U.S. patent application number 13/851886 was filed with the patent office on 2013-10-03 for system for identifying connections between perturbagens and genes associated with a skin hyperpigmentation condition.
This patent application is currently assigned to The Procter & Gamble Company. The applicant listed for this patent is THE PROCTER & GAMBLE COMPANY. Invention is credited to Robert Lloyd BINDER, Tomohiro HAKOZAKI, Jun XU, Wenzhu ZHAO.
Application Number | 20130261024 13/851886 |
Document ID | / |
Family ID | 48048316 |
Filed Date | 2013-10-03 |
United States Patent
Application |
20130261024 |
Kind Code |
A1 |
HAKOZAKI; Tomohiro ; et
al. |
October 3, 2013 |
System for Identifying Connections Between Perturbagens and Genes
Associated with a Skin Hyperpigmentation Condition
Abstract
A system for identifying connections between perturbagens and
genes associated with a skin hyperpigmentation condition. The
system includes a computer readable medium having a plurality of
instances stored thereon, and a skin hyperpigmentation-relevant
gene expression signature. Each instance includes an instance list
of rank-ordered identifiers of differentially expressed genes, and
the hyperpigmentation-relevant gene expression signature includes a
gene expression signature list of identifiers representing
differentially expressed genes associated with a hyperpigmentation
condition or differentially expressed genes associated with a
benchmark skin-lightening agent. The system also includes a
programmable computer with computer-readable instructions that
allow the computer to accessing the instances and a
hyperpigmentation-relevant gene expression signature stored on the
computer readable medium, comparing the hyperpigmentation-relevant
gene expression signature to the plurality of the instances, and/or
assigning a connectivity score to each of the plurality of
instances.
Inventors: |
HAKOZAKI; Tomohiro;
(Cincinnati, OH) ; ZHAO; Wenzhu; (Mason, OH)
; BINDER; Robert Lloyd; (Montgomery, OH) ; XU;
Jun; (Mason, OH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE PROCTER & GAMBLE COMPANY |
Cincinnati |
OH |
US |
|
|
Assignee: |
The Procter & Gamble
Company
Cincinnati
OH
|
Family ID: |
48048316 |
Appl. No.: |
13/851886 |
Filed: |
March 27, 2013 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
61618115 |
Mar 30, 2012 |
|
|
|
Current U.S.
Class: |
506/15 ; 506/39;
702/20 |
Current CPC
Class: |
C12Q 1/6881 20130101;
C12N 15/1072 20130101; G16B 25/00 20190201; G01N 33/5044 20130101;
C12Q 1/6883 20130101; C12Q 2600/148 20130101; C12Q 2600/158
20130101; G16B 20/00 20190201 |
Class at
Publication: |
506/15 ; 506/39;
702/20 |
International
Class: |
G06F 19/18 20060101
G06F019/18; G06F 19/20 20060101 G06F019/20; G01N 33/50 20060101
G01N033/50 |
Claims
1. A system for identifying connections between perturbagens and
genes associated with a skin hyperpigmentation condition,
comprising: (a) at least one computer readable medium having stored
thereon a plurality of instances, and a skin
hyperpigmentation-relevant gene expression signature, wherein the
instances and the gene expression signature are derived from one of
a human keratinocyte cell, a human fibroblast cell, a human
melanocyte cell, or a human melanoma cell, wherein each instance
comprises an instance list of rank-ordered identifiers of
differentially expressed genes, and wherein the
hyperpigmentation-relevant gene expression signature comprises one
or more gene expression signature lists of identifiers representing
differentially expressed genes associated with a hyperpigmentation
condition or differentially expressed genes associated with a
benchmark skin-lightening agent; (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 a
hyperpigmentation-relevant gene expression signature stored on the
computer readable medium; (ii) comparing the
hyperpigmentation-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.
2. The system of claim 1, further comprising a microarray scanner
for receiving a sample comprising human keratinocyte, fibroblast,
melanocyte or melanoma cells; and a second programmable computer
for transmitting gene expression data from the scanner to the first
programmable computer.
3. The system of claim 1, further comprising an array of
perturbagens for application to the human keratinocyte, fibroblast,
melanocyte or melanoma cells.
4. The system of claim 3, wherein the hyperpigmentation-relevant
gene expression signature comprises one or more gene expression
signature lists of identifiers representing differentially
expressed genes associated with a hyperpigmentation condition, and
the system further comprises a data architecture which allows the
system to identify at least one putative skin active agent having
potential efficacy in treating the skin hyperpigmentation
condition.
5. The system of claim 4, wherein the programmable computer assigns
a connectivity score to each of the plurality of instances and if
the instance has a negative connectivity score, a perturbagen
associated with the instance is identified as a putative skin
active agent having potential efficacy in the treatment of the
hyperpigmentation condition, and if the instance has a positive
connectivity score, a perturbagen associated with the instance is
identified as a putative skin active agent having potential
efficacy as a skin-darkening agent.
6. The system of claim 4, wherein the programmable computer assigns
a connectivity score to each of the plurality of instances and if
an instance has a positive connectivity score, a perturbagen
associated with the instance is identified as a putative skin
active agent having potential efficacy in the treatment of the
hyperpigmentation condition, and if an instance has a negative
connectivity score, a perturbagen associated with the instance is
identified as a putative skin active agent having potential
efficacy as a skin-darkening agent.
7. The system of claim 4, wherein hyperpigmentation condition
results from one or more of a post-inflammatory response, age,
exposure to UVB radiation, endocrine induction of pigmentation, and
a host skin predisposition.
8. The system of claim 3, wherein the hyperpigmentation-relevant
gene expression signature comprises one or more gene expression
signature lists of identifiers representing differentially
expressed genes associated with a benchmark skin-lightening agent,
and the system further comprises a data architecture which allows
the system to identify at least one putative skin active agent
having potential efficacy in treating a skin hyperpigmentation
condition.
9. The system of claim 8, wherein the programmable computer assigns
a connectivity score to each of the plurality of instances and if
an instance has a positive connectivity score, a perturbagen
associated with the instance is identified as a putative skin
active agent having potential efficacy in the treatment of a
hyperpigmentation condition, and if an instance has a negative
connectivity score, a perturbagen associated with the instance is
identified as a putative skin active agent having potential
efficacy as a skin-darkening agent.
10. The system of claim 8, wherein the benchmark skin-lightening
agent comprises an agent selected from the group consisting of a
melanocyte stimulation inhibitor, an anti-inflammatory agent, an
alpha-MSH pigment induction antagonist, a melanophage dermal
residence time suppressor, a melanin synthesis-associated enzyme,
an adrenergic beta receptor inhibitor, an antioxidant, a melanosome
transport inhibitor, a vitamin B3 compound, hexamidine
diisothionate, N-acetyl-glucosamine, an N-acyl amino acid compound,
hydroquinone, a retinoid compound, hexyldecanol, and combinations
thereof.
11. The system of claim 1, wherein the rank-ordered list of
identifiers is arranged so that an identifier associated with each
gene that is not differentially expressed is positioned between the
identifier associated with the most up-regulated gene and the
identifier associated with the most down-regulated gene.
12. The system of claim 1, wherein the hyperpigmentation condition
has an etiology associated with one or more of activation of
melanocyte stimulation, inflammation, activation of alpha-MSH
pigment induction, increased melanophage dermal residence time,
activation of an enzyme involved in a melanin synthesis pathway,
and activation of melanosome transport.
13. The system of claim 1, wherein the identifiers are selected
from the group consisting of gene names, gene symbols, and
microarray probe set IDs.
14. The system of claim 1, wherein the gene expression signature is
derived from a human keratinocyte and the signature comprises genes
associated with the identifiers set forth in at least one Table
selected from the group consisting of Tables B through N.
15. The system of claim 1, wherein gene expression signature is
derived from a human fibroblast and the signature comprises genes
associated with the identifiers set forth in at least one of Tables
O and P.
16. The system of claim 1, wherein the plurality of instances
comprises between about 50 and about 50,000 instances.
17. The system of claim 1, wherein the connectivity score is a
value between +2 and -2.
18. A computer readable medium, comprising: (a) 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 (b) 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
B through P wherein each of the one or more gene expression
signature lists comprises between about 10 and about 400
identifiers.
19. The computer readable medium of claim 18, further comprising
computer readable instructions for reading the digital file.
Description
BACKGROUND OF THE INVENTION
[0001] Skin pigment irregularities are common across ethnic and
racial groups and are often considered cosmetically disfiguring.
Disorders of pigment production and distribution occur as a
function of intensity and duration of UV radiation exposure, life
style habits, chronological age, endocrine functioning and disease
state and are found ubiquitously in older populations. Hence there
is a widespread demand for skin pigment modifying, skin lightening
and skin tone enhancing products for the cosmetic market.
[0002] The color of normal human skin is due primarily to varying
amounts and distribution of melanin, hemoglobin, and carotenoids.
Of these pigments, melanin is of primary significance to cosmetic
skin treatment protocols. Melanin is produced by specialized cells
in the skin called melanocytes through a complicated series of
chemical and enzymatic reactions, mainly involving the copper and
manganese containing enzyme tyrosinase. Once synthesized, the
melanin granules are packaged into melanosomes and transferred via
the cellular dendrites (extensions) of the melanocyte to the
surrounding keratinocytes, the most abundant cell type in the
epidermis. The rate of melanin synthesis, and the subsequent
transfer of melanin by melanocytes via their dendrites, appears to
be influenced by ultraviolet light exposure. Melanosomes
transferred to the outer layer of the skin are responsible for the
darkening of the skin, with the degree of darkening being
associated with skin type, sun exposure, and/or certain
dermatological conditions.
[0003] Two types of melanin are present in human skin: (1)
eumelanin, which is the dark brown-black pigment found in most
skin, hair, and eyes, and whose production is stimulated by
exposure to ultraviolet light, and (2) pheomelanin, which is a
yellow-orange pigment found mainly in the skin of very fair-skinned
people, particularly those with red hair. The perceived color of
skin is determined by the ratio of eumelanins to pheomelanins, and
to a smaller extent on blood within the dermis.
[0004] The pigmentation pathway has been elucidated in detail.
Summarily, melanin forms through a series of oxidative reactions
involving the amino acid tyrosine in the presence of the enzyme
tyrosinase. Tyrosinase converts tyrosine to dihydroxyphenylalanine
(DOPA) and then to dopaquinone. Subsequently, dopaquinone is
converted to dopachrome through auto-oxidation, and finally to
dihydroxyindole or dihydroxyindole-2-carboxylic acid (DHICA), which
polymerize to form eumelanin. The latter reactions occur in the
presence of dopachrome tautomerase and DHICA oxidase. In the
presence of sulfur-containing cysteine or glutathione, dopaquinone
is converted to cysteinyl DOPA or glutathione DOPA; subsequently,
pheomelanin is formed.
[0005] A variety of skin hyperpigmentation disorders are known and
etiology is diverse, overlapping in many cases, and often not fully
understood. For example, melanosis or melasma is a condition
characterized by the development of sharply demarcated blotchy,
brown spots usually in a symmetric distribution over the cheeks,
forehead, and sometimes on the upper lip and neck. This condition
frequently occurs during pregnancy (melasma gravidarum or "mask of
pregnancy"), and at menopause. Also, this condition is frequently
found among those taking oral contraceptives, and is occasionally
found among nonpregnant women who are not taking oral
contraceptives, and sometimes among men. A pattern of similar
facial hyperpigmentation is associated with a chronic liver disease
called chloasma. A common condition associated with aging skin is
the development of dark spots sometimes referred to as "age spots"
or "liver spots." Other forms of hyperpigmentation can be caused by
UV irradiation, in particular UVB radiation which up-regulates the
production of tyrosinase resulting in skin "tanning," or may result
from a genetic predisposition for the condition, or may come about
in association with a skin inflammatory event or during the course
of wound healing.
[0006] Vitiligo is a form of hypopigmentation in which cutaneous
melanocytes are either ablated or fail to produce sufficient
pigment. Ideally treatment would restore lost pigmentation in
vitiligo-affected skin, but this approach has met with little
success via topical interventions and formulations. Although
cosmetic camouflage with dihydroxyacetone sunless-tanning lotions
provides some darkening of hypo-pigmented areas, it also tends to
darken surrounding normal skin, substantially maintaining the
undesirable contrast. Hence, a more favored cosmetic approach is to
reduce the normal pigmentation of the unaffected skin to reduce
contrast and produce a tone evening effect.
[0007] Several proven targets for pigmentation control are known,
but these have generally been derived from an understanding of the
pigmentation process. Hydroquinone (parahydroxy-benzene), for
example, is a widely used skin lightening agent that is known to
provide a satisfactory cosmetic result, however its use strictly
for cosmetic purposes is discouraged due to its association with a
variety of disorders, including diabetes, hypertension, ochronosis,
periorbitary dyschromia, infectious dermatosis, contact eczema,
extended dermatophytosis, and necrotizing cellulites (see, e.g.,
Raynaud E. et al., Ann Dermatol Venereol 128(6-7):720-724, 2001).
Hydroquinone has also shown genotoxic and mutagenic activities
(see, e.g., Jagetia G. C. et al, Toxicol Lett 121(1):15-20, 2001).
Due to concerns over toxicity and carcinogenic effects, the United
States limits treatment solutions to a 2% or lower concentration
and the FDA has proposed a ban on all over-the-counter
preparations, while hydroquinone is currently banned in Europe as a
skin lightening or depigmenting agent.
[0008] Kojic acid, Azelaic acid and certain-hydroxy acids such as
glycolic acid, have demonstrated skin-lightening effects, but
reports of localized irritation and inflammation are common. The
prenylated flavonol artocarpin has shown some efficacy for
skin-lightening in the context of ultraviolet-induced skin
pigmentation (Shimizu K. et al., Planta Med 68(1):79-81, 2002).
[0009] Recently, a more detailed genomic and proteomic
understanding of melanogenesis, the melanocyte,
melanocyte-keratinocyte interaction, and the melanocyte-fibroblast
interaction has revealed potentially hundreds of proteins and other
effectors involved in the pigmentation process and in the etiology
of hyperpigmentation disorders, which may provide additional
targets. There is a need in the cosmetic arts both for generating
potential skin lightening agents and for effective and efficient
screening methods for identifying putative skin active agents with
efficacy and safety in the cosmetic treatment of hyperpigmentation
and pigmentation disorders.
[0010] Traditionally scientists have focused on the development and
provision of safe and effective topical compositions formulated to
lighten skin and such an approach has been useful for treating
localized epidermal hyperpigmentation and for masking areas of skin
hypopigmentation. There remains a need, however, for safe and
effective agents capable of delivery through topical application to
reduce the degree of skin pigmentation in both contexts.
