Reliable Rate Measurements for Active and Passive Hepatic Uptake Using Plated Human Hepatocytes

Abstract

Transporter-mediated hepatic uptake is proven to be the rate-determining step in the systemic clearance of several drugs. Therefore, accurate measurement of active and passive uptake clearances in vitro is critical to facilitate pharmacokinetics and drug-drug interaction predictions. Here, we evaluated the plated human hepatocytes (PHH) and studied the effect of incubation temperature and inhibitor concentration on uptake measurements, in order to reliably estimate hepatic uptake components. Uptake rates measured using PHH, at 37°C without and with rifamycin SV, were comparable with those obtained from suspension hepatocytes and sandwich-cultured hepatocytes for a set of 10-13 compounds. Apparent permeability across monolayers of low-efflux Madin-Darby canine kidney cells was measured at 4, 10, and 37°C. Of the 23 compounds evaluated, 13 compounds showed >2-fold reduction in passive permeability at 4°C compared to 37°C, inferring that low-temperature incubations may underestimate passive uptake. Inhibition studies using transporter-transfected cells suggested that ∼20 μM rifamycin SV completely inhibited organic anion-transporting polypeptides (OATPs), while no significant inhibition was noted for other hepatic uptake transporters. On the basis of inhibition profiles, the contribution of active versus passive and OATP versus non-OATP transport to the PHH uptake was discerned for various endogenous substrates and statins. With the exception of fluvastatin, the statins studied were predominantly transported by OATPs in PHH and the non-OATP transporters, such as Na-taurocholate co-transporting polypeptide, played a minimal role. In conclusion, PHH is useful for uptake measurements, and rifamycin SV employed at different concentrations can reliably estimate active and passive uptake and characterize OATP-dependent active uptake.

A Study on Pharmacokinetics of Bosentan with Systems Modeling, Part 2: Prospectively Predicting Systemic and Liver Exposure in Healthy Subjects

“…All active rates, CL HEP,pass , K M,HEP,uptake , and K M,HEP,efflux are estimated by fitting observed experimental data. In the presence of 1 mM inhibitor RSV, all active processes are fixed at zero based on a previous study (Bi et al, 2017). Although RSV is usually used as a transporter inhibitor, in-house data show that at 1 mM, P450-mediated metabolism is inhibited as well.…”

In Vitro–In Vivo Extrapolation of OATP1B-Mediated Drug–Drug Interactions in Cynomolgus Monkey

“…In comparison with the increased success of quantitative prediction of CYP-mediated DDIs, the confidence in the successful prediction of OATP-mediated DDIs using in vitro data is still low (Gertz et al, 2013;Prueksaritanont et al, 2014;Jones et al, 2015;Bi et al, 2017;Yoshida et al, 2017). Therefore, the objective of our study was to evaluate the IVIVE of OATP inhibition potential using cynomolgus monkey as a preclinical model and to gain confidence in mechanistic translational approach to predict clinical DDIs mediated by these transporters.…”

Organic Anion Transporter 2–Mediated Hepatic Uptake Contributes to the Clearance of High-Permeability–Low-Molecular-Weight Acid and Zwitterion Drugs: Evaluation Using 25 Drugs

“…Methods adopted for uptake studies are similar to those previously reported by our group (Bi et al, 2017(Bi et al, , 2018aMathialagan et al, 2017). Stock solutions of all compounds were made in DMSO.…”

“…At the end of incubation (2 minutes), the cellular uptake was terminated by washing the cells four times with ice-cold transport buffer, and then the cells were lysed with 0.2 ml of 1% NP-40 in water (radiolabeled compounds) or methanol containing internal standard (nonlabeled compounds). Transporter functionality was validated using in vitro probe substrates: 0.5 mM [ 3 H]-cGMP (OAT2) or 0.5 mM rosuvastatin (OATP1B1) as described previously (Bi et al, 2017;Mathialagan et al, 2018). Intracellular accumulation was determined either by mixing the cell lysate with scintillation fluid followed by liquid scintillation analysis (PerkinElmer Life Sciences) for radiolabeled compounds or by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis for nonlabeled compounds.…”

“…Determination of Uptake Clearance Using Short-Term Cultured Plated Human Hepatocytes. The hepatic uptake assay was performed using short-term cultured, cryopreserved human hepatocytes as described previously (Bi et al, 2017(Bi et al, , 2018a. Briefly, plateable cryopreserved human hepatocytes (Hu8246 lot; Thermo Fisher Scientific) were thawed in a water bath at 37°C and placed on ice.…”