title: New insights into drug absorption: studies with sirolimus.
abstract: Sirolimus is a recently marketed immunosuppressant that, in common with cyclosporine and tacrolimus, exhibits a low average oral bioavailability (approximately 20%). Likewise, sirolimus is a substrate for the major drug-metabolizing enzyme cytochrome P450 3A4 (CYP3A4) and the efflux transporter P-glycoprotein (P-gp), both of which are expressed in close proximity in epithelial cells lining the small intestine. Using CYP3A4-expressing Caco-2 cell monolayers, we examined the interplay between metabolism and transport on the intestinal first-pass extraction of sirolimus. Modified Caco-2 cells metabolized [14C]sirolimus to the same CYP3A4-mediated metabolites as human small intestinal and liver microsomes. [14C]Sirolimus also degraded to the known ring-opened product, seco-sirolimus. A ring-opened dihydro species (M2) was, surprisingly, the major product detected in cells at all sirolimus concentrations examined (2-100 micromol/L) and in incubations with human liver and intestinal homogenates but not in corresponding microsomes. M2 formation was NADPH-dependent but unaffected by prototypical CYP3A4 inhibitors. Although M2 was formed from purified seco-sirolimus (20 micromol/L) in the homogenates, it was not detected in cells when seco-sirolimus was added to the apical compartment because seco-sirolimus was essentially impermeable to the apical membrane. Sirolimus, seco-sirolimus (basolaterally dosed), and M2 were all secreted across the apical membrane, and secretion of each was inhibited by the P-gp inhibitor LY335979 (zosuquidar trihydrochloride). Along with CYP3A4-mediated metabolism and P-gp-mediated efflux, a novel elimination pathway was identified that may also contribute to the first-pass extraction, and hence low oral bioavailability, of sirolimus. This new insight into the intestinal elimination of sirolimus, which was not identified using traditional drug metabolism/transport screening methods, may represent another source for the limited absorption of sirolimus.
authors.affiliation: General Clinical Research Center and Division of Pharmacotherapy, University of North Carolina, Chapel Hill, North Carolina 27599-7600, USA. mpaine@med.unc.edu
authors.forename: Mary F
authors.initials: MF
authors.lastname: Paine
authors.forename: Louis Y
authors.initials: LY
authors.lastname: Leung
authors.forename: Paul B
authors.initials: PB
authors.lastname: Watkins
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism; D001692:Biological Transport; D018938:Caco-2 Cells; D002250:Carbon Radioisotopes; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D003986:Dibenzocycloheptenes / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007166:Immunosuppressive Agents / Q000378:metabolism* / Q000493:pharmacokinetics; D007408:Intestinal Absorption; D010539:Permeability; D011804:Quinolines / Q000494:pharmacology; D020123:Sirolimus / Q000378:metabolism* / Q000493:pharmacokinetics; D014807:Vitamin D / Q000031:analogs & derivatives* / Q000494:pharmacology
chemical_list: C116441:2-methyl-1,25-dihydroxyvitamin D3; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D002250:Carbon Radioisotopes; D003986:Dibenzocycloheptenes; D007166:Immunosuppressive Agents; D011804:Quinolines; D014807:Vitamin D; C095179:zosuquidar trihydrochloride; D003577:Cytochrome P-450 Enzyme System; C104464:CYP3A protein, human; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D020123:Sirolimus