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Comparison of the Emit immunoassay with HPLC for therapeutic drug monitoring of mycophenolic acid in pediatric renal-transplant recipients on mycophenolate mofetil therapy.

Mycophenolate mofetil (MMF) [4] is widely used for main tenance immunosuppressive therapy both in adult (1) and pediatric renal-transplant recipients (2). MMF is rapidly metabolized in vivo to its active constituent, mycophenolic acid (MPA), a reversible, noncompetitive inhibitor of inosine monophosphate dehydrogenase. Inhibition of inosine monophosphate dehydrogenase II in activated lymphocytes causes a reduction in intracellular guanine nucleotide pools and leads to an arrest of lymphocyte proliferation. MPA is extensively bound to albumin, with a protein-binding range of 97-99% in patients with healthy renal and liver function (3-5). The primary metabolite of MPA is the phenolic MPA glucuronide (MPAG) 7-O-MPAG. Two further metabolites have been identified in humans, namely the acyl glucuronide (AcMPAG) and the phenolic glucoside of MPA (6). Of these three metabolites, only the acyl glucuronide is capable of inhibiting human inosine monophosphate dehydrogenase II (7) and proliferation of human mononuclear leukocytes (8) in vitro.

The pharmacokinetic (PK) profile of MPA shows large interindividual variability. PK monitoring of MPA with the aim of optimizing the dosage of this drug to achieve adequate immunosuppression with minimized risk of graft rejection or toxicity has therefore been under investigation (9). In adult renal-transplant recipients on cyclosporin A, MMF, and steroids, PK/pharmacodynamic (PD) relationships between the area under the curve (AUC) of MPA or predose MPA concentrations and the risk of acute rejection have been established on the basis of measurements by reversed-phase HPLC with ultraviolet detection (9-11). Low MPA-AUC values estimated with an abbreviated sampling schedule and a validated HPLC procedure were found to be associated with cardiac allograft rejection in heart transplant recipients (12). A commercial, Emit-based immunoassay is available for the determination of MPA. In general, the Emit assay has the advantage of being less laborious and time-consuming and therefore better suited for routine drug monitoring. In a study involving heart-transplant recipients receiving a combination therapy of tacrolimus and MMF, low plasma predose MPA concentrations determined by the Emit procedure were found to be associated with an increased incidence of acute rejection (13). Several studies revealed a systematic positive bias between the results obtained with the Emit assay and those found with HPLC (14-16). This bias is primarily attributable to cross-reactivity of the immunosuppressive metabolite AcMPAG with the antibody used in the Emit MPA assay (16). In contrast, 7-O-MPAG does not display any notable cross-reactivity in this assay.

The interpretation of the PK/PD relationship of MPA in different patient populations can be influenced by several factors, such as the nature of the organ transplanted, the age of the patient, use of concomitant immunosuppressive therapy, protein binding, the presence of active metabolites and the assay used. The purpose of this study, therefore, was to investigate the following: (a) the clinical utility of the Emit assay in comparison with HPLC for identifying patients at risk of acute graft rejection and MMF-related side effects in pediatric renal-transplant recipients on an immunosuppressive regimen with cyclosporin A, MMF, and corticosteroids; (b) the method dependent therapeutic ranges for MPA-[AUC.sub.0-12] values and predose MPA concentrations in this patient population; and (c) the clinical effectiveness of an abbreviated AUC estimation protocol in comparison to the full MPA-AUC and single time point MPA concentrations.

Materials and Methods

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/[m.sup.2] methylprednisolone on the day of transplant surgery, which was then tapered to 60 mg/[m.sup.2] for the first, 30 mg/[m.sup.2] for the second, 15 mg/[m.sup.2] for the third, 12 mg/[m.sup.2] for the fourth, 9 mg/[m.sup.2] for the fifth, and 6 mg/[m.sup.2] for the sixth week after transplantation and 4 mg/[m.sup.2] thereafter. Cyclosporin A (microemulsion formulation) was administered in a dose of 500 mg/[m.sup.2] per day given in two divided doses for 24 h starting 6 h after surgery. Thereafter, doses (~300 mg/[m.sup.2] per day) were adjusted to achieve 12-h trough concentrations of 150-250 [micro]g/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 [micro]g/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/[m.sup.2] 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[degrees]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 (bade-Behring) on a CobasMira 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 ([c.sub.0]); time to maximum concentration [[t.sub.max] (h)]; maximum concentration [[c.sub.max] (mg/L)]; AUC from 0 to 12 h ([AUC.sub.0-12]; mg x h/L) with the linear trapezoidal rule; and the evening predose (i.e., the 12-h) concentration [[c.sub.12] (mg/L)]. An abbreviated three-point AUC based on the sampling times 0 min, 75 min, and 4 h after MMF dosing (MPA-[AUC.sub.0,75 min, 4 h]) was calculated according to a previously published algorithm (25): estimated AUC = 11.8 + 3.71 X [c.sub.0] + 1.33 X [c.sub.75 min] + 3.9 X [c.sub.4 h]. In addition, an algorithm based on an empiric equation with a limited sampling strategy up to 2 h postdose (MPA-[AUC.sub.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 x [10.sup.12]/L. Leukopenia was defined as a granulocyte count <2000/[micro]L and graded according to its severity: mild (1600-1999 granulocytes/[micro]L); moderate (1000-1599 granulocytes/[micro]L); or severe ([less than or equal to] 999 granulocytes/[micro]L). 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 signedrank test was used. For comparison of more than two groups, one-way AMOVA 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).

