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.

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.

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.

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.

Advances in the stable isotope-mass spectrometric measurement of DNA synthesis and cell proliferation.

Methods for measuring rates of DNA synthesis, and thus cell proliferation, in humans had not been available until recently. We (D. C. Macallan, C. A. Fullerton, R. A. Neese, K. Haddock, S. S. Park, and M. K. Hellerstein, 1998, Proc. Natl. Acad. Sci. USA 95, 708-713) recently developed a stable isotope-mass spectrometric technique for measuring DNA synthesis by labeling the deoxyribose (dR) moiety of purine deoxyribonucleotides through the de novo nucleotide synthesis pathway. The original analytic approach had limitations, however. Here, we describe technical improvements that increase yield, stability, sensitivity, and reproducibility of the method. The purine deoxyribonucleoside, deoxyadenosine (dA), is directly isolated from hydrolysates of DNA by using an LC18 SPE column. Two derivatives were developed for analyzing the dR moiety of dA alone (without the base), an aldonitrile-triacetate derivative, and a reduced pentose-tetraacetate (PTA) derivative. The PTA derivative in particular exhibited greater stability (no degradation after several weeks), greater GC/MS signal, and much less abundance sensitivity of isotope ratios (i.e., less dependence of mass isotopomer abundances on the amount of material injected into the mass spectrometer source), compared to previous derivatives of dA. The need for complex, multidimensional abundance corrected standard curves was thereby avoided. Using the PTA derivative, dR enrichments from DNA of fully turned over cells of rodents with 2H2O enrichments in body water of 2.2-2.8% were 9.0-9.5%, and less than 1.0 microg DNA (ca. 2 x 10(5) cells) was required for reproducible analyses. In summary, these methodologic advances allow measurement of stable isotope incorporation into DNA and calculation of cell proliferation and death rates in vivo in humans and experimental animals, with fewer cells, greater reproducibility, and less labor. Many applications of this approach can be envisioned.

De novo lipogenesis, lipid kinetics, and whole-body lipid balances in humans after acute alcohol consumption.

BACKGROUND: Acute alcohol intake is associated with changes in plasma lipid concentrations and whole-body lipid balances in humans. The quantitative roles of hepatic de novo lipogenesis (DNL) and plasma acetate production in these changes have not been established, however. OBJECTIVE: We used stable-isotope mass spectrometric methods with indirect calorimetry to establish the metabolic basis of changes in whole-body lipid balances in healthy men after consumption of 24 g alcohol. DESIGN: Eight healthy subjects were studied and DNL (by mass-isotopomer distribution analysis), lipolysis (by dilution of [1,2,3,4-(13)C(4)]palmitate and [(2)H(5)]glycerol), conversion of alcohol to plasma acetate (by incorporation from [1-(13)C(1)]ethanol), and plasma acetate flux (by dilution of [1-(13)C(1)]acetate) were measured. RESULTS: The fractional contribution from DNL to VLDL-triacylglycerol palmitate rose after alcohol consumption from 2 +/- 1% to 30 +/- 8%; nevertheless, the absolute rate of DNL (0.8 g/6 h) represented <5% of the ingested alcohol dose; 77 +/- 13% of the alcohol cleared from plasma was converted directly to acetate entering plasma. Acetate flux increased 2.5-fold after alcohol consumption. Adipose release of nonesterified fatty acids into plasma decreased by 53% and whole-body lipid oxidation decreased by 73%. CONCLUSIONS: We conclude that the consumption of 24 g alcohol activates the hepatic DNL pathway modestly, but acetate produced in the liver and released into plasma inhibits lipolysis, alters tissue fuel selection, and represents the major quantitative fate of ingested ethanol.

VLDL-triglyceride production after alcohol ingestion, studied using [2-13C1] glycerol.

We used [2-13C1]glycerol to characterize very low density lipoprotein (VLDL)-triglyceride kinetics and intrahepatic glycerol metabolism in normal men (n = 4) after alcohol (EtOH) ingestion. [2-13C1]glycerol was infused before and after the consumption of 48 g EtOH or a placebo. Three additional subjects also received [1-13C1]acetate in addition to the [2-13C1]glycerol with EtOH treatment. Incorporation of tracer into the glycerol or fatty acid moiety of VLDL-triglyceride was measured by gas chromatography-mass spectrometry and used to calculate VLDL-triglyceride production rates. Intrahepatic triose-phosphate enrichments were also calculated based on mass isotopomer distribution analysis of plasma glucose. There was no difference in VLDL-triglyceride production rates after 48 g EtOH (11.9 +/- 3.7 mg/kg/h) or placebo (14.7 +/- 3. 3 mg/kg/h). The VLDL-triglyceride rate constants calculated by kinetic modeling using the glycerol and acetate tracers in the combined isotope infusion subjects were very closely correlated (r 2 = 0.94). The peak VLDL-glycerol enrichments after EtOH were 22.5 +/- 3.3% versus 7.6 +/- 0.8% after placebo (P < 0.001), while intrahepatic triose-phosphate enrichments were 19.8 +/- 1.3% and 13. 1 +/- 1.2% (P < 0.001), respectively. Moreover, the calculated asymptotic VLDL-glycerol enrichments (representing the hepatic alpha-glycerol phosphate enrichment) were significantly higher after EtOH than placebo. The higher ratio of VLDL-glycerol to triose-phosphate labeling after EtOH suggests a metabolic block at glycerol 3-phosphate dehydrogenase. We conclude that consumption of 48 g EtOH does not increase VLDL-triglyceride production in normal men but does cause accumulation of tracer in hepatic alpha-glycerol phosphate.

The inhibition of gluconeogenesis following alcohol in humans.

Accurate quantification of gluconeogenic flux following alcohol ingestion in overnight-fasted humans has yet to be reported. [2-13C1]glycerol, [U-13C6]glucose, [1-2H1]galactose, and acetaminophen were infused in normal men before and after the consumption of 48 g alcohol or a placebo to quantify gluconeogenesis, glycogenolysis, hepatic glucose production, and intrahepatic gluconeogenic precursor availability. Gluconeogenesis decreased 45% vs. the placebo (0.56 +/- 0.05 to 0.44 +/- 0.04 mg. kg-1. min-1 vs. 0.44 +/- 0.05 to 0.63 +/- 0.09 mg. kg-1. min-1, respectively, P < 0. 05) in the 5 h after alcohol ingestion, and total gluconeogenic flux was lower after alcohol compared with placebo. Glycogenolysis fell over time after both the alcohol and placebo cocktails, from 1.46-1. 47 mg. kg-1. min-1 to 1.35 +/- 0.17 mg. kg-1. min-1 (alcohol) and 1. 26 +/- 0.20 mg. kg-1. min-1, respectively (placebo, P < 0.05 vs. baseline). Hepatic glucose output decreased 12% after alcohol consumption, from 2.03 +/- 0.21 to 1.79 +/- 0.21 mg. kg-1. min-1 (P < 0.05 vs. baseline), but did not change following the placebo. Estimated intrahepatic gluconeogenic precursor availability decreased 61% following alcohol consumption (P < 0.05 vs. baseline) but was unchanged after the placebo (P < 0.05 between treatments). We conclude from these results that gluconeogenesis is inhibited after alcohol consumption in overnight-fasted men, with a somewhat larger decrease in availability of gluconeogenic precursors but a smaller effect on glucose production and no effect on plasma glucose concentrations. Thus inhibition of flux into the gluconeogenic precursor pool is compensated by changes in glycogenolysis, the fate of triose-phosphates, and peripheral tissue utilization of plasma glucose.