Regulatory Toxicology and Pharmacology 56 (2010) 237246

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Commentary

Enhancing the utility of alanine aminotransferase as a reference standard biomarker for drug-induced liver injury
Josef S. Ozer a,*, Raj Chetty b, Gerry Kenna c, Joe Palandra a, Yiqun Zhang a, Anne Lanevschi c,1, Nandan Koppiker d, Bernard E. Souberbielle e, Shashi K. Ramaiah f
a

Pzer Global Research and Development, Pharmacokinetics, Dynamics, and Metabolism, 700 Chestereld Parkway West, T2A, Chestereld, MO 63017, USA AstraZeneca Research and Development, Clinical Pharmacology, Drug Metabolism and Pharmacokinetics, CPU, Alderley Park, Maccleseld, SK10 4TG, Cheshire, UK AstraZeneca Research and Development, Safety Assessment, Alderley Park, Maccleseld, SK10 4TG, Cheshire, UK d Pzer, Emerging Markets Business Unit, NY, NY 10017, USA e Pzer Global Research and Development, Clinical Research, CT13 9N5 Sandwich, UK f Pzer Global Research and Development, Drug Safety Research & Development, Chestereld, MO 63017, USA
b c

a r t i c l e

i n f o

a b s t r a c t
Drug-induced liver injury (DILI) is the most frequent cause of discontinuation of new chemical entities during development. DILI can either be intrinsic/predictable or an idiosyncratic type. These two forms of DILI are contrasted in their manifestation and diagnosis. Even with regulatory guidance (FDA, 2009), there is still a gap in our ability to identify predictable DILI, both specically and sensitively. Alanine aminotransferase (ALT) is the principal reference standard biomarker to diagnose DILI, yet its current application in preclinical to clinical translation for decision-making purposes has imperfections: (1) analytical ALT assay uniformity across industry would be aided by common analytical processes; (2) assessment of ALT toxicological performance in a large preclinical analysis would help to establish a true threshold of elevation for predictable DILI and improve translational use across various stages of pharmaceutical development and nally, (3) clinical evaluation of ALT elevations prospectively and retrospectively is recommended to dene and manage variations in clinical study subjects including rising body mass index (BMI) range and ALT upper limit of normal (ULN) in the broader population over time. The emergence of new hepatotoxicity biomarkers necessitates a parallel and equivalent assessment to the aminotransferases in a regulatory qualication model. 2009 Elsevier Inc. All rights reserved.

Article history: Received 6 July 2009 15 January 2010 18 January 2010 Available online 10 November 2009 Keywords: ALT AST BMI DILI Hepatotoxicity Hys law ULN, upper limit of normal

1. Introduction This perspective highlights both the successes and imperfections of ALT in its current use to assess drug-induced liver injury or DILI. We outline the advantages and impact of a broadly applied analytical validation of ALT and enhanced clinical assessment that would add value to decision making for drug development (Table 1). Although ALT as a biomarker has limitations, it is recognized as the principal preclinical and clinical biomarker driving diagnosis of DILI, which is a leading cause of attrition throughout drug discovery and development, of unsuccessful drug registration, and of post-marketing drug withdrawal (Kaplowitz, 2005; Lee, 2003; FDA, 2009). In man, the most frequent patterns of drug hepatotoxicity are hepatocellular (affecting hepatocytes), cholestatic (affecting the biliary system) and mixed hepatocellular/cholestatic (Kaplowitz, 2005; Lee, 2003). Use of the terminology predictable

DILI versus idiosyncratic DILI will be used throughout this article, while noting that signicant and important considerations differ between the two types of injuries. A key objective of the process to increase the utility of aminotransferase activities would be to reduce the incidence of DILI-causing compounds that progress through safety assessment, into clinical trials, and ultimately to the market. Additionally, the incidence of DILI should be detected earlier in the drug pipeline, but there is a major gap in our available safety biomarker tool kit to optimally achieve this goal. Thus, key gaps in our understanding of DILI that we seek to address include: (1) the complexity and differences in diagnosing DILI in animals and man, (2) dening causality of drug administration in human idiosyncratic DILI, (3) the impact of animal subject variability in susceptibility to DILI, (4) and dening the limitations in the specicity of ALT detection of DILI in preclinical and clinical studies. 1.1. Predictable and idiosyncratic DILI share common attributes and key differences Each type of drug-induced liver injury shares some common attributes, but have key differences. Idiosyncratic DILI is largely

* Corresponding author. Fax: +1 636 247 1543. E-mail address: josef.ozer@pzer.com (J.S. Ozer). 1 Present address: Televetdiagnostics Ltd., 2 Somerset Close, Congleton CW12 1SE, UK. 0273-2300/$ - see front matter 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.yrtph.2009.11.001

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Table 1 Summarized recommendations to enhance the utility of AST and ALT as reference standards of DILI. Regulatory Historical biomarkers (ALT, AST, bilirubin) to be benchmarks in qualication of new biomarkers Consider inuence of systemic non-liver diseases on aminotransferase and/or bilirubin changes Patients with underlying cirrhotic conditions or chronic hepatitis C infection could be inuenced by reduced hepatocellular cellular mass, and therefore the ALT >3 ULN threshold limits rule may need to be applied appropriately. Whether pre-existing liver disease predisposes subjects to drug-induced liver injury and guidelines for these patients to be considered as a special case Clinical ALT or AST >3 ULN to be conrmed by similarly assessed elevations in GLDH or ALT mass assay like ELISA (Biomarker Rule of Two) when such assays are validated or available, respectively. Histopathology and biopsy not readily available as an anchor for aminotransferase elevations in the clinic Blood collection and banking from human subjects for DNA sampling uniformly in pharmaceutical drug development for eventual future retrospective pharmacogenetic analysis Single clinical laboratory ALT methodology per trial to ensure assay uniformity in Phase I & II studies Utilization of baseline ALT values as a reference may increase the monitoring window for patient safety at an individual level in a way that broad denition ranges of ULN may not currently provide Preclinical Histopathology to be a principal reference standard for historical and new biomarkers Histopathology and aminotransferases considered equivalent weighted reference standard biomarkers, although the responses can be dissimilar in certain contexts ALT or AST >3 ULN to be conrmed by GLDH or ALT mass assay like ELISA (Biomarker Rule of Two) when assays are validated and available, respectively. An ALT and AST elevation predictable threshold for injury to be dened using exploratory and retired developmental compounds (anchored to histopathology) in ROC analyses Analytical Ultimately, a single analytical ALT enzymatic method to be validated and recognized by industry The universality of PLP supplementation to enzymatic ALT assays needs to be assessed. ULN ranges based on broad population analysis (universal) and compared with historical reference ranges generated from an individual clinical laboratory Individual pretest and vehicle groups to be allowed as references for biomarker changes Various revised ULN for distinct populations to be used prospectively as exploratory references for analysis in studies to dene susceptible patient populations for DILI Normalized (fold >ULN) and ALT (U/I) data should be reported to regulators if data comes from different clinical laboratories and or methods for a developmental program

