Hepatic OATP1B zonal distribution: Implications for rifampicin-mediated drug-drug interactions explored within a PBPK framework.

OATP1B facilitates the uptake of xenobiotics into hepatocytes and is a prominent target for drug-drug interactions (DDIs). Reduced systemic exposure of OATP1B substrates has been reported following multiple-dose rifampicin; one explanation for this observation is OATP1B induction. Non-uniform hepatic distribution of OATP1B may impact local rifampicin tissue concentrations and rifampicin-mediated protein induction, which may affect the accuracy of transporter- and/or metabolizing enzyme-mediated DDI predictions. We incorporated quantitative zonal OATP1B distribution data from immunofluorescence imaging into a PBPK modeling framework to explore rifampicin interactions with OATP1B and CYP substrates. PBPK models were developed for rifampicin, two OATP1B substrates, pravastatin and repaglinide (also metabolized by CYP2C8/CYP3A4), and the CYP3A probe, midazolam. Simulated hepatic uptake of pravastatin and repaglinide increased from the periportal to the pericentral region (approximately 2.1-fold), consistent with OATP1B distribution data. Simulated rifampicin unbound intracellular concentrations increased in the pericentral region (1.64-fold) compared to simulations with uniformly distributed OATP1B. The absolute average fold error of the rifampicin PBPK model for predicting substrate maximal concentration (Cmax) and area under the plasma concentration-time curve (AUC) ratios was 1.41 and 1.54, respectively (nine studies). In conclusion, hepatic OATP1B distribution has a considerable impact on simulated zonal substrate uptake clearance values and simulated intracellular perpetrator concentrations, which regulate transporter and metabolic DDIs. Additionally, accounting for rifampicin-mediated OATP1B induction in parallel with inhibition improved model predictions. This study provides novel insight into the effect of hepatic OATP1B distribution on site-specific DDI predictions and the impact of accounting for zonal transporter distributions within PBPK models.

Effects of PFAS on human liver transporters: implications for health outcomes.

Per- and polyfluoroalkyl substances (PFAS) have become internationally recognized over the past three decades as persistent organic pollutants used in the production of various consumer and industrial goods. Research efforts continue to gauge the risk that historically used, and newly produced, PFAS may cause to human health. Numerous studies report toxic effects of PFAS on the human liver as well as increased serum cholesterol levels in adults. A major concern with PFAS, also dubbed "forever chemicals", is that they accumulate in liver and kidney and persist in serum. The mechanisms responsible for their disposition and excretion in humans are poorly understood. A better understanding of the interaction of PFAS with liver transporters, as it pertains to the disposition of PFAS and other xenobiotics, could provide mechanistic insight into human health effects and guide efforts toward risk assessment of compounds in development. This review summarizes the current state of the literature on the emerging relationships (e.g., substrates, inhibitors, modulators of gene expression) between PFAS and specific hepatic transporters. The adaptive and toxicological responses of hepatocytes to PFAS that reveal linkages to pathologies and epidemiological findings are highlighted. The evidence suggests that our understanding of the molecular landscape of PFAS must improve to determine their impact on the expression and function of hepatocyte transporters that play a key role in PFAS or other xenobiotic disposition. From here, we can assess what role these changes may have in documented human health outcomes.

Physiologically Based Pharmacokinetic (PBPK) Model Predictions of Disease Mediated Changes in Drug Disposition in Patients with Nonalcoholic Fatty Liver Disease (NAFLD).

