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.
Hepatic efflux transporters include numerous well-known and emerging proteins localized to the canalicular or basolateral membrane of the hepatocyte that are responsible for the excretion of drugs ...into the bile or blood, respectively. Altered function of hepatic efflux transporters due to drug-drug interactions, genetic variation, and/or disease states may lead to changes in xenobiotic exposure in the hepatocyte and/or systemic circulation. This review focuses on transport proteins involved in the hepatocellular efflux of drugs and metabolites, discusses mechanisms of altered transporter function as well as the interplay between multiple transport pathways, and highlights the importance of considering intracellular unbound concentrations of transporter substrates and/or inhibitors. Methods to evaluate hepatic efflux transport and predict the effects of impaired transporter function on systemic and hepatocyte exposure are discussed, and the sandwich-cultured hepatocyte model to evaluate comprehensively the role of hepatic efflux in the hepatobiliary disposition of xenobiotics is characterized.
Sandwich-cultured hepatocytes (SCH) are a powerful in vitro tool that can be utilized to study hepatobiliary drug transport, species differences in drug transport, transport protein regulation, ...drug-drug interactions, and hepatotoxicity. This review provides an up-to-date summary of the SCH model, including a brief history of, and introduction to, the use of SCH, as well as methodology to evaluate hepatobiliary drug disposition. A summary of the literature that has utilized this model to examine the interplay between drug-metabolizing enzymes and transport proteins, drug-drug interactions at the transport level, and hepatotoxicity as a result of altered hepatic transport also is provided.
Organic solute transporter alpha/beta (OSTα/β) is a heteromeric solute carrier protein that transports bile acids, steroid metabolites and drugs into and out of cells. OSTα/β protein is expressed in ...various tissues, but its expression is highest in the gastrointestinal tract where it facilitates the recirculation of bile acids from the gut to the liver. Previous studies established that OSTα/β is upregulated in liver tissue of patients with extrahepatic cholestasis, obstructive cholestasis, and primary biliary cholangitis (PBC), conditions that are characterized by elevated bile acid concentrations in the liver and/or systemic circulation. The discovery that OSTα/β is highly upregulated in the liver of patients with nonalcoholic steatohepatitis (NASH) further highlights the clinical relevance of this transporter because the incidence of NASH is increasing at an alarming rate with the obesity epidemic. Since OSTα/β is closely linked to the homeostasis of bile acids, and tightly regulated by the nuclear receptor farnesoid X receptor, OSTα/β is a potential drug target for treatment of cholestatic liver disease, and other bile acid-related metabolic disorders such as obesity and diabetes. Obeticholic acid, a semi-synthetic bile acid used to treat PBC, under review for the treatment of NASH, and in development for the treatment of other metabolic disorders, induces OSTα/β. Some drugs associated with hepatotoxicity inhibit OSTα/β, suggesting a possible role for OSTα/β in drug-induced liver injury (DILI). Furthermore, clinical cases of homozygous genetic defects in both OSTα/β subunits resulting in diarrhea and features of cholestasis have been reported. This review article has been compiled to comprehensively summarize the recent data emerging on OSTα/β, recapitulating the available literature on the structure-function and expression-function relationships of OSTα/β, the regulation of this important transporter, the interaction of drugs and other compounds with OSTα/β, and the comparison of OSTα/β with other solute carrier transporters as well as adenosine triphosphate-binding cassette transporters. Findings from basic to more clinically focused research efforts are described and discussed.
Drug-induced liver injury (DILI) remains a major concern in drug development from a patient safety perspective because it is the leading cause of acute liver failure. One mechanism of DILI is altered ...bile acid homeostasis and involves several hepatic bile acid transporters. Functional impairment of some hepatic bile acid transporters by drugs, disease, or genetic mutations may lead to toxic accumulation of bile acids within hepatocytes and increase DILI susceptibility. This review focuses on the role of hepatic bile acid transporters in DILI. Model systems, primarily in vitro and modeling tools, such as DILIsym, used in assessing transporter-mediated DILI are discussed. Due to species differences in bile acid homeostasis and drug-transporter interactions, key aspects and challenges associated with the use of preclinical animal models for DILI assessment are emphasized. Learnings are highlighted from three case studies of hepatotoxic drugs: troglitazone, tolvaptan, and tyrosine kinase inhibitors (dasatinib, pazopanib, and sorafenib). The development of advanced in vitro models and novel biomarkers that can reliably predict DILI is critical and remains an important focus of ongoing investigations to minimize patient risk for liver-related adverse reactions associated with medication use.
