(19) *EP003302716B1*

(11) EP 3 302 716 B1

(12) EUROPEAN PATENT SPECIFICATION

(45) Date of publication and mention (51) Int Cl.: of the grant of the patent: A61Q 17/04 (2006.01) G01N 33/50 (2006.01) (2006.01) (2006.01) 14.10.2020 Bulletin 2020/42 A61Q 19/08 A61Q 19/00 C12Q 1/68 (2018.01) (21) Application number: 16729482.6 (86) International application number: (22) Date of filing: 08.06.2016 PCT/US2016/036401

(87) International publication number: WO 2016/200905 (15.12.2016 Gazette 2016/50)

(54) METHODS FOR IDENTIFYING CIRCADIAN RHYTHM-DEPENDENT COSMETIC AGENTS FOR SKIN CARE COMPOSITIONS VERFAHREN ZUR IDENTIFIZIERUNG VON BIORHYTHMUSABHÄNGIGEN KOSMETISCHEN MITTELN FÜR HAUTPFLEGEZUSAMMENSETZUNGEN PROCÉDÉS D’IDENTIFICATION D’AGENTS COSMÉTIQUES DÉPENDANT DU RYTHME CIRCADIEN POUR DES COMPOSITIONS DE SOIN DE LA PEAU

(84) Designated Contracting States: • OSBORNE, Rosemarie AL AT BE BG CH CY CZ DE DK EE ES FI FR GB Cincinnati, Ohio 45202 (US) GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR (74) Representative: P&G Patent Belgium UK N.V. Procter & Gamble Services Company S.A. (30) Priority: 08.06.2015 US 201562172498 P Temselaan 100 1853 Strombeek-Bever (BE) (43) Date of publication of application: 11.04.2018 Bulletin 2018/15 (56) References cited: WO-A1-2010/079285 JP-A- 2010 098 965 (73) Proprietor: The Procter & Gamble Company US-A1- 2009 220 481 US-A1- 2010 028 317 Cincinnati, OH 45202 (US) US-A1- 2015 071 895

(72) Inventors: • GEYFMAN MIKHAIL ET AL: "Clock genes, hair • MULLINS, Lisa, Ann growth and aging", AGING, NEW YORK, NY, US, Cincinnati, Ohio 45202 (US) vol. 2, no. 3, 1 March 2010 (2010-03-01), pages • TAMURA, Makio 122-128, XP002630052, ISSN: 0160-2721 Cincinnati, Ohio 45202 (US) [retrieved on 2010-03-18]

Note: Within nine months of the publication of the mention of the grant of the European patent in the European Patent Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the Implementing Regulations. Notice of opposition shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). EP 3 302 716 B1

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Description ing factors as either intrinsic or extrinsic, although these are interdependent, reflected for example by the fact that [0001] Skin is a complex, multi-layered and dynamic extrinsic factors may accelerate intrinsic aging. One ex- system that provides a protective covering defining the ample of the complex interplay of factors involves free interactive boundary between an organism and the en- 5 radicals, which are both generated internally through nor- vironment. It is the largest organ of the body and is vitally mal metabolic processes and produced as a conse- important to both our health and our self-image. The skin quence of external factors, including UVR exposure. As comprises three principal layers, the epidermis, the der- a result of the age-associated decline in protective inter- mis, and a layer of subcutaneous fat. The majority of cells nal antioxidant mechanisms, free radicals can reach in the epidermis are keratinocytes that produce a family 10 higher and sustained levels in cells and alter both proteins of proteins called keratins. The epidermis itself may be and DNA in skin. Levels of altered protein and DNA may divided into multiple layers with the outermost layer re- accumulate causing damage, sometimes referred to as ferred to as the stratum corneum, and the innermost layer oxidative stress. In addition, ongoing accumulation of referred to as the basal layer. All epidermal cells originate damage secondary to internally-generated free radicals from the basal layer and undergo a process known as 15 combined with those generated from UVR and other ex- differentiation as they gradually displace outward to the ternal assaults (surfactants, allergens, and other irritants) stratum corneum, where they fuse into squamous sheets can promote a chronic inflammatory state, which accel- and are eventually shed. In healthy, normal skin, the rate erates the aging process. For example, proteolytic en- of production is about the same as the rate of shedding zymes may be produced, resulting in collagen degrada- (desquamation). Fully mature keratinocytes function to 20 tion. In some instances, activated inflammatory cells re- protect the skin from UV light damage, and help effectu- sulting from elevations in circulating pro-inflammatory ate immune response to environmental stimuli. mediators (e.g., prostaglandins, cytokines, histamines) [0002] The dermis, which lies just beneath the epider- can produce reactive oxygen species that cause oxida- mis, is composed largely of the protein collagen, which tive damage to nucleic acids, cellular proteins, and lipids. accounts for up to 75% of the weight of the dermis and 25 Accumulated damage caused by reactive oxygen spe- is responsible for the resilience and elasticity of skin. Col- cies may stimulate a host of cytokine cascades that re- lagen bundles are held together by elastin fibers running sults in photo-aging and photo-carcinogenesis, all of through the dermis. Fibroblasts, which are the primary which can be tied to the appearance of aging skin. cells found in the dermis, function to synthesize collagen [0006] The changes caused by oxidative stress may and the dermis ground substance, which is an extracel- 30 compromise skin’s elasticity, firmness and structure, lular matrix comprising glycoproteins and gly- contributing to areas of collapse and irregularity and ul- cosaminoglycans that enmeshes fibrillar and cellular timately manifesting as fine lines, wrinkles, and texture components of the dermis. Networks of tiny blood vessels problems. There are many commercially available skin run through "rete pegs" in the dermis, bringing nutrients, care products available to consumers that are directed vitamins and oxygen to the epidermis via diffusion. 35 to improving the health and/or physical appearance of [0003] Beneath the dermis lies the hypodermis, which skin. Many such products are directed to delaying, min- comprises subcutaneous fat that cushions the dermis imizing, or even eliminating changes typically associated from underlying tissues such as muscle and bones. The with improving the appearance of aging skin. Such prod- fat is contained in adipose cells embedded in a connec- ucts typically advertise the use of one or more of cosmetic tive tissue matrix. This layer may also house the hair 40 skin-care agents known for use in improving the health follicles when they are in the growing phase. and/or appearance of skin. Accordingly, there remains a [0004] Thus, skin is a multilayered complex organ com- need to identify skin-care actives that can improve the prising a wide variety of cellular types and structures. appearance of aging skin. Skin aging is likewise a complex multi-factorial process [0007] Successful identification of new anti-aging cos- that results from unrepaired cellular and tissue damage 45 metic agents has proven to be difficult due to the multi- leading to impaired functional capacity. The aging proc- cellular, multi-factorial processes associated with skin ess in skin is the result of both intrinsic and extrinsic fac- aging. In addition, many desirable cosmetic agents may tors occurring over decades. Skin is subject to many of comprise a mixture of compounds with effects and inter- the same intrinsic aging processes as other organs, but actions that may not be fully understood. An additional is also exposed to solar radiation, pollution, cigarette 50 challenge for cosmetic formulators is that cosmetics must smoke, and other extrinsic factors that can contribute to be safe for over-the-counter consumer use. Convention- premature skin aging or photo-aging. There have been al in vitro studies of biological responses to potential cos- major advances in the understanding of the aging proc- metic agents involve screening hundreds or even thou- ess with the identification of cellular pathways and genes sands of potential agents in various cell types before an associated with longevity and aging. However, as with 55 agent that gives a desired result can be identified and aging in general, an integrated understanding of skin ag- moved into a next stage of testing. This problem may be ing has not been developed. further compounded when employing screening tech- [0005] Skin researchers have categorized age-induc- niques such as connectivity mapping or other known

2 3 EP 3 302 716 B1 4 gene response analysis techniques. Such studies can treatments. There is also a need to identify additional be hindered by the complex or weakly detectable gene cosmetic agents that provide similar or improved benefits responses typically induced and/or caused by cosmetic as compared to existing products but which are easier agents. Such weak responses arise, in part, due to the to formulate, produce, and/or market. great number of genes and gene products involved, and 5 cosmetic agents may affect multiple genes in multiple SUMMARY ways. Moreover, the degree of bioactivity of cosmetic agents may differ for each gene and be difficult to quan- [0011] Accordingly, the present invention provides tify. novel methods useful for the screening and generation [0008] Until now, skin studies typically have not taken 10 of potential cosmetic agents that work in synchronization into account the circadian rhythm of the skin and how the with the circadian rhythm of the skin for the treatment of circadian rhythm of the skin affects the efficacy of the aged skin. Through gene expression profiling and bioin- cosmetic agents. Patent application JP2010098965A formatics analysis, the present inventors have deter- discloses a screening assay for cosmetic material that mined that it is possible to derive novel and unique gene takes into account the circadian rythm of the skin. Like 15 signatures for use in developing novel screening meth- other organs, the skin is subject to the influence of bio- ods for identifying cosmetic test agents as effective for logic rhythms. Biologic rhythms are physiologic changes providing a circadian rhythm-dependent, DNA repair occurring over time with a reproducible waveform. Cel- benefit to human skin. These unique gene signatures lular functions of the skin, including DNA repair, do not may serve as indicators of previously unidentified path- occur at random times with equal probability. Instead, 20 ways associated with DNA repair, and thus can provide these cellular activities are regulated by an endogenous opportunities for identifying new classes of cosmetic clock having a circadian rhythm (24 hours). The body agents. accepts environmental cues such as the presence and [0012] In some instances, the methods allow for the absence of daylight, to help synchronize the endogenous screening of cosmetic test agents that work in synchro- clocks of various cells and systems throughout the body. 25 nization with the circadian rhythm of the skin, for example, As such, the circadian cycle consists of light and dark the dark cycle of the skin’s circadian cycle. These novel cycles that typically coincide with the phases of solar day. methods also allow for identification of new cosmetic For example, the light cycle may correspond to the hours agents that can be screened for their selective treatment of about 6:00 AM to 6:00 PM, and the dark cycle may of skin aging conditions and for the specific targeting of correspond to the hours of about 6:00 PM to about 6:00 30 particular skin cell types, such as keratinocytes or fibrob- AM. lasts. Thus, the invention provides methods uniquely suit- [0009] Circadian rhythms allow skin cells to anticipate ed for desired treatment targets. Additionally, these changes in the environment that could potentially affect methods are particularly useful as they may serve as the cells, and adapt accordingly. In subject with good indicators of previously unidentified pathways associated health and with properly functioning circadian rhythms, 35 with DNA repair, and thus can provide opportunities for skin cells function in a synchronized manner and carry identifying new classes of cosmetic agents. out their various functions at an optimal time. For exam- [0013] Accordingly, Accordingly, a screening method ple, the circadian rhythms of humans suggest that during for identifying a cosmetic test agent as effective for pro- the day, the skin promotes various protective functions viding a circadian rhythm-dependent, DNA repair benefit with regard to the environment. The circadian rhythms 40 to human skin is provided. The method comprises: (a) of humans suggests that at night, the skin promotes cel- contacting a skin tissue sample with a cosmetic test lular renewal and various metabolic synthesis processes. agent; (b) generating a transcriptional profile for the skin Although it is generally known that the efficacy of skin tissue sample, wherein the transcriptional profile com- care treatments can be optimized in accordance time of prises data related to the transcription of at least two day of administration and it is also been shown that both 45 genes selected from APITD1, ACTR5, AP5Z1, APEX1, the circadian clock and biological rhythms are affected APEX2, APLF, APTX, ATXN3, BCCIP, BRCA1, by the aging process, there are currently no methods C11orf30, CDC14B, CHEK1, CUL4A, DCLRE1C, DTL, available for identifying new anti-aging cosmetic agents EPC2, EXO5, EYA3, FAM175A, FAN1, FANCC, FANCF, that work in synchronization with the circadian rhythm of FANCG, FANCL, FIGNL1, GADD45A, GTF2H1, H2AFX, the skin to maximize anti-aging efficacy. 50 INTS3, KIAA0430, KIAA0101, KIN, MCM9, MDC1, [0010] Thus, although many skin care agents are MGME1, MORF4L2, MRE11A, MSH2, MUM1, NEIL1, known, an ongoing need exists for improved, sensitive, NEIL3, NONO, NPM1, NSMCE1, OGG1, PARPBP, and predicative screening methods to accurately identify PIF1, PML, PMS1, PMS2, POLD1, POLD2, POLE, new cosmetic agents that not only provide for the treat- POLK, POLR2F, RAD50, RAD51B, RAD51C, RAD52, ment of chronologically aged and/or photo-aged skin, but 55 RBM14, RECQL5, RFC4, RFC5, RPA2, SFPQ, SFR1, that also can more specifically target particular skin cell SMARCA5, SMC4, SMG1, SWI5, TICRR, TP53BP1, types and can work in synchronization with the circadian TP73, UBE2T, UBE2W, USP28, USP47, WDR33, rhythm of the skin to maximize efficacy and optimize ZFYVE26; (c) comparing the transcriptional profile for

