Biomarkers for the Diagnosis and Management of Drug-Induced Liver Injury

• ABSTRACT
• CURRENT STATUS OF DILI BIOMARKERS
• PROMISING AVENUES FOR DISCOVERY OF NEW DILI BIOMARKERS
• Genetics
• Serum Adducts
• Lymphocyte Transformation Test
• Anti-Liver Antibodies
• Metabolomics
• Transcriptomics
• Proteomics
• NEXT STEPS
• ABBREVIATIONS
• REFERENCES

ABSTRACT

There is a pressing need for new clinical tests that will help physicians distinguish drug-induced liver injury (DILI) from other, more common causes of liver injury, and that can identify which specific drug is the culprit when DILI occurs in the setting of polypharmacy. In situations where there are few alternative treatments, new tests are needed that can differentiate patients with DILI who would develop progressive liver injury if treatment is not stopped from patients who can safely continue drug therapy via “adaptation.” Although there has been little progress in developing and validating such tests, new insights into the mechanisms underlying DILI suggest that the desired biomarkers probably exist and can be discovered through the application of new technologies for blood and possibly urine analyses. Such discovery efforts will require the establishment of well-annotated serum and urine banks from prospective clinical trials of drugs capable of causing progressive liver injury.

Because available clinical laboratory tests are not ideal biomarkers, the diagnosis of drug-induced liver injury (DILI) remains generally one of exclusion and thus is typically time and resource intensive. (In the context of medical treatment, a biomarker has been defined as “a characteristic that is objectively measured and evaluated as an indicator of ….. responses to a therapeutic intervention.”[1]) Also, because DILI is a relatively uncommon adverse drug reaction, it is often not possible to confidently identify the specific culprit when DILI is suspected in a patient who is being treated with multiple medications. Finally, in many instances, DILI will resolve despite continuing treatment with the offending drug(s) due to a process termed “adaptation.”[2] No current biomarkers can distinguish between patients who are capable of adaptation with continued treatment and patients who will experience progressive liver injury unless treatment is discontinued. Biomarkers that could predict adaptation would be very useful in the treatment of conditions such as tuberculosis, in which DILI is a relatively common occurrence but for which there are limited treatment options.

This article reviews the promises and challenges in developing and validating biomarkers that could address these needs and thereby improve the diagnosis and management of DILI. The clinical and histological presentation of DILI can mimic most types of liver disease, and it is likely that the optimal biomarkers will differ depending on the type of DILI. Hepatocellular liver injury is generally the DILI of greatest concern to patients and physicians. This is because hepatocellular DILI can evolve quickly and be life-threatening before the development of detectable jaundice. For this reason, hepatocellular DILI is the major focus of this review.

CURRENT STATUS OF DILI BIOMARKERS

The biomarkers most commonly used to detect and manage hepatocellular injury are serum alanine aminotransferase (ALT) and total bilirubin levels. Serum ALT is more liver-specific than aspartate aminotransferase (AST)[3] and is a very sensitive detector of hepatocellular necrosis. However, serum ALT cannot distinguish liver cell necrosis due to DILI from necrosis resulting from other causes, such as viral hepatitis. Moreover, serum ALT levels may increase substantially due to events other than hepatocyte necrosis, including hepatic glycogen accumulation in poorly controlled diabetes,[4] [5] hepatocyte autophagy in anorexia nervosa,[6] and hepatic steatosis.[7]

Transcriptional activation of the ALT gene has been reported in cultured human hepatocytes exposed to peroxisome proliferator-activated receptor (PPAR) agonists[8] but has not been shown to occur in vivo. These observations may, in part, explain why frequent and fairly high serum ALT elevations observed in clinical trials do not reliably predict the potential to cause progressive liver injury. For example, ∼25% of Alzheimer's disease patients receiving treatment with tacrine experienced serum ALT elevations >3 times the upper limit of normal (ULN), and 2% exhibited ALT >20 times the ULN in clinical trials.[9] However, ALT elevations caused by tacrine, even very high ALT elevations, can resolve completely during continued treatment with the drug (Fig. [1]). Moreover, the postmarketing experience with tacrine has not suggested a high risk for progressive liver injury. Similarly, although most statins cause ALT elevations in a subset of treated patients, statin use has not been clearly associated with an increased risk of serious DILI.[10]