Nonalcoholic steatohepatitis (NASH), the progressive form of nonalcoholic fatty liver disease, is increasing in prevalence. NASH‐related alterations in hepatic protein expression (e.g., transporters) ...and in overall physiology may affect drug exposure by altering drug disposition and elimination. The aim of this study was to build a physiologically‐based pharmacokinetic (PBPK) model to predict drug exposure in NASH by incorporating NASH‐related changes in hepatic transporters. Morphine and morphine‐3‐glucuronide (M3G) were used as model compounds. A PBPK model of morphine with permeability‐limited hepatic disposition was extended to include M3G disposition and enterohepatic recycling (EHR). The model captured the area under the plasma concentration‐time curve (AUC) of morphine and M3G after intravenous morphine administration within 0.82‐fold and 1.94‐fold of observed values from 3 independent clinical studies for healthy adult subjects (6, 10, and 14 individuals). When NASH‐related changes in multidrug resistance‐associated protein 2 (MRP2) and MRP3 were incorporated into the model, the predicted M3G mean AUC in NASH was 1.34‐fold higher compared to healthy subjects, which is slightly lower than the observed value (1.63‐fold). Exploratory simulations on other physiological changes occurring in NASH (e.g., moderate decreases in glomerular filtration rate and portal vein blood flow) revealed that the effect of transporter changes was most prominent. Additionally, NASH‐related transporter changes resulted in decreased morphine EHR, which could be important for drugs with extensive EHR. This study is an important first step to predict drug disposition in complex diseases such as NASH using PBPK modeling.
Drug transporter expression and function at the blood–brain barrier is altered in Alzheimer’s disease (AD). However, the impact of AD on the expression of transporters and metabolizing enzymes in ...peripheral tissues has received little attention. The current study evaluated the expression of drug transporters and metabolizing enzymes in the small intestine and liver from 8- to 9-month-old female wild-type (WT) and APPswe/PSEN 1dE9 (APP/PS1) transgenic mice, a widely used AD model, using a quantitative targeted absolute proteomics (QTAP) approach. Furthermore, the general morphological appearance of the liver was assessed by immunohistochemistry, and lipid content was visualized using Oil Red O staining. The small intestines of APP/PS1 mice exhibited a significant 2.3-fold increase in multidrug resistance-associated protein 2 (Mrp2), a 1.9-fold decrease in monocarboxylate transporter 1 (Mct1), and a 3.6-fold increase in UDP-glucuronosyltransferase (Ugt) 2b5 relative to those from WT mice based on QTAP analysis. While the liver from APP/PS1 mice exhibited no changes in drug transporter expression, there was a 1.3-fold elevation in cytochrome P450 (Cyp) 51a1 and a 1.2-fold reduction in Cyp2c29 protein expression, and this was associated with morphological alterations including accumulation of hepatocyte lipids. These studies are the first to demonstrate that the protein expression of transporters and metabolizing enzymes important in oral drug absorption are modified in a mouse model of familial AD, which may lead to altered disposition of some orally administered drugs in AD.
Abstract
Drug-induced liver injury (DILI) is a significant clinical issue, affecting 1–1.5 million patients annually, and remains a major challenge during drug development—toxicity and safety ...concerns are the second-highest reason for drug candidate failure. The future prevalence of DILI can be minimized by developing a greater understanding of the biological mechanisms behind DILI. Both qualitative and quantitative analytical techniques are vital to characterizing and investigating DILI. In vitro assays are capable of characterizing specific aspects of a drug’s hepatotoxic nature and multiplexed assays are capable of characterizing and scoring a drug’s association with DILI. However, an even deeper insight into the perturbations to biological pathways involved in the mechanisms of DILI can be gained through the use of omics-based analytical techniques: genomics, transcriptomics, proteomics, and metabolomics. These omics analytical techniques can offer qualitative and quantitative insight into genetic susceptibilities to DILI, the impact of drug treatment on gene expression, and the effect on protein and metabolite abundance. This review will discuss the analytical techniques that can be applied to characterize and investigate the biological mechanisms of DILI and potential predictive biomarkers.
