Abstract

Cyclosporine A (CsA) and rifampin are potent inhibitors of organic anion transporting polypeptide (OATP) 1B1 and are widely used to assess the risk for drug-drug interactions. CsA displays preincubation time-dependent, long-lasting inhibition of OATP1B1 in vitro and in rats in vivo, and a proposed mechanism is the trans-inhibition by which CsA inhibits OATP1B1 from the inside of cells. The current study aimed to experimentally validate the proposed mechanism using human embryonic kidney 293 cells stably expressing OATP1B1. The uptake of CsA reached a plateau following an approximate 60-minute incubation, with the cell-to-buffer concentration ratio of 3930, reflective of the high-affinity, high-capacity intracellular binding of CsA. The time course of CsA uptake was analyzed to estimate the kinetic parameters for permeability clearance and intracellular binding. When the OATP1B1-mediated uptake of [³H]estradiol-17β-glucuronide was measured following preincubation with CsA for 5 to 120 minutes, apparent K_i values became lower with longer preincubation. Our kinetic modeling incorporated the two reversible inhibition constants [K_i,trans and K_i,cis for the inhibition from inside (trans-inhibition) and outside (cis-inhibition) of cells, respectively] and estimated K_i,trans value of CsA was smaller by 48-fold than the estimated K_i,cis value. Rifampin also displayed preincubation time-dependent inhibition of OATP1B1, albeit the extent of enhancement was only twofold. The current study provides experimental evidence for the preincubation time-dependent shift of apparent K_i values and a mechanistic basis for physiologically based pharmacokinetic modeling that incorporates permeability clearance, extensive intracellular binding, and asymmetry of K_i values between the inside and outside of cells.