SYSTEMS AND METHODS FOR IDENTIFYING COSMETIC AGENTS FOR HAIR/SCALP CARE COMPOSITIONS

This application claims benefit to U.S. Provisional Application Ser. No. 61/656,218 filed Jun. 6, 2012, which is incorporated by reference herein in its entirety.

Connectivity mapping is a well-known hypothesis generating and testing tool having successful application in the fields of operations research, computer networking and telecommunications. The undertaking and completion of the Human Genome Project, and the parallel development of very high throughput high-density DNA microarray technologies enabling rapid and simultaneous quantization of cellular mRNA expression levels, resulted in the generation of an enormous genetic database. At the same time, the search for new pharmaceutical actives via in silico methods such as molecular modeling and docking studies stimulated the generation of vast libraries of potential small molecule actives. The amount of information linking disease to genetic profile, genetic profile to drugs, and disease to drugs grew exponentially, and application of connectivity mapping as a hypothesis testing tool in the medicinal sciences ripened.

The general notion that functionality could be accurately determined for previously uncharacterized genes, and that potential targets of drug agents could be identified by mapping connections in a data base of gene expression profiles for drug-treated cells, was spearheaded in 2000 with publication of a seminal paper by T. R. Hughes et al. [“Functional discovery via a compendium of expression profiles” Cell 102, 109-126 (2000)], followed shortly thereafter with the launch of The Connectivity Map (C-map Project by Justin Lamb and researchers at MIT (“Connectivity Map: Gene Expression Signatures to Connect Small Molecules, Genes, and Disease”, Science, Vol. 313, 2006.) In 2006, Lamb's group began publishing a detailed synopsis of the mechanics of C-map construction and installments of the reference collection of gene expression profiles used to create the first generation C-map and the initiation of an on-going large scale community C-map project.

The basic paradigm of predicting novel relationships between disease, disease phenotype, and drugs employed to modify the disease phenotype, by comparison to known relationships has been practiced for centuries as an intuitive science by medical clinicians. Modern connectivity mapping, with its rigorous mathematical underpinnings and aided by modern computational power, has resulted in confirmed medical successes with identification of new agents for the treatment of various diseases including cancer. Nonetheless, certain limiting presumptions challenge application of C-map with respect to diseases of polygenic origin or syndromic conditions characterized by diverse and often apparently unrelated cellular phenotypic manifestations. According to Lamb, the challenge to constructing a useful C-map is in the selection of input reference data which permit generation of clinically salient and useful output upon query. For the drug-related C-map of Lamb, strong associations comprise the reference associations, and strong associations are the desired output identified as hits.

Noting the benefit of high-throughput, high density profiling platforms which permit automated amplification, labeling hybridization and scanning of 96 samples in parallel a day, Lamb nonetheless cautioned: “[e]ven this much firepower is insufficient to enable the analysis of every one of the estimated 200 different cell types exposed to every known perturbagen at every possible concentration for every possible duration . . . compromises are therefore required” (page 54, column 3, last paragraph). Hence, Lamb confined his C-map to data from a very small number of established cell lines. This leads to heightened potential for in vitro to in vivo mismatch, and limits information to the context of a particular cell line. Selection of cell line, therefore, may be critical to the utility of a resulting C-map.

Lamb stresses that particular difficulty is encountered if reference connections are extremely sensitive and at the same time difficult to detect (weak), and Lamb adopted compromises aimed at minimizing numerous, diffuse associations. Since the regulatory scheme for drug products requires high degrees of specificity between a purported drug agent and disease state, and modulation of disease by impacting a single protein with a minimum of tangential associations is desired in development of pharmaceutical actives, the Lamb C-map is well-suited for screening for potential pharmaceutical agents despite the Lamb compromises.

The connectivity mapping protocols of Lamb would not be predicted, therefore, to have utility for hypothesis testing/generating in the field of cosmetics. Cosmetic formulators seek agents or compositions of agents capable of modulating multiple targets and having effects across complex phenotypes and conditions. Further, the phenotypic impact of a cosmetic agent must be relatively low by definition, so that the agent avoids being subject to the regulatory scheme for pharmaceutical actives. Nonetheless, the impact must be perceptible to the consumer and preferably empirically confirmable by scientific methods. Gene transcription/expression profiles for cosmetic conditions are generally diffuse, comprising many genes with low to moderate fold differentials. Cosmetic agents, therefore, provide more diverse and less acute effects on cellular phenotype and generate the sort of associations expressly taught by Lamb as unsuitable for generating connectivity maps useful for confident hypothesis testing.