Organic cation transporters (OCTs) are members of the solute carrier 22 family of transporter proteins that are involved in absorption, distribution, and excretion of organic cations. OCT3 is ...localized in the apical (AP) membrane of enterocytes, but the literature is ambiguous about OCT1 (mOct1) localization, with some evidence suggesting a basolateral (BL) localization in human and mouse enterocytes. This is contrary to our preliminary findings showing AP localization of OCT1 in Caco-2 cell monolayers, an established model of human intestinal epithelium. Therefore, this study aims at determining the localization of OCT1 (mOct1) in Caco-2 cells, and human and mouse enterocytes. Functional studies using OCT1-specific substrate pentamidine showed transporter-mediated AP but not BL uptake in Caco-2 cells and human and mouse intestinal tissues. OCT1 inhibition decreased AP uptake of pentamidine by ∼50% in all three systems with no effect on BL uptake. A short hairpin RNA-mediated OCT1 knockdown in Caco-2 cells decreased AP uptake of pentamidine by ∼50% but did not alter BL uptake. Immunostaining and confocal microscopy in all three systems confirmed AP localization of OCT1 (mOct1). Our studies unequivocally show AP membrane localization of OCT1 (mOct1) in Caco-2 cells and human and mouse intestine. These results are highly significant as they will require reinterpretation of previous drug disposition and drug-drug interaction studies where conclusions were drawn assuming BL localization of OCT1 in enterocytes. Most importantly, these results will require revision of the regulatory guidance for industry in the United States and elsewhere because it has stated that OCT1 is basolaterally localized in enterocytes.
Compared with the significant number of studies reporting altered abundance and function of drug transporters at the blood–brain barrier (BBB) in Alzheimer’s disease (AD), the impact of AD on the ...abundance of intestinal drug transporters and the subsequent effects on oral drug absorption have received little attention. We have reported the altered abundance of some small intestinal drug transporters in a familial mouse model of AD; however, whether this leads to altered oral drug absorption is unknown. The current study examined plasma concentrations of caffeine and diazepam (markers for transcellular passive transport), digoxin (P-glycoprotein substrate), and valsartan (multidrug resistance-associated protein 2 substrate) following oral administration to 8–10 month old female wild-type (WT) and APPswe/PSEN1dE9 (APP/PS1) transgenic mice, a commonly used mouse model of familial AD. The plasma exposure of valsartan and digoxin was significantly (p < 0.05) lower in APP/PS1 animals compared with WT mice, whereas the plasma concentrations of the passive transcellular markers caffeine and diazepam did not significantly differ between the two genotypes. To assess whether the reduced oral absorption of valsartan and digoxin was due to decreased intestinal transport, the ex vivo transport of the previously mentioned drugs and mannitol (a marker of paracellular transport) across the jejunum of WT and APP/PS1 mice was assessed over 120 min. In line with the in vivo absorption studies, the permeability of caffeine and diazepam did not significantly differ between WT and APP/PS1 mice. The permeability of 3H-digoxin through the APP/PS1 mouse jejunum was lower than that measured through the WT jejunum; the average amount (relative to dose applied) permeating the tissue over 120 min was 0.22 ± 0.11% (mean ± SD) for the APP/PS1 jejunum and 0.85 ± 0.3% for the WT jejunum. A 1.9-fold reduction in the average amount of valsartan permeating the jejunum of APP/PS1 mice relative to that of WT mice was also detected. Although no apparent morphological alterations were observed in the jejunal tissue of APP/PS1 mice, the permeability of 14C-mannitol across the jejunum from APP/PS1 mice was lower than that across the WT jejunum (P app= 10.7 ± 3.7 × 10–6 and 6.0 ± 3.4 × 10–6 cm/s, respectively), suggesting tightened paracellular junctions in APP/PS1 mice. These studies are the first to demonstrate, in APP/PS1 mice, reduced intestinal permeability and the absorption of drugs commonly prescribed to people with AD for their comorbidities. If these findings translate to people with AD, then modified dosing regimens may be necessary for selected drugs to ensure that their plasma concentrations remain in the effective range.
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