Refining Liver Safety Risk Assessment: Application of Mechanistic Modeling and Serum Biomarkers to Cimaglermin Alfa (GGF2) Clinical Trials.

Cimaglermin alfa (GGF2) is a recombinant human protein growth factor in development for heart failure. Phase I trials were suspended when two cimaglermin alfa-treated subjects experienced concomitant elevations in serum aminotransferases and total bilirubin, meeting current US Food and Drug Administration criteria for a serious liver safety signal (i.e., "Hy's Law"). We assayed mechanistic biomarkers in archived clinical trial serum samples which confirmed the hepatic origin of the aminotransferase elevations in these two subjects and identified apoptosis as the major mode of hepatocyte death. Using a mathematical model of drug-induced liver injury (DILIsym) and a simulated population, we estimated that the maximum hepatocyte loss in these two subjects was <13%, which would not result in liver dysfunction sufficient to significantly increase serum bilirubin levels. We conclude that the two subjects should not be considered Hy's Law cases and that mechanistic biomarkers and modeling can aid in refining liver safety risk assessment in clinical trials.

Systems pharmacology modeling of drug-induced hyperbilirubinemia: Differentiating hepatotoxicity and inhibition of enzymes/transporters.

Elevations in serum bilirubin during drug treatment may indicate global liver dysfunction and a high risk of liver failure. However, drugs also can increase serum bilirubin in the absence of hepatic injury by inhibiting specific enzymes/transporters. We constructed a mechanistic model of bilirubin disposition based on known functional polymorphisms in bilirubin metabolism/transport. Using physiologically based pharmacokinetic (PBPK) model-predicted drug exposure and enzyme/transporter inhibition constants determined in vitro, our model correctly predicted indinavir-mediated hyperbilirubinemia in humans and rats. Nelfinavir was predicted not to cause hyperbilirubinemia, consistent with clinical observations. We next examined a new drug candidate that caused both elevations in serum bilirubin and biochemical evidence of liver injury in rats. Simulations suggest that bilirubin elevation primarily resulted from inhibition of transporters rather than global liver dysfunction. We conclude that mechanistic modeling of bilirubin can help elucidate underlying mechanisms of drug-induced hyperbilirubinemia, and thereby distinguish benign from clinically important elevations in serum bilirubin.

Blood gene expression profiling of an early acetaminophen response.

Acetaminophen can adversely affect the liver especially when overdosed. We used whole blood as a surrogate to identify genes as potential early indicators of an acetaminophen-induced response. In a clinical study, healthy human subjects were dosed daily with 4 g of either acetaminophen or placebo pills for 7 days and evaluated over the course of 14 days. Alanine aminotransferase (ALT) levels for responders to acetaminophen increased between days 4 and 9 after dosing, and 12 genes were detected with expression profiles significantly altered within 24 h. The early responsive genes separated the subjects by class and dose period. In addition, the genes clustered patients who overdosed on acetaminophen apart from controls and also predicted the exposure classifications with 100% accuracy. The responsive genes serve as early indicators of an acetaminophen exposure, and their gene expression profiles can potentially be evaluated as molecular indicators for further consideration.

Drug-induced liver injury: Advances in mechanistic understanding that will inform risk management.

Drug-induced liver injury (DILI) is a major public health problem. Intrinsic (dose-dependent) DILI associated with acetaminophen overdose is the number one cause of acute liver failure in the US. However, the most problematic type of DILI impacting drug development is idiosyncratic, occurring only very rarely among treated patients and often only after several weeks or months of treatment with the offending drug. Recent advances in our understanding of the pathogenesis of DILI suggest that three mechanisms may underlie most hepatocyte effects in response to both intrinsic and idiosyncratic DILI drugs: mitochondrial dysfunction, oxidative stress, and alterations in bile acid homeostasis. However, in some cases hepatocyte stress promotes an immune response that results in clinically important idiosyncratic DILI. This review discusses recent advances in our understanding of the pathogenesis of both intrinsic and idiosyncratic DILI as well as emerging tools and techniques that will likely improve DILI risk identification and management.

Elucidating Differences in the Hepatotoxic Potential of Tolcapone and Entacapone With DILIsym(®), a Mechanistic Model of Drug-Induced Liver Injury.