The present inventors surprisingly discovered that useful connectivity maps could be developed from cosmetic active—cellular phenotype—gene expression data associations in particular with respect to hair care cosmetics. Specifically, certain aspects of the present invention are based on the surprising discovery that selection of human cells such as fibroblasts, keratinocytes, melanocytes or dermal papilla cells are relevant cell lines and that data from such cells has resulted in construction of connectivity maps useful for hypothesis generating and testing relating to cosmetic agents in treatment of specific hair biology conditions such the appearance of gray hair, chronogenetic alopecia, senile alopecia, androgenetic alopecia, loss of hair diameter or hair breakage/fragile hair; for example, BJ fibroblasts are a better cell line than tert-keratinocytes for the identification of Monoamine Oxidase B (MAOB) inhibitors to improve hair growth. Melanocytes are cells of a better cell line for evaluating material to delay the appearance of gray hair, while a combination of cells appeared most suitable for other specific hair biology conditions. For example, a set of biological signatures were generated and combined from different cell lines and clinical data was generated from samples with multiple cell types to capture different aspect for hair growth and healthy fiber quality. Therefore, it could not be accurately predicted that data from one cell type (such as a fibroblast cell or a keratinocyte cell), or any combination thereof, could be used to construct a connectivity map effective for generating and testing hypotheses relating to cosmetic actives and genes associated with a specific hair biology condition.

Hair is a complex, multi-layered and dynamic system that provides a protective covering from elements and acts to disperse products from glands in acting as an interactive boundary between an organism and the environment. It is also vitally important to both individual health and self image. For example, a significant industry has developed to assist individuals with conditions of hair loss (alopecia) as well as to deal with excessive hair growth. In fact, a large array of hair conditions and disorders have been characterized and include alopecia, androgenic alopecia, alopecia greata, permanent alopecia, anagen growth state disorders, anagen effluvium, bulb disorders, bulge disorders, catagen and regression disorders, club hair, hirsutism, hypertrichosis, lanugo hair, miniaturization, telogen disorders, telogen effluvium, terminal hair, and vellus hair as non-limiting examples. FIG. 1 illustrates basic hair anatomy.

Due to the complexity of hair and its interaction with skin, a basic discussion of each is herein included. This discussion is necessary as various treatments of hair biology conditions include application of products or methods related to the hair itself or to the skin surrounding the hair or parts of the hair. For example, various hair treatments include methods and uses of products such as Rogaine®, Propecia®, hair transplants, hair electrolysis, and laser hair removal.

Though the intricacies of hair growth disorders is complex and requires additional research and breakthroughs, basic hair anatomy is well known, and has been previously described. For example a review of hair biology has been written by Ralf Paus and George Cotsarelis (see among other places, Paus R and Cotsarelis G. The Biology of Hair Follicles (Review Article). Mechanisms of Disease, Vol. 341 (7), 1999, pp. 491-497).

A hair contains a hair shaft that extends primarily out from the human skin surface, and having a distal portion recessing into the epidermis of the skin. Outlining the anatomy simplistically, within the skin is the hair follicle, bulb, and papilla. The hair shaft contains keratin. Within the skin, blood vessels nourish cells in the hair bulb and cellular materials including hormones can be transferred via such networks of the vasculature. Hair color is also controlled largely by pigment cells producing melanin in the hair follicle.

More generally, hair follicles cover the vast majority of the body surface. There are approximately 5 million hair follicles on the body with 100,000 on the scalp, with a density of up to roughly 300 to 500 hairs per square centimeter on the scalp. The great significance of the hair follicle requires an outlining of additional follicle details. The hair follicle can be divided anatomically into multiple parts, including the bulb consisting of the dermal papilla and matrix, the suprabulbar area from the matrix to the insertion of the arrector pili muscle, the isthmus that extends from the insertion of the arrector pili muscle to the sebaceous gland, and the infundibulum that extends from the sebaceous gland to the follicular orifice. The lower portion of the hair follicle consists of multiple portions: the dermal papilla, matrix, hair shaft (consisting from inward to outward the medulla, cortex, and cuticle), inner root sheath (IRS) consisting of the inner root sheath cuticle, Huxley's layer, Henle's layer, and the outer root sheath. The base of the follicle is invaginated by the dermal papilla, which has a capillary loop that passes through the papilla. Signal transduction and communication between the dermal papilla and the matrix cells influence how long and how thick the hair shaft will grow. The melanocytes within the matrix also produce the pigment in the hair shaft.

As indicated earlier, the hair shaft contains keratin. As for the hair medulla, this is only partially keratinized and therefore appears amorphous and may not always be present. The hair cortex cells lose their nuclei during their upward growth and do not contain any keratohyaline or trichohyaline granules. The keratin of the cortex is hard in contrast to the IRS and epidermis, which are soft. The cuticle is firmly anchored to the IRS cuticle. The cuticle of the IRS consists of a single layer of flattened overlapping cells that point downward and interlock tightly with the upward angled cells of the hair shaft cuticle. Huxley's layer is composed of two cell layers, whereas the outer Henle's layer is only one cell thick. Just before the isthmus, the IRS becomes fully keratinized but disintegrates at the level of the isthmus. Although the IRS is not present in the emerging hair shaft, the IRS serves as a strong scaffold in the lower portion of the hair follicle.

