BaricitinibisaselectiveorallybioavailableJAK1/JAK2inhibitorwithIC₅₀of5.9nMand5.7nM,respectively.

IC50:5.9nM(JAK1),5.7nM(JAK2),>400(JAK3),53nM(Tyk2)^[1]

Incell-basedassays,Baricitinib(INCB028050)provestobeapotentinhibitorofJAKsignalingandfunction.InPBMCs,BaricitinibinhibitsIL-6-stimulatedphosphorylationofthecanonicalsubstrateSTAT3(pSTAT3)andsubsequentproductionofthechemokineMCP-1withIC₅₀valuesof44nMand40nM,respectively.InisolatednaiveT-cells,INCB028050alsoinhibitspSTAT3stimulatedbyIL-23(IC₅₀=20nM).Importantly,thisinhibitionpreventedtheproductionoftwopathogeniccytokines(IL-17andIL-22)producedbyTh17cells-asubtypeofhelperTcellswithdemonstrableinflammatoryandpathogenicproperties-withanIC₅₀valueof50nM.Instarkcontrast,thestructurallysimilarbutineffectiveJAK1/2inhibitorsINCB027753andINCB029843hasnosignificanteffectinanyoftheseassayssystemswhentestedatconcentrationsupto10μM^[1].

Baricitinib(INCB028050)treatment,compareswithvehicle,inhibitstheincreaseinhindpawvolumesduringthe2wkoftreatmentby50%atadoseof1mg/kgand>95%atdosesof3or10mg/kg.Becausebaselinepawvolumemeasurementsaretakenontreatmentday0-inanimalswithsignificantsignsofdisease-itispossibletohave>100%inhibitioninanimalsshowingmarkedimprovementinswelling^[1].Baricitinib(0.7mg/day)treatedmiceexhibitssubstantiallyreducedinflammationasassessedbyH&Estaining,reducedCD8infiltration,andreducedMHCclassIandclassIIexpressionwhencomparedwithvehicle-controltreatedmice.CD8⁺NKG2D⁺cells,criticaleffectorsofdiseaseinmurineandhumanalopeciaareata(AA),aregreatlydiminishedinBaricitinibtreatedmicecomparewithvehiclecontroltreatedmice^[2].

• [1].FridmanJS,etal.SelectiveinhibitionofJAK1andJAK2isefficaciousinrodentmodelsofarthritis:preclinicalcharacterizationofINCB028050.JImmunol.2010May1;184(9):5298-307.

  [2].JabbariA,etal.ReversalofAlopeciaAreataFollowingTreatmentWiththeJAK1/2InhibitorBaricitinib.EBioMedicine.2015Feb26;2(4):351-5.

  [3].KhanIM,etal.IntermuscularandperimuscularfatexpansioninobesitycorrelateswithskeletalmuscleTcellandmacrophageinfiltrationandinsulinresistance.IntJObes(Lond).2015Nov;39(11):1607-18.

Enzymeassaysareperformedusingahomogeneoustime-resolvedfluorescenceassaywithrecombinantepitopetaggedkinasedomains(JAK1,837-1142;JAK2,828-1132;JAK3,718-1124;Tyk2,873-1187)orfull-lengthenzyme(cMETandChk2)andpeptidesubstrate.Eachenzymereactionisperformedwithorwithouttestcompound(11-pointdilution),JAK,cMET,orChk2enzyme,500nM(100nMforChk2)peptide,ATP(attheK_mspecificforeachkinaseor1mM),and2.0%DMSOinassaybuffer.ThecalculatedIC₅₀valueisthecompoundconcentrationrequiredforinhibitionof50%ofthefluorescentsignal.AdditionalkinaseassaysareperformedatCerepusingstandardconditionsat200nM.Enzymestestedincluded:Abl,Akt1,AurA,AurB,CDC2,CDK2,CDK4,CHK2,c-kit,EGFR,EphB4,ERK1,ERK2,FLT-1,HER2,IGF1R,IKKα,IKKβ,JNK1,Lck,MEK1,p38α,p70S6K,PKA,PKCα,Src,andZAP70^[1].MCEhasnotindependentlyconfirmedtheaccuracyofthesemethods.Theyareforreferenceonly.

Baricitinib(INCB028050)isdissolvedinstocksolutions,andthendilutedwithappropriatemediabeforeuse^[1].

