Minor immunophilin binding of tacrolimus and sirolimus metabolites

Objectives: We have previously identified three minor immunophilins of mole cular weights 37 kDa, 14 kDa, and 5-8 kDa capable of binding tacrolimus and sirolimus. Design and methods: When tested against pure preparations of five sirolimus metabolites, the 14 kDa protein had almost no cross-reactivity, the 37 kDa protein cross-reacted from a high of 23.2% to <10% and the 5-8 kDa protein cross-reacted from <10% to 46.4%. When the 5-8 kDa immunophilin was tested in whole blood samples to assess interference in clinical samples, the dem ethylated sirolimus metabolites showed about 25% less crossreactivity while the hydroxylated metabolites reacted about the same. Results: Since MLC data on sirolimus metabolites to date indicates that the ir pharmacologic potencies are less than or equal to 10% of the parent, the 14 kDa immunophilin appears to be the best candidate for a sirolimus radio receptor assay. The 5-8 kDa immunophilin is newly identified and its cross- reactivity with tacrolimus metabolites had not been assessed. Binding of th e 5-8 kDa immunophilin to pure preparations of three tacrolimus metabolites showed virtually no binding of the protein to 13-O-demethyl and 31-O-demet hyl tacrolimus and binding to 15-O-demethyl tacrolimus at 121% relative to parent tacrolimus. Cross-reactivity of 15-O-demethyl tacrolimus with the 5- 8 kDa protein was then assessed in whole blood samples, and it bound at a l evel of 163%. MLC data indicates that 31-O-demethyl tacrolimus is equipoten t to parent tacrolimus in immunosuppressive activity, while the 13-O-demeth yl and 15-O-demethyl have negligible immunosuppressive activity. Conclusions: Therefore, the 5-8 kDa immunophilin would have limitations in a radioreceptor assay for tacrolimus. In addition, we have evidence that th e 5-8 kDa immunophilin is a subunit of a 52 kDa immunophilin previously ide ntified by our group, and the cross-reactivity of the 5-8 kDa immunophilin with these metabolites is similar to that found previously with the 52 kDa, indicating that the two proteins could be related. Copyright (C) 2000 The Canadian Society of Clinical Chemists.