Obeticholic acid, a selective farnesoid X receptor agonist, regulates bile acid homeostasis in sandwich-cultured human hepatocytes.

Farnesoid X receptor (FXR) is a master regulator of bile acid homeostasis through transcriptional regulation of genes involved in bile acid synthesis and cellular membrane transport. Impairment of bile acid efflux due to cholangiopathies results in chronic cholestasis leading to abnormal elevation of intrahepatic and systemic bile acid levels. Obeticholic acid (OCA) is a potent and selective FXR agonist that is 100-fold more potent than the endogenous ligand chenodeoxycholic acid (CDCA). The effects of OCA on genes involved in bile acid homeostasis were investigated using sandwich-cultured human hepatocytes. Gene expression was determined by measuring mRNA levels. OCA dose-dependently increased fibroblast growth factor-19 (FGF-19) and small heterodimer partner (SHP) which, in turn, suppress mRNA levels of cholesterol 7-alpha-hydroxylase (CYP7A1), the rate-limiting enzyme for de novo synthesis of bile acids. Consistent with CYP7A1 suppression, total bile acid content was decreased by OCA (1 µmol/L) to 42.7 ± 20.5% relative to control. In addition to suppressing de novo bile acids synthesis, OCA significantly increased the mRNA levels of transporters involved in bile acid homeostasis. The bile salt excretory pump (BSEP), a canalicular efflux transporter, increased by 6.4 ± 0.8-fold, and the basolateral efflux heterodimer transporters, organic solute transporter α (OSTα ) and OSTß increased by 6.4 ± 0.2-fold and 42.9 ± 7.9-fold, respectively. The upregulation of BSEP and OSTα and OSTß, by OCA reduced the intracellular concentrations of d8 -TCA, a model bile acid, to 39.6 ± 8.9% relative to control. These data demonstrate that OCA does suppress bile acid synthesis and reduce hepatocellular bile acid levels, supporting the use of OCA to treat bile acid-induced toxicity observed in cholestatic diseases.

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.

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.

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

Tolvaptan is a vasopressin V(2)-receptor antagonist that has shown promise in treating Autosomal Dominant Polycystic Kidney Disease (ADPKD). Tolvaptan was, however, associated with liver injury in some ADPKD patients. Inhibition of bile acid transporters may be contributing factors to drug-induced liver injury. In this study, the ability of tolvaptan and two metabolites, DM-4103 and DM-4107, to inhibit human hepatic transporters (NTCP, BSEP, MRP2, MRP3, and MRP4) and bile acid transport in sandwich-cultured human hepatocytes (SCHH) was explored. IC(50) values were determined for tolvaptan, DM-4103 and DM-4107 inhibition of NTCP (∼41.5, 16.3, and 95.6 µM, respectively), BSEP (31.6, 4.15, and 119 µM, respectively), MRP2 (>50, ∼51.0, and >200 µM, respectively), MRP3 (>50, ∼44.6, and 61.2 µM, respectively), and MRP4 (>50, 4.26, and 37.9 µM, respectively). At the therapeutic dose of tolvaptan (90 mg), DM-4103 exhibited a C(max)/IC(50) value >0.1 for NTCP, BSEP, MRP2, MRP3, and MRP4. Tolvaptan accumulation in SCHH was extensive and not sodium-dependent; intracellular concentrations were ∼500 µM after a 10-min incubation duration with tolvaptan (15 µM). The biliary clearance of taurocholic acid (TCA) decreased by 43% when SCHH were co-incubated with tolvaptan (15 µM) and TCA (2.5 µM). When tolvaptan (15 µM) was co-incubated with 2.5 µM of chenodeoxycholic acid, taurochenodeoxycholic acid, or glycochenodeoxycholic acid in separate studies, the cellular accumulation of these bile acids increased by 1.30-, 1.68-, and 2.16-fold, respectively. Based on these data, inhibition of hepatic bile acid transport may be one of the biological mechanisms underlying tolvaptan-associated liver injury in patients with ADPKD.

Evaluation of the endothelin receptor antagonists ambrisentan, bosentan, macitentan, and sitaxsentan as hepatobiliary transporter inhibitors and substrates in sandwich-cultured human hepatocytes.

