Regulatory Toxicology and Pharmacology 56 (2010) 237–246

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Regulatory Toxicology and Pharmacology
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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

Pfizer Global Research and Development, Pharmacokinetics, Dynamics, and Metabolism, 700 Chesterfield Parkway West, T2A, Chesterfield, MO 63017, USA AstraZeneca Research and Development, Clinical Pharmacology, Drug Metabolism and Pharmacokinetics, CPU, Alderley Park, Macclesfield, SK10 4TG, Cheshire, UK AstraZeneca Research and Development, Safety Assessment, Alderley Park, Macclesfield, SK10 4TG, Cheshire, UK d Pfizer, Emerging Markets Business Unit, NY, NY 10017, USA e Pfizer Global Research and Development, Clinical Research, CT13 9N5 Sandwich, UK f Pfizer Global Research and Development, Drug Safety Research & Development, Chesterfield, 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 specifically 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 finally, (3) clinical evaluation of ALT elevations prospectively and retrospectively is recommended to define 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 qualification model. Ó 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 Hy’s 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 significant 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) defining causality of drug administration in human idiosyncratic DILI, (3) the impact of animal subject variability in susceptibility to DILI, (4) and defining the limitations in the specificity 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@pfizer.com (J.S. Ozer). 1 Present address: Televetdiagnostics Ltd., 2 Somerset Close, Congleton CW12 1SE, UK. 0273-2300/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.yrtph.2009.11.001

Holt and Ju. Pachkoria et al. it is essential to exclude other possible causes rather than drug administration (FDA. respectively. Thus.. 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.238 J. environmental exposures. 2003. 1978. 2009). 2006). The biomarker attributes to define predictable DILI are not precisely characterized. with some notable distinctions. 2006. Goldkind and Laine. 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. 2009). Ozer et al. 2. A mortality or transplantation rate of up to 10–15% and in certain cases even more than 50% has been observed in patients with idiosyncratic liver injury induced by drug treatment (Lewis. Eventually. 2009). These recommendations were not adhered to consistently by all prescribing physicians leading to instances of drug-induced patient deaths and liver transplantations. Both forms of DILI share common histopathologic attributes and are well detected by ALT elevations. 2009). Of par- . Temple’s Corollary of Hy’s law is defined as combined elevations in serum ALT >3Â ULN and bilirubin >2. regulators have not approved drug applications where incidence of Hy’s law was observed in late staged trials. even for pharmaceutical consortia. 1968. although the responses can be dissimilar in certain contexts ALT or AST >3Â ULN to be confirmed by GLDH or ALT mass assay like ELISA (Biomarker Rule of Two) when assays are validated and available. but not universally accepted definitions. 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. 2006). AST. 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. Historically. The manifestation of two idiosyncratic DILI instances and even one case may suggest serious consideration for the withdrawal of a product from market (FDA. 2006. 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 >3Â ULN to be confirmed by similarly assessed elevations in GLDH or ALT mass assay like ELISA (Biomarker Rule of Two) when such assays are validated or available. Temple. 2006. In cases where Hy’s law clinical chemistry elevations are met. 1978. a life-saving indication. An ALT and AST elevation predictable threshold for injury to be defined using exploratory and retired developmental compounds (anchored to histopathology) in ROC analyses Analytical Ultimately. 2008).. but this must also be viewed in the context of the risk–benefit of the compound. although exceptions are noted (Bosentan) (Temple. 2006). Susceptibility to idiosyncratic drug-induced liver injury lacks certain dose and duration dependent features typical of toxicity responses to drug treatment (Gupta and Lewis. Nevertheless. Regulatory Historical biomarkers (ALT. Lewis.0Â ULN and indicates significant potential to manifest idiosyncratic DILI (Zimmerman. / Regulatory Toxicology and Pharmacology 56 (2010) 237–246 Table 1 Summarized recommendations to enhance the utility of AST and ALT as reference standards of DILI. e.g. Senior. 2005. idiosyncratic DILI must also be a diagnosis of exclusion even when Hy’s law is met. respectively. 2008). epigenetic. and in the context of the frequency of this toxic reaction in the target population for those administered the drug. Hussaini and Farrington. elevated bilirubin is a marker of severe liver dysfunction and not cholestasis.S. 1999. 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 definition 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. In this case. further dialogue and investigations to achieve a well described and consensus definition of predictable DILI is highly recommended. 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 define 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. elevation in serum ALT >3Â ULN and/or preclinical histopathologic evidence of hepatocellular necrosis and/ or biliary changes (including repair) are generally accepted. Watkins et al. 2006.. Idiosyncratic DILI refers to injury dependent upon individual’s constitution including but not limited to potential genetic. yet a lack of appropriate preclinical models make this a challenging problem to solve. Dosedependent and reproducible hepatotoxicity observed either preclinically or clinically is referred to hereafter as predictable drug-induced liver injury as described by Zimmerman. Lucena et al. which is dose-dependent and reproducible in preclinical safety assessment studies.000 in patients dosed with troglitazone is a comparator for other compounds that induce idiosyncratic DILI (Senior. An acute liver failure incidence of 1:10. Thus. 2007). bilirubin) to be benchmarks in qualification of new biomarkers Consider influence of systemic non-liver diseases on aminotransferase and/or bilirubin changes Patients with underlying cirrhotic conditions or chronic hepatitis C infection could be influenced by reduced hepatocellular cellular mass. 2006. which is a primary goal to assess in this report (Table 1). Some drugs cause intrinsic (predictable) DILI. 2006. Discovery of novel prognostic biomarkers of idiosyncratic DILI Identification of biomarkers that are prognostic for idiosyncratic DILI response are in great need. Lewis. and or immunological responses (FDA. and therefore the ALT >3Â ULN threshold limits rule may need to be applied appropriately. 2008. product withdrawal from the market was the resulting outcome (Senior. 2008) in the absence of ALP elevation (FDA.

