Inosine monophosphate dehydrogenase activity in paediatrics: age-related regulation and response to mycophenolic acid
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- Rother, A., Glander, P., Vitt, E. et al. Eur J Clin Pharmacol (2012) 68: 913. doi:10.1007/s00228-011-1203-4
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Since many drug targets and metabolizing enzymes are developmentally regulated, we investigated a potential comparable regulation of inosine 5’-monophosphate dehydrogenase (IMPDH) activity that has recently been advocated as a pharmacodynamic biomarker of mycophenolic acid (MPA) effects in the paediatric population. Since the field of pharmacodynamic monitoring of MPA is evolving, we also analyzed the response of IMPDH activity on MPA in children vs adolescents after renal transplantation.
We analyzed IMPDH activity in peripheral blood mononuclear cells (PBMCs) in 79 healthy children aged 2.0–17.9 years in comparison to 106 healthy adults. Pharmacokinetic/pharmacodynamic profiles of MPA and IMPDH over 6 or 12 h after mycophenolate mofetil dosing were performed in 17 paediatric renal transplant recipients. IMPDH activity was measured by HPLC and normalized to the adenosine monophosphate (AMP) content of the cells, MPA plasma concentrations were measured by HPLC.
Inosine 5’-monophosphate dehydrogenase activity displayed a high inter-individual variability (coefficient of variation 40.2%) throughout the entire age range studied. Median IMPDH did not differ significantly in healthy pre-school children (82 [range, 42–184] μmol/s/mol AMP), school-age children (61 [30–153]), adolescents (83 [43–154]) and healthy adults (83 [26–215]). Similar to adults, IMPDH activity in children and adolescents was inversely correlated with MPA plasma concentration.
In conclusion, our data do not show a pronounced developmental regulation of IMPDH activity in PBMCs in the paediatric population and there is a comparable inhibition of IMPDH activity by MPA in children and adolescents after renal transplantation.
KeywordsIMPDH activityDevelopmental regulationPharmacodynamicsPaediatric renal transplantationMycophenolic acid
Area under the enzyme activity–time curve
Akaike information criterion
Maximal possible IMPDH inhibition
Minimum IMPDH activity
Analysis of variance
Area under the concentration–time curve
Body surface area
- BMI SDS
Body mass index standard deviation score
Apparent drug clearance
Maximum MPA concentration
Administered MPA content
End-stage renal disease
High-performance liquid chromatography
Inosine 5’-monophosphate dehydrogenase
Peripheral blood mononuclear cells
Single nucleotide polymorphism
Time to minimum IMPDH activity
Time of maximum MPA concentration in a dosing interval
Mycophenolate mofetil (MMF) has been approved for maintenance immunosuppressive therapy both in adult and in paediatric renal transplant recipients. Mycophenolic acid (MPA), the active moiety of MMF, reversibly inhibits inosine 5’-monophosphate dehydrogenase (IMPDH), the rate-limiting enzyme in the de novo guanine nucleotide synthesis in proliferating T and B lymphocytes .
Previous clinical studies on the efficacy and safety of MMF in paediatric renal transplant recipients have documented that the incidence of acute rejection is markedly lower in those patients who received an MPA compound compared with those who have received either azathioprine or no antimetabolite . The overall efficacy and safety of MMF appear to be similar in paediatric and adult renal transplant recipients [3, 4].
It is generally accepted that the action of and response to a drug are subject to developmental changes, e.g. caused by age-dependent differences in body composition, integument, expression of key enzymes, development of renal function, and others as reviewed in Kearns et al. . However, little is known about the effect of human ontogeny on pharmacodynamics. Data on warfarin , cyclosporine  and midazolam [8, 9] indicate that there is a true developmental change in the interaction between the drug and its specific receptor or in the relation between the plasma level and the pharmacological effect of a given drug.
Since only limited data on the activity of IMPDH in PBMCs in the paediatric population  and on its suppression by MPA  are available, we performed the present comprehensive pharmacological analysis on a potential age-related regulation of IMPDH activity in PBMCs in the paediatric population and on the response of IMPDH to MPA in children vs adolescents after renal transplantation, being the primary objectives of this study. The analysis of IMPDH activity in response to MPA is also interesting for the evolving field of pharmacodynamic monitoring of MPA [11–14]. In addition, we sought to analyze whether genetic variants of the IMPDH gene contribute to the large between-subject variability in IMPDH activity [15–19] as a secondary objective.