PURPOSE: This study was designed to verify a virtual population representing patients with nonalcoholic fatty liver disease (NAFLD) to support the implementation of a physiologically based pharmacokinetic (PBPK) modeling approach for prediction of disease-related changes in drug pharmacokinetics. METHODS: A virtual NAFLD patient population was developed in GastroPlus (v.9.8.2) by accounting for pathophysiological changes associated with the disease and proteomics-informed alterations in the abundance of metabolizing enzymes and transporters pertinent to drug disposition. The NAFLD population model was verified using exemplar drugs where elimination is influenced predominantly by cytochrome P450 (CYP) enzymes (chlorzoxazone, caffeine, midazolam, pioglitazone) or by transporters (rosuvastatin, 11C-metformin, morphine and the glucuronide metabolite of morphine). RESULTS: PBPK model predictions of plasma concentrations of all the selected drugs and hepatic radioactivity levels of 11C-metformin were consistent with the clinically-observed data. Importantly, the PBPK simulations using the virtual NAFLD population model provided reliable estimates of the extent of changes in key pharmacokinetic parameters for the exemplar drugs, with mean predicted ratios (NAFLD patients divided by healthy individuals) within 0.80- to 1.25-fold of the clinically-reported values, except for midazolam (prediction-fold difference of 0.72). CONCLUSION: A virtual NAFLD population model within the PBPK framework was successfully developed with good predictive capability of estimating disease-related changes in drug pharmacokinetics. This supports the use of a PBPK modeling approach for prediction of the pharmacokinetics of new investigational or repurposed drugs in patients with NAFLD and may help inform dose adjustments for drugs commonly used to treat comorbidities in this patient population.

Alterations in zonal distribution and plasma membrane localization of hepatocyte bile acid transporters in patients with NAFLD.

BACKGROUND: NAFLD is highly prevalent with limited treatment options. Bile acids (BAs) increase in the systemic circulation and liver during NAFLD progression. Changes in plasma membrane localization and zonal distribution of BA transporters can influence transport function and BA homeostasis. However, a thorough characterization of how NAFLD influences these factors is currently lacking. This study aimed to evaluate the impact of NAFLD and the accompanying histologic features on the functional capacity of key hepatocyte BA transporters across zonal regions in human liver biopsies. METHODS: A novel machine learning image classification approach was used to quantify relative zonal abundance and plasma membrane localization of BA transporters (bile salt export pump [BSEP], sodium-taurocholate cotransporting polypeptide, organic anion transporting polypeptide [OATP] 1B1 and OATP1B3) in non-diseased (n = 10), NAFL (n = 9), and NASH (n = 11) liver biopsies. Based on these data, membrane-localized zonal abundance (MZA) measures were developed to estimate transporter functional capacity. RESULTS: NAFLD diagnosis and histologic scoring were associated with changes in transporter membrane localization and zonation. Increased periportal BSEPMZA (mean proportional difference compared to non-diseased liver of 0.090) and decreased pericentral BSEPMZA (-0.065) were observed with NASH and also in biopsies with higher histologic scores. Compared to Non-diseased Liver, periportal OATP1B3MZA was increased in NAFL (0.041) and NASH (0.047). Grade 2 steatosis (mean proportional difference of 0.043 when compared to grade 0) and grade 1 lobular inflammation (0.043) were associated with increased periportal OATP1B3MZA. CONCLUSIONS: These findings provide novel mechanistic insight into specific transporter alterations that impact BA homeostasis in NAFLD. Changes in BSEPMZA likely contribute to altered BA disposition and pericentral microcholestasis previously reported in some patients with NAFLD. BSEPMZA assessment could inform future development and optimization of NASH-related pharmacotherapies.

Membrane transporters in drug development and as determinants of precision medicine.

The effect of membrane transporters on drug disposition, efficacy and safety is now well recognized. Since the initial publication from the International Transporter Consortium, significant progress has been made in understanding the roles and functions of transporters, as well as in the development of tools and models to assess and predict transporter-mediated activity, toxicity and drug-drug interactions (DDIs). Notable advances include an increased understanding of the effects of intrinsic and extrinsic factors on transporter activity, the application of physiologically based pharmacokinetic modelling in predicting transporter-mediated drug disposition, the identification of endogenous biomarkers to assess transporter-mediated DDIs and the determination of the cryogenic electron microscopy structures of SLC and ABC transporters. This article provides an overview of these key developments, highlighting unanswered questions, regulatory considerations and future directions.

Hepatic Bile Acid Transporters and Drug-induced Hepatotoxicity.

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.

Application of Pharmacokinetic Modeling to Characterize Hepatobiliary Disposition of Imaging Agents and Alterations due to Liver Injury in Isolated Perfused Rat Livers.

