Comparison of the Emit Immunoassay with HPLC for Therapeutic Drug Monitoring of Mycophenolic Acid in Pediatric Renal-Transplant Recipients on Mycophenolate Mofetil Therapy

patients

This study was an open-label longitudinal evaluation of the PK/PD relationship of MPA in pediatric renal-transplant recipients. The inclusion and exclusion criteria have been described in previous reports (5)(17). The study protocol was approved by the local ethics committee of each contributing center. The analyses of the clinical results of the study have been published elsewhere (18) as have the PK/PD results according to MPA measurements with a validated HPLC procedure (19). The present comparison involved a subgroup of 50 patients [31 males, 19 females; mean age, 11.8 years (range, 3.2–16.0 years)], in whom both Emit and HPLC data were collected. All patients were Caucasian. Forty of the patients had primary transplant function; 10 had delayed graft function defined as the requirement for dialysis in the first 3 weeks posttransplant. No graft loss occurred in the delayed graft function group. Immunosuppressive therapy consisted of 300 mg/m² methylprednisolone on the day of transplant surgery, which was then tapered to 60 mg/m² for the first, 30 mg/m² for the second, 15 mg/m² for the third, 12 mg/m² for the fourth, 9 mg/m² for the fifth, and 6 mg/m² for the sixth week after transplantation and 4 mg/m² thereafter. Cyclosporin A (microemulsion formulation) was administered in a dose of 500 mg/m² per day given in two divided doses for 24 h starting 6 h after surgery. Thereafter, doses (∼300 mg/m² per day) were adjusted to achieve 12-h trough concentrations of 150–250 μg/L, as measured by whole-blood monoclonal fluorescence polarization immunoassay on a TDx analyzer (TDx mFPIA; Abbott), in the first 3 months posttransplant; thereafter, 12-h trough concentrations of 100–200 μg/L were targeted. The mean cyclosporin A dose and the respective 12-h predose concentrations at the four PK sampling time points are listed in Table 1⇓ . Serum creatinine (intra- and interassay CVs, 3% and 4.5%, respectively) was measured by a CX7 (Beckman Instruments). The glomerular filtration rate was estimated with the formula of Schwartz et al. (20), stratified according to the age and gender of the patients.

dosage of mmf

MMF was administered orally in a dose of 600 mg/m² twice a day up to a maximum of 2 g/day. This dose was based on a preliminary report of a dose-finding study in pediatric renal-transplant recipients (21). If the dose could not be administered exactly by use of 250-mg capsules, MMF capsules were opened and the exact dose for each individual child was refilled into gelatin capsules comparable to those produced by the MMF manufacturer. Body surface area was calculated by the formula of DuBois and DuBois (22).

pk protocol

Patients were studied after informed (parental) consent was obtained. Blood samples for PK assessments were drawn on days 7 and 21 posttransplant (“initial phase”) and 3 and 6 months posttransplant (“stable phase”). It was mandatory that all patients had at least 2 full days of the same MMF dose given twice a day before PK investigations. The study was performed in an inpatient environment, starting in the morning. Patients were required to fast from 2200 the night before sampling until after the 75-min sample had been obtained on the following morning. Blood samples were collected at the following times: before dosing and 20, 40, and 75 min and 2, 4, 6, 8, and 12 h after dosing. All blood samples were collected in tubes containing EDTA as an anticoagulant. For determination of MPA concentrations, plasma was separated and stored at −20 °C until analysis.

measurement of mpa by hplc and emit

The procedure for the determination of MPA in plasma by HPLC has been described in detail elsewhere (5)(23). Plasma MPA concentrations were also measured by the Emit MPA immunoassay (Dade-Behring) on a Cobas-Mira analyzer according to the manufacturer’s instructions, as described previously (24). A cross-check of the calibrators used for the HPLC and Emit assays revealed no calibrator bias (<5%) between the two procedures.