It is noteworthy that nearly 80% of the patients with idiosyncratic DILI recover with drug discontinuation (Chalasani et al. Similarly. hypotension. post-operative jaundice (Mastoraki et al. 2. some patients in efficacy trials will have limited medical concerns outside the therapeutic indication being tested. idiosyncratic DILI can not be consistently resolved with revised clinical dosing once observed. although observing Hy’s law at extremely high treatment doses in early range finder toxicity studies presents complex considerations for further development. 2006.. 2002). sepsis (Thiele. connective tissue disorders often involve the liver (Gitlin. The cost of this proposed collection is a fraction of the cost to withdrawal just a single major pharmaceutical product for idiosyncratic DILI. Kaplowitz. 2009 guidance document suggests that the application of Hy’s law may differ in the presence of existing liver disease such as fatty liver. 2004) and elevated aminotransferases have been noted in a number of connective tissue disorders. and lastly the absence of other putative causes for these elevations in the individual. Recruitment for proof of concept trials is challenging for numerous reasons and the global nature of patient recruitment is an indicator of the difficulty to recruit well defined populations for compound evaluation. 1994).S. and an associated rise in unconjugated bilirubin in 24–81% of patients depending on the severity of heart failure (Dunn et al. Since idiosyncratic DILI occurs infrequently and only in susceptible drug treated patients. chronic hepatitis B or C.J. total parenteral nutrition (Spiliotis and Kalfarentzos. 2003. a twofold elevation in ALT (over baseline values) was observed with higher incidence in placebo patients. 2005).. / Regulatory Toxicology and Pharmacology 56 (2010) 237–246 239 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. Abraham et al.0Â ULN (without initial findings of cholestasis. and at least within the context of a voluntary informed consent basis. 1986). the FDA. As a matter of good clinical practice. improving detection and prognosis of idiosyncratic DILI during clinical development cannot be overcome without some modest adjustments to the current safety assessment paradigm. 2005). NASH and viral hepatitis) and drug administration could induce more severe level of injury in these patients in an instance of DILI (FDA. Interpretations of Hy’s law relative to individuals and groups in a drug trial There are criteria of Hy’s law that indicate the potential development of idiosyncratic DILI. 2009). Not all. The first criterion consists of ALT/ AST >3Â ULN in subjects compared to the control group in a trial. little prognostic power to identify the onset of rare idiosyncratic DILI events (FDA. 2009). and in patients on drugs that treat liver disease or those on drugs that inhibit bilirubin glucuronidation. which should be cost-shared among the different stakeholders in this process. Designating a Hy’s 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). bilirubin >2. which would divert resources from the current drug development paradigm. designating Hy’s law events as causal for idiosyncratic DILI becomes more complex due to either hepatic or extrahepatic disorders in the general population. 2009) and this approach is supported by the Research Opportunities section of the Guidance (FDA. Thus. 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). In addition to ruling out non-drug related cholestasis while applying Hy’s law. if any. Rarely.1. Thus. Predictable DILI is often reversible with discontinued dosing at modest injury levels (ALT . and inflammatory bowel diseases (Chapman and Angus. Clearly. NASH. although exceptions are being increasingly noted. The most difficult and costly aspect of this approach is the long term storage of the samples. and an important cause of compound attrition and/or project delay during drug discovery and development. 2008).. In a prospective study of statin use in chronic liver disease patients. The main limitations of biomarkers of predictable DILI are a lack of specificity and by extension.g. Moreover. 2002). 3. A predicted safe margin of candidate compound exposure can be modeled for dosing in first-in human (FIH) studies in conjunction with other parameters such as pharmacokinetic/pharmacodynamic (PK/PD) data.. Creatine kinase and lactate dehydrogenase (LDH) elevation support muscle injury diagnosis initially. and therefore the >3Â ULN ALT threshold may need reconsideration for this patient group (Table 1). 2007). this objective is achieved in a majority of cases. shows attributes of dose independence. such as antiretroviral agents. AST/ALT ratio of 4:1 changes to 1:1 from day 1 to 4 after insult with a persistent >4Â ULN aminotransferase elevation (Nathwani et al. FDA. Predictable DILI is a frequent organ toxicity finding in preclinical test species. Preclinical safety testing strategies would need to incorporate novel genetic and study designs to model idiosyncratic DILI preclinically.g. 2009). Overall. 2005). 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. 1973). The DILIN network is collecting and banking samples consistent with our proposal (Fontana et al. Whether predictable DILI has any definitive relationship to the development of idiosyncratic DILI is hypothetical.e. Ozer et al. conventional preclinical safety testing is not considered the most suitable avenue for the detection this injury (Lee. patients with skeletal muscle breakdown and disease show aminotransferase elevations >4Â ULN (Nathwani et al. or congestive heart failure (Temple. yet these markers subside after a few days. Predictable DILI Documenting predictable DILI in preclinical studies enables determination of safe dose-level(s) in subsequent chronic studies (i. Observing Hy’s law preclinically in Good Laboratory Practice (GLP) studies is rare and would likely halt further development of a lead compound candidate. other preexisting diseases should be similarly considered. The degree of aminotransferase elevations in patients with idiosyncratic DILI that have underlying cirrhotic conditions or chronic hepatitis C infection could be influenced by reduced hepatocellular cellular mass. including viral induced hepatitis. 2009 guidance on application of Hy’s law to DILI briefly discusses the influence of systemic non-liver diseases on aminotransferase and/or bilirubin changes. Hepatic reserve will be more limited in these patients (e. 2009). second. 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). concomitant use of another hepatotoxin or toxic drug. certain hematological diseases (Omata et al... e.. Cardiovascular causes such as right heart failure and hypotension may cause significant rises in ALT (ischemic hepatitis). 1997. one month or more). 2007). indicating the comparative complexity of benchmarking hepatotoxic drug safety in patients with pre-existing liver disease (Lewis et al. no elevation in ALP).. 2007). while clinically significant liver dysfunction is a comparatively less frequent finding during clinical trials. and shows a high mortality/transplantation risk (>10%).. As post-marketing trials and marketing of a compound occurs. FDA.

In clinical practice. sorbitol dehydrogenase. Improved dynamic range Improved specificity Lagging biomarker Labile biomarker Predictive potential Predictive potential Dual platform Metabolic. Hepatocellular DILI defined by a ratio of ALT to ALP (alkaline phosphatase) > 5 (relative ULN applied) would indicate specificity compared to other organ injuries. 2000). The guidance is mainly based on historic biomarker performance of ALT and AST. while end-stage liver disease and limiting PLP concentrations were ruled out as possible causes for decreased ALT function (Solter et al. rigorous. Aminotransferase elevations with preclinical histological findings are used for prognosis of liver toxicity projections to clinical study planning regardless of associated bilirubin increases. SDH. marked inter-individual variability is an additional characteristic feature of predictable DILI for drugs tested in man. In the absence of a formal qualification of new biomarkers in conjunction with the aminotransferases. 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. 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. Companies are encouraged and may voluntarily use additional exploratory markers and methodological approaches to govern an internal decision regarding injury events. Are aminotransferase elevations without concomitant bilirubin increases a sufficient 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. In addition. 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 qualification process (Table 1). 2008). Ozer et al. industry. 2001). liver biopsy may be performed when an explained sustained abnormal liver function test is observed (Skelly et al. malate dehydrogenase. 2008.. and better concordance with both rodent and nonrodent species and man (71%) (Olson et al. Ozer et al. 2000). (Tajiri and Shimizu. We suggest that individual developmental project teams. PNP. MDH. a lack of a common. (2) compared ALT data from enzymatic assays with dissimilar reagent conditions (e. ELISA. Furthermore. EIA. with limited concordance between rodent and man (43%).g. 1998).. glutamate dehydrogenase. For the identification of the potential to manifest idiosyncratic DILI. additional predictable DILI biomarkers would add value to AST to monitor injury induced by compounds that mask ALT activity.. if possible.. Limits of historical evidence used to support biomarker guidance includes: (1) some reported preclinical data may have lacked histopathologic observations by trained and board certified pathologists and may have lacked internal peer-review of data. In the postmarketing setting... An International Life Sciences Institue (ILSI) study examined 150 compounds from 221 human toxicity events and 12 pharmaceutical sponsors (Olson et al. For the clinical setting. Regulatory guidance recommends ALT thresholds indicative of adverse injury. Liver biopsy is associated with morbidity. A unification of biomarker performance is needed that includes ALT and complementary novel biomarkers that translate across preclinical and clinical platforms. This type of false negative event has not been extensively documented. AST elevations above ULN in the presence of ALT masking is a profound biomarker to diagnose DILI (Table 2). 