Characterization of Organic Anion Transporting Polypeptide (OATP) Expression and Its Functional Contribution to the Uptake of Substrates in Human Hepatocytes

Abstract

Since the substrate specificities of OATP1B1, 1B3, and 2B1 are broad and overlapping, the contribution of each isoform to the overall hepatic uptake is of concern when assessing transporter-mediated drug–drug interactions (DDIs) or genetic polymorphism impact in the clinic. Herein, we quantitatively measured OATP proteins in cryopreserved hepatocytes, sandwich-cultured human hepatocytes (SCHH), and the liver, and examined the relationship with functional uptake of OATP substrates in an effort to identify the OATP isoform(s) contributing to the hepatic uptake of pitavastatin. The modulation of OATP expression in SCHH was found to be lot-dependent. However, OATP protein measurements averaged from 5 lots of SCHH were comparable to that of suspended hepatocytes. All three OATP transporters in suspended hepatocytes and SCHH were significantly lower than those in the liver. In SCHH, the uptake of CCK-8 and pravastatin was found to be associated with the expression of OATP1B3 and OATP1B1, respectively. In suspended hepatocytes, OATP1B1 appeared to show a positive trend with respect to the uptake of pitavastatin, which suggests a selective contribution of OATP1B1 to pitavastatin transport and thus an OATP quantitative protein expression–activity relationship. While the passive diffusion of rosuvastatin in SCHH was consistent across hepatocyte lots, the passive diffusion of pitavastatin varied over a broad range (>4-fold) in suspended hepatocytes and was inversely correlated with transporter-mediated uptake, presumably due to cell membrane alterations imparted by cryopreservation. Collectively, SCHH maintains OATP protein expression and membrane integrity and, if feasible when considering research goals, would be considered a superior tool for the characterization of in vitro transport parameters without the complication of membrane leakage.

Hepatic Uptake of Atorvastatin: Influence of Variability in Transporter Expression on Uptake Clearance and Drug-Drug Interactions

“…Our measured transporter abundances were comparable to previously reported hepatic expression data, albeit we found higher levels of OATP1B1 (Kimoto et al, 2012;Ohtsuki et al, 2012;Bi et al, 2013;Prasad et al, 2014). We speculate that this could be a result of the sample preparation used in our study (Wisniewski et al, 2009), including the membrane fractionation, solubilization, and tryptic digestion.…”

Prediction of the Pharmacokinetics of Pravastatin as an OATP Substrate Using Plateable Human Hepatocytes With Human Plasma Data and PBPK Modeling

“…Although there is no clear guidance on the uptake rate required, the initial screen and selection of the hepatocyte lot is critical for success. The expression level of OATP1B1 and OATP1B3 (18.3 and 1.7 fmol/million cells) in the hepatocyte lot used in the study is within the range of the literature reported level (3.4–23.2 for OATP1B1 and 1.5–6.5 for OATP1B3) 27, 28, 29, 30. Because pravastatin undergoes only minimal metabolism and the incubation time was short, metabolism in hepatocyte is likely not an issue in this study.…”

Interindividual Variability in Hepatic Organic Anion-Transporting Polypeptides and P-Glycoprotein (ABCB1) Protein Expression: Quantification by Liquid Chromatography Tandem Mass Spectroscopy and Influence of Genotype, Age, and Sex

“…However, the expression of MRP2 and BCRP was significantly different (1.54 6 0.64 vs. 0.56 6 0.21 and 0.14 6 0.04 vs. 0.70 6 0.22 fmol/mg membrane protein, respectively; P , 0.0001) (Deo et al, 2012;Prasad et al, 2013). Collectively, our transporter data are comparable or modestly lower than those reported previously in a much smaller sample size Bi et al, 2012;Karlgren et al, 2012;Kimoto et al, 2012;Ohtsuki et al, 2012;Tucker et al, 2012). Based on Western blotting, interindividual variability in OATP1B1, OATP1B3, OATP2B1, and P-gp expression have been reported to be 19, 85, 17 (n = 117), and 20-fold (n = 110), respectively (Meier et al, 2006;Nies et al, 2013).…”

“…We (Deo et al, 2012;Prasad et al, 2013) and others Bi et al, 2012;Kimoto et al, 2012;Ohtsuki et al, 2012;Schaefer et al, 2012;Tucker et al, 2012) have reported data on the expression of some of these hepatic transporters. Here we extended these studies to determine 1) the interindividual variability in expression of OATP1B1 (SLCO1B1), OATP1B3 (SLCO1B3), OATP2B1 (SLCO2B1), and P-gp (ABCB1) in a large set (n = 64) of human liver samples; and 2) the influence of genotype, age, and sex on such expression.…”