independent of treatment dose-level and -duration while predictable DILI is dose-dependent and largely reversible with discontinuation of dosing. Both forms of DILI share common histopathologic attributes and are well detected by ALT elevations, with some notable distinctions. Some drugs cause intrinsic (predictable) DILI, which is dose-dependent and reproducible in preclinical safety assessment studies. The current safety assessment process ensures that a vast majority of compounds progressing into later stages of development have minimal potential to cause intrinsic DILI. Dosedependent and reproducible hepatotoxicity observed either preclinically or clinically is referred to hereafter as predictable drug-induced liver injury as described by Zimmerman, 1978. The biomarker attributes to dene predictable DILI are not precisely characterized, which is a primary goal to assess in this report (Table 1). Nevertheless, elevation in serum ALT >3 ULN and/or preclinical histopathologic evidence of hepatocellular necrosis and/ or biliary changes (including repair) are generally accepted, but not universally accepted denitions. Thus, further dialogue and investigations to achieve a well described and consensus denition of predictable DILI is highly recommended. Idiosyncratic DILI refers to injury dependent upon individuals constitution including but not limited to potential genetic, epigenetic, environmental exposures, and or immunological responses (FDA, 2009). Susceptibility to idiosyncratic drug-induced liver injury lacks certain dose and duration dependent features typical of toxicity responses to drug treatment (Gupta and Lewis, 2008). Temples Corollary of Hys law is dened as combined elevations in serum ALT >3 ULN and bilirubin >2.0 ULN and indicates signicant potential to manifest idiosyncratic DILI (Zimmerman, 1968, 1978, 1999; Lewis, 2006; Senior, 2006; Temple, 2006; Watkins et al., 2008) in the absence of ALP elevation (FDA, 2009). In this case, elevated bilirubin is a marker of severe liver dysfunction and not cholestasis. In cases where Hys law clinical chemistry elevations are met, it is essential to exclude other possible causes rather than drug administration (FDA, 2009). Thus, idiosyncratic DILI must also be a diagnosis of exclusion even when Hys law is met.

Idiosyncratic DILI is likely to be related in part to hypersensitivity or immune reactions and a consequence of complex genetic factors such as single nucleotide polymorphism (SNPs) in P450 cytochromes in selected individuals within the dosed population (Ju, 2005; Holt and Ju, 2006; Lucena et al., 2008; Pachkoria et al., 2008). A mortality or transplantation rate of up to 1015% and in certain cases even more than 50% has been observed in patients with idiosyncratic liver injury induced by drug treatment (Lewis, 2006; Hussaini and Farrington, 2007). An acute liver failure incidence of 1:10,000 in patients dosed with troglitazone is a comparator for other compounds that induce idiosyncratic DILI (Senior, 2003; Goldkind and Laine, 2006; Lewis, 2006).

2. Discovery of novel prognostic biomarkers of idiosyncratic DILI Identication of biomarkers that are prognostic for idiosyncratic DILI response are in great need, yet a lack of appropriate preclinical models make this a challenging problem to solve, even for pharmaceutical consortia. The manifestation of two idiosyncratic DILI instances and even one case may suggest serious consideration for the withdrawal of a product from market (FDA, 2009), but this must also be viewed in the context of the riskbenet of the compound, e.g. a life-saving indication, and in the context of the frequency of this toxic reaction in the target population for those administered the drug. Historically, regulators have not approved drug applications where incidence of Hys law was observed in late staged trials, although exceptions are noted (Bosentan) (Temple, 2006). Bromfenac and troglitazone were both approved by the FDA with rigorous recommendations for limited term use in patients since liver injury was a cause of concern. These recommendations were not adhered to consistently by all prescribing physicians leading to instances of drug-induced patient deaths and liver transplantations. Eventually, product withdrawal from the market was the resulting outcome (Senior, 2006). Of par-

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ticular concern was the late onset of idiosyncratic DILI after start of troglitazone dosing (average of four months) and progression of liver injury despite discontinuation of drug (Chitturi and George, 2002). Thus, idiosyncratic DILI can not be consistently resolved with revised clinical dosing once observed, shows attributes of dose independence, and shows a high mortality/transplantation risk (>10%). It is noteworthy that nearly 80% of the patients with idiosyncratic DILI recover with drug discontinuation (Chalasani et al., 2008). As a matter of good clinical practice, and at least within the context of a voluntary informed consent basis, we recommend blood collection from human subjects for DNA sampling uniformly in pharmaceutical drug development in all clinical studies for eventual future retrospective pharmacogenetic analysis (Table 1). Not all, if any, sample will be analyzed prospectively but they will be analyzed retrospectively if a DILI event occurs (or for that matter in any other cases of emerging safety concern) with the aim to determine the possible genetic component of this safety signal. The cost of this proposed collection is a fraction of the cost to withdrawal just a single major pharmaceutical product for idiosyncratic DILI. The most difcult and costly aspect of this approach is the long term storage of the samples, which should be cost-shared among the different stakeholders in this process. The DILIN network is collecting and banking samples consistent with our proposal (Fontana et al., 2009) and this approach is supported by the Research Opportunities section of the Guidance (FDA, 2009). Since idiosyncratic DILI occurs infrequently and only in susceptible drug treated patients, conventional preclinical safety testing is not considered the most suitable avenue for the detection this injury (Lee, 2003; Kaplowitz, 2005). Observing Hys law preclinically in Good Laboratory Practice (GLP) studies is rare and would likely halt further development of a lead compound candidate, although observing Hys law at extremely high treatment doses in early range nder toxicity studies presents complex considerations for further development. Preclinical safety testing strategies would need to incorporate novel genetic and study designs to model idiosyncratic DILI preclinically, which would divert resources from the current drug development paradigm. Thus, improving detection and prognosis of idiosyncratic DILI during clinical development cannot be overcome without some modest adjustments to the current safety assessment paradigm, and considerable research investment is anticipated and needed in order to discover a novel and suitable preclinical model of idiosyncratic DILI that is widely applicable to early pharmaceutical development (Table 1). 2.1. Interpretations of Hys law relative to individuals and groups in a drug trial There are criteria of Hys law that indicate the potential development of idiosyncratic DILI. The rst criterion consists of ALT/ AST >3 ULN in subjects compared to the control group in a trial; second, bilirubin >2.0 ULN (without initial ndings of cholestasis, e.g. no elevation in ALP); and lastly the absence of other putative causes for these elevations in the individual, including viral induced hepatitis, concomitant use of another hepatotoxin or toxic drug, hypotension, or congestive heart failure (Temple, 2006; FDA, 2009). Moreover, the FDA, 2009 guidance document suggests that the application of Hys law may differ in the presence of existing liver disease such as fatty liver, NASH, chronic hepatitis B or C, and in patients on drugs that treat liver disease or those on drugs that inhibit bilirubin glucuronidation, such as antiretroviral agents. Hepatic reserve will be more limited in these patients (e.g. NASH and viral hepatitis) and drug administration could induce more severe level of injury in these patients in an instance of DILI (FDA, 2009). In a prospective study of statin use in chronic liver disease

