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DRUG-INDUCED LIVER DISEASE

CONTENTS

Preface xiWilliam M. Lee

Mechanisms of Drug-Induced Liver Disease 459Basuki K. Gunawan and Neil Kaplowitz

Drug-induced liver injury depends initially on development ofhepatocyte stress and cell death, which can be induced directly byparent drugs or by toxic metabolites. Hepatocyte stress can lead to

activation of built-in death programs for apoptosis or necrosis.Subsequently, the innate immune systems participation isrecruited. The interplay between proinflammatory and anti-inflammatory components of innate immune system determinesthe outcome of drug-induced liver injury. Both environmentalfactors and genetic differences in cellular responses to stress andthe innate immune response may account for different susceptibil-ities between individuals to drug-induced liver injury.

Causality Assessment of Drug-Induced Hepatotoxicity:

Promises and Pitfalls 477Max A. Shapiro and James H. Lewis

Drug-induced liver injury is the leading cause of acute liver failurein the United States, but the ability to ascribe hepatic injuryconfidently to a specific drug remains a challenging and oftendifficult pursuit. This article explores the ongoing challengesinherent in what is currently a clinical process of eliminationmade in the attempt of assigning causality in drug-induced liverinjury. In particular, it points out the shortcomings and pitfalls thatoften limit the applicability of the causality-assessment method-

ologies currently in use.

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Drug Hepatotoxicity from a Regulatory Perspective 507John R. Senior

This article summarizes problems of drug-induced liver injury(DILI), as seen from the perspective of the Food and DrugAdministration (FDA). After brief consideration of the scope ofFDA activities and processes of new drug development and reviewfor possible approval of products for clinical use and marketing,some of the perceived current problems in detection, confirmation,close observation, differential diagnosis, and follow-up of cases ofpossible DILI in controlled clinical trials are described. Readers areinvited to consider possible solutions to the many problems of DILI,propose ways to support research in the field, and keep abreast ofprogress by visiting the web site at www.fda.gov/cder/livertox.

Acetaminophen Hepatotoxicity 525Anne M. Larson

Acetaminophen is a commonly used antipyretic and analgesicagent. It is safe when taken at therapeutic doses; however,overdose can lead to serious and even fatal hepatotoxicity. Theinitial metabolic and biochemical events leading to toxicity havebeen well described, but the precise mechanism of cell injury anddeath is unknown. Prompt recognition of overdose, aggressivemanagement, and administration ofN-acetylcysteine can minimize

hepatotoxicity and prevent liver failure and death. Liver trans-plantation can be lifesaving for those who develop acute liverfailure.

Hepatotoxicity Due to Antibiotics 549Julie E. Polson

Antimicrobial drugs are important causative agents in idiosyn-cratic drug-induced liver injury (DILI). As with idiosyncratic DILIin general, antibiotic-induced liver injury is rare but difficult to

diagnose and almost impossible to predict. Diagnosis requiresawareness of possible causal agents, vigilance in monitoringsymptoms and sometimes biochemical tests, attention to carefulhistory taking and establishing temporal association, and exclusionof competing etiologies. In most instances, patients with antibiotic-associated DILI recover if the offending agent is withdrawn in atimely fashion.

Nonsteroidal Anti-Inflammatory DrugInduced Hepatotoxicity 563Guruprasad P. Aithal and Christopher P. Day

Nonsteroidal anti-inflammatory drugs are among the mostcommon drugs associated with drug-induced liver injury, withan estimated incidence of between 3 and 23 per 100 000 patientyears. Nimesulide, sulindac, and diclofenac seem to be associatedwith the highest risk and the only risk factor consistently identified

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is the concomitant use of other hepatotoxic drugs. Diclofenac-induced liver injury is a paradigm for drug-related hepatotoxicity.Recent studies suggest that genetic factors favoring the formationand accumulation of the reactive acylglucuronide metabolite ofdiclofenac and an enhanced immune response to the metabolite-

protein adducts are associated with increased susceptibility tohepatotoxicity.

Herbal Hepatotoxicity 577Leonard B. Seeff

There is appropriate concern about the potential risk forhepatotoxicity from herbal products because they are unregulatedand therefore not standardized with regard to their contents. Thisis particularly the case for the crude herbals that are commonlyformulated as a mixture, so that their ingredients may beambiguous and even contain harmful contaminants. Presentedhere is an overview of the more commonly recognized herbalproducts that have been reported to be associated with liver injury.Although many of them are clearly implicated, there are some,particularly those identified solely through an occasional casereport, for which the relationship is uncertain.

Lipid-Lowering Agents That Cause Drug-Induced

Hepatotoxicity 597Sidharth S. Bhardwaj and Naga Chalasani

The effort to reduce cardiovascular risk factors, including hyper-lipidemia, has led to the increased use of lipid-lowering agents.Hyperlipidemic patients often have underlying fatty liver disease,however, and thus may have elevated and fluctuating liverbiochemistries. Therefore, caution should be applied beforeattributing elevated liver tests to lipid-lowering agents. Dataindicate that patients who have chronic liver disease andcompensated cirrhosis should not be precluded from receiving

statins to treat hyperlipidemia. Several recent studies and expertopinion currently fully endorse statin use in patients who havenonalcoholic fatty liver disease and other chronic liver disease ifclinically indicated.

Drug-Induced Liver Injury Associated with HIV Medications 615Mamta K. Jain

Antiretroviral therapy (ART) for HIV infection frequently has beenassociated with elevated liver enzyme levels. Determining the

cause of elevated liver enzyme levels in patients who have HIV isdifficult because ART usually consists of three different drugs,patients may be taking additional hepatotoxic medications andpatients who have HIV often suffer from other liver diseases.Several agents, however, are recognized as having noteworthy andspecific patterns of toxicity. This article reviews the different HIV

CONTENTS vii


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drug classes, incidence of elevated liver enzyme values by classand by individual drug, risk factors, specific toxicities, and possiblemechanisms of injury.

Cancer Chemotherapy I: Hepatocellular Injury 641Edmundo A. Rodriguez-Frias and William M. Lee

Although hepatotoxicity is a frequent concern with all medica-tions, chemotherapeutic agents are more often implicated incausing liver damage than most other drug classes. In manyinstances, these reactions are considered dose related becausecytotoxic therapy directed at rapidly growing cancer cells mayreadily impact hepatocytes even though they are dividing moreslowly. Because the stakes (remission of cancer) are high, so arethe risks that the oncologist and the patient are willing to assume.The dose of many chemotherapeutic agents is limited by the toxiceffects on the lungs, bone marrow, kidneys, and gastrointestinalsystem, including the liver. An awareness of the toxic potential ofeach chemotherapeutic agent is necessary before initiation of newoncologic treatments.

Cancer Chemotherapy II: Atypical Hepatic Injuries 663Edmundo A. Rodriguez-Frias and William M. Lee

Although chemotherapy generally is accompanied by regulartesting for liver enzyme abnormalities, atypical reactions mayoccur that escape ordinary detection, because hepatocyte injury isnot the primary event. The presence of fatty liver, mitochondrialchanges, and even biliary abnormalities can be associated withnormal or nearly normal liver enzyme levels. This article discussesunique aspects of liver damage associated with cancer chemo-therapy. These unique reactions merit special attention and aspecial vigilance from clinicians.

