Limited Sampling Strategy for the Estimation of Mycophenolic Acid and its Acyl Glucuronide Metabolite Area under the Concentration-Time Curve in Japanese Lung Transplant Recipients.

PURPOSE
The dose of mycophenolate mofetil (MMF) used to prevent rejection after lung transplantation is often adjusted based on the 12-hour area under the concentration-time curve (AUC0-12) of mycophenolic acid (MPA). A limited sampling strategy (LSS) is useful to define the pharmacokinetic (PK) profiles of MPA and mycophenolic acid acyl glucuronide (AcMPAG). Therefore, this study aimed to design a LSS based on multiple linear regression for estimating the AUC0-12 of MPA and AcMPAG at the minimum blood sampling points in Japanese lung transplant patients with concomitant tacrolimus.


METHODS
Forty-five lung transplantation recipients were enrolled in a PK study of MPA, mycophenolic acid glucuronide (MPAG), and AcMPAG. The plasma MPA, MPAG, and AcMPAG concentrations were determined just before and at 0.5, 1, 2, 4, 8, and 12 hours after dosing. The AUC0-12 of MPA and AcMPAG was calculated using a linear trapezoidal rule from the plasma concentration of each blood sampling time. LSS was used to develop models for estimated AUC in the model group (n = 23) and was evaluated in the validation group (n = 22).


RESULTS
The best three time-point equation was 4.04 + 1.64·C1 + 3.08·C4 + 5.17·C8 for MPA, and -0.13 + 3.01·C1 + 3.51·C4 + 5.74·C8 for AcMPAG. The prediction errors (PE) and the absolute prediction errors (APE) were within the clinically acceptable ± 5% and 15% range, respectively (MPA: PE = 2.00%, APE = 11.66%, AcMPAG: PE = 0.98%, APE = 14.69%). The percentage of estimated AUC0-12 within ± 15% of the observed AUC0-12 was 77.27% for MPA and 81.82% for AcMPAG.


CONCLUSION
LSS using three time-point (C1, C4, and C8) provides the most reliable and accurate simultaneous estimation of the AUC0-12 of MPA and AcMPAG in Japanese lung transplant patients.


INTRODUCTION
Mycophenolate mofetil (MMF) is rapidly hydrolyzed in vivo to the immunosuppressant mycophenolic acid (MPA), which reversibly inhibits inosine 5′-monophosphate dehydrogenase, an enzyme involved in the de novo synthesis of guanosine in lymphocytes (1,2). Subsequently, MPA is predominantly metabolized to a pharmacologically inactive mycophenolic acid glucuronide (MPAG) and pharmacologically active mycophenolic acid acyl glucuronide (AcMPAG) by uridine diphosphate glucuronosyltransferase (3). On the other hand, MPAG is hydrolyzed back to MPA during enterohepatic recirculation, and its contribution to the total MPA exposure is approximately 40% (4).
MMF is administered in combination with a calcineurin inhibitor, such as tacrolimus or cyclosporine, and steroid to reduce the risk of rejection after lung transplantation (5). Several studies have reported that the 12-hour area under the concentration-time curve (AUC0-12) of MPA is a useful pharmacokinetic parameter for predicting clinical efficacy and rejection (6,7). Therefore, the dose of MMF is often adjusted based on the AUC0-12 of MPA.
Recently, Zegarska et al. have been reported that AcMPAG concentrations in liver transplant recipients is related to the development of bacterial infection (8). In addition, Yoshimura et al. have been reported that the cutoff values of AcMPAG AUC0-24 for successful gastrointestinal acute graft-versushost disease prevention in hematopoietic stem cell transplant patients were 15.6 μg·hr/mL (9). Accordingly, therapeutic drug monitoring (TDM) of AcMPAG is considered important in ensuring the safety and effectiveness of MMF treatment in both clinical practice and research.
The continuous measurement of MPA and AcMPAG AUC0-12 based on multiple blood sampling points increases the patient's burden. For this reason, a limited sampling strategy (LSS) estimating the AUC0-12 based on a limited number of blood samples is essential for defining the PK profiles of MPA and AcMPAG. However, LSS that simultaneously evaluates the AUC0-12 of MPA and AcMPAG has not been reported.
This study aimed to design a LSS based on multiple linear regression for estimating the AUC0-12 of MPA and AcMPAG at the minimum blood sampling points in Japanese lung transplant patients with concomitant tacrolimus.

