max

/EC

0.001 0.01 0.1 1 10

% De cr ease in O bser ve d Mida zo lam AUC

100

120

= 0. 95

0.1 1

% Decrease in O bserved Midazolam AUC

100

120

= 0. 94

RIS

0.01 0.1 1 10

% Decrease in Midazolam AUC

100

120

= 0.93

Corning®HepatoCells:AnInVitroScreeningToolforPredicting

ClinicalCYP3A4Induction

RongjunZuo,

FengLi, 

SwetaParikh,

MercyanneAndes,

KirstenCooper,

EnneAkor,

GeorgeZhang,

JinLiu,

RonaldA.

Faris,

CharlesL.Crespi,and

ChristopherJ.Patten

TwoOakPark,CorningIncorporated,LifeSciences,Bedford,MA01730USA

6 HenshawStreet,CorningIncorporated,Life Sciences,Woburn,MA01801USA

1ScienceCenterDrive,CorningIncorporated,LifeSciences,Corning,NY14831USA

836NorthStreetBuilding300Suite3401,CorningIncorporated,LifeSciences,Tewksbury,MA01876USA

Conclusions

• CorningHepatoCellsshowedconcentration‐dependentresponsestoagroup

ofknownclinicalCYP3A4inducer swithverygoodcorrelation(R

>0.9).

• All3lotsofHepatoCells behavedsimilarly toprimaryhumanhepatocytesin

termsofestimationofinductionparameter(i.e.,RIS),withmuchsmallerlot‐

to‐lotvariations.

• PredictedDDI(AUCchange)estimatedusingRISdatageneratedwithinvitro

datafromHepatoCells correlatedverywell(R

>0.9)withclinicalclearance

dataofCYP3A4substrates.

• HepatoCells canbeusedasapotentialinvitroscreeningtoolforprediction

ofclinicalCYP3A4induction.

Abstract

Induction‐mediateddrug‐druginteractionsneedtobecarefullycharacterizedin

vitro fordrugcandidat es inordertodetermineand/orpredicttheirinduction

potentialinpatients.Currently,boththe��FDAandEMArecommendusing

primaryhumanhepatocytesasaninvitro testsystemforthispurpose.Several

modelsrangingfromsimpleto

complex,suchasbasicmodels(C

max

/EC

,relative

inductionscore,R

)andmechanisticmodels(nexteff ectmodelandPBPK

model),arebeingproposedtopredictclinicalCYP3A4inductionusinginvitro

datafromprimaryhumanhepatocytes.Inlightofthelargelot‐to‐lotvariation

inherentinprimaryhumanhepatocytes,CorningHepatoCells(derivedfrom

primaryhumanhepatocytes) wasevaluatedforits

applicabilityasan invitro

screeningtoolforpredictingclinicalCYP3A4inducers.Threelotsofcellswere

treatedwithagroupofknownclinicalstrong,moderate/weakinducers,andnon‐

inducersatmorethan8concentrationseach.Bothenzymaticactivity

(testosterone6β hydroxylaseactivity)andmRNAexpressionweremeasuredas

endpointsusingLC

‐MS/MSandRT‐PCR,respectively.EC

andE

max

were

estimatedfromconcentration‐dependentinductionresponsecurves.ARelative

InductionScore(RIS)model(aswellasother2basicmodels,C

max

/EC

andR

)

wasemployedusingEC

andE

max

dataforthemodeldrugs,andresultingRIS

datawereevaluatedfortheabilitytopredictpotentialclinicalCYP3A4

inducers/non‐inducers.Itwasfoundthatall3modelscorrelatedverywell

>0.9)withclinicalclearancedataofCYP3A4substrates.Theresultsalso

showedthatall3lotsofCorning®HepatoCellsbehavedsimilarlytoprimary

humanhepatocytesintermsofprediction,withonlyminorlot‐to‐lotvariations

forthe3lotsofcells.Inconclusion,CorningHepatoCellscanbeusedasa



potentialinvitro screeningtoolforpredictionofclinicalCYP3A4induction.

MaterialsandMethods

Materials.CryopreservedCorningHepat oCellsandCorninghepatocyte

maintenancemedium(CorningCat.No.354882)wereobtainedfromCorningLife

Sciences.AlltestcompoundswereobtainedfromSigma‐Aldrich.Compoundstock

solutionswerepreparedbydissolvingcompoundinDMSOandseriallydilutingthe

solutionsinDMSO.Finalworkingsolutionswerepreparedby

dilutingthestock

solutions1000Xinculturemedium.

