An intracellular drug interaction does not explain the suboptimal viral response in patients treated with the nucleoside-only regimen of TDF, ABC, and 3TC.

BackgroundInhibition of the transporter-mediated hepatobiliary elimination of bile salts is a putative mechanism for liver toxicity observed with some endothelin receptor antagonists (ERAs).MethodsSandwich-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.ResultsThe 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).ConclusionsSignificant 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.

A novel analytical procedure has been developed for the analysis of intracellular nucleotide triphosphates. Positive ion electrospray ionization tandem mass spectrometry (MS/MS) was interfaced to ion‐pairing high‐performance liquid chromatography (HPLC) utilizing a mobile phase containing 10 mM ammonium phosphate, pH 6.4, with 2 mM tetrabutylammonium hydroxide and 15% acetonitrile. The methodology was developed to support the analysis of the 5′‐triphosphate anabolite of the antiviral agent (−)‐FTC ((2R,5S)‐5‐fluoro‐1‐[2‐(hydroxymethyl‐1,3‐oxathiolan‐5‐yl]cytosine) in human peripheral blood mononuclear cells (PBMCs). In this procedure, all nucleotides were extracted from PBMCs with aqueous methanol, isolated with high recovery using a novel ion‐pairing solid phase extraction procedure, and then analyzed directly with LC/MS/MS with a 10‐min analysis time. A calibration curve was generated representing (−)‐FTC 5′‐triphosphate ((−)‐FTCTP) concentration over the range of 0.083 to 83 picomol/106 cells (approximately 0.08 to 80 picomoles on‐column). Linear regression analysis with 1/x2 weighting yielded a coefficient of determination (r2) of greater than 0.999. The back‐calculated concentrations of all calibration standards had relative errors within the range of +5 to −3%. A preliminary assessment of intra‐assay precision and accuracy, analyte stability, and LC/MS system stability indicated a robust method capable of being validated with a limit of quantitation estimated conservatively at 0.08 picomol/106 cells (approximately 0.08 picomoles on‐column; signal‐to‐noise (S/N) = 5). The general method developed here should be adaptable to all purine‐ and pyrimidine‐based nucleotide applications. This report provides a detailed discussion on the key HPLC, MS, and sample preparation procedures that hold the potential for even greater nucleotide sensitivity. Copyright © 2000 John Wiley & Sons, Ltd.

ABSTRACT: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.

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 β-tauromuricholic acid; α/β 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 ± 5.85 μM in CTL rat and 183 ± 55.6 μM in CTL human SCH, while medium concentrations of the total BAs measured were 1.16 ± 0.210 μM in CTL rat SCH and 9.61 ± 6.36 μM in CTL human SCH. Treatment of cells for 24 h 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.