[0011] Skin pigmentation and the broader cosmetic concept of skin
tone, are therefore highly complex conditions with multiple and
overlapping etiologies, which manifest in part as a function of
individual predisposition, and which therefore pose a significant
treatment challenge. There is a need in the art for methods of
identifying potential skin pigment modifying agents, and in
particular skin-lightening agents, and for evaluating the efficacy
of putative skin active agents using screening methods that are
substantially independent of mechanism of action or etiology of the
pigment condition. The present investigators therefore undertook an
investigation into the application of a relatively new technology
known as "connectivity mapping" to the search for new skin-active
agents with efficacy in the treatment of hyperpigmentation
disorders and related skin conditions.
[0012] Connectivity mapping is a well-known hypothesis generating
and testing tool having successful application in the fields of
operations research, telecommunications, and more recently in
pharmaceutical drug discovery. 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.
[0013] 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 (-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,
which is available under the "supporting materials" hyperlink at
http://www.sciencemag.org/content/313/5795/1929/suppl/DC1.
[0014] 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.
[0015] 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). Lamb,
however, took the position that cell type did not ultimately
matter, and confined his C-map to data from a very small number of
established cell lines out of efficiency and standardization
concerns. Theoretically this leads to heightened potential for in
vitro to in vivo mismatch, and limits output information to the
context of a particular cell line. If one accepts the Lamb precept
that cell line does not matter then this limitation may be
benign.
[0016] However, agents suitable as pharmaceutical agents and agents
suitable as cosmetic agents are categorically distinct, with the
former defining agents selected for specificity and which are
intended to have measurable effects on structure and function of
the body, while the latter are selected for effect on appearance
and may not affect structure and function of the body to a
measurable degree. Cosmetic agents tend to be substantially
non-specific with respect to effect on cellular phenotype, and
administration to the body is generally limited to application on
or close to the body surface.
[0017] In constructing C-maps relating to pharmaceutical agents,
Lamb stresses that particular difficulty may be 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.
[0018] The connectivity mapping protocols of Lamb would not be
predicted, however, to have utility for hypothesis
testing/generating in the field of cosmetics or for a primarily
cosmetic disorder where symptoms may be diffuse, systemic and
relatively mild. In complete contravention of the goal of
pharmaceutical active discovery, 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.
[0019] Successful identification of skin lightening agents has
proven to be difficult due to the multi-cellular, multi-factorial
processes involved in etiology of the hyperpigmentation condition
itself. Conventional in vitro studies of biological responses to
potential skin-lightening agents can be hindered by the complex or
weakly detectable responses typically induced and/or caused by the
putative skin active or potential skin active agents. Such weak
responses arise, in part, due to the great number of genes and gene
products involved, and the fact that skin-active 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.
[0020] The value of a connectivity map approach to discover
functional connections among cosmetic phenotypes such as
hyperpigmented skin, gene expression perturbation, and cosmetic
agent action is counter-indicated by the progenors of the
drug-based C-map. The relevant phenotypes are very complex, the
genetic perturbations are numerous and weak, and cosmetic agent
action is likewise diffuse and by definition, relatively weak. It
is unclear whether statistically valid data may be generated from
cosmetic C-maps and it is further unclear whether a cell line
exists which may provide salient or detectable cosmetic data.
SUMMARY OF THE INVENTION
[0021] Accordingly, the present inventors have developed a C-map
approach that surprisingly enables discovery of skin tone agents
having efficacy for disorders of skin pigmentation.
[0022] The present inventors discovered that useful connectivity
maps could be developed from cosmetic active--cellular
phenotype--gene expression data associations in particular with
respect to hyperpigmentation actives and cosmetic agents, despite
the highly diffuse, systemic and low-level effects these sorts of
actives generally engender. Although the Lamb team asserted that
results should be substantially independent of cell-type, the
present inventors surprisingly discovered that selection of cell
line affects the utility of the C-map for hypothesis generating and
testing relating to skin pigmentation actives and treatment of
hyperpigmentation disorders. In particular, keratinocyte cells,
rather than melanocyte or melanoma cells, exhibited a more robust
transcriptional profile when treated with skin-lightening agents,
and there was little to no thematic overlap between cell types
treated with the same benchmark skin active agent (shown in Example
8).
[0023] Accordingly, the present invention provides novel methods,
systems and models useful for generating potential new skin-active
agents efficacious for the treatment of skin conditions such as
hyperpigmentation. Through careful selection of cell type, and by
generation of a reference collection of gene-expression profiles
for known skin-active agents and recognized skin disorders, the
present inventors were surprisingly able to create connectivity map
architecture useful for testing and generating hypotheses about
skin-active agents and hyperpigmentation skin disorders.
[0024] The present invention provides embodiments which broadly
include methods and systems for determining relationships between a
skin condition/disorder of interest and one or more skin-active
agents, one or more genes associated with the skin disorder
condition, and physiological themes implicated by the skin
condition and/or affected by a skin-active agent. The inventive
methods may be used to identify skin-active agents without detailed
knowledge of the mechanisms of biological processes associated with
a skin disorder or condition of interest, all of the genes
associated with such a condition, or the cell types associated with
such a condition.
[0025] One aspect of the invention provides methods for
constructing a data architecture for use in identifying connections
between perturbagens and genes associated with skin tone,
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 keratinocyte, fibroblast,
melanocyte and melanoma 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 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 an instance on at least one computer readable medium, wherein
the instance is a keratinocyte, fibroblast melanocyte or melanoma
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 perturbagen of step (a) is different
qualitatively or quantitatively for each instance. In specific
embodiments each instance is repeated twice in C-map testing.
Example 4 illustrates generating a benchmark skin tone agent
signature using fibroblasts to create signatures and using
keratinocytes to create signatures.
[0026] The data architecture may be mined to identify relationships
between perturbagens, genotypes and phenotypes and may also be used
as an in silico tool for generating new actives with potential
efficacy for treatment of a cosmetic condition. The data
architecture may be implemented for this purpose through the use of
a query, which is an input to the C-map wherein the output is based
on connectivity scores to the query. In one embodiment, a method
for implementing the data architecture to identify at least one
putative skin active agent having potential efficacy in treating a
skin pigmentation condition is provided. The method comprises
querying the data architecture with a pigmentation-relevant gene
expression signature, wherein querying comprises comparing the
pigmentation-relevant gene expression signature to each stored cell
instance, and further wherein the pigmentation-relevant expression
signature represents genes differentially expressed in cells
derived from skin affected with a skin hyperpigmentation condition
or genes differentially expressed in cells treated with at least
one benchmark skin active agent having known efficacy in treating a
skin hyperpigmentation condition. Cell instances are derived from a
keratinocyte, fibroblast, melanocyte or melanoma cell line and the
pigmentation-relevant gene expression signature is derived from a
corresponding cell line. Example 1 illustrates development of a
hyperpigmentation condition expression signature.
[0027] Other embodiments are direct to methods for generating a
hyperpigmentation condition gene expression signature for use in
identifying connections between perturbagens and genes associated
with a skin pigmentation condition. Methods comprise: (a) providing
a gene expression profile for a reference sample of human skin
cells not affected with a pigmentation condition; (b) generating a
gene expression profile for at least one sample of human skin cells
from a subject exhibiting the hyperpigmentation 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; and (e)
storing the one or more gene expression signature lists on at least
one computer readable medium.
[0028] Another embodiment provides methods for generating a
benchmark skin pigmentation-modifying gene expression signature for
use in identifying connections between perturbagens and genes
associated with a skin pigmentation condition, the method
comprising: (a) generating a gene expression profile for a human
skin cell sample treated with at least one benchmark skin
pigmentation modifying agent, wherein the benchmark skin
pigmentation modifying agent is suspended in a vehicle composition,
(b) generating a gene expression profile for a human skin cell
sample treated with the vehicle composition; (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; and (e) storing the one or more gene
expression signature lists on at least one computer readable
medium. Gene expression signatures and immobilized arrays of probes
corresponding to the genes constituting the inventive signatures
are also provided.
[0029] In some aspects a single benchmark skin active agent may be
used to generate a benchmark signature (see Example 2) and in other
aspects a composite signature may be generated by treating a cell
sample with more than one agent (see Examples 3, 6, and 7). A
composite signature can be added in two ways: cells can be treated
with each agent separately, the signature can be generated by
comparing regulated genes from all agents (together), looking for
genes regulated in the same direction by all agents; secondarily,
agents can be mixed together prior to treatment of cells. In
another embodiment, a composite benchmark signature may be
generated for a skin-lightening agent (Example 2), and another
generated for a skin darkening agent. The signature for the
skin-lightening agent may be further tweaked by eliminating any
gene from the signature that also appears in the signature of the
skin-darkening agent, regulated in the same direction, or vice
versa. The inventors discovered that such composite signatures are
particularly useful for mining C-map for agents capable of
modifying skin pigment in the desired direction.
[0030] Systems for identifying connections between perturbagens and
genes associated with a skin hyperpigmentation condition are also
provided. The systems comprise: (a) at least one computer readable
medium having stored thereon a plurality of instances, and a skin
hyperpigmentation-relevant gene expression signature, wherein the
instances and the gene expression signature are derived from one of
a human keratinocyte cell, a human fibroblast cell, a human
melanocyte cell, or a human melanoma cell, wherein each instance
comprises an instance list of rank-ordered identifiers of
differentially expressed genes, and wherein the
hyperpigmentation-relevant gene expression signature comprises one
or more gene expression signature lists of identifiers representing
differentially expressed genes associated with a hyperpigmentation
condition or differentially expressed genes associated with a
benchmark skin-lightening agent; (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 a
hyperpigmentation-relevant gene expression signature stored on the
computer readable medium; (ii) comparing the
hyperpigmentation-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.
[0031] A computer readable medium aspect is also disclosed wherein
a data architecture comprising a first digital file is 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
B through P wherein each of the one or more gene expression
signature lists comprises between about 10 and about 400
identifiers. Instructions for reading the digital file may be
included.
[0032] These and additional objects, embodiments, and aspects of
the invention will become apparent by reference to the Figures and
Detailed Description below.
BRIEF DESCRIPTION OF THE FIGURES
[0033] FIG. 1 sets forth pigmentation control targets and some
benchmark skin active agents as depicted in Table A.
[0034] FIG. 2 sets forth identifiers for the genes constituting a
gene expression signature for hyperpigmented skin. Table B sets
forth identifiers for the 200 most significantly up-regulated genes
and Table C sets forth identifiers for the 200 most significantly
down-regulated genes.
[0035] FIG. 3 is a schematic illustration of a computer system
suitable for use with the present invention;
[0036] FIG. 4 is a schematic illustration of an instance associated
with a computer readable medium of the computer system of FIG.
3;
[0037] FIG. 5 is a schematic illustration of a programmable
computer suitable for use with the present invention;
[0038] FIG. 6 is a schematic illustration of an exemplary system
for generating an instance;
[0039] FIG. 7 is a schematic illustration of a comparison between a
gene expression signature and an instance, wherein there is a
positive correlation between the lists;
[0040] FIG. 8 is a schematic illustration of a comparison between a
gene expression signature and an instance, wherein there is a
negative correlation between the lists; and
[0041] FIG. 9 is a schematic illustration of a comparison between a
gene expression signature and an instance, wherein there is a
neutral correlation between the lists.
[0042] FIG. 10 sets forth identifiers as shown in Table D,
Hexamidine Benchmark Signature; 100 up-regulated.
[0043] FIG. 11 sets forth identifiers as shown in Table E,
Hexamidine Benchmark Signature; 100 top down-regulated.
[0044] FIG. 12 sets forth identifiers as shown in Table F, NAG
Benchmark Signature; 39 significantly up-regulated.
[0045] FIG. 13 sets forth identifiers as shown in Table G, NAG
Benchmark Signature, most significantly down-regulated.
[0046] FIG. 14 sets forth identifiers as shown in Table H,
Niacinamide Benchmark Signature, 100 top up-regulated.
[0047] FIG. 15 sets forth identifiers as shown in Table I,
Niacinamide Benchmark Signature, 100 top down-regulated.
[0048] FIG. 16 sets forth identifiers as shown in Table J,
Sepiwhite Benchmark Signature; 100 top up-regulated.
[0049] FIG. 17 sets forth identifiers as shown in Table K,
Sepiwhite Benchmark Signature; 100 top down-regulated.
[0050] FIG. 18 sets forth identifiers as shown in Table L,
Composite "Skin Tone" Benchmark Signature; approximately 40
up-regulated+58 down-regulated.
[0051] FIG. 19 sets forth identifiers as shown in Table M, RA
benchmark signature in tKC cell--200 upregulated.
[0052] FIG. 20 sets forth identifiers as shown in Table N, RA
benchmark signature in tKC, 200 down-regulated.
[0053] FIG. 21 sets forth identifiers as shown in Table O, RA
Benchmark Signature in BJ fibroblasts, 200 up-regulated.
[0054] FIG. 22 sets forth identifiers as shown in Table P, RA
Benchmark Signature in BJ fibroblast, 200 down-regulated.
[0055] FIG. 23 sets in Table Q, Average C-map scores for some
representative potential skin lightening agents with the Retinoic
Acid Keratinocyte RA.sub.--200 Signature.
[0056] FIG. 24 Shows Table R, showing a comparison of the
predictiveness of different C-map signatures for predicting the
activity of compounds in the mouse B16 melanoma cell melanogenesis
assay.
[0057] FIG. 25, Shows Table S, with data outlining the
responsiveness of tert Keratinocytes, melanocytes, and melanoma
cells to skin tone benchmarks.
DETAILED DESCRIPTION OF THE INVENTION
[0058] The present invention will now be described with occasional
reference to the specific embodiments of the invention. This
invention may, however, be embodied 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 the
invention to those skilled in the art.
[0059] 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.
[0060] 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.
[0061] As used herein, the term "cosmetic agent" means any
substance, as well as any component thereof, which may be rubbed,
poured, sprinkled, sprayed, introduced into, or otherwise applied
to a mammalian body or any part 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
dermatologically acceptable carrier suitable for topical
application to skin. 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 skin tissue;
(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 skin tissue and are
discovered, using the provided methods and systems, to induce or
cause at least one previously unknown effect (positive or negative)
on the skin tissue; 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
skin tissue.
[0062] Some examples of cosmetic agents or cosmetically actionable
materials can be found in: the PubChem database associated with the
National Institutes of Health, USA
(http://pubchem.ncbi.nlm.nih.gov); the Ingredient Database of the
Personal Care Products Council
(http://online.personalcarecouncil.org/jsp/Home.jsp); and the 2010
International Cosmetic Ingredient Dictionary and Handbook,
13.sup.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 (URL: http://www.cosmeticsdatabase.com); 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 (URL: http://www.gnpd.com);
and from suppliers of cosmetic ingredients and botanicals.
[0063] 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, panethenol, 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.