Heparins are a class of biologicals that can also cause ALT elevations but rarely, if ever, cause clinically important liver injury.[11] In these instances, it is unclear whether the ALT elevations reflect true liver injury that virtually always resolves with continued drug exposure, or whether processes other than toxicity are involved. However, it is clear that with drugs capable of causing progressive liver injury, many or most ALT elevations observed during treatment will also resolve with continued therapy. In this instance, it appears that hepatocyte injury, including necrosis, is occurring during transient ALT elevations, but that in some and perhaps most patients, the liver undergoes adaptation such that the injury resolves despite continued drug exposure. This phenomenon of adaptation has been observed with drugs capable of causing acute liver failure, including isoniazid,[12] troglitazone,[13] and ximelagatran.[14] Adaptation during continued exposure to toxicants has been observed in rodents, even when severe liver necrosis is present.[15] It appears that this adaptation involves both upregulation and downregulation of multiple gene products, including drug metabolizing enzymes[16] and transporters.[17] Changes in transporter regulation have also been observed in human liver during recovery from DILI,[18] suggesting that similar mechanisms of adaptation may be operative in rodents and man.

Hy Zimmerman[19] first noted that a patient who presents with jaundice as a result of hepatocellular DILI has at least a 10% chance of developing acute liver failure regardless of which drug has caused the hepatocellular injury. This observation, which has been referred to as “Hy's Law,” has been confirmed in recent reports.[20] [21] Combining the two biomarkers ALT and bilirubin is therefore a much greater predictor of patient outcome during a hepatocellular injury than is serum ALT alone. However, bilirubin and ALT are not optimal biomarkers in this regard because during a hepatocellular injury, the bilirubin rises only after there has been a very substantial loss of functional hepatocytes placing the patient in danger of liver failure. The ideal biomarker would predict whether the patient would adapt before the injury represented a potentially serious health threat.

PROMISING AVENUES FOR DISCOVERY OF NEW DILI BIOMARKERS

Genetics

It is now well established that genetic factors partially account for interindividual differences in susceptibility to DILI.[22] However, the genetic associations observed to date have generally been weak and will probably not be useful in the diagnosis or management of DILI. A recently described exception may be the association between the human leukocyte antigen (HLA) B*5701 haplotype and flucloxacillin liver injury.[23] Having this HLA haplotype confers an ∼80-fold increase in susceptibility to liver injury from this drug. Nonetheless, only ∼1 in 500 individuals with the HLA B*5701 haplotype (which has a prevalence of greater than 5% in white populations) will develop liver injury when treated with flucloxacillin. Pretreatment screening may therefore not be cost effective. On the other hand, knowing the HLA genotype could be helpful in establishing the diagnosis of DILI and in identifying flucloxacillin as the culprit in a patient with DILI who is receiving multiple drugs. There are two major networks that have banks of genomic DNA obtained from patients who have experienced DILI: the Drug-Induced Liver Injury Network (DILIN)[24] and the Severe Adverse Events Consortium.[25] Each has begun genetic analyses, and these efforts should go a long way toward assessing the potential of genetic biomarkers of DILI.

