US 20130261 006A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2013/0261006 A1 HAKOZAKI et al. (43) Pub. Date: Oct. 3, 2013
(54) METHOD OF GENERATINGA Publication Classification HYPERPGMENTATION CONDITION GENE EXPRESSION SIGNATURE (51) Int. Cl. CI2O I/68 (2006.01) (71) Applicant: THE PROCTER & GAMBLE (52) U.S. Cl. COMPANY, Cincinnati, OH (US) CPC ...... CI2O I/6881 (2013.01) USPC ...... 506/7:506/16 (72) Inventors: Tomohiro HAKOZAKI, Cincinnati, OH (US); Wenzhu ZHAO, Mason, OH (57) ABSTRACT (US); Robert Lloyd BINDER, A method of generating a hyperpigmentation condition gene Montgomery, OH (US); Jun XU, Mason, expression signature for use in identifying connections OH (US) between perturbagens and genes associated with a skin pig mentation condition. The method includes providing a gene Assignee: The Procter & Gamble Company, expression profile for a reference sample of human skin cells (73) not affected with a pigmentation condition; generating a gene Cincinnati, OH (US) expression profile for a sample of human skin cells from a Subject exhibiting the hyperpigmentation condition; compar (21) Appl. No.: 13/851,873 ing the expression profiles to determine a gene expression signature that includes a set of differentially expressed genes; (22) Filed: Mar. 27, 2013 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 Related U.S. Application Data gene expression signature lists; and storing the one or more (60) Provisional application No. 61/618,115, filed on Mar. gene expression signature lists on at least one computer read 30, 2012. able medium. Patent Application Publication Oct. 3, 2013 Sheet 1 of 73 US 2013/026100.6 A1
FIG. : Table A Pigmentation control targets and some Benchmark Agents Pigmentation Control Target Effective Agent Examples Examples Tyrosinase inhibition Hydroquinone, resorcinols, koic acid, arbutin, deoxy-arbutin, ascorbic acid (vitamin C) Tyrosinase copper chelation Ellagic acid, kojic acid Inhibition of tyrosinase Glucosamine, N-acetylglucosamine, tunicamycin Melanosome transfer Niacinamide, protease inhibitors Inhibit binding of alpha-MSH to N-undecylenoyl-phenylalanine melanocyte Down regulation of tyrosinase Retinoid (trans-retinoic acid, retino and its esters, retinaldehyde) Antioxidant Vitamin C compounds, vitamin E, sulfhydryl compounds Anti-inflammatory agent Hydrocortisone, phytosterol, glycyrrhetinic acid, tranexamic Increase epidermal turnover acid,Retinoids, chamomile salicylic extract acid, alpha-hydroxy acids, alpha-keto...... acids,-- adenosine monophosphate Patent Application Publication Oct. 3, 2013 Sheet 2 of 73 US 2013/026100.6 A1
FIG. 2: TABLES B and C
20745 at 214333 x isocitrate dehydrogenase 3 (NAD+) gamma 207365 x a ubiquitin specific peptidase 34
202396 at TCERC ption elongation regulator 1
20358 at LYST sosomal trafficking regulator ochondrial ribosomal protein L34 / Dutt 7U.4 u.uu J asein kinase I, gamma 2 -AL530441 0.
eli division cycle 42 (GTP binding protein, 25kDa) M35543 ansducer of ERBB2, ------v-- ...... v. - - - - - M. v.------W------v- W --w---mus ------v-u------
SRY (sex determining region Y)-box 15 217759 at tripartite motif-containing 44 216859 x 212254s at DST 211464 x at CASP6
206765 at KC 200055 at TAF10 associated factor 30kDa 21 0547 x at ICA .-- -- islet cell autoantigen 1. 69kDa -3- 201471 sat SQSTM1 205555 s at MSX2 212143 NM_002847.0.00396
214715. nc finger protein 160 m------...------m-m-r-...-
kelch domain containin g 4. 8-M.------
testis derived transcript (3 LIM domains) M_015641000474
202720 at 202178 at M_002744 000500 22076 is at TAOK3 NM_016281 000500 : BF439472 000503 Patent Application Publication Oct. 3, 2013 Sheet 3 of 73 US 2013/026100.6 A1
224.54 x Heterogeneous nuclear ribonucleoprotein D-like r protein 24
NM_016623.000539 202468 is - N Ll catenin (cadherin-associated protein), alpha-like 1 ---m 22 1737 at guanine nucleotide binding protein (G protein) alpha 12 '. 0.005 S1 like ( yeast)
homeobox D4 mu------m-, --
upstream binding protein (LBP-1a) sulin-like growth factor binding protein 3 M31159 000592
FKIAA 1598 . . . 'S''' - '''S'm ' protein kinase, AMP-activated, beta 2 non-catalytic subunit eroxisome biogenesis factor l
marylacetoacetate hydrolase domain containing 2B
enase (lipoamide) alpha 1 integrin, beta (fibronectin receptor, beta polypeptide, antigen CO29 includes MDF2, MSK12) BG500301 O.00649. 201227s at NDUFB8 NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 8, 19kDa NM_005040.00652 220602_s at s solute carrier familymm mm. 2 (facilitated glucose/fructose transporter), NM_0250840.00652.
member 5 '' ' ' ' ' ' ' ' m arbonyl reductase 3. poly (ADP-ribose) polymerase family, member 2 guanine nucleotide binding protein-like 3 (nucleolar) line 1 (Opitz/BBB syndrome) hromosome 5 open reading frame 13 -u-m-m-m-m-m-m-r ------golgi associated, gamma adaptin ear containing, ARF binding
protein
asparaginyl-tRNA synthetase etallothionein pseudogene 3 219231 at methylguanosine synthase homolog (S. cerevisiae) 215228 at scient helix loop helix 2
2 9790s at NPR3 peptide receptor C) NM_0009080.00821 ter-rig-r---210424s at GOLGA8A golgi autoantigen, golgin subfamily a 8A AF163441 000827. 210424s at GOLGA8B golgi autoantigen, golgin subfamily a, 8B AF16344 0.00827. sema domain, immunoglobulin domain (g), transmembrane domain (TM) and short cytoplasmic domain. (semaphorin) 4C M_017789 000835 slocated promoter region (to activated MET oncogene) 'WWW K0231.
209306 s a SWAP70. SWAP-70 protein------A1139569------000837 Patent Application Publication Oct. 3, 2013 Sheet 4 of 73 US 2013/0261006 A1
ise (STE24 homolog, S. cerevisiae) 857 000853. 0.00858
c fin ger protein 57
pindlin family, member 2B indlin family, member 2A F356353 000886.
sprouty homolog4 (Drosophila) WA8843 0.00898 zinc finger and BTB domain containing 1 0.00899 ctin filament) muscle ine, alpha -
apoptotic chromatin condensation inducer 1
t CCDC101 coiled-coil domain containing 101 OX4 -- SRY (sex determining region Y)-box 4. BG528420 000
201810 sat SH3BP5 SH3-domain binding protein 5 (BTK associated) AL562152 000931 221 188s at CIDEB cell death-inducing DFFA-like effector b 213140 s at SS18Ll ----- synovial sarcoma translocation gene on chromosome 18-like 1 i 218612 s at TSSC4 - tumor suppressing subtransferable candidate 4 - transcription factor AP-2 gamma (activating enhancer binding i 205286 at protein 2 gamma) U85658 ------(),00978
let cell autoantigen 1 , 69kDa w- V 004968 0.00978 tyrosine 3-monooxygenase/tryptophan 5-monooxygenase
200639_s_at YWHAZ tactivation protein, Zeta polypeptide 201804 x at TBCB tubulin folding cofactor B
actor VIII associated (intronic transcript) 220051 at PRSS21 protease, serine, 21 (testisin) 21.0153 s at ME2 malic enzyme 2, NAD(+)-dependent, mitochondrial 202123 s at bl Abelson murine leukemia viral oncogene homolog 1 20701 lSat PTK7 protein tyrosine kinase 7
r scaffold homolog (S. cerevisiae) NM_015700 001083
polymerase (DNA directed), gamma 2, accessory subunit NM_007215 001108 - - omain containing 4B 01 126
sema domain, immunoglobulin domain (Ig), short basic domain, secreted, (semaphorin) 3F , ticulocalbin 3, EF-hand calcium binding domain ------desmocollin 2
domain family. member 2 protocadherin 7 Patent Application Publication Oct. 3, 2013 Sheet 5 of 73 US 2013/0261006 A1
homeobox D1
ondin 2, extracellular matrix protein dicarbonyl/L-xylulose reductase NM_016286001188. leoporin 43kDa NM_0246470.01 195
222136 x at ZNF43 zinc finge ------217752 s at CNDP2 CNDP dipeptidase 2 (metallopeptidase M20 family) 204078------at SC65 ------synaptonemal complex - protein------SC65 microtubule associated monoxygenase, calponin andLIM domain ----...- ...-- | ------218376 sat MICAL1 containing NM_022765 0.01.237
208820207060 at PTK2EN2 engrailedPTK2 protein homeobox2 tyrosine kinase 2 - A057,3900279M001427. ---202265 ------at BMI1 polycomb ring finger oncogene ... --. zinc finger protein 419 219826------at M_02469100.335
RRP9, small subunit (SSU) processome component, homolog (yeast) - M-004704001341 rf34 chromosome I open reading frame 34 C004399 001353 221974 at - IIIPW Imprinted in Prader-Willi syndroine w770748 00135 amyloid beta (A4) precursor protein-binding, family A, member 210720s at APBA2BP binding protein AB039947 0.01365; 204525 at PHF14 PHD finger protein 14 NM_014660 001366 rimatogenesis associated
g protein 1 (liprin beta1) 200775 s at HNRPK heterogeneous nuclear ribonucleoprotein K i golgi associated, gamma adaptin ear containing, ARF binding GAI : protein RNA, cDNA DKFZp547C126 (from clone DKFZp547C126). AL3595 215828 ------...-as------.
211609 x SMD4 proteasome (prosone, macropain)26S subunit, non-ATPase, 4, U5 1007 0.01444------43977 at TMEM161A transmembrane protein 161A Al660497 (0.01453
218783 INTS7 gratorwr-in-its------...------|x-c------east-user?:-----...-au------was--wa?------issi-ell-----es-is------complex subunit 7 AL133049 001 - tAB002391 0.01470
Pase, Class I, type 8B, member 1
osin VA (heavy chain 12, myoxin) complement component 2 T --- 200001 at calpain, small subunit 1 - 216973 s at HOXB7 homeobox B7 ------65588 at LoC388796 hypothetical LOC388796 IAA827892 001552. 214092 x at SFRS14 splicing factor, arginine/serine-rich 4. ------...... ' ... ------A1928.127 0.01565, 208994s at PPIG 638762 0.01572 202047 s at CBx6 A458128 0.01595. Patent Application Publication Oct. 3, 2013 Sheet 6 of 73 US 2013/0261006 A1
212977 at 2086l3_s_at FLNB lamin B, beta (actin binding protein 278)
osphoribosylglycinamide formyltransferase. -- osphoribosylglycinamide synthetase, 210005 at GART D32051 218095 is: NM_018475 0.01705
212911 at DNAJC16 DnaJ (Hsp40) homolog, subfamily C, member 16 ----AB023179 ------,n-,----. 0.01706 218984 at PUS7 pseudouridylate synthase 7 homolog (S. cerevisiae) NM_0190420.01712, 124; 0.0172 casein kinase 1, epsilon 202332 at ------8940.01741 -: open reading frame 138 ----- 218940------at ------245580.01752------2021 15 s at NOC2L nucleolar complex associated 2 homolog (S. cerevisiae) M 01 56580.01762 203468 at CDK10 cyclin-dependent kinase (CDC2-like) 10 ------NM_003674-001774 01804 14
y : 0.01829.
A767436------0.01831 :
cathepsin B M_001908001846.
motor neuron and pancreas homeobox i A738662 0.01868
213359 at HNRPD RNA binding protein .37kDa) W74620 ------w-us001887. 220668_s_at DNMT3B DNA (cytosine-5-)-methyltransferase 3beta NM_006892001902 214218s at XIST x (inactive) specific transcript ------ul 212237 at ASXLI additional sex combs like I (Drosophila) 214123 s at C4orf10
Y - Y------0.01935------214787 at DENND4A DENN/MADD domain containing 4A 0.01955; protein tyrosine phosphatase, receptor type, f polypeptide - i 210235_s at PPFIAl (PTPRF), interacting protein (liprin), alpha I U22815 0.01961 211729 x at BLVRA biliverdin reductase A BC005902
solute carrier family 11 (proton-coupled divalent metalion
osphatase, non-receptor type 18 (brain-derive
methionyl-tRNA synthetase inhibited by benzinidazole ----- actor 4, p.1077p130 --
212761 at ------anscription factor------7-like 2 (T-cell------specific, HMG-box) A1949687 0.02051
202528 at GALE UDP-galactose-4-epimerase NM_000403.002056. Patent Application Publication Oct. 3, 2013 Sheet 7 of 73 US 2013/026100.6 A1
rnithine decarboxylase antizyme 1 sterol binding protein-like A 201850 at capping protein (actin filament), gelsolin-like 208.806 at CHD3 chromodomain helicase DNA binding protein 3 ir go--- abaptin, RAB GTPase binding effector protein 2 chemokine (C-X-C motif) ligand 14 206377 at 203083 at NM 2022.4521 7805 at LSS sterol synthase (2,3-oxidosqualene-lanosterol cyclase) Awos451
201401 s at ADRBK1 adrenergic, beta, receptor kinase v-maf musculoaponeurotic fibrosarcoma oncogene homolog F - i 205193 at MAFF (avian) NM_012323 0.00006 21622--- m----au------s at PUM2 pumiliopum homolog 2 (Drosophila) D87078 0.00011 207206 S
RAB40A, member RAS oncogene family 217797 at uitin-fold modifier conjugating enzyme
201288 at ARHGDB Rho GDP dissociation inhibitor (GDI) beta 219416 at SCARA3 scavengerScavenger receptor class A, member 3 220892 s a osphoserine aminotransferase I 00053 X-ray repair complementing defective repair in Chinese hamster 208643 s at XRCC5 cells 5 (double-strand-break rejoining; Ku autoantigen, 80kDa) ----- 0.00065 218841 at ASB8 ankyrin repeat and SOCS box-containing 8 ------...- ' hosphate isomerase 1 201777 s at KIAA0494 KIAA0494 205568 at NM 0209800, 212165 at AF070537 21265 at 2094.57 at 213764 sat MFAP5 microfibrillar associated protein 5 212588 at PTPRC protein tyrosine phosphatase, receptor type, C Yo0062 000143 Patent Application Publication Oct. 3, 2013 Sheet 8 of 73 US 2013/026100.6 A1
213030s at 219627 at ZNF767 zinc finger family member 767 202776 a oxynucleotidyltransferase, terminal, interactin g protein 2 - -- vacuolar protein sorting 13 homolog A (S. cerevisiae) ''S'-- nuclear receptor subfamily 2, group C, member 2 bonuclease, RNase A family, 4
cyclic nucleotide gated channel alpha 1 ubiquinol-cytochrome creductase complex (7.2 kD) NM_013387 000185 somal protein L13a
ariadne homolog, ubiquitin-conjugating enzyme E2 binding 208485 x at 212433 x at RPS2 208258 is at growth arrest specific 2 like 1 - 21 6304 xat iYME1-like 1 (S. cerevisiae) ... 'W' S'm
201572------x- x 218191 s at LMBRD1 211654 x at HLA-DQB1 203801 at wp-coro------orMRPS1.4 208370_s_at 204502 at SAMHD AM domain and HD domain 0.00291 210299 sat FHL our and a half LIM domains 1 v-erb-b2 erythroblastic leukemia viral oncogene homolog 3 i 202454s at W.(avian) W.W. NM_001.98
Notch homolog 2 (Drosophila) AF308601 000307 217837_s acuolar protein sorting 24 homolog ( S. cerevisiae) X 205883 at
203000 at STMN2 - BF967657 000327 205504 at NM00006100033 202294 at 206114 at
213549 at PD DK5 regulatory subunit associated protein 1-like 1 poly(rc) binding protein 2 exocyst complex component 2 protein tyrosine phosphatase, non-receptor type 1 interleukin 1, beta
dCMP deaminase icle-associated membrane protein 3 (cellubrevin) 20714 at lymphocyte antigen 6 complex, locus G6C 202371 at TCEAL4 transcription elongation factor A (SID-like 4 21 7977 at selenoprotein X, Patent Application Publication Oct. 3, 2013 Sheet 9 of 73 US 2013/026100.6 A1
ring finger protein 5
to CTAGE family, member 5
protein 44-like 216996 s at FASTKD2 FAST kinase domains 2 205590 at RASGRP S guanyl releasing protein (calcium and DAG regulated)
adaptor protein, phosphotyrosine interaction. PH domain and 218218 at APPL2 leucine zipper containing 2 21287s_s_atc2lorf25 chromosome 21 open reading frame 25 ydroxysteroid (1 1-beta) dehydrogenase ...... S...... W.
ondrial proces
adaptor-related protein complex 3, beta 1 subunit
inding protein 3 ... S. ------210375 at PTGER3 prostaglandin E receptor 3 (subtype EP3) 209373 at mal, T-cell differentiation protein-like
65 000506 209566 at AL080184 000512 Inhibitor of DNA binding 4 dominant negative helix-loop-helix 209292 at protein 202445 sat ------Ju-J.------Notch homolog 2 (Drosophila) 212573 at group D, member
thyroid hormone receptor, alpha (erythroblastic leukemia viral (v- erb-a) oncogene homolog, avian) X72631 0.00534: ' prostaglandin-endoperoxide synthase 1 (prostaglandin G/H 205128 x_at PTGS synthase and cyclooxygenase) NM_009620.00543 206628 at SLC5A1 solute carrier family 5 (sodium/glucose cotransporter), member NM_000343 000557. 204718 : EPH receptor B6 R ----8 v-x '8w - 8X -- 216245 at terleukin receptor antagonist 212438 215342 s at RABGAP1L RAB GTP
206265 sat GPLD1 w density lipoprotein receptor-related protein 10
ho GTPase activating protein 25 -M uu------mud---au------uclear factor (erythroid-derived 2-like 3. 213531 is at RAB3GAP1 RAB3 GTPase activating protein subunit I (catalytic) 212326 at VPS 3D vacuolar protein sorting 13 homolog D (S. cerevisiae) 0.0059 Patent Application Publication Oct. 3, 2013 Sheet 10 of 73 US 2013/026100.6 A1
204454 at LDOC1 210954 s at TSC22D2 204782 at 212716 s at EIF3K eukaryotic translation initiation factor 3, subunit K. 216570 x at LOC64647 217954s at PHF3 213497 at 212738 at
218034 at FIS1 NM 016068 000716. 202995 s at FBL M_006486.000721 214719 at is lute carrier family 46, member 3 217272 s at SERPINB13 serpin peptidase inhibitor, clade B (ovalbumin), member 13 AJ001698 208,577 at histone cluster 1, H3c NM_0035310.00748
iLRRC23
205121 at SGCB coglycan, beta (43kDa dystrophin-associated glycoprotein) NM_0002320.00772---
217485 x at PMS2L D38435 000775 202959 at M methylmalony Coenzyme A mutase 213446s at IQGAP1 IQ motif containing GTPase activating protein 205898 at CX3CRI chemokine (C-X3-C motif) receptor 207143 at CDK6 cyclin-dependent kinase 6 212376 s at EP400 E1A binding protein p400 prostaglandin-endoperoxide synthase 1 (prostaglandin GiH 215813_s_at PTGS1 synthase and cyclooxygenase) 218820 at --mmomosome 'm' 14-m-- open reading------...-m-----...-- frame 32 ------...--...----...-...-...------...-- 218495 at ubiquitously-expressed transcript - 218736 sat palmdelphin - 21206 at 218266 s at FREQ frequenin homolog (Drosophila) 212135 s ------a ATPase, Cah transporting, plasma membrane 4 212468 at perm associated antigen 9 UTP18, small subunit (SSU) processome component, homolog 203721s at UTP18 (yeast) NM_0160010.00890. Patent Application Publication Oct. 3, 2013 Sheet 11 of 73 US 2013/0261006 A1
2 10790s at ARIA SAR1 gene homolog A (S. cerevisiae) 203687 at enokine (C-X3-C motif) ligand 212649 at DHX29 DEAH (Asp-Glu-Ala-His) box polypeptide 29 isocitrate dehydrogenase 3 (NAD+) beta 210418 s at IDH3B dihydrolipoamide S-acetyltransferase (E2 component of pyruvate 21 2568 s at DLAT dehydrogenase complex) -- ale-e anced an 1 -- - - , , ,------203045 at NINJ ninjurin - - - - NM_004 1480.00940 202675 at SDHB succinate dehydrogenase complex, subunit B, iron sulfur (Ip) NM_0030000.00943 CFLAR CASP8 and FADD-like apoptosis regulator ORO2A coronin, actin binding protein, 2A B-cell translocation gene I. anti-proliferative AL535380 galactoside ding, soluble, 9 (ga NM_00958 ------solute carrier family 25 (mitochondrial carrier, adenine nucleotide 200657 at SLC25A5 translocator), member 5 NM_001 1520.00990 208066 s at GTF2B 'a-t------ou--au-Lux-.general transcription factor IB NM_0015140.00994 nuclear factor of activated T-cells, cytoplasmic, calcineurin... ii., --- 210555_s_at NFATC3 dependent 3 U85430 ma-a-a-a-was-Ya------,218143 s at SCAMP2 mbrane protein 2 ------. 211596 s at immunoglobulin-like domains 1 203989 x at F2R coagulation factor II (thrombin) receptor NM_001992.001012
213455 at i. AM114A1 family with sequence similarity 14, member AI w87466 0.01032 207018 is at RAB27B, member RAS oncogene family -- NM_0041630.01 209877 at SNCG Synuclein, gamma (breast cancer-specific protein 1) AFO 10126 0.01.039 210785 S at Clorf38 chromosome 1 open reading frame 38 AB035482 0.01046 platelet-activating factor acetylhydrolase, isoform Ib, alpha ;
2008 16s at AFAH1B1 subunit 45kDa ------NM 000430 00107 202992 a mplement component 7 - M 0005870.01081 22787 at chromosome 6 open reading frame 120 BF431618 001082 orf106 chromosome 1 open reading frame 106 --- NM_0182650.01 107 factor Y, alpha 217833w-ren-wox------or-out-cross-ov-----. at SYNCRIP synaptotagmin binding, cytoplasmic RNA interacting------protein AL520908 001109- s aldehyde dehydrogenase 5 family, member Al (succinate : semialdehyde dehydrogenase) AL031230 001110 vav 3 guanine nucleotide exchange factor ------NM_006. 1300
fibronectin leucine rich transmembrane protein 2
ivisinin-like period homolog 201988 s at CREBL2 cAMP responsive element binding protein-like 2 BF438056 Patent Application Publication Oct. 3, 2013 Sheet 12 of 73 US 2013/0261006 A1
213400 s at TBLIX transducin (beta)-like IX linked 213386 at C9orf125------s-s-s-s------chromosome 9 open reading frame 125
202429s------...... u. at PPP3CA isoform . . . 220797 at METT10D methyltransferase 10 domain containing 220413 at SLC39A2 solute carrier family 39 (zinc transporter), member 2 NM_014579 001230
guanine nucleotide binding protein (G protein), alpha 15 (Gq NM_002068 0.01.238 GNA15 class) ------art tumor necrosis factor, alpha-induced protein 8 C005352------0.01 par-3 partitioning defective 3 homolog (C. elegans) AF196185 0.01244. ------r-rrorleukotriene A4 hydrolase J02959 0.01262 MID1 interacting protein 1 (gastrulation specific G12 homolog 218251 at MIDIIPI (zebrafish) NM_0212420.0262: 202022 at ALDOC aldolase C, fructose- NM_0051650,01265
207178s at FRK fyn-related kinase ------NM_0020310.01277
203240 at FCGBP Fc fragment of IgG binding protein
308857y Patent Application Publication Oct. 3, 2013 Sheet 13 of 73 US 2013/026100.6 A1
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Patent Application Publication Oct. 3, 2013 Sheet 16 of 73 US 2013/026100.6 A1
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FIG. 10: Table D chmark Signature; 100
201689 sat TPD52 203370_s at PDLIM7 PDZ and LIM domain 7 (enigma) ------phosphatidylinositol glycan anchor biosynthesis, class A (paroxysmal nocturnal hemoglobinuria) NM_002641 00 FAR CASP8 and FAD -like apoptosistre. regulator ...... AF005775...... 0.0. D24 CD24 molecule M58664 0.0 204214 s at RAB32 RAB32, member RAS oncogene family NM_006834 00 vating transcription factor 3 NM_001674 -:
NM0022280,0000 osphoribosyl pyrophosphate synthetase 2016 17 x at aldesmon I NM_0043420.00002 209091s at SH3GLBI SH3 domain GRB2-like endophilin B1 AF263293 000003 201690s at TPD52 tumor protein D52 AA524023 000003 209344 at TPM4------...------s-s tropomyosin 4 BC002827 000005 : pleckstrinhomology domain containing, family C (with FERM domain) member 1 0.00005 acyl-Coenzyme A binding domain containing 3 220327 at vestigial like 3 (Drosophila) 205525 at CALDI caldesmon I NM_0184950.00007 at COI 4A6 collagen, type IV, alpha 6 -...------8 205992_s_at IL15 interleukin 15 ------e, macropain) activator subunit 4
------s association (RalGDS/AF-6) domain family 0.00011
at shock 70kDa protein 1B NM_0053450.00011
------'at shock 70kDa protein IA --- or--rouvas work
20475 x_at NM_0049490,00014
221994 at PDLIM5 PDZ and LIM domain 5 AA196325 000015 209028 is at ABI interactor 1 202147 s at IFRD1 terferon related developmental regulator 1 221 12 Syntaxin 12 ------eat shock 70kDa protein IB 201058 s at MYL9 myosin, light chain.9, regulatory ...----...------205020 s at ARLAA ADP-ribosylation factor-like 4A 22002) at TMC7 transmembrane channel-like 7 208960s at KLF6 --- Kruppel-like factor 6 -, - . . . . . ------21 8303.x a lysine-rich coiled-coil 1 207517 at
21676 s at IFNGR1 interferon gamma receptor I 212373 at FEMIB fem-1 homolog b (C. elegans) Patent Application Publication Oct. 3, 2013 Sheet 21 of 73 US 2013/0261006 A1
related RAS viral (r-ras) oncogene homolog noncogene
203303 at DYNLT3 dynein, light chain, Tctex-type 3
IFRD terferon-related developmental regulator 21269 s at SH3BGRL3 SH3 domain binding glutamic acid-rich protein like 3 phosphodiesterase 6D, cGMP-specific, rod, delta 207574 sat GADD45B growth arrest and DNA-damage inducible, beta 202552 s at CRIM1 cysteine rich transmembrane BMP regulator (chordin-like) ;
219290 x at DAPP dual adaptor of phosphotyrosine and 3-phosphoinositides 204485 s : target of mybi (chicken)-like 1 212099 at RHOB ras homolog gene family, member B AI263909 208394 x at ESMI endothelial cell-specific molecule NM0070360. 212763 at odulin regulated spectrin-associated protein 1-like 1 AW59321 202375 at . SEC24 related gene family, member D (S. cerevisiae) chromatin modifying protein 2B
myristoylated alanine-rich protein kinase C substrate actin, gainina 2, Smooth muscle, enteric Corf164-sata-ra-'sa-va-valurua-e-r-ul----Laurus-au-uawa-e-...-es---a-a-a-a--a chromosome 1 open reading ------frame 164 SLC35D2 solute carrier family 35, member D2 208456_s_at RRAS2 related RAS viral (r-ras) oncogene homolog2 203855 at WDR47 WD repeat domain 47 204094 is at TSC22D2 TSC22 domain family, member 2 213102 at ACTR3 208407_s ciated protein), delta 1 208865 at CSNK1A1 casein kinase I, alpha 1. BG534.245 200996 at ACTR3 IARP3 actin-related protein 3 homolog (yeast) NM_005721 210306 at L3MBTL 218556 at ORMDL2
omosome 11 open reading frame 24
- procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 A1912583
Ras suppr Rho guanine nucleotide exchange factor (GEF)3
hromosome 9 open reading frame.
