Is Calcineurin Activity Useful as a Biomarker to Optimize Cyclosporine A Therapy in Renal Transplant Recipients?
R.R. Press(1,2), H.H. van Rossum(3), B.A. Ploeger(4,5), J. den Hartigh(1), J. van Pelt(3), H.-J. Guchelaar(1), J.W. de Fijter(2) and M. Danhof(4,5)
Departments of (1) Clinical Pharmacy and Toxicology, (2)Nephrology, (3)Clinical Chemistry, Leiden University Medical Center, Leiden, The Netherlands;(4)Leiden Amsterdam Center for Drug Research (LACDR), Leiden, The Netherlands;(5)Leiden Experts on Advanced Pharmacokinetics and Pharmacodynamics (LAP&P Consultants BV), Leiden, The Netherlands.
Objectives: The occurrence of acute rejection episodes and clinical nephrotoxicity relates to cyclosporine A (CsA) exposure in renal transplant recipients[1]. Due to the large inter-individual variability in CsA exposure therapeutic drug monitoring (TDM) is mandatory. However, still patients suffer from acute rejection episodes and acute and chronic nephrotoxicity. To further reduce these clinical events, insight into the individual susceptibility for CsA therapy is warranted. A biomarker other than drug concentration could be a useful addition to explain differences in clinical response between patients[2]. Specifically, the measurement of the phosphatase-activity of calcineurin, the target enzyme of CsA, has potential to serve as a basis to further individualize CsA therapy. Therefore, in the present study the pharmacokinetic-pharmacodynamic (PK-PD) relationship between CsA exposure and the activity of the calcineurin enzyme was evaluated. The aim of the study was to determine whether this biomarker complies with the conditions to be useful in clinical practice. First, the biomarker should describe the between patient variability in clinical response to CsA better compared to exposure measurements. Second, variability in PD parameters such as potency (EC50) or efficacy (Emax) should relate to clinical outcome, such as rejection episodes or nephrotoxicity.
Methods: Renal transplant recipients (n=95) were followed for 6 months after transplantation. These patients received quadruple immunosuppressive therapy with basiliximab, mycophenolate-sodium, prednisolone and CsA. The initial CsA dose of 4 mg/kg b.i.d was adjusted to a target AUC[0-12h] of 5400 μg*h/L in the first 6 weeks after transplantation and 3250 μg*h/L thereafter as part of a strict TDM strategy. In this context CsA concentrations were measured together with calcineurin activity in the mornings of weeks 1, 4, 8, 16 and 26. During weeks 1 and 26, a 6-hour profile of both CsA concentrations and calcineurin phosphatase activity after dose intake was obtained. On the other occasions a limited sampling strategy was applied, routinely measuring on t = 0, 2 and 3 h to estimate the AUC of CsA as described by Cremers et al[3]. Furthermore, baseline calcineurin activity was determined prior to the transplantation. Calcineurin phosphatase activity was determined in the white blood cell fraction with a spectrophotometric-assay based on phosphate quantification as described by Sellar et al[4]. Calcineurin activity was expressed in two ways, as enzyme activity per million white blood cells and per mg protein. CsA concentrations were determined in whole blood with a fluorescence polarization immunoassay (Axsym-Abbott). Non-linear-mixed-effects-modelling was used for data analysis (NONMEM VI)[5]. A PK-PD analysis was performed after obtaining accurate PK parameter estimates from the combination of this dataset with that of a densely sampled population of 33 renal transplant recipients (Press et al., submitted TDM 2009). Moreover, a series of covariates were collected to evaluate their effect on the PK and PD. These covariates include demographics (age, bodyweight, sex), prednisolone dose, white blood cell fraction differentiation (monocytes, lymphocytes, granulocytes (basophil, neutrophil, eosinophil)), amount of intracellular protein, and creatinin values. Finally, the occurrence of acute rejection episodes in the first 6 months after transplantation was obtained, while at 6 months a protocol biopsy was performed to identify subclinical rejection.
