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WO2023227746A2 - Signature moléculaire de lentigo actinique associée à la gestion de vésicules - Google Patents

Signature moléculaire de lentigo actinique associée à la gestion de vésicules Download PDF

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Publication number
WO2023227746A2
WO2023227746A2 PCT/EP2023/064124 EP2023064124W WO2023227746A2 WO 2023227746 A2 WO2023227746 A2 WO 2023227746A2 EP 2023064124 W EP2023064124 W EP 2023064124W WO 2023227746 A2 WO2023227746 A2 WO 2023227746A2
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Prior art keywords
skin
expression
genes
chosen
gene
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WO2023227746A3 (fr
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Emilie WARRICK
Françoise Bernerd
Charlène GAYRARD
Christine Duval
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LOreal SA
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LOreal SA
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/33—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing oxygen
    • A61K8/35—Ketones, e.g. benzophenone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/49—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds
    • A61K8/494—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds with more than one nitrogen as the only hetero atom
    • A61K8/4953—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds with more than one nitrogen as the only hetero atom containing pyrimidine ring derivatives, e.g. minoxidil
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/67—Vitamins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
    • A61K8/73—Polysaccharides
    • A61K8/735—Mucopolysaccharides, e.g. hyaluronic acid; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/92—Oils, fats or waxes; Derivatives thereof, e.g. hydrogenation products thereof
    • A61K8/922—Oils, fats or waxes; Derivatives thereof, e.g. hydrogenation products thereof of vegetable origin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening the skin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/148—Screening for cosmetic compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention is in the cosmetics field, and it relates to the skin. It falls more generally within the context of the characterization of pigmentary spots on the skin and the treatment of these spots.
  • Skin colour is mainly due to the presence of a pigment, melanin, in the epidermis.
  • Melanin is synthesized by specific dendritic cells located in the basal layer of the epidermis, known as melanocytes. Melanogenesis takes place in organelles, the melanosomes, which, loaded with melanin, are transferred to the neighbouring epidermal cells, the keratinocytes, via the dendrites.
  • Skin colour, or constitutive pigmentation varies according to individuals depending on the amount of melanin produced and also on the chemical nature of the melanins.
  • Melanins are macromolecules formed from tyrosine (eumelanin) or from tyrosine and cysteine (pheomelanin). The synthesis mechanisms involve enzymes, the main ones of which are tyrosinase and tyrosinase-related protein (Tyrp-1). Naturally, skin pigmentation is stimulated by exposure to the sun, this is the tanning phenomenon.
  • pigmentation process is modified, and which can result in pigmentation defects, hypopigmentations (vitiligo, albinism) or, conversely, in excess pigmentation, hyperpigmentations.
  • benign hyperpigmentation disorders characterized by abnormal accumulation (outside the case of tanning) of melanin, mention may be made of actinic lentigo, melasma, pigmentary sequelae of acne, post-inflammatory pigmentation, meadow dermatitis, or benign facial dychromia.
  • Actinic lentigo also called senile or solar lentigo, age spots or liver spots, or else commonly known as “senile keratoma”, “graveyard daisies” or “graveyard flowers”, is by far the most frequent of the pigmentation lesions. Lesions of this type appear on areas of skin which have been exposed to light, such as the face, the back of the hands, the upper limbs and in particular the dorsal face of the forearms, the back, and in particular the upper back. They generally affect individuals starting from the age of 40.
  • actinic lentigines are represented by benign pigmented maculae which are dark to light brown in colour, and the edges of which are distinct but irregular. They are very variable in size and can extend from a few millimetres to more than two centimetres.
  • Pigmentary incontinence with the presence of melanin and melanophages may also exist in the dermis.
  • Benign skin pigmentation disorders are generally considered to be unsightly. Because of the proven association between the appearance of actinic lentigines and chronic exposure to the sun, prevention of their appearance generally involves the topical application of “photoprotective” products such as sunscreens. In the case of “curative” treatments, many processes, mainly for cosmetic purposes, have therefore been developed in an attempt to eliminate them or reduce their presence. The elimination or reduction of the presence of these disorders is normally based on the application of depigmenting treatments based on reducing the melanin synthesis activity in the melanocytes. Depigmenting molecules interfere with one or more steps of melanogenesis. One of the approaches of the main products used at the current time is based on the inhibition of tyrosinase, one of the key enzymes in the melanogenesis process. The aim of these treatments is to reduce or even stop pigment synthesis.
  • the main known depigmenting substances are hydroquinone and derivatives thereof, kojic acid, arbutin, iminophenols, ascorbic acid and derivatives thereof, the combination of carnitine and quinone, aminophenol derivatives, benzothiazole derivatives, natural extracts, and corticoids.
  • Exfoliants for increasing desquamation, thereby more readily eliminating the melanin present in the stratum corneum, are also often combined with these active agents.
  • Another non-cosmetic treatment method consists in destroying the lesions by physical or chemical means using lasers or peeling treatments. However, these are quite intensive procedures which do not tackle the aetiology of the disorder. In the majority of cases, the actinic lentigines reappear some time after the treatment.
  • the depigmenting substances may notably display a certain level of instability, poor efficiency at low concentration, or biological activity impacting on other undesirable functions.
  • the depigmenting substances may notably display a certain level of instability, poor efficiency at low concentration, or biological activity impacting on other undesirable functions.
  • due to the fact that they do not target the root cause of the pigmentation disorder there is a great heterogeneity of response to the various depigmenting treatments.
  • one given benign skin pigmentation disorder such as actinic lentigo or melasma
  • This variability may be observed between lesions in different individuals, but also in the same individual, between different lesions.
  • melanogenesis is not the major biological mechanism involved in the appearance of pigmentary spots and notably actinic lentigo (Warrick et al., Br. J. Dermatol. 2017, Aoki, Br. J. Dermatol. 2007).
  • the present inventors have demonstrated for the first time the involvement of genes related to the management (notably the transport, fusion, accumulation, retention and degradation) of vesicles, preferably melanosomes, in pigmentary disorders resulting in pigmentary spots of the skin.
  • certain genes related to the management of vesicles preferably of the melanosomes, have significantly different transcription levels between healthy skin and skin resulting from a pigmentary spot, thus highlighting the link between deregulation of the management of vesicles, preferably melanosomes, within the skin, preferably the epidermis, and modulation of pigmentation notably leading to the appearance of pigmentary spots.