AUS81 endonuclease homolog (S. cere 202695 s at S serine/threonine kinase 17a NM_004760.000137-i. : 202381 at ADAM9 ADAM metallopeptidase domain9 (meltringamma) NM_003816 000143
209379_s_at KIAA1128 KIAA1128 AF24.1785 000168 istone deacetylase 9 205659 at HDAC9 ------Patent Application Publication Oct. 3, 2013 Sheet 22 of 73 US 2013/026100.6 A1
200985 is at CD59 218088 s at RRAGC 36920 at MT W myotubularin ... 201328 at ETS2 v-ets erythroblastosis virus E26 oncogene homolog2 (avian) 8. serpin peptidase inhibitor, clade E (nexin, plasminogen activator 202627 is a inhibitor type l), member 1 201688 s a tumor protein D52 BG3890.15 20518 at zinc finger protein 193 NM_0062990. 00208. SP8 and FADD ike apoptosis regulator 202269 x : uanylate binding protein I, interferon-inducible, 67kDa uclear receptor binding factor 2
S100 calcium binding protein A14
Kruppel-like factor 6
202822 at LIM domain containing preferred translocation partner in lipoma BF221852 0.00241
206504 at cytochrome P450 family 24, subfamily A. polypeptide NM_000782.000263. ASP8 and FADD like apoptosis regulator W V calmodulin 1 (phosphorylase kinase, delta) ---- 0 000271 KDEL (Lys-Asp-Glu-Leu) endoplasmic reticulum protein : 204017 at KDELR3 retention receptor 3 NM_006855 000273 Patent Application Publication Oct. 3, 2013 Sheet 23 of 73 US 2013/0261006 A1
FIG. 11: Table E Hexamidine Benchmark Signature; 100 top down-regulated
BC00002
BC005838 0.0
213476 x ------.S.-melanocortin --, --, -, ------receptor (alpha melanocyte-...----- stimulating------hormone
--- VDR tamin D3) receptor 213762 x at RBMX RNA binding motif protein, X-linked 204255------wi------s at VDR vitamin D (1,25-dihydroxyvitamin D3) receptor 205677s at DLEU1 deleted in lymphocytic leukemia, 1 206157 at PTX3 t ..., nduced by IL-1 beta 20137 is at CUL3 0.00006 201376 s at HNRPF heterogeneous nuclear ribonucleoprotein F 0.00008 SLFN12 chlafen family member 12 IF6 erferon, gamma-inducible protein 16 m -
211750------x at TUBAic tubulin,w ... alpha-- or co- ic or ...... sc. ------to-o-o-o: 204359 at FLRT2 fibronectin leucine rich transmembrane protein 2 phosphatase and tensin homolog (mutated in multiple advanced 211711s at PTEN cancers ) 0.00016: 208985 sat EIF3J eukaryotic translation initiation factor 3. subunit J 0.00016 ATAD ATPase family, AAA domain containing 2 ------0.00017. at HOXA9 homeobox A9 ------0.00017 solute carrier family 25 ( mitochondrial carrier, ornithine transporter) member 15 M_014252 000017 ------' '...--, -,-,-,-----, ucleoprotein DI polypeptide 16kDa Co01721 000018 218093 s at ANKRD10 ankyrin repeat domain 10 NM_017664 0.00018, 20965 at apoptosis antagonizing transcription factor 0.00019 217990 at 209191 at TUBB6 tubulin, beta 6 209251.x at TUBAIC tubulin, alpha ic 214651s at HOXA9 homeobox A9 813 0.0002
204159 at CDKN2C cyclin-dependent kinase inhibitor 2c (p18, inhibits CDK4) NM_001262 0.00023 14 at FGFBP1 fibroblast growth factor binding protein NM_005130 000026.
general transcription factor ITC, polypeptide 4,90kDa 0.00027 chromosome 1 open reading frame 112 NM. 0.00028 heterogeneous nuclear ribonucleoprotein H3 ( 2H9) - - protein phosphatase D magnesium-dependent, delta isoform 214431 at GMPS guanine monphosphate synthetase NM_003875 0.00032 Patent Application Publication Oct. 3, 2013 Sheet 24 of 73 US 2013/0261006 A1
asparagine-linked glycosylation 13 homolog (S. cerevisiae) NM_018466 dyskeratosis congenita I, dyskerin --w------w-law-a------wana-K---AJO 10395 204544 at HPS5 Hermansky-Pudlak syndromes NM_007216 205284 at KIAA033 KIAA0133 ------NM_014777 202633 ------rat TOPBPI topoisomerase (DNA) II binding protein NM_007027 .00044. 212774 at ZNF238 zinc finger protein 238 AJ223321 .00046: 202453 is at GTF2H1 general transcription factor IIH, polypeptide 1,62kDa NM_005316 000046 21 1951 at NOLC1 nucleolar and coiled body phosphoprotein i D21262 0.00047. 217850re------at GNL3 guanine nucleotide binding protein-like 3 (nucleolar) NM_014366 0.00053 ------...---aminoadipate-semialdehyde dehydrogenase phosphopantetheinyl airs.------: 202170 sat AASDHPPT transferase AF151057 0.00053
217919 sat MRPL42 mitochondrial ribosomal protein L42 BE782148 ------si000056 Art in, T. 2469 is at CPSF6 cleavage and polyadenylation specific factor 6, 68kDa ------...--...... --w-sixAU149367
-...------2639 xat ; UBA1B AL581768 000058 8891 at DUSP6 dual specificity phosphatase 6 BC003 143 000059 826 at FKBP1 FK506 binding protein 15, 133kDa AB014574 0.00060 ------i SWI/SNF related, Tatrix associated, actin dependent regulator------of
213859_x at SMARCA5 chromatin, subfamily a, member 5 ------a- AI652586 wi-wa-num-www.i0.00060 205061s at EXOSC9 exosome component 9. NM_005033 000061
21072x------at TUBAIB tubulin, alpha------ib ------a------BC00648 000062 f
a------suu-----a-----ws------w------x----...---21 1953_s_at RANBP5 RAN binding protein 5 ...------...------a-AU148466 000064
204772 s at TTF1 transcription termination factor, RNA polymerase I NM007344-- a------sur 0.00065 212721n------e. at SFRS12arror------ow-saw-ene-sca-Maes---all-u-uasi-rum--n------ex-au splicing factor, arginine?serine-rich 12 A1810380 000065 204084s at CLN5 ceroid-lipofuscinosis, neuronal 5 AI91 1687 --- 0.00066 splicing factor proline/glutamine rich (polypyrimidine tract binding ------201586 s FPQ protein associated) --S------or------0.00066 : :------202983 at helicase like transcription factor 202658 at PEXITB------peroxisomal biogenesis factor 11B M003846 S. will NUP98 nucleoporin 98kDa TTU41815 0.00068, ------
O S2ST1x-r-----suu-ay-luvuus. heparan sulfate 2-O-sulfotransferase 1 Aw151887------0.00070. PSMA5 oteasone (prosome, macropain) subunit, alpha type, 5 NM002790 . 208892- is at swaxxAix-www-rDUSP6 dual var------aspecificity phosphatase------6 BC003143
:---n203436 we-a-we-s-s-s-s-s-s-s-s-s-s------was-s-s-s-s-s-s-s---axial------at RPP30 ribonuclease PIMRP30kDa subunit 218133 s at NIF3L NTF3 NGG1 interacting factor 3-like 1 (S. pombe) M021824 218842 at C-to------...------RPAP3 RNA polymerase II associated protein 3 NM_024604 0.00079 209773 s ------M.-as---as-a-vis-m-ul-mow-wa--as-a-a-a-a-a-vra-a-a-ma-ra-i-v-a-a-a-a-smac------iat RRM2 ribonucleotide reductase M2 polypeptide BC001886 0.00085 ------su------> ------...------s-s-s-s-s-s-s------prostaglandin-endoperoxide synthase 1 (prostaglandin G/H synthase 215813-s at PTGS1 and cyclooxygenase) S3629 0.00090. 20604 at ISL ISL LIM homeobox i ------NM_002.202 00009. 203689 s at FMR1 fragilex mental retardation IT ul-ll-ul------204662...I.T.T. at ------CP10 CP110P110 protein 219862 sat NARF nuclear prelamin A recognition factor Patent Application Publication Oct. 3, 2013 Sheet 25 of 73 US 2013/0261006 A1
laminin, beta 1 ------...------GTPase activating protein (SH3 domain) binding protein 2 AU149503 C proline synthetase co-transcribed homolog (bacterial) AA176833 DEAH (Asp-Glu-Ala-His) box polypeptide 5 - BC002574 213133 s. Aw).37404 21 1367s at CASP1 convertase) 219987 at LOC72819 Hypothetical protein Loc728.193 E856385 DX3X DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, X-linked BG492602 000122 splicing factor, arginine?serine rich 1 (splicing factor 2, alternate
ne maintenance complex component 10 ull-length cDNA clone CSODK002YF13 of HeLa cells Cot 25. 213294 at normalized of Homo sapiens (human) AV755522 209585 s at MINPP1 multiple inositol polyphosphate histidine phosphatase, 1 AF084943 000133 amylo- 6 glucosidase, 4-alpha-glucanotransferase (glyco gen 203566 s at AGL debranching enzyme, glycogen storage disease type III) SM 000645 218423 x_at --- vacuolar protein sorting 54 homolog (S. cerevisiae) NM_01.65 16 non-ATPase, 4 AB033605
omodomain helicase DNA - 206122 at SOX15 SRY (sex determining region Y-box 15 ------21978 at QTRTD queuine tRNA-ribosyltransferase domain containing NM_024638 0.00152. Patent Application Publication Oct. 3, 2013 Sheet 26 of 73 US 2013/026100.6 A1
FIG. 2: Table F hmark Signature; 39 significantly
interleukin 9 receptor
211316 x at CFLAR CASP8 and FADD-like apoptosis regulator 16 00073 211092 sat F2 ------, urofibromin 2 (bilateral acoustic neurona) 27 0.00977, 21912.2 s at THGIL tRNA-histidine guanylyltransferase 1-like (S. cerevisiae) NM_017872 0.00990. N OP9 constitutive photomorphogenic homolog subunit 5 201652 at COPS5 Arabidopsis) 209885 at RHOD ras homolog gene family, member D 205637s at SH3GL3 -domain GRB2-like 3 'S 208.29s at 221355 at olinergic receptor, nicotinic, gamma
218260 at mosome 9 open reading frame 58 209438 at phosphorylase kinase, alpha 2 (liver) 209329 xat A HIG1 domain family, member 2A milar to 60S ribosomal protein L21 M-phase phosphoprotein 10 (U3 small nucleolar 'ribonucleoprotein) heat shock 70kDa protein 2 solute carrier family 24 (sodium/potassium/calcium exchanger),
213051 at 219759 at eukocyte-derived argini 209099 x jagged (Alagile syndrome) v W 213679 at C30A tetratricopeptide repeat domain 30A 219706 a
21.8057 203936 s at MMP9 219406 at
xchange factor
coiled-coil domain containing 9 205232 is telet-activating factor acetylhydrolase 2, 40kDa ------3------
200972 at TSPAN3 tetraspanin 3 AF034.176 Human mRNA (Tripodis and Ragoussis) Homo 212608s at sapiens cDNA clone ntcons contig w85912 0.03931: Patent Application Publication Oct. 3, 2013 Sheet 27 of 73 US 2013/0261006 A1
prohibitin 2 NM_007273 0.04080 ------as------s:------/w------
218609s at NUDT2 nudix (nucleoside diphosphate linked moiety X)-type motif 2 214861 at JMJD2C jumonji domain containing 2C ------AI341811NM_0011610,04750 0.04757 : ------phosphoribosylformylglycinamidine synthase (FGAR 213302 at PFAS amidotransferase) AL044326 0.04928 Patent Application Publication Oct. 3, 2013 Sheet 28 of 73 US 2013/026100.6 A1
FIG. 13: Table G
NAG Benchmark Signature, most significantly down-regulated
21868621.0735 iss at RHBDF1 rhomboidcarbonic anhydrase 5 homolog XII 1 (Drosophila) 21 1487 x osomal protein S17
218176 at m melanoma antigen family F, 1 W. ------. ----mm ------m-m----...-a-3------219885 at schlafen family member 12 protein tyrosine phosphatase, receptor type, fpolypeptide 202066 at PPFIA 1 (PTPRF), interacting protein (liprin), alpha 1 ALNACT 218871 ondroitin sulfate GalNAcT-2 '' la- W.J.M.W. otein phosphatase 2 (formerly 2A), catalytic subunit, beta ? 201375 s form ---...-----...------NM_0041560.01239 212215 at rolyl endopeptidase-like / 203537 at hosphoribosyl pyrophosphate synthetase-associated protein 2 01544 oid-lipofuscinosis, neuronal 5 204084 s : 1545, forkhead box J3 AK027075 chromosome 6 open reading frame 106 NM_024294-0.
201761 at MTHFD2 methenyltetrahydrofolate cyclohydrolase 217783 s at YPEL5 yippee-like 5 (Drosophila) NM_01606 00211: 217975 at WBP5 ww domain binding protein 5 NM_0163030. 217922 at MAN1A2 mannosidase, alpha, class 1A, member 2 ------212644s at C14orf32 chromosome 14 open reading frame 32 T 212585 at OSBPL8 oxysterolxysterol binding protein-like 8 ...----...----...------21828 at NFYB clear transcription factor Y, beta TROVE domain family, member 2
213027 at TROVE2
209806 at HIST1H2B histone cluster 1, H2bk TT 202014 at tein phosphatase 1, regulatory (in 143300.02517
206662 at GLRX lutaredoxin (thioltransferase) 2 19307 at renyl (decaprenyl) diphosphate synthase, subunit 2 PRP19/PSO4 pre-mRNA processing factor 19 homolog (S. 203103_s cerevisiae) NM_014502 0.02765 cyclin-dependent kinase 7 (MO15 homolog, Xenopus laevis, cdk ------activating kinase) serine incorporator 3 22216.2 s at ADAMTS1 ADAM metallopeptidase with thrombospondin type 1 motif, 1. acuolar protein sorting 54 homo log (S. cerevisiae) golgi phosphoprotein 3 (coat-protein) NM_022NM_022.300.0345 PTPRF interacting protein, binding protein (liprin beta 1) N35896
u osyltransferase 2 (secretor status included) 201453 x at RHEB Ras homolog enriched in brain 212450 at KIAA0256 KIAA0256 gene product Patent Application Publication Oct. 3, 2013 Sheet 29 of 73 US 2013/026100.6 A1
Suppression of tumorigenicity 7
inc finger protein 672 221864 at ORAI3 ORAI calcium release-activatedrelease-acti calcium modulator 3 2 1697 -X at PNo1 partner of NOB homolog ( S. cerevisiae) inhibitor of DNA binding 3, dominant negative helix-loop-helix
pro tein --- 204622 x nuclear recept -- grou A. member 2 phosphoinositide-3-kinase, catalytic, beta polypeptide BC003393 0.04925. Patent Application Publication Oct. 3, 2013 Sheet 30 of 73 US 2013/026100.6 A1
F.G. 14: Table H Niacinamide Benchmark Signature, 100 top up-regulated
I263909
202207 at 2 16237 s at
538 s at HSPA2 heat shock 70kDa protein 2 205525 at CALDI caldesmon kelch-like 22 (Drosophila) ------
209344 at TPM4 KDEL (Lys-Asp-Glu-Leu) endoplasmic reticulum protein ---- s 204017 at KDELR3 retention receptor 3 NM_0068550,00050 glycosylphosphatidylinositol anchor attachment protein 1 : |
homolog (yeast) BC006383 0.00050, harrest and DNA-damage inducible, beta & 207517 at aminin, gamma 2 209068 at eneous nuclear ribonucleoprotein D-like 204686 at
5A (56kDa with KKE/D repeat)
mesoderm specific transcript homolog (mouse) phosphopantothenoylcysteine decarboxylase erythrocyte membrane protein band 4.9 (dematin) 209067 s at HNRPDL eterogeneous nuclear ribonucleoprotein D-like 205020 s at ADP ribosylation factor-like 4A 205796 at TCP1L t-complex 1 (mouse)-like -. 202269 x at GBPI anylate binding protein 1, interferon-inducible, 67kDa tumor protein p63 212535 at MEF2A myocyte enhancer factor 2A Patent Application Publication Oct. 3, 2013 Sheet 31 of 73 US 2013/026100.6 A1
heterogeneous nuclear ribonucleoprotein H3 (2H9) 212461 at AZIt antizyme inhibitor pleckstrin homology domain containing, family C (with FERM 209210 sat PLEKHC1 domain) member 1 202246 is at CDK4 cyclin-dependent kinase 4 218719_s------W-S. - - -- ...... Ns- complex subunit 3 (Psf3 homolog) 21 1275 s glycogenin 1 52164 at C11orf24 chromosome 11 open reading frame 24 FGF receptor activating protein
stearoyl-CoA desaturase 5 ike factor 6 late kinase 2 (Hallervorden-Spatz syndrome) YROXD1 pyridine nucleotide-disulphide oxidoreductase domain Ingiopoietin-like 4 218146 at lycosyltransferase 8 domain containing 1 2186.52s at dylinositol glycan anchor biosynthesis, class G integrator complex subun
BRCA1 interacting protein C-terminal helicase 1. R1, 20S rRNA accumulation, homolog (S. cerevisiae) - archaemetzincins-2 217873 at CA calcium binding protein 39 216870 x at DLEU2 deleted in lymphocytic leukemia, 2 ----- AF264787 215766 at AL096729
NM_014255 000308 EC24 related gene family, member D (S. cerevisiae) AU143984 000311 7 at mitochondrial ribosomal protein L15 NM_014 175000312 : TAF10 RNA polymerase II, TATA box binding protein (TBP)- 200055 at TAF10 associated factor, 30kDa NM_006284 0.00321 212829 at PIP4K2A phosphatidylinositol-5-phosphate 4-kinase, type II, alpha ------u-na-na--as-s-s-s------BE878277 000327 214240 at alanin AL556409 000328.
345. 202581 at HSPAIB heat shock 70kDa protein IB t 218358 at cysteine-rich with EGF-like domains 2 s protein 492
212225 at ukaryotic translation initiation factor 221994 at 3. stomatin (EPB72)-like 1 Y16522 0.00414 206351s at PEX10 peroxisome biogenesis factor 10 NM_0026170.00418 ; guanine nucleotide binding protein (G protein), alpha inhibiting 201181 at GNAI3 activity polypeptide 3 NM_0064960.00438 Patent Application Publication Oct. 3, 2013 Sheet 32 of 73 US 2013/026100.6 A1
neurofibromin 2 (bilateral acoustic neuroma) ukaryotic translation initiation factor 4A, isoform 3 PRPF4 PRP4 pre mRNA processing factor 4 homolog (yeast) 218303_x at KRCC1 lysine-rich coiled-coil 1 202208 s ADP-ribosylation factor-like 4C 203370 sat PDLIM7 PDZ and LIM domain 7 (enigma) T 202695 sat STK17A serine/threonine kinase 17a ------IRS7B dehydrogenase/reductase (SDR family) member 7B 20034s at RPL5 Tribosomal protein L5 TTT 202726 at LIGI ligase I, DNA, ATP-dependent modulin (phosphorylase kinase, delta) astic ataxia of Charlevoix-Saguenay (sacsin)
nc finger protein 180 hromosome 16 oper reading frame 42 -- 22m 1543 s at ERLIN2 R lipid raft associated 2 201362 at IVNSIABP fluenza virus NS1A binding protein stigial like 3 (Drosophila) 6 Kruppel-like factor 6 205584 at hromosome X open reading frame 45 S'''''''''' -...-...-
hromosome 8 open reading frame 33 PDS5, regulator of cohesion maintenance, homolog A (S. 213984 at PDS5A cerevisiae) v------v---m-au-8- AW9912.19------r------a------E-3 202787 s. at MAPKAPK3 mitogen-activated protein kinase-activated protein kinase 3 U43784 0 00632 Patent Application Publication Oct. 3, 2013 Sheet 33 of 73 US 2013/0261006 A1
FIG. 15: Table Niacinamide Benchmark Signature, 100 top down-regulated
208831 x a pentenyl-diphosphated 20162 IN BG292233 00.
0.0,
205014 at FGFBP1 fibroblast growth factor binding protein NM_005.1300.00001 201627s at INSIG1 insulin induced gene --- M_0055420.00001 219836 at ZBED2 zinc finger, BED-type containing 2 NM_0245080.00002 8. Heterogeneous nuclear ribonucleoprotein D (AU-rich element 213359 at HNRPD 205822 sat HMGCS1 202679------at
2-sul------209674 at 208796 is a ------a-. 202068 s 219885 at NM_018042 213562------s at SQLE ------.- 222209 sat TM 205282 at NM_004630.00012 ------...------211559 s at ------L49506 000013 218686 s at RHBDF rhomboids homologi (Drosophila) TNM 02:24500.00014 206861s at CGGBP1 CGG triplet repeat binding protein 1 217783 s at YPEL5 yippee-like 5 (Drosophila) solute carrier family 7. (cationic amino acid transporter, y+ 209921 at SLC7A11 system) member i AB040875 201625 s at SIG insulin induced gene 20222 KIAA0907 ------
ionine adenosyltransferase II, beta .usrex-ri--- farnesyltransfera e 1 i Solute carrier family 2 (facilitated glucose transporter), member 3 : 22 1751 at SLC2A3P pseudogene AL565516 0.00025
203910 at ARHGAP29 Rho GTPase activating protein 29 NM0048150.00027 213017 at ABHD3 abhydrolase domain containing 3
retin dehydrogenase all-trans/9-cis/11-cis) low density lipoprotein receptor related protein 8, apolipoprotein 208433 s at LRP8 e receptor NM 0175220.00037 Patent Application Publication Oct. 3, 2013 Sheet 34 of 73 US 2013/026100.6 A1
NAD(P) dependent steroid dehydrogenase-like 206247 at MHC class I polypeptide-related sequence B 212622 at transmembraneru------m------m-m------protein 4 B ------
209363 s at diator complex subunit 21 ehydrocholesterol reduc ase 201790 21929s at SAP30-like T G protein-coupled receptor 87 retinoldehydrogenase 11 (all-trans/9-cis/11-cis) 213094 at --Sa-a-a-a-a-a-a------G protei n-coupled receptor 126 --m-m- -- r 205788 s c finger CCCH-type containing 11 A 205904 at MHC class I polypeptide-related sequence A X-X 200832 s at SCD AB032261 000098. 218842 at RPAP3 NM_0246040.00114 209362 at 216607 s at cytochrome P450, family 51, subfamily A, polypeptide U40053 0.0022. 208647 at FDFT farnesyl-diphosphate farnesyltransferase f A872727 0.00122 solute carrier family 7, (cationic amino acid transporter, y-- 217678 at tem) member 11 201346 at ectin receptor 2 221582 at
222262 s at ETNK -:------&-xx------216060s at DAAM1 ishevelled associated activator of morphogenesis vK02 1890 0.001 as homolog gene family. member F (in filopodia) ...'....W. S. W.' alpha thalassemia/mental retardation syndrome X-linked (RAD54 i s U72937 0.00185 5 12760. O.OO 93 RAB5B, member RAS oncogene family AF267863 0.00201, diazepam binding inhibitor (GABA receptor modulator, acyl Coenzyme A binding protein) 0.00216 ADAM metallopeptidase domain 10 olymerase (RNA) | polypeptide D, 16 kDa lute carrier family 19 (thiamine transporter), member 2 22 1255 s at TMEM93 transmembrane protein 93.
210290 at inc finger protein 174 BC00116 0.00345 Patent Application Publication Oct. 3, 2013 Sheet 35 of 73 US 2013/0261006 A1
ELOVL family member 6, elongation of long chain fatty acids 210868 s at ELOVL6 (FEN 1/Elo2, SUR4/Elo3-like, yeast) 204119 s at 203367 at 27975 at WBPS ww domain binding protein 5 NM 0163030.00408. 200620 at TMEM59 ansmembrane protein 59 ------NM 004872 .00433
gri------e-r-to-sil------.205376 at INPP4B ositol polyphosphate-4-phosphatase, type II, 105kDa NM_0038660.00436 212.192 at KCTD12 potassium channel tetrameri------sation domain containing 12 AI718937
R low density lipoprotein receptor (familial hypercholesterolemia) A1861942 000457 ORAI calcium release-activated calcium modulator 3 - quinolinate phosphoribosyltransferase (nicotinate-nucleotide
QPRT pyrophosphorylase (carboxylating)) 204044 at 215093 at NSDHL NAD(P) dependent steroid dehydrogenase-like ------ubiqui jugating
plakophilin 4 solute carrier family 25 (mitochondrial carrier, phosphate carrier), 204342 at LC25A24 member 24 NM_0133860. 202284 s c yclin-dependent kinase inhibitor 1A (p21 y Cip1 201807 at acuolar protein sorting 26 honolog A (S. pombe) ------201955205000 at cyclin C -Glu-Ala-Asp)------box polypeptide 3, Y-linked 2025.36 at CHMP2B chromatin modifying protein 2B 212589 at RRAS2 related RAS viral (rras) oncogene homolog2 206662 at glutaredoxin (thioltransferase) 217127 at CTH cystathionase (cystathionine gamma-lyase) 210266 s at TRIM33 tripartite motif-containing 33 Patent Application Publication Oct. 3, 2013 Sheet 36 of 73 US 2013/0261006 A1
FIG. 6: Table Sepiwhite Benchmark Signature; 100 top u
chloride channel, calcium activated, family member 2 YD domain containing ion transport regulator 3 inducible protein 3
------s-s-s
chro s
S.
------hioredoxin interacting protein NM_0064720.00006 217869 at hydroxysteroid (17-beta) dehydrogenase 12 NM_01614 000007 218983 at complement component 1, r subcomponent-like NM_016 213725 x YLT 1. xylosyltransferase I s fibronectin 1 high mobility group AT-hook 2 endothelin receptor type A ------Aw001777 000019 203882 at ISGF3G interferon stimulated transcription factor 3, gamma 48kDa NM_0060840.00021 219655 at C7orf10 chromosome 7 open reading frame 10 NM_024728.0.00031 2013 12 s at SH3BGRL SH3 domain binding glutamic acid-rich protein like NM_003022.( 00036, 218990 s. at SPRR3 small proline-rich protein 3 NM_005416 000036,
SEC63 homolog (S. cerevisiae) NM_007214 000044
:, receptor-type, M_014547 000049 PTPRZ polypeptide 1 M 002851 000050. protein-Lisoaspartate (D-aspartate) O-methyltransferase domain : AB028973 0.00053 AL049782 000054 AL136658 0.00r small proline-rich protein 1A NM 0.03125 small proline-rich protein IB (corni?in) NM_003 --- Transcribed locus ------'gos------sterol-C5-desaturase (ERG3 delta-5 desaturase homolog, S. cerevisiae)-likeG protein-coupled receptor 77 D858 0.000850.00084. box protein 3 217188s at C14orfi chromosome 14 open reading frame 215729s at VGLLl vestigial like 1 (Drosophila) BE 22 1208 s at C 11orf61 chromosome 1 open reading frame 61 Patent Application Publication Oct. 3, 2013 Sheet 37 of 73 US 2013/0261006 A1
201814 at TBC1D5 TBC1 domain family, member 5 218644 at PLEK2 2024.80s at ------death effector domain containing
small proline-rich protein 1A 28280 x at HIST2H2AA4 histone cluster 2, H2a34 218280 x at HIST2H2AA3 histone cluster 2, H2aa3 --- 16.000137 3. TAF12 RNA polymerase II, TATA box binding protein (TBP)- 209463 is at TAF12 associated factor, 20kDa - D50544 0.001.47 ------f guanine nucleotide binding protein (G protein), alpha inhibiting 209576 at activity polypeptide 209264204967 isat at TSPAN4ROOM2 tetraspaninshroom family 4 member 2 -- - ... ------AF05484100E649 000185 32069 at N4BP1 Nedd4 binding protein AB014515 000192 - RAD54 homolog B (S. cerevisiae) NM_012415 0002 abhydrolase domain containing 5 peptidase inhibitor 3, skin-derived (SKALP) monoamine oxidase A 212282 at ------a smembrane protein ------matrix metallopeptidase 9 (gelatinase B,92kDa gelatinase. 203936 s at MMP9 92kDa type IV collagenase) 217988------iss---s------...------...- at CCNBIIPI cyclin B1 interacting protein 213459 at RPL37A ribosomal protein L37a 200821 at lysosomal-associated membrane protein 2 22 787 at mosome 6 open reading frame 12 inablastoma binding protein 8 chromosome X open reading frame 5
cKusick-Kau yndrome SHC (Src homology 2 domain containing) transforming protein 1 AI809967 000278 histone deacetylase 9 NM_014707000285 homolog gene fa mily, member D BC001.338 0.00303 214290 s at HIST2H2AA3 histone cluster 2, H2aa3 A313324 0.003 2. 214290s at HIST2H2AA4 histone cluster 2, H2aa4 - finger, FYVE domain containing 21 ite carrier family 33 (acetyl CoA transporter), member 1 BE464756 -oxoguanine------DNA glycosylase NM_01682 0.00340
oredoxin------interacting protein------AA82232
dehyde dehydrogenase 7 family, member Al BC00255 0.00349.
chromosome 20 open reading frame 42 AA469071 000380 emopamil binding protein ( sterol isomerase ) -- A v702405 Patent Application Publication Oct. 3, 2013 Sheet 38 of 73 US 2013/0261006 A1
ci rep oss mp men g p defic
mplementation group 1 (includes overlapping antisense
203720 s at ERCC1 uence) NM_001983 000388------
219979 s at C11orf73 chromosome 11 open reading frame 73 204955 at SRPX sushi-repeat-containing protein, X-linked - recombination Signal binding protein for immunoglobulin kappa ------. ------.....-- NM_015874 000425 20778.5------s at RBPJ ubiquitin-conjugating enzyme E2. Ji (UBC6 homolog, yeast) NM 0160210.00431
zinc finger protein 302 . NM_0184430.00432
Fas (TNF receptor superfamily, member 6) 3.
DP-glucose ceramide glucosyltransferase-like 2 ataxia telangiectasia mutated
thioredoxin interacting protein
chromosome 5 open reading frame 21 201308 s at 11-Sep septin 11 n i s acetyl-Coenzyme A acetyltransferase 2 (acetoacetyl Coenzyme : 209608_s_at ACAT2 : tRNA-histidine guanylyltransferase 1-like (S. cerevisiae) -- NM_017872 0.00491 219122_s------v------...------...------translocase of inner mitochondrial membrane 23 homolog B 28 9 at TIMM23B (yeast) i M_0063270.00497 translocase of inner mitochondrial membrane 23 homolog i NM_006.3270.00497 218119 at TIMM23 (yeast)tyrosine 3-monooxygenase/tryptophan 5-monooxygenase - U28936
218677 at is 219543------at PBLD 201617 x at CALDI caldesmon I 201185 at HTRAI HtrA serine peptidase I ------203729 a elial membrane protein 3
21 1801-x_ mitofusin i geranylgeranyldiphosphate------synthase I AW299507 0.00668, Patent Application Publication Oct. 3, 2013 Sheet 39 of 73 US 2013/0261006 A1
FIG. 7: Table K hite Benchmark Signature;
217767 at LocG53879 imilar to Complement C3 precursor
208002 s at ACOT7 acyl CoA thioesterase 7 --- 212501 at CEBPB --- (CAAT?enhancer binding protein (C/EBP). beta 0.00002, 209774 x at CXCL2
gase, modifier subunit
-...----, -i-. . . . . chemokine (CXC motif) ligand 1 (melanoma growth I 204470 at CXCL1 stimulating activity, alpha) NM_00151.10.00005 : AHA1 , activator of heat shock 90kDa protein ATPase homologi t 201491 at AHSA 1 (yeast) NM_0121110.00013 211940 x at H3F3A H3 histone, family 3A ------v------...-e-...------Yix------w-BE869922 0.
201839 s at TACSTD1 tumor-associated calcium signal transducer M_0023540.00022 206157 at PTx3 . i gene, rapidly induced by IL-1 beta 209674 at cryptochrome 1 (photolyase------like) ------
201289 at CYR61 cysteine-rich, angiogenic inducer, 61 NM_0015540.00046 201381 x at CACYBP calcyclin binding protein - 7356 000047. aldo-keto reductase family 1, member C1 (dihydrodiol dehydrogenase 1, 20-alpha (3-alpha)-hydroxysteroid 204151_x at AKR1C dehydrogenase) NM_001353 0.00057 201843 s at EFEMP EGF containing fibulin-like extracellular matrix protein 1 NM_004 1050.00063
205067 at IL1B interleukin I, beta NM_0005760.00068.------2.
