ModelingofAc-veTransportandMetabolismforinvitroSuspendedandSandwichHepatocyteAssaysU-lizing

MembranePlus™JamesMullin

PrincipalScien1stSimula1onsPlus,Inc.

Overview•  MembranePlus™–aso:warepla<ormforsimula1onofdrug

transportincellassays.

•  DevelopmentofHepatocyteTransportModel–  Sandwichandplatedhepatocytes.–  Suspendedhepatocytes.

•  ModelCaseStudies–  SodiumTaurocholatetransport–  Sta1ns(uptake+metabolism)–  IVIVEwithcombina1onofMembranePlusandGastroPlus

MembranePlus

•  Instant permeability output from molecular structure or experimental data. •  Unbound Intracellular concentrations (Membrane, cytosol, lysosome, etc.) •  In vitro Km and Vmax for enzymes and transporters •  Parameter sensitivity analysis (on logP, shaking rate, pH etc.) •  Sandwich and suspended hepatocyte model (version 2 imminent) •  DDI predictions (Coming Soon in Version 3)

Data Analysis

Assay Prediction •  Permeability •  Intracellular concentrations (Membrane, cytosol, lysosome) •  Virtual Trials (coming soon)

MembranePlus–SoCwareOverview

Applicable only to cell-based assays

ADMET PredictorTM Values

ApicalSolu/on

ApicalMembrane

Cytosol

BasolMembrane

BasolateralSolu-on

FilterSupport

MembraneEntry/Exit

MembraneEntry/Exit

ParacellularDiffusion

Transporters

Metabolism

Diffusion

Diffusion

FilterpermeabilityDU

Proteinbinding

DBDU

ProteinbindingDB

Loss(plas-cbinding,nonspecificloss)

Loss(plas-cbinding,nonspecificloss)

Lysosomaltrapping

Evapora-on

ApicalUWL

BasolateralUWL

Transporters

Overview of Transwell Mechanisms

ProteinbindingDBDU

MembranePlus–MembraneEntry/ExitRateStructureBasedModel

•  Observedvs.Predictedon44trainingdatasets( ) ( ) HBAoCHBDHBDHCRNGMClogPCInterceptVlog i ×+−+×+×+= 43_21

Membraneentryandexitratesforanionsandca1onsaredeterminedbasedonlogDvs.pHprofile

Allexperimental1mepointsto2hr

lysosome

Lysosome membrane

Vi and Vo correspond to the membrane/water entry and exit rate

pH = 4-5

pH = 7.4

MembranePlus–LysosomalTrappingModel

Cell

KazmiF.,DrugMetab.Disp.41(3):897(2013)

LipophilicAminesLogP>1pKa>6.5

EnzymesandTransporters-Equa-ons•  Kine1csofcarrier-mediatedtransportandmetabolismiscalculated

usingMichaelis-Mentenkine1cs:

•  Vmaxunits:µM/s(µmol/L/s)•  Kmunits:µM(µmol/L)•  Generalunitsconverterallowsconver1ngtheseintodifferenttypesof

units•  Transportertypes:InfluxandEfflux•  Transporterloca1ons:ApicalandBasolateral

∑ ⎟⎟⎠

⎞⎜⎜⎝

⎛

+

×=

i uim

ui

metab cKcVintracell

intracellmaxυ

∑ ⎟⎟⎠

⎞⎜⎜⎝

⎛

+

×=

ibufferu

im

bufferu

i

influx cKcVmaxυ

∑ ⎟⎟⎠

⎞⎜⎜⎝

⎛

+

×=

i uim

ui

efflux cKcVintracell

intracellmaxυ

SandwichandSuspendedHepatocyteModel

•  Newtoversion2.0(expectedMay/June2017)

•  Modelnon-lineardruguptakeandmetabolismin:–  SandwichHepatocytes–  PlatedHepatocytes–  SuspendedHepatocytes

•  Inclusionofintracellularproteinbinding(freefrac1on)

BasolateralSolu/on

BasolMembrane

Cytosol

BasolMembrane

MembraneEntry/Exit

MembraneEntry/Exit

Metabolism

Diffusion

Proteinbinding

DUDBLoss(plas-cbinding,nonspecificloss)

Lysosomaltrapping

Evapora-on

BasolateralUWL

Transporters

Overview of Sandwich Hepatocyte Mechanisms

WellPlateBo=om

ApicalMem.

