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

Plasma proteins or human serum albumin (HSA) have been reported to increase the in vitro intrinsic uptake clearance (CL_int,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 CL_int,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 (HEK293 transfected cells) and passive CL_int,uptake (MOCK HEK293 cells) of 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 vs. HBSS buffer. 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 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 CL from in vitro uptake studies.