Tolcapone and entacapone are catechol-O-methyltransferase (COMT) inhibitors developed as adjunct therapies for treating Parkinson's disease. While both drugs have been shown to cause mitochondrial dysfunction and inhibition of the bile salt export protein (BSEP), liver injury has only been associated with the use of tolcapone. Here we used a multiscale, mechanistic model (DILIsym(®)) to simulate the response to tolcapone and entacapone. In a simulated population (SimPops™) receiving recommended doses of tolcapone (200 mg t.i.d.), increases in serum alanine transaminase (ALT) >3× the upper limit of normal (ULN) were observed in 2.2% of the population. In contrast, no simulated patients receiving recommended doses of entacapone (200 mg 8× day) experienced serum ALT >3× ULN. Further, DILIsym(®) analyses revealed patient-specific risk factors that may contribute to tolcapone-mediated hepatotoxicity. In summary, the simulations demonstrated that differences in mitochondrial uncoupling potency and hepatic exposure primarily account for the difference in hepatotoxic potential for tolcapone and entacapone.

Blood transcript immune signatures distinguish a subset of people with elevated serum ALT from others given acetaminophen.

The diagnosis of drug-induced liver injury is hindered by the limited utility of clinical chemistries. We have shown that hepatotoxicants can produce peripheral blood transcriptome "signatures" (PBTS) in rodents and humans. In this study, 42 adults were treated with acetaminophen (APAP; 1 g every 6 hours) for seven days, followed by three days of placebo. Eleven subjects received only placebo. After five days, 12 subjects (30%) had increases in serum alanine aminotransferase (ALT) levels ("responders"). PBTS of 707 and 760 genes, respectively, could distinguish responders and nonresponders from placebos. Functional analysis of the responder PBTS revealed increased expression of genes involved in TH2-mediated and innate immune responses, whereas the nonresponders demonstrated increased gene expression consistent with a tolerogenic immune response. Taken together, these observations suggest that the clinical subjects with transient increases in serum ALT failed to maintain or intensify a hepatic tolerogenic immune response.

Comparative analysis of portal hepatic infiltrating leucocytes in acute drug-induced liver injury, idiopathic autoimmune and viral hepatitis.

Drug-induced liver injury (DILI) is often caused by innate and adaptive host immune responses. Characterization of inflammatory infiltrates in the liver may improve understanding of the underlying pathogenesis of DILI. This study aimed to enumerate and characterize leucocytes infiltrating liver tissue from subjects with acute DILI (n = 32) versus non-DILI causes of acute liver injury (n = 25). Immunostains for CD11b/CD4 (Kupffer and T helper cells), CD3/CD20 (T and B cells) and CD8/CD56 [T cytotoxic and natural killer (NK) cells] were evaluated in biopsies from subjects with acute DILI, either immunoallergic (IAD) or autoimmune (AID) and idiopathic autoimmune (AIH) and viral hepatitis (VH) and correlated with clinical and pathological features. All biopsies showed numerous CD8(+) T cells and macrophages. DILI cases had significantly fewer B lymphocytes than AIH and VH and significantly fewer NK cells than VH. Prominent plasma cells were unusual in IAD (three of 10 cases), but were associated strongly with AIH (eight of nine) and also observed in most with AID (six of nine). They were also found in five of 10 cases with VH. Liver biopsies from subjects with DILI were characterized by low counts of mature B cells and NK cells in portal triads in contrast to VH. NK cells were found only in cases of VH, whereas AIH and VH both showed higher counts of B cells than DILI. Plasma cells were associated most strongly with AIH and less so with AID, but were uncommon in IAD.

MITOsym®: A Mechanistic, Mathematical Model of Hepatocellular Respiration and Bioenergetics.