BACKGROUND: Inhibition of the transporter-mediated hepatobiliary elimination of bile salts is a putative mechanism for liver toxicity observed with some endothelin receptor antagonists (ERAs). METHODS: Sandwich-cultured human hepatocytes were used to study the hepatobiliary distribution and accumulation of exogenous taurocholate, ERAs and endogenous bile acids. The molecular mechanisms for findings in hepatocytes or clinical observations were further explored using either vesicular assays (efflux transporters) or transfected cell-lines (uptake transporters). Inhibition constants (IC50) were measured for the human hepatobiliary transporters bile salt export pump (BSEP), sodium taurocholate cotransporting polypeptide (NTCP), multidrug resistance protein 2 (MRP2), P-glycoprotein (Pgp), breast cancer resistance protein (BCRP), organic anion-transporting polypeptide 1B1 (OATP1B1) and OATP1B3. RESULTS: The ERAs showed dose-dependent reductions in exogenous taurocholate cellular accumulation in human hepatocytes, with macitentan having the greatest effect. Consistent with their effects on bile acids, the ERAs inhibited bile transporters. IC50 values for OATP1B1 and OATP1B3 ranged from 2 µM for macitentan to 47 µM for ambrisentan. Macitentan and bosentan also inhibited NTCP with IC50 values of 10 and 36 µM, respectively. Similar to previously reported findings with sitaxsentan, BSEP inhibition was observed for bosentan and macitentan with IC50 values of 42 and 12 µM, respectively. In contrast, ambrisentan showed little or no inhibition of these transporters. Other transporters tested were weakly inhibited by the ERAs. Accumulation in hepatocytes was also a factor in the effects on bile transport. Macitentan demonstrated the greatest accumulation in human hepatocytes (∼100x) followed by sitaxsentan (∼40x), bosentan (∼20x) and ambrisentan (∼2x). CONCLUSIONS: Significant differences in the inhibition of hepatic transporters were observed between the evaluated ERAs in vitro. Macitentan had the highest level of cellular accumulation and caused the greatest effects on bile acid distribution in human hepatocytes followed by sitaxsentan and bosentan. Ambrisentan showed a low potential to affect bile acids.

Combination lopinavir and ritonavir alter exogenous and endogenous bile acid disposition in sandwich-cultured rat hepatocytes.

Inhibition of the bile salt export pump (BSEP) can cause intracellular accumulation of bile acids and is a risk factor for drug-induced liver injury in humans. Antiretroviral protease inhibitors lopinavir (LPV) and ritonavir (RTV) are reported BSEP inhibitors. However, the consequences of LPV and RTV, alone and combined (LPV/r), on hepatocyte viability, bile acid transport, and endogenous bile acid disposition in rat hepatocytes have not been examined. The effect of LPV, RTV, and LPV/r on cellular viability and the disposition of [(3)H]taurocholic acid (TCA) and [(14)C]chenodeoxycholic acid (CDCA) was determined in sandwich-cultured rat hepatocytes (SCRH) and suspended rat hepatocytes. Lactate dehydrogenase and ATP assays revealed a concentration-dependent effect of LPV and RTV on cellular viability. LPV (5 µM), alone and combined with 5 µM RTV, significantly decreased [(3)H]TCA accumulation in cells + bile of SCRHs compared with control. LPV/r significantly increased [(3)H]TCA cellular accumulation (7.7 ± 0.1 pmol/mg of protein) compared with vehicle and 5 µM LPV alone (5.1 ± 0.7 and 5.0 ± 0.5 pmol/mg of protein). The [(3)H]TCA biliary clearance was reduced significantly by LPV and RTV and further reduced by LPV/r. LPV and RTV did not affect the initial uptake rates of [(3)H]TCA or [(14)C]CDCA in suspended rat hepatocytes. LPV (50 µM), RTV (5 µM), and LPV/r (5 and 50 µM/5 µM) significantly decreased the accumulation of total measured endogenous bile acids (TCA, glycocholic acid, taurochenodeoxycholic acid, glycochenodeoxycholic acid, and α/ß-tauromuricholic acid) in SCRH. Quantification of endogenous bile acids in SCRH may reveal important adaptive responses associated with exposure to known BSEP inhibitors.

