Culture Period-Dependent Changes in the Uptake of Transporter Substrates in Sandwich-Cultured Rat and Human Hepatocytes

Abstract

ABSTRACT:Sandwich-cultured hepatocytes (SCH) are a useful tool for evaluating hepatobiliary drug transport in vitro. Some studies have investigated the in vitro-in vivo correlations of the biliary clearance of drugs using SCH. In most cases, the biliary clearance observed in vivo correlated well with the predicted clearance, but the predicted absolute values were underestimated when based on in vitro experiments with SCH. We hypothesized that the down-regulated function of uptake transporters is one of the causes of this underestimation. Therefore, the uptake of taurocholate, digoxin, pravastatin, and rosuvastatin was investigated in sandwich-cultured rat hepatocytes (SCRH) cultured for 5, 24, 48, and 96 h, and the predicted hepatic clearance from in vitro uptake clearance (CL H, vitro ) was calculated with a dispersion model. In SCRH cultured for 96 h, the saturable uptake of taurocholate, digoxin, pravastatin, and rosuvastatin decreased to 7.5, 3.3, 64, and 23%, respectively, of their uptake in hepatocytes cultured for 5 h, and a better prediction of in vivo hepatic clearance (CL H, vivo ) was achieved when based on CL H, vitro of 5-h-cultured hepatocytes. These results suggest that the uptake activity is considerably reduced in cell culture, even in a sandwich-culture format. In a similar study, we also examined taurocholate and rosuvastatin in sandwich-cultured human hepatocytes (SCHH). Unlike in SCRH, the saturable uptake of these compounds did not differ markedly in SCHH cultured for 5 or 96 h. Thus, the uptake activity in SCHH was maintained relatively well compared with that in SCRH.

Hepatic Basolateral Efflux Contributes Significantly to Rosuvastatin Disposition I: Characterization of Basolateral Versus Biliary Clearance Using a Novel Protocol in Sandwich-Cultured Hepatocytes

“…Namely, OATP expression and function in rat SCH is decreased by 4-to 5-fold over days in culture (Kotani et al, 2011;Tchaparian et al, 2011). Therefore, RSV CL Uptake likely is underestimated in rat SCH (Table 1); in contrast, OATP-mediated CL Uptake in human SCH appears to be maintained over days in culture (Kotani et al, 2011). Bcrp and Mrp2 expression have been reported to increase and decrease, respectively, in rat SCH compared with liver tissue (Li et al, 2009a(Li et al, ,b, 2010.…”

“…Transporter expression and function have been reported to change over time in rat SCH. Namely, OATP expression and function in rat SCH is decreased by 4-to 5-fold over days in culture (Kotani et al, 2011;Tchaparian et al, 2011). Therefore, RSV CL Uptake likely is underestimated in rat SCH (Table 1); in contrast, OATP-mediated CL Uptake in human SCH appears to be maintained over days in culture (Kotani et al, 2011).…”

A Perspective on the Prediction of Drug Pharmacokinetics and Disposition in Drug Research and Development

“…SCH involves culturing primary hepatocytes between two layers of gelled matrix in a sandwich configuration, allowing the hepatocytes to form a bile canalicular network (Bi et al, 2006). While down-regulation of uptake transporters in sandwichcultured rat hepatocytes has been a concern for the reliability of parameters obtained sandwich-cultured human hepatocytes (SCHH) has proven to maintain the expression of both transporters and major CYP enzymes Kimoto et al, 2011;Kotani et al, 2011). This model can be used to estimate the initial hepatic uptake and biliary secretion .…”

Predicting when Biliary Excretion of Parent Drug is a Major Route of Elimination in Humans

“…These procedures are difficult, uncommon, and variable. In vitro predictions can be carried out in sandwichcultured rat or human hepatocytes, which preserve cell polarity and bile canaliculi (15,16), but uptake transporter expression is not well preserved in sandwich-cultured rat hepatocytes (17) and biliary clearance can be rate limited by uptake (18). Several studies have demonstrated that in vitro measures correlate with, but underpredict, in vivo biliary clearance (19)(20)(21).…”