[0064] Non-limiting examples of agents herein utilized are
described in detail below, such as for vitamin B3 compounds, N-acyl
amino acid compounds, and retinoid compounds. In some embodiments,
the vitamin B compound is a B3 compound having the formula:
##STR00001##
wherein R is --CONH.sub.2 (i.e., niacinamide), --COOH (i.e.,
nicotinic acid) or --CH.sub.2OH (i.e., nicotinyl alcohol);
derivatives thereof; and salts of any of the foregoing. Exemplary
derivatives include nicotinic acid esters, including
non-vasodilating esters of nicotinic acid (e.g., tocopheryl
nicotinate, myristyl nicotinate). Examples of suitable vitamin
B.sub.3 compounds are well known in the art and are commercially
available from a number of sources (e.g., the Sigma Chemical
Company, ICN Biomedicals, Inc., and Aldrich Chemical Company).
[0065] Some embodiments of the compositions of the present
invention comprise a safe and effective amount of one or more
N-acyl amino acid compounds. The amino acid can be one of any of
the amino acids known in the art. The N-acyl amino acid compounds
of the present invention correspond to the formula:
##STR00002##
wherein R can be a hydrogen, alkyl (substituted or unsubstituted,
branched or straight chain), or a combination of alkyl and aromatic
groups. A list of possible side chains of amino acids known in the
art are described in Stryer, Biochemistry, 1981, published by W.H.
Freeman and Company. R.sup.1 can be C.sub.1 to C.sub.30, saturated
or unsaturated, straight or branched, substituted or unsubstituted
alkyls; substituted or unsubstituted aromatic groups; or mixtures
thereof.
[0066] Preferably, the N-acyl amino acid compound is selected from
the group consisting of N-acyl Phenylalanine, N-acyl Tyrosine,
their isomers, their salts, and derivatives thereof. The amino acid
can be the D or L isomer or a mixture thereof. N-acyl Phenylalanine
corresponds to the following formula:
##STR00003##
wherein R.sup.1 can be C.sub.1 to C.sub.30, saturated or
unsaturated, straight or branched, substituted or unsubstituted
alkyls; substituted or unsubstituted aromatic groups; or mixtures
thereof.
[0067] N-acyl Tyrosine corresponds to the following formula:
##STR00004##
wherein R.sup.1 can be C.sub.1 to C.sub.30, saturated or
unsaturated, straight or branched, substituted or unsubstituted
alkyls; substituted or unsubstituted aromatic groups; or mixtures
thereof.
[0068] A particularly useful compound in the present invention is
N-undecylenoyl-L-phenylalanine. This agent belongs to the broad
class of N-acyl Phenylalanine derivatives, with its acyl group
being a C11 mono-unsaturated fatty acid moiety and the amino acid
being the L-isomer of phenylalanine. N-undecylenoyl-L-phenylalanine
corresponds to the following formula:
##STR00005##
As used herein, N-undecylenoyl-L-phenylalanine is commercially
available under the tradename Sepiwhite.RTM. from SEPPIC,
France.
[0069] Some embodiments of the present invention include retinoid
compounds. As used herein, "Retinoid Compounds" include all natural
and/or synthetic analogs of Vitamin A or retinol-like compounds
which possess the biological activity of Vitamin A in the skin as
well as the geometric isomers and stereoisomers of these compounds.
The Retinoid Compound includes, but is not limited to, retinol,
retinol esters (e.g., C.sub.2-C.sub.22 alkyl esters of retinol,
including retinyl palmitate, retinyl acetate, retinyl proprionate),
retinal, and/or retinoic acid (including all-trans retinoic acid
and/or 13-cis-retinoic acid). In some embodiments, the Retinoid
Compound is retinoic acid. These compounds are well known in the
art and are commercially available from a number of sources, e.g.,
Sigma Chemical Company (St. Louis, Mo.), and Boerhinger Mannheim
(Indianapolis, Ind.). Other Retinoid Compounds which may be useful
herein are described in U.S. Pat. No. 4,677,120, issued Jun. 30,
1987 to Parish et al.; U.S. Pat. No. 4,885,311, issued Dec. 5, 1989
to Parish et al.; U.S. Pat. No. 5,049,584, issued Sep. 17, 1991 to
Purcell et al.; U.S. Pat. No. 5,124,356, issued Jun. 23, 1992 to
Purcell et al.; and Reissue 34,075, issued Sep. 22, 1992 to Purcell
et al.
[0070] As used herein, the term "putative skin active agent" means
a cosmetic agent as herein defined that has shown promise through
preliminary screens as effecting a specific change in skin biology
related to pigmentation but that has not yet been tested for
effectiveness through the methods herein described for constructing
a data architecture for use in identifying connections between
perturbagens and genes associated with skin tone, comprising
[0071] As used herein, the term "skin-active agent" is a subset of
cosmetic agents as defined herein and includes generally any
substance, as well as any component thereof, intended to be applied
to the skin for the purpose of effectuating a treatment of an
undesirable skin condition or disorder, or for achieving a
desirable skin status. Examples relating to skin tone include skin
pigmentation disorders, including disorders of hyperpigmentation,
such as ephelides (freckles), lentigines including age spots (solar
lentigos), post-inflammatory hyperpigmentation, Cafe au lait
macules, Addisons disease and other systemic disease effects,
hemochromatosis, melasma (mask of pregnancy and other hormonal
related pigment disorders) and acanthosis nigricans, as well as
phototoxia and medicinal-induced alternations in pigmentation.
Examples of disorders of hypopigmentation include Vitiligo and skin
trauma-related ablation of melanocytes in circumscribed areas. In
some case a cosmetic consumer merely desires a change in
pigmentation status of skin as determined by some cultural
standard, such as skin lightening among some dark skinned people
and skin darkening or tanning among some light skinned people.
[0072] Although the term "skin tone" is most often thought of with
respect to skin pigmentation and evenness of coloration, "skin
tone" may also include other characteristics of skin that
contribute to a consumer perception of overall tone. For example,
pore size and distribution, and skin texture are also generally
considered attributes of overall skin tone.
[0073] Categorical examples of skin-active agents include skin
pigment modifying agents, steroidal anti-inflammatory agents,
non-steroidal anti-inflammatory agents, pediculocides, sensates,
enzymes, vitamins, hair growth actives, sunscreens, and
combinations thereof. Cosmetic compositions according to the
instant invention may contain skin-active agents.
[0074] Many processes and proteins are known to be involved in the
pigmentary process; there is a wide array of targets against which
to screen for pigmentation control agents. Among the many targets
are inhibitors of melanocyte stimulation (e.g., antioxidants,
anti-inflammatory agents), cell receptor antagonists (e.g.,
alpha-MSH antagonists), inhibitors of melanin synthesis enzymes
(e.g., tyrosinase, TRP-1, TRP-2), inhibitors of melanosome
transport within the melanocyte and transfer to the keratinocyte
(e.g., PAR-2 antagonists), and activators of melanin degradation
within the keratinocyte.
[0075] Skin active agents which modify skin pigmentation are known
in the art. Certain substances may only be considered cosmetic in
severely reduced concentrations since there are side effects which
suggest undesirable systemic activity beyond the cosmetic concern
being addressed. These include hydroquinone, trans-retinoic acid,
and corticosteroids. Generally, a classic target for cosmetic
formulation is inhibition of tyrosinase, the first enzyme in the
conversion of tyrosine to melanin. A wide array of compounds, such
as kojic acid, arbutin, ascorbic acid, ellagic acid, sulfhydryl
compounds, and resorcinols, are effective tyrosinase inhibitors, as
is a more recently discussed deoxy-arbutin. However, since several
of these materials also have other effects, it is difficult to
directly connect a specific mechanism to the observed effect on
pigmentation. Table A provides a short list of the many known
targets and a few agents effective against them.
[0076] Generally, a "benchmark skin active agent" refers to any
chemical, compound, environmental factor, small or large molecule,
extract, formulation, or combinations thereof that is known to
induce or cause a superior effect (positive or negative) on skin
tissue. In accordance with the present invention, agents having
known efficacy in either skin-lightening or skin darkening contexts
are also herein referred to as "Benchmark Agents."
[0077] Non-limiting examples of benchmark skin active agents are
set forth in Table A, along with the corresponding theorized
pigmentation control target.
[0078] Newer benchmark skin active agents include niacinamide and
glucosamine (in particular, its derivative N-acetyl glucosamine
[NAG]), which have recently been shown to be effective in reducing
melanin production in culture. In vitro, glucosamine reduces
production of melanin by inhibiting activation of tyrosinase, while
niacinamide inhibits melanosome transfer from melanocytes to
keratinocytes. Cosmetic moisturizer formulations containing
niacinamide alone are effective in reducing the appearance of
hyperpigmented spots in vivo and the addition of NAG to the formula
yields greater effectiveness. Another benchmark pigmentation
control agent is N-undecylenoyl-L-phenylalanine, which has been
reported to inhibit biding of alpha-MSH to the melanocyte in vitro
and is effective as a component of cosmetic moisturizer
formulations in clinical testing.
[0079] The terms "gene expression signature," and "gene-expression
signature" refer to a rationally derived list, or plurality of
lists, of genes representative of a skin tissue condition or a skin
agent. In specific contexts, the skin agent may be a benchmark skin
active agent or a potential skin agent. Thus, the gene expression
signature may serve as a proxy for a phenotype of interest for skin
tissue. 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
compared to 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.
[0080] 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 a skin condition such as age spots, or
may include a gene expression signature derived from a
physiological process associated with a skin condition. A C-map may
be queried with perturbagens, gene expression signatures, skin
disorders, thematic signatures, or any data feature or combination
of data features or associations that comprise the data
architecture.
[0081] The term "instance," as used herein, refers to data from a
gene expression profiling experiment in which skin cells 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, the
skin cells, and the microarray.
[0082] 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 in the present invention. In
some embodiments, the perturbagen is applied to human cells and the
gene expression data derived from the gene expression profiling
experiment may be stored as an instance in a data architecture.
Human cells in accordance with the invention may be keratinocyte,
melanocyte, fibroblast, or melanoma cells. 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.
[0083] The term "dermatologically acceptable," as used herein,
means that the compositions or components described are suitable
for use in contact with human skin tissue.
[0084] 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).
[0085] 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.
[0086] As used herein, the term "hyperpigmentation gene expression
signature" refers to a gene expression signature derived from gene
expression profiling of a hyperpigmentation condition.
[0087] As used herein, the term "connectivity score" refers to a
derived value representing the degree to which an instance
correlates to a query.
[0088] 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.
[0089] 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.
[0090] 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.; Illumina, Inc.; GE
Healthcare, Inc.; Applied Biosystems, Inc.; Beckman Coulter, Inc.;
etc.
[0091] 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.
[0092] In accordance with one aspect of the present invention,
provided are devices, systems and methods for implementing a
connectivity map utilizing one or more query signatures associated
with a pigmentation or pigmentation-related 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 of full thickness skin biopsies of skin
exhibiting a skin condition 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. An example of a gene expression signature
includes a hyperpigmentation gene expression signature, an example
of which is described more fully hereafter. A query signature may
be derived from transcriptional profiling of a keratinocyte,
fibroblast, melanocyte, or melanoma cell line exposed to benchmark
skin-active agents such as skin-lightening agents. In other
embodiments, the query signature may be a benchmark gene expression
signature wherein a skin-lightening benchmark signature is further
refined by comparing it to a skin-darkening benchmark signature and
genes having similar directional regulation are eliminated. In
further embodiments a cell is treated with more than one benchmark
skin active agent to derive a benchmark composite signature, and in
specific embodiments, the cell is treated with a plurality of
benchmark skin active agents wherein the selected agents include
those acting from different mechanisms known to underpin skin
pigmentation. In other specific embodiments a general benchmark
skin tone signature may be generated by treating a cell with a
plurality of benchmark skin active agents including agents
comprising benchmark skin active agents for skin pigmentation, skin
pore size and distribution, and/or skin texture. These query
signatures may be used singularly or in combination. In specific
embodiments a composite signature has been shown to provide
advantages in predicting gene changes for chemicals affecting tone
versus signatures from single chemicals.
[0093] In accordance with another aspect 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 an epidermal or
dermal cell line, including for example keratinocyte, fibroblast,
melanocyte and melanoma cells. Instances from more complex cell
culture systems may also be used, such as skin organotypic cultures
containing the targeted cell or ex vivo human skin. Instances from
a plurality of cell lines may be used with the present
invention.
[0094] In accordance with yet another aspect of the present
invention, provided are devices, systems and methods for
identification of relationships between a skin condition, e.g. skin
hyperpigmentation condition query signature and a plurality of
instances, where the query signature may be a gene expression
signature or a physiological theme expression signature. 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 skin condition of
interest, leading to the identification of new cosmetic agents for
treating the skin condition or new uses of known cosmetic
agents.
I. Systems and Devices
[0095] Referring to FIGS. 3, 5 and 6, some examples of systems and
devices in accordance with the present invention for use in
identifying relationships between perturbagens, skin pigmentation
conditions, and genes associated with the skin pigmentation
condition will now be described. System 10 comprises one or more of
computing devices 12, 14, a computer readable medium 16 associated
with the computing device 12, and communication network 18.
[0096] The computer readable medium 16, which may be provided as a
hard disk drive, comprises a digital file 20, such as a database
file, comprising a plurality of instances 22, 24, and 26 stored in
a data structure associated with the digital file 20. The plurality
of instances may be stored in relational tables and indexes or in
other types of computer readable media. The instances 22, 24, and
26 may also be distributed across a plurality of digital files, a
single digital file 20 being described herein however for
simplicity.
[0097] The digital file 20 can be provided in wide variety of
formats, including but not limited to a word processing file format
(e.g., Microsoft Word), a spreadsheet file format (e.g., Microsoft
Excel), and a database file format. Some common examples of
suitable file formats include, but are not limited to, those
associated with file extensions such as *.xls, *.xld, *.xlk, *.xll,
*.xlt, *.xlxs, *.dif, *.db, *.dbf, *.accdb, *.mdb, *.mdf, *.cdb,
*.fdb, *.csv, *sql, *.xml, *.doc, *.txt, *.rtf, *.log, *.docx,
*.ans, *.pages, *.wps, etc.
[0098] Referring to FIG. 4, in some embodiments the instance 22 may
comprise an ordered listing of microarray probe set IDs, wherein
the value of N is equal to the total number of probes on the
microarray used in analysis. Common microarrays include Affymetrix
GeneChips and Illumina BeadChips, both of which comprise probe sets
and custom probe sets. To generate the reference gene profiles
according to the invention, preferred chips are those designed for
profiling the human genome. Examples of Affymetrix chips with
utility in the instant invention include model Human Genome HG-U133
Plus 2.0 and HG-U219. A specific Affymetrix chip employed by the
instant investigators is HG-U133A2.0, however it will be understood
by a person or ordinary skill in the art that any chip or
microarray, regardless of proprietary origin, is suitable so long
as the probe sets of the chips used to construct a data
architecture according to the invention are substantially
similar.