Results

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.sub.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 [c.sub.12] 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.sub.0-12] values by Emit and the respective glomerular filtration rate (Fig. 1).

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.sub.0-12] values derived from Emit data at 3 months posttransplant ranged from 29.2 to 147 mg x 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 [c.sub.0] (113%), [c.sub.12] (85%), and [AUC.sub.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).

[FIGURE 1 OMITTED]

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 (co) and the respective MPA-[AUC.sub.0-12] or the 12-h evening trough concentration ([c.sub.12]; Table 3). Although the [c.sub.0] concentration may have been taken at a more variable time after the last dose than the [c.sub.12] concentration, which was sampled exactly 12 h after a supervised dose, the correlations of [c.sub.12] with the full AUC were not superior to those of [c.sub.0]. There was also only a moderate correlation between the MPA peak concentration ([c.sub.max]) and the respective MPA-[AUC.sub.0-12]. The abbreviated MPA-[AUC.sub.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.sub.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.sub.0-12] and predose ([c.sub.12]) concentrations were calculated according to the Emit measurements (Fig. 2).

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.sub.0-12] and MPA-[c.sub.12] 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.sub.0-12] and MPA-[c.sub.12] 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 x 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.sub.0-12] values <37.5 mg x h/L compared with 15% with MPA-[AUC.sub.0-12] values >37.5 mg x h/L. The cyclosporin A dose [rejecters, 7.5 (4.2-13) mg x [kg.sup.-1] x [day.sup.-1]; nonrejecters, 8.0 (3.1-14) mg x [kg.sup.-1] x [day.sup.-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].

[FIGURE 2 OMITTED]

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.sub.0-12] and [c.sub.12] 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, [c.sub.0], [c.sub.12], [AUC.sub.0-12], and the abbreviated AUC estimation [AUC.sub.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.sub.0-12] by HPLC of 33.8 mg x h/L had a diagnostic sensitivity of 80% and a diagnostic specificity of 57%. The corresponding value by Emit was 36.1 mg x h/L. For the PK variable [c.sub.12], 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%.

[FIGURE 3 OMITTED]

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).

Discussion

This is the first report in pediatric renal-transplant recipients demonstrating that the Emit assay appears to be comparable to the HPLC methodology for assessing the risk of acute rejection. We have shown that both MPA-[AUC.sub.0-12] and predose MPA trough concentrations are significantly associated with the risk of acute rejection in this patient population irrespective of which methodology is used to measure plasma MPA. However, because of the cross-reactivity of the antibody used in the Emit assay with the active metabolite acyl-MPAG, the decision thresholds for the Emit are higher than those reported with HPLC. From our data, an MPA-[AUC.sub.0-12] of 36.1 mg x h/L derived from Emit measurements will have a clinical efficacy comparable to a HPLC-derived threshold of 33.8 mg x h/L. In the case of predose MPA plasma concentrations, an Emit value of 1.4 mg/L is comparable to a HPLC value of 1.2 mg/L. The HPLC methodology is generally laborious and time-consuming, and therefore is less suited for routine clinical monitoring than immunoassay procedures. In contrast to the immunoassay, sample pretreatment is necessary for the HPLC determination of MPA. In our experience, up to 50 samples can be analyzed during 24 h by our HPLC method in combination with an autosampling device. The same number of samples can be analyzed within 2-3 h by the Emit procedure. Immunoassays, however, are often less specific than HPLC-based procedures because of the cross-reactivity of the antibody with structurally similar drug metabolites that may be pharmacologically inactive. In the case of the Emit immunoassay for MPA, cross-reactivity occurs with only one of the known metabolites of MPA, AcMPAG (6, 24), and this metabolite is potentially pharmacologically active (7, 8). The Emit assay, therefore, has the theoretical advantage to measure both MPA and its pharmacologically active metabolite. One could therefore have expected the PK variables derived from the Emit measurements to be superior to the HPLC measurements for the discrimination of acute rejection episodes. However, this was not the case. The fact that the cross-reactivity of the acyl glucuronide is concentration dependent (24) and that the AcMPAG-[AUC.sub.0-12] values are relatively low in relation to the MPA-[AUC.sub.0-12] values (mean 16% of MPA-[AUC.sub.0-12]) (29) may explain these findings.