2009 guidance emphasizes criterion to identify idiosyncratic DILI. 4-hydroxyphenylpyruvate dioxygenase. and to a lesser extent key aspects of predictable DILI. even acute liver failure does not always warrant a liver biopsy (Polson and Lee. Luminex Enzymatic Enzymatic EIA EIA Enzymatic. Serum ALT elevations must exceed a defined threshold to be considered an adverse injury for a given dose of compound. immunoprecipitation. 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. aspartate aminotransferase. but how does clinical qualification of new liver biomarkers become fully enabled with limited histological diagnosis in the qualification 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). even with moderate clinical symptoms. GSTa glutathione-S-transferase alpha. are largely poorly understood and considered to be multifactorial (Lee. IP. while frequent enough to be observed at any point along the compound development pipeline. PON1.. and contract research organizations (CROs). historical and reported evidence is used by regulators to govern recommended aminotransferase thresholds. The reasons for this variability. The manifestation of predictable DILI shows variability between preclinical species and humans (Olson et al. but this is mostly a limited concern. AST. 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). although administration of subchronic doses of microcystin-Leu-Arg decreases ALT synthesis. 2005). purine nucleoside phosphorlyase. 2000). paraoxonase. and hence for individual susceptibility. Comments to the regulatory guidance for DILI FDA. 2009). Enzymatic EIA Enzymatic Enzymatic Enzymatic 4. Therefore. primate) and man (63%). fulfilling Hy’s law provides compelling evidence of clinical injury to regulators. GLDH. and a rare but significant risk of mortality. hpd. and (3) and by extension. newer chemical entities (NCEs) have the potential to mask elevations in ALT activity. Nonspecific 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. Hence regulatory guidance must align with clinical practice where histological diagnosis is limited by the availability of clinical samples. alanine aminotransferase isoform 2. although histological correlation might be expected if clinical samples were not a limiting factor in the clinical setting.240 J. vitamin B/pyridoxal-50 phosphate/PLP inclusion or lack thereof).. 2008). 2003). The FDA 2000 non-clinical assessment indicates that ALT or AST >3–5Â ULN be considered an adverse event in the absence of con- . 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. and uniform aminotransferase analytical validation across academia. EIA IP. Generally. / Regulatory Toxicology and Pharmacology 56 (2010) 237–246 Table 2 Biomarker and platform abbreviations are indicated: ALT2. nonrodent preclinical species (dog. (Rockey et al.. however large molecular weight and stable macro-AST enzyme activity has been identified in the absence of associated DILI (Caropreso et al.S. liver biopsy is prohibitive for most programs for ethical and logistical reasons. Muscle form No available assay Functional biomarker Historical Historical Enzymatic EIA. 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 <8Â ULN elevations). Preclinical data are only partially prognostic of human toxicity.

This document also indicates that ALT rises even up to 20Â ULN are observed for drugs like tacrine. 2007. vomiting.. / Regulatory Toxicology and Pharmacology 56 (2010) 237–246 241 cordant histopathology data.S. 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). immunodepletion and activity assays are coupled to detect activity independently from each isoform (Lindblom et al.1. 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). liquid chromatography–tandem mass spectrometry (LC/ MS/MS) techniques can be potentially employed to measure plasma levels of these modulators in a multiplex approach. Miyoshi et al. ATT therapy is often reintroduced successfully with resolution of initial ALT elevations. 2009:     ALT or AST >8Â ULN. 1994b). Vesicle exocytosis (zeiosis or blebbing) could be linked to subtle plasma ALT elevations in the absence of histopathologic injury. We propose that measurement of ALT protein levels by LC/MS/MS or immunoassay techniques when ALT >3Â ULN would provide additional analytical evidence that elevations result from ALT leakage and not enzyme induction. elevations of ALT enzyme cofactors of metabolism or ALT induction events. being an enzymatic activity assay.. Thus. Although ALT is a historic standard for characterizing liver injury. The following scenarios are likely considerations when discontinuation of treatment is recommended by FDA. 4. right upper quadrant pain or tenderness. Carbamoyl-phosphate inhibits ALT or AST and PLP-dependent enzymes in vitro. although there are no reports that substantiate this view. indicating than subtle elevations of serum ALT can occur in the absence of injury. PLP levels below Lower Limit of Quantitation (LLOQ) were observed in patients with liver disease. fever. This inconsistency over which ALT level indicates true liver injury largely results from lack of sufficient evidence to determine critical threshold limits for ALT partly from ethical risks for continued clinical dosing in the range of suspected injury thresholds. although limits to the amount of NADH included is an unfortunate consequence of this assay modification. 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... 2007). Induction of serum ALT activity correlated with ALT transcriptional elevations (less than twofold) with lipid lowering fenofibrate dosing in rats (Kobayashi et al. 2009). Assessment to define the threshold of ALT activity elevation that truly corresponds with adverse liver injury has not yet been rigorously evaluated. cross-validation of the ALT methodology or a common SOP among laboratories within a developmental program is desired.. 2008). might be modulated by substrate concentration or cofactors in a subtle manner that has not yet been defined or well characterized analytically. Ozer et al. The American Thoracic Society’s official statement for anti-turberculosis therapy (ATT)-induced DILI recommends treatment cessation at ALT >3Â ULN in the presence of hepatitis or jaundice and ALT >5Â ULN in the absence of symptoms (Saukkonen et al. 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). Detailed study of the aminotransferases in conjunction with new biomarker qualifications (e. 2006)... Whether these proposed ALT mass based assays in development would be as sensitive as enzyme activity is currently unknown (data not shown). Thus.J. In order to assess the effects of the modulators on ALT activity. 1994a).. Uniform agreement and practices across industry regarding PLP supplementation is encouraged (Table 1). 2008) (Table 2). 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).. Aminothiols (including cysteine) and vitamin B derivatives either inhibit ALT or PLP-ALT regeneration in vitro (Pagani et al. Recommended analytical approaches to enhance validation of ALT activity Might subtle modulations of ALT activity be caused by mechanisms other than hepatic injury? ALT. which would be correlated to corresponding histopathology and aminotransferase activity levels (exogenously sourced PLP would be excluded here) from individual samples. 2006). which is an increasing practice in industry. rash or eosinophilia. yet the physiological and toxicological significance remains to be fully elucidated in vivo (Pagani et al. Hepatocyte blebbing has been linked to toxicant exposures that show histopathologic injury (Barriault et al. nausea. Better defined ALT and AST thresholds will allow both regulators and pharmaceutical companies to greatly improve management of DILI in the drug development process. Subtle elevations in serum ALT activity (ALT >3Â ULN) may correspond to increased ALT concentration in serum or such increases may correspond to other influencing factors: ALT isoform modulations. a lack . it may have logistical challenges for outsourcing sample analysis. it has imperfections. single instance ALT or AST >5Â ULN for more than 2 weeks. Distinguishing between serum ALT1 and ALT2 isoforms may improve specificity compared to ALT leakage from the liver since the isoforms are differentially expressed in organs (Lindblom et al. yet more uniformity should be considered. thus limiting assay performance including dynamic range (Lustig et al. PLP supplementation is not universally practiced. 1994. multiple readings ALT or AST >3Â ULN and TBL >2Â ULN or INR > 1. 1986). These practices are already commonly practiced across pharmaceutical companies. which induces potential variability among reported data since supplementation infrequently increases ALT measurements. the threshold of ALT used for decision-making purposes may be up to 8Â above ULN as a stopping criterion (FDA. by extension. 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). 2009). Ultimately..g. Although ALT assay coherence throughout a single developmental program is a proposed goal. Depending upon the views of clinical directors.. 2009). 5. A more recent non-binding FDA draft recommendation indicates ALT or AST >5Â ULN for >2 weeks or ALT >8Â ULN in a single instance are considered adverse signals (FDA. PLP is the biologically active form of vitamin B6 that is utilized physiologically and is a cofactor for ALT or AST activity. While each isoform shows enzymatic activity in recombinant systems (Liu et al. 1977). 1988). Whether this assay will translate from preclinical species to man is being investigated. which excluded cirrhotic patients and may have impacted ALT or AST measurements (Anon. 1996). Regulatory qualification of novel biomarkers that add information to predictable DILI assessment There are benefits of a better understanding of ALT and AST as principal markers of predictable DILI in conjunction with new biomarker qualifications. Liu et al.5 ALT or AST >3Â ULN with the appearance of worsening of fatigue. which did not cause incidence of severe idiosyncratic DILI (Goldkind and Laine. This approach is being adapted to high throughput use and undergoing validation.