  • the melanosome is an intracellular organelle in the form of a vesicle, said vesicle being specialized in the production of melanin, a pigment protecting human skin from solar radiation. It is produced in the melanocytes. Surrounded by a lipid membrane, its shape varies from round to elongated. In human skin, melanosomes do not remain in the melanocytes, but are transported towards the keratinocytes along arms resembling the dendrites of neurons (the melanocytes are of neural origin), in which they move, borne by kinesins, along a system composed of microtubules, actin, intermediate filaments and associated proteins. This transport may be triggered or intensified by certain factors, notably by the intensity of ultraviolet radiation (tanning).
  • keratinocytes distribute melanosomes above their nucleus, protecting it from damage by UV radiation.
  • the number of melanosomes produced by the melanocyte, their size, their concentration of melanins and the nature of these melanins (eumelanin or pheomelanin), and also the way in which the keratinocyte distributes them above its nucleus, are hereditary characteristics that influence the pigmentation of human skin.
  • FR2974370, FR2974372, FR2974373 and FR2974371 although showing molecular deregulations or functional dysfunctions in actinic lentigo, notably with the involvement of genes of the biological signalling pathway TGFp-SMAD, genes involved in the remodelling of the extracellular matrix, genes coding for proteoglycans and extracellular glycoproteins, said documents do not disclose genes related to the management of vesicles, notably of the melanosomes.
  • a first subject of the present invention is a method for characterizing an apparent or suspected skin pigmentary spot in a human being, comprising the measurement of the expression levels and then comparison of the expression levels measured, in samples of skin from said spot and from adjacent non-lesioned skin, of at least one gene involved in the management of vesicles, preferably of the melanosomes, chosen from the list consisting of the genes JAKMIP2, ARC, KIF21A, SLITRK6, NRN1 , SCIN, SYNPO2, TNNT1 , SNTB1 , ARHGAP29, RAB3IP, RHOBTB3, RASIP1 , ASGR1 , CHMP4C, SVIP, LRMP, NAV3 and PCLO.
  • a second subject of the present invention is a method for evaluating the efficacy of a treatment for skin pigmentary spots, comprising the measurement of the expression levels and then comparison of the expression levels measured, in sample of skin from said pigmentary spot, before and after treatment, of at least one gene involved in the management of vesicles, preferably of the melanosomes, chosen from the list consisting of the genes JAKMIP2, ARC, KIF21A, SLITRK6, NRN1 , SCIN, SYNPO2, TNNT1 , SNTB1 , ARHGAP29, RAB3IP, RHOBTB3, RASIP1 , ASGR1 , CHMP4C, SVIP, LRMP, NAV3 and PCLO.
  • Another subject of the present invention is a method for the in vitro screening of an active agent for preventing and/or treating skin pigmentary spots, said method comprising the following steps: i. placing a cell model representative of the skin in contact with at least one active agent to be tested, or exposing said cell model to a physical treatment to be tested; ii.
  • vesicles preferably of the melanosomes, chosen from the list consisting of the genes JAKMIP2, ARC, KIF21A, SLITRK6, NRN1 , SCIN, SYNPO2, TNNT1 , SNTB1 , ARHGAP29, RAB3IP, RHOBTB3, RASIP1 , ASGR1 , CHMP4C, SVIP, LRMP, PCLO and NAV3 or the level of expression or activity of the expression product of said chosen gene, and iii. comparing said measurement with a reference measurement.
  • the invention also relates to a cosmetic method for treating or preventing a non- pathological skin pigmentary spot in human skin, comprising the application to the skin of a composition comprising at least one active agent modulating the level of expression or activity of at least one gene involved in the management of vesicles preferably of the melanosomes, in which said gene is chosen from the list consisting of the genes JAKMIP2, ARC, KIF21A, SLITRK6, NRN1 , SCIN, SYNPO2, TNNT1 , SNTB1 , ARHGAP29, RAB3IP, RHOBTB3, RASIP1 , ASGR1 , CHMP4C, SVIP, LRMP, PCLO and NAV3.
  • the invention also relates to the cosmetic use of a modulator of the level of expression or activity of the expression product of at least one gene involved in the management of vesicles, preferably of the melanosomes, chosen from the list consisting of the genes JAKMIP2, ARC, KIF21A, SLITRK6, NRN1 , SCIN, SYNPO2, TNNT1 , SNTB1 , ARHGAP29, RAB3IP, RHOBTB3, RASIP1 , ASGR1 , CHMP4C, SVIP, LRMP, PCLO and NAV3 in the treatment of non-pathological skin pigmentary spots.
  • a modulator of the level of expression or activity of the expression product of at least one gene involved in the management of vesicles preferably of the melanosomes, chosen from the list consisting of the genes JAKMIP2, ARC, KIF21A, SLITRK6, NRN1 , SCIN, SYNPO2, TNNT1 , SNTB1
  • the present invention also relates to a method for preparing a cell model suitable for screening active agents for preventing and/or treating skin pigmentary spots, comprising the following steps: i. collecting keratinocytes and optionally melanocytes; and then ii. transfecting said keratinocytes collected in step i.
  • siRNA capable of repressing the level of expression of at least one gene involved in the management of vesicles, preferably of the melanosomes, chosen from the list consisting of the genes JAKMIP2, KIF21A, SLITRK6, SCIN, TNNT1 , SNTB1 , ARHGAP29, RAB3IP, RHOBTB3, ASGR1 , CHMP4C, SVIP, PCLO and NAV3; and/or transfecting said keratinocytes collected in step i.
  • an expression vector preferably a plasmid, of at least one gene involved in the management of vesicles, preferably of the melanosomes, chosen from the list consisting of the genes ARC, NRN1 , SYNPO2, RASIP1 and LRMP, and then iii. culturing the keratinocytes transfected in step ii. or co-culturing the keratinocytes transfected in step ii. with the melanocytes collected in step i.
  • an expression vector preferably a plasmid, of at least one gene involved in the management of vesicles, preferably of the melanosomes, chosen from the list consisting of the genes ARC, NRN1 , SYNPO2, RASIP1 and LRMP
  • Another subject is a cell model suitable for screening active agents for preventing and/or treating skin pigmentary spots, which may be obtained or which is directly obtained according to the preparation method of the invention.