The incidence of the MMF-related adverse events diarrhea, vomiting, or abdominal pain was not associated with MPA PK variables, consistent with previous findings in adult renal-transplant recipients (10). The adverse events leukopenia and/or infections were also not associated with any of the PK variables derived from total MPA measurements. This finding is an agreement with the results of the Randomized Concentration Controlled Study in adult renal-transplant recipients (10), but in contrast to the recent report of Mourad et al. (30) from an unicenter trial in 31 adult renal-transplant recipient. In this study, a MPA concentration 30 min after the oral dose of MMF ([c.sub.30]) was associated with an increased risk for MMF-related side effects. This difference may be attributable to the high preponderance of leukopenia (60% of all side effects) in the latter study presumably as a consequence of induction therapy with anti-thymocyte globulin, whereas in our study without induction of antithymocyte globulin, the incidence of leukopenia was rather low (four episodes during the observation period of 6 months). Recently, Mourad et al. (31) observed a relationship between total plasma MPA concentrations and toxicity of either gastrointestinal (42%) or hematologic origin (58%) in a patient population on a tacrolimus-based immunosuppressive regimen. However, because the incidence of gastrointestinal side effects is higher in patients on tacrolimus vs cyclosporin A (32) and PK variables of MPA are differentially influenced by coadministration of tacrolimus vs cyclosporin A (33, 34), the report of Mourad et al. (31) is not necessarily contradictory to our observations. In this context, it is noteworthy that data from our group (19) and other investigators (35) suggest that measurement of free MPA by HPLC appears to be more appropriate for the assessment of the toxic risk of MMF regarding leukopenia and/or infections.

There is currently only limited information on the clinical utility of the Emit immunoassay. Meiser et al. (13) used serial daily measurements of predose MPA concentrations determined with the Emit assay (target range, 2.5-4.5 mg/L) to individualize MMF dosage in heart-transplant recipients on concomitant therapy with tacrolimus. In comparison to a previous fixed dose regimen of MMF, they found a reduced incidence of acute rejection in the collective with dosage individualization. Similarly, Yamani et al. (36), in a study of 215 heart-transplant recipients, observed in the presence of therapeutic cyclosporin A or tacrolimus blood concentrations a significantly decreased incidence of acute rejecuon when MPA trough concentrations determined by Emit was [greater than or equal to] 2 mg/L compared to samples with MPA trough concentrations <2 mg/L.

The results of our study illustrate that the Emit immunoassay will be applicable for monitoring MMF therapy in renal transplantation. It has to be emphasized that the value of therapeutic drug monitoring of MPA has not been formally tested. However, given the association between MPA PK variables and the risk of acute rejection, therapeutic drug monitoring of MPA in the initial post-transplant phase could be helpful in selected high-risk patients. It would be reasonable to aim at achieving MPA-AUC values or predose MPA concentrations somewhat higher than those achieved in the lower percentiles by individual adjustment of the MMF dose. For such an approach, the percentiles specific to patients and immunosuppressive regimens for MPA-AUC and MPA predose concentrations from this study, according to both HPLC and Emit immunoassay measurements, could serve as guidelines for optimization of MMF therapy in pediatric renal-transplant recipients. Data from this and other studies indicate that a consensus is arising that for minimizing the risk of rejection after transplantation, total MPA-AUC values in the early posttransplant stages should be maintained within the therapeutic window of 30-60 mg x h/L, according to HPLC data, or 35 to 70 mg x h/L, according to Emit data. The target predose concentration should be maintained within the range of 1-3.5 mg/L, according to HPLC data, and 1.3-4.5 mg/L, according to Emit data. Because some fluctuations are to be expected as a consequence of the enterohepatic circulation of MPA, extreme values or a large change should be verified by repeat measurement before a change in the MMF dose is undertaken.