Returning once again to the hair follicle, the hair follicle is significant in hair development and cycling of hair follicles involves three main stages, including anagen, catagen, and telogen. The anagen phase is known as the growth phase, and a hair can spend several years in this phase. The catagen phase is a transitional phase occurring over a few weeks, with hair growth slowing and the follicle shrinking. The telogen phase is a resting phase where, over months, hair growth stops and the old hair detaches from the follicle; a new hair begins the growth phase and pushes the old hair out.

As indicated, an intricate relationship exists between hair and skin. Regarding the skin, the skin comprises three principal layers, the epidermis, the dermis, and a layer of subcutaneous fat. The majority of cells in the epidermis are keratinocytes that produce a family of proteins called keratins. Keratins contribute to the strength of the epidermis. The epidermis itself may be divided into multiple layers with the outermost layer referred to as the stratum corneum, and the innermost layer referred to as the basal layer. All epidermal cells originate from the basal layer and undergo a process known as differentiation as they gradually displace outward to the stratum corneum, where they fuse into squamous sheets and are eventually shed. In healthy, normal skin, the rate of production equals the rate of shedding (desquamation).

The differentiating epidermal cells form distinct though naturally blended layers. As the cells displace outward, they flatten and become joined by spiny processes forming the stratum spinosum, or spinous layer. The cells manufacture specialized fats called sphingolipids, and begin to express keratins associated with terminal differentiation. As keratin is produced, it is incorporated into the cellular matrix, strengthening the skin and providing structural support to the outer layers. As the cells migrate further outward and develop characteristic granules that contain proteins which contribute to the aggregation of keratins; they now form part of the granular layer. Cells lose their nuclei in the outer part of this layer, and the granules release their contents contributing to cornification. Vesicles containing lipids discharge into the spaces between the cells, creating a barrier structure that has been suggested to function like bricks (cells) and mortar (lipids). As the cells rise into the outermost layer of the epidermis—the stratum corneum, sometimes called the horny layer or the cornified layer—they take the form of flattened discs, tightly packed together. These flattened cells, called corneocytes, are effectively dead. The lipids of the epidermis play an important role in maintaining skin health, as they help the stratum corneum to regulate water loss while providing a virtually impermeable hydrophobic barrier to the environment. Fully mature keratinocytes function to protect the skin from UV light damage, and help effectuate immune responses to environmental stimuli.

The dermis, which lies just beneath the epidermis, is composed largely of the protein collagen. Most of the collagen is organized in bundles which run horizontally through the dermis and which are buried in a jelly-like material called the ground substance. Collagen accounts for up to 75% of the weight of the dermis, and is responsible for the resilience and elasticity of skin. The collagen bundles are held together by elastic fibers running through the dermis. The fibers are comprised of a protein called elastin, and make up less than 5% of the weight of the dermis. Fibroblasts function to synthesize collagen and the dermis ground substance, including components glycoproteins and glycosaminoglycans such as hyaluronic acid (which is able to bind water). The junction between the epidermis and the dermis is not straight but undulates—more markedly so in some areas of the body than others. A series of finger-like structures called rete pegs project up from the dermis, and similar structures project down from the epidermis. These projections increase the area of contact between the layers of skin, and help to prevent the epidermis from being sheared off. As skin ages, the projections get smaller and flatter. Networks of tiny blood vessels run through the rete pegs, bringing nutrients, vitamins and oxygen to the epidermis, although the epidermis itself is avascular and nourished by diffusion from the rete pegs. The dermis also contains the pilobaceous units comprising hair follicles and sebaceous glands, apocrine and eccrine sweat glands, lymphatic vessels, nerves, and various sensory structures, including the mechano-sensing Pacinian and Meissner's corpuscles.

Beneath the dermis lies the hypodermis, which comprises subcutaneous fat that cushions the dermis from underlying tissues such as muscle and bones. The fat is contained in adipose cells embedded in a connective tissue matrix. This layer may also house the hair follicles when they are in the growing phase.

Thus, skin is a multilayered complex organ comprising a wide variety of cellular types and structures, including epidermal and dermal connective tissue with blood and lymphatic vessels, the pilosebaceous units, glands, nerves, various sensory structures, the hypodermal adipose tissue, and the elastic fascia beneath the hypodermis. In turn, these structures are composed of a number of different cell types including keratinocytes, melanocytes, neuroendocrine Merkel cells, sebocytes, fibroblasts, endothelial cells, pericytes, neurons, adipocytes, myocytes and resident immunocytes including Langerhans cells, other dendritic cells, T cells and mast cells. Two of the main cell lineages in the skin are epithelial cells, which in general form the linings of the body and the parenchyma of many organs and glands, and mesenchymal cells, which form connective tissue, blood vessels and muscle. Dermal fibroblasts are mesenchymal cells, and keratinocytes are epithelial cells, which comprise most of the structure of the epidermis.

Thus hair and skin are intricate components that work in a complex manner to regulate hair health. As stated, there are a significant number of hair disorders, and there are many hair care products available to consumers which are directed to improving the health and/or physical appearance of hair. Despite current treatments, an ongoing need exists to identify cosmetic agents that can provide new or improved benefits to hair. There is also a need to identify additional cosmetic agents that provide similar or improved benefits as compared to existing products but which are easier to formulate, produce, and/or market.

