Novel in vitro and in silico models for the prediction of chemical toxicity

Dominic Williams

The University of LiverpoolThe University of Liverpool

dom@liv.ac.uk

Adverse Drug Reactions

patient morbidity & mortality

4th – 6th leading cause of death in USA1

precludes otherwise effective drug therapy

drug withdrawal (4% 1974 - 1994)2drug withdrawal (4%, 1974 - 1994)

€2B/p.a. in vivo toxicity testing 3

Drug attrition

Liver, skin, blood, cardiovascular

1Lazarou et al., 19982Jefferys et al., 19943Andersen et al., 2009

Lessons for the future

Inform on mechanism and pathogenesis

Inform Medicinal Chemists

Inform Clinicians

Inform Regulators

I f th P bli h t i f iblInform the Public – what is feasible

Develop better biomarkers

Improve in vitro models

Classification of Adverse Drug Reactions

ON TARGET ADRs • Predictable from the known primary or secondary

pharmacology of the drug

• Exaggeration of the pharmacological effect of the druggg p g g

• Clear dose-dependent relationship

OFF TARGET ADRs • Not predictable from a knowledge of the basic pharmacology

of the drug

• Exhibit marked inter-individual susceptibility (idiosyncratic)Exhibit marked inter individual susceptibility (idiosyncratic)

• Complex dose dependence

ADR += f1Chemistry

of drug f2Biology of individual

Drug-Induced Liver Injury

Leading cause of acute liver failure1Leading cause of acute liver failure Drugs cause 58% of all ALF

High morbidity & mortality2g y y 20% survival without transplant

Main reason for late stage termination or withdrawal2

76 drugs found to be significant cause of hepatotoxicity across 3 DILI Registries (US, Sweden, Spain)3

C f li i j 5 / iCause of liver injury ≥ 5 cases/registry

1 Lee AASLD, 2009; 2 Verma & Kaplowitz 2009; 3 Suzuki et al., 2010

Drugs withdrawn from major markets due to hepatotoxicity

Drug: Therapeutic area:- Alpidem* AnxiolyticAspirin (children) NSAID -Aspirin (children) NSAID - Bendazac* NSAIDBenoxaprofen NSAID -- Bromfenac* NSAID

*O NH2

Chlormezanone* Anxiolytic -- Dilavelol* Anti-hypertensiveEbrotidine* H2 receptor antagonist -- Fipexide* Stimulant

HO

NH

OH

Fipexide StimulantNefazodone* Anti-depressant -- Nimesulide NSAIDNomifensine Anti-depressant -

h i i i- Oxyphenisatin Laxative Pemoline* ADHD -- Perhexilene Anti-anginalTemafloxacin* Anti-infective -Temafloxacin Anti infective - Tolcapone* Anti-parkinson’sTolrestat* Anti-diabetic -- Troglitazone* Anti-diabetic T fl i * A ibi iTrovafloxacin* Antibiotic -Ximelagatran- Anti-coagulant- Zimeldine Anti-depressant

* Need et al., Nat Genetics 2005

Drug Metabolism: Pharmacology

DRUGCellular

accumulationRESPONSE

accu u a o

Concentration in

Plasma

Concentration in

ORGANS CELLS ORGANELLESPhase I/IIDrug

PlasmaORGANS – CELLS - ORGANELLES

Stablemetabolites Dispositionmetabolites

Absorption

Metabolism

Excretioni

Drug plasma levelPharmacological

Drug & MetabolitesPharmacological &

Excretion exposureg

Toxicological exposure

Pathogenic Mechanisms of DILI

Acute fatty liver with lactic acidosisAcute hepatic necrosisAcute fatty liver with lactic acidosisAcute hepatic necrosis

DRUG

pAcute liver failureAcute viral hepatitis-like liver injuryAutoimmune-like hepatitis

pAcute liver failureAcute viral hepatitis-like liver injuryAutoimmune-like hepatitis