200738s at PGKl phosphoglycerate kinase I M_000291 000080: tubulin foldi cofactor A
2095.14s at RAB27A BE502030 205476 at CCL20 NM_00459 201338 ------
218309 at CAMK2N1 M_018584------000167 205774 at 20418 is at ZBTB43 zinc finger and BTB domain containing 43 T90308 000214
204078 at synaptonemal complex protein SC65 ------NM_0064550.00227 - ATP synthase,
208746 x. at ATP5L ------208700s_at TKT transketolase (Wernicke-Korsakoff syndrome) ------guanine nucleotide binding protein (G protein), beta polypeptide 2008.52 x at GNB2 3TT "" " " " NM_005273 000240 Patent Application Publication Oct. 3, 2013 Sheet 40 of 73 US 2013/0261006 A1
(prosome, macropain) subunit, alpha type, 5 NM0027900 ethanolamine kinase NM_0186380.00246 (HC class I polypeptide-related sequence B ------s "ctin, galactoside-binding, soluble, 8 (galectin 8) ------207345 at FST follistatin
eckstrin homology-like domain, family A, member 1 : plasminogen activator, urokinase 4 219933 at GLRX2 glutaredoxin 2 protein tyrosine phosphatase, receptor type, fpolypeptide (PTPRF), interacting protein (liprin), alpha thanolamine binding protein l oteasome (prosome, macropain) subunit, beta type, 2
mping translocation breakpoint tripartite motif-containing 16 204341------at TRIM16L tripartite motif containing 16-like 221563 at DUSP10 dual specificity phosphatase 10 203234 at uridine phosphorylasel ------ATP synthase, H+ transporting, mitochondrial F0 complex,
subunit C3 (subunit 9) metallothionei ------small nuclear ribonucleoprotein polypeptides B and B1 J04564 000440 2 8026 at CCDC56 coiled-coil domain containing 56 --- NM_014019 000459: nor necrosis factor, alpha-induced protein 3 NM 006290 00046 1.
cysteine and histidine-rich domain (CHORD)-containing 1 NM 012124 0.00476. pool------. ------motif, --- 000483 NM 0181830.00558 BC004489 000571 NM004500000577 i
AF151056 000645 glycine cleavage system protein H (aminomethyl carrier) AW237404 0.00656 olute carrier family 2 (facilitated glucose transporter), member 6 NM 017585 000673, UDP-glucose dehydrogenase NM_0033590. NM
214866 at PLAUR plasminogen activator, urokinase receptor 0.00802.------
210758 at PSIP1 PC4 and SFRS1 interacting protein 1 AFO98482 o 222062 at IL27RA interleukin 27 receptor, alpha AI983115 0.008 16. 201243 s at ATPB1 ATPase. Na+/K+ transporting, beta 1 polypeptide NM 0.01677 000818 Patent Application Publication Oct. 3, 2013 Sheet 41 of 73 US 2013/026100.6 A1
S. ribosomal protein S5 217753 s at RPS26 ribosomal protein s26 201152 s at MBNLI muscleblind-like (Drosophila) v-maf musculoaponeurotic fibrosarcoma oncogene homolog F
vian) AL021977 001018. deoxyhypusine synthase ' ' U26266 001058.
proteasome (prosome, macropain) subunit, beta type, 9 (large multifunctional peptidase 2) ------W-u------ru-um.3.------m-m-----...-mm------a (glucosamine) 3-O-sulfotransferase 2 mru's 202120 x at AP2S1 adaptor-related protein complex 2, sigma 1 subunit NM004069 001161; 208784s at KLHDC3 kelch domain containing 3 BCOO1793 0.01167.
broblast growth factor 2 (basic) NM002006 0.01.194 aior histocompatibility complex, class I, AT S.
otein phosphatase 1, regulatory (inhibitor) subunit 15A
546 0.01258 NM_0058050.01281
homolog 1 (S. cerevisiae) Patent Application Publication Oct. 3, 2013 Sheet 42 of 73 US 2013/026100.6 A1
F.G. 18: Table L Composite "Skin Tone' Benchmark Signature; approximately 40 up-regulated
8 old shock domain protein 201160s at TendoplasmicCSDA A reticulum AL55690 201694201216------is-Klas-s-s-s-s-s-s------was------xx satat EGR1ERP29 proteinearly growth 29 response I ------NM_00681NM_001964 rum----3-mw-. 5136 7 Endothelin converting 201749 at ECE1 enzyme 1 BF969352 286 386 1.34: SW ---->------| programmed cell death 4 ------' 's--...------(neoplastic transformation 202730s at PDCD4 inhibitor) NM_014456. 2054: 2352 1.18 BMP and activin membrane-bound inhibitor : homolog 203304 at BAMBI (Xenopus laevis) NM 012342 ww domain binding 203597 s at WBP4. protein 4 :AI734228 888 1098 1.19 203 831 at 3F D v2 rara-m-m-ma a XXXYsra------999 1206 123 NM_004619 2011 1.33 204352 at TRAF5 *------v-rel reticuloendotheliosis viral oncogene homolog B, nuclear factor of kappa light polypeptide gene enhancer in B-cells 3 205205 at RELB 207624 sat RPGR GTPase regulator ras homolog gene family,
TP53member activated D protein AB007457BC001338 977 1167. 1.14.
solute carrier family5 8.
(sodium iodide symporter), ber 5 D87920. tubiquitin protein ligase E3 f component n-recognin 4 AB00793 w Ribosomal protein L27a BE737027 . BE32772
lass I, A S. pulmonary- S.--- 213936 x at SFTPB associated prote Patent Application Publication Oct. 3, 2013 Sheet 43 of 73 US 2013/0261006 A1
214317 x at RPS9 ribosomal protein S9 BE348997 23563. 29560. 1.22 CDNA clone : 21439.5 x at IMAGE:48.38699 A33.559 :
21506 x at DST dystonin BCOO4912 215450 at 216609 at TXN Thioredoxin 217903 at STRNA protein 4 NM G protein-coupled receptor 21815 1x at GPR172A 172A NM 02453 : r 218388 at PGLS phosphogluconolactonase NM 012088 chromosome 13 open C13orf23 reading frame 23 NM_025 138 508 DnaJ (Hsp40) homolog, 218435 at DNAJC15 subfamily C, member 15 NM_013238 RAB 17, member RAS 218931 at RAB 17 oncogene family NM_022449 578 678 1.12 : ypothetical protein ------219254 at FL22222 FLJ22222 NM_024648 248 219916 s at RNF39 ring finger protein 39 NM_025236. 363 g leucine rich repeat 220219 s at LRRC37A containing 37A NM_018001. 166. 209 153 eucine rich repeat 166
166 4 O 484 1.13 NM 007071 ------
H3 domain binding glutamic acid-rich protein 221269 s at SH3BGRL3 like 3 NM_03286 chromosome 9 open : s 221865 at C9orf91 ading frame 91 BF969986 432 528, 1.3 22 1943 x. RPL38 bosomal protein L38 AW303136 sulfotransferase family, cytosolic, 1A, phenol 22.2094 at SULT1A3 preferring, member 3 AI58O112 186 286 sulfotransferase family, --- cytosolic, 1A, phenol
22.2094 west-gross-s-s-s-s-s-s-s-s-s-s-sousat SULT1A4 s preferring, memberre 4 AI5801-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o: 12 186 286 1.6 acetylserotonin O 36553 at ASMTL methyltransferase-like AA669799 750 363 58 down-regulated below. Patent Application Publication Oct. 3, 2013 Sheet 44 of 73 US 2013/0261006 A1
initiation factor 1A, X 201017 at EIF1AX linked BG 1496.98 un------a------ee------eukaryotic translation ------arms------{ 201437 s at EIF4E initiation factor 4E NM_001968 4499 201534 sat it itin-like 3 703: 1.34 cytochrome b5 type B (outer mitochondrial 201634 s at CYB5B membrane) NM_030579. 6244. 5584. 1.09 COP9 constitutive photomorphogenic s : homolog(Arabidopsis) subunit 5 NM_006837 7929 201652 at COPS5 ------7453 1.05 myristoylated alanine-rich i 201669 s at MARCKS protein kinase C substrate NM_002356 4402; 3864 1.1 ------chromosome 3 open s i
------is------,201678s at C3orf57 -si------reading frame 37 NM_020187.------wv-wi------2061 1776. 1.17------sparc/osteonectin, cwcv. and kazal-like domains f
202524 s at SPOCK2 proteoglycan (testican)2 NM_014767 - V -- 232; - 205;------1.26; 203008_x at iTXNDC9 thioredoxincontaining 9 domain NM_005.783 12747 11685. 1.1 : Down syndrome critical 203635 at DSCR3 region gene 3 NM_006052. 878 748; 1.22. ------CDNA clone ------oo------: ------. 203812 at IMAGE:5922621 --- ABO 11538 209; 137; 1.74 ------chromosome 1 open ------s------204700 x at 1orf107 reading frame 107 tripartite motif-containing NM_014388 ------, -si-----ite------1665 1455,------1.13
204804------sul-L------soul-----w-sau-we-in-M.------sustar at TRIM21 21 L-...------, i.v.------si-...+...:-...-L. NM_003141 naternal embryonic 204825 at MELK leucine zipper kinase Mo14791 8178 1.05 RNA binding motif protei 20511.5 s at RBM19 cholinergic19 receptor, 407 371, 1.12.
nicotinic, alpha 5 654, 1.19 H-domain containing 1 receptor accessory protein 208873 s at REEPs 5 BC000232 ------4454, ------
210218 s at SP100 SP100 nuclear antigen U36501 507 ------> suppressor of : i s 21 0242 x at ST20 tumorigenicity 20 AF249277 1019. 995 1.15 fascin homolog 1, actin- : bundling protein i (Strongylocentrotus i. 210933 is at FSCN1 purpuratus) BC004908 3865, Patent Application Publication Oct. 3, 2013 Sheet 45 of 73 US 2013/0261006 A1
serine/threonine kinase 3 21 1078 s at STK3 (STE20 homolog, yeast) Z25422 580 212092 at PEG10 ------paternally expressed 10 BE858.180 817, GrpE-like 1, mitochondrial 212432 at GRPEL (E. coli) AL542571 5747; G patch domain containing : 212485 at GPATCH8 8 AU 46596 1243 DnaJ (Hsp40) homolog, 212490 at DNAJCS subfamily C, member 8 AA843895 549 autism susceptibility ------212599 at AUTS2 candidate 2 AK025298 223 05 ubiquitin-conjugating enzyme E2N (UBC13 212751 at UBE2N : homolog, yeast) BG290646 3625 3.133 18. polymerase (DNA - directed), delta 3. 212836 at POLD3 accessory subunit D26018 696. 1409. 1.23 ess-s-s-s-s similar to Aspartate ------3. --- : mitochondrialaminotransferase. precursor (Transaminase A) (Glutamate oxaloacetate 216545 at LOC645,538 transaminase 2) AL049710 228 178 1.51 216993 s at COL11A2 collagen, type XI, alpha2 U32169 340 285 124 : : tyrosine 3 monooxygenase/tryptophan 5-monooxygenase activation protein, beta 217717 s at YWHAB polypeptide BF246499 | 15355 SERPINEI mRNA binding 217724 at SERBP1 protein -i. 15265 1.22 . mitogen-activated protein or-to 217808 s at MAPKAP kinase associated protein NM_024117 217864 S_
21798 is at FXC1 1240rvivoros. 117 : 1175 1.19 267865 2921 212s 22 -s-s-s-s-s-s-s-s-s-s-siris-storic-criss-sous-s-s-s-s-s-s-s-s-calcium/calmodulin -s-s-s-s-s-s-s-s ------.
218309 at CAMK2N1 inhibitor 1 ATP/GTP binding protein 218480 at AGBLS like 5 NM eukaryotic translation initiation factor 2B, subunit 218488 at EIF2B3 3 gamma, 58kDa 218514 at Patent Application Publication Oct. 3, 2013 Sheet 46 of 73 US 2013/026100.6 A1
------
218650 at DG critical region gene 8 775 573. 487; 1.28
218926 at MYNN myoneurin NM_018657 . DKN2A interacting ---...------...--- g 1199, 998 1.18 218929 at CDKN2AIP protein NM_017632, 1636. 1271, 1.2 chromosome 2 open s | 21900s at C2Orf43 reading frame 43 900 785 1.19
s
enhancer of mRNA decapping 3 homolog (S. i. erevisiae) 219522 at FJX1 NM_014344 s 219539 at GEMING serum/glucocorticoidassociated protein 6 NM_024775 . . . . 38.31 ...... M...... 3555. 1.19 regulated kinase family, - 220038 at SGK3 member 3 NM_013257. 779 karyopherin alpha 3 : 221502 at importin alpha 4) AL120704 5124 UTP14, U3 small nucleolar ribonucleoprotein. : 221514 at UTP14A homolog A (yeast) : I glutamate-rich WD repeat t BC001149 1776. 1527, 1.12. 22 1549 at GRWD1 containing 1 AF337808 1385, 1152, 1.17 s mediator complex subunit 3 g
221650s at ED18 salariassassissars&ain:18 BC002694 s chromosome 5 open : 221823 at ------awaii-xesC5orf30 woma-ir-is-soever-in- readingorsososolois-s-s-s-s-s-os-is-gons--asseross-s-surgos-os-sur-so-so frame 30 AL565741 NFKB inhibitor interacting s: 222105 s at NKIRAS2 Ras-like 2 : AA452565 s
synapse defective, Rho s GTPase, homolog 1 (C. 44702 at SYDE1 elegans) R77097 Patent Application Publication Oct. 3, 2013 Sheet 47 of 73 US 2013/0261006 A1
FIG. 19: Table M
cronym NetAffx Title acyl-CoA synthetase long-chain family member 3 ATP-binding cassette, sub-family A (ABCI), member 1
dehydrogenase/reductase (SDR family) member 3 DnaJ (Hsp40) honolog, subfamily B, member 1 arbohydrate (chondroitin 4) sulfotransferase 11 niacin receptor 2 ---...-- phosphorylase 1
SRY (sex determining region Y-box 9 (sex determining region Y)-box 4.
tissue factor pathway inhibitor------2 i 0.6745:
DnaJ (Hsp40) homolog, subfamily B, member 1 0.6956: ------DNA-damage regulated autophagy modulator 1 i 0.6939: ------it
hyaluronannoic acid synthase induced 2.14 0.7514
homolog gene family, member B
ADP-ribosylation factor-like 4C endothelial cell-specific molecule I formin homology 2 domain containing 3 36.209277 at TFPI2 tissue factor pathway inhibitor 2 inhibitor of DNA binding t, dominant negative helix-loop 37,208937_s_at ID1 helix protein 0.8465i apolipoprotein B mRNA editing enzyme, catalytic 38,206632 s at APOBEC3B polypeptide-like 3B 16078. Patent Application Publication Oct. 3, 2013 Sheet 48 of 73 US 2013/026100.6 A1
insulin-like growth factor binding protein 3 nuclear receptor interacting protein 1 Kruppel-like factor 6 intercellular adhesion molecule 1 ...... ATP-binding cassette, sub-family A (ABC1), member 1
icotinamide phosphoribosyltransferase microtubule associated monoxygenase, calponin and LIMIT domain containing 1 lasmi ogen activator, urokinase leckstrin homology-like domain, family A, member 1 carnitine palmitoyltransferase 2 F-hand domain family, member D2 cium/calmodulin-dependent protein kinase II inhibitor 1 0.4016 broblast activation protein, alpha acher Collins-Franceschetti syndrome 1 nnexin A W.
rcellular adhesion molecule 1 12143 sat IGFBP3. - s sulin-like growth factor binding protein 3 1538 is at HSPA2 heat shock 70kDa protein 2 8 mmSv. 201889 at - - mily with sequence similarity 3, member C plasminogen activator, urokinase neuroepithelial cell transforming 1 like factor 6 as minogen activator, tissue FAIP3 interacting protein '' naj (Hsp40) homolog, subfamily A, member BCL2-interacting killer (apoptosis-inducing) ehydrogenase 1 family, member A3 74,202208 uropilin (NRP) and toiloid (TLL)-like 2 MAP kinase interacting serine/threonine kinase 2 hep g EGF-like growth factor Patent Application Publication Oct. 3, 2013 Sheet 49 of 73 US 2013/026100.6 A1
92kDa type IV collagenase) is at GLUL glutamate-ammonia ligase (glutamine synthetase) 0.3824 related C3 botulinum toxin substrate 2 (rho family, small 82213603 s at RAC2 GTP binding protein Rac2) 0.4568 02600 s at NRIP1 nuclear receptor interacting protein 12916 84202786 at STK39 serine threoninethre kinase 39 (STE20/SPS1 homolog, yeast) 0.528 85,206200 sat ANXA1 annexin A11 ...... SMAD family member 3
integrin, beta 6 dual specificity phosphatase 1
MDSi and EV complex locus suppressor of cytokine si gnaling 2 ------was------.S.------ou ww.laamu....mora-Wm----v-S-S-3-m-----...-au--m.
smen brane bound O-acyltransferase domain containing 2 .--
asic helix-loop-helix family, member e40 ---. . ... ---...------... ------: AS domain containing serine/threonine kinase 0.3592 secreted frizzled-related protein 0.3867 ium binding and coiled-coil domai n 2 0.3057. 96209108 at TSPAN6 tetraspanin 6 97220465 at hypothetical LOC30054 min B, beta 99.211113 s at ABCG1 ATP-binding cassette, sub-family G (WHITE), member 100,210786 sat FLI1 riend leukemia virus integration 1 steroid-5alpha-reductase, alpha polypeptide 1 (3-oxo-5 alpha-steroid delta 4-dehydrogenase alpha 1) ocyst complex component 7
hydrolase domain containing 3 ------W------
12 217966 s at
mediator of DNA-damage checkpoint transmembrane protein with EGF-like and two follistatin-like M-Wu-3------m--3-o-o-W- domains 1 0.6406 ------8.:------x-xx-x-xx-x-xx-a::-sexmyristoylated is alanine-rich--a-e------a-a-a-a------protein kinase C substrate------ex O.4037 17967 s at FAM29A family with sequence similarity 129, member A 0.3821 Patent Application Publication Oct. 3, 2013 Sheet 50 of 73 US 2013/0261006 A1
201834 at I 125 217996 at PHLDA 1 pleckstrin homology-like domain, family A, member 1 0.4136 126.204639 at ADA adenosine deaminase 0.7075, 127210276 s at TRIOBP nucleolar ein 12 o549s 128216237 s at minichromosome maintenance complex component 5 0.4018. 129201466 s at JUN jun oncogene 0.4221 . er family 25, member 37 132,202949 s at FHL2 four and a half LIM domains 2 13320918 s at TUBA1A tubulin, alpha la 134.212186 at ACACA- - - - acetyl-Coenzyme A carboxylase alpha - -, -, - ...... 3520 147 s at TIMP3
catenin (cadherin-associated protein), delta 1
acyl-CoA synthetase long chain family member 3
inichromosome maintenance complex component 2 ------i- to integrin, alpha 2 (CD49B, alpha 2 subunit of VLA2
ding cassette, sub-family G (WHITE), member 1 pyruvate dehyrogenase phosphatase catalytic subunit 1 0.3194 S-like antigen 1562 0517 s at AKAP12 A kinase (PRKA) anchor protein 12 s :- at PAQR3 progestin and adipoq receptor family member III 0.2703. fibroblast growth factor 2 (basic) 0.7506. hemokine (C-X-C motif) ligand 3 ---- 1, apoptosis-inducing factor --- 161202581 at HSPA 1A 1622 12298 at NRP1 Patent Application Publication Oct. 3, 2013 Sheet 51 of 73 US 2013/026100.6 A1
phosphoribosyl pyrophosphate synthetase-associated protein ------
- formin binding protein 1-like ------m 168 219058 xat TINAGL1 tubulointerstitial nephritis antigen-like 169202584 at NFX1 nuclear transcription factor, X-box binding IT 170217999 sat PHLDA 1 pleckstrin homology-like domain, family A, member 1 171202392 s at PISD chromosome 22 open reading frame 30 172203991 s -- lysine (K). specific demethylase 6A 173203837 at MAP3K5 mitogen-activated protein kinase kinase kinase 5
serpin peptidase inhibitor, clade E (nexin, plasminogen ERPINE1 activator inhibitor type I), member 1 RNA binding motif protein 19 P-ribosylation factor-like 4C ' ' ' ' ' ' ' ' ' 206972 s at GPR161 G protein coupled receptor 161 178210118 s at IL1A interleukin 1, alpha s--- *- ::::::::::::-...------LYix----xx-x-x------is 179205847 at PRSS22 protease, serine, 22 1802.13392 at IQCK IQ motif containing K platelet-derived growth factor beta polypeptide (simian 181 204200 s at PDGFB sarcoma viral (v-sis) oncogene homolog) LPIN1 lipin 6 adenine-specific DNA methyltransferase I (putative) RAB8E, member RAS oncogene family
CASP8 and FADD-like apoptosis regulator AUS augmin-like complex, subunit 5
0.3422 binding protein (li 0.4981
FK506 binding protein 1A, 12kDa 0.2669 amyloid beta (A4) precursor protein-binding, family B, member 2 K3 homeobox '' ' ' - cadherin 4, type I, R-cadherin (retinal) 5208779 x at DDR1 discoidin domain receptor tyrosine kinase 1 196213572 s at SERPINB1 serpin peptidase inhibitor, clade B (ovalbumin), member 1972 12729 at discs, large homolog 3 (Drosophila) 198204475 at MMP1 matrix metallopeptidase 1 (interstitial collagenase) 0.9297 199371 17 at ARHGAP8 Rho GTPase activating protein 8 0.4329 200217202 s at GLUL glutamate-ammonia------ligase (glutamine synthetase) 0.5098, Patent Application Publication Oct. 3, 2013 Sheet 52 of 73 US 2013/0261006 A1
F.G. 20: Table N RA benchmark signature in thC, 200 down-regulated
Log 2 fold
changel tRA to i
4214587 at ollagen, type VIII, alpha 1 ------ter 2, H2be 5202708s------at 6204653 at ------drenergic, ------beta-2-, receptor, surface mbrane protein 40
r jointed box (Drosop solute carrier family 38, member 2 ------3 zinc finger protein 238 ------
------12,202769 at ------cyclin G2
202 0.6733 rate 5-dioxygenase 2 0.2883
AHNAK nucleoprotein 2 0.8134 myeloid/lymphoid O mixed-lineage leukemia (trithorax
25,218440 at 26 201368 at nc finger protein 36, C3H type-like 2 -0.691, 27 --- inc finger, BED-type containing 2 28.204602 at KKi dickkopf homolog 1 (Xenopus laevis) -
29 201739 at GK - -- serum/gluc ------solute carrier family 7, (cationic amino acid transporter, y+ 31221563 at DUSP10 dualsystem) specificity member phosphatase 1 | 10 - -1.11291,0269. 32.201109s at THBS1 thrombospondin -1.2465 33218319 at PELI1 pellino homolog 1 (Drosophila) -0.3831 : prostaglandin-endoperoxide synthase 1 (prostaglandin G/H i 34,215813_s_at PTGS1 synthase and cyclooxygenase) -0.6398 Patent Application Publication Oct. 3, 2013 Sheet 53 of 73 US 2013/0261006 A1
ema domain, immunoglobulin domain (Ig), short basic i
35,35666 at SEMA3F domain, secreted, (semaphorin) 3F -0.5912 36.204359 at FLRT2 fibronectin leucine rich transmembrane protein 2 16162 37209815 at PTCH1 patched honolog 1 (Drosophila) ------0.5571 solute carrier family 7, (cationic amino acid transporter. y+ 38.209921 at System) member 11
39.205157s at eratin 17 protein phosphatase 1A (formerly 2C), magnesium 40203966 s at dependent, alpha isoform 41215071s at istone cluster 1, H2ac S.---, -, -, - - - -- 42203640 at musclehind-like 2 (Drosophila) 43,202874 sat ATPase, H+ transporting, lysosomal 42kDa VI subunit CIT 44213568 atv. odd skipped related 2 (Drosophila) 45.209674 at
trophoblast glycoprotein thioredoxin interacting protein
nosine deaminase, RN A specific, B1 (RED1 homolog rat solute carrier family 7 (cationic amino acid transporter, y+ 50216092 s at SLC7A8 system), member 8 51218093 s at ANKRD10 ankyrin repeat domain 10 52,218886 at PAKIP1 PAK1 interacting protein 1 9101 at CTGF connective tissue growth factor 54,218973 at EFTUD elongation factor Tu GTP binding domain containing 1 55.219250 s at --- bronectin leucine rich transmembrane protein 3 56.219284 at HSPBAP ISPB (heat shock 27kDa) associated protein 1 : prostaglandin-endoperoxide synthase 1 (prostaglandin G/H 57.205128 x at PTGS1 synthase and cyclooxygenase) 58,209758 s at MFAP5 microfibrillar associated protein 5 TCF4 transcription factor 4 dual specificity phosphatase 10
utamate-cysteine ligase, catalytic subunit
NA-damage-inducible transcript 3
ectodermal-neural cortex (with BTB like domain) Amphiregulin RABGGTB ab geranylgeranyltrans rase, beta subunit
------bosomal protein L31 ------
: protein tyrosine phosphatase-like (pro line instead of catalytic 67212640 at PTPLB ------arginine), member b s 68209946 at VEGFC vascular endothelial growth factor C 69,204930s at BNIP1 BCL2/adenovirus E1B 19kDa interacting protein - - - - 70200790 at ODC1 ornithine decarbox 7 SMPDL3A Patent Application Publication Oct. 3, 2013 Sheet 54 of 73 US 2013/0261006 A1
zinc finger protein 318 0.3655 ------TAF5 RNA polymerase II, TATA box binding protein (TBP)-
associated factor, l00kDa -0.3 104. mitogen-activated protein kinase binding protein------0.551
KIAA0907
gap junction protein, beta
CD55 molecule, decay accelerating factor for complement CD55 (Cromer blood group) -1. 1921,
FAM60A family with sequence similarity 60, member A -0.2787
laminin, alpha 2. . UNC84A
chromosome 7 open reading frame 20
AT rich interactive domain 5B (MRF-like) -0.9359. inositol polyphosphate-5-phosphatase, 145kDa -0.2639
922055.34 at------PCDH7 protocadherin 7 0.489
protein phosphatase IB (formerly 2C), magnesium-dependent, : 3 209296 at beta isoform ual specificity phosphatase 5
--- x at FNI fibronectin dual-specificity tyrosine-(Y)-phosphorylation regulated kinase : 202971s at DYRK2 2 -0.3726. ------203778 at ------MANBA mannosidase, beta -A, -- - -- lysosomal ------0.3569------100206332 is at IFI16 air-:------as------ax-ruz-i-sa-a-a-a-a-interferon, gamma-inducible ------was---ix--o-wri------all------, protein 16 -, -r -0.3091irrus i CD55 molecule, decay accelerating factor for complement 101 201926_s_at CD55 (Cromer blood group) 102217312 s at COL7A1 collagen, type VII, alpha 103.204204 at olute carrier family 31 (copper transporters), member 2 104219492 at CHIC2 ysteine-rich hydrophobic domain 2
105205000 at DDX3Y DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, Y-linked 0.3032 s at JAG1 - Alagille syndrome) 10721868is at SDF2L stromal cell-derived factor 2-like I
O 204686 at insulin receptor substrate J -- reductase I
110.209185 s at IRS2 insulin receptor substrate 2 Patent Application Publication Oct. 3, 2013 Sheet 55 of 73 US 2013/026100.6 A1
fibronectin 1
solute carrier family 7, (cationic amino acid transporter, y+ system) member 11 ymine-DNA glycosylase glutamate-cysteine------ligase, ------catalytic subunit ------i transmembrane protein 45A ---...------0. xysterol binding protein-like 10
palladin, cytoskeletal associated protein -0.2632
hexokinase 2 3476; 19208.527 x at HIST1H2BE histone cluster 1, H2be IL5RA interleukin 15 receptor, alpha
dosulfine alpha --- - : : ------, - D repeat domain 26 - chromosome 7 open reading frame 64
AXL receptor tyrosine kinase
polymerase (RNA) I polypeptide C, 30kDa broblast growth factor binding protein
ate transporter), member 2 138 CAMSAPIL calmodulin regulated spectrin-associated protein 1-like
amin C, gamma syncoilin, intermediate filament protein
SH3 domain and tetratricopeptide repeats 2 stathionase (cystathionine gamma-lyase)
pre-B-cell leukemia homeobox2 ST6 (alpha-N-acetyl-neuramin yl-2,3-beta-galactosyl- 3)-N- 147220979 s at ST66ALNAC5re------in-i------as-a-i-u------acetylgalactosaminide alpha-2,6-sialyltransferase 5
CTD (carboxy-terminal domain, RNA polymerase II, polypeptide A) small phosphatase-like serine/threonine kinase 3 (STE20 homolog, yeast) 150202364al------x-r------at MXI. MAX interactor 1 151204584 at LICAM L1 cell adhesion molecule Patent Application Publication Oct. 3, 2013 Sheet 56 of 73 US 2013/026100.6 A1
ethanolamine kinase 1 -0.3293.
jagged 1 (Alagile syndrome) -0.3677.
12 tripartite motif-containing 2
------PH finger rotei 15
sequence similarity 117, member A
UGT1A10 UDPglucuronosyltransferase 1 family, polypeptide AI 159220262s at DLK2 delta-like 2 homolog (Drosophila) 0.2829. 160217599 s at MDFC MyoD family inhibitor domain containing -0.3258.
161219885 at schlafen family member 12 -0.4383. 162216309 x_at JRK jerky homolog (mouse) -0.6743 stei e-rich with EGF-like domains 2
-0.4052 -0.4477 167202973 x at FAM13A family with sequence similarity 13, member A -0.3649.-0.3615 16821 1986 at AHNAK AHNAK nucleoprotein i -0.3579. YSL bystin-like -0.2825.
FK506 binding protein 14, 22 kDa -0.3488
ansmembrane and coiled-coil domains 3 : --- -0.4286 ---
B9 protein domain 0.5494 protein phosphatase 2 (formerly 2A), regulatory subunit B",
-- --.
--- protocadherin 7
------RAS protein activator like 2 ------0.6331
m scleblind-like 2 (Drosophila)
-0.8339
vimentin------0.3397
FUBPI far upstream element (FUSE) binding protein -0.2702 3637 s at MIDI midline 1 (Opitz/BBB syndrome) 182212667 at SPARC secreted protein, acidic, cysteine-rich (osteonectin) 183 207347 at ERCC6 excision repair cross-complementinggroup 6 rodent repair deficiency 184205415 s at ATXN3 ataxin 3 185215767 at ZNF804A zinc finger protein 804A 186219529 at CLIC3 chloride intracellular channel 3 ------! recombination si gnal binding protein for immunoglobulin 18721 1974-X at RBPJ kappa J region Com ------m-m-l-----
solute carrier family 16, member 1 (monocarboxylic acid rter 1) aldo-keto reductase family 1, member C3 (3-alpha 189209 160 at A hydroxysteroid dehydrogenase, type II) 190204014 at DUSP4 dual specificity phosphatase 4 -0.4 Patent Application Publication Oct. 3, 2013 Sheet 57 of 73 US 2013/0261006 A1
inc finger, MIZ-type containing 1 -0.309 frizzled homolog 2 (Drosophila) 0.9973
Rap guanine nucleotide exchange factor (GEF)2 oncogene re ted to SRC, FGR, YES eine ch transmembrane BMP regulator (chord - ike) ------
D3e molecule, epsilon associated protein inin, garnina 2 ------
------iss------no-rix------or-s-s-s-s------...s.------.aldo-keto reductase family 1, member C2 (dihydrodiol i dehydrogenase 2; bile acid binding protein; 3-alpha 200209699_x at AKR1C2 hydroxysteroid dehydrogenase, type III) : -0.6079 Patent Application Publication Oct. 3, 2013 Sheet 58 of 73 US 2013/0261006 A1
FIG. 21: Table O
RA Benchmark Signature88: i. in BJ fibroblasts, 200 up-regulated
Rank Probe set ID Acronym NetAffx Title G protein-coupled receptor, family C. group 5, i : 12031.08 at GPRC5A member A 1.3625 3-lo ------s-s-s-s-s------cysteine-rich secretory protein LCCL domain i CRISPLD2 containing 2 0.9181
XN latexin ---- 2.0417 transglutaminase 2 (Cpolypeptide, protein- : glutamine-gamma-glutamyltransferase) chemokine (C-C motif) ligand 2 transducin-likefibroblas enhancer of split 1 (E(sp1) homolog, - TLE1 Drosophila) CHST11 carbohydrate (chondroitin 4) sulfotransferase I endothelial cell-specific molecule 1 -. 102 12774 at finger protein 238 11 200832 sat stearoyl-CoA desaturase (delta-9-desaturase)
------...------12.209505 at NR2F1 uclear receptor subfamily 2, group F, member T 4 - ---
17212183 at
18220890s at DEAD (Asp-Glu-Ala-Asp) box polypeptide 47 19217220 at 202 1277 at mily with sequence similarity 171, member Al 0.4845 : 21 05479_s at PLAU plasminogen activator, urokinase & retinoic acid receptor responder (tazarotene induced
A-damage regulated ophagy modulator 24, 03372 s at - pressor of cytokine signaling 2 25218284 at SMAD3
8.----26,208868 a------is at clear receptor interacting protein family with sequence similarity 65, member B lator of G-prote n si gnalin g2,
30209278 s at TFPI2 tissue factor pathway inhibitor 2
322024.81 at w-...--who-wl:33208131-s 'ar-s-s-s:--ka-i-m-m-s------. at PTGIS prostaglandin I2 (prostacyclin)e (SDR synthasefamily) mem Patent Application Publication Oct. 3, 2013 Sheet 59 of 73 US 2013/0261006 A1
bradykinin receptor B1 1.4604 ipoma HMGIC fusion partner-like 2 1.01 metallothionein 1X
ceptor interactin 42. 43 201309 x_at chromosome 5 open reading frame 13 44208966 xat terferon, gamma inducible protein 16 45204595 s at stanniocalcin mesoderm specific transcript homolog (mouse) ------1 culin 4B
- 1B interleukin 1, beta 50 201596 x at keratin 18 51.212143 s at IGFBP3 insulin-like growth factor binding protein 3 0.3455 52205532 s at CDH6 cadherin 6, type 2. K-cadherin (fetal kidney) 1.3836.
------53213497 main at Mx--w- ABTB2------54.202637s at at adhesion molecule 1 suppressor of cytokine signaling 2 --- vitamin D (1,25-dihydroxyvitamin D3) receptor 57 12624s at CHN1 ------chimerin (chimaerin) 1 ---, 58.210002 at GATA6
64-40148 at APBB2 1310-s at CSORF13 66,212093------s at MTUS1
B, member 2 68.2094.88 s at RF RNA binding protein with multiple splicing 69.202949 s at FHL2 four and a half LIM domains 2 wingless-type MMTV integration site family, member 5A ANS N-acetylneuraminic acid synthase 0.531 ------
lysophosphatidic acid receptor 6 shevelled associated activator of morphogenesis 2 B-cell CLL/lymphomafivminhoma 6is Patent Application Publication Oct. 3, 2013 Sheet 60 of 73 US 2013/026100.6 A1
MTTF metallothionein F ------melanophilin
----- family with sequence similarity 65, member B
kinase (PRKA) anchor protei
ted protein 2,62kDa 0.3248 EPAS endothelial PAS domain protein staglandin E synthase ---w-cover-toMYOz1 myozenin 1 NR2F1 nuclear receptor subfamily 2, group F, member 1 --- 8269 at RNASEN ribonuclease type III, nuclear 90215997 s at CUL4B culin 4B 91.202446 s at PLSCR1 phospholipiddapper, antagonist scramblase of beta I catenin, homolog T 979 at DACT1 ------rrier family 16, member 4 (monocarboxylic sporter 5) 93.205234 at SLC6A4------rferon induced protein with tetratricopeptide 94203153 at FIT .repeats 1 t 0.3926 95.209487 at RBPMS RNA binding protein with multiple splicing 0.3471. ------peptidase domain containing associated with muscle : PAMR regeneration 1 SEC14-like 2 (S. cerevisiae) ------0.9893. dehydrogenase I family, member Al 0.4935 ------serpin peptidase inhibitor, clade B covalbumin), -- 99.209723 at SERPINB9 member 9 0.7879, ---100,201416 ru-irawa-a-saucas-a--ms---it-a-misr-e-r-w-u-la------n-in-a-vu-al...a at SOX4 SRY (sex determining------region Y)-box 4 ------as------0.9518 NA------binding protein with multiple ------Splicing 0.4986 101.207836 s at ...... ------218025 sat , , , - PECI --- peroxisomal D3, D2-enoyl-CoA isomerase 0.2676, v myb myeloblastosis viral oncogene homolog ------103.204798 at MYB (avian) -3-m-- RARB nudixretinoic (nucleoside acid receptor, diphosphate beta linked moiety- X).------105.212181s at NUDT4IST3H2A typehistone motif4 cluster 3. H2a -- 0.5044
. . . . . ular retinoic acid binding p ------() 2248 at ELF 74-like factor 1 (ets domain transcription factor) --- platelet-derived growth factor receptor, alpha 111 203131 at PDGFRA polypeptide ------0.5284 Patent Application Publication Oct. 3, 2013 Sheet 61 of 73 US 2013/026100.6 A1
glucuronidase, beta? immunoglobulin lambda-like polypeptide pseudogene matrix Gla protein
metallothionein IH 0.8472 polo-like kinase 2 (Drosophila) sterile alpha motif domain containing 9
r ydrolase domain containing 2 ------le phosphorylase I neuroepithelial cell transforming 1 tyrosy-DNA phosphodiesterase I
mechanistic target of rapamycin (serine/threonine ---...------| 121202288 at kinase) 122204255 is at VDR vitamin D (1,25-dihydroxyvitamin D3) receptor 123,202036_s_at- - - secreted frizzled-related protein 1 : 124216336 xat metallothionein IE metallothionein pseudogene 2 63 growth differentiation factor 15 tein kinase, AMP-activated, gamma 2 non
regnancy specific beta-l-glycoprotein 1
bhydrolase domain containing 5 ------
5 domain containing 4A --- ; RY (sex determining region Y)-box 4 potassium inwardly-rectifying channel, subfamily J, member 2 0.9798 ocyte membrane protein band 4.1 like 4B a on wated protein kinase kinase kinase 8 paired box 3 --- -- Rho-related BTB domain containing 3 140207069 sat SMAD family member 6
141203629 s at component of oligomeric golgi complex 5 MP3 TIMP metallopeptidase inhibitor 3 143,219283 at C1GALTICI CIGALT1-specific chaperone 1442) 1711s at PTEN phosphatase and tensin homolog --- COX11 homolog, cytochrome c oxidase assembly ------ACSL3COX) --- oteinyl-CoA (yeast) synthetase long-chain family member 3 147205085 at ORC1L origin recognition complex, subunit 1-like (yeast) 148 213260 at FOXC1 149201860s: at PLAT 150204146 at RAD51API RAD51 associated protein 1 - Patent Application Publication Oct. 3, 2013 Sheet 62 of 73 US 2013/026100.6 A1
in 10
0.87 0.5473 insulin-like growth factor binding protein 3 ------IGFBP3.------0.324 exin All 0.3222.
158210612------as at ------w:---- naptojanin 2 ...... 0.6355 159200701 at NPC2 liemann-Pick disease, type C2 0.2681
160215774_s_at------...------. 0.5239 1612105.2 s at VEGFA vascular endothelial growth factor A ------0.8526 ------162215182x at null 0.4776. 163206240 s at ZNF136 inger protein 136 16420873 at HNRNPULI eterogeneous nuclear ribonucleoprotein U-like t ------...... 0.2701 IM1 dimethyladenosine transferase 1-like (S.------iron------, 1652.10802 s at DIMTFL s-as------..ws.------w-s. 166206675 s at SKIL
------s------1672.16180 s at SYNJ2
oviral IAP repea wingless-type MMTV integration site family, 172213425 at WNTSA member 5A 173218820 at C14ORF132 chromosome 14 open reading frame 132 0.597; ORAI calcium release-activated calcium modulator - 2 174218812_s_at IORAI2 - - - , , , , , , ------; : -es---slam-les------1752,785 - 176203394 sat hairy and enhancer of split------1. (Drosophila) as-s-s-a-ul-u-vee---as-as-a----ruravy---0.553 r------calcineurin-like phosphoesterase domain containing------17721861.0 s at CPPED i 178217783 s at 1792 1966 at Cl4ORF10.-- mosome 14 open reading frame 104 ' - - - - 180202393 s at KLF1 ppel-like factor 10 mago-nashi homolog 2, proliferation-associated IMAGOH2 (Drosophila) W.W. --
utaminyl-peptidesue factor pathway cyclotransferase inhibitor 2 0.3 184205398 s at 185203851 at 186212786 at 187206084 at protein tyrosine phosphatase, receptor type, R ------...--0.7848 SH3 and cysteine rich domain ------0.4111. Patent Application Publication Oct. 3, 2013 Sheet 63 of 73 US 2013/0261006 A1
: -->a------1902 1990 at FGD6 FYVE. RhoGEF and PH domain containing 6 ------...--1912210 COLEC12 collectin sub-family member r------us------girl------wa-...------amyloid beta (A4) precursor protein-binding, famil
192212970 at APBB2 B, member 2 ------'ra------193220 186s at PCDH24 protocadherin 24 in crewswimwww.rwinill-ul-ulu...ww.w---aww.------...amyloid beta (A4) precursor------protein-binding, family i ------194212972_x T.I.T.T. at APBB2- IT------B, member ------sur-wa 2 0.6666, 195203098 at CDYL chronodomain protein, Y-like 0.357 1965.1158 at FAM174B w-ul-win...-----sa-family with -sequence - -c.c.------...-ar-s-s-s-s---irrin- similarity 174, member B - is 1972.19759 at ERAP2 endoplasmic reticulum aminopeptidase 2 0.561. - - - ATP-binding cassette, sub-family A (ABC). 198,203504: ---now-sture--two-lug-i-swer.-rewar---out-wr.t. sat ABCA1 Emember ------, 1 ------0.9099 y t integrin, alpha 4 (antigen CD49D, alpha 4 subunit of i ------199205885_s is:------4------...------...wa. at ITGA4 VLA-4 receptor) - 0.2878, 200 201149 s at TIMP3 TIMP metallopeptidase inhibitor------wi...... w------i-...------wor-a: 3 0.5748 Patent Application Publication Oct. 3, 2013 Sheet 64 of 73 US 2013/0261006 A1
F.G. 22: Table P RA Benchmark Signature in BJ fibroblast, 200 down-regulated
Log2FC tRA to Rank Probe set ID Acronym NetAffx Title ------DMSO 1210120s at RANBP3 TRAN binding protein 3 -0.4156 i ------. solute carrier family 2 (facilitated glucose transporter),------2 216236_s_at SLC2A14 member 14 -0.5463 3,206924 at Illi interleukin ------0.5997,
--Mirror-i-ur-raorussex-:--4204933 s at TNFRSF11B------tumor necrosis factor receptor superfamily, member 11b -0.9241
5,213496 at LPPR4 plasticity related gene X------0.47 18: ------was------6,209288 is at CDC42EP3 CDC42 effector protein (Rho GTPase binding)3 -0.3328. ------7204011 at SPRY2 sprouty homolog2 (Drosophila) 2-ax-----z------mn------0.6182 ------e-...------...------8222162 s at ADAMTS1 ADAM metallopeptidase with thrombospondin type 1 motif, 1 ... 1095 !------la------x------wu------
9201423 sat CUL4A cullin 4A18-saw88-'ko-y:saw:------...sw-wall:-wa------simm:ww.8.x: ww:...---- 0.29 - -r-,-----suu-a-vu-ee------recisix-wor-in-rural---www.10209386 at TM4SFI transmembrane 4 L six family member 1 Mew.------ee 11204948 s at FST follistatin ------:005
------1220984.1 s at leucine rich repeat neuronal 3
13215034s at TM4SFI transmembraneamc-mara war--va-wa-n-mina-Murawc-user-rx 4 L six family member------1 ------07015 re-arc------as------14213906 at v-myb myeloblastosis viral oncogene homolog (avian)-like 1 -0.6493; mix-ur-a- swor--aw...aue------ee-no-no------willow 15 215336 at A kinase (PRKA) anchor protein 11 ------viru-w- ww.cv----was--8ws------vuunun-0.7124 : r ro o : 16206157 at PTX3 pentraxin-related------suck-u...... -lx gene, rapidly induced by '------mak-r-in----...------&-----, IL-1 beta i -0.7367 -m-www.uk 17210310s at FGF5 fibroblast growth factor 5 -0.3979. 18204014 at DUSP4 dual specificity phosphatase 4 -0.7517, i transducin-like enhancer of split 4 (E(spi) homolog. 19204872 at TLE4 Drosophila) s -0.6695 2022 1528_s_at ELMO2 engulfment and cell motility 2 -0.5656. 21203794 at CDC42BPA CDC42 binding protein kinase alpha (DMPK-like) ------&x.-0.3263; ------it-ou---it-a-low------22.212915 at PDZRN3 PDZ domain containing ring finger--wxw------wire-w.ox.ac.uw-awar-----ax-Xa-we-two-www.www. 3 ------r ------as-s-s-s-s-s-s-s-la------wn---assaul
car. 23208961s at KLF6 Kruppel-like factor 6 ------24206814 at ---NGF t nerve growth- factor- (beta polypeptide) ------
25212745 s at BBS4 ardet-Biedl syndrome 4 26203476 at TPBG 27,208025 sat 4 Zuev4-s-at rivatiz. influenza virus NS1A bit ding protein irr-i----its-ii28 201363 sat IVNSIABP co-e-r-cr--- -0.2912, 29218706 s at GRAMD3 GRAM domain containing 3 ------w-sk-ea------a-row-sau-0.3717
T30212230:---arrows-we-a-sw-----eas------w-sur-w-s.------at --- PPAP2B phosphatidic acid phosphatase type 2B ------s -03096,
-sul-wall-scalwica31 201395 alwaxaca-k-k-k-well at RBM5 awa-ur-cy--- RNA binding motif protein 5 0.2941
-----somiecess-well-e-or-way------32209387s at TM4sFi s---warx-r-virus---a-w-s.------transmembrane 4 L six family member 1 E.
3360474------Ysarcus------it-arms-smarca--ce----v-crew- at FERMT1 fermitinawarcs-x-x8:a::...'...was--w:-: family homolog 1 (Drosophila) 0.3397 34202708 s at HIST2H2BE histone cluster 2, H2bes---a-sixes------Asia---&-a-we------x-x-x------wiwa-e-w-s.------xxis-six stria------ee-war:-0.3994 ------potassium intermediatelsmall conductance calcium-activated
35,220116 at KCNN2 channel, subfamily N. member 2 -0.5252 36221696 s at ST serine/threonine tyrosine kinase 1 - -0.7139, Patent Application Publication Oct. 3, 2013 Sheet 65 of 73 US 2013/026100.6 A1
absent in melanoma 1 iesterase 1C, cal nodulin-dependent 70kDa -07434 ghtless I homolog (Drosophila) 0.3338. 40209840s at LRRN3 leucine rich repeat neuronal 3 1.01.04 590 at RASGRP1 RAS guanyl releasing protein 1 (calcium and DAG regulated) -O.749 42 08482 at SSTR M- somatostatin receptor m -09081 43219973 at ARSJ arylsulfatase family, member J M gremlin 1. cysteine knot superfamily. homolog ( Xenopus ---
nily, member 11b 0. ST3 beta-galactoside alpha-2.3 sialyltransferase 5
203708 at
h mobility group box
potassium channel, subfamily K. member 2 t Rap/RangAP
inhibin, beta A homeobox p/p300 interacting transactivator, with Glu/Asp-rich boxy-terminal domain, 2 66215498 s 67208727s at
RAsp21 protein activator (GTPase activating protein)
lponin and LIM 75218376 s at MICAL -----ma.u-33---33-3-3-m- Patent Application Publication Oct. 3, 2013 Sheet 66 of 73 US 2013/026100.6 A1
gremlin 1, cysteine knot superfamil homolog (Xenopus laevis)
membrane associated ring finger (C3HC4)3 al specificity phosphatase 5
------.
deleted in lymphocytic leukemia 2 (non-prot -w'''''m 'm' chemokine (C-XC motif) ligand 12 ( stromal cell-derived factor I)
early growth response 1
-- rotein 38 ATPase, Ca++ transporting, plasma membrane 4 HLA-B associated transcript 2 ulin-like growth factor 2 mRNA binding protein 3 adenomatous polyposis coli Jo-luma- disabled homolog2, mitogen-responsive phosphoprotein ( Drosophila)
B11 family interacting protein
dual specificity phosphatase 10 ----...------mm-l serpin peptidase inhibitor, clade B (ovalbumin), member 2
atural killer-tumor recognition sequence disabled homolog 2, mitogen-responsive phosphoprotein 98.201279_s_at DAB2 Drosophila) inhibitor of DNA binding 2, dominant negative helix-loop 99.213931 at ID2 helix protein 100211040 x at GTSE1 102.2073 at CSNK2AI 102.214682 at Loc39949 g protein -0.6861 103220606 s at C17ORF48 chromosome 7 open reading frame 48 0.3041
104220738s at RPS6KA6 ribosomal protein S6 kinase,90kDa, polypeptide 6 -0.6137 105 219696 at ENN/MADD domain containing IB T 106204004 at RKC, apoptosis, WT1, regulator ------m-m-m-m-m-m-- m se, Ca++ transporting plasma membrane 1 ; 108203736 s. at PPFIBP. PTPRF interacting protein, binding protein (liprin beta I) -0.3131. 109214668 at C13ORF1 hromosome 3 open reading frame LIMprotein W''S . ------a-a-a-u------I 10200965.s at ABLIM1------fins-related tyrosine kinase I (vascular endothe?ial growth
--- factor/vascular permeability factor receptor) -0.3405--8-8 yocyte enhancer factor 2c -0.514
translocation (myeloid leukemia associated) pseudoge Patent Application Publication Oct. 3, 2013 Sheet 67 of 73 US 2013/0261006 A1
smoothelin Guanine nucleotide binding protein (G protein), beta -
RAS p21 protein activator (GTPase activating protein) .
YKT6 v-SNARE homolog (S. cerevisiae) ------ELK1, member of ETS oncogene family fibroblast growth factor 5 solute carrier family 2 (facilitated glucose transporter), member 14
kyrin repeat domain 12
disabled homolog 2, mitogen-responsive phosphoprotein : (Drosophila) KDMSD lysine (K)-specific demethylase 5D 129204361s at SKAP2 src kinase associated phosphoprotein 2 130202464 S at PFKFB3 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 exis-ou--ms----...-:-8-3------: -. T ------. nephroblastoma overexpressed gene -- is at PRRX1 paired related homeobox 1 133219431 at ARHGAP10 Rho GTPase activating protein 10 ARP2 actin-related protein 2 homolog (yeast)
arrier family 35, member El : ms-related tyrosine kinase 1 (vascular endothelial growth --- factor/vascular permeabilit factor receptor)
141212098 at LOC151162 hypothetical LOC151162 142.212218s at FASN fatty acid synthase
43 202080 s at TRAK1 trafficking protein, kinesin binding i-2 and S-phase expressed .. ------sarcoglycan, delta (35kDa dystrophin-associated glycoprotein) -- bromodomain containing 2 147.219254 at C17ORF101 chromosome 17 open reading frame 101 14s 222190 s. at C16ORF58 - hromosome 16 open reading frame 58 1492.13939 s at RUFY3 RUN and FYVE domain containing 3 s : 150202948 at interleukin 1 receptor, type I -0.3306 151222108 at AMIGO2 adhesion molecule with Ig-like domain 2 -0.4444 152203387 s at TBC1D4 TBC1 domain family, member 4 0.3451 ------
19635 at ZNF606 inc finger protein 606 i------...------0.2757, Patent Application Publication Oct. 3, 2013 Sheet 68 of 73 US 2013/0261006 A1
154204720s at DnaJ (Hsp40) homolog, subfamily C, member 6 155220305 at MAVs mitochondrial antiviral signaling protein -0.3294. p-or-to------protein phosphatase 3. (formerly 2B), regulatory subunit B, s 156.204506 at alpha isoform s -0.5595
157218970s at CUTC cutC copper transporter homolog (E. coli) -0.2703 158210311 at FGF5 fibroblast growth factor 5 59 09203 s at BICD2 bicaudal D homolog 2 (Drosophila) ...... ------160209209_s at FERMT2 fermitin family homolog 2 (Drosophila) - - - - - 1612032.42 s at PDLIM5 PDZ and LIM domain 5
162206237 s at NRG1 neuregulin ------1959959 at FAM110B family with sequence similarity 110, member B 219563 at 14ORF139 - chromosome 14 open reading framei 39 ------
165204190 at USPL ubiquitin specific peptidase like 166216933 x at APC adenomatous polyposis coli 167209946 at VEGFC vascular endothelial growth factor C 168206307 s at 169212365 at 170215177 s at integrin, alpha 6 171202342 s at TRIM2 tripartite motif-containing 2 1722.03386 at TBC1D4 TBC1 domain family, member 4 173213672 at MARS methionyl-tRNA synthetase ------eceptor (G protein-coupled) activity modifyin g protein insulin-like growth factor binding protein 5 (BCL2-related)
CD55 molecule, decay accelerating factor for complement 178.201925 s at CD55 (Cromer blood group) -0.3644 17921862 x at CFLAR CASP8 and FADD-like apoptosis regulator -0.2666 I-----Tour-180212364 at ------ow.------0.2759. 181205798 at IL7R interleukin 7 receptor transducin-like enhancer of split 4 (Eispi) honolog. 1822.16997_x at TLE4 Drosophila) PPP1R3 tein phosphatase 1. regulatory (inhi itor) subunit 3C 184221276185202674 iss at SYNCLMO7 syncoilin,LIM domain intermediate 7 filament protein ------.S.------s-s-s-s-s------186202647s at NRAS neuroblastoma RAS viral (v-ras) oncogene homolog protein-coupled receptor 172A sociated membrane protein 4 ffector protein (Rho GTPase binding)3 tripartite motif-containing 45 0.7815 microphthalmia-associated transcription factor
solute carrier family 35, member F5 -- - SPTLC2 serine palmitoyltransferase, long chain base subunit 2
237 x at PAWR PRKC, apoptosis, WT1, regulator Patent Application Publication Oct. 3, 2013 Sheet 69 of 73 US 2013/0261006 A1
chromosome 3 open reading frame 18 kyrin repeat and SOCS box-containing 6
tein phosphatase 1, regulatory (inhibitor) subunit 12A 1982.1828s at TNIK RAF2 and NCK interacting kinase
protein phosphatase 3 (formerly 2B), catalytic subunit, beta 199202432 at PPP3CB isoform 20020965.5 s at TMEM47 transmembrane protein 47 Patent Application Publication Oct. 3, 2013 Sheet 70 of 73 US 2013/0261006 A1
FIG. 23: Table Q Average CMap scores for some representative potential skin-lightening agents with the Retinoic Acid Keratinocyte RA 200 Signature
Cell Average Chemical Line Concentration CMap score All-trans retinoic acid thC 1804 Mvo-inositol 128 Hexyldecanol O. 10% Chlorhexidine tkC | 10 uM Patent Application Publication Oct. 3, 2013 Sheet 71 of 73 US 2013/0261006 A1
FIG. 24: Table R Comparison of the predicti veness of different C-map signatures for predicting the activity of compounds in the mouse B 16 melanoma cell melanogenesis assay
Average C-map Score Chemical Composite Hexamidine NAG Niacinamide Sepiwhite Sepiwhite MSH Benchmark 0.95 Nicotinamide t Benchmark .. 7 ().75 Hexamidine disethionate Benchmark 0.93 O.32 N-acetyl-D-glucosamine Benchmark O.69 ------09 | Ennodin Inhibitor 0.97 chlorhexidine diacetate -- Inhibitor 0.9 au- 0.33 - Usnic Acid Inhibitor O.9 0.88 O.32 0.88 O.63 Nordihydroguaiaretic acid Inhibitor (.87 0.75 from Larrea divaricata (creosote bush) Tergitol NP 10 inhibitor 0.87 --alom-ul- 0.57 3,4,4-Trichlorocarbanilide Inhibitor 0.86 U-753.02 Inhibitor 0.81 0.55 0.65 AA-86 Inhibitor m 0.53 Boswellin CG Inhibitor Ricinoleic acid Inhibitor ------Nw-Nitro-L-arginine methyl Inhibitor i ester HCl : SB 28795 Inhibitor 0.62 : chlorhexidine inhibitor 0.4s 0.43 Tetrahydrocurcumin CG Inhibitor 0.44 HYDROQUINONE inhibitor FH 6-Hydroxy-1,3-benzoxathiol- Inhibitor 2-One 6-HYDROXYINDOLE Inhibitor Acetyl tributyl citrate Inhibitor TT |- Berberine Chloride inhibitor f ------
Brij98 ------X-X- inhibitor I ------Copper(II) D-gluconate inhibitor Oley alchol Inhibitor Piroctone olamine Pluronic LO Tannic acidi Inhibitor tert-BUTY inhibit (r HYDROQUINONE inhibitor Patent Application Publication Oct. 3, 2013 Sheet 72 of 73 US 2013/026100.6 A1
Total inhibitors and benchmarks identified Note: no C-map score means that the chemical was not a hit (i.e. in the top 200 instances of the corresponding signature with a score >=0.30). Patent Application Publication Oct. 3, 2013 Sheet 73 of 73 US 2013/0261006 A1
FIG. 25: Table S - - I T Number of probe sets with significant (<0.05) p
Skin Tone Benchmark values compared to DMSO controls Skin one Benchmar Concentration Tert- HRL | HeMnMP Materials keratinocyteskeratinocy melanomamel melanocytes
Hexamidine 5 M : 4263 1497 2359
disothionate L i Myo-inositol 5082 s 960 36.87
N-acetyl-glucosamine NDP-MSH I 73 Niacinamide 100 uM - 298 - 1345 1312 Sepiwhite - O LM 245 883 230 US 2013/026100.6 A1 Oct. 3, 2013
METHOD OF GENERATINGA found among those taking oral contraceptives, and is occa HYPERPGMENTATION CONDITION GENE sionally found among nonpregnant women who are not tak EXPRESSION SIGNATURE ing oral contraceptives, and sometimes among men. A pattern of similar facial hyperpigmentation is associated with a BACKGROUND OF THE INVENTION chronic liver disease called chloasma. A common condition 0001 Skin pigment irregularities are common across eth associated with aging skin is the development of dark spots nic and racial groups and are often considered cosmetically sometimes referred to as “age spots” or “liver spots.” Other disfiguring. Disorders of pigment production and distribution forms of hyperpigmentation can be caused by UV irradiation, occur as a function of intensity and duration of UV radiation in particular UVB radiation which up-regulates the produc exposure, life style habits, chronological age, endocrine func tion of tyrosinase resulting in skin "tanning,” or may result tioning and disease state and are found ubiquitously in older from a genetic predisposition for the condition, or may come populations. Hence there is a widespread demand for skin about in association with a skin inflammatory event or during pigment modifying, skin lightening and skin tone enhancing the course of wound healing. products for the cosmetic market. 0006 Vitiligo is a form of hypopigmentation in which 0002 The color of normal human skin is due primarily to cutaneous melanocytes are either ablated or fail to produce varying amounts and distribution of melanin, hemoglobin, Sufficient pigment. Ideally treatment would restore lost pig and carotenoids. Of these pigments, melanin is of primary mentation in vitiligo-affected skin, but this approach has met significance to cosmetic skin treatment protocols. Melanin is with little Success via topical interventions and formulations. produced by specialized cells in the skin called melanocytes Although cosmetic camouflage with dihydroxyacetone Sun through a complicated series of chemical and enzymatic reac less-tanning lotions provides some darkening of hypo-pig tions, mainly involving the copper and manganese containing mented areas, it also tends to darken Surrounding normal skin, enzyme tyrosinase. Once synthesized, the melanin granules Substantially maintaining the undesirable contrast. Hence, a are packaged into melanosomes and transferred via the cel more favored cosmetic approach is to reduce the normal lular dendrites (extensions) of the melanocyte to the sur pigmentation of the unaffected skin to reduce contrast and rounding keratinocytes, the most abundant cell type in the produce a tone evening effect. epidermis. The rate of melanin synthesis, and the Subsequent 0007. Several proven targets for pigmentation control are transfer of melanin by melanocytes via their dendrites, known, but these have generally been derived from an under appears to be influenced by ultraviolet light exposure. Mel standing of the pigmentation process. Hydroquinone anosomes transferred to the outer layer of the skin are respon (parahydroxy-benzene), for example, is a widely used skin sible for the darkening of the skin, with the degree of dark lightening agent that is known to provide a satisfactory cos ening being associated with skin type, Sun exposure, and/or metic result, however its use strictly for cosmetic purposes is certain dermatological conditions. discouraged due to its association with a variety of disorders, 0003. Two types of melanin are present in human skin: (1) including diabetes, hypertension, ochronosis, periorbitary eumelanin, which is the dark brown-black pigment found in dySchromia, infectious dermatosis, contact eczema, extended most skin, hair, and eyes, and whose production is stimulated dermatophytosis, and necrotizing cellulites (see, e.g., by exposure to ultraviolet light, and (2) pheomelanin, which Raynaud E. et al., Ann Dermatol Venereol 128(6-7):720-724, is a yellow-orange pigment found mainly in the skin of very 2001). Hydroquinone has also shown genotoxic and fair-skinned people, particularly those with red hair. The per mutagenic activities (see, e.g., Jagetia G. C. etal, Toxicol Lett ceived color of skin is determined by the ratio of eumelanins 121(1):15-20, 2001). Due to concerns over toxicity and car to pheomelanins, and to a smaller extent on blood within the cinogenic effects, the United States limits treatment solutions dermis. to a 2% or lower concentration and the FDA has proposed a 0004. The pigmentation pathway has been elucidated in ban on all over-the-counterpreparations, while hydroquinone detail. Summarily, melanin forms through a series of oxida is currently banned in Europe as a skin lightening or depig tive reactions involving the amino acid tyrosine in the pres menting agent. ence of the enzyme tyrosinase. Tyrosinase converts tyrosine to dihydroxyphenylalanine (DOPA) and then to 0008 Kojic acid, Azelaic acid and certain-hydroxy acids dopaquinone. Subsequently, dopaquinone is converted to Such as glycolic acid, have demonstrated skin-lightening dopachrome through auto-oxidation, and finally to dihy effects, but reports of localized irritation and inflammation droxyindole or dihydroxyindole-2-carboxylic acid are common. The prenylated flavonol artocarpin has shown (DHICA), which polymerize to form eumelanin. The latter Some efficacy for skin-lightening in the context of ultraviolet reactions occur in the presence of dopachrome tautomerase induced skin pigmentation (Shimizu K. et al., Planta Med and DHICA oxidase. In the presence of sulfur-containing 68(1):79-81, 2002). cysteine or glutathione, dopaquinone is converted to cysteinyl 0009 Recently, a more detailed genomic and proteomic DOPA or glutathione DOPA: subsequently, pheomelanin is understanding of melanogenesis, the melanocyte, melano formed. cyte-keratinocyte interaction, and the melanocyte-fibroblast 0005. A variety of skin hyperpigmentation disorders are interaction has revealed potentially hundreds of proteins and known and etiology is diverse, overlapping in many cases, other effectors involved in the pigmentation process and in and often not fully understood. For example, melanosis or the etiology of hyperpigmentation disorders, which may pro melasma is a condition characterized by the development of vide additional targets. There is a need in the cosmetic arts sharply demarcated blotchy, brown spots usually in a sym both for generating potential skin lightening agents and for metric distribution over the cheeks, forehead, and sometimes effective and efficient screening methods for identifying on the upper lip and neck. This condition frequently occurs putative skin active agents with efficacy and safety in the during pregnancy (melasma gravidarum or “mask of preg cosmetic treatment of hyperpigmentation and pigmentation nancy'), and at menopause. Also, this condition is frequently disorders. US 2013/026100.6 A1 Oct. 3, 2013
0010 Traditionally scientists have focused on the devel mapping, with its rigorous mathematical underpinnings and opment and provision of safe and effective topical composi aided by modern computational power, has resulted in con tions formulated to lighten skin and Such an approach has firmed medical Successes with identification of new agents been useful for treating localized epidermal hyperpigmenta for the treatment of various diseases including cancer. None tion and for masking areas of skin hypopigmentation. There theless, certain limiting presumptions challenge application remains a need, however, for safe and effective agents capable of C-map with respect to diseases of polygenic origin or of delivery through topical application to reduce the degree of syndromic conditions characterized by diverse and often skin pigmentation in both contexts. apparently unrelated cellular phenotypic manifestations. 0011 Skin pigmentation and the broader cosmetic con According to Lamb, the challenge to constructing a useful cept of skin tone, are therefore highly complex conditions C-map is in the selection of input reference data which permit with multiple and overlapping etiologies, which manifest in generation of clinically salient and useful output upon query. part as a function of individual predisposition, and which For the drug-related C-map of Lamb, strong associations therefore pose a significant treatment challenge. There is a comprise the reference associations, and strong associations need in the art for methods of identifying potential skin pig are the desired output identified as hits. ment modifying agents, and in particular skin-lightening 0015 Noting the benefit of high-throughput, high density agents, and for evaluating the efficacy of putative skin active profiling platforms which permit automated amplification, agents using screening methods that are substantially inde labeling hybridization and Scanning of 96 samples in parallel pendent of mechanism of action or etiology of the pigment a day, Lamb nonetheless cautioned: "even this much fire condition. The present investigators therefore undertook an power is insufficient to enable the analysis of every one of the investigation into the application of a relatively new technol estimated 200 different cell types exposed to every known ogy known as “connectivity mapping to the search for new perturbagen at every possible concentration for every pos skin-active agents with efficacy in the treatment of hyperpig sible duration ... compromises are therefore required’ (page mentation disorders and related skin conditions. 54, column 3, last paragraph). Lamb, however, took the posi 0012 Connectivity mapping is a well-known hypothesis tion that cell type did not ultimately matter, and confined his generating and testing tool having Successful application in C-map to data from a very small number of established cell the fields of operations research, telecommunications, and lines out of efficiency and standardization concerns. Theo more recently in pharmaceutical drug discovery. The under retically this leads to heightened potential for in vitro to in taking and completion of the Human Genome Project, and the Vivo mismatch, and limits output information to the context of parallel development of very high throughput high-density a particular cell line. If one accepts the Lamb precept that cell DNA microarray technologies enabling rapid and simulta line does not matter then this limitation may be benign. neous quantization of cellular mRNA expression levels, 0016. However, agents suitable as pharmaceutical agents resulted in the generation of an enormous genetic database. At and agents suitable as cosmetic agents are categorically dis the same time, the search for new pharmaceutical actives via tinct, with the former defining agents selected for specificity in silico methods such as molecular modeling and docking and which are intended to have measurable effects on struc studies stimulated the generation of vast libraries of potential ture and function of the body, while the latter are selected for Small molecule actives. The amount of information linking effect on appearance and may not affect structure and func disease to genetic profile, genetic profile to drugs, and disease tion of the body to a measurable degree. Cosmetic agents tend to drugs grew exponentially, and application of connectivity to be substantially non-specific with respect to effect on cel mapping as a hypothesis testing tool in the medicinal Sciences lular phenotype, and administration to the body is generally ripened. limited to application on or close to the body Surface. 0013 The general notion that functionality could be accu 0017. In constructing C-maps relating to pharmaceutical rately determined for previously uncharacterized genes, and agents, Lamb stresses that particular difficulty may be that potential targets of drug agents could be identified by encountered if reference connections are extremely sensitive mapping connections in a database of gene expression pro and at the same time difficult to detect (weak), and Lamb files for drug-treated cells, was spearheaded in 2000 with adopted compromises aimed at minimizing numerous, dif publication of a seminal paper by T. R. Hughes et al. “Func fuse associations. Since the regulatory scheme for drug prod tional discovery via a compendium of expression profiles' ucts requires high degrees of specificity between a purported Cell 102, 109-126 (2000), followed shortly thereafter with drug agent and disease state, and modulation of disease by the launch of The Connectivity Map (-map Project by Justin impacting a single protein with a minimum of tangential Lamb and researchers at MIT (“Connectivity Map: Gene associations is desired in development of pharmaceutical Expression Signatures to Connect Small Molecules, Genes, actives, the Lamb C-map is well-suited for screening for and Disease'. Science, Vol 313, 2006.) In 2006, Lamb's group potential pharmaceutical agents despite the Lamb compro began publishing a detailed synopsis of the mechanics of 1SCS. C-map construction and installments of the reference collec 0018. The connectivity mapping protocols of Lamb would tion of gene expression profiles used to create the first gen not be predicted, however, to have utility for hypothesis test eration C-map and the initiation of an on-going large scale ing/generating in the field of cosmetics or for a primarily community C-map project, which is available under the “Sup cosmetic disorder where symptoms may be diffuse, systemic porting materials' hyperlink at http://www.sciencemag.org/ and relatively mild. In complete contravention of the goal of content/313/5795/1929/suppl/DC1. pharmaceutical active discovery, cosmetic formulators seek 0014. The basic paradigm of predicting novel relation agents or compositions of agents capable of modulating mul ships between disease, disease phenotype, and drugs tiple targets and having effects across complex phenotypes employed to modify the disease phenotype, by comparison to and conditions. Further, the phenotypic impact of a cosmetic known relationships has been practiced for centuries as an agent must be relatively low by definition, so that the agent intuitive Science by medical clinicians. Modern connectivity avoids being Subject to the regulatory scheme for pharmaceu US 2013/026100.6 A1 Oct. 3, 2013 tical actives. Nonetheless, the impact must be perceptible to useful for testing and generating hypotheses aboutskin-active the consumer and preferably empirically confirmable by sci agents and hyperpigmentation skin disorders. entific methods. Gene transcription/expression profiles for cosmetic conditions are generally diffuse, comprising many 0024. The present invention provides embodiments which genes with low to moderate fold differentials. Cosmetic broadly include methods and systems for determining rela agents, therefore, provide more diverse and less acute effects tionships between a skin condition/disorder of interest and on cellular phenotype and generate the sort of associations one or more skin-active agents, one or more genes associated expressly taught by Lamb as unsuitable for generating con with the skin disorder condition, and physiological themes nectivity maps useful for confident hypothesis testing. implicated by the skin condition and/or affected by a skin 0019. Successful identification of skin lightening agents active agent. The inventive methods may be used to identify has proven to be difficult due to the multi-cellular, multi skin-active agents without detailed knowledge of the mecha factorial processes involved in etiology of the hyperpigmen nisms of biological processes associated with a skin disorder tation condition itself. Conventional in vitro studies of bio or condition of interest, all of the genes associated with Such logical responses to potential skin-lightening agents can be a condition, or the cell types associated with Such a condition. hindered by the complex or weakly detectable responses typi cally induced and/or caused by the putative skin active or 0025. One aspect of the invention provides methods for potential skin active agents. Such weak responses arise, in constructing a data architecture for use in identifying connec part, due to the great number of genes and gene products tions between perturbagens and genes associated with skin involved, and the fact that skin-active and cosmetic agents tone, comprising: (a) providing a gene expression profile for may affect multiple genes in multiple ways. Moreover, the a control human cell, wherein the control cell is from a human degree of bioactivity of cosmetic agents may differ for each cell line selected from the group consisting of keratinocyte, gene and be difficult to quantify. fibroblast, melanocyte and melanoma cell lines; (b) generat 0020. The value of a connectivity map approach to dis ing a gene expression profile for a human cell exposed to at cover functional connections among cosmetic phenotypes least one perturbagen, wherein the cell is selected from the Such as hyperpigmented skin, gene expression perturbation, same cell line as the control cell; (c) identifying genes differ and cosmetic agent action is counter-indicated by the pro entially expressed in response to the at least one perturbagen genors of the drug-based C-map. The relevant phenotypes are by comparing the gene expression profiles of (a) and (b); (d) very complex, the genetic perturbations are numerous and creating an ordered list comprising identifiers representing weak, and cosmetic agent action is likewise diffuse and by the differentially expressed genes, wherein the identifiers are definition, relatively weak. It is unclear whether statistically ordered according to the differential expression of the genes; valid data may be generated from cosmetic C-maps and it is (e) storing the ordered list as an instance on at least one further unclear whether a cell line exists which may provide computer readable medium, wherein the instance is a kerati salient or detectable cosmetic data. nocyte, fibroblast melanocyte or melanoma instance accord ing to the selection in (a); and (f) constructing a data archi SUMMARY OF THE INVENTION tecture of stored instances by repeating (a) through (e), 0021. Accordingly, the present inventors have developed a wherein the at least one perturbagen of step (a) is different C-map approach that Surprisingly enables discovery of skin qualitatively or quantitatively for each instance. In specific tone agents having efficacy for disorders of skin pigmenta embodiments each instance is repeated twice in C-map test tion. ing. Example 4 illustrates generating a benchmark skin tone 0022. The present inventors discovered that useful con agent signature using fibroblasts to create signatures and nectivity maps could be developed from cosmetic active— using keratinocytes to create signatures. cellular phenotype gene expression data associations in 0026. The data architecture may be mined to identify rela particular with respect to hyperpigmentation actives and cos tionships between perturbagens, genotypes and phenotypes metic agents, despite the highly diffuse, systemic and low and may also be used as an in silico tool for generating new level effects these sorts of actives generally engender. actives with potential efficacy for treatment of a cosmetic Although the Lamb team asserted that results should be sub condition. The data architecture may be implemented for this stantially independent of cell-type, the present inventors Sur purpose through the use of a query, which is an input to the prisingly discovered that selection of cell line affects the C-map wherein the output is based on connectivity Scores to utility of the C-map for hypothesis generating and testing the query. In one embodiment, a method for implementing the relating to skin pigmentation actives and treatment of hyper data architecture to identify at least one putative skin active pigmentation disorders. In particular, keratinocyte cells, agent having potential efficacy in treating a skin pigmentation rather than melanocyte or melanoma cells, exhibited a more condition is provided. The method comprises querying the robust transcriptional profile when treated with skin-lighten data architecture with a pigmentation-relevant gene expres ing agents, and there was little to no thematic overlap between sion signature, wherein querying comprises comparing the cell types treated with the same benchmark skin active agent pigmentation-relevant gene expression signature to each (shown in Example 8). stored cell instance, and further wherein the pigmentation 0023. Accordingly, the present invention provides novel relevant expression signature represents genes differentially methods, systems and models useful for generating potential expressed in cells derived from skin affected with a skin new skin-active agents efficacious for the treatment of skin hyperpigmentation condition or genes differentially conditions such as hyperpigmentation. Through careful expressed in cells treated with at least one benchmark skin selection of cell type, and by generation of a reference col active agent having known efficacy in treating a skin hyper lection of gene-expression profiles for known skin-active pigmentation condition. Cell instances are derived from a agents and recognized skin disorders, the present inventors keratinocyte, fibroblast, melanocyte or melanoma cell line were Surprisingly able to create connectivity map architecture and the pigmentation-relevant gene expression signature is US 2013/026100.6 A1 Oct. 3, 2013
derived from a corresponding cell line. Example 1 illustrates least one computer readable medium having stored thereon a development of a hyperpigmentation condition expression plurality of instances, and a skin hyperpigmentation-relevant signature. gene expression signature, wherein the instances and the gene 0027. Other embodiments are direct to methods for gen expression signature are derived from one of a human kera erating a hyperpigmentation condition gene expression sig tinocyte cell, a human fibroblast cell, a human melanocyte nature for use in identifying connections between pertur cell, or a human melanoma cell, wherein each instance com bagens and genes associated with a skin pigmentation prises an instance list of rank-ordered identifiers of differen condition. Methods comprise: (a) providing a gene expres tially expressed genes, and wherein the hyperpigmentation sion profile for a reference sample of human skin cells not relevant gene expression signature comprises one or more affected with a pigmentation condition; (b) generating a gene gene expression signature lists of identifiers representing dif expression profile for at least one sample of human skin cells ferentially expressed genes associated with a hyperpigmen from a Subject exhibiting the hyperpigmentation condition, tation condition or differentially expressed genes associated (c) comparing the expression profiles of (a) and (b) to deter with a benchmark skin-lightening agent; (b) a programmable mine a gene expression signature comprising a set of genes computer comprising computer-readable instructions that differentially expressed in (a) and (b); (d) assigning an iden cause the programmable computer to execute one or more of tifier to each gene constituting the gene expression signature the following: (i) accessing the plurality of instances and a and ordering the identifiers according to the direction of dif hyperpigmentation-relevant gene expression signature stored ferential expression to create one or more gene expression on the computer readable medium; (ii) comparing the hyper signature lists; and (e) storing the one or more gene expres pigmentation-relevant gene expression signature to the plu sion signature lists on at least one computer readable medium. rality of the instances, wherein the comparison comprises 0028. Another embodiment provides methods for gener comparing each identifier in the gene expression signature list ating a benchmark skin pigmentation-modifying gene with the position of the same identifier in the instance list for expression signature for use in identifying connections each of the plurality of instances; and (iii) assigning a con between perturbagens and genes associated with a skin pig nectivity score to each of the plurality of instances. mentation condition, the method comprising: (a) generating a 0031. A computer readable medium aspect is also dis gene expression profile for a human skin cell sample treated closed wherein a data architecture comprising a first digital with at least one benchmark skin pigmentation modifying file is stored in a spreadsheet file format, a word processing agent, wherein the benchmark skin pigmentation modifying file format, or a database file format suitable to be read by a agent is Suspended in a Vehicle composition, (b) generating a respective spreadsheet, word processing, or database com gene expression profile for a human skin cell sample treated puter program, the first digital file comprising data arranged with the vehicle composition; (c) comparing the expression to provide one or more gene expression signature lists com profiles of (a) and (b) to determine a gene expression signa prising a plurality of identifiers when read by the respective ture comprising a set of genes differentially expressed in (a) spreadsheet, word processing, or database computer pro and (b); (d) assigning an identifier to each gene constituting gram; and wherein each identifier is selected from the group the gene expression signature and ordering the identifiers consisting of a microarray probe set ID, a human gene name, according to the direction of differential expression to create a human gene symbol, and combinations thereofrepresenting one or more gene expression signature lists; and (e) storing a gene set forth in any of Tables B through P wherein each of the one or more gene expression signature lists on at least one the one or more gene expression signature lists comprises computer readable medium. Gene expression signatures and between about 10 and about 400 identifiers. Instructions for immobilized arrays of probes corresponding to the genes reading the digital file may be included. constituting the inventive signatures are also provided. 0032. These and additional objects, embodiments, and 0029. In some aspects a single benchmark skin active aspects of the invention will become apparent by reference to agent may be used to generate a benchmark signature (see the Figures and Detailed Description below. Example 2) and in other aspects a composite signature may be generated by treating a cell sample with more than one agent BRIEF DESCRIPTION OF THE FIGURES (see Examples 3, 6, and 7). A composite signature can be 0033 FIG. 1 sets forth pigmentation control targets and added in two ways: cells can be treated with each agent Some benchmark skin active agents as depicted in Table A. separately, the signature can be generated by comparing regu 0034 FIG. 2 sets forth identifiers for the genes constitut lated genes from all agents (together), looking for genes ing a gene expression signature for hyperpigmented skin. regulated in the same direction by all agents; secondarily, Table B sets forth identifiers for the 200 most significantly agents can be mixed together prior to treatment of cells. In up-regulated genes and Table C sets forth identifiers for the another embodiment, a composite benchmark signature may 200 most significantly down-regulated genes. be generated for a skin-lightening agent (Example 2), and 0035 FIG. 3 is a schematic illustration of a computer another generated for a skin darkening agent. The signature system suitable for use with the present invention; for the skin-lightening agent may be further tweaked by 0036 FIG. 4 is a schematic illustration of an instance eliminating any gene from the signature that also appears in associated with a computer readable medium of the computer the signature of the skin-darkening agent, regulated in the system of FIG. 3; same direction, or vice versa. The inventors discovered that 0037 FIG. 5 is a schematic illustration of a programmable Such composite signatures are particularly useful for mining computer suitable for use with the present invention; C-map for agents capable of modifying skin pigment in the 0038 FIG. 6 is a schematic illustration of an exemplary desired direction. system for generating an instance; 0030 Systems for identifying connections between per 0039 FIG. 7 is a schematic illustration of a comparison turbagens and genes associated with a skin hyperpigmenta between a gene expression signature and an instance, wherein tion condition are also provided. The systems comprise: (a) at there is a positive correlation between the lists: US 2013/026100.6 A1 Oct. 3, 2013
0040 FIG. 8 is a schematic illustration of a comparison 0060. As used interchangeably herein, the terms “connec between a gene expression signature and an instance, wherein tivity map' and “C-map' refer broadly to devices, systems, there is a negative correlation between the lists; and articles of manufacture, and methodologies for identifying 0041 FIG. 9 is a schematic illustration of a comparison relationships between cellular phenotypes or cosmetic con between a gene expression signature and an instance, wherein ditions, gene expression, and perturbagens, such as cosmetic there is a neutral correlation between the lists. actives. 0042 FIG. 10 sets forth identifiers as shown in Table D, 0061. As used herein, the term "cosmetic agent’ means Hexamidine Benchmark Signature: 100 up-regulated. any Substance, as well as any component thereof, which may 0043 FIG. 11 sets forth identifiers as shown in Table E, be rubbed, poured, sprinkled, sprayed, introduced into, or Hexamidine Benchmark Signature; 100 top down-regulated. otherwise applied to a mammalian body or any part thereof. 0044 FIG. 12 sets forth identifiers as shown in Table F. NAG Benchmark Signature; 39 significantly up-regulated. Cosmetic agents may include Substances that are Generally 004.5 FIG. 13 sets forth identifiers as shown in Table G, Recognized as Safe (GRAS) by the US Food and Drug NAG Benchmark Signature, most significantly down-regu Administration, food additives, and materials used in non lated. cosmetic consumer products including over-the-counter 0046 FIG. 14 sets forth identifiers as shown in Table H, medications. In some embodiments, cosmetic agents may be Niacinamide Benchmark Signature, 100 top up-regulated. incorporated in a cosmetic composition comprising a derma 0047 FIG. 15 sets forth identifiers as shown in Table I, tologically acceptable carrier Suitable for topical application Niacinamide Benchmark Signature, 100 top down-regulated. to skin. A cosmetic agent includes, but is not limited to, (i) 0048 FIG. 16 sets forth identifiers as shown in Table J, chemicals, compounds, Small or large molecules, extracts, Sepiwhite Benchmark Signature; 100 top up-regulated. formulations, or combinations thereof that are known to 0049 FIG. 17 sets forth identifiers as shown in Table K, Sepiwhite Benchmark Signature; 100 top down-regulated. induce or cause at least one effect (positive or negative) on 0050 FIG. 18 sets forth identifiers as shown in Table L, skin tissue; (ii) chemicals, compounds, Small molecules, Composite “Skin Tone' Benchmark Signature; approxi extracts, formulations, or combinations thereof that are mately 40 up-regulated--58 down-regulated. known to induce or cause at least one effect (positive or 0051 FIG. 19 sets forth identifiers as shown in Table M, negative) on skin tissue and are discovered, using the pro RA benchmark signature in tkC cell-200 upregulated. vided methods and systems, to induce or cause at least one 0052 FIG. 20 sets forth identifiers as shown in Table N. previously unknown effect (positive or negative) on the skin RA benchmark signature in tkC, 200 down-regulated. tissue; and (iii) chemicals, compounds, Small molecules, 0053 FIG. 21 sets forth identifiers as shown in Table O, extracts, formulations, or combinations thereof that are not RA Benchmark Signature in BJ fibroblasts, 200 up-regulated. 0054 FIG.22 sets forth identifiers as shown in Table PRA known have an effect on skin tissue and are discovered, using Benchmark Signature in BJ fibroblast, 200 down-regulated. the provided methods and systems, to induce or cause an 0055 FIG. 23 sets in Table Q, Average C-map scores for effect on skin tissue. Some representative potential skin lightening agents with the 0062 Some examples of cosmetic agents or cosmetically Retinoic Acid Keratinocyte RA 200 Signature. actionable materials can be found in: the PubChem database 0056 FIG. 24 Shows Table R, showing a comparison of associated with the National Institutes of Health, USA (http:// the predictiveness of different C-map signatures for predict pubchem.ncbi.nlm.nih.gov); the Ingredient Database of the ing the activity of compounds in the mouse B16 melanoma Personal Care Products Council (http://online-personalcare cell melanogenesis assay. council.org/jsp/Home.jsp); and the 2010 International Cos 0057 FIG. 25, Shows Table S, with data outlining the responsiveness of tert Keratinocytes, melanocytes, and mela metic Ingredient Dictionary and Handbook, 13" Edition, noma cells to skin tone benchmarks. published by The Personal Care Products Council; the EU Cosmetic Ingredients and Substances list; the Japan Cosmetic DETAILED DESCRIPTION OF THE INVENTION Ingredients List; the Personal Care Products Council, the SkinDeep database (URL: http://www.cosmeticsdatabase. 0058. The present invention will now be described with com); the FDA Approved Excipients List; the FDA OTC List: occasional reference to the specific embodiments of the invention. This invention may, however, be embodied in dif the Japan Quasi Drug List; the US FDA Everything Added to ferent forms and should not be construed as limited to the Food database; EU Food Additive list; Japan Existing Food embodiments set forth herein. Rather, these embodiments are Additives, Flavor GRAS list; US FDA Select Committee on provided so that this disclosure will be thorough and com GRAS Substances; US Household Products Database; the plete, and to fully convey the scope of the invention to those Global New Products Database (GNPD) Personal Care, skilled in the art. Health Care, Food/Drink/Pet and Household database (URL: 0059. Unless otherwise defined, all technical and scien http://www.gmpd.com); and from Suppliers of cosmetic ingre tific terms used herein have the same meaning as commonly dients and botanicals. understood by one of ordinary skill in the art to which this invention pertains. The terminology used in the description of 0063. Other non-limiting examples of cosmetic agents the invention herein is for describing particular embodiments include botanicals (which may be derived from one or more only and is not intended to be limiting of the invention. As of a root, stem bark, leaf, seed or fruit of a plant). Some used in the description of the invention and the appended botanicals may be extracted from a plant biomass (e.g., root, claims, the singular forms “a” “an,” and “the are intended to stem, bark, leaf, etc.) using one more solvents. Botanicals include the plural forms as well, unless the context clearly may comprise a complex mixture of compounds and lack a indicates otherwise. distinct active ingredient. Another category of cosmetic US 2013/026100.6 A1 Oct. 3, 2013 agents are vitamin compounds and derivatives and combina tions thereof, such as a vitamin B3 compound, a vitamin B5 O H compound, a vitamin B6 compound, a vitamin B9 compound, RCNH-C-COOH a vitamin A compound, a vitamin C compound, a vitamin E compound, and derivatives and combinations thereof (e.g., CH 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 wherein R' can be C to Cao Saturated or unsaturated, straight described in detail below, such as for vitamin B3 compounds, or branched, substituted or unsubstituted alkyls; substituted N-acyl amino acid compounds, and retinoid compounds. In or unsubstituted aromatic groups; or mixtures thereof. Some embodiments, the vitamin B compound is a B3 com 0067 N-acyl Tyrosine corresponds to the following for pound having the formula: mula:
O H O RCNH-C-COOH N CH2 wherein R is CONH. (i.e., niacinamide), —COOH (i.e., nicotinic acid) or —CH-OH (i.e., nicotinyl alcohol); deriva tives thereof, and salts of any of the foregoing. Exemplary derivatives include nicotinic acid esters, including non-va Sodilating esters of nicotinic acid (e.g., tocopheryl nicotinate, OH myristyl nicotinate). Examples of Suitable vitamin B com pounds are well known in the art and are commercially avail wherein R' can be C to Cao Saturated or unsaturated, straight or branched, substituted or unsubstituted alkyls; substituted able from a number of sources (e.g., the Sigma Chemical or unsubstituted aromatic groups; or mixtures thereof. Company, ICN Biomedicals, Inc., and Aldrich Chemical 0068 A particularly useful compound in the present Company). invention is N-undecylenoyl-L-phenylalanine. This agent 0065. Some embodiments of the compositions of the belongs to the broad class of N-acyl Phenylalanine deriva present invention comprise a safe and effective amount of one tives, with its acyl group being a C11 mono-unsaturated fatty or more N-acyl amino acid compounds. The amino acid can acid moiety and the amino acid being the L-isomer of pheny be one of any of the amino acids known in the art. The N-acyl lalanine. N-undecylenoyl-L-phenylalanine corresponds to amino acid compounds of the present invention correspond to the following formula: the formula:
O H O H CH=CH-(CH2)CNH-C-COOH RCNH-C-COOH CH R wherein R can be a hydrogen, alkyl (substituted or unsubsti tuted, 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, As used herein, N-undecylenoyl-L-phenylalanine is commer 1981, published by W.H. Freeman and Company. R' can be cially available under the tradename Sepiwhite(R) from SEP C to Co., Saturated or unsaturated, straight or branched, PIC, France. substituted or unsubstituted alkyls; substituted or unsubsti 0069. Some embodiments of the present invention include tuted aromatic groups; or mixtures thereof. retinoid compounds. As used herein, “Retinoid Compounds” include all natural and/or synthetic analogs of Vitamin A or 0066 Preferably, the N-acyl amino acid compound is retinol-like compounds which possess the biological activity selected from the group consisting of N-acyl Phenylalanine, of Vitamin A in the skin as well as the geometric isomers and N-acyl Tyrosine, their isomers, their salts, and derivatives stereoisomers of these compounds. The Retinoid Compound thereof. The amino acid can be the D or L isomer or a mixture includes, but is not limited to, retinol, retinol esters (e.g., thereof. N-acyl Phenylalanine corresponds to the following C-C alkyl esters of retinol, including retinyl palmitate, formula: retinyl acetate, retinyl proprionate), retinal, and/or retinoic US 2013/026100.6 A1 Oct. 3, 2013
acid (including all-trans retinoic acid and/or 13-cis-retinoic the melanocyte and transfer to the keratinocyte (e.g., PAR-2 acid). In some embodiments, the Retinoid Compound is ret antagonists), and activators of melanin degradation within the inoic acid. These compounds are well known in the art and are keratinocyte. commercially available from a number of Sources, e.g., 0075 Skin active agents which modify skin pigmentation Sigma Chemical Company (St. Louis, Mo.), and Boerhinger are known in the art. Certain Substances may only be consid Mannheim (Indianapolis, Ind.). Other Retinoid Compounds ered cosmetic in severely reduced concentrations since there which may be useful herein are described in U.S. Pat. No. are side effects which suggest undesirable systemic activity 4,677,120, issued Jun. 30, 1987 to Parish et al.; U.S. Pat. No. beyond the cosmetic concern being addressed. These include 4.885,311, issued Dec. 5, 1989 to Parish et al.; U.S. Pat. No. hydroquinone, trans-retinoic acid, and corticosteroids. Gen 5,049,584, issued Sep. 17, 1991 to Purcellet al.; U.S. Pat. No. erally, a classic target for cosmetic formulation is inhibition 5,124.356, issued Jun. 23, 1992 to Purcell et al.; and Reissue of tyrosinase, the first enzyme in the conversion of tyrosine to 34,075, issued Sep. 22, 1992 to Purcellet al. melanin. A wide array of compounds, such as kojic acid, 0070. As used herein, the term “putative skin active agent' arbutin, ascorbic acid, ellagic acid, Sulfhydryl compounds, means a cosmetic agent as herein defined that has shown and resorcinols, are effective tyrosinase inhibitors, as is a promise through preliminary Screens as effecting a specific more recently discussed deoxy-arbutin. However, since sev change in skin biology related to pigmentation but that has not eral of these materials also have other effects, it is difficult to yet been tested for effectiveness through the methods herein directly connect a specific mechanism to the observed effect described for constructing a data architecture for use in iden on pigmentation. Table A provides a short list of the many tifying connections between perturbagens and genes associ known targets and a few agents effective against them. ated with skin tone, comprising 0076 Generally, a “benchmark skin active agent” refers to any chemical, compound, environmental factor, Small or 0071. As used herein, the term “skin-active agent' is a large molecule, extract, formulation, or combinations thereof Subset of cosmetic agents as defined herein and includes that is known to induce or cause a Superior effect (positive or generally any substance, as well as any component thereof, negative) on skin tissue. In accordance with the present inven intended to be applied to the skin for the purpose of effectu tion, agents having known efficacy in either skin-lightening ating a treatment of an undesirable skin condition or disorder, or skin darkening contexts are also herein referred to as or for achieving a desirable skin status. Examples relating to “Benchmark Agents.” skin tone include skin pigmentation disorders, including dis 0077. Non-limiting examples of benchmark skin active orders of hyperpigmentation, such as ephelides (freckles), agents are set forth in Table A, along with the corresponding lentigines including age spots (Solar lentigos), post-inflam theorized pigmentation control target. matory hyperpigmentation, Café au lait macules. Addisons 0078 Newer benchmark skin active agents include niaci disease and other systemic disease effects, hemochromatosis, namide and glucosamine (in particular, its derivative N-acetyl melasma (mask of pregnancy and other hormonal related glucosamine NAG), which have recently been shown to be pigment disorders) and acanthosis nigricans, as well as pho effective in reducing melanin production in culture. In vitro, totoxia and medicinal-induced alternations in pigmentation. glucosamine reduces production of melanin by inhibiting Examples of disorders of hypopigmentation include Vitiligo activation of tyrosinase, while niacinamide inhibits melano and skin trauma-related ablation of melanocytes in circum Some transfer from melanocytes to keratinocytes. Cosmetic scribed areas. In some case a cosmetic consumer merely moisturizer formulations containing niacinamide alone are desires a change in pigmentation status of skin as determined effective in reducing the appearance of hyperpigmented spots by some cultural standard, Such as skin lightening among in vivo and the addition of NAG to the formula yields greater Some dark skinned people and skin darkening or tanning effectiveness. Another benchmark pigmentation control among some light skinned people. agent is N-undecylenoyl-L-phenylalanine, which has been 0072 Although the term “skin tone' is most often thought reported to inhibit biding of alpha-MSH to the melanocyte in of with respect to skin pigmentation and evenness of colora vitro and is effective as a component of cosmetic moisturizer tion, “skintone' may also include other characteristics of skin formulations in clinical testing. that contribute to a consumer perception of overall tone. For 007.9 The terms “gene expression signature.” and “gene example, pore size and distribution, and skin texture are also expression signature” refer to a rationally derived list, or generally considered attributes of overall skin tone. plurality of lists, of genes representative of a skin tissue condition or a skin agent. In specific contexts, the skin agent 0073 Categorical examples of skin-active agents include may be a benchmark skin active agent or a potential skin skin pigment modifying agents, steroidal anti-inflammatory agent. Thus, the gene expression signature may serve as a agents, non-steroidal anti-inflammatory agents, pedicu proxy for a phenotype of interest for skin tissue. A gene locides, sensates, enzymes, Vitamins, hair growth actives, expression signature may comprise genes whose expression, Sunscreens, and combinations thereof. Cosmetic composi relative to a normal or control state, is increased (up-regu tions according to the instant invention may contain skin lated), whose expression is decreased (down-regulated), and active agents. combinations thereof. Generally, a gene expression signature 0074. Many processes and proteins are known to be for a modified cellular phenotype may be described as a set of involved in the pigmentary process; there is a wide array of genes differentially expressed in the modified cellular pheno targets against which to Screen for pigmentation control type compared to the cellular phenotype. A gene expression agents. Among the many targets are inhibitors of melanocyte signature can be derived from various sources of data, includ stimulation (e.g., antioxidants, anti-inflammatory agents), ing but not limited to, from in vitro testing, in Vivo testing and cell receptor antagonists (e.g., alpha-MSH antagonists), combinations thereof. In some embodiments, a gene expres inhibitors of melanin synthesis enzymes (e.g., tyrosinase, sion signature may comprise a first list representative of a TRP-1, TRP-2), inhibitors of melanosome transport within plurality of up-regulated genes of the condition of interestand US 2013/026100.6 A1 Oct. 3, 2013 a second list representative of a plurality of down-regulated wave, and a memory Stick. Examples of Volatile memory genes of the condition of interest. include, but are not limited to, random access memory 0080. As used herein, the term "query” refers to data that (RAM), synchronous RAM (SRAM), dynamic RAM is used as an input to a Connectivity Map and against which a (DRAM), synchronous DRAM (SDRAM), double data rate plurality of instances are compared. A query may include a SDRAM (DDR SDRAM), and direct RAM bus RAM gene expression signature associated with a skin condition (DRRAM). Examples of non-volatile memory include, but Such as age spots, or may include a gene expression signature are not limited to, read only memory (ROM), programmable derived from a physiological process associated with a skin read only memory (PROM), erasable programmable read condition. A C-map may be queried with perturbagens, gene only memory (EPROM), and electrically erasable program expression signatures, skin disorders, thematic signatures, or mable read only memory (EEPROM). A memory can store any data feature or combination of data features or associa processes and/or data. Still other computer readable media tions that comprise the data architecture. include any suitable disk media, including but not limited to, 0081. The term “instance.” as used herein, refers to data magnetic disk drives, floppy disk drives, tape drives, Zip from a gene expression profiling experiment in which skin drives, flash memory cards, memory sticks, compact disk cells are dosed with a perturbagen. In some embodiments, the ROM (CD-ROM), CD recordable drive (CD-R drive), CD data comprises a list of identifiers representing the genes that rewriteable drive (CD-RW drive), and digital versatile ROM are part of the gene expression profiling experiment. The drive (DVD ROM). identifiers may include gene names, gene symbols; microar 0085. As used herein, the terms “software' and “software ray probe set IDs, or any other identifier. In some embodi application” refer to one or more computer readable and/or ments, an instance may comprise data from a microarray executable instructions that cause a computing device or experiment and comprises a list of probe set IDs of the other electronic device to perform functions, actions, and/or microarray ordered by their extent of differential expression behave in a desired manner. The instructions may be embod relative to a control. The data may also comprise metadata, ied in one or more various forms like routines, algorithms, including but not limited to data relating to one or more of the modules, libraries, methods, and/or programs. Software may perturbagen, the gene expression profiling test conditions, the be implemented in a variety of executable and/or loadable skin cells, and the microarray. forms and can be located in one computer component and/or 0082. The term “perturbagen, as used herein, means any distributed between two or more communicating, co-operat thing used as a challenge in a gene expression profiling ing, and/or parallel processing computer components and experiment to generate gene expression data for use in the thus can be loaded and/or executed in serial, parallel, and present invention. In some embodiments, the perturbagen is other manners. Software can be stored on one or more com applied to human cells and the gene expression data derived puter readable medium and may implement, in whole or part, from the gene expression profiling experiment may be stored the methods and functionalities of the present invention. as an instance in a data architecture. Human cells in accor I0086. As used herein, the term “hyperpigmentation gene dance with the invention may be keratinocyte, melanocyte, expression signature” refers to a gene expression signature fibroblast, or melanoma cells. Any substance, chemical, com derived from gene expression profiling of a hyperpigmenta pound, active, natural product, extract, drug e.g. Sigma tion condition. Aldrich LOPAC (Library of Pharmacologically Active Com I0087 As used herein, the term “connectivity score” refers pounds) collection. Small molecule, and combinations to a derived value representing the degree to which an thereof used as to generate gene expression data can be a instance correlates to a query. perturbagen. A perturbagen can also be any other stimulus 0088. As used herein, the term “data architecture” refers used to generate differential gene expression data. For generally to one or more digital data structures comprising an example, a perturbagen may also be UV radiation, heat, organized collection of data. In some embodiments, the digi osmotic stress, pH, a microbe, a virus, and Small interfering tal data structures can be stored as a digital file (e.g., a spread RNA. A perturbagen may be, but is not required to be, any sheet file, a text file, a word processing file, a database file, cosmetic agent. etc.) on a computer readable medium. In some embodiments, 0083. The term “dermatologically acceptable,” as used the data architecture is provided in the form of a database that herein, means that the compositions or components described may be managed by a database management system (DBMS) are suitable for use in contact with human skin tissue. that is be used to access, organize, and select data (e.g., 0084 As used herein, the term “computer readable instances and gene expression signatures) stored in a data medium” refers to any electronic storage medium and base. includes but is not limited to any volatile, nonvolatile, remov I0089. As used herein, the terms “gene expression profil able, and non-removable media implemented in any method ing” and "gene expression profiling experiment” refer to the or technology for storage of information Such as computer measurement of the expression of multiple genes in a biologi readable instructions, data and data structures, digital files, cal sample using any suitable profiling technology. For Software programs and applications, or other digital informa example, the mRNA expression of thousands of genes may be tion. Computer readable media includes, but are not limited determined using microarray techniques. Other emerging to, application-specific integrated circuit (ASIC), a compact technologies that may be used include RNA-Seq or whole disk (CD), a digital versatile disk (DVD), a random access transcriptome sequencing using NextGen sequencing tech memory (RAM), a synchronous RAM (SRAM), a dynamic niques. RAM (DRAM), a synchronous DRAM (SDRAM), double (0090. As used herein, the term “microarray” refers data rate SDRAM (DDR SDRAM), a direct RAM bus RAM broadly to any ordered array of nucleic acids, oligonucle (DRRAM), a read only memory (ROM), a programmable otides, proteins, Small molecules, large molecules, and/or read only memory (PROM), an electronically erasable pro combinations thereofon a Substrate that enables gene expres grammable read only memory (EEPROM), a disk, a carrier sion profiling of a biological sample. Non-limiting examples US 2013/026100.6 A1 Oct. 3, 2013 of microarrays are available from Affymetrix, Inc.; Agilent agent, exposed to an epidermal or dermal cell line, including Technologies, Inc., Illumina, Inc.; GE Healthcare, Inc.; for example keratinocyte, fibroblast, melanocyte and mela Applied Biosystems, Inc.; Beckman Coulter, Inc., etc. noma cells. Instances from more complex cell culture sys 0091. Unless otherwise indicated, all numbers expressing tems may also be used, such as skin organotypic cultures quantities of ingredients, properties such as molecular containing the targeted cell or ex vivo human skin. Instances weight, reaction conditions, and so forth as used in the speci from a plurality of cell lines may be used with the present fication and claims are to be understood as being modified in invention. all instances by the term “about'. Additionally, the disclosure 0094. In accordance with yet another aspect of the present of any ranges in the specification and claims are to be under invention, provided are devices, systems and methods for stood as including the range itself and also anything Sub identification of relationships between a skin condition, e.g. Sumed therein, as well as endpoints. All numeric ranges are skin hyperpigmentation condition query signature and a plu inclusive of narrower ranges; delineated upper and lower rality of instances, where the query signature may be a gene range limits are interchangeable to create further ranges not expression signature or a physiological theme expression sig explicitly delineated. Unless otherwise indicated, the numeri nature. For example, it may be possible to ascertain pertur cal properties set forth in the specification and claims are bagens that give rise to a statistically significant activity on a approximations that may vary depending on the desired prop statistically significant number of genes associated with a erties sought to be obtained in embodiments of the present skin condition of interest, leading to the identification of new invention. Notwithstanding that numerical ranges and param cosmetic agents for treating the skin condition or new uses of eters setting forth the broad scope of the invention are known cosmetic agents. approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical I. Systems and Devices values, however, inherently contain certain errors necessarily (0095 Referring to FIGS. 3, 5 and 6, some examples of resulting from error found in their respective measurements. systems and devices in accordance with the present invention 0092. In accordance with one aspect of the present inven for use in identifying relationships between perturbagens, tion, provided are devices, systems and methods for imple skin pigmentation conditions, and genes associated with the menting a connectivity map utilizing one or more query sig skin pigmentation condition will now be described. System natures associated with a pigmentation or pigmentation 10 comprises one or more of computing devices 12, 14, a related condition. The query signatures may be derived in computer readable medium 16 associated with the computing variety of ways. In some embodiments, the query signatures device 12, and communication network 18. may be gene expression signatures derived from gene expres 0096. The computer readable medium 16, which may be sion profiling of full thickness skin biopsies of skin exhibiting provided as a hard disk drive, comprises a digital file 20, Such a skin condition of interest compared to a control. The gene as a database file, comprising a plurality of instances 22, 24. expression profiling can be carried out using any Suitable and 26 stored in a data structure associated with the digital file technology, including but not limited to microarray analysis 20. The plurality of instances may be stored in relational or NextGen sequencing. An example of a gene expression tables and indexes or in other types of computer readable signature includes a hyperpigmentation gene expression sig media. The instances 22, 24, and 26 may also be distributed nature, an example of which is described more fully hereafter. across a plurality of digital files, a single digital file 20 being A query signature may be derived from transcriptional pro described herein however for simplicity. filing of a keratinocyte, fibroblast, melanocyte, or melanoma (0097. The digital file 20 can be provided in wide variety of cell line exposed to benchmark skin-active agents such as formats, including but not limited to a word processing file skin-lightening agents. In other embodiments, the query sig format (e.g., Microsoft Word), a spreadsheet file format (e.g., nature may be a benchmark gene expression signature Microsoft Excel), and a database file format. Some common wherein a skin-lightening benchmark signature is further examples of suitable file formats include, but are not limited refined by comparing it to a skin-darkening benchmark sig to, those associated with file extensions such as *.xls, *.xld, nature and genes having similar directional regulation are *.xlk, *.xll, *.xlt, *.xlxs, *.dif, *.db, *.dbf, *.accdb, *.imdb, eliminated. In further embodiments a cell is treated with more *.mdf, *.cdb, *.fdb, *.csv, *sql, *.xml, *.doc, *...txt, * rtf, than one benchmark skin active agent to derive a benchmark *...log, *.docx, ...ans, *.pages, *.wps, etc. composite signature, and in specific embodiments, the cell is 0.098 Referring to FIG. 4, in some embodiments the treated with a plurality of benchmark skin active agents instance 22 may comprise an ordered listing of microarray wherein the selected agents include those acting from differ probe set IDs, wherein the value of N is equal to the total ent mechanisms known to underpin skin pigmentation. In number of probes on the microarray used in analysis. Com other specific embodiments a general benchmark skin tone mon microarrays include Affymetrix GeneChips and Illu signature may be generated by treating a cell with a plurality mina BeadChips, both of which comprise probe sets and of benchmark skin active agents including agents comprising custom probe sets. To generate the reference gene profiles benchmark skin active agents for skin pigmentation, skin according to the invention, preferred chips are those designed pore size and distribution, and/or skin texture. These query for profiling the human genome. Examples of Affymetrix signatures may be used singularly or in combination. In spe chips with utility in the instant invention include model cific embodiments a composite signature has been shown to Human Genome HG-U133 Plus 2.0 and HG-U219. A specific provide advantages in predicting gene changes for chemicals Affymetrix chip employed by the instant investigators is affecting tone versus signatures from single chemicals. HG-U133A2.0, however it will be understood by a person or 0093. In accordance with another aspect of the present ordinary skill in the art that any chip or microarray, regardless invention, provided are devices, systems, and methods for of proprietary origin, is Suitable so long as the probe sets of implementing a connectivity map utilizing one or more the chips used to construct a data architecture according to the instances derived from a perturbagen, Such as a cosmetic invention are Substantially similar. US 2013/026100.6 A1 Oct. 3, 2013
0099 Instances derived from microarray analyses utiliz 0101. As previously described, the data stored in the first ing Affymetrix GeneChips may comprise an ordered listing and second digital files may be stored in a wide variety of data of gene probe set IDs where the list comprises 22,000+IDs. structures and/or formats. In some embodiments, the data is The ordered listing may be stored in a data structure of the stored in one or more searchable databases, such as free digital file 20 and the data arranged so that, when the digital databases, commercial databases, or a company's internal file is read by the software application 28, a plurality of proprietary database. The database may be provided or struc character strings are reproduced representing the ordered list tured according to any model known in the art, such as for ing of probe set IDs. While it is preferred that each instance example and without limitation, a flat model, a hierarchical comprise a full list of the probesetIDs, it is contemplated that model, a network model, a relational model, a dimensional model, oran object-oriented model. In some embodiments, at one or more of the instances may comprise less than all of the least one searchable database is a company's internal propri probe set IDs of a microarray. It is also contemplated that the etary database. A user of the system 10 may use a graphical instances may include other data in addition to or in place of user interface associated with a database management system the ordered listing of probe set IDs. For example, an ordered to access and retrieve data from the one or more databases or listing of equivalent gene names and/or gene symbols may be other data sources to which the system is operably connected. substituted for the ordered listing of probeset IDs. Additional In some embodiments, the first digital file 20 is provided in data may be stored with an instance and/or the digital file 20. the form of a first database and the second digital file 30 is In some embodiments, the additional data is referred to as provided in the form of a second database. In other embodi metadata and can include one or more of cell line identifica ments, the first and second digital files may be combined and tion, batch number, exposure duration, and other empirical provided in the form of a single file. data, as well as any other descriptive material associated with 0102. In some embodiments, the first digital file 20 may an instance ID. The ordered list may also comprise a numeric include data that is transmitted across the communication value associated with each identifier that represents the network 18 from a digital file 36 stored on the computer ranked position of that identifier in the ordered list. readable medium 38. In one embodiment, the first digital file 0100 Referring again to FIGS. 3, 4 and 5, the computer 20 may comprise gene expression data obtained from a cell readable medium 16 may also have a second digital file 30 line (e.g., a fibroblast cell line and/or a keratinocyte cell line) stored thereon. The second digital file 30 comprises one or as well as data from the digital file 36, Such as gene expression more lists 32 of microarray probe set IDs associated with one data from other cell lines or cell types, gene expression sig or more pigmentation-relevant gene expression signatures. natures, perturbagen information, clinical trial data, scientific The listing 32 of microarray probe set IDs typically com literature, chemical databases, pharmaceutical databases, and prises a much smaller list of probe set IDs than the instances other such data and metadata. The digital file 36 may be of the first digital file 20. In some embodiments, the list provided in the form of a database, including but not limited comprises between 2 and 1000 probe set IDs. In other to Sigma-Aldrich LOPAC collection, Broad Institute C-MAP embodiments the list comprises greater than 10, 50, 100, 200, collection, GEO collection, and Chemical Abstracts Service or 300 and/or less than about 800, 600, or about 400 probe set (CAS) databases. IDs. The listing 32 of probe set IDs of the second digital file 0103) The computer readable medium 16 (or another com 30 comprises a list of probe set IDs representing up, and/or puter readable media, Such as 16) may also have stored down-regulated genes selected to represent a skin tone con thereon one or more digital files 28 comprising computer dition of interest. In some embodiments, a first list may rep readable instructions or Software for reading, writing to, or resent the up-regulated genes and a second list may represent otherwise managing and/or accessing the digital files 20, 30. the down-regulated genes of the gene expression signature. The computer readable medium 16 may also comprise soft The listing(s) may be stored in a data structure of the digital ware or computer readable and/or executable instructions that file 30 and the data arranged so that, when the digital file is cause the computing device 12 to perform one or more steps read by the software application 28, a plurality of character of the methods of the present invention, including for strings are reproduced representing the list of probe set IDs. example and without limitation, the step(s) associated with Instead of probe set IDs, equivalent gene names and/or gene comparing a gene expression signature stored in digital file 30 symbols (or another nomenclature) may be substituted for a to instances 22, 24, and 26 stored in digital file 20. In some list of probe set IDs. Additional data may be stored with the embodiments, the one or more digital files 28 may form part gene expression signature and/or the digital file 30 and this is of a database management system for managing the digital commonly referred to as metadata, which may include any files 20, 28. Non-limiting examples of database management associated information, for example, cell line or sample systems are described in U.S. Pat. Nos. 4.967,341 and 5,297, Source, and microarray identification. Examples of listings of 279. probe set IDs for a skin hyperpigmentation gene expression 0104. The computer readable medium 16 may form part of signature, specifically wherein the skin hyperpigmentation or otherwise be connected to the computing device 12. The condition is age spots, is set forth in FIG. 2, Tables B (the 200 computing device 12 can be provided in a wide variety of most up-regulated genes) and C (the 200 most down-regu forms, including but not limited to any general or special lated genes in a skin hyperpigmentation gene expression sig purpose computer Such as a server, a desktop computer, a nature). In some embodiments, one or more skin hyperpig laptop computer, a tower computer, a microcomputer, a mini mentation condition gene expression signatures may be computer, and a mainframe computer. While various comput stored in a plurality of digital files and/or stored on a plurality ing devices may be suitable for use with the present invention, of computer readable media. In other embodiments, a plural a generic computing device 12 is illustrated in FIG. 5. The ity of gene expression signatures (e.g., 32, 34) may be stored computing device 12 may comprise one or more components in the same digital file (e.g., 30) or stored in the same digital selected from a processor 40, system memory 42, and a sys file or database that comprises the instances 22, 24, and 26. tem bus 44. The system bus 44 provides an interface for US 2013/026100.6 A1 Oct. 3, 2013
system components including but not limited to the system connected by other interfaces, such as a parallel port, an IEEE memory 42 and processor 40. The system bus 36 can be any 1394 serial port, a game port, a universal serial bus (USB) of several types of bus structures that may further intercon port, an IR interface, etc. The computing device 12 may drive nect to a memory bus (with or without a memory controller), a separate or integral display device 54, which may also be a peripheral bus, and a local bus using any of a variety of connected to the system bus 44 via an interface, such as a commercially available bus architectures. Examples of a local video port 56. bus include an industrial standard architecture (USA) bus, a 0109 The computing devices 12, 14 may operate in a microchannel architecture (MSA) bus, an extended ISA networked environment across network 18 using a wired (EISA) bus, a peripheral component interconnect (PCI) bus, a and/or wireless network communications interface 58. The universal serial (USB) bus, and a small computer systems network interface port 58 can facilitate wired and/or wireless interface (SCSI) bus. The processor 40 may be selected from communications. The network interface port can be part of a any suitable processor, including but not limited to, dual network interface card, network interface controller (NIC), microprocessor and other multi-processor architectures. The network adapter, or LAN adapter. The communication net processor executes a set of stored instructions associated with work 18 can be a wide area network (WAN) such as the one or more program applications or software. Internet, or a local area network (LAN). The communication 0105. The system memory 42 can include non-volatile network 18 can comprise a fiber optic network, a twisted-pair memory 46 (e.g., read only memory (ROM), erasable pro network, a T1/E1 line-based network or other links of the grammable read only memory (EPROM), electrically eras T-carrier/E carrier protocol, or a wireless local area or wide able programmable read only memory (EEPROM), etc.) and/ area network (operating through multiple protocols such as or Volatile memory 48 (e.g., random access memory (RAM)). ultra-mobile band (UMB), long term evolution (LTE), etc.). A basic input/output system (BIOS) can be stored in the Additionally, communication network 18 can comprise base non-volatile memory 38, and can include the basic routines stations for wireless communications, which include trans that help to transfer information between elements within the ceivers, associated electronic devices for modulation/de computing device 12. The volatile memory 48 can also modulation, and Switches and ports to connect to a backbone include a high-speed RAM such as static RAM for caching network for backhaul communication Such as in the case of data. packet-switched communications. 0106 The computing device 12 may further include a storage 44, which may comprise, for example, an internal II. Methods for Creating a Plurality of Instances hard disk drive HDD,e.g., enhanced integrated drive elec 0110. In some embodiments, the methods of the present tronics (EIDE) or serial advanced technology attachment invention may comprise populating at least the first digital file (SATA) for storage. The computing device 12 may further 20 with a plurality of instances (e.g., 22, 24, 26) comprising include an optical disk drive 46 (e.g., for reading a CD-ROM data derived from a plurality of gene expression profiling or DVD-ROM48). The drives and associated computer-read experiments, wherein one or more of the experiments com able media provide non-volatile storage of data, data struc prise exposing, for example, keratinocyte cells (or other skin tures and the data architecture of the present invention, com cells such as human skin equivalent cultures or ex vivo cul puter-executable instructions, and so forth. For the computing tured human skin) to at least one perturbagen. For simplicity device 12, the drives and media accommodate the storage of of discussion, the gene expression profiling discussed here any data in a suitable digital format. Although the description after will be in the context of a microarray experiment. of computer-readable media above refers to an HDD and 0111 Referring to FIG. 6, one embodiment of a method of optical media such as a CD-ROM or DVD-ROM, it should be the present invention is illustrated. The method 58 comprises appreciated by those skilled in the art that other types of exposing a keratinocyte cell to a perturbagen 64. The pertur media which are readable by a computer, Such as Zip disks, bagen may be dissolved in a carrier, such as dimethylsulfox magnetic cassettes, flash memory cards, cartridges, and the ide (DMSO). After exposure, mRNA is extracted from the like may also be used, and further, that any such media may cells exposed to the perturbagen and reference cells 66 (e.g., contain computer-executable instructions for performing the keratinocyte cells) which are exposed to only the carrier. The methods of the present invention. mRNA 68,70, 72 may be reverse transcribed to cDNA 64,76, 0107. A number of software applications can be stored on 78 and marked with different fluorescent dyes (e.g., red and the drives 44 and Volatile memory 48, including an operating green) if a two color microarray analysis is to be performed. system and one or more software applications, which imple Alternatively, the samples may be prepped for a one color ment, in whole or part, the functionality and/or methods microarray analysis, and further a plurality of replicates may described herein. It is to be appreciated that the embodiments be processed if desired. The cDNA samples may be co-hy can be implemented with various commercially available bridized to the microarray 80 comprising a plurality of probes operating systems or combinations of operating systems. The 82. The microarray may comprise thousands of probes 82. In central processing unit 40, in conjunction with the Software some embodiments, there are between 10,000 and 50,000 applications in the Volatile memory 48, may serve as a control gene probes 82 present on the microarray 80. The microarray system for the computing device 12 that is configured to, or is scanned by a scanner 84, which excites the dyes and mea adapted to, implement the functionality described herein. Sures the amount fluorescence. A computing device 86 may 0108. A user may be able to enter commands and infor be used to analyze the raw images to determine the expression mation into the computing device 12 through one or more levels of a gene in the cells 60, 62 relative to the reference cells wired or wireless input devices 50, for example, a keyboard, 66. The scanner 84 may incorporate the functionality of the a pointing device. Such as a mouse (not illustrated), or a touch computing device 86. The expression levels include: i) up screen. These and other input devices are often connected to regulation e.g., greater binding of the test material (e.g., the central processing unit 40 through an input device inter cDNA 74, 76) to the probe than the reference material (e.g., face 52 that is coupled to the system bus 44 but can be cDNA 78), or ii) down-regulation e.g., greater binding of US 2013/026100.6 A1 Oct. 3, 2013
the reference material (e.g., cDNA 78) to the probe than the number of regulated genes will depend on the strength of the test material (e.g., cDNA 74, 76), iii) expressed but not effect of the perturbagen associated with the instance. Other differentially e.g., similar binding of the reference material arrangements within the list 84 may be provided. For (e.g., cDNA 78) to the probe than the test material (e.g., example, the probe IDs associated with the down-regulated cDNA 74.76), and iv) no detectable signal or noise. The up genes may be arranged at the top of the list 84. This instance and down-regulated genes are referred to as differentially data may also further comprise metadata such as perturbagen expressed. Microarrays and microarray analysis techniques identification, perturbagen concentration, cell line or sample are well known in the art, and it is contemplated that other Source, and microarray identification. microarray techniques may be used with the methods, devices 0.115. In some embodiments, one or more instances com and systems of the present invention. For example, any Suit prise at least about 1,000, 2,500, 5,000, 10,000, or 20,000 able commercial or non-commercial microarray technology identifiers and/or less than about 30,000, 25,000, or 20,000 and associated techniques may used. Good results have been identifiers. In some embodiments, the database comprises at obtained with Affymetrix GeneChip(R) technology and Illu least about 50, 100, 250, 500, or 1,000 instances and/or less mina BeadChipTM technology. One illustrative technique is than about 50,000, 20,000, 15,000, 10,000, 7,500, 5,000, or described in the Examples, “Generally Applicable' methods 2,500 instances. Replicates of an instance may be created, and section. However, one of skill in the art will appreciate that the the same perturbagen may be used to derive a first instance present invention is not limited to the methodology of the from keratinocyte cells and a second instance from another example and that other methods and techniques are also con skin cell type, Such as fibroblasts, melanocytes, melanoma or templated to be within its scope. complex tissue, for example ex vivo human skin. 0112 In a very specific embodiment, an instance consists 0116. The present inventors have discovered that instances of the rank ordered data for all of the probe sets on the derived with a cell type. Such as keratinocyte cells, are more Affymetrix HG-U133A2.0 GeneChip wherein each probe on predictive than other cell types when used in combination the chip has a unique probe set Identifier. The probe sets are with a skin lightening benchmark agent expression signature rank ordered by the fold change relative to the controls in the derived from the same cell type. In other words, better results same C-map batch (single instance/average of controls). The are achieved if the cell type used to generate the query signa probe set Identifiers are rank-ordered to reflect the most up ture is the same as the instance cell type. While this cell regulated to the most down-regulated. consistency guide may appear predictable, what is Surprising 0113 Notably, even for the non-differentially regulated is that with respect to benchmark agent signatures the present genes the signal values for a particular probe set are unlikely inventors Surprisingly discovered that certain benchmark skin to be identical for the instance and control so a fold change active agents have greaterpredictive efficacy with certain cell different from 1 will be calculated that can be used for com types over others. For example, as set forth in Examples 4 and prehensive rank ordering. In accordance with methods dis 5, the present inventors compared Retinoic Acid benchmark closed by Lamb et al. (2006), data are adjusted using 2 thresh gene expression signatures derived from BJ fibroblast cells olds to minimize the effects of genes that may have very low and keratinocyte cells, it was Surprisingly discovered that noisy signal values, which can lead to spurious large fold those derived from keratinocyte cells yield better results with changes. The thresholding is preferably done before the rank respect to a potential agent hit list based on connectivity with ordering. An example for illustrative purposes includes a the query signature. process wherein a first threshold is set at 20. If the signal for III. Methods for Deriving Hyperpigmentation Gene a probeset is below 20, it is adjusted to 20. Ties for ranking are Expression Signatures broken with a second threshold wherein the fold changes are recalculated and any values less than 2 are set to 2. For any 0117 Some methods of the present invention comprise remaining ties the order depends on the specific Sorting algo identifying a gene expression signature that represents the rithm used but is essentially random. The probe sets in the up-regulated and down-regulated genes associated with a middle of the list do not meaningfully contribute to an actual skin condition of interest, in particular with skin tone or connectivity Score. hyperpigmentation. 0114. The rank ordered data are stored as an instance. The 0118. The pathogenesis of a skin pigmentation condition probes may be sorted into a list according to the level of gene typically involves complex processes involving numerous expression regulation detected, wherein the list progresses known and unknown extrinsic and intrinsic factors, as well as from up-regulated to marginal or no regulation to down responses to such factors that are subtle over a relatively short regulated, and this rank ordered listing of probe IDs is stored period of time but non-subtle over a longer period of time. as an instance (e.g., 22) in the first digital file 20. Referring to This is in contrast to what is typically observed in drug devel FIG. 4, the data associated with an instance comprises the opment and drug screening methods, wherein a specific tar probe ID 80 and a value 82 representing its ranking in the list get, gene, or mechanism of action is of interest. Due to the (e.g., 1, 2, 3, 4... N, where N represents the total number of unique screening challenges associated with a skin pigmen probes on the microarray). The ordered list 84 may generally tation condition, the quality of the gene expression signature comprise approximately three groupings of probe IDs: a first representing the condition of interest can be important for grouping 86 of probe IDs associated with up-regulated genes, distinguishing between the gene expression data actually a second group 88 of probe IDs associated with genes with associated with a response to a perturbagen from the back marginal regulation or no detectable signal or noise, and a ground expression data. third group 90 of probe IDs associated with down-regulated 0119. One challenge in developing gene expression signa genes. The most up regulated genes are at or near the top of the tures for skin tone and pigmentation-related skin disorders is list 84 and the most down-regulated genes are at or near the that the number of genes selected needs to be adequate to bottom of the list84. The groupings are shown for illustration, reflect the dominant and key biology but not so large as to but the lists for each instance may be continuous and the include many genes that have achieved a level of statistical US 2013/026100.6 A1 Oct. 3, 2013
significance by random chance and are non-informative. tion of interest compared to a control. For generation of an Thus, query signatures should be carefully derived since the exemplary hyperpigmentation gene expression signature, predictive value may be dependent upon the quality of the biopsies are taken from forearm age spots and compared to gene expression signature. non-affected forearm skin sampled from the same Subject. 0120. One factor that can impact the quality of the query 0.124. In other embodiments of the present invention, a signature is the number of genes included in the signature. gene expression signature may be derived from a gene expres The present inventors have found that, with respect to a cos sion profiling analysis of keratinocyte, melanocyte, fibroblast metic data architecture and connectivity map, too few genes or melanoma cells treated with one or more benchmark skin can result in a signature that is unstable with regard to the active agents, in particular a skin-lightening agent, to repre highest scoring instances. In other words, Small changes to sent cellular perturbations leading to improvement in the skin the gene expression signature can result in significant differ tissue condition treated with that benchmark skin active ences in the highest scoring instance. Conversely, too many agent, wherein the signature comprises a plurality of genes genes may tend to partially mask the dominant biological up-regulated and down-regulated by the benchmark skin responses and will include a higher fraction of genes meeting active agent in cells in vitro. As one illustrative example, statistical cutoffs by random chance—thereby adding unde microarray gene expression profile data where the pertur sirable noise to the signature. The inventors have found that bagen is the known skin lightening agent Niacinamide may be the number of genes desirable in a gene expression signature analyzed using the present invention to determine from the is also a function of the strength of the biological response rank-ordered instances in the query results, the perturbagens associated with the condition and/or the number of genes associated with the highest scoring instances. needed to meet minimal values (e.g., a p-value less than about 0.125. A composite benchmark signature according to the 0.05 or less than about 1.0, or in accordance with applicable invention is a signature derived from a cell treated with more statistical principles) for statistical significance. Hence, what than one benchmark skin active agent (described in Examples is considered an ideal number of genes will vary from condi 3, 6, and 7). The actives may be selected to reflect more than tion to condition. When the biology is weaker, such as is the one mechanism of action in skin, or may be selected to reflect case typically with cosmetic condition phenotypes, fewer more than one attribute of general skin tone. genes than those which may meet the statistical requisite for 0.126 Inafurther specific embodiment, a benchmark skin inclusion in the prior art, may be used to avoid adding noisy lightening gene expression signature is compared to a bench genes. mark skin-darkening gene expression signature and genes not 0121 For example, the present inventors have determined differentially regulated between the two are eliminated from that where gene expression profiling analysis of a skin con the signature intended to be the query signature. Non-limiting dition yields from between about 2,000 and 4,000 genes examples of skin-darkening agents according to this embodi having a statistical p-value of less than 0.05 and approxi ment include alpha-melanocyte-stimulating hormone mately 1000 genes having a p-value of less than 0.001, a very (a-MSH) and any related melanocortin1 receptoragonists or strong biological response is indicated. A moderately strong stimulant thereof. biological response may yield approximately 800-2000 genes have a statistical p-value of less than 0.05 combined IV. Methods for Comparing a Plurality of Instances to One or with approximately 400-600 genes have a p-value of less than More Pigmentation Gene Expression Signatures 0.001. In these cases, a gene expression signature comprising I0127. Referring to FIG. 7 and FIG. 8, a method for que between about 100 and about 600 genes appears ideal. rying a plurality of instances with one or more hyperpigmen Weaker biology may be better represented by a gene expres tation-relevant gene signatures will now be described. sion signature comprising fewer genes, such as between Broadly, the method comprises querying a plurality of about 20 and 100 genes. instances with one or more hyperpigmentation-relevant gene 0122) While a gene expression signature may representall signatures and applying a statistical method to determine how significantly regulated genes associated with a skin condition strongly the signature genes match the regulated genes in an of interest; typically it represents a Subset of Such genes. The instance. Positive connectivity occurs when the genes in the present inventors have discovered that hyperpigmentation up-regulated signature list are enriched among the up-regu gene expression signatures comprising between about 50 and lated genes in an instance and the genes in the down-regulated about 400 genes of approximately equal numbers of up-regu signature list are enriched among the down-regulated genes in lated and/or down-regulated genes are stable, reliable, and an instance. On the other hand, if the up-regulated genes of can provide predictive results. For example, a suitable gene the signature are predominantly found among the down-regu expression signature may have from about 100-150 genes, lated genes of the instance, and vice versa, this is scored as 250-300 genes, 300-350 genes, or 350-400 genes. In a very negative connectivity. FIG. 7 schematically illustrates an specific embodiment, a hyperpigmentation gene expression extreme example of a positive connectivity between signature signature includes the 100 most up- and down-regulated 90 and the instance 104 comprising the probe IDs 102, genes. However, one of skill in the art will appreciate that wherein the probe IDs of the instance are ordered from most gene expression signatures comprising fewer or more genes up-regulated to most down-regulated. In this example, the are also within the scope of the various embodiments of the probe IDs 100 (e.g., XXX XXs, X, X7, Xs) of the gene invention. For purposes of depicting a gene expression sig signature90, comprising an up list97 and a down list99, have nature, the probe set IDs associated with the genes are pref a one to one positive correspondence with the most up-regu erably separated into a first list comprising the most up lated and down-regulated probe IDs 102 of the instance 104, regulated genes and a second list comprising the most down respectively. Similarly, FIG. 8 schematically illustrates an regulated, as set forth in FIG. 2, Tables B and C. extreme example of a negative connectivity between signa 0123 Gene expression signatures may be generated from ture 94 and the instance 88 comprising the probe IDs 90. full thickness skin biopsies from skin having the skin condi wherein the probe IDs of the instance are ordered from most US 2013/026100.6 A1 Oct. 3, 2013
up-regulated to most down-regulated. In this example, the lation, Kendall's Tau correlation, and the Gamma statistic. probe IDs of the up list 93 (e.g., X, X X X) correspond Generally, in order to eliminate a requirement that all profiles exactly with the most down-regulated genes of the instance be generated on the same microarray platform, a non-para 88, and the probe IDs of the downlist 95 (e.g., Xs, X, X, Xs) metric, rank-based pattern matching strategy based on the correspond exactly to the most up-regulated probe IDs of the Kolmogorov-Smirnov statistic (see M. Hollander et al. “Non instance 88. FIG. 9 schematically illustrates an extreme parametric Statistical Methods’; Wiley, New York, ed. 2, example of neutral connectivity, wherein there is no consis 1999) (see, e.g., pp. 178-185). It is noted, however, that where tent enrichment of the up- and down-regulated genes of the all expression profiles are derived from a single technology signature among the up- and down-regulated genes of the platform, similar results may be obtained using conventional instance, either positive or negative. Hence the probe IDs 106 measures of correlation, for example, the Pearson correlation (e.g., X, XX X Xs, X, X-7, Xs) of a gene signature 108 coefficient. (comprising an up list 107 and a down list 109) are scattered 0.130. In specific embodiments, the methods and systems with respect to rank with the probe IDs 110 of the instance of the present invention employ the nonparametric, rank 112, wherein the probe IDs of the instance are ordered from based pattern-matching strategy based on the Kolmogorov most up-regulated to most down-regulated. While the above Smirnov statistic, which has been refined for gene profiling embodiments illustrate process where the gene signature data by Lamb's group, commonly known in the art as Gene comprises a both an up list and a down list representative of Set Enrichment Analysis (GSEA) (see, e.g., Lamb et al. 2006 the most significantly up-and down-regulated genes of a skin and Subramanian, A. et al. (2005) Proc. Natl. AcadSci U.S.A., condition, it is contemplated that the gene signature may 102, 15545-15550). For each instance, a down score is cal comprise only an up list or a down list when the dominant culated to reflect the match between the down-regulated biology associated with a condition of interest shows gene genes of the query and the instance, and an up score is calcu regulation in predominantly one direction. lated to reflect the correlation between the up-regulated genes 0128. In some embodiments, the connectivity score can be of the query and the instance. In certain embodiments the a combination of an up-score and a down score, wherein the down score and up score each may range between -1 and +1. up-score represents the correlation between the up-regulated The combination represents the strength of the overall match genes of a gene signature and an instance and the down-score between the query signature and the instance. represents the correlation between the down-regulated genes I0131 The combination of the up score and down score is of a gene signature and an instance. The up score and down used to calculate an overall connectivity score for each score may have values between +1 and -1. For an up score instance, and in embodiments where up and down score (and down score) a high positive value indicates that the ranges are set between -1 and +1, the connectivity score corresponding perturbagen of an instance induced the expres ranges from -2 to +2, and represents the strength of match sion of the microarray probes of the up-regulated (or down between a query signature and the instance. The sign of the regulated) genes of the gene signature, and a high negative overall score is determined by whether the instance links value indicates that the corresponding perturbagen associated positivity or negatively to the signature. Positive connectivity with the instance repressed the expression of the microarray occurs when the perturbagen associated with an instance probes of the up-regulated (or down-regulated) genes of the tends to up-regulate the genes in the up list of the signature gene signature. The up-score can be calculated by comparing and down-regulate the genes in the down list. Conversely, each identifier of an up list of a gene signature comprising the negative connectivity occurs when the perturbagen tends to up-regulated genes (e.g., Tables B, D, F, H and lists 93, 97. reverse the up and down signature gene expression changes, and 107) to an ordered instance list, while the down-score can The magnitude of the connectivity score is the sum of the be calculated by comparing each identifier of a down list of a absolute values of the up and down scores when the up and gene signature comprising the down-regulated genes (see, down scores have different signs. A high positive connectivity e.g., Tables C, E, G, I and down lists 95, 99, and 109) to an score predicts that the perturbagen will tend to induce the ordered instance list. In these embodiments, the gene signa condition that was used to generate the query signature, and a ture comprises the combination of the up list and the down high negative connectivity score predicts that the perturbagen list. will tend to reverse the condition associated with the query 0129. In some embodiments, the connectivity score value signature. A Zero score is assigned where the up and down may range from +2 (greatest positive connectivity) to -2 scores have the same sign, indicating that a perturbagen did (greatest negative connectivity), wherein the connectivity not have a consistent impact the condition signature (e.g., score (e.g., 101, 103, and 105) is the combination of the up up-regulating both the up and down lists). score (e.g., 111,113,115) and the down score (e.g., 117, 119, 0.132. According to Lamb et al. (2006), there is no stan 121) derived by comparing each identifier of a gene signature dard for estimating statistical significance of connections to the identifiers of an ordered instance list. In other embodi observed. Lamb teaches that the power to detect connections ments the connectivity range may be between +1 and -1. may be greater for compounds with many replicates. Repli Examples of the scores are illustrated in FIGS. 7, 8 and 9 as cating in this context means that the same perturbagen is reference numerals 101, 103, 105, 111, 113, 115, 117, 119, profiled multiple times. Where batch to batch variation must and 121. The strength of matching between a signature and an be avoided, a perturbagen should be profiled multiple times in instance represented by the up scores and down scores and/or each batch. However, since microarray experiments tend to the connectivity score may be derived by one or more have strong batch effects it is desirable to replicate instances approaches known in the art and include, but are not limited in different batches (i.e., experiments) to have the highest to, parametric and non-parametric approaches. Examples of confidence that connectivity Scores are meaningful and repro parametric approaches include Pearson correlation (or Pear ducible. son r) and cosine correlation. Examples of non-parametric 0.133 Each instance may be rank ordered according to its approaches include Spearman's Rank (or rank-order) corre connectivity score to the query signature and the resulting US 2013/026100.6 A1 Oct. 3, 2013
rank ordered list displayed to a user using any suitable soft tation conditions and disorders, and in particular those relat ware and computer hardware allowing for visualization of ing to hyperpigmentation. Main factors in the development of data. conditions of hyperpigmentation are exposure to certain envi 0134. In some embodiments, the methods may comprise ronmental conditions and hormonal changes. In general, the identifying from the displayed rank-ordered list of instances number of active melanocytes per unit area of skin decreases (i) the one or more perturbagens associated with the instances with age (10-20% decline per decade), and there are more of interest (thereby correlating activation or inhibition of a active melanocytes in chronically Sun-exposed skin than in plurality of genes listed in the query signature to the one or non-exposed skin. This increased number of active melano more perturbagens); (ii) the differentially expressed genes cytes in Sun-damaged skin indicates the influence of chronic associated with any instances of interest (thereby correlating UV exposure (e.g., on face, hands, and arms) in stimulating Such genes with the one or more perturbagens, the skin tissue melanogenic activity. Since chronic UV exposure also alters condition of interest, or both); (iii) the cells associated with dermal fibroblast function in aging skin and since fibroblasts any instance of interest (thereby correlating such cells with appear to play a regulatory role in melanin production, dermal one or more of the differentially expressed genes, the one or damage from Sunlight may contribute to the production of more perturbagens, and the skin tissue condition of interest); hyperpigmentation in exposed aging skin. or (iv) combinations thereof. The one or more perturbagens 0.138 Post-inflammatory hyperpigmentation (PIH) results associated with an instance may be identified from the meta from inflammation of the skin and disproportionately affects data stored in the database for that instance. However, one of people with darker skin. Inflammation induced pigmentation skill in the art will appreciate that perturbagen data for an is often seen associated with acne lesions, ingrown hairs, instance may be retrievably stored in and by other means. scratches, insect bites, and Surfactant damage. As an example Because the identified perturbagens statistically correlate to of the latter, exposure of human forearm skin to the harsh activation or inhibition of genes listed in the query signature, surfactant sodium lauryl sulfate (SLS) under patch for a few and because the query signature is a proxy for a skin condition hours will produce erythema within a day. Over the course of of interest, e.g. a hyperpigmentation condition, the identified 1-2 weeks after this SLS exposure, hyperpigmentation will perturbagens may be candidates for new cosmetic agents, result, particularly in darker skin, but it will occur even in new uses of known cosmetic agents, or to validate known Caucasian skin. Topical treatment with anti-inflammatory agents for known uses relevant to the hyperpigmentation agents is known to ameliorate this. A non-limiting example is condition. phytosterol. 0135) In some embodiments, the methods of the present I0139 Exposure of skin to sunlight is the most common invention may further comprise testing the selected candidate cause of skin hyperpigmentation and is increasingly believed cosmetic agent, using in vitro assays and/or in vivo testing, to that it is a subset of PIH caused by a post-inflammatory validate the activity of the agent and usefulness as a cosmetic response to UV damage to skin. The inflammatory response agent. Any suitable invitro test method can be used, including may be the result of an obvious acute inflammatory event such those known in the art, and most preferably in vitro models as sunburn or due to repeated sub-erythemal exposures to UV. developed in accordance with the present invention. For While in the latter, there may not be visible erythema, histo example, MatTek human skin equivalent cultures or other logically, such exposed skin has elevated inflammatory cell skin equivalent cultures may be treated with one or a combi content, yielding a 'subclinical inflammatory process. This nation of perturbagens selected for mimicry of the skin con explanation is Supported by the fact that topical treatment dition of interest with respect to regulation of the genes con with anti-inflammatory agents immediately after UVB expo stituting a physiological theme pattern for the skin condition Sure prevents induction of delayed tanning. of interest. In some embodiments, evaluation of selected 0140 Inflammation may result in hyperpigmentation agents using in vitro assays may reveal, confirm, or both, that through several mechanisms. Among them is direct stimula one or more new candidate cosmetic agents may be used in tion of melanocytes by inflammatory mediators such as IL-1- conjunction with a known cosmetic agent (or a combination alpha. Reactive oxygen species such as Superoxide and nitric of known cosmetic agents) to regulate a skin condition of oxide generated in damaged skin (e.g., from UV exposure) or interest. released as by-products from inflammatory cells are also 0.136 Clinical testing can be useful to confirm putative known stimulators of melanocytes. Additionally, damage skin-pigmentation modifying efficacy. Clinical methods induced in epidermal cells can lead to release of endocrine include live expert grading, chromameter, and color image inducers of pigmentation Such as alpha-melanin stimulating capture and analysis. A new useful clinical measurement tool hormone (MSH). The resulting hyperpigmentation induced in assessing effectiveness is based on the principle of non by all these effects is adaptive since it appears to provide some contact SIAscopyTM, a recently described method to measure measure of protection against Subsequent insult since mela skin melanin content and distribution. It rapidly captures nin is known to have both UV absorption and reactive oxygen facial maps of skin chromophores, permitting determination species scavenging capacity. of the content and distribution of melanin in any spot or any 0.141. The melanin produced during an inflammatory area of the skin. Clinical testing on various body sites such as event also can enter the dermis where it is engulfed by mac forearm, face, chest, and back have been reported, and all rophages, producing “melanophages. These cells are often have utility in evaluating technology. Any thoroughly con retained in the upper dermis for prolonged periods since trolled clinical evaluation is expensive and therefore practi removal of dermal melanin apparently is a very slow process. cality limits testing to only the most promising candidates. Thus, post-inflammatory hyperpigmentation can be a very long-lived problem for the skin. V. Hyperpigmentation Disorders 0.142 Solar (Actinic) Lentigos are hyperpigmented spots 0.137 The present invention provides methods for identi also known as lentigines, age spots, and liver spots. They fying putative skin active agents for the treatment of pigmen develop as a result of chronic exposure of skin to UV radiation US 2013/026100.6 A1 Oct. 3, 2013 and occur on Sun-exposed parts of the body (in particular, the hormonal component, likely progesterone, since episodes of hands, arms, face, upper chest, and shoulders). Chronic expo melasma are often associated with pregnancy and the use of sure of skinto UV results in chronic inflammation, such as the hormonal birth control. There may also be an estrogen com epidermal endothelin cascade. The dark appearance of age ponent since estrogen receptor expression is increased in spots results from excessive melanin in the region, and may melasma. result from overproduction of melanin in the hyperactive 0.148. In melasma lesions, there is excess melanin present melanocytes, longer retention of melanin in aging epidermis in both the epidermis and upper dermis, associated with due to the slower turnover of this tissue layer, longer retention extravascular macrophages. Since there is only a slight of melanin in keratinocytes within rete ridges, and dermal increase in the number of melanocytes, the abnormality melanin-containing melanophages, which have been appears to be in function of the skin cells, in particular, observed histologically to lie beneath the lentigines. There is increase expression of factors in keratinocytes, fibroblasts, reduced wound healing with age at least in part due to reduced and melanocytes of the involved skin. In contrast to PIH, there clearance of materials from dermis apparently due to vascular is no apparent inflammatory phase involved in its develop and lymphatic changes, so that the residence time of mel ment. Additionally, there is likely a genetic compound pre anophages in dermis may be lengthened in older populations. disposing individuals to melasma, although the specific 0143 Certain observations suggest that there is a change genetic basis for it is not defined. in the genetic and phenotypic expression within an age spotas 014.9 The pigmentation process is complex as evidenced compared to cells in Surrounding non-affected skin. For particularly by recent revelations from genomic and pro example, within lesional lentigo skin, the rete ridges are teomic analysis. There are approximately 1,500 gene prod greatly exaggerated, extending deeper into the dermis. This ucts (proteins) expressed in melanosomes of all developmen deep penetration runs counter to the general observation of tal stages, with 600 of them being expressed at any given time, flattening of the convoluted dermal-epidermal junction with and with 100 of them apparently unique to the melanosome. aging, evidenced by the diminution of the rete ridges. In Solar Added to this are many other proteins (membrane-associated, lentigines, the basement membrane is also perturbed, which cytoskeletal, transport, etc.) involved in pigmentation in both likely contributes to melanin entering the dermis to result in the melanocyte and the keratinocyte, indicating the complex melanophage formation. ity of the pigmentary process. While the basic process (e.g., 0144. Moreover, the expression levels of several melano stimulation of melanocytes and conversion of tyrosine to genesis-associated genes are known to be increased in actinic melanin) is well Studied, there are many regulatory elements lentigos. There is an accentuation of the epidermal endothelin that have emerged from recent research involved in signaling, inflammatory cascade, together with decreased proliferation in the transport of melanosomes within the melanocyte, and and differentiation of lesional keratinocytes. Many of these the transfer of melanosomes to the keratinocyte. changes appear to be permanent since these spots persist even when further UV exposure is avoided. VI. Cosmetic Compositions and Personal Care Products 0145 While lentigos appear to be permanent, their mela 0150 Generally, skin-active agents identified for the nin content and thus their intensity will vary seasonally. For enhancement of skin tone or for treatment of pigment-related example, in evaluation of women with facial hyperpigmented skin conditions may be applied in accordance with cosmetic spots in October versus December (in Kobe, Japan or Cincin compositions and formulation parameters well-known in the nati, Ohio, USA), there is a marked reduction in the size of art. Various methods of treatment, application, regulation, or spots over that time period, Suggesting that the lack of con improvement may utilize the skin care compositions com tinued exposure to Sunlight in winter leads to gradual reduc prising skin-active agents identified according to the inven tion in melanin production (seasonal fading) even in hyper tive methods. pigmented spots. Additionally, in a separate examination of 0151. Skin hyperpigmentation as a cosmetic concern is facial spots in March versus May (in Cincinnati, Ohio, USA), generally treated by topical formulation administration, so a marked increase in the size of spots is observed, consistent that depigmentation is restricted to hyperpigmented areas and with the expected increased pigmentation due to increased normal skin is left unaffected by the drug. Sun exposure in spring (seasonal darkening). 0152 Because of the desirability of providing various cos 0146 From a consumer appearance standpoint, hyperpig metic skin anti-aging benefits to a consumer, it may be ben mented spots and uneven pigmentation are important in the eficial to incorporate test agents or compounds identified by perception of age. In a series of studies, facial images were one or more of the screening methods described herein into a digitally modified to remove all age-defining textural features cosmetic composition Suitable for topical application to skin. (e.g., facial furrows, folds, lines, wrinkles) leaving only pig That is, it may be desirable to include the test agent as an mentation as the variable. Studies have shown that when ingredient in the cosmetic composition. In certain embodi using naive judge evaluation and computer image analysis of ments, the cosmetic composition may include a dermatologi the facial images, pigmentation features can contribute to up cal acceptable carrier, the test agent, and one or more optional to 20 years in perceived age of individuals. ingredients of the kind commonly included in the particular 0147 The hyperpigmentary disorder generally referred to cosmetic compositing being provided. as melasma is not well understood. It occurs typically as 0153 Dermatologically acceptable carriers should be safe symmetrical lesions on the face, primarily in darker skin type for use in contact with human skin tissue. Suitable carriers females at puberty or later in life. Sunlight exposure is likely may include water and/or water miscible solvents. The cos a factor in the development of melasma since it occurs on the metic skin care composition may comprise from about 1% to face (a Sun-exposed body site) and since the condition wors about 95% by weight of water and/or water miscible solvent. ens in the Summer. Most melasma Sufferers have a hypersen The composition may comprise from about 1%, 3%. 5%, sitivity to ultraviolet radiation, and even brief exposures to 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, Sunlight can stimulate hyperpigmentation. There is also a 60%, 65%, 70%, 75%, 80%, or 85% to about 90%, 85%, 80%, US 2013/026100.6 A1 Oct. 3, 2013
75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, prise from about 0.05%, 0.1%, 0.2%, 0.3%, 0.5%, or 1% to 25%, 20%, 15%, 10%, or 5% water and/or water miscible about 20%, 10%, 5%, 3%, 2%, or 1% emulsifier. Emulsifiers solvents. Suitable water miscible solvents include monohy may be nonionic, anionic or cationic. Non-limiting examples dric alcohols, dihydric alcohols, polyhydric alcohols, glyc of emulsifiers are disclosed in U.S. Pat. No. 3,755,560, U.S. erol, glycols, polyalkylene glycols such as polyethylene gly Pat. No. 4,421,769, and McCutcheons, Emulsifiers and col, and mixtures thereof. When the skin care composition is Detergents, 2010 Annual Ed., published by M. C. Publishing in the form of an emulsion, water and/or water miscible Co. Other suitable emulsifiers are further described in the Solvents are carriers typically associated with the aqueous Personal Care Product Council's International Cosmetic phase. Ingredient Dictionary and Handbook, Thirteenth Edition, 0154 Suitable carriers also include oils. The skin care 2006, under the functional category of "Surfactants—Emul composition may comprise from about 1% to about 95% by Sifying Agents.” weight of one or more oils. The composition may comprise 0.161 Linear or branched type silicone emulsifiers may from about 0.1%, 0.5%, 1%, 2%. 5%, 10%, 15%, 20%, 25%, also be used. Particularly useful polyether modified silicones 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, include KF-6011, KF-6012, KF-6013, KF-6015, KF-6015, 80%, 85%, or 90% to about 90%, 85%, 80%, 75%, 70%, 65%, KF-6017, KF-6043, KF-6028, and KF-6038 from Shin Etsu. 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, Also particularly useful are the polyglycerolated linear or 10%. 5%, or 3% of one or more oils. Oils may be used to branched siloxane emulsifiers including KF-6100, KF-6104. solubilize, disperse, or carry materials that are not suitable for and KF-6105 from Shin Etsu. Emulsifiers also include emul water or water soluble solvents. Suitable oils include sili Sifying silicone elastomers. Suitable silicone elastomers may cones, hydrocarbons, esters, amides, ethers, and mixtures be in the powder form, or dispersed or solubilized in solvents thereof. The oils may be volatile or nonvolatile. Such as Volatile or nonvolatile silicones, or silicone compat 0155 Suitable silicone oils include polysiloxanes. Com ible vehicles such as paraffinic hydrocarbons or esters. Suit mercially available polysiloxanes include the polydimethyl able emulsifying silicone elastomers may include at least one siloxanes, which are also known as dimethicones, examples polyalkyl ether or polyglycerolated unit. of which include the DM-Fluid series from Shin-Etsu, the 0162 Structuring agents may be used to increase viscos Vicasil(R) series sold by Momentive Performance Materials ity, thicken, solidify, or provide solid or crystalline structure Inc., and the Dow Corning.R. 200 series sold by Dow Corning to the skin care composition. Structuring agents are typically Corporation. Specific examples of suitable polydimethylsi grouped based on solubility, dispersibility, or phase compat loxanes include Dow Corning.R. 200 fluids (also sold as ibility. Examples of aqueous or water structuring agents Xiameter R. PMX-200 Silicone Fluids) having viscosities of include polymeric agents, natural or synthetic gums, polysac 0.65, 1.5, 50, 100, 350, 10,000, 12,500 100,000, and 300,000 charides, and the like. In one embodiment, the composition centistokes. may comprises from about 0.0001%, 0.001%, 0.01%, 0.05%, 0156 Suitable hydrocarbon oils include straight, 0.1%, 0.5%, 1%, 2%, 3%, 5% to about 25%, 20%, 10%, 7%, branched, or cyclic alkanes and alkenes. The chain length 5%, 4%, or 2%, by weight of the composition, of one or more may be selected based on desired functional characteristics Structuring agents. such as volatility. Suitable volatile hydrocarbons may have 0163 Polysaccharides and gums may be suitable aqueous between 5-20 carbon atoms or, alternately, between 8-16 phase thickening agents. Suitable classes of polymeric struc carbon atoms. turing agents include but are not limited to carboxylic acid O157. Other suitable oils include esters. The suitable esters polymers, polyacrylamide polymers, Sulfonated polymers, typically contained at least 10 carbon atoms. These esters high molecular weight polyalkylglycols or polyglycerins, include esters with hydrocarbyl chains derived from fatty copolymers thereof, hydrophobically modified derivatives acids or alcohols (e.g., mono-esters, polyhydric alcohol thereof, and mixtures thereof. Silicone gums are another oil esters, and di- and tri-carboxylic acid esters). The hydrocar phase structuring agent. Another type of oily phase structur byl radicals of the esters hereof may include or have ing agent includes silicone waxes. Silicone waxes may be covalently bonded thereto other compatible functionalities, referred to as alkyl silicone waxes which and are semi-solids Such as amides and alkoxy moieties (e.g., ethoxy or ether or solids at room temperature. Other oil phase structuring linkages, etc.). agents may be one or more natural or synthetic waxes such as 0158 Other suitable oils include amides. Amides include animal, vegetable, or mineral waxes. compounds having an amide functional group while being 0164. The skin care compositions may be generally pre liquid at 25° C. and insoluble in water. Suitable amides pared by conventional methods such as known in the art of include N-acetyl-N-butylaminopropionate, isopropyl N-lau making compositions and topical compositions. Such meth roylsarcosinate, and N,N-diethyltoluamide. Other suitable ods typically involve mixing of ingredients in or more steps to amides are disclosed in U.S. Pat. No. 6,872,401. a relatively uniform state, with or without heating, cooling, 0159) Other suitable oils include ethers. Suitable ethers application of vacuum, and the like. Typically, emulsions are include Saturated and unsaturated fatty ethers of a polyhydric prepared by first mixing the aqueous phase materials sepa alcohol, and alkoxylated derivatives thereof. Exemplary rately from the fatty phase materials and then combining the ethers include Cao alkyl ethers of polypropylene glycols, two phases as appropriate to yield the desired continuous and di-Cs so alkyl ethers. Suitable examples of these materi phase. The compositions are preferably prepared such as to als include PPG-14 butyl ether, PPG-15 stearyl ether, dioctyl optimize stability (physical stability, chemical stability, pho ether, dodecyloctyl ether, and mixtures thereof. tostability, etc.) and/or delivery of active materials. The com 0160 The skin care composition may comprise an emul position may be provided in a package sized to store a suffi sifier. An emulsifier is particularly suitable when the compo cient amount of the composition for a treatment period. The sition is in the form of an emulsion or if immiscible materials size, shape, and design of the package may vary widely. are being combined. The skin care composition may com Certain package examples are described in U.S. Pat. Nos. US 2013/026100.6 A1 Oct. 3, 2013
D570,707; D391,162: D516,436; D535,191, D542,660; GeneChip(R), which was then washed, stained and scanned D547, 193: D547,661; D558,591; D563,221; 2009/0017080: using the protocol provided by Affymetrix. 2007/0205226; and 2007/0040306. Example 1 EXAMPLES 0170 This Example illustrates use of C-map to identify 0.165. The present invention will be better understood by connections between perturbens and genes associated with a reference to the following examples which are offered by way pigmentation condition, wherein the pigmentation condition of illustration not limitation. is a hyperpigmentation condition. Specifically an analysis of tissue from the arms of individuals showing the hyperpig Generally Applicable C-Map Methodology mentation 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 Generating Instances specific statistical comparisons, filtering, and sorting. The 0166 Individual experiments (referred to as batches) gen expression signature can then be used to implement a data erally comprise 30 to 96 samples analyzed using Affymetrix architecture by providing a C-map query to identify relation GeneChip(R) technology platforms, containing 6 replicates of ships between perturbens and genes associated with a hyper the vehicle control (e.g., DSMO), 2 replicate samples of a pigmentation pigmentation condition. positive control that gives a strong reproducible effect in the cell type used, and samples of the test material/perturbagen. Deriving a Hyperpigmentation Condition Expression Replication of the test material is done in separate batches due Signature to batch effects. In vitro testing was performed in 6-well 0171 RNA isolated from clinical samples was analyzed plates to provide sufficient RNA for GeneChip(R) analysis (2-4 using the Affymetrix HG-U133 Plus 2.0 GeneChips, which ug total RNA yield/well). contain 54,613 probe sets complementary to the transcripts of 0167 Human telomerized keratinocytes (tkC) were more than 20,000 genes. However, instances in the provided obtained from the University of Texas, Southwestern Medical database used were derived from gene expression profiling Center, Dallas, Tex... thC cells were grown in EpiLife(R) media experiments using Affymetrix HG-U133A 2.0 GeneChips, with 1x Human Keratinocyte Growth Supplement (Invitro containing 22.214 probe sets, which are a Subset of those gen, Carlsbad, Calif.) on collagen I coated cell culture flasks present on the Plus 2.0 GeneChip. Therefore, in developing and plates (Becton Dickinson, Franklin Lakes, N.J.). Kerati gene expression signatures from the clinical data, the probe nocytes were seeded into 6-well plates at 20,000 cells/cm 24 sets were filtered for those included in the HG-U133A 2.0 hours before chemical exposure. Human skin fibroblasts (BJ gene chips. cell line from ATCC, Manassas, Va.) were grown in Eagle's 0172 A statistical analysis of the microarray data is per Minimal Essential Medium (ATCC) supplemented with 10% formed to derive a plurality of hyperpigmentation gene fetal bovine serum (HyClone, Logan, Utah) in normal cell expression signatures which may comprise a statistically rel culture flasks and plates (Corning, Lowell, Mass.). BJ fibro evant number of the up-regulated and down-regulated genes. blasts were seeded into 6-well plates at 12,000 cells/cm24 In certain embodiments a hyperpigmentation gene expression hours before chemical exposure. signature includes between 10 and 400 up-regulated and/or (0168 All cells were incubated at 37° C. in a humidified between 10 and 400 down-regulated genes. In more specific incubator with 5% CO. At t=-24 hours cells were embodiments a hyperpigmentation gene expression signature trypsinized from T-75 flasks and plated into 6-well plates in includes the 50 most statistically relevant up-regulated genes basal growth medium. At t=0 media was removed and alone or in combination with the 50 most statistically relevant replaced with the appropriate dosing solution as per the down-regulated genes. Regulation is determined in compari experimental design. Dosing Solutions were prepared the pre son to gene expression in normal cells. vious day in sterile 4 ml Falcon snap cap tubes. Pure test 0173 a. Filtering based on Absent/Margin/Present materials may be prepared at a concentration of 1-200 uM, Calls. This filter creates a list of potential genes for and botanical extracts may be prepared at a concentration of inclusion in the gene expression signature. For example, 0.001 to 1% by weight of the dosing solution. After 6 to 24 a suitable filter may be that at least 50% of the samples hours of chemical exposure, cells were viewed and imaged. in one treatment group must have a Present call for each The wells were examined with a microscope before cell lysis probe set. Present calls are derived from processing the and RNA isolation to evaluate for morphologic evidence of raw GeneChip data and provide evidence that the gene toxicity. If morphological changes were sufficient to suggest transcript complementary to a probe set that is actually cytotoxicity, a lower concentration of the perturbagen was expressed in the biological sample. The probes that are tested. Cells were then lysed with 350 ul/well of RLT buffer absent from all samples are likely to be just noisy mea containing B-mercaptoethanol (Qiagen, Valencia, Calif.), surements. This step is important to filter out probe sets transferred to a 96-well plate, and stored at -20°C. that do not contribute meaningful data to the signature. 0169 RNA from cell culture batches was isolated from the For hyperpigmentation gene expression signatures, the RLT buffer using Agencourt(R) RNAdvance Tissue-Bind mag data was filtered for probe sets with at least 50% Present netic beads (Beckman Coulter) according to manufacturers calls provided by the Affymetrix MAS 5 software. instructions. 1 jug of total RNA per sample was labeled using 0.174 b. Filtering According to a Statistical Measure. Ambion Message AmpTM II Biotin Enhanced kit (Applied For example, a Suitable statistical measure may be p-val Biosystems Incorporated) according to manufacturers ues from a t-test, ANOVA, correlation coefficient, or instructions. The resultant biotin labeled and fragmented other model-based analysis. As one example, p-values cRNA was hybridized to an Affymetrix HG-U133A 2.0 may be chosen as the statistical measure and a cutoff US 2013/026100.6 A1 Oct. 3, 2013 19
value of p=0.05 may be chosen. Limiting the signature WHITE (Sepiwhite is the purported tradename of the agent list to genes that meet Some reasonable cutoff for statis known as undecylenoyl phenylalanine) are applied as tical significance compared to an appropriate control is described below to tert-Keratinocyte cells to generate the important to allow selection of genes that are character benchmark skin pigmentation-modifying gene expression istic of the biological state of interest. This is preferable signature. to using a fold change value, which does not take into (0179 A. Hexamidine (hex). account the noise around the measurements. The t-sta 0180 A tert-Keratinocyte (tkC) cell line is used to con tistic was used to select the probe sets in the signatures duct the genomics study with an Affy U133A chip. (a) Probe because it is signed and provides an indication of the selection method for up-regulated probes: 1. mean expression directionality of the gene expression changes (i.e. up- or value of hex treated 200, 2. ratio of present calls of hex down-regulated) as well as statistical significance. treated chips=50%, 3. up regulated by hex, but down regu (0175 c. Sorting the Probe Sets. All the probe sets are lated by MSH (a skin darkening agent), 4. hex treated p-0.05, Sorted into sets of up-regulated and down-regulated sets top 100 probes ranked by p value; (b) Probe selection method using the statistical measure. For example, if a t-test was for down regulated probes, 1... mean expression value of used to compute p-values, the values (positive and nega DMSO controld-200, 2. ratio of present calls of DMSO con tive) of the t-statistic are used to sort the list since p-val trol chips-50%, 3. down-regulated by hex, but up-regulated ues are always positive. The sorted t-statistics will place by MSH (a skin darkening agent), 4. hex treated p-0.05, top the sets with the most significant p-values at the top and 100 probes ranked by p value. The illustrative signatures are bottom of the list with the non-significant ones near the set forth as FIGS. 10 and 11, Tables D and E, respectively. middle. 0181 B. N-acetyl-glucosamine (NAG). 0176 d. Creation of the Gene expression signature. 0182 thCC cell line, Affy U133A chip; Probe selection Using the filtered and sorted list created, a suitable num method for up-regulated probes: 1. mean expression value of ber of probe sets from the top and bottom are selected to NAG treated-200, 2. ratio of present calls of NAG treated create a gene expression signature that preferably has chips-50%, 3. up regulated by NAG, but down regulated by approximately the same number of sets chosen from the MSH (a skin darkening agent), 4. NAG treated p-0.05, 39 top as chosen from the bottom. For example, the gene probes are selected. Probe selection method for down-regu expression signature created may have at least about 10, lated probes: 1. mean expression value of DMSO con 50, 70, 100, 200, or 300 and/or less than about 800, 600, trold-200, 2. ratio of present calls of DMSO control 400 or about 100 genes corresponding to a probe set on chips-50%, 3. down regulated by NAG, but up regulated by the chip. The number of probe sets approximately cor MSH (a skin darkening agent), 4. NAG treated p-0.05, 43 responds to the number of genes, but a single gene may probes are selected. The illustrative signatures are set forth as be represented by more than one probe set. It is under FIGS. 12 and 13, Tables F and G, respectively. stood that the phrase “number of genes' as used herein, corresponds generally with the phrase “number of probe 0183 C. Niacinamide. sets. 0.184 The Niainamide signature was generated by filter 0177. An exemplary Hyperpigmentation Condition Sig ing analogous to that used for hexamidine or NAG was used. nature according to the invention is provided, wherein the The illustrative signatures are set forth as FIGS. 14 and 15, hyperpigmentation condition is Solar Lentigines (Age Tables H and I, respectively. Spots). Data is generated from an arm age spot genomics 0185. D. Sepiwhite. study, full spottissue vs. full normal tissue comparison. Probe 0186 The Sepiwhite signature was generated by filtering selection method for up-regulated probes: 1. mean expression analogous to that used for hexamidine or NAG was used. The value of spottissue-200, 2. ratio of present call of spot tissue illustrative signatures are set forth as FIGS. 16 and 17, Tables chips-50%, 3. probe is significantly up regulated, p<0.05, 4. J and K, respectively. top 50, 100 and 200 probes ranked by p values, are selected respectively. Three signatures are generated using top up and Example 3 down regulated probes cut at 50, 100 and 200, respectively. 0187. This Example illustrates use of C-map and the gen C-Map hit evaluation: hits are selected based on their average eration of a signature as described in Example 2; however weight from three signatures. Probe selection method for Example 3 outlines development of a composite “skin tone' down-regulated probes: 1. mean expression value of normal signature where Niacinaminde, Sepiwhite, NAG, and Hexa tissue-200, 2. ratio of present call of normal tissue midine are used together to generate a signature. This chips-50%, 3. probe is significantly down regulated, p<0. Example illustrates generation of an exemplary composite 05, 4. Top 50, 100 and 200 probes ranked by p values, are “skin tone' Signature comprised of four benchmark skin selected respectively. The illustrative signature is set forth as lightening agents: Niacinamide, Sepiwhite, NAG, and Hexa FIG. 2, Tables B and C. midine. Chips Used for Signature Generation: DMSO control Example 2 chips used for Signature generation: Conditions for Signature Generation: 1. a probe must have 10% present call among 0178. This Example provides support for the use of bench Control chips or BenchMark chips, 2. The average signal for mark signatures in a C-map query to generate putative agents. an up-regulated probe on the treated chip must be >200, 3. The Example specifically outlines generation of a benchmark The average signal for a down-regulated probe on the control skin pigmentation-modifying gene expression signature. As chip must be >200. 4. A probe must be up or down-regulated described herein, the benchmark skin pigmentation-modify cross all benchmark chips. Table Headers: Average signal of ing gene expression signature was generated using methods all control chips, AvgFC, Avg.SignalTreated: Average signal such as filtering as described in Example 1. More specifically, of all treated chips, Average fold change, Avg.SignalControl. Hexamidine, N-acetylglucosamine, Niacinamide, or SEPI The illustrative signature is set forth as FIG. 18, Table L. US 2013/026100.6 A1 Oct. 3, 2013 20
0188 This Example supports embodiments outlining how CMap scores for Some skin lightening agents with the Ret a composite signature may be generated by treating a cell inoic Acid Keratinocyte RA 200 Signature are shown in sample with more than one agent. As indicated earlier, a FIG. 23, Table Q. composite signature can be added in two ways: cells can be treated with each agent separately, the signature can be gen Example 6 erated by comparing regulated genes from all agents (to gether), looking for genes regulated in the same direction by 0191 This Example provides evidence of the advantages all agents; secondarily, agents can be mixed together prior to ofusing composite signatures and illustrates clinical affirma treatment of cells. In another embodiment, a composite tion of the C-map model for predicting efficacy of skin benchmark signature may be generated for a skin-lightening lightening agents. This Example Supports embodiments agent, and another generated for a skin darkening agent. The related to composite signatures (as described at the end of signature for the skin-lightening agent may be further Example 3). An illustrative “Skin Tone' Signature developed tweaked by eliminating any gene from the signature that also from a genomics study using a composite skin-lightening appears in the signature of the skin-darkening agent, regu benchmark agent signature is derived from Niacinamide, lated in the same direction, or vice versa. The inventors dis Hexamidine, Sepiwhite, and NAG. The signature is used to covered that such composite signatures are particularly useful query C-map and generate a list of potential skin-lightening for mining C-map for agents capable of modifying skin pig agents. A top hit, Chlorhexidine Diactate (CD) is entered into ment in the desired direction. clinical testing for confirmation of efficacy. The control for clinical efficacy is a 5% Niacinamide--1% Sepiwhite formu lation in the control vehicle. Example 4 0.192 Primary endpoints are changes in color spot area 0189 This Example illustrates use of C-map and the gen fraction (image analysis) and melanin spot area fraction eration of a signature. More specifically, the signature was (NC2) from baseline. Secondary endpoints are changes from generated through application of Retinoic acid to fibroblasts baseline in L*a*b (color image analysis), mean melaningray and keratinocytes. This Example illustrates a method forgen scale (NC2), and melanin evenness (NC2). Texture area frac erating a benchmark skin tone agent signature according to tion and pore area fraction are also evaluated to explore the invention in each of two different cell types for compari impact on other aspects relating to overall skin tone. Statisti son of the C-map hit evaluations, wherein the benchmark skin cal significant Superiority to the vehicle at one of these time active agent is Retinoic acid (“RA) and the cell types are (a) points is a project Success criteria. Statistically significant fibroblast, and (b) keratinocyte. Superiority to the high efficacy benchmark skin active agent to vehicle performance is the clinical Success criteria. (a) Tert-keratinocytes (tkC) RA Signatures. Cells were 0193 Study Design: The experimental protocol included a treated with 1 uM tRA for 6 hr, tested in triplicate, with 9-week (1-week preconditioning & 8-week treatment), ran triplicate DMSO controls, and analyzed on HG-U133A domized, double-blind, round robin, vehicle-controlled, GeneChips; Signatures were generated like for BJ fibroblasts, split-face tone benefit study. The subject population included below; the signature KC RA 200 consists of 100 most sig 330 Chinese females, 25-55 years old with hyperpigmented nificant up- and 100 most significant down-regulated probe spots. 318 subjects completed the entire study. Pre-condition sets; the signature KC RA 400 consists of 200 most signifi ing was achieved with application of Nature Science Deep cant up-and 200 most significant down-regulated probe sets. Purify cleanser and study-specific moisturizer for a week. (b) Fibroblast RA Signatures. The selected cell type is BJ Olay Complete SPF 15 UV Moisturizing Lotion is concomi fibroblast. Cells were treated with 1 uMtRA for 6 hr, tested in tantly used during pre-conditioning and treatment. 0.5g each triplicate, with triplicate DMSO controls, and analyzed on test product perhalf-face (forehead to jaw line; ~4 mg/cm) is HG-U133A GeneChips. Present calls.>0 for naive, DMSO dosed 2x a day (morning/evening). Color SAF and treatment and RA (9 samples total). Mean signald-200 for DMSO OR area, Melanin SAF, gray scale, evenness by NC2, and addi RA samples t-test p-0.05: Filtered for minimum fold change tional measurement of Fine Lines and Wrinkle and Texture up or down of 1.2: Used log fold change to establish direc are by REAL 3.01 A. Data collection points include baseline, tionality and sorted up and down lists by t-test p value. The and ends of weeks 4, 6 and 8. The tested hypothesis is that signature BJ RA 200 consists of 100 most significant up there is no difference in clinical endpoint versus the bench and 100 most significant down-regulated probe sets: The mark composition treatment. Study site is Kuntai Clinical signature BJ RA 400 consists of 200 most significant up Center, Beijing, China and study time frame is February 2010 and 200 most significant down-regulated probe sets. The (pre-conditioning) to April 2010. illustrative signatures are set forth as FIGS. 19, 20, 21, AND 0194 0.05% Chlorhexidine Diacetate in SC-99 vehicle 22, Tables M, N, O and Prespectively. (7% glycerin) was a top connectivity hit using the tone com posite benchmark signature query set forth as Example 3 and Example 5 is tested for clinical efficacy with respect to four different tone criteria against a known high efficacy benchmark composi 0190. This example summarizes representative potential skin-lightening agents and C-map query results for the bench tion of 5% Niacinamide and 1% Sepiwhite in SC-99 vehicle. mark skin active agent all-trans-retinoic acid according to the Results: invention. C-map was queried using the Retinoic Acid/Kera tinocyte 200 benchmark signature. The average C-map scores 0.195 According to the spot area fraction color test: 0.05% for the top scoring known skin lightening agents are tabled. CD showed significantly fewer spots when compared to Retinoic acid had the highest score of the materials tested vehicle at week 8. In the spot area fraction NC2 test, CD because it was used to generate the signature. The data shown showed a significantly reduced fraction when compared to the are for teleomerized human keratinocytes (tkC). Average vehicle at both weeks 6 and 8. In the NC2 Melanin evenness US 2013/026100.6 A1 Oct. 3, 2013
test, CD demonstrated superiority to the vehicle at weeks 6 with melanocytes. More specifically, in this Example, six skin and 8, and with respect to Basal skin tone, CD demonstrated tone benchmark materials were applied to each of three cell superiority at week 8. Surprisingly, CD also demonstrated types (tert-keratinocytes, melanocyes, and melanoma cells), superior efficacy in the Pore area and Texture area fractions and with four of six tested materials tert-keratinocytes when compared to the vehicle at week 8, Suggesting that it is showed the greatest response (as indicated in FIG. 25, Table a good candidate for overall skin tone and texture enhance S which shows that with four of the six tested materials, the ment. number of probe sets with significant P-values compared to DMSO controls was greatest for tert-keratinocytes). As can Example 7 be seen in Table S, The six tested skin tone benchmark mate rials included: Haxamidine diisothionate, Myo-inositol, 0196. This Example provides support for the unexpected N-acetyl-glucosamine, NDP-MSH, Niacinamide, and Sepi effectiveness of composite signature use with the C-map white. For completeness, details of the exact types of melano technology, with this Example illustrating a comparison of cytes and melanoma cells, as well as the cell culturing con expression signature efficacy in predicting inhibitors; specifi ditions and result analysis are provided herein below. cally, from 33 various chemicals identified as melanogenesis 0199 HEMn primary neonatal medium pigment melano inhibitors in the mouse B16 melanoma cell assay (FIG. 24. cytes were obtained from Invitrogen, Carlsbad, Calif. and Table R). The composite signature was unexpectedly more were cultured in Medium 254 from Invitrogen. HBL mela effective at identifying inhibitors than any of the individual noma cells were obtained from the Laboratory of Oncology signatures (such as for Niacinamide, NAG, Hexamidine, or and Experimental Surgery, Institut Bordet, Université Libre Sepiwhite). For this analysis C-map hits were defined as de Bruxelles, Belgium and were cultured in F-10 Nutrient materials occurring in the top 200 instances (from the same Mixture (Ham) from Invitrogen supplemented with 10% fetal pool of 2266 instances) with a scorele0.30. Result Summary bovine serum (HyClone, Logan, Utah). Human telomerized of correctly predicted melanogenesis inhibitors: Composite keratinocytes (tert-keratinocytes) were obtained from the signature: 20, Niacinamide: 1. NAG: 1, Hexamidine: 2, and Sepiwhite: 9. The C-map scores shown in Table Rare average University of Texas, Southwestern Medical Center, Dallas, scores across the instances of the chemicals. A maximum Tex. and were grown in EpiLife(R) media with 1x Human positive C-map score is 2.0 indicating perfect positive con Keratinocyte Growth Supplement (Invitrogen). All cells were nectivity. The individual materials do not show perfectly high incubated at 37° C. in a humidified incubator with 5% CO2. scores linking to themselves because of replicate variability, 0200 Cells were seeded into 6-well plates a 24 hours which is more evident for materials with relatively weak before chemical exposure, and the skin tone benchmark effects on gene expression. Surprisingly, for this set of mate chemicals listed in the table below were added to culture rials the composite signature in 3 of 4 cases gave better scores medium dissolved in DMSO. The final concentration of with the benchmark materials than the individual benchmark DMSO was 0.1%, and cells treated just with DMSO served as signatures. The process of generating the benchmark signa controls. After 6 hours of chemical exposure cells were then ture may select for the most consistently regulated probe sets, lysed with 350 ul/well of RLT buffer containing B-mercapto which may account for this result. ethanol (Qiagen, Valencia, Calif.), transferred to a 96-well 0.197 As indicated in Example 3, this Example (7) sup plate, and stored at -20° C. RNA from cell culture batches ports embodiments outlining how a composite signature may was isolated from the RLT buffer using Agencourt(R) RNAd be generated by treating a cell Sample with more than one vance Tissue-Bind magnetic beads (Beckman-Coulter, Brea agent. As indicated earlier, a composite signature can be Calif.92821) according to manufacturers instructions. 1 ug added in two ways: cells can be treated with each agent of total RNA per sample was labeled using Ambion Message separately, the signature can be generated by comparing regu AmpTM II Biotin Enhanced kit (Life Technologies, Grand lated genes from all agents (together), looking for genes Island, N.Y. 14072) according to manufacturers instructions. regulated in the same direction by all agents; secondarily, The resultant biotin labeled and fragmented cRNA was agents can be mixed together prior to treatment of cells. In hybridized to an Affymetrix HG-U133A 2.0 GeneChip(R), another embodiment, a composite benchmark signature may which was then washed, stained and scanned using the pro be generated for a skin-lightening agent, and another gener tocol provided by Affymetrix. ated for a skin darkening agent. The signature for the skin 0201 Regarding the results and analysis of the testing: lightening agent may be further tweaked by eliminating any Two sample t-tests were performed on each treatment to gene from the signature that also appears in the signature of compare with the DMSO control. The number of probe sets the skin-darkening agent, regulated in the same direction, or with significant p-values (<0.05) are summarized in the table vice versa. The inventors discovered that such composite below. Each GeneChip(R) contains 22215 probes sets. Using a signatures are particularly useful for mining C-map foragents significance level of 0.05, 1111 probe sets (95% confidence interval of 1047 to 1174) are expected to be significant by capable of modifying skin pigment in the desired direction. chance alone. This estimate is somewhat conservative since Example 8 there may be multiple probe sets for the same gene. 0202 In summary, the tert-keratinocytes were generally 0198 This Example provides support to illustrate that it is the most responsive cells to the skin tone benchmark materi believed that keratinocyte cells, rather than melanocyte or als. There were more significantly regulated probesets for 4/6 melanoma cells, have exhibited a more robust transcriptional skin tone benchmark materials in the tert-keratinocytes com profile when treated with skin-lightening agents. Kerati pared to either HeMnMP melanocytes or HBL melanoma nocytes have been preliminarily shown to be easier to grow cells. The tert-keratinocytes have an additional advantage than melanocytes and have increased responsiveness Such over the second most responsive cells, HeMnMP melano that keratinocytes may be able to be used to detect active cytes, in that they grow Substantially faster and are more chemicals over a wider range of concentrations than testing practical cell line for routine screening. US 2013/026100.6 A1 Oct. 3, 2013 22
0203 Every document cited herein is hereby incorporated 6. An immobilized array of oligonucleotides which hybrid herein by reference in its entirety unless expressly excluded ize to the genes constituting the gene expression signature or otherwise limited. The citation of any document is not an according of claim 1. admission that it is prior art with respect to any invention 7. A method of generating a gene expression signature for disclosed or claimed herein or that it alone, or in any combi use in identifying connections between perturbagens and nation with any other reference or references, teaches, Sug genes associated with a skin pigmentation condition, the gests or discloses any such invention. Further, to the extent method comprising: any meaning or definition of a term in this document conflicts a. generating a gene expression profile for a human skin with any meaning or definition of the same term in a docu cell sample treated with at least one benchmark skin ment incorporated by reference, the meaning or definition pigmentation modifying agent, wherein the benchmark assigned to that term in this document shall govern. skin pigmentation modifying agent is suspended in a 0204. The values disclosed herein are not to be understood vehicle composition; as being strictly limited to the exact numerical values recited. b. generating a gene expression profile for a human skin Instead, unless otherwise specified, each Such value is cell sample treated with the vehicle composition; intended to mean both the recited value and a functionally c. comparing the expression profiles of (a) and (b) to deter equivalent range Surrounding that value. mine a gene expression signature comprising a set of 0205 The present invention should not be considered lim genes differentially expressed in (a) and (b): ited to the specific examples described herein, but rather d. assigning an identifier to each gene constituting the gene should be understood to cover all aspects of the invention. expression signature and ordering the identifiers accord Various modifications, equivalent processes, as well as ing to the direction of differential expression to create numerous structures and devices to which the present inven one or more gene expression signature lists; and tion may be applicable will be readily apparent to those of e. Storing the one or more gene expression signature lists on skill in the art. Those skilled in the art will understand that at least one computer readable medium. various changes may be made without departing from the 8. The method of claim 7, wherein the human skin cells scope of the invention, which is not to be considered limited derive from an epidermal or a dermal skin layer and are to what is described in the specification. selected from the group consisting of keratinocyte, fibroblast, What is claimed is: melanoma and melanocyte cells. 1. A method of generating a hyperpigmentation condition 9. The method of claim 8, wherein the benchmark skin gene expression signature for use in identifying connections pigment modifying agent comprises an agent selected from between perturbagens and genes associated with a skin pig the group consisting of a melanocyte stimulation inhibitor, an mentation condition, the method comprising: anti-inflammatory agent, an alpha-MSH pigment induction a. providing a gene expression profile for a reference antagonist, a melanophage dermal residence time Suppressor, sample of human skin cells not affected with a pigmen a melanin synthesis-associated enzyme inhibitor, a melano tation condition; Some transport inhibitor, a vitamin B3 compound, hexami b. generating a gene expression profile for at least one dine diisothionate, Myo-inositol, N-acetyl-glucosamine sample of human skin cells from a Subject exhibiting the (NAG), NDP-MSH, an N-acyl amino acid compound, a ret hyperpigmentation condition, inoid compound, hexyldecanol, hydroquinone and combina c. comparing the expression profiles of (a) and (b) to deter tions thereof. mine a gene expression signature comprising a set of 10. The method of claim 9, wherein the skin cell is a genes differentially expressed in (a) and (b): keratinocyte and the signature comprises genes associated d. assigning an identifier to each gene constituting the gene with the identifiers set forth in at least one Table selected from expression signature and ordering the identifiers accord the group consisting of Table D, E, F, G, H, I, M, and N. ing to the direction of differential expression to create 11. The method of claim 10, wherein the benchmark skin one or more gene expression signature lists; and lightening agent is a vitamin B compound, the skin cell is a e. Storing the one or more gene expression signature lists on keratinocyte cell, and the signature comprises genes associ at least one computer readable medium. ated with the identifiers set forth in Table H or Table I. 2. The method of claim 1, wherein the human skin cells 12. The method of claim 10, wherein the benchmark skin derive from an epidermal ordermal skin layer and are selected lightening agent is Hexamidine diisothionate, the skin cell is from the group consisting of keratinocyte, fibroblast, and a keratinocyte cell, and the signature comprises genes asso melanocyte cells. ciated with the identifiers set forth in Table D or Table E. 3. The method of claim 1, wherein the hyperpigmentation 13. The method of claim 10, wherein the benchmark skin condition has an etiology associated with one or more of lightening agent is N-acetyl-glucosamine, the skin cell is a activation of melanocyte stimulation, inflammation, activa keratinocyte cell, and the signature comprises genes associ tion of alpha-MSH pigment induction, increased melanoph ated with the identifiers set forth in Table For Table G. age dermal residence time, activation of an enzyme involved 14. The method of claim 10, wherein the benchmark skin in a melanin synthesis pathway, and activation of melano lightening agent is an N-acyl amino acid compound, the skin Some transport. cell is a keratinocyte cell, and the signature comprises genes 4. The method of claim 1, wherein the identifiers are associated with the identifiers set forth in Table J or Table K. selected from the group consisting of gene names, gene sym 15. The method of claim 10, wherein the benchmark skin bols, and microarray probe set IDs. lightening agent is a retinoid compound, the skin cell is a 5. The method of claim 1, wherein the hyperpigmentation keratinocyte, and the signature comprises genes associated condition is solar lentigines and the signature comprises with the identifiers set forth in Table M or Table N. genes associated with the identifiers set forth in Table Band 16. The method of claim 9, wherein the benchmark skin Table C. lightening agent is a retinoid compound and the skin cell is a US 2013/026100.6 A1 Oct. 3, 2013 23 fibroblast cell, and the signature comprises genes associated with the identifiers set forth in Table O or Table P. 17. The method of claim 9, wherein the at least one bench mark skin modifying agent comprises a skin lightening agent selected from the group consisting of a vitamin B3 com pound, an N-acyl amino acid compound, N-Acetyl Glu cosamine and Hexamidine, and the signature comprises iden tifiers set forth in Table L. 18. The method of claim 7, wherein the benchmark skin pigmentation modifying agent comprises a skin lightening agent, the method further comprising: (a)(2) through (e)(2) repeating steps (a) through (e) except that the benchmark skin modifying agent comprises a skin darkening agent, and (f) comparing the gene expression signature list from (e)(2) to the gene expression signature list from (e) and elimi nating from the gene expression signature list from (e) any identifier corresponding to a gene appearing on the (e)(2) list and having the same direction of differential expression as the gene on the (e) list. 19. The method of claim 7, wherein the identifiers are selected from the group consisting of gene names, gene sym bols, and microarray probe set IDs. k k k k k