Serum Adducts

Several studies have suggested that finding acetaminophen-protein adducts in serum can both confirm the diagnosis of DILI and confidently identify acetaminophen as the cause for the DILI.[26] [27] [28] [29] The underlying concept of this biomarker, which is well established in rodents, is that liver injury due to acetaminophen is reliably preceded by the formation of covalent adducts between intrahepatocyte proteins and the major reactive metabolite formed in the liver from acetaminophen (N-acetyl-p-benzoquinone imine [NAPQI]).[30] The assumption is that finding acetaminophen-protein adducts in the serum can only occur in the setting of acetaminophen DILI. Acetaminophen-protein adducts have a much longer half life in serum than acetaminophen or its primary metabolites and may therefore have diagnostic value well after other acetaminophen-derived products are no longer detectable.[29] Enthusiasm for the assay has been diminished somewhat by the recent demonstration that low levels of acetaminophen adducts are detectable in the serum of healthy adult volunteers taking therapeutic doses of acetaminophen and who have normal ALT levels (i.e., no evidence for liver injury) (Laura James, personal communication). It remains unclear what would happen if an individual consuming therapeutic doses of acetaminophen experienced an unrelated liver injury, such as viral hepatitis, but it seems likely that the circulating levels of these adducts would rise substantially and potentially falsely implicate acetaminophen as the cause of the liver injury. Additional studies will therefore be necessary to better define the limitations of the adduct assay.

It is unclear whether circulating drug-protein adducts are present in many or most types of DILI. Acetaminophen behaves as a dose-dependent hepatotoxin and is administered in relatively large doses (up to 4 g daily). The toxic metabolite NAPQI may reflect well more than 5% of the total metabolism in an overdose situation, and toxicity is associated with substantial covalent binding to hepatocyte proteins.[31] However, with most other drugs capable of causing DILI, the reaction is “idiosyncratic” and occurs at much lower daily doses of the drug. In addition, toxic metabolites (when involved) often represent a more minor fraction of total metabolism[32] such that circulating adducts, if present, may be technically difficult to identify and quantify.

Lymphocyte Transformation Test

The lymphocyte transformation test is performed by culturing a patient's lymphocytes in the presence of the drug suspected to have caused the adverse reaction.[33] A positive test is the occurrence of lymphocyte proliferation, which can be measured several ways.[34] Proliferation reflects the existence of a subset of T lymphocytes that recognize and are activated by the drug. The assumption is that a positive response reflects an immunological mechanism for the DILI, and this is supported by the observation that the test is most frequently positive when DILI is accompanied by fever, rash, and/or peripheral eosinophilia.[35] [36] Nonetheless, lymphocyte transformation has been observed in some patients who have experienced DILI in the absence of hypersensitivity findings.[36] This is consistent with recent reports of associations between certain HLA haplotypes and DILI, which does not have hypersensitivity features, including hepatocellular injury due to ximelagatran,[37] flucloxacillin[23] and ticlopidine.[38]

The lymphocyte transformation test is commercially available in Japan, where it appears to be widely used to establish causality links between drugs and liver injury. It is rarely used in DILI causality assessment outside of Japan, and there have been very few studies using the technique in recent literature. Nonetheless, the technique appears promising in view of the growing recognition that non-immunoallergic DILI may involve acquired immune responses. It has been shown that the sensitivity of the assay can be increased by adding prostaglandin inhibitors. Removing regulatory T cells might also improve sensitivity of the assay. The DILIN is proceeding with an ancillary study to critically assess the value of the lymphocyte transformation test in causality assessment and prognosis.

Anti-Liver Antibodies

Patients with liver injury associated with several drugs characteristically have circulating antibodies to liver and kidney endoplasmic reticulum, termed anti-liver-kidney microsomal (LKM) antibodies.[39] [40] These antibodies result from an acquired immune response to a new antigen created by covalent binding between a reactive metabolite and a hepatocellular protein. Anti-LKM antibodies frequently react with cytochromes P450 (CYPs) but may also react with other drug-metabolizing enzymes.[41] [42] [43] Anti-enzyme antibodies have been found in DILI due to several drugs, including tienilic acid,[44] dihydralazine,[45] and halothane.[46] The current concept is that a highly reactive metabolite covalently binds to, or otherwise damages, the enzyme that produced it.[47] Antibodies are formed if this altered enzyme is antigenic and gets outside the hepatocyte, where it can be picked up by antigen-presenting cells. Because the antibodies recognize different proteins with liver injury due to different drugs (i.e., the bioactivating enzymes differ depending on drug structure), anti-liver antibodies should, in some instances, be useful in causality assessment. The DILIN has created a serum bank from patients who have experienced DILI, and it should be possible for the first time to determine the prevalence and clinical usefulness of these autoantibodies.

