FURTHER METABOLISM OF FK506 (TACROLIMUS) - IDENTIFICATION AND BIOLOGICAL-ACTIVITIES OF THE METABOLITES OXIDIZED AT MULTIPLE SITES OF FK506

To characterize the metabolic pathway of FK506 (tacrolimus), FK506 or its 31-O-desmethyl metabolite was incubated with liver microsomes prep ared from dexamethasone-treated rats in the presence of a NADPH-genera ting system under aerobic conditions. Besides the four oxidized metabo lites already reported, four new metabolites were isolated and identif ied by HPLC, mass spectrometry, and NMR spectroscopy, and their biolog ical activities were examined. The di-demethylated metabolites at the 15- and 31-, 13- and 31-, and 13- and 15-methoxy groups of FK506, were designated respectively as M-V, M-VI, and NI-VII. The fourth, M-VIII, was the metabolite produced after O-demethylation at the 31-methoxy g roup and formation of a fused 10-membered ring structure through the 1 9- to 22-carbon of the macrolide ring after oxidation of the 19-methyl group, and of the 36- and 37-vinyl group of FK506. The immunosuppress ive activity of the isolated metabolites was estimated in a mouse mixe d lymphocyte reaction system and the IC50 values for M-V, M-VI, M-VII, M-VIII, and FK506 were >1000, 8.78, >1000, 15.27, and 0.11 ng/ml, res pectively. Reactivity of the metabolites with mouse anti-FKBOB monoclo nal antibody was studied and immunocrossreactivity of M-V was 92.3% of FK506, but no reactivity was observed for M-VI, M-VII, and M-VIII. FK 506 thus was metabolized at multiple sites by rat hepatic microsomes a nd the metabolites formed (M-V similar to M-VIII) exhibited weak or ne gligible immunosuppressive activity.