Use of transporter knockdown Caco-2 cells to investigate the in vitro efflux of statin drugs.

The objective of the present study was to determine the efflux transporters responsible for acid and lactone statin drug efflux using transporter knockdown Caco-2 cells. The bidirectional transport was determined in Caco-2 cell monolayers in which the expression of P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), or multidrug resistance associated protein 2 (MRP2) was knocked down by transduction with lentivirus containing human transporter-targeted small hairpin RNAs (shRNAs). Cells transduced with lentivirus containing nontargeted shRNA served as the vector control. Atorvastatin, lovastatin, and rosuvastatin displayed extremely low apical-to-basolateral (A-to-B) transport, which made the Papp,A-B values too unreliable to calculate the efflux ratio. Thus, transport comparisons were performed using the B-to-A permeability (Papp,B-A) values. Presented in the order of vector control, P-gp, BCRP, and MRP2 knockdown Caco-2 cells, the Papp,B-A values (×10−6, cm/s) were 28.1 ± 1.3, 8.6 ± 2.9, 20.3 ± 1.8, and 21.5 ± 1.6 for atorvastatin; 96.1 ± 7.1, 25.3 ± 3.5, 57.3 ± 9.8, and 48.2 ± 2.3 for fluvastatin; and 14.1 ± 1.9, 4.6 ± 1.7, 5.8 ± 0.7, and 6.6 ± 1.8 for rosuvastatin, respectively. Lovastatin and simvastatin showed no efflux in the vector control or knockdown cell monolayers in either lactone or acid forms. Results indicate that atorvastatin, fluvastatin, and rosuvastatin were transported by P-gp, BCRP, and MRP2. On the other hand, neither the lactone nor the resulting acid of lovastatin and simvastatin was transported by P-gp, BCRP, or MRP2. The current study demonstrated that the transporter knockdown Caco-2 cells are useful tools for studying drug-transporter interactions and should help eliminate some of the ambiguity associated with the identification of drug-transporter interactions based on chemical inhibitors alone.