Major active components in grapefruit, orange, and apple juices responsible for OATP2B1-mediated drug interactions.

We aimed to explore the major active components in grapefruit juice (GFJ), orange juice (OJ), and apple juice (AJ) that are responsible for OATP2B1-mediated drug interactions, by means of in vitro studies using Xenopus oocytes expressing OATP2B1 with a typical OATP2B1 substrate, estrone-3-sulfate. All three juices inhibited OATP2B1-mediated estrone-3-sulfate uptake with half-maximum inhibition (IC50 ) values of 0.222% (GFJ), 0.807% (OJ), and 2.27% (AJ). Eight major flavonoids (naringin, naringenin, hesperidin, hesperetin, phloridzin, phloretin, quercetin, and kaempferol) contained in the juices inhibited OATP2B1-mediated estrone-3-sulfate uptake with IC50 values of 4.63, 49.2, 1.92, 67.6, 23.2, 1.31, 9.47, and 21.3 µM, respectively. When the concentration-IC50 ratios ([C]/IC50 ) of these flavonoids in GFJ, OJ, and AJ were calculated, values of [C]/IC50 ≥ 100 were obtained for naringin in GFJ and hesperidin in OJ. No flavonoid in AJ showed a ratio higher than unity. However, significant inhibition of OATP2B1 was observed with a mixture of phloridzin, phloretin, hesperidin, and quercetin at the concentrations present in AJ. In conclusion, our results indicate that naringin and hesperidin are the major OATP2B1 inhibitors in GFJ and OJ, respectively, whereas a combination of multiple components appears to be responsible for OATP2B1 inhibition by AJ.

A role of prostaglandin transporter in regulating PGE2 release from human bronchial epithelial BEAS-2B cells in response to LPS.

Naturally occurring prostaglandin E2 (PGE2) plays a role in inflammatory responses through eicosanoid signaling pathways. PGE2 is impermeable to cell membranes at physiological pH and needs solute carrier across the membranes; however, it remains unclear how intercellular concentrations of PGE2 are regulated under the condition of inflammation. We aimed to clarify a role of organic anion-transporting polypeptide 2A1 (OATP2A1/SLCO2A1), also known as prostaglandin transporter (PGT), in PGE2 release from cells. Human bronchial epithelial BEAS-2B cells were treated with lipopolysaccharide (LPS), and PGT inhibitors were tested to evaluate contribution of PGT to PGE2 release by assessing its extracellular concentration and characterizing PGT-mediated PGE2 efflux in Xenopus laevis oocytes. As a result, LPS elevated mRNA expression of a pro-inflammatory cytokine IL6 and extracellular concentration of PGE2 in human bronchial epithelial BEAS-2B cells. PGT inhibitors tested (e.g. bromocresol green (BCG), bromosulfophthalein (BSP), and PGB1) significantly inhibited efflux of PGE2 from oocytes expressing PGT. Similarly, the amount of released PGE2 from the BEAS-2B cells decreased in the presence of BCG and BSP by 45 and 44% respectively while TGBz increased the concentration by 71%, suggesting that PGT mediates the release. In conclusion, these results imply a role of PGT in regulating intra- and extracellular concentrations of PGE2 in response to cells under inflammatory conditions.

Major active components in grapefruit, orange, and apple juices responsible for OATP2B1-mediated drug interactions.

We aimed to explore the major active components in grapefruit juice (GFJ), orange juice (OJ), and apple juice (AJ) that are responsible for OATP2B1-mediated drug interactions, by means of in vitro studies using Xenopus oocytes expressing OATP2B1 with a typical OATP2B1 substrate, estrone-3-sulfate. All three juices inhibited OATP2B1-mediated estrone-3-sulfate uptake with half-maximum inhibition (IC(50) ) values of 0.222% (GFJ), 0.807% (OJ), and 2.27% (AJ). Eight major flavonoids (naringin, naringenin, hesperidin, hesperetin, phloridzin, phloretin, quercetin, and kaempferol) contained in the juices inhibited OATP2B1-mediated estrone-3-sulfate uptake with IC(50) values of 4.63, 49.2, 1.92, 67.6, 23.2, 1.31, 9.47, and 21.3 µM, respectively. When the concentration-IC(50) ratios ([C]/IC(50) ) of these flavonoids in GFJ, OJ, and AJ were calculated, values of [C]/IC(50) ≥ 100 were obtained for naringin in GFJ and hesperidin in OJ. No flavonoid in AJ showed a ratio higher than unity. However, significant inhibition of OATP2B1 was observed with a mixture of phloridzin, phloretin, hesperidin, and quercetin at the concentrations present in AJ. In conclusion, our results indicate that naringin and hesperidin are the major OATP2B1 inhibitors in GFJ and OJ, respectively, whereas a combination of multiple components appears to be responsible for OATP2B1 inhibition by AJ.

Long-lasting inhibitory effect of apple and orange juices, but not grapefruit juice, on OATP2B1-mediated drug absorption.

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 time-dependent 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.

Functional pleiotropy of organic anion transporting polypeptide OATP2B1 due to multiple binding sites.

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.

Intestinal absorption of HMG-CoA reductase inhibitor pitavastatin mediated by organic anion transporting polypeptide and P-glycoprotein/multidrug resistance 1.

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.