Differential Modulation of Cytochrome P450 Activity and the Effect of 1-Aminobenzotriazole on Hepatic Transport in Sandwich-Cultured Human Hepatocytes

Abstract

ABSTRACT:Sandwich-cultured human hepatocytes (SCHH) have been widely used for in vitro assessments of biliary clearance. However, the modulation of metabolism enzymes has not been fully evaluated in this system. The present study was therefore undertaken to determine the activity of cytochrome P450 (P450) 1A2, 2C8, 2C9, 2C19, 2D6, and 3A and to evaluate the impact of 1-aminobenzotriazole (ABT) on hepatic uptake and biliary excretion in SCHH. The SCHH maintained integrity and viability as determined by lactate dehydrogenase release and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium assays conducted over the culture period. Although all assessed P450 activity decreased in day 2 SCHH, the extent of the decrease and the subsequent rebound in activity varied across the different isoforms. Day 5 CYP1A2 activity was approximately 2.5-fold higher than day 1 activity, whereas the CYP3A and CYP2C9 activities were 90 and 60% of the day 1 levels, respectively. In contrast, the initial CYP2C8, CYP2C19, and CYP2D6 activity losses did not rebound over the 5-day culture period. Furthermore, ABT was not found to have an effect, whether directly or indirectly as a P450 inactivator, with respect to the hepatic transport of rosuvastatin, atrovastatin, and midazolam in SCHH. Taken together, these results suggest that the SCHH model is a reliable tool to characterize hepatic uptake and biliary excretion. Due to the differential modulation of P450 activity, SCHH may not be considered a suitable tool for metabolic stability assessments with compounds predominantly cleared by certain P450 enzymes.

Interaction between HIV protease inhibitors (PIs) and hepatic transporters in sandwich cultured human hepatocytes: implication for PI‐based DDIs

“…To eliminate hepatocyte metabolism of the PIs, all SCHH were pre‐incubated with 5 mM ABT for 1 h before conducting the transport studies, and the lack of hepatocyte metabolism of the 3 H‐PIs via LC/UV coupled with radioactivity fractionation was confirmed. Previous studies conducted in human liver microsomes and hepatocytes showed that pre‐incubation with ABT (> 2 mM), a potent CYP inactivator, can effectively abolish the activities of multiple CYPs . In addition, ABT does not appear to be an inhibitor of the transporters expressed in the human hepatocytes, as Kimoto et al .…”

A Perspective on the Prediction of Drug Pharmacokinetics and Disposition in Drug Research and Development

“…Data obtained from specific chemical inhibitor studies in human liver microsomes (HLM) are also used to corroborate the estimates of f m obtained with rCYPs. The CL int estimate obtained in HLM is generally compared with CL int determined in human hepatocytes incubated with and without 1-aminobenzotriazole to determine if other metabolic pathways could contribute to reducing the estimates of f m CYP (Emoto et al, 2010;Kimoto et al, 2012a). It is important to recognize that the predicted clinical DDI (AUCR) is very sensitive when the estimates of f m by one particular metabolic route or enzyme exceed 0.8-0.9, and it is therefore important to account for all possible metabolic pathways (Fig.…”

Analysis of the Metabolic Pathway of Bosentan and of the Cytotoxicity of Bosentan Metabolites Based on a Quantitative Modeling of Metabolism and Transport in Sandwich-Cultured Human Hepatocytes

“…The hepatotoxic effect of Ro 47-8634 also tended to disappear in human hepatocytes pretreated with 1-ABT, although the recovery was not statistically significant. This may arise through the weak inactivation potential of 1-ABT for CYP2C9 compared with other P450 isoforms (Kimoto et al, 2012). On the other hand, ETM-mediated specific CYP3A inhibition did not affect the cytotoxicity induced by Ro 47-8634.…”