Successful identification of hair-related cosmetic agents has proven to be difficult due to the multi-cellular, multi-factorial processes that occur in and around hair. In addition, many desirable cosmetic agents may comprise a mixture of compounds with effects and interactions that may not be fully understood. This is often the case with a botanical or other natural extract that may affect many cellular/pathways. An additional challenge for cosmetic formulators is that cosmetics must be very safe and adverse effects generally are not acceptable. Further, while much is known about hair biology, there is much that is still poorly understood or unknown. Conventional in vitro studies of biological responses to potential cosmetic agents involve testing hundreds or thousands of potential agents in various cell types before an agent that gives the desired result can be identified and moved into a next stage of testing. However, such studies can be hindered by the complex or weakly detectable responses typically induced and/or caused by cosmetic agents. Such weak responses arise, in part, due to the great number of genes and gene products involved, and cosmetic agents may affect multiple genes in multiple ways. Moreover, the degree of bioactivity of cosmetic agents may differ for each gene and be difficult to quantify.

The value of a connectivity map approach to discover functional connections among cosmetic phenotypes of hair biology, gene expression perturbation, and cosmetic agent action is counter-indicated by the progenitors of the drug-based C-map. The relevant phenotypes are very complex, the gene expression perturbations are numerous and weak, and cosmetic agent action is likewise diffuse and by definition, relatively weak. It has thus far been unclear whether statistically valid data could be generated from cosmetic C-maps and whether a cell line existed to provide salient or detectable cosmetic data.

Surprisingly, the present inventors have provided a C-map approach that is generalizable and biologically relevant for identification of potential cosmetic actives, and demonstrate that the C-map concept is viable by (as a non-limiting example) use of benchmark cosmetic actives to query the reference data and by identification of new cosmetic actives.

Accordingly, the present invention provides novel methods and systems useful for generating potential new actives for the treatment of hair biology conditions. In particular, by careful selection of cell type, and by generation of a reference collection of gene-expression profiles for known cosmetic actives, the present inventors were surprisingly able to create a connectivity map useful for testing and generating hypotheses about cosmetic actives and cosmetic conditions. The present investigators confirmed the validity of connectivity mapping as a tool for identifying cosmetic agents efficacious in specific hair biology conditions. Potentially efficacious cosmetic agents were identified using gene expression signatures derived from clinical as well as through in vitro experiments of simple cell culture systems.

The present inventors discovered that it is possible to derive unique hair biology-relevant gene expression signatures for use in a connectivity map. The present inventors have also surprisingly discovered methods that utilize a plurality of unique hair biology-relevant gene expression signatures in a connectivity map to identify useful cosmetic hair care agents.

Embodiments herein described broadly include methods and systems for determining relationships between a hair biology condition of interest and one or more cosmetic agents, one or more genes associated with the hair biology condition, and one or more cells associated with the hair biology condition. Such methods may be used to identify cosmetic agents without detailed knowledge of the mechanisms of biological processes associated with a hair biology condition of interest, all of the genes associated with such a condition, or the cell types associated with such a condition.

According to one embodiment of the invention, herein described is a method for constructing a data architecture for use in identifying connections between perturbagens and genes associated with one or more hair biology conditions, comprising: (a) providing a gene expression profile for a control human fibroblast or keratinocyte cell; (b) generating a gene expression profile for a human fibroblast or keratinocyte cell exposed to at least one perturbagen; (c) identifying genes differentially expressed in response to the at least one perturbagen by comparing the gene expression profiles of (a) and (b); (d) creating an ordered list comprising identifiers representing the differentially expressed genes, wherein the identifiers are ordered according to the differential expression of the genes; (e) storing the ordered list as a fibroblast or keratinocyte instance on at least one computer readable medium; and (f) constructing a data architecture of stored fibroblast or keratinocyte instances by repeating (a) through (e), wherein the at least one perturbagen of step (a) is different for each fibroblast or keratinocyte instance.

Specific embodiments herein described include a method for formulating a hair care composition by identifying connections between perturbagens and genes associated with one or more hair biology conditions, comprising: (a) accessing a plurality of instances stored on at least one computer readable medium, wherein each instance is associated with a perturbagen and a hair-related cell type and wherein each instance comprises an ordered list comprising a plurality of identifiers representing a plurality of up-regulated and a plurality of down regulated genes; (b) accessing at least one hair biology-related gene expression signature stored on the at least one computer readable medium, wherein the at least one hair biology-related gene expression signature comprises one or more lists comprising a plurality of identifiers representing a plurality of up-regulated genes and a plurality of down-regulated genes associated with a hair biology-related condition; (c) comparing the at least one hair biology-related gene expression signature to the plurality of the instances, wherein the comparison comprises comparing each identifier in the one or more gene expression signature lists with the position of the same identifier in the ordered lists for each of the plurality of instances; (d) assigning a connectivity score to each of the plurality of instances; and (e) formulating a hair care composition comprising a dermatologically acceptable carrier and at least one perturbagen, wherein the connectivity score of the instance associated with the at least one perturbagen has a negative correlation.