[0099] Instances derived from microarray analyses utilizing
Affymetrix GeneChips may comprise an ordered listing of gene probe
set IDs where the list comprises 22,000+IDs. The ordered listing
may be stored in a data structure of the digital file 20 and the
data arranged so that, when the digital file is read by the
software application 28, a plurality of character strings are
reproduced representing the ordered listing of probe set IDs. While
it is preferred that each instance comprise a full list of the
probe set IDs, it is contemplated that one or more of the instances
may comprise less than all of the probe set IDs of a microarray. It
is also contemplated that the instances may include other data in
addition to or in place of the ordered listing of probe set IDs.
For example, an ordered listing of equivalent gene names and/or
gene symbols may be substituted for the ordered listing of probe
set IDs. Additional data may be stored with an instance and/or the
digital file 20. In some embodiments, the additional data is
referred to as metadata and can include one or more of cell line
identification, batch number, exposure duration, and other
empirical data, as well as any other descriptive material
associated with an instance ID. The ordered list may also comprise
a numeric value associated with each identifier that represents the
ranked position of that identifier in the ordered list.
[0100] Referring again to FIGS. 3, 4 and 5, the computer readable
medium 16 may also have a second digital file 30 stored thereon.
The second digital file 30 comprises one or more lists 32 of
microarray probe set IDs associated with one or more
pigmentation-relevant gene expression signatures. The listing 32 of
microarray probe set IDs typically comprises a much smaller list of
probe set IDs than the instances of the first digital file 20. In
some embodiments, the list comprises between 2 and 1000 probe set
IDs. In other embodiments the list comprises greater than 10, 50,
100, 200, or 300 and/or less than about 800, 600, or about 400
probe set IDs. The listing 32 of probe set IDs of the second
digital file 30 comprises a list of probe set IDs representing up,
and/or down-regulated genes selected to represent a skin tone
condition of interest. In some embodiments, a first list may
represent the up-regulated genes and a second list may represent
the down-regulated genes of the gene expression signature. The
listing(s) may be stored in a data structure of the digital file 30
and the data arranged so that, when the digital file is read by the
software application 28, a plurality of character strings are
reproduced representing the list of probe set IDs. Instead of probe
set IDs, equivalent gene names and/or gene symbols (or another
nomenclature) may be substituted for a list of probe set IDs.
Additional data may be stored with the gene expression signature
and/or the digital file 30 and this is commonly referred to as
metadata, which may include any associated information, for
example, cell line or sample source, and microarray identification.
Examples of listings of probe set IDs for a skin hyperpigmentation
gene expression signature, specifically wherein the skin
hyperpigmentation condition is age spots, is set forth in FIG. 2,
Tables B (the 200 most up-regulated genes) and C (the 200 most
down-regulated genes in a skin hyperpigmentation gene expression
signature). In some embodiments, one or more skin hyperpigmentation
condition gene expression signatures may be stored in a plurality
of digital files and/or stored on a plurality of computer readable
media. In other embodiments, a plurality of gene expression
signatures (e.g., 32, 34) may be stored in the same digital file
(e.g., 30) or stored in the same digital file or database that
comprises the instances 22, 24, and 26.
[0101] As previously described, the data stored in the first and
second digital files may be stored in a wide variety of data
structures and/or formats. In some embodiments, the data is stored
in one or more searchable databases, such as free databases,
commercial databases, or a company's internal proprietary database.
The database may be provided or structured according to any model
known in the art, such as for example and without limitation, a
flat model, a hierarchical model, a network model, a relational
model, a dimensional model, or an object-oriented model. In some
embodiments, at least one searchable database is a company's
internal proprietary database. A user of the system 10 may use a
graphical user interface associated with a database management
system to access and retrieve data from the one or more databases
or other data sources to which the system is operably connected. In
some embodiments, the first digital file 20 is provided in the form
of a first database and the second digital file 30 is provided in
the form of a second database. In other embodiments, the first and
second digital files may be combined and provided in the form of a
single file.
[0102] In some embodiments, the first digital file 20 may include
data that is transmitted across the communication network 18 from a
digital file 36 stored on the computer readable medium 38. In one
embodiment, the first digital file 20 may comprise gene expression
data obtained from a cell line (e.g., a fibroblast cell line and/or
a keratinocyte cell line) as well as data from the digital file 36,
such as gene expression data from other cell lines or cell types,
gene expression signatures, perturbagen information, clinical trial
data, scientific literature, chemical databases, pharmaceutical
databases, and other such data and metadata. The digital file 36
may be provided in the form of a database, including but not
limited to Sigma-Aldrich LOPAC collection, Broad Institute C-MAP
collection, GEO collection, and Chemical Abstracts Service (CAS)
databases.
[0103] The computer readable medium 16 (or another computer
readable media, such as 16) may also have stored thereon one or
more digital files 28 comprising computer readable instructions or
software for reading, writing to, or otherwise managing and/or
accessing the digital files 20, 30. The computer readable medium 16
may also comprise software or computer readable and/or executable
instructions that cause the computing device 12 to perform one or
more steps of the methods of the present invention, including for
example and without limitation, the step(s) associated with
comparing a gene expression signature stored in digital file 30 to
instances 22, 24, and 26 stored in digital file 20. In some
embodiments, the one or more digital files 28 may form part of a
database management system for managing the digital files 20, 28.
Non-limiting examples of database management systems are described
in U.S. Pat. Nos. 4,967,341 and 5,297,279.
[0104] The computer readable medium 16 may form part of or
otherwise be connected to the computing device 12. The computing
device 12 can be provided in a wide variety of forms, including but
not limited to any general or special purpose computer such as a
server, a desktop computer, a laptop computer, a tower computer, a
microcomputer, a mini computer, and a mainframe computer. While
various computing devices may be suitable for use with the present
invention, a generic computing device 12 is illustrated in FIG. 5.
The computing device 12 may comprise one or more components
selected from a processor 40, system memory 42, and a system bus
44. The system bus 44 provides an interface for system components
including but not limited to the system memory 42 and processor 40.
The system bus 36 can be any of several types of bus structures
that may further interconnect to a memory bus (with or without a
memory controller), a peripheral bus, and a local bus using any of
a variety of commercially available bus architectures. Examples of
a local bus include an industrial standard architecture (ISA) bus,
a microchannel architecture (MSA) bus, an extended ISA (EISA) bus,
a peripheral component interconnect (PCI) bus, a universal serial
(USB) bus, and a small computer systems interface (SCSI) bus. The
processor 40 may be selected from any suitable processor, including
but not limited to, dual microprocessor and other multi-processor
architectures. The processor executes a set of stored instructions
associated with one or more program applications or software.
[0105] The system memory 42 can include non-volatile memory 46
(e.g., read only memory (ROM), erasable programmable read only
memory (EPROM), electrically erasable programmable read only memory
(EEPROM), etc.) and/or volatile memory 48 (e.g., random access
memory (RAM)). A basic input/output system (BIOS) can be stored in
the non-volatile memory 38, and can include the basic routines that
help to transfer information between elements within the computing
device 12. The volatile memory 48 can also include a high-speed RAM
such as static RAM for caching data.
[0106] The computing device 12 may further include a storage 44,
which may comprise, for example, an internal hard disk drive [HDD,
e.g., enhanced integrated drive electronics (EIDE) or serial
advanced technology attachment (SATA)] for storage. The computing
device 12 may further include an optical disk drive 46 (e.g., for
reading a CD-ROM or DVD-ROM 48). The drives and associated
computer-readable media provide non-volatile storage of data, data
structures and the data architecture of the present invention,
computer-executable instructions, and so forth. For the computing
device 12, the drives and media accommodate the storage of any data
in a suitable digital format. Although the description of
computer-readable media above refers to an HDD and optical media
such as a CD-ROM or DVD-ROM, it should be appreciated by those
skilled in the art that other types of media which are readable by
a computer, such as Zip disks, magnetic cassettes, flash memory
cards, cartridges, and the like may also be used, and further, that
any such media may contain computer-executable instructions for
performing the methods of the present invention.
[0107] A number of software applications can be stored on the
drives 44 and volatile memory 48, including an operating system and
one or more software applications, which implement, in whole or
part, the functionality and/or methods described herein. It is to
be appreciated that the embodiments can be implemented with various
commercially available operating systems or combinations of
operating systems. The central processing unit 40, in conjunction
with the software applications in the volatile memory 48, may serve
as a control system for the computing device 12 that is configured
to, or adapted to, implement the functionality described
herein.
[0108] A user may be able to enter commands and information into
the computing device 12 through one or more wired or wireless input
devices 50, for example, a keyboard, a pointing device, such as a
mouse (not illustrated), or a touch screen. These and other input
devices are often connected to the central processing unit 40
through an input device interface 52 that is coupled to the system
bus 44 but can be connected by other interfaces, such as a parallel
port, an IEEE 1394 serial port, a game port, a universal serial bus
(USB) port, an IR interface, etc. The computing device 12 may drive
a separate or integral display device 54, which may also be
connected to the system bus 44 via an interface, such as a video
port 56.
[0109] The computing devices 12, 14 may operate in a networked
environment across network 18 using a wired and/or wireless network
communications interface 58. The network interface port 58 can
facilitate wired and/or wireless communications. The network
interface port can be part of a network interface card, network
interface controller (NIC), network adapter, or LAN adapter. The
communication network 18 can be a wide area network (WAN) such as
the Internet, or a local area network (LAN). The communication
network 18 can comprise a fiber optic network, a twisted-pair
network, a Tl/El line-based network or other links of the
T-carrier/E carrier protocol, or a wireless local area or wide area
network (operating through multiple protocols such as ultra-mobile
band (UMB), long term evolution (LTE), etc.). Additionally,
communication network 18 can comprise base stations for wireless
communications, which include transceivers, associated electronic
devices for modulation/demodulation, and switches and ports to
connect to a backbone network for backhaul communication such as in
the case of packet-switched communications.
II. Methods for Creating a Plurality of Instances
[0110] In some embodiments, the methods of the present invention
may comprise populating at least the first digital file 20 with a
plurality of instances (e.g., 22, 24, 26) comprising data derived
from a plurality of gene expression profiling experiments, wherein
one or more of the experiments comprise exposing, for example,
keratinocyte cells (or other skin cells such as human skin
equivalent cultures or ex vivo cultured human skin) to at least one
perturbagen. For simplicity of discussion, the gene expression
profiling discussed hereafter will be in the context of a
microarray experiment.
[0111] Referring to FIG. 6, one embodiment of a method of the
present invention is illustrated. The method 58 comprises exposing
a keratinocyte cell to a perturbagen 64. The perturbagen may be
dissolved in a carrier, such as dimethyl sulfoxide (DMSO). After
exposure, mRNA is extracted from the cells exposed to the
perturbagen and reference cells 66 (e.g., keratinocyte cells) which
are exposed to only the carrier. The mRNA 68, 70, 72 may be reverse
transcribed to cDNA 64, 76, 78 and marked with different
fluorescent dyes (e.g., red and green) if a two color microarray
analysis is to be performed. Alternatively, the samples may be
prepped for a one color microarray analysis, and further a
plurality of replicates may be processed if desired. The cDNA
samples may be co-hybridized to the microarray 80 comprising a
plurality of probes 82. The microarray may comprise thousands of
probes 82. In some embodiments, there are between 10,000 and 50,000
gene probes 82 present on the microarray 80. The microarray is
scanned by a scanner 84, which excites the dyes and measures the
amount fluorescence. A computing device 86 may be used to analyze
the raw images to determine the expression levels of a gene in the
cells 60, 62 relative to the reference cells 66. The scanner 84 may
incorporate the functionality of the computing device 86. The
expression levels include: i) up-regulation [e.g., greater binding
of the test material (e.g., cDNA 74, 76) to the probe than the
reference material (e.g., cDNA 78)], or ii) down-regulation [e.g.,
greater binding of the reference material (e.g., cDNA 78) to the
probe than the test material (e.g., cDNA 74, 76)], iii) expressed
but not differentially [e.g., similar binding of the reference
material (e.g., cDNA 78) to the probe than the test material (e.g.,
cDNA 74. 76)], and iv) no detectable signal or noise. The up- and
down-regulated genes are referred to as differentially expressed.
Microarrays and microarray analysis techniques are well known in
the art, and it is contemplated that other microarray techniques
may be used with the methods, devices and systems of the present
invention. For example, any suitable commercial or non-commercial
microarray technology and associated techniques may used. Good
results have been obtained with Affymetrix GeneChip.RTM. technology
and Illumina BeadChip.TM. technology. One illustrative technique is
described in the Examples, "Generally Applicable" methods section.
However, one of skill in the art will appreciate that the present
invention is not limited to the methodology of the example and that
other methods and techniques are also contemplated to be within its
scope.
[0112] In a very specific embodiment, an instance consists of the
rank ordered data for all of the probe sets on the Affymetrix
HG-U133A2.0 GeneChip wherein each probe on the chip has a unique
probe set Identifier. The probe sets are rank ordered by the fold
change relative to the controls in the same C-map batch (single
instance/average of controls). The probe set Identifiers are
rank-ordered to reflect the most up-regulated to the most
down-regulated.
[0113] Notably, even for the non-differentially regulated genes the
signal values for a particular probe set are unlikely to be
identical for the instance and control so a fold change different
from 1 will be calculated that can be used for comprehensive rank
ordering. In accordance with methods disclosed by Lamb et al.
(2006), data are adjusted using 2 thresholds to minimize the
effects of genes that may have very low noisy signal values, which
can lead to spurious large fold changes. The thresholding is
preferably done before the rank ordering. An example for
illustrative purposes includes a process wherein a first threshold
is set at 20. If the signal for a probe set is below 20, it is
adjusted to 20. Ties for ranking are broken with a second threshold
wherein the fold changes are recalculated and any values less than
2 are set to 2. For any remaining ties the order depends on the
specific sorting algorithm used but is essentially random. The
probe sets in the middle of the list do not meaningfully contribute
to an actual connectivity score.
[0114] The rank ordered data are stored as an instance. The probes
may be sorted into a list according to the level of gene expression
regulation detected, wherein the list progresses from up-regulated
to marginal or no regulation to down-regulated, and this rank
ordered listing of probe IDs is stored as an instance (e.g., 22) in
the first digital file 20. Referring to FIG. 4, the data associated
with an instance comprises the probe ID 80 and a value 82
representing its ranking in the list (e.g., 1, 2, 3, 4 . . . N,
where N represents the total number of probes on the microarray).
The ordered list 84 may generally comprise approximately three
groupings of probe IDs: a first grouping 86 of probe IDs associated
with up-regulated genes, a second group 88 of probe IDs associated
with genes with marginal regulation or no detectable signal or
noise, and a third group 90 of probe IDs associated with
down-regulated genes. The most up regulated genes are at or near
the top of the list 84 and the most down-regulated genes are at or
near the bottom of the list 84. The groupings are shown for
illustration, but the lists for each instance may be continuous and
the number of regulated genes will depend on the strength of the
effect of the perturbagen associated with the instance. Other
arrangements within the list 84 may be provided. For example, the
probe IDs associated with the down-regulated genes may be arranged
at the top of the list 84. This instance data may also further
comprise metadata such as perturbagen identification, perturbagen
concentration, cell line or sample source, and microarray
identification.