HumanPBMCsareisolatedbyleukapheresisfollowedbyFicoll-Hypaquecentrifugation.ForthedeterminationofIL-6-inducedMCP-1production,PBMCsareplatedat3.3×10⁵cellsperwellinRPMI1640+10%FCSinthepresenceorabsenceofvariousconcentrationsofINCB028050(1nM,10nM,100nM,1μM,and10μM).Followingpreincubationwithcompoundfor10minatroomtemperature,cellsarestimulatedbyadding10ng/mLhumanrecombinantIL-6toeachwell.Cellsareincubatedfor48hat37°C,5%CO₂.SupernatantsareharvestedandanalyzedbyELISAforlevelsofhumanMCP-1.TheABIlityofINCB028050toinhibitIL-6-inducedsecretionofMCP-1isreportedastheconcentrationrequiredfor50%inhibition(IC₅₀).ProliferationofBa/F3-TEL-JAK3cellsisperformedover3dusingCell-TiterGlo^[1].MCEhasnotindependentlyconfirmedtheaccuracyofthesemethods.Theyareforreferenceonly.

Baricitinib(INCB028050)issUSPendedin0.5%methylcellulose(Rat)^[1].

Rat^[1]
Femalerats(n=6pergenderpergroup)aregivenadoseof10mg/kgBaricitinibandgivenbyoralgavageat10mL/kg.Thefirstthreeratsarebledat0(predose),2,8,and24h,andthesecondthreeratsarebled1,4,and12hafterdosing.EDTAisusedastheanticoagulant,andsamplesarecentrifugedtoobtainplasma.AnanalyticalmethodforthequantificationofINCB028050hasbeendevelopedandusedtoanalyzesamplesfromtoxicologystudies.Themethodcombinesaproteinprecipitationextractionwith10%methanolinacetonitrileandLC/MS/MSanalysis.Themethodhasdemonstratedalinearassayrange1-5000nMusing0.1mLofstudysamples.DataareprocessedusingAnalyst1.3.1.Astandardcurveisdeterminedfrompeakarearatioversusconcentrationusingaweightedlinearregression(1/x2).
Mice^[2]
TheC3H/HeJgraft-recipientmousemodelofAAisusedfortheseexperiments.Briefly,alopecicskinfromaC3H/HeJmousethatspontaneouslydevelopedhairlossisgraftedonto8-10weekoldC3H/HeJmicefreeofdisease.Atthetimeofgrafting,anosmoticpumpthatadministeredapproximately0.7mg/dayofBaricitiniborplaceboisimplanted.Osmoticpumpsarechangedmonthly.Atime-to-eventsurvivalanalysisforintervalcensoreddataisperformed.ThesurvivalandintervalpackagesinRareusedtoperformlog-ranktests.Thehypothesisthatthesurvivaldistributionsareequalinthe(n=10)Baricitinib-treatedmiceand(n=10)placebo-treatedmiceisrejectedatthe5%levelusingSun"sscoretoperformanexactlog-ranktwo-sampletestwiththep-valueof0.0035.MCEhasnotindependentlyconfirmedtheaccuracyofthesemethods.Theyareforreferenceonly.

• [1].FridmanJS,etal.SelectiveinhibitionofJAK1andJAK2isefficaciousinrodentmodelsofarthritis:preclinicalcharacterizationofINCB028050.JImmunol.2010May1;184(9):5298-307.

  [2].JabbariA,etal.ReversalofAlopeciaAreataFollowingTreatmentWiththeJAK1/2InhibitorBaricitinib.EBioMedicine.2015Feb26;2(4):351-5.

  [3].KhanIM,etal.IntermuscularandperimuscularfatexpansioninobesitycorrelateswithskeletalmuscleTcellandmacrophageinfiltrationandinsulinresistance.IntJObes(Lond).2015Nov;39(11):1607-18.

371.42

C₁₆H₁₇N₇O₂S

1187594-09-7

RoomtemperatureincontinentalUS;mayvaryelsewhere

DMSO:25mg/mL

Baricitinibissuspendedin0.5%methylcellulose^[3].

*"<1 mg/ml"="" means="" slightly="" soluble="" or="" insoluble.="" "≥"="" means="" soluble,="" but="" saturation="">

• [1].FridmanJS,etal.SelectiveinhibitionofJAK1andJAK2isefficaciousinrodentmodelsofarthritis:preclinicalcharacterizationofINCB028050.JImmunol.2010May1;184(9):5298-307.

  [2].JabbariA,etal.ReversalofAlopeciaAreataFollowingTreatmentWiththeJAK1/2InhibitorBaricitinib.EBioMedicine.2015Feb26;2(4):351-5.

  [3].KhanIM,etal.IntermuscularandperimuscularfatexpansioninobesitycorrelateswithskeletalmuscleTcellandmacrophageinfiltrationandinsulinresistance.IntJObes(Lond).2015Nov;39(11):1607-18.