In yet more specific embodiments, described herein is a method for generating a gene expression signature for use in identifying connections between perturbagens and genes associated with one or more hair biology conditions, comprising: (a) providing a gene expression profile for a reference sample of human hair-related cells; (b) generating a gene expression profile for at least one sample of human hair-related cells from a subject exhibiting at least one hair biology condition, (c) comparing the expression profiles of (a) and (b) to determine a gene expression signature comprising a set of genes differentially expressed in (a) and (b); (d) assigning an identifier to each gene constituting the gene expression signature and ordering the identifiers according to the direction of differential expression to create one or more gene expression signature lists; (e) storing the one or more gene expression signature lists on at least one computer readable medium.

In other specific embodiments herein described, is a system for identifying connections between perturbagens and genes associated with one or more hair biology conditions, comprising: (a) at least one computer readable medium having stored thereon a plurality of instances, and at least one hair biology-relevant gene expression signature, wherein the instances and the gene expression signature are derived from a human dermal fibroblast cell, wherein each instance comprises an instance list of rank-ordered identifiers of differentially expressed genes, and wherein the at least one hair biology-relevant gene expression signature comprises one or more gene expression signature lists of identifiers representing differentially expressed genes associated with a hair biology condition; (b) a programmable computer comprising computer-readable instructions that cause the programmable computer to execute one or more of the following: (i) accessing the plurality of instances and the at least one hair biology-relevant gene expression signature stored on the computer readable medium; (ii) comparing the at least one hair biology-relevant gene expression signature to the plurality of the instances, wherein the comparison comprises comparing each identifier in the gene expression signature list with the position of the same identifier in the instance list for each of the plurality of instances; and (iii) assigning a connectivity score to each of the plurality of instances.

In yet additional specific embodiments herein described, is a gene expression signature consisting of genes selected from the genes set forth in Tables C and D.

In yet additional specific embodiments herein described, is a gene expression signature consisting of genes selected from the genes set forth in Tables E and F.

Additional specific embodiments herein described include a computer readable medium, comprising: a data architecture comprising a digital file stored in a spreadsheet file format, a word processing file format, or a database file format suitable to be read by a respective spreadsheet, word processing, or database computer program, the first digital file comprising data arranged to provide one or more gene expression signature lists comprising a plurality of identifiers when read by the respective spreadsheet, word processing, or database computer program; and wherein each identifier is selected from the group consisting of a microarray probe set ID, a human gene name, a human gene symbol, and combinations thereof representing a gene set forth in any of Tables A-R and T-U, wherein each of the one or more gene expression signature lists comprises between about 50 and about 600 identifiers.

Additional specific embodiments herein described include a method for constructing a data architecture for use in identifying connections between perturbens and genes associated with improving hair biology, comprising: (a) providing a gene expression profile for a control human cell, wherein the control cell is from a human cell line selected from the group consisting of fibroblast, keratinocyte, melanocyte, and dermal papilla cell lines; (b) generating a gene expression profile for a human cell exposed to at least one perturbagen, wherein the cell is selected from the same cell line as the control cell; (c) identifying genes differentially expressed in response to at least one perturbagen by comparing the gene expression profiles of (a) and (b); (d) creating an ordered list comprising identifiers representing the differentially expressed genes, wherein the identifiers are ordered according to the differential expression of the genes; (e) storing the ordered list as an instance on at least one computer readable medium, wherein the instance is a fibroblast, keratinocyte, melanocyte, or dermal papilla instance according to the selection in (a); and (f) constructing a data architecture of stored instances by repeating (a) through (e), wherein the at least one perturben of step (b) is different qualitatively or quantitatively for each instance.

Additional specific embodiments herein described include a method for constructing a data architecture for use in identifying connections between perturbagens and genes associated with one or more hair biology conditions, comprising: (a) providing a gene expression profile for a control human keratinocyte cell; (b) generating a gene expression profile for a human keratinocyte cell exposed to at least one perturbagen; (c) identifying genes differentially expressed in response to the at least one perturbagen by comparing the gene expression profiles of (a) and (b); (d) creating an ordered list comprising identifiers representing the differentially expressed genes, wherein the identifiers are ordered according to the differential expression of the genes; (e) storing the ordered list as a keratinocyte instance on at least one computer readable medium; and (f) constructing a data architecture of stored keratinocyte instances by repeating (a) through (e), wherein the at least one perturbagen of step (a) is different for each keratinocyte instance.

These and additional objects, embodiments, and aspects of the invention will become apparent by reference to the Figures and Detailed Description below.

