaning of the present application any level of expression of said marker used for reference. For example, a reference expression level can be obtained by measuring the level of expression of the marker of interest in a child skin model under particular conditions. Those skilled in the art will be able to choose these particular conditions according to their objective during the implementation of the invention.
According to another embodiment, the reference level of expression of a biological marker is the level of expression of said marker obtained in a child skin model, brought into contact with a formulation or the reference asset, and exposed to desiccation conditions.
When the reference expression level is an expression level obtained in a skin model exposed to desiccation conditions, those skilled in the art will readily understand that the desiccation conditions of the skin model used in the method of the present invention. The invention and the model used to obtain a reference expression level are preferably identical. Thus, preferably, the drying conditions used, in particular the humidity level in the atmosphere, as well as the duration of the exposure used in the method of the invention and the model used to obtain a reference expression level are preferentially identical.
For example, one skilled in the art can use as a reference formulation any known formulation of the prior art for its effect in the prevention or treatment of dry skin.
Preferentially, the reference formulation is an oil-in-water emulsion, such as those described below. More preferably, the reference formulation is formulation A, which contains avocado perseoses. These are C7 sugars obtained from avocado. Perseoses of avocado and their extraction processes are described in, inter alia, WO 2004/112742, WO 2005/105123, WO 2005/115421, WO 2008/025847, WO 2011/073281, WO 2014/017049, WO 2014 / 122326, WO 2015/044230, etc. PRODUCT A: EMULSION E / H type Cold Cream
PRODUCT B: EMULSION E / H type Cold Cream
PRODUCT C: EMULSION E / H type Cold Cream
PRODUCT D: EMULSION E / H type Cold Cream
PRODUCT E: COLD CREAM MILK
Product F: Cold Cream Wash Gel
According to another preferred embodiment, the level of reference expression of a biological marker is the level of expression of said marker obtained in a skin model obtained from a cutaneous sample from a child, said model being untreated with the formulation or the asset of interest, and not exposed to desiccation conditions.
According to another preferred embodiment, the level of reference expression of a biological marker is the level of expression of said marker obtained in a skin model obtained from a cutaneous sample from a child, said model being untreated with the formulation or the asset of interest, but exposed to desiccation conditions.
According to another embodiment, the reference expression level of a biological marker is the level of expression of said marker obtained in a skin model obtained from a cutaneous sample from a child treated with the formulation or the asset of interest, and not exposed to desiccation conditions. Those skilled in the art will furthermore easily understand that the comparison of step d) is preferably carried out between measurements of expression levels obtained for skin models obtained from cutaneous samples from children, of structures histological similar, even identical. By "similar histological structures" is meant within the meaning of the present application that the relative proportions of the cell types included in the skin models compared are similar. Thus, it is preferable that the relative proportions of the cell types included in the skin model of step a) do not differ by more than 5% from the relative proportions of the cell types included in the skin model used to obtain the reference expression level of step d). By "relative proportion of a cell type" is meant in the sense of the present application the ratio of the number of cells corresponding to this cell type on the number of total cells included in the skin model. Thus, it is for example preferable that the proportion of keratinocytes on the total cell number in the skin model of step a) does not differ by more than 5% of the proportion of keratinocytes on the total number of cells in the skin model used to obtain the reference expression level of step d). By "identical histological structures" is meant within the meaning of the present application that the relative proportions of the cell types included in the skin models compared are identical. Within the meaning of the present invention, the relative proportions of the cell types included in the nipple 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 skin. reference expression of step d) when they do not differ by more than 0.1%. Advantageously, the proportion of keratinocytes on the number of total cells in the skin model of step a) does not differ by more than 0.1% of the proportion of keratinocytes