Cell-to-Medium Concentration Ratio Overshoot in the Uptake of Statins by Human Hepatocytes in Suspension, but Not in Monolayer: Kinetic Analysis Suggesting a Partial Loss of Functional OATP1Bs

Wooin Lee,Aya Kiriake,Nora Lee,Naoki Ishiguro,Hiroyuki Kusuhara,Renato J Scialis,Xiaoyan Chu,Larry Tremaine,Yuichi Sugiyama,Hong Shen,Ralf Lotz,Ryota Kikuchi,Kazuya Maeda,Kiyoe Morita,Satoshi Koyama,Emi Kimoto

doi:10.1208/s12248-020-00512-6

Abstract

Suspended human hepatocytes (SHH) have long been used in assessing hepatic drug uptake, while plated human hepatocytes in short-term monolayer culture (PHH) have gained use in recent years. This study aimed to cross-evaluate SHH and PHH in measuring the hepatic uptake mediated by organic anion transporting polypeptide 1Bs (OATP1Bs). We compared the time courses of cell-to-medium (C/M) concentration ratios and initial uptake clearance values of the OATP1B substrates (pitavastatin, rosuvastatin, cerivastatin, pravastatin, dehydropravastatin, and SC-62807) between SHH and PHH. For all compounds except cerivastatin, the C/M ratios in SHH displayed an apparent overshoot (an initial increase followed by a decrease) during the 180-min uptake experiment, but not in PHH. Based on the literature evidence suggesting the possible internalization of OATP1Bs in primary hepatocytes, separate experiments measured the drug uptake after varying lengths of pre-incubation in the drug-free medium. The initial uptake clearances of pitavastatin and rosuvastatin declined in SHH beyond an apparent threshold time of 20-min drug-free pre-incubation, but not in PHH. Kinetic modeling quantitatively captured the decline in the active uptake clearance in SHH, and more than half of the active uptake clearances of pitavastatin and rosuvastatin were prone to loss during the 180-min uptake experiment. These results suggested a partial, time-delayed loss of the functional OATP1Bs in SHH upon prolonged incubation. Our results indicate that PHH is more appropriate for experiments where a prolonged incubation is required, such as estimation of unbound hepatocyte-to-medium concentration ratio (Kp,uu) at the steady-state.

Highlights

The liver is a major organ responsible for the elimination of many drugs
Our results indicate that Plated human hepatocytes (PHH) is more appropriate for experiments where a prolonged incubation is required, such as estimation of unbound hepatocyte-tomedium concentration ratio (Kp,uu) at the steady-state
The time courses for the drug uptake in Suspended human hepatocytes (SHH) are shown in Figs. 2 and 3 and Figs

Summary

Introduction

The liver is a major organ responsible for the elimination of many drugs. The accurate assessment of hepatic clearance is essential in predicting the pharmacokinetic profiles of drug candidates in humans. In the case of anionic molecules, entry to hepatocytes often relies on uptake transporters, which can be the rate-determining step in the overall hepatic drug elimination [1]. It has become a standard practice to assess anionic drug candidates for their uptake clearance at the physiological pH, and to apply the in vitro uptake clearance to the extended clearance concept for prediction of the overall hepatic clearance in humans [1]. In evaluating hepatic uptake clearance, the available in vitro systems include human primary hepatocytes in suspension (SHH) or short-term monolayer culture (PHH) and more complex three-dimensional cultures (e.g., hepatocyte spheroids, liver-on-a-chip) [4,5,6]. Human primary hepatocytes in the SHH or PHH format continue to be the standard and practical in vitro systems in quantifying the hepatic uptake of drugs

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