Mechanisms of drug-induced liver injury: the role of hepatic transport proteins

The objectives of this research were to investigate mechanisms of drug-induced liver injury (DILI) that involve drug-bile acid (BA) interactions at hepatic transporters, and develop a novel strategy to reliably predict human DILI. Troglitazone (TGZ), an antidiabetic withdrawn from the market due to severe DILI, was employed as a model hepatotoxic drug. Pharmacokinetic modeling of taurocholic acid (TCA, a model BA) disposition data from human and rat sandwich-cultured hepatocytes (SCH) revealed that species differences exist in TCA hepatocellular efflux pathways; in human SCH, TCA biliary excretion predominated, whereas biliary and basolateral excretion contributed equally to TCA efflux in rat SCH. This finding explains, in part, why rats are less susceptible to DILI compared to humans after administration of drugs that inhibit BA biliary excretion. The present study also revealed for the first time that TGZ sulfate (TS), a major TGZ metabolite, inhibits BA basolateral efflux in addition to biliary excretion. These findings support the hypothesis that TS is an important mediator of altered hepatic BA disposition; increased hepatic TS exposure due to impaired canalicular transport function might predispose a subset of patients to hepatotoxicity. A novel in vitro model system, rat SCH lacking selected canalicular transporters [breast cancer resistance protein (Bcrp) and multidrug resistance-associated protein 2 (Mrp2)] was established to test this hypothesis; biliary excretion of hepatically-generated TS was not significantly altered, suggesting that alternate transporters can excrete TS into bile, and loss of Bcrp and/or Mrp2 function would not necessarily be risk factors for increased hepatocellular TS accumulation in rats. To translate experimental data to in vivo humans, a mechanistic model that incorporated TGZ/TS disposition, BA physiology/pathophysiology, hepatocyte life cycle, and liver injury biomarkers was developed; intracellular BA concentrations and toxicity measured in SCH were used to link BA homeostasis and hepatotoxicity. This mechanistic model adequately predicted the incidence, delayed presentation, and species differences in TGZ hepatotoxicity. This dissertation research revealed a number of important and novel findings that improve our understanding about mechanisms underlying BA-mediated DILI, and establish a framework to integrate biological information and experimental data to evaluate DILI mechanisms and predict hepatotoxic potential of chemical entities.Doctor of Philosoph

Evidence-based selection of training compounds for use in the mechanism-based integrated prediction of drug-induced liver injury in man

The current test systems employed by pharmaceutical industry are poorly predictive for drug-induced liver injury (DILI). The ‘MIP-DILI’ project addresses this situation by the development of innovative preclinical test systems which are both mechanism-based and of physiological, pharmacological and pathological relevance to DILI in humans. An iterative, tiered approach with respect to test compounds, test systems, bioanalysis and systems analysis is adopted to evaluate existing models and develop new models that can provide validated test systems with respect to the prediction of specific forms of DILI and further elucidation of mechanisms. An essential component of this effort is the choice of compound training set that will be used to inform refinement and/or development of new model systems that allow prediction based on knowledge of mechanisms, in a tiered fashion. In this review, we focus on the selection of MIP-DILI training compounds for mechanism-based evaluation of non-clinical prediction of DILI. The selected compounds address both hepatocellular and cholestatic DILI patterns in man, covering a broad range of pharmacologies and chemistries, and taking into account available data on potential DILI mechanisms (e.g. mitochondrial injury, reactive metabolites, biliary transport inhibition, and immune responses). Known mechanisms by which these compounds are believed to cause liver injury have been described, where many if not all drugs in this review appear to exhibit multiple toxicological mechanisms. Thus, the training compounds selection offered a valuable tool to profile DILI mechanisms and to interrogate existing and novel in vitro systems for the prediction of human DILI

Characterization of drug disposition in humans using novel in vitro methodologies based on the Extended Clearance Model

