Inhibition of Organic Anion Transporting Polypeptides (OATP1B1 and 1B3) by Betulinic Acid: Effects of Pre-incubation and Albumin in the Media

Abstract

The transporters of the organic anion transporting polypeptide 1B subfamily (OATP1B1 or OATP1B3, OATP1B1/3) can influence the hepatic uptake and elimination of clinically important drugs such as statins. While it was reported that common plant-derived pentacyclic triterpenoids interact with OATP1B1/3, detailed investigations have been lacking for betulinic acid (BA) including its potential risk for drug-herb interactions. In our current study, we investigated the interactions of BA and its closely related pentacyclic triterpenoids (ursolic acid, UA

oleanolic acid, OA

close similarities in their structures and high-affinity plasma protein binding) with OATP1B1/3 and rat Oatp1b2 using in vitro and in vivo models. Different experimental conditions were employed including co-incubation and pre-incubation (incubation with inhibitors, followed by washout). BA, UA and OA effectively inhibited the uptake of fluorescent probes and atorvastatin by OATP1B1/3 or rat Oatp1b2 in vitro. In order to examine the in vivo relevance of OATP1B1/3 inhibition by BA, the pharmacokinetic profiles were examined in rats that received co-administration of BA (4 or 20 mg/kg) intravenously. The in vivo exposure of atorvastatin was altered only in rats that received the BA dose of 20 mg/kg. Additional in vitro experiments were carried out to further probe the nature of OATP1B1/3 inhibition by BA, OA or UA. We found that pre-incubation with BA, OA or UA led to a sustained inhibition of the OATP1B3 activity at least up to 2.5 hrs. The inhibited OATP1B3 activity however recovered rapidly in the media containing 10% fetal bovine serum. The addition of albumin to the media was found to decrease intracellular concentrations of BA and to expedite the recovery of OATP1B3 activity following pre-incubation. When asunaprevir and cyclosporin A (previously reported to inhibit OATP1B3 upon pre-incubation) were used, the addition of albumin to the media shortened the recovery time with asunaprevir, but not with cyclosporin A. Overall, our results show that BA inhibits OATP1B transporters in vitro, and has the potential to incur hepatic transporter-mediated drug interactions in vivo, especially with high doses of BA. Our results identify BA as another OATP1B3 inhibitor with pre-incubation effect and suggest that the pre-incubation effect and its duration can be impacted by altered equilibrium of inhibitors between intracellular and extracellular space (e.g. albumin in the media).