pk analysis

The following PK data for MPA were determined: predose concentration (c₀); time to maximum concentration [t_max (h)]; maximum concentration [c_max (mg/L)]; AUC from 0 to 12 h (AUC_0–12; mg · h/L) with the linear trapezoidal rule; and the evening predose (i.e., the 12-h) concentration [c₁₂ (mg/L)]. An abbreviated three-point AUC based on the sampling times 0 min, 75 min, and 4 h after MMF dosing (MPA-AUC_{0, 75 min, 4 h}) was calculated according to a previously published algorithm (25): estimated AUC = 11.8 + 3.71 × c₀ + 1.33 × c_{75 min} + 3.9 × c_{4 h}. In addition, an algorithm based on an empiric equation with a limited sampling strategy up to 2 h postdose (MPA-AUC_0–2) was taken for the calculation of the full AUC (11). The PK analysis was performed with the computer program BiAS (Epsilon-Verlag).

acute rejection episodes

The clinical diagnosis of rejection was made without any knowledge of the MPA concentration. Thirteen of 50 patients experienced at least one acute rejection episode during the 6-month study period; two of these patients had two rejection episodes, leading to a total number of 15 acute rejection episodes. Eleven of 15 acute rejection episodes were confirmed by biopsy; histologic examination and classification of a core biopsy was performed according to the Banff criteria (26). If a biopsy was logistically impossible or clinically contraindicated, the diagnosis of “presumed rejection” was established on the basis of clinical judgment (supported by one or more of the following clinical findings: increased body temperature, graft swelling, graft tenderness, increase in serum creatinine of >20% from the baseline concentration, or oliguria). The acute rejection episodes occurred 34 (median) days (range, 8–170 days) after renal transplantation; 12 of 15 rejections occurred within the first 70 days posttransplant.

adverse event monitoring

Adverse events, defined as an abnormal change in physical signs, symptoms, or laboratory values whether or not deemed causally related to the study medication, were recorded throughout the study when reported by a patient or noted by an investigator. Thrombocytopenia was defined as a thrombocyte count <150 × 10¹²/L. Leukopenia was defined as a granulocyte count <2000/μL and graded according to its severity: mild (1600–1999 granulocytes/μL); moderate (1000–1599 granulocytes/μL); or severe (≤999 granulocytes/μL). Diarrhea was graded as follows: mild diarrhea (n = 7), transient diarrhea lasting up to 2 days; moderate diarrhea (n = 3), tolerable diarrhea lasting longer than 2 days; and severe diarrhea (n = 1), intolerable diarrhea requiring therapy. Infections were classified as moderate when requiring specific antibiotic or antiviral therapy and as severe when requiring hospitalization. The following infections were recorded: herpes labialis (n = 5), herpes zoster (n = 1), oral mucocutaneous candidiasis (n = 1), urinary tract infection (n = 8), cytomegalovirus pneumonia (n = 1), cytomegalovirus colitis (n = 1), bacterial septicemia (n = 3), pneumonia (n = 1), pharyngitis (n = 1), febrile viral infection (n = 4).

statistics

The Shapiro–Wilk test was used to confirm normal distribution of data (27). Because not all values were gaussian, data in Table 1⇑ and 2⇓ are given as median (range). For comparison between two groups, the Wilcoxon signed-rank test was used. For comparison of more than two groups, one-way ANOVA on repeated measurements followed by all pairwise comparison (Student-Newman-Keuls test) was used. Correlations between variables were assessed by univariate linear regression analysis. Differences of P <0.05 were considered to be statistically significant.

