Susceptibility Factors in IdiosyncraticSusceptibility Factors in IdiosyncraticDrug-Induced Liver InjuryDrug-Induced Liver Injury

Steven YeeSteven YeeMolecular and Cellular Toxicology SectionMolecular and Cellular Toxicology Section

National Institutes of HealthNational Institutes of Healthyees@nhlbi.nih.govyees@nhlbi.nih.gov

8 June 20048 June 2004

U.S. Department of Healthand Human Services

National Institutes of Health

National Heart, Lung, and Blood Institute

• Occurs in small fraction of individualsOccurs in small fraction of individuals

• Difficult to predictDifficult to predict

• Major clinical problem; often life-threateningMajor clinical problem; often life-threatening • Leading cause of acute liver failureLeading cause of acute liver failure

• Reason drugs removed from clinical Reason drugs removed from clinical development and widespread usedevelopment and widespread use

Drug-Induced Liver InjuryDrug-Induced Liver Injury

Examples of Drugs Withdrawn Due to Examples of Drugs Withdrawn Due to Liver DiseaseLiver Disease

IproniazidIproniazid19561956

Ibufenac (in Europe only)Ibufenac (in Europe only)19751975

TicrynafenTicrynafen1979 1979

BenoxaprofenBenoxaprofen1982 1982

Perhexiline (in France)Perhexiline (in France) 19851985

Dilevalol (in Portugal, Ireland)Dilevalol (in Portugal, Ireland)19901990

BromfenacBromfenac 19981998

Troglitazone Troglitazone 20002000

SerzoneSerzone20042004

• Mechanism often involves drug metabolitesMechanism often involves drug metabolites– Affect critical biochemical functionsAffect critical biochemical functions– Specific immune responsesSpecific immune responses

• Only a few drugs demonstrate these Only a few drugs demonstrate these underlying causesunderlying causes

• Tissue susceptibility: imbalance between Tissue susceptibility: imbalance between protoxicants and protectantsprotoxicants and protectants

– Environmental factorsEnvironmental factors– Genetic polymorphismGenetic polymorphism

Putative MechanismsPutative Mechanisms

Hepatotoxicants Potentiated by Exposure to Hepatotoxicants Potentiated by Exposure to Small Doses of Lipopolysaccharide (LPS)Small Doses of Lipopolysaccharide (LPS)

XenobioticsXenobioticsCClCCl44

GalactosamineGalactosamine

EthanolEthanol

T2-toxinT2-toxin

CadmiumCadmium

HalothaneHalothane

LeadLead

Allyl AlcoholAllyl Alcohol

Aflatoxin BAflatoxin B11

ChlorpromazineChlorpromazine

RanitidineRanitidine

SourceSourceFormal Formal et al.et al., 1960, 1960

Galanos Galanos et al.et al., 1979, 1979

Nolan Nolan et al.et al., 1980, 1980

Tai and Petska, 1988Tai and Petska, 1988

Cook Cook et al.et al.., 1974., 1974

Lind Lind et al.et al., 1984, 1984

Honchel Honchel et alet al., 1991., 1991

Sneed Sneed et al.et al., 1997, 1997

Barton Barton et al.et al., 2000, 2000

Buchweitz Buchweitz et al.et al., 2002, 2002

Luyendyk Luyendyk et al.et al., 2003, 2003

LPSLPS MacrophagesMacrophagesNeutrophilsNeutrophils

Endothelial CellsEndothelial CellsEpithelial CellsEpithelial Cells

TLR4TLR4

CytokinesCytokines Coagulation FactorsCoagulation FactorsPlatelet Activating FactorPlatelet Activating Factor Complement ActivationComplement ActivationLeukotrienesLeukotrienes Arachidonic Acid MetabolitesArachidonic Acid MetabolitesProstaglandinsProstaglandins Reactive Oxygen SpeciesReactive Oxygen SpeciesNitric OxideNitric Oxide

Cellular Level Events of LPSCellular Level Events of LPS

Monocrotaline (MCT)Monocrotaline (MCT)