The question arises as to which PK variable of MPA is best suited for therapeuuc drug monitoring of MMF therapy in clinical practice. In this study, the variables MPA-[AUC.sub.0-12], MPA-[AUC.sub.0, 75 min, 4 h] MPA [c.sub.12], and MPA co were comparable for assessing the risk of acute rejection episodes, whereas MPA-[AUC.sub.0-2] had a somewhat poorer predictive value (Table 4). In two previous studies in adult renal-transplant recipients, the MPA-AUC was a better predictor of outcome than the predose concentration (11, 37). Predose concentrauons are more convenient than measurements of AUC, which are complex and affect the cost of routine clinical monitoring. However, the predose [c.sub.0] concentration may be sampled at a more variable time after the last dose. Hence, the calculation of AUC with a limited sampling strategy of up to 4 h after MMF dosing (MPA-[AUC.sub.0, 75 min, 4 h]) appears be a more precise approach for the assessment of the risk of acute rejection than the predose [c.sub.0] concentration.

In conclusion, we have established that the Emit assay is comparable to the HPLC methodology for assessing this risk of acute rejection in pediatric renal-transplant recipients on an immunosuppressive triple-drug therapy with cyclosporin A, MMF, and corticosteroids. We have established putative therapeutic ranges for these PK variables according to the Emit immunoassay measurements in this specific patient populauon. These data could facilitate the therapeutic drug monitoring of MPA for optimization of MMF efficacy by steering patients away from the extreme values of MPA PK variables.

Contributing investigators (in alphabetical order of the centers in Germany): M. Boswald and W. Rascher (Erlangen); M. Schroder and J. Dippell (Frankfurt); L. B. Zimmerhackl, A. Staskewitz, and M. Brandis (Freiburg); L. Weber, N. Wagner, and B. Tonshoff (coordinators; Heidelberg); U. John, L. Patzer, and J. Misselwitz (Jena); U. Querfeld (Koln); C. Greiner and H. Eichstatt (Leipzig); G. Klaus and H. W. Seyberth (Marburg); B. Klare (Munchen); M. Bulla and E. Kuwertz-Broking (Munster); and M. Holder and H. Leichter (Stuttgart, Germany). This study was supported by a research grant from Hoffmann-La Roche AG (Grenzach-Wyhlen, Germany). M.S. was supported by a grant from the Volkswagen-Stiftung. The Emit assays were a gift from Dade Behring, Germany. We thank Tanja Schneider and Christa Scholz for excellent technical assistance.

References

(1.) Mele TS, Halloran PF. The use of mycophenolate mofetil in transplant recipients. Immunopharmacology 2000;47:215-45.

(2.) Benfield MR, Stablein D, Tejani A. Trends in immunosuppressive therapy: a report of the North American Pediatric Renal Transplant Cooperative Study (NAPRTCS). Pediatr Transplant 1999;3:27-32.

(3.) Bullingham RES, Nicholls A, Hale M. Pharmacokinetics of mycophenolate mofetil (RS 61443). A short review. Transplant Proc 1996;28:925-9.

(4.) Nowak J, Shaw LM. Mycophenolic acid binding to human serum albumin: characterization and relation to pharmacodynamics. Clin Chem 1995;41:1011-7.

(5.) Weber LT, Shipkova M, Lamersdorf T, Niedmann PD, Wiesel M, Mandelbaum A, et al. Pharmacokinetics of mycophenolic acid (MPA) and determinants of MPA free fraction in pediatric and adult renal transplant recipients. J Am Soc Nephrol 1998;9:1511-20.

(6.) Shipkova M, Armstrong VW, Wieland E, Niedmann PD, Schutz E, Brenner-Weiss G, et al. Identification of glucoside and carboxyl-linked glucuronide conjugates of mycophenolic acid in plasma of transplant recipients treated with mycophenolate mofetil. Br J Pharmacol 1999;126:1075-82.

(7.) Schutz E, Shipkova M, Armstrong VW, Wieland E, Oellerich M. Identification of a pharmacologically active metabolite of mycophenolic acid in plasma of transplant recipients treated with mycophenolate mofetil. Clin Chem 1999;45:419-22.

(8.) Shipkova M, Wieland E, Schutz E, Wiese C, Niedmann PD, Oellerich M, et al. The acyl glucuronide metabolite of mycophenolic acid inhibits the proliferation of human mononuclear leukocytes. Transplant Proc 2001;33:1080-1.