Endogenous bile acid disposition in rat and human sandwich-cultured hepatocytes.

Sandwich-cultured hepatocytes (SCH) are used commonly to investigate hepatic transport protein-mediated uptake and biliary excretion of substrates. However, little is known about the disposition of endogenous bile acids (BAs) in SCH. In this study, four endogenous conjugated BAs common to rats and humans [taurocholic acid (TCA), glycocholic acid (GCA), taurochenodeoxycholic acid (TCDCA), and glycochenodeoxycholic acid (GCDCA)], as well as two BA species specific to rodents (α- and ß-tauromuricholic acid; α/ß TMCA), were profiled in primary rat and human SCH. Using B-CLEAR® technology, BAs were measured in cells+bile canaliculi, cells, and medium of SCH by LC-MS/MS. Results indicated that, just as in vivo, taurine-conjugated BA species were predominant in rat SCH, while glycine-conjugated BAs were predominant in human SCH. Total intracellular BAs remained relatively constant over days in culture in rat SCH. Total BAs in control (CTL) cells+bile, cells, and medium were approximately 3.4, 2.9, and 8.3-fold greater in human than in rat. The estimated intracellular concentrations of the measured total BAs were 64.3±5.9 µM in CTL rat and 183±56 µM in CTL human SCH, while medium concentrations of the total BAs measured were 1.16±0.21 µM in CTL rat SCH and 9.61±6.36 µM in CTL human SCH. Treatment of cells for 24h with 10 µM troglitazone (TRO), an inhibitor of the bile salt export pump (BSEP) and the Na⁺-taurocholate cotransporting polypeptide (NTCP), had no significant effect on endogenous BAs measured at the end of the 24-h culture period, potentially due to compensatory mechanisms that maintain BA homeostasis. These data demonstrate that BAs in SCH are similar to in vivo, and that SCH may be a useful in vitro model to study alterations in BA disposition if species differences are taken into account.

Automated applications of sandwich-cultured hepatocytes in the evaluation of hepatic drug transport.

Predictions of the absorption, distribution, metabolism, excretion, and toxicity of compounds in pharmaceutical development are essential aspects of the drug discovery process. B-CLEAR is an in vitro system that uses sandwich-cultured hepatocytes to evaluate and predict in vivo hepatobiliary disposition (hepatic uptake, biliary excretion, and biliary clearance), transporter-based hepatic drug-drug interactions, and potential drug-induced hepatotoxicity. Automation of predictive technologies is an advantageous and preferred format in drug discovery. In this study, manual and automated studies are investigated and equivalence is demonstrated. In addition, automated applications using model probe substrates and inhibitors to assess the cholestatic potential of drugs and evaluate hepatic drug transport are examined. The successful automation of this technology provides a more reproducible and less labor-intensive approach, reducing potential operator error in complex studies and facilitating technology transfer.

Differential disposition of chenodeoxycholic acid versus taurocholic acid in response to acute troglitazone exposure in rat hepatocytes.

Inhibition of bile acid (BA) transport may contribute to the hepatotoxicity of troglitazone (TRO), a peroxisome proliferator-activated receptor gamma agonist. Typically, studies use taurocholic acid (TCA) as a model substrate to investigate effects of xenobiotics on BA disposition. However, TRO may differentially affect the transport of individual BAs, potentially causing hepatocyte accumulation of more cytotoxic BAs. The effects of TRO on the disposition of [(14)C]-labeled chenodeoxycholic acid ([(14)C]CDCA), an unconjugated cytotoxic BA, were determined in suspended hepatocytes and sandwich-cultured hepatocytes (SCH) from rats. (E)-3-[[[3-[2-(7-chloro-2-quinolinyl)ethenyl]phenyl][[3-(dimethylamino)-3-oxopropyl]thio]methyl]thio]-propanoic acid (MK571), a multidrug resistance-associated protein (MRP) inhibitor, was included to evaluate involvement of MRPs in CDCA disposition. Accumulation in cells + bile of total [(14)C]CDCA species in SCH was sixfold greater than [(3)H]TCA and unaffected by 1 and 10µM TRO; 100µM TRO and 50µM MK571 ablated biliary excretion and significantly increased intracellular accumulation of total [(14)C]CDCA species. Results were similar in Mrp2-deficient TR(-) rat hepatocytes. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis revealed that taurine- and glycine-conjugated CDCA, in addition to unconjugated CDCA, accumulated in hepatocytes during the 10-min incubation. In suspended rat hepatocytes, initial [(14)C]CDCA uptake was primarily Na(+)-independent, whereas initial [(3)H]TCA uptake was primarily Na(+)-dependent; TRO and MK571 decreased [(14)C]CDCA uptake to a lesser extent than [(3)H]TCA. Unexpectedly, MK571 inhibited Na(+)-taurocholate cotransporting polypeptide and bile salt export pump. Differential effects on uptake and efflux of individual BAs may contribute to TRO hepatotoxicity. Although TCA is the prototypic BA used to investigate the effects of xenobiotics on BA transport, it may not be reflective of other BAs.