PURPOSE: MITOsym, a new mathematical model of hepatocellular respiration and bioenergetics, has been developed in partnership with the DILIsym® model with the purpose of translating in vitro compound screening data into predictions of drug induced liver injury (DILI) risk for patients. The combined efforts of these two models should increase the efficiency of evaluating compounds in drug development in addition to enhancing patient care. METHODS: MITOsym includes the basic, essential biochemical pathways associated with hepatocellular respiration and bioenergetics, including mitochondrial oxidative phosphorylation, electron transport chain activity, mitochondrial membrane potential, and glycolysis; also included are dynamic feedback signals based on perturbation of these pathways. The quantitative relationships included in MITOsym are based primarily on published data; additional new experiments were also performed in HepG2 cells to determine the effects on oxygen consumption rate as media glucose concentrations or oligomycin concentrations were varied. The effects of varying concentrations of FCCP on the mitochondrial proton gradient were also measured in HepG2 cells. RESULTS: MITOsym simulates and recapitulates the reported dynamic changes to hepatocellular oxygen consumption rates, extracellular acidification rates, the mitochondrial proton gradient, and ATP concentrations in the presence of classic mitochondrial toxins such as rotenone, FCCP, and oligomycin. CONCLUSIONS: MITOsym can be used to simulate hepatocellular respiration and bioenergetics and provide mechanistic hypotheses to facilitate the translation of in vitro data collection to predictions of in vivo human hepatotoxicity risk for novel compounds.

Systems pharmacology modeling predicts delayed presentation and species differences in bile acid-mediated troglitazone hepatotoxicity.

Troglitazone (TGZ) causes delayed, life-threatening drug-induced liver injury in some patients but was not hepatotoxic in rats. This study investigated altered bile acid homeostasis as a mechanism of TGZ hepatotoxicity using a systems pharmacology model incorporating drug/metabolite disposition, bile acid physiology/pathophysiology, hepatocyte life cycle, and liver injury biomarkers. In the simulated human population, TGZ (200-600 mg/day × 6 months) resulted in delayed increases in serum alanine transaminase >3× the upper limit of normal in 0.3-5.1%, with concomitant bilirubin elevations >2× the upper limit of normal in 0.3-3.6%, of the population. By contrast, pioglitazone (15-45 mg/day × 6 months) did not elicit hepatotoxicity, consistent with clinical data. TGZ was not hepatotoxic in the simulated rat population. In summary, mechanistic modeling based only on bile acid effects accurately predicted the incidence, delayed presentation, and species differences in TGZ hepatotoxicity, in addition to predicting the relative liver safety of pioglitazone. Systems pharmacology models integrating physiology and experimental data can evaluate drug-induced liver injury mechanisms and may be useful to predict the hepatotoxic potential of drug candidates.

Mechanistic Modeling Reveals the Critical Knowledge Gaps in Bile Acid-Mediated DILI.

Bile salt export pump (BSEP) inhibition has been proposed to be an important mechanism for drug-induced liver injury (DILI). Modeling can prioritize knowledge gaps concerning bile acid (BA) homeostasis and thus help guide experimentation. A submodel of BA homeostasis in rats and humans was constructed within DILIsym, a mechanistic model of DILI. In vivo experiments in rats with glibenclamide were conducted, and data from these experiments were used to validate the model. The behavior of DILIsym was analyzed in the presence of a simulated theoretical BSEP inhibitor. BSEP inhibition in humans is predicted to increase liver concentrations of conjugated chenodeoxycholic acid (CDCA) and sulfate-conjugated lithocholic acid (LCA) while the concentration of other liver BAs remains constant or decreases. On the basis of a sensitivity analysis, the most important unknowns are the level of BSEP expression, the amount of intestinal synthesis of LCA, and the magnitude of farnesoid-X nuclear receptor (FXR)-mediated regulation.

A mechanistic model of drug-induced liver injury AIDS the interpretation of elevated liver transaminase levels in a phase I clinical trial.

Entolimod (CBLB502) is a Toll-like receptor 5 agonist in development as a single-dose countermeasure against total body irradiation. Efficacy can be assessed from animal studies, but the "Animal Rule" does not apply to safety assessment. Marked elevations of serum aminotransferases (exceeding 1,000 IU/l) were observed in some human subjects receiving Entolimod in a safety study, threatening its continued development. The percentage of total hepatocytes undergoing necrosis in these subjects was estimated using a mechanistic, multiscale, mathematical model (DILIsym). The simulations suggested that no subject in the safety study experienced more than a modest loss of hepatocytes (<5%), which was comparable to estimates from a study of healthy volunteers receiving treatment with heparins. The predicted hepatocyte loss with Entolimod was lower than that required to cause liver dysfunction or that is routinely excised from volunteers donating for autologous liver transplantation and did not likely represent a serious health risk.

