Clinical Science (2004) 107, 69–74 (Printed in Great Britain)
Mycophenolate mofetil treatment following
renal transplantation decreases GTP
concentrations in mononuclear leucocytes
Piotr. JAGODZINSKI∗ , Slawomir LIZAKOWSKI∗ , Ryszard T. SMOLENSKI†1 ,
Ewa M. SLOMINSKA†, David GOLDSMITH‡, H. Anne SIMMONDS§ and
Boleslaw RUTKOWSKI∗
∗
Department of Nephrology, Transplantology and Internal Medicine, Medical University of Gdansk, Debinki 7 Str., 80-211
Gdansk, Poland, †Department of Biochemistry, Medical University of Gdansk, Debinki 7 Str., 80-211 Gdansk, Poland,
‡Department of Renal Medicine, Guy’s Hospital, London SE1 9RT, U.K., and §Purine Research Unit, Guy’s Hospital,
London SE1 9RT, U.K.
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MMF (mycophenolate mofetil) has been proven to provide an effective immunosuppression by noncompetitive selective reversible inhibition of IMPDH (inosine monophosphate dehydrogenase), the
enzyme playing a crucial role in GTP biosynthesis. However, the exact metabolic changes induced
by inhibition of IMPDH in target cells of the immune system have been the subject of recent
debate. The aim of the present study was to evaluate whether MMF treatment produced sustained
changes in the guanosine nucleotide pool of MNLs (mononuclear leucocytes) in vivo. Sixty-two
renal failure patients were divided into three groups: chronic renal failure patients undergoing
haemodialysis (CRF-HD; n = 20) and two groups of patients after renal transplantation, the first
on AZA (azathioprine; TN-AZA; n = 23) and the second treated with MMF (TN-MMF; n = 19). In
addition, MNLs from 25 healthy subjects were analysed as controls. Anion-exchange HPLC was
used to quantify purine and pyrimidine nucleotides in MNLs. We report a significant decrease in
GTP and the total MNL guanine nucleotide pool in the TN-MMF group (P < 0.05) compared with
control, CRF-HD and TN-AZA groups, although no significant differences were found between
any of the other groups. Adenine nucleotide concentrations in MNLs were decreased in the
TN-AZA group, but not in the TN-MMF group compared with the CRF-HD group and controls.
There were no differences in CTP concentrations, but UTP concentrations were decreased in
the CRF-HD, TN-AZA and TN-MMF groups compared with controls. MMF caused a significant
and sustained decrease in the guanine nucleotide pool in MNLs from renal transplant recipients.
This decrease contrasts with the elevation in GTP reported in erythrocytes of MMF-treated
patients.
INTRODUCTION
MPA (mycophenolic acid) is the active metabolite of
MMF (mycophenolate mofetil), the novel immunosup-
pressive drug now replacing AZA (azathioprine) in the
prevention of acute rejections in solid organ transplantations. Three large double-blind randomized trials in
kidney transplant recipients [1–3] have shown that
Key words: GTP, kidney transplantation, mononuclear leucocyte, mycophenolic acid, purine nucleotide, pyrimidine nucleotide.
Abbreviations: AZA, azathioprine; CRF-HD, chronic renal failure patients undergoing haemodialysis; IMPDH, inosine
monophosphate dehydrogenase; MMF, mycophenolate mofetil; MNL, mononuclear leucocyte; MPA, mycophenolic acid.
1 Present address: Heart Science Centre, Imperial College London, Harefield Hospital, Harefield UB9 6JH, U.K.
Correspondence: Dr Piotr Jagodzinski (e-mail [email protected]).
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Table 1 Clinical features of patients enrolled in the study
Values are means +
− S.D. GN, glomerulonephritis; DN, diabetic nephropathy; HN, hypertension nephropathy; TUB/INT, tubulointerstitial
nephritis; POLYC, polycystic kidney disease.
