Isolation of two immunosuppressive metabolites after in vitro metabolism of rapamycin.

Rapamycin was incubated with human liver microsomes and an NADPH regenerating system, the metabolites were purified by semipreparative HPLC, and their structures were elucidated by direct chemical ionization and FAB-MS. At least six fractions were isolated containing rapamycin metabolites, indicating that rapamycin is metabolized by the human liver cytochrome P-450 system. One of these metabolites was identified as 41-O-demethyl-rapamycin. A second metabolite was hydroxylated in a yet unknown position. These two metabolites retained immunosuppressive activity in a phytohemagglutinin-stimulated human lymphocyte assay with IC50S of 1 and 1.5 nmol/liter, respectively. Rapamycin was metabolized by rat small intestinal microsomes to at least two metabolites, indicating extra-hepatic metabolism of rapamycin.