5% of the placebo patients in a set of Phase I trials had an ALT value >2Â ULN (Rosenzweig et al. / Regulatory Toxicology and Pharmacology 56 (2010) 237–246 of concordance exists for the true injury threshold to use when translating a program from preclinical to clinical assessment. 1998). Kundrotas and Clement. Thus.500 normal subjects for this analysis. and 20% of these subjects showed no apparent cause for the ALT elevations (Friedman et al. 2007). 2008). Authors suggest that two ALT readings be taken to track asymptomatic individuals with elevations. This underscores how ALT ULN variation is seemingly elevating over time in a portion of the United States population. normalized data are compared across study centers from the trial. Most of the elevations were persistent over a several week period. and hepatotoxic medications also influenced ALT levels.. which overlaps with multiple dosing Phase I schedules.. BMI of obese individuals (>30 kg/m2) correlates with increased serum ALT values of at least 2Â ULN and in a stratified manner with overweight groups.. ROC analyses usually bundle all positive hepatobiliary injury (histopathology) samples into a single bin regardless of severity grade.3. 2006a. A systematic analysis of ULN is a complex undertaking. 2008). where obesity continues to be a significant and worsening health issue. BMI. 1995). unpublished observations). Elevated ALT values in high BMI patients may necessitate a separate ULN be established as a reference for these patients (Alatalo et al.. Over 9000 . yet exclusion of certain related biochemical parameters (e. Body mass index influences 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 nonspecific screening of HCV infections in donors (Klein.. The FDA. still yielded approximately 17. 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 unification of ALT assay approaches is initiated by industry. Epi-phenomenon of the military basic training environment was considered a possible influencing factor.S. where alcohol showed an influence of only twofold (Robinson and Whitehead. The new ULN. 2006). 7. Exclusion of 94% of these test subjects with acute illnesses. 0.. although clinical chemistry method differences in the populations studied might impact the extent of the polymorphic influence (Yuan et al. while cholesterol. Females had lower ALT values than males. 5. BMI increased the relative probability of an elevated ALT value nearly ninefold. Seven different clinical definitions 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. which excludes many safe blood donations from being utilized (Ramesh et al. two-thirds of 100 identified asymptomatic blood donors showed persistent or intermittent ALT elevations over a six month period... 5. 1990). 2008). 1987.. Abnormally elevated ALT values occurred mostly during the second week of treatment..0–1. The ALT upper limit of normal needs rigorous analytical definition and subsequent validation We recommend that a universal ALT upper limit of normal be sufficiently defined by an analytical validation process (Table 1). Currently.b). 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. In another blood donor screening study.000 subjects (Clark et al.. 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. was 37. or hepatic disease undefined by a current marker or endpoint. The most critical independent associated variables were gender and obesity. In male obese subjects. Would the population affected by a revised ALT ULN show an increased susceptibility or risk for predictable or idiosyncratic DILI? Importantly. increased weight.05% of nearly 20. Repeating measurements increased the probability of an elevated ALT measurement..000 inductees showed unexplained ALT elevations. 1989). We predict that patients which show ALT values approaching the current 1. Since ALT elevations can occur in the absence of observed liver injury. the ALT upper limit of normal is driven by the historic values from the laboratory that runs the assay (Watkins et al. 1986). In trials for new chemical entities. 1993). Ozer et al. Use of preclinical samples with hepatocellular necrosis grade 1 severity is an optimal comparator in a receiver operator curve (ROC) analysis. glucose and lipids) would capture most subjects with increased BMI. Unexplained ALT elevations were also observed in large general populations exceeding 20. this newly defined patient population can be prospectively examined in future trials or for biomarkers qualifications.5 ULN range prior to compound administration will be at an increased risk of DILI incidence with drug treatment that induces hepatotoxicity (AZ. compared to 52 U/I (Kariv et al. We recognize that considerable investigative efforts are needed for this type of study. 1993). and in other developed countries. but 25% of the elevations were a single instance. glucose. 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. liver diseases and systemic diseases. Blood donors show ALT elevations in nearly 18% of the population with independent association with males. The prevalence of elevated baseline ALT values in the US has surprisingly doubled from 1988–1994 to 1999–2002 and was associated with Non-Alcoholic Fatty Liver Disease or NAFLD risk factors.... 2006).. Moderate alcohol use has a greater influence on ALT elevation when BMI increases ALT >2Â ULN (Alatalo et al. Employing a single laboratory method (with similar reagents and analyzer) to run ALT samples for a biomarker qualification application is recommended and would address concerns regarding variations in ALT activity levels between assay methods (Table 1). Variation might be attributed to non-Gaussian distribution.242 J. A large scale study of healthy people indicates that the ALT ULN is actually lower than the historically accepted value (Kariv et al. Since many asymptomatic individuals with elevated ALT were found to resolve below ULN within months on retesting (Friedman et al. 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. 2003). 2008). 5. triglycerides.2.. Genome-wide association (GWA) approaches defined two loci (CPN1-ERLIN1-CHUK and PNPLA3-SAMM50) in humans that influence elevations of serum ALT and AST activities. One phenotype increases the incidence of ALT activities just above ULN in one third of carriers (Yuan et al. we recommend that comparable criterion be considered for patients in a clinical trial paradigm were liver injury is being monitored. which should be considered more fully in a clinical development trial. muscle leakage of ALT.5 U/I. In one study. histopathology assessment is a critical standard to examine in a qualification paradigm. corresponding to the upper 95 percentile of the normal population. 1999).1. 2008). 1987). 1998). Even moderate obesity adversely influences ALT levels (Nomura et al. 2009 guidance provides similar recommendation regarding the frequency of observed ALT elevations in control compared to treated trial populations. while excluding HCV antisera and excessive alcohol consumption (Ioannou et al. Body mass index (BMI) data was not available. Approximately.g. waist size and even transfusion site (Papatheodoridis et al.

