European Journal of Clinical Pharmacology

, Volume 68, Issue 6, pp 913–922

Inosine monophosphate dehydrogenase activity in paediatrics: age-related regulation and response to mycophenolic acid

Authors

  • A. Rother
    • Department of Paediatrics IUniversity Children’s Hospital Heidelberg
  • P. Glander
    • Department of NephrologyCharité Universitätsmedizin Berlin
  • E. Vitt
    • Department of Paediatric NephrologyChildren’s Hospital of the Ludwig-Maximilians University of Munich
  • D. Czock
    • Department of Clinical Pharmacology and PharmacoepidemiologyUniversity Hospital Heidelberg
  • N. von Ahsen
    • Department of Clinical ChemistryUniversity of Göttingen
  • V. W. Armstrong
    • Department of Clinical ChemistryUniversity of Göttingen
  • M. Oellerich
    • Department of Clinical ChemistryUniversity of Göttingen
  • K. Budde
    • Department of NephrologyCharité Universitätsmedizin Berlin
  • R. Feneberg
    • Department of Paediatrics IUniversity Children’s Hospital Heidelberg
  • B. Tönshoff
    • Department of Paediatrics IUniversity Children’s Hospital Heidelberg
    • Department of Paediatric NephrologyChildren’s Hospital of the Ludwig-Maximilians University of Munich
    • Paediatric NephrologyUniversity Children’s Hospital, Dr. von Haunersche Kinderklinik
Pharmacodynamics

DOI: 10.1007/s00228-011-1203-4

Cite this article as:
Rother, A., Glander, P., Vitt, E. et al. Eur J Clin Pharmacol (2012) 68: 913. doi:10.1007/s00228-011-1203-4

Abstract

Purpose

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.

Methods

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.

Results

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.

Conclusions

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.

Keywords

IMPDH activityDevelopmental regulationPharmacodynamicsPaediatric renal transplantationMycophenolic acid

Abbreviations

A

IMPDH activity

AEC

Area under the enzyme activity–time curve

AIC

Akaike information criterion

Alow

Maximal possible IMPDH inhibition

Amin

Minimum IMPDH activity

AMP

Adenosine monophosphate

ANOVA

Analysis of variance

AUC

Area under the concentration–time curve

BSA

Body surface area

BMI SDS

Body mass index standard deviation score

C

MPA concentration

CL/F

Apparent drug clearance

Cmax

Maximum MPA concentration

D

Administered MPA content

ESRD

End-stage renal disease

Freq

Frequency

H

Sigmoidicity parameter

HPLC

High-performance liquid chromatography

IMPDH

Inosine 5’-monophosphate dehydrogenase

MPA

Mycophenolic acid

MMF

Mycophenolate mofetil

PBMCs

Peripheral blood mononuclear cells

PD

Pharmacodynamic

PK

Pharmacokinetic

RTx

Renal transplantation

SD

Standard deviation

SNP

Single nucleotide polymorphism

tAmin

Time to minimum IMPDH activity

tCmax

Time of maximum MPA concentration in a dosing interval

XMP

Xanthosine 5’-monophosphate

Introduction

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 [1].

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 [2]. 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. [5]. However, little is known about the effect of human ontogeny on pharmacodynamics. Data on warfarin [6], cyclosporine [7] 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 [10] and on its suppression by MPA [11] 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 [1114]. In addition, we sought to analyze whether genetic variants of the IMPDH gene contribute to the large between-subject variability in IMPDH activity [1519] 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 [20] and reflect a relevant target population of our investigation.

Inosine 5’-monophosphate dehydrogenase activity data of healthy children were compared with data collected from 106 healthy adult blood donors between October 2002 and June 2008. Demographics of healthy control subjects are given in Table 1.
Table 1

Demographics of healthy individuals

 

Number of individuals

Age

Gender

Median (range)

Male/female n; (% female)

Healthy children

 

79

7.2

(2.1–17.9)

46/33

(42)

Age group

2 to 5.9 years

26

3.9

(2.1–5.4)

18/8

(31)

6 to 11.9 years

31

7.2

(6.1–11.8)

18/13

(42)

12 to 17.9 years

22

15.0

(12.7–17.9)

10/12

(55)

Healthy adults

 

106

30.8

(18.7–67.3)

79/27

(25)

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.