Embodiments of the present invention will now be described. Embodiments of this invention may, however, be provided in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and to fully convey the scope of specific embodiments of the invention to those skilled in the art.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in the description of the invention herein is for describing particular embodiments only and is not intended to be limiting of the invention. As used in the description of the invention and the appended claims, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

As used interchangeably herein, the terms “connectivity map” and “C-map” refer broadly to devices, systems, articles of manufacture, and methodologies for identifying relationships between cellular phenotypes or cosmetic conditions, gene expression, and perturbagens, such as cosmetic actives.

As used herein, the term “cosmetic agent” means any substance, as well any component thereof, intended to be rubbed, poured, sprinkled, sprayed, introduced into, or otherwise applied to a mammalian body or any part thereof for purposes of cleansing, beautifying, promoting attractiveness, altering the appearance, or combinations thereof. Cosmetic agents may include substances that are Generally Recognized as Safe (GRAS) by the US Food and Drug Administration, food additives, and materials used in non-cosmetic consumer products including over-the-counter medications. In some embodiments, cosmetic agents may be incorporated in a cosmetic composition comprising a carrier suitable for topical application. A cosmetic agent includes, but is not limited to, (i) chemicals, compounds, small or large molecules, extracts, formulations, or combinations thereof that are known to induce or cause at least one effect (positive or negative) on hair; (ii) chemicals, compounds, small molecules, extracts, formulations, or combinations thereof that are known to induce or cause at least one effect (positive or negative) on hair and are discovered, using the provided methods and systems, to induce or cause at least one previously unknown effect (positive or negative) on the hair; and (iii) chemicals, compounds, small molecules, extracts, formulations, or combinations thereof that are not known have an effect on skin tissue and are discovered, using the provided methods and systems, to induce or cause an effect on hair.

Some examples of cosmetic agents or cosmetically actionable materials can be found in the PubChem database associated with the National Institutes of Health, USA; the Ingredient Database of the Personal Care Products Council; and the 2010 International Cosmetic Ingredient Dictionary and Handbook, 13^th Edition, published by The Personal Care Products Council; the EU Cosmetic Ingredients and Substances list; the Japan Cosmetic Ingredients List; the Personal Care Products Council, the SkinDeep database; the FDA Approved Excipients List; the FDA OTC List; the Japan Quasi Drug List; the US FDA Everything Added to Food database; EU Food Additive list; Japan Existing Food Additives, Flavor GRAS list; US FDA Select Committee on GRAS Substances; US Household Products Database; the Global New Products Database (GNPD) Personal Care, Health Care, Food/Drink/Pet and Household database; and from suppliers of cosmetic ingredients and botanicals.

Other non-limiting examples of cosmetic agents include botanicals (which may be derived from one or more of a root, stem bark, leaf, seed or fruit of a plant). Some botanicals may be extracted from a plant biomass (e.g., root, stem, bark, leaf, etc.) using one more solvents. Botanicals may comprise a complex mixture of compounds and lack a distinct active ingredient. Another category of cosmetic agents are vitamin compounds and derivatives and combinations thereof, such as a vitamin B3 compound, a vitamin B5 compound, a vitamin B6 compound, a vitamin B9 compound, a vitamin A compound, a vitamin C compound, a vitamin E compound, and derivatives and combinations thereof (e.g., retinol, retinyl esters, niacinamide, folic acid, panthenol, ascorbic acid, tocopherol, and tocopherol acetate). Other non-limiting examples of cosmetic agents include sugar amines, phytosterols, hexamidine, hydroxy acids, ceramides, amino acids, and polyols.

The terms “gene expression signature,” “gene-expression signature,” and “hair biology-related gene expression signature” refer to a rationally derived list, or plurality of lists, of genes representative of a hair biology condition or a hair biology agent. In specific contexts, the hair biology agent may be a benchmark hair biology agent or a potential hair biology agent. Thus, the gene expression signature may serve as a proxy for a phenotype of interest for a hair-related cell type or types. A gene expression signature may comprise genes whose expression, relative to a normal or control state, is increased (up-regulated), whose expression is decreased (down-regulated), and combinations thereof. Generally, a gene expression signature for a modified cellular phenotype may be described as a set of genes differentially expressed in the modified cellular phenotype over the cellular phenotype. A gene expression signature can be derived from various sources of data, including but not limited to, from in vitro testing, in vivo testing and combinations thereof. In some embodiments, a gene expression signature may comprise a first list representative of a plurality of up-regulated genes of the condition of interest and a second list representative of a plurality of down-regulated genes of the condition of interest.

As used herein, the term “benchmark hair biology agent” refers to any chemical, compound, small or large molecule, extract, formulation, or combinations thereof that is known to induce or cause a superior effect (positive or negative) on hair-related cell types. Non-limiting examples of positive benchmark hair biology agents include Minoxidil, Latanoprost, ZPT (zinc pyrithione), ATRA (all trans retinoic acid), a combination of caffeine and Niacinamide and Panthenol, adenosine, apigenin, Finasteride, Cyclosporin A (CSP A), FK506, Bimatoprost, Spironolactone or Cyproterone acetate, RU58841, carnitine tartrate, Aminexil, 6-Benzylaminopurine, melatonin, carpronium chloride, MG132, NEOSH101, AS101, Roxithromycin. Non-limiting negative benchmarks hair biology agents include Vaniqa® (Eflornithine, a drug used to slow the growth of unwanted hair on the face in women, usually around the lips or under the chin.), as well as DHT (Dihydrotestosterone or 5α-Dihydrotestosterone, the primary contributing factor in male pattern baldness).