No genetic locus has yet been identified showing screening potential to avoid the onset of immunological reactions that induce idiosyncratic DILI. improved biomarkers of hepatocellular jaundice are critical to parse out ALT elevations that infrequently progress to acute liver failure and idiosyncratic DILI. although absence of uric acid changes does not preclude a diagnosis of metabolic syndrome.. ALT elevations in patients using marketed products can guide development of future products 6. a patient with a low ALT baseline value that reveals a 3Â elevation relative to baseline with compound administration would initiate a flagged monitoring event that would precede possible subsequent elevation >3Â ULN. 5. 2007). patient with a high ALT baseline value (ULN to 1.. By identifying the associated MHC class I allele HLA-BÃ5701 in the HIV patient pool eliminates toxic epidermal necrosis (TEN) or Stevens–Johnson Syndrome with avoidance of abacavir treatment (Phillips and Mallal. Clearly. Similar experience was shown with tacrine. 2004). 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. 2007). Patients with metabolic syndrome have three to ninefold elevated risk for development of type II diabetes (Bauduceau et al. ALT and uric acid both are biomarkers that associate and elevate with the development of metabolic syndrome (Kang et al. 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. Ozer et al. high arterial blood pressure. 2006).000 (Chitturi and George. ALT elevations resolved with continued treatment. No reports associate serum uric acid elevations with hepatoxic injury. factors in addition to ALT eleva- . This proposed monitoring strategy would enhance the safety window compared to current standards with a modest adjustment of current practices.. hypertension. uric acid shows no known relationship to liver injury. Additional drugs show similar potential to induce mild elevations of aminotransferases in the absence of severe DILI: aspirin. dyslipidemia. Abacavir induced hypersensitivity syndrome (AHS) has potential to induce mortality in <8% of treated patients (Phillips and Mallal. although ALT elevations were more frequent and at higher levels compared to statin therapy. use of normal western diets is anticipated to reduce the incidence of unexplained ALT elevations compared to use of high fat diets. 2009).5Â ULN values (Kuffner et al. glucose abnormalities. although no hepatocellular jaundice observations were made. and/or dyslipidemia with waist measurement adds significant value to this metric (Despres. notably troglitazone and bromfenac (Goldkind and Laine. In light of these issues. Whereas. HLA-BÃ1502 identified in patients of similar ancestry has potential to reduce carbamazepine-induced skin reactions by screening before commencement of therapy (Sheffield and Phillimore. ALT elevations were observed in osteoarthritis trials where subjects were given large daily doses of APAP (up to 3. Subtle ALT elevations during clinical trials History reveals programs that were successfully brought forward despite clinical ALT elevations. LC/MS and LC/ MS/MS methods have been developed to measure serum uric acid (Dai et al. Valproic acid (VPA) induced liver injury affects a limited segment of the population. 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. 2008). and less successful attempts to market other therapeutics with a cause of concern. an ethnic group which showed the greatest prevalence of hepatic steatosis and cirrhosis susceptibility (Romeo et al. In clinical trials. The incidence of metabolic syndrome varies by country.S. 2009). 2008). 2007). hydroxul-methylglutaryl coenzyme A-reductase inhibitors or statins (FDA.. multiple drug therapy patients.. Second generation cholinesterase inhibitors show less ALT elevations and improved dosing thereby replacing tacrine. These ALT elevations are observed in individuals that show three characteristics of metabolic syndrome (Kang et al.4... / Regulatory Toxicology and Pharmacology 56 (2010) 237–246 243 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. 2009). ethnicity. 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. while moderate alcohol consumption has a synergistic influence on observed ALT elevations (Alatalo et al. Upwards of 25% of adults in the United States have metabolic syndrome according to World Health Organization (WHO) criterion (Ford et al. unaccompanied by signs of liver injury. 2008).J. 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. and individuals with mitochondrial disorders among other conditions (Chitturi and George. a combination of genetic and epigenetic factors.5 ULN) should be tracked during monitoring for >3Â ULN from the onset of the study. While the development of metabolic syndrome may share risk factors with non-alcoholic fatty liver disease. Plasma insulin and other components of metabolic syndrome show high correlation to simple waist measurement. More than 17% of the patients had an ALT value that exceeded ULN and 4% showed >1.. 2002). which is usually associated with abdominal and liver fat content (Bauduceau et al. 2007). Subtle elevations of ALT are observed with Acetaminophen (APAP) dosing in humans in the absence of reported hepatotoxicity or liver failure.. Using this approach.. 2006). heparin. 2009). although the risk elevates to 1:500 in young children. 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. 2002). Hyperalimentation induced by high fat diet within a one month period induced a body weight increase of 5–15% and ALT elevations above the reference limits in more than 50% of the subjects (Kechagias et al. such as tacrine or statins. Higher body mass index (BMI) associated with metabolic syndrome appears to associate with ALT >2 ULN. The high incidence of metabolic syndrome in the United States population underscores the complexity of new candidate drug evaluations in populations considered normal for first-in human studies.. Concordance of moderate fasting hyperglycemia. 2001). VPA has a low liver injury risk overall at 1:37. tacrine. 2008). Thus. and insulin resistance. and nutritional intake while cumulative risk has been observed in numerous studies over a 10-year window (Bauduceau et al. we propose that patients’ ALT elevations may be considered relative to their own predosing baselines (or alternatively to historical ranges) when analyzing aminotransferase elevations. 2009). 6. 2008).1. The HLA-BÃ5801 allele is associated with allopurinol-induced cutaneous rash in patients of Han Chinese ancestry and screening prior to treatment can significantly reduce incidence of this potentially fatal skin reaction (Sheffield and Phillimore.. Broader experience with additional therapeutics in this class validated that liver injury is not a cause of concern. Simvastain was brought forward with 2% rate of ALT >3Â ULN in clinical trials. including diet are most likely.9 g/ day). While genetic polymorphisms might contribute to unexplained ALT increases. 2006).