(9.) Shaw LM, Nicholls A, Hale M, Armstrong VW, Oellerich M, Yatscoff R, et al. Therapeutic monitoring of mycophenolic acid. A consensus panel report. Clin Biochem 1998;31:317-22.

(10.) van Gelder T, Hilbrands LB, Vanrenterghem Y, Weimar W, de Fijter JW, Squifflet JP, et al. A randomized double-blind, multicenter plasma concentration controlled study of the safety and efficacy of oral mycophenolate mofetil for the prevention of acute rejection after kidney transplantation. Transplantation 1999;68:261-6.

(11.) Hale MD, Nicholls AJ, Bullingham RE, Hene R, Hoitsma A, Squifflet JP, et al. The pharmacokinetic-pharmacodynamic relationship for mycophenolate mofetil in renal transplantation. Clin Pharmacol Ther 1998;64:672-83.

(12.) DeNofrio D, Loh E, Kao A, Korecka M, Pickering FW, Craig KA, et al. Mycophenolic acid concentrations are associated with cardiac allograft rejection. J Heart Lung Transplant 2000;1911:1071-6.

(13.) Meiser BM, Pfeiffer M, Schmidt D, Reichenspurner H, Ueberfuhr P, Paulus D, et al. Combination therapy with tacrolimus and mycophenolate mofetil following cardiac transplantation: importance of mycophenolic acid therapeutic drug monitoring. J Heart Lung Transplant 1999;18:143-9.

(14.) Schutz E, Shipkova M, Armstrong VW, Niedmann PD, Weber L, Tonshoff B, et al. Therapeutic drug monitoring of mycophenolic acid: comparison of HPLC and immunoassay reveals new MPA metabolites. Transplant Proc 1998;30:1185-7.

(15.) Beal JL, Jones CE, Taylor PJ, Tett SE. Evaluation of an immunoassay (EMIT) for mycophenolic acid in plasma from renal transplant recipients compared with a high-performance liquid chromatography assay. Ther Drug Monit 1998;20:685-90.

(16.) Shipkova M, Schutz E, Armstrong VW, Niedmann PD, Wieland E, Oellerich M. Overestimation of mycophenolic acid by EMIT correlates with MPA metabolite. Transplant Proc 1999;31:1135-7.

(17.) Weber LT, Lamersdorf T, Shipkova M, Niedmann PD, Wiesel M, Zimmerhackl LB, et al. Area under the plasma concentration-time curve for total, but not for free, mycophenolic acid increases in the stable phase after renal transplantation: a longitudinal study in pediatric patients. German Study Group on Mycophenolate Mofetil Therapy in Pediatric Renal Transplant Recipients. Ther Drug Monit 1999;2:498-506.

(18.) Staskewitz A, Kirste G, Tonshoff B, Weber LT, Boswald M, Burghard R, et al. Mycophenolate mofetil in pediatric renal transplantation without induction therapy: results after 12 months of treatment. Transplantation 2001;71:638-44.

(19.) Weber LT, Shipkova M, Armstrong VW, Wagner N, Schutz E, Mehls O, et al. The pharmacokinetic-pharmacodynamic relationship for total and free mycophenolic acid in pediatric renal transplant recipients: a report of the German Study Group on Mycophenolate Mofetil Therapy. J Am Soc Nephrol 2002, in press.

(20.) Schwartz GJ, Brion LP, Spitzer A. The use of plasma creatinine concentration for estimating glomerular filtration rate in infants, children, and adolescents. Pediatr Clin North Am 1987;34:571-90.

(21.) Ettenger RB, Warshaw B, Mentser M, Potter D, Moulton L, Marik J, et al. Mycophenolate mofetil (MMF) in pediatric renal transplantation [Abstract]. Pediatr Nephrol 1996;10:C39.

(22.) DuBois D, DuBois EF. A formula to estimate the approximate surface area if height and weight be known. Arch Intern Med 1916;17:863-71.

(23.) Shipkova M, Niedmann PD, Armstrong VW, Schutz E, Wieland E, Shaw LM, et al. Simultaneous determination of mycophenolic acid and its glucuronide in human plasma using a simple high-performance liquid chromatography procedure. Clin Chem 1998; 44:1481-3.

(24.) Shipkova M, Schutz E, Armstrong VW, Niedmann PD, Oellerich M, Wieland E. Determination of the acyl glucuronide metabolite of mycophenolic acid in human plasma by HPLC and Emit. Clin Chem 2000;46:365-72.

(25.) Oellerich M, Shipkova M, Schutz E, Wieland E, Weber L, Tonshoff B, 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.

