Pharmacokinetics and pharmacodynamics of mycophenolic acid in Nagase analbuminemic rats: Evaluation of protein binding effects using the modeling and simulation approach

Article abstract of DOI:10.1016/j.dmpk.2015.10.004

This study aimed to examine the pharmacokinetics and pharmacodynamics of mycophenolic acid (MPA) in Nagase analbuminemic rats (NARs) to evaluate the effect of protein binding on the associated inosine-5-monophosphate dehydrogenase (IMPDH) activity. Free fractions of MPA in the control rats and NARs were 2.09 and 24.8%, respectively. Pharmacokinetic and pharmacodynamic parameters simultaneously obtained by the nonlinear mixed effects modeling program NONMEM explained reasonably well the concentrations of MPA and MPA glucuronide as well as IMPDH activity in both rats. NARs showed a higher clearance and a smaller volume of distribution based on the free MPA concentration than the controls did, besides the increase in free fraction. The half-maximal inhibitory concentration based on free MPA was estimated as 163 ng/mL for both rats. Simulations based on the obtained pharmacokinetic and pharmacodynamic parameters showed that the area under the IMPDH activity-time curve decreased non-linearly according to the increase in free fraction of MPA. In conclusion, the experimental data obtained from NARs followed by the modeling and simulation approach quantitatively clarified that the free MPA concentration was suitable for the biomarker of immunosuppressive effect of MPA. Dose adjustments based on the total MPA may cause unnecessary overexposure to MPA in patients with hypoalbuminemia.

Full text of DOI:10.1016/j.dmpk.2015.10.004

Drug Metabolism and Pharmacokinetics 30 (2015) 441e448
Contents lists available at ScienceDirect
Drug Metabolism and Pharmacokinetics
journal homepage: http://www.journals.elsevier.com/drug-metabolism-and-
pharmacokinetics
Regular article
Pharmacokinetics and pharmacodynamics of mycophenolic acid in Nagase
analbuminemic rats: Evaluation of protein binding effects using the modeling and
simulation approach
,
,
b
,
,
Kazuaki Yoshimura a b, Ikuko Yano a *, Misaki Kawanishi a b, Shunsaku Nakagawa b,
Atsushi Yonezawa b, Kazuo Matsubara b
a Department of Clinical Pharmacy and Education, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan
b Department of Clinical Pharmacology and Therapeutics, Kyoto University Hospital, Sakyo-ku, Kyoto 606-8507, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
This study aimed to examine the pharmacokinetics and pharmacodynamics of mycophenolic acid (MPA)
in Nagase analbuminemic rats (NARs) to evaluate the effect of protein binding on the associated inosine-
5ʹ-monophosphate dehydrogenase (IMPDH) activity. Free fractions of MPA in the control rats and NARs
were 2.09 and 24.8%, respectively. Pharmacokinetic and pharmacodynamic parameters simultaneously
obtained by the nonlinear mixed effects modeling program NONMEM explained reasonably well the
concentrations of MPA and MPA glucuronide as well as IMPDH activity in both rats. NARs showed a
higher clearance and a smaller volume of distribution based on the free MPA concentration than the
controls did, besides the increase in free fraction. The half-maximal inhibitory concentration based on
free MPA was estimated as 163 ng/mL for both rats. Simulations based on the obtained pharmacokinetic
and pharmacodynamic parameters showed that the area under the IMPDH activity-time curve decreased
non-linearly according to the increase in free fraction of MPA. In conclusion, the experimental data
obtained from NARs followed by the modeling and simulation approach quantitatively clarified that the
free MPA concentration was suitable for the biomarker of immunosuppressive effect of MPA. Dose ad-
justments based on the total MPA may cause unnecessary overexposure to MPA in patients with
hypoalbuminemia.
Received 14 September 2015
Received in revised form
9 October 2015
Accepted 25 October 2015
Available online 3 November 2015
Keywords:
Mycophenolic acid
Inosine-5ʹ-monophosphate dehydrogenase
Nagase analbuminemic rat
Nonlinear mixed effects modeling
UDP-Glucuronosyltransferase
Copyright © 2015, The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.
1. Introduction
immunosuppressants such as a calcineurin inhibitor, tacrolimus or
cyclosporine.
Mycophenolic acid (MPA) selectively inhibits inosine-5ʹ-mono-
phosphate dehydrogenase (IMPDH), which catalyzes the rate-
limiting step in the de novo synthesis of guanine nucleotide and
suppresses the proliferation of B and T lymphocytes [1]. MPA is
clinically used to suppress acute rejection after solid organ trans-
plantation as well as graft-versus-host disease in hematopoietic
stem cell transplantation [2,3], in the combination with other
MPA is metabolized by uridine 5ʹ-diphospho-glucuronosyl-
transferase (UGT) isoforms in the liver to an inactive MPA glucu-
ronide (MPAG), which is mainly eliminated through the urine [3].
The pharmacokinetics of MPA is affected by changes in hepatic or
renal functions [4] and shows a large inter- and intraindividual
variability [5]. Therefore, patients who have had transplants fol-
lowed by MPA administration should be managed by therapeutic
drug monitoring of plasma MPA concentrations. The consensus
target exposure range of MPA has been proposed as an area under
the concentration-time curve (AUC) from 0 to 12 h of 30e60 mg h/
mL based on the total plasma concentration in patients with renal
transplants [6]. The target exposure range of MPA is possibly
influenced by changes in plasma albumin concentrations, since the
free fraction of MPA was 1e2% [7].
* Corresponding author. Department of Clinical Pharmacy and Education, Grad-
uate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto, 606-
8501, Japan.
E-mail addresses: yoshimura.kazuaki.52r@st.kyoto-u.ac.jp (K. Yoshimura),
iyano@kuhp.kyoto-u.ac.jp
(I.
Yano),
misaki.kawanishi@shionogi.co.jp
IMPDH activity in peripheral blood mononuclear cells (PBMCs)
(M. Kawanishi), nakashun@kuhp.kyoto-u.ac.jp (S. Nakagawa), ayone@kuhp.kyoto-
u.ac.jp (A. Yonezawa), kmatsuba@kuhp.kyoto-u.ac.jp (K. Matsubara).
has been suggested as
a
surrogate biomarker for the
http://dx.doi.org/10.1016/j.dmpk.2015.10.004
1347-4367/Copyright © 2015, The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.