NN

CHCH22OO

OOO=CO=C

CC C=OC=O

CCCC

OHOH

CHCH33 CHCH33

OHOHHH33CCHH

MonocrotalineMonocrotaline

Crotalaria SpectabilisCrotalaria Spectabilis(Rattlebox)(Rattlebox)

NN

CHCH22OO

OOO=CO=C

CC C=OC=O

CCCC

OHOH

CHCH33 CHCH33

OHOHHH33CCHH

MCTMCT

NN

CHCH22OO

OOO=CO=C

CC C=OC=O

CCCC

OHOH

CHCH33 CHCH33

OHOHHH33CCHH

MonocrotalineMonocrotalinePyrrolePyrrole(MCTP)(MCTP)

CYP 3ACYP 3A

CovalentCovalentBinding toBinding toCellular Cellular

MacromoleculesMacromolecules

Cell DeathCell Death

MCT ToxicityMCT Toxicity

MCT/LPS Treatment ProtocolMCT/LPS Treatment Protocol

0 hr 4 hr X hr0 hr 4 hr X hr

MCT or MCT or VehVeh(i.p.)(i.p.)

LPS orLPS orVehVeh(i.v.)(i.v.)

1. Plasma1. Plasma2. Liver2. Liver

• • Male Sprague-Dawley RatsMale Sprague-Dawley Rats

Hours After MCT Administration

0 12 24 36 48 60 72 84 96

Pla

sma

AL

T A

ctiv

ity

(U/L

)

0

200

400

600

800

1000

1200a

a

aa

a

a

Veh/VehVeh/LPSMCT/VehMCT/LPS

Modest Inflammation EnhancesModest Inflammation EnhancesMCT ToxicityMCT Toxicity

LPS 4 hours after MCTLPS 4 hours after MCT

LPS Lowers the Threshold for MCT ToxicityLPS Lowers the Threshold for MCT Toxicity

MCT (mg/kg)

0 50 100 150 200

Pla

sma

AL

T A

ctiv

ity

(U/L

)

0

250

500

750

1000

1250

1500

aa

a

a

MCT/Veh MCT/LPS

Kupffer Cell Inactivation Reduces Kupffer Cell Inactivation Reduces HepatotoxicityHepatotoxicity

Pla

sma

TN

F-

(p

g/m

l)0

20

40

60

80

100

120

Veh/Veh MCT/LPS

aVehGdCl3

b

Pla

sma

AL

T A

ctiv

ity

(U/L

)

0

200

400

600

800

1000

1200

Veh/Veh MCT/LPS

a,b

aVehGdCl3

TNF-TNF- Depletion Attenuates MCT/LPS Depletion Attenuates MCT/LPS HepatotoxicityHepatotoxicity

Pla

sma

TN

F-

(p

g/m

l)0

20

40

60

80

100

Veh/Veh MCT/LPS

a

b

CSATS

Pla

sma

AL

T A

ctiv

ity

(U/L

)

0

200

400

600

800

1000

1200

Veh/Veh MCT/LPS

a,b

aCSATS

Neutrophil Depletion AttenuatesNeutrophil Depletion Attenuates MCT/LPS HepatotoxicityMCT/LPS Hepatotoxicity

Nu

mb

er o

f P

MN

s/H

PF

0

10

20

30

40

Veh/Veh MCT/LPS

a

a,b

CSNAS

Pla

sma

AL

T A

ctiv

ity

(U/L

)

0

200

400

600

800

1000

Veh/Veh MCT/LPS

a

b

CSNAS

Inactivation of the Coagulation System Inactivation of the Coagulation System Decreases Liver InjuryDecreases Liver Injury

Pla

sma

AL

T A

ctiv

ity

(U/L

)

0

300

600

900

1200

Veh/Veh MCT/LPS

a

a,b

VehHep

Pla

sma

AL

T A

ctiv

ity

(U/L

)

0

300

600

900

1200

1500

Veh/Veh MCT/LPS

a

a,b

VehWarf

Pla

sma

Fib

rin

og

en (

mg

/dl)

0

100

200

300

400

Veh/Veh MCT/LPS

a

VehWarf

Pla

sma

Fib

rin

og

en (

mg

/dl)

0

100

200

300

400

Veh/Veh MCT/LPS

a

VehHep

SummarySummary

Sinusoid

SEC

Space of Disse

HPC

MCT

LPS

PMN

PMN

CINC-1

TNF

[Early Injury]

[Injury]

[Late Injury]MCT MCTP

MCT MCTP

Kupffer Cell

[Early Injury][Activated SEC]

MCT

[Activated/ Sensitized HPC]

Kupffer Cell

PMN

Approximate Time (hr)

0 64 8

CoagulationSystem Activation

Thrombin Fibrin Clot

Toxic Factors (proteases, etc.)