DRUG&

Bland cholestasisCholestatic hepatitisCirrhosis

Bland cholestasisCholestatic hepatitisCirrhosis

METABOLITESImmuno-allergic hepatitisNodular regenerationNon-alcoholic fatty liver

Immuno-allergic hepatitisNodular regenerationNon-alcoholic fatty liverSinusoidal obstruction syndromeVanishing bile duct syndromeSinusoidal obstruction syndromeVanishing bile duct syndrome

DILI can present with multiple:DILI can present with multiple:

varying phenotypes

clinical & histopathological features

A i l ‘h i i i ’ i lik l

Tujios & Fontana, Nat Rev Gastroenterol Hepatol; 2011

A single ‘hepatotoxicity signature’ is unlikely

Well characterised patients provide mechanistic clues

Pathogenic Mechanisms of DILISM

EC

CLEARANCE

DRUGMETABOLITE

REACTIVE METABOLITE

mitochondrialysosome

Organelle impairmentphospholipidosismicrovesicular steatosishepatocyte apoptosis

organelle impairmentbioaccumulation

REACTIVE METABOLITEinhibition of biliary efflux

CLEARANCE

hepatocyte apoptosishepatocyte necrosis

Intrahepatichypersensitivityi ll i t i itIntrahepatic

cholestasisimmunoallergic toxicity

Drug Metabolism: Toxicology

DRUG Cellularaccumulation

• Lysosome• Mitochondria• Mitochondria• BSEP

Phase I/II/III bioactivation

Chemicallyreactive

metabolites

Stablemetabolites

• Inhibition of protein function• apoptosis/necrosis

• Recognition by immune systeml t/ l tmetabolites

bioinactivation

• covalent/non-covalent

Excretion

Drug Metabolism: Toxicology

DRUG Cellularaccumulation

• Lysosome• Mitochondria• Mitochondria• BSEP

Phase I/II/III bioactivation

Chemicallyreactive

metabolites

InhibitionOf

P450s

• Heme complex formation• Protein alkylationmetabolitesP450s

bioinactivation

Excretion

Ideal working relationship betweenchemistry & drug metabolismy g

detoxicationbi ti tibioactivationcell defenceapoptosisnecrosis

innate immunityadaptive immunity

Park et al., Nat. Rev. Drug Disc. 2011

Improve translation = Improved Drug Safety

Improved Translation In vitro mechanistic evaluation of

Chemistry of the drug

hazard/risk

g

I i

f1Chemistry

of drug

Biology of the system

Improving drug safety

science + f Biology of

Variability

+ f3 model system

yof the

patient + f2Biology of individual

Improved Translation

Requirement for novel, translational, in vitro models for hepatotoxicity

Hepatic drug toxicity is a big problem for pharmaceutical industry:the physiological gap between incubations and liverthe lack of physiological integration for amplification/adaptationinability to assess how minor chemical stress leads to major toxicity in some people lack of consideration of systemic effects

However, systemic disposition and toxicity is an issue across the whole chemical industryBiocides, pesticides, food additives, cosmetic ingredients, consumer products etc.

US National Research Council: ‘Toxicity testing in the 21st Century: A vision & a strategy’Use human cells to predict human toxicityReduce animal useReduce animal use

Require novel in vitro models, based on human cells, to quantitatively assess chemical hazardhazard

Improved in vitro to in vivo extrapolation in chemical safety risk assessment for systemic toxicity

Interdisciplinary collaboration between:

Engineer Bioanalysis

between:

Mathematical modellersChemical/tissue engineers Engineer BioanalysisChemical/tissue engineersToxicologistsSimCyp

MathematicallyModel

Develop a zonated hepatic hollow fibre bioreactor for chemical safety assessment

Safe humanin vivo dose

Replicating liver physiology for toxicology

Bile canaliculus

N BSEPSER

M

Paracetamol (mouse)