3 5 EP 3 302 716 B1 6 the skin tissue sample to a control transcriptional profile; across different embodiments, and materials, features, and (d) identifying the cosmetic test agent as effective structures and/or characteristics may be omitted or sub- for providing a circadian rhythm-dependent, DNA repair stituted from what is described. Thus, embodiments and benefit to human skin when the transcriptional profile for aspects described herein may comprise or be combina- the skin tissue sample and the control transcriptional pro- 5 ble with elements or components of other embodiments file are concordant. and/or aspects despite not being expressly exemplified [0014] These and additional objects, embodiments, in combination, unless otherwise stated or an incompat- and aspects of the invention will become apparent by ibility is stated. reference to the Figures and Detail Description below. [0017] All ingredient percentages are by weight of the The scope of the invention is defined by the appended 10 corresponding composition, unless specifically stated claims. otherwise. All ratios are weight ratios, unless specifically stated otherwise. All ranges are inclusive and combina- BRIEF DESCRIPTION OF THE FIGURES ble. The number of significant digits conveys neither a limitation on the indicated amounts nor on the accuracy [0015] 15 of the measurements. All numerical amounts are under- stood to be modified by the word "about" unless otherwise FIGS. 1A and 1B illustrate that global gene expres- specifically indicated. Unless otherwise indicated, all sion profiles revealed rhythmic patterns of expres- measurements are understood to be made at approxi- sion in a number of Gene Ontology categories, in- mately 25 °C and at ambient conditions, where "ambient cluding DNA repair genes. 20 conditions" means conditions under about 1 atmosphere FIG. 2 illustrates that several key genes controlling of pressure and at about 50% relative humidity. All nu- DNA repair and several metabolic processes dis- meric ranges are inclusive of narrower ranges; delineat- played strong circadian rhythmicity. FIG. 2A illus- ed upper and lower range limits are interchangeable to trates the interaction network of representative DNA create further ranges not explicitly delineated. repair genes. FIG. 2B illustrates representative DNA 25 [0018] The transcriptional profiles herein can com- repair genes expression changes. prise, consist essentially of, or consist of, data related to FIG. 3 illustrates representative data for exemplary the genes in a subject gene signature (e.g., in the form DNA repair genes in the epidermis. FIG. 3A illus- of gene identifiers and direction of regulation) as well as trates the expression change (%) of exemplary other optional components described herein (e.g., meta- genes in the epidermis due to aging (expression 30 data). As used herein, "consisting essentially of’ means change % from average of 20s). FIG. 3B illustrates that a transcriptional profile includes data related to the the expression change (%) of expression of repre- transcription of only select genes from a subject gene sentative DNA repair genes in keratinocytes in vitro signature or gene expression profile, but may also in- with GSE treatment (0.01%). FIG. 3C illustrates the clude additional data only if the additional data is not re- circadian pattern of representative DNA repair genes 35 lated to the transcription of genes not included in the sub- in the epidermis from full thickness punch biopsies. ject gene signature, and which do not materially alter the FIG. 4 illustrates representative data for exemplary basic and novel characteristics of the claimed composi- DNA repair genes in the dermis. FIG. 4A illustrates tions or methods. As used in the description and the ap- the expression change (%) of exemplary genes in pended claims, the singular the dermis due to aging (expression change % from 40 forms "a," "an," and "the" are intended to include the plu- average of 20s). FIG. 4B illustrates the expression ral forms as well, unless the context clearly indicates oth- change (%) of expression of representative DNA re- erwise. pair genes in fibroblasts in vitro with GSE treatment [0019] The genes disclosed herein correspond to their (0.01%). FIG. 4C illustrates the circadian pattern of respective known sequences as of June 8, 2015. representative DNA repair genes in the dermis from 45 full thickness punch biopsies. Definitions

DETAILED DESCRIPTION [0020] "Circadian clock" means the endogenous cycle of about 24 hours that regulates the activities of a cell. [0016] Reference within the specification to "embodi- 50 [0021] "Circadian rhythm-dependent benefit" means a ment(s)" or the like means that a particular material, fea- benefit to keratinous tissue (including a DNA repair ben- ture, structure and/or characteristic described in connec- efit) that works in synchronization with the circadian tion with the embodiment is included in at least one em- rhythm of human skin tissue. bodiment, optionally a number of embodiments, but it [0022] "Circadian rhythm gene signature" means a does not mean that all embodiments incorporate the ma- 55 gene signature derived from gene expression profiling of terial, feature, structure, and/or characteristic described. the circadian rhythm patterns of keratinous tissue. Furthermore, materials, features, structures and/or char- [0023] "Cosmetic agent" or "cosmetic test agent" mean acteristics may be combined in any suitable manner any substance, as well any component thereof, intended

4 7 EP 3 302 716 B1 8 to be rubbed, poured, sprinkled, sprayed, introduced into, In some embodiments, a gene signature may comprise or otherwise applied to a mammalian body or any part a first list representative of a plurality of up-regulated thereof. Cosmetic agents may include substances that genes of the condition of interest and a second list rep- are Generally Recognized as Safe (GRAS) by the US resentative of a plurality of down-regulated genes of the Food and Drug Administration, food additives, and ma- 5 condition of interest. terials used in non-cosmetic consumer products includ- [0028] "Intrinsic aging gene signature" means a gene ing over-the-counter medications. In some embodi- signature derived from gene expression profiling of an ments, cosmetic agents may be incorporated in a cos- intrinsic aging skin condition. metic composition comprising a dermatologically accept- [0029] "Intrinsic aging skin condition" means a skin ag- able carrier suitable for topical application to skin. Some 10 ing condition that derives, in whole or part, from chrono- non-limiting examples of cosmetic agents or cosmetically logical aging of the skin. "Intrinsically-aged" skin thus re- actionable materials can be found in: the PubChem da- fers to skin that has been chronologically aged, and has tabase associated with the National Institutes of Health, been substantially protected from exposure to sunlight USA; the Ingredient Database of the Personal Care Prod- and/or ultraviolet light. ucts Council; and the 2010 International Cosmetic Ingre- 15 [0030] "Keratinous tissue," means keratin-containing dient Dictionary and Handbook, 13th Edition, published layers disposed as the outermost protective covering of by The Personal Care Products Council; the EU Cosmet- mammals which includes, but is not limited to, skin, hair, ic Ingredients and Substances list; the Japan Cosmetic nails, cuticles, horns, claws, beaks, and hooves. With Ingredients List; the Personal Care Products Council, the respect to skin, the term refers to one or all of the dermal, SkinDeep database; the FDA Approved Excipients List; 20 hypodermal, and epidermal layers, which includes, in the FDA OTC List; the Global New Products Database part, keratinous tissue. (GNPD); and from suppliers of cosmetic ingredients and [0031] "Microarray" means any ordered array of nucle- botanicals. ic acids, oligonucleotides, proteins, small molecules, [0024] "Dark cycle of the circadian clock" means the large molecules, and/or combinations thereof on a sub- time period in which a subject sleeps. This is usually at 25 strate that enables gene expression profiling of a biolog- night (absence of daylight). However, for certain sub- ical sample. Some non-limiting examples of microarrays jects, the dark cycle can occur during the day, for example are available from Affymetrix, Inc.; Agilent Technologies, a person who works at night and sleeps during the day. Inc.; Ilumina, Inc.; GE Healthcare, Inc.; Applied Biosys- Conversely, "light cycle" is the cycle of the circadian clock tems, Inc.; and Beckman Coulter, Inc. during which a subject is typically awake. 30 [0032] "Photo-aging gene signature" means a gene [0025] "Dermatologically acceptable" means that the signature derived from gene expression profiling of a compositions or components described are suitable for photo-aging skin condition. use in contact with human skin tissue. [0033] "Photo-aging skin condition" means a skin ag- [0026] "Gene expression profiling" and "gene expres- ing condition that derives, in whole or part, from exposure sion profiling experiment" mean a measurement of the 35 to sunlight and/or ultraviolet light (e.g., UVR, UVA, UVB, expression of multiple genes in a biological sample using and/or UVC). "Photo-aged" skin thus refers to skin that any suitable profiling technology. For example, the mR- has been exposed to sunlight and/or ultraviolet light. NA expression of thousands of genes may be determined [0034] "Safe and effective amount" means an amount using microarray techniques. Other emerging technolo- of a compound or composition sufficient to significantly gies that may be used include RNA-Seq or whole tran- 40 induce a positive benefit, preferably a positive skin or feel scriptome sequencing using NextGen sequencing tech- benefit, including independently or in combinations the niques. benefits disclosed herein, but low enough to avoid seri- [0027] "Gene signature," means a rationally derived ous side effects, i.e., to provide a reasonable benefit to list, or plurality of lists, of genes representative of a skin risk ratio, within the scope of sound judgment of the tissue condition or response to a cosmetic agent. In spe- 45 skilled artisan. cific contexts, the cosmetic agent may be a benchmark [0035] "Skin" means the outermost protective covering skin agent or a potential skin agent. Thus, the gene sig- of mammals that is composed of cells such as keratino- nature may serve as a proxy for a phenotype of interest cytes, fibroblasts and melanocytes. Skin includes an out- for skin tissue. A gene signature may comprise genes er epidermal layer and an underlying dermal layer. Skin whose expression, relative to a normal or control state, 50 may also include hair and nails as well as other types of is increased (up-regulated), whose expression is de- cells commonly associated with skin, such as, for exam- creased (down-regulated), and combinations thereof. ple, myocytes, Merkel cells, Langerhans cells, macro- Generally, a gene signature for a modified cellular phe- phages, stem cells, sebocytes, nerve cells and adi- notype may be described as a set of genes differentially pocytes. expressed in the modified cellular phenotype over the 55 [0036] "Skin aging" means a human skin tissue condi- cellular phenotype. A gene signature can be derived from tion resulting from the expression or repression of genes, various sources of data, including but not limited to, from environmental factors (e.g., sun exposure, UVA and/or in vitro testing, in vivo testing and combinations thereof. UVB exposure, smoking), intrinsic factors (e.g. endog-

5 9 EP 3 302 716 B1 10 enous free radical production or cellular senescence) or and quantification of the biomarker. Suitable examples interactions there between that produces one or more of of methods of quantifying gene expression are disclosed fine lines and/or wrinkles, dry skin, inflamed skin, rough in U.S. Publication No. 2012/0283112; U.S. application skin, sallow skin, telangectasia, sagging skin, enlarged Ser. Nos. 13/851,858, 13/851,864, 13/851,873, and pores, and combinations thereof. 5 13/851,886; and U.S. Ser. No. 13/966,418, filed by Mills, [0037] "Skin-care" means regulating and/or improving et al., on Aug. 15, 2012. a skin condition. Some non-limiting examples of "skin- [0041] A non-limiting example of gene expression pro- care products" include skin creams, moisturizers, lotions, filing involves a method of measuring gene expression and body washes. "Skin-care composition" means a and comparing the gene expression measurements to composition that regulates and/or improves skin condi- 10 reference gene expression measurements (e.g., taken tion. from a control sample comprises exposing test cells (e.g., [0038] "Topical application" means to apply or spread keratinocytes and/or other skin cell) to a test agent, such the compositions of the present invention onto the sur- as a cosmetic test agent. The test agent may be dissolved face of the keratinous tissue. in a suitable carrier such as dimethyl sulfoxide (DMSO). [0039] Novel gene signatures have now been discov- 15 Optionally, reference cells, which are typically the same ered which can be used to identify or evaluate the poten- type of cell as the test cells but which are only exposed tial efficacy of a test agent for providing a skin-care ben- to the carrier (i.e., no test agent), may be used as a con- efit. In addition, novel methods useful for the screening trol. After exposure to the test agent and/or control, mR- and generation of potential cosmetic agents that work in NA is extracted from the test cells and reference cells. synchronization with the circadian rhythm of the skin for 20 The mRNA extracted from the cells may, optionally, be the treatment of aged skin are provided herein. These reverse transcribed to cDNA and marked with fluorescent novel methods exploit the newly discovered gene signa- dye(s) (e.g., red and green if a two color microarray anal- tures to identify or evaluate the potential ability of a test ysis is to be performed). In some instances, the cDNA agent to provide a targeted benefit to particular portion samples may be prepped for a one color microarray anal- of skin or type of skin cell. Thus, the novel methods allow 25 ysis, and a plurality of replicates may be processed if for the identification or evaluation of cosmetic agents that desired. The cDNA samples may be co-hybridized to the may provide maximize efficacy and allow for improved microarray comprising a plurality of probes (e.g., tens, skin-care treatments. The genes of interest disclosed hundreds, or thousands of probes). In some embodi- herein, and subsets thereof, may be used as gene panels ments, each probe on the microarray has a unique probe to identify cosmetic agents that can provide a circadian- 30 set identifier. The microarray is scanned by a scanner, rhythm dependent DNA repair benefit to the epidermal which excites the dyes and measures the amount fluo- and/or dermal layer of photo-aged and/or intrinsically- rescence. A computing device analyzes the raw images aged skin. Furthermore, these unique gene signatures to determine the amount of cDNA present, which is rep- may serve as indicators of previously unidentified path- resentative of the expression levels of a gene. The scan- ways associated with DNA repair, and thus can provide 35 ner may incorporate the functionality of the computing opportunities for identifying new classes of cosmetic device. Typically, gene expression data collected by the agents. system may include: i) up-regulation of gene expression [0040] Gene expression may be detected and/or (e.g., greater binding of the test material (e.g., cDNA) to measured in a variety of ways. In certain embodiments, probes compared to reference material (e.g., cDNA)), ii) the method comprises measuring messenger ribonucleic 40 down-regulation of gene expression (e.g., reduced bind- acid ("mRNA") encoded by one or more genes of interest ing of the test material (e.g., cDNA) to probes than the in a gene signature. Optionally, the method may include test material (e.g., cDNA)), iii) non-fluctuating gene ex- reverse transcribing mRNA encoded by one or more of pression (e.g., similar binding of the test material (e.g., the genes and measuring the corresponding comple- cDNA) to the probes compared to the reference material mentary DNA ("cDNA"). Any suitable quantitative nucleic 45 (e.g., cDNA)), and iv) no detectable signal or noise. The acid assay may be used herein. For example, conven- up- and down-regulated genes may be referred to as tional quantitative hybridization, Northern blot, and "differentially expressed." Differentially expressed genes polymerase chain reaction procedures may be used for may be further analyzed and/or grouped together (e.g., quantitatively measuring the amount of an mRNA tran- via known statistical methods) to identify genes that are script or cDNA in a biological sample. Optionally, the mR- 50 representative of a particular skin condition or biological NA or cDNA may be amplified by polymerase chain re- response to a cosmetic test agent. action (PCR) prior to hybridization. The mRNA or cDNA [0042] The unique gene signatures developed for the sample is then examined by, e.g., hybridization with oli- methods herein are determined by bioinformatics analy- gonucleotides specific for mRNAs or cDNAs encoded by sis and comparison of gene expression profiles of skin one or more of the genes of the panel, optionally immo- 55 aging conditions of interest, the gene expression profile bilized on a substrate (e.g., an array or microarray). Bind- of the circadian rhythm pattern of human skin tissue, and ing of the biomarker nucleic acid to oligonucleotide DNA repair genes. Gene signatures used for generating probes specific for the biomarker(s) allows identification the unique gene signatures of interest may be generated,

