Simultaneous determination of everolimus and cyclosporine concentrations by HPLC with ultraviolet detection.

In the clinical practice of organ transplantation everolimus (RAD) is used in combination with cyclosporine (CsA), the most common antirejection agent. Both drugs show a narrow therapeutic window, which requires strict monitoring of their blood concentration. Simple methods for simultaneous measurement of RAD and CsA concentration are needed. As we have recently developed an HPLC-UV assay for RAD determination, we decided to implement it to allow concomitant measurement of CsA. The within- and between-day coefficients of variation of the measurement were less than 12.1% for RAD and 9.8% for CsA. The within- and between-day inaccuracy of quality control samples were less than 9.7% for RAD and less than 4.9% for CsA. The method was found accurate and precise and useful for simultaneous therapeutic monitoring of the two drugs.

Comparison of different cyclosporine immunoassays to monitor C0 and C2 blood levels from kidney transplant recipients: not simply overestimation.

BACKGROUND: Immunoassays used for the measurement of cyclosporine (CsA) usually show cross-reactivity for CsA metabolites, usually resulting in unacceptable bias. METHODS: To assess the performance of different immunoassays, CsA concentrations were analyzed in 132 samples using ACMIA, EMIT-VIVA, CEDIA-PLUS, and HPLC. Samples were collected from kidney transplant patients monitored with the traditional blood CsA trough level (C0, n=73) and the new sampling at 2-h post CsA dosing (C2, n=59). RESULTS: Overall, the correlations between HPLC and other methods were good (r values ranging from 0.85 to 0.97). The use of C2 concentrations to monitor CsA exposure were associated with an overall better performance of all the immunoassays as compared with C0 values. However, none of the immunoassays agreed with the guidelines proposed in the Lake Louis Consensus Conference. Of note, the CEDIA-PLUS was the only that provided a linear relationship with HPLC for both sampling times. A false positive case associated with ACMIA was also documented in blood samples from a patient withdrawn from CsA for 1 month. CONCLUSION: These data suggest that the performance of some of the most used immunoassays is not satisfactory, eventually leading to incorrect therapeutic decision guided by erroneous CsA monitoring.

High-performance liquid chromatography with ultraviolet detection for therapeutic drug monitoring of everolimus.

We developed and validated a high-performance liquid chromatography-ultraviolet (HPLC-UV) method for determining everolimus concentrations in human whole blood. Sample preparation involved a solid-phase extraction after protein precipitation. The separation of everolimus from internal standard (IS) and endogenous components was achieved using an isocratic elution on an octyl column. The method showed a linear relationship between peak height ratios and blood concentrations in the range of 1-200 ng/mL (r(2)=0.9997). The observed intra- and inter-day assay imprecision had a coefficient of variation (CV)=12.8%, and inaccuracy was 11.4%. The method was found to be precise, accurate, and sensible making it useful for routine therapeutic monitoring of everolimus.

Therapeutic drug monitoring of sirolimus: effect of concomitant immunosuppressive therapy and optimization of drug dosing.

Sirolimus (SRL) is a new immunosuppressant which shares a common metabolic pathway with several other immunosuppressive agents. This leads to potential pharmacokinetic interactions that might affect SRL blood levels with relevant clinical consequences. As a validated laboratory, 2658 SRL trough samples (corresponding to 495 kidney transplant recipients treated with different immunosuppressive regimens) from more than 40 Italian Transplant Units were analyzed. We found that dose-normalized SRL trough levels were significantly higher in patients treated with cyclosporine (CsA) and SRL (4.15 +/- 2.23 ng/mL/mg SRL), compared with patients treated with mycophenolate mofetil (MMF) and SRL (3.26 +/- 1.86 ng/mL/mg SRL; p < 0.01) or with MMF, steroids and SRL (2.52 +/- 1.73 ng/mL/mg SRL; p < 0.01). Mean intra- and interpatient variabilities were 19% and 47%, respectively. Both parameters are significantly affected by the time postsurgery, with the first week post transplantation being associated with the greatest variability. As additional analysis, a simple dose-adjustment formula has been proposed as a useful tool to guide SRL dose changes. The proposed equation has been able to predict SRL concentration after a dose change in 73% of the tested samples. These findings suggest that different immunosuppressants significantly interfere with SRL bioavailability. Strategies aimed at reducing variability in SRL exposure may have a positive clinical impact.