Kyunghee Yang

Risk factors for development of cholestatic drug-induced liver injury: inhibition of hepatic basolateral bile acid transporters multidrug resistance-associated proteins 3 and 4.

Impaired hepatic bile acid export may contribute to development of cholestatic drug-induced liver injury (DILI). The multidrug resistance-associated proteins (MRP) 3 and 4 are postulated to be compensatory hepatic basolateral bile acid efflux transporters when biliary excretion by the bile salt export pump (BSEP) is impaired. BSEP inhibition is a risk factor for cholestatic DILI. This study aimed to characterize the relationship between MRP3, MRP4, and BSEP inhibition and cholestatic potential of drugs. The inhibitory effect of 88 drugs (100 μM) on MRP3- and MRP4-mediated substrate transport was measured in membrane vesicles. Drugs selected for investigation included 50 BSEP non-inhibitors (24 non-cholestatic; 26 cholestatic) and 38 BSEP inhibitors (16 non-cholestatic; 22 cholestatic). MRP4 inhibition was associated with an increased risk of cholestatic potential among BSEP non-inhibitors. In this group, for each 1% increase in MRP4 inhibition, the odds of the drug being cholestatic increased by 3.1%. Using an inhibition cutoff of 21%, which predicted a 50% chance of cholestasis, 62% of cholestatic drugs inhibited MRP4 (P < 0.05); in contrast, only 17% of non-cholestatic drugs were MRP4 inhibitors. Among BSEP inhibitors, MRP4 inhibition did not provide additional predictive value of cholestatic potential; almost all BSEP inhibitors were also MRP4 inhibitors. Inclusion of pharmacokinetic predictor variables (e.g., maximal unbound concentration in plasma) in addition to percent MRP4 inhibition in logistic regression models did not improve cholestasis prediction. Association of cholestasis with percent MRP3 inhibition was not statistically significant, regardless of BSEP-inhibition status. Inhibition of MRP4, in addition to BSEP, may be a risk factor for the development of cholestatic DILI.

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.

Hepatic Basolateral Efflux Contributes Significantly to Rosuvastatin Disposition I: Characterization of Basolateral Versus Biliary Clearance Using a Novel Protocol in Sandwich-Cultured Hepatocytes

Transporters responsible for hepatic uptake and biliary clearance (CLBile) of rosuvastatin (RSV) have been well characterized. However, the contribution of basolateral efflux clearance (CLBL) to hepatic and systemic exposure of RSV is unknown. Additionally, the appropriate design of in vitro hepatocyte efflux experiments to estimate CLBile versus CLBL remains to be established. A novel uptake and efflux protocol was developed in sandwich-cultured hepatocytes (SCH) to achieve desired tight junction modulation while maintaining cell viability. Subsequently, studies were conducted to determine the role of CLBL in the hepatic disposition of RSV using SCH from wild-type (WT) and multidrug resistance-associated protein 2 (Mrp2)-deficient (TR−) rats in the absence and presence of the P-glycoprotein and breast cancer resistance protein (Bcrp) inhibitor elacridar (GF120918). RSV CLBile was nearly ablated by GF120918 in TR− SCH, confirming that Mrp2 and Bcrp are responsible for the majority of RSV CLBile. Pharmacokinetic modeling revealed that CLBL and CLBile represent alternative elimination routes with quantitatively similar contributions to the overall hepatocellular excretion of RSV in rat SCH under baseline conditions (WT SCH in the absence of GF120918) and also in human SCH. Membrane vesicle experiments revealed that RSV is a substrate of MRP4 (Km = 21 ± 7 µM, Vmax = 1140 ± 210 pmol/min per milligram of protein). Alterations in MRP4-mediated RSV CLBL due to drug-drug interactions, genetic polymorphisms, or disease states may lead to changes in hepatic and systemic exposure of RSV, with implications for the safety and efficacy of this commonly used medication.