6 11 EP 3 302 716 B1 12 for example, from full thickness skin biopsies or other to 40 years old. The subjects can be male or female. In donor skin tissue (e.g., surgical waste) from skin, which some embodiments, the circadian rhythm gene signature exhibits a skin aging condition of interest, compared to may be derived by comparing the four time points. In a control. The gene signatures that can be used to pro- other embodiments, the biopsies can be obtained from vide the presently-disclosed novel screening method for 5 sun protected sites or may be obtained from sun exposed identifying a test agent as effective for providing a circa- sites. dian rhythm-dependent, DNA-repair benefit to human [0046] Gene signatures may also be derived from a skin can involve analysis of RNA from full or partial human gene expression profiling analysis of fibroblast and/or ke- skin tissue samples or from simple cell types removed ratinocyte cells treated with a positive control skin agent from such samples (such as through laser capture micro- 10 to represent cellular perturbations leading to improve- dissection or physical cell or cell layer removal or other ment in the skin tissue condition treated with that bench- ways known in the art). Dermal and epidermal layers may mark skin agent, said signature comprising a plurality of be removed and analyzed separately or together. Addi- genes up-regulated and down-regulated by the bench- tionally, cell lines can be used to generate such gene mark skin agent in cells in vitro. As one illustrative exam- expression profiles and resulting signatures, such as ke- 15 ple, microarray gene expression profile data where the ratinocyte cell lines or fibroblast cell lines. Profiles can positive control agent is the known skin anti-aging agent be generated from such individual cells, layers, or from Golden Silk Extract ("GSE") (INCI name: Glycerin, Hy- multiple cells, layers, parts (or in whole) of the human drolyzed Silk. CAS No. 56-81-5) may be analyzed using skin tissue sample or samples. the present method. Thus, a list of genes strongly up- [0043] Two gene signature types for skin aging include 20 regulated and strongly down-regulated in response to an intrinsic aging gene signature and a photo-aging gene challenge with GSE, or any other control agent or bench- signature, which may be derived by comparing gene ex- mark skin agent, can be derived. Said list of genes (a pression data from a full thickness skin biopsy from skin proxy for skin anti-aging) can be used in combination with having the condition of interest and a control. Examples the gene expression profiles of skin aging conditions of 2 and 3 below describe in greater detail non-limiting meth- 25 interest and the gene expression profile of the circadian ods for deriving these gene signatures. Generally, for a rhythm pattern of human skin tissue to provide unique photo-aging gene signature, biopsies may be taken from and new gene signature which can serve as query sig- sun exposed skin (e.g., extensor forearm, cheek, fore- natures to screen for skin anti-aging agents that will pro- head) and sun protected skin (e.g., buttocks, armpit, up- vide maximize efficacy and allow for the optimization of per inner arm, upper inner thigh) of a plurality of older 30 treatments. Furthermore, these unique gene signatures subjects. The subjects may vary in age, but one age may serve as indicators of previously unidentified path- range is between about 45 years of age and 70 years of ways associated with DNA repair, and thus can provide age. A gene expression profiling analysis of the biopsy opportunities for identifying new classes of cosmetic samples may be performed and one or more photo-aging agents. It is to be appreciated that numerous genes may gene signatures derived from a statistical analysis of the 35 be analyzed during gene profiling, but it is important to results. In some instances, a photo-aging gene signature recognize that only the genes identified in the various may be derived by comparing a sun exposed site of an gene signatures discussed below are selected for further older individual (e.g., 45 to 80 y.o.) to a sun exposed site analysis in the transcriptional profiles of the present of a younger individual (e.g., 18 to 25 y.o.) method. [0044] Generally, for an intrinsic aging gene signature, 40 [0047] Non-limiting aspects and examples of various biopsies may be taken from sun protected sites (e.g., embodiments of skin screening methods will now be de- buttocks) of a plurality of older and younger subjects. The scribed. In certain embodiments, the methods allow for subjects may vary in age, but one age range is between the screening of cosmetic agents that work in synchro- about 45 years of age and 80 years of age for the older nization with the circadian rhythm of the skin. In more subjects and 18 years of age and 25 years of age for the 45 particular embodiments, the methods allow for the younger subjects. A gene expression profiling analysis screening of cosmetic agents that work in synchroniza- of the biopsy samples may be performed and one or more tion with a dark cycle of the skin’s circadian cycle. These intrinsic aging gene signatures derived from a statistical novel methods also allow for identification or evaluation analysis of the microarray results. In some embodiments, of cosmetic agents that can be screened for their selec- a photo-aging gene signature may be derived by com- 50 tive treatment of skin aging conditions and for the specific paring a sun protected site of an older individual (e.g., targeting of particular skin cell types, such as keratinoc- 45 to 80 y.o.) to a sun protected site of a younger indi- ytes or fibroblasts. Examples of skin aging conditions in- vidual (e.g., 18 to 25 y.o.) clude intrinsic skin aging conditions, which are generally [0045] Generally, for the gene signature of the circa- age-dependent, and photo-aged skin conditions, which dian rhythm of human skin tissue, biopsies may be taken 55 are a form of skin aging where the skin is exposed to UV from healthy volunteers at four time points: 12 midnight, radiation. 6 am, 12 noon, and 6 pm. The subjects can vary in age, [0048] In some instances, a screening method for iden- but one range is 20 to 74 years old. Another range is 20 tifying or evaluating the potential effectiveness of a cos-

7 13 EP 3 302 716 B1 14 metic test agent for providing a circadian rhythm-depend- that expression of the genes in the respective transcrip- ent, DNA repair benefit to human skin is provided. For tional profiles is regulated in generally opposite direc- example, the method may comprise: (a) contacting a skin tions, then the test agent can be identified as having po- tissue sample (e.g., full thickness tissue sample or por- tential effectiveness for providing a circadian rhythm-de- tion thereof) with a cosmetic test agent; (b) generating a 5 pendent, DNA repair benefit to human skin transcriptional profile for the skin tissue sample ("test pro- [0051] In some instances, the transcriptional profile for file"), wherein the test profile comprises data related to the skin tissue sample contacted with a cosmetic test the transcription of at least two genes selected from agent (a first skin tissue sample) can be compared to a APITD1, ACTR5, AP5Z1, APEX1, APEX2, APLF, APTX, second skin tissue sample contacted with a control agent. ATXN3, BCCIP, BRCA1, C11orf30, CDC14B, CHEK1, 10 In such an embodiment, the cosmetic test agent can be CUL4A, DCLRE1C, DTL, EPC2, EXO5, EYA3, identified as effective for providing a circadian rhythm- FAM175A, FAN1, FANCC, FANCF, FANCG, FANCL, dependent, DNA repair benefit to human skin when a FIGNL1, GADD45A, GTF2H1, H2AFX, INTS3, comparison of the transcriptional profiles of the first and KIAA0430, KIAA0101, KIN, MCM9, MDC1, MGME1, second skin tissue samples indicates regulation of at MORF4L2, MRE11A, MSH2, MUM1, NEIL1, NEIL3, NO- 15 least two genes selected from one of the gene signatures NO, NPM1, NSMCE1, OGG1, PARPBP, PIF1, PML, above in a direction corresponding to a circadian rhythm- PMS1, PMS2, POLD1, POLD2, POLE, POLK, POLR2F, dependent, DNA repair benefit to human skin.. RAD50, RAD51B, RAD51C, RAD52, RBM14, RECQL5, [0052] In some instances, it may be desirable to select RFC4, RFC5, RPA2, SFPQ, SFR1, SMARCA5, SMC4, a gene signature wherein the transcription profile for the SMG1, SWI5, TICRR, TP53BP1, TP73, UBE2T, 20 human skin tissue sample comprises data related to the UBE2W, USP28, USP47, WDR33, and ZFYVE26; (c) transcription of at least two genes selected from CUL4A, comparing the transcriptional profile for the skin tissue EYA3, FAN1, NONO, OGG1, PMS2, RFC4, WDR33, sample to a control transcriptional profile; and (d) identi- KIN, NPM1, RAD50, APEX1, BCCIP, DTL, MGME1, fying the cosmetic test agent as having potential effec- NSMCE1, PIF1, POLK, RAD51C, SFR1, UBE2W, tiveness for providing a circadian rhythm-dependent, 25 ZFYVE26, CHEK1, GADD45A, MSH2, PARPBP, RFC5, DNA repair benefit to human skin when the transcription- SMC4, UBE2T, APITD1, CHEK1, KIAA0101, ATXN3, al profile for the skin tissue sample, relative to the control and BRCA1. In a particularly suitable example, the tran- transcriptional profile, corresponds to regulation (i.e., up- scriptional profile may comprise data related to the tran- regulation or downregulation) of the at least two genes scription of at least two genes selected from H2AFX, in a direction indicative of a circadian rhythm-dependent, 30 NSMCE1, RAD51C, UBE2T, and WDR33. DNA repair benefit to human skin. For example, if the [0053] In some instances, it may be desirable to iden- test transcriptional profile and a positive control transcrip- tify or evaluate the potential effectiveness of a cosmetic tional profile (i.e., a control transcriptional profile gener- test agent for providing a circadian rhythm-dependent, ated by contacting a test sample with a positive control) DNA repair benefit to intrinsically-aged human skin. For are concordant, meaning that expression of the genes in 35 example, the method may comprise: (a) contacting a skin the respective transcriptional profiles is regulated in the tissue sample with a cosmetic test agent; (b) generating same direction, then the test agent can be identified as a transcriptional profile for the skin tissue sample, where- exhibiting potential effectiveness for providing said ben- in the transcriptional profile comprises data related to the efit. transcription of at least two genes selected from APITD1, [0049] In the foregoing example, the test profile may 40 APEX1, APTX, ATXN3, BRCA1, C11orf30, CDC14B, consist essentially of data related to the transcription of CHEK1, DCLRE1C, DTL, EPC2, EXO5, EYA3, at least two genes selected from gene signature. That is, FAM175A, FANCF, GADD45A, GTF2H1, H2AFX, the transcriptional profile does not include data related KIAA0101, KIN, MORF4L2, NEIL3, NPM1, NSMCE1, to the transcription of other genes not listed. OGG1, PARPBP, PIF1, PML, POLD2, RAD50, RAD51B, [0050] While the above example and some of the ex- 45 RAD51C, RFC4, SMARCA5, SMC4, TP53BP1, UBE2T, amples that follow utilize a positive control, it is to be UBE2W, and WDR33; (c) comparing the transcriptional appreciated that embodiments wherein a transcriptional profile for the skin tissue sample to a control transcrip- profile is generated using a vehicle control or a negative tional profile; and (d) identifying the cosmetic test agent control are also contemplated herein, and it is within the as exhibiting potential effectiveness for providing a cir- skill of the ordinary artisan to determine whether a test 50 cadian rhythm-dependent, DNA repair benefit to intrinsi- agent has potential effectiveness for providing a circadi- cally-aged human skin when the transcriptional profile an rhythm-dependent, DNA repair benefit to human skin for the skin tissue sample, relative to the control tran- by comparing a test transcriptional profile to a control scriptional profile, corresponds to regulation of the genes transcriptional profile generated with a vehicle or nega- in a direction indicative of said benefit.. In this example, tive control. For example, if the test transcriptional profile 55 it may be desirable to generate a transcriptional profile and a negative control transcriptional profile (i.e., a con- comprising data related the transcription of at least two trol transcriptional profile generated by contacting a test genes selected from a subset of the foregoing list of sample with a negative control) are discordant, meaning genes, such as APTX, C11orf30, FANCF, H2AFX,