As used herein, “hair biology condition” is a state of the hair existence capable of improvement; in various non-limiting embodiments this could include pathologies or disorders to which study or application of formulations are aimed to alter that state. Non-limiting examples include dandruff, alopecia, unwanted hair loss, unwanted hair growth, hair thinning, loss of hair diameter, premature hair graying, hair fragility, curl or lack of curl.

As used herein, “hair-related cells” or “hair related cell types” refer to cells or types of cells that are either directly part of a hair (such as a cell shaft), or that are intricately associated with the hair such as to be necessary for homeostatic hair conditions or that involve hair growth. Non-limiting examples of hair related cell types include dermal papilla cells, keratinocytes including inner and outer root sheath cells, dermal fibroblasts, melanocytes, hair/skin stem cells. Induced pluripotent stem cells (IPSC) can be induced in specific embodiments described herein into “hair-related cells” In specific, non-limiting examples, induced pluripotent stem cells (IPSC) can be induced into a human cell or human cell line selected from the group consisting of dermal papilla cells, keratinocytes including inner and outer root sheath cells, dermal fibroblasts, melanocytes, hair/skin stem cells.

As used herein, the term “query” refers to data that is used as an input to a Connectivity Map and against which a plurality of instances are compared. A query may include a gene expression signature associated with one or both of a hair biology condition and a benchmark hair biology agent.

The term “instance,” as used herein, refers to data from a gene expression profiling experiment in which hair-related cell types are dosed with a perturbagen. In some embodiments, the data comprises a list of identifiers representing the genes that are part of the gene expression profiling experiment. The identifiers may include gene names, gene symbols, microarray probe set IDs, or any other identifier. In some embodiments, an instance may comprise data from a microarray experiment and comprises a list of probe set IDs of the microarray ordered by their extent of differential expression relative to a control. The data may also comprise metadata, including but not limited to data relating to one or more of the perturbagen, the gene expression profiling test conditions, cells of the hair-related cell types, and the microarray.

The term “keratinous tissue,” as used herein, refers to keratin-containing layers disposed as the outermost protective covering of mammals which includes, but is not limited to, skin, hair, nails, cuticles, horns, claws, beaks, and hooves. With respect to skin, the term refers to one or all of the dermal, hypodermal, and epidermal layers, which includes, in part, keratinous tissue.

The term “perturbagen,” as used herein, means anything used as a challenge in a gene expression profiling experiment to generate gene expression data for use with embodiments of the present invention. In some embodiments, the perturbagen is applied to fibroblast and/or keratinocyte cells and the gene expression data derived from the gene expression profiling experiment may be stored as an instance in a data architecture. Any substance, chemical, compound, active, natural product, extract, drug [e.g. Sigma-Aldrich LOPAC (Library of Pharmacologically Active Compounds) collection], small molecule, and combinations thereof used as to generate gene expression data can be a perturbagen. A perturbagen can also be any other stimulus used to generate differential gene expression data. For example, a perturbagen may also be UV radiation, heat, osmotic stress, pH, a microbe, a virus, and small interfering RNA. A perturbagen may be, but is not required to be, any cosmetic agent.

The term “dermatologically acceptable,” as used herein, means that the compositions or components described are suitable for use in contact with human skin tissue without undue toxicity, incompatibility, instability, allergic response, and the like.

As used herein, the term “computer readable medium” refers to any electronic storage medium and includes but is not limited to any volatile, nonvolatile, removable, and non-removable media implemented in any method or technology for storage of information such as computer readable instructions, data and data structures, digital files, software programs and applications, or other digital information. Computer readable media includes, but are not limited to, application-specific integrated circuit (ASIC), a compact disk (CD), a digital versatile disk (DVD), a random access memory (RAM), a synchronous RAM (SRAM), a dynamic RAM (DRAM), a synchronous DRAM (SDRAM), double data rate SDRAM (DDR SDRAM), a direct RAM bus RAM (DRRAM), a read only memory (ROM), a programmable read only memory (PROM), an electronically erasable programmable read only memory (EEPROM), a disk, a carrier wave, and a memory stick. Examples of volatile memory include, but are not limited to, random access memory (RAM), synchronous RAM (SRAM), dynamic RAM (DRAM), synchronous DRAM (SDRAM), double data rate SDRAM (DDR SDRAM), and direct RAM bus RAM (DRRAM). Examples of non-volatile memory include, but are not limited to, read only memory (ROM), programmable read only memory (PROM), erasable programmable read only memory (EPROM), and electrically erasable programmable read only memory (EEPROM). A memory can store processes and/or data. Still other computer readable media include any suitable disk media, including but not limited to, magnetic disk drives, floppy disk drives, tape drives, Zip drives, flash memory cards, memory sticks, compact disk ROM (CD-ROM), CD recordable drive (CD-R drive), CD rewriteable drive (CD-RW drive), and digital versatile ROM drive (DVD ROM).