HypoperfusionSinusoid

SEC

Space of Disse

HPC

MCT

LPS

PMN

PMN

CINC-1

TNF

[Early Injury]

[Injury]

[Late Injury]MCT MCTPMCT MCTP

MCT MCTPMCT MCTP

Kupffer Cell

Kupffer Cell

[Early Injury][Activated SEC]

MCT

[Activated/ Sensitized HPC]

Kupffer Cell

Kupffer Cell

PMN

Approximate Time (hr)Approximate Time (hr)

0 64 8

CoagulationSystem Activation

Thrombin Fibrin Clot

Toxic Factors (proteases, etc.)

Hypoperfusion

Does a loss in Does a loss in hepatoprotective factors hepatoprotective factors

result in drug-induced liver result in drug-induced liver disease?disease?

DrugDrugLiverLiver

ReactiveReactiveMetabolitesMetabolites

ProteinProteinAdductsAdducts

CellularCellularHomeostasisHomeostasis

AlteredAltered

(Inhibition)(Inhibition)

ResponseResponse

ToxicityToxicity ProtectionProtectionIL-6, IL-10, COX-2Stress Proteins

ProtectiveProtectiveFactorsFactors

ExtensiveExtensiveLiver InjuryLiver InjuryAnd DeathAnd Death

Model CompoundModel Compound

• Many of the protective factors discovered through Many of the protective factors discovered through research on acetaminophen (APAP) toxicityresearch on acetaminophen (APAP) toxicity

• AcetaminophenAcetaminophen– Clinically relevant; analgesic, antipyreticClinically relevant; analgesic, antipyretic– Over 50,000 ER visits per yearOver 50,000 ER visits per year– 450 death per year450 death per year

• SuicideSuicide• Accidental ingestionAccidental ingestion• “ “Therapeutic misadventures”Therapeutic misadventures”

– Unlike with drug idiosyncrasy, it is well characterized Unlike with drug idiosyncrasy, it is well characterized and reproducible in animalsand reproducible in animals

• Bioactivation to Bioactivation to NN-acetyl--acetyl-pp-benzoquinone imine -benzoquinone imine (NAPQI)(NAPQI)

APAP-Induced Liver InjuryAPAP-Induced Liver Injury

HN CH3

O

OH

ACETAMINOPHEN

N CH3

O

O

NAPQI

Sulfation Glucuronidation

DetoxDetox

GSHDetox

CysteinylConjugate

Protein Adducts

Mitochondria Dysfunction

Reactive Oxygen Species

LIVERINJURY

P450 2E1P450 1A2P450 3A4

Depleted

Serum Cytokines After Administration ofSerum Cytokines After Administration of300 mg APAP/kg to C57BL/6 Mice300 mg APAP/kg to C57BL/6 Mice

Time (hr)

4 8 12 240

3000

6000

9000

12000

15000ALT

IU/L

4 8 12 240

100

200

300IL-4

pg/m

L

4 8 12 240

100

200

300IL-10

pg/m

L

4 8 12 240

100

200

300

400

500IL-13

pg/m

LIL-6

4 8 12 240

40

80

120

160

200

pg/

mL

Bourdi (2002), Hepatology, 35:289Bourdi (2002), Hepatology, 35:289

Development of Hepatotoxicity in Mice Given Development of Hepatotoxicity in Mice Given IL-13 Neutralizing Antibody 2 Hours beforeIL-13 Neutralizing Antibody 2 Hours before

200 mg APAP/Kg 200 mg APAP/Kg

Ser

um

AL

T (

IU/L

)