8 15 EP 3 302 716 B1 16

KIAA0101, KIN, NPM1, NSMCE1, RAD50, RAD51C, tify or evaluate the potential effectiveness of a cosmetic SMARCA5, SMC4, UBE2T, and WDR33. In this exam- test agent for providing a circadian rhythm-dependent, ple, it may be more desirable to use 0.1% GSE as a DNA repair benefit to the epidermis and/or the cells found positive control and generate a transcriptional profile for therein. For example, a full thickness skin sample may the skin sample comprising data related to the transcrip- 5 be separated into epidermis and dermis layer, such that tion of at least two genes selected from APITD1, CHEK1, that the epidermis may be analyzed separately. Addition- KIAA0101, NSMCE1, PIF1, RAD50, RAD51C, ATXN3, ally or alternatively, keratinocytes may be cultured and BRCA1, DTL, PARPBP, RFC4, SMC4, UBE2T, and tested according to the present method.. In such an em- WDR33. In this example, it may be even more desirable bodiment, the method may comprise: (a) contacting a to use 0.01% GSE as a positive control and generate a 10 sample of keratinocytes with a cosmetic test agent; (b) transcriptional profile for the skin sample comprising data generating a transcriptional profile for the sample of ke- related to the transcription of at least two genes selected ratinocytes, wherein the transcriptional profile comprises from EYA3, KIN, NPM1, RAD50, RFC4, and WRD33.In data related to the transcription of at least two genes some instances, it may be desirable to identify or evaluate selected from APTX, C11orf30, CDC14B, CHEK1, the potential effectiveness of a cosmetic test agent for 15 EPC2, EYA3, FANCF, GTF2H1, H2AFX, KIN, NEIL3, providing a circadian rhythm-dependent, DNA repair NSMCE1, PIF1, RAD50, RAD51C, SMARCA5, UBE2T, benefit to photo-aged human skin. For example, the and WDR33; (c) comparing the transcriptional profile for method may comprise: (a) contacting a skin tissue sam- the sample of keratinocytes to a control transcriptional ple with a cosmetic test agent; (b) generating a transcrip- profile; and (d) identifying the test agent as exhibiting tional profile for the skin tissue sample, wherein the tran- 20 potential effectiveness for providing a circadian rhythm- scriptional profile comprises data related to the transcrip- dependent, DNA repair benefit to the epidermal layer of tion of at least two genes selected from ACTR5, AP5Z1, human skin when the transcriptional profile for the kerat- APEX1, APEX2, APLF, APTX, ATXN3, BCCIP, BRCA1, inocyte sample, relative to the control transcriptional pro- C11orf30, CDC14B, CHEK1, CUL4A, DTL, EPC2, file, corresponds to regulation of the genes in a direction EYA3, FAM175A, FAN1, FANCC, FANCF, FANCG, 25 indicative of said benefit. FANCL, FIGNL1, GADD45A, GTF2H1, H2AFX, INTS3, [0055] In some instances, it may be desirable to iden- KIAA0430, KIAA0101, KIN, MCM9, MDC1, MGME1, tify or evaluate the potential effectiveness of a cosmetic MORF4L2, MRE11A, MSH2, MUM1, NEIL1, NEIL3, NO- test agent for providing a circadian rhythm-dependent, NO, NPM1, NSMCE1, OGG1, PARPBP, PIF1, PMS1, DNA repair benefit to an epidermal layer of intrinsically- PMS2, POLD1, POLD2, POLE, POLK, POLR2F, 30 aged human skin. For example, the method may com- RAD50, RAD51B, RAD51C, RAD52, RBM14, RECQL5, prise: (a) contacting a test sample (e.g., keratinocytes or RFC4, RFC5, RPA2, SFPQ, SFR1, SMARCA5, SMC4, epidermal tissue) with a cosmetic test agent; (b) gener- SMG1, SWI5, TICRR, TP53BP1, TP73, UBE2T, ating a transcriptional profile for the test sample, wherein UBE2W, USP28, USP47, WDR33, and ZFYVE26; (c) the transcriptional profile comprises data related to the comparing the transcriptional profile for the skin tissue 35 transcription of at least two genes selected from APITD1, sample to a control transcriptional profile; and (d) identi- APTX, C11orf30, CDC14B, CHEK1, DCLRE1C, EPC2, fying the cosmetic test agent as exhibiting potential ef- EXO5, EYA3, FANCF, GADD45A, GTF2H1, H2AFX, fectiveness for providing a circadian rhythm-dependent, KIAA0101, KIN, NEIL3, NPM1, NSMCE1, PIF1, RAD50, DNA repair benefit to photo-aged human skin when the RAD51B, RAD51C, SMARCA5, SMC4, UBE2T, and transcriptional profile for the skin tissue sample, relative 40 WDR33; (c) comparing the transcriptional profile for the to the control transcriptional profile, corresponds to reg- test sample to a control transcriptional profile; and (d) ulation of the genes in a direction indicative of said ben- identifying the cosmetic test agent as exhibiting potential efit. In this example, it may also be desirable to use 0.01% effectiveness for providing a circadian rhythm-depend- GSE as a positive control and generate a transcriptional ent, DNA repair benefit to the epidermal layer of intrinsi- profile comprising data related the transcription of at least 45 cally-aged human skin when the transcriptional profile two genes selected from APEX1, BCCIP, CUL4A, DTL, for the test sample, relative to the control transcriptional EYA3, FAN1, MGME1, NONO, NSMCE1, OGG1, PIF1, profile, corresponds to regulation of the genes in a direc- PMS2, POLK, RAD50, RAD51C, SFR1, UBE2W, tion indicative of said benefit. In this example, it may be ZFYVE26, CHEK1, GADD45A, MSH2, PARPBP, RFC4, desirable to use 0.1% GSE as a positive control and gen- RFC5, RPA1, SMC4, UBE2T, and WDR33. In this ex- 50 erate a transcriptional profile for the test sample compris- ample, it may be particularly desirable to use 0.1% GSE ing data related to the transcription of at least two genes as a positive control and generate a transcriptional profile selected from APITD1, CHEK1, KIAA0101, NSMCE1, comprising data related the transcription of at least two PIF1, RAD50, and RAD51C. In this example, it may be genes selected from APEX1, CHEK1, DTL, EYA3, FAN1, more desirable to use 0.01% GSE as a positive control H2AFX, MORF4L2, MSH2, NONO, NSMCE1, POLD2, 55 and generate a transcriptional profile for the test sample RAD51C, RFC4, SWI5, TP53BP1, UBE2T, UBE2W, and comprising data related to the transcription of EYA3. WDR33. [0056] In some instances, it may be desirable to iden- [0054] In some instances, it may be desirable to iden- tify or evaluate the potential effectiveness of a cosmetic

9 17 EP 3 302 716 B1 18 test agent for providing a circadian rhythm-dependent, foregoing disclosed genes of interest may serve as indi- DNA repair benefit to an epidermal layer of photo-aged cators of previously unidentified pathways associated human skin. For example, the method may comprise: (a) with DNA repair, and thus can provide opportunities for contacting a test sample (e.g., keratinocytes or epidermal identifying new classes of cosmetic agents. tissue) with a cosmetic test agent; (b) generating a tran- 5 [0058] In some instances, the present screening meth- scriptional profile for the test sample, wherein the tran- od may be used to identify or evaluate the potential ef- scriptional profile comprises data related to the transcrip- fectiveness of a cosmetic test agent for providing a cir- tion of at least two genes selected from ACTR5, AP5Z1, cadian rhythm-dependent, DNA repair benefit to a dermal APEX1, APLF, ATXN3, BCCIP, BRCA1, C11orf30, layer of human skin. For example, the method may com- CDC14B, CHEK1, CUL4A, DTL, EPC2, EYA3, 10 prise: (a) contacting a test sample (e.g., fibroblasts or FAM175A, FAN1, FANCF, FANCG, FANCL, FIGNL1, dermal tissue) with a cosmetic test agent; (b) generating GTF2H1, H2AFX, INTS3, KIAA0430, KIN, MCM9, a transcriptional profile for the test sample, wherein the MDC1, MGME1, MORF4L2, MRE11A, MSH2, MUM1, transcriptional profile comprises data related to the tran- NEIL1, NEIL3, NONO, NSMCE1, OGG1, PIF1, PMS1, scription of at least two genes selected from APEX1, AP- PMS2, POLD1, POLD2, POLE, POLK, RAD50, 15 TX, ATXN3, BRCA1, C11orf30, DTL, FAM175A, FANCF, RAD51C, RAD52, RBM14, RECQL5, RFC4, SFR1, H2AFX, KIAA0101, KIN, MORF4L2, NPM1, NSMCE1, SMARCA5, SMG1, SWI5, TICRR, TP53BP1, UBE2T, OGG1, PARPBP, PML, POLD2, RAD50, RAD51C, UBE2W, USP47, WDR33, and ZFYVE26; (c) comparing RFC4, SMARCA5, SMC4, TP53BP1, UBE2T, UBE2W, the transcriptional profile for the test sample to a control and WDR33; (c) comparing the transcriptional profile for transcriptional profile; and (d) identifying the cosmetic 20 the test sample to a control transcriptional profile; and test agent as exhibiting potential effectiveness for pro- (d) identifying the cosmetic test agent as exhibiting po- viding a circadian rhythm-dependent, DNA repair benefit tential effectiveness for providing a circadian rhythm-de- to the epidermal layer of photo-aged human skin when pendent, DNA repair benefit to the dermal layer of human the transcriptional profile for the test sample, relative to skin when the transcriptional profile for the test sample, the control transcriptional profile, corresponds to regula- 25 relative to the control transcriptional profile, corresponds tion of the genes in a direction indicative of said benefit. to regulation of the genes in a direction indicative of said In this example, it may be desirable to use a positive benefit. In this example it may be desirable to generate control (e.g., GSE) and generate a transcriptional profile a transcriptional profile for the test sample comprising for the test sample comprising data related to the tran- data related to the transcription of at least two genes scription of at least two genes selected from APEX1, BC- 30 selected from APEX1, APTX, DTL, H2AFX, KIAA0101, CIP, CUL4A, DTL, EYA3, FAN1, MGME1, NONO, MORF4L2, NSMCE1, PARPBP, POLD2, RAD51C, NSMCE1, OGG1, PIF1, PMS2, POLK, RAD50, RFC4, SMC4, TP53BP1, UBE2T, UBE2W, and WDR33. RAD51C, SRF1, UBE2W, and ZFYVE26. In this exam- [0059] In some instances, it may be desirable to iden- ple, it may be more desirable to use 0.1% GSE as a tify or evaluate the potential effectiveness of a cosmetic positive control and generate a transcriptional profile for 35 test agent for providing a circadian rhythm-dependent, the test sample comprising data related to the transcrip- DNA repair benefit to a dermal layer of intrinsically-aged tion of at least two genes selected from APEX1, BCCIP, human skin. For example, the method may comprise: (a) CUL4A, DTL, FAN1, MGME1, NSMCE1, PIF1, PMS2, contacting a sample of fibroblasts with a cosmetic test POLK, RAD50, RAD51C, SFR1, UBE2W, and agent; (b) generating a transcriptional profile for the sam- ZFYVE26. In this example, it may be even more desirable 40 ple of fibroblasts, wherein the transcriptional profile com- to use 0.01% GSE as a positive control and generate a prises data related to the transcription of at least two transcriptional profile for the test sample comprising data genes selected from APEX1, APTX, ATXN3, BRCA1, related to the transcription of at least two genes selected C11orf30, DTL, FAM175A, FANCF, H2AFX, KIAA0101, from CUL4A, EYA3, FAN1, NONO, OGG1, and PMS2. KIN, MORF4L2, NPM1, NSMCE1, OGG1, PARPBP, [0057] In some instances, the transcriptional profile for 45 PML, POLD2, RAD50, RAD51C, RFC4, SMARCA5, a sample of keratinocytes contacted with a cosmetic test SMC4, TP53BP1, UBE2T, UBE2W, and WDR33; (c) agent (a first sample of keratinocytes) can be compared comparing the transcriptional profile for the sample of to a second sample of keratinocytes contacted with a fibroblasts to a control transcriptional profile; and (d) iden- control agent. In such an embodiment, the cosmetic test tifying the cosmetic test agent as exhibiting potential ef- agent can be identified as exhibiting potential effective- 50 fectiveness for providing a circadian rhythm-dependent, ness for providing a circadian rhythm-dependent, DNA DNA repair benefit to the dermal layer of intrinsically- repair benefit to the epidermal layer of intrinsically-aged aged human skin when the transcriptional profile for the human skin when the transcriptional profiles of the first sample of fibroblasts, relative to the control transcription- and second samples of keratinocytes, relative to one an- al profile, corresponds to regulation of the genes in a other, correspond to regulation of at least two genes of 55 direction indicative of said benefit. In this example, it may interest in a direction indicative of said benefit. The dis- be desirable to generate a transcriptional profile for the closed genes of interest in the above gene signatures test sample comprising data related to the transcription may be suitable for use in this example. Additionally, the of at least two genes selected from ATXN3, BRAC1, DTL,