BACKGROUND: Understanding the impact of altered hepatic uptake and/or efflux on the hepatobiliary disposition of the imaging agents [99mTc]Mebrofenin (MEB) and [153Gd]Gadobenate dimeglumine (BOPTA) is important for proper estimation of liver function. METHODS: A multi-compartmental pharmacokinetic (PK) model describing MEB and BOPTA disposition in isolated perfused rat livers (IPRLs) was developed. The PK model was simultaneously fit to MEB and BOPTA concentration-time data in the extracellular space, hepatocytes, bile canaliculi, and sinusoidal efflux in livers from healthy rats, and to BOPTA concentration-time data in rats pretreated with monocrotaline (MCT). RESULTS: The model adequately described MEB and BOPTA disposition in each compartment. The hepatocyte uptake clearance was much higher for MEB (55.3 mL/min) than BOPTA (6.67 mL/min), whereas the sinusoidal efflux clearance for MEB (0.000831 mL/min) was lower than BOPTA (0.0127 mL/min). The clearance from hepatocytes to bile (CLbc) for MEB (0.658 mL/min) was similar to BOPTA (0.642 mL/min) in healthy rat livers. The BOPTA CLbc was reduced in livers from MCT-pretreated rats (0.496 mL/min), while the sinusoidal efflux clearance was increased (0.0644 mL/min). CONCLUSION: A PK model developed to characterize MEB and BOPTA disposition in IPRLs was used to quantify changes in the hepatobiliary disposition of BOPTA caused by MCT pretreatment of rats to induce liver toxicity. This PK model could be applied to simulate changes in the hepatobiliary disposition of these imaging agents in rats in response to altered hepatocyte uptake or efflux associated with disease, toxicity, or drug-drug interactions.

Effect of mTOR inhibitors on sodium taurocholate cotransporting polypeptide (NTCP) function in vitro.

The sodium taurocholate cotransporting polypeptide (NTCP; gene name SLC10A1) is the primary hepatic basolateral uptake transporter for conjugated bile acids and the entry receptor for the hepatitis B and D virus (HBV/HDV). Regulation of human NTCP remains a knowledge gap due to significant species differences in substrate and inhibitor selectivity and plasma membrane expression. In the present study, various kinase inhibitors were screened for inhibition of NTCP function and taurocholate (TCA) uptake using NTCP-transfected HuH-7 cells. This study identified everolimus, an mTOR inhibitor and macrocyclic immunosuppressive drug, as an NTCP inhibitor with modest potency (IC50 = 6.7-8.0 µM). Further investigation in differentiated HuH-7 cells expressing NTCP and NTCP-overexpressing Flp-In T-REx 293 cells revealed that the mechanism of action of everolimus on NTCP is direct inhibition and mTOR-independent. Structural analogs of everolimus inhibited NTCP-mediated TCA uptake, however, functional analogs did not affect NTCP-mediated TCA transport, providing further evidence for direct inhibition. This work contributes to the growing body of literature suggesting that NTCP-mediated bile acid uptake may be inhibited by macrocyclic peptides, which may be further exploited to develop novel medications against HBV/HDV.

Considerations for Physiologically Based Modeling in Liver Disease: From Nonalcoholic Fatty Liver (NAFL) to Nonalcoholic Steatohepatitis (NASH).