ProteinbindingDBDU

•  Collagenisassumedtonotaffecttransportprocesses•  Modelisalsoapplicableforplatedhepatocyteswhenbilevolumeisnotconsidered

BilePocketBile

Pocket

BileEntry/Exit

BileTransporters

Incuba/onMedia

Membrane

Cytosol

Metabolism

DiffusionLayerLoss

(plas-cbinding,nonspecificloss)

LysosomalTrapping

Evapora-on

Overview of Suspended Hepatocyte Mechanisms

MembraneEntry/Exit

TransportersNoCellMembraneDifferen-a-on

Proteinbinding

DBDU

ProteinbindingDBDU

PhysiologicalSeUngs

•  Platedhepatocytescanbemodeledbyseingbilevolumeandareapercentagesto0.•  Bilepocketscontractandopen/releasedrugtothemediathusafirstorderbileefflux

rateisavailable

CaseStudy1:SodiumTaurocholateUptakeIntoBile

SodiumTaurocholate

•  ExperimentaldatafromQualystGuo,ISSX2014

•  Allproper1esexceptKmvaluesfromADMETPredictor

Property ValueS+IS(mg/mL)@pH2.78 0.0184S+pKa(Acid) 1.1S+SF 906S+pH 2.78S+logP 0.82S+Peff(x10-4cm/s) 0.36DiffCoef(x10-5cm/s) 0.53S+fumic 0.911

AP 7.2 = ADMET Predictor v. 7.2 S+ stands for properties predicted with Simulations Plus models S+Sw = solubility in pure water

ModelofSodiumTaurocholateBileCanalicularUptakeinSandwich

Hepatocytes

•  Guo,2014ISSXPoster

OSTalpha/beta

Na+TaurocholateModelAssump-ons

•  Assump1ons–  NoproteinbindingforNaTaurocholate–  Nos1rring–  Completemonolayerofcells(100%viability)–  ADMETPredictorvaluesforproper1esandtransportmodel

parameterscalculatedusingcalibra1on

•  LiteraturevaluesforKmwereusedasastar1ngpointforbuildingthemodel.

•  Kmvaluesaresimilaracrossspecies–  Swi:-Mol-Pharm-2010-7(2)-491–500

CellAssayInputsFeedSolu1onConc. 1,2.5 µM

BSA 4 %Wellsize 24 wellVolume 0.3 mLCellVol. 6.46 pL

CellLayerThickness 18.6 micronCellDen 0.4 Mcell/well

Transporter Km(µM) Cells LiteratureSourceOSTalpha/beta 25.8 Human Swi:-Mol-Pharm-2010-7(2)-491–500OverallUptake 19 Rat Schwarz-Eur-J-Biochem-1975-55-617-623

NTCP 6 Human J-Exp-Biol-2001-204-1673-1686BSEP 5 Rat J-Exp-Biol-2001-204-1673-1686

TCAResults1µMDonorExperimentalKmValues

Transporter Type Km(Exp)(µM) Vmax(µmol/s/L)BSEP Efflux 5 6.83E-03

OSTalpha/beta Efflux 25.8 9.63E-02NTCP Influx 6 3.88E-02

•  ExperimentalKmvaluesu1lizedfromliteratureandVmaxvaluesfittodata.

EffluxPhase

•  Wehaveobtainedsuperiormodelresultswithnewdatasetsbuttheycannotbeshownun1lpublished.

CaseStudy2:Quan-fica-onofInfluxTransportvs.Metabolism

Sta-nCompounds

SuspendedHepatocytes

Quan-fytheRela-veImportanceofInfluxTransport(OATP1B1)vs.Metabolism

•  Mediaandwholecellconcentra1ondataforatorvasta1n,cerivasta1n,andindomethacin.

•  Usedasimplecompartmentalmodeltoextractclearancevalues

Paine,DMD(2008)36:1365–1374

Atorvasta-n

•  Solubility–  S+Sol=0.18mg/mL@pH4.5–  Sol(waterexp)=0.11mg/mL–  Sol(pH1.2)=0.01mg/mL–  Sol(pH7.4)=0.70mg/mL

•  S+SF=285–  SF(Exp)=70

•  S+LogP4.28–  LogPexp=4.18(biobyte)

•  S+pKa(Acid)=4.71,11.05–  pKaExp=4.51(Schonherr_Eur-J-Pharm-Biopharm_2015-155-170)

•  OATP1B1Substrate(Km=0.77µM)Vildhede_DrugMetab_Disp_2014_42.7_1210-1218

•  Caco-2Papp(MembranePlus)=4.80E-05cm/s•  S+Metabolism(2C9)

