Clinical Chemistry / CYCLOSPORINE METABOLISM IN TRANSPLANT RECIPIENTS

Cyclosporine is used in the prevention of allograft rejection. Owing to its narrow therapeutic index, regular monitoring of the whole blood levels of cyclosporine is required. We observed that immunoassays measured significantly higher cyclosporine levels than did high-performance liquid chromatography (HPLC) over time after transplantation. As cyclosporine metabolites crossreact even with immunoassays, this observation might be due to alterations of the cyclosporine metabolism. We analyzed cyclosporine metabolite concentrations in the early and in the late posttransplantation periods in 127 patients after kidney, bone marrow, heart-lung, and liver transplantation by HPLC and determined whole blood levels of cyclosporine by 4 immunoassays (enzyme-multiplied immunoassay [EMIT], cloned enzyme donor immunoassay [CEDIA], AxSYM [Abbott Laboratories, Chicago, IL], and TDx [Abbott Laboratories]). Despite reduced dose, we found significantly higher cyclosporine concentrations measured by the EMIT, AxSYM, and TDx assays in various patient groups. These results are due to the increased metabolite/cyclosporine ratio in the late posttransplantation period. In particular, the metabolites AM1 and AM19 increased significantly over time in bone marrow transplant recipients. Therefore, cyclosporine levels measured by immunoassays should be interpreted with caution. Cyclosporine is a potent immunosuppressant drug that is used in the prevention of allograft rejection and treatment of certain autoimmune diseases.1 Its use is limited by severe toxic effects and a narrow therapeutic index. Therefore, to avoid adverse effects and to ensure effective immunosuppression despite intraindividual and interindividual variability in pharmacokinetics and metabolism, regular monitoring of the whole blood levels of cyclosporine is required.2 Cyclosporine is metabolized extensively in the liver by the cytochrome P-450 oxidase. The primary metabolites are the monohydroxylated AM1 (M-17) and AM9 (M-1) and the Ndemethylated AM4n (M-21). Further oxidation of AM1 and AM9 results in the dihydroxylated AM19 (M-8), AM49 (M10), and AM69 (M-16).3,4 The distribution of cyclosporine and its main metabolites in whole blood are reported to be as follows: cyclosporine (27%), AM1 (24%), AM9 (14%).5 Among different transplant groups (kidney, bone marrow, heart-lung, and liver), the highest quantities of cyclosporine metabolites (AM1 and AM9) were observed in patients after liver and heart transplantation, whereas only in liver recipients were the blood levels of AM1 found to be significantly (P < .02) higher than the parent drug levels.6 Cyclosporine metabolites cross-react even with the newly introduced specific monoclonal immunoassays (fluorescence polarization immunoassay [FPIA]/AxSYM [Abbott Laboratories, Chicago, IL], modified enzyme-multiplied immunoassay [EMIT], and cloned enzyme donor immunoassay [CEDIA]).7,8 By spiking a whole blood pool from healthy donors with the major cyclosporine metabolites AM1 and AM9, Hamwi et al9 found that the monoclonal FPIA/TDx (Abbott Laboratories) showed the highest cross-reactivity (7.1%) toward AM1, followed by FPIA/AxSYM (5.5%) and CEDIA (4.5%), Clinical Chemistry / ORIGINAL ARTICLE Am J Clin Pathol 2000;114:536-543 537 © American Society of Clinical Pathologists whereas the EMIT showed no detectable cross-reactivity with AM1. Furthermore, all assays displayed a cross-reactivity toward AM9; the highest cross-reactivity was seen with CEDIA (23%), followed by FPIA/TDx (18.2%) and FPIA/AxSYM (13.7%), and the lowest with EMIT (9.2%).9 Hamwi et al9 also showed that these immunoassays measured significantly (P < .001) higher cyclosporine concentrations than those measured by high-performance liquid chromatography (HPLC). In the comparison studies between each of the 4 monoclonal immunoassays (CEDIA, EMIT, FPIA/AxSYM, and FPIA/TDx) for cyclosporine and the reference method, HPLC, the slopes of the regression lines were 1.25 for CEDIA, 1.12 for the EMIT, 1.15 for FPIA/AxSYM, and 1.4 for the FPIA/TDx.9 Furthermore, these differences depended not only on the assay used but also on the transplanted organ.9 In addition, the FPIA/TDx immunoassay measured significantly (P < .015) higher cyclosporine levels over time after transplantation than did HPLC.9 Cross-reactivity of immunoassays with metabolites when determining cyclosporine levels may result in inadequate immunosuppression. For example, the main metabolite AM1 shows only 10% to 20% of the parent compound’s activity.10 Therefore, exact knowledge of the metabolism of cyclosporine in different patient groups during the posttransplantation period is required for accurate interpretation of the results measured by monoclonal immunoassays. The aims of the present study were to analyze by HPLC the metabolite patterns in the early (3 months or less after transplantation) and late (>3 months after transplantation) posttransplantation periods in different patient groups after kidney, bone marrow, heart-lung, and liver transplantation and to compare these results with results obtained by 4 immunoassays. Materials and Methods