Patients
This study was a single-center prospective study, was performed in Tohoku University Hospital from December 2016 to December 2017. The inclusion criteria were as follows: age ≥18 years, after lung transplantation, use of MMF, and the ability and willingness to provide written informed consent. We did not set the exclusion criteria. The chronic diseases that lead to lung transplantation were lymphangioleiomyomatosis (n=16), interstitial pneumonia (n=9), pulmonary hypertension (n=7), bronchiectasis (n=3), Eisenmenger syndrome (n=3), pulmonary emphysema (n=2), diffuse panbronchiolitis (n=2), cystic fibrosis (n=1), and other pulmonary disease (n=2). Forty-five transplant recipients were enrolled in a study investigating the pharmacokinetics of MPA, MPAG, and AcMPAG. This study protocol was approved by the Ethics Committee of Tohoku University Graduate School of Medicine (approval number: 2017-1-096). All patients were provided written informed consent.

Immunosuppression regimen
All patients received MMF, tacrolimus, and prednisolone as a basic triple immunosuppressive regimen in lung transplantation. MMF (CellCept ® ; Chugai Pharmaceutical Co., Ltd., Tokyo, Japan) was administered by intubation from the day 2 after surgery, and it was switched to oral administration after tube was withdrawn. MMF was administered at 1000 mg/day (body weight < 60 kg) or 1500 mg/day (body weight ≥ 60 kg). The dose of MMF was adjusted so that the white blood cell count was 4000 or more. The dose was reduced if abdominal symptoms were present. Tacrolimus was adjusted to maintain a target concentration of 10 to 14 ng/mL within 6 months after transplantation, 9 to 13 ng/mL from 7 months to 1 year after transplantation, and 8 to 10 ng/mL afterward. Prednisolone was orally administered at 1 mg/kg/day after transplantation, and then tapered to a fixed maintenance dose of 5 mg/day by 6 months after transplantation.

STATISTICAL ANALYSIS
According to the previous report (12)(13)(14), the patients were randomly assigned to two groups at a 1: 1 ratio: the model group (n = 23) and the validation group (n = 22). An AUC0-12 prediction formula was derived using multiple regression analysis with the model group AUC0-12 as a dependent variable and the plasma concentration at each blood sampling time as an explanatory variable. A maximum of three concentrations were used for the clinically convenient limited sampling strategy. In the validation group, the predictive performance of the LSS was analyzed with linear regression, correlation coefficient (r), prediction error (PE) (%), absolute prediction error (APE) (%), and the percentage of estimated AUC within ± 15% of the observed AUC as previous reported (15). The two error parameters were calculated using the following equations: where n is the number of patients. According to the previous report (15)(16)(17), the acceptable percentage limits of PE and APE were defined to be ± 5% and 15%, respectively. The Bland-Altman test was used to evaluate the agreement between the observed and estimated AUC, and the fixed range was defined as the mean ± 1.96SD. Continuous variables (expressed as mean ± SD) were compared using the t test, and categorical variables were compared using the Fisher's exact test. The significance level was set at P < 0.05. We used SAS Version 9.4 (SAS Institute Inc., Cary, NC, USA) for statistical analysis.

RESULTS
Forty-five patients (18 males and 27 females) were included in this study and the patients were randomly assigned to two groups at a 1: 1 ratio: the model group (n = 23) and the validation group (n = 22). Patient characteristics are summarized in Table  1. The mean age was 44.4 ± 11.6 years and the mean body weight was 51.5 ± 11.6 kg. There were no significant differences in sex, laboratory test results, concomitant medications affecting the pharmacokinetics of MPA (proton pump inhibitor, magnesium oxide, ciprofloxacin), MMF dose, pharmacokinetic parameters of MPA and metabolites, tacrolimus dose, or the post-transplant period between the model group and the validation group.
The mean plasma concentration-time profiles of MPA, MPAG, and AcMPAG in the model and validation groups are shown in Figure 1. These curves followed a similar tendency, whereby the peak was reached after 2 hours for MPA and 4 hours for MPAG and AcMPAG. Among these compounds, MPA and AcMPAG are related to clinical outcomes; therefore, we focused on both MPA and AcMPAG.  Multiple linear regression analyses of the MPA and AcMPAG AUC0-12 are shown in Table 2. The highest correlation coefficient between MPA AUC0- 12 and plasma MPA concentrations was at C1, C4, and C8 for three time points (MPA AUC0-12 = 4.04 + 1.64·C1 + 3.08·C4 + 5.17·C8, r = 0.923, P < 0.001).