Sex (female/male)
Age (years)
Serum creatinine (µmol/l)
Serum urea (mmol/l)
Clinical features (n)
GN
DN
HN
TUB/INT
POLYC
Other
Control (n = 25)
CHF-HD (n = 20)
TN-AZA (n = 23)
TN-MMF (n = 19)
12/13
37.7 +
− 26.2
100 +
− 18.2
3.99 +
− 1.01
5/15
47.8 +
− 15.5
845 +
− 164.7
21.71 +
− 5.32
10/13
43 +
− 14.6
254.8 +
− 209
14.3 +
− 9.11
8/11
46 +
− 11.6
345.4 +
− 336
19.75 +
− 11.86
–
–
–
–
–
–
6
2
2
3
2
5
8
4
2
1
0
8
7
3
1
1
0
7
addition of MMF to cyclosporin and steroids results in
a decrease of acute rejection episodes in the first few
months following transplantation when compared with
AZA-treated patients. The mechanism of MPA action has
been investigated in detail [1–4]. MPA is a potent inhibitor of IMPDH (inosine monophosphate dehydrogenase) [4], which catalyses the conversion of IMP (inosine
5 monophosphate) into XMP (xanthosine 5 monophosphate), an intermediate metabolite in the synthesis of
guanosine nucleotides. An adequate supply of guanosine
nucleotides is crucial to many aspects of cellular metabolism and depletion of GTP pools by inhibition of
de novo purine biosynthesis has been shown to result in
decreased activation of T-lymphocytes [5]. Pharmacological depletion of guanine nucleotides inhibits DNA
synthesis by arresting cells in the G1 -phase [6]. As proliferating lymphocytes almost exclusively use the de novo
pathway of purine synthesis, these cells are particularly
sensitive to the inhibitory action of MPA. MPA has a
more potent effect on lymphocytes as it more completely
inhibits the type II isoform of IMPDH (lymphocytes)
than type I (other cells) [4,7].
Recently it was reported that long-term MMF treatment can increase erythrocyte and serum IMPDH activity [8,9], as noted previously for another IMPDH inhibitor ribavirin [10,11]. This finding is curious, since we
[12] and others [11] have demonstrated previously that,
although both ATP and GTP concentrations in erythrocytes increase with decline in renal function, they return
to control values following successful transplantation.
However, we found no difference in either ATP or GTP
concentrations in resting T-lymphocytes of renal failure
patients compared with controls [10,12]. The aim of the
present study was to evaluate whether MMF treatment
also affects the nucleotide pools of MNLs (mononuclear
leucocytes) in renal transplant recipients, which, if so,
may have implications for its long-term efficacy.
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METHODS
Subjects
Sixty-two patients and 25 healthy volunteers were enrolled in the study. Patients were divided into three
groups: 20 chronic renal failure patients undergoing haemodialysis (CRF-HD) and two groups of patients
after successful renal transplantation, the first taking
AZA (TN-AZA; n = 23 patients) and the second taking
MMF (TN-MMF; n = 19 patients). This immunosuppressive regimen included steroids as an intravenous
bolus of 250 mg of methylprednisolone at 0, 1 and
2 days, followed by 125 mg of methylprednisolone on
day 3. Prednisone was then given orally at 0.5 mg/kg
of body weight with decreasing doses. The target dose
after 6 months was 10 mg. Cyclosporin was given at an
initial dose of 2 × 5 mg/kg of body weight orally. Further
doses were adjusted according to blood measurement of
cyclosporine level. Target level was 250 ng/ml in the first
6 months, then 200–250 ng/ml up to the end of first year,
160–180 ng/ml in the second year and 120–140 ng/ml in
subsequent years. Dosage of cyclosporin was adjusted to
achieve the target level before the next dose of cyclosporin
and > 1700 ng/ml 2 h after cyclosporin administration.
Either AZA or MMF was given as the third drug. AZA
was administered at 3 mg · kg−1 of body weight · 24 h−1
dose under control of blood morphology. MMF was
given at 2 × 0.5 g doses on the first day post-transplant
and then 2 × 0.75 g on the second day, followed by 2 ×
1.0 g doses.
The relevant patient details are compared with controls
in Tables 1 and 2. The study was approved by the Local
Ethics Committee and all the subjects signed an informed
consent form. Blood was drawn before patients had taken
their medication on the day of the study. MPA levels were
not assayed in this study.
Mycophenolate mofetil and mononuclear leucocyte GTP levels
Table 2 Clinical details of patients enrolled in the study
after renal transplantation
Values are means +
− S.D. Cumulative dose is the total dose prescribed from
engraftment to time of study.