ROC plots of sensitivity vs 1 − specificity were generated to determine whether a particular PK variable could discriminate patients with an acute rejection from those who experienced no rejection. Areas under the ROC curves and the 95% confidence interval limits were calculated with the method of Hanley and McNeil (28). The ROC curve analysis was carried out with Analyze-It software (Ver. 1.44; Analyze-It Software).

comparison of hplc and immunoassay

The PK variables for MPA as calculated from either the HPLC or the Emit assay data are shown in Table 2⇑ . Both in the initial and the stable phase, the PK variables calculated from data measured by the Emit assay were consistently higher than those calculated from the HPLC data. The median Emit MPA-AUC_0–12 values were 11.6% higher (1 week posttransplant), 17.4% higher (3 weeks), 10.3% higher (3 months), and 6.2% higher (6 months) than those obtained by HPLC. The Emit MPA c₁₂ values were 28.6% higher (1 week), 23.3% higher (3 weeks), 23.9% higher (3 months), and 16.7% higher (6 months) than the corresponding HPLC values. The overestimation of the Emit assay is primarily attributable to the cross-reactivity of the acyl glucuronide metabolite with the MPA antibody (23). Because the kidneys clear this metabolite, it could be expected that the overestimation by Emit is greater in patients with reduced renal function compared with the rest of the study population. Indeed, we observed an inverse curvilinear relationship between the proportional bias of MPA-AUC_0–12 values by Emit and the respective glomerular filtration rate (Fig. 1⇓ ).

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Figure 1.

Relationship between the proportional bias of MPA-AUC_0–12 values by Emit and the respective glomerular filtration rate (GFR).

There was an inverse curvilinear relationship (r = 0.55; P <0.01) that could be best described by the equation: y = 168.7 − (1.1597x) + (0.0061x²).

pk results

In agreement with our previous finding, there was a large interindividual variation of PK data, despite the fact that all patients were receiving the same body surface- adjusted MMF dosage (Table 2⇑ ). For example, MPA-AUC_0–12 values derived from Emit data at 3 months posttransplant ranged from 29.2 to 147 mg · h/L. The interindividual CV was comparable in the initial (21%) and stable phase (22%) posttransplant. Whereas the PK variables at 1 week and 3 weeks posttransplant were not significantly different, there was an increase in the PK variables c₀ (113%), c₁₂ (85%), and AUC_0–12 (97%) according to the HPLC data between the 3-week and the 3-month sampling times; the respective increases in the PK variables according to the Emit data were comparable (Table 2⇑ ). There was no further statistically significant increase of MPA PK variables between the 3- and 6-month sampling times, in agreement with our previous report in a smaller cohort of patients (17).

Because a full MPA-AUC that requires at least eight blood samples during a 12-h dose interval is impractical in clinical routine practice, we investigated whether a single-time point MPA concentration or an abbreviated AUC derived from a limited number of samples correlated with the respective full MPA-AUC. Only a moderate correlation was observed between either the predose MPA trough concentration (c₀) and the respective MPA-AUC_0–12 or the 12-h evening trough concentration (c₁₂; Table 3⇓ ). Although the c₀ concentration may have been taken at a more variable time after the last dose than the c₁₂ concentration, which was sampled exactly 12 h after a supervised dose, the correlations of c₁₂ with the full AUC were not superior to those of c₀. There was also only a moderate correlation between the MPA peak concentration (c_max) and the respective MPA-AUC_0–12. The abbreviated MPA-AUC_{0, 75 min, 4 h} gave a reasonable correlation with the full AUC that was somewhat superior to that seen between the abbreviated profile MPA-AUC_0–2 and the full AUC (Table 3⇓ ). There was a good agreement between the abbreviated AUC values as calculated from either the HPLC data or Emit data with the respective full AUC (Table 3⇓ ).

To provide the context for an assessment of PK variables in an individual patient in relation to the pediatric population distribution, 5th to 95th percentiles for MPA-AUC_0–12 and predose (c₁₂) concentrations were calculated according to the Emit measurements (Fig. 2⇓ ).

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Figure 2.

Time course of MPA-AUC_0–12 (A) and predose trough (c₁₂) plasma concentrations (B) derived from Emit measurements in the first 6 months after grafting in pediatric renal-transplant recipients.