(26.) Solez K, Axelsen RA, Benediktsson H, Burdick JF, Cohen AH, Colvin RB, et al. International standardization of criteria for the histologic diagnosis of renal allograft rejection: the Banff working classification of kidney transplant pathology. Kidney Int 1993;44: 411-22.

(27.) Zar JH. Biostatistical analysis. Englewood Cliffs, NJ: Prentice Hall, 1984.

(28.) Hanley JA, McNeil BJ. A method of comparing the areas under receiver operating characteristic curves derived from the same cases. Radiology 1983;148:839-43.

(29.) Weber LT, Shipkova M, Schutz E, Mehls O, Oellerich M, Armstrong VW, et al. Pharmacokinetics of the pharmacologically active acyl glucuronide metabolite (AcMPAG) of mycophenolic acid (MPA) in pediatric renal transplant recipients [Abstract]. Pediatr Transplant 2000;4:67-8.

(30.) Mourad M, Malaise J, Chaib Eddour D, De Meyer M, Konig J, Schepers R, et al. Correlation of mycophenolic acid pharmacokinetic parameters with side effects in kidney transplant patients treated with mycophenolate mofetil. Clin Chem 2001;47:88-94.

(31.) Mourad M, Malaise J. Chaib Eddour D, De Meyer M, Konig J, Schepers R, et al. Pharmacokinetic basis for the efficient and safe use of low-dose mycophenolate mofetil in combination with tacrolimus in kidney transplantation. Clin Chem 2001;47:1241-8.

(32.) Mayer AD, Dmitrewski J, Squifflet JP, Besse T, Grabensee B, Klein B, et al. Multicenter randomized trial comparing tacrolimus (FK506) and cyclosporine in the prevention of renal allograft rejection: a report of the European Tacrolimus Multicenter Renal Study Group. Transplantation 1997;64:436-43.

(33.) Zucker K, Tsaroucha A, Olson L, Esquenazi V, Tzakis A, Miller J. Evidence that tacrolimus augments the bioavailability of mycophenolate mofetil through the inhibition of mycophenolic acid glucuronidation. Ther Drug Monit 1999;21:35-43.

(34.) Van Gelder T, Klupp J, Barten MJ, Christians U, Morris RE. Coadministration of tacrolimus and mycophenolate mofetil does not increase mycophenolic acid (MPA) exposure, but coadministration of cyclosporine inhibits the enterohepatic recirculation of MPA, thereby decreasing its exposure [Abstract]. Transplantation 2000;69(Suppl):S192.

(35.) Kaplan B, Gruber SA, Nallamathou R, Katz SM, Shaw LM. Decreased protein binding of mycophenolic acid associated with leukopenia in a pancreas transplant recipient with renal failure. Transplantation 1998;65:1127-9.

(36.) Yamani MH, Starling RC, Goormastic M, Van Lente F, Smedira N, McCarthy P, et al. The impact of routine mycophenolate mofetil drug monitoring on the treatment of cardiac allograft rejection. Transplantation 2000;69:2326-30.

(37.) Takahashi K, Ochiai T, Uchida K, Yasumura T, Ishibashi M, Suzuki S, et al. Pilot study of mycophenolate mofetil (RS-61443) in the prevention of acute rejection following renal transplantation in Japanese patients. RS-61443 Investigation Committee. Japan Transplant Proc 1995;27:1421-4.

LUTZ T. WEBER, [1] MARIA SHIPKOVA, [2] VICTOR W. ARMSTRONG, [2] NATALIE WAGNER, [1] EKKEHARD SCHUTZ, [2] OTTO MEHLS, [1] LOTHAR B. ZIMMERHACKL, [3] MICHAEL OELLERICH, [2] AND BURKHARD TONSHOFF [1] *

[1] Division of Pediatric Nephrology, University Children's Hospital, Im Neuenheimer Feld 150, 69120 Heidelberg, Germany.

[2] Department of Clinical Chemistry, Georg-August University Gottingen, 37070 Gottingen, Germany.

[3] University Children's Hospital Freiburg, 79106 Freiburg, Germany.

[4] Nonstandard abbreviations: MMF, mycophenolate mofetil; MPA, mycophenolic acid; PK, pharmacokinetic; PD, pharmacodynamic; AUC, area under the curve; MPAG, MPA glucuronide; AcMPAG, MPA acyl glucuronide; [c.sub.0], concentration immediately before dosing; [c.sub.max], maximum (peak) concentration; and [c.sub.12], concentration 12 h after dosing.

* Author for correspondence. Fax 49-6221-564203; e-mail Burkhard_ Toenshoff@med.uni-heidelberg.de.

