COMPARISON OF BINDING CHARACTERISTICS OF 4 RAPAMYCIN METABOLITES TO THE 14 AND 52 KDA IMMUNOPHILINS WITH THEIR PHARMACOLOGICAL 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 (M LC) assay. Methods: Four RAPA metabolites were isolated by HPLC from t he urine of renal transplant patients. Each metabolite was evaluated a t 40 mu g/L for its pharmacologic activity using the MLC assay. The re sults of the MLC assay were compared to those obtained using the radio receptor assay (RRA), which measured the binding characteristics of eq ual concentrations of the metabolites to the 14 and 52 kDa minor immun ophilins. Results: Each of the four RAPA metabolites showed low immuno suppressive 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 mino r immunophilin, with 21% of parent binding to the 14 kDa protein and 2 5% of parent binding to the 52 kDa protein. RM2, 3, and 4 bound to bot h proteins with less than or equal to 2% of parent binding. Conclusion : We have demonstrated that the RRA for these four RAPA metabolites sh ows little cross-reactivity. There is no commercially available immuno assay for RAPA at present The RRA, therefore, provides an excellent wa y to rapidly assess efficacy/toxicity of RAPA in patients receiving th e drug.