4

Perivenous / Centrilobular

M

Hepatic

Central vein

Perivenous / Centrilobular↓ Oxygen↓ HormonesGlycolysisChemical detoxification

Hepatocytes

sinusoids

1

2

3

Periportal↑ Oxygen

Chemical detoxificationLipogenesis

Kupffer cell

3 ↑ Oxygen↑ HormonesGluconeogenesisUreagenesisHepatic arteriole

Portal vein Bile ductBile duct

PP

CL

PP

CL

PP

CL

PP

CL Methapyrilene(rat)

PP

CL

PP

CL

PP

CL

PP

CL

Design an in vitro hepatic sinusoid

Plasma-likecompartment

A hollow fibre bioreactor

Liver Sinusoidcompartment

Centrilobular-like region

Periportal-like region

Bile-likeBile likecompartment

Cell numberViability

GlucoseAlbumin

Oxygen levelPressuresViability

MorphologyUreaGlycogen

PressuresFlow rates

Design an in vitro hepatic sinusoid

End viewof fibresf f

t ill

hepatocytes

extra-capillary space

hollow fibre membrane

media

A single fibre:

Defining Operating Characteristics

Engineers / Mathematicians:Mathematicians / Modellers:

Scalable in silico PBPK modelScaffold design & production

tertiary systemspinning conditionsdope additives

Scalable in silico PBPK modelIn silico sinusoid composed of:

HepG2freshly isolated rat hepatocytes

Toxicologists / Modellers:

dope additivespost-spinning treatment

Choice of & scaffold characterisationasymmetric / symmetric wall

freshly isolated human hepatocytes

2D baseline characteristics of cell type pore sizefibre dimensionsporosity

Fluid transport / lumen pressures

Quantitatively assess how 3D environment maintains or improves

Fluid transport / lumen pressuresAlbumin permeation & foulingNutrient & oxygen transportCell seeding / confluence environment maintains or improves

functional drug metabolism & toxicityMass transfer limitations of traditional scaffolds

f3Biology of

model system

Quantitative Bioanalytical Endpoints

Paracetamol provides functional enzymatic coverage:Paracetamol provides functional enzymatic coverage: CYP’s 2E1, 1A2, 2A6, 3A4, 2D6Glucuronidation & sulphationMRP2, 3, 4 & BCRP, ,

Incorporation of bioactivation & covalent binding

Demonstrates zone specific toxicity

Toxicity induces inflammatory cytokine and toxicity biomarker release

Weighting of results to in vivo (rat, chronic infusion) or fresh human hepatocyte data

Considerable literature data well characterised compound

Allows evaluation of biology / pharmacology within the model system e.g. bioreactor

Paracetamol (APAP; acetaminophen)

• Recommended dose - 4g. Toxic dose >4g

• Most common form DILI in US & UK

• 400-500 deaths/yr, 70-100,000 hospital visits/yr

• Centrilobular damage

• Pharmacophore = Toxicophore

Lee W.B. AASLD 2009• Excellent translational ‘tool’

• Evaluation of novel models

Paracetamol (APAP; acetaminophen)

DetoxicationGLUCURONIDE

SULPHATE

DetoxicationGLUCURONIDE

SULPHATE

• Recommended dose - 4g. Toxic dose >4g

• Most common form DILI in US & UK

BioactivationBioactivation• 400-500 deaths/yr, 70-100,000 hospital

visits/yr

GSH

Overdose

GSH

Overdose• Centrilobular damage

• Pharmacophore = Toxicophore

COVALENT BINDING TOXICITYCOVALENT BINDING TOXICITY• Excellent translational ‘tool’

• Evaluation of novel models

Baseline 2D Operating CharacteristicsFreshly isolated rat hepatocytes (12x106 cells)Cultured rat hepatocytesFreshly isolated human hepatocyte (resection)

ll li

Toxicity

Hep G2 cell line

Metabolism

16%2%

79%

3%

I Paracetamol glucuronide

Parent compound (500M) disappearanceI Paracetamol-glucuronide

II Cysteinyl-paracetamolIII ParacetamolIV Paracetamol-sulphateV Paracetamol-glutathione

h l

disappearance

VI 3-methoxy-paracetamolVII NAC-paracetamol

Values are the mean ± SEM, n=4

Baseline 2D Operating CharacteristicsFreshly isolated rat hepatocytesCultured rat hepatocytes (2x106 cells; monolayer & sandwich culture)Freshly isolated human hepatocyte (resection)

ll li

Toxicity

Hep G2 cell line

Metabolism (72h)78%

Parent compound disappearance

4nm

(mA

U)