patients, a twofold elevation in ALT (over baseline values) was observed with higher incidence in placebo patients, indicating the comparative complexity of benchmarking hepatotoxic drug safety in patients with pre-existing liver disease (Lewis et al., 2007). The degree of aminotransferase elevations in patients with idiosyncratic DILI that have underlying cirrhotic conditions or chronic hepatitis C infection could be inuenced by reduced hepatocellular cellular mass, and therefore the >3 ULN ALT threshold may need reconsideration for this patient group (Table 1). FDA, 2009 guidance on application of Hys law to DILI briey discusses the inuence of systemic non-liver diseases on aminotransferase and/or bilirubin changes. In addition to ruling out non-drug related cholestasis while applying Hys law, other preexisting diseases should be similarly considered. Cardiovascular causes such as right heart failure and hypotension may cause signicant rises in ALT (ischemic hepatitis), and an associated rise in unconjugated bilirubin in 2481% of patients depending on the severity of heart failure (Dunn et al., 1973). Similarly, connective tissue disorders often involve the liver (Gitlin, 1997; Abraham et al., 2004) and elevated aminotransferases have been noted in a number of connective tissue disorders, certain hematological diseases (Omata et al., 1986), sepsis (Thiele, 2002), total parenteral nutrition (Spiliotis and Kalfarentzos, 1994), post-operative jaundice (Mastoraki et al., 2007), and inammatory bowel diseases (Chapman and Angus, 2007). Rarely, patients with skeletal muscle breakdown and disease show aminotransferase elevations >4 ULN (Nathwani et al., 2005). Creatine kinase and lactate dehydrogenase (LDH) elevation support muscle injury diagnosis initially, yet these markers subside after a few days. AST/ALT ratio of 4:1 changes to 1:1 from day 1 to 4 after insult with a persistent >4 ULN aminotransferase elevation (Nathwani et al., 2005). Designating a Hys law events as causal of idiosyncratic DILI are likely to increase in complexity due to extrahepatic disorders in the general population and we recommend a more comprehensive analysis of this potential (Table 1). Clearly, some patients in efcacy trials will have limited medical concerns outside the therapeutic indication being tested, although exceptions are being increasingly noted. Recruitment for proof of concept trials is challenging for numerous reasons and the global nature of patient recruitment is an indicator of the difculty to recruit well dened populations for compound evaluation. As post-marketing trials and marketing of a compound occurs, designating Hys law events as causal for idiosyncratic DILI becomes more complex due to either hepatic or extrahepatic disorders in the general population.

3. Predictable DILI Documenting predictable DILI in preclinical studies enables determination of safe dose-level(s) in subsequent chronic studies (i.e. one month or more). A predicted safe margin of candidate compound exposure can be modeled for dosing in rst-in human (FIH) studies in conjunction with other parameters such as pharmacokinetic/pharmacodynamic (PK/PD) data. Overall, this objective is achieved in a majority of cases. Predictable DILI is a frequent organ toxicity nding in preclinical test species, and an important cause of compound attrition and/or project delay during drug discovery and development, while clinically signicant liver dysfunction is a comparatively less frequent nding during clinical trials. The main limitations of biomarkers of predictable DILI are a lack of specicity and by extension, little prognostic power to identify the onset of rare idiosyncratic DILI events (FDA, 2009). Whether predictable DILI has any denitive relationship to the development of idiosyncratic DILI is hypothetical. Predictable DILI is often reversible with discontinued dosing at modest injury levels (ALT

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J.S. Ozer et al. / Regulatory Toxicology and Pharmacology 56 (2010) 237246 Table 2 Biomarker and platform abbreviations are indicated: ALT2, alanine aminotransferase isoform 2; AST, aspartate aminotransferase; GLDH, glutamate dehydrogenase; GSTa glutathione-S-transferase alpha; hpd, 4-hydroxyphenylpyruvate dioxygenase; MDH, malate dehydrogenase; PNP, purine nucleoside phosphorlyase; PON1, paraoxonase; SDH, sorbitol dehydrogenase; EIA, ELISA; IP, immunoprecipitation; Ozer et al., 2008. Biomarker candidate GLDH GSTa AST SDH Regucalcin Cytochrome c Arginase I ALT2 Hpd/serum protein F PON1 MDH PNP Added value to ALT activity Yes Yes Yes Yes Pending Pending Unknown Unknown Unknown Unknown Unknown Unknown Comment Platform

<8 ULN elevations), while frequent enough to be observed at any point along the compound development pipeline. Nonspecic and extrahepatic elevation of ALT can occur and additional biomarkers of predictable DILI would add value to ALT in the discrimination of such spurious signals (Ozer et al., 2008). Hepatocellular DILI dened by a ratio of ALT to ALP (alkaline phosphatase) > 5 (relative ULN applied) would indicate specicity compared to other organ injuries. (Tajiri and Shimizu, 2008). An International Life Sciences Institue (ILSI) study examined 150 compounds from 221 human toxicity events and 12 pharmaceutical sponsors (Olson et al., 2000). Preclinical data are only partially prognostic of human toxicity, with limited concordance between rodent and man (43%), nonrodent preclinical species (dog, primate) and man (63%), and better concordance with both rodent and nonrodent species and man (71%) (Olson et al., 2000). Generally, this study showed liver toxicity to have among the highest termination rate for compounds with lower concordance rates for translation of toxicity compared to other toxicities and no correlation to therapeutic class. The manifestation of predictable DILI shows variability between preclinical species and humans (Olson et al., 1998). Furthermore, marked inter-individual variability is an additional characteristic feature of predictable DILI for drugs tested in man. The reasons for this variability, and hence for individual susceptibility, are largely poorly understood and considered to be multifactorial (Lee, 2003).