Materials and methods
Reference populations for IMPDH activity
Whole blood samples for the measurement of IMPDH activity were collected from otherwise healthy children, who required a routine blood examination. Exclusion criteria were acute or chronic diseases, current or prior infectious episodes in the preceding 3 days before sampling, and the use of any medication beside vitamin or mineral supplementation. The time lag of 3 days to an infectious episode before sampling was considered sufficient to exclude any potential influence on IMPDH activity. Mild infections do not affect IMPDH activity (P. Glander, personal communication). Between May 2007 and August 2008, 79 healthy children were enrolled in the study into the following three age groups: pre-school children (aged 2.0 to 5.9 years), school-aged children (aged 6.0 to 11.9 years) and adolescents (aged 12.0 to 17.9 years). All participants were white. Collection of blood samples in healthy children younger than 2 years of age was considered unethical owing to the small circulating blood volume. In addition, roughly 95% of paediatric renal transplant recipients are 2 years of age or older  and reflect a relevant target population of our investigation.
Demographics of healthy individuals
Number of individuals
Male/female n; (% female)
2 to 5.9 years
6 to 11.9 years
12 to 17.9 years
Study population for MPA pharmacokinetics and IMPDH activity profiles
Seventeen children and adolescents, aged 2.5 to 18.9 years, were enrolled between June 2007 and July 2008 from a total of 23 patients having received a renal transplant during this time period. Exclusion criteria were a leukocyte count of ≤2,500/μL, a haemoglobin concentration of ≤5 g/dL, severe gastrointestinal disease, thus criteria when patients had to be withdrawn from MMF for clinical reasons, as well as age <2 years or ≥19 years, or treatment with antacids, cholestyramine, iron supplements, or probenecid. Six patients were not included because of age (n = 2), MMF intolerance (n = 1), or impossibility of adequate blood sampling (n = 3). All patients had primary transplant function without the requirement for dialysis post-transplant.
Characteristics of the patient group for pharmacokinetic/pharmacodynamic monitoring
2.0 to 11.9 years
12.0 to 18.9 years
Number of patients, n
Days post-transplant, days
16 ± 4
17 ± 4
Age at RTx, years
6.4 ± 3.5
16.6 ± 2.0
Gender male/female, n
Donor living/deceased, n
Patients previously undergoing transplantation, n
MMF morning dose, mg/m2 BSA
390 ± 134
393 ± 149
Tacrolimus trough level, μg/L
10.3 ± 2.1
10.4 ± 2.8
38.4 ± 2.8
38.6 ± 5.2
15.4 ± 6.0
11.9 ± 4.4
Creatinine clearance, mL/min per 1.73 m2 BSA
105 ± 40
70 ± 21
Pre-transplant IMPDH activity was measured in 7 children and 9 adolescents with ESRD on the waiting list for renal transplantation (median, 1 day; range, 0–275 days before transplantation). PK/PD profiles during the first 6 hours after MMF intake obtained in the early period post-transplant at 16 ± 4 days were analysed in 8 children, full 12-h PK/PD profiles in 9 adolescents. In 5 out of 9 adolescent patients, a second 12-h PK/PD profile was obtained in the stable period post-transplant at 7.8 ± 1.8 weeks.
The study was approved by the respective Ethics Committees of the Medical Faculties of Heidelberg and Munich; it was designed and conducted in agreement with the principles as stated in the Declaration of Helsinki. All parents/guardians, patients and healthy volunteers had given written informed consent prior to study entry.
MMF (CellCeptTM, Roche Pharma AG, Grenzach-Wyhlen, Germany) was administered either as capsule or suspension in a dose of 1200 mg/m2 BSA per day in two divided doses; on day 14 post-transplant, the dose was reduced to 600 mg/m2 BSA per day.
Tacrolimus (Prograf™, Astellas Pharma GmbH, Munich, Germany) was administered at an initial dose of 0.3 mg/kg body weight per day in two divided doses in patients <40 kg and at 0.2 mg/kg in patients ≥40 kg. Tacrolimus doses were adjusted to achieve 12-hour trough levels of 8-12 ng/mL on day 0-21 post-transplant and 5-10 ng/mL thereafter. Methylprednisolone (UrbasonTM, Sanofi-Aventis GmbH, Frankfurt, Germany) was given at an initial intravenous bolus of 300 mg/m2 BSA at least 1 hour prior to reperfusion and then tapered to 48 mg/m2 BSA p.o. at day 1 post-transplant, 32 mg/m2 BSA at day 2 to 7, 24 mg/m2 BSA for the second, 16 mg/m2 BSA for the third and fourth, 8 mg/m2 BSA for the fifth and sixth week after grafting, and 4 mg/m2 BSA thereafter.