[0115] In some embodiments, one or more instances comprise at least
about 1,000, 2,500, 5,000, 10,000, or 20,000 identifiers and/or
less than about 30,000, 25,000, or 20,000 identifiers. In some
embodiments, the database comprises at least about 50, 100, 250,
500, or 1,000 instances and/or less than about 50,000, 20,000,
15,000, 10,000, 7,500, 5,000, or 2,500 instances. Replicates of an
instance may be created, and the same perturbagen may be used to
derive a first instance from keratinocyte cells and a second
instance from another skin cell type, such as fibroblasts,
melanocytes, melanoma or complex tissue, for example ex vivo human
skin.
[0116] The present inventors have discovered that instances derived
with a cell type, such as keratinocyte cells, are more predictive
than other cell types when used in combination with a skin
lightening benchmark agent expression signature derived from the
same cell type. In other words, better results are achieved if the
cell type used to generate the query signature is the same as the
instance cell type. While this cell-consistency guide may appear
predictable, what is surprising is that with respect to benchmark
agent signatures the present inventors surprisingly discovered that
certain benchmark skin active agents have greater predictive
efficacy with certain cell types over others. For example, as set
forth in Examples 4 and 5, the present inventors compared Retinoic
Acid benchmark gene expression signatures derived from BJ
fibroblast cells and keratinocyte cells, it was surprisingly
discovered that those derived from keratinocyte cells yield better
results with respect to a potential agent hit list based on
connectivity with the query signature.
III. Methods for Deriving Hyperpigmentation Gene Expression
Signatures
[0117] Some methods of the present invention comprise identifying a
gene expression signature that represents the up-regulated and
down-regulated genes associated with a skin condition of interest,
in particular with skin tone or hyperpigmentation.
[0118] The pathogenesis of a skin pigmentation condition typically
involves complex processes involving numerous known and unknown
extrinsic and intrinsic factors, as well as responses to such
factors that are subtle over a relatively short period of time but
non-subtle over a longer period of time. This is in contrast to
what is typically observed in drug development and drug screening
methods, wherein a specific target, gene, or mechanism of action is
of interest. Due to the unique screening challenges associated with
a skin pigmentation condition, the quality of the gene expression
signature representing the condition of interest can be important
for distinguishing between the gene expression data actually
associated with a response to a perturbagen from the background
expression data.
[0119] One challenge in developing gene expression signatures for
skin tone and pigmentation-related skin disorders is that the
number of genes selected needs to be adequate to reflect the
dominant and key biology but not so large as to include many genes
that have achieved a level of statistical significance by random
chance and are non-informative. Thus, query signatures should be
carefully derived since the predictive value may be dependent upon
the quality of the gene expression signature.
[0120] One factor that can impact the quality of the query
signature is the number of genes included in the signature. The
present inventors have found that, with respect to a cosmetic data
architecture and connectivity map, too few genes can result in a
signature that is unstable with regard to the highest scoring
instances. In other words, small changes to the gene expression
signature can result in significant differences in the highest
scoring instance. Conversely, too many genes may tend to partially
mask the dominant biological responses and will include a higher
fraction of genes meeting statistical cutoffs by random
chance--thereby adding undesirable noise to the signature. The
inventors have found that the number of genes desirable in a gene
expression signature is also a function of the strength of the
biological response associated with the condition and/or the number
of genes needed to meet minimal values (e.g., a p-value less than
about 0.05 or less than about 1.0, or in accordance with applicable
statistical principles) for statistical significance. Hence, what
is considered an ideal number of genes will vary from condition to
condition. When the biology is weaker, such as is the case
typically with cosmetic condition phenotypes, fewer genes than
those which may meet the statistical requisite for inclusion in the
prior art, may be used to avoid adding noisy genes.
[0121] For example, the present inventors have determined that
where gene expression profiling analysis of a skin condition yields
from between about 2,000 and 4,000 genes having a statistical
p-value of less than 0.05 and approximately 1000 genes having a
p-value of less than 0.001, a very strong biological response is
indicated. A moderately strong biological response may yield
approximately 800-2000 genes have a statistical p-value of less
than 0.05 combined with approximately 400-600 genes have a p-value
of less than 0.001. In these cases, a gene expression signature
comprising between about 100 and about 600 genes appears ideal.
Weaker biology may be better represented by a gene expression
signature comprising fewer genes, such as between about 20 and 100
genes.
[0122] While a gene expression signature may represent all
significantly regulated genes associated with a skin condition of
interest; typically it represents a subset of such genes. The
present inventors have discovered that hyperpigmentation gene
expression signatures comprising between about 50 and about 400
genes of approximately equal numbers of up-regulated and/or
down-regulated genes are stable, reliable, and can provide
predictive results. For example, a suitable gene expression
signature may have from about 100-150 genes, 250-300 genes, 300-350
genes, or 350-400 genes. In a very specific embodiment, a
hyperpigmentation gene expression signature includes the 100 most
up- and down-regulated genes. However, one of skill in the art will
appreciate that gene expression signatures comprising fewer or more
genes are also within the scope of the various embodiments of the
invention. For purposes of depicting a gene expression signature,
the probe set IDs associated with the genes are preferably
separated into a first list comprising the most up-regulated genes
and a second list comprising the most down-regulated, as set forth
in FIG. 2, Tables B and C.
[0123] Gene expression signatures may be generated from full
thickness skin biopsies from skin having the skin condition of
interest compared to a control. For generation of an exemplary
hyperpigmentation gene expression signature, biopsies are taken
from forearm age spots and compared to non-affected forearm skin
sampled from the same subject.
[0124] In other embodiments of the present invention, a gene
expression signature may be derived from a gene expression
profiling analysis of keratinocyte, melanocyte, fibroblast or
melanoma cells treated with one or more benchmark skin-active
agents, in particular a skin-lightening agent, to represent
cellular perturbations leading to improvement in the skin tissue
condition treated with that benchmark skin active agent, wherein
the signature comprises a plurality of genes up-regulated and
down-regulated by the benchmark skin active agent in cells in
vitro. As one illustrative example, microarray gene expression
profile data where the perturbagen is the known skin lightening
agent Niacinamide may be analyzed using the present invention to
determine from the rank-ordered instances in the query results, the
perturbagens associated with the highest scoring instances.
[0125] A composite benchmark signature according to the invention
is a signature derived from a cell treated with more than one
benchmark skin active agent (described in Examples 3, 6, and 7).
The actives may be selected to reflect more than one mechanism of
action in skin, or may be selected to reflect more than one
attribute of general skin tone.
[0126] In a further specific embodiment, a benchmark
skin-lightening gene expression signature is compared to a
benchmark skin-darkening gene expression signature and genes not
differentially regulated between the two are eliminated from the
signature intended to be the query signature. Non-limiting examples
of skin-darkening agents according to this embodiment include
alpha-melanocyte-stimulating hormone (a-MSH) and any related
melanocortin 1 receptor agonists or stimulant thereof.
IV. Methods for Comparing a Plurality of Instances to One or More
Pigmentation Gene Expression Signatures
[0127] Referring to FIG. 7 and FIG. 8, a method for querying a
plurality of instances with one or more hyperpigmentation-relevant
gene signatures will now be described. Broadly, the method
comprises querying a plurality of instances with one or more
hyperpigmentation-relevant gene signatures and applying a
statistical method to determine how strongly the signature genes
match the regulated genes in an instance. Positive connectivity
occurs when the genes in the up-regulated signature list are
enriched among the up-regulated genes in an instance and the genes
in the down-regulated signature list are enriched among the
down-regulated genes in an instance. On the other hand, if the
up-regulated genes of the signature are predominantly found among
the down-regulated genes of the instance, and vice versa, this is
scored as negative connectivity. FIG. 7 schematically illustrates
an extreme example of a positive connectivity between signature 90
and the instance 104 comprising the probe IDs 102, wherein the
probe IDs of the instance are ordered from most up-regulated to
most down-regulated. In this example, the probe IDs 100 (e.g.,
X.sub.1, X.sub.2 X.sub.3, X.sub.4, X.sub.5, X.sub.6, X.sub.7,
X.sub.8) of the gene signature 90, comprising an up list 97 and a
down list 99, have a one to one positive correspondence with the
most up-regulated and down-regulated probe IDs 102 of the instance
104, respectively. Similarly, FIG. 8 schematically illustrates an
extreme example of a negative connectivity between signature 94 and
the instance 88 comprising the probe IDs 90, wherein the probe IDs
of the instance are ordered from most up-regulated to most
down-regulated. In this example, the probe IDs of the up list 93
(e.g., X.sub.1, X.sub.2 X.sub.3, X.sub.4) correspond exactly with
the most down-regulated genes of the instance 88, and the probe IDs
of the down list 95 (e.g., X.sub.5, X.sub.6, X.sub.7, X.sub.8)
correspond exactly to the most up-regulated probe IDs of the
instance 88. FIG. 9 schematically illustrates an extreme example of
neutral connectivity, wherein there is no consistent enrichment of
the up- and down-regulated genes of the signature among the up- and
down-regulated genes of the instance, either positive or negative.
Hence the probe IDs 106 (e.g., X.sub.1, X.sub.2 X.sub.3, X.sub.4,
X.sub.5, X.sub.6, X.sub.7, X.sub.8) of a gene signature 108
(comprising an up list 107 and a down list 109) are scattered with
respect to rank with the probe IDs 110 of the instance 112, wherein
the probe IDs of the instance are ordered from most up-regulated to
most down-regulated. While the above embodiments illustrate process
where the gene signature comprises a both an up list and a down
list representative of the most significantly up- and
down-regulated genes of a skin condition, it is contemplated that
the gene signature may comprise only an up list or a down list when
the dominant biology associated with a condition of interest shows
gene regulation in predominantly one direction.
[0128] In some embodiments, the connectivity score can be a
combination of an up-score and a down score, wherein the up-score
represents the correlation between the up-regulated genes of a gene
signature and an instance and the down-score represents the
correlation between the down-regulated genes of a gene signature
and an instance. The up score and down score may have values
between +1 and -1. For an up score (and down score) a high positive
value indicates that the corresponding perturbagen of an instance
induced the expression of the microarray probes of the up-regulated
(or down-regulated) genes of the gene signature, and a high
negative value indicates that the corresponding perturbagen
associated with the instance repressed the expression of the
microarray probes of the up-regulated (or down-regulated) genes of
the gene signature. The up-score can be calculated by comparing
each identifier of an up list of a gene signature comprising the
up-regulated genes (e.g., Tables B, D, F, H and lists 93, 97, and
107) to an ordered instance list, while the down-score can be
calculated by comparing each identifier of a down list of a gene
signature comprising the down-regulated genes (see, e.g., Tables C,
E, G, I and down lists 95, 99, and 109) to an ordered instance
list. In these embodiments, the gene signature comprises the
combination of the up list and the down list.
[0129] In some embodiments, the connectivity score value may range
from +2 (greatest positive connectivity) to -2 (greatest negative
connectivity), wherein the connectivity score (e.g., 101, 103, and
105) is the combination of the up score (e.g., 111, 113, 115) and
the down score (e.g., 117, 119, 121) derived by comparing each
identifier of a gene signature to the identifiers of an ordered
instance list. In other embodiments the connectivity range may be
between +1 and -1. Examples of the scores are illustrated in FIGS.
7, 8 and 9 as reference numerals 101, 103, 105, 111, 113, 115, 117,
119, and 121. The strength of matching between a signature and an
instance represented by the up scores and down scores and/or the
connectivity score may be derived by one or more approaches known
in the art and include, but are not limited to, parametric and
non-parametric approaches. Examples of parametric approaches
include Pearson correlation (or Pearson r) and cosine correlation.
Examples of non-parametric approaches include Spearman's Rank (or
rank-order) correlation, Kendall's Tau correlation, and the Gamma
statistic. Generally, in order to eliminate a requirement that all
profiles be generated on the same microarray platform, a
non-parametric, rank-based pattern matching strategy based on the
Kolmogorov-Smirnov statistic (see M. Hollander et al.
"Nonparametric Statistical Methods"; Wiley, New York, ed. 2, 1999)
(see, e.g., pp. 178-185). It is noted, however, that where all
expression profiles are derived from a single technology platform,
similar results may be obtained using conventional measures of
correlation, for example, the Pearson correlation coefficient.
[0130] In specific embodiments, the methods and systems of the
present invention employ the nonparametric, rank-based
pattern-matching strategy based on the Kolmogorov-Smirnov
statistic, which has been refined for gene profiling data by Lamb's
group, commonly known in the art as Gene Set Enrichment Analysis
(GSEA) (see, e.g., Lamb et al. 2006 and Subramanian, A. et al.
(2005) Proc. Natl. Acad Sci U.S.A, 102, 15545-15550). For each
instance, a down score is calculated to reflect the match between
the down-regulated genes of the query and the instance, and an up
score is calculated to reflect the correlation between the
up-regulated genes of the query and the instance. In certain
embodiments the down score and up score each may range between -1
and +1. The combination represents the strength of the overall
match between the query signature and the instance.
[0131] The combination of the up score and down score is used to
calculate an overall connectivity score for each instance, and in
embodiments where up and down score ranges are set between -1 and
+1, the connectivity score ranges from -2 to +2, and represents the
strength of match between a query signature and the instance. The
sign of the overall score is determined by whether the instance
links positivity or negatively to the signature. Positive
connectivity occurs when the perturbagen associated with an
instance tends to up-regulate the genes in the up list of the
signature and down-regulate the genes in the down list. Conversely,
negative connectivity occurs when the perturbagen tends to reverse
the up and down signature gene expression changes, The magnitude of
the connectivity score is the sum of the absolute values of the up
and down scores when the up and down scores have different signs. A
high positive connectivity score predicts that the perturbagen will
tend to induce the condition that was used to generate the query
signature, and a high negative connectivity score predicts that the
perturbagen will tend to reverse the condition associated with the
query signature. A zero score is assigned where the up and down
scores have the same sign, indicating that a perturbagen did not
have a consistent impact the condition signature (e.g.,
up-regulating both the up and down lists).
[0132] According to Lamb et al. (2006), there is no standard for
estimating statistical significance of connections observed. Lamb
teaches that the power to detect connections may be greater for
compounds with many replicates. Replicating in this context means
that the same perturbagen is profiled multiple times. Where batch
to batch variation must be avoided, a perturbagen should be
profiled multiple times in each batch. However, since microarray
experiments tend to have strong batch effects it is desirable to
replicate instances in different batches (i.e., experiments) to
have the highest confidence that connectivity scores are meaningful
and reproducible.