An updated review on drug‐induced cholestasis: Mechanisms and investigation of physicochemical properties and pharmacokinetic parameters

Drug-induced cholestasis is an important form of acquired liver disease and is associated with significant morbidity and mortality. Bile acids are key signaling molecules, but they can exert toxic responses when they accumulate in hepatocytes. This review focuses on the physiological mechanisms of drug-induced cholestasis associated with altered bile acid homeostasis due to direct (e.g., bile acid transporter inhibition) or indirect (e.g., activation of nuclear receptors, altered function/expression of bile acid transporters) processes. Mechanistic information about the effects of a drug on bile acid homeostasis is important when evaluating the cholestatic potential of a compound, but experimental data often are not available. The relationship between physicochemical properties, pharmacokinetic parameters, and inhibition of the bile salt export pump among 77 cholestatic drugs with different pathophysiological mechanisms of cholestasis (i.e., impaired formation of bile vs. physical obstruction of bile flow) was investigated. The utility of in silico models to obtain mechanistic information about the impact of compounds on bile acid homeostasis to aid in predicting the cholestatic potential of drugs is highlighted.

Exploring BSEP inhibition-mediated toxicity with a mechanistic model of drug-induced liver injury

Inhibition of the bile salt export pump (BSEP) has been linked to incidence of drug-induced liver injury (DILI), presumably by the accumulation of toxic bile acids in the liver. We have previously constructed and validated a model of bile acid disposition within DILIsym®, a mechanistic model of DILI. In this paper, we use DILIsym® to simulate the DILI response of the hepatotoxic BSEP inhibitors bosentan and CP-724,714 and the non-hepatotoxic BSEP inhibitor telmisartan in humans in order to explore whether we can predict that hepatotoxic BSEP inhibitors can cause bile acid accumulation to reach toxic levels. We also simulate bosentan in rats in order to illuminate potential reasons behind the lack of toxicity in rats compared to the toxicity observed in humans. DILIsym® predicts that bosentan, but not telmisartan, will cause mild hepatocellular ATP decline and serum ALT elevation in a simulated population of humans. The difference in hepatotoxic potential between bosentan and telmisartan is consistent with clinical observations. However, DILIsym® underpredicts the incidence of bosentan toxicity. DILIsym® also predicts that bosentan will not cause toxicity in a simulated population of rats, and that the difference between the response to bosentan in rats and in humans is primarily due to the less toxic bile acid pool in rats. Our simulations also suggest a potential synergistic role for bile acid accumulation and mitochondrial electron transport chain (ETC) inhibition in producing the observed toxicity in CP-724,714, and suggest that CP-724,714 metabolites may also play a role in the observed toxicity. Our work also compares the impact of competitive and noncompetitive BSEP inhibition for CP-724,714 and demonstrates that noncompetitive inhibition leads to much greater bile acid accumulation and potential toxicity. Our research demonstrates the potential for mechanistic modeling to contribute to the understanding of how bile acid transport inhibitors cause DILI.

Species differences in hepatobiliary disposition of taurocholic acid in human and rat sandwich-cultured hepatocytes: implications for drug-induced liver injury.

The bile salt export pump (BSEP) plays an important role in bile acid excretion. Impaired BSEP function may result in liver injury. Bile acids also undergo basolateral efflux, but the relative contributions of biliary (CLBile) versus basolateral efflux (CLBL) clearance to hepatocellular bile acid excretion have not been determined. In the present study, taurocholic acid (TCA; a model bile acid) disposition was characterized in human and rat sandwich-cultured hepatocytes (SCH) combined with pharmacokinetic modeling. In human SCH, biliary excretion of TCA predominated (CLBile = 0.14 ± 0.04 ml/min per g liver; CLBL = 0.042 ± 0.019 ml/min per g liver), whereas CLBile and CLBL contributed approximately equally to TCA hepatocellular excretion in rat SCH (CLBile = 0.34 ± 0.07 ml/min per g liver; CLBL = 0.26 ± 0.07 ml/min per g liver). Troglitazone decreased TCA uptake, CLBile, and CLBL; membrane vesicle assays revealed for the first time that the major metabolite, troglitazone sulfate, was a noncompetitive inhibitor of multidrug resistance–associated protein 4, a basolateral bile acid efflux transporter. Simulations revealed that decreased CLBile led to a greater increase in hepatic TCA exposure in human than in rat SCH. A decrease in both excretory pathways (CLBile and CLBL) exponentially increased hepatic TCA in both species, suggesting that 1) drugs that inhibit both pathways may have a greater risk for hepatotoxicity, and 2) impaired function of an alternate excretory pathway may predispose patients to hepatotoxicity when drugs that inhibit one pathway are administered. Simulations confirmed the protective role of uptake inhibition, suggesting that a drug’s inhibitory effects on bile acid uptake also should be considered when evaluating hepatotoxic potential. Overall, the current study precisely characterized basolateral efflux of TCA, revealed species differences in hepatocellular TCA efflux pathways, and provided insights about altered hepatic bile acid exposure when multiple transport pathways are impaired.