DISCUSSION
In this study, we showed that the AUC0-12 can be predicted with high accuracy by LSS using plasma MPA and AcMPAG concentrations following MMF administration in Japanese lung transplantation patients. Both immunoassays and chromatographic methods are available for therapeutic drug monitoring of MPA. Although immunoassays are widely used in clinical laboratories due to ease of adopting such methods on automated analyzers, immunoassay such as a particle-enhanced turbidimetric inhibition immunoassay (PETINIA) shows cross-reactivity with AcMPAG. In fact, we have revealed an average positive bias of 26.3% in the PETINIA compared to that with LC-MS/MS in lung transplant patients (18). Furthermore, MPAG and AcMPAG can only be measured by LC-MS/MS. Therefore, plasma MPA, MPAG, and AcMPAG concentrations were quantified by LC-MS/MS in this study.
The pharmacokinetics of MPA and its metabolites are reported to be affected by the administration of concomitant drugs, and differ between lung and heart transplant patients (19)(20)(21)(22)(23). Therefore, the development of the LSS of MPA and AcMPAG should consider concomitant medication as well as the transplanted organ. Proton pump inhibitor or magnesium oxide reduces the solubility of MMF and decreases the drug exposure of mycophenolic acid (24,25). In addition, ciprofloxacin reduces plasma MPA concentration because of noncompetitive inhibition of deconjugation of MPAG by intestinal β-glucuronidase (26). However, there were no significant differences in baseline characteristics of patients including concomitant drugs between the model group and the validation group. The mean MPA, MPAG, and AcMPAG concentration-time profiles of all patients, the model group, and the validation group followed the same trend.
LSS in lung transplantation patients was previously reported by Ting et al (12). Those authors developed LSS by two time points (C0 and C2) in the same number of patients for cyclosporine and tacrolimus in combination; however, the correlation coefficient of the two time points (C0 and C2) was 0.699 in this study, and a good correlation was not observed (data not shown). Cyclosporine inhibits the biliary transporter ATP-binding cassette, subfamily C, member 2 (ABCC2), resulting in reduced enterohepatic re-circulation of MPAG/MPA (18)(19)(20), but tacrolimus has no such effect. For this reason, pharmacokinetics of MPA, MPAG, and AcMPAG are significantly different between cyclosporine and tacrolimus (29). All patients received tacrolimus, suggesting that the influence of C8 or C12 derived from the enterohepatic re-circulation of MPAG/MPA was greater than that reported by . Tacrolimus is used more commonly after lung transplantation due to its effect at reducing the risk of bronchiolitis obliterans syndrome and low levels of rejection as well as control of persistent rejection (32,33). Therefore, the formula used here to estimate AUC0-12 may be useful to adjust the dose of MMF in patients treated with tacrolimus after lung transplantation.
Blood sampling 8 hours after MMF administration is not practical because it restrains outpatients' activities for a long time. The same multiple regression method was performed with the variables within 2 hours post-dose (C0, C0.5, C1, and C2), but the correlation coefficient of the three time points was not sufficient (data not shown). When the trough concentration is included at blood sampling points within 4 hours, there was a good correlation with the three time-point equation (C0, C2, and C4) (MPA: r = 0.820, P < 0.001, AcMPAG: r = 0.969, P < 0.001). However, the APE exceeded 15%, and should therefore be carefully evaluated. Recently, we developed a LC-MS/MS method for the quantification of MPA, MPAG, and AcMPAG in dried blood spot samples (10). The dried blood spot method makes it possible to collect blood sample 8 hours after MMF administration without restraining the outpatients. Therefore, application of the dried blood spot method to outpatients may permit TDM using three-point blood sampling (C1, C4, and C8).
This study has some limitations that should be considered. All cases were Japanese lung transplant patients taking tacrolimus, and it is not evaluated by other races. In additon, no collected datapoint between 4 and 8 hours after MMF intake potentially related to the enterohepatic re-circulation of MPAG/MPA was not collected. Moreover, the PK profiles were obtained at approximately 4 years post-transplant, and genetic polymorphism for MPA and AcMPAG metabolism were not analyzed. We also need to consider using population PK modeling to make AUC estimation based on limited sampling more robust and enhance its prediction performance.

CONCLUSION
We established a formula to estimate the AUC0-12 of MPA and AcMPAG by LSS in Japanese lung transplant patients with concomitant tacrolimus. The best three time-point equation was 4.04 + 1.64·C1 + 3.08·C4 + 5.17·C8 for MPA, and -0.13 + 3.01·C1 + 3.51·C4 + 5.74·C8 for AcMPAG. It could be a useful tool to utilized for clinical practice and research.

CONFLICT OF INTEREST
No conflicts of interest to disclose.