TN-AZA (n = 23)
Treatment time (months)
Warm ischaemia time (min)
Cold ischaemia time (h)
Immunosuppressant cumulative dose (g)
AZA
MMF
Cyclosporin
TN-MMF (n = 19)
13 +
− 23
31 +
− 7.8
21 +
− 9.5
7.1 +
− 9.9
31.9 +
− 11.9
22.7 +
−8
27.4 +
− 45
–
91.6 +
− 134.4
–
253.6 +
− 439.5
55.5 +
− 65.3
Preparation of MNLs
MNLs were separated from heparinized blood, diluted
one to two times in RPMI 1640 medium and subjected
to Histopaque density gradient centrifugation (700 g,
30 min) at room temperature. MNLs were collected
from the interface after centrifugation and washed once
in RPMI 1640. The pellet was resuspended in 1 ml of
RPMI 1640 and spun in a microcentrifuge at 100 g for
10 min. The supernatant was removed and the pellet
was resuspended in 50 µl of RPMI 1640 and frozen in
− 70 ◦ C.
Extraction and measurement
of MNL nucleotides
Nucleotide extraction was performed directly before the
measurements. A solution of 1.3 M perchloric acid was
added to microcentrifuge tubes containing frozen MNLs
at a ratio of 1:1. Deproteinized samples were centrifuged
for 5 min at 13 500 g. Each supernatant was transferred
to a fresh microcentrifuge tube, and 3 M potassium phosphate solution was added to obtain pH = 6. The samples
were centrifuged and the supernatant was used for
nucleotide measurements.
An HPLC system with UV detection (Merck-Hitachi)
was used to measure nucleotide concentrations. All measurements were performed according to the method
described previously by us [12]. Briefly, a 4.6 mm anionexchange Phenomenex column packed with 3 µm
Hypersil-NH2 -NH2 was used. Buffer A was 5 mM
potassium phosphate and buffer B was 0.5 M potassium
phosphate/1.0 M KCl. The system was run in a gradient
mode starting with 100 % buffer A, increasing to 100 %
B in 26 min, followed by 100 % buffer B for 2 min. The
column was then re-equilibrated with 100 % buffer A for
5 min. The chromatographic identification of nucleotides
was based on comparison with the retention times of
known standards. Quantitative analysis was done by
comparison of peak areas on the chromatogram with
external standards. Nucleotide levels were expressed
per 106 MNLs. Number of MNLs for this calculation
was obtained by analysis of the same MNL suspension
Figure 1 GTP (a) and GTP + GDP (b) concentrations in MNLs
from study patients and controls
Control, healthy volunteers (n = 25); CRF-HD, n = 20; TN-AZA, n = 23; TN-MMF,
n = 19. All concentrations are expressed as pmol/106 MNLs.
prior to the extraction using the Sysmex haematological
autoanalyser. Serum creatinine and urea concentration
were measured using routine laboratory methods.
Statistical analysis
Statistical analysis was performed using a standard software package (Statistica, Stat-Soft). Data are expressed
as means +
− S.D. A P value < 0.05 was considered to
be statistically significant. Distribution of variables was
evaluated using Shapiro–Wilk’s test. Between groups, differences of variables were assessed by ANOVA. Association between one dependent and more than two
independent variables was assessed by linear regression
or by multiple regression analysis as appropriate.
RESULTS
Guanosine nucleotide concentrations
in MNLs
GTP concentrations were decreased in MNLs from
all transplanted patients. The decrease was statistically
significant for the TN-MMF group (P < 0.05), in contrast
with the TN-AZA group (Figure 1a), compared with
the MNLs from the healthy and CRF-HD patients (Figure 1b).
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Figure 3 Concentrations of ATP, ADP and AMP (a) and UTP
and CTP (b) in the MNLs from study patients and controls
Values represent means +
− S.D. Control (healthy volunteers), n = 25; CRF-HD,
n = 20; TN-AZA, n = 23; TN-MMF, n = 19. All concentrations are expressed as
pmol/106 MNLs.
Figure 2 Correlation between MNL guanine nucleotide
concentration and time of MMF treatment (a) and lack of
change in guanine nucleotide pool in MMF-treated patients
irrespective of the time of MMF usage (b)
All concentrations are expressed as pmol/106 MNLs.
Long-term MMF treatment
The MMF-treated patients after kidney transplantation
were divided into two subgroups: one taking MMF
for less than 6 months and the second for more than
6 months. No difference in guanosine nucleotide concentrations was observed between the two groups (Figure 2b). Moreover, no correlation was found between
the time after renal transplantation (time of immunosuppression using MMF) and concentration of purine and
pyrimidine nucleotides (Figure 2a).
nucleotide pool in TN-AZA patients was significantly
lower than in other groups (Figure 3a). Moreover, we
found significantly higher UTP concentrations in MNLs
of healthy volunteers (Figure 3b) compared with the
patient groups. There were no differences in the AMP,
GDP, and CTP pools between groups.
