Comparison of binding characteristics of four rapamycin metabolites to the 14 and 52 kDa immunophilins with their pharmacologic activity measured by the mixed-lymphocyte culture assay

Objectives: To compare the binding characteristics of four rapamycin (RAPA) metabolites to the 14 and 52 kDa minor immunophilins with their pharmacologic activity, as measured by the mixed-lymphocyte culture (MLC) assay. Methods: Four RAPA metabolites were isolated by HPLC from the urine of renal transplant patients. Each metabolite was evaluated at 40 μg/L for its pharmacologic activity using the MLC assay. The results of the MLC assay were compared to those obtained using the radioreceptor assay (RRA), which measured the binding characteristics of equal concentrations of the metabolites to the 14 and 52 kDa minor immunophilins. Results: Each of the four RAPA metabolites showed low immunosuppressive activity by MLC. RM2 showed the highest activity, with 9% of parent RAPA activity. RM1, 3, and 4 showed 2%, 8%, and 4% activity, respectively. Only RM1 was found to bind significantly to either minor immunophilin, with 21% of parent binding to the 14 kDa protein and 25% of parent binding to the 52 kDa protein. RM2, 3, and 4 bound to both proteins with 2% of parent binding. Conclusion: We have demonstrated that the RRA for these four RAPA metabolites shows little cross-reactivity. There is no commercially available immunoassay for RAPA at present. The RRA, therefore, provides an excellent way to rapidly assess efficacy/toxicity of RAPA in patients receiving the drug.