Safety and efficacy of drugs depend on their exposure in the body, which is determined by dose and bioavailability, but also by drug disposition as a result of tissue distribution and elimination processes. Knowledge about drug disposition in humans is therefore critical for the successful development of new drugs, with clinical information being unavailable at early development stages. To overcome this limitation, the pharmacokinetic properties of new drug candidates are routinely characterized using cell-based in vitro methods and in vitro-in vivo extrapolation (IVIVE) models. However, the assessment of drug distribution and elimination remains challenging. It was therefore the aim of this thesis to 1) establish a mechanistic in vitro model to study the hepatic distribution of unbound drug and to validate the model by predicting the clinical risk of drug-induced cholestasis, 2) investigate the applicability of additional in vitro methods for the determination of hepatic distribution of unbound drug, and 3) develop an in vitro model for the prediction of total (hepatic and renal) drug clearance and elimination pathway contributions in humans. Knowledge about the drug distribution into tissues and the corresponding unbound intracellular drug concentrations is of particular interest in the context of intracellular drug effects related to toxicity, pharmacokinetics, and pharmacodynamics. For instance, prediction of drug-induced cholestasis due to inhibition of the intrahepatic bile salt export pump (BSEP) is commonly conducted using the unbound systemic drug exposure as a surrogate for the unbound intrahepatic concentration following the “free-drug hypothesis”. However, this assessment offers limited translatability to the clinical cholestasis risk since the effective unbound intrahepatic drug concentration is affected by active transport and/or metabolic processes. To improve such evaluations of intrahepatic drug interactions, the determination of the liver-to-blood partition coefficient for unbound drug at steady-state (Kpuu) was established based on in vitro measurements of active and passive sinusoidal uptake permeability, sinusoidal efflux permeability, hepatic metabolism, and biliary secretion according to the Extended Clearance Model (ECM). Following successful validation of the ECM-based Kpuu approach by in vitro-in vivo correlation in rats, human Kpuu data of 18 drug compounds were used to calculate unbound intrahepatic drug concentrations based on clinical drug exposure. This assessment significantly improved the translation of BSEP inhibition in vitro data to human and allowed the prediction of the clinical cholestasis frequency. Moreover, usefulness of the ECM as a drug classification system and for the quantitative evaluation of genetic and physiological risk factors for the development of cholestasis was demonstrated. The determination of unbound intrahepatic drug concentrations using the ECM-based hepatic Kpuu is therefore expected to improve early risk assessment of drug-induced cholestasis as well as of other intrahepatic drug interactions. The ECM-based determination of Kpuu was successfully established and validated. However, this approach is labor and cost-intensive. A second project therefore aimed at comparing alternative in vitro Kpuu determination methods for the previously investigated compound set. For this purpose, three straightforward approaches were selected that rely on separate in vitro measurements of the liver-to-blood partition coefficient for total drug at steady-state (Kp) and the unbound fraction in hepatocytes (fuhep). Kp was generally determined in hepatocellular drug accumulation experiments in the absence of intrinsic metabolic and biliary clearance processes, whereas fuhep was either measured in hepatocellular drug accumulation experiments on ice (temperature method), using homogenized hepatocytes in equilibrium dialysis experiments (homogenization method), or calculated from the distribution coefficient logD7.4 using an empirical model (logD7.4 method). All investigated methods indicated deviations to ECM-derived Kpuu data, which were closely linked to the pharmacokinetic and physicochemical compound properties, namely the extent of intrinsic hepatic clearance, logD7.4, and molecular weight. The usefulness of the alternative Kpuu determination methods is therefore limited, with the ECM remaining the preferred approach for an integrated assessment of hepatic Kpuu. Nevertheless, the alternative methods can provide valid fuhep data if the physicochemical compound properties are considered for the selection of the appropriate method. During drug development, hepatic drug clearance is routinely predicted using in vitro approaches such as the ECM. In contrast, appropriate in vitro models for the prediction of renal drug clearance are lacking. Thus, the assessment of total clearance for new drug candidates is strongly limited. To overcome this drawback, an empirical in vitro model was established that provides estimates of the relative hepatic metabolic, biliary, and renal elimination pathway contributions in humans, based on in vitro sinusoidal uptake permeability data. This assessment subsequently allows the extrapolation of hepatic into total drug clearance. Under consideration of ECM-based hepatic clearances, the model provided accurate predictions of total human clearance for 10 developmental compounds. Moreover, it was demonstrated that the Extended Clearance Concept Classification System (ECCCS) is applicable to evaluate the relevance of metabolic, biliary, and renal drug elimination, which provides useful guidance for the design of follow-up enzyme and transporter phenotyping studies. Thus, the established model allows a simple and highly reliable assessment of total drug clearance and relative elimination pathway contributions in humans based solely on hepatic in vitro data, facilitating a tailor-made pharmacokinetic assessment during early drug development

