The purpose of the present study was to determine whether a physiologic plasma concentration of a-ketoglutarate (aKG) influences the kinetic interaction of ligands with organic anion transporter 1 (OAT1). The effect of extracellular aKG on the kinetics of para-aminohippurate (PAH) and cidofovir transport was examined along with its effect on the potency of 10 drugs in five different classes (uricosuric, nonsteroidal anti-inflammatories, loop diuretics, angiotensin II receptor antagonists, and b-lactam antibiotics) to inhibit OAT1 expressed in Chinese hamster ovary cells. Extracellular aKG competitively inhibited PAH and cidofovir transport with K i values (∼5 mM) approximating its unbound plasma concentration (determined by equilibrium dialysis). When PAH was the substrate, extracellular aKG (5 mM) significantly increased IC 50 values for some inhibitors (up to 4-fold), such as probenecid, but not for others (an inhibitor-dependent effect). For some inhibitors, a significant increase in IC 50 value was observed when cidofovir was the substrate, but not PAH (a substrate-dependent effect). A significant increase in IC 50 value was also observed for inhibition of PAH transport by probenecid in renal basolateral membrane vesicles (5.2-fold). The substrate-and inhibitor-dependent effect of extracellular aKG on ligand interactions with OAT1 highlights the complexity of the OAT1 ligand-binding surface. The effect of extracellular aKG on the potency of OAT1 inhibition should be considered when assessing drug-drug interaction potential at the transporter.

The mechanism by which drugs inhibit organic anion transporter 1 (OAT1) was examined. OAT1 was stably expressed in Chinese hamster ovary (CHO) cells, and para-aminohippurate (PAH) and 6-carboxyfluorescein were the substrates. Most compounds (10 of 14) inhibited competitively, increasing the Michaelis constant (K m ) without affecting the maximal transport rate (J max ). Others were mixed-type (lowering J max and increasing K m ) or noncompetitive (lowering J max only) inhibitors. The interaction of a noncompetitive inhibitor (telmisartan) with OAT1 was examined further. Binding of telmisartan to OAT1 was observed, but translocation was not. Telmisartan did not alter the plasma membrane expression of OAT1, indicating that it lowers J max by reducing the turnover number. PAH transport after telmisartan treatment and its washout recovered faster in the presence of 10% fetal bovine serum in the washout buffer, indicating that binding of telmisartan to OAT1 and its inhibitory effect are reversible. Together, these data suggest that telmisartan binds reversibly to a site distinct from substrate and stabilizes the transporter in a conformation unfavorable for translocation. In the absence of an exchangeable extracellular substrate, PAH efflux from CHO-OAT1 cells was relatively rapid. Telmisartan slowed PAH efflux, suggesting that some transporter-mediated efflux occurs independent of exchange. Although drug-drug interaction predictions at OAT1 assume competitive inhibition, these data show that OAT1 can be inhibited by other mechanisms, which could influence the accuracy of drug-drug interaction predictions at the transporter. Telmisartan was useful for examining how a noncompetitive inhibitor can alter OAT1 transport activity and for uncovering a transport mode independent of exchange.

Renal tubular drug secretion mediated by the organic anion transporter 1 (OAT1) is a potential site of drug‐drug interactions. A number of marketed drugs inhibit OAT1, but the mechanism by which they do so is not typically known. The purpose was to investigate the predominant mechanism by which therapeutic drugs inhibit OAT1. OAT1 was expressed in Chinese hamster ovary cells and 3H‐para‐aminohippurate was used as the substrate. The Kreb’s cycle intermediate alpha‐ketoglutarate (αKG) competitively inhibited OAT1 with a Ki value (5.4 μM) approximating its unbound plasma concentration. In the presence of αKG (5 μM) the potency of inhibition caused by probenecid, furosemide and ibuprofen decreased ~2‐fold (~2‐fold increase in IC50 values), whereas the potency of inhibition caused by telmisartan was unaffected. This led us to speculate that probenecid, furosemide and ibuprofen interact competitively with αKG and 3H‐para‐aminohippurate while telmisartan is a non‐competitive inhibitor. Probenecid, furosemide and ibuprofen all competitively inhibited OAT1 with Ki values of 10.5 μM, 17.8 μM and 3.1 μM, respectively. In contrast, telmisartan inhibited OAT1 by reducing the maximal transport rate without affecting the Michaelis constant, indicating a non‐competitive type inhibition (IC50 of value of 0.33 μM). Interestingly, wash‐out experiments showed that the inhibitory effect of telmisartan is quasi‐irreversible. These data show that OAT1 inhibition can occur through different kinetic mechanisms. The mechanism by which inhibitors reduce OAT1 activity could influence the magnitude of drug‐drug interactions at OAT1‐mediated renal tubular drug secretion. Supported by NSHRF. Grant Funding Source: Supported by Nova Scotia Health Research Foundation

There are discrepancies in the literature regarding ligand selectivity of organic anion transporter 2 (OAT2). The purpose of this study was to re‐examine the interaction of ligands with both transcript variants of OAT2 since it is not known whether they are functionally distinct. The two splice variants of human OAT2 (OAT2‐TV1 and OAT2‐TV2) were stably transfected into three cell lines (CHO, HEK and MDCK), and the interaction of previously identified substrates and inhibitors was assessed. Expression of both transcript variants was confirmed at the mRNA level, but only OAT2‐TV1 protein was detected at the cell surface. Uptake of penciclovir was ~1.5‐20 fold higher in OAT2‐TV1 cells compared to control, but para‐aminohippurate (PAH), estrone sulfate (ES), succinate and glutarate transport was not observed. Eight Krebs cycle intermediates (1mM) failed to cis‐inhibit penciclovir uptake. Conversely, cis‐inhibition by a structurally diverse set of xenobiotic drugs, including anions, cations and neutral compounds suggests that OAT2‐TV1 is broadly selective. Treatment of cells with sulfo‐NHS‐SS biotin inhibited penciclovir transport. Co‐treatment of cells with furosemide along with sulfo‐NHS‐SS biotin ameliorated transport activity, suggesting that lysine residue(s) line the ligand binding surface. These results show that OAT2‐TV2 is non‐functional in mammalian cells, likely due to a trafficking defect. Additionally, lack of mediated transport or cis‐inhibition by several previously reported compounds highlights the necessity to re‐examine ligand selectivity of OAT2‐TV1 to better understand its importance in pharmacokinetics. Supported by CIHR and NSHRF.