2006). Mitochondrial damage. 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. in vitro approaches can aid our understanding of DILI mechanism. and BMI did not correlate and treatment continuation with jaundice was associated with fatality (Anand et al.. Even in patient trials in later stages. 2002).. This includes clean baseline liver function (such as exclusion of subjects with ALT >1. although additional tools are needed to complement these approaches.5Â ULN) and absence of a history of liver disease. Unfortunately. Practical guidelines and an algorithm for diagnosis and early treatment of DILI have been proposed for diagnostic review of injury prognosis (Tajiri and Shimizu. the decision to withdraw the subject is based on caution with a common view to avoid any risk to the subject on study. but the primary clinician and patient history are the most critical drivers for this decision. 6.. Regucalcin may predict transient ALT elevation outcomes. and >20 aminotransferase elevations in treated patients be compared to control groups in trials.. 2002). NAT-2 genetic analysis to define slow acetylator phenotypes is complex and requires many restriction endonuclease digestions. Ozer et al. 2008).. Thus. while age. Clinically. or even among consortia members. 6. 2005). although a delay of several weeks in the bilirubin signal may arise. 2006). 1Â 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. There is no universal and agreed upon ALT elevation threshold across development programs..to greater accumulation of the toxic mono-acetyl hydrazine (MAH) metabolite. slow acetylators show a more frequent ALT elevation with isoniazid treatment from metabolism of acetylisoniazid. even within a single company. the length of monitoring is a complex issue considering concerns for containing health costs. 1999). 2008).2. 6. A parallel assessment of ALT. It is standard practice in some pharmaceutical company Phase I healthy volunteer trials for subjects to be taken off study with a finding of ALT or AST >3Â ULN. This is a difficult decision for the study director to make. The Ca2+-binding protein regucalcin regulates calcium metabolism in kidney and liver. Cellular imaging predictions of primary human hepatocyte culture are modestly predictive of clinical outcomes. sex. total bilirubin. making the utility of this approach limited to screen for DILI comprehensively. oxidative stress. while the serum activity is diagnostic (Miller et al. although the evidence is controversial since fast acetylators make higher levels and show greater clearance of this toxic metabolite (Saukkonen et al. 2006). 2009). 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 qualification (Table 2). FDA. indicating that elevations are permissible in the absence of bilirubin injury (>2 ULN). With regards to the clinical trial setting. but assays are not commercially available to evaluate in a qualification paradigm. 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. 2003). Thus. 2007). Do transient rises in ALT predict severe DILI at a later time? FDA. so translational approaches are limited until additional biomarkers of liver injury are discovered and tested in the clinical setting (Table 2). and ALP values is recommended during all monitoring of ALT elevations in clinical trials to judge the potential for serious idiosyncratic DILI (FDA. Regucalcin is 93% conserved between rat and human. 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.244 J. While these studies are with a limited set of toxicants. 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. the path forward to reach an agreed universal ALT threshold will be challenging since preclinical models of these events will be difficult to reproduce. 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 briefly monitored to indicate a transient response if the elevation is just above 3Â ULN. Nevertheless. It has been proposed that pre-existing liver disease does not predispose subjects to drug-induced liver toxicity (Zimmerman. another important point is that recruitment of patients and healthy volunteers is based on certain minimum entry criteria. urinary biomarkers including metabolites could be a source of predictive markers of DILI. The incidence of transient ALT rises in a patient pool is much greater in frequency compared to those patients that progress to severe DILI. Regucalcin elevations early post-dose are nearly twentyfold greater compared to one month after dosing (Yamaguchi et al. intake of acetaminophen/paracetamol (Watkins et al. whereas regucalcin serum elevations were observed as long as 30-days post-toxicant treatment (Yamaguchi et al. even in the absence of associated bilirubin increases.3. More frequent isoniazid dosing (2Â weekly with rifampin) for ATT is associated with greater DILI incidence compared to 1Â weekly therapy with rifapentine. Serum regucalcin elevations appear in acute D-galactosamine and lipopolysaccharide (GalN/LPS)-induced liver injury in mice (Lv et al.. >5. 2009 guidance document recommends repeated measurement of elevated aminotransferase activities that cause concern (48–72 h after initial detection and repeatedly for several weeks thereafter). In a clinical trial. 2009). frequent monitoring is recommended in these subjects with ALT elevations (FDA.. while hepatic over expression of regucalcin suppresses cell death and apoptosis (Yamaguchi. and/or exercise (Pettersson et al.. 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. / Regulatory Toxicology and Pharmacology 56 (2010) 237–246 tions influence susceptibility to severe DILI. which is considered permissible for continued dosing of a drug candidate. inpatient hospitalization for 2 weeks or more. Hepatitis B infection and silent chronic liver disease correlated to ATT induced DILI. AST. In a post-marketing scenario. Nevertheless.4. 2009 guidance recommends that incidence of >3. Elevated predose production .. Urinary cyt c show predictive response prior to the onset of aminotransferase elevations and histopathologic observations of injury. >10. 2008). 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.S. Cytochrome c (cyt c) is a serum and urinary biomarker of liver injury induced by acetoaminophen (APAP) and D-galactosamine (GalN) treatment. assuming that the initial degree of ALT elevation is not itself sufficient cause of concern to the clinician to remove the patient from treatment. alcohol use. In CCl4 treated rats showed twofold ALT and AST elevations 3-days post-treatment. 2008). taking into consideration the potential risks to patient safety with continued dosing and the appropriate level of ALT elevation.

Dis.N. Gastroenterol. Klein. 107. Alatalo. Lett. Although this seminal pharmacometabonomic study in humans found a clear association between an individual’s predose urinary metabolite profile and the post-dose urinary fate of acetaminophen. With ever-increasing regulatory oversight. Available from: <http://www.. clinical features. E. J.).P. Ford. et al. 2005. Natl. indicating that there are a number of well defined approaches that can add value to their applications in the drug development setting. Med. Drug-Induced Liver Injury Network (DILIN) prospective study: rationale. Textbook of Hepatology. 2006. 2009. Re-evaluation of serum alanine aminotransferase upper normal limit and its modulating factors in a large-scale population study. Curr. A systematic review of NSAIDs withdrawn from the market due to hepatotoxicity: lessons learned from the bromfenac experience. Ann.. B. Pyridoxine and its metabolism in chronic liver disease.. Proc. 154. Angus. et al. Clement. We thank Christina Hunt for reading the paper and making suggestions on genetic factors and Holly Jordan for the rationale for biomarker qualification. Pharmacol. pharmaceutical companies are now addressing the issue of drug-induced liver injury more rigorously now than before. 