Effect of a Common Genetic Variant (p.V444A) in the Bile Salt Export Pump on the Inhibition of Bile Acid Transport by Cholestatic Medications

The bile salt export pump (BSEP) is the primary canalicular transporter responsible for the secretion of bile acids from hepatocytes into bile canaliculi, and inhibition of this transporter has been associated with drug-induced liver injury (DILI). A common variant (rs2287622; p.V444A) in the gene encoding BSEP has been associated with an increased risk of cholestatic DILI. Although p.444V BSEP (reference) and p.444A BSEP (variant) do not differ in their transport kinetics of taurocholic acid (TCA), transport of the more abundant glycocholic acid (GCA) has not been investigated. Importantly, differences in the susceptibility of p.444V and p.444A BSEP to inhibition by drugs causing cholestatic DILI have not been investigated. To address these issues, the transport kinetics of GCA were evaluated by incubating membrane vesicles expressing either p.444V or p.444A BSEP with GCA over a range of concentrations (1, 10, 25, 50, and 100 μM). The abilities of commonly used cholestatic medications to inhibit the transport of TCA and GCA by the reference and variant proteins were compared. Resulting data indicated that GCA transport kinetics for reference and variant BSEP followed Michaelis-Menten kinetics and were not statistically different [ Vmax values of 1132 ± 246 and 959 ± 256 pmol min-1 (mg of protein)-1, respectively, and Km values of 32.7 ± 18.2 and 45.7 ± 25.5 μM, respectively]. There were no statistically significant differences between the reference and variant BSEP in the inhibition of TCA or GCA transport by the cholestatic drugs tested. In conclusion, differential inhibition of TCA or GCA transport cannot account for an association between the variant BSEP and the risk for cholestatic DILI due to the drugs tested

Influence of Seeding Density and Extracellular Matrix on Bile Acid Transport and Mrp4 Expression in Sandwich-Cultured Mouse Hepatocytes

This study was undertaken to examine the influence of seeding density, extracellular matrix and days in culture on bile acid transport proteins and hepatobiliary disposition of the model bile acid taurocholate. Mouse hepatocytes were cultured in a sandwich configuration on six-well Biocoat™ plates with an overlay of Matigel™ (BC/MG) or gelled-collagen (BC/GC) for 3 or 4 days at seeding densities of 1.0, 1.25 or 1.5 × 106 cells/well. The lower seeding densities of 1.0 and 1.25 × 106 cells/well resulted in good hepatocyte morphology and bile canalicular network formation, as visualized by 5- (and 6)-carboxy-2′,7′dichlorofluorescein accumulation. In general, taurocholate cellular accumulation tended to increase as a function of seeding density in BC/GC; cellular accumulation was significantly increased in hepatocytes cultured in BC/MG compared to BC/GC at the same seeding density on both days 3 and 4 of culture. In general, in vitro intrinsic biliary clearance of taurocholate was increased at higher seeding densities. Levels of bile acid transport proteins on days 3 and 4 were not markedly influenced by seeding density or extracellular matrix except for multidrug resistance protein 4 (Mrp4), which was inversely related to seeding density. Mrp4 levels decreased ~2- to 3-fold between seeding densities of 1.0 × 106 and 1.25 × 106 cells/well regardless of extracellular matrix; an additional ~3- to 5-fold decrease in Mrp4 protein was noted in BC/GC between seeding densities of 1.25 × 106 and 1.5 × 106 cells/well. Results suggest that seeding density, extracellular matrix and days in culture profoundly influence Mrp4 expression in sandwich-cultured mouse hepatocytes. Primary mouse hepatocytes seeded in a BC/MG configuration at densities of 1.25 × 106 cells/well or below, and cultured for 3 days, yielded optimal transport based on the probes studied. This work demonstrates the applicability of the sandwich-cultured model to mouse hepatocytes

Hepatocellular Disposition and Transporter Interactions with Tolvaptan and Metabolites in Sandwich-Cultured Human Hepatocytes

Tolvaptan is a selective V2-receptor antagonist primarily metabolized by CYP 3A. The present study investigated the hepatocellular disposition of tolvaptan and the generated tolvaptan metabolites, DM-4103 and DM-4107, as well as the potential for drug-drug interactions (DDIs) with metabolic and transport proteins in sandwich-cultured human hepatocytes (SCHH). Tolvaptan was incubated with SCHH and quantified by liquid chromatography–tandem mass spectrometry. Pioglitazone, verapamil, MK-571, and elacridar were used as inhibitors to investigate mechanisms of transport and metabolism of tolvaptan and metabolites. Taurocholate (TCA), pravastatin, digoxin, and metformin were used as transporter probes to investigate which transport proteins were inhibited by tolvaptan and metabolites. Cellular accumulation of tolvaptan (0.15–50 μM), DM-4103, and DM-4107 in SCHH was concentration-dependent. Tolvaptan accumulation (15 μM) in SCHH was not altered markedly by 50 μM pioglitazone, verapamil, MK-571, or 10 μM elacridar. Coincubation of tolvaptan with pioglitazone, verapamil, MK-571, and elacridar reduced DM-4107 accumulation by 45.6, 79.8, 94.5, and 23.0%, respectively, relative to control. Coincubation with increasing tolvaptan concentrations (0.15–50 μM) decreased TCA (2.5 μM) cell+bile accumulation and the TCA biliary excretion index (BEI; from 76% to 51%), consistent with inhibition of the bile salt export pump (BSEP). Tolvaptan (15 μM) had no effect on the cellular accumulation of 2.5 μM pravastatin or metformin. Digoxin cellular accumulation increased, and the BEI of digoxin decreased from 23.9 to 8.1% in the presence of 15 μM tolvaptan, consistent with inhibition of P-glycoprotein. In summary, SCHH studies revealed potential metabolic- and transporter-mediated DDIs involving tolvaptan and metabolites