  • Another subject of the present invention is a cell model suitable for screening active agents for preventing and/or treating skin pigmentary spots, comprising keratinocytes and optionally melanocytes, in which said keratinocytes exhibit repression of at least one gene involved in the management of vesicles, preferably of the melanosomes chosen from the list consisting of the genes JAKMIP2, KIF21A, SLITRK6, SCIN, TNNT1 , SNTB1 , ARHGAP29, RAB3IP, RHOBTB3, ASGR1 , CHMP4C, SVIP, PCLO and NAV3, and/or in which said keratinocytes exhibit overexpression of at least one gene involved in the management of vesicles, preferably of the melanosomes, chosen from the list consisting of the genes ARC, NRN1 , SYNPO2, RASIP1 and LRMP.
  • the invention also relates to an in vitro method for screening active agents for preventing and/or treating skin pigmentary spots, said method comprising the following steps: i. placing a cell model comprising keratinocytes and optionally melanocytes, in which said keratinocytes exhibit repression of at least one gene involved in the management of vesicles, preferably of the melanosomes, chosen from the list consisting of the genes JAKMIP2, KIF21A, SLITRK6, SCIN, TNNT1 , SNTB1 , ARHGAP29, RAB3IP, RHOBTB3, ASGR1 , CHMP4C, SVIP, PCLO and NAV3, and/or in which said keratinocytes exhibit overexpression of at least one gene involved in the management of vesicles, preferably of the melanosomes, chosen from the list consisting of the genes ARC, NRN1 , SYNPO2, RASIP1 , and LRMP, in contact with at least one active agent
  • vesicles preferably of the melanosomes, chosen from the list consisting of the genes JAKMIP2, ARC, KIF21A, SLITRK6, NRN1 , SCIN, SYNP02, TNNT1 , SNTB1 , ARHGAP29, RAB3IP, RH0BTB3, RASIP1, ASGR1 , CHMP4C, SVIP, LRMP and PCLO and NAV3, or the level of expression or activity of the expression product of said chosen gene; and iii. comparing said measurement with a reference measurement.
  • level of expression of a gene means, preferentially, the level of transcription and/or the level of translation of said gene, more preferentially the level of transcription of said gene.
  • the evaluation of the transcription level of the chosen gene it can be performed in various ways well known to those skilled in the art, directly or else after reverse transcription.
  • the transcription level may notably be evaluated using RNA chips or DNA chips sold commercially for this purpose. A possible evaluation method is described in the experimental section.
  • vehicle means a more or less spherical structure formed by a biological membrane, notably a lipid bilayer, closed on itself, present in the cells of the skin, preferably in the keratinocytes.
  • the vesicles are intracellular transport vesicles; even more preferentially, the vesicles are melanosomes.
  • vesicle management or “melanosome management” means the transport, fusion, accumulation, retention and degradation of said vesicles or melanosomes.
  • One subject of the present invention is method for characterizing an apparent or suspected skin pigmentary spot in a human being, comprising the measurement of the expression levels and then comparison of the expression levels measured, in samples of skin from said spot and from adjacent non-lesioned skin, of at least one gene involved in the management of vesicles, preferably of the melanosomes, chosen from the list consisting of the genes JAKMIP2, ARC, KIF21A, SLITRK6, NRN1 , SCIN, SYNPO2, TNNT1 , SNTB1 , ARHGAP29, RAB3IP, RHOBTB3, RASIP1, ASGR1 , CHMP4C, SVIP, LRMP, NAV3 and PCLO.
  • Such a method makes it possible, inter alia, to confirm the nature of the pigmentary spot in the case where it is already apparent, for example visually to the naked eye.
  • the method also makes it possible to predict the appearance of a spot, when the latter is not yet observed but only suspected, or to conclude that a skin is prone to the formation of skin spots, or subject to pigmentation defects, for example when no spots are yet apparent.
  • the skin pigmentary spots concerned are preferably hyperpigmentation spots, also known as hyperpigmentary spots, corresponding to an excess of pigment.
  • Benign hyperpigmentary disorders that may be envisaged within the context of the invention are notably characterized by an abnormal accumulation (apart from tanning) of melanin, and may be chosen from actinic lentigo, melasma, pigmentary sequelae of acne, post-inflammatory pigmentation, meadow dermatitis, or benign facial dyschromias, and pigmentation imperfections or irregularities rendering the complexion or the skin colour non- uniform.
  • the pigmentary spots are actinic lentigo.
  • the pigmentary spots concerned are preferentially pigmentary spots of human skin.
  • the levels of expression of at least one of the genes of the invention are measured in skin from an actual or suspected spot, and in adjacent non-lesioned skin.
  • the levels are measured in skin samples taken from within the spot and from an adjacent non-lesioned area.
  • the samples are, for instance, skin biopsies, shaves, ex vivo skin samples, suction bubbles.
  • biopsies a few millimetres in diameter are sufficient, for example a biopsy 2 mm or 3 mm in diameter. Total excision of a lesion may also be considered.
  • the non-lesioned area is preferably an adjacent area as close as possible to the spot, but at a sufficient distance for the sample taken to contain no cells possibly belonging to the pigmentary spot.
  • the non-lesioned adjacent area is an area having an exposure to light and to the sun comparable to those of the area exhibiting the pigmentary spot.
  • the non-lesioned area may originate from a symmetrical area on the other part of the subject, having perfectly identical position; for example, in the case of a spot on the left hand, the non-lesioned skin may be the corresponding area on the right hand. In this case, the non-lesioned area is not strictly speaking an adjacent area.
  • the term “non-lesioned” refers to an area which does not have any pigmentary spots, nor any pigmentation irregularities, preferably an area which is homogeneous in terms of pigmentation.
  • non-lesioned area serves as a reference, it must in any event be as comparable as possible to the area of the spot, but free of pigmentation defects.
  • the method according to the invention involves comparing the levels of expression of at least one of the genes of the invention. Therefore, it may be sufficient to quantitatively or qualitatively evaluate the difference between the two expression levels, without individually evaluating and quantifying each of the expression levels.
  • the method according to the invention is preferably performed in vitro or else ex vivo.
