ISOLATION, IDENTIFICATION, AND BIOLOGICAL-ACTIVITIES OF OXIDATIVE METABOLITES OF FK506, A POTENT IMMUNOSUPPRESSIVE MACROLIDE LACTONE

To characterize structures and biological activities of FK506 metaboli tes, FK506 was incubated with liver microsomes prepared from phenobarb ital-treated rats in the presence of NADPH generating system under aer obic condition. Oxidative metabolites formed in the reaction medium we re isolated and identified. Purified samples were analyzed by HPLC, ma ss spectrometry, and NMR spectroscopy. M-I, M-II, and M-III were the 0 -demethylated metabolites at the 13-, 31-, and 15-positions of FK506, respectively, and M-IV was the monohydroxylated metabolite at the 12-p osition. M-I was the dominant metabolite in this reaction system. M-II and M-III retained the tetrahydropyrane ring in their structures like FK506, but M-I and M-IV had rearranged structures in which the tetrah ydropyrane ring was changed to a tetrahydrofuran ring. Measuring the i mmunosuppressive activity in the mouse mixed lymphocyte reaction syste m, IC50 values for M-I, M-II, M-III, M-IV, and FK506 were 1.65, 0.23, >127, 5.52, and 0.15 nM, respectively. Reactivity of the metabolites w ith mouse anti-FK506 monoclonal antibody was studied and immunocross-r eactivity of M-I, M-II, M-III, and M-IV with the antibody were nil, 10 9.0, 90.5, and 8.8% of FK506, respectively. These results indicate tha t rat hepatic microsomes oxidatively metabolize FK506 to four metaboli tes, and some of them exhibit pharmacological activity.