on the total number of cells in the skin model used. to obtain the reference expression level of step d). Those skilled in the art will understand just as easily that the comparison of step d) is preferably carried out between measurements of expression levels obtained for skin models that are of similar or even identical size, volume or weight. . Thus, it is preferable that 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 skin. obtaining the reference expression level of step d). More preferably, the size, and the volume and weight of the skin model of step a) do not differ by more than 5% from the size, volume and weight of the skin model used to obtain the level of the skin. reference expression of step c). Even more preferably, 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, volume and weight of the skin model used for the treatment. obtaining the reference expression level of step d).
Alternatively, if the skin models differ by more than 5% in size, volume and weight, those skilled in the art can normalize the level obtained in step c) and the reference level of step d ) using a normalization factor.
This normalization factor may for example be a directly accessible physical marker such as the mass of cells of the sample, or the mass of a cellular constituent, such as the mass of cellular DNA or the mass of cellular proteins.
It may also be advantageous to use as a normalizing factor the level of expression of a gene that is expressed at the same level in all or substantially all cells of the body. In other words, according to a particular embodiment of the present invention, the level of expression of a household gene is used as normalization factor. According to another embodiment, the level obtained in step c) and the reference level of step d) are normalized using the level of expression, not of housekeeping genes, but of proteins encoded by those -this. A household gene is a gene expressed in all cell types, which encodes a protein having a basic function necessary for the survival of all cell types. A list of human housekeeping genes can be found in Eisenberg et al. (Trends in Genet, 19: 362-365, 2003). The household genes according to the invention include, for example, the genes RPS28, GAPDH, B2M, TFRC, YWHAZ, RP LO, 18S, GUSB, UBC, TBP, GAPDH, PPIA, POLR2A, ACTB, PGK1, HPRT1, IP08 and HMBS. Those skilled in the art will thus easily be able to evaluate the effectiveness of the formulation of interest as a function of the comparison of step d).
According to another aspect, the subject of the invention is a kit for carrying out a method according to the invention, comprising the means necessary for measuring the level of expression of at least one marker chosen from lipids, NMF, markers of cutaneous inflammation, markers of barrier function and markers preferentially expressed in stem cells. Preferably, the lipid is a ceramide, the marker of the barrier function is chosen from DSG, SCEL, PADI1, CASP14, LOR, TGM1 and CLDN1, and the marker preferably expressed in the stem cells is chosen from ΔΝρ63, KRT15, KRT19, BIRC5, and N0TCH1.
According to a particular embodiment, the kit according to the invention further comprises the means necessary for measuring the level of expression of a combination of biological markers chosen from the group comprising or consisting of: at least one marker of the skin inflammation and at least one barrier marker as defined above; or at least one marker of cutaneous inflammation and at least one marker preferentially expressed in the stem cells, as defined above; or • at least one marker of the barrier and at least one marker preferentially expressed in the stem cells, as defined above.
In a more preferred embodiment, said combination comprises at least one cutaneous inflammation marker and at least one barrier marker and at least one marker preferably expressed in the stem cells, as defined above.
The following examples are provided by way of illustration and are not, unless otherwise indicated, intended to be limiting.
EXAMPLES
Example 1 Validation of the Phenotype "Dry Skin"
In order to validate the modeling of a dry skin phenotype, the production of NMF (Natural Moisturizing Factor) and ceramides by the epidermis incubated in a dry atmosphere was evaluated. 1. Material and methods
Reconstructed epidermis was performed with keratinocytes from a 1-year-old donor.
Epidermal reconstruction was performed according to the model derived from the Poumay et al method (Arch Dermatol Res 2004; 296: 203-11). After 2 days of immersion culture, the reconstructed epidermis or RHE (Reconstructed Human Epidermis) was cultured at the air / liquid interface for 11 days. On day 11, the epidermis were incubated for 48 hours in a humid incubator for the control epidermis (normal condition: 37 ° C., 5% CO 2 and relative humidity> 99%) or in a drying oven (37 ° C., 5% CO 2 and relative humidity <25%). At the end of the incubation, the amounts of NMF and ceramides produced by the epidermis were evaluated.