  • the pigmentary spot is a non-pathological, benign spot, notably as opposed to pathological lesions such as naevi; it may be a skin pigmentation irregularity.
  • a method according to the invention involves comparing the levels of expression of at least one gene involved in the management of vesicles, preferably of the melanosomes, chosen from the list consisting of the genes JAKMIP2, KIF21A, SLITRK6, SCIN, SYNPO2, TNNT1 and LRMP.
  • a method according to the invention involves comparing the levels of expression of at least two different genes from among the genes of the invention, preferably at least three different genes, or even five different genes. It is also possible to compare the expression levels of at least six or even at least eight different genes.
  • genes are preferably chosen from the following lists of the eight preferred genes: JAKMIP2, KIF21A, SLITRK6, SCIN, SYNPO2, TNNT1 and LRMP. All the paired combinations, among the eight preferred genes, are preferential combinations for the implementation of the invention.
  • the suspected or observed spot is a hyperpigmentary spot if the measured expression level is: i. higher in the skin sample from the spot relative to the level in the adjacent non-lesioned skin sample, if the gene is chosen from ARC, NRN1 , SYNPO2, RASIP1 and LRMP, and/or ii.
  • the gene is chosen from JAKMIP2, KIF21A, SLITRK6, SCIN, TNNT1 , SNTB1 , ARHGAP29, RAB3IP, RHOBTB3, ASGR1 , CHMP4C, SVIP, NAV3 and PCLO.
  • the present inventors have demonstrated the overexpression of the genes ARC, NRN1 , SYNPO2, RASIP1 , and LRMP in skin from hyperpigmentary spots, notably actinic lentigo, relative to the level of expression in adjacent non-lesioned skin. They also demonstrated the under-expression of the genes JAKMIP2, KIF21A, SLITRK6, SCIN, TNNT1 , SNTB1 , ARHGAP29, RAB3IP, RHOBTB3, ASGR1 , CHMP4C, SVIP, NAV3 and PCLO in skin from hyperpigmentary spots, notably actinic lentigo, relative to the level of expression in adjacent non-lesioned skin.
  • the term “higher” or “lower” means a difference in expression levels that is statistically significant, higher than the noise and reproducible.
  • the difference in expression level is at least 10%, i.e. if the expression level of a gene of the invention in the non-lesioned skin is set to 1 , the modulation level is at least 1.1 for a gene overexpressed in the lesioned skin and not more than 0.9 for a gene underexpressed in the lesioned skin.
  • the level of expression of one of the genes of the invention is compared within the skin of a Caucasian individual.
  • the level of expression of one of the genes of the invention is compared within the skin of an Asian individual.
  • said individual is at least forty years old, preferably at least fifty years old, or even sixty years old.
  • the individual under consideration in the context of the present invention is preferably female.
  • the level of expression of one or more genes of the invention may be compared within skin samples of said individual.
  • the method described is also a test method for predicting the formation of skin spots in a subject.
  • the level of expression of at least one, and preferably several, genes of the invention is compared in a skin sample to its level of expression in normal skin. A significant modulation of the level of expression of said gene(s) relative to the level of expression of said gene(s) in normal skin leads to the conclusion that the skin of the test subject is prone to the formation of skin spots.
  • expression level in normal skin means either the level of expression of said gene(s) in the skin of the same subject in a body area known to be free of spots, for example areas not exposed to solar radiation, or areas not prone to the formation of pigmentary spots, or the average level of expression of said gene(s) in the skin of persons free of spots and preferably having the same type of skin as the test subject.
  • the present invention also relates to a method for evaluating the efficacy of a treatment for skin pigmentary spots, comprising the measurement of the expression levels and then comparison of the expression levels measured in a sample of skin from said pigmentary spot, before and after treatment, of at least one gene involved in the management of vesicles, preferably of the melanosomes, chosen from the list consisting of the genes JAKMIP2, ARC, KIF21A, SLITRK6, NRN1 , SCIN, SYNPO2, TNNT1 , SNTB1 , ARHGAP29, RAB3IP, RHOBTB3, RASIP1 , ASGR1 , CHMP4C, SVIP, LRMP, NAV3 and PCLO.
  • the treatment evaluated may be a treatment aimed at reducing pigmentary spots or any other modulation of pigmentation, notably to even out skin tone, homogenize skin colour.
  • the comparison of the expression levels measured of the chosen gene(s) is performed within the skin from a pigmentary spot, or within a corresponding sample, before and after treatment. There is therefore no comparison to an expression level in healthy, non-lesioned skin.
  • a given treatment is considered to be effective for the treatment of a hyperpigmentary spot if the measured expression level is: lower after treatment relative to the expression level before treatment, if the gene is chosen from ARC, NRN1 , SYNPO2, RASIP1 and LRMP, and/or higher after treatment relative to the expression level before treatment, if the gene is chosen from JAKMIP2, KIF21A, SLITRK6, SCIN, TNNT1 , SNTB1 , ARHGAP29, RAB3IP, RHOBTB3, ASGR1 , CHMP4C, SVIP, NAV3 and PCLO.
  • a treatment will be considered to have no effect if the expression levels of the chosen gene, before and after treatment, are substantially identical, or else if the differences observed are not significant.
  • the treatment is considered to be effective for the treatment of a pigmentary spot or irregularity, if, for the majority of the genes tested and preferably for all of the genes tested, taken individually, the treatment is considered to be effective.
  • the treatment should preferably have no effect, but must not have the opposite effect.
  • the method for evaluating the efficacy of a treatment for skin pigmentary spots involves characterizing a pigmentary spot or irregularity according to the method of the invention, before and after treatment, and comparing the differences in expression levels observed between the lesioned skin of the pigmentary spot or irregularity and healthy skin.
  • the treatment is considered to be effective if the difference between the levels of expression of the chosen gene(s) in the lesioned skin from the spot relative to the healthy skin, preferably adjacent, is less after treatment than it was before treatment.
  • the treatment is efficacious when, for the majority of the chosen genes, and preferably for all of the chosen genes, taken individually, it can be concluded that the treatment is efficacious.
  • the comparison of the levels of expression of the chosen gene(s) is performed on skin samples taken from the pigmentary spot.
  • the treatment under consideration evaluated by means of the method of the invention, is not limited to a particular type of treatment.
