CYP3A5 genotype does not influence the blood concentration of tacrolimus measured with the Abbott immunoassay.

To the Editor: Therapeutic drug monitoring of immunosuppressants is well established as an aid to optimizing patient management after allograft transplantation. Most laboratories measure cyclosporin and tacrolimus by immunoassays. However, measured values may be affected by the presence of circulating metabolites because of the limited specificity of the antibodies used in these assays. Cyclosporin is metabolized by the cytochrome P450 3A4 (CYP3A4) and cytochrome P450 3A5 (CYP3A5) enzymes. Studies on the biotransformation of cyclosporin by CYP3A4 and CYP3A5 have demonstrated disparate patterns of metabolite profiles between the 2 enzymes for this substrate. CYP3A4 catalyzes the formation of 3 primary cyclosporin metabolites; 2 are monohydroxylated (AM1 and AM9), and the third is demethylated (AM4N). CYP3A5 produces only the AM9 metabolite (1 ). This may have important implications for immunoassay measurements of cyclosporin, as polymorphic expression of the CYP3A5 gene leads to some individuals not producing the CYP3A5 enzyme (2). In addition, the CYP3A5 enzyme has a lower catalytic activity for cyclosporin compared with CYP3A4. However, the preferential catalysis exhibited by these enzymes is not the same for all substrates. Preferential production of particular metabolites could occur with tacrolimus. Genetic studies have shown that expressors of CYP3A5 require significantly larger doses of tacrolimus to attain therapeutic blood concentrations of this drug (3 ). These studies have been based on blood concentrations determined with an immunoassay (MEIA tacrolimus II immunoassay for the IMx analyzer; Abbott Diagnostics). If CYP3A4 and CYP3A5 were to produce different metabolite profiles from tacrolimus, then the degree of interference in the immunoassay would differ for expressors and nonexpressors of CYP3A5. To investigate this possibility, we compared tacrolimus results, obtained with the Abbott immunoassay, with those of an HPLC–tandem mass spectrometry (MS/MS) assay, using whole blood samples taken from expressors (homozygous CYP3A5*1*1 and heterozygous CYP3A5*1*3) and nonexpressors (homozygous CYP3A5*3*3) of the CYP3A5 gene. Predose blood samples collected from kidney transplant patients receiving tacrolimus were analyzed. The samples were collected at least 3 months posttransplantation. A total of 136 samples taken from 105 patients were grouped into expressors (n 62 samples from 32 patients) and nonexpressors (n 74 samples from 73 patients) of CYP3A5. The samples were analyzed by the Abbott immunoassay on the day of receipt, then stored frozen at 20 °C until analyzed by HPLC-MS/MS. Tacrolimus analysis was performed with the Abbott immunoassay and by an in-house HPLC-MS/MS method. The HPLC-MS/MS assay was used as the comparison method. For direct comparison, the Abbott calibrators and controls were used for both methods to minimize any calibration bias. The DNA was exFig. 1. Tacrolimus blood concentrations in CYP3A5 nonexpressors (A) and CYP3A5 expressors (B), as measured by Abbott IMx immunoassay and LC-MS/MS.