The purpose of this study was to examine whether the presence of multiple binding sites can explain the pleiotropy of substrate recognition by OATP2B1, using Xenopus oocytes expressing OATP2B1. OATP2B1-mediated uptake of estrone-3-sulfate apparently exhibited biphasic saturation kinetics, with Km values of 0.10 ± 0.05 and 29.9 ± 12.1 µM and Vmax values of 14.1 ± 6.4 and 995 ± 273 fmol/min/oocyte for high- and low-affinity sites, respectively. Contribution analysis revealed that transport of estrone-3-sulfate mediated by high- and low-affinity sites on OATP2B1 could be evaluated at the concentrations of 0.005 and 50 µM, respectively. pH-dependence study of OATP2B1-mediated estrone-3-sulfate uptake suggested that high- and low-affinity sites show different pH sensitivity. When the inhibitory effect of 12 compounds on estrone-3-sulfate uptake by high- and low-affinity sites on OATP2B1 was examined, 4 compounds appeared to be inhibitors of the high-affinity site on OATP2B1. Two other compounds appeared to be inhibitors for the low-affinity site and four others were inhibitory at both sites. These results indicated the presence of multiple binding sites on OATP2B1 with different affinity for drugs. Accordingly, it is likely that drug-drug and drug-beverage interactions occur only when two drugs share the same binding site on OATP2B1.
The purpose of this study was to investigate the involvement of organic anion transporting polypeptide (OATP/Oatp) and P-glycoprotein (P-gp)/multidrug resistance 1 (MDR1/Mdr1) in intestinal absorption of pitavastatin. Pitavastatin was found to be a substrate for human OATP1A2, OATP2B1, and MDR1 and rat Oatp1a5, Oatp2b1, and Mdr1a in experiments using transporter-expressing Xenopus oocytes and LLC-PK1 cell systems. Naringin inhibited Oatp1a5- and Mdr1a-mediated transport of pitavastatin with IC(50) values of 18.5 and 541 µM, respectively. The difference in the IC(50) values of naringin for Oatp1a5 and Mdr1a-mediated pitavastatin transport may account for the complex concentration-dependent effect of naringin on the intestinal absorption of pitavastatin. Rat intestinal permeability of pitavastatin measured by the in situ closed-loop perfusion method was indeed significantly reduced by 200 µM naringin, but was significantly increased by 1000 µM naringin. Furthermore, the permeability was significantly increased by elacridar, a potent inhibitor of Mdr1, while the permeability was significantly decreased in the presence of both elacridar and naringin, suggesting that Oatp1a5 and Mdr1a are both involved in intestinal absorption of pitavastatin. Our present results indicate that OATP/Oatp and MDR1/Mdr1 play roles in the intestinal absorption of pitavastatin as influx and efflux transporters, respectively.
Enzyme-based grapefruit juice (GFJ)-drug interactions are mainly due to mechanism-based irreversible inhibition of metabolizing enzyme CYP3A4 by GFJ components, but the transporter organic anion transporting polypeptide (OATP)2B1 is also a putative site of interaction between drugs and fruit juices (FJ) in the absorption process. Here we aimed to investigate the effect of preincubation with FJ on OATP2B1-mediated transport of drugs in vitro. When OATP2B1-expressing Xenopus oocytes were preincubated with GFJ, orange juice (OJ), or apple juice (AJ), AJ induced a remarkable decrease in OATP2B1-mediated estrone-3-sulfate uptake in a concentration-dependent manner (IC 50 = 1.5%). A similar but less potent effect was observed with OJ (IC 50 = 21%), whereas GFJ had no effect. Similar results were obtained in preincubation studies using fexofenadine. Preincubation with OJ and AJ resulted in timedependent inhibition of OATP2B1. Again, AJ had the more potent effect; its action lasted for at least 240 minutes, suggesting that AJ irreversibly inhibits OATP2B1-mediated drug uptake. Kinetic analysis revealed that coincubation and preincubation with AJ reduced OATP2B1-mediated estrone-3-sulfate uptake via competitive and noncompetitive mechanisms, respectively. Thus, OATP2B1 is functionally impaired through both competitive and long-lasting inhibition mechanisms by AJ and OJ, but not GFJ. Interestingly, although GFJ but not AJ is able to irreversibly inhibit CYP3A4, in the case of OATP2B1, AJ but not GFJ has a long-lasting inhibitory effect. Accordingly, complex FJ-drug interactions may occur in vivo, and their clinical significance should be examined.
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