Development of a Cell-Based High-Throughput Assay to Screen for Inhibitors of Organic Anion Transporting Polypeptides 1B1 and 1B3

Abstract

The two organic anion transporting polypeptides (OATPs) 1B1 and 1B3 are expressed at the sinusoidal membrane of hepatocytes. They have a broad and overlapping substrate specificity and transport many endobiotics and drugs. Specific inhibitors are required to determine the contribution of each OATP to the hepatocellular uptake of common substrates. We have developed a cell-based high-throughput assay to screen chemical libraries in order to identify such inhibitors for OATP1B1 and OATP1B3. We have used OATP1B1- or OATP1B3-expressing Chinese Hamster Ovary cells on 96-well plates and determined uptake of fluorescein-methotrexate (FMTX). We validated the assay with known inhibitors and screened the well characterized Prestwick library of 1120 drugs. Along with several known OATP inhibitors including rifampicin, cyclosporine A and mifepristone we identified some new inhibitors. For inhibitors that seemed to be able to distinguish between OATP1B1- and OATP1B3-mediated FMTX uptake IC50 values were determined. Estropipate (estrone-3-sulfate stabilized with piperazine) was the most selective OATP1B1 inhibitor (IC50 = 0.06 μM vs. 19.3 μM for OATP1B3). Ursolic acid was the most selective OATP1B3 inhibitor (IC50 = 2.3 μM vs. 12.5 μM for OATP1B1). In conclusion, this cell-based assay should allow us to identify even more specific inhibitors by screening larger chemical libraries.

Expression of organic anion-transporting polypeptides 1B1 and 1B3 in ovarian cancer cells: Relevance for paclitaxel transport

“…This K m is in the range of the mean plasma concentration in patients (0.85 ± 0.21 µM) after 24 h of infusion [41]. Our conclusion that paclitaxel could be a better substrate for OATP1B1than OATP1B3 is supported by very recent experiments of Gui and coworkers as they found that paclitaxel (2.5 µM) inhibited the uptake of the OATP1B1/1B3 substrate fluorescein-methotrexate by 93% in OATB1B1-but only by 53% in OATP1B3-expressing CHO cells [42]. Differences in estrogen-responsive and estrogen-independent ovarian cancer cell lines also suggest that alterations in mRNA expression levels may also be observed in tumor tissue samples from patients.…”

Drug–Drug Interaction Studies: Regulatory Guidance and An Industry Perspective

“…Consistent with this, our recent studies (63) using estrone sulfate and estropipate, potent prototypical inhibitors for OATP1B1 (66), also yielded different IC 50 values for the uptake of pitavastatin (IC 50 , 0.6 and 0.8 μM, respectively) as compared to that of atorvastatin (IC 50 >50, >10 μM, respectively). Further complicating this is the fact that currently we have little understanding of the kinetics of drug-transporter interactions.…”

A Novel Fluorescence Resonance Energy Transfer-Based Screen in High-Throughput Format To Identify Inhibitors of Malarial and Human Glucose Transporters

“…Distribution ratios (i.e., the ratio of intracellular concentration of radiolabel relative to the extracellular concentration) were calculated as described previously (14). IC 50 s were calculated using nonlinear regression analysis and are tabulated in Table 2.…”

“…Current assays for transporter inhibition in a high-throughput format generally employ radiolabeled substrate or cell death of a transporter-overexpressing cell line as a readout (13,14). Both formats have significant limitations.…”