Inhibitory Effects and Duration of Structurally Related Pentacyclic Triterpenoids on the Activity of the Hepatic Influx Transporter Organic Anion Transporting Polypeptide 1B3 (OATP1B3)
Authors: Oh Yunseok , Min Jee Sun , Ryoo Gongmi , Park Jieun , Chun Se‐Eun , Han Songhee , Bae Soo Kyung , Lee Wooin 
Published: November 2023

BackgroundMany pentacyclic triterpenoids have shown promising anti‐diabetic potential along with hepatoprotective and anti‐inflammatory effects. Some of triterpenoids are also shown to interact with the major hepatic influx transporters such as OATP1B1 and OATP1B3. In particular, it was previously reported that ursolic acid and oleanolic acid with close structural and chemical similarities can effectively inhibit the transport activity of OATP1B1 and OATP1B3. However, little was known whether other common pentacyclic triterpenoids such as betulinic acid and betulin will display similar interactions with OATP1B1 or OATP1B3 and may pose a potential risk for drug interactions.MethodsWe first examined the inhibitory effects of four pentacyclic triterpenoids (betulinic acid, betulin, ursolic acid, oleanolic acid) on the cellular retention of fluorescein using HEK293 cells stabling expressing OATP1B3. In addition, the duration of the inhibitory effects of these compounds on the fluorescein retention was examined by comparing the results between co‐incubation and pre‐incubation followed by extensive washes. Cyclosporin A and rifampicin were used as controls for long‐lasting and short‐acting inhibition, respectively. In order to determine whether the tested triterpenoids are substrates of OATP1B3, analytical assays were developed using HPLC interfaced with mass spectrometry.ResultsThe kinetic parameters for fluorescein uptake (Km, 35.5 ± 3.4 μM; Vmax, 96.9 ± 3.9 pmol/(mg protein •min)) were comparable to those reported in the literature. Consistent with the previous reports, both ursolic acid and oleanolic acid (0.1 – 10 μM) inhibited cellular uptake of fluorescein by OATP1B3 in a concentration‐dependent manner. Betulinic acid (0.1 – 10 μM) also inhibited cellular uptake of fluorescein in a concentration‐dependent manner but betulin did not in the same concentration ranges. When the duration of their inhibitory effects on OATP1B3 was examined, all three compounds (betulinic acid, ursolic acid, oleanolic acid) retained their inhibitory effects even after extensive washes (washed twice with ice‐cold phosphate buffer and stored at 4 °C for 30 min prior to uptake measurements); their IC50 values did not differ between co‐incubation and pre‐incubation followed by washout. The inhibitory effects of these compounds (at 1 μM) were not observed when cells were kept in normal growth media for 2.5 h prior to uptake measurements, suggesting a shorter recovery time than cyclosporin A.ConclusionsOur results indicated that the pre‐incubation with betulinic acid, oleanolic acid and ursolic acid (with a carboxylate functional group) can inhibit the transport activity of OATP1B3 while betulin is not. We are currently investigating whether these compounds are substrates for OATP1B3. These results may provide structural insights underlying selective interactions between OATP1B3 and triterpenoid‐based compounds and implications regarding a potential risk for transporter‐mediated drug interactions.