2009). R.... or PSTC and IMI and its membership companies. 38. Drug Discov. 1093–1102.W. 2137–2148. Lewis. 76–82. Acknowledgments J. 2006. et al. Alanine aminotransferase screening of blood donors: pro. Risk factors of hepatotoxcity during anti-tuberculosis treatment. Shelli Schomaker. et al.. L. N. 2004. Disclaimer The personal views expressed in this article by the authors may not be understood nor quoted as being made on behalf of or reflecting the position of the FDA. 34. Kaplowitz. 213–220.K.. 474–485. 15. Technol.. 4. Prevalence and long-term course of macro-aspartate aminotransferase in children.. In: Gitlin. 33.. FDA. E. 2005. Kang. Ann. Anand. Despres. and patients affected by such condition. Gastroenterol. References Abraham. Connective tissue diseases and the liver. (Eds. Increasing prevalence of the metabolic syndrome among U. A. 28.. N. Sci. Conflict of interest The authors declare that there are no conflicts of interest. Expert Opin. 2009. Nat. 8.S.pdf>. indicating where there is scope for improvement. AAPS J. Fast-food-based hyper-alimentation can induce rapid and profound elevation of serum alanine aminotransferase in healthy subjects.. 1973. Hepatotoxicity of commonly used drugs: nonsteroidal anti-inflammatory drugs. E. Idiosyncratic drug-induced liver injury: an overview. Farrington. 2006. Engl. S. The effect of gastrointestinal diseases on the liver and biliary tract. 489–499. Sci. S.W. R. 22.. Pharmacoepidemiol. Dig. Liver Int. Life Sci. et al.gov/downloads/Drugs/ GuidanceComplianceRegulatoryInformation/Guidances/UCM174090. Hepatology 43. 2006. Med. et al. 98. 55. Kariv. Opin. J. R. et al. Drug-induced hepatotoxicity. 1994. Sci. 8. et al.K.e1-4 (Epub). J. Dis. and other predictors of severe drug-induced hepatotoxicity: putting risk-benefit into perspective. 2008. George. 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. 1145–1151. Toxicol. 22. 349. J. pp. Ioannou. W. L. Gupta. G. Gitlin. 2009). Drug Saf. P..H. J. Pearson Professional Limited. pp. Acad. Gut 57. 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 qualification of such markers.. Laine. 377–387. Friedman.. Health consequences of visceral obesity. D. design and conduct. et al. et al. Protection by indomethacin against the lethality and hepatotoxicity of phalloidin in mice. Toxicol. Mechanisms of drug-induced liver injury. N. Am.J. / Regulatory Toxicology and Pharmacology 56 (2010) 237–246 245 of endogenous colonic p-cresol correlates inversely to a reduction of an individual’s ability to sulfonate acetaminophen (APAP) by acting as a competitive substrate and leading to reduced urinary excretion of APAP (Clayton. Ju. and James Mayne for helpful suggestions on regulatory and qualification considerations. 32. Goldkind. Clin. 134–136.D. Am. Chromatogr. Evaluation of blood donors with elevated serum alanine aminotransferase levels. et al. medical. white blood cell count. 7. 445–450. EMEA.S. Aliment.A.C were equal contributors (co-1st authors) to this paper. 1977. 2003. academic... L. Ther. Denise Robinson-Gravatt. M. N. Rev. Hussaini. 15. Conventional biochemical tools like ALT and AST have limitations and are imperfect as biomarkers of DILI. further studies in animal models will allow better mechanistic understanding of metabolism upon induction of DILI. 71..W. 174–189. 857. Ozer et al. This perspective addresses some of these gaps and highlights the need for better use of available diagnostic standards. 115– 135. 101. J. regulators. N. Review article: the use of potentially hepatotoxic drugs in patients with liver disease. 2008. Am. 26. Retrospective analysis of transient elevations in alanine aminotransferase during long-term treatment with acetaminophen in osteoarthritis clinical trials.. 960–967. et al. 2007. Barriault. 2001.. Should we have more definitions of metabolic syndrome or simply take waist measurement? Diabetes Metab.. S. Diabetes Care 27. P. Drug Discov. 45–49..fda. 2008. Liver Dis. The Liver and Systemic Disease. 1097–1103. Res.. and outcomes from a prospective study of drug-induced liver injury in the United States. 2444–2449. Holt.C. We thank Bill Mattes. 1997.I. E48–E54. et al.. Endocr. 1622–1633. Clark. 1021–1041. Am. 744–748. A. Elevated serum alanine aminotransferase activity and calculated risk of coronary heart disease in the United States. Clinical chemistry and histopathology anchors are recommended to characterize predictive DILI biomarker activities. 33.. Nutr. J. 221–229. 2006. 265. 363–367. Effect of moderate alcohol consumption on liver enzymes increases with increasing body mass index. 88. Hepatic manifestations of autoimmune rheumatic diseases. 55–68. Anon. and Wendy Bailey for helpful discussions in the PSTC HWG PPMG working group.N.. Effects of fenofibrate on plasma and hepatic transaminase activities and hepatic transaminase gene expression in rats. Drug Saf. Pharmacometabonomic identification of a significant host– microbiome metabolic interaction affecting human drug metabolism. USA 106. L. 2007. Gastroenterology 135. Serum alanine aminotransferase (ALT) elevation in asymptomatic US Air Force basic trainee blood donors. 2007. antidiabetic agents. . anticonvulsants. 2008. ‘Hy’s law’. Int. J. Dai. et al. The prevalence and predictors of elevated serum aminotransferase activity in the United States in 1999–2002. 649–654. Kechagias. C.M. Sci. 673–684. and regulatory leaders to advance and improve the science of liver safety assessment in preclinical development and clinical practice. 2003. Kuffner. 534–541. J. 257–269. Y.). Causes. 123–129.. 2002. Mike Aleo.P. Opin. J..H. Ann. MJAFI 62. antihypertensives. 2006b. J.. 6. N. Biomed. 169–183. The liver in congestive heart failure: a review. 2006a. J. 1993. 287–295. 35.H. Immunological mechanisms of drug-induced liver injury. Bauduceau. 2007.. 1924-1934. and R.J. J. Pediatr. Clayton. Fontana. et al. especially metabolic or lipidomic markers (Ozer and Teitelbaum. C. clinicians. S. et al.. et al. 2008.S. et al.H. the ‘Rezulin Rule’. 63.O. et al. Idiosyncratic drug hepatotoxicity. Med. et al. 14728–14733. T. psychotropic drugs.S. J. Chapman. Caropreso. Kundrotas. Ioannou. G. 2004. Controversies in transfusion medicine.A. G. Chalasani.. J. lipid-lowering agents. We thank Jennifer Colangelo. Med. 1934. 1987. (Ed. Semin. B Analyt. In: Rodes... 38–43. Pharmacoepidemiol. et al. Nutr. H. Drug Saf. Curr. and uric acid in their association with metabolic syndrome: a study of Korean adults. Med. X. Rev. C. We encourage continued dialogue among pharmaceutical. 2145–2150. Drug Saf. Lewis. Adults. Lee. Devel.. 137–144. The prevalence and etiology of elevated aminotransferase levels in the United States.G.. 2006.. 333–339.. Determination of serum uric acid using high-performance liquid chromatography (HPLC)/isotope dilution mass spectrometry (ID-MS) as a candidate reference method. Hong Kong.. Guidance for Industry: Drug-Induced Liver Injury: Premarketing Clinical Evaluation 2009. 1990. Transfusion 30.J. Chitturi. Kobayashi.M. 2009.. M. Comparison of alanine aminotransferase. Conclusions Drug-induced liver injury is a significant hurdle for the pharmaceutical industry. Rheum. Oxford Blackwell. Dunn. Ju. 2009.

