Vol. 284, Issue 3, 1074-1081, March 1998

In Vitro Immunosuppressive Activity, Isolation from Pig Liver Microsomes and Identification by Electrospray ms-ms of a New FK-506 C₁₉-C₂₀ Epoxide Metabolite

G. Lhoëst, R. Dieden, R.K. Verbeeck, N. Maton, A. Ingendoh and D. Latinne

Department of Pharmaceutical Sciences (G.L., R.D., R.K.V., N.W., A.L.), UCL, Pharmacokinetics and Metabolism Unit-FATC, Laboratory of Mass Spectrometry, Brussels, Belgium, and Experimental Immunology Unit (D.L.), UCL, Brussels, Belgium

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 immunosuppressive 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 them with known immunosuppressive agents via mixed lymphocyte reaction is of clinical importance because some metabolites may retain the pharmacological 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 the isolation from pig liver microsomes, and the identification by electrospray ms-ms, of the FK-506 C₁₉-C₂₀ epoxide metabolite. We found that this new metabolite exhibits reduced in vitro immunosuppressive activity compared with FK-506 and has approximately the same immunosuppressive potency as other known immunosuppressive drugs, such as cyclosporin A and IMM-125, a hydroxyethyl derivative of D-serine cyclosporin A. We were able to demonstrate that after incubation of the FK-506 metabolite in human mixed lymphocyte reaction cultures for 6 days, the compound was stable under the conditions used for cell culture as evidenced 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 cannot be due to a toxic effect.