A systematic in vitro investigation of the inhibitor preincubation effect on multiple classes of clinically relevant transporters

Preincubation of a drug uptake transporter with its inhibitor in a cell-based assay may result in the apparent enhancement of the inhibitory potency. Limited data is available on whether this potentiation of transporter inhibition by preincubation (PTIP) takes place with clinically relevant solute-carrier transporters other than OATP1B1/3. Therefore, PTIP was examined systematically using OATP1B1/3, OAT1/3, OCT1/2, and MATE1/2-K cell lines. IC 50 values were determined with or without 3 hours of preincubation, and compounds with a PTIP ≥2.5x were further characterised by assessing the time course of transport inhibition potency and cellular concentration. The extent of potentiation was correlated with the physicochemical properties of individual inhibitors. PTIP was observed for OCTs as well as OATPs but not for OATs or MATEs, and most instances of PTIP persisted after controlling for toxicity and non-specific binding. In certain cases, preincubation in excess of 2 hours was required to attain full inhibitory potency. For 5/30 drugs examined, preincubation had the potential to change the in vitro drug-drug interaction risk prediction from "no risk" to "risk" based on current regulatory criteria. Molecular weight and LogD7.4, as well as the ratio of passive cellular accumulation and cellular uptake rate (K p,passive /PS inf ) correlated with PTIP; thus, low cellular permeation and a slow build-up of unbound intracellular inhibitor concentration may contribute to PTIP. Taken together, our data suggest that PTIP is partly determined by the physicochemical properties of the perpetrator drug, and preincubation may affect the in vitro predicted DDI risk for OCTs as well as OATPs. SIGNIFICANCE STATEMENT During the development of a novel pharmaceutical drug, in vitro studies are conducted to assess the risk of potential adverse interactions between existing medications a patient may already be taking and the novel compound. The exact way these in vitro assays are performed may influence the outcome of risk assessment. Here we suggest that the interaction risk may be underestimated unless specific assay protocols are modified to include an additional incubation step that allows the test drug to accumulate inside the cells, and demonstrate that adding this step is particularly important for large and hydrophobic drug molecules.