Patients were enrolled into two age groups: pre-pubertal children (aged 2.0 to 11.9 years, n = 8) and adolescents (aged 12.0 to 18.9 years, n = 9). Patient characteristics and biochemistry data on the day of pharmacokinetic (PK)/pharmacodynamic (PD) sampling early post-transplant (days 8–23 post-transplant) are given in Table 2.
Table 2

Characteristics of the patient group for pharmacokinetic/pharmacodynamic monitoring

 

Age

2.0 to 11.9 years

12.0 to 18.9 years

P value

Number of patients, n

8

9

 

Days post-transplant, days

16 ± 4

17 ± 4

n.s. a

Age at RTx, years

6.4 ± 3.5

16.6 ± 2.0

 

BMI SDSLMS

−0.26 (−2.14–1.63)

−0.73 (−2.09–0.63)

n.s. b

BSA, m2

0.68 (0.58–1.25)

1.27 (1.17–1.82)

0.001 b

Gender male/female, n

7/1

4/5

n.s. c

Donor living/deceased, n

3/5

3/6

n.s. c

Patients previously undergoing transplantation, n

0

3

n.s. c

MMF morning dose, mg/m2 BSA

390 ± 134

393 ± 149

n.s. a

Tacrolimus trough level, μg/L

10.3 ± 2.1

10.4 ± 2.8

n.s. a

Albumin, g/L

38.4 ± 2.8

38.6 ± 5.2

n.s. a

Leucocytes, 109/L

15.4 ± 6.0

11.9 ± 4.4

n.s. a

Creatinine clearance, mL/min per 1.73 m2 BSA

105 ± 40

70 ± 21

0.035 a

Data are expressed as mean ± SD, BMI SDSLMS (body mass index standard deviation score) and BSA (body surface area) are given as median (range). BMI SDSLMS was calculated by the LMS method [38]. BSA was calculated by the Mosteller formula [39]. Creatinine clearance was estimated with the Schwartz equation [40] in patients <18 years and with the Cockcroft–Gault equation [41] in patients ≥18 years

aStudent’s t test

bMann–Whitney U test

cFisher’s exact test

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.

Immunosuppressive therapy

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 [21]. 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 [22]. 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% [22]. 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.

IMPDH genotyping

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 [16], and rs11761662 (source: HapMap data) and IMPDH2 rs4974081 (source: HapMap data), L263F [15], and rs11706052 [18]. 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) [23].

Statistical analysis

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 [24] was performed using PowerMarker V3.25 [25]. Associations between genetic polymorphisms and IMPDH activity were tested using HAPSTAT software [26]. Pharmacokinetic and pharmacodynamic analyses were performed using WinNonlin Professional Version 5.2 (Pharsight Corporation, Mountain View, CA, USA) and Microsoft Excel 2003.

Sample size considerations for primary objectives:
  • 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.

Results

IMPDH activity in healthy individuals

The IMPDH activity in both paediatric and adult control subjects displayed a large inter-individual variability (coefficient of variation, 40.2%; Fig. 1). There were no age-related differences in IMPDH activity in these healthy individuals (r2 = 0.009, p = 0.19; Fig. 2). We did not observe a difference in median IMPDH activity with respect to gender. Median baseline IMPDH activity was comparable in healthy children and adults (78 [30 to 184] μmol/s/mol AMP vs. 83 [26 to 215] μmol/s/mol AMP) and did not differ significantly in healthy pre-school children (82 [range, 42–184) μmol/s/mol AMP), school-aged children (61 [30–153] μmol/s/mol AMP) and adolescents (83 [43–154] μmol/s/mol AMP).
https://static-content.springer.com/image/art%3A10.1007%2Fs00228-011-1203-4/MediaObjects/228_2011_1203_Fig1_HTML.gif
Fig. 1

Inter-individual variability of Inosine 5’-monophosphate dehydrogenase (IMPDH) activity in peripheral blood mononuclear cells (PBMCs) of 79 healthy children and adolescents and 106 healthy adults

https://static-content.springer.com/image/art%3A10.1007%2Fs00228-011-1203-4/MediaObjects/228_2011_1203_Fig2_HTML.gif
Fig. 2

IMPDH activity in PBMCs as a function of chronological age in 79 healthy children and 106 healthy adults. There was no statistically significant correlation (r2 = 0.009, p = 0.19)

Pharmacokinetics of MPA

PK parameters of MPA in children and adolescents early post-transplant are summarised in Table 3. The MPA plasma concentrations during the first 6 hours after MMF intake were comparable between children and adolescents (Table 3). The PK profiles of MPA in both age groups were characterised by an early and sharp increase of MPA concentration, the maximum concentration being reached at 0.5 hours after MMF administration in all patients except for one child (1 hour). A small second peak of MPA plasma concentration 6 to 12 hours after MMF administration was observed in five of nine adolescents, consistent with the known enterohepatic recirculation.
Table 3

Comparison of pharmacokinetic and pharmacodynamic parameters in children and adolescents in the early period after renal transplantation

 

Age group

2 to 11.9 years

12 to 18.9 years

P valuea

MPA concentration, mg/L

  C0

1.9 (0.1–4.4)

0.8 (0.2–3.4)

n.s.