442
K. Yoshimura et al. / Drug Metabolism and Pharmacokinetics 30 (2015) 441e448
immunosuppressive effects of MPA, and pre-transplant IMPDH
activity is associated with rejection in patients with renal trans-
plants [8]. In a recent study, the area under the effect-time curve
(AUEC) of IMPDH activity on day 21 after hematopoietic stem cell
transplantation was found to be associated with cytomegalovirus
reactivation, non-relapse mortality, and overall mortality [9].
Therefore, the measurement of IMPDH activity in addition to
monitoring the AUC of total plasma MPA is considered an effective
predictor of the clinical outcome of MPA therapy. However, little is
known about the quantitative relationship between the free frac-
tion of MPA in the plasma and IMPDH activity.
Nagase analbuminemic rats (NARs) are an established animal
model for human familial analbuminemia [10]. NARs were used to
examine the effect of decreased protein binding on the pharma-
cokinetics and pharmacodynamics of drugs having a high protein
binding property, such as bumetanide [11], azosemide [12], and
methotrexate [13]. NARs have also been utilized in the investigation
of the toxicokinetics and toxicodynamics of clofibrate [14]. In this
study, we constructed the simultaneous pharmacokinetic and
pharmacodynamic model of MPA, and analyzed the experimental
data obtained from NARs as well as control rats by the nonlinear
mixed effects modeling method. The simulation study based on the
obtained pharmacokinetic and pharmacodynamic parameters
quantitatively evaluated the effect of protein binding on the IMPDH
activity, as a biomarker of the pharmacodynamics of MPA.
MPA and MPAG concentrations were calculated by multiplying
their respective total concentrations by the average of the free
fraction of each drug for each group. All procedures were conducted
in compliance with the Guidelines for Animal Experiments of the
Kyoto University.
2.3. Analysis of cytochrome P450 enzyme (CYP) and UGT mRNA
expressions using real-time polymerase chain reaction (PCR)
The total RNA was extracted from the liver using the RNeasy
mini kit (Qiagen Inc., Valencia, CA, USA) according to the manu-
facturer's instruction and further quantitated using the BioSpec-
nano spectrophotometer (Shimadzu, Kyoto, Japan) at a wave-
length of 260 nm. The total RNA (1 mg) was reverse-transcribed
using a high capacity cDNA reverse transcription kit (Applied Bio-
systems). The rat cDNAs were mixed with the forward and reverse
primers of the target CYPs (CYP1A2, 2B2, 2C11, and 3A2), UGTs
(UGT1A1, 1A2, 1A6, 1A7, and 1A8) or GAPDH and SYBR Green PCR
master mix (Applied Biosystems). After an initial denaturation at
95 ꢀC for 10 min, the amplification was performed by denaturation
at 95 ꢀC for 15 s and extension at 60 ꢀC for 60 s, repeated for 50
cycles. The expression level of each mRNA was quantified by
measuring the fluorescence intensity using the StepOnePlus real-
time PCR system (Applied Biosystems) and was expressed as a ra-
tio of GAPDH. The primer designs were referenced from various
published articles [15e21], while the primer sequences and PCR
product sizes were confirmed using primer-BLAST (http://www.
ncbi.nlm.nih.gov/tools/primer-blast/).
2. Materials and methods
2.1. Chemicals
2.4. MPAG formation in rat liver microsomes
MPA and MPAG were purchased from Wako Pure Chemical In-
dustries Ltd. (Osaka, Japan) and Analytical Services International
Ltd. (London, UK), respectively. Xanthosine-5ʹ-monophosphate
(XMP) disodium salt was obtained from Santa Cruz Biotechnology
Inc. (Santa Cruz, CA, USA). Adenosine-5ʹ-monophosphate (AMP)
sodium salt, inosine-5ʹ-monophosphate disodium salt from yeast
