Is the Protein-Mediated Uptake of Drugs by Organic Anion Transporting Polypeptides a Real Phenomenon or an Artifact?

Abstract

Plasma proteins or human serum albumin (HSA) have been reported to increase the in vitro intrinsic uptake clearance (CLint,uptake) of drugs by hepatocytes or organic anion transporting polypeptide (OATP)-transfected cell lines. This so-called protein-mediated uptake effect (PMUE) is thought to be due to an interaction between the drug-protein complex and the cell membrane causing an increase in the unbound drug concentration at the cell surface, resulting in an increase in the apparent CLint,uptake of the drug. To determine if the PMUE on OATP-mediated drug uptake is an artifact or a real phenomenon, we determined the effect of 1%, 2%, and 5% HSA on OATP1B1-mediated [human embryonic kidney (HEK)293 transfected cells] and passive CLint,uptake (mock HEK293 cells) on a cocktail of five statins. In addition, we determined the non-specific binding (NSB) of the statin-HSA complex to the cells/labware. The increase in uptake of atorvastatin, fluvastatin, and rosuvastatin in the presence of HSA was completely explained by the extent of NSB of the statin-HSA complex, indicating that the PMUE for these statins is an artifact. In contrast, this was not the case for OATP1B1-mediated uptake of pitavastatin and passive uptake of cerivastatin, suggesting that the PMUE is a real phenomenon for these drugs. Additionally, the PMUE on OATP1B1-mediated uptake of pitavastatin was confirmed by a decrease in its unbound IC50 in the presence of 5% HSA versus Hank's balanced salt solution buffer (HBSS). These data question the utility of routinely including plasma proteins or HSA in uptake experiments and the previous findings on PMUE on OATP-mediated drug uptake. SIGNIFICANCE STATEMENT: Here we report, for the first time, that the protein-mediated uptake effect (PMUE) on organic anion transporting polypeptide (OATP)-transported drugs could be an artifact of the non-specific binding (NSB) of the drug-albumin complex to cells/labware. Future experiments on PMUE must take into consideration such NSB. In addition, mechanisms other than PMUE need to be explored to explain the underprediction of in vivo OATP-mediated hepatic drug clearance from in vitro uptake studies.