An in vitro assay to assess transporter-based cholestatic hepatotoxicity using sandwich-cultured rat hepatocytes.

Drug-induced cholestasis can result from the inhibition of biliary efflux of bile acids in the liver. Drugs may inhibit the hepatic uptake and/or the biliary efflux of bile acids resulting in an increase in serum concentrations. However, it is the intracellular concentration of bile acids that results in hepatotoxicity, and thus serum concentrations may not necessarily be an appropriate indicator of hepatotoxicity. In this study, sandwich-cultured rat hepatocytes were used as an in vitro model to assess the cholestatic potential of drugs using deuterium-labeled sodium taurocholate (d(8)-TCA) as a probe for bile acid transport. Eight drugs were tested as putative inhibitors of d(8)-TCA uptake and efflux. The hepatobiliary disposition of d(8)-TCA in the absence and presence of drugs was measured by using liquid chromatography/tandem mass spectrometry, and the accumulation (hepatocytes and hepatocytes plus bile), biliary excretion index (BEI), and in vitro biliary clearance (Cl(biliary)) were reported. Compounds were classified based on inhibition of uptake, efflux, or a combination of both processes. Cyclosporine A and glyburide showed a decrease in total (hepatocytes plus bile) accumulation, an increase in intracellular (hepatocytes only) accumulation, and a decrease in BEI and Cl(biliary) of d(8)-TCA, suggesting that efflux was primarily affected. Erythromycin estolate, troglitazone, and bosentan resulted in a decrease in accumulation (total and intracellular), BEI, and Cl(biliary) of d(8)-TCA, suggesting that uptake was primarily affected. Determination of a compound's relative effect on bile acid uptake, efflux, and direct determination of alterations in intracellular amounts of bile acids may provide useful mechanistic information on compounds that cause increases in serum bile acids.

Characterization of sandwich-cultured hepatocytes as an in vitro model to assess the hepatobiliary disposition of copper.

Sandwich-cultured hepatocytes (SCH) from rats (SCRH), dogs (SCDH), and humans (SCHH) were used as an in vitro model to assess the hepatobiliary disposition of copper (Cu). The expression of Cu transporters, ceruloplasmin synthesis, Cu uptake, and biliary excretion and species differences in drug-induced alterations in Cu disposition were determined in SCH from all species. Western blot analysis verified basolateral Cu uptake transporter 1 (CTR1) and canalicular Cu efflux transporter (ATP7B) expression: enzyme-linked immunosorbent assay verified synthesis/secretion of ceruloplasmin (major Cu binding protein found in blood). Endogenous Cu in SCRH, SCDH, and SCHH were 17.2 +/- 7.00, 490 +/- 44.8, and 43.5 +/- 15.8 ng/well, respectively. The hepatobiliary disposition of Cu as measured by uptake (increase in intracellular Cu in comparison to endogenous levels) and biliary excretion (increase in Cu in wash solutions obtained from hepatocytes exposed to calcium-free versus standard buffer) was determined as a function of Cu concentration and incubation time. In general, an increase in Cu concentration or incubation time resulted in an increase in Cu uptake and/or biliary excretion; however, the extent to which they affected Cu disposition was species dependent. 5-(1,1-Dioxido-1,2-thiazinan-2-yl)-N-(4-fluorobenzyl)-8-hydroxy-1,6-naphthyridine-7-carboxamide (L-000870810) (an anti-HIV compound, the development of which was halted due to an observed Cu-specific toxicity in the liver and kidneys of dogs after long-term exposure) showed no effect on Cu disposition in SCRH; however, it increased the biliary excretion of Cu in SCDH and SCHH. This is the first report to demonstrate the utility of SCH as a model to assess hepatobiliary disposition of Cu in an in vitro system.

