Therapeutic monitoring of immunosuppressive drugs

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Victor W Armstrong
PhD
apl Professor
Department of Clinical Chemistry
Centre for Internal Medicine
Georg-August-Universität Göttingen
Göttingen
Germany
E:[email protected]

Successful organ transplantation is dependent upon administration of pharmacological immunosuppressants for the prophylaxis of acute organ rejection. On account of their narrow therapeutic indices and their inter- and intraindividual pharmacokinetic variability, therapeutic drug monitoring (TDM) is an important adjunct to optimising therapy with these agents.

Several consensus documents have been published on TDM of immunosuppressive drugs.(1–4) A joint working group of the Scientific Division of the International Federation of Clinical Chemistry (www.ifcc.org) and the International Association for Therapeutic Drug Monitoring and Clinical Toxicology (www.iatdmct.org) is currently updating these documents(5) and is preparing reference articles on best practice for monitoring these drugs.

Ciclosporin
Ciclosporin and tacrolimus are the cornerstones of most immunosuppressive protocols. To maintain effective immunosuppression and minimise adverse side-effects, dosing is guided by monitoring predose (trough or C(0)) blood levels to within time- and organ-dependent therapeutic ranges.(1,6) Most centres use commercially available semiautomated immunoassays with a relatively high specificity for the parent compound for routine monitoring. However, they still display a spectrum of cross-reactivity towards various drug metabolites,(7) and there is no international standardisation of the various immunoassays.

This can be illustrated in the case of ciclosporin through analysis of the results obtained from an international quality control scheme (www.bioanalytics.co.uk). Figure 1 displays the mean relative values obtained for six commercially available immunoassays and specific high-performance liquid chromatography (HPLC) methods using ciclosporin- supplemented drug-free blood samples (Figure 1a) or samples from patients under treatment with ciclosporin (Figure 1b). These various analytical methods obviously differ in both their accuracy and their specificity for the measurement of the parent drug in any one sample. Thus, substantial diversity can be observed between the results from any two methods. This between-method difference itself is not constant and depends on such factors as transplant type, the time after transplantation and liver function.

Sirolimus
Sirolimus is the most recent immunosuppressive drug to be approved for use in renal transplant recipients. Evidence suggests that sirolimus is a critical-dose drug requiring drug concentration monitoring of predose concentrations to optimise clinical efficiency and minimise toxicity.(8) In its approval of the use of sirolimus, the Committee for Proprietary Medicinal Products of the EU stated that optimal therapy requires TDM in all patients. An automated immunoassay was available for quantification of sirolimus in the earlier pivotal clinical studies. This immunoassay was, however, withdrawn. One major drawback was a substantial overestimation of sirolimus in patients’ samples when compared with a specific HPLC procedure.

Sirolimus is therefore currently monitored using either HPLC with UV detection or liquid chromatography in combination with mass spectrometric detection. The necessity to monitor sirolimus and the closely related drug everolimus has led to the development of highly specific liquid chromatography– tandem mass spectrometric procedures (LC-MS/MS)(9,10) that have a much shorter turnaround time than the conventional HPLC methods. Furthermore, several immunosuppressive drugs can be quantified in the same run using a standard pretreatment protocol. However, it is important to note that, despite their inherent specificity, LC-MS/MS are subject to confounding variables, including ion suppression and in-source fragmentation.(11) Ion suppression is caused by the presence of less volatile compounds in the sample matrix that can change the efficiency of droplet formation or droplet evaporation, which in turn affects the amount of charged ion in the gas phase that ultimately reaches the detector. In-source fragmentation can result from the transformation of drug conjugate metabolites to the respective target analytes during the ionisation process. Careful validation of these methods is therefore essential.

Mycophenolate mofetil
Mycophenolate mofetil is a prodrug used in combination with ciclosporin or tacrolimus that is rapidly hydrolysed to its active constituent mycophenolic acid (MPA) in vivo. HPLC methods and an EMIT immunoassay can be used to measure MPA in plasma.(12) The latter also cross-reacts with an active metabolite of MPA.(13) Pharmacokinetic/ pharmacodynamic studies have demonstrated a correlation between MPA-AUC and the risk of acute rejection in kidney and heart transplant recipients.(14,15) TDM may help to minimise the risk of acute rejection, whereby the appropriate strategy, three-point AUC or predose MPA concentration, is still under debate.