The experiments were repeated 3 times; for each of these 3 tests, 3 replicates were made and analyzed.
Ceramide analysis
Epidermal lipids were extracted by shaking the epidermis from a mixture of organic solvents for 2 hours at room temperature. A solid / liquid extraction treatment was then carried out in order to isolate the ceramides from the other lipids constituting the epidermis.
The presence of ceramides having a sphingoid base of shingosine [S], dihydrosphingosine [DS] and phytosphingosine [P] type with an even chain length ranging from 16 to 22 carbon atoms was investigated by LC / MS method.
The ceramide content was normalized by the amount of total protein (BCA assay).
Analysis of NMF elements
The reconstructed epidermis was extracted with stirring for 2 hours at room temperature from an aqueous mixture in the presence of a nonionic surfactant to promote the extraction of markers of interest.
The catabolites of filaggrin were assayed by an LC / UV method for screening urocanic acid (UCA) under its two isomers (cis and trans) as well as L-pyrrolidone carboxylic acid (PCA).
The NMF content was normalized by the amount of total protein (BCA assay). 2. Results and conclusion
Ceramides are important structural components of the epidermis. They are one of the constituents of the lipid matrix of the stratum corneum. The role of this matrix is essential in the regulation of water permeability. It is indeed a hydrophobic barrier that regulates the flow of water through the SC. The lipid matrix is composed of an equimolar mixture of ceramides (45 to 50% of the total weight), cholesterol (20-25%) and free fatty acids (10-15%). Ceramides are derived from the transformation of sphingomyelin by sphingomyelinases and glucoceramides by 6-glucocerebrosidase.
The major function of the NMF is to maintain an optimal water level in the stratum corneum. A well-known source of NMF constituents is filaggrin. Incubation of 1-year reconstructed epidermis in a dry oven induced significant inhibition of ceramide and NMF content in the epidermis (Table 1).
Decreasing the amount of NMF and ceramides is one of the main features of dry skin, known and described in the literature; the established model consisting of the incubation of reconstructed epidermis in a dry atmosphere is therefore well representative of the cutaneous dryness phenotype.
Table 1: NMF content and ceramides in 1-year reconstructed epidermis incubated in normal condition (control) or in dry incubator (dry atmosphere)
Example 2 Characterization of Infant Epidermis Response Versus Adult Epidermis 1. Materials and Methods
Reconstructed epidermis was performed, as described above, with the keratinocytes of a donor of 1 year on the one hand and with the keratinocytes of a donor of 19 years on the other hand; the keratinocytes of these two donors from a preputial sample. On day 10, the epidermis was incubated for 24 hours in a humid incubator for the control epidermis (normal condition: 37 ° C., 5% CO 2 and relative humidity> 99%) or in a drying oven (37 ° C., 5% CO 2 and relative humidity <25%).
After 24h incubation, the gene expression of the markers of inflammation, hydration, barrier function and stem cells was evaluated by qRT-PCR (real-time quantitative PCR).
After 48 hours of incubation, the viability and morphology of the epidermis were studied.
Evaluation of the viability of reconstructed epidermis
The viability of the epidermis was evaluated by an MTT reduction test in n = 2: the epidermis were incubated in the presence of MTT (tetrazolium salt) whose transformation into blue crystals of formazan is proportional to the activity of the succinate dehydrogenase (mitochondrial enzyme). After dissociation of the cells and solubilization of formazan by addition of isopropanol / HCl, the optical density (OD), representative of the number of living cells and their metabolic activity, was measured at 540 nm.
Evaluation of morphology of reconstructed epidermis
The morphology of the reconstructed epidermis was evaluated in n = 2 by histological analysis after Hematoxylin / Eosin staining.
The epidermis was fixed and embedded in paraffin, cross sections were made by microtome. The sections were deparaffinized and stained with hematoxylin / eosin. Hematoxylin stains the nuclei of the cells in blue / violet; Eosin stains the cytoplasm in pink more or less bright. The analysis of histological sections in microscopy made it possible to evaluate and quantify certain morphological parameters such as the thickness of the layer of living cells.