Correlation between nucleotide
concentration and clinical features
We have analysed a possible correlation between the
concentration of each of the nucleotides measured or
their total pools and concentration of creatinine and urea,
haemodialysis treatment or cyclosporin concentration.
No statistically significant correlation was found between
nucleotide concentrations and concentrations of uraemic
markers or any other clinical parameters indicated.
Concentration of other purine and
pyrimidine nucleotides in MNLs
DISCUSSION
No significant differences in the nucleotide concentrations for either purines or pyrimidines were observed
between healthy controls and the CRF-HD group. By
contrast, the ATP pool in MNLs taken from the TNAZA and TN-MMF groups was decreased, as noted by
us previously [10] using MMF in T-lymphocytes in vitro.
The lower ATP concentration was followed by a decrease
in ADP in TN-AZA group. Thus the total adenine
The findings of the present study confirm that MMF
treatment produces the anticipated decrease in guanosine nucleotide concentrations in peripheral blood
MNLs following inhibition of IMPDH by its active
metabolite MPA. The novel and important finding is
that this GTP depletion, which occurs immediately after
commencement of MMF therapy, is sustained in the long
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Mycophenolate mofetil and mononuclear leucocyte GTP levels
term. This sustained decrease in GTP in MMF-treated
transplant recipients is important for two reasons.
First, Langman et al. [13] demonstrated that the
peak concentration of MPA achieved at 1 h after MMF
dosing in renal transplant recipients resulted in approx.
40 % inhibition of IMPDH activity in whole blood and
a significantly lower concentration of guanine nucleotides in lymphocytes of these MMF-treated patients.
Moreover, there was a clear correlation between a decrease in MPA concentration in serum and restoration of
IMPDH activity. Such an increase should be followed by
a corresponding elevation in guanosine nucleotides.
Secondly, recent reports have documented the induction of IMPDH activity in both erythrocytes and
serum of patients on MMF [8]. Weigel et al. [9] have
shown also that long-term administration of MMF to
heart transplant patients with normal renal function was
associated with elevation of GTP concentration in the
erythrocytes of these patients, subsequent to induction
of IMPDH activity. In the accompanying paper [13a],
we report similar findings in renal transplant patients,
where plasma GTP levels from two groups of patients on
MMF- or AZA-based antimetabolite therapy were compared. A similar elevation of erythrocyte GTP concentrations was reported earlier in immunodeficient
children treated with another IMPDH inhibitor ribavirin
[14]. Interestingly, there was no difference in either
ATP or GTP concentrations in MNLs of our chronic
renal failure group compared with controls, a finding
noted by us in an earlier study [12]. This contrasts with
the considerable elevation of both ATP and GTP found
in erythrocytes from the same renal failure patients compared with controls, which return to normal following
successful transplantation, confirming a considerable difference between nucleotide metabolism in erythrocytes
compared with MNLs.
The elevation in erythrocyte GTP in MMF-treated
patients raised the distinct possibility that, if long-term
MMF therapy were to induce IMPDH activity, then
antirejection protection would eventually become less
effective. Clearly, our present results showing lower
GTP concentrations in MMF-treated renal transplant
recipients, both immediately and sustained long-term, do
not support this possibility. Thus there is no correlation
between the concentration of guanosine nucleotides in
MNLs and the length of MMF treatment. The explanation for these differences must lie in the fact that
the changes in IMPDH activity during long-term MMF
treatment were observed only in serum and erythrocytes,
where the type I isoform of IMPDH predominates. MPA,
however, is a more potent inhibitor of type II IMPDH,
which is up-regulated in stimulated lymphocytes [15] and
can, thus, explain all the observed results. Further work in
this field should attempt to measure MPA levels (not done
in this or the accompanying study [13a]), IMPDH activity
and GTP levels in the same patients before and after the
commencement of MMF therapy to fully characterize the
relationships between these parameters.
Our present observations in MNLs are supported
by clinical data from a 3 year MMF multi-centre study
[16], which showed good results of long-term MMF
antirejection therapy with no need for a dose increase.
The present study demonstrates conclusively that stable
low guanosine MNL nucleoside concentrations can be
sustained long term and are likely to be responsible for
the good clinical outcome of MMF-treated patients after
renal transplantation.
ACKNOWLEDGEMENTS
This work was supported by the Polish State Committee
for Scientific Research (W-731), and the Dr Hadwen Trust
for Humane Research.
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Received 9 October 2003/11 December 2003; accepted 14 January 2004
Published as Immediate Publication 14 January 2004, DOI 10.1042/CS20030332
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