Inhibition of Human Hepatic Bile Acid Transporters by Tolvaptan and Metabolites: Contributing Factors to Drug-Induced Liver Injury?

Tolvaptan is a vasopressin

Evaluation of 99mTechnetium-Mebrofenin and 99mTechnetium-Sestamibi as Specific Probes for Hepatic Transport Protein Function in Rat and Human Hepatocytes

This study characterized 99mTc-Mebrofenin (MEB) and 99mTc-Sestamibi (MIBI) hepatic transport and preferential efflux routes (canalicular vs. basolateral) in rat and human sandwich-cultured hepatocytes (SCH)

Integration of Preclinical and Clinical Data with Pharmacokinetic Modeling and Simulation to Evaluate Fexofenadine as a Probe for Hepatobiliary Transport Function

The suitability of fexofenadine as a probe substrate to assess hepatobiliary transport function in humans was evaluated by pharmacokinetic modeling/simulation and in vitro/in situ studies using chemical modulators

Knocking Down Breast Cancer Resistance Protein (Bcrp) by Adenoviral Vector-Mediated RNA Interference (RNAi) in Sandwich-Cultured Rat Hepatocytes: A Novel Tool To Assess the Contribution of Bcrp to Drug Biliary Excretion

BCRP transports numerous drugs/derived metabolites and toxins, and exhibits overlapping substrate specificity with P-glycoprotein (P-gp) and multidrug resistance-associated protein 2 (MRP2). Assessing the contribution of BCRP to drug/metabolite biliary excretion in the intact hepatocytes remains a challenge. Current studies were designed to develop a novel in vitro tool to specifically assess the contribution of Bcrp to drug biliary excretion. Adenoviral vectors expressing short hairpin (sh) RNA targeting Bcrp (Ad-si01Bcrp) or a non-target control (Ad-siNT) were packaged and infected into sandwich-cultured rat hepatocytes (SCRH). Protein levels were detected by immunoblot. Biliary excretion index (BEI) and in vitro biliary clearance (Clbiliary) of nitrofurantoin (BCRP substrate) and digoxin (P-gp substrate) were compared among non-infected, Ad-siNT- and Ad-si01Bcrp-infected cells in SCRH. shRNA targeting Bcrp efficiently knocked down Bcrp in SCRH, while levels of other transport proteins (P-gp, Mrp2, Bsep, Mrp4 and Oatp1a1) were unaffected. In SCRH exhibiting Bcrp knockdown, cellular accumulation of nitrofurantoin was increased markedly and nitrofurantoin BEI and in vitro Clbiliary were decreased to 11% and 14% of control, respectively. Digoxin values were unaffected by knockdown of Bcrp. Results indicated that Bcrp contributed predominantly to nitrofurantoin biliary excretion, but played a negligible role in digoxin biliary excretion. In summary, Bcrp knockdown in SCRH is the first in vitro model utilizing intact hepatocytes to assess the contribution of Bcrp to the biliary excretion of drugs. This approach may be useful in predicting drug-drug interactions in biliary excretion and the consequence of impaired BCRP function on the hepatic exposure of drugs/derived metabolites

Methods To Evaluate Biliary Excretion of Drugs in Humans: An Updated Review

Determining the biliary clearance of drugs in humans is very challenging because bile in not readily accessible due to the anatomy of the hepatobiliary tract. The collection of bile usually is limited to post-surgical patients with underlying hepatobiliary disease. In healthy subjects, feces typically are used as a surrogate to quantify the amount of drug excreted via non-urinary pathways. Nevertheless, it is very important to characterize hepatobiliary elimination because this is a potential site of drug interactions that might result in significant alterations in systemic or hepatic exposure. In addition to the determination of in vivo biliary clearance values of drugs, the availability of in vitro models that can predict the extent of biliary excretion of drugs in humans may be a powerful tool in the pre-clinical stages of drug development. In this review, recent advances in the most commonly used in vivo methods to estimate biliary excretion of drugs in humans are outlined. Additionally, in vitro models that can be employed to investigate the molecular processes involved in biliary excretion are discussed to present an updated picture of the new tools and techniques that are available to study the complex processes involved in hepatic drug transport

Use of Tc-99m Mebrofenin as a Clinical Probe to Assess Altered Hepatobiliary Transport: Integration of In Vitro, Pharmacokinetic Modeling, and Simulation Studies

Transport of the hepatobiliary scintigraphy agent Tc-99m mebrofenin (MEB) was characterized, and simulation studies were conducted to examine the effects of altered hepatic transport on MEB pharmacokinetics in humans