  C0.5

8.4 (3.5–28.7)

14.7 (11.1–28.3)

n.s.

  C1

6.0 (3.3–14.0)

8.0 (4.7–10.0)

n.s.

  C2

3.0 (2.0–5.7)

2.6 (1.3–4.0)

n.s.

  C3

3.3 (0.6–5.4)

1.8 (0.6–3.6)

n.s.

  C6

1.1 (0.1–3.6)

1.6 (0.4–2.6)

n.s.

  tCmax, h

0.5 (0.5–1.0)

0.5 (0.5)

n.s.

  AUC0–6h, h∙mg/L

21.2 (10.3–40.3)

22.4 (16.5–26.6)

n.s.

IMPDH activity, μmol/s/mol AMP

  A0

71 (27–103)

79 (43–138)

n.s.

  A0.5

23 (5–66)

20 (13–42)

n.s.

  A1

34 (13–44)

39 (12–64)

n.s.

  A2

50 (33–67)

52 (23–103)

n.s.

  A3

46 (19–63)

44 (17–85)

n.s.

  A6

50 (34–71)

47 (22–102)

n.s.

  Apre-transplant

84 (76–141)

81 (61–133)

n.s.

  tAmin, h

0.5 (0.5–3.0)

0.5 (0.5–2.0)

n.s.

  AEC0–6h, h∙μmol/s/mol AMP

284 (181–354)

304 (148–515)

n.s.

  Maximum IMPDH inhibition, %

64 (29–87)

73 (52–90)

n.s.

Data are expressed as median; the range is given in parenthesis

aMann–Whitney U test

The comparison of PK profiles in the early vs stable period post-transplant revealed higher maximal MPA concentrations (Cmax) in the stable period (median, 22.6 mg/L; range, 15.6–32.4 mg/L) than in the early period post-transplant (median, 14.0 mg/L; range, 8.2–15.3 mg/L; p = 0.043); Fig. 3). Similarly, a 1.9-fold increase in median AUC0–12 h was observed between the early (28.8 h∙mg/L; range, 20.4–38.9 h∙mg/L) and the stable period (53.8 h∙mg/L; range, 36.0–63.4 h∙mg/L; p = 0.043), owing to a 1.8-fold decline in MPA clearance (median CL/F early period, 11.5 L/h; range, 9.5–13.8 L/h; stable period, 6.5 L/h; range, 5.0–10.3 L/h; p = 0.043).
https://static-content.springer.com/image/art%3A10.1007%2Fs00228-011-1203-4/MediaObjects/228_2011_1203_Fig3_HTML.gif
Fig. 3

Mean 12-h MPA plasma concentration–time profile vs mean IMPDH activity–time profile in PBMCs in the early and stable period after renal transplantation in five adolescents. Although the median MPA-AUC0–12 h in the stable period post-transplant was 1.9-fold higher than in the early period, the corresponding median AEC0–12 h decreased by only 21%

Pharmacodynamics of MPA

Individual IMPDH activity–time courses were explored graphically and showed high between-patient variability with both single-nadir and multiple-nadir profiles. Nevertheless, IMPDH activity was inversely correlated with MPA plasma concentration, with a maximal inhibition of IMPDH activity at the time of maximal MPA plasma concentration (tCmax and tAmin) in 12 out of 17 patients (Table 3). The comparison of IMPDH activity and of its response to MPA in children and adolescents early post-transplant did not reveal any significant difference (Table 4). Although the median MPA-AUC0–12 h in the stable period post-transplant was 1.9-fold higher than in the early period, the corresponding median AEC0–12 h decreased only by 21% in the stable period (575 h∙μmol/s/mol AMP; range, 481–765 h∙μmol/s/mol AMP) compared with the early period (727 h∙μmol/s/mol AMP; range, 505–1,113 h∙μmol/s/mol AMP; p = 0.35; Fig. 3).
Table 4

Paediatric genotype data for polymorphisms in IMPDH1 and IMPDH2

 