Improved dynamic range Improved specicity Lagging biomarker Labile biomarker Predictive potential Predictive potential Dual platform Metabolic, Muscle form No available assay Functional biomarker Historical Historical

Enzymatic EIA, Luminex Enzymatic Enzymatic EIA EIA Enzymatic, EIA IP, Enzymatic EIA Enzymatic Enzymatic Enzymatic

4. Comments to the regulatory guidance for DILI FDA, 2009 guidance emphasizes criterion to identify idiosyncratic DILI, and to a lesser extent key aspects of predictable DILI. The guidance is mainly based on historic biomarker performance of ALT and AST. Serum ALT elevations must exceed a dened threshold to be considered an adverse injury for a given dose of compound. Regulatory guidance recommends ALT thresholds indicative of adverse injury. Companies are encouraged and may voluntarily use additional exploratory markers and methodological approaches to govern an internal decision regarding injury events. In the absence of a formal qualication of new biomarkers in conjunction with the aminotransferases, historical and reported evidence is used by regulators to govern recommended aminotransferase thresholds. Limits of historical evidence used to support biomarker guidance includes: (1) some reported preclinical data may have lacked histopathologic observations by trained and board certied pathologists and may have lacked internal peer-review of data, (2) compared ALT data from enzymatic assays with dissimilar reagent conditions (e.g. vitamin B/pyridoxal-50 phosphate/PLP inclusion or lack thereof), and (3) and by extension, a lack of a common, rigorous, and uniform aminotransferase analytical validation across academia, industry, and contract research organizations (CROs). We suggest that individual developmental project teams, if possible, maintain a common ALT analytical approach or SOP from FIH through Phase II in light of potential for variations from use of different assay reagent sources or analyzer manufacturers (Table 1). In addition, newer chemical entities (NCEs) have the potential to mask elevations in ALT activity, but this is mostly a limited concern. This type of false negative event has not been extensively documented, although administration of subchronic doses of microcystin-Leu-Arg decreases ALT synthesis, while end-stage liver disease and limiting PLP concentrations were ruled out as possible causes for decreased ALT function (Solter et al., 2000). AST elevations above ULN in the presence of ALT masking is a profound biomarker to diagnose DILI (Table 2), however large molecular weight and stable macro-AST enzyme activity has been identied in the absence of associated DILI (Caropreso et al., 2009). Therefore, additional predictable DILI biomarkers

would add value to AST to monitor injury induced by compounds that mask ALT activity. For the identication of the potential to manifest idiosyncratic DILI, fullling Hys law provides compelling evidence of clinical injury to regulators, although histological correlation might be expected if clinical samples were not a limiting factor in the clinical setting. Hence regulatory guidance must align with clinical practice where histological diagnosis is limited by the availability of clinical samples. Liver biopsy is associated with morbidity, and a rare but signicant risk of mortality, (Rockey et al., 2009) and therefore a liver biopsy is not performed in most clinical development programs for ethical and logistical reasons when a patient shows abnormal liver function tests, even with moderate clinical symptoms. In clinical practice, even acute liver failure does not always warrant a liver biopsy (Polson and Lee, 2005). In the postmarketing setting, liver biopsy may be performed when an explained sustained abnormal liver function test is observed (Skelly et al., 2001). Are aminotransferase elevations without concomitant bilirubin increases a sufcient standard biomarker to monitor for predictable DILI in the clinic? We view aminotransferases as the logical clinical reference biomarker or principal standard for predictable DILI, but how does clinical qualication of new liver biomarkers become fully enabled with limited histological diagnosis in the qualication process? The combination of ALT/AST with a new generation of biomarker tools is anticipated to improve performance over the current regulatory recommendation (Table 2). Utility and guidance for use of this multi-biomarker approach would be surely enhanced by initiation of a rigorous study that would be supported by a regulatory qualication process (Table 1). Aminotransferase elevations with preclinical histological ndings are used for prognosis of liver toxicity projections to clinical study planning regardless of associated bilirubin increases. Histopathology is the principal preclinical tool that is routinely performed to detect liver injury throughout drug development and is not simply a process triggered by aminotransferase elevations. For the clinical setting, liver biopsy is prohibitive for most programs for ethical and logistical reasons. A unication of biomarker performance is needed that includes ALT and complementary novel biomarkers that translate across preclinical and clinical platforms. A historical comparison of regulatory documents indicates variable levels of ALT elevation considered as a cause of concern for predictable DILI in the absence of a concomitant bilirubin signal. The FDA 2000 non-clinical assessment indicates that ALT or AST >35 ULN be considered an adverse event in the absence of con-

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cordant histopathology data. A more recent non-binding FDA draft recommendation indicates ALT or AST >5 ULN for >2 weeks or ALT >8 ULN in a single instance are considered adverse signals (FDA, 2009). The following scenarios are likely considerations when discontinuation of treatment is recommended by FDA, 2009:     ALT or AST >8 ULN; single instance ALT or AST >5 ULN for more than 2 weeks; multiple readings ALT or AST >3 ULN and TBL >2 ULN or INR > 1.5 ALT or AST >3 ULN with the appearance of worsening of fatigue, nausea, vomiting, right upper quadrant pain or tenderness, fever, rash or eosinophilia.

We recommend that a comprehensive analytical validation of aminotransferases activity will aid in the determination of the true threshold with a predictable DILI event (Table 1). The American Thoracic Societys ofcial statement for anti-turberculosis therapy (ATT)-induced DILI recommends treatment cessation at ALT >3 ULN in the presence of hepatitis or jaundice and ALT >5 ULN in the absence of symptoms (Saukkonen et al., 2006). ATT therapy is often reintroduced successfully with resolution of initial ALT elevations. Detailed study of the aminotransferases in conjunction with new biomarker qualications (e.g. GLDH and GSTa) is an appropriate initial phase to conduct an analytical validation of the aminotransferases since most large companies are active members of the key consortiums (Table 2). Better dened ALT and AST thresholds will allow both regulators and pharmaceutical companies to greatly improve management of DILI in the drug development process. 4.1. Recommended analytical approaches to enhance validation of ALT activity Might subtle modulations of ALT activity be caused by mechanisms other than hepatic injury? ALT, being an enzymatic activity assay, might be modulated by substrate concentration or cofactors in a subtle manner that has not yet been dened or well characterized analytically. PLP is the biologically active form of vitamin B6 that is utilized physiologically and is a cofactor for ALT or AST activity. Incorporation of PLP into the aminotransferase assays is recommended by the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) and National Reference System for the Clinical Laboratory (Vanderlinde, 1986), although limits to the amount of NADH included is an unfortunate consequence of this assay modication, thus limiting assay performance including dynamic range (Lustig et al., 1988). PLP supplementation is not universally practiced, which induces potential variability among reported data since supplementation infrequently increases ALT measurements. Uniform agreement and practices across industry regarding PLP supplementation is encouraged (Table 1). To reduce the potential for ALT assay variability among pharmaceutical trials we recommend that measurements be performed by a single laboratory during a trial and extended further for additional Phase I and II studies for a single developmental program if possible (Table 1). These practices are already commonly practiced across pharmaceutical companies, yet more uniformity should be considered. Although ALT assay coherence throughout a single developmental program is a proposed goal, it may have logistical challenges for outsourcing sample analysis, which is an increasing practice in industry. Thus, cross-validation of the ALT methodology or a common SOP among laboratories within a developmental program is desired. Ultimately, a similar ALT analytical method across all industry is a goal to enhance the utility of commonly used thresholds of elevation for prognosis of predictable and idiosyncratic DILI (Table 1). PLP levels below Lower Limit of Quantitation (LLOQ) were observed in patients with liver disease, which excluded cirrhotic