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.

Sandwich-Cultured Hepatocytes as a Tool to Study Drug Disposition and Drug-Induced Liver Injury.

Sandwich-cultured hepatocytes (SCH) are metabolically competent and have proper localization of basolateral and canalicular transporters with functional bile networks. Therefore, this cellular model is a unique tool that can be used to estimate biliary excretion of compounds. SCH have been used widely to assess hepatobiliary disposition of endogenous and exogenous compounds and metabolites. Mechanistic modeling based on SCH data enables estimation of metabolic and transporter-mediated clearances, which can be used to construct physiologically based pharmacokinetic models for prediction of drug disposition and drug-drug interactions in humans. In addition to pharmacokinetic studies, SCH also have been used to study cytotoxicity and perturbation of biological processes by drugs and hepatically generated metabolites. Human SCH can provide mechanistic insights underlying clinical drug-induced liver injury (DILI). In addition, data generated in SCH can be integrated into systems pharmacology models to predict potential DILI in humans. In this review, applications of SCH in studying hepatobiliary drug disposition and bile acid-mediated DILI are discussed. An example is presented to show how data generated in the SCH model were used to establish a quantitative relationship between intracellular bile acids and cytotoxicity, and how this information was incorporated into a systems pharmacology model for DILI prediction.

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.

Prediction of altered bile acid disposition due to inhibition of multiple transporters: An integrated approach using sandwich-cultured hepatocytes, mechanistic modeling and simulation

Transporter-mediated alterations in bile acid disposition may have significant toxicological implications. Current methods to predict interactions are limited by the interplay of multiple transporters, absence of protein in the experimental system, and inaccurate estimates of inhibitor concentrations. An integrated approach was developed to predict altered bile acid disposition due to inhibition of multiple transporters using the model bile acid taurocholate (TCA). TCA pharmacokinetic parameters were estimated by mechanistic modeling using sandwich-cultured human hepatocyte data with protein in the medium. Uptake, basolateral efflux, and biliary clearance estimates were 0.63, 0.034, and 0.074 mL/min/g liver, respectively. Cellular total TCA concentrations (Ct,Cells) were selected as the model output based on sensitivity analysis. Monte Carlo simulations of TCA Ct,Cells in the presence of model inhibitors (telmisartan and bosentan) were performed using inhibition constants for TCA transporters and inhibitor concentrations, including cellular total inhibitor concentrations ([I]t,cell) or unbound concentrations, and cytosolic total or unbound concentrations. For telmisartan, the model prediction was accurate with an average fold error (AFE) of 0.99–1.0 when unbound inhibitor concentration ([I]u) was used; accuracy dropped when total inhibitor concentration ([I]t) was used. For bosentan, AFE was 1.2–1.3 using either [I]u or [I]t. This difference was evaluated by sensitivity analysis of the cellular unbound fraction of inhibitor (fu,cell,inhibitor), which revealed higher sensitivity of fu,cell,inhibitor for predicting TCA Ct,Cells when inhibitors exhibited larger ([I]t,cell/IC50) values. In conclusion, this study demonstrated the applicability of a framework to predict hepatocellular bile acid concentrations due to drug-mediated inhibition of transporters using mechanistic modeling and cytosolic or cellular unbound concentrations.