  • It may be a treatment with a chemical molecule, an active agent, a natural extract, notably an essential oil, a nucleic acid, notably an RNA such as siRNA or mRNA, a protein complex or any other molecule or combination of molecules.
  • a chemical molecule an active agent
  • a natural extract notably an essential oil
  • a nucleic acid notably an RNA such as siRNA or mRNA
  • a protein complex or any other molecule or combination of molecules.
  • the tested treatment may be directed towards attenuating a skin spot or causing it to disappear, modulating the skin’s pigmentation, unifying the complexion, making the skin colour homogeneous.
  • Treatments which are particularly preferred in the context of the present invention are cosmetic treatments, more particularly topical cosmetic treatments.
  • the pigmentary spot under consideration is a non-pathological pigmentary spot or irregularity, for example actinic, solar or senile lentigo.
  • the levels of expression of more than one gene are preferably compared.
  • the chosen gene is one from among the genes JAKMIP2, KIF21A, SLITRK6, SCIN, SYNPO2, TNNT1 and LRMP, and any paired or threesome combinations of the genes in this list are particularly preferred.
  • the skin sample preferably originates from a Caucasian or Asian human being, preferably at least 40 years old, preferably at least 50 years old, or even at least 60 years old.
  • the method for evaluating the efficiency of the treatment according to the present invention is preferably performed in vitro or ex vivo. It is desirable for the skin sample before treatment and after treatment to be taken from the same pigmentation spot, or else from a very close pigmentation spot if the size of the spot does not make it possible to easily obtain samples before and after treatment. in vitro method for screening active agents for preventing and/or treating skin pigmentary spots
  • the present invention also relates to an in vitro method for screening active agents for preventing and/or treating skin pigmentary spots, said method comprising the following steps: i. placing a cell model representative of the skin in contact with at least one active agent to be tested, or exposing said cell model to a physical treatment to be tested; ii.
  • vesicles preferably of the melanosomes, chosen from the list consisting of the genes JAKMIP2, ARC, KIF21A, SLITRK6, NRN1 , SCIN, SYNPO2, TNNT1 , SNTB1 , ARHGAP29, RAB3IP, RHOBTB3, RASIP1 , ASGR1 , CHMP4C, SVIP, LRMP, PCLO and NAV3, or the level of expression or activity of the expression product of said chosen gene, and iii. comparing said measurement with a reference measurement.
  • the term “representative skin cell model” means a model comprising at least skin cells such as keratinocytes and optionally melanocytes.
  • the levels of expression of at least one of the genes of the invention can be evaluated by reference or after standardization with the level of expression of other genes, the level of expression of which is supposed to be substantially identical in the spot and in the area of non-lesioned skin chosen.
  • genes for standardization are well known to those skilled in the art and may depend on the area of the body where the spot is located.
  • genes may be mentioned as being suitable for the standardization of the levels of expression of the genes of the invention encoding: ribosomal protein L13a (RPL13A), beta- 2-microglobulin (B2M), ribosomal protein S9 (RPS9), ribosomal protein S28 (RPS28) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
  • RPL13A ribosomal protein L13a
  • B2M beta- 2-microglobulin
  • RPS9 ribosomal protein S9
  • RPS28 ribosomal protein S28
  • GPDH glyceraldehyde-3-phosphate dehydrogenase
  • a reference measurement may be a quantitative or qualitative value relating to the level of expression of said gene(s), or the level of expression or activity of the expression product of said chosen gene(s) determined in a sample in the absence of the active agent or physical treatment being tested.
  • a reference measurement may be obtained by repeating the steps of a process of the invention, and notably steps i. and ii. of a method according to the invention as defined previously, in the absence of active agents or of physical treatments to be tested.
  • a comparison of the test measurement with a reference measurement, and an observation of a deviation or an absence of deviation between the two measurements makes it possible to draw information regarding the effect of the active agent or of the physical treatment tested.
  • the quantitative or qualitative determination of the level of expression of said gene(s), or the level of expression or activity of the expression product of said gene(s) may be performed by any method known to those skilled in the art, and notably as described previously.
  • the cell model may be of any type considered to be suitable by those skilled in the art. These may notably be monoculture or coculture cell models, or else three-dimensional models of reconstructed skin, or else skin cultured ex vivo. Cell models representative of pigmentation spots, more particularly of actinic lentigines, which can be used in the context of this method, also exist.
  • Such cell models do not need to mimic pigmentary spots (or lentigo) as a whole, but must mimic biological events, morphological characteristics or pigmentation characteristics observed in pigmentary spots, notably lentigo.
  • Such in vitro models are well known to those skilled in the art.
  • said cell model is a model comprising keratinocytes and melanocytes.
  • the in vitro method described above involves comparison of the levels of expression of at least two genes, preferably at least three, five, six, or eight genes of the invention, as explained for the other evaluation methods according to the invention.
  • the invention also relates to an in vitro method for screening active agents for preventing and/or treating skin pigmentary spots, said method comprising the following steps: i. placing a cell model comprising keratinocytes and optionally melanocytes, in which said keratinocytes exhibit repression of at least one gene involved in the management of vesicles, preferably of the melanosomes, chosen from the list consisting of the genes JAKMIP2, KIF21A, SLITRK6, SCIN, TNNT1 , SNTB1 , ARHGAP29, RAB3IP, RHOBTB3, ASGR1 , CHMP4C, SVIP, PCLO and NAV3, and/or in which said keratinocytes exhibit overexpression of at least one gene involved in the management of vesicles, preferably of the melanosomes, chosen from the list consisting of the genes ARC, NRN1 , SYNPO2, RASIP1 and LRMP, in contact with at least one active agent to be
  • vesicles preferably of the melanosomes, chosen from the list consisting of the genes JAKMIP2, ARC, KIF21A, SLITRK6, NRN1 , SCIN, SYNPO2, TNNT1 , SNTB1 , ARHGAP29, RAB3IP, RHOBTB3, RASIP1 , ASGR1 , CHMP4C, SVIP, LRMP, PCLO and NAV3, or the level of expression or activity of the expression product of said chosen gene; and iii. comparing said measurement with a reference measurement.
  • the term “repressed gene” means a gene whose expression level is lower than the average level of expression of said gene in the skin of persons free of pigmentary spots.