Received September 24, 2001; accepted December 12, 2001.
Table 1. Cyclosporin A dose and whole-blood predose concentrations in
50 pediatric renal-transplant recipients at the four PK sampling time
points posttransplant. (a)

 Cyclosporin A PK sampling time

 1 week 3 weeks

Dose, mg x [kg.sup.-1] x
 [day.sup-1] 7.85 (3.13-17.7) 6.98 (3.81-20.7)
Predose concentration,
 [micro]g/L 185 (31-452) 193 (126-306)

 Cyclosporin A PK sampling time

 3 months 6 months

Dose, mg x [kg.sup.-1] x
 [day.sup-1] 5.90 (2.65-13.4) 5.70 (3.06-13.8)
Predose concentration,
 [micro]g/L 172 (73-258) 144 (85-199)

(a) Data are median (range).

Table 2. Comparison of the PK variables for MPA at the four PK
sampling time points posttransplant as calculated from either the HPLC
or the Emit data. (a)

 MPA PK variable 1 week

 HPLC Emit

[c.sub.0], mg/L 1.01 (b) 1.242 (c)
 (0.11-2.66) (0.25-4.44)
[c.sub.12], mg/L 1.12 (b) 1.442 (c)
 (0.11-4.85) (0.28-6.04)
[c.sub.max], mg/L 9.74 (b) 10.72 (c)
 (1.50-32.1) (2.23-35.8)
[AUC.sub.0-12], mg x h/L 33.6 (b) 37.52 (c)
 (3.12-60.6) (6.26-76.9)

 MPA PK variable 3 weeks

 HPLC Emit

[c.sub.0], mg/L 0.96 (b) 1.30 (b,d)
 (0.06-2.54) (0.00-2.72)
[c.sub.12], mg/L 0.86 (b,d) 1.062 (c)
 (0.00-7.54) (0.00-8.04)
[c.sub.max], mg/L 14.2 (b) 14.3 (b)
 (3.53-45.7) (3.82-50.6)
[AUC.sub.0-12], mg x h/L 32.2 (b) 37.82 (c)
 (12.8-58.4) (6.26-76.9)

 MPA PK variable 3 months

 HPLC Emit

[c.sub.0], mg/L 2.07 (c) 2.38 (c,d)
 (0.35-11.2) (0.56-13.6)
[c.sub.12], mg/L 1.59 (c) 1.97 (c,d)
 (0.28-6.89) (0.55-8.20)
[c.sub.max], mg/L 24.9 (c) 25.0 (c)
 (7.70-53.5) (6.64-60.9)
[AUC.sub.0-12], mg x h/L 63.3 (c) 69.8 (c,d)
 (28.6-139) (29.2-147)

 MPA PK variable 6 months

 HPLC Emit

[c.sub.0], mg/L 2.92 (c) 3.08 (c,d)
 (0.18-12.6) (0.24-14.4)
[c.sub.12], mg/L 2.27 (b) 2.65 (c,d)
 (0.25-9.44) (0.52-9.62)
[c.sub.max], mg/L 26.0 (c) 27.4 (c,d)
 (9.40-52.5) (5.89-53.2)
[AUC.sub.0-12], mg x h/L 65.7 (c) 69.8 (c,d)
 (21.3-117) (26.9-152)

(a) Data are median (range).

(b,c) Intratest comparison was performed by one-way ANOVA on repeated
measurements followed by all pairwise comparison (Student-Newman-Keuls
test). Values sharing common superscripts are not significantly
different, whereas values without common superscripts are
significantly different (P <0.05). Intertest comparison was performed
by Wilcoxon signed-rank test.

(c) Emit vs HPLC, P <0.01.

Table 3. Correlation of the MPA PK variables [c.sub.0], [c.sub.12],
and [c.sub.max] and the abbreviated profiles MPA-[AUC.sub.0, 75 min, 4
h] and MPA-[AUC.sub.0-2] with the full-time MPA-[AUC.sub.0-12], as
calculated from either the HPLC or EMIT data, in 50 pediatric
renal-transplant recipients.

 PK variable HPLC Emit

MPA [c.sub.0] r = 0.66; P <0.0001 r = 0.68; P <0.0001
MPA [c.sub.12] r = 0.54; P <0.0001 r = 0.53; P <0.0001
MPA [csub.max] r = 0.71; P <0.0001 r = 0.73; P <0.0001
MPA-[AUC.sub.0,
 75 min, 4 h] r = 0.90; P <0.0001 r = 0.92; P <0.0001
[AUC.sub.0-2] r = 0.87; P <0.0001 r = 0.89; P <0.0001

Table 4. Areas under the ROC curves for PK variables of MPA as
calculated from either the HPLC or the Emit data to discriminate
between patients with (n = 10) or without an acute rejection episode
during the first 70 days posttransplant.