Pharmacokinetic and pharmacodynamic protocol
For a 12-hour PK/PD profile, blood samples were drawn immediately before MMF dosing (pre-dose) and at 0.5, 1, 1.5, 2, 3, 4, 6, 8 and 12 hours after MMF dosing in the morning. In children, aged 2.0 to 11.9 years, an abbreviated PK/PD profile during the first 6 hours after MMF dosing was obtained because of the lower blood volume in these patients. Patients fasted from 10 pm the night before sampling until the 1.5 hours sample had been obtained. Patients were then allowed to have a light breakfast. It was mandatory that all patients had at least two full days of the same MMF dose given twice a day prior to PK investigations.
Measurement of MPA and IMPDH activity
Blood samples were transferred within 30 minutes to the laboratory, where 0.5 ml plasma samples for quantification of MPA were frozen at -20°C. PBMCs were separated by Ficoll-density centrifugation from a 2.5 ml whole blood sample and stored at -80°C until analysis. MPA plasma concentrations were determined by a validated liquid chromatography tandem mass spectrometry assay as described previously . The same volumes were used with this assay to determine MPA concentrations in samples from children and adults to avoid any influence on assay performance data.
IMPDH activity was measured using a modified, non-radioactive assay based on the incubation of lysed PBMCs with inosine 5’-monophosphate followed by chromatographic determination of produced xanthosine 5’-monophosphate (XMP). The newly generated XMP was normalised to the adenosine monophosphate (AMP) concentration of the cells, which was quantified in the same high-performance liquid chromatography run as XMP. A detailed description and validation of this procedure have been previously published . Using AMP concentrations for normalisation of the newly generated XMP results in a smaller variability when compared with that of other normalisation factors used previously, such as protein concentrations and cell counts. The within-run and within-day variabilities of the method are <11% . Furthermore this modified assay consists of only one analytical operation without the potential burden of another previous operation (e.g. cell count) or mistaken samples.
Genomic DNA was isolated from frozen anticoagulated blood (2,5 ml) using spin columns (Macherey Nagel, Düren, Germany). Single nucleotide polymorphisms (SNPs) were amplified by polymerase chain reaction and genotyped using hybridisation probes on a Roche LightCycler 480 (Roche Diagnostics, Mannheim, Germany). The following seven SNPs were chosen for analysis: IMPDH1 rs11766743, rs7802305 (source: HapMap data), rs2278293 , and rs11761662 (source: HapMap data) and IMPDH2 rs4974081 (source: HapMap data), L263F , and rs11706052 . In addition to the samples from healthy children blood samples from 26 children, aged 2.4 to 17.8 years, with end-stage renal disease (ESRD), 18/26 undergoing dialysis treatment, were obtained during routine blood sampling for genotyping.
Pharmacokinetic and pharmacodynamic analysis
Mycophenolate mofetil pharmacokinetic parameters were derived from individual plasma concentration–time profiles by using standard non-compartmental equations. Pre-dose concentration before drug administration (C0), C0.5 to C6 values, tCmax (time of maximum concentration (Cmax) in a dosing interval), and the area under the concentration–time curve (AUC0–6 h) were compared in children and adolescents early post-transplant. Cmax, AUC0–12 h, and the apparent drug clearance (CL/F) in adolescents was compared in the early and stable periods post-transplant. The AUC was calculated by the linear trapezoidal method, CL/F after oral administration of dose D was calculated as CL/F = D/AUC0–12 h, where D was the administered MPA content (370 mg after administration of 500 mg MMF).
For pharmacodynamic analysis, pre-transplant IMPDH activity (Apre-transplant), IMPDH activity before drug administration (A0, 12 hours after the previous dose), A0.5 to A6 values, tAmin (time to minimum enzyme activity [Amin]), and relative IMPDH inhibition ((1 - Amin / A0) * 100) were compared in children and adolescents. The respective areas under the enzyme activity–time curve (AEC0–6 h or AEC0–12 h) were calculated by the linear trapezoidal method.
In addition, IMPDH activities were plotted vs MPA concentrations. Furthermore, the individual concentration–activity relationships were analysed using four variants of the general inhibitory Emax model: A = Apre-transplant – Alow * CH / (CH + IC50H). Parameters were the maximal possible IMPDH inhibition (Alow), the concentration IC50 leading to a half-maximum IMPDH suppression, and the sigmoidicity parameter H. Comparison between model variants was made by visual inspection of the results and the Akaike information criterion (AIC) .