[0133] Each instance may be rank ordered according to its
connectivity score to the query signature and the resulting rank
ordered list displayed to a user using any suitable software and
computer hardware allowing for visualization of data.
[0134] In some embodiments, the methods may comprise identifying
from the displayed rank-ordered list of instances (i) the one or
more perturbagens associated with the instances of interest
(thereby correlating activation or inhibition of a plurality of
genes listed in the query signature to the one or more
perturbagens); (ii) the differentially expressed genes associated
with any instances of interest (thereby correlating such genes with
the one or more perturbagens, the skin tissue condition of
interest, or both); (iii) the cells associated with any instance of
interest (thereby correlating such cells with one or more of the
differentially expressed genes, the one or more perturbagens, and
the skin tissue condition of interest); or (iv) combinations
thereof. The one or more perturbagens associated with an instance
may be identified from the metadata stored in the database for that
instance. However, one of skill in the art will appreciate that
perturbagen data for an instance may be retrievably stored in and
by other means. Because the identified perturbagens statistically
correlate to activation or inhibition of genes listed in the query
signature, and because the query signature is a proxy for a skin
condition of interest, e.g. a hyperpigmentation condition, the
identified perturbagens may be candidates for new cosmetic agents,
new uses of known cosmetic agents, or to validate known agents for
known uses relevant to the hyperpigmentation condition.
[0135] In some embodiments, the methods of the present invention
may further comprise testing the selected candidate cosmetic agent,
using in vitro assays and/or in vivo testing, to validate the
activity of the agent and usefulness as a cosmetic agent. Any
suitable in vitro test method can be used, including those known in
the art, and most preferably in vitro models developed in
accordance with the present invention. For example, MatTek human
skin equivalent cultures or other skin equivalent cultures may be
treated with one or a combination of perturbagens selected for
mimicry of the skin condition of interest with respect to
regulation of the genes constituting a physiological theme pattern
for the skin condition of interest. In some embodiments, evaluation
of selected agents using in vitro assays may reveal, confirm, or
both, that one or more new candidate cosmetic agents may be used in
conjunction with a known cosmetic agent (or a combination of known
cosmetic agents) to regulate a skin condition of interest.
[0136] Clinical testing can be useful to confirm putative
skin-pigmentation modifying efficacy. Clinical methods include live
expert grading, chromameter, and color image capture and analysis.
A new useful clinical measurement tool in assessing effectiveness
is based on the principle of noncontact SIAscopy.TM., a recently
described method to measure skin melanin content and distribution.
It rapidly captures facial maps of skin chromophores, permitting
determination of the content and distribution of melanin in any
spot or any area of the skin. Clinical testing on various body
sites such as forearm, face, chest, and back have been reported,
and all have utility in evaluating technology. Any thoroughly
controlled clinical evaluation is expensive and therefore
practicality limits testing to only the most promising
candidates.
V. Hyperpigmentation Disorders
[0137] The present invention provides methods for identifying
putative skin active agents for the treatment of pigmentation
conditions and disorders, and in particular those relating to
hyperpigmentation. Main factors in the development of conditions of
hyperpigmentation are exposure to certain environmental conditions
and hormonal changes. In general, the number of active melanocytes
per unit area of skin decreases with age (10-20% decline per
decade), and there are more active melanocytes in chronically
sun-exposed skin than in non-exposed skin. This increased number of
active melanocytes in sun-damaged skin indicates the influence of
chronic UV exposure (e.g., on face, hands, and arms) in stimulating
melanogenic activity. Since chronic UV exposure also alters dermal
fibroblast function in aging skin and since fibroblasts appear to
play a regulatory role in melanin production, dermal damage from
sunlight may contribute to the production of hyperpigmentation in
exposed aging skin.
[0138] Post-inflammatory hyperpigmentation (PIH) results from
inflammation of the skin and disproportionately affects people with
darker skin. Inflammation induced pigmentation is often seen
associated with acne lesions, ingrown hairs, scratches, insect
bites, and surfactant damage. As an example of the latter, exposure
of human forearm skin to the harsh surfactant sodium lauryl sulfate
(SLS) under patch for a few hours will produce erythema within a
day. Over the course of 1-2 weeks after this SLS exposure,
hyperpigmentation will result, particularly in darker skin, but it
will occur even in Caucasian skin. Topical treatment with
anti-inflammatory agents is known to ameliorate this. A
non-limiting example is phytosterol.
[0139] Exposure of skin to sunlight is the most common cause of
skin hyperpigmentation and is increasingly believed that it is a
subset of PIH caused by a post-inflammatory response to UV damage
to skin. The inflammatory response may be the result of an obvious
acute inflammatory event such as sunburn or due to repeated
sub-erythemal exposures to UV. While in the latter, there may not
be visible erythema, histologically, such exposed skin has elevated
inflammatory cell content, yielding a "subclinical" inflammatory
process. This explanation is supported by the fact that topical
treatment with anti-inflammatory agents immediately after UVB
exposure prevents induction of delayed tanning.
[0140] Inflammation may result in hyperpigmentation through several
mechanisms. Among them is direct stimulation of melanocytes by
inflammatory mediators such as IL-1-alpha. Reactive oxygen species
such as superoxide and nitric oxide generated in damaged skin
(e.g., from UV exposure) or released as by-products from
inflammatory cells are also known stimulators of melanocytes.
Additionally, damage induced in epidermal cells can lead to release
of endocrine inducers of pigmentation such as alpha-melanin
stimulating hormone (MSH). The resulting hyperpigmentation induced
by all these effects is adaptive since it appears to provide some
measure of protection against subsequent insult since melanin is
known to have both UV absorption and reactive oxygen species
scavenging capacity.
[0141] The melanin produced during an inflammatory event also can
enter the dermis where it is engulfed by macrophages, producing
"melanophages." These cells are often retained in the upper dermis
for prolonged periods since removal of dermal melanin apparently is
a very slow process. Thus, post-inflammatory hyperpigmentation can
be a very long-lived problem for the skin.
[0142] Solar (Actinic) Lentigos are hyperpigmented spots also known
as lentigines, age spots, and liver spots. They develop as a result
of chronic exposure of skin to UV radiation and occur on
sun-exposed parts of the body (in particular, the hands, arms,
face, upper chest, and shoulders). Chronic exposure of skin to UV
results in chronic inflammation, such as the epidermal endothelin
cascade. The dark appearance of age spots results from excessive
melanin in the region, and may result from overproduction of
melanin in the hyperactive melanocytes, longer retention of melanin
in aging epidermis due to the slower turnover of this tissue layer,
longer retention of melanin in keratinocytes within rete ridges,
and dermal melanin-containing melanophages, which have been
observed histologically to lie beneath the lentigines. There is
reduced wound healing with age at least in part due to reduced
clearance of materials from dermis apparently due to vascular and
lymphatic changes, so that the residence time of melanophages in
dermis may be lengthened in older populations.
[0143] Certain observations suggest that there is a change in the
genetic and phenotypic expression within an age spot as compared to
cells in surrounding non-affected skin. For example, within
lesional lentigo skin, the rete ridges are greatly exaggerated,
extending deeper into the dermis. This deep penetration runs
counter to the general observation of flattening of the convoluted
dermal-epidermal junction with aging, evidenced by the diminution
of the rete ridges. In solar lentigines, the basement membrane is
also perturbed, which likely contributes to melanin entering the
dermis to result in melanophage formation.
[0144] Moreover, the expression levels of several
melanogenesis-associated genes are known to be increased in actinic
lentigos. There is an accentuation of the epidermal endothelin
inflammatory cascade, together with decreased proliferation and
differentiation of lesional keratinocytes. Many of these changes
appear to be permanent since these spots persist even when further
UV exposure is avoided.
[0145] While lentigos appear to be permanent, their melanin content
and thus their intensity will vary seasonally. For example, in
evaluation of women with facial hyperpigmented spots in October
versus December (in Kobe, Japan or Cincinnati, Ohio, USA), there is
a marked reduction in the size of spots over that time period,
suggesting that the lack of continued exposure to sunlight in
winter leads to gradual reduction in melanin production (seasonal
fading) even in hyperpigmented spots. Additionally, in a separate
examination of facial spots in March versus May (in Cincinnati,
Ohio, USA), a marked increase in the size of spots is observed,
consistent with the expected increased pigmentation due to
increased sun exposure in spring (seasonal darkening).
[0146] From a consumer appearance standpoint, hyperpigmented spots
and uneven pigmentation are important in the perception of age. In
a series of studies, facial images were digitally modified to
remove all age-defining textural features (e.g., facial furrows,
folds, lines, wrinkles) leaving only pigmentation as the variable.
Studies have shown that when using naive judge evaluation and
computer image analysis of the facial images, pigmentation features
can contribute to up to 20 years in perceived age of
individuals.
[0147] The hyperpigmentary disorder generally referred to as
melasma is not well understood. It occurs typically as symmetrical
lesions on the face, primarily in darker skin type females at
puberty or later in life. Sunlight exposure is likely a factor in
the development of melasma since it occurs on the face (a
sun-exposed body site) and since the condition worsens in the
summer. Most melasma sufferers have a hypersensitivity to
ultraviolet radiation, and even brief exposures to sunlight can
stimulate hyperpigmentation. There is also a hormonal component,
likely progesterone, since episodes of melasma are often associated
with pregnancy and the use of hormonal birth control. There may
also be an estrogen component since estrogen receptor expression is
increased in melasma.
[0148] In melasma lesions, there is excess melanin present in both
the epidermis and upper dermis, associated with extravascular
macrophages. Since there is only a slight increase in the number of
melanocytes, the abnormality appears to be in function of the skin
cells, in particular, increase expression of factors in
keratinocytes, fibroblasts, and melanocytes of the involved skin.
In contrast to PIH, there is no apparent inflammatory phase
involved in its development. Additionally, there is likely a
genetic compound predisposing individuals to melasma, although the
specific genetic basis for it is not defined.
[0149] The pigmentation process is complex as evidenced
particularly by recent revelations from genomic and proteomic
analysis. There are approximately 1,500 gene products (proteins)
expressed in melanosomes of all developmental stages, with 600 of
them being expressed at any given time, and with 100 of them
apparently unique to the melanosome. Added to this are many other
proteins (membrane-associated, cytoskeletal, transport, etc.)
involved in pigmentation in both the melanocyte and the
keratinocyte, indicating the complexity of the pigmentary process.
While the basic process (e.g., stimulation of melanocytes and
conversion of tyrosine to melanin) is well studied, there are many
regulatory elements that have emerged from recent research involved
in signaling, in the transport of melanosomes within the
melanocyte, and the transfer of melanosomes to the
keratinocyte.
VI. Cosmetic Compositions and Personal Care Products
[0150] Generally, skin-active agents identified for the enhancement
of skin tone or for treatment of pigment-related skin conditions
may be applied in accordance with cosmetic compositions and
formulation parameters well-known in the art. Various methods of
treatment, application, regulation, or improvement may utilize the
skin care compositions comprising skin-active agents identified
according to the inventive methods.
[0151] Skin hyperpigmentation as a cosmetic concern is generally
treated by topical formulation administration, so that
depigmentation is restricted to hyperpigmented areas and normal
skin is left unaffected by the drug.
[0152] Because of the desirability of providing various cosmetic
skin anti-aging benefits to a consumer, it may be beneficial to
incorporate test agents or compounds identified by one or more of
the screening methods described herein into a cosmetic composition
suitable for topical application to skin. That is, it may be
desirable to include the test agent as an ingredient in the
cosmetic composition. In certain embodiments, the cosmetic
composition may include a dermatological acceptable carrier, the
test agent, and one or more optional ingredients of the kind
commonly included in the particular cosmetic compositing being
provided.
[0153] Dermatologically acceptable carriers should be safe for use
in contact with human skin tissue. Suitable carriers may include
water and/or water miscible solvents. The cosmetic skin care
composition may comprise from about 1% to about 95% by weight of
water and/or water miscible solvent. The composition may comprise
from about 1%, 3%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,
55%, 60%, 65%, 70%, 75%, 80%, or 85% to about 90%, 85%, 80%, 75%,
70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or
5% water and/or water miscible solvents. Suitable water miscible
solvents include monohydric alcohols, dihydric alcohols, polyhydric
alcohols, glycerol, glycols, polyalkylene glycols such as
polyethylene glycol, and mixtures thereof. When the skin care
composition is in the form of an emulsion, water and/or water
miscible solvents are carriers typically associated with the
aqueous phase.
[0154] Suitable carriers also include oils. The skin care
composition may comprise from about 1% to about 95% by weight of
one or more oils. The composition may comprise from about 0.1%,
0.5%, 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,
60%, 65%, 70%, 75%, 80%, 85%, or 90% to about 90%, 85%, 80%, 75%,
70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%,
5%, or 3% of one or more oils. Oils may be used to solubilize,
disperse, or carry materials that are not suitable for water or
water soluble solvents. Suitable oils include silicones,
hydrocarbons, esters, amides, ethers, and mixtures thereof. The
oils may be volatile or nonvolatile.
[0155] Suitable silicone oils include polysiloxanes. Commercially
available polysiloxanes include the polydimethylsiloxanes, which
are also known as dimethicones, examples of which include the
DM-Fluid series from Shin-Etsu, the Vicasil.RTM. series sold by
Momentive Performance Materials Inc., and the Dow Corning.RTM. 200
series sold by Dow Corning Corporation. Specific examples of
suitable polydimethylsiloxanes include Dow Corning.RTM. 200 fluids
(also sold as Xiameter.RTM. PMX-200 Silicone Fluids) having
viscosities of 0.65, 1.5, 50, 100, 350, 10,000, 12,500 100,000, and
300,000 centistokes.
[0156] Suitable hydrocarbon oils include straight, branched, or
cyclic alkanes and alkenes. The chain length may be selected based
on desired functional characteristics such as volatility. Suitable
volatile hydrocarbons may have between 5-20 carbon atoms or,
alternately, between 8-16 carbon atoms.
[0157] Other suitable oils include esters. The suitable esters
typically contained at least 10 carbon atoms. These esters include
esters with hydrocarbyl chains derived from fatty acids or alcohols
(e.g., mono-esters, polyhydric alcohol esters, and di- and
tri-carboxylic acid esters). The hydrocarbyl radicals of the esters
hereof may include or have covalently bonded thereto other
compatible functionalities, such as amides and alkoxy moieties
(e.g., ethoxy or ether linkages, etc.).
[0158] Other suitable oils include amides. Amides include compounds
having an amide functional group while being liquid at 25.degree.
C. and insoluble in water. Suitable amides include
N-acetyl-N-butylaminopropionate, isopropyl N-lauroylsarcosinate,
and N,N,-diethyltoluamide. Other suitable amides are disclosed in
U.S. Pat. No. 6,872,401.