Nonalcoholic fatty liver disease (NAFLD), representing a clinical spectrum ranging from nonalcoholic fatty liver (NAFL) to nonalcoholic steatohepatitis (NASH), is rapidly evolving into a global pandemic. Patients with NAFLD are burdened with high rates of metabolic syndrome-related comorbidities resulting in polypharmacy. Therefore, it is crucial to gain a better understanding of NAFLD-mediated changes in drug disposition and efficacy/toxicity. Despite extensive clinical pharmacokinetic data in cirrhosis, current knowledge concerning pharmacokinetic alterations in NAFLD, particularly at different stages of disease progression, is relatively limited. In vitro-to-in vivo extrapolation coupled with physiologically based pharmacokinetic and pharmacodynamic (IVIVE-PBPK/PD) modeling offers a promising approach for optimizing pharmacologic predictions while refining and reducing clinical studies in this population. Use of IVIVE-PBPK to predict intra-organ drug concentrations at pharmacologically relevant sites of action is particularly advantageous when it can be linked to pharmacodynamic effects. Quantitative systems pharmacology/toxicology (QSP/QST) modeling can be used to translate pharmacokinetic and pharmacodynamic data from PBPK/PD models into clinically relevant predictions of drug response and toxicity. In this review, a detailed summary of NAFLD-mediated alterations in human physiology relevant to drug absorption, distribution, metabolism, and excretion (ADME) is provided. The application of literature-derived physiologic parameters and ADME-associated protein abundance data to inform virtual NAFLD population development and facilitate PBPK/PD, QSP, and QST predictions is discussed along with current limitations of these methodologies and knowledge gaps. The proposed methodologic framework offers great potential for meaningful prediction of pharmacological outcomes in patients with NAFLD and can inform both drug development and clinical practice for this population.

Developing evidence-based resources for evaluating postgraduate trainees in the biomedical sciences.

Postgraduate trainees elevate the academic strength of institutions by conducting research, promoting innovation, securing grant funding, training undergraduate students, and building alliances. Rigorous and systematic program evaluation can help ensure that postgraduate training programs are achieving the program's intended outcomes. The purpose of this project was to develop evidence-based evaluation tools that could be shared across federally funded biomedical training programs to enhance program evaluation capacity. This manuscript describes the evidence-based process used to determine program evaluation needs of these programs at a research-intensive university. Using a multi-phased sequential exploratory mixed methods approach, data were collected from trainees, employers, leaders, and program directors. Data analyses included document analysis of program plans, inductive coding of focus groups and interviews, and descriptive analysis of surveys. Two overarching categories-Trainee Skills and Program Characteristics-were identified including six themes each. Program directors prioritized communication, social and behavioral skills, and collaboration as the trainee skills that they needed the most help evaluating. Furthermore, program directors prioritized the following program characteristics as those that they needed the most help evaluating: training environment, trainee outcomes, and opportunities offered. Surveys, interview scripts, and related resources for the categories and themes were developed and curated on a publicly available website for program directors to use in their program evaluations.

A novel differentiated HuH-7 cell model to examine bile acid metabolism, transport and cholestatic hepatotoxicity.

Hepatic cell lines serve as economical and reproducible alternatives for primary human hepatocytes. However, the utility of hepatic cell lines to examine bile acid homeostasis and cholestatic toxicity is limited due to abnormal expression and function of bile acid-metabolizing enzymes, transporters, and the absence of canalicular formation. We discovered that culturing HuH-7 human hepatoma cells with dexamethasone (DEX) and 0.5% dimethyl sulfoxide (DMSO) for two weeks, with Matrigel overlay after one week, resulted in a shorter and improved differentiation process. These culture conditions increased the expression and function of the major bile acid uptake and efflux transporters, sodium taurocholate co-transporting polypeptide (NTCP) and the bile salt export pump (BSEP), respectively, in two-week cultures of HuH-7 cells. This in vitro model was further characterized for expression and function of bile acid-metabolizing enzymes, transporters, and cellular bile acids. Differentiated HuH-7 cells displayed a marked shift in bile acid composition and induction of cytochrome P450 (CYP) 7A1, CYP8B1, CYP3A4, and bile acid-CoA: amino acid N-acyltransferase (BAAT) mRNAs compared to control. Inhibition of taurocholate uptake and excretion after a 24-h treatment with prototypical cholestatic drugs suggests that differentiated HuH-7 cells are a suitable model to examine cholestatic hepatotoxicity.

Clinical Relevance of Hepatic and Renal P-gp/BCRP Inhibition of Drugs: An International Transporter Consortium Perspective.

