An Integrated In Vitro Organ Platform to Evaluate Cholestasis

By James McKim, Ph.D., DABT

Disclosure Statement

• TheFounderandPresidentofIONTOX,LLC• ConsultantforBioIVT-Qualyst

Presentation Outline

• Backgroundcholestasis• Bileacidhomeostasis• AOPforcholestatictoxicity• OptimalinvitroIntestineandlivermodel• EvaluatingcholestaticprocessesinanIntegratedorganmodel

What is Cholestatic Liver Injury?

• Cholestatic and mixed cholestatic/hepatocellular injury representing 50% of all drug induced liver toxicities.

• Dysfunction in bile acid synthesis, transport, or signaling pathways that control bile acid homeostasis.

• Acute or chronic• Mixed hepatocellular/cholestatic injury or biliary• Disrupted bile flow à pure intrahepatic cholestasis• Obstructive cholangiopathy with injury in bile duct epithelium

Understanding Bile Acid Homeostasis

Hepatic Signaling Mechanisms That Control Bile Acid Concentrations

CYP7A1

CholesterolBileAcidSynthesis

MRP3/4

OATPs

NTCP

BSEPNTCP CA

CDCA

Suppression

Induction

OSTα/β

Ileum

Hepatocyte

OSTα/β

FXR

FXR

FGF19

I-BABP

Na+

Na+

PGP

Na+

FGFR4

BileAcids

ConjugatedBileAcids

IntestinalBileAcids

ASBT

• Bile acid disposition is tightly regulated by the Farnesoid X Receptor (FXR)

• FXR activitation leads to:– Increased FGF19– Suppression of CYP7A1– Induction of BSEP, MDR3,

OSTα/β• Potential for a drug to impact

multiple pathways

Bile Acid Pathway Regulation Significance(normal conditions)

Uptake – NTCP/OATPs No FXR Regulation Extensive hepatic uptake

Canalicular Efflux - BSEP FXR Induced 75 % of total clearance

Basolateral Efflux – MRP3/4 No FXR Regulation 25 % of total clearance

Basolateral Efflux - OSTα/β FXR Induced Not significant

Synthesis – CYP7A1 FXR Suppressed < 5% of bile acid pool daily

Jackson et al. (2016) Basolateral efflux transporters: A potentially important pathway for the prevention of cholestatic hepatoxicity. Appl In Vitro Toxicol, 2 207-216.

The Adaptive Response: Synergistic Effects

• TheAdaptiveResponsecanbeinitiatedby:

• BSEPinhibition• Increasedintracellularconcentrationsofbileacids

• IncreasedintracellularconcentrationsplusinhibitionofBSEPleadstoasynergisticeffectontheadaptiveresponse

µM)

CsA (1

µM)

CsA (1

0 µM)

CsA (2

0 µM)

CDCA (30

(30 µM

)

CDCA

µM) +

CsA (1

(30 µM

)

CDCA

µM) +

CsA (1

0

(30 µM

)

CDCA

µM) +

CsA (2

0

0255075

100125150175200

OST

b m

RNA

Cont

ent

(Rel

ativ

e Fo

ld C

hang

e)

An Adverse Outcome Pathway for Cholestatic Liver Toxicity

Vinken M.(2013)Toxicology312158-165

Cholestasisisacomplexprocess:multiplemechanismsareinvolvedintheregulationofbileacidconcentrationsinthehepatocyte

CurrentapproacheshavefocusedonBSEPinhibitionasapredictorforcholestasisCholestatic

Hepatotoxicity

BSEPInhibition

↑ OSTα/βExpression

↓ CYP7A1 ExpressionFXR

Activation

Bile AcidAccumulation

SHPActivation

An In Vitro Liver Model for Cholestasis

• Human or rat primary hepatocytes in sandwich culture• Bile pockets• Uptake and efflux transporters present and functioning• FXR signaling pathways intact