–  Experimental(3A4)(Km=44.9µM)Park_Xenobio'ca_2008_38.9_1240-1251

•  Solubility–  S+Sol=0.2mg/mL@pH4.92

•  S+SF=280.7•  S+LogP3.04

–  LogDexp=3.26@pH5(biobyte)

•  S+pKa(Acid/Base)=4.39,5.39•  OATP1B1/1A1?Substrate(Km=5.86µM)

Xenobio-Metabol-Dispos-2000-15(3)-219-225

•  Caco-2Papp(MembranePlus)=7.5E-08cm/s

•  S+Metabolism(3A4)(Km=11.8µM)

Cerivasta-n

AP 7.2 = ADMET Predictor v. 7.2 S+ stands for properties predicted with Simulations Plus models S+Sw = solubility in pure water

ModelResults

Media

Cells

Media

Cells

Media

Cells

Media

Cells

Atorvasta1n

Cerivasta1n

Lipidbilayerpermeabilitymodelwasfitduetopoorpredic1onfromstructure

ModelResults,Cont.

Cerivasta-nResults

Vmax3A4(umol/s/L) 3.74E-02

VmaxOATP1B1(umol/s/L) 295.28

Clmet(uL/min/Mcell) 20(17Lit.)

Clupt(uL/min/Mcell) 704(413Lit.)

Atorvasta-nResults

Vmax3A4(umol/s/L) 0.119

VmaxOATP1B1(umol/s/L) 31.9

Clmet(uL/min/Mcell) 63(68Lit.)

Clupt(uL/min/Mcell) 471(375Lit.)

Paine,DMD(2008)36:1365–1374

Metabolic

Influx

Metabolic

Influx

Metabolism

Rate(uL/min/106cells)

Influ

xRa

te(u

L/min/106cells)

Metabolism

Rate(uL/min/106cells)

Influ

xRa

te(u

L/min/106cells)

ModelComparison•  Themechanis1cmodelinMembranePlusachievedsimilarresultasthe

simplercompartmentalmodelwithfewerfitedparameters

*Paine,DMD(2008)36:1365–1374

Compartmenalmodel*

MembranePlus

Intracellularvolume Fited Systemparameter

Cellmembranevolume Fited Systemparameter

Membrane/waterpar11oning(kmem)

Fited Predictedfromcompoundproper1es

Ac1veuptake Fited Fited

Passivediffusion Fited Predictedfromcompoundproper1es(atorvasta1nonly)

Metabolism Fited Fited

CaseStudy3:InVitrotoInVivoExtrapola-onUsingMembranePlusandGastroPlus

PropafenoneMetabolismSuspendedHepatocytes

•  ReportsindicateitisaCYP2D6substrateandhassaturabledosedependentkine1cs.

•  HaveIVdatatocomparepredictedKmandVmaxto.

Property ValueS+Sw(mg/mL)@pH10.28 0.49S+pKa(Base) 9.47S+SF 114S+logP 3.03S+Peff(x10-4cm/s) 1.76DiffCoef(x10-5cm/s) 0.65

AP 7.2 = ADMET Predictor v. 7.2 S+ stands for properties predicted with Simulations Plus models S+Sw = solubility in pure water

PropafenoneHumanHepatocyteData

•  100rpm•  2mLincuba1on•  1Mcell/mL

Komura, Drug metabolism and disposition 33.6 (2005): 726-732.

FittoInVitroData

•  Km=0.0146µM•  Vmax=9.27E-02

µmol/s/Lcytosol

•  VmaxConvertsto:•  2.17E-02nmol/min/

Mcell

Kmcalculatedwaslowerthanthepaperwhichisindica1veofthetrueintracellularKmbasedonunboundintracellularconcentra1on

InvivoKm/VmaxConversionGastroPlus

Predic-onof70mgIVBolusDose•  PBPKmodelwithdefaultparameters(Lukacovamethod)in

GastroPluswiththeinvitroextrapolatedCYP2D6clearance

Data:Hollmann,CardiacArrhythmias,Springer125-132

Conclusion

•  MembranePlushasmul1plecapabili1estoassess–  Passiveandac1vedrugtransportinvariousassays–  Interplaybetweenmetabolismandpassive/ac1vedrugtransport:

•  SandwichHepatocytes•  PlatedHepatocytes•  SuspendedHepatocytes

–  Drugdisposi1oninBile•  SandwichHepatocytes

–  Drugconcentra1onwithincellularstructures•  Lysosomes,lipidbilayers

–  ExtractrelevantparametersforIVIVEinGastroPlus

–  Addi1onalvalida1onwithdatasetsandso:warereleaseimminent