Pharmacokinetics, Pharmacodynamics and Drug Metabolism
Sodium fluorescein is a probe substrate for hepatic drug transport mediated by OATP1B1 and OATP1B3

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Abstract

The aim of this study was to characterize the in vitro hepatic uptake kinetics of sodium fluorescein (NaFluo) and identify the transporters involved. NaFluo exhibited saturable uptake kinetics in suspended rat and human hepatocytes as reflected by Km values of 22.5 and 14.1 µM, and Vmax values of 98.3 and 5.8 pmol/(million cells • min), respectively. Coincubation with known inhibitors (e.g. rifampicin) of organic anion transporting polypeptide (OATP/Oatp; SLCO gene family) significantly decreased NaFluo uptake in hepatocytes. In contrast, neither inhibitors/substrates of the organic cation transporter or organic anion transporter family nor depletion of extracellular sodium resulted in significant inhibition of NaFluo uptake. To explore the contribution of individual uptake transporters, NaFluo uptake was determined in Chinese hamster ovary cells transfected with OATP1B1, OATP1B3, and OATP2B1. Transporter‐mediated uptake of NaFluo was observed in OATP1B1‐ and OATP1B3‐transfected cells (Km = 4.2 and 10.9 µM; Vmax = 30.9 and 135 [pmol/(mg protein • min)], respectively). NaFluo can be used as a probe substrate to study Oatp/OATP1B‐mediated drug interactions in fluorescence‐based in vitro transport assays of rat and human liver. Labeling of drugs or bile salts with a fluorescein moiety can be expected to result in fluorescent conjugates with substantially altered hepatic uptake characteristics as compared with the unconjugated compounds. © 2011 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 100:5018–5030, 2011

Section snippets

INTRODUCTION

In addition to hepatic metabolism and biliary excretion, hepatic drug uptake is an important aspect of hepatobiliary drug disposition of endogenous and exogenous compounds. In the last decennia, several transporters influencing drug clearance have been identified.1 In the basolateral hepatocyte membrane, members of the organic anion transporting polypeptide family [OATP (human), Oatp (rat); SLCO/Slco gene family] support the sodium‐independent uptake of bile salts [e.g., taurocholate (TC)] in

Materials

Amprenavir (APV) was kindly provided by GlaxoSmithKline (Middlesex, UK). Ritonavir (RTV), indinavir (IDV) sulfate, saquinavir (SQV) mesylate, and nelfinavir mesylate were donated by Hetero Drugs Limited (Hyderabad, India). Atazanavir (ATV) was obtained from Bristol–Myers Squibb (New Brunswick, New Jersey) and darunavir (DRV) was obtained through the National Institutes of Health AIDS Reagent Program. CGamF was kindly provided by Professor Alan Hofmann (University of California, San Diego).

Determination of NaFluo Uptake Kinetics in Suspended Rat and Human Hepatocytes

Preliminary experiments on time dependency of NaFluo uptake in suspended rat hepatocytes demonstrated linearity during the first 90 s of the uptake process (data not shown). Initial uptake rates were concentration dependent and could be described by a model incorporating Michaelis–Menten type kinetics including the Hill factor n (Fig. 1a). Mean (± SD, n = 3 batches of hepatocytes) estimates for Km and Vmax were 22.5 ± 4.4 µM and 98.3 ± 8.7 pmol/(million cells • min) and Hill factor 1.7 ± 0.6.

DISCUSSION

The aim of this study was to characterize the uptake mechanism and kinetics of NaFluo in suspended rat and human hepatocytes and in CHO cells transfected with hNTCP/rNtcp or the human hepatic OATP isoforms (OATP1B1, OATP1B3, and OATP2B1).

Our results support saturable NaFluo uptake in both rat and human hepatocytes, indicative of the involvement of one or more uptake transporters. Kinetic analysis of the uptake data revealed that the Km value for NaFluo uptake is comparable between rat and human

Acknowledgements

Tom De Bruyn received a PhD scholarship from the Agency for Innovation by Science and Technology, Flanders. This study was supported by grants from the following independent funders: “Fonds voor Wetenschappelijk Onderzoek” Flanders and “Onderzoeksfonds” of the Katholieke Universiteit Leuven, Belgium.

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