• Other in vitro liver models may also work• Spheroids• HepaRG

Blood

Organicanions/cation

MRP3

ABCG2(BCRP)

ABCG5ABCG8

Cholesterol

MDR3

MRP2

BSEP

MDR1

Na+Bilesalts

OATPsOATP-1B1OATP-1B3OATP-2B1

NTCP

OCT1

OAT2 ConjugatesGSH,glucuronide

LipidCation

phospholipids

glucuronideMRP4 MRP5 MRP6

SulfoconjugatesBilesalts cGMP

Organicanions

CNT1/2

Bile

Sulfoconjugates

10

SLC47A1MATE1

Movement of Bile Acids Depends on Transporters

Characterization of EpiIntestinal™ Model

• Presence of ASBT

20

21

22

23

24

25

26

27

Control Cyclo Trog CycloCDCA

TrogCDCA

CtValue

TreatmentGroups

qPCRIndicatingBasalExpressionofASBT

GAPDH

ASBT

0

1

2

3

4

Cyclo Trog CycloCDCA TrogCDCA

FoldIn

ductionvsCon

trol

TreatmentGroups

GeneExpressioninIntestine

FGF19CYP7A1ASBT

Inhibition of ASBT Reduces Bile Acid Uptake

0

25

50

75

100

125

30µMgCDCA

%GCD

CAUptake

EffectofFluvonGCDCAUptakeat4hr

-Fluv

+Fluv

Fluvastatin 500 µM in 50 µL

Experimental Hypothesis

• Integratedorganmodelscannotonlyrecapitulatesingleorganresponses,butcanaddimportantinformationondrugkinetics,bioavailability,andinter-organmetabolismwhichwouldimprovepredictionsofsystemiceffectsinvivo.

Drugs Known to Disrupt Bile Acid Homeostasis

• Cyclosporin A • Immunosuppressant drug widely used in clinic• Liver toxicity due to bile acid toxicity is rare• potent BSEP inhibitor

• IC50 of 0.5 µM

• Troglitazone• First drug for treatment of type II diabetes• Associated with liver toxicity• BSEP and FXR inhibitor (parent and metabolites) • Can cause C-DILI in clinic

Physical-Chemical Properties Can Change Pharmacokinetics In Vitro and In Vivo

MW = 1202 Da, cLogP = 4.0, PSA = 279 A2

Bioavailability variable = 10-89%

PSA > 140 A2 low membrane permeability

MW = 441.54 Da, cLogP = 4.6, PSA = 110.6 A2

Bioavailability 40-50%

Experimental Design• Hµ-DMOP™ IntestineàLiver

• EpiIntestinal™ (MatTek Corp)• Human Hepatocytes Sandwich Culture

• Apply single dose test drugs• CyclosporineA(30µM)• Troglitazone(300µM)

• 6 hr after adding drug to intestine• AddCDCA(30µM)toliver

• Collect samples• Intestinebasolateral• Livermedia• Simulatedblood(perfusate)

• At 24 hr collect tissues• Cytotoxicity• Geneexpression(FGF19,OST,CYP7A1)

Details of Integrated Organ Plate Setup

Multiple Biological Compartments Can be Sampled

EpiIntestinal™ Air-Liquid Interface

Time-Dependent Uptake of Test Drugs

PlateTroglitazonePermeability=58%

PlateCyclosporinePermeability=90%300 µM Stock6.6 µg in 50 µL

30 µM Stock1.8 µg in 50 µL

3.4 µM

1 µM

Drug Levels in Liver Were at or Below the Lower Limit of Detection

0 1 0 2 0 3 00

1 0 0

2 0 0

3 0 0

4 0 0

L iv e r M e d ia

S a m p le T im e (H o u rs )

