The inhibitor preincubation effect is universal to SLC transporter assays and is only partially eliminated in the presence of extracellular protein.

Abstract

Variation in the methodology of in vitro transporter inhibition assays causes wide divergence in reported IC50/Ki data. Notably, although potentiation of transporter inhibition by preincubation (PTIP) has been described, current guidelines do not specifically recommend inhibitor preincubation, only encourage sponsors to follow emerging literature. To clarify how generally preincubation should be considered in transporter inhibition studies, and whether PTIP can be solely explained by protein binding of the respective inhibitors, we performed in vitro inhibition assays on SLC and ABC transporters scarcely or not covered in prior research, and examined the effect of extracellular protein in preincubation and washout experiments. In SLC assays without extracellular protein, a 30-minute preincubation caused significant >2-fold change of IC50 in 21/33 transporter-inhibitor combinations involving 19 evolutionarily disparate transporters. The preincubation effect correlated with inhibitor properties like protein binding and aqueous solubility. In vesicular transport assays of MDR1, BCRP, MRP2, and BSEP, sizable PTIP was observed for only 2/23 combinations, and preincubation was practically inconsequential in BCRP or MDR1 monolayer assays. In SLC assays, PTIP partly persisted in the presence of 5% albumin, indicating that the absence of extracellular protein does not fully explain PTIP. The presence of protein, however, complicated the interpretation of results. Overall, while preincubating without protein may overpredict inhibitory potency, adding protein compromises clarity, and omitting preincubation altogether may miss clinically relevant inhibitors. Therefore, we propose that protein-free preincubation should be considered in all SLC inhibition assays. ABC inhibition seems less commonly affected by preincubation, but conclusions require further investigation. Significance Statement Drugs may inhibit transporter proteins in the body, which may precipitate drug interactions. In vitro transporter inhibition assays help predict such drug interactions. Some inhibitors act more potently when preincubated with the transporter prior to the assay. Here we argue that this effect is not a mere in vitro artefact due to the lack of plasma proteins and should be considered in all uptake inhibition assays to model the worst-case scenario. Preincubation in efflux transporter inhibition assays is likely dispensable.