patients and may have impacted ALT or AST measurements (Anon, 1977). Carbamoyl-phosphate inhibits ALT or AST and PLP-dependent enzymes in vitro, yet the physiological and toxicological signicance remains to be fully elucidated in vivo (Pagani et al., 1994b). Aminothiols (including cysteine) and vitamin B derivatives either inhibit ALT or PLP-ALT regeneration in vitro (Pagani et al., 1994a). In order to assess the effects of the modulators on ALT activity, liquid chromatographytandem mass spectrometry (LC/ MS/MS) techniques can be potentially employed to measure plasma levels of these modulators in a multiplex approach, which would be correlated to corresponding histopathology and aminotransferase activity levels (exogenously sourced PLP would be excluded here) from individual samples. We propose that measurement of ALT protein levels by LC/MS/MS or immunoassay techniques when ALT >3 ULN would provide additional analytical evidence that elevations result from ALT leakage and not enzyme induction. Whether these proposed ALT mass based assays in development would be as sensitive as enzyme activity is currently unknown (data not shown). Vesicle exocytosis (zeiosis or blebbing) could be linked to subtle plasma ALT elevations in the absence of histopathologic injury, although there are no reports that substantiate this view. Hepatocyte blebbing has been linked to toxicant exposures that show histopathologic injury (Barriault et al., 1994; Miyoshi et al., 1996). Induction of serum ALT activity correlated with ALT transcriptional elevations (less than twofold) with lipid lowering fenobrate dosing in rats (Kobayashi et al., 2009), indicating than subtle elevations of serum ALT can occur in the absence of injury. Distinguishing between serum ALT1 and ALT2 isoforms may improve specicity compared to ALT leakage from the liver since the isoforms are differentially expressed in organs (Lindblom et al., 2007; Liu et al., 2008) (Table 2). While each isoform shows enzymatic activity in recombinant systems (Liu et al., 2008), immunodepletion and activity assays are coupled to detect activity independently from each isoform (Lindblom et al., 2007). This approach is being adapted to high throughput use and undergoing validation. Whether this assay will translate from preclinical species to man is being investigated. Further efforts to improve this approach are merited since ALT2 leakage from skeletal muscle may be related to possible muscle wasting or disease in elderly patients and not liver injury (Table 2).

5. Regulatory qualication of novel biomarkers that add information to predictable DILI assessment There are benets of a better understanding of ALT and AST as principal markers of predictable DILI in conjunction with new biomarker qualications. Although ALT is a historic standard for characterizing liver injury, it has imperfections. Subtle elevations in serum ALT activity (ALT >3 ULN) may correspond to increased ALT concentration in serum or such increases may correspond to other inuencing factors: ALT isoform modulations, elevations of ALT enzyme cofactors of metabolism or ALT induction events. Assessment to dene the threshold of ALT activity elevation that truly corresponds with adverse liver injury has not yet been rigorously evaluated. Depending upon the views of clinical directors, the threshold of ALT used for decision-making purposes may be up to 8 above ULN as a stopping criterion (FDA, 2009). This document also indicates that ALT rises even up to 20 ULN are observed for drugs like tacrine, which did not cause incidence of severe idiosyncratic DILI (Goldkind and Laine, 2006). This inconsistency over which ALT level indicates true liver injury largely results from lack of sufcient evidence to determine critical threshold limits for ALT partly from ethical risks for continued clinical dosing in the range of suspected injury thresholds. Thus, by extension, a lack

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of concordance exists for the true injury threshold to use when translating a program from preclinical to clinical assessment. Since ALT elevations can occur in the absence of observed liver injury, histopathology assessment is a critical standard to examine in a qualication paradigm. Use of preclinical samples with hepatocellular necrosis grade 1 severity is an optimal comparator in a receiver operator curve (ROC) analysis. ROC analyses usually bundle all positive hepatobiliary injury (histopathology) samples into a single bin regardless of severity grade. 5.1. The ALT upper limit of normal needs rigorous analytical denition and subsequent validation We recommend that a universal ALT upper limit of normal be sufciently dened by an analytical validation process (Table 1). Currently, the ALT upper limit of normal is driven by the historic values from the laboratory that runs the assay (Watkins et al., 2008). It is possible for data from a large clinical study to come from multiple sources with different ULN values and data are normalized and reported as fold-change above ULN. Thus, normalized data are compared across study centers from the trial. A systematic analysis of ULN is a complex undertaking. A large scale study of healthy people indicates that the ALT ULN is actually lower than the historically accepted value (Kariv et al., 2006). The most critical independent associated variables were gender and obesity, while cholesterol, triglycerides, glucose, and hepatotoxic medications also inuenced ALT levels. Exclusion of 94% of these test subjects with acute illnesses, liver diseases and systemic diseases, still yielded approximately 17,500 normal subjects for this analysis. The new ULN, corresponding to the upper 95 percentile of the normal population, was 37.5 U/I, compared to 52 U/I (Kariv et al., 2006). Females had lower ALT values than males. Body mass index (BMI) data was not available, yet exclusion of certain related biochemical parameters (e.g. glucose and lipids) would capture most subjects with increased BMI. Estimates are that up to 14% of the normal population would be affected by a revised and lower ULN threshold for ALT (Piton et al., 1998). Seven different clinical denitions of ULN have been reported in an industry white paper and guidance on the use of these different ULN thresholds is recommended herein (Piton et al., 1998). Would the population affected by a revised ALT ULN show an increased susceptibility or risk for predictable or idiosyncratic DILI? Importantly, this newly dened patient population can be prospectively examined in future trials or for biomarkers qualications. We predict that patients which show ALT values approaching the current 1.01.5 ULN range prior to compound administration will be at an increased risk of DILI incidence with drug treatment that induces hepatotoxicity (AZ, unpublished observations). 5.2. Body mass index inuences ALT ULN values in the general population Selection of blood donors requires testing for transmissible diseases and ALT is a well accepted surrogate marker for nonspecic screening of HCV infections in donors (Klein, 1990). BMI of obese individuals (>30 kg/m2) correlates with increased serum ALT values of at least 2 ULN and in a stratied manner with overweight groups, which excludes many safe blood donations from being utilized (Ramesh et al., 1995). Even moderate obesity adversely inuences ALT levels (Nomura et al., 1986). In male obese subjects, BMI increased the relative probability of an elevated ALT value nearly ninefold, where alcohol showed an inuence of only twofold (Robinson and Whitehead, 1989). Moderate alcohol use has a greater inuence on ALT elevation when BMI increases ALT >2 ULN (Alatalo et al., 2008). Elevated ALT values in high BMI patients may necessitate a separate ULN be established as a reference for these patients (Alatalo et al., 2008).

