Organic cation transporter (OCT) 2 mediates the entry step for organic cation secretion by renal proximal tubule cells and is a site of unwanted drug-drug interactions (DDIs). But reliance on decision tree-based predictions of DDIs at OCT2 that depend on IC values can be suspect because they can be influenced by choice of transported substrate; for example, IC values for the inhibition of metformin versus MPP transport can vary by 5- to 10-fold. However, it is not clear whether the substrate dependence of a ligand interaction is common among OCT2 substrates. To address this question, we screened the inhibitory effectiveness of 20 M concentrations of several hundred compounds against OCT2-mediated uptake of six structurally distinct substrates: MPP, metformin,,,-trimethyl-2-[methyl(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino]ethanaminium (NBD-MTMA), TEA, cimetidine, and 4-4-dimethylaminostyryl--methylpyridinium (ASP). Of these, MPP transport was least sensitive to inhibition. IC values for 20 structurally diverse compounds confirmed this profile, with IC values for MPP averaging 6-fold larger than those for the other substrates. Bayesian machine-learning models of ligand-induced inhibition displayed generally good statistics after cross-validation and external testing. Applying our ASP model to a previously published large-scale screening study for inhibition of OCT2-mediated ASP transport resulted in comparable statistics, with approximately 75% of "active" inhibitors predicted correctly. The differential sensitivity of MPP transport to inhibition suggests that multiple ligands can interact simultaneously with OCT2 and supports the recommendation that MPP not be used as a test substrate for OCT2 screening. Instead, metformin appears to be a comparatively representative OCT2 substrate for both in vitro and in vivo (clinical) use.

Organic cation (OC) transporter 2 (OCT2) mediates the first step in the renal secretion of many cationic drugs: basolateral uptake from blood into proximal tubule cells. The impact of this process on the pharmacokinetics of drug clearance as estimated using a physiologically-based pharmacokinetic approach relies on an accurate understanding of the kinetics of transport because the ratio of the maximal rate of transport to the Michaelis constant (i.e., J/ K) provides an estimate of the intrinsic clearance (Cl) used in in vitro-in vivo extrapolation of experimentally determined transport data. Although the multispecificity of renal OC secretion, including that of the OCT2 transporter, is widely acknowledged, the possible relationship between relative affinity of the transporter for its diverse substrates and the maximal rates of their transport has received little attention. In this study, we determined the J and apparent Michaelis constant (K) values for six structurally distinct OCT2 substrates and found a strong correlation between J and K; high-affinity substrates [K values <50 M, including 1-methyl-4-phenylpyridinium, or 1-methyl-4-phenylpyridinium (MPP), and cimetidine] displayed systematically lower J values (<50 pmol cm min) than did low-affinity substrates (K >200 M, including choline and metformin). Similarly, preloading OCT2-expressing cells with low-affinity substrates resulted in systematically larger-stimulated rates of MPP uptake than did preloading with high-affinity substrates. The data are quantitatively consistent with the hypothesis that dissociation of bound substrate from the transporter is rate limiting in establishing maximal rates of OCT2-mediated transport. This systematic relationship may provide a means to estimate Cl for drugs for which transport data are lacking.

Organic cation transporter 2 (OCT2) clears the blood of cationic drugs. Efforts to understand OCT2 selectivity as a means to predict the potential of new molecular entities (NMEs) to produce unwanted drug-drug interactions typically assess the influence of the NMEs on inhibition of transport. However, the identity of the substrate used to assess transport activity can influence the quantitative profile of inhibition. Metformin and 1-methyl-4-phenylpyridinium (MPP), in particular, display markedly different inhibitory profiles, with IC50 values for inhibition of MPP transport often being more than fivefold greater than IC50 values for the inhibition of metformin transport by the same compound, suggesting that interaction of metformin and MPP with OCT2 cannot be restricted to competition for a single binding site. Here, we determined the kinetic basis for the mutual inhibitory interaction of metformin and MPP with OCT2 expressed in Chinese hamster ovary cells. Although metformin did produce simple competitive inhibition of MPP transport, MPP was a mixed-type inhibitor of metformin transport, decreasing the maximum rate of mediated substrate transport and increasing the apparent Michaelis constant ( Ktapp) for OCT2-mediated metformin transport. Furthermore, whereas the IC50 value for metformin’s inhibition of MPP transport did not differ from the Ktapp value for metformin transport, the IC50 value for MPP’s inhibition of metformin transport was less than its Ktapp value for transport. The simplest model to account for these observations required the influence of a distinct inhibitory site for MPP that, when occupied, decreases the translocation of substrate. These observations underscore the complexity of ligand interaction with OCT2 and argue for use of multiple substrates to obtain the needed kinetic assessment of NME interactions with OCT2.

OATP1B1 and OATP1B3 are two drug transporters that mediate the uptake of multiple endo- and xenobiotics, including many drugs, into human hepatocytes. Numerous inhibitors have been identified, and for some of them, it is not clear whether they are also substrates. Historically radiolabeled substrates or LC-MS/MS methods were needed to test for transported substrates, both of which can be limiting in time and money. However, the competitive counterflow (CCF) assay originally described for OCT2 and, more recently, for OCT1, OATP2B1, and OATP1A2 does not require radiolabeled substrates or LC-MS/MS methods and, as a result, is a more cost-effective approach to identifying substrates of multidrug transporters. We used a CCF assay based on the stimulated efflux of the common model substrate estradiol-17β-glucuronide (E17βG) and tested 30 compounds for OATP1B1- and OATP1B3-mediated transport. Chinese Hamster Ovary (CHO) cells stably expressing OATP1B1 or OATP1B3 were preloaded with 10 nM [3H]-estradiol-17β-glucuronide. After the addition of known substrates like unlabeled estradiol-17β-glucuronide, estrone-3-sulfate, bromosulfophthalein, protoporphyrin X, rifampicin, and taurocholate to the outside of the preloaded CHO cells, we observed efflux of [3H]-estradiol-17β-glucuronide due to exchange with the added compounds. Of the tested 30 compounds, some organic cation transporter substrates like diphenhydramine, metformin, and salbutamol did not induce [3H]-estradiol-17β-glucuronide efflux, indicating that the two OATPs do not transport them. However, 22 (for OATP1B1) and 16 (for OATP1B3) of the tested compounds resulted in [3H]-estradiol-17β-glucuronide efflux, suggesting that they are OATP substrates. Among these compounds, we further tested clarithromycin, indomethacin, reserpine, and verapamil and confirmed that they are substrates of the two OATPs. These results demonstrate that the substrate spectrum of the well-characterized organic anion transporting polypeptides includes several organic cations. Furthermore, as for other drug uptake transporters, the CCF assay is an easy-to-use screening tool to identify novel OATP substrates.