Quantification of biliary excretion and sinusoidal excretion of 5(6)-carboxy-2′,7′-dichlorofluorescein (CDF) in cultured hepatocytes isolated from Sprague Dawley, Wistar and Mrp2-deficient Wistar (TR⁻) rats

Highlights

• A method to measure sinusoidal and canalicular efflux of CDF was presented.

• Biliary efflux of CDF in Sprague Dawley and Wistar rats shows bi-phasic kinetics.

• The high affinity component was attributed to Mrp2 efflux.

• The low affinity to multiple binding sites on Mrp2.

• Transporters on the sinusoidal membrane also played a role in CDF efflux.

Abstract

Hepatic efflux of drug candidates is an important issue in pre-clinical drug development. Here we utilise a method which quantifies and distinguishes efflux of drugs at the canalicular and sinusoidal membranes in rat hepatocyte cultures. Bi-phasic kinetics of transport of 5(6)-carboxydichlorofluorescein (CDF) at the canalicular membrane was demonstrated in Sprague Dawley (SD) and Wistar (W) rat hepatocytes. The high affinity component (Km = 3.2 ± 0.8 μM (SD), 9.0 ± 3.1 μM (W)) was attributed to Mrp2-mediated transport, the low affinity component (Km = 192.1 ± 291.5 μM (SD), 69.2 ± 36.2 μM (W)) may be attributed to transport involving a separate Mrp2 binding site. Data from membranes (Hill coefficient (h) = 2.0 ± 0.5) and vesicles (h = 1.6 ± 0.2) expressing Mrp2 and from SD (h = 1.6 ± 0.4) and Wistar (h = 4.0 ± 0.6) hepatocytes suggests transport involves more than one binding site. In TR⁻ hepatocytes, CDF efflux was predominantly over the sinusoidal membrane (Km = 100.7 ± 36.0 μM), consistent with low abcc2 (Mrp2) expression and compensatory increase in abcc3 (Mrp3) expression. This report shows the potential of using this in vitro method to model changes in biliary excretion due to alterations in transporter expression.

Introduction

Understanding the role of drug transport proteins in the absorption, distribution and elimination of drugs is an important component in our awareness of the safety and efficacy of drugs. Whilst the study of drug transport proteins in vivo are prohibitively complex, in vitro techniques, by contrast, permit detailed mechanistic investigations to elucidate the role of transport proteins in the uptake and efflux of drugs across membranes, and the likelihood of drug–drug interactions arising through concomitantly administered drugs competing through common transport processes. In vitro studies commonly in use for the study of drug transport activities typically include the use of singularly expressed transport proteins and isolated hepatocytes either in suspension or in culture (Ho et al., 2006, Jigorel et al., 2005, Kim et al., 1998, Roelofsen et al., 1994). The use of membrane and vesicle preparations allows the study of drug transport across membranes by single transport proteins, but not the complex interplay between transport proteins involved in the uptake and efflux of drugs across cellular membranes. In contrast to membrane and vesicle studies, the use of isolated hepatocytes in culture is the preferred method for the study of drug transport as they form distinct bile canalicular structures and express a range of drug-metabolising enzymes and transport proteins permitting detailed mechanistic investigations linking these two processes in ADME studies (Johnson et al., 2006, LeCluyse et al., 1994a, LeCluyse et al., 2000).

A method for the quantification of Mrp2-mediated efflux in rat hepatocyte cultures has been described previously by Liu et al., 1999b, Liu et al., 1999c. The fluorescent Mrp2 substrate, 5(6)-carboxy-2′,7′-dichlorofluorescein (CDF) is used to visualise bile canalicular integrity. CDF is added to the cells in culture as the 5(6)-carboxy-2′,7′-dichlorofluorescein diacetate (CDF-DA) pro-substrate, and is hydrolysed by intracellular esterases to the fluorescent product, CDF, which is eliminated by the efflux transport protein Mrp2 into the canalicular space. In the absence of calcium ions, the bile canaliculae are no longer intact and CDF diffuses from the canniculi space into the incubation media (Liu et al., 1999b). The Liu et al., 1999a, Liu et al., 1999b, Liu et al., 1999c method uses the measurement of CDF to determine biliary excretion by quantifying the difference between accumulation of CDF in cells and bile in the presence of calcium ions, and accumulation in cells alone in the absence of calcium ions. Determination of the biliary excretion index (BEI) and biliary clearance (CL_bile) using this method allows extrapolation to the in vivo situation (Annaert et al., 2001, Liu et al., 1999a). However, the method described by Liu et al., 1999a, Liu et al., 1999b, Liu et al., 1999c has a limitation. The method only quantifies biliary efflux and takes no account of efflux across the sinusoidal membrane. This limitation has been overcome by modifying the method of Liu et al., in order to measure efflux directly (Ellis et al., 2008, Jemnitz et al., 2010). In this study, our aim was to use the modified method to distinguish and quantify canalicular (biliary) efflux and sinusoidal (urinary) efflux of the model Mrp2 substrate, CDF in sandwich cultured hepatocytes.