The prevalence of elevated baseline ALT values in the US has surprisingly doubled from 19881994 to 19992002 and was associated with Non-Alcoholic Fatty Liver Disease or NAFLD risk factors, while excluding HCV antisera and excessive alcohol consumption (Ioannou et al., 2006a,b). Blood donors show ALT elevations in nearly 18% of the population with independent association with males, increased weight, BMI, waist size and even transfusion site (Papatheodoridis et al., 2007). This underscores how ALT ULN variation is seemingly elevating over time in a portion of the United States population, and in other developed countries, where obesity continues to be a signicant and worsening health issue. 5.3. Unexplained ALT elevations and their impact upon drug development The United States military has a huge database of ALT data on new trainees (Kundrotas and Clement, 1993). Approximately, 0.05% of nearly 20,000 inductees showed unexplained ALT elevations. Variation might be attributed to non-Gaussian distribution, muscle leakage of ALT, or hepatic disease undened by a current marker or endpoint. Authors suggest that two ALT readings be taken to track asymptomatic individuals with elevations. Most of the elevations were persistent over a several week period, but 25% of the elevations were a single instance. In another blood donor screening study, two-thirds of 100 identied asymptomatic blood donors showed persistent or intermittent ALT elevations over a six month period, and 20% of these subjects showed no apparent cause for the ALT elevations (Friedman et al., 1987). Epi-phenomenon of the military basic training environment was considered a possible inuencing factor, which should be considered more fully in a clinical development trial. Since many asymptomatic individuals with elevated ALT were found to resolve below ULN within months on retesting (Friedman et al., 1987; Kundrotas and Clement, 1993), we recommend that comparable criterion be considered for patients in a clinical trial paradigm were liver injury is being monitored. In trials for new chemical entities, we suggest that the frequency of subtle ALT elevations should exceed what had been reported for blood donor screening programs before discontinuation of a developmental program is triggered. Unexplained ALT elevations were also observed in large general populations exceeding 20,000 subjects (Clark et al., 2003). In one study, 7.5% of the placebo patients in a set of Phase I trials had an ALT value >2 ULN (Rosenzweig et al., 1999). Repeating measurements increased the probability of an elevated ALT measurement. Abnormally elevated ALT values occurred mostly during the second week of treatment, which overlaps with multiple dosing Phase I schedules. The FDA, 2009 guidance provides similar recommendation regarding the frequency of observed ALT elevations in control compared to treated trial populations. Employing a single laboratory method (with similar reagents and analyzer) to run ALT samples for a biomarker qualication application is recommended and would address concerns regarding variations in ALT activity levels between assay methods (Table 1). A pilot case study of cross-laboratory ALT assay harmonization should be examined to determine if patient safety is improved before a more in depth consideration of a unication of ALT assay approaches is initiated by industry. We recognize that considerable investigative efforts are needed for this type of study. Genome-wide association (GWA) approaches dened two loci (CPN1-ERLIN1-CHUK and PNPLA3-SAMM50) in humans that inuence elevations of serum ALT and AST activities, although clinical chemistry method differences in the populations studied might impact the extent of the polymorphic inuence (Yuan et al., 2008). One phenotype increases the incidence of ALT activities just above ULN in one third of carriers (Yuan et al., 2008). Over 9000

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nonsynonymous variants were assayed by chip-based oligonucleotide hybridization in the Dallas Heart Study and a PNLA3 variant associated with high fat content and ALT elevations in Hispanics, an ethnic group which showed the greatest prevalence of hepatic steatosis and cirrhosis susceptibility (Romeo et al., 2008). No genetic locus has yet been identied showing screening potential to avoid the onset of immunological reactions that induce idiosyncratic DILI. Abacavir induced hypersensitivity syndrome (AHS) has potential to induce mortality in <8% of treated patients (Phillips and Mallal, 2009). By identifying the associated MHC class I allele HLA-B5701 in the HIV patient pool eliminates toxic epidermal necrosis (TEN) or StevensJohnson Syndrome with avoidance of abacavir treatment (Phillips and Mallal, 2009). The HLA-B5801 allele is associated with allopurinol-induced cutaneous rash in patients of Han Chinese ancestry and screening prior to treatment can signicantly reduce incidence of this potentially fatal skin reaction (Shefeld and Phillimore, 2009). HLA-B1502 identied in patients of similar ancestry has potential to reduce carbamazepine-induced skin reactions by screening before commencement of therapy (Shefeld and Phillimore, 2009). The approach used for abacavir and associated examples might also be applied to facilitate the development and operationalization of potential genetic tests for idiosyncratic DILI. We propose that patient samples be broadly banked from trials to be applied retrospectively at a later time to discover genetic tests after detection of DILI injury. While genetic polymorphisms might contribute to unexplained ALT increases, a combination of genetic and epigenetic factors, including diet are most likely. Hyperalimentation induced by high fat diet within a one month period induced a body weight increase of 515% and ALT elevations above the reference limits in more than 50% of the subjects (Kechagias et al., 2008). In clinical trials, use of normal western diets is anticipated to reduce the incidence of unexplained ALT elevations compared to use of high fat diets. 5.4. ALT elevations that indicate the development of metabolic syndrome Metabolic syndrome is a constellation of risk factors for cardiovascular disease and diabetes including central obesity, dyslipidemia, glucose abnormalities, hypertension, and insulin resistance. The incidence of metabolic syndrome varies by country, ethnicity, and nutritional intake while cumulative risk has been observed in numerous studies over a 10-year window (Bauduceau et al., 2007). Upwards of 25% of adults in the United States have metabolic syndrome according to World Health Organization (WHO) criterion (Ford et al., 2004). Patients with metabolic syndrome have three to ninefold elevated risk for development of type II diabetes (Bauduceau et al., 2007). Plasma insulin and other components of metabolic syndrome show high correlation to simple waist measurement, which is usually associated with abdominal and liver fat content (Bauduceau et al., 2007). Concordance of moderate fasting hyperglycemia, high arterial blood pressure, and/or dyslipidemia with waist measurement adds signicant value to this metric (Despres, 2001). ALT and uric acid both are biomarkers that associate and elevate with the development of metabolic syndrome (Kang et al., 2008). Higher body mass index (BMI) associated with metabolic syndrome appears to associate with ALT >2 ULN, while moderate alcohol consumption has a synergistic inuence on observed ALT elevations (Alatalo et al., 2008). These ALT elevations are observed in individuals that show three characteristics of metabolic syndrome (Kang et al., 2008). Serum uric acid was associated with metabolic syndrome in a cross-sectional population study where abdominal obesity was the main factor driving uric acid variance (Onat et al., 2006), although absence of uric acid changes does not preclude a diagnosis of metabolic syndrome. LC/MS and LC/