Gene Differential Expression Analysis The expression of the markers was evaluated by qRT-PCR on the messenger RNAs extracted from the RHEs of each treatment. The gene expression analysis was performed in n = 2 using a PCR array containing 30 genes of interest and 2 reference genes (housekeeping genes). The total RNAs of each sample were extracted using TriPure Isolation Reagent® according to the protocol recommended by the supplier. The quantity and quality of the RNAs were evaluated by capillary electrophoresis (Bioanalyzer, Agilent). Complementary DNAs (cDNA) were synthesized by reverse transcription of the RNAs in the presence of oligo (dT) and the enzyme "Transcriptor Reverse Transcriptase". The obtained cDNA was quantified by spectrophotometry, and then the amounts of cDNA were adjusted.
The PCR reactions were performed by quantitative PCR with the Roche Molecular System Inc. ("light cycler") system and according to the procedure recommended by the supplier. The reaction mixture for each sample was: cDNA, primers of the different markers used, reaction mixture containing the enzyme Taq DNA polymerase, SYBR Green I marker (DNA intercalator) and MgCl 2. Fluorescence incorporation into the amplified DNA is measured continuously during PCR cycles. The quantitative analysis of the results is based on the collection of threshold cycles (or Ct for threshold cycle). The threshold cycle corresponds to the point where the fluorescence emission signal is statistically and significantly higher than the background noise. The threshold cycle is directly correlated to the number of initial copies of the target DNA.
Table 1 lists the genes that have been studied.
The relative gene expression (ER) value is expressed in arbitrary units according to the following formula: ER = (i / 2numberofcydes) x 106
Table 2: Classification and name of genes studied
2. Results a. Morphology and viability of the epidermis Incubation in a dry atmosphere did not significantly alter the morphology of the epidermis observed by microscopy after staining with hematoxylin / eosin (Figure 1); Moreover, the viability of the epidermis was similarly affected in the epidermis 1 year and adult epidermis (respectively 30% and 29% inhibition, Table 3). However, the measurement of the thickness of the living cell layer was more strongly impacted in 1-year epidermis (-18%) compared with adult epidermis in which no decrease was observed on this parameter (Table 3). .
These observations tend to show an increased sensitivity of 1-year-old epidermis to desiccant stress.
Table 3: Analysis of viability and thickness of the living cell layer
b. Expression analysis of aspecific and neurogenous inflammation markers Incubation in a dry atmosphere induced an increase in the level of expression of markers of nonspecific and neurogenic inflammation, whether in reconstructed 1-year epidermis or in adult reconstructed epidermis (Table 4).
This stimulation is, however, much greater in the case of reconstructed infant epidermis (+ 355%) compared to adult epidermis (+ 114%).
This suggests increased sensitivity of infant epidermals whose inflammatory and neuro-sensory response is exacerbated in response to desiccant stress.
Table 4: Level of Gene Expression of Inflammation Markers (relative expression in% compared to Control grown under normal conditions)
vs. Expression analysis of stem cell marker genes
Stem cells of tissues in permanent renewal are classically defined as being rare and relatively quiescent cells. They have a unique ability to self-renew and regenerate tissue to ensure homeostasis and the integrity of the tissue in which they reside.
Among the epidermal stem cells, interfollicular stem cells located in the basal layer constitute the main epidermal reservoir in stem cells. These cells reside in an anatomical and functional microenvironment, the niche, which helps maintain their characteristics, especially when physiological conditions change. The interfollicular stem cells and their niches are involved in maintaining the integrity and regeneration of the epidermis. Stem cells can only be identified by following several markers. We thus assessed the level of expression of different gene markers characteristic of stem cells in the "dry skin" model. Incubation in a dry atmosphere induced a significant decrease (-45%) in the mean expression level of the pool of stem cell markers studied (Table 5) in the 1-year epidermis, whereas no inhibition of the expression these markers were observed in adult epidermis (+ 12%).
This tends to show an increased vulnerability of this cellular capital in the epidermis of 1 year.
Table 5: Level of Gene Expression of Stem Cell Markers (Relative expression as% versus Control 1 year)
d. Analysis of the expression of barrier and hydration marker genes Incubation in a dry atmosphere induced a decrease in the level of expression of barrier markers, whether in reconstructed epidermis of 1 year or in reconstructed adult epidermis (Table 6).
However, this inhibition is greater in the case of reconstructed infant epidermis (-57%) compared to adult epidermis (-38%).
This suggests a greater vulnerability of markers of barrier function in infant epidermis.
Table 6: Level of Gene Expression of Barrier and Hydration Markers (Relative expression in% versus 1 year control)