Results: CsA displayed variable and delayed absorption which could be described with a transit compartment, using a first-order rate constant describing the transfer from the dose compartment into the transit compartment and subsequently into the central compartment. Disposition and elimination was described by a two compartment model with first-order elimination. The PK parameters were allometrically scaled to bodyweight to the power of 0.75 and 1 for clearance and volume of distribution respectively. Furthermore, CsA disposition was affected by concomitant prednisolone administration, with a prednisolone dose over 20 mg resulting in a 15% higher apparent clearance and a 50% lower absorption rate constant. The PD relationship clearly was a direct-effect and could best be described with an Emax-model in the form: E = E0 * [1 - (Emax * CsA conc.) / (IC50 + CsA conc.)], with E0 defined as the baseline activity, Emax as the maximum effect, IC50 as the concentration at the half-maximal effect and finally CsA concentration as the concentration of cyclosporine A. The baseline activity (E0) with a median of 210 pmol/min/106 white blood cells or 12 pmol/min/mg protein showed considerable within subject variability of 28% for both activities, which was much greater than the between subject variability. A maximum inhibition (Emax) of 65% of the baseline activity and an IC50 of 150 μg/L were estimated, using both the activity expressed per million cells and per mg protein. The considerable within subject variability (inter-occasion variability) in the baseline hampered the identification of between subject variability in Emax and IC50. The cause of the inter-occasion variability could result from the biological system itself, from the calcineurin assay or it could be related to variability in the composition of the white blood cell sample. The biomarker was measured in white blood cell samples consisting of lymphocytes, granulocytes and monocytes. Each of these subsets could have a different activity of calcineurin. Moreover, they each have different protein contents in the cell. The amount of intracellular protein ranged from 93 mg to 407 mg with a median of 193 mg. Therefore, the covariate intracellular protein was used to explain part of the within subject variability and was structurally related to baseline activity (E0). The relationship was linear with an increase of 2.4% upon a 10 mg increase in amount of protein for activities expressed per million white blood cells. Finally, only 13 acute rejection episodes were observed in this AUC-controlled population, while 11 biopsies demonstrated subclinical acute rejection at 6 months after transplantation. The fact that clinical events still occur, despite exposure monitoring, indicates differences in susceptibility for CsA which provides a basis for biomarker use. However, the lack of observed inter-individual variability in the efficacy and potency parameters hindered the correlation with clinical outcome.
Conclusion and discussion: A clear concentration versus effect relationship between CsA and calcineurin activity was observed. However, the variability in the biomarker between occasions was too large to identify interindividual variability in efficacy and potency of CsA to inhibit calcineurin. Therefore, differences in susceptibility for CsA could not be related to this biomarker. Still, this biomarker seems relevant from a mechanistic point of view. Therefore, further optimization of this biomarker resulting in a reduction in the within subject variability might result in a clinically relevant biomarker. Indeed, such a biomarker can be used to identify which patients are more or less susceptible to CsA therapy compared to other. This could be achieved by improving the sample preparation procedure or by measuring calcineurin activity at the target site, i.e. calcineurin activity in T-cells.
References:
[1] Mahalati K, Belitsky P, Sketris I, et al. Neoral monitoring by simplified sparse sampling area under the concentration-time curve: its relationship to acute rejection and cyclosporine nephrotoxicity early after kidney transplantation. Transplantation 1999 Jul 15;68(1):55-62.
[2] Danhof M, Alvan G, Dahl SG, et al. Mechanism-based pharmacokinetic-pharmacodynamic modeling-a new classification of biomarkers. Pharm Res 2005 Sep;22(9):1432-7.
[3] Cremers SC, Scholten EM, Schoemaker RC, et al. A compartmental pharmacokinetic model of cyclosporin and its predictive performance after Bayesian estimation in kidney and simultaneous pancreas-kidney transplant recipients. Nephrol Dial Transplant 2003 Jun;18(6):1201-8.
[4] Sellar KJ, van Rossum HH, Romijn FP, et al. Spectrophotometric assay for calcineurin activity in leukocytes isolated from human blood. Anal Biochem 2006 Nov 1;358(1):104-10.
[5] NONMEM Users Guide (1989-2006) [computer program]. Icon Development Solutions; 2006.
