Purpose: To identify novel time-dependent (TD) inhibitors of uptake transporters in vitro.
Methods: HEK293 cells overexpressing uptake transporters OATP1B1/1B3, OAT1/3, OCT1/2, and MATE1/2-K were used to determine IC₅₀ values of corresponding inhibitors with or without 3 hours of preincubation. A total of 64 transporter-inhibitor combinations were analyzed. A shift in IC50 greater than 2.5-fold (i.e., IC₅₀ with preincubation ≤ 0.4 x IC₅₀ without preincubation) was considered relevant. In addition, transwell permeability of the inhibitors across a low efflux MDCK cell (MDCK-LE) monolayer was measured.
Results: TD inhibition was observed, albeit with different frequencies, across all classes of uptake transporters investigated. The proportion of inhibitors tested positive with at least one member of a cognate transporter pair was 3/5 for OATP1B1/1B3, 1/10 for OAT1/3, 6/9 for OCT1/2, and 1/8 for MATE1/2-K. In particular, ledipasvir, an antiviral previously not recognized as an OCT inhibitor, was shown to potently suppress both OCT1 and 2 upon preincubation (IC₅₀ with preincubation: 0.15 µM and 74.3 µM, respectively). MDCK-LE permeability of the inhibitors ranged between 0.012 and 16.9*10^-6 cm/s, and compounds with low to medium P_app (≤ 5*10^-6 cm/s) were more likely to show TD behavior, as such compounds were involved in 10/15, or 66.7%, of observed cases of TD inhibition. However, the association between MDCK-LE permeability and TD effect was not statistically significant. Among inhibitors that were non-substrates of their respective transporters, the magnitude of IC₅₀ shift correlated positively with cLogP (Spearman's r = 0.43, P=0.008) and molecular weight (r = 0.67, P<0.0001).
Conclusion: Since TD behavior was seen not only in OATPs but also in OATs, OCTs, and MATEs, the phenomenon of TD transporter inhibition seems to extend beyond OATPs. Non-substrate inhibitors with high hydrophobicity and high molecular weight were more liable to exhibiting a TD effect, while TD inhibition was less typical of high MDCK-LE permeability compounds.