and nicotinamide adenine dinucleotide (NADþ) were from Nacalai
Tesque Inc. (Kyoto, Japan). All the chemicals used were of the
highest grade available.
Liver microsomes from control rats and NARs were prepared
according to a commonly used procedure [22]. MPAG formation by
liver microsomes was studied by the previously reported method
[23] with a slight modification. Liver microsomes (1 mg) was
incubated in buffer containing 75 mM Tris-hydrochloric acid (pH
7.45) and 10 mM magnesium chloride. The total volume was 50
and the final concentration of MPA was 5e640 g/mL. After pre-
treated with alamethicin (50 g/mg) for 15 min on ice, reactions
mL,
m
m
were initiated by the addition of uridine 5’-diphospho-glucuronic
acid (3 mM final concentration) and were allowed to proceed at
37 ꢀC for 30 min. Reactions were stopped by the addition of two
volumes of the internal standard methanol solution. The amount of
MPAG formed was measured, and kinetic parameters, the
2.2. In vivo pharmacokinetic and pharmacodynamic study
Male Sprague-Dawley rats (control) and NARs (8- to 10-week-
old and 9- to 11-week-old, respectively) were obtained from Japan
SLC, Inc. (Osaka, Japan). Both groups of rats were anesthetized with
intraperitoneal injections of 40 mg/kg pentobarbital sodium. A
polyethylene tube was inserted into the femoral artery and vein.
Then, MPA dissolved in 10% cremophor and 10% ethanol in saline
was intravenously infused for 1 h via the femoral vein at doses of
0.5 or 5 and 5 or 15 mg/kg in the control and NAR groups,
respectively. Blood samples (0.2 mL) were collected sequentially
before and at 0.5, 1, 1.25, 1.5, 2, 2.5, 3, and 4 h after the start of MPA
administration and were stored at 4 ꢀC until completion of blood
collection. The blood samples were centrifuged at 14,000 g for
MichaeliseMenten constant (Km;
mg/mL) and the maximum ve-
locity (Vmax; ng/mg protein per 30 min), were determined using a
non-linear least squares method (WinNonlin 6.4; Pharsight,
Mountain View, CA).
2.5. Analytical methods
Concentrations of MPA and MPAG were analyzed using a reverse
phase liquid chromatography-tandem mass spectrometry accord-
ing to the previously reported method [24]. The limits of deter-
mination were 2 and 20 ng/mL for MPA and MPAG, respectively.
The PBMC samples were used to measure IMPDH activity ac-
cording to the previously reported method [24] with a slight
5 min to obtain the plasma, and then plasma samples (100
mL) were
acidified by adding 2 L of 10% acetic acid for the assay of MPA and
m
MPAG. The PBMCs, which were obtained from the residual blood
samples by centrifuging at 1000 g for 15 min using Ficoll-Paque
Premium 1.084 (GE Healthcare Bio-Sciences AB, Uppsala, Swe-
den), were frozen at ꢁ20 ꢀC until measurements of IMPDH activity
were performed. The free fraction of MPA and MPAG in both groups
of rats was determined using plasma from blood samples collected
15 min after MPA administration. The plasma samples were ultra-
filtrated (Amicon Ultra 30 K centrifugal filter devices, Merck Milli-
pore Ltd., Carrigtwohill, Ireland) at 14,000 g for 10 min. The free
modification. Briefly, thawed PBMCs diluted with 200
were vortexed and centrifuged at 1000 g for 2 min. The reaction
was initiated by adding 50 L of supernatant to 52 L of incubation
mL of water
m
m
mixture consisting of 1.6 mM phosphate buffer (pH 7.4), 3.9 mM
potassium chloride, 4.8 mM inosine-5ʹ-monophosphate disodium
salt, and 0.6 mM NADþ as final concentrations. The mixture was
incubated at 37 ꢀC for 120 min. The reaction was stopped by the
addition of 5 mL of 2.5 M perchloric acid followed by 10 mL of 3 M

