CYP3A-LIKE CYTOCHROME P450-MEDIATED METABOLISM AND POLARIZED EFFLUX OF CYCLOSPORINE-A IN CACO-2 CELLS - INTERACTION BETWEEN THE 2 BIOCHEMICAL BARRIERS TO INTESTINAL TRANSPORT

Transport of cyclosporin A (CsA) across Caco-2 cells is modulated by i ts directional efflux, mediated by a p-glycoprotein-like pump (Augusti jns et al., Biochem, Biophys. Res. Comm. 197:360-365, 1994). In additi on to this unidirectional flux, oxidative metabolism of CsA by cytochr ome P450 is likely to influence the absorption of this cyclic peptide across intestinal mucosa, Thus, metabolism of CsA in the in vitro Caco -2 cell culture system was investigated. Formation of several metaboli tes was observed during the course of CsA transport across Caco-2 cell monolayers. Results from LC/MS/MS experiments revealed that the major metabolite was 1 eta-hydroxy CsA (M-17), one of the three major metab olites produced by CYP3A4 present in both the liver and small intestin e in humans. Preincubation of Caco-2 cell monolayers with troleandomyc in, a specific inhibitor for the microsomal CYP3A protein, reduced the formation of the metabolite M-17, suggesting that an enzyme that func tionally resembles CYP3A is responsible for the formation of this meta bolite. However, formation of only the M-17 metabolite suggests that t he isozyme present in the Caco-2 cells is distinct from CYP3A4, which also catalyzes the formation of significant quantities of the metaboli tes (9) gamma-hydroxy cyclosporin A (M-1) and N-4-desmethyl cyclospori n A (M-21) from CsA. Interestingly, the amount of M-17 accumulating on the apical (AP) side was much greater than that on the basolateral (B L) side during the AP BL transport of CsA across Caco-2 cell monolayer s. This is consistent with p-glycoprotein pump-mediated efflux of the metabolite to the apical side. Furthermore, formation of the M-17 meta bolite on the AP side of cell monolayers during the AP --> BL transpor t of CsA was much greater than that during the BL --> AP transport. Th is result suggests that the p-glycoprotein efflux pump causes an incre ase in the metabolism of CsA during the course of its AP --> BL transp ort by effectively slowing down the transport of CsA molecules across Caco-2 cells, Thus, Caco-2 cells serve as an excellent model to dissec t the relative roles played by p-glycoprotein-mediated efflux and CYP3 A-catalyzed oxidation in modulating the overall absorption of CsA and other such compounds.