Strategies for early prediction and timely recognition of drug-induced liver injury: The case of cyclin-dependent kinase 4/6 inhibitors

The idiosyncratic nature of drug-induced liver injury (Dili) represents a current challenge for drug developers, regulators and clinicians. The myriad of agents (including medications, herbals, and dietary supplements) with recognized Dili potential not only strengthens the importance of the post-marketing phase, when urgent withdrawal sometimes occurs for rare unanticipated liver toxicity, but also shows the imperfect predictivity of pre-clinical models and the lack of validated biomarkers beyond traditional, non-specific liver function tests. After briefly reviewing proposed key mechanisms of Dili, we will focus on drug-related risk factors (physiochemical and pharmacokinetic properties) recently proposed as predictors of Dili and use cyclin-dependent kinase 4/6 inhibitors, relatively novel oral anticancer medications approved for breast cancer, as a case study to discuss the feasibility of early detection of Dili signals during drug development: published data from pivotal clinical trials, unpublished post-marketing reports of liver adverse events, and pharmacokinetic properties will be used to provide a comparative evaluation of their liver safety and gain insight into drug-related risk factors likely to explain the observed differences

Managing the challenge of drug-induced liver injury: a roadmap for the development and deployment of preclinical predictive models

Drug-induced liver injury (DILI) is a patient-specific, temporal, multifactorial pathophysiological process that cannot yet be recapitulated in a single in vitro model. Current preclinical testing regimes for the detection of human DILI thus remain inadequate. A systematic and concerted research effort is required to address the deficiencies in current models and to present a defined approach towards the development of new or adapted model systems for DILI prediction. This Perspective defines the current status of available models and the mechanistic understanding of DILI, and proposes our vision of a roadmap for the development of predictive preclinical models of human DILI

Preclinical models of idiosyncratic drug-induced liver injury (iDILI): Moving towards prediction

Idiosyncratic drug-induced liver injury (iDILI) encompasses the unexpected harms that pre- scription and non-prescription drugs, herbal and dietary supplements can cause to the liver. iDILI remains a major public health problem and a major cause of drug attrition. Given the lack of biomarkers for iDILI prediction, diagnosis and prognosis, searching new models to predict and study mechanisms of iDILI is necessary. One of the major limitations of iDILI preclinical assessment has been the lack of correlation between the markers of hepatotoxicity in animal toxicological studies and clinically significant iDILI. Thus, major advances in the understanding of iDILI susceptibility and pathogenesis have come from the study of well-phenotyped iDILI patients. However, there are many gaps for explaining all the complexity of iDILI susceptibility and mechanisms. Therefore, there is a need to optimize preclinical hu- man in vitro models to reduce the risk of iDILI during drug development. Here, the current experimental models and the future directions in iDILI modelling are thoroughly discussed, focusing on the human cellular models available to study the pathophysiological mechanisms of the disease and the most used in vivo animal iDILI models. We also comment about in silico approaches and the increasing relevance of patient-derived cellular models.This work was supported by grants of Instituto de Salud Carlos III cofounded by Fondo Europeo de Desarrollo Regional-FEDER (contract numbers: PI18/01804, PI19-00883, PT20/00127, UMA18-FEDERJA-194, PY18-3364, Spain) and grants of Consejería de Salud de Andalucía cofounded by FEDER (contract number: PEMP-0127-2020, Spain). M.V.P. holds a Sara Borrell (CD21/00198, Spain) research contract from ISCIII and Consejería de Salud de Andalucía. C.L.G. holds a Juan de la Cierva Incorporación (IJCI-2017-31466, Spain) research contract from Ministerio de Ciencia del Gobierno de España. SCReN and CIBERehd are funded by ISCIII (Spain). This publication is based upon work from COST Action “CA17112dProspective European Drug-Induced Liver Injury Network” supported by COST (European Cooperation in Science and Technology); www.cost.eu. The figures in this review were created with Biorender.com