10 19 EP 3 302 716 B1 20

KIN, NPM1, PARPBP, RAD50, RAD51C, RFC4, SMC4, scriptional profiles of the first and second samples of fi- UBE2T, and WDR33. In this example, it may be more broblasts, relative to one another, correspond to regula- desirable to use 0.1% GSE as a positive control and gen- tion of at least two genes of interest in a direction indic- erate a transcriptional profile for the test sample compris- ative of said benefit. The disclosed genes of interest in ing data related to the transcription of at least two genes 5 the above gene signatures may be suitable for use in this selected from ATXN3, BRCA1, DTL, PARPBP, RAD51C, example. Additionally, the foregoing disclosed genes of RFC4, SMC4, UBE2T, and WDR33. In this example, it interest may serve as indicators of previously unidentified may be even more desirable to use 0.01% GSE as a pathways associated with DNA repair, and thus can pro- positive control and generate a transcriptional profile for vide opportunities for identifying new classes of cosmetic the test sample comprising data related to the transcrip- 10 agents. tion of at least two genes selected from KIN, NPM1, [0062] Also disclosed are methods of providing a cir- RAD50, RFC4, and WRD33.. cadian rhythm-dependent, DNA repair benefit to the skin [0060] In some instances, it may be desirable to iden- of a subject. The methods generally describe applying a tify or evaluate the potential effectiveness of a cosmetic composition comprising an effective amount of Golden test agent for providing a circadian rhythm-dependent, 15 Silk Extract during a dark cycle of the subject’s circadian DNA repair benefit to a dermal layer of photo-aged hu- cycle. In certain embodiments, the composition further man skin. The method comprises: (a) contacting a sam- comprises a dermatologically acceptable carrier. In some ple of fibroblasts with a cosmetic test agent; (b) generat- embodiments, the composition comprises from about ing a transcriptional profile for the sample of fibroblasts, 0.01% to about 0.1% by weight of Golden Silk Extract. wherein the transcriptional profile comprises data related 20 In particular embodiments, the composition comprises to the transcription of at least two genes selected from about 0.01% Golden Silk Extract. In other particular em- APEX1, APEX2, APTX, CHEK1, DTL, EYA3, FAN1, bodiments, the composition comprises about 0.1% Gold- FANCC, GADD45A, H2AFX, KIAA0101, MORF4L2, en Silk Extract. In some embodiments, the composition MSH2, NONO, NPM1, NSMCE1, PARPBP, POLD2, is applied for the entirety of the dark cycle of the subject’s POLR2F, RAD51B, RAD51C, RFC4, RFC5, RPA2, SF- 25 circadian cycle. PQ, SMC4, SWI5, TP53BP1, TP73, UBE2T, UBE2W, [0063] Generally, the cosmetic test agents identified USP28, and WDR33; (c) comparing the transcriptional by the presently-disclosed methods may be applied in profile for the sample of fibroblasts to a control transcrip- accordance with cosmetic compositions and formulation tional profile; and (d) identifying the cosmetic test agent parameters well-known in the art. Various methods of as exhibiting potential effectiveness for providing a cir- 30 treatment, application, regulation, or improvement may cadian rhythm-dependent, DNA repair benefit to the der- utilize the skin care compositions comprising skin-active mal layer of photo-aged human skin when the transcrip- agents identified according to the inventive methods. tional profile for the sample of fibroblasts, relative to the [0064] Because of the desirability of providing various control transcriptional profile, corresponds to regulation cosmetic skin anti-aging benefits to a consumer, it may of the genes in a direction indicative of said benefit. In 35 be beneficial to incorporate cosmetic test agents or com- this example, it may be desirable to generate a transcrip- pounds identified by one or more of the screening meth- tional profile comprising data related to the transcription ods described herein into a cosmetic composition suita- of at least two genes selected from CHEK1, DTL, FAN1, ble for topical application to skin. That is, it may be de- GADD45A, MSH2, PARPBP, RAD51C, RFC4, RFC5, sirable to include the cosmetic test agent as an ingredient RPA2, SMC4, UBE2T, and WDR33. In this example, it 40 in the cosmetic composition. In certain embodiments, the may be more desirable to use 0.1% GSE as a positive cosmetic composition may include a dermatological ac- control and generate a transcriptional profile for the sam- ceptable carrier, the test agent, and one or more optional ple of fibroblasts comprising data related to the transcrip- ingredients of the kind commonly included in the partic- tion of at least two genes selected from CHEK1, DTL, ular cosmetic compositing being provided. "Dermatolog- FAN1, GADD45A, MSH2, PARPBP, RAD51C, RFC5, 45 ically acceptable carrier" means that a carrier that is suit- RPA2, SMC4, UBE2T, and WDR33. In this example, it able for topical application to keratinous tissue, has good may be even more desirable to use 0.01% GSE as a aesthetic properties, is compatible with the ingredients positive control and generate a transcriptional profile for in the composition, and will not cause any unreasonable the sample of fibroblasts comprising data related to RFC5 safety or toxicity concerns. Suitable carriers may include and WDR33. 50 water and/or water miscible solvents. The cosmetic skin [0061] In some instances, the transcriptional profile for care composition may comprise from about 1% to about a sample of fibroblasts contacted with a cosmetic test 95% by weight of water and/or water miscible solvent. agent (a first sample of fibroblasts) can be compared to Suitable water miscible solvents include monohydric al- a second sample of fibroblasts contacted with a control cohols, dihydric alcohols, polyhydric alcohols, glycerol, agent. In such an embodiment, the cosmetic test agent 55 glycols, polyalkylene glycols such as polyethylene glycol, can be identified as exhibiting potential effectiveness for and mixtures thereof. When the skin care composition is providing a circadian rhythm-dependent, DNA repair in the form of an emulsion, water and/or water miscible benefit to the dermal layer of human skin when the tran- solvents are carriers typically associated with the aque-

11 21 EP 3 302 716 B1 22 ous phase. strips, patches, electrically-powered patches, wound [0065] Suitable carriers also include oils. The skin care dressing and adhesive bandages, hydrogels, film-form- composition may comprise from about 1% to about 95% ing products, facial and skin masks (with and without in- by weight of one or more oils. Oils may be used to solu- soluble sheet), make-up such as foundations, eye liners, bilize, disperse, or carry materials that are not suitable 5 and eye shadows, and the like. The composition may be for water or water soluble solvents. Suitable oils include provided in a package sized to store a sufficient amount silicones, hydrocarbons, esters, amides, ethers, and mix- of the composition for a treatment period. The size, tures thereof. The oils may be volatile or nonvolatile. shape, and design of the package may vary widely. Cer- [0066] The compositions of the present invention may tain package examples are described in U.S. Pat. Nos. contain a variety of other ingredients provided that they 10 D570,707; D391,162; D516,436; D535,191; D542,660; do not unacceptably alter the benefits of the invention. D547,193; D547,661; D558,591; D563,221; When present, compositions of the present invention 2009/0017080; 2007/0205226; and 2007/0040306. may contain from about 0.0001% to about 50%; from [0070] Non-limiting example of various aspects of the about 0.001% to about 20%; or, alternately, from about methods described herein are provided below. The ex- 0.01% to about 10%, by weight of the composition, of the 15 amples are given solely for the purpose of illustration and optional components. The amounts listed herein are only are not intended to be construed as limiting the invention, to be used as a guide, as the optimum amount of the as many variations thereof are possible. optional components used in a composition will depend on the specific active selected since their potency does Example 1 vary considerably. Hence, the amount of some optional 20 components useful in the present invention may be out- Testing potential cosmetic test agents side the ranges listed herein. [0067] The optional components, when incorporated [0071] Individual experiments (referred to as batches) into the composition, should be suitable for use in contact generally include 30 to 96 samples analyzed using Af- with human skin tissue without undue toxicity, incompat- 25 fymetrix GeneChip® technology platforms, containing 6 ibility, instability, allergic response, and the like. The com- replicates of the vehicle control (e.g., DSMO), 2 replicate positions of the present invention may include optional samples of a positive control that gives a strong repro- components such as anti-acne actives, desquamation ducible effect in the cell type used (e.g., all trans-retinoic actives, anti-cellulite agents, chelating agents, flavo- acid for fibroblast cells), and samples of the test material. noids, tanning active, non-vitamin antioxidants and rad- 30 Replication of the test material is done in separate batch- ical scavengers, hair growth regulators, anti-wrinkle ac- es due to batch effects. In vitro testing was performed in tives, anti-atrophy actives, minerals, phytosterols and/or 6-well plates to provide sufficient RNA for GeneChip® plant hormones, N-acyl amino acid compounds, antimi- analysis (2-4 mg total RNA yield/well). crobial or antifungal actives, and other useful skin care [0072] Human telomerized keratinocytes (tKC) were actives, non-limiting examples of skin-care agents that 35 obtained from the University of Texas, Southwestern may be suitable for use in the present composition are Medical Center, Dallas, Tex. tKC cells were grown in described in US 2006/0275237, US 2004/0175347 and EpiLife® media with 13 Human Keratinocyte Growth The International Cosmetic Ingredient Dictionary and Supplement (Invitrogen, Carlsbad, Calif.) on collagen I Handbook, Thirteenth Edition. coated cell culture flasks and plates (Becton Dickinson, [0068] The skin care compositions may be generally 40 Franklin Lakes, N.J.). Keratinocytes were seeded into 6- prepared by conventional methods such as known in the well plates at 20,000 cells/cm2 24 hours before chemical art of making compositions and topical compositions. exposure. Human skin fibroblasts (BJ cell line from Such methods typically involve mixing of ingredients in ATCC, Manassas, Va.) were grown in Eagle’s Minimal or more steps to a relatively uniform state, with or without Essential Medium (ATCC) supplemented with 10% fetal heating, cooling, application of vacuum, and the like. Typ- 45 bovine serum (HyClone, Logan, Utah) in normal cell cul- ically, emulsions are prepared by first mixing the aqueous ture flasks and plates (Corning, Lowell, Mass.). BJ fibrob- phase materials separately from the fatty phase materials lasts were seeded into 6-well plates at 12,000 cells/cm2 and then combining the two phases as appropriate to 24 hours before chemical exposure. yield the desired continuous phase. The compositions [0073] As a non-limiting example, all cells were incu- are preferably prepared such as to optimize stability 50 bated at 37° C. in a humidified incubator with 5% CO2. (physical stability, chemical stability, photostability, etc.) At t=-24 hours cells were trypsinized from T-75 flasks and/or delivery of active materials. and plated into 6-well plates in basal growth medium. At [0069] The compositions may be in various product t=0 media was removed and replaced with the appropri- forms that include, but are not limited to, solutions, sus- ate dosing solution as per the experimental design. Dos- pensions, lotions, creams, gels, toners, sticks, pencil, 55 ing solutions were prepared the previous day in sterile 4 sprays, aerosols, ointments, cleansing liquid washes and ml Falcon snap cap tubes. Pure test materials may be solid bars, shampoos and hair conditioners, pastes, prepared at a concentration of 1-200 mM, and botanical foams, powders, mousses, shaving creams, wipes, extracts may be prepared at a concentration of 0.001 to