A traditional means of searching for anti-liver antibodies is to separate liver proteins by polyacrylamide gel electrophoresis, transfer the separated proteins to nitrocellulose sheets, and then determine if the serum contains antibodies to any liver proteins (i.e., the Western blotting technique). A problem with this technique is that the liver proteins are denatured in the process and may therefore not contain the antigenic sites present in vivo. Alternatively, the antigen targets may be present but below the level of detection with this technique. Another approach is to determine whether serum contains antibodies that react with commercially available arrays that contain thousands of proteins and protein fragments.[48] A limitation here is that there do not appear to be commercially available arrays derived from liver.

Metabolomics

The processes involved in initiation of DILI and adaptation to DILI are likely to influence the metabolism of many endogenous substances. For example, progressive mitochondrial injury has been demonstrated for fialuridine in the liver of animals and humans[49] and in cell culture for other drugs such as nefazadone[50] and troglitazone.[51] This could lead to characteristic changes in serum or urine metabolome long before mitochondrial function has deteriorated to the point of cytotoxicity detectable by serum ALT increases. Moreover, these changes might distinguish DILI from other types of liver injury. Some changes might be common to most or all forms of DILI because processes like mitochondrial damage could be “downstream” of molecule-specific events, such as reactive metabolite formation. On the other hand, some changes may be drug or drug-class specific.

Both high-resolution nuclear magnetic resonance (NMR) and mass spectral techniques can now determine the “fingerprint” of thousands of endogenous metabolites present in urine or serum.[52] It is possible to use these technologies to simultaneously quantitate thousands of metabolites in serum or urine obtained before, during, and after liver injury. Pattern recognition software can then identify characteristic changes that may distinguish different types of DILI, or that may identify patients most susceptible to DILI. An important point is that these techniques can identify metabolite “signatures” without knowing the identity of the metabolites (but which can subsequently be determined).

In one example of this approach,[53] the urinary metabolome was analyzed before and after outbred rats were administered a toxic single dose of acetaminophen. Using NMR technology, they were able to identify a pattern of endogenous metabolites in the baseline (pretreatment) urinary metabolome that correlated with the extent of liver injury observed in each rat after treatment with acetaminophen. The concept of using the serum or urine metabolome to predict outcome from exposure to a drug was termed “pharmacometabonomics” by these investigators.[53] The pharmacometabonomic approach has just recently been applied to DILI in humans. In one recent study,[54] unbiased analysis of the serum metabolome revealed that patients who are susceptible to ximelagatran DILI tended to have lower serum pyruvate than nonsusceptible patients, and that treatment with ximelagatran tended to cause a further drop in serum pyruvate in the susceptible patients. Mechanisms linking this observation to DILI have not been reported.

In another study of healthy adult volunteers treated with 4 g of acetaminophen daily, the metabolite pattern in the baseline urine did not predict which subjects would develop elevations in serum ALT.[55] However, changes in the urinary metabolome determined soon after the start of acetaminophen dosing (but before development of ALT elevations) did correlate with the ALT elevations subsequently observed. The urine breakdown products of NAPQI were noted to be higher in those who would experience ALT elevations versus those who would not. However, acetaminophen metabolites alone were not significantly predictive. The approach of defining predictive endogenous and exogenous metabolites early in the course of drug treatment was termed “early intervention pharmacometabonomics.”[55] Future studies should determine whether this approach can be used to identify patients who will develop liver injury during treatment with other drugs that cause DILI. These studies suggest that metabolomic approaches, perhaps combined with genetic tests, have promise in aiding the diagnosis and management of DILI.