The role of P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) in drug-drug interactions (DDIs) and limiting drug absorption as well as restricting the brain penetration of drugs with certain physicochemical properties is well known. P-gp/BCRP inhibition by drugs in the gut has been reported to increase the systemic exposure to substrate drugs. A previous International Transporter Consortium (ITC) perspective discussed the feasibility of P-gp/BCRP inhibition at the blood-brain barrier and its implications. This ITC perspective elaborates and discusses specifically the hepatic and renal P-gp/BCRP (referred as systemic) inhibition of drugs and whether there is any consequence for substrate drug disposition. This perspective summarizes the clinical evidence-based recommendations regarding systemic P-gp and BCRP inhibition of drugs with a focus on biliary and active renal excretion pathways. Approaches to assess the clinical relevance of systemic P-gp and BCRP inhibition in the liver and kidneys included (i) curation of DDIs involving intravenously administered substrates or inhibitors; (ii) in vitro-to-in vivo extrapolation of P-gp-mediated DDIs at the systemic level; and (iii) curation of drugs with information available about the contribution of biliary excretion and related DDIs. Based on the totality of evidence reported to date, this perspective supports limited clinical DDI risk upon P-gp or BCRP inhibition in the liver or kidneys.

Regulation of Drug Transport Proteins-From Mechanisms to Clinical Impact: A White Paper on Behalf of the International Transporter Consortium.

Membrane transport proteins are involved in the absorption, disposition, efficacy, and/or toxicity of many drugs. Numerous mechanisms (e.g., nuclear receptors, epigenetic gene regulation, microRNAs, alternative splicing, post-translational modifications, and trafficking) regulate transport protein levels, localization, and function. Various factors associated with disease, medications, and dietary constituents, for example, may alter the regulation and activity of transport proteins in the intestine, liver, kidneys, brain, lungs, placenta, and other important sites, such as tumor tissue. This white paper reviews key mechanisms and regulatory factors that alter the function of clinically relevant transport proteins involved in drug disposition. Current considerations with in vitro and in vivo models that are used to investigate transporter regulation are discussed, including strengths, limitations, and the inherent challenges in predicting the impact of changes due to regulation of one transporter on compensatory pathways and overall drug disposition. In addition, translation and scaling of in vitro observations to in vivo outcomes are considered. The importance of incorporating altered transporter regulation in modeling and simulation approaches to predict the clinical impact on drug disposition is also discussed. Regulation of transporters is highly complex and, therefore, identification of knowledge gaps will aid in directing future research to expand our understanding of clinically relevant molecular mechanisms of transporter regulation. This information is critical to the development of tools and approaches to improve therapeutic outcomes by predicting more accurately the impact of regulation-mediated changes in transporter function on drug disposition and response.

Lipidomics profiles in hepatocytes from nonalcoholic steatohepatitis patients differ markedly from in vitro-induced steatotic hepatocytes.

Nonalcoholic steatohepatitis (NASH) is a severe form of liver injury that can be caused by a variety of stimuli and has a significant mortality rate. A common technique to induce in vitro steatosis involves culturing primary human hepatocytes (PHH) in fatty acid-enriched media. This study compared the lipidome of PHH cultured in fatty acid-enriched media to hepatocytes from patients with NASH and healthy controls. Hepatocytes from NASH patients displayed increased total cellular abundance of glycerolipids and phospholipids compared to healthy control hepatocytes. PHH cultured in fatty acid-enriched media demonstrated increased glycerolipids. However, these culture conditions did not induce elevated phospholipid levels. Thus, culturing PHH in fatty acid-enriched media has limited capacity to emulate the environment of hepatocytes in NASH patients.

New Pharmacokinetic Parameters of Imaging Substrates Quantified from Rat Liver Compartments.