MS/MS methods have been developed to measure serum uric acid (Dai et al., 2007). No reports associate serum uric acid elevations with hepatoxic injury. Voluntary and exploratory serum uric acid measurements in conjunction with subtle clinical ALT rises may add to our understanding between the relationship between metabolic syndrome and liver injury. While the development of metabolic syndrome may share risk factors with non-alcoholic fatty liver disease, uric acid shows no known relationship to liver injury. The high incidence of metabolic syndrome in the United States population underscores the complexity of new candidate drug evaluations in populations considered normal for rst-in human studies. ALT ULN recommendations from the manufacturer of enzymatic reagents for ALT may come from an analysis done with a population much different from the geographical location of the study. In light of these issues, we propose that patients ALT elevations may be considered relative to their own predosing baselines (or alternatively to historical ranges) when analyzing aminotransferase elevations. Using this approach, a patient with a low ALT baseline value that reveals a 3 elevation relative to baseline with compound administration would initiate a agged monitoring event that would precede possible subsequent elevation >3 ULN. Whereas, patient with a high ALT baseline value (ULN to 1.5 ULN) should be tracked during monitoring for >3 ULN from the onset of the study. This proposed monitoring strategy would enhance the safety window compared to current standards with a modest adjustment of current practices.

6. ALT elevations in patients using marketed products can guide development of future products 6.1. Subtle ALT elevations during clinical trials History reveals programs that were successfully brought forward despite clinical ALT elevations, such as tacrine or statins, and less successful attempts to market other therapeutics with a cause of concern, notably troglitazone and bromfenac (Goldkind and Laine, 2006). Simvastain was brought forward with 2% rate of ALT >3 ULN in clinical trials, although no hepatocellular jaundice observations were made. Broader experience with additional therapeutics in this class validated that liver injury is not a cause of concern. Similar experience was shown with tacrine, although ALT elevations were more frequent and at higher levels compared to statin therapy. Second generation cholinesterase inhibitors show less ALT elevations and improved dosing thereby replacing tacrine. Clearly, improved biomarkers of hepatocellular jaundice are critical to parse out ALT elevations that infrequently progress to acute liver failure and idiosyncratic DILI. Subtle elevations of ALT are observed with Acetaminophen (APAP) dosing in humans in the absence of reported hepatotoxicity or liver failure. ALT elevations were observed in osteoarthritis trials where subjects were given large daily doses of APAP (up to 3.9 g/ day). More than 17% of the patients had an ALT value that exceeded ULN and 4% showed >1.5 ULN values (Kuffner et al., 2006). ALT elevations resolved with continued treatment, unaccompanied by signs of liver injury. Additional drugs show similar potential to induce mild elevations of aminotransferases in the absence of severe DILI: aspirin, tacrine, heparin, hydroxul-methylglutaryl coenzyme A-reductase inhibitors or statins (FDA, 2009). Valproic acid (VPA) induced liver injury affects a limited segment of the population. VPA has a low liver injury risk overall at 1:37,000 (Chitturi and George, 2002), although the risk elevates to 1:500 in young children, multiple drug therapy patients, and individuals with mitochondrial disorders among other conditions (Chitturi and George, 2002). Thus, factors in addition to ALT eleva-

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tions inuence susceptibility to severe DILI. Cellular imaging predictions of primary human hepatocyte culture are modestly predictive of clinical outcomes. Mitochondrial damage, oxidative stress, and intracellular glutathione levels can be measured to achieve a true positive predictive rate of up to 60% with a low false positive rate less than 5% (Xu et al., 2008). Thus, in vitro approaches can aid our understanding of DILI mechanism, although additional tools are needed to complement these approaches. More frequent isoniazid dosing (2 weekly with rifampin) for ATT is associated with greater DILI incidence compared to 1 weekly therapy with rifapentine. Unfortunately, 1 weekly isoniazid dosing led to failure/relapse of turberculosis treatment resulting from the presence of N-acetyl transferase 2 (NAT-2) phenotypes that rapidly metabolize drug leading to poor PK (AUC) and co-therapy (rifamycin) mono-resistance (Weiner et al., 2003). NAT-2 genetic analysis to dene slow acetylator phenotypes is complex and requires many restriction endonuclease digestions, making the utility of this approach limited to screen for DILI comprehensively. Nevertheless, slow acetylators show a more frequent ALT elevation with isoniazid treatment from metabolism of acetylisoniazid- to greater accumulation of the toxic mono-acetyl hydrazine (MAH) metabolite, although the evidence is controversial since fast acetylators make higher levels and show greater clearance of this toxic metabolite (Saukkonen et al., 2006). Hepatitis B infection and silent chronic liver disease correlated to ATT induced DILI, while age, sex, alcohol use, and BMI did not correlate and treatment continuation with jaundice was associated with fatality (Anand et al., 2006). 6.2. Do transient rises in ALT predict severe DILI at a later time? FDA, 2009 guidance document recommends repeated measurement of elevated aminotransferase activities that cause concern (4872 h after initial detection and repeatedly for several weeks thereafter), since changes in aminotransferase values can be rapid and any delays in prompt measurement might lead to missing the rapid progression of the initial abnormality as the herald of a severe DILI reaction that could possibly follow. The incidence of transient ALT rises in a patient pool is much greater in frequency compared to those patients that progress to severe DILI. Clinically, a patient administered a medication that led to a transient ALT elevation should be monitored for a reappearance of the ALT elevation or persistent elevation, assuming that the initial degree of ALT elevation is not itself sufcient cause of concern to the clinician to remove the patient from treatment. In a post-marketing scenario, the length of monitoring is a complex issue considering concerns for containing health costs, but the primary clinician and patient history are the most critical drivers for this decision. In a clinical trial, ALT elevations may lead a patient to be removed from the trial depending upon the degree of an elevation or may lead a patient to be briey monitored to indicate a transient response if the elevation is just above 3 ULN. This is a difcult decision for the study director to make, taking into consideration the potential risks to patient safety with continued dosing and the appropriate level of ALT elevation, which is considered permissible for continued dosing of a drug candidate. There is no universal and agreed upon ALT elevation threshold across development programs, even within a single company, or even among consortia members. Nevertheless, the path forward to reach an agreed universal ALT threshold will be challenging since preclinical models of these events will be difcult to reproduce, so translational approaches are limited until additional biomarkers of liver injury are discovered and tested in the clinical setting (Table 2). A parallel assessment of ALT, AST, total bilirubin, and ALP values is recommended during all monitoring of ALT elevations in clinical trials to judge the potential for serious idiosyncratic DILI (FDA, 2009).

