Multiple transport systems mediate the hepatic uptake and biliary excretion of the metabolically stable opioid peptide [D-penicillamine2,5]enkephalin.

Rapid and extensive biliary excretion of [D-penicillamine2,5]enkephalin (DPDPE) in rats as the unchanged peptide suggests that multiple transport proteins may be involved in the hepatobiliary disposition of this zwitterionic peptide. Although DPDPE is a P-glycoprotein substrate, the role of other transport proteins in the hepatic clearance of DPDPE has not been established. Furthermore, the ability of various experimental approaches to quantitate the contribution of a specific hepatic uptake or excretion process when multiple transport systems are involved has not been addressed. 3H-DPDPE uptake in suspended Wistar rat hepatocytes was primarily (>95%) due to temperature-dependent transport mechanisms; similar results were obtained in suspended hepatocytes from Mrp2-deficient (TR-) rats. Pharmacokinetic modeling revealed that saturable and linear processes were involved in 3H-DPDPE uptake in hepatocytes. The use of transport modulators suggested that hepatic uptake of 3H-DPDPE was mediated by Oatp1a1, Oatp1a4, and likely Oatp1b2. Accumulation of 3H-DPDPE in sandwich-cultured (SC) hepatocytes was rapid; uptake of 3H-DPDPE in SC rat hepatocytes from control and TR- rats was similar. However, the biliary excretion index and biliary clearance decreased by 83 and 85%, respectively, in TR- SC rat hepatocytes, indicating that DPDPE is an Mrp2 substrate. Rate constants for uptake and excretion of 3H-DPDPE in SC rat hepatocytes were determined by pharmacokinetic modeling; data were consistent with basolateral excretion of 3H-DPDPE from the hepatocyte. These results demonstrate the complexities of hepatobiliary disposition when multiple transport mechanisms are involved for a given substrate and emphasize the necessity of multi-experimental approaches for the comprehensive resolution of these processes.