Index 677

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FORTHCOMING ISSUES

November 2007

Hepatitis B VirusIra Jacobson, MD,Guest Editor

February 2008

Cholestasis

Donald Jensen, MD,Guest Editor

May 2008

Hepatitis C Virus

K. Rajender Reddy, MD,and David E. Kaplan, MD,Guest Editors

RECENT ISSUES

May 2007

Liver Transplantation

Paul Martin, MD,Guest Editor

February 2007

Non-Alcoholic Steatohepatitis and Non-AlcoholicFatty Liver Diseases

Zobair M. Younossi, MD,Guest Editor

November 2006

Hepatitis C Virus Update

David R. Nelson, MD,Guest Editor

THE CLINICS ARE NOW AVAILABLE ONLINE!

Access your subscription at:www.theclinics.com


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Preface

Guest Editor

Drug-induced liver injury (DILI) continues to play an important role in

the evolution of modern medical care. DILI cases are by their natureiatrogenic but also often represent self-inflicted wounds, whether as a drug

overdose such as acetaminophen or as an idiosyncratic reaction. Hepatotox-

icity caused by drugs continues to feature prominently in consideration for

drug approval by the United States Food and Drug Administration (FDA)

and is the primary reason for drugs failing to be approved or being with-

drawn after initial approval. Recent examples include ximelagatran, a highly

anticipated anticoagulant that probably would have replaced warfarin if not

for its failure to be approved in the United States and its subsequent volun-

tary withdrawal in Europe. Other drugs withdrawn or significantly limitedin recent years include troglitazone and bromfenac (both withdrawn),

trovafloxacin, nefazodone, and telithromycin (limited use, strong warnings).

These unexpected calamities affect patients because the typical DILI case is

severe and often fatal or requires liver transplantation. These events also

impact the pharmaceutical industry. Drug approval becomes a hard-won

prize that is becoming ever more elusive and costly. Difficulties in obtaining

approval affect the cost of products that are approved as well. Despite many

recent advances in the understanding of drug-induced liver injury, it still is

not possible to identify safe new products readily and effectively.Against this background, I present this edition ofClinics in Liver Disease,

an in-depth look at many of the current hot topics in the field of DILI. Each

is directed toward a particular area of current interest and provides new in-

sights not available when this topic was covered previously. It begins with an

William M. Lee, MD

1089-3261/07/$ - see front matter 2007 Elsevier Inc. All rights reserved.

doi:10.1016/j.cld.2007.06.011 liver.theclinics.com

Clin Liver Dis 11 (2007) xixii


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incisive discussion of mechanisms of drug-induced liver injury from Dr. Neil

Kaplowitz, followed by a careful review of the unwieldy topic of assessing

causality by Dr. James Lewis. Analyzing the drug approval process andexplaining it to the lay public was the task of Dr. John Senior, the FDAs

inhouse hepatology consultantdwho better to take on the task?

After these introductory articles of general interest, I asked experts to re-

view some of the specific drug classes of most interest and the drug-induced

liver damage they cause. These are the problem drug classes most commonly

encountered by the practitioner. Each class has its own signature form of in-

jury and its own specific associated issues. Acetaminophen poisoning is the

primary cause of acute liver failure in the United States, actually exceeding

all idiosyncratic drugs by about threefold in terms of numbers of directlyrelated deaths. The drug classes most commonly associated with liver injury

are antibiotics and nonsteroidal anti-inflammatory drugs (NSAIDS). DILI

caused by these agents is nicely covered by Drs. Polson and Drs. Aithal

and Day, respectively. The NSAID article includes much new information

concerning pharmacogenomics. The 3-hydroxy-3-methylglutaryl coenzyme

A reductase inhibitors, otherwise known as statins, is a topic of great

current interest, and no one is better qualified to review this topic than

Dr. Chalasani, who has written many of the primary articles in the field.

Another topic confusing to the public is the use of herbal medicationsand their risks. Dr. Seeff kindly has taken on the task of reviewing all we

know in this area. More drug toxicity issues are raised by cancer chemother-

apy and highly active antiretroviral (HAART) drugs than by any other

classes. Dr. Jain has developed a highly practical but detailed review of

the issues surrounding HAART therapy, and Dr. Rodriguez and I have

tackled the problem of liver injury during oncologic treatments.

All in all, I think drug-induced liver injury has become, in the last 5 years,

an exciting field as exemplified by the articles included here. I wish to thank

all those who labored so diligently to help create this issue, including mostspecifically the authors and their assistants as well as Kerry Holland and

Norman Gitlin. Any blame comes to me, while any credit goes to them

for what I personally think is a dynamite issue of Clinics in Liver Disease

that will be of interest to the primary care provider, specialists, and the

public at large, in short, anyone trying to make sense of this fascinating

field.

William M. Lee, MD

Division of Digestive & Liver DiseasesUniversity of Texas Southwestern Medical School

5959 Harry Hines Boulevard, HP.4.420

Dallas, TX 75390-8887

E-mail address: [email protected]

xii PREFACE
mailto:[email protected]:[email protected]


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Mechanisms of Drug-Induced

Liver Disease

Basuki K. Gunawan, MD*, Neil Kaplowitz, MDResearch Center for Liver Disease, Keck School of Medicine, University of Southern

California, 2011 Zonal Avenue, HMR 101, Los Angeles, CA 90033, USA

Drug-induced liver disease is of great importance because it is the leading

cause of acute liver failure in the United States[1]. The most common cause

for drug-induced liver disease resulting in acute liver failure is acetamino-

phen (APAP), with about half the cases reported to be accidental [1].

Drug-induced liver disease is also a major reason for withdrawal of drugs

during drug development and clinical use with major medical and economic

consequences [2]. The understanding of the mechanism of drug-inducedliver injury is of great importance and may lead to prevention and better

treatments.

Types of drug-induced liver injury

Drug-induced liver injury can be predictable; it is normally dose-

dependent, reproducible in animal models, and presents after a short latency

(hours to a few days). Drugs that cause this type of liver injury are usually

identified during initial toxicology studies. These findings typically preclude

further use as medication. Most drug-induced liver injuries, however, are

unpredictable or idiosyncratic, although they may or may not be dose-

dependent or reproducible in animal models [24]. This idiosyncratic type

can be classified as allergic, presenting with fever, rash, eosinophilia, and

rapidly recurring positive challenge; or as nonallergic, presenting without

allergic features[2,5]. Idiosyncratic drug reactions occur in only a small per-

centage of individuals who are exposed to the drug, ranging from 0.01% to

1%, and hence signify the uniqueness of the susceptible individuals [2,3,5].

Both genetic and environmental factors likely play a role in determining

* Corresponding author.

E-mail address: [email protected](B.K. Gunawan).



Clin Liver Dis 11 (2007) 459475


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the occurrence of this idiosyncratic type. The factors involved in drug-

induced liver disease are listed in Box 1and can be viewed as a sequential

cascade.Detailed discussion of all these factors is beyond the limits of this article.

Some of the key pathogenetic factors are highlighted.