Hepatobiliary imaging is increasingly used by pharmacologists to quantify liver concentrations of transporter-dependent drugs. However, liver imaging does not quantify concentrations in extracellular space, hepatocytes, and bile canaliculi. Our study compared the compartmental distribution of two hepatobiliary substrates gadobenate dimeglumine [BOPTA; 0.08 liver extraction ratio (ER)] and mebrofenin (MEB; 0.93 ER) in a model of perfused rat liver. A gamma counter placed over livers measured liver concentrations. Livers were preperfused with gadopentetate dimeglumine to measure extracellular concentrations. Concentrations coming from bile canaliculi and hepatocytes were calculated. Transporter activities were assessed by concentration ratios between compartments and pharmacokinetic parameters that describe the accumulation and decay profiles of hepatocyte concentrations. The high liver concentrations of MEB relied mainly on hepatocyte and bile canaliculi concentrations. In contrast, the three compartments contributed to the low liver concentrations obtained during BOPTA perfusion. Nonlinear regression analysis of substrate accumulation in hepatocytes revealed that cellular efflux is measurable ∼4 minutes after the start of perfusion. The hepatocyte-to-extracellular concentration ratio measured at this time point was much higher during MEB perfusion. BOPTA transport by multidrug resistance associated protein 2 induced an aquaporin-mediated water transport, whereas MEB transport did not. BOPTA clearance from hepatocytes to bile canaliculi was higher than MEB clearance. MEB did not efflux back to sinusoids, whereas BOPTA basolateral efflux contributed to the decrease in hepatocyte concentrations. In conclusion, our ex vivo model quantifies substrate compartmental distribution and transport across hepatocyte membranes and provides an additional understanding of substrate distribution in the liver. SIGNIFICANCE STATEMENT: When transporter-dependent drugs target hepatocytes, cellular concentrations are important to investigate. Low concentrations on cellular targets impair drug therapeutic effects, whereas excessive hepatocyte concentrations may induce cellular toxicity. With a gamma counter placed over rat perfused livers, we measured substrate concentrations in the extracellular space, hepatocytes, and bile canaliculi. Transport across hepatocyte membranes was calculated. The study provides an additional understanding of substrate distribution in the liver.

Novel Bile Acid-Dependent Mechanisms of Hepatotoxicity Associated with Tyrosine Kinase Inhibitors.

Drug-induced liver injury (DILI) is the leading cause of acute liver failure and a major concern in drug development. Altered bile acid homeostasis via inhibition of the bile salt export pump (BSEP) is one mechanism of DILI. Dasatinib, pazopanib, and sorafenib are tyrosine kinase inhibitors (TKIs) that competitively inhibit BSEP and increase serum biomarkers for hepatotoxicity in ∼25-50% of patients. However, the mechanism(s) of hepatotoxicity beyond competitive inhibition of BSEP are poorly understood. This study examined mechanisms of TKI-mediated hepatotoxicity associated with altered bile acid homeostasis. Dasatinib, pazopanib, and sorafenib showed bile acid-dependent toxicity at clinically relevant concentrations, based on the C-DILI assay using sandwich-cultured human hepatocytes (SCHH). Among several bile acid-relevant genes, cytochrome P450 (CYP) 7A1 mRNA was specifically upregulated by 6.2- to 7.8-fold (dasatinib) and 5.7- to 9.3-fold (pazopanib), compared with control, within 8 hours. This was consistent with increased total bile acid concentrations in culture medium up to 2.3-fold, and in SCHH up to 1.4-fold, compared with control, within 24 hours. Additionally, protein abundance of sodium taurocholate co-transporting polypeptide (NTCP) was increased up to 2.0-fold by these three TKIs. The increase in NTCP protein abundance correlated with increased function; dasatinib and pazopanib increased hepatocyte uptake clearance (CLuptake) of taurocholic acid, a probe bile acid substrate, up to 1.4-fold. In conclusion, upregulation of CYP7A1 and NTCP in SCHH constitute novel mechanisms of TKI-associated hepatotoxicity. SIGNIFICANCE STATEMENT: Understanding the mechanisms of hepatotoxicity associated with tyrosine kinase inhibitors (TKIs) is fundamental to development of effective and safe intervention therapies for various cancers. Data generated in sandwich-cultured human hepatocytes, an in vitro model of drug-induced hepatotoxicity, revealed that TKIs upregulate bile acid synthesis and alter bile acid uptake and excretion. These findings provide novel insights into additional mechanisms of bile acid-mediated drug-induced liver injury, an adverse effect that limits the use and effectiveness of TKI treatment in some cancer patients.

Identification of Key Amino Acids that Impact Organic Solute Transporter α/ß (OSTα/ß).