12 23 EP 3 302 716 B1 24

1% by weight of the dosing solution. In certain embodi- to isolate subpopulations of cells from the tissue sections. ments, GSE (Golden Silk Extract) was used as the control For example, laser capture microdissection can be used test agent at concentrations from about 0.01% to about to isolate the epidermal layer or dermal layer of the skin. 0.1%. In specific embodiments, GSE was used at about A suitable, non-limiting example of a method of collecting 0.01%, while in other particular embodiments, GSE was 5 biopsy samples and sectioning, sorting, and/or staining used at about 0.1%. After 6 to 24 hours of chemical ex- the samples in more fully described in U.S. Provisional posure, cells were viewed and imaged. The wells were App. No. 61/798,208 filed by Osoria, et al., on Mar. 15, examined with a microscope before cell lysis and RNA 2013. In such an example, the epidermis and dermis lay- isolation to evaluate for morphologic evidence of toxicity. ers of the section biopsy samples were separated with a If morphological changes were sufficient to suggest cy- 10 PALM Microbeam IVTM brand Laser-capture Microdis- totoxicity, a lower concentration of the test agent was section ("LCM") system (available from Carl Zeiss Micro- tested. Cells were then lysed with 350 ul/well of RLT buff- Imaging GmgH, Germany) in accordance with the man- er containing β-mercaptoethanol (Qiagen, Valencia, Cal- ufacturer’s instructions. Of course, it is to be appreciated if.), transferred to a 96-well plate, and stored at -20° C. that any suitable means of extracting RNA from a tissue [0074] RNA from cell culture batches was isolated from 15 sample known in the art may be used. the RLT buffer using Agencourt® RNAdvance Tissue- [0077] RNA was quantified using a NanoDrop spectro- Bind magnetic beads (Beckman Coulter) according to photometer (Thermo Scientific, Waltham, Mass.) and manufacturer’s instructions. 1 mg of total RNA per sample quality was confirmed using an Agilent (Santa Clara, Cal- was labeled using Ambion Message Amp™ II Biotin En- if.) 2100 BioAnalyzer. Total RNA was converted to Gene- hanced kit (Applied Biosystems Incorporated) according 20 Chip targets using the Enzo BioArray labeling procedure to manufacturer’s instructions. The resultant biotin la- (Enzo Life Sciences, Farmingdale, N.Y.) and protocol beled and fragmented cRNA was hybridized to an Af- provided. All biotin-labeled GeneChip targets were hy- fymetrix HG-U133A 2.0 GeneChip®, which was then bridized to Affymetrix Human Genome HG-U133 Plus washed, stained and scanned using the protocol provid- 2.0 GeneChips overnight, which were then washed, ed by Affymetrix. Alternatively, cRNA was analyzed using 25 stained and scanned using the protocol provided by Af- Affymetix HG-U219 gene arrays. fymetrix. Of course, it is to be appreciated that any suit- able means of quantifying and qualifying RNA from a Example 2 tissue sample known in the art may be used. [0078] The samples can be analyzed on the Affymetrix Deriving a Photo-Aging Gene signature 30 HG-U133 Plus 2.0 GeneChips, which contain 54,613 probe sets complementary to the transcripts of more than [0075] A clinical survey study to obtain biopsy speci- 20,000 genes. However, instances in the provided data- mens for use in the investigation of gene expression pat- base used were derived from gene expression profiling terns associated with sun light-mediated skin aging (pho- experiments using Affymetrix HG-U133A 2.0 Gene- to-aging) was performed. Baseline gene expression pat- 35 Chips, containing 22,214 probe sets, which are a subset terns were examined in sun-protected and sun-exposed of those present on the Plus 2.0 GeneChip. Therefore, skin from young and aged women to examine gene ex- in developing gene signatures from the clinical data, the pression profiles associated with photo-aging. A total of probe sets were filtered for those included in the HG- 3 full thickness skin biopsies (-4 mm) were taken from U133A 2.0 gene chips. Alternatively, the extracted RNA sun-protected (buttocks) and sun-exposed (extensor 40 was run on a GeneTitan U219 brand microarray to iden- forearm and face) body sites from each of female volun- tify the genes that were expressed. Forearm and buttock teers (aged 20 to 74 years). There were approximately samples were processed on the same day using the 25 subjects in each decile form 20 to 74 years old. The same manufacturing lot of GeneChips. older women were selected to have moderate to severe [0079] Using, generally the following selection proc- forearm photo-damage. Biopsies were flash frozen in liq- 45 ess, a statistical analysis of the microarray data was per- uid nitrogen and stored at -80° C. until RNA isolation. formed to derive a plurality of photo-aging gene signa- [0076] RNA was extracted from full thickness biopsies tures comprising up-regulated and down-regulated or from laser capture microdissected skin samples. In genes. certain samples, the frozen skin biopsies were homoge- nized in Trizol (Invitrogen) and RNA extracted using the 50 Filtering based on Absent/Margin/Present Calls. protocol provided by Invitrogen. Since the tissue samples were from full thickness biopsies, RNA was extracted [0080] This filter creates a list of potential genes for from a variety of cell types within the full-thickness skin inclusion in the gene signature. For example, a suitable sample, including keratinocytes, fibroblasts, melano- filter may be that at least 50% of the samples in one cytes, endothelial cells, pericytes, nerves, smooth mus- 55 treatment group must have a Present call for each probe cle, sebocytes, adipocytes, and immunocytes). RNA was set. Present calls are derived from processing the raw further purified using RNEasy spin columns (Qiagen). In GeneChip data and provide evidence that the gene tran- other samples, laser capture microdissection was used script complementary to a probe set that is actually ex-

13 25 EP 3 302 716 B1 26 pressed in the biological sample. The probes that are mens for use in the investigation of gene expression pat- absent from all samples are likely to be just noisy meas- terns associated with chronological (intrinsic) skin aging urements. This step is important to filter out probe sets was performed. Baseline gene expression patterns were that do not contribute meaningful data to the signature. examined in sun-protected skin from young and aged For both photo-aging and intrinsic aging gene signatures, 5 women to examine gene expression profiles associated the data was filtered for probe sets with at least 10% with intrinsic aging. A total of 3 full thickness skin biopsies Present calls provided by the Affymetrix MAS 5 software. (-4 mm) were taken from sun-protected (buttocks) body sites from each of female volunteers (aged 20 to 74 Filtering According to a Statistical Measure. years). There were approximately 25 subjects in each 10 decile form 20 to 74 years old. Biopsies were flash frozen [0081] For example, a suitable statistical measure may in liquid nitrogen and stored at -80° C. until RNA isolation. be p-values from a t-test, ANOVA, correlation coefficient, Using, generally the same sorting process as set forth in or other model-based analysis. As one example, p-val- Example 2, RNA was extracted, purified, quantified and ues may be chosen as the statistical measure and a cutoff qualified, and analyzed. Similar to Example 2, a statistical value of p=0.05 may be chosen. Limiting the signature 15 analysis of the microarray data was performed to derive list to genes that meet some reasonable cutoff for statis- intrinsic aging gene signatures. tical significance compared to an appropriate control is important to allow selection of genes that are character- Example 4 istic of the biological state of interest. This is preferable to using a fold change value, which does not take into 20 Development of Circadian Rhythm Gene signature account the noise around the measurements. The t-sta- tistic was used to select the probe sets in the signatures [0085] A clinical survey study to obtain biopsy speci- because it is signed and provides an indication of the mens for use in the investigation of gene expression pat- directionality of the gene expression changes (i.e. up- or terns associated with establish the circadian transcrip- down-regulated) as well as statistical significance. 25 tome in healthy human skin over a 24 hour period. Biop- sies were taken from 20 healthy male subjects ages Sorting the Probe Sets. 21-55, Fitzpatrick Skin Type I-III, with normal sleep pat- terns. Total RNA was isolated from epidermal and dermal [0082] All the probe sets are sorted into sets of up- compartments of full thickness 2 mm volar forearm punch regulated and down-regulated sets using the statistical 30 biopsies taken at 6 hr. intervals: 12 midnight, 6 AM, 12 measure. For example, if a t-test was used to compute noon, and 6 PM. Peripheral blood and instrumental p-values, the values (positive and negative) of the t-sta- measures of the skin were also collected at 6 hr. intervals, tistic are used to sort the list since p-values are always and saliva samples collected every 3 hrs. Gene expres- positive. The sorted t-statistics will place the sets with sion data presented are from epidermis only. Biopsies the most significant p-values at the top and bottom of the 35 were flash frozen in liquid nitrogen and stored at -80° C. list with the non-significant ones near the middle. until RNA isolation. Using, generally the same sorting process as set forth in Example 2, RNA was extracted, Creation of the Gene signature. purified, quantified and qualified, and analyzed. Similar to Example 2, a statistical analysis of the microarray data [0083] Using the filtered and sorted list created, a suit- 40 was performed to derive intrinsic aging gene signatures. able number of probe sets from the top and bottom are The significance was evaluated by ANOVA model with- selected to create a gene signature that preferably has out intercept on the normalized expression value of each approximately the same number of sets chosen from the sample by percent change from day average. top as chosen from the bottom. For example, the gene signature created may have at least about 10, 50, 100, 45 Example 5 200, or 300 and/or less than about 800, 600, or about 400 genes corresponding to a probe set on the chip. The DNA Repair Theme and Network Analysis number of probe sets approximately corresponds to the number of genes, but a single gene may be represented [0086] Theme analysis was used a tool to better un- by more than one probe set. It is understood that the 50 derstand the connection and results from the photo-ag- phrase "number of genes" as used herein, corresponds ing, intrinsic aging, and circadian rhythm studies and generally with the phrase "number of probe sets." DNA-repair (DNA repair process). A suitable, non-limit- ing example of a method of theme analysis in more fully Example 3 described in U.S. Pub. No. 20120283112 filed by Binder, 55 et al., on Feb. 22, 2012. In short, the method uses an Deriving an Intrinsic Aging Gene signature ontology of controlled vocabulary terms developed by the Gene Ontology (GO) Consortium that describes the [0084] A clinical survey study to obtain biopsy speci- biological process, molecular functions and cellular com-

14 27 EP 3 302 716 B1 28 ponents associated with gene products. Analysis in- document conflicts with any meaning or definition of the volves statistical comparison of a regulated list of genes same term in a document cited, the meaning or definition and a larger list of all the expressed genes, to determine assigned to that term in this document shall govern. if genes annotated to specific GO terms are significantly enriched in the regulated list. Such analysis can be per- 5 formed using Theme Extractor proprietary software and Claims an algorithm that calculates the p value for each ontology term. In this case, the terms involved DNA repair and/or 1. A screening method for evaluating or identifying the DNA repair process. Additionally, for the circadian rhythm potential effectiveness of a cosmetic test agent for gene signature, theme analysis can be conducted by the 10 providing a circadian rhythm-dependent, DNA repair Database for Annotation, Visualization and Integrated benefit to human skin, comprising: Discovery on genes that are significantly differentiated to either up or down from day average at each time point a. contacting a test sample comprising skin cells (p value < .01 and .05). When multiple probe sets for a with a cosmetic test agent; gene having less than the threshold p-value, represent- 15 b. generating a transcriptional profile for the test ative single probe sets are selected by the quality of probe sample, wherein the transcriptional profile con- sets and expression value. The overrepresented biolog- sists essentially of data related to the transcrip- ical processes were identified when False Discovery tion of at least two genes selected from APITD1, Rate (FDR) is less than 10% at some time point. For ACTR5, AP5Z1, APEX1, APEX2, APLF, APTX, network analysis for the circadian rhythm, associations 20 ATXN3, BCCIP, BRCA1, C11orf30, CDC14B, with the DNA repair process. CHEK1, CUL4A, DCLRE1C, DTL, EPC2, [0087] The various embodiments of the presently-dis- EXO5, EYA3, FAM175A, FAN1, FANCC, FAN- closed screening methods recite various gene groupings CF, FANCG, FANCL, FIGNL1, GADD45A, that display a significant negative age correlation (ex- GTF2H1, H2AFX, INTS3, KIAA0430, pression becomes lower with age, p-value < 0.01), dis- 25 KIAA0101, KIN, MCM9, MDC1, MGME1, play the circadian rhythm of interest (expression is sig- MORF4L2, MRE11A, MSH2, MUM1, NEIL1, nificantly elevated at midnight or early morning compar- NEIL3, NONO, NPM1, NSMCE1, OGG1, ing to day average, p-value < 0.05; or significantly lower PARPBP, PIF1, PML, PMS1, PMS2, POLD1, at noon comparing to day average (p-value is less than POLD2, POLE, POLK, POLR2F, RAD50, 0.05); are elevated at both midnight and early morning; 30 RAD51B, RAD51C, RAD52, RBM14, RECQL5, and display a strong circadian pattern (p-value is less RFC4, RFC5, RPA2, SFPQ, SFR1, SMARCA5, than 0.05 by one-way ANOVA test) in the circadian SMC4, SMG1, SWI5, TICRR, TP53BP1, TP73, study)), in human skin tissue, including the epidermis and UBE2T, UBE2W, USP28, USP47, WDR33, and dermis layers. The sequences for the genes listed in the ZFYVE26; various groupings are the sequences listed in the Gen- 35 c. comparing the transcriptional profile for the Bank® genetic sequence database housed by the Na- test sample to a control transcriptional profile; tional Center for Biotechnology Information ("NCBI") as and of June 8, 2015. The sequences for the genes listed in d. identifying the cosmetic test agent as exhib- the various groupings can display from about 80% to iting potential effectiveness for providing a cir- about 100% homology with the gene sequences listed in 40 cadian rhythm-dependent, DNA repair benefit the NCBI’s GenBank® database. In certain embodi- to human skin when the transcriptional profile ments, the methods recite various gene groupings that for the test sample, relative to the control tran- also are significantly upregulated by GSE (about 0.01% scriptional profile, corresponds to regulation of or about 0.1%) either in 6 hour or 24 hour point in kerat- the at least two genes in a direction indicative inocytes and/or fibroblasts. 45 of said benefit. [0088] The dimensions and values disclosed herein are not to be understood as being strictly limited to the 2. The method of claim 1, wherein the circadian rhythm- exact numerical values recited. Instead, unless other- dependent, DNA repair benefit is for photo-aged hu- wise specified, each such dimension is intended to mean man skin, and the transcriptional profile consists es- both the recited value and a functionally equivalent range 50 sentially of data related to the transcription of at least surrounding that value. For example, a dimension dis- two genes selected from APEX1, CHEK1, DTL, closed as "40 mm" is intended to mean "about 40 mm." EYA3, FAN1, H2AFX, MORF4L2, MSH2, NONO, [0089] The citation of any document is not an admis- NSMCE1, POLD2, RAD51C, RFC4, SWI5, sion that it is prior art with respect to any invention dis- TP53BP1, UBE2T, UBE2W, and WDR33. closed or claimed herein or that it alone, or in any com- 55 bination with any other reference or references, teaches, 3. The method of claim 1, wherein the circadian rhythm- suggests or discloses any such invention. Further, to the dependent, DNA repair benefit is for intrinsically- extent that any meaning or definition of a term in this aged human skin, and the transcriptional profile con-

