In this study, we explored clofazimine (CFZ) as a potential substrate of uptake and efflux transporters that might be involved in CFZ disposition, using transporter gene overexpressing cell lines in vitro. The intracellular concentrations of CFZ were significantly increased in the presence of selective inhibitors of P-gp and BCRP, which include verapamil, cyclosporine-A, PSC-833, quinidine, Ko143, and daunorubicin. In a bidirectional transport assay using transwell cultures of cell lines overexpressing P-gp and BCRP, the mean efflux ratios of CFZ were found to be 4.17 ± 0.63 and 3.37 ± 1.2, respectively. The Km and maximum rate of uptake (Vmax) were estimated to be 223.3 ± 14.73 μM and 548.8 ± 87.15 pmol/min/mg protein for P-gp and 381.9 ± 25.07 μM and 5.8 ± 1.22 pmol/min/mg protein for BCRP, respectively. Among the uptake transporters screened, the CFZ uptake rate was increased 1.93 and 3.09-fold in HEK293 cell lines overexpressing OAT1 and OAT3, respectively, compared to the control cell lines, but no significant uptake was observed in cell lines overexpressing OCT1, OCT2, OATP1B1, OATP1B3, OATP2B1, or NTCP. Both OAT1- and OAT3-mediated uptake was inhibited by the selective inhibitors diclofenac, probenecid, and butanesulfonic acid. The Km and Vmax values of CFZ were estimated to be 0.63 ± 0.15 μM and 8.23 ± 1.03 pmol/min/mg protein, respectively, for OAT1 and 0.47 ± 0.1 μM and 17.81 ± 2.19 pmol/min/mg protein, respectively, for OAT3. These findings suggest that CFZ is a novel substrate of BCRP, OAT1, and OAT3 and a known substrate of P-gp in vitro.
Keywords: antituberculosis; clofazimine; membrane transporter; substrate characterization.