Organic solute transporter α/ß (OSTα/ß) is a bidirectional bile acid transporter localized on the basolateral membrane of hepatic, intestinal, and renal epithelial cells. OSTα/ß plays a critical role in intestinal bile acid reabsorption and is upregulated in hepatic diseases characterized by elevated bile acids, whereas genetic variants in SLC51A/B have been associated with clinical cholestasis. OSTα/ß also transports and is inhibited by commonly used medications. However, there is currently no high-resolution structure of OSTα/ß, and structure-function data for OSTα, the proposed substrate-binding subunit, are lacking. The present study addressed this knowledge gap and identified amino acids in OSTα that are important for bile acid transport. This was accomplished using computational modeling and site-directed mutagenesis of the OSTα subunit to generate OSTα/ß mutant cell lines. Out of the 10 OSTα/ß mutants investigated, four (S228K, T229S, Q269E, Q269K) exhibited decreased [3H]-taurocholate (TCA) uptake (ratio of geometric means relative to OSTα/ß wild type (WT) of 0.76, 0.75, 0.79, and 0.13, respectively). Three OSTα/ß mutants (S228K, Q269K, E305A) had reduced [3H]-TCA efflux % (ratio of geometric means relative to OSTα/ß WT of 0.86, 0.65, and 0.79, respectively). Additionally, several OSTα/ß mutants demonstrated altered expression and cellular localization when compared with OSTα/ß WT. In summary, we identified OSTα residues (Ser228, Thr229, Gln269, Glu305) in predicted transmembrane domains that affect expression of OSTα/ß and may influence OSTα/ß-mediated bile acid transport. These data advance our understanding of OSTα/ß structure/function and can inform future studies designed to gain further insight into OSTα/ß structure or to identify additional OSTα/ß substrates and inhibitors. SIGNIFICANCE STATEMENT: OSTα/ß is a clinically important transporter involved in enterohepatic bile acid recycling with currently no high-resolution protein structure and limited structure-function data. This study identified four OSTα amino acids (Ser228, Thr229, Gln269, Glu305) that affect expression of OSTα/ß and may influence OSTα/ß-mediated bile acid transport. These data can be utilized to inform future investigation of OSTα/ß structure and refine molecular modeling approaches to facilitate the identification of substrates and/or inhibitors of OSTα/ß.

Analytical and Omics-Based Advances in the Study of Drug-Induced Liver Injury.

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.

Pregnancy-Related Hormones Increase UGT1A1-Mediated Labetalol Metabolism in Human Hepatocytes.

Pregnancy-related hormones (PRH) are recognized as important regulators of hepatic cytochrome P450 enzyme expression and function. However, the impact of PRH on the hepatic expression and function of uridine diphosphate glucuronosyltransferases (UGTs) remains unclear. Using primary human hepatocytes, we evaluated the effect of PRH exposure on mRNA levels and protein concentrations of UGT1A1, UGT2B7, and other key UGT enzymes, and on the metabolism of labetalol (a UGT1A1 and UGT2B7 substrate commonly prescribed to treat hypertensive disorders of pregnancy). Sandwich-cultured human hepatocytes (SCHH) from female donors were exposed to the PRH estradiol, estriol, estetrol, progesterone, and cortisol individually or in combination. We quantified protein concentrations of UGT1A1, UGT2B7, and four additional UGT1A isoforms in SCHH membrane fractions and evaluated the metabolism of labetalol to its glucuronide metabolites in SCHH. PRH exposure increased mRNA levels and protein concentrations of UGT1A1 and UGT1A4 in SCHH. PRH exposure also significantly increased labetalol metabolism to its UGT1A1-derived glucuronide metabolite in a concentration-dependent manner, which positively correlated with PRH-induced changes in UGT1A1 protein concentrations. In contrast, PRH did not alter UGT2B7 mRNA levels or protein concentrations in SCHH, and formation of the UGT2B7-derived labetalol glucuronide metabolite was decreased following PRH exposure. Our findings demonstrate that PRH alter expression and function of UGT proteins in an isoform-specific manner and increase UGT1A1-mediated labetalol metabolism in human hepatocytes by inducing UGT1A1 protein concentrations. These results provide mechanistic insight into the increases in labetalol clearance observed in pregnant individuals.