15 29 EP 3 302 716 B1 30

sists essentially of data related to the transcription KIN, MORF4L2, MSH2, NONO, NPM1, NSMCE1, of at least two genes selected from APITD1, APEX1, OGG1, PARPBP, PML, POLD2, POLR2F, RAD50, APTX, ATXN3, BRCA1, C11orf30, CDC14B, RAD51B, RAD51C, RFC4, RFC5, RPA2, SFPQ, CHEK1, DCLRE1C, DTL, EPC2, EXO5, EYA3, SMC4, SMARCA5, SWI5, TP53BP1, TP73, UBE2T, FAM175A, FANCF, GADD45A, GTF2H1, H2AFX, 5 UBE2W, USP28, and WDR33. KIAA0101, KIN, MORF4L2, NEIL3, NPM1, NSMCE1, OGG1, PARPBP, PIF1, PML, POLD2, 9. The method of claim 8, wherein the circadian rhythm- RAD50, RAD51B, RAD51C, RFC4, SMARCA5, dependent, DNA repair benefit is for a dermal layer SMC4, TP53BP1, UBE2T, UBE2W, and WDR33. of photo-aged human skin, and the transcriptional 10 profile consists essentially of data related to the tran- 4. The method of claim 1, wherein the test sample is scription of at least two genes selected from APEX1, selected from full-thickness human skin tissue, ke- APEX2, APTX, CHEK1, DTL, EYA3, FAN1, FANCC, ratinocytes, and fibroblasts. GADD45A, H2AFX, KIAA0101, MORF4L2, MSH2, NONO, NPM1, NSMCE1, PARPBP, POLD2, 5. The method of claim 4, wherein the circadian rhythm- 15 POLR2F, RAD51B, RAD51C, RFC4, RFC5, RPA2, dependent, DNA repair benefit is for an epidermal SFPQ, SMC4, SWI5, TP53BP1, TP73, UBE2T, layer of human skin, the test sample is keratinocytes, UBE2W, USP28, and WDR33. and the transcriptional profile consists essentially of data related to the transcription of at least two genes 10. The method of claim 8, wherein the circadian rhythm- selected from APITD1, APEX1, APTX, ATXN3, 20 dependent, DNA repair benefit is for a dermal layer BRCA1, C11orf30, CDC14B, CHEK1, CUL4A, of intrinsically-aged human skin, and the transcrip- DCLRE1C, DTL, EPC2, EYA3, EXO5, FAM175A, tional profile consists essentially of data related to FAN1, FANCF, GADD45A, GTF2H1, H2AFX, the transcription of at least two genes selected from KIAA0101, KIN, MORF4L2, NEIL3, NONO, NPM1, APEX1, APTX, ATXN3, BRCA1, C11orf30, DTL, NSMCE1, OGG1, PARPBP, PIF1, PML, PMS2, 25 FAM175A, FANCF, H2AFX, KIAA0101, KIN, POLD2, RAD50, RAD51B, RAD51C, RFC4, MORF4L2, NPM1, NSMCE1, 0001, PARPBP, PML, SMARCA5, SMC4, TP53BP1, UBE2T, UBE2W, and POLD2, RAD50, RAD51C, RFC4, SMARCA5, WDR33. SMC4, TP53BP1, UBE2T, UBE2W, and WDR33.

6. The method of claim 5, wherein the circadian rhythm- 30 11. The method of any one of claims 1 to 10, wherein dependent, DNA repair benefit is for an epidermal the control profile is generated by contacting a sec- layer of photo-aged human skin, and the transcrip- ond test sample with a positive control and the tran- tional profile consists essentially of data related to scriptional profile and the control profile are concord- the transcription of at least two genes selected from ant. CUL4A, EYA3, FAN1, NONO, OGG1, and PMS2. 35 12. The method of claim 11, wherein the test sample is 7. The method of claim 5, wherein the circadian rhythm- full-thickness human skin tissue subjected to laser dependent, DNA repair benefit is for an epidermal capture microdissection. layer of intrinsically-aged human skin, and the tran- scriptional profile consists essentially of data related 40 13. The method of claim 12, wherein human skin tissue to the transcription of at least two genes selected is separated into an epidermal layer and a dermal from APITD1, APEX1, APTX, ATXN3, BRCA1, layer by the laser capture microdissection. C11orf30, CDC14B, CHEK1, DCLRE1C, DTL, EPC2, EXO5, EYA3, FAM175A, FANCF, 14. The method of any one of claims 1 to 13, further GADD45A, GTF2H1, H2AFX, KIAA0101, KIN, 45 comprising: isolating RNA from the test sample, us- MORF4L2, NEIL3, NPM1, NSMCE1, OGG1, ing the isolated RNA to create cRNA, labeling the PARPBP, PIF1, PML, POLD2, RAD50, RAD51B, cRNA, and hybridizing the labeled cRNA to a micro- RAD51C, RFC4, SMARCA5, SMC4, TP53BP1, array. UBE2T, UBE2W, and WDR33. 50 8. The method of claim 4, wherein the circadian rhythm- Patentansprüche dependent, DNA repair benefit is for a dermal layer of human skin, the test sample is fibroblasts, and the 1. Screening-Verfahren zum Bewerten oder Identifizie- transcriptional profile consists essentially of data re- ren der potentiellen Wirksamkeit eines kosmeti- lated to the transcription of at least two genes se- 55 schen Testmittels zum Bereitstellen einer biorhyth- lected from APEX1, APEX2, APTX, ATXN3, BRCA1, musabhängigen DNA-Reparatur-Nutzwirkung an C11orf30, CHEK1, DTL, EYA3, FAM175A, FAN1, menschliche Haut, umfassend: FANCC, FANCF, GADD45A, H2AFX, KIAA0101,

16 31 EP 3 302 716 B1 32

a. Inkontaktbringen einer Testprobe, umfas- UBE2T, UBE2W und WDR33. send Hautzellen, mit einem kosmetischen Test- mittel; 4. Verfahren nach Anspruch 1, wobei die Testprobe b. Generieren eines Transkriptionsprofils für die ausgewählt ist aus menschlichem Hautgewebe vol- Testprobe, wobei das Transkriptionsprofil im 5 ler Dicke, Keratinozyten und Fibroblasten. Wesentlichen aus Daten besteht, die die Tran- skription von mindestens zwei Genen betreffen, 5. Verfahren nach Anspruch 4, wobei die biorhythmus- ausgewählt aus APITD1, ACTR5, AP5Z1, abhängige DNA-Reparatur-Nutzwirkung für eine APEX1, APEX2, APLF, APTX, ATXN3, BCCIP, epidermale Schicht von menschlicher Haut ist, die BRCA1, C11orf30, CDC14B, CHEK1, CUL4A, 10 Testprobe Keratinozyten ist, und das Transkriptions- DCLRE1C, DTL, EPC2, EXO5, EYA3, profil im Wesentlichen aus Daten besteht, die die FAM175A, FAN1, FANCC, FANCF, FANCG, Transkription von mindestens zwei Genen betreffen, FANCL, FIGNL1, GADD45A, GTF2H1, H2AFX, ausgewählt aus APITD1, APEX1, APTX, ATXN3, INTS3, KIAA0430, KIAA0101, KIN, MCM9, BRCA1, C11orf30, CDC14B, CHEK1, CUL4A, MDC1, MGME1, MORF4L2, MRE11A, MSH2, 15 DCLRE1C, DTL, EPC2, EYA3, EXO5, FAM175A, MUM1, NEIL1, NEIL3, NONO, NPM1, FAN1, FANCF, GADD45A, GTF2H1, H2AFX, NSMCE1, OGG1, PARPBP, PIF1, PML, PMS1, KIAA0101, KIN, MORF4L2, NEIL3, NONO, NPM1, PMS2, POLD1, POLD2, POLE, POLK, NSMCE1, OGG1, PARPBP, PIF1, PML, PMS2, POLR2F, RAD50, RAD51B, RAD51C, RAD52, POLD2, RAD50, RAD51B, RAD51C, RFC4, RBM14, RECQL5, RFC4, RFC5, RPA2, SFPQ, 20 SMARCA5, SMC4, TP53BP1, UBE2T, UBE2W und SFR1, SMARCA5, SMC4, SMG1, SWI5, TIC- WDR33. RR, TP53BP1, TP73, UBE2T, UBE2W, USP28, USP47, WDR33 und ZFYVE26; 6. Verfahren nach Anspruch 5, wobei die biorhythmus- c. Vergleichen des Transkriptionsprofils für die abhängige DNA-Reparatur-Nutzwirkung für eine Testprobe mit einem Kontroll-Transkriptions- 25 epidermale Schicht von lichtgealterter menschlicher profil; und Haut ist, und das Transkriptionsprofil im Wesentli- d. Identifizieren des kosmetischen Testmittels chen aus Daten besteht, die die Transkription von als potentiell Wirksamkeit für das Bereitstellen mindestens zwei Genen betreffen, ausgewählt aus einer biorhythmusabhängigen DNA-Reparatur- CUL4A, EYA3, FAN1, NONO, OGG1 und PMS2. Nutzwirkung an menschliche Haut vorweisend, 30 wenn das Transkriptionsprofil für die Testprobe, 7. Verfahren nach Anspruch 5, wobei die biorhythmus- bezogen auf das Kontroll-Transkriptionsprofil, abhängige DNA-Reparatur-Nutzwirkung für eine einer Regulierung der mindestens zwei Gene in epidermale Schicht von intrinsisch gealterter eine Richtung entspricht, die auf die Nutzwir- menschlicher Haut ist, und das Transkriptionsprofil kung hindeutet. 35 im Wesentlichen aus Daten besteht, die die Trans- kription von mindestens zwei Genen betreffen, aus- 2. Verfahren nach Anspruch 1, wobei die biorhythmus- gewählt aus APITD1, APEX1, APTX, ATXN3, abhängige DNA-Reparatur-Nutzwirkung für lichtge- BRCA1, C11orf30, COC14B, CHEK1, DCLRE1C, alterte menschliche Haut ist, und das Transkriptions- DTL, EPC2, EXO5, EYA3, FAM175A, FANCF, profil im Wesentlichen aus Daten besteht, die die 40 GADD45A, GTF2H1, H2AFX, KIAA0101, KIN, Transkription von mindestens zwei Genen betreffen, MORF4L2, NEIL3, NPM1, NSMCE1, OGG1, PAR- ausgewählt aus APEX1, CHEK1, DTL, EYA3, FAN1, PBP, PIF1, PML, POLD2, RAD50, RAD51B, H2AFX, MORF4L2, MSH2, NONO, NSMCE1, RAD51C, RFC4, SMARCA5, SMC4, TP53BP1, POLD2, RAD51C, RFC4, SWI5, TP53BP1, UBE2T, UBE2T, UBE2W und WDR33. UBE2W und WDR33. 45 8. Verfahren nach Anspruch 4, wobei die biorhythmus- 3. Verfahren nach Anspruch 1, wobei die biorhythmus- abhängige DNA-Reparatur-Nutzwirkung für eine abhängige DNA-Reparatur-Nutzwirkung für intrin- dermale Schicht von menschlicher Haut ist, die Test- sisch gealterte menschliche Haut ist, und das Tran- probe Fibroblasten ist, und das Transkriptionsprofil skriptionsprofil im Wesentlichen aus Daten besteht, 50 im Wesentlichen aus Daten besteht, die die Trans- die die Transkription von mindestens zwei Genen kription von mindestens zwei Genen betreffen, aus- betreffen, ausgewählt aus APITD1, APEX1, APTX, gewählt aus APEX1, APEX2, APTX, ATXN3, ATXN3, BRCA1, C11orf30, CDC14B, CHEK1, BRCA1, C11orf30, CHEK1, DTL, EYA3, FAM175A, DCLRE1C, DTL, EPC2, EXO5, EYA3, FAM175A, FAN1, FANCC, FANCF, GADD45A, H2AFX, FANCF, GADD45A, GTF2H1, H2AFX, KIAA0101, 55 KIAA0101, KIN, MORF4L2, MSH2, NONO, NPM1, KIN, MORF4L2, NEIL3, NPM1, NSMCE1, OGG1, NSMCE1, OGG1, PARPBP, PML, POLD2, PARPBP, PIF1, PML, POLD2, RAD50, RAD51B, POLR2F, RAD50, RAD51B, RAD51C, RFC4, RFC5, RAD51C, RFC4, SMARCA5, SMC4, TP53BP1, RPA2, SFPQ, SMC4, SMARCA5, SWI5, TP53BP1,

