Quantitative Expression Profile of Hepatobiliary Transporters in Sandwich Cultured Rat and Human Hepatocytes

Abstract

As sandwich cultured (SC) hepatocytes can repolarize to form bile canalicular networks, allowing active excretion of compounds in a vectorial manner, the model has been widely used for assessing the transporter related complexity of ADME/tox issues. A lack of quantitative information on transporter expression during cell culture has made in vitro to in vivo extrapolation of hepatobiliary transport difficult. In the present study, using our newly developed LC-MS/MS absolute quantitative methods, we determined the quantitative expression profile of three biliary transporters in SC rat and human hepatocytes. A significant shift of hepatobiliary transporter proteins was observed both in human and rat sandwich cultures. A decrease of BSEP/Bsep protein and an increase of BCRP/Bcrp protein were detected in both rat and human hepatocytes over time in culture. Interestingly, Mrp2 in rat hepatocytes was significantly diminished, while MRP2 constantly increased in human hepatocytes during the cell culture. Consequently, the interspecies difference between rat and human in absolute amount of MRP2/Mrp2 was minimized over time in culture. Following the sandwich culture, the species difference of hepatobiliary transporter protein between human and rat at day 5 post SC was diminished (MRP2/Mrp2), identical (BSEP/Bsep) or reversed (BCRP/Bcrp), compared to the in vivo situation. In addition, the absolute protein amount of BCRP/Bcrp or MRP2/Mrp2 was proportionally correlated with the intrinsic biliary clearance estimated in various lots of SC rat and human hepatocytes. The results revealed that absolute protein amount is a key determinant for hepatobiliary clearance and could provide fundamental support on extrapolation of biliary secretion from in vitro to in vivo.

Quantitative Targeted Proteomics for Membrane Transporter Proteins: Method and Application

“…In SIL methods such as the absolute quantification (AQUA) method (30), the IS is commonly spiked during or postdigestion and is the most feasible and rapid approach. Indeed, SIL peptides are among the most common IS used to evaluate the expression of drug transporters (5)(6)(7)18,19,(46)(47)(48)(49)(50)(51). A comparison of the SIL peptide with SILAM approach was recently carried out in the LC-MS/ MS method development in our laboratory (37).…”

“…Furthermore, the modulation of liver specific transporter expressions could also be species specific (64-66), which could limit the predictive power of a model. With the targeted quantitative proteomics methods aforementioned, Li et al characterized MRP2, bile salt export pump (BSEP), and breast cancer resistance protein (BCRP) expressions in sandwich cultured hepatocytes (SCH) to compare proteins across the cell culture periods and with in vivo expression (6,46,47). They observed a 40% loss of rat Mrp2 protein, but not human MRP2 protein, in cryopreserved hepatocytes as compared to that in the liver.…”

“…Interestingly, a decrease in BSEP/Bsep and an increase in BCRP/Bcrp were observed in human and rat sandwich cultures, while in the same culture Mrp2 was decreased in rat and MRP2 in human hepatocyte was increased. As a result, following the sandwich culture, species difference between human and rat was diminished for MRP2/ Mrp2 and reversed for BCRP/Bcrp (46). Through integrating a scaling factor of hepatobiliary transporter levels between in vitro SCH and in vivo, the prediction of biliary secretion in rats was improved (5).…”

“…Understanding the difference of membrane protein expression is the key to bridge the gap between in vitro models and interspecies that underlie interexperimental variation and to extrapolate in vitro data to in vivo (5,6,18,46,47,75). Appreciation of interspecies differences of proteins involved in drug elimination and their expression levels can increase the confidence in data interpretation.…”

Characterization of Digoxin Uptake in Sandwich-Cultured Human Hepatocytes

“…Sandwich-cultured hepatocytes form intact bile canalicular networks, and they maintain functional expression levels of uptake and efflux transporter proteins for several days (Hoffmaster et al, 2004;Li et al, 2009). We previously reported the ability to use cryopreserved human hepatocytes in sandwich culture (Bi et al, 2006) and have shown that uptake and efflux transporter activities are similar to those in fresh hepatocytes.…”