Co

nc

en

tra

tio

n i

n n

g/m

L T rog lita zone

C y c lo

DevelopmentofhighlysensitiveanalyticalmethodsKeytokineticdata83nM

680nM

Valuesareestimatesbasedonextrapolationsoutsidethecalibrationcurve

The Adaptive Response of Liver Was Activated in Presence of Drug plus Bile Acid

0

5

10

15

20

25

30

35

40

CDCA Cyclo Trog Cyclo+CDCA Trog+CDCA

FoldIn

ductionvsCon

trol

TreatmentGroups

GeneExpressioninLiverFGF19

CYP7A1

OSTα

OSTß

Bile acid alone had little effectDrug alone had little effect

Drug plus BA Increase in FGF19Increase in OSTSuppression of CYP7A1

Adaptive response reduced by Trog

Liver Cytotoxicity Only Observed When Adaptive Response is Inhibited

300 µM + 30 µM

HepatocellularToxicityonlyobservedwhenTheliveradaptiveresponseisblocked

BSEPinhibitionandFXRantagonism

Cholestatic DILI: Hepatocellular Injury

Need to integrate multiple mechanisms

Initiating Insult• BSEP Inhibition

Secondary Insult• FXR Antagonism

and/or• Basolateral Efflux

Inhibition HepatocyteHepatocyte

Nucleus

HepatocyteHepatocyte

Nucleus

FXRAntagonism

FXRActivation

MRP3/4

NTCPNa+

OSTα/β

Compounds can Increase the Intracellular Concentration of Bile Acids through:• BSEP Inhibition plus• Basolateral Efflux Inhibition (MRP3/4 and/or OST𝛼/𝛽) and/or• FXR Antagonism

FXRActivation

Jackson et al. (2016) Basolateral efflux transporters: A potentially important pathway for the prevention of cholestatic hepatoxicity. Appl In Vitro Toxicol, 2 207-216.

Target Organ Dose is Much Lower Than in Single Organ Models

• CyclosporineA• Therapeuticbloodconcentration• Toxicbloodconcentration

• Troglitazone• Therapeuticbloodconcentration=• Toxicbloodconcentration=

What New Information Can we Obtain from Integrated Organ Systems?

• Permeability(absorption)• Estamate Bioavailability• Firstpassmetabolism• Enterohepaticcirculation• Targetorgandoseismoreaccurate• Lowertargetorgandosesmorecloselymimicstheinvivosituation

Developing In Vitro Physiologically Based Pharmacokinetic Models

(Currently,nometabolismwasincludedinthelungorliver)Intestine

Conclusions• Wellcharacterizedintegratedorgansystemscanprovidevaluablekineticandexposuredata.

• ThetestsystemusedhasdemonstratedthatKeyhepaticintracellularsignalingpathwayscontrollingbileacidhomeostasisarefunctioning.

• Bileacidtransporter(ASBT)isfunctioninginMatTekEpiIntestinal.• BileacidtoxicitywasobservedonlywithTroglitazone(C-DILIpositive)• Thedatarevealthattargetorgandosesaremuchlowerthanappliedaconditionthatmoreaccuratelymimicstheinvivosituation.

• PBPKmodelsarepossiblewiththistypeofdata.

Acknowledgements• IONTOX

• Nicholas Hibbard• Bonnie Corpus• Jamin Willoughby

• BioIVT-Qualyst• Jonathan Jackson• Chris Black• Ken Brouwer

Summary and Closing Remarks

Thankyoutoallofthespeakers

An Adverse Outcome Pathway for Cholestatic Liver Toxicity

Vinken M.(2013)Toxicology312158-165

Cholestasisisacomplexprocess:multiplemechanismsareinvolvedintheregulationofbileacidconcentrationsinthehepatocyte

CurrentapproacheshavefocusedonBSEPinhibitionasapredictorforcholestasisCholestatic

Hepatotoxicity

BSEPInhibition

↑ OSTα/βExpression

↓ CYP7A1 ExpressionFXR

Activation

Bile AcidAccumulation

SHPActivation