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TP73, UBE2T, UBE2W, USP28 und WDR33. comprenant des cellules de peau avec un agent de test cosmétique ; 9. Verfahren nach Anspruch 8, wobei die biorhythmus- b. la génération d’un profil transcriptionnel pour abhängige DNA-Reparatur-Nutzwirkung für eine l’échantillon de test, dans lequel le profil trans- dermale Schicht von lichtgealterter menschlicher 5 criptionnel est constitué sensiblement de don- Haut ist, und das Transkriptionsprofil im Wesentli- nées se rapportant à la transcription d’au moins chen aus Daten besteht, die die Transkription von deux gènes choisis parmi APITD1, ACTR5, mindestens zwei Genen betreffen, ausgewählt aus AP5Z1, APEX1, APEX2, APLF, APTX, ATXN3, APEX1, APEX2, APTX, CHEK1, DTL, EYA3, FAN1, BCCIP, BRCA1, C11orf30, CDC14B, CHEK1, FANCC, GADD45A, H2AFX, KIAA0101, MORF4L2, 10 CUL4A, OCLRE1C, DTL, EPC2, EXO5, EYA3, MSH2, NONO, NPM1, NSMCE1, PARPBP, POLD2, FAM175A, FAN1, FANCC, FANCF, FANCG, POLR2F, RAD51B, RAD51C, RFC4, RFC5, RPA2, FANCL, FIGNL1, GADD45A, GTF2H1, H2AFX, SFPQ, SMC4, SWI5, TP53BP1, TP73, UBE2T, INTS3, KIAA0430, KIAA0101, KIN, MCM9, UBE2W, USP28 und WDR33. MDC1, MGME1, MORF4L2, MRE11A, MSH2, 15 MUM1, NEIL1, NEIL3, NONO, NPM1, 10. Verfahren nach Anspruch 8, wobei die biorhythmus- NSMCE1, OGG1, PARPBP, PIF1, PML, PMS1, abhängige DNA-Reparatur-Nutzwirkung für eine PMS2, POLD1, POLD2, POLE, POLK, dermale Schicht von intrinsisch gealterter mensch- POLR2F, RAD50, RAD51B, RAD51C, RAD52, licher Haut ist, und das Transkriptionsprofil im We- RBM14, RECQL5, RFC4, RFC5, RPA2, SFPQ, sentlichen aus Daten besteht, die die Transkription 20 SFR1, SMARCA5, SMC4, SMG1, SWI5, TI- von mindestens zwei Genen betreffen, ausgewählt CRR, TP53BP1, TP73, UBE2T, UBE2W, aus APEX1, APTX, ATXN3, BRCA1, C11orf30, DTL, USP28, USP47, WDR33 et ZFYVE26 ; FAM175A, FANCF, H2AFX, KIAA0101, KIN, c. la comparaison du profil transcriptionnel pour MORF4L2, NPM1, NSMCE1, OGG1, PARPBP, l’échantillon de test à un profil transcriptionnel PML, POLD2, RAD50, RAD51C, RFC4, SMARCA5, 25 témoin ; et SMC4, TP53BP1, UBE2T, UBE2W und WDR33. d. l’identification de l’agent de test cosmétique comme présentant une efficacité potentielle 11. Verfahren nach einem der Ansprüche 1 bis 10, wobei pour fournir un effet bénéfique de réparation das Kontrollprofil durch Inkontaktbringen einer zwei- d’ADN, dépendant du rythme circadien à la peau ten Testprobe mit einer Positivkontrolle generiert 30 humaine lorsque le profil transcriptionnel pour wird, und das Transkriptionsprofil und das Kontroll- l’échantillon de test, par rapport au profil trans- profil übereinstimmend sind. criptionnel témoin, correspond à une régulation des au moins deux gènes dans une direction 12. Verfahren nach Anspruch 11, wobei die Testprobe caractéristique dudit effet bénéfique. menschliches Hautgewebe voller Dicke ist, das einer 35 Lasermikrodissektion unterzogen wird. 2. Procédé selon la revendication 1, dans lequel l’effet bénéfique de réparation d’ADN, dépendant du ryth- 13. Verfahren nach Anspruch 12, wobei menschliches me circadien est pour une peau humaine vieillie par Hautgewebe durch die Lasermikrodissektion in eine rayonnement, et le profil transcriptionnel est consti- epidermale Schicht und eine dermale Schicht ge- 40 tué sensiblement de données se rapportant à la trennt wird. transcription d’au moins deux gènes choisis parmi APEX1, CHEK1, DTL, EYA3, FAN1, H2AFX, 14. Verfahren nach einem der Ansprüche 1 bis 13, ferner MORF4L2, MSH2, NONO, NSMCE1, POLD2, umfassend: Isolieren von RNA aus der Testprobe, RAD51C, RFC4, SWI5, TP53BP1, UBE2T, UBE2W Verwenden der isolierten RNA, um cRNA zu erzeu- 45 et WDR33. gen, Markieren der cRNA und Hybridisieren der mar- kierten cRNA an ein Mikroarray. 3. Procédé selon la revendication 1, dans lequel l’effet bénéfique de réparation d’ADN, dépendant du ryth- me circadien est pour une peau humaine intrinsè- Revendications 50 quement vieillie, et le profil transcriptionnel est cons- titué sensiblement de données se rapportant à la 1. Procédé de criblage pour évaluer ou identifier l’effi- transcription d’au moins deux gènes choisis parmi cacité potentielle d’un agent de test cosmétique pour APITD1, APEX1, APTX, ATXN3, BRCA1, C11orf30, fournir un effet bénéfique de réparation d’ADN, dé- CDC14B, CHEK1, DCLRE1C, DTL, EPC2, EXO5, pendant du rythme circadien à la peau humaine, 55 EYA3, FAM175A, FANCF, GADD45A, GTF2H1, comprenant : H2AFX, KIAA0101, KIN, MORF4L2, NEIL3, NPM1, NSMCE1, OGG1, PARPBP, PIF1, PML, POLD2, a. la mise en contact d’un échantillon de test RAD50, RAD51B, RAD51C, RFC4, SMARCA5,

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SMC4, TP53BP1, UBE2T, UBE2W et WDR33. PML, POLD2, POLR2F, RAD50, RAD51B, RAD51C, RFC4, RFC5, RPA2, SFPQ, SMC4, 4. Procédé selon la revendication 1, dans lequel SMARCA5, SWI5, TP53BP1, TP73, UBE2T, l’échantillon de test est choisi parmi un tissu cutané UBE2W, USP28 et WDR33. humain sur toute son épaisseur, des kératinocytes 5 et des fibroblastes. 9. Procédé selon la revendication 8, dans lequel l’effet bénéfique de réparation d’ADN, dépendant du ryth- 5. Procédé selon la revendication 4, dans lequel l’effet me circadien est pour une couche dermique de peau bénéfique de réparation d’ADN, dépendant du ryth- humaine vieillie par rayonnement, et le profil trans- me circadien est pour une couche épidermique de 10 criptionnel est constitué sensiblement de données peau humaine, l’échantillon de test est des kératino- se rapportant à la transcription d’au moins deux gè- cytes, et le profil transcriptionnel est constitué sen- nes choisis parmi APEX1, APEX2, APTX, CHEK1, siblement de données se rapportant à la transcrip- DTL, EYA3, FAN1, FANCC, GADD45A, H2AFX, tion d’au moins deux gènes choisis parmi APITD1, KIAA0101, MORF4L2, MSH2, NONO, NPM1, APEX1, APTX, ATXN3, BRCA1, C11orf30, 15 NSMCE1, PARPBP, POLD2, POLR2F, RAD51B, CDC14B, CHEK1, CUL4A, DCLRE1C, DTL, EPC2, RAD51C, RFC4, RFC5, RPA2, SFPQ, SMC4, SWI5, EYA3, EXO5, FAM175A, FAN1, FANCF, TP53BP1, TP73, UBE2T, UBE2W, USP28 et GADD45A, GTF2H1, H2AFX, KIAA0101, KIN, WDR33. MORF4L2, NEIL3, NONO, NPM1, NSMCE1, OGG1, PARPBP, PIF1, PML, PMS2, POLD2, 20 10. Procédé selon la revendication 8, dans lequel l’effet RAD50, RAD51B, RAD51C, RFC4, SMARCA5, bénéfique de réparation d’ADN, dépendant du ryth- SMC4, TP53BP1, UBE2T, UBE2W et WDR33. me circadien est pour une couche dermique de peau humaine intrinsèquement vieillie, et le profil trans- 6. Procédé selon la revendication 5, dans lequel l’effet criptionnel est constitué sensiblement de données bénéfique de réparation d’ADN, dépendant du ryth- 25 se rapportant à la transcription d’au moins deux gè- me circadien est pour une couche épidermique de nes choisis parmi APEX1, APTX, ATXN3, BRCA1, peau humaine vieillie par rayonnement, et le profil C11orf30, DTL, FAM175A, FANCF, H2AFX, transcriptionnel est constitué sensiblement de don- KIAA0101, KIN, MORF4L2, NPM1, NSMCE1, nées se rapportant à la transcription d’au moins deux OGG1, PARPBP, PML, POLD2, RAD50, RAD51C, gènes choisis parmi CUL4A, EYA3, FAN1, NONO, 30 RFC4, SMARCA5, SMC4, TP53BP1, UBE2T, OGG1 et PMS2. UBE2W et WDR33.

7. Procédé selon la revendication 5, dans lequel l’effet 11. Procédé selon l’une quelconque des revendications bénéfique de réparation d’ADN, dépendant du ryth- 1 à 10, dans lequel le profil témoin est généré par me circadien est pour une couche épidermique de 35 mise en contact d’un deuxième échantillon de test peau humaine intrinsèquement vieillie, et le profil avec un témoin positif, et le profil transcriptionnel et transcriptionnel est constitué sensiblement de don- le profil témoin sont concordants. nées se rapportant à la transcription d’au moins deux gènes choisis parmi APITD1, APEX1, APTX, 12. Procédé selon la revendication 11, dans lequel ATXN3, BRCA1, C11orf30, CDC14B, CHEK1, 40 l’échantillon de test est un tissu cutané humain sur DCLRE1C, DTL, EPC2, EXO5, EYA3, FAM175A, toute son épaisseur soumis à une microdissection FANCF, GADD45A, GTF2H1, H2AFX, KIAA0101, par capture au laser. KIN, MORF4L2, NEIL3, NPM1, NSMCE1, OGG1, PARPBP, PIF1, PML, POLD2, RAD50, RAD51B, 13. Procédé selon la revendication 12, dans lequel le RAD51C, RFC4, SMARCA5, SMC4, TP53BP1, 45 tissu cutané humain est séparé en une couche épi- UBE2T, UBE2W et WDR33. dermique et une couche dermique par la microdis- section par capture au laser. 8. Procédé selon la revendication 4, dans lequel l’effet bénéfique de réparation d’ADN, dépendant du ryth- 14. Procédé selon l’une quelconque des revendications me circadien est pour une couche dermique de peau 50 1 à 13, comprenant en outre : l’isolement d’ARN de humaine, l’échantillon de test est des fibroblastes, l’échantillon de test, l’utilisation de l’ARN isolé pour et le profil transcriptionnel est constitué sensible- créer de l’ARNc, le marquage de l’ARNc, et l’hybri- ment de données se rapportant à la transcription dation de l’ARNc marqué à une micromatrice. d’au moins deux gènes choisis parmi APEX1, APEX2, APTX, ATXN3, BRCA1, C11orf30, CHEK1, 55 DTL, EYA3, FAM175A, FAN1, FANCC, FANCF, GADD45A, H2AFX, KIAA0101, KIN, MORF4L2, MSH2, NONO, NPM1, NSMCE1, OGG1, PARPBP,

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This list of references cited by the applicant is for the reader’s convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard.

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