IN-VITRO IMMUNOSUPPRESSIVE ACTIVITY, ISOLATION FROM PIG-LIVER MICROSOMES AND IDENTIFICATION BY ELECTROSPRAY MS-MS OF A NEW FK-506 C-19-C-20EPOXIDE METABOLITE

In order to mediate their effects, cyclosporin A and FK-506 must each bind with high affinity to a cytosolic target protein that belongs to the immunophilin group. FK-506 forms complexes with the FK-506 binding protein FKBP, mainly FKBP-12, and these complexes possess immunosuppr essive activity through their ability to interact with another target, the abundant serine threonine phosphatase calcineurin. Evaluating the immunosuppressive activities of the FK-506 metabolites by comparing t hem with known immunosuppressive agents via mixed lymphocyte reaction is of clinical importance because some metabolites may retain the phar macological activity of the parent drug or exhibit cytotoxic effects. FK-506 is metabolized by the cytochrome P-450-dependent mixed-function oxygenase system in different animal species, and we are reporting th e isolation from pig liver microsomes, and the identification by elect rospray ms-ms, of the FK-506 C-19-C-20 epoxide metabolite. We found th at this new metabolite exhibits reduced in vitro immunosuppressive act ivity compared with FK-506 and has approximately the same immunosuppre ssive potency as other known immunosuppressive drugs, such as cyclospo rin A and IMM-125, a hydroxyethyl derivative of D-serine cyclosporin A . We were able to demonstrate that after incubation of the FK-506 meta bolite in human mixed lymphocyte reaction cultures for 6 days, the com pound was stable under the conditions used for cell culture as evidenc ed by electrospray-ms data. A weak direct cytotoxic effect (< 30% cell death) was observed only at the highest concentrations (2500 and 5000 ng/ml), which shows that the mixed lymphocyte reaction inhibition can not be due to a toxic effect.