Estimation of Abbreviated Cyclosporine A Area under the Concentration-Time Curve in Allogenic Stem Cell Transplantation after Oral Administration

Measurements of Cyclosporine (CsA) systemic exposure permit its dose adjustment in allogenic stem cell transplantation recipients to prevent graft-versus-host disease. CsA LSSs were developed and validated from 60 ASCT patients via multiple linear regressions. All whole-blood samples were analyzed by fluorescence polarization immunoassay (FPIA-Axym). The 10 models that have used CsA concentrations at a single time point did not have a good fit with AUC0–12 (R2 < 0.90). C 2 and C 4 were the time points that correlated best with AUC0–12 h, R2 were respectively 0.848, and 0.897. The LSS equation with the best predictive performance (bias, precision and number of samples) utilized three sampling concentrations was AUC0–12 h = 0.607 + 1.569 × C 0.5 + 2.098 × C 2 + 3.603 × C 4 (R2 = 0.943). Optimal LSSs equations which limited to those utilizing three timed concentrations taken within 4 hours post-dose developed from ASCT recipient's patients yielded a low bias <5% ranged from 1.27% to 2.68% and good precision <15% ranged from 9.60% and 11.02%. We propose an LSS model with equation AUC0–12 h = 0.82 + 2.766 × C 2 + 3.409 × C 4 for a practical reason. Bias and precision for this model are respectively 2.68% and 11.02%.


Introduction
Cyclosporine is one of the most common immunosuppressants used in allogenic stem cells transplant (ASCT) both in children and adults [1,2]. This population had more digestive problems compared to solid transplant patient; consisting of mucositis and diffuse inflammation of the intestinal tract related to the preparative regimen. In addition, they frequently develop digestive graft-versus-host disease (GVHD) and intestinal viral disease affecting the absorption of CsA [3][4][5]. In most case cyclosporine is administered intravenously from day −1 to days 30-50 then orally until day 180 [6].
Individualization of cyclosporine (CsA) dosage using therapeutic drug monitoring remains an indispensable tool for management of CsA therapy [7,8]. A number of clinical trials have employed C 2 monitoring in solid organ transplantations and demonstrated improvements in rejection rates and acute toxicity compared to trough-level (C 0 ) monitoring [9,10].
In ASCT Barkholt et al. [11] have not found a correlation between C 2 and either severs GVHD or infection in ASCT patients with C 0 guided CsA dosing. Furukawa et al., 2010 [12], demonstrate that in ASCT patients receiving twicedaily 3 h intravenous administrations, the concentration of CsA at 2 h (C 2 ) and 2 h and 50 min (C 3 ) were best single predictors of exposure (AUC 0-12 h ) while the trough concentration correlates poorly. Inoue et al. [13] were observed two close relationships between AUC 0-12 h and the C 3 for infusion and between AUC 0-12 h and the C 8 for oral administration.
The area under a blood drug concentration-time curve (AUC) is an important index in therapeutic drug monitoring  [14]. ASCT outcomes may be optimized by cyclosporine dose adjustment according to systemic exposure (measured by AUC) rather than trough concentration.
The aim of this study was to develop LSSs for predicting cyclosporine AUC 0-12 h to provide a practical method for a more accurate therapeutic (TDM) after oral dosing in patients undergoing ASCT and validated selected models.
CsA (Sandimmun) was started by continuous intravenous (i.v.) infusion from day 1 to 15-21 days after transplantation, and then patients were randomized to oral CsA (equoral or neoral) at twice IV dose. After that all patients received oral CsA (mean of 300 mg/day). The daily dose was taken in two equally divided doses at 12 h intervals.

Inclusion and Exclusion
Criteria . After the conditioning regimes, patients were included in the study, if they were aged between 3 and 50 years, clinically stable after a first ASCT, and receiving oral CsA as graft-versus-host disease prophylaxis. Patients should have stable serum creatinine (<2 baseline value) and no history of hepatic dysfunction (bilirubin and aminotransferases <2 normal value).
Patients excluded if they developed graft-versus-host disease or microangiopathy, received another drug which interferes with CsA pharmacokinetics, and showed evidence of noncontrol digestive problem, renal (2 × creatinine baseline value), or hepatic dysfunction (bilirubin and aminotransferases >2 × normal value).

Blood Sampling and Measurements of Cyclosporine Concentrations.
After written informed consent, blood samples (3 mL) were collected in tubes containing EDTA anticoagulant from the patients at time 0 (C 0 ; pre dose) and at 0.5 h (C 0.5 ), 1 h (C 1 ), 1.5 h (C 1.5 ), 2 h (C 2 ), 3 h (C 3 ), 4 h (C 4 ), 6 h (C 6 ), 8 h (C 8 ), and 12 h (C 12 ) after steady-state morning Cs dose. All whole-blood samples were analyzed by fluorescence polarization immunoassay (FPIA-Axym) in Laboratory of Clinical Pharmacology of the National Center of Pharmacovigilance. Precision data of immunoassay was demonstrated previously by comparison of the two methods for CsA therapeutic monitoring HPLC and AxSYM. Result showed that the correlation coefficient is 0.99 [15].