Thawing,plating,andculturingofthecells.Onday1,HepatoCellswerethawed

ina37Cwaterbath.Afterremovingthecryo‐freezingmedia,thecellpelletwas

resuspendedinculturemediumcontaining10%FBS,thenthecellswereseededin

96‐wellCorningBioCoat™CollagenI‐coatedplateatadensityof80,000cellsper

well.CorningMatrigel®matrixwasaddedtocellmonolayerataconcentrationof

0.25mg/mL24hoursafterseeding.Afterbeingdosedwithtestcompoundsat

differentconcentrationsforaconsecutive3days,thecells

werewashedwith��

freshculturemediumandincubatedwith200µMtestosteronefor1hourto

measureenzymeactivity.Themetabolite6β‐hydroxytestosteronewasmeasured

byLC‐MS/MS.Afterenzymeassay,mRNAwasisolatedfromcellsusingaQiagen

RNeasy®96‐kit.CYP3A4mRNAexpressionlevelwasdeterminedusingApplied

Biosystems

two‐stepprotocolusinga7300Real‐timePCRsystem.

Dataanalysis.EC

andE

max

weredeterminedfromconcentration‐dependent

inductionresponsecurvesbyfittingthecurvestoasigmoidal3parameter

functionofSigmaPlot™(SystatSoftwareInc.).RISandR

werecalculatedusing

unboundC

max

fromtheliterature

1,3

andequationsdescribedbelow:RIS=(E

max

x

max,ub

)/(EC

+C

max,ub

),R

=1/(1+dxE

max

xC

max,ub

/(EC

+C

max,ub

))withd=1.

Acalibrationcurvewasgeneratedbyplottingtheinductionparamter(e.g.,RIS)

againstobservedmidazolamAUCchangeusingtheHill3parameterfunctionof

SigmaPlot.PredictionaccuracyusingHepatoCellswasevaluatedbyassesingthe

correlationbetweenpredictedAUCchangewithobservedAUCchange,andwas

compared

withthatofprimaryhumanhepatocytes.

References

1. USFDADraftGuidanceforIndustry:DrugInteractionStudies(2012).

2. EMAGuidanceonInvestigationofDrugInteraction(2013).

3. Fahmi OD,etal.DrugMetab Dispos 40:2204‐2211(2012).

4. Fahmi OD,etal.DrugMetab Dispos 38:1605‐1611(2010).

Figure1:ExamplesofConcentration‐dependentInductionResponseCurvesUsedto

DetermineE

max

andEC

DE F

Phenytoin,  M

1 10 100 1000

Fold (enz ym atic activity)

= 0.987

Phenytoin,  M

1 10 100 1000

Fold (enz ym atic activity)

= 0.996

Phenytoin,  M

1 10 100 1000

Fold (enzymatic activity)

= 0.985

Rifampicin, M

0.1 1 10 100

Fold (enzy mat ic activity )

Carbamazepine,



0.1 1 10 100

Fold (enzymatic activity)

Phenobarbitol,



1 10 100 1000

Fold (enzymatic activity)

Phenobarbitol,



1 10 100 1000

Fold (enzymatic a ctivity)

Terbinafine,



0.1 1 10 100

Fold (enzymatic activity)

Sulfinpyrazone,



0.1 1 10 100

Fold (enzymatic activity)

Table2.ThreeprototypelotsofCorningHepatoCellsgenerat edsimilarRISdata

asprimaryhumanhepatocytes;however,HepatoCellsshowedmuchsmaller

variancethanprimaryhumanhepatocytes(average%CVis18.2%forHepatoCells,

and53.2%forprimaryhumanhepatocytes).

Figure4.CalibrationcurvewasfittedtoaHill3parametermodel(SigmaPlot),

andthecorrespondingequationwasusedtocalculatepredictedAUCchange

usingin vitro inductionparameterRIS.ThepredictedAUCchangewasthen

plottedagainstobservedAUCchangetodeterminetheaccuracyofprediction.

CorningHepatoCellshaveshownsimilarpredictionaccuracyasprimaryhuman

hepatocytes(forbothcellmodels,predictedAUCchanges

formostofthe

compoundsfallwith20%ofprediction).