Total

Healthy

ESRD

Genotype

Gene

Marker

Genotype

Count

Frequency

Count

Frequency

Count

Frequency

Fisher’s exact test p

IMPDH1

rs11766743

Wild type

67

0.64

49

0.62

18

0.69

 
  

Heterozygous

33

0.31

26

0.33

7

0.27

 
  

Homozygous

5

0.05

4

0.05

1

0.04

 
 

0.85

IMPDH1

rs7802305

Wild type

41

0.39

29

0.37

12

0.46

 
  

Heterozygous

50

0.48

38

0.48

12

0.46

 
  

Homozygous

14

0.13

12

0.15

2

0.08

 
 

0.61

IMPDH1

rs2278293

Wild type

30

0.29

23

0.29

7

0.27

 
  

Heterozygous

52

0.50

39

0.49

13

0.50

 
  

Homozygous

23

0.22

17

0.22

6

0.23

 
 

1

IMPDH1

rs11761662

Wild type

75

0.71

53

0.67

22

0.85

 
  

Heterozygous

27

0.26

23

0.29

4

0.15

 
  

Homozygous

3

0.03

3

0.04

0

0.00

 
 

0.28

IMPDH2

rs4974081

Wild type

59

0.56

44

0.56

15

0.58

 
  

Heterozygous

41

0.39

30

0.38

11

0.42

 
  

Homozygous

5

0.05

5

0.06

0

0.00

 
 

0.55

IMPDH2

rs11706052

Wild type

84

0.80

67

0.85

17

0.65

 
  

Heterozygous

20

0.19

11

0.14

9

0.35

 
  

Homozygous

1

0.01

1

0.01

0

0.00

 
 

0.05

Total counts and genotype counts in healthy controls and ESRD patients are presented

To further characterise the pharmacodynamics of MPA, we plotted IMPDH values against MPA plasma concentrations (Fig. 4) and calculated individual IC50 values using inhibitory Emax models. The simple Emax model (with H set at 1) and the sigmoid Emax model with full inhibition at high concentrations turned out to be the most appropriate models. Although the fit of some profiles was improved by using the sigmoid model, the mean AIC value was nearly unchanged (0.83 ± 5.0 vs 0.76 ± 6.3 for the simple and the sigmoid models respectively). Two model variants with incomplete inhibition at high concentrations turned out to be inferior as indicated by the AIC values (2.9 ± 5.0 and 4.6 ± 5.5 respectively). We found no significant differences in IC50 between children (median, 5.4 mg/L; range, 0.8–30.6 mg/L; median H, 1.0) and adolescents (median, 4.4 mg/L; range, 0.8–12.0 mg/L; median H, 0.9) early post-transplant, but in adolescents the median IC50 was higher in the stable period (9.6 mg/L; range, 1.1–17.1 mg/L; median H, 0.5; p = 0.043).
https://static-content.springer.com/image/art%3A10.1007%2Fs00228-011-1203-4/MediaObjects/228_2011_1203_Fig4_HTML.gif
Fig. 4

IMPDH activity as a function of MPA plasma concentration in children (aged 2.0–11.9 years, n = 8, triangles) and adolescents (aged 12.0–18.9 years, n = 9, circles) in the early period after renal transplantation. The insert illustrates the derived PK–PD relationship for children (broken curve) and adolescents (continuous curve)

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.

Discussion

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 [1517, 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 [29]. Free MPA is supposed to reflect the pharmacologically active moiety [30]. 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 [14]. 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 [11]. Fukuda et al. [11] 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 [31]. 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 [22] in the present study that, in contrast to the former assay [32], 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 [33]. 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 [11] and adult renal transplant recipients [13] 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 [34]. 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 [35].

The determination and monitoring of IMPDH activity have recently been advocated as a pharmacodynamic biomarker of MPA effects [11]. In addition, pre-transplant IMPDH activity has been associated with clinical outcome in adult renal transplant recipients [36]. It is currently being debated whether the determination of pre-transplant IMPDH activity is sufficient to guide MMF dosing for improving outcome [36] or whether pre-dose IMPDH activity [14] 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 [29], an integrated approach that combines pharmacokinetic monitoring of MPA and pharmacodynamic monitoring of IMPDH has the potential to optimise immunosuppressive therapy with MMF.

Acknowledgements

We gratefully acknowledge the expert technical assistance of Sandra Hartung and Ulrike Hügel.

Funding source

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.

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© Springer-Verlag 2012