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

Ellis, L.C.J. and Grant, M.H. and Hawksworth, G.M. and Weaver, R.J. (2014) Quantification of biliary excretion, sinusoidal excretion and diffusion of 5(6)-carboxy-2',-7'-dichlorofluorescein (CDF) in hepatocytes isolated from Sprague Dawley, Wistar and Mrp2 (TR-) deficient rats. Toxicology in Vitro, 28 (6). 1165–1175. ISSN 0887-2333

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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.