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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.
VOLUME 11 NUMBER 3 AUGUST 2007 v
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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
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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
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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).
1089-3261/07/$ - see front matter 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.cld.2007.06.001 liver.theclinics.com
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
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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).
1089-3261/07/$ - see front matter 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.cld.2007.06.003 liver.theclinics.com
Clin Liver Dis 11 (2007) 477505
mailto:[email protected]:[email protected] -
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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
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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
483CAUSALITY ASSESSMENT IN DILI
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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