6.3. Clinical considerations for ALT elevations that lead to patient withdrawal from a drug trial The decision to withdraw a patient from a prescribed drug either in routine clinical practice or in a clinical trial setting is a major decision point in evaluating drug-induced hepatotoxicity. It is standard practice in some pharmaceutical company Phase I healthy volunteer trials for subjects to be taken off study with a nding of ALT or AST >3 ULN, even in the absence of associated bilirubin increases. This is a standard approach even though it is relatively well known that aminotransferase elevations to this degree is not uncommon in healthy volunteers due to various factors like diet, inpatient hospitalization for 2 weeks or more, intake of acetaminophen/paracetamol (Watkins et al., 2006), and/or exercise (Pettersson et al., 2008). FDA, 2009 guidance recommends that incidence of >3, >5, >10, and >20 aminotransferase elevations in treated patients be compared to control groups in trials, indicating that elevations are permissible in the absence of bilirubin injury (>2 ULN), although a delay of several weeks in the bilirubin signal may arise. Thus, frequent monitoring is recommended in these subjects with ALT elevations (FDA, 2009). The risk-averse nature governing possible premature withdrawal of subjects in early clinical studies does not readily allow a better understanding of the course of the liver injury because patient safety is paramount in drug development both to regulators and sponsors. Even in patient trials in later stages, the decision to withdraw the subject is based on caution with a common view to avoid any risk to the subject on study. With regards to the clinical trial setting, another important point is that recruitment of patients and healthy volunteers is based on certain minimum entry criteria. This includes clean baseline liver function (such as exclusion of subjects with ALT >1.5 ULN) and absence of a history of liver disease. It has been proposed that pre-existing liver disease does not predispose subjects to drug-induced liver toxicity (Zimmerman, 1999). Practical guidelines and an algorithm for diagnosis and early treatment of DILI have been proposed for diagnostic review of injury prognosis (Tajiri and Shimizu, 2008). 6.4. Additional biomarkers that may show potential to predict the onset of predictable DILI Several new biomarkers that are predictive of DILI show promise for additional investigations to assess the merits for potential qualication (Table 2). The Ca2+-binding protein regucalcin regulates calcium metabolism in kidney and liver, while hepatic over expression of regucalcin suppresses cell death and apoptosis (Yamaguchi, 2005). Serum regucalcin elevations appear in acute D-galactosamine and lipopolysaccharide (GalN/LPS)-induced liver injury in mice (Lv et al., 2007). Regucalcin may predict transient ALT elevation outcomes. In CCl4 treated rats showed twofold ALT and AST elevations 3-days post-treatment, whereas regucalcin serum elevations were observed as long as 30-days post-toxicant treatment (Yamaguchi et al., 2002). Regucalcin elevations early post-dose are nearly twentyfold greater compared to one month after dosing (Yamaguchi et al., 2002). Regucalcin is 93% conserved between rat and human, but assays are not commercially available to evaluate in a qualication paradigm. Cytochrome c (cyt c) is a serum and urinary biomarker of liver injury induced by acetoaminophen (APAP) and D-galactosamine (GalN) treatment. Urinary cyt c show predictive response prior to the onset of aminotransferase elevations and histopathologic observations of injury, while the serum activity is diagnostic (Miller et al., 2008). While these studies are with a limited set of toxicants, urinary biomarkers including metabolites could be a source of predictive markers of DILI. Elevated predose production

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of endogenous colonic p-cresol correlates inversely to a reduction of an individuals ability to sulfonate acetaminophen (APAP) by acting as a competitive substrate and leading to reduced urinary excretion of APAP (Clayton, 2009). Although this seminal pharmacometabonomic study in humans found a clear association between an individuals predose urinary metabolite prole and the post-dose urinary fate of acetaminophen, further studies in animal models will allow better mechanistic understanding of metabolism upon induction of DILI. Clinical chemistry and histopathology anchors are recommended to characterize predictive DILI biomarker activities, especially metabolic or lipidomic markers (Ozer and Teitelbaum, 2009). 7. Conclusions Drug-induced liver injury is a signicant hurdle for the pharmaceutical industry, regulators, clinicians, and patients affected by such condition. With ever-increasing regulatory oversight, pharmaceutical companies are now addressing the issue of drug-induced liver injury more rigorously now than before. Diagnosing and managing DILI in the preclinical and clinical settings are challenging and there is an immediate need for better processes and tools to characterize liver toxicity potential of compounds at various stages of development. Conventional biochemical tools like ALT and AST have limitations and are imperfect as biomarkers of DILI, indicating that there are a number of well dened approaches that can add value to their applications in the drug development setting. This perspective addresses some of these gaps and highlights the need for better use of available diagnostic standards, indicating where there is scope for improvement. In addition this article has focused also on the unmet need for improved and novel biomarkers of liver toxicity (Table 2) and the role of regulatory agencies in enabling qualication of such markers. We encourage continued dialogue among pharmaceutical, academic, medical, and regulatory leaders to advance and improve the science of liver safety assessment in preclinical development and clinical practice. Conict of interest The authors declare that there are no conicts of interest. Disclaimer The personal views expressed in this article by the authors may not be understood nor quoted as being made on behalf of or reecting the position of the FDA, EMEA, or PSTC and IMI and its membership companies. Acknowledgments J.O. and R.C were equal contributors (co-1st authors) to this paper. We thank Christina Hunt for reading the paper and making suggestions on genetic factors and Holly Jordan for the rationale for biomarker qualication. We thank Bill Mattes, Shelli Schomaker, and Wendy Bailey for helpful discussions in the PSTC HWG PPMG working group. We thank Jennifer Colangelo, Mike Aleo, Denise Robinson-Gravatt, and James Mayne for helpful suggestions on regulatory and qualication considerations. References
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Further reading
Antoine, D.J. et al., 2008. Understanding the role of reactive metabolites in druginduced hepatotoxicity: state of the science. Expert Opin. Drug Metab. Toxicol. 4, 14151427. Ju, C., Pohl, L.R., 2005. Tolerogenic role of Kupffer cells in immune-mediated adverse drug reactions. Toxicology 209, 109112. Pauli-Magnus, C., Meier, P.J., 2006. Hepatobiliary transporters and drug-induced cholestasis. Hepatology 44, 778787. Tuschl, G. et al., 2008. Primary hepatocytes as a model to analyze species-specic toxicity and drug metabolism. Expert Opin. Drug Metab. Toxicol. 4, 855 870.