Data are the median and the 5th to 95th percentiles of the total patient population (n = 50). All patients had received the full MMF dose of 600 mg/m² twice per day for at least 2 days before PK sampling. ○, episode of an acute rejection. The respective acute rejection episodes are graphically assigned to the nearest PK sampling period preceding the clinical event. The median time period between an acute rejection episode and the preceding PK sampling period was 8 days (range, 0–85 days).

pk/pd analysis regarding acute rejections

There were a total of 15 acute rejection episodes in 13 of the 50 patients during the 6-month study period. The MPA-AUC_0–12 and MPA-c₁₂ values determined by Emit for the patients with an acute rejection in relation to the respective percentiles of the whole study population are shown in Fig. 2⇑ . In the initial posttransplant phase, both the AUC_0–12 and MPA-c₁₂ values were distributed in the lower PK percentiles (Fig. 2⇑ ). Nine of 12 patients with rejections in the initial posttransplant phase had MPA-AUC values by Emit below the median of 37.5 mg · h/L before experiencing an acute rejection episode. The comparison with those patients who neither suffered from an acute rejection nor an adverse event yielded a relative risk of acute rejection of 42% with MPA-AUC_0–12 values <37.5 mg · h/L compared with 15% with MPA-AUC_0–12 values >37.5 mg · h/L. The cyclosporin A dose [rejecters, 7.5 (4.2–13) mg · kg⁻¹ · day⁻¹; nonrejecters, 8.0 (3.1–14) mg · kg⁻¹ · day⁻¹] and the 12-h predose whole-blood concentrations [rejecters, 187 (76–330) mg/L; nonrejecters, 190 (88–331) mg/L] were not significantly different between the two groups [data are median (range) of the respective mean data calculated from the PK-sampling time points at 1 week and 3 weeks posttransplant].

To establish which PK value is the best predictor for the risk of acute rejection, ROC curves were computed for each of the four PK variables. For these calculations, the mean values from the 1- and 3-week sampling time points were taken for each variable and patient. Diagnostic sensitivities (true-positive results) were calculated for each individual PK value as the fraction of those patients with an acute rejection who had a value below this discrimination threshold. The corresponding diagnostic specificities (false-negative results) were calculated as the fraction of patients with no rejection episode who had a value below this decision threshold. The ROC plots of sensitivity vs 1 − specificity for the PK variables AUC_0–12 and c₁₂ by HPLC and Emit are shown in Fig. 3⇓ , the statistical comparison of the areas under the ROC curves is given in Table 4⇓ . For the HPLC data, c₀, c₁₂, AUC_0–12, and the abbreviated AUC estimation AUC_{0, 75 min, 4 h} were able to discriminate patients with acute rejections from patients with no rejection. The Emit data gave comparable results. As expected, the corresponding decision thresholds, below which there is an enhanced risk of acute rejection, were higher for the Emit data than those noted for the HPLC data. An AUC_0–12 by HPLC of 33.8 mg · h/L had a diagnostic sensitivity of 80% and a diagnostic specificity of 57%. The corresponding value by Emit was 36.1 mg · h/L. For the PK variable c₁₂, a concentration of 1.2 mg/L determined by HPLC and 1.4 mg/L determined by Emit gave a sensitivity of 80% and a specificity of 60%.

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Figure 3.

ROC curves illustrating the ability of the pharmacokinetic variables MPA-AUC_0–12 (□) and predose trough (c₁₂) concentration (▪) to discriminate between patients with (n = 10) and without (n = 40) an acute rejection episode during the first 70 days posttransplant.

The PK variables were calculated from the HPLC (A) or Emit (B) data. The solid line represents the theoretical ROC curve for no discrimination between the two groups.

pk/pd analysis regarding adverse events

PK variables of total MPA, irrespective whether derived from HPLC or Emit measurements, were not discriminatory for leukopenia and/or infections, neither in the initial (Table 5⇓ ) nor the stable posttransplant phase (Table 6⇓ ). There was also no association between the incidence of diarrhea, anemia, or thrombocytopenia and any of the PK values derived from measurements of MPA (results not shown).