As used herein, the terms “software” and “software application” refer to one or more computer readable and/or executable instructions that cause a computing device or other electronic device to perform functions, actions, and/or behave in a desired manner. The instructions may be embodied in one or more various forms like routines, algorithms, modules, libraries, methods, and/or programs. Software may be implemented in a variety of executable and/or loadable forms and can be located in one computer component and/or distributed between two or more communicating, co-operating, and/or parallel processing computer components and thus can be loaded and/or executed in serial, parallel, and other manners. Software can be stored on one or more computer readable medium and may implement, in whole or part, the methods and functionalities of the present invention.

As used herein, the term “connectivity score” refers to a derived value representing the degree to which an instance correlates to a query.

As used herein, the term “data architecture” refers generally to one or more digital data structures comprising an organized collection of data. In some embodiments, the digital data structures can be stored as a digital file (e.g., a spreadsheet file, a text file, a word processing file, a database file, etc.) on a computer readable medium. In some embodiments, the data architecture is provided in the form of a database that may be managed by a database management system (DBMS) that is be used to access, organize, and select data (e.g., instances and gene expression signatures) stored in a database.

As used herein, the terms “gene expression profiling” and “gene expression profiling experiment” refer to the measurement of the expression of multiple genes in a biological sample using any suitable profiling technology. For example, the mRNA expression of thousands of genes may be determined using microarray techniques. Other emerging technologies that may be used include RNA-Seq or whole transcriptome sequencing using NextGen sequencing techniques.

As used herein, the term “microarray” refers broadly to any ordered array of nucleic acids, oligonucleotides, proteins, small molecules, large molecules, and/or combinations thereof on a substrate that enables gene expression profiling of a biological sample. Non-limiting examples of microarrays are available from Affymetrix, Inc.; Agilent Technologies, Inc.; Ilumina, Inc.; GE Healthcare, Inc.; Applied Biosystems, Inc.; Beckman Coulter, Inc.; etc.

Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth as used in the specification and claims are to be understood as being modified in all instances by the term “about.” Additionally, the disclosure of any ranges in the specification and claims are to be understood as including the range itself and also anything subsumed therein, as well as endpoints. All numeric ranges are inclusive of narrower ranges; delineated upper and lower range limits are interchangeable to create further ranges not explicitly delineated. Unless otherwise indicated, the numerical properties set forth in the specification and claims are approximations that may vary depending on the desired properties sought to be obtained in embodiments of the present invention. Notwithstanding that numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical values, however, inherently contain certain errors necessarily resulting from error found in their respective measurements.

In accordance with one aspect of specific embodiments of the present invention, provided are devices, systems and methods for implementing a connectivity map utilizing one or more query signatures associated with a hair biology condition. The query signatures may be derived in variety of ways. In some embodiments, the query signatures may be gene expression signatures derived from gene expression profiling biopsies of a hair sample of interest compared to a control. The gene expression profiling can be carried out using any suitable technology, including but not limited to microarray analysis or NextGen sequencing. Query signatures may be used singularly or in combination.

In accordance with another aspect of specific embodiments of the present invention, provided are devices, systems, and methods for implementing a connectivity map utilizing one or more instances derived from a perturbagen, such as a cosmetic agent, exposed to a fibroblast (e.g., BJ fibroblasts) and/or keratinocyte cell line. Instances from more complex cell culture systems may also be used, such as organotypic cultures containing both keratinocytes and fibroblasts and optionally other cell types such as melanocytes or cells from cultured ex vivo samples such as follicles or hair bearing skin. Instances from a plurality of cell lines may be used with the present invention.

In accordance with yet another aspect of specific embodiments of the present invention, provided are devices, systems and methods for identification of relationships between a hair biology-related query signature and a plurality of instances. For example, it may be possible to ascertain perturbagens that give rise to a statistically significant activity on a statistically significant number of genes associated with a hair condition of interest, leading to the identification of new cosmetic agents for treating a hair condition or new uses of known cosmetic agents.

As indicated previously, additional specific embodiments herein described include a computer readable medium, comprising: a data architecture comprising a digital file stored in a spreadsheet file format, a word processing file format, or a database file format suitable to be read by a respective spreadsheet, word processing, or database computer program, the first digital file comprising data arranged to provide one or more gene expression signature lists comprising a plurality of identifiers when read by the respective spreadsheet, word processing, or database computer program; and wherein each identifier is selected from the group consisting of a microarray probe set ID, a human gene name, a human gene symbol, and combinations thereof representing a gene set forth in any of Tables A-R and T-U, wherein each of the one or more gene expression signature lists comprises between about 50 and about 600 identifiers. Tables A-R and T-U are herein provided below: