US 20130261024A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2013/0261024 A1 HAKOZAKI et al. (43) Pub. Date: Oct. 3, 2013
(54) SYSTEM FOR IDENTIFYING CONNECTIONS (52) U.S. CI. BETWEEN PERTURBAGENS AND GENES CPC ...... G06F 19/18 (2013.01); G0IN33/5044 ASSOCATED WITH A SKN (2013.01); G06F 19/20 (2013.01) HYPERPGMENTATION CONDITION USPC ...... SO6/15: 506/39: 702/20 (71) Applicant: THE PROCTER & GAMBLE COMPANY, Cincinnati, OH (US) (57) ABSTRACT (72) Inventors: Tomohiro HAKOZAKI, Cincinnati, OH (US); Wenzhu ZHAO, Mason, OH (US); Robert Lloyd BINDER, Montgomery, OH (US); Jun XU, Mason, A system for identifying connections between perturbagens OH (US) and genes associated with a skin hyperpigmentation condi tion. The system includes a computer readable medium hav (73) Assignee: The Procter & Gamble Company, ing a plurality of instances stored thereon, and a skin hyper Cincinnati, OH (US) pigmentation-relevant gene expression signature. Each instance includes an instance list of rank-ordered identifiers (21) Appl. No.: 13/851,886 of differentially expressed genes, and the hyperpigmentation relevant gene expression signature includes a gene expression (22) Filed: Mar. 27, 2013 signature list of identifiers representing differentially Related U.S. Application Data expressed genes associated with a hyperpigmentation condi tion or differentially expressed genes associated with a (60) Provisional application No. 61/618,115, filed on Mar. benchmark skin-lightening agent. The system also includes a 30, 2012. programmable computer with computer-readable instruc tions that allow the computer to accessing the instances and a Publication Classification hyperpigmentation-relevant gene expression signature stored (51) Int. Cl. on the computer readable medium, comparing the hyperpig G06F 9/18 (2006.01) mentation-relevant gene expression signature to the plurality G06F 9/20 (2006.01) of the instances, and/or assigning a connectivity score to each GOIN33/50 (2006.01) of the plurality of instances.
Patent Application Publication Oct. 3, 2013 Sheet 1 of 73 US 2013/0261024 A1
FIG. : Table A Pigmentation ControlPigmentation Target controlEffective targets Agent and Examples some Benchmark Agents ...... ------...... Examples s Tyrosinase inhibition - . . . Hydroquinone, resorcinols, kojic acid, arbutin, deoxy-arbutin, ascorbic acid (vitamin C) Inhibition of tyrosinase Glucosamine, N-acetylglucosamine, tunicamycin glycosylation s Melanosome transfer iacinamide, protease inhibitors Inhibit binding of alpha-MSH to N-undecylenoyl-phenylalanine melanocyte Down regulation of tyrosinase Retinoid (trans-retinoic acid, retinol and its esters, retinaldehyde) Antioxidant Vitamin C compounds, vitamin E, sulfhydryl compounds Anti-inflammatory agent Hydrocortisone, phytosterol, glycyrrhetinic acid, tranexamic acid, chamomile extract Increase epidermal turnover Retinoids, Salicylic acid, alpha-hydroxy acids, alpha-keto acids, adenosine monophosphate s. Patent Application Publication Oct. 3, 2013 Sheet 2 of 73 US 2013/0261024 A1
FG. 2: TABLES B and C
Table B Age Spot Signature: top 200 up-regulated
203941 at INTS9 integrator complex subunit 9 M_0182500.00012, 204736s at CSPG4 chondroitin sulfate proteoglycan 4 NM_001897.0.00033
218567 x at DPP3 dipeptidyl-peptidase 3 NM_005700000051 location ( myeloid leukemia-as sociated) 20745 at myostatin 214333 x at IDH3G isocitrate dehydrogenase 3 (NAD+) gamma U69268 207365 x at USP34 ubiquitin specific peptidase 34 2 17953 at PHF3 PHD finger protein 3 217586 x 215200 xat 202396 at transcription elongation regulator 1 203518 at ySOsonal trafficking regulator 22 1692_s_at 202573 at asein kinase , gamma 2 AL530441 -W---a--- I division cycle 42 (GTP binding protein, 25kDa) 202704 at
caspase 6, apoptosis related cysteine peptidase U20537 potassium inwardly-rectifying channel, subfamily J, member 2 AF153820 000287 TAF10 RNA polymerase II, TATA box binding protein (TBP).
Insh homeobox2 IGFBP3 insulin-like growth factor binding protein 3 BF340228 0.0039 - protein tyrosine phosphatase, receptor type, N polypeptide 2
M_002847 0.00396
LAMC laminin, gamma (formerly LAMB2)
214715 x AK024789 0.00436 221219 s at KLHDC4 NM_0175660.00437 202720 at TES NM_015641000474
202178 at PRKCZ 213582 at ATPase, Class VI, type 11A Patent Application Publication Oct. 3, 2013 Sheet 3 of 73 US 2013/0261024 A1
eterogeneous nuclear ribonucleoprotein D-like
203247s------...-- 4 zinc finger protein 24 203714 is tubulin folding cofactor ET y with sequence similarity 49, member B 06623 000539 zinc finger, MYM-type 4 catenin (cadherin-associated protein), alpha-like I S. uanine nucleotide binding protei n (G protein) alpha 12
BCS-like (yeast) 218082 s at UBP1 20095 is at IGFBP3 221802 s at 598 'S'm ------ma 214474 at protein kinase, AMP-activated, beta 2 non-catalytic subunit NM_00539. eroxisome biogenesis factor 222055 at acetoacetate hydrolase domain contai 222055 at marylacetoacetate hydrolase domain containing 2B -vm------a Wm. --- vate dehydrogenase (lipoamide) alpha ------&------&NM_000284.000636,
grin, beta (fibronectin receptor. beta polypeptide, antigen
solute carrier family 2 (facilitated glucose/fructose transporter). member 5 arbonyl reductase 3 poly(ADP-ribose) polymerase family, member 2
anine nucleotide binding protein-like 3 (nucleolar) --...--
line 1 (Opitz/BBB syndrome) hromosome 5 open reading frame 13
olgi associated, gamma adaptin ear containing, ARFbinding i
tRNA synthetase 3 Metallothionein Ipseudogene 3 2 1923 at TGSi trimethylguanosine synthase homolog (S. cerevisiae) 215228 at ient helix loop helix 2 202124s at TRAK2 trafficking protein, kinesin binding 2 --":------natriuretic peptide receptor Ciguanylate cyclase C ( atrionatriuretic ptide receptor C) gi autoantigen, golgin subfamily a, 8 - W - - - - - a ------W-8----''' ------210424. 219039 at SEMA4C NM_017789 000835 215220s at TPR translocated promoter region (to activated MET oncogene) AK02311 1 000835 209306 s at SWAP70 SWAP-70 protein : Patent Application Publication Oct. 3, 2013 Sheet 4 of 73 US 2013/0261024 A1
212987 at FBXO9 211417 x at GGT1 AF356353 0.
------211704.s ------at SPIN2A spindlin family, member 2A ------AF356353 000886 221489 sat SPRY4. sprouty homolog4 (Drosophila) WA8843 0.00898 213376 at ZBTB1 zinc finger and BTB domain containing 1 AI656.706 0.00899 2083.74s capping protein (actin filament) musc 201715 s at ACIN1 apoptotic chromatin condensation inducer 1 NM_0 48117 at CCDC101 coiled-coil domain containing 101 IAA160496 0.00907 SRY (sex determining region Y)-box 4. ------918
SH3-domain binding protein 5 (BTK associated) :
Il death-inducing DFFA-like effector b
synovial sarcoma translocation gene on chromosome 18-like 1 AB014593 0.00957, tumor suppressing subtransferable candidate 4 NM_0057060.00959. transcription factor AP-2 gainina (activating enhancer binding
antigen 1,69kDa tyrosine 3 monooxygenase/tryptophan 5-monooxygenase
activation protein, Zeta polypeptide tubulin folding cofactor B ectodysplasin A
TBK1 binding protein 1 MO1494 ation factor VIII associated (intronic transcript) 1 220051 at PRSS21 protease, serine, 21 (testisin) 210153 sat ME2 ------L-s-va-...------...-...-...------...----...------...--&-ir-iris-us-rmalic enzyme 2, NAD(+)-dependent, mitochondrial 202123s at ABL vabl Abelson murine leukemia viral oncogene homolog -
PTK7 protein tyrosine kinase 7
201403 s at 20581 at ------au-- d), gamma2, accessory subunit NM_007 .. DENND4B DENN/MADD domain containing 4B NM_014856 0.01 126 sema domain, immunoglobulin domain (Ig), short basic domain, Patent Application Publication Oct. 3, 2013 Sheet 5 of 73 US 2013/0261024 A1
Affy ID 214604 at thorneobox Dl
218638 sa spondin 2, extracellular matrix protein 217973 at dicarbonyl/L-xylulose reductase 219007 at N nucleoporin 43kDa NM_0246470.01.195. zinc finger protein 43 -- - -
222136 x AK022905 001206 CNDP dipeptidase 2 (metallopeptidase M20 family) NM_0182350.01211------synaptonemal protein SC65 0.06455 0.01.233 microtubule associated monoxygenase, calponin and LIM dom intaining 1 NM_022765 0.01.237 PTK2 protein tyrosine kinase 2 AL037339 001279 engrailed homeobox2 NM_0014270.01282 202265 at MI polycomb ring finger oncogene 219826 at zinc finger protein 419 NM 02469 10,033 RRP9, small subunit (SSU) processome component, homolog 204133 at RRP9 (yeast) NM_004704001341 2 10652 sat 1orf34 chromosome 1 open re ading frame 34 - C004399.00 353 - IPW Imprinted in Prader-Willi syndrome W770748 001355 amyloid beta (A4) precursor protein-binding, family A, member 2 210720 sat APBA2BP binding protein AB039947 001365
204525 at PHF14 PHD finger protein 14 NM_0146600,01366 ermatogenesis ass modulator transmembrane protein 97. - at PPFIBPI PTPRF interacting protein, binding protein (liprin beta 1) N 200775 sat HNRPK heterogeneous nuclear ribonucleoprotein K BC : golgi associated, gamma adaptin ear containing, ARF binding 50277 at GGA protein l - AW001443 001428 215828 at MRNA; cDNA DKFZp547C126 (from clone DKFZp547C126) A 9 0.014.42 at PSMD4 proteasome (prosome, macropain)26S subunit, non-ATPase, 4
keratin 16 (focal non-epidermol ytic palmoplantar keratoderma) - Pase, Class 1, type 8B, member 1 -
207688s at TNHBC inhibin, beta C NM_0055380.01511, 217409 at MYo5A myosin VA (heavy chain 12, myoxin) Z22957 0.01511
complement compon ; calpain, small subunit 216973 s at ---, -vily 65588 at ypothetical LOC388796 ------issions:------AA8278920.
splicing factor, arginine/serine-rich 14 A1928.127 0.01565.
Al638762 Patent Application Publication Oct. 3, 2013 Sheet 6 of 73 US 2013/0261024 A1
matrix metallopeptidase 14 (membrane-inserted)
-C motif) receptor 7
208613 s at- FLNB filamin B, beta (actin binding protein 278) i phosphoribosylglycinamide formyltransferase, ------: phosphoribosylglycinamide synthetase, 210005 at GART phosphoribosylaminoimidazole synthetase D32051 0.01694; 218095 sat TMEM165 transmembrane protein 165 NM_018475 0.01705.
21291 at DNAJC16 DnaJ (Hsp40) homolog, subfamily C. member 16 AB023179 0.01706 218984 at PUS7 pseudouridylate synthase 7 homolog (S. cerevisiae) NM 0190420.01712, 202912 at ADM a-M---- 202332 at CSNKE casein kinase 1, epsilon NM_001894.00174 i.
218940 at C14orf138 chromosome 14 open reading frame 138 NM_024558 0.01752 202115 s at NOC2L nucleolar complex associated 2 homolog (S. ce. M_015658.00762. 203468 at CDK10 cyclin-dependent kinase (CDC2-like) 10 ------NM_003674-001774
33322 at SFN NM000303001814 212561 at RAB6IP1 RAB6i AA349595 0.01829 222.69 x at SH2D3A 212401s at CDC2L2 200839 sat CTSB--. cathepsin B NM_001908 0.0 1846. 214614 at MNX1 motor neuron and pancreas homeobox i A738662 0.01868. 202128 at KIAA0317 KIAA0317 NM_0148210.01870. Heterogeneous nuclear ribonucleoprotein D (AU-rich element (213359 at HNRPD RNA binding protein 1,37kDa) 220668 sat DNMT3B DNA (cytosine-5-)-methyltransferase 3beta 214218s at XIST x (inactive)-specific transcript 212237 at ASXLI additional sex combs like 1 (Drosophila)
A126492 )01917 .01919. 935.
protein tyrosine phosphatase, receptor type, fpolypeptide (PTPRF), interacting protein (liprin), alpha 1 U22815 0.01961: ------r------r------
iliverdin reductase A BC005902 0.01962. ute carrier family 11 (proton-coupled divalent metalion , inenber 2 :
213672 at MAR 209974 sat BUB3 BUB3 budding uninhibited by benzimidazoles 3 homolog (yeast) 202248 at 2F transcription factor 4. p107/p130 binding
anscription factor 7-like 2 (T-cell specific, HMG-box) 2 2761 at ------202528 at GALE DP-galactose 4-epimerase M_000403.002056. Patent Application Publication Oct. 3, 2013 Sheet 7 of 73 US 2013/0261024 A1
carboxylase antizyme 1 20948.5 s at OSBPLA oxysterol binding protein-like A 201850 at APG apping protein (actin filament), gelsolin-like 208.806 at HD3 chromodomain helicase DNA binding protein 3 74694 sat s abaptin, RAB GTPase binding effector protein 2 --- 218002 s at CXCL14 chemokine (C-X-C motif) ligand 14 206377 at orkhead box F2 203083 at THBS2 thrombospondin2
217805 at ILF3 interleukin enhancer binding factor 3.90kDa 2022.45 at LSS lanosterolosterol synthase (2,3-oxidosqualene-lanosterol(2,3-oxidosqualene-lanost cyclase) AWO8451C
Table C
Age Spot Signature: top 200 down-regulated
i (avian) NM 012323 0.00006 21622 s at PUM2 pumilio homolog 2 (Drosophila) D87078 0.00011 207206 s at ALOX12 arachidonate 12-lipoxygenase NM 0006970.00013 209602 s at GATA3 GATA binding protein 3 203642 s at COBLLI 'm' W. W. ''''m S. Sm' NM_0149000.00025 210832x at PTGER3 prostaglandin Ereceptor 3 (subtype EP3) D38298 (0.00037 217589 at RAB40A RAB40A, member RAS oncogene family ------&------is 217797 at UFC ubiquitin-fold modifier conjugating enzyme 201288 at ARHGDB Rho GDP dissociation inhibitor (GDI) beta 219416 at SCARA3 nger receptor class A member 3 220892 s a PSAT hosphoserine aminotransferase --S-am-r-us--- 3-m-m-m-m------3-3-mm-3- X-ray repair complementing defective repair in Chinese hamster 208643 s at XRCC5 cells 5 (double-strand-break rejoining; Ku autoantigen, 80kDa) J04977 218841 at , kyrin repeat and SOCS box-containing 8 NM_024095 000067 200822.x josephosphate isomerase I ------201777s at KIAA0494 IAA0494 BC002525 205568 at AQP9 aquaporin 9 221 65 at TMEM133A ansmembrane protein 183A AF070537 'O.00088 21265 at TMEM183B transmembrane protein 183B ''m't 206156 at GJB5 WPW.W.'WWWW domain containing E3 ubiquitin protein ligase I DUSP5 s ual specificity phosphatase 5
209976 s at CYP2E1 cytochrome P450 family 2, subfamily E. polypeptide 1 AFi 82276 000133. 213764 sat MFAPS microfibrillar associated protein 5 212588 at PTPRC protein tyrosine phosphatase, receptor type, C Y00062 00014 Patent Application Publication Oct. 3, 2013 Sheet 8 of 73 US 2013/0261024 A1
213030 s at PLXNA2 219627 at ZNF767ZN zinc finger family member 767 202776. xynucleotidyltransferase, terminal, interacting rotein 2
213686 at WPS3A vacuolar protein sorting 13 homologarm-m------W------A (S. cerevisiae) AI186145 000167 206038 s at NR2C2 clear receptor subfamily 2, group C, member 2 NM_003298 0.00172 rix-xx-x-x-r&ls------...,x- 205158 at nuclease, RNase A family, 4 clic nucleotide gated channel alpha 1
somal protein L13a ariadne honolog, ubiquitin-conjugating enzyme E2 binding protein, 1 (Drosophila) AR CASP8 and FADD-like apoptosis regulator .. bosomal protein S2 m
somal protein L18a owth arrest specific 2 like 1 ME1-like 1 (S. cerevisiae)
chondrial ribosomal protein s 4. --ma--- regulator of calcineurin 1 0.00290
SAM domain and HD domain NM_0154740.0029 f LIM domains v-erb b2 erythroblastic leukemia viral oncogene homolog 3
vacuolar protein sorting 24 homolog (S. cerevisiae) 205883 at ZBTB16 inc finger and BTB domain containing 16 203000 at STMN2 stathm in-like 2 BF967657 205504 at Bruton agammaglobulinenia tyrosine kinase 202294 at stromal antigen 206114 at EPHA4 EPH receptor A4 213549 at PDZD8 PDZ domain containing 8 CDK5 regulatory subunit associated protein 1-like 1 213264 at N'- poly(rc) binding protein 2 219349 sat exocyst complex component 2 202716 at protein tyrosine phosphatase, non-r 205067 at interleukin 1, beta
20137 s at DCTD dCMP deaminase 211749s at VAMP3 vesiclesicle associated membrane proteinpro 3 (cellubrevin)m-m-m-3-...-...-J.------207114 at lymphocyte antigen 6 complex, locus G6C 20237 at re--- transcription elongation factor A (SID-like 4 217977 at selenoprotein X, Patent Application Publication Oct. 3, 2013 Sheet 9 of 73 US 2013/0261024 A1
215549 x : 218024 at AST kinase domains 2 --six------RAS guanyl releasing protein (calcium and DAG-regulated) N adaptor protein, phosphotyrosine interaction, PH domain and 218218 at APPL2 leucine Zipper containing 2 NM_0181710.00439 212875 s a C2 orf25 chromosome 21 open reading frame 25 APO01745 0004:43 oid (11-beta) dehydrogenase 1 ''''''' - m S''' 'W. peptidase (mitochondrial processing) beta 203141s at AP3B1 adaptor-related protein complex 3, beta 1 subunit GATA binding protein 3 20375 at rostaglandin E receptor 3 (subtype EP3) x83858y - Y - Y - 20937 at al, T-cell differentiation protein-like BC003179 : LSM14B, SCD6 homologE r------s:-r-s-s-s-s-s-sw------six------ex-s-s-s-s---wic------...--KIAA0999 protein RAB11 family interacting protein (class D ----...-am--a.m.------21394.5 s at NUP210 Nucleoporin 210kDa AA909765 0.00506, 209566 at INSIG2 insulin induced gene 2 AL080184 0.005 12. Inhibitor of DNA binding 4 dominant negative helix-loop-helix 209292 at ID4 0.00515, -mm otch homolog2 (Drosophi la) -- - ''' ------83-M8--u --- endonuclease domain containing 1 --- al-m------a-...-a-um. 31637s at NR1D1 nuclear receptor subfamily l, group D, member 1 M thyroid hormone receptor, alpha (erythroblastic leukemia viral (v x 31637 s at THR erb-a) oncogene homolog, avian) 72631. 0.00534 prostaglandin-endoperoxide synthase (prostaglandin G/H 205128 x-at PTGS synthase and cyclooxygenase) NM_0009620.00543 206628 at Solute carrier family 5 (sodium/glucose cotransporter), member 1 204718 at EPHB6 EPH receptor B6 216245 at interleukin receptor antagonist 212438 at cid binding protein RY-I 215342 is at AB GTPase activating protein 1-like glycosylphosphatidylinositol specific phospholipase D1 201412 at LRP10 low density lipoprotein receptor-related protein 10
49 at ARHGAP25 Rho GTPase activating protein 25
FE23 nuclear factor (erythroid-derived 2)-like 3 RAB3GAPP1 RAB3 GTPase activating protein subunit I (catalytic) 597; 212326 at Ps 13D vacuolar protein sorting 13 homolog D (S. cerevisiae) AB007922 000597 Patent Application Publication Oct. 3, 2013 Sheet 10 of 73 US 2013/0261024 A1
leucine zipper, down-regulated in mW.m. m...... 210954s at TSC22D2 TSC22 domain family, member 2 204782 at 0.00624 212716 s at EIF3K eukaryotic translation initiation factor 3, subunit K 216570 x at LOC646417 similar to 60S ribosomal protein L29 (P23) 27954is at mum F3 PHD finger protein 3 -m-m-m-m-m-m----, -m-m-m-mow-m-m-m-m-vu- - -m-m-m-m-mo v- 'a v V v - V - - - -u- uv as wo 213497 at ABTB2 ankyrin repeat and BTB (PO7) domain containing 2 WW- S. 212738 at ARHGAP19 Rho GTPase activating protein 19
NM_014741.000679. NM_0177360.00699.
RHGAP19 Rho GTPase activating protein 19
218494 is at LC2A4RG...' SLC2A4 regulator 214289 at SMB Proteasome (prosome, Inacropain) subunit, beta type, 1 ission 1 ( mitochondrial outer membrane) homolog (S. cerevisiae) 21 8034 at 214719 at SLC45A3 solute carrier family 46, member 3 ------217272 s at SERPINB 13 Serpin peptidase inhibitor, clade B (ovalbumin), member 13 0.00746;
glycoprotein)
212376 s at EP400 E1A binding protein p4 t prostaglandin-endoperoxide synthase 1 (prostaglandin G/H 215813s at PTGS1 synthase and cyclooxygenase) 218820 at C14orf132 re-ri-o-o-o-o-o-o- 218495 at UXT ubiquitously-expressed transcript ------are:------a------218736 s at PALMD palmdelphin ''''''''''WS''' '. NM_0177340.00858. UBX domai in col
frequenin homolog(Drosophila) transporting, plasma membrane 4 m.' 212468 at SPAG9 sociated antigen 9 AK02351 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 2013/0261024 A1
203687 at CX3CL1 chemokine (C-X3-C motif) ligand 1 NM_0029960.00896 212649 at DHX29 DEAH (Asp-Glu-Ala-His) box polypeptide 29 AL079292 : 2104 18s at IDH3B isocitrate dehydrogenase 3 (NAD+) beta AF023265 r – or riordihydrolipoamide re-re------S-acetyltransferase (E2 component------of pyruvate 212568 s at DLAT dehydrogenase complex) BF978872 000924, 218061 at MEA1 male-enhanced antigen 1 NM_014623 000927 203045 at ninjurin 1 202675------surror------sous--aw.--wi------as-s-so at succinate dehydrogenase complex, subunit B, iron------sulfur (Ip) ------NM_0030000.00943. 2 1862 x at CFLAR ------CASP8 and FADD-like apoptosis regulator 205538 at CORO2A -- coronin, actin binding protein, 2A 200920s------at BTGl ------cell translocation gene I. anti-proliferative - ing, soluble, 9 (ga ------solute carrier family 25 (mitochondrial carrier, adenine nucleotide - 200657208066 ats at SLC25A5GTF2B generaltranslocator), transcription member factor 5 IB NM_0015140.00994 s nuclear factor of activated T-cells, cytoplasmic, calcineurin- t
2105.55 s at NFATC3 dependent 3 ------U85430------AMP2 secretory carrier membrane protein 2 ------
leucine-rich repeats and immunoglobulin-like domains 1
coagulation factor II (thrombin) receptor family with sequence similarity 114, member Al
AB27B RAB27B. member RAS oncogene family ------synuclein, gamma (breast cancer-specific protein l) AF010 126 0.0039. 210785 s at Corris chromosome 1 open reading frame 38 AB035482 0.01046 platelet-activating factor acetylhydrolase, isoform Ib, alpha 200816 s AFAHIBI subunit 45kDa. NM_000430 00107 202992 at omplement component 7 -- 221787 at 6orf120 chromosome 6 open reading frame 120 31618 0.01082 ------s------219010 at Clorf106 chromosome 1 open reading frane 106 01826 204109s at NFYA --- nuclear transcription factor Y, alpha - : 217833 at SYNCRIP synaptotagmin binding, cytoplasmic RNA interacting protein 5-si-o-o:------8:------sur------...--&------aldehyde dehydrogenase 5 family, member Al (succinate r 203608 at ALDH5Al semialdehyde dehydrogenase) AL031230 001110: 218807 at VAV3 vav 3 guanine nucleotide exchange factor NM_00613001111 20462 at protein kinase (cAMP-dependent, catalytic) inhibitor alpha NM_006823 0.01 123 lymerase (RNA) II (DNA directed) polype ptide C, 33kDa 205220 at protein-coupled receptor 109B
2 - onectin leucine rich transmembra 203798 s at ------visinin-like l - - am -----
202861 at PER1 eriod homolog 1 (Drosophila) 201988 s at CREBL2 cAMP responsive element binding protein-like 2. BF438056 0.0183. Patent Application Publication Oct. 3, 2013 Sheet 12 of 73 US 2013/0261024 A1
203675 at 203934 at kinase insert domain receptor (a type 212931 at transcription factor 20 (AR1) .
transducin (beta)-like IX-linked chromosome 9 open reading frame 125 0.01216. 209580s at MBD4 methyl-CpG binding domain protein 4 AFI 14784 0.01221 ------protein phosphatase 3 (formerly 2B), catalytic------subunit, alpha ------202429s at PPP3CA isoform AL353950 0.01222 220797 at METT10D methyltransferase 10 domain containing NM_0240860.01223 25416 s at STOML2 stomatin (EPB72)-like 2 AC004472 0.01224 220413 at SLC39A2 solute carrier family 39 (zinc transporter), member 2 NM_014579 001230 205349 at NM_002068 0.01.238 210260s at TNFAIP8 tumor necrosis factor, alpha-induced protein 8 BC005352 001243 221527 s at PARD3 par-3 partitioning defective 3 homolog (C. elegans) IAF1961850.01244. 20877 is at LTA4H leukotriene A4 hydrolase 02959 o01262. MIDI interacting protein (gastrulation specific G12 homolog ------, NM_0212420.01262,
NM_0051650,01265 207178 s at FRK ?yn-related kinase ------NM_0020310.01.277 203240 at FCGBP Fc fragment of IgG binding protein NM_003890 001280
3O8857 v. Patent Application Publication Oct. 3, 2013 Sheet 13 of 73 US 2013/0261024 A1
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FIG. 10: Table D
Hexamidine Benchmark Signature; 100 up-regulated
217873 at AB39 201689 s at TPD52 tumor protein D52 203370_s phosphatidylinositolPDZ and LIM domain glycan 7 (enigina) anchor biosynthesis,------class A --- PIG (paroxysmal nocturnal hemoglobinuria) 205281s------at NM_002641;------, ------0.0 209939 x CFLAR CASP8 and FADD-like apoptosis regulator F005775 0.0 208651 x at CD24 CD24 molecule ------M58664 0.0
er RAS oncogene family ------M_0068
M_0043420.00002 63293 000003 2016 209344 at TPM4 BC002827 000005 pleckstrinhomology domain containing, family C (with FERM s 209210 s. at domain) member 1 ------20 --- A binding domain containing 3 220327 at VGLL3 vestigial like 3 (Drosophila) 205525 ------at CALDI caldesmon 1 210945 at COL4A6 205992_s_at IL5 insulin induced gene 2
proteasome (prosome, macropain) activator subunit 4
adenylate cyclase 7 NM_00140.00010 as association (RalGDS/AF-6) domain family NM_0071820.00011 W at HSPAIB Theat shock 70kDa protein B 20475200800s x atat HSPADSC2 1A desmocollinheat shock 70kDa 2 protein A ------221994 at PDLIM5
209028 s at ABI ab interactor 202147 s at IFRD1 interferon-related developmental regulator 1 212112s. 202581 at HSPAB heat shock 70kDa protein IB 201058's at MYL9 myosin, light chain 9, regulatory
205020-s at ARL4A ADP-ribosylation factor-like 4A - 22002 at TMC7 transmembrane channel-like 7 ------ouma 208960s at KLF6 Kruppel-like factor 6 2183.03.x at KRCC1 lysine-rich coiled-coil 1 207517 at LAMC2 laminingamma 2 NM_0188910.00025 21 1676 s at IFNGR1 -- interferon gamma receptor AF056979 000026. 212373 at FEM1B fem-1 homolog b (C. elegans) Aw139179 000031 Patent Application Publication Oct. 3, 2013 Sheet 21 of 73 US 2013/0261024 A1
2 2647 at NM 0062700.00035 213281 at 218290 at PLEKHJ1 pleckstrin homology domain containing, family J member 1 NM_0180490.00036 203303 at LT3 dynein, light chain, Tctex-type 3 NM_006.5200.00038 202146------at D interferon-related developmental regulator 1 221269 s at H3BGRL3 SH3 domain binding glutamic acid-rich protein like 3 204091 at PDE6D phosphodiesterase 6D, coMP specific, rod, delta era.------owth arrest and DNA-damage inducible, beta w-lu-'os-s-s-s-s-s------...tv/-...-
cysteine rich transmembrane BMP regulator 1 (chordin-like) NM_016441 00049 dual adaptor of phosphotyrosine and 3-phosphoinositides NM_014395 0.0005
0054860.00056.
212763 at I-like 1 202375 at SEC24 related gene family, member D (S cerevisiae) 202538s at CHMP2B chromatin modifying protein 2B
201669 s
202274 at 219988s at Clorf164 chromosome open reading frame 164 :- - - - - 213083 at SLC35D2 solute carrier family 35, member D2 AJ005866 208456-- s at------...- RRAS2 ...------related RAS viral (rras) oncogene homolog2 203855 at D repeat domain 47
208865 at CSNKIA asein kinase I, alpha I RP3 actin-related protein 3 homolog (yeast) 210306 at L3MBTL (3)mbt-like (Drosophila) U89358
218556 at ORMDL2------ORM1-like 2 (S. cerevisiae) NM_014 1820. 219929_s_at ZFYVE21 zinc finger, FYVE domain containing 21 NM_02407 10.00104. 52164 at C11orf24 chromosome 11 ------open reading frame 24 - 0.00105. 202619 is at PLOD2 procollagen-lysine,2-oxoglutarate 5-dioxygenase 2 214085 x at GLIPR1 201980s at RSUI
NMUsvy ubiquitin-conjugating enzyme E2B (RAD6 homolog) AI768723 000 125 C24 related gene family, member D (S. cerevisiae)------AU143984 218463 s at MUS81 -- MUS81 endonuclease homolog (S. cerevisiae) serine/threonine kinase 17a 202381202695 ats at STK17AADAM9 ADAM metallopeptidase domain 9 (meltringamma) NM_003816 000143 209379 s at KIAA1128 KIAA1128 ------AF24.1785 000168 205659 at HDAC9 histone deacetylase 9 NM_014707 00075 Patent Application Publication Oct. 3, 2013 Sheet 22 of 73 US 2013/0261024 A1
200985 s at CD59 CD59 molecule, complement regulatory protein 218088 s at RRAGC R ------a--as-a-S-S------a------a-a-a-a-a-ra-a- S2 vets erythroblastosis virus E26 oncogene homolog2(avian) serpin peptidase inhibitor. clade E (nexin, plasminogen activato 202627 s at SERPINE1 inhibitor type 1), member 201688 s at TPD52 tumor protein D52 zinc finger protein 193 CASP8 and FADD-like apoptosis regulator uanylate binding protein I, interferon-inducible, 67kDa
nuclear receptor binding factor 2 SOOA 4 S100 calcium binding protein A14
Kruppel-like factor 6
21 1984 at CALM1 calmodulin (phosphorylase kinase, delta) KDEL (Lys-Asp-Glu-Leu) endoplasmic reticulum protein 204017 at KDELR3 retention receptor 3 NM_006855 Patent Application Publication Oct. 3, 2013 Sheet 23 of 73 US 2013/0261024 A1
FIG. 11: Table E Hexanidine Benchmark Signature; 100 top down-regulated
melanocortin receptor (alpha melanocyte stimulating hormone receptor)
PTX3 pentraxin-related gene, rapidly induced by IL i beta NM_002852 000
cullin 3
zinc finger, BED-type containing 5 NM_02121 1 000006,
heterogeneous nuclear ribonucleoprotein F AI591354------0.00008
interferon, gamma-inducible protein 16 TT ------s tubulin, alpha ic
rich transmembrane protein 2 phosphatase and tensin homolog (mutated in multiple advanced cancers 1) eukaryotic translation initiation factor 3, subunity
ily, AAA domain containing 2
-
solute carrier family 25 (mitochondrial carrier, ornithine SLC25A15 transporter) member 15 NM_014252 000017,
SNRPD small nuclear ribonucleoprotein D1 polypeptide 16kDa BCOO 1721 0.00018 K W. 10 ankyrin repeat domain - ... --...----- vyi o
apoptosis antagonizing transcription factor
2C cyclin-dependent kinase inhibitor 2c (p18, inhibits CDK4) NM_001262 000023 205014 at broblast growth factor binding protein i NM_005 130 T 0.00026.
factor IIIC, polypeptide 4,90kDa NM 012204 00027
chromosome 1 open reading frame 12 -- - eterogeneous nuclear ribonucleoprotein H3 (2H9)
ID magnesium-dependent, delta isoform
Patent Application Publication Oct. 3, 2013 Sheet 24 of 73 US 2013/0261024 A1
asparagine ------dyskeratosis ------congenita 1. dyskerin 204544 at HPS5 Hermansky-Pudlak syndrome 5 205284 at KIAA0133 KIAA0133 202633 at TOPBP1 topoisomerase (DNA) II binding protein NM_007027 000044
212774------at ZNF238 Zinc fingerprotein 238 AJ223321re-as-a-----...-au-...} 000046 202453s at GTF2H1 general transcription factor ITH, polypeptide I. 62kDa NM_005316 0.00046 21 1951 at NOLC1 nucleolar and coiled-body phosphoprotein 1 D21262 0.00047, 217850re--gs at GNL3 guanineut---al-Maws-4------as-r-M.------nucleotide binding protein-like 3 (nucleolar) NM_014366 --- 0.00053 ------, -- aminoadipate semialdehyde dehydrogenase phosphopantetheinyl - --- 2021.70s at AASDHPPT transferase AF151057 0.00053, 217919 sat MRPL42 mitochondrial ribosomal protein L42 ------>------ciBE782148 0.00056 202469 s at CPSF6 cleavage and polyadenylation specific factor 6, 68kDa AU149367 000057 206332-s at IFI16 interferon, gamma inducible protein 16 NM_005531 0.00053 22639 x at TUBAIB tubulin, alpha b. AL581768 000058 208891 at DUSP6 dual specificity phosphatase 6 ------BC003143 000059 31826 at FKBP15 FK506 binding protein 15, 133kDa AB014574 0.00060. -----sur-...------SWI/SNF related, matrix associated, actin dependent regulator of ------213859 x_at SMARCA5 chromatin, subfamily a member 5 AI652586 205061-swww.arwaluw-wallow at are EXOSC9war-cut:--ww.ru-www.war-wet-wor--awar.-----woo, exosome component 9. i-o,----...--as-s-s-saw-wow.------is-wirer------NM_005033 -
M-vam211072.x at TUBAIB tubulin, alpha1b BC006481 000062 21 1953 s -at RANBP5 RAN binding protein 5 AU148466 000064. 204772 is at TTF1 transcription termination factor, RNA polymerase I NM007344 0.00065 212721r------sa------ee-as-Ms->s------almark---n------ee-a------at SFRS12 isplicing factor, arginine?serine-rich 2 A1810380 000065 204084 sat CLNs ceroid-lipofuscinosis, neuronal 5 A1911687 0.00066 splicing factor proline/glutamine-rich (polypyrimidine tract bindin g & i 201586_s_at SFPQ protein associated) NM_005066 0.00066 202983-- - - at ---or-rHLTF helicase like transcription factor A760760rtantán T. 000067.nnnn,7 202658--Inter-reus-s:------as-au------we-as-s------exes------n-s------at PEX 11B peroxisomal biogenesis factor 11B NM_003846 0.00067 210793_s_at------>;------sur------w------au-cetwo-a- NUP98 nucleoporin 98kDa U41815 0.00068, 203284s at HS2STI heparan sulfate 2-O-sulfotransferase I Aw151887 0.00070; 2012201274 -----, at PSMA5 SMA -r-,proteasome ------e-r- (prosome,prosome or--race.------macropain) subunit, alpha type, 5 NM002NM_002790 0.00072, 208892 is at DUSP6 dual specificity phosphatase 6 -----isBC003143 w-. wall-e----. it03436 to at co-or-for-7-----it------RPP30 ribonuclease P/MRP 30kDa subunit ------IF3 NGG1 interacting factor 3-like 1 (S. pombe) 218133 s at NIF3L - RNA polymerase II associated protein 3 ------s .00 218842 at RPAP3 ...Yasi-ca---was------well-ar-cross------Xs------&-w-wow 209773 s at RRM2 ribonucleotide reductase M2 polypeptide BC001886 0.00085 --mir...wi-reuwser-in-us&sex-axim-ruler-wirin- prostaglandin-endoperoxide------es------synthase 1 (prostaglandin G/H synthase 215813_s_at PTGS1 and cyclooxygenase) S36219 s:-...--Aka'wakwe-Xxx-exe-x-xx-air.206104 at ISL1 LLM------arer------al- homeobox I m------NM002.202 00009.
203689 sat FMR1 fragilex mental retardation 1 AI743037 000093.
204662 at CP10 CPI 10 protein 219862 is at NARF nuclear prelamin A recognition factor ------ea--- NM 012336 0.00097. Patent Application Publication Oct. 3, 2013 Sheet 25 of 73 US 2013/0261024 A1
WM_002291 208840s at G3BP2 GTPase activating protein (SH3 domain) binding protein 2 AU149503 0.00098 209384 at PROSC proline synthetase co-transcribed homolog (bacterial) AA176833 0.00100
201386s at DHX 15 DEAH (Asp-Glu-Ala-His)-- box polypeptide 15 AF279891 ------000100
ring finger and WD repeat domain 3 0.00102. SH glycine cleavage system protein H... (amin lethyl carrier) caspase l, apoptosis related cysteine peptidase (interleukin 1, beta, 211367_s_at CASP1 convertase) U13699 219987 at LOC728.193 Hypothetical protein LOC728.193 NM_024534 signal recognition particle 72 kDa BE856385 DEAD (Asp-Glu-Ala Asp) box polypeptide 3, X-linked BG492602 plicing factor, arginine/serine-rich (splicing factor 2, alternate 208863 s at SFRS1 plicing factor) |M72709 220651s at MCM10. inichromosome maintenance complex component 10 Full-length cDNA clone CSODK002YF 13 of HeLa cells Cot 25 normalized of Homo sapiens (human) 0.001.33 multiple inositol polyphosphate histidine phosphatase, 1 0.0033; amylo 1,6-glucosidase, 4-alpha-glucanotransferase (glycogen ; 203566 s at AGL debranching enzyme, glycogen storage disease type III) NM_000645 000135, 218423 x at VPS54 vacuolar protein sorting 54 homolog (S. cerevisiae) NM_016516 000139 teasome (prosome, macropain)26S non-ATPase, 4 ------RNA binding motif protein 8A BG289199 0.00139
superkiller viralicidic activity 2-like 2 (S. cerevisiae) D29641 000142. PDSS2 prenyl (decaprenyl) diphosphate synthase, subunit 2 NM_020381 000143 2024.86 at G3 ATPase family gene 3-like 2 (yeast) NM006796 0.00 modomain helicase 1ng p UU14s,
206122 at SRY (sex determining region Y)-box 5 M006942 000146 21978 at queuine tRNA ribosyltransferase domain containing 1 NM_024638 000152, Patent Application Publication Oct. 3, 2013 Sheet 26 of 73 US 2013/0261024 A1
FIG. 2: Table F NAG Benchmark Signature; 39 significantly up-regulated
n 9 receptor
ilateral CO RNA-histidine guanylyltransferase 1-like (S. cerevisiae) COP9 constitutive photomorphogenic homolog subunit 5 201652 at COPS5 209885 at is homolog gene family, member D 205637s at SH3GL3 SH3-domain GRB2-like 3 S. m. 20829s at TH tyrosine hydroxylase 221355 at CHRNG olinergicor ------receptor, nicotinic, ganna 218260 at 209438 at
similar to 60S ribosomal protein L21
M-phase phosphoprotein 1 0 (U3 small nucleolar W' - O ribonucleoprotein) m -- W - 212885 at 0.02001--:lexes---ist 21538 is a eat shock 70kDa protein 2
209099 x at JAG1 ------&-exe-lyas- M-M1 23679 at AL049329 0.02639 219706 at C20orf29 212396 s at KIAA0090 aphase promoting complex subunit 5 Ox4 neighbor matrix metallopeptidase 9 (gelatinase B,92kDa gelatinase. 203936 s at MMP9 219406 at C chromosome 1 open reading frame 50 212274 at 28807 at av 3 guanine nucleotide exchange factor 206257 at coiled-coil domain containing 9 205232 s at platelet-activating factor acetylhydrolase 2, 40kDa 200972 at 22608s at w85912 0.03931: Patent Application Publication Oct. 3, 2013 Sheet 27 of 73 US 2013/0261024 A1
nudix (nucleoside diphosphate linked moiety x)-type motif 2 - NM_001161004750 214861 at JMJD2C jumonji domain containing 2C ------A34181 0.04757 phosphoribosylformylglycinamidine synthase (FGAR
amidotransferase) AL04432 Patent Application Publication Oct. 3, 2013 Sheet 28 of 73 US 2013/0261024 A1
FIG. 13: Table G NAG Benchmark Signature, most significantly down-regulated
210735 s at CA12 carbonic anhydrase XII ...... 218686 s at RHBDF1 omboid 5 homolog 1 (Drosophila) 211487 x at RPS17 ribosomal protein S17 y member 12 219885 at SLFN12 protein tyrosine phosphatase, receptor type. f polypeptide 202066 at PPFIA (PTPRF), interacting protein (liprin), alpha 1. AA195259 000932. GALNACT- - 2887 1.x at chondroitin sulfate GalN AcT-2 'S'm NM_018590 0.00936 protein phosphatase 2 (formerly 2A), catalytic subunit, beta i 201375 sat PPP2CB isoform 0.01.239. 212215 at t 203537 at yrophosphate synthetase-associated protein 2 204084s at ceroid-lipofuscinosis. neuronal 5 273 10s at forkhead box J3 205457 at hromosome 6 open reading frame 106 214657------:-us :: at TncRNA Trophoblast-derived noncoding RNA ------8 methylenetetrahydrofolate dehydro genase (NADP+ dependent)2. 201761 at MTHFD2 methenyltetrahydrofolate cyclohydrolase NM OO66360,01993 217783 s at YPEL5 yippee-like 5 (Drosophila) NMolé06 00215 217975 at WBP5 TWW domain binding protein 5 M_016303 002119. AN1A2 mannosidase, alpha, class 1A, member 2 AL157902 002156. orf32 chromosome 14 open reading frame 32 671747 0.02257 212585 at OSBPL8 oxysterol binding protein-likes BF970829.002269 218128 at AU151875 o02283
NM_01433.0002517
219307 at prenyl (decaprenyl) diphosphate synthase, subunit 2 NM_02038 f PRP19/PSO4 pre-mRNA processing factor 19 homolog (S.T. : 203103 s at PRPF19 'cerevisiae) - NM_014502 0.02765 -mm-m-m-----Wu--m--- cyclin-dependent kinase 7 (Mo15 homolog, Xenopus laevis,w------cdk-, activating kinase) 0.02926 serine incorporator 3
ng protein, binding protein (liprin beta1) 210608_s at FUT2 . fucosyltransferase 2 (Secreto Stats 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 2013/0261024 A1
207524 at ST7 21806.8 s at ZNF672 zinc finger 221864 at ORAI3 ORAI calcium release-activated calcium modulator 3 AW517464 0.04438 2 1697 x at PNOl partner of NOB I homolog (S. cerevisiae) AF349314 0.04561 ------inhibitor of DNA binding 3, dominant negative helix-loop-helix
207826_s_at ID3 protein NM002 167 0.0457. nuclear receptor subfamily 4, group A, member 2 NM_0061860,04685 212688 at PIK3CB phosphoinositide-3-kinase, catalytic, beta polypeptide BC003393 0.04925 Patent Application Publication Oct. 3, 2013 Sheet 30 of 73 US 2013/0261024 A1
F.G. 14: Table H.
ide Benchmark Signature, 100t
high mobility group AT-hook 2 licing factor, arginine/serine-rich 7, 35kDa ------RMT1 TRM1 tRNA methyltransferase 1 homolog (S. cerevisiae) 202393 s at KLF10 Kruppel-like factor 10 ------207618 s
200800 s at HSPAB heat shock 70kDa protein IB 202207 at ARL4C ADP-ribosylation factor-like 4C 216237 s. A307529 000014 205466 s at H. 208083 s at integrin, beta 6 21 1538 s at heat shock 70kDa protein 2 205525 at CALD caldesmon 49329 at 22 kelch-like 22 (Drosophila) 200749 at AN, member RAS oncogene family 210511s at INHBA inhibin, beta A 204610-s at CCDC85B coiled-coil domain containing 85B tissue specific transplantation antigen P35B
tropomyosin 4 KDE (Lys-Asp-Glu-Leu) endoplasmic reticulum protein ention receptor 3 ycosylphosphatidylinositol anchor attachment protein 21 1060 207574 s at GADD45B growth arrest and DNA-damage inducible, bet
207517 at LAMC2 laminin, gamma2 &------a------geneous nuclear ribonucleoprotein D-like
insulin receptor substrate
jagged 1 (Alagile syndrome)
202016 at M NM_0024020,00086,
219066 at PPCDC phosphopantothenoylcysteine decarboxylase NM_02823 000093. throcyteInembrane protein band 4.9 (dematin) heterogeneous nuclear ribonucleoprotein D-like 205020 s at ARIAA ADP-ribosylation factor-like 4A mplex 11 (m ise)-like 202269 x at GBP guanylate binding protein I, interferon-inducible, 67kDa BC002666 000129
207382 at NM_003722 000133. 212 ''''''''' ' ' ' ' ' ' ' ' m ' ' AA142929 000139 Patent Application Publication Oct. 3, 2013 Sheet 31 of 73 US 2013/0261024 A1
eterogeneous nuclear ribonucleoprotein H 212461 at AZIN1 antizyme inhibitor 1
pleckstrin homology domain containing, family C (with FERM
domain) member 1
t3 (Psf3 homolog)
A065135 000178 41858 at FRAG1 FGF receptor activating 049261 000179
208960s220232 at at KLF6 Kruppel-like factor 6 BE675435 000193 218809 at PANK2 pantothenate kinase 2 (Hallervorden-Spatz syndrome) NM_0249600.00196. 219802 at PYROXD1 pyridine nucleotide-disulphide oxidoreductase domain NM_024854.000198 221009_s giopoietin-like 4 218146 at 8D glycosyltransferase 8 domain containing 1 21 865----- 2 s at PIGG 12 integratorphosphatidyli complex nositolglycan subunit 12anchor biosynthesis.---...-----e...&-sis---axl.--.3-li------class G BRCA1 interacting protein C-terminal helicase 1 A accumulation, homo
CAB39 calcium binding protein 39 216870------x at DLEU2 ------ic leukemia, 215766 at GSTAI Glutathione S-transferase Ai
204346 s at RASSF1 Ras association (RalGDS/AF-6) domain family 207850 at CXCL3 -C motif) ligand 3 202857 at TMEM4 transmembrane protein 4 ------
215.209------at SEC24D------, SEC24 related gene family, member------D ------(S. cerevisiae) AU143984a------000311
RPL15 itochondrial ribosomal protein L15 ------NM ------014175 000312--- TAF10 RNA polymerase II, TATA box binding protein (TBP)- TAF10 associated factor, 30kDa NM_006284.000321 PIP4K2A phosphatidylinositol-5-phosphate 4-kinase, type II, alpha BE878277 0.00327
-...... ------stone deacetylase 9 202581 at 218358 at 215532 x_a 204295 at 221994 at PDLIM5 PDZ and LIM domain 5 AA196325 0004
33736 at STOMLl stomatin (EPB72)-like I ------Y16522 0.00414. 635 is at PEXIO eroxisome biogenesis factor iO ------M 002617 000418. : guanine nucleotide binding protein (G protein), alpha inhibiting 201181 at GNAI3 tivity polypeptide 3 NM_006496 0.00438. Patent Application Publication Oct. 3, 2013 Sheet 32 of 73 US 2013/0261024 A1
urofibr 27------n 2 ( eral acoustic neuroma) 1303 at karyotic translation initiation factor 4A, isoform 3 4 pre-mRNA processing factor 4 homolog (yeast)
lysine-rich coiled-coil 1
ADP-ribosylation factor-like 4C BC001051 000474 and LIM domain 7 (enigma)
serine/threonine kinase 7a
dehydrogenase/reductase (SDR family) member 7B
ribosomal protein L5
ligase I, DNA, ATP-dependent calmodulin 1 (phosphorylase kinase, delta)
spastic ataxia of Charlevoix-Saguenay (sacsin)
finger protein 180 chromosome 16 open reading frame 42
ER lipid raft associated 2 ------fluenza virus NS1A binding
at
205584 at X------m------m------X------S---- 21887 s_at ( m-m-m-m------. chromosome 8 open reading frame 33 PDS5, regulator of cohesion maintenance, homolog A (S.
213984 at cerevisiae) 202787 s. Patent Application Publication Oct. 3, 2013 Sheet 33 of 73 US 2013/0261024 A1
F.G. 15: Table I Niacinamide Benchmark Signature, 100 top down-regulated
Av704962 00
BC005247 00, 201626 at insulin induced gene I BG292233 0.0 201 170 s. main containing, class B, 2 21981 at LIPGT 205014 at FGFBP1 201627s at INSIGI 219836 at ZBED2 213359 at HNRPD HMGCs 209674------at CRY1 208796 sat CCNG1 202068 s at LDLR low density lipoprotein receptor (familial hypercholesterolemia) 202769 at CCNG2 cycli
eptor-related protein 8, apolipop NM_0046310,00012 211559 sat CCNG2 cyclin G2 L49506 000013
218686 s at RHBDF1 rhomboids homologi (Drosophila) NM_0224.500.00014 20686) is at CGGBP CGG triplet repeat binding protein NM_0036630.00014. 217783 sat YPEL5 yippee-like 5 (Drosophila) NM_0160610.00015 solute carrier family 7, (cationic amino acid transporter, y+ . 209921 at SLC7A1 1 system) member 11 ------n, al------0.00016 201625 sat INSIGI insulin induced gene 202220 at KIAA0907 KIAA0907 27993 s at MAT2B methionine adenosyltransferase II, beta 00018. 210950s at FDFT1 farnesyl-diphosphate farnesyltransferase one--- Solute carrier family 2 (facilitated glucose transporter), member 3 i ; 221751 at SLC2A3P pseudogene AL565516 0.00025. 20390 at ARHGAP29 Rho GTPase activating protein 29 NM_0048150.00027 bhydrolase do ai ning 3
208433 s at LRP8 e receptor NM_0175220.00037 Patent Application Publication Oct. 3, 2013 Sheet 34 of 73 US 2013/0261024 A1
209279_s_at NSDHL NAD(P) dependent steroid dehydrogenase-like 206247 at MICB HC class I polypeptide-related sequence B NM 212622 at ansmembrane protein 4 1B ediator complex subunit 21 7-dehydrocholesterol reductase 219936 s at GPR87 G protein-coupled receptor 87
217776 at RDH11 retinol dehydrogenase l l (all-trans/9-cis/11-cis)
otein-coupled receptor 126 205788 s at ZC3H11A zinc finger CCCH-type containing 11A MHC class I polypeptide-related sequence A yrin repeat domain 10 212623 at TMEM4B ansmembrane protein 4 B 200832s at SCD stearoyl-CoA desaturase (delta-9-desaturase) 218842 at 209362 at 216607 s at CYP51A1 ic 208647 at esyl-diphosphate farnesyltransferase 1 lute carrier family 7, (cationic amino acid transporter, y+ 217678 at em) member 201346 at iponectin receptor 2 M_02455 1000135 221582 at HIST2H2A histone cluster 3, H2a BC001193 000135
tin-related protein 6 homolog (yeast) '''''''
M55580 NM_002.9700. 222262s at 216060s at DAAM1 ------219045 at RHOF s linked (RAD54 208861s------&------s------at ATRX fattyhonolog, acid desaturaseS. cerevisiae) - U72937
RAB5B, member RAS oncogene family 3. diazepam binding inhibitor (GABA receptor modulator, acyl 2093.89 x at DBI Coenzyme A binding protein) DAM10 ADAM metallopeptidase domain 10 218258 lymerase (RNA) I polypeptide D, 16kDa 20968 at lute carrier family 19 (thiamine transporter), member 2 221255 sat mbrane protein 93 2008.63 s at RAB11A, member RAS oncogene family 213988s at SAT1 spermidine?spermine N1-acetyltransferase 1 BE971383 o00335 210290 at ZNF174 - zinc finger protein 174 ' ' - ' ' ' ' ' ' Patent Application Publication Oct. 3, 2013 Sheet 35 of 73 US 2013/0261024 A1
i. sJ ELOVL family member 6, elongation of long chain fatty acids
210868------is at------ELOVL6 (FEN 1/Elo2, SUR4/Elo.3-like,------v------yeast) ------BC00305 0.00359 glutamate-cysteine li gase. modifier subunit NM_0020610.00363
27975 at WBP5 -----'l'-...------...--ww domain binding protein- - - 5- - 200620 at TMEM59 transmembrane 59 205376 at INPP4B inositol polyphosp hosphatase, type II, 105kDa 202067-s212 192 at at LDLRKCTD12 lowpotassium density channel lipoprotein tetramerisation receptor (familial domain hypercholesterolemia containing 12 AI718937 ORAI3 ORAI calcium release-activated calcium modulator 3 quinolinate phosphoribosyltransferase (nicotinate-nucleotide 204044 at pyrophosphorylase (carboxylating)) 2all ------a------sis-->15093 at N ADP) dependent ------. steroid dehydrogenase-like ubiquitin-conjugating enzyme E2G 1 (UBC7 homolog, yeast)
wD repeat and FYVE domain containing 3 ------PKP4 plakophilin 4 ------solute carrier family 25 (mitochondrial carrier, phosphate carrier), NM_004896.000546
201955 at 202536 at chromatin modifying protein 2B 0. 212589 at RRAS2 related RAS viral (rras) oncogene homolog2 AI753792 to,00581 206662 at LRX gl aredoxin (thioltransferase)
27127 at CTH cystathionase (cystathionine gamma-lyase) L354872 000590, 210266_s_at TRIM33 tripartite motif-containing 33 AF220137 000598 Patent Application Publication Oct. 3, 2013 Sheet 36 of 73 US 2013/0261024 A1
FIG. 16: Table J Sepiwhite Benchmark Signature; 100 top up-regulated
202708------. s at HIST2H2BE histone cluster ------2, H2be ------..w.------, 217528 at chloride channel, calcium activated, family member 2 2024.89 domain containing ion transport regulator 3 210609 s at TP53.3 tumor protein p53 inducible protein 3 20865 x at CD24 CD24 molecule 000002 221922 at GPSM2 G protein signaling modulator 2 (AGS3-like, C. elegans) AW195581 000003
221211 s at C21orf7 in-a-lalichromosome a 21 open reading------frame 7 NM_0201520.00005 uroplakin 1B - -- AB002155 000006 hioredoxin interacting protein
fibronectin 208025 s high mobility group AT-hook 2 -- 204464 sat EDNRA endothelin receptor type A ------78047 s at hypothetical protein LOC729580 --m------.S. ------membrane protein 45A --- - ...... '. -- 203882 at ISGF3G interferon stimulated transcription factor 3, gamma 48kDa 296.55 at C7orf10 chromosome 7 open reading frame 10 2013 12s at SH3BGRL SH3 domain binding glutamic acid-rich protein like 218990s at SPRR3 small proline-rich protein 3 NM_005416 000036,
SEC63 homolog (S. cerevisiae) ignal transducer and activator of transcription 1.9 kDa 0073150.00046 ANSCI MANSC domain containing 1
tropomodulin3 (ubiquitous) protein tyrosine phosphatase, receptor-type, Z polypeptide 1 protein-L-isoaspartate (Daspartate) O methyltransferase domain 212406_s_at PCMTD2 containin g 2 215318 at CG012 hypothetical gene CG012 202562 s at C14orfi chromosome 14 open reading frame 205064 at SPRR1A small proline-rich protein A 205064 at SPRRIB small proline-rich protein 1B (cornifin) 222316 at ranscribed locus sterol-C5-desaturase (ERG3 delta-5-desaturase homolog, S. 21 1423 s at SCSDL cerevisiae)-like 221958 is at GPR177 G protein-coupled receptor 177 ------218432 at FBXO3 F-box protein 3
215729s21788 at VGLL4orf vestigialomosome like 114 (Drosophila) open reading frame ---, -,-,-,-,-,-,------E542323 o.0006. 221208 s at C11orf61 chromosome 11 open reading frame 61 NM_024630.00116. Patent Application Publication Oct. 3, 2013 Sheet 37 of 73 US 2013/0261024 A1
amily, member CUE domain containing 2 TBC1 domain family, member 5
28280 x at HIST2H2AA3 histone cluster 2, H2aa3 ------NM_003516 000137...------s - TAF12 RNA polymerase II, TATA box binding protein (TBP)- s i
2094.63------s at TAF12 associated factor, 20kDa 50544------...--- 000147 guanine nucleotide binding protein (G protein), alpha inhibiting 209576 at GNAI1 activity polypeptide
219494 at AD54 homolog B (S. cerevisiae) 218739 at 203691 at PI3 204388s at smembrane protein 97 matrix metallopeptidase 9 (gelatinase B.92kDa gelatinase, 203936 S------NM_004994.000211 27988 at NM_021178.000213
203344 is NM_0028940.00247
219969 at NM_0183600.00257 NM_018848------000270.
NM_014707000285 0.00303 A313324 000312
0.00312-. 1. solute carrier family 33 (acetyl-CoA transporter), member 1 BEA64756 000326
8-oxoguanine DNA glycosylase NM_0168210.00340 NIP thioredoxin interacting protein AA8223 20895 at ALDH7A1 aldehyde dehydrogenase 7 family, member AI BC0025 15 0.00349 60474 at C20orf42 chromosome 20 open reading frame 42 AA469071 000380
213787s --- emopamil binding protein (sterol isomerase) Av702405 000385 Patent Application Publication Oct. 3, 2013 Sheet 38 of 73 US 2013/0261024 A1
--- excision repair cross-complementing3. rodent repair :::::::::::deficiency, i complementation group 1 (includes overlapping antisense 203720 s at ERCC1 219979rol ------...------s at C1 lorf73 hromosome ------11 open reading frame 73 ------a-w------containing protein. X-linked recombination signal binding protein for immunoglobulin kappa J region NM_015874 000425
N M 01602 1 0.0043
finger protein 215719 x at Fas (TNF receptor superfamily, member 6) 218801 at UGCGL2 UDP-glucose ceramide glucosyltransferase-like 2
212672 at ATM ------ataxiatelangiectasia mutated
201009 s at TXNIP thioredoxin interacting protein 212936 at C5orf21 chromosome 5 open reading frame 21 tin acetyl-Coenzyme A acetyltransferase 2 (acetoacetyl Coenzyme
209608_s_at A thiolase) 219122 s at tRNA-hi stidine guanyl yltransferase 1 -like (S. cerevisiae) translocase of inner mitochondrial membrane 23 homolog B :
218119 at TIMM23B (yeast) translocase of inner mitochondrial membrane 23 homolog 218119 at TIMM23 (yeast) tyrosine 3-monooxygenase/tryptophan 5-monooxygenase
219763 at DENNDIA 218677 at S100A14 S100 calcium binding protein A14 219543 at PBLD phenazine biosynthesis-like protein domain containing - 201617 x at CALDI caldesmon 1 201185203729 at EMP3 202321 at GGPS1 geranylgeranyld Patent Application Publication Oct. 3, 2013 Sheet 39 of 73 US 2013/0261024 A1
F.G. 17: Table K Sepiwhite Benchmark Signature; 100 top down-regulated 217767 at LOC653879 is milar to Complement C3 precursor 208002 y CoA thioesterase 7
CAAT?enhancer binding protein (C/EBP). beta 0.00002 209774 x at CXCL2 chemokine (CX-C motif) ligand 2 M57731------000002 ----- 203925 at lig St. modifier subunit------201426 s at 206343 s at NRG1 : emokine (CX-C motif) ligand I (melanoma growth
stimulating activity, alpha) ------HA1, activator of heat shock 90kDa protein ATPase homolog 1
201839s 1 tumor-associated calcium signal transducer II 206157_a pentraxin-related gene, rapidly induced by IL beta -
yptochrome 1 (photolyase-like) 204948 s at FST NM_0134090,00041
201289 at CYR61 NM_0015540.00046 , x at CACYBP
dehydrogenase 1, 20-alpha (3-alpha)-hydroxysteroid dehydrogenase)
EGF-containing fibulin-like extracellular matrix protein 1.
interleukin l, beta
phosphoglycerate kinase 1
tubulin folding cofactor A
muscleblind-like (Drosophila)
terogeneous nuclear ribonucleoprote RAB27A, member RAS oncogene famil emokine (C-C motif) li gand , general transcription factor IIIA ------
218309 at CAMK2N1 calcium/calmodulin-dependent protein kinase II inhibitor - - - - 5 205774 at F12 coagulation factor XII (Hageman factor). NM 000505.000211 204181 sat ZBTB43 zinc finger and BTB domain containing 43 T90308 000214
204078 at SC65 synaptonemal complex protein SC65 transaldolase 1 ATP synthase, H+ transporting, mitochondrial F0 complex, subunit G AF070655 0.00231. 208700 s at TKT transketolase (Wernicke-Korsakoff syndrome) L12711 000237 guanine nucleotide binding protein (G protein), beta polypeptide 200852 x at GNB2 2 NM_005273 0.00240 Patent Application Publication Oct. 3, 2013 Sheet 40 of 73 US 2013/0261024 A1
NM_0027900. ---,-,-,-,-- 'm' NM_0186380.00246 206247 at MICB MHC class I polypeptide-related sequence B NM_0059310.00267
208934 s at ---LGALS8 lectin, galactoside-binding,------soluble,------... - -8 - -(galectin 8) AF342815 000268 27997------at PHLDA pleckstrinhomology-like domain, family A. member 2 1668 s at PLAU plasminogen activator, urokinase 219933 at GLRX2 glutaredoxin 2 ------M_016066 000338 protein tyrosine phosphatase, receptor type, f polypeptide PPFIA (PTPRF), interacting protein (liprin), alpha 1. 202066------...-a, -si-no-,at i.e.------...------...-----...- 210825s at PEBP1 phosphatidylethanolamine binding protein 1
hromosome 14 open reading frame 32 0.00351
200048 s at JTB jumping translocation breakpoint 204341 at TRIM16 - - - - ipartite motif-containing 16 ------NM_0064700.00362 204341 at TRIM16L taining 16-like 221563 at DUSP10 dual specificity phosphatase 10 N36770 000381 203234 at UPPI uridine phosphorylase 1 NM_003364,000389. ATP synthase, H+ transporting, mitochondrial F0 complex, co------wi------207507_s at ATP5G3 subunit C3 (subunit 9) NM_001689,0.00410 204326 x at MT1X metallothionein 1X 00426 ------20882 1 at SNRPB 0.00440: 218026 at CCDC56 NM_014019.000459------202644s at TNFAIP3
istidine-rich domain (CHORD)-containing 1
SBNo 207332 s at TFRC
205449 at SAC3D ------AF15,056 H glycine cleavage system protein H (aminomethyl carrier) AW237404 000656 - solute carrier family 2 (facilitated g transporter), member IM 017585 000673, 203343 at UGDH UDP-glucose dehydrogenase ------' ------NM_0033590.00693. i s 205292 s. at HNRNPA2B1 heterogeneous nuclear ribonucleoprotein A2/B1
202252 RAB 13, member RAS oncoger i 214866 at PLAUR plasminogen activator, irokinase receptor i
0.00802. PSIP1 PC4 and SFRS1 interacting protein 1 L27RA interleukin 27 receptor, alpha 201243 s at ATPB ATPase, Na+/K+ transporting, beta 1 polypeptide Patent Application Publication Oct. 3, 2013 Sheet 41 of 73 US 2013/0261024 A1
217753 s at RPS26 ribosomal protein S26 201152 s at MBNLI muscleblind-like (Drosophila)
v-maf musculoaponeurotic fibrosarcoma oncogene homolog F (avian) deoxyhypusine synthase -mumm-3-3-3-3--- - -
proteasome (prosoine, macropain) subunit, beta type, 9 (large multifunctional peptidase 2) (glucosamine) 3-O sulfotransferase 2 --- adaptor related protein complex 2, sigma 1 subunit
'kelch domain containing 3
I Tat interactive protein 2, 30kDa CDV3 homolog (mouse) A928407 0.0194 fibroblast growth factor 2 (basic) NM_002006 0.01.194 mo jor histocompatibility complex, class I. A - - 202014 at protein phosphatase 1. regulatory (inhibitor) subunit 5A v-u-v-W
oredoxin reductase I ------. ...------m------
suppressor of Ty 4 homolog 1 (S. cerevisiae) Patent Application Publication Oct. 3, 2013 Sheet 42 of 73 US 2013/0261024 A1
FIG. 18: Table L Composite "Skin Tone' Benchmark Signature; approximately 40 up-regulated
+ 58 down-regulated
old shock domain protein 201160s at CSDA A AL556190 1670: 1. endoplasmic reticulum 201216 at protein 29 NM_00681 201694 s at ly growth response NM_001964. 5136 7069 dothelin converting : & 201749 at BF969352 programmed cell death 4 (neoplastic transformation 202730s at inhibitor) NM_014456, membrane bound inhibitor homolog 203304 at (Xenopus laevis) NM_012342 WW domain binding protein 4 203597 s at WBP4 (formin binding protein 21) AI734228 1098 1.19 20383 at R3HDM2 R3H domain containing 2 NM_014925, 9 1206 123
F receptor-associated
204352 at TRAF5 ctor 5 NM 004619, 182 ------201 1.33 rel reticuloendotheliosis iviral oncogene homolog B, nuclear factor of kappa light polypeptide
205205 at RELB NM_006509. 435 1.34 207624s at GTPase regulator NM_000328 312 1.27
ras homolog gene family, 209885 at RHOD member D BC001338 4531 210886 x : TP53 activated protein AB007457 977 ute carrier family 5 8. (sodium iodide symporter), ember 5 D87920 jubiquitin protein ligase E3 mponent n-recognin 4 AB007931 bosomal protein L27a
213932 x at HLA-A complex, classi, A. A1923492 : surfactant, pulmonary- m i 213936 x at SFTPB associated protein B AW276646 Patent Application Publication Oct. 3, 2013 Sheet 43 of 73 US 2013/0261024 A1
214317 x ------...------at RPS9 ribosomal protein S9 BE348997 23.563 29560. 122
CDNA clone ; 214395 x at IMAGE:483.8699 484 646 1.28 215016 x at DST dystonin 2500 2590. 1.09 215450 at 5 8 5 9 s 7 32 1.6 216609 at TXN Thioredoxin 33.54 4269 1.2
striatin, calmodulin binding 217903 at STRN4 protein 4 NM_013403 330 G protein-coupled receptor 218151 x at GPR172A 172A NM O245.31 2345
218388-vs.------s------...------at PGLS phosphogluconolactonase NM_012088 ------2785------3513, 1.14 - chromosome 13 open 218420 s at C13orf23 reading frame 23 NM_025138 508 567. 1.2 i DnaJ (Hsp40) homolog, 3. 218435 at DNAJC15 subfamily C. member 15 NM_013238 242 2.28 RAB 17, member RAS S. 2 18931 at RAB 17 oncogene family NM_022449 s7 8 678 1.12 hypothetical protein 219254 at FL22222 FLJ22222 NM_024648 34 21 219916 s at RNF39 ring finger protein 39 NM_025236 489 1.29
220219 s at LRRC37A containing 37A NM_018001 leucine rich repeat RC37A2 containing 37, member A2 NM_018001 leucine rich repeat : 2202 19_s_at LRRC37A3 containing 37, member A3 NM_01 800 1 HERV-H LTR-associating 220387 s at HHLA3 3. NM_007071 410 484 1.13 SH3 domain binding & glutamic acid-rich protein 22 1269 s at SH3BGRL3 like 3 NM_031286 9634; 11906 1.16 chromosome 9 open ------221865 at C9orts. reading frame 91 22 1943 x at RPL38 ibosomal protein sulfotransferase family, . cytosolic, 1A, phenol 22.2094 at SULT1A3 preferring, member 3 A5801 12 186 286; 1.6 sulfotran sferase family, : 2 cytosolic, 1A, phenol preferring, member 4 A580 12 186 222094 at ------re-r-r, acetylserotonin O- i 3. 36553 at ASMTL methyltransferase-like AA669799 750
58 down-regulated below...... 8
Patent Application Publication Oct. 3, 2013 Sheet 44 of 73 US 2013/0261024 A1
eukaryotic translation initiation factor 1A, X : 201017 at EIF1AX linked --- BG149698 2960 2165; 1.3
eukaryotic translation 201437 s at EIF4E initiation factor 4E --- NM_001968 ------4499 1.09 201534s at UBL3 ubiquitin like 3 AF044221 : cytochrome b5 type B (outer mitochondrial 201634s at CYB5B membrane) NM_030579. COP9photomorphogenic constitutive homolog subunit 5 201652 at COPS5 (Arabidopsis) NM_006837. myristoylated alanine-rich 201669 s at MARCKS protein kinase C substrate NM_002356 ------: chromosome 3 open 201678s at C3orf37 reading fraine 37 NM_020187 sparc/osteonectin, CWCW and kazal-like domains 202524s at SPOCK2 proteoglycan (testican)2 NM 014767 i ioredoxin domain 203008_x at TXNDC9 ntaining 9 NM_005783. 12747 11685, 1.1 Down syndrome critical 203635 at DSCR3 region gene 3 203812 at CDNA clone 204700 x_at Clorf107 reading frame 107 : tripartite motif containing 204804 at TRIM21 21 ------939, 1.23 i maternal embryonic 204825 at MELK leucine zipper kinase NM_014791, 8469 : RNA binding motif protein : 205115s at RBM19 19 NM_016.96------to-r------cholinergic receptor,
206533 at - i.CHRNAs nicotinic, alpha 5 ------654, 1.19 ------al2081 12 x at EHD1 EH-domain containing NM_006795 1146; 1.27 receptor accessory protein 5 3790. I. 17. SP 100 nuclear antigen : suppressor of 210242 x_at ST20 tufiorigenicity20 995, I.15. fascin homolog 1, actin bundling protein (Strongylocentrotus 210933s at FSCN purpuratus) 2769 1.21 Patent Application Publication Oct. 3, 2013 Sheet 45 of 73 US 2013/0261024 A1
serine/threonine kinase 3 21 1078 s at STK3 (STE20 homolog, yeast) Z25.422 212092 at PEG10 paternally expressed 10 BE358180
GrpE-like 1, mitochondrial RPEL (E. coli) AL542571 G patch domain containin 212485 at PATCHS 8 DnaJ (Hsp40) homolog,
212490 at NAJC8 subfamily C, member 8 autism susceptibility : candidate 2 212599 at ubiquitin-conjugating AK025298 enzyme E2N (UBC13 212751 at UBE2N. '' homolog, yeast) BG290646 s polymerase (DNA
directed), delta 3. accessory subunit D26018 similar to Aspartate aminotransferase. mitochondrial precursor (Transaminase A)
(Glutamate oxaloacetate transaminase 2) AL049710 pha 2 U3269 tyrosine 3 monooxygenase/tryptophan 5-monooxygenase activation protein, beta 21.7717 s at YWHAB polypeptide BF246499 15355 - SERPINE1 mRNA binding 217724 at SERBP1 protein 1 AF 31807 17928 15265 mitogen-activated protein -s-s-s-s-s-s-s------217808s at MAPKAP! kinase associated protein 1 NM_024117 2180 132 2 NS3 sin 3 JM_022748 protein inhibitor of 217864 S at PIAS] activated STAT. NM 016166 M_012.192
218187s at Csor? 33 reading frame 33 023080 218294 is at NUP50 nucleoporin 50kDa AF267865 : calcium/calmodulin- s dependent protein kinase II 218309 at CAMK2N1 inhibitor NM_018584 s ATP/GTP binding protein
218480 at AGBLS like 5 NM_021831
initiation factor 2B. subunit 218488 at gamma, 58kDa 218514 at chromosome 17 open Patent Application Publication Oct. 3, 2013 Sheet 46 of 73 US 2013/0261024 A1
reading frame 71 DiGeorge syndrome 218650 at DGCRs critical region gene 8 487. 1.28 218926 at VYNN W.W. W., W.W. 998 1.18 3. DKN2A interacting 218929 at CDKN2AIP rotein NM_0176332
chromosome 2 open s 219008 at C2Orf43 reading frame 43 NM_021925 :
enhancer of mRNA ...
219207 at NM_025083 . . four jointed box s1. 3 219522 at (Drosophila) NM_014344s 219539 at ssociated protein 6 NM_024775
serum/glucocorticoid regulated kinase family, 220038 at 22 1502 at U3 small nucleolar ribonucleoprotein. : 221514 at UTP14A homolog A (yeast) BC001149 glutamate-rich WD repeat 221549 at GRWD1 containing AF337808 mediator complex subunit BC002694
---...s.ngw.onis-s-s-s s : interacting
2-a-seriosis-s-s-s-sous-assessories-s-s-swarais222105 s at NKIRAS2 Ras-likesalar-sa-s-s-s-s-s-s-s-s-s-s-s-s-s-s-s-s-s-s-s-s-s-s-s-s-s-s-s-s-s-s-s-s-s-a- 2 AA452565 synapse defective J, Rho GTPase, homolog 1 (C. Patent Application Publication Oct. 3, 2013 Sheet 47 of 73 US 2013/0261024 A1
FIG. 19: Table M RA benchmark signature in trC cell - 200 upregulated 3.
acyl-CoA synthetase long-chain family member 3 A (ABC1), member 1 eparin-binding EGF-like growth factor
dehydrogenase/reductase (SDR family) member 3
DnaJ (Hsp40) homolog, subfamily B. member irbohydrate (chondroitin 4) sulfotransferase 11 0.8533 acin receptor 2
interleukin l, beta - 1.6479 SRY (sex determining region Y)-box 9 ------
Y (sex determining region Y)-box 4 sue factor pathway inhibitor 2
ressor of cytokine signaling renylcysteine carboxyl methyltransf ase
gh mobility group AT-hook 1 loxidase-like 2 19,202 TRIO and F-actin binding protein Kruppel-like factor 10 LL/lymphona 3 aquaporin 3 (Gill blood group)
Acyl-CoA synthetase long-chain family member 3 deleted in liver cancer DnaJ (Hsp40) homolog, subfamily B, member 1
DNA-damage regulated autophagy modulator 1
eir ligase homolog (mouse)
nsporting, beta polypeptide - -
0.7514
ras homolog gene family, member B m - --
formin homology 2 domain containing 3 tissue factor pathway inhibitor 2 37208937 s at ID helix protein 0.8465 -- apolipoprotein B mRNA editing enzyme, catalytic 38.206632 s at APOBEC3B polypeptide-like 3B 1.6078 Patent Application Publication Oct. 3, 2013 Sheet 48 of 73 US 2013/0261024 A1
ruppel-like factor 6 43,202638 s at ICAM1 intercellular adhesion molecule 1 4. 203505 at ABCA) ATP-binding cassette, sub-family A (ABC1), member 1 46.212268 at SERPINB1 serpin peptidase inhibitor, clade B (ovalbumin), member 1 --- 47.204541 at SEC14-like 2 (S. cerevisiae) '' 7739 sat icotinamide phosphoribosyltransferase am ; microtubule associated monoxygenase, calponin and LIM 49,218376 is at MICAL1 domain containing 1 0.4038, 50211668 is at PLAU plasminogen activator, urokinase 0.8499 51217997 at PHLDAI pleckstrin homology-like domain, family A, member 0.4622.
carnitine palmitoyltransferase 2
EF-hand domain family, member D2 calcium/calmodulin dependent protein kinase II inhibitor 0.40 fibroblast activation protein, alpha
kinase kinase kinase kinase 4 olute carrier family 25 (carnitine/acylcarnitine transloc
eat shock 70kDa protein 2 65 201889 at family with sequence similarity 3, member C plasminogen activator, urokinase
roepithelial cell transforming 1
Kruppel-like factor 6
plasminogen activator, tissue FAIP3 interacting protein 71 200880 at DNAJAI DnaJ (Hsp40) homolog, subfamily A, member 1 ------.72205780 ----...-----...----...-a-...----- at BIK CL2 interacting killer (apoptosis-inducing) 73203180 at ldehyde dehydrogenase 1 family, member A3
ADP-ribosylation factor-like 4C sphingosine kinase 1
-- neuropilin (NRP) and tolloid (TLL)-like 2
18205s at MKNK2 MAP kinase interacting serine/threonine kinase 2 eparin-binding EGF-like growth factor 3S associated phosphoprotein 2 matrix metallopeptidase 9 (gelatinase B, 92kDa gelatinase, Patent Application Publication Oct. 3, 2013 Sheet 49 of 73 US 2013/0261024 A1
92kDa type V collagenase) 1500s at GLULT lutamate-ammonia ligase (glutamine synthetase) iras-related C3 botulinum toxin substrate 2 (rho family. small ---...--...-- 82 2 3603. S. at RAC2 GTP bindingprotein Rac2) 0.4568 83------...- 202600 s at NRIP1 nuclear receptor interacting protein ------...------...s------12916 84.202786 at serine threonine kinase 39 (STE20/SPS1 homolog, yeast) 0.528 3Xi A11 86218284 at SMAD3 MAD family member 3
87208083 s at ITGB6
ual specificity phosphatase 1
MDSi and EVII complex locus
suppressor of cytokine signaling 2
membrane bound O-acyltransferase domain containing 2 p elix family. member e PAS domain containing serine/threonine kinase secreted frizzled-related protein r inding and coiled-coil domain 2
ATP-binding cassette, sub-family G (WHITE), member 1
------Friend leukemia virus integration l 'm steroid-5alpha-reductase, alpha polypeptide 1 (3-oxo-5 alpha-steroid delta 4-dehydrogenase alpha 1) : exocyst complex component 7 abhydrolase domain containing 3
ts homologous factor
hesion inolecule
ucine Zipper protein
108 221509 at DENR density-regulated protein 109203072 at MYOIE
syndecan 4 mediator of DNA-damage checkpoint transmembrane protein with EGF-like and two follistatin-like t 205122 at TMEFF1 domains 1 0.6406 19201669 sat MARCKS myristoylated alanine-rich protein kinase C substrate 0.4037 20217967 s at FAM129A fami 0.382 Patent Application Publication Oct. 3, 2013 Sheet 50 of 73 US 2013/0261024 A1
ca-122 201930 at minichromosome maintenance complex component 6
123201834 at PRKABI protein kinase. AMP-activated, beta | non-catalytic------subunit ------S0.2963 2295s at CTSH cathepsin H 0.6305 125,217996 at PHLDAI pleckstrin homology-like domain, family A, member 0.4136. 126.204639 at ADA adenosine deanuinase 127210276 s. minichromosome maintenance complex component 5 - family 25, member 37 --
- alf LIM domains 2 ---, -,-,-,-,-,-,-,------133209 iss rt134.212186 at 35201147 s 136204361s 13728501 at 138202859 x : 139208862 is associated protein), delta
protein-coupled receptor 61
pase 6, apoptosis-related cystein
cyl-CoA synthetase long chain family member 3
143,202.07 s at MCM2 inichromosome maintenance complex component 2 i i integrin, alpha 2 (CD49B, alpha 2 subunit of VLA2 144205032 at ITGA2 receptor)
1452.10229s at CSF2 146 218613 at PSD3 147.201416 at Sox4 148 217875 s at PMEPA1 1492.13916 at ZNF20 150219631 at LRP12
151217272 s at SERPINBI 152204567 s at ABCG TP-binding- cassette, sub-family G (WHITE), member- 154153 21------8273 s a PDP1 pyruvate dehyrogenase phosphatase catalytic subunit------1 kinase (PRKA) anchor protein 12
: 10792 x at SIVA s-inducing factor 161202581 at HSPA 1A heat shock 70kDa protein 1A 212298 at NRP1 iin Patent Application Publication Oct. 3, 2013 Sheet 51 of 73 US 2013/0261024 A1
- olute carrier family 22, member 14
phosphoribosyl pyrophosphate synthetase-associated protein 1
uclear transcription factor, x-box binding
pleckstrinhomology-like domain, family A, member 1 chromosome 22 open reading frame 30 lysine (K)-specific demethylase 6A 173203837 at MAP3K5 mitogen-activated protein kinase kinase kinase 5 serpin peptidase inhibitor, clade E (nexin, plasminogen activator inhibitor type 1), member
i protein coupled receptor 161
rleukin 1, alpha protease, Serine, 22 213392 at IQCK IQ motif containing K platelet-derived growth factor beta polypeptide (simian lar
N6AMT1 N-6 adenine-specific DNA methyltransferase I (putative) 8B RAB8E, member RAS oncogene family FLAR CASP8 and FADD-like apoptosis regulator HAUS5 HAUS augmin like complex, subunit 5 inding protein (liprin beta1)
amyloid beta (A4) precursor protein binding, family B. rember 2 -:X------is-r NK3 homeobox i ------&-six------sw-st:------x-xx-wl X:------cadherin 4, type I, R-cadherin (retinal) irvotic translation initiation factor 5
iscoidin domain receptor tyrosine kinase 1 valbumin), member
198204475 at MMP1 matrix metallopeptidase 1 (interstitial collagenase) 199371 17 at ARHGAP8 Rho GTPase activating protein 8 -: 8 - ...... 200217202 s at GLUL glutamate-ammonia ligase (glutainine synthetase) Patent Application Publication Oct. 3, 2013 Sheet 52 of 73 US 2013/0261024 A1
FIG. 20: Table N
RA benchmark signature in thC, 200 down-regulated
ilog i change tRA to
e Symbol NetAffx Title ------
DMSO -08928
ethanolamine kinase 1 0.2843. arrier family 38, member 2
'n, type VIII, alpha 1
202708_s_at histone cluster 2, H2be - transcription factor AP-2 alpha (activating enhancer binding protein 2 alpha) adrenergic, beta-2-, receptor, Surface transmembrane protein 40 four jointed box 1 (Drosophila)
solute carrier family 38, member 2
ZNF238 zinc finger protein 238 13 2011oss at THBS1 thrombospondin - 14217579 xat ------...----...------15222162 S tallopeptidase with thrombospondin type 1 motif
coronin, actin binding protein, C growth arrest specific 6 AMIGO2 adhesion molecule with Ig-like domain 2
coagulation factor II (thrombin) receptor-like I 21202619 sat PLOD2 procollagen-lysine,2-oxoglutarateyclin G2 5-dioxygenase 2 - - - -, - ...... -0.28830.6733 22212992 at AHNAK2 AHNAKnucleoprotein 2 0.834 i-r------s myeloid/lymphoid or mixed-lineage leukemia (trithorax 23,204917 is at MLLT3
nc finger, BED-type containing 2 27 219836 at ZBEI ------so------s-s------0.6671
------1-en-unio-a--are--as-s-s-s-s28.204602 at DKK dickkopf homolog 1 (Xenopus laevis) 0.7933 29201739 at SGKI serum glucocorticoid regulated kinase 1 -0.7944
- solute carrier family 7, (cationic amino acid transporter, y + system) member 1 | - - - -1,0269. dual specificity phosphatase 10 -1.1129 thrombospondin - 12465 28319 at PELII pellino homolog 1 (Drosophila) ------: -0.3831 ------prostaglandin-endoperoxide ------synthase ------1 (prostaglandin G/H 34215813 s at PTGS1 synthase and cyclooxygenase) -0.6398 Patent Application Publication Oct. 3, 2013 Sheet 53 of 73 US 2013/0261024 A1
sema domain, immunoglobulin domain (Ig), short basic 35,35666 at domain, secreted, (semaphorin) 3F
36.204359 at fibronectin leucine rich transmembrane protein 2 ------.37209815 at PTCH1 patched honolog 1 (Drosophila) Solute carrier family 7, (cationic amino acid transporter, y + 38209921 at system) member 11 - - 3 9 205157s at protein phosphatase 1A (formerly 2C), magnesium 40 203966 s at ependent, alpha isoform ------tone cluster 1, H2ac 42203640 at 2 muscleblind-like 2 (Drosophila) ----- 43.202874 sat ATP6V1C1 ATPase, H+ transporting, lysosomal 42kDa, VI subunit Ci 44213568 at dd skipped related 2 (Drosophila) 0.520s 45.209674 at cryptochrome 1 (photolyase-like) . ------m3-mm------Sm3--0.288.1
46203946------sat rginase, type II -0.3651 47,203476 at 48201010s at TXNIP oredoxin interacting protein 0.484. 49203865_s_at ADARB1 adenosine deaminase, RNA-specific, B1 (RED1 homolog rat) -0.6168 solute carrier family 7 (cationic amino acid transporter, y + 50216092 s at SLC7A8 - system), member 8 ANKRD10 ankyrin repeat domain 10
AKIP1 PAK1 interacting protein
elongation factor Tu GTP binding domain containing 1
-- fibronectin leucine rich transmembrane protein 3 56.219284 at HSPBAP1 HSPB (heat shock 27kDa) associated protein prostaglandin-endoperoxide synthase 1 (prostaglandin G/H 57.205 128 x_at PTGS1 synthase and cyclooxygenase) 58,209758 s at MFAP5 microfibrillar associated protein 5 59.212386 at TCF4 transcription factor 4 ------eror------ir-i60215501s at 'pro-con-writer------vu-arc------was--on---DUSP10 --- hosphatase o ------:
63 201341 at ENCI ectodermal neural cortex (with BTB-like domain) 64 215564 at AREG Amphiregulin
65209180 at RABGGTB Rab geranylgeranyltransferase, beta subunit 66200962 at RPL3 ribosomal protein L31 --~------protein tyrosine phosphatase-like (proline instead------of catalytic
67212640 at PTPLB arginine), member b 68209946 at VEGFC
69,204930s at BNIP1 790 at ODC1
72213624 at SMPDL3A sphingomyelin phosphodiesterase, acid-like 3A Patent Application Publication Oct. 3, 2013 Sheet 54 of 73 US 2013/0261024 A1
732O3521s at ZNF318 inc finger protein 318 -0.365 TAF5 RNA polymerase II, TATA box binding protein (TBP). 7421.0053 at associated factor, 100kDa 13394 at mitogen-activated protein kinase binding protein 76202220 at KIAA0907
77206156 at gap junction protein, beta 5.31.1kDa -0.3179 78.207574 sat growth arrest and DNA-damage-inducible, beta -0.4058 : : serpin peptidase inhibitor, clade E (nexin, plasminogen 79.212190 at activator inhibitor type 1), member 2
integrin. alpha 6
neuregulin 14702 at fibronectin -r- or------. ------, - - - CD55 molecule, decay accelerating factor for complement (Cromer blood group) - ith sequence similarity 60, member - 86,208782 at 87.220318 at
co-or------, inositol polyphosphate-5-phosphatase, 145kDa ow-r------vi-virg------0.9359.-0.2639------. 92.205534 at PCDH7 protocadherin 7 -0.489 protein phosphatase B (formerly 2C), magnesium dependent, 93 209296 at PPMB beta isoform -0.2956 94.2094.57 at ---v---> ------dual specificity phosphatase 5 -0.3584;
CR4-NOT transcription complex, subunit 8 HIV-1 Tat interactive protein 2, 30kDa
1 specificity tyrosine (Y)-phosphorylation regulated kinase 99,203778 at MANBA mannosidase, beta A, lysosomal -0.3726.03569 100206332 is at IFI16 interferon, gamma-inducible protein 16 -0.3091 TrrissCD55 molecule,maiornia decayaravarajaratino accelerating factorfart for ------complement
lute carrier family 31 (copper transporters), member 2 -0. steine rich hydrophobic domain 2
DDX3Y DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, Y-linked 16268 is at JAG1 jagged 1 (Alagile syndrome) 10721868 is at SDF2L stromal cell-derived factor 2-like 108.204686 at IRS1 insulin receptor substrate
109.201266 at TXNRD1 thioredoxin reductase 1 110 091 5 s at IRS2 sulin receptor substrate 2 ------Patent Application Publication Oct. 3, 2013 Sheet 55 of 73 US 2013/0261024 A1
solute carrier family 7, (cationic amino acid transporter, y+ 112.207528 s at SLC7All system) member 11 ------113203743 s at TDG ------a------as-s-s------sis------thymine-DNA glycosylase ! 14202923 s at GCLC glutamate-cysteine ligase, catalytic subunit
1152.19410 at TMEM45A transmembrane protein 45A 16219073 s at OSBPL10 Xysterol binding protein-like 10 ... --
alladin, cytoskeletal associated protein (hexokinase 2 118202934 at -----ms------119208.527 x at ihistone cluster 1.H2be -- interleukin 15 receptor, alpha
------: ------0.525.
12922.15966s s at C7ORF64 130202686 s at AXL receptor tyrosine kinase larginase, type II -0.5513-0.2754 ------soul------uregulin 1 minin, alpha 2
lute carrier family 26 (sulfate transporter), member 2 -0.4357. almodulin regulated spectrin-associated protein 1-like 1 -0305 -guanine nucleotide exchangefactor -- chromatin modifying protein B
143,221276 s at incoilin, intermediate filament protein 144219710 at SH3TC2 SH3 domain and tetratricopeptide repeats 2 tathionase (cystathionine gamma-lyase) 146 202876 is at PBX2 pre-B-cell leukemia homeobox2 | ST6 (alpha-N-acetyl-neuraminyl-2,3-beta-galactosy I-1 3)-N- 147220979 is at ST6GALNAC5 acetylgalactosaminide alpha-2,6-sialyltransferase 5 -0.8082
:
CTD (carboxy-terminal domain, RNA polymerase II, 148201906 s at CTDSPL polypeptide A) small phosphatase-like ------serine/threonine kinase 3 (STE20 homolog, yeast) 150202364 at -- MAX interactor 1 151204584 at L1 cell adhesion molecule Patent Application Publication Oct. 3, 2013 Sheet 56 of 73 US 2013/0261024 A1
se-la-ul-wiss-----...svim-wit152222262s ----- at -- 153 209099 x at agged 1 (Alagile syndrome) 0.3677. -- - - - tripartite motif containing 2 ------: PHD finger protein 15 156221249 s at FAM117A family with sequence similarity 117, member A 158204532 x at UGT1A10 UDPglucuronosyltransferase 1 family, polypeptide Al -0.5 159220262s at DLK2 delta-like 2 homolog (Drosophila) 160217599 sat MyoD family inhibitor domain containing 1612 19885 at schlafen family member 12 ... --- 162216309-x_at JRK jerky homolog (mouse) ; 163218358 at CRELD2 cysteine-rich with EGF like domains 2 -0.4017,
164,210765 at CSEIL CSE1 chromosome segregation 1-like (yeast) -0.4052 rogrammed cell death 4 (neoplastic transformation inhibitor) -0.4477 dimethylarginine dimethylaminohydrolase 2 i ------a------0.3615, 167202973 x at FAM13A family with sequence similarity 13, member A s -0.3649 16821 1986 at AHNAK AHNAK nucleoprotein | -0.3579. 169203612 at bystin-like ...... 0.2825
0.9163
protein phosphatase 2 (formerly 2A), regulatory subunit B", ------174209633 at PPP2R3A alpha -0.284 175203889 at SCG5 secretogranin V (7B2 protein) -1.2842, ------protocadherin 7 -0.6.199 RAS prot 0.6331,
a upstream element (FUSE) binding protein -0.2702 dline 1 (Opitz/BBB syndrome) -0.3946 182212667 at ------secreted protein, acidic, cysteine-rich (osteonectin)------0.3021
i excision repair cross-complementing rodent repair deficiency,
------...s.186219529 at C loride intracellular channel 3 -0.4138. recombination signal binding protein for immunoglobulin & 18721 1974 x at RBPJ kappa J region -0.2718 ------
| solute carrier family 16, member 1 (monocarboxylic acid 188209900 s_at SLC16A1 transporter I) ------aldo-keto reductase family 1, member C3 (3-alpha 189209160 at AKR1C3 hydroxysteroid dehydrogenase, type II) 190204014 at DUSP4 dual specificity phosphatase 4 Patent Application Publication Oct. 3, 2013 Sheet 57 of 73 US 2013/0261024 A1
198204136 at COL7A 199207147 at DLX2 distal-less homeobox2 0.631. aldo-keto reductase family 1, member C2 (dihydrodiol dehydrogenase 2; bile acid binding protein; 3-alpha :
200------209699 xat AKR1C2 ------hydroxysteroid dehydrogenasc, type III) -0.6079II. Patent Application Publication Oct. 3, 2013 Sheet 58 of 73 US 2013/0261024 A1
FIG. 21: Table O
RA Benchmark Signature in BJ fibroblasts, 200 up-regulated
i Rank Probe set ID Acronym NetAffx Title DMSO G protein-coupled receptor, family C, group 5, 1203108 at GPRC5A cysteine-richmember A secretory protein LCCL domain 222 1541 at CRISPLD2 containing 2 s 3218729 at LXN latexin 20417 4 201042 at transglutaminaseglutamine-gamma-glutamyltransferase) 2 (Cpolypeptide, protein- - 0.9431.
5216598. -- chemokine (C-C motif gand 2 ------...------fibroblast------, -ss------activation protein, alpha transducin-li ke enhancer of split 1. (Esp1) homolog, 7203221 at TLE1 Drosophila)r 8219634 at CHST11 carbohydrate (chondroitin 4) sulfotransferase 11 -a-vu- 9208394 x at ESM1 endothelial cell-specific molecule 1 102 12774 at ZNF238 |zinc finger protein 238 ------11 200832------s at re-or-e------SCD earoyl-CoA desaturase (delta-9-desaturase) 12.209505 at NR2F1 uclear receptor subfamily 2, group F, member 1 13203738 at CSORF22 chromosome 5 open reading frame 22
------SFRP1 secreted frizzled related protein 1 T. nudix (nucleoside diphosphate linked moiety X). 19217220 at ------...--20212771 at ogen activator, urokinase
oic acid receptor responder (tazarotene induced)
NA-damage regulated autophagy modulator
ppressor of cytokine signaling 2 SMAD family member 3 0.
PL1 GABA(A) receptor-associated protein like 1. - : nuclear receptor interacting protein s ------...------
family with sequence sinilarity 65,member B
regulator of G-protein signaling 2. 24kDa tissue factor pathway inhibitor 2
adykinin receptor B2
dehydrogenase/reductase (SDR family) member 3
prostaglandin I2 (prostacyclin) synthase Patent Application Publication Oct. 3, 2013 Sheet 59 of 73 US 2013/0261024 A1
34,205005 s at 35 201829 at NET neuroepithelial cell transforming 1 - 36204597 x at STC1 stanniocalcin
BDKRB1 bradykinin receptor B
12658 at LHFPL2 lipoma HMGIC fusion partner-like 2 ------
4140204326 (202599 satx at MTIXNRP1 nuclearmetallothionein receptor IXinteracting protein 1 - 43 201309 x at C5ORF3 44208966 x at
45204595-e--. s at STC 46.202016 at MEST 47.202213 s at CUL4B 48,202859_x at ILs 4939402 at 50,201596. xat 51.212143 s at insulin-like growth factor binding protein 3 - 52.205532 s at cadherin 6, type 2. K-cadherin (fetal kidney) 3.21. 497 at epeat and BTB (POZ) domain containin
· (1,25-dihydroxyvitamin D3) receptor 57212624s at CHN1 chimerin (chimaerin)
58.210002 at GATA6 GATA binding protein 6 59.212859 x at MTIE metallothionein 1E 0.3369. 60 218129 sat : : uclear transcription factor Y, beta ------0.45 17 ------1211668 sat 62.205006 s at ------0.861 7 63 0.8897 64,40148 at APBB2 B, member 2 65.20 1310s at CSORF3 chromosome 5 open reading frame 13 66 2 mitochondrial turnor suppressor ------amyloid a------as-a-s------'sa------as-a-a-a------so-s-s------beta (A4) precursor protein-binding, family 67 213419 at B, member 2 68.2094.88 s ple splicing
69202949s at FHL2 four and a half LIM domains 2
member 5A cetylneuraminic acid synthase ysophosphatidic acid receptor 6 dishevelled associated activator of morphogenesis 2 0.3829 B-cell CLL/lymphoma 6 0.2978 Patent Application Publication Oct. 3, 2013 Sheet 60 of 73 US 2013/0261024 A1
p-to-o-o-o-7537433 at protein inhibitor of activated STAT, 2 F - s------78.206707_x at FAM65B family with sequence similarity------,-,-,- 65, member B 79.202295 s at CTSH cathepsin H 8020517 s at------x-x------s-x-x-xx------AKAP12 A kinase (PRKA) anchor protein 12 t TIMP3 TIMP metallopeptidase inhibitor 3
------ILB- - v -- v----- " ------rleukin 1, beta CDR2 cerebellar degeneration-related protein 2,62kDa 0.3248 se domain containing ---
lial PAS domain prote prostaglandin E synthase - uclear receptor subfamily 2, group F, member 1 ...------
ibonuclease type III, nuclear :
phospholipid scramblase I - : - - - - dapper, antagonist of beta-catenin, homolog -- 92.219179 at DACTI (Xenopus laevis) s solute carrier family 16, member 4 (monocarboxylic 93.205234 at SLC16 A4 acid transporter 5) s interferon induced protein with tetratricopeptide 94203153 at repeats 95.209487 at A binding protein with mu s peptidase domain containing ass 96213661 at regeneration 1 98.21222497204541 at SEC14L2ALDH1A1 SEC1aldehyde dehydrogenase 1 family, member Al 0.49. serpin peptidase inhibitor, clade B (ovalbumin),
stro-is-a------sous-99.209723 at r SERPINB9SERF member 9 10020416 at SOX4 Y (sex determining region Y)-box 4 101.207836 s at RBPMS RNA binding protein with multiple splicing 102.2 18025-s at PECI peroxisomal D3.D2-enoyl-CoA isomerase ----- v myb myeloblastosis viral oncogene homolog MYB (avian) ic acid receptor, beta ---
nudix (nucleoside diphosphate linked moiety X} type motif 4 03:
histone cluster 3, H2a giopoietin-like 4 108214803 at 109.202575 at CRA 110212418 at ELF1 111203131 at PDGFRA polypeptide Patent Application Publication Oct. 3, 2013 Sheet 61 of 73 US 2013/0261024 A1
112213502 x at 0229l_s_at
19201830s at 12021971.5 s at
vitamin D (1,25-dihydroxyvitamin D3) receptor secreted frizzled-related protein 1 ------on---- metallothionein IE 0.2757,
127218292-s at PRKAG2 catalytic subunit 1282095 sat PSG1 pregnancy specific beta-l-glycoprotein
13805 at 1 31 221002 s at 132201669 s at MARCKS myristoylated alanine-rich protein kinase C substrate, 0. 133219749 at SH2 domain containing 4A a------134 201417 at SRY (sex determining region Y)-box 4 s 0.7638 potassium inwardly-rectifying channel, subfamily J. 135206765 at IKCNJ2 member 2 0.9798 1362201.61 s at EPB41L4B erythrocyte membrane protein band 4.1 like 4B 0.826 137205027s at MAP3K8 itogen-activated protein kinase kinase kinase 8
138207680 x at PAX3 paired box 3 s 0.3762 ------wi------202976 sat 3TB3 Rho-related BTB domain containing------3 SMAD family member 6
------on------CIGALT1C1 CIGALT1-specific chaperone 1442) 1711s at PTEN phosphatase and tensin homolog
t COX11 homolog, cytochrome c oxidase assembly 145214277 at protein (yeast) 146 201662 s at ACSL3 S. yl-CoA synthetase long-chain family member 3 147205085 at ORC1L origin recognition complex, subunit 1-like (yeast) 148.213260 at forkhead box C1 149201860s at P AT plasminogen activator, tissue 150204146 at ------RAD51 associated protein 1 --- Patent Application Publication Oct. 3, 2013 Sheet 62 of 73 US 2013/0261024 A1
152203656 at FIG4 FIG4 homolog (S. cerevisiae) 0.2 153.209409 at GRB10 growth factor receptor-bound protein 10 0.3779 154203355 s at PSD3 pleckstrin and Sec7 domain containing 3 0.87 ------155202035 s at SFRP) secreted fri zzled-related protein 0.5473. 156210095 sat IGFBP3 insulin-like growth factor binding protein 3 0.324 : 157214783 s at ANXA11 -- 0.3222. 158210612 s at SYNJ2 synaptojanin2 0.6355 ------xx------was ---a-...-s:------.:-u--ms'-'---8-a:--'l-w -:------... writer------159 00701is . at. . .NPC2 . T Niemann-Pick------disease, type C2 ------0.2681 15774s at null 0.5239;
1210512 s at VEGFA 0.8526
-r-HNRNPULI 0.2701
DIMTIL : cerevisiae) -like oncogene 1672.16180 s at SYN2 ynaptojanin 2 ossai g------.168221530s at basic helix-loop-helix------or------family, member e4} 0.9186 16920966 s at pha, class 2B, member 1 170212268 at SERPINB1 memberrpin peptidase i inhibitor, clade B (ovalbumin), 171202094 at BIRC5 baculoviral IAP repeat-containing 5 | wingless-type MMTV integration site family, 1722 13425 at WNTSA member 5A 0.507--...- 0.597 ---18820 at Cl4ORF132 chromosome------14 open reading frame 132 ------, 1. ORAI calcium release activated calcium modulator s
eu------174218812.s at riORAI2 0.5284 1752 7853 at INS3 0.4939 176203394 s at HES1 hairy and enhancer of split 1. (Drosophila) 0.5153 ------: - - - - calcineurin-like- - phosphoesterase- - domain -containin - g ------177218610 sat CPPED O.37; 178217783 sat ELS ppee-like 5 (Drosophila) 1792.19166 at C14ORF104 chromosome 14 open reading frame 104 0.
homolog2, proliferation-associated 181217692 at IMAGOH2 Drosophila) 182209277 at TFP2 tissue factor pathway inhibitor 2
183205174 sat ------glutaminyl-peptide cyclotransferase - --- i. ------184205398 s at AD3 SMAD family member 3 185203851 at IGFBP6 insulin-like growth factor binding protein 6
186212786 at CLEC16A C-type lectin domain family 16, member A 05266
187206084------at ------aPTPRR ------...--- rotein tyrosine phosphatase, receptor type, R - 0.7848 188205743 at STAC SH3 and cysteine rich domain 0.411. Patent Application Publication Oct. 3, 2013 Sheet 63 of 73 US 2013/0261024 A1
189206586 at CNR2 annabinoid receptor 2 (macrophage) 0.403 190219901 at FGD6 FYVE, RhoGEF and PH domain containing ------yaw, walkssc:-uwww.all-am-Yxx-x-wrk...------ex-ser-ul-----...----...-rew ------0.7331 221019_s_at - COLEC12Y --viruvalua-ul--ee-ucu------w:------&-----wu-ul------su.collectin sub-family member 12 0.6698 amyloid beta (A4) precursor protein-binding, family i 192212970 at APBB2 B, member 2 0.361 19322O186s------at ------...-es------wu-ti-ur-is------PCDH24 protocadherin 24 ww-as------0.7735 i------out-os------e.amyloid beta (A4) precursor protein-binding,------family
a-ra-ra-ra-relaw-ma--sur-wa-aa-re-mur194212972_x at APBB2 B, member 2 : 0.6666, 195203098 at CDYL ------lem.------...re-is-e------196 ------chromodomain protein, Y-like warlos-as-----4----woon-sur-wachsw-wowar:0.357 1965.1158 at family with sequence similarity 174, member B 0.75 re------w------iss------FAM174B ------n sail 1972.19759 at ERAP2 endoplasmic reticulum aminopeptidase 2 0.561 ------ex------e-w-a------ATP-binding cassette, sub-family A (ABC), ------198203504s at imember 1 i ().9099: integrin, alpha 4 (antigen------CD49D, alpha 4 subunit of VLA-4 receptor) : ------was------...--&---sur------e.s.l.o.o.w....svil-wa-...-----wiwxw---a-row...tv199205885 is at ITGA4 ------...... 0.2878. wur----- 200 201 149 s at TIMP3 wire-sex-sex TIMPworwer-sul-wor------ee-sww. metallopeptidase inhibitor ------all---4------a- 3 0.5748 Patent Application Publication Oct. 3, 2013 Sheet 64 of 73 US 2013/0261024 A1
F.G. 22: Table P RA Benchmark Signature in BJ fibroblast, 200 down-regulated
Rank Probe set ID Acronym NetAffx Title 1210120s------at RANBP3 TRAN binding protein solute carrier family 2 (facilitated glucose transporter). 2216236 s at SLC2A14 member 14
3206924-X-X-X------wrumwr------r-wn------at Li r 4204933_s_at TNFRSF11B...a - tumor--- necrosis------factor receptor superfamily, member 1 lb 5213496 at LPPR4 plasticity related gene I --- 6,209288_s_at CDC42EP3 CDC42 effector protein (Rho GTPase binding)3 "--,7204011 at "r"---SPRY2 sprouty homolog 2 (Drosophila)or--ri-r-
y 8222162 sat ADAMTS1 ADAMwaw.www.www.w3.inux.s.l. metallopeptidase with... ------wall-www-rw-sythrombospondin type 1 motif, 9201423 s at CUL4A culin 4A --...--3------S --- 10209386 at TM4SFI transmembrane 4 L six family member 1 ------all11204948_s_at ------w------as------als------ow------FST follistatin 12209841, s_at LRRN3 leucine rich repeat neuronal 3 13215034s at TM4SFI transmembrane 4 L six family member 0.7015 v-nyb myeloblastosis viral oncogene homolog (avian)-like 1 14213906 at MYBL ------&-----war ------r --a ------15215336 at AKAP11. A kinase (PRKA) anchor protein 11 0.724. 16206157 at PTX3 y induced by IL-1 beta -0.7367 172103 to sat FGF5 fibrobla, growth factors 0.3979; 18204014 at DUSP4 dual specificity phosphatase 4 0.7517 --- transducin-like enhancer of split 4 (E(sp 1) homolog, 3. 19204872 at TLE4 Drosophila) -0.6695 p-a-...------...--sime------2022 1528_s_at ELMO2 Fengulfment and cell Y--- motility 2 ------| --0.5656 ------21203794 at CDC42BPA CDC42 binding protein kinase alpha (DMPK-like) t -0.3263 22212915 at PDZRN3 PDZ domain containing ring fingers mm --- -0.5008, 23208961s at KLF6 Kruppel-like factor 6 0.5552. ro- a Nrr era, for a rainianiday TT in a 7A. :------24.206814 at NGF nerve growth factor (beta polypeptide) 0.374, : ---wu-we-a-sir-uxwatarzware-25,212745 s at BBS4------Bardet-Biedl syndrome a 4 26,203476 at TPBG trophoblast glycoprotein ...-a, -e-...-...----->------a------27,208025s at HMGA2 high mobility group AT-hook 2 ------six-w-----xx-aa-ra-i-herouw-----as-as--onco waiwiwu g-too------e.28 201363 s atwillwaw IVNS1ABP influenza virus NS1A binding protein -0.2912 292.8706 sat GRAMD3 GRAM domain containing 3 -x-x-xx------ti-0.3717 - - --- 30212230--in-a------view-ess------iss-cruits-sous-les-s-s-sus---or-too------...------> at PPAP2B phosphatidic acid phosphatase type 2B 0.3096,
s : s 31 201395 at RBM5 RNA binding motif protein 5 32209387su.-ya-as-classical------wow-wa-ur-y s at TM4sF1 3360474 at TFERMT1 34202708 s at HIST2H2B r ------exe-re...las------...-a-we-iss-s-s ------tassium intermediatelsmall conductance calcium-activated ------a-a------a-ra-e------35,2201.16 at KCNN2 channel, subfamily N. member 2 -0.5252 36221696 s at STYK1 serine/threonine?tyrosine kinase 1 ------Patent Application Publication Oct. 3, 2013 Sheet 65 of 73 US 2013/0261024 A1
372 12543 at AIM1 absent in melanoma 1 -0. 7 l 5 8. 207303 at PDEC phosphodiesterase 1C, calmodulin-dependent 70kDa 0. 222065 sat FLII ightless I homolog (Drosophila)
leucine rich repeat neuronal 3 ------
RAS guanyl releasing protein (calcium and DAG regulated) -0.749
somatostatin receptor 09081
ylsulfatase family, member J -0.3923 gremlin T. cysteine knot superfamily, homolog (Xenopus laevis) ... --m
bisphosphoglycerate mutase S. osphodiesterase 4B , cAMP-specific (phosphodiesterase E4 49203708 at PDE4B dunce homolog, Drosophila) 213103 at StAR related lipid transfer (START) domain containing 13 u----M3M-3- OBL-like 1 .m.-- --3------CDC42 effector protein 52209286 at ·------i lute carrier family 7, (cationic amino acid transporter, y+ 53217678 at SLC7A1 l stem) member 1 54214002 at -3-mm-33--ul-mulu -uum----- . ------You- - - --m:------55.212650 at H domain binding protein 56,204529_s_at thymocyte selection associated high mobility group box 0.863 PDZ and LIM domain 5 -0.6056. permidine?spermine N 1-acetyltransferase 1
inhibin, beta B
-----m-- H2.0-like homeobox ------
Cbp/p300-interacting transactivator, with Glu/Asp-rich ------as-c::----...------a------saw--
CITED2 carboxy-terminal domain, 2 tivated protein kinase kinase 3 ------67208 68 221 coiled-coil domain containing 99 armadillo repeat containing RAS p21 protein activator (GTPase activating protein) -0. protein kinase D ... zzled homolog6 (Drosophila) chromodomain helicase DNA binding protein 1-like praja ring finger 2 microtubule associated monoxygenase, calponin and LIM 75,218376 s at MICAL1 domain containing 1 Patent Application Publication Oct. 3, 2013 Sheet 66 of 73 US 2013/0261024 A1
gremlin , cysteine knot Superfamily, homolog (Xenopus laevis) -0.359: collagen, type IV, alpha 1 -0.4068
cruppel-like factor 6 0.8283
AT rich interactive domain 5B (MRF1-like)
40240 membrane associated ring finger (C3HC4)3 six-rulexus-X:-aux---- dual specificity phosphatase 5 protein phosphatase 2 (formerly 2A), regulatory subunit A, alpha isoform i -0.2723
intermediate filament family orphan 1 -0.4916 leucine rich repeat containing 14 852156 . deleted in lymphocytic leukemia 2 (non-protein coding) rum------mu-w chemokine (C-X-C motif) ligand I2 (stromal cell-derived | S.--- factor I) : ly growth response 1
F-box protein 38 ------WM---- W -8- ATPase, Ca++ transporting. plasma membrane 4 associated transcript2 ------m------m-----ms------
insulin-like growth factor 2 mRNA binding protein 3
92203525s at adenomatous polyposis coli disabled homolog2, mitogen-responsive phosphoprotein
erpin peptidasei
atural killer-tumor rec gnition Sequence disabled homolog2, mitogen-responsive phosphoprotein
98.201279 sat ------DAB2 99.213931 at ID2 100.21040 x at GTSE1 101212073 at 102.214682 at
ATP2B1 ATPase, Ca++ transporting, plasma membrane PPFIBP1 PTPRF interacting protein, binding protein 1 (liprin beta1) 0.31: hromosome 13 open reading frame actin binding LIM protein X- mummam fins-related tyrosine kinase 1 (vascular endothelial growth .
111 222033 s at factor/vascular permeability factor receptor) -0.3405 myocyte enhancer factor 2C ' ' ------W m -0.514 T translocation (myeloid leukemia associated) pseudogene Patent Application Publication Oct. 3, 2013 Sheet 67 of 73 US 2013/0261024 A1
smoothelin Guanine nucleotide binding protein (G protein), beta polypeptide 2-like •------OBL-like 1 117210621-s at RASA 1 RAS p21 protein activator (GTPase activating protein) 1 18204823 at neuron navigator 3 ------1192 -Ya-a-i------19627 at zinc finger family member 767 - 120 205437 at inc finger protein 21 121 YKT6 v-SNARE homolog (S. cerevisiae) 122210376 x at ELKI ELK1, member of ETS oncogene family 123208378 x at FGF5 fibroblast growth factor 5 ------solute carrier family 2 (facilitated glucose transporter), member 14 ankyrin repeat domain 12 at LIN37 in 37 homolog (C. elegans) ---- 126213526------s ------disabled homolog2, mitogen-responsive phosphoprotein ------(Drosophila) ine (K)-specific demethylase 5D src kinase associated phosphoprotein 2
135,214833 at TMEM63A ansmembrane protein 63A 3--4------on------v------war------136.212089 at in A/C s -0.2649. 137222263 at SLC35E1 solute carrier family 35, member El -0.4369 fms-related tyrosine kinase 1 (vascular endothelial growth :
) -----
trafficking protein, kinesin binding 2 and S-phase expressed 1 arcoglycan, delta (35kDa dystrophin-associated glycoprotein) I romodomain containing 2 omosome 17 open reading frame 101 chromosome 16 open reading frame 58 1492.13939 s at RUFY3 UN and FYVE domain containing 3 150202948 at IL1R terleukin I receptor, type I 1512221084-i-m------so-sow at AMIGO2
zinc finger protein 606 ------0.2757, Patent Application Publication Oct. 3, 2013 Sheet 68 of 73 US 2013/0261024 A1
155220305 at MAVs mitochondrial antiviral signaling protein T. 156204506 at iPPP3R proteialpha fisoform phosphatase 3 (formerly 2B), regulatory subunit B, 157218970s a cutC copper transporter homolog (E. coli) -0.2703 15821031 1 at fibroblast growth factor 5 -0.3899 ------ur-...------bicaudal D homolog2 (Drosophila) -0.3269
0.3959
y 110, member B 1642.19563 at 165,204.190 at iquitin specific peptidase like I ...... -- : W ------
- adenomatous polyposis coli vascular endothelial growth factor C rkhead box D1 myosin IB ------
insulin-like growth factor binding proteins
176200796 ---...-mm. ... eloid cell leukemia sequence 1 (BCL2-related) ------177221841 s at F4 Kruppel-like factor 4 (gut) ------0.5958
CD55 molecule, decay accelerating factor for complement (Cromer blood gro
interleukin 7 receptor ------transducin-like enhancer of split 4 (Esp I) honolog, 182216997_x at TLE4 Drosophila) ------183204284 at PPP1R3C protein phosphatase 1, regulatory (inhibitor) subunit 3C - 184221276 is at SYNC syncoilin, intermediate filament protein LIM domain 7 ---- blastoma RAS viral (v-ras) oncogene homolog
G protein-coupled receptor 172A 760s at VAMP4 vesicle-associated membrane protein 4 CDC42EP3 CDC42 effector protein (Rho GTPase binding)3 -ra-'sa-ra-r-s-s-s-s-s:-M-1------>tripartite motif-containingw-...------...-- 45 microphthalmia-associated transcription factor --- solute carrier family 35, member F5 - serine palmitoyltransferase, long chain base subunit 2 PRKC, apoptosis, WT1, regulator Patent Application Publication Oct. 3, 2013 Sheet 69 of 73 US 2013/0261024 A1
195219 114 at hromosome 3 open reading frame 18 ------
221657s. kyrin repeat and SOCS box-c 197201604 sat PPP1R12A rotein phosphatase 1, regulatory (inhibitor) subunit 12A -0.3374 1982 1828s at TNIK RAF2 and NCK interacting kinase -0.5138. protein phosphatase 3 (formerly 2B), catalytic subunit, beta
Patent Application Publication Oct. 3, 2013 Sheet 70 of 73 US 2013/0261024 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 trC uM | 1.804 Myo-inositol KC 20 uM 28 Hexyldecanol KC O. O% 0.61 Chlorhexidine R. O LM O.54 Patent Application Publication Oct. 3, 2013 Sheet 71 of 73 US 2013/0261024 A1
FIG. 24: Table R Comparison of the predictiveness of different C-map signatures for predicting the activity of compounds in the mouse B 16 melanoma cell melanogenesis assay
f Average C-map Score Type Composite Hexamidine I NAG | Niacinamide Sepiwhite Sepiwhite MSH Benchmark 26 0.95 Nicotinamide Benchmark 1, 17 0.75 - Hexamidine diisethionate Benchmark 0.93 O.32 -
N-acetyl-D-glucosamine Benchilark 0.69 Ennodin Inhibitor 0.97 chlorhexidine diacetate Inhibitor (.9 0.33 Usmic Acid Inhibitor 0.9 resveratrol Inhibitor 0.88 Tyrphostin AG 879 --- Inhibitor O.88 Nordihydroguaiaretic acid Inhibitor 0.87 from Larrea divaricata (creosote bush) Tergitol NP 10 Inhibitor O.87 3.44. Trichlorocarbanilide Inhibitor 0.86 U-753.02 Inhibitor O8. ().55 ().65 AA-861 Inhibitor O.8 | Boswellin CG Inhibitor -
Ricinoleic acid Inhibitor 0.76 m ! Nw-Nitro-L-arginine methyl Inhibitor O69 ester HC SB 28795 Inhibitor 0.62 chlorhexidine Inhibitor 0.45 0.43O.43 Tetrahydrocurcumin CG Inhibitor 0.44 HYDROQUINONE Inhibitor 6-Hydroxy-1,3-benzoxathiol 2-one 6-HYDROXYINDOLE Acetyl tributyl citrate
Berberine Chloride Brij98 Copper(II) D-gluconate Oley alchol inhibitor Piroctone oiamine T Inhibitor puronic of inhibitor Tannic acid Inhibitor tert-BUTYL hibitor HYDROQUINONE xymenynic acid Inhibitor Patent Application Publication Oct. 3, 2013 Sheet 72 of 73 US 2013/0261024 A1
| Total Inhibitors and benchmarks 2 2 identified u-omomommy 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 2013/0261024 A1
FIG. 2S: Table S Number of probe sets with significant (<0.05) p Skin Tone Benchmark values compared to DMSO controls n one Benchmar Concentration Tert- HBL HeMIMP Materials keratikeratinocytes melanoma melanocytes | i cells Hexamidine 5 M 4263 1497 2359
diisothionate - - Myo-inositol 20 uM 5082 960 3687 N-acetyl-glucosamine 200 M 1034 1454 NDP-MSH* | 100 nM 1448 73 Niacinamide OO M + 298 | 1345 1312 Sepiwhite O uM : 245 883 23 ()
308,872 y1 US 2013/0261024 A1 Oct. 3, 2013
SYSTEM FOR IDENTIFYING CONNECTIONS nancy'), and at menopause. Also, this condition is frequently BETWEEN PERTURBAGENS AND GENES found among those taking oral contraceptives, and is occa ASSOCATED WITH ASKN sionally found among nonpregnant women who are not tak HYPERPGMENTATION CONDITION 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 0008 Kojic acid, Azelaic acid and certain-hydroxy acids to dihydroxyphenylalanine (DOPA) and then to Such as glycolic acid, have demonstrated skin-lightening dopaquinone. Subsequently, dopaquinone is converted to effects, but reports of localized irritation and inflammation dopachrome through auto-oxidation, and finally to dihy are common. The prenylated flavonol artocarpin has shown droxyindole or dihydroxyindole-2-carboxylic acid Some efficacy for skin-lightening in the context of ultraviolet (DHICA), which polymerize to form eumelanin. The latter induced skin pigmentation (Shimizu K. et al., Planta Med reactions occur in the presence of dopachrome tautomerase 68(1):79-81, 2002). and DHICA oxidase. In the presence of sulfur-containing 0009 Recently, a more detailed genomic and proteomic cysteine or glutathione, dopaquinone is converted to cysteinyl understanding of melanogenesis, the melanocyte, melano DOPA or glutathione DOPA: subsequently, pheomelanin is cyte-keratinocyte interaction, and the melanocyte-fibroblast formed. interaction has revealed potentially hundreds of proteins and 0005. A variety of skin hyperpigmentation disorders are other effectors involved in the pigmentation process and in known and etiology is diverse, overlapping in many cases, the etiology of hyperpigmentation disorders, which may pro and often not fully understood. For example, melanosis or vide additional targets. There is a need in the cosmetic arts melasma is a condition characterized by the development of both for generating potential skin lightening agents and for sharply demarcated blotchy, brown spots usually in a sym effective and efficient screening methods for identifying metric distribution over the cheeks, forehead, and sometimes putative skin active agents with efficacy and safety in the on the upper lip and neck. This condition frequently occurs cosmetic treatment of hyperpigmentation and pigmentation during pregnancy (melasma gravidarum or “mask of preg disorders. US 2013/0261024 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 potential pharmaceutical agents despite the Lamb compro group began publishing a detailed synopsis of the mechanics 1SCS. of C-map construction and installments of the reference col 0018. The connectivity mapping protocols of Lamb would lection of gene expression profiles used to create the first not be predicted, however, to have utility for hypothesis test generation 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 2013/0261024 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 2013/0261024 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 2013/0261024 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. Cosmetic agents may include Substances that are Generally NAG Benchmark Signature; 39 significantly up-regulated. Recognized as Safe (GRAS) by the US Food and Drug 004.5 FIG. 13 sets forth identifiers as shown in Table G, Administration, food additives, and materials used in non NAG Benchmark Signature, most significantly down-regu cosmetic consumer products including over-the-counter lated. medications. In some embodiments, cosmetic agents may be 0046 FIG. 14 sets forth identifiers as shown in Table H, incorporated in a cosmetic composition comprising a derma Niacinamide Benchmark Signature, 100 top up-regulated. tologically acceptable carrier Suitable for topical application 0047 FIG. 15 sets forth identifiers as shown in Table I, to skin. A cosmetic agent includes, but is not limited to, (i) Niacinamide Benchmark Signature, 100 top down-regulated. chemicals, compounds, Small or large molecules, extracts, 0048 FIG. 16 sets forth identifiers as shown in Table J, formulations, or combinations thereof that are known to Sepiwhite Benchmark Signature; 100 top up-regulated. induce or cause at least one effect (positive or negative) on 0049 FIG. 17 sets forth identifiers as shown in Table K, skin tissue; (ii) chemicals, compounds, Small molecules, Sepiwhite Benchmark Signature; 100 top down-regulated. extracts, formulations, or combinations thereof that are 0050 FIG. 18 sets forth identifiers as shown in Table L, known to induce or cause at least one effect (positive or Composite “Skin Tone' Benchmark Signature; approxi negative) on skin tissue and are discovered, using the pro mately 40 up-regulated--58 down-regulated. vided methods and systems, to induce or cause at least one 0051 FIG. 19 sets forth identifiers as shown in Table M, previously unknown effect (positive or negative) on the skin RA benchmark signature in tkC cell 200 upregulated. tissue; and (iii) chemicals, compounds, Small molecules, 0052 FIG. 20 sets forth identifiers as shown in Table N. extracts, formulations, or combinations thereof that are not RA benchmark signature in tkC, 200 down-regulated. known have an effect on skin tissue and are discovered, using 0053 FIG. 21 sets forth identifiers as shown in Table O, the provided methods and systems, to induce or cause an RA Benchmark Signature in BJ fibroblasts, 200 up-regulated. effect on skin tissue. 0054 FIG.22 sets forth identifiers as shown in Table PRA 0062 Some examples of cosmetic agents or cosmetically Benchmark Signature in BJ fibroblast, 200 down-regulated. actionable materials can be found in: the PubChem database 0055 FIG. 23 sets in Table Q, Average C-map scores for associated with the National Institutes of Health, USA (http:// Some representative potential skin lightening agents with the pubchem.ncbi.nlm.nih.gov); the Ingredient Database of the Retinoic Acid Keratinocyte RA 200 Signature. Personal Care Products Council (http://online-personalcare 0056 FIG. 24 Shows Table R, showing a comparison of council.org/jsp/Home.jsp); and the 2010 International Cos the predictiveness of different C-map signatures for predict metic Ingredient Dictionary and Handbook, 13" Edition, ing the activity of compounds in the mouse B16 melanoma published by The Personal Care Products Council; the EU cell melanogenesis assay. Cosmetic Ingredients and Substances list; the Japan Cosmetic 0057 FIG. 25, Shows Table S, with data outlining the Ingredients List; the Personal Care Products Council, the responsiveness of tert Keratinocytes, melanocytes, and mela SkinDeep database (URL: http://www.cosmeticsdatabase. noma cells to skin tone benchmarks. com); the FDA Approved Excipients List; the FDA OTC List: the Japan Quasi Drug List; the US FDA Everything Added to DETAILED DESCRIPTION OF THE INVENTION Food database; EU Food Additive list; Japan Existing Food 0058. The present invention will now be described with Additives, Flavor GRAS list; US FDA Select Committee on occasional reference to the specific embodiments of the GRAS Substances; US Household Products Database; the invention. This invention may, however, be embodied in dif Global New Products Database (GNPD) Personal Care, ferent forms and should not be construed as limited to the Health Care, Food/Drink/Pet and Household database (URL: embodiments set forth herein. Rather, these embodiments are http://www.gmpd.com); and from Suppliers of cosmetic ingre provided so that this disclosure will be thorough and com dients and botanicals. plete, and to fully convey the scope of the invention to those 0063. Other non-limiting examples of cosmetic agents skilled in the art. include botanicals (which may be derived from one or more 0059. Unless otherwise defined, all technical and scien of a root, stem bark, leaf, seed or fruit of a plant). Some tific terms used herein have the same meaning as commonly botanicals may be extracted from a plant biomass (e.g., root, understood by one of ordinary skill in the art to which this stem, bark, leaf, etc.) using one more solvents. Botanicals invention pertains. The terminology used in the description of may comprise a complex mixture of compounds and lack a the invention herein is for describing particular embodiments distinct active ingredient. Another category of cosmetic only and is not intended to be limiting of the invention. As agents are vitamin compounds and derivatives and combina used in the description of the invention and the appended tions thereof, such as a vitamin B3 compound, a vitamin B5 claims, the singular forms “a” “an,” and “the are intended to compound, a vitamin B6 compound, a vitamin B9 compound, include the plural forms as well, unless the context clearly a vitamin A compound, a vitamin C compound, a vitamin E indicates otherwise. compound, and derivatives and combinations thereof (e.g., US 2013/0261024 A1 Oct. 3, 2013 retinol, retinyl esters, niacinamide, folic acid, panethenol, ascorbic acid, tocopherol, and tocopherol acetate). Other O H non-limiting examples of cosmetic agents include Sugar RCNH-C-COOH amines, phytosterols, hexamidine, hydroxy acids, ceramides, amino acids, and polyols. CH 0064. Non-limiting examples of agents herein utilized are described in detail below, such as for vitamin B3 compounds, N-acyl amino acid compounds, and retinoid compounds. In Some embodiments, the vitamin B compound is a B3 com pound having the formula: wherein R' can be C to Cao Saturated or unsaturated, straight or branched, substituted or unsubstituted alkyls; substituted or unsubstituted aromatic groups; or mixtures thereof. 0067 N-acyl Tyrosine corresponds to the following for O' mula:
O H RCNH-C-COOH wherein R is CONH. (i.e., niacinamide), —COOH (i.e., CH2 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, myristyl nicotinate). Examples of Suitable vitamin B com pounds are well known in the art and are commercially avail OH able from a number of sources (e.g., the Sigma Chemical Company, ICN Biomedicals, Inc., and Aldrich Chemical wherein R' can be C to Cao Saturated or unsaturated, straight Company). or branched, substituted or unsubstituted alkyls; substituted 0065. Some embodiments of the compositions of the or unsubstituted aromatic groups; or mixtures thereof. present invention comprise a safe and effective amount of one 0068 A particularly useful compound in the present or more N-acyl amino acid compounds. The amino acid can invention is N-undecylenoyl-L-phenylalanine. This agent be one of any of the amino acids known in the art. The N-acyl belongs to the broad class of N-acyl Phenylalanine deriva amino acid compounds of the present invention correspond to tives, with its acyl group being a C11 mono-unsaturated fatty acid moiety and the amino acid being the L-isomer of pheny the formula: lalanine. N-undecylenoyl-L-phenylalanine corresponds to the following formula: O H RCNH-C-COOH O H
R CH=CH-(CH2)CNH-C-COOH CH 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 2013/0261024 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 2013/0261024 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 2013/0261024 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 2013/0261024 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 2013/0261024 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 (ISA) 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 2013/0261024 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 2013/0261024 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 2013/0261024 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 2013/0261024 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 2013/0261024 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 2013/0261024 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 2013/0261024 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 2013/0261024 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 control >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 control 50, 70, 100, 200, or 300 and/or less than about 800, 600, >200, 2. ratio of present calls of DMSO control chips >=50%, 400 or about 100 genes corresponding to a probe set on 3. down regulated by NAG, but up regulated by MSH (a skin the chip. The number of probe sets approximately cor darkening agent), 4. NAG treated p-0.05, 43 probes are responds to the number of genes, but a single gene may selected. The illustrative signatures are set forth as FIGS. 12 be represented by more than one probe set. It is under 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 spot tissue >200, 2. ratio of present call of spottissue illustrative signatures are set forth as FIGS. 16 and 17, Tables chips >=50%, 3. probe is significantly up regulated, p<0.05, J and K, respectively. 4. 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 chips midine are used together to generate a signature. This >=50%, 3. probe is significantly down regulated, p<0.05, 4. Example illustrates generation of an exemplary composite Top 50, 100 and 200 probes ranked by p values, are selected “skin tone' Signature comprised of four benchmark skin respectively. The illustrative signature is set forth as FIG. 2, lightening agents: Niacinamide, Sepiwhite, NAG, and Hexa 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 2013/0261024 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 signal >=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 2013/0261024 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, Universite 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 score 0.30. Result Summary of bovine serum (HyClone, Logan, Utah). Human telomerized correctly predicted melanogenesis inhibitors: Composite sig keratinocytes (tert-keratinocytes) were obtained from the nature: 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 2013/0261024 A1 Oct. 3, 2013 22
0203 Every document cited herein is hereby incorporated ond programmable computer for transmitting gene expres herein by reference in its entirety unless expressly excluded sion data from the scanner to the first programmable or otherwise limited. The citation of any document is not an computer. admission that it is prior art with respect to any invention 3. The system of claim 1, further comprising an array of disclosed or claimed herein or that it alone, or in any combi perturbagens for application to the human keratinocyte, fibro nation with any other reference or references, teaches, Sug blast, melanocyte or melanoma cells. gests or discloses any such invention. Further, to the extent 4. The system of claim3, wherein the hyperpigmentation any meaning or definition of a term in this document conflicts relevant gene expression signature comprises one or more with any meaning or definition of the same term in a docu gene expression signature lists of identifiers representing dif ment incorporated by reference, the meaning or definition ferentially expressed genes associated with a hyperpigmen assigned to that term in this document shall govern. tation condition, and the system further comprises a data 0204. The values disclosed herein are not to be understood architecture which allows the system to identify at least one as being strictly limited to the exact numerical values recited. putative skin active agent having potential efficacy in treating Instead, unless otherwise specified, each Such value is the skin hyperpigmentation condition. intended to mean both the recited value and a functionally 5. The system of claim 4, wherein the programmable com equivalent range Surrounding that value. puter assigns a connectivity score to each of the plurality of 0205 The present invention should not be considered lim instances and if the instance has a negative connectivity score, ited to the specific examples described herein, but rather a perturbagen associated with the instance is identified as a should be understood to cover all aspects of the invention. putative skin active agent having potential efficacy in the Various modifications, equivalent processes, as well as treatment of the hyperpigmentation condition, and if the numerous structures and devices to which the present inven instance has a positive connectivity Score, a perturbagenasso tion may be applicable will be readily apparent to those of ciated with the instance is identified as a putative skin active skill in the art. Those skilled in the art will understand that agent having potential efficacy as a skin-darkening agent. various changes may be made without departing from the 6. The system of claim 4, wherein the programmable com scope of the invention, which is not to be considered limited puter assigns a connectivity score to each of the plurality of to what is described in the specification. instances and if an instance has a positive connectivity score, What is claimed is: a perturbagen associated with the instance is identified as a 1. A system for identifying connections between pertur putative skin active agent having potential efficacy in the bagens and genes associated with a skin hyperpigmentation treatment of the hyperpigmentation condition, and if an condition, comprising: instance has a negative connectivity score, a perturbagen associated with the instance is identified as a putative skin (a) at least one computer readable medium having stored active agent having potential efficacy as a skin-darkening thereon a plurality of instances, and a skin hyperpigmen tation-relevant gene expression signature, wherein the agent. instances and the gene expression signature are derived 7. The system of claim 4, wherein hyperpigmentation con from one of a human keratinocyte cell, a human fibro dition results from one or more of a post-inflammatory blast cell, a human melanocyte cell, or a human mela response, age, exposure to UVB radiation, endocrine induc noma cell, wherein each instance comprises an instance tion of pigmentation, and a host skin predisposition. list of rank-ordered identifiers of differentially 8. The system of claim3, wherein the hyperpigmentation expressed genes, and wherein the hyperpigmentation relevant gene expression signature comprises one or more relevant gene expression signature comprises one or gene expression signature lists of identifiers representing dif more gene expression signature lists of identifiers rep ferentially expressed genes associated with a benchmark resenting differentially expressed genes associated with skin-lightening agent, and the system further comprises a data a hyperpigmentation condition or differentially architecture which allows the system to identify at least one expressed genes associated with a benchmark skin putative skin active agent having potential efficacy in treating lightening agent; a skin hyperpigmentation condition. (b) a programmable computer comprising computer-read 9. The system of claim 8, wherein the programmable com able instructions that cause the programmable computer puter assigns a connectivity score to each of the plurality of to execute one or more of the following: instances and if an instance has a positive connectivity score, a perturbagen associated with the instance is identified as a (i) accessing the plurality of instances and a hyperpig putative skin active agent having potential efficacy in the mentation-relevant gene expression signature stored treatment of a hyperpigmentation condition, and if an on the computer readable medium; instance has a negative connectivity score, a perturbagen (ii) comparing the hyperpigmentation-relevant gene associated with the instance is identified as a putative skin expression signature to the plurality of the instances, active agent having potential efficacy as a skin-darkening wherein the comparison comprises comparing each agent. identifier in the gene expression signature list with the 10. The system of claim 8, wherein the benchmark skin position of the same identifier in the instance list for lightening agent comprises an agent selected from the group each of the plurality of instances; and consisting of a melanocyte stimulation inhibitor, an anti (iii) assigning a connectivity score to each of the plural inflammatory agent, an alpha-MSH pigment induction ity of instances. antagonist, a melanophage dermal residence time Suppressor, 2. The system of claim 1, further comprising a microarray a melanin synthesis-associated enzyme, an adrenergic beta scanner for receiving a sample comprising human kerati receptor inhibitor, an antioxidant, a melanosome transport nocyte, fibroblast, melanocyte or melanoma cells; and a sec inhibitor, a vitamin B3 compound, hexamidine diisothionate, US 2013/0261024 A1 Oct. 3, 2013 23
N-acetyl-glucosamine, an N-acyl amino acid compound, 16. The system of claim 1, wherein the plurality of hydroquinone, a retinoid compound, hexyldecanol, and com instances comprises between about 50 and about 50,000 binations thereof. instances. 11. The system of claim 1, wherein the rank-ordered list of 17. The system of claim 1, wherein the connectivity score identifiers is arranged so that an identifier associated with each gene that is not differentially expressed is positioned is a value between +2 and -2. between the identifier associated with the most up-regulated 18. A computer readable medium, comprising: gene and the identifier associated with the most down-regu (a) a data architecture comprising a digital file stored in a lated gene. spreadsheet file format, a word processing file format, or 12. The system of claim 1, wherein the hyperpigmentation a database file format suitable to be read by a respective condition has an etiology associated with one or more of spreadsheet, word processing, or database computer activation of melanocyte stimulation, inflammation, activa program, the first digital file comprising data arranged to tion of alpha-MSH pigment induction, increased melanoph provide one or more gene expression signature lists age dermal residence time, activation of an enzyme involved comprising a plurality of identifiers when read by the in a melanin synthesis pathway, and activation of melano respective spreadsheet, word processing, or database Some transport. computer program; and 13. The system of claim 1, wherein the identifiers are (b) wherein each identifier is selected from the group con selected from the group consisting of gene names, gene sym sisting of a microarray probe setID, a human gene name, bols, and microarray probe set IDs. a human gene symbol, and combinations thereof repre 14. The system of claim 1, wherein the gene expression senting a gene set forth in any of Tables B through P signature is derived from a human keratinocyte and the sig wherein each of the one or more gene expression signa nature comprises genes associated with the identifiers set ture lists comprises between about 10 and about 400 forth in at least one Table selected from the group consisting identifiers. of Tables B through N. 15. The system of claim 1, wherein gene expression signa 19. The computer readable medium of claim 18, further ture is derived from a human fibroblast and the signature comprising computer readable instructions for reading the comprises genes associated with the identifiers set forth in at digital file. least one of Tables O and P.