P-glycoprotein-mediated in vitro biliary excretion in sandwich-cultured rat hepatocytes

Recently, sandwich-cultured (SC) rat hepatocytes have been used as an in vi tro model to assess biliary excretion of drugs and xenobiotics. The purpose of the present study was to validate the use of SC rat hepatocytes for the in vitro assessment of P-glycoprotein (P-gp)-mediated biliary drug excreti on. The specific and fluorescent P-gp substrate rhodamine 123 (Rh123) and t he P-gp substrate digoxin were selected as model compounds. Rh123 and digox in accumulation and Rh123 efflux under standard and Ca2+-free conditions we re quantified in SC rat hepatocytes to determine substrate secretion into c analicular networks in vitro. The major role of P-gp in the biliary excreti on of these compounds was confirmed by inhibition experiments with the pote nt P-gp inhibitor GF120918. Hepatocyte culture conditions, including media type and time in culture, significantly affected Rh123 biliary excretion. P -gp expression, as assessed by Western blot, was increased with culture tim e. Dexamethasone (an in vivo inducer of P-gp) concentrations ranging from 0 .01 to 1 muM in the cell culture medium did not influence P-gp expression o r Rh123 biliary excretion. Rh123 and digoxin biliary clearance values, pred icted from SC rat hepatocyte data, were consistent with values reported in vivo and in isolated perfused rat liver studies. In conclusion, the results of this study demonstrate the utility of SC rat hepatocytes as an in vitro model to study and predict the biliary excretion of P-gp substrates.