Pharmacokinetic and Statistical Analysis
2.4.1. Pharmacokinetics. Peak CsA concentration (C max ) and the time to reach it (T max ) were recorded for each patient and were reported as the mean ± standard deviation.
Pharmacokinetic parameters were calculated using a noncompartment model. The area under the plasma concentration-time curves AUC 0-12 h was calculated from drug plasma concentration data based on trapezoid rule [16]. Step 1. Multiple linear regressions were performed to estimate abbreviated AUC (dependent variable) and each time point of CsA levels (independent variables) that best fitted the CsA AUC 0-12 h . Selected models are those who have P < 0.05 for any sampling time and high correlation coefficient (R 2 > 0.9). These analyses produced equations AUC as follows: AUC = Cst + M 1 × C 1 + M 2 × C 2 + · · · + M n × C n ; M and Cst are coefficients and n is the number of samples.

Limited
Optimal LSS equations were limited to those utilizing a maximum of four timed concentrations taken within 6 hours after dose, and with a coefficient of determination (R 2 ) > 0.9.
Step 2. To validate the equation, we used a second group of pharmacokinetic profile data obtained from ASCT patients recipients (validation group) with the same characteristics as the initial group. Predictive performances of LSS equations were evaluated by calculating the percentage bias (mean prediction error me %) and percentage precision (root-meansquared prediction error rmse %) [17,18]. 1 Model   All statistical analyses were performed using SPSS software for windows (version 11.5.0 Inc., Chicago, Illinois). Data were expressed as mean ± SD.

Results
Thirty full pharmacokinetic profiles of CsA were obtained ( Figure 1). The mean CsA dose at the time of the measurement was 113 ± 40.85 mg. The peak concentration of CsA generally accrued 2-3 hours after morning dosing. Mean trough concentration C 0 was 0.470 ± 0.178 µg·mL −1 . The mean AUC 0-12 h was 4.340 ± 1.195 µg·h·mL −1 ( Table 2).
The linear regression equations and the concentration at each time point are listed in Table 3. The 10 models that used CsA concentrations at a single time point did not have a good fit (R 2 < 0.90). C 2 and C 4 were the time points that correlated best with AUC 0-12 h , R 2 were, respectively, 0.848 and 0.897. Moreover, the correlation between AUC 0-12 h and the trough concentration (C 0 or C 12 ) was poor, R 2 = 0.391 for C 0 and R 2 = 0.786 for C 12 ( Table 3). The correlations between AUC 0-12 h and CsA concentrations at various points C 0 , C 2 , and C 4 , are demonstrated in Figures 2(a), 2(b), and 2(c). The dose of CsA per kilogram of body weight and total dose of CsA did not correlate with AUC 0-12 h (data not shown).
Pharmacokinetic data from 30 ASCT recipient's patients were used to test the LSS equations. Multiple linear regression analysis of the correlation between the estimated cyclosporine (CsA) AUC 0-12 h and full hours CsA AUC were listed in Table 4.

Discussion
Graft-versus-host disease (GVHD) remains a major limiting factor for a successful result after ASCT; it affects mortality, morbidity, and quality of life. CsA-based immunosuppression has been the most frequently used regimen for prophylaxis and treatment of GVHD in patients undergoing HSCT [19,20]. Therapeutic monitoring of CsA provides a more accurate measure of posttransplant immunosuppression and this by assessment of pharmacokinetic parameters. The area under concentration-time curve (AUC) is a more important index in therapeutic drug monitoring and in pharmacokinetics. In this investigation patients were received a mean dose of cyclosporine A (neoral or equoral) 113.0 ± 40.85 mg. A recent study demonstrates that two formulations were bioequivalent in ASCT patients [21]. The time to reach C max (T max ) in this study was 1.908 ± 0.954; this is similar to the values reported for other HSCT patients, 1.9 ± 0.8 h [22] 2.4 ± 1.1 h [3]. As previous studies have reported, T max for HSCT recipients has shown that absorption is delayed in comparison to that measured in solid organ allograft recipients [22]. It is thought that the differences are due to the presence of gastrointestinal inflammation caused by mucositis or GVHD [3].
A number of trials have employed C 2 monitoring in solid organ transplantation [23], C 2 monitoring is being increasingly employed in the management of solid organ recipients with a recent survey of renal transplant centres finding that most of the respondents were now measuring C 2 concentration [24]. In a stem cells transplant setting, where it is clear that current practice of trough-level monitoring still results in unacceptably high levels of GVHD, therefore is a scope to explore the potential of monitoring the drug (both I.V and oral) via alternative measures of exposure or activity [1]. A recent investigation demonstrate a close relationship between AUC 0-12 h and the C 8 after oral administration of CsA in allogenic haematopoietic stem cell transplantation (ASTH) [13], and between AUC 0-12 h and two concentrations C 3 and C 2 after twice-daily infusion [12,13]. Therapeutic monitoring of Tunisian ASTH patients was based on C 0 determination; this concentration is weakly correlated with AUC 0-12 h (R 2 = 0.391). That we bring to search what concentrations are well correlated with AUC and how to estimate abbreviated AUC in ASCT.
The major findings of this study are that the concentration measurements at C 2 and C 4 were the time points that correlated best with AUC 0-12 h after oral administration of CsA in allogenic stem cells transplant patients rather than the trough level. These time points provide a more accurate measure of posttransplant immunosuppression. Therefore, the target concentration of CsA in ASHT patients, after the switch to oral administration using C 2 monitoring to adjust the dose, is easily applied the same level of C 4 monitoring with oral administration.

Conclusion
Limited sampling strategy models were selected to predict the full 12-hour CsA AUC in allogenic haematopoietic stem cell transplant patients receiving CsA. We propose an LSS model with equation AUC 0-12 h = 0.82 + 2.766 × C 2 + 3.409 × C 4 , which is limited to utilizing two timed concentrations taken within 4 hours after dose and providing a low bias and a good precision, it is for a practical reason.