Drug metabolism in the liver

The liver is the principal organ for metabolism and elimination of

many drugs. Even though some drugs cause hepatotoxicity when the parent

compound directly targets specific organelles, such as mitochondria or

nuclei, most toxic drugs require metabolism to toxic metabolites [2]. Thereare three phases of drug metabolism in the liver. In phase I, drugs are

metabolized by cytochrome P-450 enzymes. This process can generate toxic

electrophilic chemicals and free radicals. In phase II, the parent drug or

metabolites are conjugated with glutathione (GSH), sulfate, or glucuronide

to produce water-soluble compounds. Consequently, the compounds can

then be excreted from the body in bile or urine. The route of elimination

is mainly determined by excretory transporters in the hepatocyte canalicular

and sinusoidal membrane (phase III). Genetic polymorphisms or environ-

mental factors, such as concomitant drugs and alcohol, can account for dif-ferences in phase I, II, and III drug metabolism between individuals and

Box 1. Factors involved in drug-induced liver disease

Exposure to toxic metabolites

Regulation and expression of phase I, II, and III drug

metabolism

Direct consequences of toxic metabolite-covalent binding,

oxidative stress Intracellular stress

Organelle stress: mitochondria, endoplasmic reticulum,

nucleus

Stress kinases

Metabolic disturbance (eg, fat accumulation)

Inhibition of bile salt export pump: either cholestasis or

possible sensitization to apoptosis by bile acid induced

targeting of death receptors to the plasma membrane

Cell death: programmed apoptosis versus programmednecrosis

Participation of mitochondrial outer membrane

permeabilization or mitochondrial permeability transition

Innate immune response

Adaptive immune response

460 GUNAWAN & KAPLOWITZ


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may be determinants of susceptibility to idiosyncratic drug-induced liver

injury by influencing the hepatic exposure to toxic metabolites.

Biochemical events leading to drug-induced liver injury

The toxic metabolites from drug metabolism can then either directly affect

the biochemistry of the liver cells leading to cell damage or elicit an immune-

mediated attack on the liver [4]. Drug metabolites can covalently bind to

proteins, lipids, and DNA, and mediate cell death by inciting biochemical

events, such as oxidative stress, GSH depletion, and redox changes, and lipid

peroxidation. Consequently, these events may directly affect the functions

of mitochondria, endoplasmic reticulum, microtubules, cytoskeleton, andnucleus leading to an overwhelming direct insult [2,4]. Alternatively, these

events may lead to activation or inhibition of signaling kinases, transcription

factors, and gene expression profiles, which may sensitize hepatocytes or

cholangiocytes to the toxic effects of the innate immune system, such as

cytokines and chemokines, which are activated by the initial liver injury.

The toxic metabolite-initiated immune-mediated attack on the liver is

mainly achieved by cytotoxic T cells through an apoptotic mechanism

mediated by Fas ligand (FasL) and granzyme B/porin[5]. The initial trigger

for activity of cytotoxic T cells involves haptenization. In hepatocytes, thedrug metabolites can covalently bind to macromolecules, such as proteins, re-

sulting in altered proteins. This process is called haptenization and it can

incite an immune-mediated attack because the hapten-protein complexes

are recognized as neoantigens[6], which are internalized and processed by

antigen-presenting cells and subsequently presented on the cell surface with

major histocompatibility complex class II, leading to activation of helper

T cells. The activation of helper T cells results in release of cytokines, which

activate cytotoxic T cells, natural killer (NK) cells, and B cells and sub-

sequently generates autoantibodies and cell-mediated immune responses[5,7,8].

Many drugs that form reactive metabolites and undergo haptenization,

however, do not cause drug-induced liver disease. It seems that hapteniza-

tion alone might be insufficient to trigger an immune-mediated attack.

One hypothesis that may explain this phenomenon is the danger hypothesis

[9]. For the immune-mediated attack to occur, a second costimulatory dan-

ger signal must present. This danger signal primes the immune system

against the haptens in genetically susceptible individuals. The danger signal

may include concomitant infection or inflammatory conditions, becausethere have been reports of increased allergic hepatotoxicity in patients

with concomitant hepatitis B and C or HIV infection or with AIDS

[1014]. Alternatively, the danger signal may be the low-grade stress or in-

jury in hepatocytes. Supporting this hypothesis is the more frequent occur-

rence of mild alanine aminotransferase abnormalities in response to drugs

that more rarely induce overt allergic injury.

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The neoantigen formed because of haptenization can trigger the produc-

tion of autoantibodies directed against both the native and modified cyto-

chrome P-450 protein [6,15]. The autoantibodies formed in liver diseasecaused by several different drugs are normally specific to the particular

cytochrome P-450 isoenzymes that metabolize the drugs (Table 1). It is

unclear, however, whether the anticytochrome P-450 autoantibodies medi-

ate an immune attack on hepatocytes. These autoantibodies are found in the

serum when the drug-induced liver disease is diagnosed and they decline and

may disappear after recovery[6]. In addition, in drug-induced liver disease

caused by halothane or tienilic acid, an antibody-dependent cell-mediated

cytotoxicity has been demonstrated in vitro [16,17]. Autoantibodies are

commonly found in patients without evidence of drug-induced liver disease,however, suggesting that their presence is not always associated with liver

injury.

Mode of cell death

The outcome of the events initiated by toxic metabolites either through

directly affecting the biochemistry in hepatocytes or immune-mediated

response is cell death. The mode of cell death may be apoptosis or necrosis.

Apoptosis involves shrinkage, nuclear disassembly, and fragmentation of

the cell into discrete bodies with intact plasma membranes. The apoptotic

cells are then rapidly phagocytosed by neighboring cells. In contrast, necrosis

involves cell swelling and lysis as a result of profound loss of mitochondrial

function and resultant ATP depletion[4,5]. The selection between apoptosis

versus necrosis depends on several factors, including ATP status [5]. A more

severe injury to mitochondria might favor necrosis, whereas a less severe in-

jury to mitochondria without profound ATP depletion might favor apoptosis

[5,18].

The initiation of cell death usually involves the participation of mitochon-

dria. The interaction of proapoptotic Bcl2 family members (Bid, Bax, Bak,

Bmf, and Bim) and antiapoptotic members of this family (Bcl-2 and Bcl-XL)

regulates the permeability of mitochondria[4]. The immune-mediated attack

on hepatocytes involves the participation of the extrinsic death system, such

as tumor necrosis factor-a(TNF-a) or FasL, which then bind to their death

receptors leading to caspase 8 activation [4,19,20]. Caspase 8 subsequently

Table 1Autoantibody targets in drug-induced liver disease

Autoantibody target Drug

CYP2C9 Tienilic acid

CYP1A2 Dihydralazine

CYP3A1 Anticonvulsants (eg, phenytoin)

CYP2E1 Halothane



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cleaves Bid leading to translocation of Bax to the mitochondria and the

aggregation of Bax and Bak[4,21]. In turn, Bax and Bak promote the perme-

abilization of the mitochondria leading to cell death [4]. In contrast, theintrinsic death system is triggered by intracellular stress caused by covalent

binding, GSH depletion, or oxidative stress, which can also activate Bcl2

family members, but the precise mechanism is not entirely clear [4]. The

key role of sustained c-Jun-N-terminal kinase (JNK) activation has been

demonstrated in both extrinsic and intrinsic cell death.

