Identification of a novel route of extraction of sirolimus in human small intestine: roles of metabolism and secretion.

Using Caco-2 cell monolayers expressing CYP3A4, we investigated the interplay between metabolism and transport on the first-pass intestinal extraction of the immunosuppressant sirolimus, a CYP3A4/P-glycoprotein (P-gp) substrate. Modified Caco-2 cells metabolized [(14)C]sirolimus to the predicted amounts of CYP3A4-mediated products based on CYP3A4 content, which was approximately 20% of that measured in human small intestinal mucosal homogenate. [(14)C]Sirolimus also degraded to the known ring-opened product, seco-rapamycin. Unexpectedly, a ring-opened dihydro metabolite (M2) was the major product detected in cells at all sirolimus concentrations examined (2-100 microM). Greater M2 formation after apical versus basolateral dosing (1.6-fold) was explained by higher intracellular content of sirolimus after apical dosing. M2 was not detected in incubations with human liver and intestinal microsomes but was readily detected with corresponding homogenates. M2 formation was NADPH-dependent but unaffected by the CYP3A4 inhibitors ketoconazole and troleandomycin. Although M2 was formed from purified seco-rapamycin (20 microM) in the homogenates, it was not detected in cells when seco-rapamycin was added to the apical compartment, because seco-rapamycin was essentially impermeable to the apical membrane. Sirolimus, seco-rapamycin (basolaterally dosed), and M2 were all actively secreted across the apical membrane, and secretion of each was inhibited by the P-gp inhibitor LY335979 [(2R)-anti-5-[3-[4-(10,11-difluoromethanodibenzo-suber-5-yl)piperazin-1-yl]-2-hydroxypropoxy]quinoline trihydrochloride]. Along with CYP3A4-mediated metabolism and P-gp-mediated secretion, we conclude that the following novel pathway, which occurs at least in the intestine, may contribute significantly to the first-pass extraction of sirolimus in humans: intracellular degradation of sirolimus to seco-rapamycin, metabolism of seco-rapamycin to M2 by an unidentified non-microsomal enzyme, and P-gp-mediated secretion of M2 and seco-rapamycin.