Inhibition of Hepatic Organic Anion-Transporting Polypeptide by RNA Interference in Sandwich-Cultured Human Hepatocytes: An In Vitro Model to Assess Transporter-Mediated Drug-Drug Interactions

Abstract

ABSTRACT:Organic anion-transporting polypeptides (OATPs), members of the SLCO/SLC21 family, mediate the transport of various endo-and xenobiotics. In human liver, OATP1B1, 1B3, and 2B1 are located at the basolateral membrane of hepatocytes and are involved in hepatic drug uptake and biliary elimination. Clinically significant drug-drug interactions (DDIs) mediated by hepatic OATPs have drawn great attention from clinical practitioners and researchers. However, there are considerable challenges to prospectively understanding the extent of OATP-mediated DDIs because of the lack of specific OATP inhibitors or substrates and the limitations of in vitro tools. In the present study, a novel RNA interference knockdown sandwich-cultured human hepatocyte model was developed and validated. Quantitative polymerase chain reaction, microarray and immunoblotting analyses, along with uptake assays, illustrated that the expression and transport activity of hepatic OATPs were reduced by small interfering (siRNA) efficiently and specifically in this model. Although OATP siRNA decreased only 20 to 30% of the total uptake of cerivastatin into human hepatocytes, it caused a 50% reduction in cerivastatin metabolism, which was observed by monitoring the formation of the two major metabolites of cerivastatin. The results suggest that coadministration of a drug that is a hepatic OATP inhibitor could significantly alter the pharmacokinetic profile of cerivastatin in clinical studies. Further studies with this novel model demonstrated that OATP and cytochrome P450 have a synergistic effect on cerivastatin-gemfibrozil interactions. The siRNA knockdown sandwich-cultured human hepatocytes may provide a new powerful model for evaluating DDIs.

Differential Modulation of Cytochrome P450 Activity and the Effect of 1-Aminobenzotriazole on Hepatic Transport in Sandwich-Cultured Human Hepatocytes

“…In total, these results are consistent with those noted in a recent review, which indicates the hepatocyte culture model maintains cell integrity and viability (Swift et al, 2010) and suggests the increase in CYP1A2 activity is unlikely to be due to cellular stress generated during long culture periods. Although the mechanism underlying the increase in CYP1A2 activity was not defined in the present study, the findings conflict with a recent study that reports a substantial decrease in all P450 enzyme activity in 5-day SCHH (Liao et al, 2010). Because CYP1A2 is a highly inducible protein, we speculate that this discrepancy can potentially be attributed to the culture conditions, and further investigation is needed to elucidate the mechanism of CYP1A2 modulation in SCHH.…”

OATP1B1-related drug–drug and drug–gene interactions as potential risk factors for cerivastatin-induced rhabdomyolysis

“…The importance of the OATP1B1 transporter in cerivastatin metabolism is highlighted in a recent publication showing that OATP1B1 inhibition by siRNA led to a 20–30% reduction in total uptake of cerivastatin into human hepatocytes, 50% reduction in formation of M-1 cerivastatin metabolite, and no change in M-23 formation [32]. Transporter– enzyme interplay is now well recognized as an important factor influencing drug metabolism [33,34].…”

Critical Human Hepatocyte-Based In Vitro Assays for the Evaluation of Adverse Drug Effects

“…It is to be noted that recent research findings have demonstrated that in addition to drug metabolizing enzyme activities, uptake and efflux transporters also play critical roles in the manifestation of adverse drug effects 10,45 . Human hepatocyte assays for the evaluation of uptake and efflux transporters have been established and are being applied towards drug development [46][47][48][49][50] . These transporter assays, when applied in conjunction with the assays described in this chapter, should aid the selection of the most appropriate drug candidates for further drug development.…”