Desmoglein 1 (DSG1) is a constitutive protein of corneodesmosomes that cohesive corneocytes within the stratum corneum.
Sciellin (SCL) is a precursor of the horny envelope.
PADI1 (Peptidyl arginine Deiminase 1) and Caspase 14 (CASP14) are two enzymes involved in the processing of filaggrin to obtain NMF. 1. Conclusion The comparative analysis of the behavior of reconstructed 1-year and adult (19 years old) epidermis in the dry skin induction model shows a higher vulnerability and susceptibility of infant epidermis to the desiccant stress induced by the skin. incubation in a dry atmosphere.
This increased vulnerability justifies the development of cosmetics for dry skin specifically adapted to the skin of the baby.
Example 3: Evaluation of products for dry skin
The previously established dry skin model of incubating reconstructed 1-year-old infant epidermis in a dry atmosphere was used to comparatively evaluate the biological efficacy of 4 dry skin care products. 1. Material and methods
Reconstructed epidermis was made, as previously described, from keratinocytes of a 1-year-old donor. On day 11, the epidermis was treated by topical application of the test products at a rate of 5 mg / cm 2 and then incubated for 6 h, 24 h or 48 h in a dry oven (dry atmosphere condition: 37 ° C, 5% C02 and humidity relative <25%), except for humidified control epidermis (37 ° C, 5% CO2 and relative humidity> 99%) and analyzed as described above:
After 6h and 24h incubation, the gene expression of markers of aspecific and neurogenic inflammation, hydration, barrier function and stem cells (Table 1) was evaluated by qRT-PCR (quantitative PCR in vitro). real time) in n = 2.
After 48 h of incubation, the viability (MTT) and the morphology (histology, hematoxylin / eosin staining) of the epidermis were studied in n = 2 and the NMF and ceramide production analyzed at n = 3.
Products under test = reference formulations as mentioned above:
Product A - Product B Product C Product D 2. Results a. Morphology and viability of the epidermis
As in the previous step of model development, incubation in a dry atmosphere did not significantly alter the morphology and viability of the epidermis.
Table 7: Analysis of the viability of the epidermis
b. Analysis of the expression of marker genes for neurogenic and nonspecific inflammation
Products A, B and C inhibit inflammation markers expressed under incubation conditions in a dry atmosphere (Table 8). These products therefore offer protection against cutaneous reactivity which can be exacerbated under these conditions. Only product D has no inhibitory effect on these markers.
Table 8: Level of Gene Expression of the Markers of Neurogenic and Nonspecific Inflammation (Relative Expression as a% Relative to the Dry Oven Control)
vs. Expression analysis of stem cell marker genes
Only product A completely restores (96% restoring) the level of expression of stem cell markers inhibited by incubation in a dry atmosphere with greater efficacy than the other products tested (Table 9).
Product A thus protects cell capital in the dry skin model.
Table 9: Level of Gene Expression of Stem Cell Markers (Relative expression in% vs. 1 year control)
d. Analysis of the expression of barrier marker genes
Under incubation conditions in a dry atmosphere, products A and B restored the level of expression of the barrier marker genes and showed a recovery efficiency (140% and 113%, respectively) higher than the other formulas tested.
Thus, product A, and to a lesser extent product B, protect the expression of altered barrier markers by incubation in a dry atmosphere.
Table 10: Gene expression level of the barrier markers (relative expression in% compared to the 1 year control)
Loricrin (LOR) is a late marker of epidermal differentiation involved in the formation of the horny envelope.
The transglutaminase 1 (TGM1) enzyme responsible for the cross-linking of different proteins for the placement of the horny envelope, ensuring the tightness / strength of the stratum corneum.
Claudine 1 (CLDN1) is one of the constitutive proteins of tight junctions, involved in the barrier function of the skin, controlling in particular the fluid flows.
Desmoglein 1 (DSG1) is a constitutive protein of corneodesmosomes that cohesive corneocytes within the stratum corneum.
Corneodesmosin is a constitutive protein of corneodesmosomes, junctions that form between corneocytes to maintain the structure of the stratum corneum and thereby participate in good desquamation of the skin [10]. e. Analysis of NMF and ceramide production
Products A and B restored the production of inhibited NMF ceramides and NMFs in the dry skin model (Table 11) while the other two products tested (C and D) showed lower efficacy: practically no effect on production of ceramides, low increase in NMF production.
Table 11: NMF content and ceramides in 1-year reconstructed epidermis incubated in normal condition (control) or in dry incubator (dry atmosphere)

