Pharmacokinetics and metabolism of sirolimus.

Sirolimus (rapamycin, Rapamune) is a potent immunosuppressive drug that received marketing approval from the US Food and Drug Administration on September 15, 1999. Research into defining its pharmacokinetic (PK) behavior, interaction with other agents, and metabolism is ongoing. It has been established that oral doses of both liquid and solid formulation are rapidly, though incompletely and variably, absorbed. Metabolism by the intestinal and hepatic CYP3A family of enzymes likely contributes to variability in absorption and low bioavailability. Sirolimus has a long terminal half-life, the AUC correlates well with trough and peak concentrations, and it exhibits a moderate degree of dose proportionality. There is significant interpatient variability in PK parameters of sirolimus, though it exhibits predictable PK behavior when used with prednisone and cyclosporine neoral. There is a decreased rejection risk with higher doses and target level attainment. Several species of sirolimus metabolites have been characterized, and are measurable in whole blood and tissue specimens. Many more species of sirolimus metabolites are detectable, but they are not quantifiable at this time. The total concentration of metabolites appears to be less than that of the parent drug when examined through the PK profile. A reference method for the quantitation of metabolites remains elusive because of a lack of proper standardization. The clinical significance of sirolimus metabolites remains to be proven.

Minor immunophilin binding of tacrolimus and sirolimus metabolites.

OBJECTIVES: We have previously identified three minor immunophilins of molecular 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 demethylated sirolimus metabolites showed about 25% less cross-reactivity while the hydroxylated metabolites reacted about the same. RESULTS: Since MLC data on sirolimus metabolites to date indicates that their pharmacologic potencies are < or =10% of the parent, the 14 kDa immunophilin appears to be the best candidate for a sirolimus radioreceptor assay. The 5-8 kDa immunophilin is newly identified and its cross-reactivity with tacrolimus metabolites had not been assessed. Binding of the 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-demethyl 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 level of 163%. MLC data indicates that 31-O-demethyl tacrolimus is equipotent to parent tacrolimus in immunosuppressive activity, while the 13-O-demethyl 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 the 5-8 kDa immunophilin is a subunit of a 52 kDa immunophilin previously identified 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.

Comparison of steady-state trough sirolimus samples by HPLC and a radioreceptor assay.

OBJECTIVES: We have previously identified a minor immunophilin of 52 kDa molecular weight capable of binding tacrolimus and sirolimus. Because immunophilins are capable of binding both parent drug and metabolites and HPLC assays are typically used to assess parent drug in clinical situations, we used this immunophilin in a radioreceptor assay (RRA) to determine if any metabolites not included in the HPLC measurement would bind to the immunophilin and be associated with thrombocytopenia in patients receiving sirolimus. DESIGN AND METHODS: We tested 51 steady-state trough whole blood samples from non-thrombocytopenic patients and 51 steady-state trough samples from thrombocytopenic patients and compared them to HPLC measurements of parent drug in the same samples. We also tested whole blood samples spiked with authentic sirolimus metabolites using RRA to ascertain the effect these metabolites have on the technique. RESULTS: We found minimal cross-reactivity in this assay for sirolimus metabolites (binding ranged from <10% to 26%), and good correlation of the radioreceptor assay with HPLC (linear regression slope 0.92, y-intercept 0.79). There was no statistically significant difference between the RRA and HPLC results in two patient groups-thrombocytopenic and non-thrombocytopenic-using the paired t-test (p<0.005) and Bland-Altman analysis. CONCLUSIONS: These findings indicate that although the RRA could be substituted for HPLC in therapeutic drug monitoring, the 52 kDa immunophilin does not offer an advantage in terms of detecting metabolites associated with thrombocytopenia. However, the RRA offers the advantages of shorter turnaround time, smaller sample volume and potential for automation.