Descriptive statistics were calculated to characterise patient demographics, baseline IMPDH activity, pharmacokinetic and pharmacodynamic parameters of various age groups and periods post-transplant, which included mean, standard deviation (SD) and coefficient of variation, in the case of non-normal statistical distribution median and range. The Shapiro–Wilks test was used to confirm normal distribution of data. Differences between groups were tested for statistical significance by two-sided Student’s t test, Fisher’s exact test, Mann–Whitney U test, Wilcoxon signed-rank test, Jonckheere–Terpstra test, and non-parametrical ANOVA (Kruskal–Wallis test, followed by Schaich–Hammerle post hoc analysis), as applicable. Age dependency of IMPDH activity in healthy individuals was analysed by linear and non-linear regression analysis. Descriptive statistics and statistical tests were performed using SPSS software Version 17.0 (SPSS, Chicago, IL, USA); p < 0.05 was considered statistically significant. The exact test for Hardy–Weinberg equilibrium  was performed using PowerMarker V3.25 . Associations between genetic polymorphisms and IMPDH activity were tested using HAPSTAT software . Pharmacokinetic and pharmacodynamic analyses were performed using WinNonlin Professional Version 5.2 (Pharsight Corporation, Mountain View, CA, USA) and Microsoft Excel 2003.
Age-related regulation of IMPDH activity: For an estimated difference in baseline IMPDH activity of 60% that was considered to be clinically relevant and 80% power for analysis 20 healthy children had to remain in each age group for statistical comparison.
Response of IMPDH to MPA: For an estimated difference in IMPDH inhibition of 30% and 80% power for analysis 6 children and adolescents after renal transplantation were necessary for statistical comparison respectively.
IMPDH activity in healthy individuals
Pharmacokinetics of MPA
Comparison of pharmacokinetic and pharmacodynamic parameters in children and adolescents in the early period after renal transplantation
2 to 11.9 years
12 to 18.9 years
MPA concentration, mg/L
IMPDH activity, μmol/s/mol AMP
AEC0–6h, h∙μmol/s/mol AMP
Maximum IMPDH inhibition, %
Pharmacodynamics of MPA
Paediatric genotype data for polymorphisms in IMPDH1 and IMPDH2
Fisher’s exact test p
Polymorphisms in IMPDH1 and IMPDH2 and IMPDH activity
All studied SNPs were in Hardy–Weinberg equilibrium (p > 0.1). The IMPDH2 L263F mutation was not observed in our population. The remaining 4 SNPs in IMPDH1 and 2 SNPs in IMPDH2 were analysed in 105 samples (Table 4).
Genotype frequencies were different between healthy controls and ESRD patients for rs11706052 in IMPDH2. However, numbers of ESRD patients (n = 26) were smaller compared with healthy controls (n = 79). Using an additive model in HAPSTAT, there were no significant associations between the six SNPs in the study and IMPDH activity when tested SNP-wise or in two haplotypes with or without controlling for health vs ESRD status as environmental variables.
This is the first study to comprehensively evaluate a potential developmental regulation of IMPDH, the target enzyme of MPA, during childhood and adolescence. We found that IMPDH activity in healthy children above the age of 2 years does not undergo pronounced developmental or gender-specific regulation.
We observed a large inter-individual variability of IMPDH activity in healthy children and adolescents comparable to that previously reported in adults [27, 28]. It is likely that genetic differences or environmental factors determine this variability. In the present study we analysed seven relevant SNPs of the IMPDH1 and IMPDH2 gene regarding their respective frequency and association with IMPDH activity. Previous studies have observed associations between various IMPDH gene polymorphisms and both IMPDH activity [15, 18] and the risk of acute rejection episodes [16, 17, 19], although data for the latter finding are conflicting [16, 19]. Interestingly, we observed in children with ESRD a higher frequency of heterozygous carriers of the IMPDH2 variant rs11706052 than in healthy children or in adults with ESRD [17, 19]. The biological significance of this finding remains to be elucidated. In our analysis we could not confirm any influence of the six SNPs under observation on IMPDH activity, but this lack of association may be due to the relatively small number of paediatric subjects studied compared with previous reports in adults [15–17, 19].