权利要求:
Claims (14)
[1" id="c-fr-0001]
A method for evaluating the in vitro efficacy of an active ingredient or formulation on preventing the effects of dehydration on the skin of a child, characterized in that said method comprises the following steps: contacting said active or said formulation with a reconstructed skin model, said model being obtained from a skin sample from a child; b) cultivating the reconstructed skin model of step a) under desiccation conditions; c) measuring the level of expression of at least one biological marker in the skin model of step b); and d) evaluating the effectiveness of said asset or said formulation as a function of the level of step c).
[2" id="c-fr-0002]
2. A method for evaluating the in vitro efficacy of an active ingredient or a formulation in reducing the effects of dehydration on the skin of a child, characterized in that said method comprises the following steps: a) cultivating the skin model reconstructed under desiccation conditions, said model being obtained from a skin sample from a child; b) contacting said asset or said formulation with the reconstructed skin model of step a); c) measuring the level of expression of at least one biological marker in the skin model of step b); and d) evaluating the effectiveness of said asset or said formulation as a function of the level of step c).
[3" id="c-fr-0003]
A method of identifying an active ingredient or formulation for preventing the effects of dehydration on the skin of a child, characterized in that said method comprises the following steps: a) contacting an asset or a a candidate formulation with a reconstructed skin model, said model being obtained from a skin sample from a child; b) cultivating the reconstructed skin model of step a) under desiccation conditions; c) measuring the level of expression of at least one biological marker in the skin model of step b); and d) determining whether said active or said candidate formulation is a formulation or an active agent for preventing the effects of dehydration on the skin of children depending on the level of step c).
[4" id="c-fr-0004]
A method of identifying an active ingredient or formulation for reducing the effects of dehydration on the skin of children, characterized in that said method comprises the following steps: a) cultivating the reconstructed skin model in desiccation conditions, said model being obtained from a skin sample from a child; b) contacting a candidate asset or formulation with the skin model of step a); c) measuring the level of expression of at least one biological marker in the skin model of step b); and d) determining whether said active or said candidate formulation is a formulation or an active agent for reducing the effects of dehydration on the skin of children as a function of the level of step c).
[5" id="c-fr-0005]
5. A process for evaluating the tolerance of an active ingredient or a formulation on dehydrated children's skin, said method comprising the following steps: a) bringing said active agent or said formulation into contact with a reconstructed skin model, said model being obtained from a skin sample from a child; b) cultivating the reconstructed skin model of step a) under desiccation conditions in the presence of the asset or formulation; c) measuring the level of expression of at least one biological marker in the skin model of step b); and d) determining whether said active agent, active ingredient or formulation is well tolerated by the skin of a child as a function of the level of step c).
[6" id="c-fr-0006]
6. Method according to any one of claims 1 to 5, characterized in that said marker is selected from lipids, NMF, inflammation markers, markers of the barrier function and markers preferentially expressed in cells strains.
[7" id="c-fr-0007]
7. Method according to any one of claims 1 to 6, characterized in that step c) comprises measuring a combination of biological markers, said combination comprising at least two markers, said markers being chosen in at least two different categories of markers as defined in claim 6.
[8" id="c-fr-0008]
8. Method according to claim 7, characterized in that said combination comprises at least one lipid and the NMF and at least one marker of the inflammation and at least one barrier marker and at least one marker preferably expressed in the stem cells .
[9" id="c-fr-0009]
9. Method according to any one of claims 1 to 8, characterized in that the lipid is a ceramide, the inflammation marker is selected from IL1A, IL8, PTGS2, NGFR, TAC1, TAC1R, TRPV1, TRPV3, MRGPRD , OSMR, PLA2G2F and F2RL1, the marker of the barrier function is selected from DSG, SCEL, PADI1, CASP14, LOR, TGM1 and CLDN1, and the marker preferably expressed in the stem cells is selected from ΔΝρ63, KRT15, KRT19, BIRC5, etNOTCHI.
[10" id="c-fr-0010]
10. Method according to any one of claims 1 to 9, characterized in that the cutaneous sample is from a donor selected from the group consisting of newborns, whose age is between 0 and 1 month, infants, between 1 month and 2 years old, and children between 2 and 16 years old.
[11" id="c-fr-0011]
11. Method according to any one of claims 1 to 10, characterized in that the skin sample is from a skin having a phototype I, II, III, IV, V or VI.
[12" id="c-fr-0012]
12. Method according to any one of claims 1 to 11, characterized in that the reconstructed skin model is chosen from suspended skin cell cultures, monolayer cutaneous skin cultures, cutaneous skin cell cultures, reconstructed skin cultures and reconstructed mucosa cultures.
[13" id="c-fr-0013]
13. The method of claim 12, characterized in that the cells of said model come from an explant skin tissue or stem cells differentiated into cutaneous cells.
[14" id="c-fr-0014]
14. Method according to any one of claims 10 to 12, characterized in that said model comprises at least fibroblasts or keratinocytes.