  • the term “overexpressed gene” means a gene whose expression level is higher than the average expression level of said gene in the skin of persons without pigmentary spots.
  • the term “qualitative or quantitative measurement of melanin” means both the determination of the presence of melanosomes, and/or the organization of melanosomes and/or the morphology of melanosomes in the keratinocytes notably using an optical or electronic microscope, but also the assay of melanin via techniques that are well known to those skilled in the art, or even the labelling of protein(s) characteristic of the presence of melanosomes, for instance the PM EL 17 protein, and assay thereof by immunofluorescence.
  • the repression or overexpression of the level of expression of one or more of the abovementioned genes may notably be achieved via any method known to those skilled in the art.
  • such cells can be obtained by transfection and homologous recombination of a nucleic acid fragment which inserts into or takes the place of the gene expressing the amino acid sequence whose expression is to be suppressed or overexpressed.
  • a given gene by transfection, for example using a lentivirus, into the cell of a nucleic acid sequence encoding an interfering RNA specific for the mRNA derived from the gene whose expression is to be suppressed.
  • the level of expression of at least one of the abovementioned genes is repressed by transfection into cells, preferably keratinocytes, of siRNA.
  • an expression vector preferably a plasmid comprising the nucleic acid sequence of the given gene and also a promoter sequence for the expression of said gene.
  • the present invention also relates to a cell model that is suitable for screening active agents for preventing and/or treating skin pigmentary spots, comprising keratinocytes and optionally melanocytes, in which said keratinocytes exhibit repression of at least one gene involved in the management of vesicles, preferably of the melanosomes chosen from the list consisting of the genes JAKMIP2, KIF21A, SLITRK6, SCIN, TNNT1 , SNTB1 , ARHGAP29, RAB3IP, RHOBTB3, ASGR1 , CHMP4C, SVIP, PCLO and NAV3, and/or in which said keratinocytes exhibit overexpression of at least one gene involved in the management of vesicles, preferably of the melanosomes, chosen from the list consisting of the genes ARC, NRN1 , SYNPO2, RASIP1 and LRMP.
  • a cell model that is suitable for screening active agents for preventing and/or treating skin pigmentary spots
  • the term “repressed gene” means a gene whose expression level is lower than the average level of expression of said gene in the skin of persons free of pigmentary spots.
  • the term “overexpressed gene” means a gene whose expression level is higher than the average expression level of said gene in the skin of persons without pigmentary spots.
  • said gene(s) are repressed by transfection of siRNA into said keratinocytes.
  • the present invention also relates to a method for preparing a cell model suitable for screening active agents for preventing and/or treating skin pigmentary spots, comprising the following steps: i. collecting keratinocytes and optionally melanocytes; and then ii. transfecting said keratinocytes collected in step i.
  • siRNA capable of repressing the level of expression of at least one gene involved in the management of vesicles, preferably of the melanosomes, chosen from the list consisting of the genes JAKMIP2, KIF21A, SLITRK6, SCIN, TNNT1 , SNTB1 , ARHGAP29, RAB3IP, RHOBTB3, ASGR1 , CHMP4C, SVIP, PCLO and NAV3; and/or transfecting said keratinocytes collected in step i.
  • an expression vector preferably a plasmid, notably a plasmid, of at least one gene involved in the management of vesicles, preferably of the melanosomes, chosen from the list consisting of the genes ARC, NRN1 , SYNPO2, RASIP1 and LRMP, and then iii. culturing the keratinocytes transfected in step ii. or co-culturing the keratinocytes transfected in step ii. with the melanocytes collected in step i.
  • the human keratinocytes and melanocytes are notably obtained from explants from surgical waste.
  • the culture media used for culturing the keratinocytes and melanocytes in step iii. are well known to those skilled in the art.
  • the keratinocytes and melanocytes are co-cultured for a period of 48h to 72h, preferably 72h.
  • the keratinocytes are cultured for a period of 48h to 72h, preferably 72h.
  • the present invention also relates to a cell model that can be obtained or that is directly obtained via the abovementioned preparation process.
  • Cosmetic method for treating or preventing a non-pathological skin pigmentary spot in human skin Cosmetic method for treating or preventing a non-pathological skin pigmentary spot in human skin
  • the present invention also relates to a cosmetic method for treating or preventing non-pathological skin pigmentary spots in human skin, comprising the application to the skin of a composition comprising at least one active agent modulating the level of expression or activity of at least one gene involved in the management of vesicles, preferably melanosomes, in which said gene is chosen from the list consisting of the genes JAKMIP2, ARC, KIF21A, SLITRK6, NRN1 , SCIN, SYNPO2, TNNT1 , SNTB1 , ARHGAP29, RAB3IP, RHOBTB3, RASIP1 , ASGR1 , CHMP4C, SVIP, LRMP, PCLO and NAV3.
  • a cosmetic method for treating or preventing non-pathological skin pigmentary spots in human skin comprising the application to the skin of a composition comprising at least one active agent modulating the level of expression or activity of at least one gene involved in the management of vesicles, preferably melanosomes, in which
  • the non-pathological skin pigmentary spots are understood as being benign spots, the removal of which is desirable only for aesthetic reasons and not for therapeutic reasons.
  • Pigmentation irregularities include imperfections in terms of pigmentation which make the complexion non-uniform or the skin colour non-homogeneous.
  • the application of said composition is preferably performed on healthy skin.
  • the present inventors have shown the important role of the genes of the invention within the dermis on pigmentary spots, and notably the link between deregulation of the level of expression of these genes and the appearance of pigmentary spots.
  • suspending or reducing the modulation of these genes may reduce or abolish the deregulations observed and thus make it possible to restore a situation as regards the genes involved in the management of vesicles, preferably of the melanosomes, notably of the transport of the melanosomes within the keratinocytes, which is compatible with the absence of pigmentary spots, thus with a uniform complexion and a homogeneous skin colour.
  • more than one gene from among the genes of the invention is preferably chosen, for example at least two genes, at least three, five, six or eight.
  • the cosmetic method is directed towards modulating the level of expression of all the genes of the invention.
  • the cosmetic method according to the invention is directed towards restoring expression levels close to those which are observed in healthy skin, for example adjacent to a pigmentary spot.