Identification, Ki determination and CoMFA analysis of nuclear receptor ligands as competitive inhibitors of OATP1B1-mediated estradiol-17β-glucuronide transport

Evidence shows that drug-drug interactions can occur at the level of drug transporters such as the organic anion transporting polypeptides (OATPs), a group of membrane solute carriers that mediate the sodium-independent transport of a wide range of amphipathic organic compounds. The polyspecific OATP1B1 is exclusively expressed at the basolateral membrane of hepatocytes and mediates uptake of amphipathic organic compounds from blood into hepatocytes. Nuclear receptors are ligand-activated transcription factors that play an important role in xenobiotic disposition and human diseases. Quite a few nuclear receptor ligands interact with transport proteins. A high-resolution three-dimensional structure is critical to understand the polyspecificity of OATP1B1 to predict and prevent adverse drug-drug interactions. Unfortunately there are no crystal structures of OATPs/Oatps available to date. Therefore, in this study we attempted to elucidate the characteristics of the substrate binding site of OATP1B1 based on small molecules interacting with it. First, we identified inhibitors of the OATP1B1 model substrate estradiol-17β-glucuronide from about forty nuclear receptor ligands. Among them, GW1929, paclitaxel and troglitazone were strong inhibitors, while 5α-androstane, 5α-androstane-3β, 17β-diol-17-hexahydrobenzoate and estradiol-3-benzoate were weak inhibitors. Then, we selected 25 compounds and performed inhibition kinetic studies to identify competitive inhibitors and determine their Ki values which ranged from submicromolar to submillimolar. Finally, we performed CoMFA analysis on the identified competitive inhibitors. The CoMFA results indicate that the substrate binding site of OATP1B1 consists of a large hydrophobic middle part with basic residues at both ends that could be very important for substrate binding

The Role of Uptake and Efflux Transporters in the Disposition of Glucuronide and Sulfate Conjugates

Glucuronidation and sulfation are the most typical phase II metabolic reactions of drugs. The resulting glucuronide and sulfate conjugates are generally considered inactive and safe. They may, however, be the most prominent drug-related material in the circulation and excreta of humans. The glucuronide and sulfate metabolites of drugs typically have limited cell membrane permeability and subsequently, their distribution and excretion from the human body requires transport proteins. Uptake transporters, such as organic anion transporters (OATs and OATPs), mediate the uptake of conjugates into the liver and kidney, while efflux transporters, such as multidrug resistance proteins (MRPs) and breast cancer resistance protein (BCRP), mediate expulsion of conjugates into bile, urine and the intestinal lumen. Understanding the active transport of conjugated drug metabolites is important for predicting the fate of a drug in the body and its safety and efficacy. The aim of this review is to compile the understanding of transporter-mediated disposition of phase II conjugates. We review the literature on hepatic, intestinal and renal uptake transporters participating in the transport of glucuronide and sulfate metabolites of drugs, other xenobiotics and endobiotics. In addition, we provide an update on the involvement of efflux transporters in the disposition of glucuronide and sulfate metabolites. Finally, we discuss the interplay between uptake and efflux transport in the intestine, liver and kidneys as well as the role of transporters in glucuronide and sulfate conjugate toxicity, drug interactions, pharmacogenetics and species differences.Peer reviewe