[0159] Other suitable oils include ethers. Suitable ethers include
saturated and unsaturated fatty ethers of a polyhydric alcohol, and
alkoxylated derivatives thereof. Exemplary ethers include
C.sub.4-20 alkyl ethers of polypropylene glycols, and di-C.sub.8-30
alkyl ethers. Suitable examples of these materials include PPG-14
butyl ether, PPG-15 stearyl ether, dioctyl ether, dodecyl octyl
ether, and mixtures thereof.
[0160] The skin care composition may comprise an emulsifier. An
emulsifier is particularly suitable when the composition is in the
form of an emulsion or if immiscible materials are being combined.
The skin care composition may comprise from about 0.05%, 0.1%,
0.2%, 0.3%, 0.5%, or 1% to about 20%, 10%, 5%, 3%, 2%, or 1%
emulsifier. Emulsifiers may be nonionic, anionic or cationic.
Non-limiting examples of emulsifiers are disclosed in U.S. Pat. No.
3,755,560, U.S. Pat. No. 4,421,769, and McCutcheon's, Emulsifiers
and Detergents, 2010 Annual Ed., published by M. C. Publishing Co.
Other suitable emulsifiers are further described in the Personal
Care Product Council's International Cosmetic Ingredient Dictionary
and Handbook, Thirteenth Edition, 2006, under the functional
category of "Surfactants--Emulsifying Agents."
[0161] Linear or branched type silicone emulsifiers may also be
used. Particularly useful polyether modified silicones include
KF-6011, KF-6012, KF-6013, KF-6015, KF-6015, KF-6017, KF-6043,
KF-6028, and KF-6038 from Shin Etsu. Also particularly useful are
the polyglycerolated linear or branched siloxane emulsifiers
including KF-6100, KF-6104, and KF-6105 from Shin Etsu. Emulsifiers
also include emulsifying silicone elastomers. Suitable silicone
elastomers may be in the powder form, or dispersed or solubilized
in solvents such as volatile or nonvolatile silicones, or silicone
compatible vehicles such as paraffinic hydrocarbons or esters.
Suitable emulsifying silicone elastomers may include at least one
polyalkyl ether or polyglycerolated unit.
[0162] Structuring agents may be used to increase viscosity,
thicken, solidify, or provide solid or crystalline structure to the
skin care composition. Structuring agents are typically grouped
based on solubility, dispersibility, or phase compatibility.
Examples of aqueous or water structuring agents include polymeric
agents, natural or synthetic gums, polysaccharides, and the like.
In one embodiment, the composition may comprises from about
0.0001%, 0.001%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 5% to about
25%, 20%, 10%, 7%, 5%, 4%, or 2%, by weight of the composition, of
one or more structuring agents.
[0163] Polysaccharides and gums may be suitable aqueous phase
thickening agents. Suitable classes of polymeric structuring agents
include but are not limited to carboxylic acid polymers,
polyacrylamide polymers, sulfonated polymers, high molecular weight
polyalkylglycols or polyglycerins, copolymers thereof,
hydrophobically modified derivatives thereof, and mixtures thereof.
Silicone gums are another oil phase structuring agent. Another type
of oily phase structuring agent includes silicone waxes. Silicone
waxes may be referred to as alkyl silicone waxes which and are
semi-solids or solids at room temperature. Other oil phase
structuring agents may be one or more natural or synthetic waxes
such as animal, vegetable, or mineral waxes.
[0164] The skin care compositions may be generally prepared by
conventional methods such as known in the art of making
compositions and topical compositions. Such methods typically
involve mixing of ingredients in or more steps to a relatively
uniform state, with or without heating, cooling, application of
vacuum, and the like. Typically, emulsions are prepared by first
mixing the aqueous phase materials separately from the fatty phase
materials and then combining the two phases as appropriate to yield
the desired continuous phase. The compositions are preferably
prepared such as to optimize stability (physical stability,
chemical stability, photostability, etc.) and/or delivery of active
materials. The composition may be provided in a package sized to
store a sufficient amount of the composition for a treatment
period. The size, shape, and design of the package may vary widely.
Certain package examples are described in U.S. Pat. Nos. D570,707;
D391,162; D516,436; D535,191; D542,660; D547,193; D547,661;
D558,591; D563,221; 2009/0017080; 2007/0205226; and
2007/0040306.
EXAMPLES
[0165] The present invention will be better understood by reference
to the following examples which are offered by way of illustration
not limitation.
Generally Applicable C-Map Methodology
Generating Instances
[0166] Individual experiments (referred to as batches) generally
comprise 30 to 96 samples analyzed using Affymetrix GeneChip.RTM.
technology platforms, containing 6 replicates of the vehicle
control (e.g., DSMO), 2 replicate samples of a positive control
that gives a strong reproducible effect in the cell type used, and
samples of the test material/perturbagen. Replication of the test
material is done in separate batches due to batch effects. In vitro
testing was performed in 6-well plates to provide sufficient RNA
for GeneChip.RTM. analysis (2-4 .mu.g total RNA yield/well).
[0167] Human telomerized keratinocytes (tKC) were obtained from the
University of Texas, Southwestern Medical Center, Dallas, Tex. tKC
cells were grown in EpiLife.RTM. media with 1.times. Human
Keratinocyte Growth Supplement (Invitrogen, Carlsbad, Calif.) on
collagen I coated cell culture flasks and plates (Becton Dickinson,
Franklin Lakes, N.J.). Keratinocytes were seeded into 6-well plates
at 20,000 cells/cm.sup.2 24 hours before chemical exposure. Human
skin fibroblasts (BJ cell line from ATCC, Manassas, Va.) were grown
in Eagle's Minimal Essential Medium (ATCC) supplemented with 10%
fetal bovine serum (HyClone, Logan, Utah) in normal cell culture
flasks and plates (Corning, Lowell, Mass.). BJ fibroblasts were
seeded into 6-well plates at 12,000 cells/cm.sup.2 24 hours before
chemical exposure.
[0168] All cells were incubated at 37.degree. C. in a humidified
incubator with 5% CO.sub.2. At t=-24 hours cells were trypsinized
from T-75 flasks and plated into 6-well plates in basal growth
medium. At t=0 media was removed and replaced with the appropriate
dosing solution as per the experimental design. Dosing solutions
were prepared the previous day in sterile 4 ml Falcon snap cap
tubes. Pure test materials may be prepared at a concentration of
1-200 .mu.M, and botanical extracts may be prepared at a
concentration of 0.001 to 1% by weight of the dosing solution.
After 6 to 24 hours of chemical exposure, cells were viewed and
imaged. The wells were examined with a microscope before cell lysis
and RNA isolation to evaluate for morphologic evidence of toxicity.
If morphological changes were sufficient to suggest cytotoxicity, a
lower concentration of the perturbagen was tested. Cells were then
lysed with 350 ul/well of RLT buffer containing
.beta.-mercaptoethanol (Qiagen, Valencia, Calif.), transferred to a
96-well plate, and stored at -20.degree. C.
[0169] RNA from cell culture batches was isolated from the RLT
buffer using Agencourt.RTM. RNAdvance Tissue-Bind magnetic beads
(Beckman Coulter) according to manufacturer's instructions. 1 .mu.g
of total RNA per sample was labeled using Ambion Message Amp.TM. II
Biotin Enhanced kit (Applied Biosystems Incorporated) according to
manufacturer's instructions. The resultant biotin labeled and
fragmented cRNA was hybridized to an Affymetrix HG-U133A 2.0
GeneChip.RTM., which was then washed, stained and scanned using the
protocol provided by Affymetrix.
Example 1
[0170] This Example illustrates use of C-map to identify
connections between perturbens and genes associated with a
pigmentation condition, wherein the pigmentation condition is a
hyperpigmentation condition. Specifically an analysis of tissue
from the arms of individuals showing the hyperpigmentation
condition Solar Lentigines (age spots) is compared to an analysis
of tissue from full normal controls, and an expression signature is
created as herein described through specific statistical
comparisons, filtering, and sorting. The expression signature can
then be used to implement a data architecture by providing a C-map
query to identify relationships between perturbens and genes
associated with a hyperpigmentation pigmentation condition.
Deriving a Hyperpigmentation Condition Expression Signature
[0171] RNA isolated from clinical samples was analyzed using the
Affymetrix HG-U133 Plus 2.0 GeneChips, which contain 54,613 probe
sets complementary to the transcripts of more than 20,000 genes.
However, instances in the provided database used were derived from
gene expression profiling experiments using Affymetrix HG-U133A 2.0
GeneChips, containing 22,214 probe sets, which are a subset of
those present on the Plus 2.0 GeneChip. Therefore, in developing
gene expression signatures from the clinical data, the probe sets
were filtered for those included in the HG-U133A 2.0 gene
chips.
[0172] A statistical analysis of the microarray data is performed
to derive a plurality of hyperpigmentation gene expression
signatures which may comprise a statistically relevant number of
the up-regulated and down-regulated genes. In certain embodiments a
hyperpigmentation gene expression signature includes between 10 and
400 up-regulated and/or between 10 and 400 down-regulated genes. In
more specific embodiments a hyperpigmentation gene expression
signature includes the 50 most statistically relevant up-regulated
genes alone or in combination with the 50 most statistically
relevant down-regulated genes. Regulation is determined in
comparison to gene expression in normal cells. [0173] a. Filtering
based on Absent/Margin/Present Calls. This filter creates a list of
potential genes for inclusion in the gene expression signature. For
example, a suitable filter may be that at least 50% of the samples
in one treatment group must have a Present call for each probe set.
Present calls are derived from processing the raw GeneChip data and
provide evidence that the gene transcript complementary to a probe
set that is actually expressed in the biological sample. The probes
that are absent from all samples are likely to be just noisy
measurements. This step is important to filter out probe sets that
do not contribute meaningful data to the signature. For
hyperpigmentation gene expression signatures, the data was filtered
for probe sets with at least 50% Present calls provided by the
Affymetrix MAS 5 software. [0174] b. Filtering According to a
Statistical Measure. For example, a suitable statistical measure
may be p-values from a t-test, ANOVA, correlation coefficient, or
other model-based analysis. As one example, p-values may be chosen
as the statistical measure and a cutoff value of p=0.05 may be
chosen. Limiting the signature list to genes that meet some
reasonable cutoff for statistical significance compared to an
appropriate control is important to allow selection of genes that
are characteristic of the biological state of interest. This is
preferable to using a fold change value, which does not take into
account the noise around the measurements. The t-statistic was used
to select the probe sets in the signatures because it is signed and
provides an indication of the directionality of the gene expression
changes (i.e. up- or down-regulated) as well as statistical
significance. [0175] c. Sorting the Probe Sets. All the probe sets
are sorted into sets of up-regulated and down-regulated sets using
the statistical measure. For example, if a t-test was used to
compute p-values, the values (positive and negative) of the
t-statistic are used to sort the list since p-values are always
positive. The sorted t-statistics will place the sets with the most
significant p-values at the top and bottom of the list with the
non-significant ones near the middle. [0176] d. Creation of the
Gene expression signature. Using the filtered and sorted list
created, a suitable number of probe sets from the top and bottom
are selected to create a gene expression signature that preferably
has approximately the same number of sets chosen from the top as
chosen from the bottom. For example, the gene expression signature
created may have at least about 10, 50, 70, 100, 200, or 300 and/or
less than about 800, 600, 400 or about 100 genes corresponding to a
probe set on the chip. The number of probe sets approximately
corresponds to the number of genes, but a single gene may be
represented by more than one probe set. It is understood that the
phrase "number of genes" as used herein, corresponds generally with
the phrase "number of probe sets."
[0177] An exemplary Hyperpigmentation Condition Signature according
to the invention is provided, wherein the hyperpigmentation
condition is Solar Lentigines (Age Spots). Data is generated from
an arm age spot genomics study, full spot tissue vs. full normal
tissue comparison. Probe selection method for up-regulated probes:
1. mean expression value of spot tissue >200, 2. ratio of
present call of spot tissue chips >=50%, 3. probe is
significantly up regulated, p<0.05, 4. top 50, 100 and 200
probes ranked by p values, are selected respectively. Three
signatures are generated using top up and down regulated probes cut
at 50, 100 and 200, respectively. C-Map hit evaluation: hits are
selected based on their average weight from three signatures. Probe
selection method for down-regulated probes: 1. mean expression
value of normal tissue >200, 2. ratio of present call of normal
tissue chips >=50%, 3. probe is significantly down regulated,
p<0.05, 4. Top 50, 100 and 200 probes ranked by p values, are
selected respectively. The illustrative signature is set forth as
FIG. 2, Tables B and C.
Example 2
[0178] This Example provides support for the use of benchmark
signatures in a C-map query to generate putative agents. The
Example specifically outlines generation of a benchmark skin
pigmentation-modifying gene expression signature. As described
herein, the benchmark skin pigmentation-modifying gene expression
signature was generated using methods such as filtering as
described in Example 1. More specifically, Hexamidine, N-acetyl
glucosamine, Niacinamide, or SEPIWHITE (Sepiwhite is the purported
tradename of the agent known as undecylenoyl phenylalanine) are
applied as described below to tert-Keratinocyte cells to generate
the benchmark skin pigmentation-modifying gene expression
signature.
[0179] A. Hexamidine (hex).
[0180] A tert-Keratinocyte (tKC) cell line is used to conduct the
genomics study with an Affy U133A chip. (a) Probe selection method
for up-regulated probes: 1. mean expression value of hex treated
>200, 2. ratio of present calls of hex treated chips >=50%,
3. up regulated by hex, but down regulated by MSH (a skin darkening
agent), 4. hex treated p<0.05, top 100 probes ranked by p value;
(b) Probe selection method for down regulated probes, 1. mean
expression value of DMSO control >200, 2. ratio of present calls
of DMSO control chips >=50%, 3. down-regulated by hex, but
up-regulated by MSH (a skin darkening agent), 4. hex treated
p<0.05, top 100 probes ranked by p value. The illustrative
signatures are set forth as FIGS. 10 and 11, Tables D and E,
respectively.
[0181] B. N-acetyl-glucosamine (NAG).
[0182] tKC cell line, Affy U133A chip; Probe selection method for
up-regulated probes: 1. mean expression value of NAG treated
>200, 2. ratio of present calls of NAG treated chips >=50%,
3. up regulated by NAG, but down regulated by MSH (a skin darkening
agent), 4. NAG treated p<0.05, 39 probes are selected. Probe
selection method for down-regulated probes: 1. mean expression
value of DMSO control >200, 2. ratio of present calls of DMSO
control chips >=50%, 3. down regulated by NAG, but up regulated
by MSH (a skin darkening agent), 4. NAG treated p<0.05, 43
probes are selected. The illustrative signatures are set forth as
FIGS. 12 and 13, Tables F and G, respectively.
[0183] C. Niacinamide.
[0184] The Niainamide signature was generated by filtering
analogous to that used for hexamidine or NAG was used. The
illustrative signatures are set forth as FIGS. 14 and 15, Tables H
and I, respectively.