Monolayer

IVI II

III

0.5mM Paracetamol12% 9%

78%

0.5%

disappearance

banc

e at

254

d h

IV

III

Increased metabolism in sandwich culture hepatocytes

87%

UV

Abs

orb Sandwich

I II

III

IV10% 2% 0.5%

Rat Cells IVIVE clearance(ml/min)

Hepatocyte 2 96Wistar Rat Cl in vivoTime (min)

I Paracetamol glucuronideII ParacetamolIII Paracetamol sulphate

Hepatocyte suspensions

2.96

Sandwich culture

1.37

Wistar Rat Cl in vivo6.6 ml/min

III Paracetamol sulphateIV Paracetamol glutathione

Values are the mean ± SEM, n=4

Monolayer culture

0.85

*Raftogianis et al., 1995; Aanderud & Bakke, 1983

Baseline 2D Operating Characteristics

10

12

gate

(M

) Paracetamol Glutathione Conjugate formation

4

6

8Rat Hepatocyte Suspensions (APAP 500µM):

Formation of APAP-GSH plateau’s after 3hthio

ne C

onju

g

0

2

4

0 1 2 3 4 5 6

Formation of APAP GSH plateau s after 3h

APA

P G

luta

t

Time (h)

789

Monolayer Sandwichate

(M

)

34567

Hepatocytes in Culture (APAP 500µM):

hion

e Co

njug

a

0123

0 20 40 60

Increased bioactivation in hepatocytes cultured with matrigel overlay

APA

P G

luta

th

Time (h)

Baseline 2D Operating CharacteristicsCultured rat hepatocytes on different polymers

120I Paracetamol glucuronide

Metabolism (24h)Parent compound

disappearance

60

80

100

120

Rem

aini

ng

Collagen PS PLGA

II ParacetamolIII Paracetamol sulphate

IV Paracetamol glutathione

disappearance

0

20

40

60

Para

ceta

mol

12%

61%

26%

0 2%00 20 40 60%

P

Time (h)

0.2%

69%Bi t i l IVIVE l 22%

9%

0.5%Biomaterial IVIVE clearance

(ml/min)

Collagen 0.73

PS t d 0 85

14%

49%37%

0 5%

PS coated 0.85

PLGA 0.66

0.5%

Baseline 2D Operating Characteristics

Toxicity in suspension (6h)

Freshly isolated rat hepatocytesCultured rat hepatocytes Freshly isolated human hepatocyte (resection)

Metabolism in fresh human hepatocyte suspensions (6h) co

ntro

l)

Trypan blue

mA

U)

Toxicity in suspension (6h)y p y ( )Hep G2 cell line

1,000

1,400

APAP 79%

hepatocyte suspensions (6h)

abili

ty (%

c

ATP

e at

254

nm (m

-200

500

0

APAP-sulphate 4%APAP-GSH 1%

APAP-glucuronide 16%

Via

Paracetamol (mM)UV

Abs

orba

nce

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.0200

Metabolite : Parent Compound Ratio

( )U

Interindividual variability

Time (min)

APAP-G APAP-S APAP-GSH

0.5mM 0.653 (±0.420)

0.139 (±0.053)

0.025 (±0.015)

y

Inter-isolation variability

Quality of resection hepatocytes

Values are the mean ± SEM, n=4

Baseline 2D Operating Characteristics

Freshly isolated rat hepatocytesMetabolism

Cultured rat hepatocytesFreshly isolated human hepatocyte Hep G2 cell line

G2 ll li i i iHepG2 cell line resistant to APAP cytotoxicityLow P450 activity (CYP3A4)Variation in enzyme activities