  • said pigmentary spot is a hyperpigmentary spot, for example an actinic, senile or solar lentigo.
  • the modulation sought in the cosmetic methods according to the invention is a total, partial or temporary inhibition of the expression of at least one gene chosen from the genes ARC, NRN1 , SYNPO2, RASIP1 and LRMP and/or an increase, possibly temporary, in the expression of at least one gene chosen from JAKMIP2, KIF21A, SLITRK6, SCIN, TNNT1 , SNTB1 , ARHGAP29, RAB3IP, RHOBTB3, ASGR1 , CHMP4C, SVIP, PCLO and NAV3.
  • the inhibition is not a total inhibition, but a partial inhibition, tending to reduce the level of expression of the chosen gene without completely inhibiting its expression.
  • the term “increase or decrease in the level of expression” includes the increase or decrease in the level of transcription of said genes, and the increase or decrease in the level of translation of said genes, and also the increase or decrease in the activity of the proteins encoded by said genes.
  • a cosmetic method according to the present invention thus comprises the application of a cosmetic composition notably comprising at least one active agent chosen from a chemical molecule, a natural extract, a nucleic acid, a peptide, or a treatment modulating the level of expression or the activity of the expression product of at least one of the genes of the invention.
  • the cosmetic composition is preferably applied topically.
  • the modulation is performed by means of the use of a modulator of one of the genes of the invention.
  • a cosmetic method according to the invention will advantageously comprise the application of a compound chosen from caffeine, calcitriol, folic acid, hyaluronic acid, palm oil, sesame oil, curcumin, and mixtures thereof.
  • a cosmetic method according to the invention is performed after the characterization of the pigmentary spot that it is desired to treat by means of a method according to the first aspect.
  • this first step makes it possible to characterize the spot and thus to detect the genes of which the expression level is strongly modulated between the area of the spot and a non-lesioned, preferably adjacent, area. It is then possible to adapt a treatment which makes it possible to act on the gene(s) of the invention which are differentially modulated within this spot, by specifically applying modulators for said genes.
  • the cosmetic method for treating or preventing a non- pathological skin pigmentary spot in human skin comprises the following steps: i. characterizing an apparent or suspected skin pigmentary spot in a human according to the characterization method according to the invention; ii. deducing from step i. whether said spot is an apparent or suspected skin pigmentary spot; iii.
  • a cosmetic composition comprising an active agent for modulating the level of expression of at least one gene involved in the management of vesicles, preferably of the melanosomes, chosen from the list consisting of the genes JAKMIP2, ARC, KIF21A, SLITRK6, NRN1 , SCIN, SYNPO2, TNNT1 , SNTB1 , ARHGAP29, RAB3IP, RHOBTB3, RASIP1, ASGR1 , CHMP4C, SVIP, LRMP and PCLO and NAV3.
  • a cosmetic composition comprising an active agent for modulating the level of expression of at least one gene involved in the management of vesicles, preferably of the melanosomes, chosen from the list consisting of the genes JAKMIP2, ARC, KIF21A, SLITRK6, NRN1 , SCIN, SYNPO2, TNNT1 , SNTB1 , ARHGAP29, RAB3IP, RHOBTB3, RASIP1, ASGR
  • the cosmetic method according to the invention may also comprise the application of one or more additional active agents aimed at reinforcing the desired effects, for example any substance described as depigmenting, keratolytic and/or desquamating agents, antioxidants, chemical or physical UV sunscreens, anti-inflammatory and/or soothing agents, deoxyribonucleic acids and derivatives thereof.
  • additional active agents aimed at reinforcing the desired effects, for example any substance described as depigmenting, keratolytic and/or desquamating agents, antioxidants, chemical or physical UV sunscreens, anti-inflammatory and/or soothing agents, deoxyribonucleic acids and derivatives thereof.
  • the cosmetic method according to the present invention is also applicable in the case of prevention of the appearance of pigmentary spots or other irregularities of skin complexion or colour, notably hyperpigmentary spots such as actinic lentigo.
  • the present invention also relates to the cosmetic use of a modulator of the level of expression or of the activity of the expression product of at least one gene involved in the management of vesicles, preferably of the melanosomes, chosen from the list consisting of the genes JAKMIP2, ARC, KIF21A, SLITRK6, NRN1 , SCIN, SYNPO2, TNNT1 , SNTB1 , ARHGAP29, RAB3IP, RHOBTB3, RASIP1 , ASGR1 , CHMP4C, SVIP, LRMP, PCLO and NAV3 in the prevention and/or treatment of non-pathological skin pigmentary spots.
  • a modulator of the level of expression or of the activity of the expression product of at least one gene involved in the management of vesicles preferably of the melanosomes, chosen from the list consisting of the genes JAKMIP2, ARC, KIF21A, SLITRK6, NRN1 , SCIN, SYNPO2, TN
  • the modulator used is an inhibitor of at least one gene chosen from ARC, NRN1 , SYNPO2, RASIP1 and LRMP, and/or an activator of at least one gene chosen from JAKMIP2, KIF21A, SLITRK6, SCIN, TNNT1 , SNTB1 , ARHGAP29, RAB3IP, RHOBTB3, ASGR1 , CHMP4C, SVIP, PCLO and NAV3.
  • the inhibitor is a partial inhibitor leading to a reduction in the level of expression or a reduction in the activity of the expression product of said gene, without completely abolishing the expression or activity of said gene.
  • Such modulators may notably be caffeine, calcitriol, folic acid, hyaluronic acid, palm oil, sesame oil, curcumin, and mixtures thereof, for use in the cosmetic treatment of hyperpigmentary spots.
  • modulators are also antisense molecules, siRNAs, and microRNAs.
  • Preferred modulators in the context of the present invention are notably caffeine, calcitriol, folic acid, hyaluronic acid, palm oil, sesame oil, curcumin, and mixtures thereof.
  • the modulators can be used in combination with other products, active agents or excipients. Preferably, they are conditioned in a form suitable for topical application, for example in the form of an ointment, a cream, notably in emulsion form, a lotion or a salve.
  • a form suitable for topical application for example in the form of an ointment, a cream, notably in emulsion form, a lotion or a salve.
  • the pigmentary spots under consideration are preferably hyperpigmentary spots and most preferentially actinic, solar or senile lentigines.