Managing the risk of drug-induced liver injury.

In clinical trials, bosentan was shown to cause significant drug-induced liver injury (DILI) in some patients. Because it is not possible to identify those at higher risk for DILI, all patients to be treated with bosentan must enroll in a program requiring documentation of liver blood testing before they can receive the drug. Because this program is costly and is perceived as onerous, a genetic test capable of identifying susceptible individuals would probably be rapidly adopted by physicians.

The effects of heparins on the liver: application of mechanistic serum biomarkers in a randomized study in healthy volunteers.

Heparins have been reported to cause elevations in serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) but have not been associated with clinically significant liver injury. The mechanisms underlying these benign laboratory abnormalities are unknown. Forty-eight healthy men were randomized to receive subcutaneous injections of unfractionated heparin (UFH; 150 U/kg), enoxaparin sodium (1 mg/kg), dalteparin sodium (120 IU/kg), or adomiparin sodium (125 IU/kg; a novel heparin) every 12 h for 4.5 days. Asymptomatic elevations in serum ALT or AST were observed in >90% of the subjects. Elevations were also observed in the levels of serum sorbitol dehydrogenase (SDH), glutamate dehydrogenase (GLDH), miR-122, high-mobility group box-1 protein (including the acetylated form), full-length keratin 18, and DNA. Keratin 18 fragments, which are apoptosis biomarkers, were not detected. Biomarker profiles did not differ significantly across heparin treatments. We conclude that heparins as a class cause self-limited and mild hepatocyte necrosis with secondary activation of an innate immune response.

Serum proteomic profiling in patients with drug-induced liver injury.

BACKGROUND: Idiosyncratic drug-induced liver injury (DILI) is a complex disorder that is difficult to predict, diagnose and treat. AIM: To describe the global serum proteome of patients with DILI and controls. METHODS: A label-free, mass spectrometry-based quantitative proteomic approach was used to explore protein expression in serum samples from 74 DILI patients (collected within 14 days of DILI onset) and 40 controls. A longitudinal analysis was conducted in a subset of 21 DILI patients with available 6-month follow-up serum samples. RESULTS: Comparison of DILI patients based on pattern, severity and causality assessment of liver injury revealed many differentially expressed priority 1 proteins among groups. Expression of fumarylacetoacetase was correlated with alanine aminotransferase (ALT; r = 0.237; P = 0.047), aspartate aminotransferase (AST; r = 0.389; P = 0.001) and alkaline phosphatase (r = -0.240; P = 0.043), and this was the only protein with significant differential expression when comparing patients with hepatocellular vs. cholestatic or mixed injury. In the longitudinal analysis, expression of 53 priority 1 proteins changed significantly from onset of DILI to 6-month follow-up, and nearly all proteins returned to expression levels comparable to control subjects. Ninety-two serum priority 1 proteins with significant differential expression were identified when comparing the DILI and control groups. Pattern analysis revealed proteins that are components of inflammation, immune system activation and several hepatotoxicity-specific pathways. Apolipoprotein E expression had the greatest power to differentiate DILI patients from controls (89% correct classification; AUROC = 0.97). CONCLUSION: This proteomic analysis identified differentially expressed proteins that are components of pathways previously implicated in the pathogenesis of idiosyncratic drug-induced liver injury.

Drug safety sciences and the bottleneck in drug development.

During a recent review of a new drug application for treatment of a chronic disease, US Food and Drug Administration (FDA) regulators agreed with the sponsor's assessment of efficacy. However, it was noted that two subjects receiving active treatment experienced abnormal liver chemistries that possibly, but not definitely, indicated a liver safety liability. The sponsor was told that a prerequisite for approval would be a new clinical trial involving 20,000 patients treated for 1 year, with 10,000 receiving the new drug and 10,000 receiving a comparator treatment. The sponsor is now faced with the substantial costs involved in undertaking such a large study, the loss of patent life during the conduct and analysis of the study, and the prospect of losing in-class market position. If the drug is ultimately approved, this detour will result in costs and potential revenue loss to the sponsor of well over $1 billion.

Case definition and phenotype standardization in drug-induced liver injury.