0

1000

2000

3000

4000

______ ______ ______ 4 8 24

Hours After APAP Administration

a

CAb/APAPIL-13 NAb/APAP

a

a

Ser

um

IL

-13

(pg

/ml)

0

25

50

75

100

______ ______ ______ 4 8 24

Hours After APAP Administration

CAb/APAPIL13NAb/APAP

a a

Ser

um

AL

T (

IU/L

)

0

3000

6000

9000

12000

______ ______ ______ 4 8 24

Hours After APAP Administration

a

WTKO

a

a

Development of Hepatotoxicity in IL-13 KO Development of Hepatotoxicity in IL-13 KO Mice Treated With 200 mg APAP/Kg Mice Treated With 200 mg APAP/Kg

Liver Histopathology at 8 hoursLiver Histopathology at 8 hours

CVCV CVCV

CVCV

CVCV

CVCV

CVCV

CVCV

CVCV

CAb/VehCAb/Veh CAb/APAPCAb/APAP

IL-13 NAb/APAPIL-13 NAb/APAP APAP - KOAPAP - KO

Nitric Oxide PathwayNitric Oxide Pathway

L-Arginine

L-Ornithine

IL-4, IL-10, IL-13IL-4, IL-10, IL-13

IL-1, IL-6, IL-1, IL-6, TNF-TNF-IFN-IFN-γγ

NOiNOSiNOS

ArginaseArginase

Citrulline

Urea

L-NILAminoguanidine (AMG)

Nitrite/Nitrate

Peroxynitrite+Superoxide+Superoxide

• Endogenous IL-13 is a hepatoprotective Endogenous IL-13 is a hepatoprotective factor in APAP-induced liver injuryfactor in APAP-induced liver injury

• Elevated serum TNF-Elevated serum TNF-α concentration – α concentration – causal role?causal role?

• Elevated NO levels – causal role?Elevated NO levels – causal role?

• Deficiency in IL-13 may increase Deficiency in IL-13 may increase susceptibility to drug-induced liver susceptibility to drug-induced liver diseasedisease

SummarySummary

Idiosyncratic Drug-Induced Liver DiseaseIdiosyncratic Drug-Induced Liver Disease

• Complex “multihit” process Complex “multihit” process

• Liver protoxicants and protectants have Liver protoxicants and protectants have a role in the overall pathogenesisa role in the overall pathogenesis

– Environmental FactorsEnvironmental Factors– Genetic polymorphismsGenetic polymorphisms

• Underproduction of hepatoprotective and Underproduction of hepatoprotective and overproduction of hepatoprotoxicant overproduction of hepatoprotoxicant

factors (i.e., imbalance) influences factors (i.e., imbalance) influences susceptibility to this susceptibility to this liver diseaseliver disease

Determination of Susceptibility FactorsDetermination of Susceptibility Factors

• Identification of liver protoxicant and protectant Identification of liver protoxicant and protectant factors results in better understanding of factors results in better understanding of mechanism and in facilitating prediction of mechanism and in facilitating prediction of drug-drug-induced liver diseaseinduced liver disease

– Inflammatory mediatorsInflammatory mediators– COX-2 productsCOX-2 products– Heat shock proteinsHeat shock proteins

• Application of new technologiesApplication of new technologies– ToxicogenomicsToxicogenomics– ProteomicsProteomics– MetabonomicsMetabonomics

AcknowledgmentsAcknowledgments

Michigan State UniversityMichigan State University

Robert RothRobert Roth

Patricia GaneyPatricia GaneyJack HarkemaJack Harkema

Chuck BartonChuck BartonJohn BuchwietzJohn BuchwietzBryan CoppleBryan CoppleShawn KinserShawn KinserJim LuyendykJim LuyendykJane MaddoxJane MaddoxRosie SneedRosie Sneed

National Institutes of HealthNational Institutes of Health

Lance PohlLance Pohl

Michael AdamsMichael AdamsHamid AmouzadehHamid AmouzadehMohammed BourdiMohammed BourdiJohn GeorgeJohn GeorgeMichael HoltMichael HoltMary Jane MassonMary Jane MassonKevin WelchKevin Welch

Santana FloresSantana Flores

Thomas Wynn, NIAIDThomas Wynn, NIAID