Identification of a Novel Route of Extraction of Sirolimus in Human Small Intestine: Roles of Metabolism and Secretion

Abstract

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 ϳ20% 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 M). 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 M) 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 Pgp-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 nonmicrosomal enzyme, and P-gp-mediated secretion of M2 and secorapamycin.

Contributions of CYP3A4, P-glycoprotein, and Serum Protein Binding to the Intestinal First-Pass Extraction of Saquinavir

“…1A), and chemical inhibition of P-gp activity markedly reduced the apparent permeability ratio (B3 A/ A3 B) at all concentrations examined (Table 1). These permeability ratios were generally higher than those reported by others (Alsenz et al, 1998;Kim et al, 1998b), which may reflect up-regulation of P-gp as a result of 1␣,25-(OH) 2 -D 3 treatment (Schmiedlin-Ren et al, 1997;Paine et al, 2002). However, P-gp content in homogenate prepared from 1␣,25-(OH) 2 -D 3 -treated monolayers was comparable with that in human small intestinal mucosal homogenates (Paine et al, 2002).…”

“…1B). Higher cellular content with apical dosing has also been reported for sirolimus in this same cell system, in which the apical-basolateral difference was postulated to result from saturation of P-gp by sirolimus after apical but not basolateral administration (Paine et al, 2002). In addition, the extent of sirolimus metabolism was greater with the apical route, as would be expected based on cellular content.…”

“…These values are considerably less than the extraction ratio predicted for the human intestine in vivo (ϳ99%) by Thummel et al (1997). One potential explanation for this discrepancy is that the modified Caco-2 cells have been shown to contain only 16 to 30% of the CYP3A4 present in human intestinal mucosal homogenates (Paine et al, 2002). Low CYP3A4 content in the cells would account for why the E i of midazolam calculated from modified Caco-2 cell data were about one-third of the average E i determined in vivo during liver transplantation surgery (15 versus 43%, respectively) (Fisher et al, 1999).…”

“…However, previous work from our laboratory demonstrated that exposure of Caco-2 cells to the hormone 1␣,25-(OH) 2 -D 3 significantly increased CYP3A4 immunoreactive protein and catalytic activity (Schmiedlin-Ren et al, 1997). As such, these modified Caco-2 cells have been used to study the interplay between transport and metabolism during the intestinal first-pass extraction of indinavir (Hochman et al, 2000) and the immunosuppressant sirolimus (Paine et al, 2002). Accordingly, we reasoned that these modified Caco-2 cells would provide an appropriate model to assess the roles of CYP3A4, P-gp, and AAG in determining the extent of the intestinal first-pass extraction of saquinavir.…”

Modeling of Intestinal Drug Absorption: Roles of Transporters and Metabolic Enzymes (For the Gillette Review Series)

“…Many drugs are substrates of both Pgp and cytochrome P450 3A (Terao et al, 1996;Wacher et al, 1998;Benet and Cummins, 2001). These are exemplified by verapamil (Saitoh and Aungst, 1995;Sandström et al, 1998); anticancer drugs such as vincristine, etoposide, daunorubicin, and paclitaxel (Leu and Huang, 1995;Sonnichsen et al, 1995;Nakayama et al, 2000;Chico et al, 2001;Wacher et al, 2001;Abraham et al, 2002); digoxin (Cavet et al, 1996;Greiner et al, 1999); indinavir, the human immunodeficiency virus protease inhibitor (Hochman et al, 2000(Hochman et al, , 2001Li et al, 2002); and immunosuppressive agents cyclosporin (Gan et al, 1996;Lown et al, 1997b), tacrolimus (Lampen et al, 1995;Hashimoto et al, 1998;Hashida et al, 2001), and sirolimus (Lampen et al, 1998;Paine et al, 2002).…”