an integrated in vitro organ platform to evaluate cholestasis€¦ · an adverse outcome pathway...
TRANSCRIPT
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An Integrated In Vitro Organ Platform to Evaluate Cholestasis
By James McKim, Ph.D., DABT
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Disclosure Statement
• TheFounderandPresidentofIONTOX,LLC• ConsultantforBioIVT-Qualyst
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Presentation Outline
• Backgroundcholestasis• Bileacidhomeostasis• AOPforcholestatictoxicity• OptimalinvitroIntestineandlivermodel• EvaluatingcholestaticprocessesinanIntegratedorganmodel
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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
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Understanding Bile Acid Homeostasis
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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.
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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)
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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
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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
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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
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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
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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
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Experimental Hypothesis
• Integratedorganmodelscannotonlyrecapitulatesingleorganresponses,butcanaddimportantinformationondrugkinetics,bioavailability,andinter-organmetabolismwhichwouldimprovepredictionsofsystemiceffectsinvivo.
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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
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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%
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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)
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Details of Integrated Organ Plate Setup
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Multiple Biological Compartments Can be Sampled
EpiIntestinal™ Air-Liquid Interface
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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
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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
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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
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Liver Cytotoxicity Only Observed When Adaptive Response is Inhibited
300 µM + 30 µM
HepatocellularToxicityonlyobservedwhenTheliveradaptiveresponseisblocked
BSEPinhibitionandFXRantagonism
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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.
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Target Organ Dose is Much Lower Than in Single Organ Models
• CyclosporineA• Therapeuticbloodconcentration• Toxicbloodconcentration
• Troglitazone• Therapeuticbloodconcentration=• Toxicbloodconcentration=
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What New Information Can we Obtain from Integrated Organ Systems?
• Permeability(absorption)• Estamate Bioavailability• Firstpassmetabolism• Enterohepaticcirculation• Targetorgandoseismoreaccurate• Lowertargetorgandosesmorecloselymimicstheinvivosituation
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Developing In Vitro Physiologically Based Pharmacokinetic Models
(Currently,nometabolismwasincludedinthelungorliver)Intestine
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Conclusions• Wellcharacterizedintegratedorgansystemscanprovidevaluablekineticandexposuredata.
• ThetestsystemusedhasdemonstratedthatKeyhepaticintracellularsignalingpathwayscontrollingbileacidhomeostasisarefunctioning.
• Bileacidtransporter(ASBT)isfunctioninginMatTekEpiIntestinal.• BileacidtoxicitywasobservedonlywithTroglitazone(C-DILIpositive)• Thedatarevealthattargetorgandosesaremuchlowerthanappliedaconditionthatmoreaccuratelymimicstheinvivosituation.
• PBPKmodelsarepossiblewiththistypeofdata.
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Acknowledgements• IONTOX
• Nicholas Hibbard• Bonnie Corpus• Jamin Willoughby
• BioIVT-Qualyst• Jonathan Jackson• Chris Black• Ken Brouwer
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Summary and Closing Remarks
Thankyoutoallofthespeakers
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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