Ciclosporin C(2) monitoring
Although monitoring of predose ciclosporin concentrations has been an important adjunct for optimising ciclosporin dosage, it has long been recognised that the predose or C(0) level does not adequately reflect total drug exposure as reflected by the area under the concentration–time curve (AUC). Measurement of a 2-hour postdose ciclosporin concentration has therefore been proposed as a better surrogate for minimising the risk of acute rejection in patients receiving the microemulsion formulation Neoral.(16,17) The rationale for this approach is based on the fact that, for this formulation, most of the variability in the total exposure occurs during the first four hours after dosing. Of the various time-points in this period, C(2) is the best surrogate for the AUC. Since the therapeutic target concentrations for C(2) are often above the dynamic range of the commercially available immunoassays, onsite validated dilution guidelines are necessary.(18)

Conclusion
Monitoring of the immunosuppressive drugs ciclosporin, tacrolimus and sirolimus as a guide to dose adjustment is now accepted practice. Knowledge of comparative assay performance is essential for the interpretation of immunosuppressive drug concentrations and for comparison of data generated at different centres. HPLC in combination with mass spectrometric detection offers an alternative for monitoring immunosuppressive drugs in clinical practice. It should, however, be considered that, when switching from the commonly used immunoassays for ciclosporin and tacrolimus to LC-MS/MS methods, the therapeutic ranges will need to be revised. Although capital costs for the LC-MS/MS equipment are high, direct operating costs are lower than those for commercial immunoassays. C(2) monitoring is a promising new option to make immunosuppression with the microemulsion formulation more efficient, particularly during the early post-transplant phase. The need for a timed blood sample outside the regular sampling schedule necessitates further organisational requirements, which may be judged differently between transplant centres.

References

1. Oellerich M, Armstrong VW, Kahan B, et al. Lake Louise Consensus Conference on cyclosporin monitoring in organ transplantation: report of the consensus panel. Ther Drug Monit 1995;17:642-54.
2. Jusko WJ, Thomson AW, Fung J, et al. Consensus document: therapeutic monitoring of tacrolimus (FK-506). Ther Drug Monit 1995;17:606-14.
3. Yatscoff RW, Boeckx R, Holt DW, et al. Consensus guidelines for therapeutic drug monitoring of rapamycin: report of the consensus panel. Ther Drug Monit 1995;17:676-80.
4. Shaw LM, Nicholls A, Hale M, et al. Therapeutic monitoring of mycophenolic acid. A consensus panel report. Clin Biochem 1998;31:317-22.
5. Holt DW, Armstrong VW, Griesmacher A, et al. International Federation of Clinical Chemistry/International Association of Therapeutic Drug Monitoring and Clinical Toxicology working group on immunosuppressive drug monitoring. Ther Drug Monit 2002;24:59-67.
6. Oellerich M, Armstrong VW, Schutz E, Shaw LM. Therapeutic drug monitoring of cyclosporine and tacrolimus. Update on Lake Louise Consensus Conference on cyclosporin and tacrolimus. Clin Biochem 1998;31:309-16.
7. Schutz E, Svinarov D, Shipkova M, Niedmann PD, et al. Cyclosporin whole blood immunoassays (AxSYM, CEDIA, and Emit): a critical overview of performance characteristics and comparison with HPLC. Clin Chem 1998;44:2158-64.
8. Napoli KL, Taylor PJ. From beach to bedside: history of the development of sirolimus. Ther Drug Monit 2001;23:559-86.
9. Streit F, Armstrong VW, Oellerich M. Rapid liquid chromatography-tandem mass spectrometry routine method for simultaneous determination of sirolimus, everolimus, tacrolimus, and cyclosporin A in whole blood. Clin Chem 2002;48:955-8.
10. Holt DW, Lee T, Jones K, Johnston A. Validation of an assay for routine monitoring of sirolimus using HPLC with mass spectrometric detection. Clin Chem 2000;46:1179-83.
11. Annesley TM. Ion suppression in mass spectrometry. Clin Chem 2003;49:1041-4.
12. Weber LT, Shipkova M, Armstrong VW, Wagner N, Schutz E, Mehls O, et al. Comparison of the Emit immunoassay with HPLC for therapeutic drug monitoring of mycophenolic acid in pediatric renal-transplant recipients on mycophenolate mofetil therapy. Clin Chem 2002;48:517-25.
13. Shipkova M, Schutz E, Armstrong VW, et al. Determination of the acyl glucuronide metabolite of mycophenolic acid in human plasma by HPLC and Emit. Clin Chem 2000;46:365-72.
14. Oellerich M, Shipkova M, Schutz E, et al. Pharmacokinetic and metabolic investigations of mycophenolic acid in pediatric patients after renal transplantation: implications for therapeutic drug monitoring. German Study Group on Mycophenolate Mofetil Therapy in Pediatric Renal Transplant Recipients. Ther Drug Monit 2000;22:20-6.
15. Shaw LM, Korecka M, DeNofrio D, Brayman KL. Pharmacokinetic, pharmacodynamic, and outcome investigations as the basis for mycophenolic acid therapeutic drug monitoring in renal and heart transplant patients. Clin Biochem 2001;34:17-22.
16. Levy G, Thervet E, Lake J, Uchida K. Patient management by Neoral C(2) monitoring: an international consensus statement. Transplantation 2002;73:S12-S18.
17. Oellerich M, Armstrong VW. Two-hour cyclosporine concentration determination: an appropriate tool to monitor neoral therapy? Ther Drug Monit 2002;24:40-6.
18. Holt DW, Johnston A, Kahan BD, et al. New approaches to cyclosporine monitoring raise further concerns about analytical techniques. Clin Chem 2000;46:872-4.