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FR2945444A1|2009-05-13|2010-11-19|Limousine D Applic Biolog Dite|Use of an agent increasing expression of nicotinic acetylcholine receptor of skin cells, as an active ingredient for preparing a cosmetic composition for improving and/or repairing the skin barrier function|

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FR2983868A1|2011-12-07|2013-06-14|Oreal|In vitro or ex vivo use of an amino acid sequence coded by nucleic acid sequence or nucleic acid sequence coding amino acid sequence, as a biomarker e.g. to identify active agents to prevent or treat impaired barrier function of scalp|

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WO2014009566A1|2012-07-13|2014-01-16|Laboratoires Expanscience|Method for identifying molecular markers of children's skin|

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WO2014170495A2|2013-04-19|2014-10-23|Laboratoires Expanscience|Use of barrier biomarkers for evaluating the effectiveness of active ingredients|

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FR3011008A1|2013-09-24|2015-03-27|Expanscience Lab|METHODS OF EVALUATING DELETER EFFECTS OF UV ON CHILD'S SKIN|

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WO2015104413A1|2014-01-10|2015-07-16|Laboratoires Expanscience|Reconstituted nipple skin model|

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FR3019186A1|2014-03-31|2015-10-02|Expanscience Lab|METHODS OF EVALUATING THE DELETE EFFECTS OF URINE ON CHILDREN'S SKIN|

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EP3735701A1|2018-01-05|2020-11-11|Johnson & Johnson Consumer Inc.|Skin barrier preparation and method therefor|

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US11229595B2|2019-05-29|2022-01-25|Johnson & Johnson Consumer Inc.|Skin barrier preparation and method therefor|

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US20210190761A1|2019-12-19|2021-06-24|Johnson & Johnson Consumer Inc.|Method for Evaluating the Efficacy of a Composition in Reducing the Effects of Cancer Therapeutics on Skin|

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EP3852117A1|2020-01-14|2021-07-21|Michel Daigne|Multi-agent system for optimising skin care with distributed artificial intelligence|

法律状态:
2016-12-07| PLFP| Fee payment|Year of fee payment: 2 |

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2017-06-23| PLSC| Publication of the preliminary search report|Effective date: 20170623 |

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2017-12-13| PLFP| Fee payment|Year of fee payment: 3 |

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2019-12-12| PLFP| Fee payment|Year of fee payment: 5 |

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2020-12-14| PLFP| Fee payment|Year of fee payment: 6 |

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2021-11-10| PLFP| Fee payment|Year of fee payment: 7 |

优先权:

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申请号 | 申请日 | 专利标题

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FR1562528A|FR3045669B1|2015-12-16|2015-12-16|METHODS OF EVALUATING THE EFFECTS OF DEHYDRATION ON CHILDREN'S SKIN|

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FR1562528|2015-12-16|FR1562528A| FR3045669B1|2015-12-16|2015-12-16|METHODS OF EVALUATING THE EFFECTS OF DEHYDRATION ON CHILDREN'S SKIN|

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US16/062,488| US20190242880A1|2015-12-16|2016-12-16|Method for evaluating the effects of dehydration on children's skin|

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PCT/EP2016/081562| WO2017103195A1|2015-12-16|2016-12-16|Method for evaluating the effects of dehydration on children's skin|

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EP16809887.9A| EP3391046A1|2015-12-16|2016-12-16|Method for evaluating the effects of dehydration on children's skin|