Drug-induced liver injury (DILI) is the most frequent reason cited for the withdrawal of approved drugs from the market and accounts for up to 15% of the cases of acute liver failure. Investigators around the globe have begun to identify and study patients with DILI; several large registries and tissue banks are being established. In order to gain the maximum scientific benefit from these efforts, the definitions and terminology related to the clinical phenotypes of DILI must be harmonized. For this purpose, an international DILI Expert Working Group of clinicians and scientists reviewed current DILI terminology and diagnostic criteria so as to develop more uniform criteria that would define and characterize the spectrum of clinical syndromes that constitute DILI. Consensus was established with respect to the threshold criteria for definition of a case as being DILI, the pattern of liver injury, causality assessment, severity, and chronicity. Consensus was also reached on approaches to characterizing DILI in the setting of chronic liver diseases, including autoimmune hepatitis (AIH).

The application of metabonomics to predict drug-induced liver injury.

The occurrence of drug-induced liver injury (DILI) presents a significant safety issue for patients and represents a major cause of regulatory action. The methods that are in current use for early detection and prediction of DILI in patients are not adequate. The liver is the major site of synthesis of endogenous metabolites, and data suggest that alterations in the profiles of endogenous metabolites ("the metabolome") may precede development of clinically overt DILI. Metabonomics involves the application of analytical technologies such as nuclear magnetic resonance and mass spectrometry to detect changes in the metabolome. In this review, we describe the emerging role of metabonomics in predicting and understanding the mechanisms underlying DILI. Recent human clinical trials of drugs, including acetaminophen (APAP) and ximelagatran, have shown that the metabonomics of biofluids (plasma and urine) collected before and immediately after dosing can identify individual patients who are likely to develop DILI. These studies support the need to include metabonomic investigations in clinical trials of potentially hepatotoxic medications.

Use of pharmaco-metabonomics for early prediction of acetaminophen-induced hepatotoxicity in humans.

Achieving the ability to identify individuals who are susceptible to drug-induced liver injury (DILI) would represent a major advance in personalized medicine. Clayton et al. demonstrated that the pattern of endogenous metabolites in urine could predict susceptibility to acetaminophen-induced liver injury in rats. We designed a clinical study to test this approach in healthy adults who received 4 g of acetaminophen per day for 7 days. Urine metabolite profiles obtained before the start of treatment were not sufficient to distinguish which of the subjects would develop mild liver injury, as indicated by a rise in alanine aminotransferase (ALT) to a level more than twice the baseline value (responders). However, profiles obtained shortly after the start of treatment, but prior to ALT elevation, could distinguish responders from nonresponders. Statistical analyses revealed that predictive metabolites included those derived from the toxic metabolite N-acetyl paraquinone imine (NAPQI), but that the inclusion of endogenous metabolites was required for significant prediction. This "early-intervention pharmaco-metabonomics" approach should now be tested in clinical trials of other potentially hepatotoxic drugs.

Blood gene expression signatures predict exposure levels.

To respond to potential adverse exposures properly, health care providers need accurate indicators of exposure levels. The indicators are particularly important in the case of acetaminophen (APAP) intoxication, the leading cause of liver failure in the U.S. We hypothesized that gene expression patterns derived from blood cells would provide useful indicators of acute exposure levels. To test this hypothesis, we used a blood gene expression data set from rats exposed to APAP to train classifiers in two prediction algorithms and to extract patterns for prediction using a profiling algorithm. Prediction accuracy was tested on a blinded, independent rat blood test data set and ranged from 88.9% to 95.8%. Genomic markers outperformed predictions based on traditional clinical parameters. The expression profiles of the predictor genes from the patterns extracted from the blood exhibited remarkable (97% accuracy) transtissue APAP exposure prediction when liver gene expression data were used as a test set. Analysis of human samples revealed separation of APAP-intoxicated patients from control individuals based on blood expression levels of human orthologs of the rat discriminatory genes. The major biological signal in the discriminating genes was activation of an inflammatory response after exposure to toxic doses of APAP. These results support the hypothesis that gene expression data from peripheral blood cells can provide valuable information about exposure levels, well before liver damage is detected by classical parameters. It also supports the potential use of genomic markers in the blood as surrogates for clinical markers of potential acute liver damage.