Despite a 1.9-fold increase in MPA exposure in the stable compared with the early period post-transplant the corresponding AEC0–12 of IMPDH decreased by only 21%. We have previously shown that in the first months post-transplant only the exposure of total MPA increases, not that of free (unbound) MPA . Free MPA is supposed to reflect the pharmacologically active moiety . Hence, the observed discrepancy between a pronounced increase in total MPA and only a marginal corresponding decline in IMPDH activity can be explained by unchanged exposure to free MPA, although this was not explicitly measured in the present study. This line of reasoning is supported by the observations of Chiarelli et al., who reported on a significant inverse correlation of the concentration of free MPA 2 h after the morning dose and IMPDH activity, but not of total MPA-C2 or –C0 . Hence, it appears that the degree of IMPDH inhibition is rather a function of the concentration of free MPA than of total MPA. These observations also imply that the immunosuppressive activity of MPA, at least towards inhibition of IMPDH activity, remains relatively constant in the first months post-transplant, despite a pronounced increase in the exposure of total MPA.
We found higher IC50 values than had been previously reported in paediatric renal transplant recipients . Fukuda et al.  report on IC50 of about 1 mg/L that is well aligned with the clinically established pre-dose MPA concentration target range of 1.0 to 3.5 mg/L in the paediatric population . The median IC50 values in our study are substantially higher and correspond with MPA-Cmax rather than MPA predose levels. This difference may be explained by the use of an improved assay for determination of IMPDH activity  in the present study that, in contrast to the former assay , is no longer potentially influenced by the MNC count that could lead to falsely low measurements of IMPDH activity.
The observed degree of maximum IMPDH inhibition after MMF intake was comparable in children and adolescents (mean maximum inhibition 64% and 72% respectively) and is in concordance with the mean maximum inhibition observed in adult (67%) renal transplant recipients . This observation is also consistent with data from clinical studies, which demonstrated comparable efficacy of MMF regarding the prevention of acute rejection episodes in paediatric renal transplant recipients [2, 4]. Data from this and previous studies in paediatric  and adult renal transplant recipients  also show that IMPDH activity returns to baseline within 4–8 h of administration of MMF. This observation raises the question whether dosing of MMF every 8 h might be more effective than the common b.i.d. regimen. The clinical necessity of full IMPDH inhibition during a dosing period in renal transplant recipients, however, is unknown, and the variability of MPA pharmacokinetics limits the understanding of which degree of IMPDH inhibition is really needed . It should, however, be mentioned in this context that most regimens on MMF induction therapy, for example in lupus nephritis, are based on t.i.d. rather than b.i.d. dosing of MMF .
The determination and monitoring of IMPDH activity have recently been advocated as a pharmacodynamic biomarker of MPA effects . In addition, pre-transplant IMPDH activity has been associated with clinical outcome in adult renal transplant recipients . It is currently being debated whether the determination of pre-transplant IMPDH activity is sufficient to guide MMF dosing for improving outcome  or whether pre-dose IMPDH activity  or maximal IMPDH inhibition is superior in identifying patients at risk of acute rejection and MMF-related side-effects. The observed similarities between the paediatric and adult populations regarding variability of IMPDH activity and suppression in response to MPA suggest that a comparable relationship between IMPDH activity and clinical outcome may also exist in the paediatric patient population. However, the present study was not designed for this specific analysis.
In conclusion, our data do not show an age-related difference in baseline IMPDH activity in PBMCs of healthy paediatric individuals and it was comparable to that of healthy adults. In addition, we were able to show a comparable inhibition of IMPDH activity by MPA in children and adolescents after renal transplantation. These pharmacological data corroborate the currently applied dosing recommendations for MMF in the paediatric population. IMPDH activity displayed a high inter-individual variability throughout the entire age range studied. In view of the known association of IMPDH activity and clinical outcome [13, 14, 36, 37] and the variable pharmacokinetics of MPA , an integrated approach that combines pharmacokinetic monitoring of MPA and pharmacodynamic monitoring of IMPDH has the potential to optimise immunosuppressive therapy with MMF.
We gratefully acknowledge the expert technical assistance of Sandra Hartung and Ulrike Hügel.
This study was supported by the Peter-Stiftung für die Nierenwissenschaft (scientific foundation to promote kidney research, particularly in children). The manuscript was not prepared or funded by a commercial organisation.
Conflicts of interest
Lutz T. Weber and Burkhard Tönshoff have received research grants from Roche Pharma AG and Novartis Pharma GmbH. Klemens Budde has received research grants and honoraria from Roche Pharma AG and Novartis Pharma GmbH.