Role of innate immune system

The innate immune system has been implicated in hepatotoxicity causedby various drugs, such as APAP, dihydralazine, and halothane[6,22]. Stud-

ies have demonstrated that the liver injury caused by hepatotoxins can be

associated with participation of increased numbers of proinflammatory

mediators, such as cytokines, chemokines, reactive oxygen intermediates,

and reactive nitrogen intermediates[22]. These proinflammatory mediators

can be directly cytotoxic (eg, hydrogen peroxide, nitric oxide, peroxynitrite);

can degrade the extracellular matrix (eg, collagenase, elastase); and can

promote cell adhesion and infiltration (eg, interleukin [IL]-1, TNF-a,

chemokines) [22]. Other mediators may indirectly damage hepatocytes bymodifying hepatocyte protein and nucleic acid biosynthesis, and cytochrome

P-450mediated metabolism[22]. In addition, the liver is selectively enriched

in Kupffer cells. Kupffer cells are the resident phagocytic macrophages in

the liver and account for 20% of nonparenchymal cells in the liver [2224].

They are well positioned to remove particulate and foreign materials from

the portal circulation because of their location in hepatic sinusoids and

predominantly in periportal and central regions of the liver lobule [22,25].

Their functions are diverse and include phagocytosis, endocytosis, immuno-

modulation, and synthesis and secretion of numerous biologically activemediators, which ultimately lead to liver injury [22,24,26]. Other cells in the

liver, such as endothelial cells and stellate cells, have also been implicated to

participate in drug-induced liver injury through the inflammatory mediators

[22]. The innate immune system has also been demonstrated to be protec-

tive; the role of the innate immune system seems to be complex (see section

on APAP hepatotoxicity). The trigger for the participation of innate im-

mune cells and mediators is believed to be caused by hepatocyte necrosis

but the mechanism is unclear[24]. It is also conceivable that nonlethal stress

in hepatocytes triggers activation of the innate immune system (eg, increasedhepatocytes expression of NKNKT cell activating ligands or decreased ex-

pression of inhibitory ligands). NK-NKT cells, which reside in the liver, may

play a key role in modulating the innate immune response by secreting in-

terferon (IFN)-g, osteopontin, IL-4, and so forth and directly killing cells

by FasL expression. An overview of the pathogenesis of drug-induced liver

disease is shown inFig. 1.



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Acetaminophen: an example of drug hepatotoxicity

APAP is the most extensively studied hepatotoxin and provides the bulkof knowledge on drug hepatotoxicity. The current understanding of the

mechanism of its hepatotoxicity is described next as an example, illustrating

some of the key aspects of the pathogenesis of drug-induced liver injury.

APAP is primarily metabolized in the liver by glucuronidation and sulfation

pathways into nontoxic metabolites that are then excreted in the urine.

A small amount of APAP is metabolized by oxidation, however, using

cytochrome P-450, mainly CYP2E1, into a highly electrophilic metabolite,

N-acetyl-p-benzoquinone-imine (NAPQI). With a small amount of APAP,

hepatic GSH subsequently detoxifies NAPQI into a nontoxic mercapturicacid and cysteine derivatives thereby preventing hepatocyte damage. With

a large amount of APAP, the glucuronidation and sulfation pathways are

saturated leading to an increased production of NAPQI by cytochrome

P-450 pathway. At the point when sufficient NAPQI is generated to deplete

hepatic GSH severely in both cytosol and mitochondria of hepatocytes, cell

death ensues[27,28].

Parent Drug

mitochondria reactive

metabolite hapten

Hepatocytes

(upstream)covalent binding

oxidative stress

dangerStress/Cell Death

(mild) adaptation

Cytokines

ChemokinesROS, RNS

NK/NKT

KupfferPMNs

Innate Immune

System

(downstream)Pro-inflammatory Anti-inflammatory

Adaptive Immune

Response

Recovery

Overt Liver

Injury

Repair

Recovery

Fig. 1. Pathogenesis of drug-induced liver disease. The development of drug-induced liver

injury depends initially on upstream development of stress and cell death, which can be induced

directly by parent drugs or reactive metabolites. This stress or cell death can then lead into

recovery, or activate downstream participation of innate immune system. The interplay between

proinflammatory and anti-inflammatory components of innate immune system determines the

ensuing outcome: overt liver injury versus repair or recovery.



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Factors that affect NAPQI production and its detoxification conse-

quently influence the threshold for APAP hepatotoxicity. CYP2E1 is the

main isoenzyme for APAP metabolism; hence, increased activity of thisisoenzyme can lower the threshold for APAP hepatotoxicity and vice versa.

CYP2E1-null mice are protected against APAP, whereas induction of the

isoenzyme by isoniazid or ethanol has been shown to increase the suscepti-

bility to APAP [2931]. It is not surprising that chronic alcoholic patients

have been reported to develop hepatotoxicity at dosages only modestly

higher than the maximum recommended[3237].

The regulation of GSH synthesis also influences APAP hepatotoxicity.

NF-E2-related factor 2 (Nrf2) is a transcription factor that regulates GSH

synthetic and detoxification enzymes[38]. Nrf2-null mice have been shownto be more susceptible to APAP hepatotoxicity [39,40]. Nrf2 activity is

repressed by a cytoplasmic protein Keap1, which binds to it and promotes

its degradation [41]. Hepatocyte-specific deletion of the Keap1 gene has

been shown to activate Nrf2 and protect against APAP hepatotoxicity

[42]. In addition, it seems that GSH synthesis is dynamically regulated as

Nrf2 is activated even with low-dose APAP [38]. The provision of GSH is

the mechanism behind the use ofN-acetylcysteine for APAP hepatotoxicity

because it is a cysteine precursor for GSH synthesis leading to replenishment

of GSH in hepatocytes.Although one fact concerning the toxic mechanism of APAP is indisput-

able, namely that as a prerequisite for toxicity, excessive NAPQI production

(exposure) and marked hepatic GSH depletion need to occur, the mechanism

of cell death downstream of NAPQI remains incompletely understood. It has

been believed that unopposed covalent binding of NAPQI to cysteine groups

on cellular proteins forming APAP-protein adducts, particularly in mito-

chondria potentiated by mitochondrial GSH depletion, leads to dysfunction

of mitochondria, oxidative stress, and nuclear damage, and ultimately cell

death ensues[27,4348]. Evidence has emerged, however, that NAPQI cova-lent binding or GSH depletion, probably by mitochondrial oxidative stress,

triggers intracellular metabolic processes (eg, signaling pathways), which

recruit intrinsic cell death machinery and promote apoptosis or necrosis.