 HPLC

 Area under the
 MPA PK variable ROC curve 95% CI (a) P

[c.sub.0], mg/L 0.67 0.49-0.85 0.03
[c.sub.12], mg/L 0.70 0.53-0.87 0.01
[csub.max], mg/L 0.57 0.37-0.78 0.24
[AUC.sub.0-12], mg x h/L 0.66 0.47-0.84 0.04
[AUC.sub.0, 75 min, 4 h] 0.72 0.54-0.90 0.009
[AUC.sub.0-2] 0.64 0.46-0.82 0.06

 Emit

 Area under the
 MPA PK variable ROC curve 95% CI P

[c.sub.0], mg/L 0.66 0.48-0.85 0.04
[c.sub.12], mg/L 0.68 0.50-0.86 0.03
[csub.max], mg/L 0.57 0.35-0.80 0.26
[AUC.sub.0-12], mg x h/L 0.64 0.45-0.84 0.04
[AUC.sub.0, 75] min, 4 h 0.72 0.53-0.91 0.01
[AUC.sub.0-2] 0.65 0.45-0.85 0.08

(a) CI, confidence interval.

Table 5. Areas under the ROC curves for PK variables of MPA as
calculated from either the HPLC or the Emit data to discriminate
between patients with or without adverse events of infections (n = 20)
and/or leukopenia (n = 2) during the first 70 days posttransplant.

 HPLC

 Area under the
MPA PK variable ROC curve 95% CI (a) P

[c.sub.0], mg/L 0.62 0.45-0.79 0.09
[csub.12], mg/L 0.63 0.45-0.81 0.07
[c.sub.max], mg/L 0.53 0.31-0.75 0.40
[AUC.sub.0-12], mg x h/L 0.54 0.35-0.73 0.34
[AUC.sub.0, 75 min, 4 h] 0.54 0.35-0.73 0.35
[AUC.sub.0-2] 0.52 0.33-0.71 0.42

 Emit

 Area under the
MPA PK variable ROC curve 95% CI P

[c.sub.0], mg/L 0.57 0.39-0.75 0.23
[csub.12], mg/L 0.59 0.41-0.77 0.18
[c.sub.max], mg/L 0.58 0.37-0.79 0.23
[AUC.sub.0-12], mg x h/L 0.61 0.42-0.79 0.13
[AUC.sub.0, 75] min, 4 h 0.59 0.40-0.78 0.17
[AUC.sub.0-2] 0.56 0.36-0.77 0.27

(a) CI, confidence interval.

Table 6. Areas under the ROC curves for PK variables of MPA as
calculated from either the HPLC or the Emit data to discriminate
between patients with or without adverse events of infections (n = 7)
and/or leukopenia (n = 2) in the stable posttransplant phase.

 HPLC

 Area under the
 MPA PK variable ROC curve 95% CI P

[c.sub.0], mg/L 0.52 0.33-0.72 0.41
[c.sub.12], mg/L 0.53 0.33-0.73 0.38
[c.sub.max], mg/L 0.58 0.38-0.77 0.22
[AUC.sub.0-12], mg x h/L 0.57 0.37-0.77 0.23
[AUC.sub.0, 75 min, 4 h] 0.64 0.45-0.83 0.09
[AUC.sub.0-2] 0.54 0.35-0.73 0.34

 Emit

 Area under the P
 MPA PK variable ROC curve 95% CI

[c.sub.0], mg/L 0.58 0.38-0.78 0.22
[c.sub.12], mg/L 0.53 0.32-0.74 0.39
[c.sub.max], mg/L 0.52 0.32-0.72 0.42
[AUC.sub.0-12], mg x h/L 0.58 0.38-0.77 0.22
[AUC.sub.0, 75] min, 4 h 0.61 0.52-0.88 0.06
[AUC.sub.0-2] 0.58 0.39-0.77 0.21
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Title Annotation:Drug Monitoring and Toxicology
Author:Weber, Lutz T.; Shipkova, Maria; Armstrong, Victor W.; Wagner, Natalie; Schutz, Ekkehard; Mehls, Ott
Publication:Clinical Chemistry
Article Type:Clinical report
Date:Mar 1, 2002
Words:7320
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