  • actinic lentigines are chosen on the back of the hand, with a minimum size of 3 mm.
  • biopsies 3 mm in diameter were performed on one of the hands of each patient.
  • one of the biopsies corresponds to the actinic lentigo (AL) lesion and the other to an adjacent area of non-lesional skin (NS) (also verified by epiluminescence).
  • A actinic lentigo
  • NS non-lesional skin
  • RNA stabilizing solution RNAIater, Qiagen reference 76154
  • the samples are cut up with a scalpel in order to facilitate homogenization, and then transferred into the lysis buffer (Qiagen lysis buffer, RLT + 10% beta-mercaptoethanol).
  • the homogenization is performed in a Potter grinder (Fisher Labosi ref. A6391000) with RNase-free polypropylene pistons (Fisher Labosi ref. A1419753).
  • the homogenization is performed with the TissueRuptor from Qiagen (ref. 990890).
  • RNA was extracted with the RNeasy micro kits (Qiagen ref.: 74004) according to the supplier’s instructions.
  • the RNA quantification is performed by means of a RiboGreen assay (Molecular Probes ref. R11490).
  • the RNA quantification is performed by spectrophotometry (Nanodrop 8000).
  • RNA quality is validated with an Agilent 2100 bioanalyser which gives the ratio of the 28S ribosomal RNA intensity to the 18S ribosomal RNA intensity, and also an RNA Integrity Number (RIN) which takes into account the RNA degradation.
  • RIN RNA Integrity Number
  • a reverse transcriptase (RT) reaction is performed in order to obtain the corresponding cDNAs.
  • RT reverse transcriptase
  • a cDNA amplification step is performed.
  • the cDNAs are then labelled with fluorochromes (Affymetrix labelling kit) and/or hybridized on Affymetrix® DNA chips which make it possible to reveal the level of expression of all the genes of the human gene (DNA biochips of Affymetrix U133A 2.0 type containing 54 000 probes, thus making it possible to study the expression of 47 000 transcripts, including 38 500 characterized genes).
  • fluorochromes Affymetrix labelling kit
  • the Affymetrix RMA software was used to generate the raw files and to perform the quality controls of the two studies before a data analysis. After revealing the specific hybridizations and processing the raw data (extraction, subtraction of the background noise, standardization), the gene expression is compared between the healthy skin and the lesioned skin.
  • T test a linear T-test was applied to the data with the aid of the ArrayStudio software in order to compare the gene expression profile of the actinic lentigo biopsy group with that of the non-lesional-skin biopsy group.
  • a list of 1973 ProbeSets significantly modulated (p ⁇ 0.05) in actinic lentigo compared with non- lesional skin was thus established for the French study and a list of 3084 ProbeSets significantly modulated in actinic lentigo compared with non-lesional skin for the Japanese study.
  • Unsupervised analysis o Suppression of the patient effect: in order to suppress the patient effect observed in the results of the differential analyses, the following algorithm was performed: each patient and each probeset, 1) subtraction of the Iog2 mean expression of the two conditions (lesional, non-lesional), 2) addition of the Iog2 mean expression of all of the patients and conditions.
  • NMF Non Negative Matrix Factorization
  • the common signature of the two studies thus includes 245 genes significantly modulated in the two studies.
  • genes of this family are predominantly under-expressed in actinic lentigo.
  • Table 1 List of genes grouped in sub-families all associated with the functional family of vesicle/melanosome regulation. The first column indicates the sub-family of genes related to vesicle/melanosome organization, the second column the gene symbol, the third column details the gene name. Columns 4 and 6 indicate the average modulation level (Fold-change (FC), geometric mean of the individual modulation levels over all study patients in Actinic Lentigo) relative to the adjacent non-lesional skin (NLS) in the French study and the Japanese study, respectively. Columns 5 and 7 show the p-values associated with these modulations in the French study and the Japanese study, respectively.
  • Example 2 Modulators of interest on the signature
  • the interest for the treatment of actinic lentigo is to have inhibitors of the level of expression of at least one gene chosen from ARC, NRN1 , SYNPO2, RASIP1 and LRMP, or activators of the level of expression of at least one gene chosen from JAKMIP2, KIF21A, SLITRK6, SCIN, TNNT1, SNTB1 , ARHGAP29, RAB3IP, RHOBTB3, ASGR1 , CHMP4C, SVIP, PCLO and NAV3.

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Abstract

La présente invention concerne une méthode de caractérisation d'une tache pigmentaire de la peau apparente ou suspecté chez un être humain, comprenant la mesure des niveaux d'expression puis la comparaison des niveaux d'expression mesurés, dans des échantillons de peau provenant de ladite tache et provenant de la peau non lésée adjacente, d'au moins un gène impliqué dans la gestion de vésicules, de préférence des mélanosomes, choisi dans la liste constituée des gènes JAKMIP2, ARC, KIF21A, SLITRK6, NRN1, SCIN, SYNPO2, TNNT1, SNTB1, ARHGAP29, RAB3IP, RHOBTB3, RASIP1, ASGR1, CHMP4C, SVIP, LRMP, NAV3 et PCLO. L'invention concerne également des méthodes d'évaluation de l'efficacité d'un traitement des taches pigmentaires, des méthodes cosmétiques de traitement des taches pigmentaires, ainsi que divers modulateurs desdits gènes et leur utilisation.
PCT/EP2023/064124 2022-05-25 2023-05-25 Signature moléculaire de lentigo actinique associée à la gestion de vésicules Ceased WO2023227746A2 (fr)

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FR2974371A1 (fr) 2011-04-22 2012-10-26 Oreal Signature moleculaire des taches pigmentaires cutanees, associee a la famille des proteoglycanes et glycoproteines extracellulaires
FR2974373A1 (fr) 2011-04-22 2012-10-26 Oreal Signature moleculaire des taches pigmentaires cutanees, associee a la matrice extracellulaire
FR2974370A1 (fr) 2011-04-22 2012-10-26 Oreal Signature moleculaire des taches pigmentaires cutanees, associee a la voie de signalisation tgf-beta - smad
FR2974372A1 (fr) 2011-04-22 2012-10-26 Oreal Signature moleculaire des taches pigmentaires cutanees, associee au remodelage de la matrice extracellulaire

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