Tacrolimus metabolite cross-reactivity in different tacrolimus assays

Objectives: Tacrolimus (FK506) is an immunosuppressive drug with great clin ical promise. There is a controversy regarding the role of tacrolimus metab olites in immunosuppression and toxicity, and immunoassays and immunophilin binding assays have not been adequately tested for metabolite cross-reacti vity. Methods are limited to HPLC and HPLC-MS for quantifying the parent dr ug. Mixed lymphocyte culture assay (MLC) is the preferred functional bioass ay for the measurement of parent drug and active metabolites but it is not practical for routine laboratory use. Due to differences in assay methods a nd reagent specificity, the concentration of tacrolimus in a given specimen may vary among different assay kit manufacturers. The objective of this st udy was to evaluate the degree of cross-reactivity or interference of the t hree first-generation tacrolimus metabolites [13-O-demethyl (M-I), 31-O-dem ethyl (M-II) and 15-O-demethyl (M-III)] among two different tacrolimus immu noassays (Immunoassay: PRO-Trac II FK506, Abbott IMx tacrolimus-II); and th e radioreceptor assays (RRA) using minor immunophilins (14, 37, and 52 kDa immunophilins) and tacrolimus binding protein (FKBP12). Methods: First-generation tacrolimus metabolites (M-I, M-II, and M-III) spi ked in drug-free whole blood were assayed with RRA using three minor immuno philins (14, 37, and 52 kDa) and two commercial immunoassay procedures (Inc star PRO-Trac ii tacrolimus, Abbott IMx tacrolimus II). The results were co mpared to previously published FKBP-12 RRA data and their immunosuppressive potency. Results and conclusion: The first generation tacrolimus metabolites (M-I, M -II, and M-III) were tested using concentrations of 10 and 20 ng/mL. The si gnificance of the metabolite interference (% of the total interference) was calculated based on the relative concentration of each metabolite present at steady-state trough concentrations in renal transplant recipients (22). Metabolite I, which has no functional immunosuppressive activity showed min imal interference compared to M-II and M-III in all assays except the 14 kD a RRA. The Incstar PRO-Trac II tacrolimus assay showed the least M-I interf erence. Metabolite-II, which has a pharmacologic potency similar to the par ent drug, showed a significant interference in the immunoassays and signifi cant interference in radioreceptor assays. Metabolite III, which is pharmac ologically inactive, produces 3-10% interference in the different assays if its presence in the blood is 6% of the parent drug. The total interference from these three metabolites was greater in the immunoassays than in the r eceptor assays. Receptor assays for tacrolimus provide results closer to th e target value than do immunoassays. (C) 1998 The Canadian Society of Clini cal Chemists.