JNK, a stress kinase protein, has been demonstrated to play a pivotal role

in murine APAP hepatotoxicity [49]. APAP induces a sustained activation

of JNK and inhibition of JNK markedly protects against APAP despite the

comparable extent of covalent binding and GSH depletion [49]. Inhibition

of JNK has also been shown to protect in one other hepatotoxicity model,

ischemia-reperfusion injury[50,51].The JNK activation is likely induced by oxidative stress, either through

redox alteration of the sequestration of JNK or other upstream kinases

by thioredoxin and GSH S-transferase or through inhibition of JNK phos-

phatase[5254]. It is unclear if the effect of APAP on this pathway is caused

by an effect on protein thiols as a direct consequence of GSH depletion,

change in the GSH/GSSG ratio, or the action of H2O2 released from



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mitochondria. JNK is presumably not activated by a TNF receptor

dependent mechanism in response to APAP because JNK inhibitor protected

against APAP in TNF receptor-1null mice[49].There are two JNK isoenzymes in liver: JNK1 and JNK2. It is unclear

which isoenzyme is responsible for the cell damage. Some work has suggested

that JNK1 may promote cell death, whereas JNK2 may promote proliferation

and survival [5557]. Recent evidence using JNK1 and JNK2 knockout

mice or selective silencing of either isoenzyme with antisense oligonucleotide,

however, shows that JNK2 plays a greater role in APAP hepatotoxicity[49].

The precise target of JNK in the pathogenesis of APAP toxicity is un-

clear. Strong candidate targets of JNK are the Bcl-2 family members and

mitochondria proteins. JNK has been shown to influence both proapoptoticand antiapoptotic Bcl-2 family members[5861]. In addition, Bax transloca-

tion to mitochondria has been observed in vivo after APAP treatment,

which was blocked by JNK inhibitor[49]. Bax translocation alone is likely

insufficient to be lethal to hepatocytes. APAP-induced mitochondrial GSH

depletion and covalent binding, however, may render mitochondria more

susceptible to JNK perturbations in the Bcl-2 family. Moreover, other

Bcl-2 family members may contribute as targets of JNK because there is

evidence that Bid, Bim, and Bax translocate to mitochondria in response

to APAP treatment[62]. In their work, the authors did not see tBid forma-tion or Bim translocation. They did observe, however, that JNK itself and

phospho-JNK translocate to mitochondria, suggesting other mitochondrial

targets (Fig. 2). Phosphorylation of Bcl-XLhas been observed in response to

JNK, which can inactivate a protective mechanism. Because the major form

APAP

CYP 2E1

NAPQI

Mitochondria ROS Sustained JNK

activation

MPT

TranslocationBax

? otherNECROSIS

mitochondrial GSH

covalent binding

P-Bcl-XL

Fig. 2. The role of JNK in APAP toxicity in hepatocytes. Oxidative stress (increased ROS)

occurs in response to effects of NAPQI on mitochondria, which leads to sustained activation

of JNK. JNK then promotes translocation of death-inducing proteins to mitochondria, includ-

ing JNK, and leads to mitochondrial permeability transition (MPT) and necrotic cell death.



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of cell death in vitro and in vivo in APAP toxicity is necrosis [63], it is likely

that the JNK-initiated events do not lead to apoptosis because of the severe

injury to mitochondria, consequent oxidative stress, and insufficient ATPlevels required to sustain propagation of the apoptosis cascade. Irrespective

of exactly how JNK leads to cell death, the fact that JNK inhibition protects

against APAP, even when administration of inhibitor is delayed until after

covalent binding and GSH depletion have reached maximal levels, strongly

supports the concept that APAP induces a type of programmed necrosis

downstream of APAP metabolism.

Another very interesting aspect of cellular injury from APAP is the possi-

ble role of toxic protein mediators (eg, calpains and DNase) from hepatocytes

undergoing necrosis leading to the injury of adjacent hepatocytes, apparentinnocent bystander effect. Limaye and colleagues[64,65]have demonstrated

that a cell-impermeable calpain inhibitor limited progression of liver injury

from APAP and CCl4. Overexpression of calpastatin, an endogenous calpain

inhibitor, also protected[65]. In addition, Dnase 1 knockout were protected

because of inhibition of progression of necrosis from the pericentral-most

hepatocytes [66]; Dnase 1 is released from wild-type hepatocytes, which

undergo necrosis[66].

Role of innate immune system in acetaminophen hepatotoxicity

The severity of APAP liver injury may be influenced by the innate

immune system. It is well established that hepatocellular necrosis can induce

an inflammatory response [24]. Recent studies have demonstrated that

chromatin protein high mobility group box-1 released from necrotic cells

can trigger inflammatory response, and neutralization of high mobility

group box-1 leads to decreased APAP-induced inflammatory cell infiltration

in the liver [67,68]. It has also been shown that APAP-induced hepatocyte

damage activates the innate immune system, which then releases inflamma-tory mediators, such as cytokines, chemokines, reactive oxygen, and nitro-

gen species, and these events contribute significantly to the severity of the

liver injury[6973]. Furthermore, different innate immune system knockout

or mutant mice have been shown to have altered susceptibility to APAP

hepatotoxicity. Knockout or mutant mice that lack IFN-g, lipopolysaccha-

ride-binding protein, Fas or FasL, or CXC chemokine receptor 2 (CXCR2)

are less susceptible to APAP hepatotoxicity[69,7476]. Knockout mice that

lack IL-10, IL-6, or C-C chemokine receptor 2, however, are more suscep-

tible [7779]. In most of these knockout mice, the altered susceptibilityto APAP hepatotoxicity is not associated with significant change in GSH

depletion or APAP covalent binding. In addition, these mice demonstrate

that in response to nontoxic dose of APAP, there is stimulation of the

expression of both proinflammatory and anti-inflammatory cytokines, and

the tendency of favoring expression of one type more than the other leads

to increased or decreased susceptibility to APAP [5].



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NK and NKT cells are examples of cells that are important components

of the innate immune system and play a pivotal role in the APAP hepato-

toxicity. These cells are abundant in the liver and account for 20% to50% of isolated liver leukocytes [24,8084]. NK cells are specialized in de-

tecting aberrant cells, such as cells that have been infected, transformed,

or stressed. NK cells subsequently destroy the cells directly or generate cy-

tokines and chemokines, which activate other components of the immune

system[25,85,86]. NKT cells recognize antigens in the context of major his-

tocompatibility complex Ilike molecule CD1d and are capable of rapidly

producing cytokines, including IFN-g and IL-4 when activated [24,87]. In

APAP hepatotoxicity, depletion of both NK and NKT cells provided signif-

icant protection[69]. This protection is associated with inhibition of mRNAexpression for IFN-g, FasL, and chemokines, and reduced neutrophil accu-

mulation in the liver [69]. These results suggest that depletion of NK and

NKT cells might prevent APAP hepatotoxicity not only by absence of direct

cytotoxicity but also by preventing the production of proinflammatory cyto-

kines and chemokines. The predominant source of IFN-g in liver injury is

NK and NKT cells [24,69]. The signals that activate NK and NKT cells

have not yet been identified but could include cytokines produced by

Kupffer cells in response to initial hepatocyte damage or altered expression

of activating or inhibitory ligands in hepatocytes in response to APAP [5].The role of Kupffer cells in APAP hepatotoxicity has been suggested by