Intracellular pharmacokinetics of tenofovir diphosphate, carbovir triphosphate, and lamivudine triphosphate in patients receiving triple-nucleoside regimens.

OBJECTIVE: To evaluate the potential for a pharmacologic mechanism to explain suboptimal virologic responses observed in a triple-nucleoside only regimen containing tenofovir disoproxil fumarate (TDF), abacavir (ABC), and lamivudine (3TC). METHODS: This was a prospective evaluation of intracellular concentrations and pharmacokinetics of tenofovir diphosphate (TFV-DP), carbovir triphosphate (CBV-TP), and lamivudine triphosphate (3TC-TP) in patients on triple-nucleoside regimens. Fifteen patients on a stable TDF plus ABC plus a third nucleoside reverse transcriptase (RT) inhibitor (3TC [n = 13], stavudine [n = 2]) regimen discontinued TDF or ABC, replacing it with a nonnucleoside RT inhibitor or protease inhibitor. Peripheral blood mononuclear cells were collected after the last dose of TDF or ABC at baseline and over 12 to 96 hours as well as at days 14 and 28 after discontinuation. Nucleotide concentrations were measured directly using liquid chromatography with tandem mass spectrometry; changes after ABC or TDF discontinuation would provide evidence of an intracellular drug interaction. RESULTS: Intracellular nucleotide concentrations of the continued drugs were unaffected when TDF or ABC was discontinued. Intracellular levels of TFV-DP exhibited less inter- and intrapatient variability than CBV-TP or 3TC-TP. TFV-DP also had persistent intracellular levels on TDF discontinuation (median half-life of 150 hours, range: 60 to >175 hours). CBV-TP concentrations fell to below the limit of detection in all patients by 72 hours after the last ABC dose in accordance with a median half-life of 18 hours (range: 12-19 hours). CONCLUSIONS: An intracellular drug interaction does not explain the suboptimal viral response in patients treated with the nucleoside-only regimen of TDF, ABC, and 3TC.

Pharmacokinetic and pharmacodynamic characteristics of emtricitabine support its once daily dosing for the treatment of HIV infection.

Emtricitabine (FTC) is a potent deoxycytidine nucleoside analogue that was recently approved for the treatment of HIV infection. Emtricitabine is activated by intracellular phosphorylation to its 5'-triphosphate (FTC5'-TP), a competitive inhibitor of the HIV reverse transcriptase (RT). Early clinical studies incorporating pharmacokinetic-pharmacodynamic (PK-PD) analyses provided a sound rationale for developing FTC as a once daily drug. A short-term open-label monotherapy trial in therapy naive HIV-infected subjects evaluated various dosage regimens of FTC, i.e., 25, 100, and 200 mg qd and/or bid, with serial measurements of plasma HIV RNA, plasma FTC, and intracellular (PBMC) FTC-5'-TP levels over the 14 days of treatment. PK data were augmented by other steady-state studies, one in healthy volunteers and the other in HIV-infected patients receiving 200 mg FTC qd, with measurements of plasma FTC and/or intracellular FTC-5'-TP levels. Correlation between anti-HIV activity and FTC-5'-TP levels was examined with dose- and concentration-response relationships determined. The once daily dosing schedule is supported by the relatively long half-lives of plasma FTC (8-10 hr) and PBMC FTC-TP (39 hr) and the high plasma FTC and PBMC FTC-5'-TP concentrations. HIV RNA suppression (PD) correlates well with PBMC FTC-5'-TP levels (PK), both reaching a plateau at doses > or = 200 mg/day. The PK and PD characteristics of FTC demonstrate that it is a once daily nucleoside RT inhibitor.