Transcriptomics

Different hepatotoxicants produce different patterns of changes in messenger (m)RNA transcripts in the rodent liver,[56] and it is reasonable to assume that the same is true in humans. Because biomarkers requiring liver biopsy would not be optimal, there have been efforts in rodents to identify which drug-specific transcripts result in secreted proteins that might be measurable in the blood. To date, these efforts have met with little success. An alternate approach has been to measure changes in whole blood transcriptome during DILI. In one rodent study,[57] changes in whole blood transcriptome provided a more sensitive and specific predictor of the extent of liver injury than serum ALT. In the same study, transcript changes were also observed in whole blood obtained from patients suffering from acetaminophen DILI. Although these studies are intriguing, the transcriptome in whole blood chiefly reflects lymphocytes, and the relationship between the liver and lymphocyte transcriptome is unknown. An exciting and recent finding is that during DILIs, liver-derived mRNAs—both microRNA[58] and mRNA[59]—are detectable in circulating cell-free plasma. It remains to be determined to what extent circulating plasma obtained during DILI contains the full liver transcriptome. This is a very promising area of research because the ability to monitor changes in human liver transcriptome from a blood sample could represent a revolution in diagnostics.

Proteomics

Techniques are rapidly developing to identify and quantify thousands of proteins in serum and, with the help of statistical techniques, identify patterns of proteins that may yield useful biomarkers for DILI. Examples of protein biomarkers that may be discovered in this way are cytokines. During treatment with isoniazid, ALT elevations accompanied by hepatitis symptoms (fatigue, nausea, right upper quadrant pain) appear to be more predictive of progressive liver injury (i.e., inability to adapt) than are asymptomatic ALT elevations.[60] Symptoms in this setting may be mediated by cytokines or other endogenous proteins that may be detectable long before symptoms appear.

NEXT STEPS

The technologies are now available to begin large-scale efforts to identify and validate new biomarkers of DILI. Registries and tissue banks created by DILIN and the Severe Adverse Events Consortium will be rich resources for biomarker discovery. However, because the subjects are enrolled only after the diagnosis of DILI is established, blood or urine is not collected from the patients before the start of treatment, nor during treatment. Such prospectively collected specimens will probably be required for discovery and validation of nongenetic biomarkers capable of predicting whether a given patient will develop DILI and what the outcome of continued treatment will be. The Institute of Medicine convened a workshop in October 2008 to begin to identify the steps that will be required to develop better biomarkers of drug safety. The recommendations[61] include prospective clinical trials with blood and urine collections from patients treated with drugs known to be capable of severe liver injury, as well as drugs that cause frequent ALT elevations but rarely cause severe liver injury. It is important that these recommendations be enacted.

ABBREVIATIONS

• ALT alanine aminotransferase

• AST aspartate aminotransferase

• CYP cytochrome P450

• DILI drug induced liver injury

• DILIN Drug-Induced Liver Injury Network

• HLA human leukocyte antigen

• NAPQI N-acetyl-p-benzoquinone imine

• NMR nuclear magnetic resonance

• PPAR peroxisome proliferator-activated receptor

• ULN upper limit of normal

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• 61 Institute of Medicine. Accelerating the development of biomarkers for drug safety: workshop summary. Available at: http://www.iom.edu/CMS/3740/24155/70596.aspx Accessed July 10, 2009
  Search in Google Scholar

Paul B WatkinsM.D. 

Director, The Hamner–UNC Institute for Drug Safety Sciences, The Hamner Institutes for Health Sciences

Six Davis Drive, P.O. Box 12137, Research Triangle Park, NC 27709

Email: pbwatkins@med.unc.edu

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Paul B WatkinsM.D. 

Director, The Hamner–UNC Institute for Drug Safety Sciences, The Hamner Institutes for Health Sciences

Six Davis Drive, P.O. Box 12137, Research Triangle Park, NC 27709

Email: pbwatkins@med.unc.edu