Substrate-Dependent Inhibition of Organic Anion Transporting Polypeptide 1B1: Comparative Analysis with Prototypical Probe Substrates Estradiol-17β-Glucuronide, Estrone-3-Sulfate, and Sulfobromophthalein

Abstract

Organic anion transporting polypeptide (OATP) 1B1 plays an important role in the hepatic uptake of many drugs, and the evaluation of OATP1B1-mediated drug-drug interactions (DDIs) is emphasized in the latest DDI (draft) guidance documents from U.S. and E.U. regulatory agencies. It has been suggested that some OATP1B1 inhibitors show a discrepancy in their inhibitory potential, depending on the substrates used in the cell-based assay. In this study, inhibitory effects of 14 compounds on the OATP1B1-mediated uptake of the prototypical substrates [ values, respectively. Also, the clinically relevant OATP inhibitors rifampin and cyclosporin A exhibited up to 12-and 6-fold variation in their IC 50 values, respectively. Regardless of the inhibitors tested, the most potent OATP1B1 inhibition was observed when [ 3 H]E 2 G was used as a substrate. Mutual inhibition studies of OATP1B1 indicated that E 2 G and E 1 S competitively inhibited each other, whereas BSP noncompetitively inhibited E 2 G uptake. In addition, BSP inhibited E 1 S in a competitive manner, but E 1 S caused an atypical kinetics on BSP uptake. This study showed substratedependent inhibition of OATP1B1 and demonstrated that E 2 G was the most sensitive in vitro OATP1B1 probe substrate among three substrates tested. This will give us an insight into the assessment of clinically relevant OATP1B1-mediated DDI in vitro with minimum potential of false-negative prediction.

Organic Anion Transporting Polypeptide (OATP)1B1 and OATP1B3 as Important Regulators of the Pharmacokinetics of Substrate Drugs

“…Izumi et al have also recently showed the inhibitory effects of 14 compounds on the OATP1B1-mediated uptake of three kinds of prototypical substrates, E 2 17βG, E-sul and bromosulfophthalein (BSP). 38) As a result, although the K i value of the same inhibitor was sometimes estimated to be very different depending on the substrates tested, regardless of the inhibitors tested, the most potent OATP1B1 inhibition was observed when E 2 17βG was used as a substrate. These results indicated that it is recommended to use the real combination of substrate and inhibitor when predicting the risk of specific DDI cases, while E 2 17βG may be used for a routine high-throughput inhibition assay to perform more conservative DDI prediction.…”

Investigation of the Impact of Substrate Selection on In Vitro Organic Anion Transporting Polypeptide 1B1 Inhibition Profiles for the Prediction of Drug-Drug Interactions

“…The stably OATP1B1-expressing human embryonic kidney 293 (HEK293) cells and the corresponding control HEK293 cells, as established previously (Izumi et al, 2013), were used in this study. The cells were maintained in Dulbecco's modified Eagle medium (Invitrogen, Carlsbad, CA) supplemented with 10% (v/v) of fetal bovine serum, penicillin (final concentration, 100 units/ml), streptomycin (100 mg/ml), and hygromycin B (80 mg/ml) in a humidified incubator containing 5% CO 2 gas at 37°C.…”

“…One ml of the cell suspension was added to each well of a poly-D-lysine-coated 24-well plate (BD Biosciences, San Jose, CA), and the cells were further cultured in the incubator for 48 hours. The uptake and inhibition studies were performed as described previously elsewhere (Izumi et al, 2013). Briefly, cell culture medium was replaced with prewarmed Krebs Henseleit (KH) buffer (118 mM NaCl, 23.1 mM NaHCO 3 , 4.83 mM KCl, 0.96 mM KH 2 PO 4 , 1.20 mM MgSO 4 , 12.5 mM HEPES, 5.0 mM glucose, and 1.53 mM CaCl 2 , pH 7.4), and the cells were preincubated for 5 minutes at 37°C.…”

“…However, it was reported that the in vitro inhibitory potencies of gemfibrozil and other inhibitors on OATP1B1 depend on the probe substrates (Noé et al, 2007;Izumi et al, 2013). Therefore, care is needed in probe substrate selection for in vitro OATP1B1 inhibition assays to avoid false-negative DDI predictions.…”

“…We previously examined the in vitro inhibitory effects of 14 compounds on OATP1B1 using three prototypical probe substrates: estradiol-17b-glucuronide (E 2 G), estrone-3-sulfate (E 1 S), and sulfobromophthalein (BSP). Among them, E 2 G provided the lowest K i value for all inhibitors examined; thus, we proposed that the use of E 2 G as an in vitro probe substrate may help mitigate the risk of false-negative DDI prediction (Izumi et al, 2013).…”