(source and culture conditions) Paracetamolbanc

e(m

AU

) Paracetamol metabolites in HepG2 cells (24h; 2 x106)

Paracetamol(source and culture conditions) ParacetamolSulphate (2%)

UV

Abs

orb

at 2

54nm

Toxicity

Time (min)

10

15

g pr

otei

n)No coating PS PLGA

5

ATP

(nm

ol/m

g

Wang et al 2002 ; J Toxicol Sci Vol 27 (2002); Hewit and Hewit Xenobiotica (2004)

00 24

A

Time (h)

In silico rat hepatocyte

All data from published literature

No assumptions in the model

Modelling directs ‘wet-lab’ research

Kim et al., 1992; McPhail et al., 1993

Allows visualization of enzyme capacity

Visualisation of enzymatic capacities

Rat Hepatocytes

100M APAP

R f S l h iRate of Sulphationlimited by:

• [APAP]A & bi di• Amount & binding affinity of sulphotransferase

Kim et al., 1992; McPhail et al., 1993

Explore different scenarios through modelling

Rat Hepatocytes

1mM APAP

Sulphation:Sulphation:• Saturated• PAPS depletion • Rate limited by PAPS

synthesissynthesis• Media [sulphate]

GSH depletion occurring; [GSH] prevents toxicity[GSH] prevents toxicity

GSH gives cell a time window for Phase II to clear APAPclear APAP

Kim et al., 1992; McPhail et al., 1993; Willson et al., 1991; Ochoa et al., 2013; Sweeny & Reinke, 1988.

Explore different scenarios through modelling

Rat Hepatocytes

5mM APAP

• GSH depleted ~200 mins• GSH depleted 200 mins• APAP-SG limited by rate of

GSH synthesis• GSH synthesis <<NAPQI

formation = Covalentformation = Covalent binding

• CYP450 activity not saturated

• [APAP] = faster &• [APAP] = faster & earlier GSH

6h shows little [APAP] media, GSH depletion insensitive toGSH depletion insensitive to changes in other Phase II pathways

Kim et al., 1992; McPhail et al., 1993; Willson et al., 1991; Ochoa et al., 2013; Sweeny & Reinke, 1988.

In silico rat hepatocyte zonation

periportalsulphationperiportal rat hepatocyte

centrilobular

l id tit il b l t h t t

Well-mixed periportal

glucuronidationcentrilobular rat hepatocyte

centrilobular

Araya et al., 1986

In silico rat hepatocyte sinusoid

bilebile

sinusoid

bile

Each individual cell has its own set of parameters

D d bi i i h d l h i i PP iDecreased bioactivation enhanced sulphation in PP regions

Expandable to include cell death & other cell types

In silico sinusoid allows PBPK refinement

Allows refinement of PBPK modelsCan be used for head-to-head evaluation of novel in vitro models of drug metabolism

liver microsomes

In vitro clearance

Scale up PBPK model

Prediction 1Whole liver clearance

Prediction 1

lPrediction 2

singlecell

Prediction 2

Prediction 3

Evaluation of in vitro models of

drug metabolismsinusoid

Summary

Collaboration with mathematical modellers has enhanced experimentation

Get more out of each experimentGet more out of each experiment

Directs experimentation to areas of importance or data deficiency

In vitro mechanistic

+f2Biology of individual

In vitro mechanistic evaluation of hazard/risk

f1Chemistry

of drug +f3Biology of

model system=

Improved in vitro recapitulation of in vivo physiology = improved predictions

R fi t f PBPK d lRefinement of PBPK models

Evaluation of novel in vitro models of drug metabolism

Thank YouYUniversity of Liverpool:Sophie ReganIan SorrellIan SorrellSteve Webb (sdw@liv.ac.uk)

University of Bath:y fMarianne Ellis

UCL:Rebecca Shipley

University of Loughborough:University of Loughborough:John WardDennis Reddyhoff

SimCyp:Iain Gardner