several studies but the results are controversial. APAP treatment causes an

increase in Kupffer cell numbers in the liver [24,8890]. Two studies found

that mice pretreated with macrophage inhibitors (gadolinium chloride and

dextran surfate) are more resistant to APAP hepatotoxicity [88,89]. It is

believed that Kupffer cells participate in injury through the production of

cytokines and reactive oxygen and nitrogen species [88,89]. A recent study

by Ju and colleagues [90], however, found that Kupffer celldepleted mice

had significantly increased susceptibility to APAP-induced liver injury.These mice were pretreated with liposome-entrapped clodronate (liposome-

clodronate), which has been shown to cause nearly complete depletion of

Kupffer cells from the liver [9092]. The discrepancy may be caused by in-

complete depletion of Kupffer cells by gadolinium chloride[90]. Ju and col-

leagues [90] also found that Kupffer cell depletion by liposome-clodronate

led to significant decreases in the levels of hepatic mRNA expression of

several hepatoregulatory cytokines and mediators, including IL-6 and

IL-10, suggesting that Kupffer cells are a significant source for production

of these cytokines. The protective role of IL-6 and IL-10 in APAP hepato-toxicity is discussed further. Moreover, one study found that the protection

afforded by Kupffer cell depletion only lasted for 2 hours [93].

Neutrophils may also contribute to APAP hepatotoxicity. It is known

that APAP treatment results in increased numbers of neutrophils in the liver

[69,94,95]. It is unclear, however, if the neutrophils play a role in the liver

injury itself, contributing to the severity of organ damage, or only serve



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to remove the debris after the liver injury has occurred. Animals depleted of

neutrophils with antibodies against neutrophils are more resistant to APAP

hepatotoxicity [74,94,96]. This protection occurs despite unaltered GSHdepletion or APAP covalent binding[96]. The neutrophils are likely to dam-

age hepatocytes through cytotoxicity and generation of reactive oxygen

intermediates and FasL expression of hepatic leukocytes [9698]. Lawson

and colleagues [95], however, demonstrated that neutrophils play a role in

removal of cell debris and do not directly contribute to the damage to the

hepatocytes. They recently demonstrated that neutrophil depletion with

anti-Gr-1 may lead to increased expression of hepatocellular metallothio-

neine, which may protect against APAP. The data available, although

suggesting a role of neutrophils, are not definitive. More importantly,however, irrespective of the uncertainty regarding the pathophysiologic

role of neutrophils, the participation of inflammatory mediators in APAP

toxicity is strongly supported by existing data from multiple laboratories.

Several proinflammatory cytokines have been demonstrated to play a role

in APAP hepatotoxicity. IFN-gnull mice have decreased susceptibility to

APAP implicating its critical role in APAP hepatotoxicity[69,74]. TNF-ais

another proinflammatory cytokine whose role is more controversial. Some

studies found that immunoneutralization of TNF-aor mouse lacking TNF

receptor-1 were protected against liver injury induced by APAP [99,100].TNF-ais produced by activated Kupffer cells and its production is increased

after APAP treatment[70,96]. The role of TNF-a in APAP hepatotoxicity

seems to be complex and controversial, however, because recent studies did

not demonstrate the protection against APAP in TNF-aor TNF receptor-1

gene-deficient mice[101103]or in a study using TNF-ainhibition[104]. In

addition, at high doses, TNF-a causes liver damage but mice lacking TNF

receptor-1 are deficient in liver growth and regeneration [105]. Opposing

injury and repair mechanisms may account for the varied findings.

Other cytokines, such as IL-6 and IL-10, provide protection againstAPAP hepatotoxicity. IL-6 null mice and IL-10 null mice are more suscep-

tible to APAP[77,78]. The susceptibility of IL-6 null mice is associated with

a deficiency in the expression of cytoprotective hepatic heat-shock proteins

[78], whereas in IL-10 null mice, the susceptibility is associated with elevated

expression of TNF-a, IL-1, IFN-g, and iNOS, suggesting that IL-10 is

anti-inflammatory[77].

Chemokines, such as keratinocyte-derived chemokine, macrophage

inflammatory protein-1a, macrophage inflammatory protein-2, monocyte

chemoattractant protein 1, and IFN-ainducible protein (IP-10) are alsobelieved to play a significant role in APAP hepatotoxicity because these che-

mokines are up-regulated in animals after APAP treatment [69,74,99,103].

Similar to data on TNF-a, it is controversial whether they are proinflamma-

tory or anti-inflammatory [24]. For example, CXCR2 is an important

chemokine receptor in controlling neutrophil migration and Ishida and col-

leagues [76] found that CXCR2-null mice are protected against APAP



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hepatotoxicity. In addition, its protection is associated with reduced neutro-

phil infiltration in the liver [76]. Mice lacking C-C chemokine receptor 2,

however, a different chemokine receptor, were more sensitive to APAP [79].A key question regarding the innate immune response is whether it leads

to modulation of direct killing of hepatocytes, perhaps sensitized by the

effects of drugs (eg, GSH depletion), or the modulation of the toxic mech-

anisms in the hepatocyte (eg, expression of protective cellular mechanisms).

Summary

It seems that the biochemical changes in hepatocytes (eg, GSH depletionand covalent binding) or immune-mediated response induced by toxic

metabolites are required to cause hepatotoxicity. Toxic metabolites may

induce intracellular stress (oxidative or organelle specific) leading to the

activation of built-in death programs for apoptosis or necrosis. The partic-

ipation of innate immune system downstream of the biochemical events

induced by toxic metabolites contributes to the severity of liver injury.

The interplay of proinflammatory and anti-inflammatory mediators may

determine whether a specific drug causes severe injury or no injury. Both

environmental factors and genetic differences in cellular responses to stressand the innate immune response may account for different susceptibilities

between individuals to drug-induced liver injury.

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Causality Assessment of Drug-Induced

Hepatotoxicity: Promises and Pitfalls

Max A. Shapiro, MD, James H. Lewis, MD*

Hepatology Section, Division of Gastroenterology, Georgetown University Hospital,

Georgetown University Medical Center, Washington, DC 20007, USA

Art is science made clear.

dJean Cocteau, 1926

Drug-induced liver injury (DILI) remains an important clinical concern,

accounting for 4% to 10% of all adverse drug reactions [1,2]. It has been

diagnosed in about 1% of general medical inpatients [3], for 10% to 33%

of patients presenting with acute hepatitis [4,5], and for 5% to 10% of con-

sultations performed in hepatology practices[6,7]. DILI is the leading causeof acute liver failure in the United States[8]with acetaminophen also top-

ping the list of drugs causing acute liver failure leading to emergency liver

transplantation [9]. DILI is among the most frequent reasons for drug-re-

lated regulatory actions [10], including nonapprovals, restriction of use,

and removal of drugs from the market [11].

Popper and colleagues [12] described DILI as a penalty for progress

nearly half a century ago, but the ability to ascribe hepatic injury confidently

to a specific drug remains a challenging and often difficult pursuit. The lack

of a specific biomarker or characteristic histologic feature to identify a drugas causal further hampers this effort and has fostered reliance on clinical as-

sessment techniques that are based largely on medical judgment and expert

opinion rather than on a truly objective means of assessing causality accu-

rately. As a result, depending on the knowledge and experience of the clini-

cian performing the diagnostic evaluation, the final assessment may lack

precision and seems at times to reduce the process to one of making a diag-

nosis of exclusion based on circumstantial evidence. The consequences of

erroneously attributing the cause of hepatic injury to a drug can be dire

* Corresponding author. Georgetown University Hospital, Room M2408, 3800 Reservoir

Road, NW, Washington, DC 20007.

E-mail address: [email protected](J.H. Lewis).



Clin Liver Dis 11 (2007) 477505


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for patients and health care providers and for the pharmaceutical industry

and regulatory bodies as well.

Although a number of thoughtful efforts have been undertaken duringthe past 2 decades to create causality-assessment instruments that attempt

to objectify clinical findings and impressions using numeric scales [1316],

none has proven infallible in creating a true reference standard to diagnose

DILI in all of its forms and disguises[17,18]. This article explores the ongo-

ing challenges inherent in what is currently a clinical process of elimination

made in the attempt of assigning causality in DILI. In particular, it points

out the shortcomings and pitfalls that often limit the applicability of the cau-

sality-assessment methodologies (CAMs) currently in use around the globe.

It is anticipated that ongoing advances in pharmacogenomics, toxicology,and understanding of the mechanisms of hepatocyte injury eventually will

improve the ability to diagnose DILI in the not-too-distant future. For

the time being, clinicians must make the best use of the available causality

methods, imperfect though they may be.

Historical basis of assessing causality

Causality assessment began as more of an art than a science. A number of

early studies explored the process by which a variety of nonorgan-specificadverse drug reactions were assessed [1924]. Arimone and colleagues [25]

recently reviewed these methods and divided the nearly 2 dozen approaches

into three main categories: expert judgment, probabilistic methodologies,

and algorithms. In general, none was considered highly satisfactory. For ex-

ample, they cited evidence that expert judgment was limited by subjectivity

and a lack of standardization leading to poor reproducibility among the ex-

perts. Probabilistic methods, mostly derived from Bayes theorem, require

precise information to model probability and therefore are not considered

appropriate for routine clinical use. The algorithmic approach to causality,although relatively simple to apply in theory, was thought to be less useful

because of potential bias in the often arbitrary weighting of each criterion

[25]. Many of the methods relying on an algorithm were based on guidelines

developed by the World Health Organization (WHO) and others [25,26],

which considered time to onset, the response to dechallenge and rechallenge,

a search for nondrug-related causes, risk factors for the reaction, and pre-

vious reports of the reaction to determine if a particular reaction is caused

by a specific drug. Arimone and colleagues[25]recently proposed a weight-

ing process based on statistical regression of modified WHO criteria thatthey applied to randomly selected adverse drug reactions affecting various

organ systems from a French pharmacovigilance database. They were able

to demonstrate good agreement between their weighted probability scores

and expert opinion (which was based on a visual analogue scale) but ac-

knowledged that further refinements in their weighting tool would be needed

before such a method could be more widely applied [25].

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As several investigators noted [21,2325], reliance on clinical judgment

alone often is inadequate for determining causality, based on the low levels

of agreement between evaluators (expert or not) and the poor correlationwith objective methods, as summarized in Table 1. As can be seen, signifi-

cant differences often exist among evaluators: the rate of complete agree-

ment between any two experts ranged from 41% to 57% [13,20]. With the

use of a CAM, however, Naranjo and colleagues[20]showed that interrater

agreement rose from 83% to 92%. Similarly, the use of a clinical diagnostic

scale improved interrater agreement to 86% in a more recent study [16].

Getting more than two evaluators to agree on the level of causality cer-

tainty, however, has proven to be quite challenging: three evaluators were

in agreement in 40% to 60% of cases [16,27], but five evaluators were inagreement in only 17% of cases [13]. Moreover, the majority of the agree-

ment occurred for causality that was at the far ends of the assessment scale

(ie, either excluded or definitely related). Classifying a reaction as pos-

sibly related or probably related proved more difficult. When clinical

judgment was compared with an objective scale, the correlation was even

less accurate. In one study, there was complete agreement with physicians

opinion in only 6% of adverse drug reactions [21]. Allowing for one level

of discrepancy improved agreement somewhat (49%) but at the risk of di-

luting the precision of the assessment[21]. These authors attributed the rel-atively poor level of agreement to the tendency of physicians in their study

to form fairly strong opinions based on only a few clinical criteria [21].

Agreement between clinical judgment and a CAM seems to improve over

time as evaluators become more familiar with scoring criteria. For example,

in a recent study of the Roussel Uclaf Causality Assessment Method (RU-

CAM) in a clinical trial setting, the kappa statistic measuring interrater

agreement improved from the first 50 cases assessed to the final 50 cases

[28,29].

Causality assessment method and drug-induced liver injury

Zimmerman and other early pioneers in the field of hepatotoxicity

employed many of the same components found in the current WHO

guideline [26] and other authors criteria for causality assessment of

drug reactions in general [19,20,25] and applied them to a common-sense

clinical approach to establishing the cause of suspected DILI [30]. Exam-

ining the circumstances of the liver injury, the host factors, the clinico-pathologic features of the reaction, its course and outcome, excluding

other causes, and comparing the reaction with the known spectrum of

injury associated with that agent formed the basis of DILI causality as-

sessment based on expert opinion that has been increasingly refined over

the years within the limitations of diagnostic tools and clinical acumen

(Box 1).

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Table 1

Clinical judgment and expert opinion in causality assessment

Study Comparison Type of reaction

Method of assess

and levels of caus

Blanc et al[27] Experts assessments All-cause ADRs Questionnaire 5 l

Naranjo et al [20] Clinicians assessments

CAM

All-cause ADRs Probability scale

Arimone et al[13] Experts assessments All-cause ADRs VAS 7 levels

Miremont et al [21] Physician opinion versus

CAM

All-cause ADRs French scale[22]

Danan et al[14] Experts assessments Hepatotoxicity RUCAM[14,15]

Maria and Victorino

[16]

Experts assessments Hepatotoxicity CDS[16]5 levels


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Aithal et al [40] Physician reports versus

CAM

Hepatotoxicity Modified CIOMS

3 levels

Aithal et al [42] Physician suspicion

versus CAM

Hepatotoxicity Modified CIOMS

and CDS[16]

Meier et al[3] Physician diagnosis

versus CAM

Inpatient hepatotoxicity WHO criteria[26

2 levels

Lewis et al [28] Physician opinion versus

CAM

Clinical trial

of hepatotoxicity

of a single agent

RUCAM[14,15]

Abbreviations:ADR, nonorgan-specific adverse drug reaction; CAM, causality method assessment; CDS, Clinica

International Organizations of Medical Sciences; RUCAM, Roussel Uclaf Causality Assessment Method; VAS, visa See individual studies for definitions.


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In an effort to improve on the early efforts at ADR causality, a number of

objective methodologic instruments have been introduced during the past 2

decades to assess DILI specifically. The two most widely used of thesemethods are the RUCAM[14,15]and the Clinical Diagnostic Scale (CDS)

[16]. A third method is being studied is the DILI Network approach to cau-

sation, which represents the latest attempt to objectify expert opinion [10].

Roussel Uclaf Causality Assessment Method

The RUCAM was developed at the request of the Council for Interna-

tional Organizations of Medical Sciences (CIOMS) by an internationally

recognized panel of experts brought together by Danan and Benichou of

the Drug Safety Department of the French pharmaceutical maker Roussel

Box 1. Elements used in the clinical assessment of acute

drug-induced liver injury

1. High index of suspicion that a drug is responsible

2. Time of onset (latency)

3. Clinical features4. Biochemical injury pattern (hepatocellular, cholestatic,

mixed); aspartate aminotransferase/alanine

aminotransferase ratio and maximum absolute

liver-associated enzyme values

5. Mechanism of injury (eg, intrinsic, hypersensitivity,

metabolic idiosyncrasy)

6. Extrahepatic symptoms

7. Clinical/biochemical course of the reaction (response to

dechallenge, rechallenge; and adaptation [drug tolerance])8. Drug serum levels, drugprotein adducts, lymphocyte

transformation tests, and others

9. Histologic findings

10. Genetic markers, polymorphisms

11. Exclusion of other causes of acute drug-induced liver injury

Serology for viral hepatitis A, B, C, D, E, cytomegalovirus,

Epstein-Barr virus, other viral causes

Metabolic, autoimmune causes

Alcoholic liver disease Shock, sepsis

Gallstones

Muscle injury

Postoperative jaundice

Pregnancy-related liver injury

482 SHAPIRO & LEWIS


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Uclaf in 1989 and 1990[14,15]. A major goal was to adapt existing methods

for assessing nonorgan-specific drug reaction to well-defined hepatic reac-

tions[31,32]. From these meetings, a consensus opinion emerged that culmi-nated in the construction of a causality assessment method based on seven

major criteria that were in common use at the time: (1) time to onset, (2)

course of the reaction, (3) risk factors for the reaction, (4) assessing the

role of concomitant therapies, (5) screening for nondrug-related causes,

(6) weighing the information known about the DILI in question, and (7) con-

firmation of the reaction by positive rechallenge or in vitro assays (Table 2).

Each criterion was assigned a score ranging from 3 to 3 corresponding

to the probability of the involvement of the drug being evaluated. Maximum

total scores ranged from 7 to 14, defining the following causal relation-ships: a causal relationship was excluded with a score of zero, unlikely with

a score of 1 or 2, possible with a score of 3 to 5, probable with a score of 6 to

8, and highly probable with a score higher than 8. The panel distinguished

between hepatocellular reactions and those that were cholestatic or mixed

based on the ratio of aminotransferase levels to alkaline phosphatase

(AP). Reproducibility of the scoring system was assessed by an independent

team that reviewed a series of 50 case reports of acute DILI received by the

Bordeaux regional pharmacovigilance center. All four of these independent

experts were in agreement in only 37% of cases, but three experts agreed in74% of cases, and two experts agreed in 99% of cases [14]. The scoring sys-

tem then was validated by comparing 49 case reports of drug-induced acute

liver injury with positive rechallenge from the literature and 28 well-matched

controls. Sensitivity of 86% and specificity of 89%, a positive predictive

value of 93%, and a negative predictive value of 78% were achieved, with

no overlap between the cases and the controls [15].

Since the RUCAM first appeared, it has been used most widely outside

the United States to validate published case reports and case series

[33,34]. Lee and colleagues[29]described the successful application of RU-CAM scoring to detect the hepatotoxicity of a new agent undergoing clinical

trial testing when no prior information about the potential reaction existed.

The Maria and Victorino clinical diagnostic scale

The complexity of the RUCAM prompted Maria and Victorino[16]from

Portugal to propose and validate a somewhat simpler scoring system to assess

DILI. These authors constructed a CDS, based on a modification of the RU-CAM criteria, that also used the time to onset and time course of the reaction,

the exclusion of alternative causes, the response to re-exposure (by intentional

or accidental rechallenge), and previous reports in the literature implicating

the drug. They added a fifth criterion based on the presence of extrahepatic

manifestations of DILI, specifically fever, rash, eosinophilia, arthralgias,

and cytopenia. The CDS then was validated against the opinion of three

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Table 2

Comparison of criteria used in RUCAM versus CDS for acute drug-induced liver injury

Assessment component

and injury pattern RUCAM[14,15]

Points

awarded CD

Time to onset from start of drugHepatocellular type 590 days (IT), 115 days (ST) 2 45

!5 or O90 days (IT), 15 (ST) 1 !4

Cholestatic or mixed type 590 days (IT), 190 days (ST) 2 !4

!5 or O90 days (IT),

O15 days (ST)

1

Time to onset from cessation of drug

Hepatocellular type !15 days 1 !7

Cholestatic or mixed type !30 days 1 O

Time to enzyme normalization

after cessation of drug

Hepatocellular type Decrease O 50% within 8 daysDecrease O 50% within 30 days

Decrease O 50% after 30 days

Persistence

32

0

2

VaU

!6

O6

Cholestatic or mixed type Decrease O 50% within 180 days

Decrease ! 50% within 180 days

Persistence

2

1

0

Va

U

!

O

Risk factors

Alcohol use 1 N/

Pregnancy (in cholestatic/mixed only) 1

Age O55 1

Concomitant drug use

None or incompatible timing 0 N/

Compatible or suggestive timing 1

Known hepatotoxin with suggestive timing 2


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Evidence of role (positive

rechallenge or validated test)

3

Alternative causes (viral hepatitis,

biliary obstruction, alcoholic

or other liver disease, recent hypotension,

other causes)All causes reasonably ruled out

Most causes ruled out

!3 causes not ruled out

R3 causes not ruled out

Nondrug cause highly probable

2

1

0

2

3

Co

Pa

Po

d

Pro

d

Extrahepatic manifestations

Fever, rash, arthralgia,

eosinophilia O6%, cytopenia

N/A R

23

1

NoPreviously reported

hepatotoxicity of drug

In product information

Published reports

Reaction unknown

2

1

0

Pu

No

No

Response to re-exposure

Intentional or accidental Positive

Compatible

Negative

3

1

2

Po

Ne


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Table 2 (continued)

Assessment component

and injury pattern RUCAM[14,15]

Points

awarded CD

Other

Toxic plasma concentration of the drug

or validated lab test

Positive

Negative

3

3

N/

Total point range 7 to 14

Probability of drug causality Highly probable/definite O8

Probable 68

Possible 35

Unlikely 12

Excluded !1

Abbreviations:IT, initial treatment; N/A, not assessed; ST, subsequent treatment; ULN, upper limit of normal.


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experts in drug-induced hepatotoxicity, a decision-tree analysis, and the re-

sults of immunologic testing of 50 cases of suspected DILI. Scores ranged

from6 to20 and were broken down into subgroups based on the probabil-ity of a true causal relationship: a relationship was excluded by a score less

than 6, was unlikely with a score of 6 to 9, possible with a score of 10 to 13,

probable with a score of 14 to 17, and definite with a score above 17.

Although the Maria and Victorino CDS is similar to the RUCAM, it

varies in several significant ways, with differences in the time to onset of

the reaction and the course after drug has been stopped. Also, it reduces

the probability of a suspected drug

35575737 drug induced liver disease 2007

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