Purpose: The purpose of this study was to elucidate the mechanisms by which an HMG-CoA reductase inhibitor, atorvastatin (an organic acid with a pKa of 4.46), was transported in the secretory and absorptive directions across Caco-2 cell monolayers.
Methods: Caco-2 cells were grown on polycarbonate membrane inserts in 6-well Snapwell plates (Costar). The permeability of radiolabeled compounds across Caco-2 cell monolayers was determined using a side-by-side diffusion apparatus (NaviCyte) and an automated liquid handler (Hamilton Microlab 2200). The apical uptake of 14C-atorvastatin was also determined in Caco-2 cells. Cyclosporin A (20 microM) was present in the uptake media to block potential P-glycoprotein-mediated atorvastatin efflux.
Results: Polarized permeation of atorvastatin was observed with the basolateral-to-apical (B-to-A) permeability being 7-fold greater than the A-to-B permeability (35.6 x 10(-6) and 4.9 x 10(-6) cm/s, respectively). The secretion of atorvastatin was a saturable process with an apparent Km of 115 microM. The B-to-A permeability of atorvastatin was significantly reduced by cyclosporin A (10 microM), verapamil (100 microM), and a P-glycoprotein specific monoclonal antibody, UIC2(10 microg/ml) (43%, 25%, and 13%, respectively). Furthermore, both CsA and verapamil significantly increased the A-to-B permeability of atorvastatin by 60%; however, UIC2 did not affect the A-to-B permeability of atorvastatin. CsA uncompetitively inhibited the B-to-A flux of atorvastatin with a Ki of 5 microM. In addition, atorvastatin (100 microM) significantly inhibited the B-to-A permeability of vinblastine by 61%. The apical uptake of atorvastatin increased 10.5-fold when the apical pH decreased from pH 7.4 to pH 5.5 while the pH in the basolateral side was fixed at pH 7.4. A proton ionophore, carbonylcyanide p-trifluoro-methoxyphenylhydrazone (FCCP) significantly decreased atorvastatin uptake. In addition, atorvastatin uptake was significantly inhibited by benzoic acid, nicotinic acid, and acetic acid each at 20 mM (65%, 14%, and 40%, respectively). Benzoic acid competitively inhibited atorvastatin uptake with a Ki of 14 mM. Similarly, benzoic acid, nicotinic acid, and acetic acid significantly, inhibited the A-to-B permeability of atorvastatin by 71%, 21%, and 66%, respectively.
Conclusion: This study demonstrated that atorvastatin was secreted across the apical surface of Caco-2 cell monolayers via P-glycoprotein-mediated efflux and transported across the apical membrane in the absorptive direction via a H(+)-monocarboxylic acid cotransporter (MCT). In addition, this study provided the first evidence that negatively charged compounds, such as atorvastatin, can be a substrate for P-glycoprotein.