TestCompound

Concentration

Range,µM

LotPR2 LotPR3A LotPR3B

max

(Fold

)

(µM)

2

max

(Fold)

(µM)

2

max

(Fold)

(µM)

2

Rifampicin 0.049‐50 16.1 1.2 0.95 10.1 0.6 0.96 25.0 1.4 0.95

Phenytoin 0.49‐500 9.6 39.5 0.99 7.9 34.3 1.00 15.5 51.9 0.98

Carbamazepine 0.24‐250 5.0 18.0 0.96 3.9 14.2 0.93 3.5 23.4 0.90

Phenobarbitol 1.95‐2000 8.9 247.7 0.98 6.2 211.0 0.99 14.8 330.7 0.98

Terbinafine 0.1‐100 4.4 2.2 0.95 3.3 1.3 0.92 6.9 4.2 0.98

Sulfinpyrazone 0.2‐200 8.6 4.6 0.93 8.0 6.2 0.95 15.3 9.2 0.96

Probenecid 0.1‐100 31.3 40.7 0.94 40.5 52.8 0.93 36.5 43.8 0.96

Pioglitazone 0.1‐100 36.5 5.4 0.97 39.5 5.5 0.98 62.4 9.8 0.96

Dexamethasone 0.24‐250 34.1 43.5 0.97 27.7 28.0 0.98 42.1 42.8 0.95

Nifedipine 0.1‐100 9.9 4.1 0.96 10.3 3.9 0.98 11.0 4.9 0.98

Omeprazole 0.1‐100 19.4 3.8 0.97 14.3 3.8 0.97 21.4 5.1 0.98

Figure1.Concentration‐dependentCYP3A4inductionresponsecurveswereplottedusingfold

inductiondataofenzymaticactivityfromagroupofknowinducers(mRNAdatashowedsimilar

concentrationdependentresponsecurve).Examplesareshownherefor6modelcompounds

usingonelotofCorningHepatoCells.AllcurvesarefittedusingSigmaPlot

sigmoidal3parameter

function,withR

allgreaterthan0.9.

Figure2:ConsistentPerformancebetween3DifferentLotsofCorningHepatoCells

Figure2.Threedifferentlotsof��CorningHepatoCellsshowedsimilar concentration‐dependent

inductionresponsetomodelcompounds,suggestingconsistentperformanceofHepatoCells.

(A)LotPR2;(B)LotPR3A;(C) LotPR3B.

Table1:E

max

andEC

DeterminationUsingConcentration‐dependentInduction

ResponseCurves

Table1.EC

andE

max

weredeterminedasdescribedinMaterialsandMethods.All3lotsof

CorningHepatoCellsshowedgoodfittingwithR

>0.9forallcompoundstested.

Figure3:CalibrationCurvesUsing3DifferentInVitro Induction

Parameters

Figure3.Calibrationcurvesusing

3differentinvitroinduction 

parametersand%decreasein

observedmidazolamAUCall

showedgoodcorrelationwith

>0.9.

3A.RIScalibration

3B.R

calibration

3C.C

max

/EC

calibration

Compounds

Observed

AUCchange

RISfrom

CorningHepatoCells

RISfrom

PrimaryHumanHepatocyte

AverageRIS %CV

LotPR2 LotPR3A LotPR3B Lot295 Lot312 Lot318 HepatoCells PHH HepatoCells PHH

Dexamethasone 19 0.0042 0.0053 0.0053 0.001 0.001 0.0004 0.0050 0.0008 12.7% 43.3%

Terbinafine* 25 0.047 0.059 0.040 0.027 0.024 0.007 0.0485 0.0193 20.1% 55.8%

Nifedipine* 4 0.048 0.052 0.045 0.019 0.007 0.011 0.0484 0.0123 7.4% 49.5%

Pleconaril* 35 0.087 0.095 0.101 0.025 0.029 0.011 0.0944 0.0217 7.5% 43.6%

Omeprazole ‐25 0.189 0.139 0.154 0.008 0.019 0.019 0.161 0.0153 16.0% 41.4%

Pioglitazone* 26 0.190 0.199 0.178 0.012 0.011 0.025 0.189 0.0160 5.5% 48.8%

Troglitazone* 65 0.513 0.355 0.650 0.12 0.2 0.03 0.506 0.1167 29.2% 72.9%

Phenobarbitol 61 0.902 0.729 1.158 0.88 2.4 1.6 0.930 1.63 23.2% 46.7%

Carbamazepine* 94 1.25 1.074 0.723 1.1 1.1 9.3 1.02 3.83 26.5% 123.5%

Phenytoin* 94 1.50 1.381 1.908 1 1.3 1.8 1.60 1.37 17.3% 29.6%

Rifampicin* 97 10.93 8.175 16.201 7 11 6.4 11.77 8.13 34.7% 30.7%

Table2:ComparisonofInductionParameter(RIS)between

CorningHepatoCellsandPrimaryHumanHepatocytes

Figure4:CorrelationAnalysisofObservedMidazolamVictimDrug

AUCChange(%)vs.PredictedAUCChange(%)fromRIS

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