E. F. et al. Toxicol. Decreased hepatic ALT synthesis is an outcome of subchronic microcystin-LR toxicity. 1986. 1996. Efficacy and safety of high-dose pravastatin in hypercholesterolemic patients with well-compensated chronic liver disease: results of a prospective. D. M. Biochim.. purification. G. Life Sci. double-blind. Care Med.. Pagani. Watkins. P. Isoforms of alanine aminotransferases in human tissues and serum – differential tissue expression using novel antibodies. Ozer. Yuan. 1461–1465. N. J.246 J.. 2006. The regulation of aminotransferase activity by carbamoylphosphate. 135–161. 54. 778–787... 1986. Drug-induced liver diseases. J. Genet. R.). Acta 1204. et al. .).. S.. Biophys. Thiele. Omata. 2008. Crit. Med. et al. 241–243. Pharmacoepidemiol. T. Understanding the role of reactive metabolites in druginduced hepatotoxicity: state of the science. 1341–1347. J.. J. Methods 60. Hepatology 41. et al. H. 21. et al. 2008. Hepatology 48. et al. C. Polson. 383–387.. et al. 253–259. M.J. The regulation of alanine and aspartate aminotransferase by different aminothiols and by vitamin B-6 derivatives. Saukkonen. Clin. Lee.. Sheffield. 2009.I.J. / Regulatory Toxicology and Pharmacology 56 (2010) 237–246 Robinson.. pp. Solter. Respir. Papatheodoridis. Pharmacol. Gastroenterol... Gastroenterology 110. Y. Hepatology 49.B. J. 1999. Lucena. K. et al.. Evaluation of commercially formulated aspartate aminotransferase and alanine aminotransferase activity determinations by the Scandinavian committee on enzymes and IFCC methods as modified for use with automated enzyme analysers. 1999. Baltimore (MD).. 2005. 283–290. 535–538. 2006. J. V. 2008. Appl. Genet. Aminotransferase elevations in healthy adults receiving 4 grams of acetaminophen daily: a randomized controlled trial. 349–354. 371–389.. 2003. Ramesh. Drugs 16. P. Lab Sci. J. Primary hepatocytes as a model to analyze species-specific toxicity and drug metabolism. World J. Drug Metab. R..V. 2009. 55–65. 15. 174. Toxicol. Am.).C. et al. The spectrum of hepatotoxicity. et al. Hum. et al. X. 2007.. 2007. W. 61–67. 13. 2001.. 15. 2007. P. 1994a. Purif. L.H. Toxicol. H. In: Schiff.. 2008. Mastoraki. Cellular imaging predictions of clinical drug-induced liver injury.. Mol. J. 2006. Identification of novel molecular candidates for acute liver failure in plasma of BALB/c murine model. Philadelphia. High prevalence of elevated liver enzymes in blood donors: associations with male gender and central adiposity. P. 815–828. Clin. Rev. Biochem. Drug Metab. 6. et al. Hepatol.. 2000. Analysis of IL-10. Hepatol. Int. Ju. J. Schiff’s Diseases of the Liver. Piton. J. 2009.. 2008. J. Appl. Meier... multicenter trial. Crit. 83. S. 1998. Toxicol. Nathwani. Phillips.J. Effect of body mass and other factors on serum liver enzyme levels in men attending for well population screening. Total parenteral nutrition-associated liver dysfunction. Transfuse Med. Vanderlinde. 1994.. Potential role of regucalcin as a specific biochemical marker of chronic liver injury with carbon tetrachloride administration in rats. Mallal. Lett. Tajiri.. Rosenzweig. Gastroenterol.D. 1680–1689. Skelly. M. Elsevier Science. 1453–1463. Transaminase elevation on placebo during phase I trials: prevalence and significance. Nutrition 10. (Ed. 19–23.. 855– 870. et al. 79–93. 321–322. 2008. R. 2006. Hypertens. Zimmerman. Lippincott-Raven... Hepatology 46. 28. 2008. Lindblom. Senior. MULTIVIRC group. Onat. et al. et al.. 25–45. Pagani. et al. Olson.. et al. Regulatory Perspectives. J. Zimmerman. J. 6774–6785. D. J. Novel prodromal biomarkers that are symptomatic of injury require histopathologic or clinical chemistry anchors to characterize activity.. Proteome Res. Calpain activation in plasma membrane bleb formation during tert-butyl hydroperoxide-induced rat hepatocyte injury. 1994b. Ann. Lv. 250–256. pp. Miller.. K. Further reading Antoine. J..J. Hepatic manafestations of systemic disease and other disorders of the liver. J.. et al. Temple. Review of pyridoxal phosphate and the transaminases in liver disease. The current state of serum biomarkers of hepatotoxicity. D. Marcel Dekker. 30. M.. J. A. Clin. L. 1998. Spiliotis. 281–287. Expert Opin. 60.. Watkins.E. G. 2008. 2008. 14. D. 273–274.. Drug-induced liver disease. R. Hepatobiliary transporters and drug-induced cholestasis. A. 1179–1197. Findings on liver biopsy to investigate abnormal liver function tests in the absence of diagnostic serology. IL-4 and TNF-alpha polymorphisms in drug-induced liver injury (DILI) and its outcome.R. 2005... Toxicol. Hepatology 48.R. Expert Opin. J. et al. et al. Pachkoria. Dis. Hepatobiliary Pancreat. Pohl. Zimmerman. 255–260. Biochem. 1017–1044. et al. Cytochrome c: a non-invasive biomarker of drug-induced liver injury. Clin. Liver function in moderate obesity – study in 534 moderately obese subjects among 4613 male company employees. 2005. 2746–2752. 2006. Yamaguchi. 935–952. Gastrointestinal and Liver Disease. Weiner. H. Obes.. et al. Concordance of the toxicity of pharmaceuticals in humans and in animals. 164. 247–256. Am. 35. Int. 380–382. 520– 528. 4. Yamaguchi. 48. Protein Expr. and initial characterization of human alanine aminotransferase (ALT) isoenzyme 1 and 2 in High-five insect cells. Ann. 588–596. et al. Xu. Hepatol. Br. Ozer. 4. 19.L. Phillimore. Hepatology 41.J.M. Mol. Int. Successful translation of pharmacogenetics into the clinic: the abacavir example. L. (Ed... How can ‘Hy’s law’ help the clinician? Pharmacoepidemiol. Ozer et al. Pathological spectrum of liver diseases in sickle cell disease.. Care Med.. 1–9. Using controlled clinical trials to learn more about acute drug-induced liver injury. H.E. Expression.. 1603–1619. E. et al. In: Kaplowitz.R. et al. 2009. Biol. 2008.B. et al. randomized. Hepatology 27.. J. J. Tolerogenic role of Kupffer cells in immune-mediated adverse drug reactions. Miyoshi. 393–400. 194–205. et al. Arch. A.J. Biochem.J.. Whitehead. 167.. 2008. 10. P. Clin. 32. Toxicol. Toxicology 209.. Dis. T. 2007. 1988. 6. Tuschl. Eur. Nat..J. 2007. 109–112.P. Pauli-Magnus.S. Drug-induced liver disease. 31. Olson. 973–1064. 1978..M. Med. Liu. Hy’s law: predicting serious hepatotoxicity. F. Pettersson. et al. 1995. Pharmacol. Perspect. 107–114. Factors associated with serum alanine transaminase activity in healthy subjects: consequences for the definition of normal values. Serum alanine aminotransferase in skeletal muscle diseases. 2003. Clin. et al. M. Romeo. J. Practical guidelines for diagnosis and early management of drug-induced liver injury. 56–67. J. 65. Genetic variation in PNPLA3 confers susceptibility to nonalcoholic fatty liver disease. Population-based genome-wide association studies reveal six loci influencing plasma levels of liver enzymes. 1968. 2000.. 16. et al. Toxicol. M. 87–93.. Biochem. Role of regucalcin in maintaining cell homeostasis and function. Am. et al. 66–77. H. Postoperative jaundice after cardiac surgery. Cell. Lustig. 466. Biochem. A. R. Relationship of serum alanine aminotransferase (ALT) to body manss index (BMI) in blood donors: the need to correct ALT for BMI in blood donor screening. Dig. Rockey.. C. AASLD position paper: the management of acute liver failure. et al. Toxicology 245. 1989. 49. 97–105. (Eds. Mol. V.. 2002. 1897–1904. Lewis. et al. 216–220. 1213–1219. 12.A. 1415–1427. In: Feldman. Senior.J. Br.. 2002. S. Toxicol. 775–783. 2008. M. Muscular exercise can cause highly pathological liver function tests in healthy men. Serum uric acid is a determinant of metabolic syndrome in a population-based study. The predictivity of the toxicity of pharmaceuticals in humans from animal data – an interim assessment. 1986. et al. 102–103. 5. Am. for selection of blood donors. H. 235–239. et al. 241. placebo-controlled. et al. Respir. 105. Ther. et al. 195–199.. Regul. 1055–1062. NY. Pharmacol. Nomura. Shimizu. Pharmacol. 2006. An official ATS statement: hepatotoxicity of antituberculosis therapy. Hepatology 44. Teitelbaum. Sci. et al. 19. Glutathione S-transferase m1 and t1 null genotypes increase susceptibility to idiosyncratic drug-induced liver injury. Sci. J.S. J. 26 (Pt 5). and for patients with chronic hepatitis C. Pharmacol.. Jama 296. 225–231. Drug Saf. P. Clinical use of pharmacogenomic tests in 2009. 40. Drug Saf. Liver biopsy. 2005. Diagn. Kalfarentzos. Biophys.... H.. 2008. 15. Low isoniazid concentrations and outcome of tuberculosis treatment with once-weekly isoniazid and rifapentine.

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