[0185] D. Sepiwhite.
[0186] The Sepiwhite signature was generated by filtering analogous
to that used for hexamidine or NAG was used. The illustrative
signatures are set forth as FIGS. 16 and 17, Tables J and K,
respectively.
Example 3
[0187] This Example illustrates use of C-map and the generation of
a signature as described in Example 2; however Example 3 outlines
development of a composite "skin tone" signature where
Niacinaminde, Sepiwhite, NAG, and Hexamidine are used together to
generate a signature. This Example illustrates generation of an
exemplary composite "skin tone" Signature comprised of four
benchmark skin-lightening agents: Niacinamide, Sepiwhite, NAG, and
Hexamidine. Chips Used for Signature Generation: DMSO control chips
used for Signature generation: Conditions for Signature Generation:
1. a probe must have 10% present call among Control chips or
BenchMark chips, 2. The average signal for an up-regulated probe on
the treated chip must be >200, 3. The average signal for a
down-regulated probe on the control chip must be >200, 4. A
probe must be up or down-regulated cross all benchmark chips. Table
Headers: Average signal of all control chips, AvgFC,
AvgSignalTreated: Average signal of all treated chips, Average fold
change, AvgSignalControl. The illustrative signature is set forth
as FIG. 18, Table L.
[0188] This Example supports embodiments outlining how a composite
signature may be generated by treating a cell sample with more than
one agent. As indicated earlier, a composite signature can be added
in two ways: cells can be treated with each agent separately, the
signature can be generated by comparing regulated genes from all
agents (together), looking for genes regulated in the same
direction by all agents; secondarily, agents can be mixed together
prior to treatment of cells. In another embodiment, a composite
benchmark signature may be generated for a skin-lightening agent,
and another generated for a skin darkening agent. The signature for
the skin-lightening agent may be further tweaked by eliminating any
gene from the signature that also appears in the signature of the
skin-darkening agent, regulated in the same direction, or vice
versa. The inventors discovered that such composite signatures are
particularly useful for mining C-map for agents capable of
modifying skin pigment in the desired direction.
Example 4
[0189] This Example illustrates use of C-map and the generation of
a signature. More specifically, the signature was generated through
application of Retinoic acid to fibroblasts and keratinocytes. This
Example illustrates a method for generating a benchmark skin tone
agent signature according to the invention in each of two different
cell types for comparison of the C-map hit evaluations, wherein the
benchmark skin active agent is Retinoic acid ("RA") and the cell
types are (a) fibroblast, and (b) keratinocyte.
(a) Tert-keratinocytes (tKC) RA Signatures. Cells were treated with
1 uM tRA for 6 hr, tested in triplicate, with triplicate DMSO
controls, and analyzed on HG-U133A GeneChips; Signatures were
generated like for BJ fibroblasts, below; the signature
KC_RA.sub.--200 consists of 100 most significant up- and 100 most
significant down-regulated probe sets; the signature
KC_RA.sub.--400 consists of 200 most significant up- and 200 most
significant down-regulated probe sets. (b) Fibroblast RA
Signatures. The selected cell type is BJ fibroblast. Cells were
treated with 1 uM tRA for 6 hr, tested in triplicate, with
triplicate DMSO controls, and analyzed on HG-U133A GeneChips.
Present calls >0 for naive, DMSO and RA (9 samples total). Mean
signal >=200 for DMSO OR RA samples t-test p<0.05; Filtered
for minimum fold change up or down of 1.2; Used log fold change to
establish directionality and sorted up and down lists by t-test p
value. The signature BJ_RA.sub.--200 consists of 100 most
significant up- and 100 most significant down-regulated probe sets;
The signature BJ_RA.sub.--400 consists of 200 most significant up-
and 200 most significant down-regulated probe sets. The
illustrative signatures are set forth as FIGS. 19, 20, 21, AND 22,
Tables M, N, O and P respectively.
Example 5
[0190] This example summarizes representative potential
skin-lightening agents and C-map query results for the benchmark
skin active agent all-trans-retinoic acid according to the
invention. C-map was queried using the Retinoic Acid/Keratinocyte
200 benchmark signature. The average C-map scores for the top
scoring known skin lightening agents are tabled. Retinoic acid had
the highest score of the materials tested because it was used to
generate the signature. The data shown are for teleomerized human
keratinocytes (tKC). Average CMap scores for some skin lightening
agents with the Retinoic Acid Keratinocyte RA.sub.--200 Signature
are shown in FIG. 23, Table Q.
Example 6
[0191] This Example provides evidence of the advantages of using
composite signatures and illustrates clinical affirmation of the
C-map model for predicting efficacy of skin-lightening agents. This
Example supports embodiments related to composite signatures (as
described at the end of Example 3). An illustrative "Skin Tone"
Signature developed from a genomics study using a composite
skin-lightening benchmark agent signature is derived from
Niacinamide, Hexamidine, Sepiwhite, and NAG. The signature is used
to query C-map and generate a list of potential skin-lightening
agents. A top hit, Chlorhexidine Diactate (CD) is entered into
clinical testing for confirmation of efficacy. The control for
clinical efficacy is a 5% Niacinamide+1% Sepiwhite formulation in
the control vehicle.
[0192] Primary endpoints are changes in color spot area fraction
(image analysis) and melanin spot area fraction (NC2) from
baseline. Secondary endpoints are changes from baseline in L*a*b
(color image analysis), mean melanin gray scale (NC2), and melanin
evenness (NC2). Texture area fraction and pore area fraction are
also evaluated to explore impact on other aspects relating to
overall skin tone. Statistical significant superiority to the
vehicle at one of these time points is a project success criteria.
Statistically significant superiority to the high efficacy
benchmark skin active agent to vehicle performance is the clinical
success criteria.
[0193] Study Design: The experimental protocol included a 9-week
(1-week preconditioning & 8-week treatment), randomized,
double-blind, round robin, vehicle-controlled, split-face tone
benefit study. The subject population included 330 Chinese females,
25-55 years old with hyperpigmented spots. 318 subjects completed
the entire study. Pre-conditioning was achieved with application of
Nature Science Deep Purify cleanser and study-specific moisturizer
for a week. Olay Complete SPF 15 UV Moisturizing Lotion is
concomitantly used during pre-conditioning and treatment. 0.5 g
each test product per half-face (forehead to jaw line; .about.4
mg/cm.sup.2) is dosed 2.times. a day (morning/evening). Color SAF
and treatment area, Melanin SAF, gray scale, evenness by NC2, and
additional measurement of Fine Lines and Wrinkle and Texture are by
REAL 3.01A. Data collection points include baseline, and ends of
weeks 4, 6 and 8. The tested hypothesis is that there is no
difference in clinical endpoint versus the benchmark composition
treatment. Study site is Kuntai Clinical Center, Beijing, China and
study time frame is February 2010 (pre-conditioning) to April
2010.
[0194] 0.05% Chlorhexidine Diacetate in SC-99 vehicle (7% glycerin)
was a top connectivity hit using the tone composite benchmark
signature query set forth as Example 3 and is tested for clinical
efficacy with respect to four different tone criteria against a
known high efficacy benchmark composition of 5% Niacinamide and 1%
Sepiwhite in SC-99 vehicle.
Results:
[0195] According to the spot area fraction color test: 0.05% CD
showed significantly fewer spots when compared to vehicle at week
8. In the spot area fraction NC2 test, CD showed a significantly
reduced fraction when compared to the vehicle at both weeks 6 and
8. In the NC2 Melanin evenness test, CD demonstrated superiority to
the vehicle at weeks 6 and 8, and with respect to Basal skin tone,
CD demonstrated superiority at week 8. Surprisingly, CD also
demonstrated superior efficacy in the Pore area and Texture area
fractions when compared to the vehicle at week 8, suggesting that
it is a good candidate for overall skin tone and texture
enhancement.
Example 7
[0196] This Example provides support for the unexpected
effectiveness of composite signature use with the C-map technology,
with this Example illustrating a comparison of expression signature
efficacy in predicting inhibitors; specifically, from 33 various
chemicals identified as melanogenesis inhibitors in the mouse B16
melanoma cell assay (FIG. 24, Table R). The composite signature was
unexpectedly more effective at identifying inhibitors than any of
the individual signatures (such as for Niacinamide, NAG,
Hexamidine, or Sepiwhite). For this analysis C-map hits were
defined as materials occurring in the top 200 instances (from the
same pool of 2266 instances) with a score 0.30. Result Summary of
correctly predicted melanogenesis inhibitors: Composite signature:
20, Niacinamide: 1, NAG: 1, Hexamidine: 2, and Sepiwhite: 9. The
C-map scores shown in Table R are average scores across the
instances of the chemicals. A maximum positive C-map score is 2.0
indicating perfect positive connectivity. The individual materials
do not show perfectly high scores linking to themselves because of
replicate variability, which is more evident for materials with
relatively weak effects on gene expression. Surprisingly, for this
set of materials the composite signature in 3 of 4 cases gave
better scores with the benchmark materials than the individual
benchmark signatures. The process of generating the benchmark
signature may select for the most consistently regulated probe
sets, which may account for this result.
[0197] As indicated in Example 3, this Example (7) supports
embodiments outlining how a composite signature may be generated by
treating a cell sample with more than one agent. As indicated
earlier, a composite signature can be added in two ways: cells can
be treated with each agent separately, the signature can be
generated by comparing regulated genes from all agents (together),
looking for genes regulated in the same direction by all agents;
secondarily, agents can be mixed together prior to treatment of
cells. In another embodiment, a composite benchmark signature may
be generated for a skin-lightening agent, and another generated for
a skin darkening agent. The signature for the skin-lightening agent
may be further tweaked by eliminating any gene from the signature
that also appears in the signature of the skin-darkening agent,
regulated in the same direction, or vice versa. The inventors
discovered that such composite signatures are particularly useful
for mining C-map for agents capable of modifying skin pigment in
the desired direction.
Example 8
[0198] This Example provides support to illustrate that it is
believed that keratinocyte cells, rather than melanocyte or
melanoma cells, have exhibited a more robust transcriptional
profile when treated with skin-lightening agents. Keratinocytes
have been preliminarily shown to be easier to grow than melanocytes
and have increased responsiveness such that keratinocytes may be
able to be used to detect active chemicals over a wider range of
concentrations than testing with melanocytes. More specifically, in
this Example, six skin tone benchmark materials were applied to
each of three cell types (tert-keratinocytes, melanocyes, and
melanoma cells), and with four of six tested materials
tert-keratinocytes showed the greatest response (as indicated in
FIG. 25, Table S which shows that with four of the six tested
materials, the number of probe sets with significant P-values
compared to DMSO controls was greatest for tert-keratinocytes). As
can be seen in Table S, The six tested skin tone benchmark
materials included: Haxamidine diisothionate, Myo-inositol,
N-acetyl-glucosamine, NDP-MSH, Niacinamide, and Sepiwhite. For
completeness, details of the exact types of melanocytes and
melanoma cells, as well as the cell culturing conditions and result
analysis are provided herein below.
[0199] HEMn primary neonatal medium pigment melanocytes were
obtained from Invitrogen, Carlsbad, Calif. and were cultured in
Medium 254 from Invitrogen. HBL melanoma cells were obtained from
the Laboratory of Oncology and Experimental Surgery, Institut
Bordet, Universite' Libre de Bruxelles, Belgium and were cultured
in F-10 Nutrient Mixture (Ham) from Invitrogen supplemented with
10% fetal bovine serum (HyClone, Logan, Utah). Human telomerized
keratinocytes (tert-keratinocytes) were obtained from the
University of Texas, Southwestern Medical Center, Dallas, Tex. and
were grown in EpiLife.RTM. media with 1.times. Human Keratinocyte
Growth Supplement (Invitrogen). All cells were incubated at
37.degree. C. in a humidified incubator with 5% CO2.
[0200] Cells were seeded into 6-well plates a 24 hours before
chemical exposure, and the skin tone benchmark chemicals listed in
the table below were added to culture medium dissolved in DMSO. The
final concentration of DMSO was 0.1%, and cells treated just with
DMSO served as controls. After 6 hours of chemical exposure cells
were then lysed with 350 ul/well of RLT buffer containing
.beta.-mercaptoethanol (Qiagen, Valencia, Calif.), transferred to a
96-well plate, and stored at -20.degree. C. RNA from cell culture
batches was isolated from the RLT buffer using Agencourt.RTM.
RNAdvance Tissue-Bind magnetic beads (Beckman-Coulter, Brea Calif.
92821) according to manufacturer's instructions. 1 ug of total RNA
per sample was labeled using Ambion Message Amp.TM. II Biotin
Enhanced kit (Life Technologies, Grand Island, N.Y. 14072)
according to manufacturer's instructions. The resultant biotin
labeled and fragmented cRNA was hybridized to an Affymetrix
HG-U133A 2.0 GeneChip.RTM., which was then washed, stained and
scanned using the protocol provided by Affymetrix.
[0201] Regarding the results and analysis of the testing: Two
sample t-tests were performed on each treatment to compare with the
DMSO control. The number of probe sets with significant p-values
(<0.05) are summarized in the table below. Each GeneChip.RTM.
contains 22215 probes sets. Using a significance level of 0.05,
1111 probe sets (95% confidence interval of 1047 to 1174) are
expected to be significant by chance alone. This estimate is
somewhat conservative since there may be multiple probe sets for
the same gene.
[0202] In summary, the tert-keratinocytes were generally the most
responsive cells to the skin tone benchmark materials. There were
more significantly regulated probe sets for 4/6 skin tone benchmark
materials in the tert-keratinocytes compared to either HeMnMP
melanocytes or HBL melanoma cells. The tert-keratinocytes have an
additional advantage over the second most responsive cells, HeMnMP
melanocytes, in that they grow substantially faster and are more
practical cell line for routine screening.
[0203] Every document cited herein is hereby incorporated herein by
reference in its entirety unless expressly excluded or otherwise
limited. The citation of any document is not an admission that it
is prior art with respect to any invention disclosed or claimed
herein or that it alone, or in any combination with any other
reference or references, teaches, suggests or discloses any such
invention. Further, to the extent any meaning or definition of a
term in this document conflicts with any meaning or definition of
the same term in a document incorporated by reference, the meaning
or definition assigned to that term in this document shall
govern.
[0204] The values disclosed herein are not to be understood as
being strictly limited to the exact numerical values recited.
Instead, unless otherwise specified, each such value is intended to
mean both the recited value and a functionally equivalent range
surrounding that value.
[0205] The present invention should not be considered limited to
the specific examples described herein, but rather should be
understood to cover all aspects of the invention. Various
modifications, equivalent processes, as well as numerous structures
and devices to which the present invention may be applicable will
be readily apparent to those of skill in the art. Those skilled in
the art will understand that various changes may be made without
departing from the scope of the invention, which is not to be
considered limited to what is described in the specification.
* * * * *
References