K. Yoshimura et al. / Drug Metabolism and Pharmacokinetics 30 (2015) 441e448
443
potassium monohydrogen phosphate. The XMP and internal AMP
in the supernatants were then determined by mass spectrometric
measurements. The limits of determination were 50 and 100 ng/mL
for XMP and AMP, respectively. The IMPDH activity was calculated
based on the XMP produced, which was normalized to the intra-
cellular AMP.
simultaneously. The pharmacokinetic parameters estimated were
the total body clearance of free MPA (CLf), inter-compartment
clearance of free MPA (Qf), total body clearance of free MPAG
(CLfMPAG), central and peripheral volume of distribution of free
MPA (Vc,f and Vp,f, respectively), and volume of distribution of free
MPAG (VfMPAG). Each pharmacokinetic parameter of the total MPA
or MPAG plasma concentrations were obtained by multiplying
each parameter of the respective free concentrations by the free
fraction of MPA or MPAG. In the mathematical calculation, the
MPAG concentrations were converted to their equivalent MPA
concentrations by dividing by 1.55. The pharmacodynamic pa-
rameters estimated were the half-maximal inhibitory concentra-
tion of free MPA (IC50,f) as well as the Hill coefficient and baseline
IMPDH activity (IMPDH0).
Simultaneous pharmacokinetic and pharmacodynamic pa-
rameters were estimated using the nonlinear mixed effects
modeling program NONMEM 7.2 with the ADVAN 7 subroutine by
the first-order conditional estimation method with interaction
[26]. The exponential and proportional error models were used
for the interindividual and residual variability, respectively. In
addition, each pharmacokinetic parameter “X” of the NAR group
was modeled by multiplying the pharmacokinetic parameter “X”
of the control by the factor (Fx); for example, CLf of NARs was
obtained by multiplying CLf of the control by FCLf. The statistical
significance of each parameter such as the Fx, interindividual
variability, and Hill coefficient was compared between two
2.6. Pharmacokinetic and pharmacodynamic analysis
To characterize the differences in the time-course of MPA,
pharmacokinetic and pharmacodynamic parameters in each rat
were obtained by the non-compartmental analysis using the pro-
gram WinNonlin 6.4. In addition, the simultaneous population
pharmacokinetic and pharmacodynamic model analysis was per-
formed to extract the changed pharmacokinetic and pharmaco-
dynamic parameters in NARs and to simulate the effect of protein
binding on the pharmacokinetics and pharmacodynamics of MPA
(Supplemental Fig. 1). The MPA concentration was fitted to a 2-
compartment model and the MPA was metabolized to MPAG by
a first-order process. The MPA was assumed to completely convert
to MPAG [25], and its concentration was described using a 1-
compartment model with first-order elimination. The IMPDH ac-
tivity was modeled using a sigmoid maximum inhibitory model
based on the central free MPA concentration. The constructed
model was parameterized based on the concentrations of free MPA
and MPAG in order to fit both the control and NARs data
A
B
40
2.0
30
20
10
0
1.5
1.0
0.5
0.0
0
1
2
3
4
0
1
2
3
4
Time (h)
Time (h)
C
D
20
15
15
10
5
10
5
0
0
0
1
2
3
4
0
1
2
3
4
Time (h)
Time (h)
Fig. 1. Time-course of total MPA (A), free MPA (B), total MPAG (C), and free MPAG (D) concentrations. Each concentration was obtained after intravenous infusion of MPA at 5 mg/kg
for 1 h in controls (open cycle) and NARs (closed cycle). Data are expressed as mean SEM of five rats.

444
K. Yoshimura et al. / Drug Metabolism and Pharmacokinetics 30 (2015) 441e448
models using the chi-squared test: for one model, the parameter
of interest is freely estimated and for the other, the parameter
should be set to an identical value such as zero or one. If the
objective function value was not reduced by more than 7.88 in the
former model compared to the latter model, the parameter to be
estimated was fixed at zero or one (P < 0.005 with a freedom of 1
by chi-squared test).
3. Results
3.1. Time-course of MPA and MPAG concentrations and IMPDH
activity
The body weight of the NAR group was significantly lower than
that of the controls at 236 6 and 332 16 g, respectively. The free
fractions were significantly higher in the NARs than in the controls
at 24.8
2.6 and 2.09
0.05% for MPA, and 59.3
2.8 and
2.7. Validation of model
23.9 4.0% for MPAG, respectively. There was no difference in the
total protein concentrations measured by the Biuret method
(5.18
The goodness-of-fit and visual predictive check plots were used
for the internal validation of the final model. The goodness-of-fit
plots consisted of observed versus population or individual pre-
dicted concentrations. The visual predictive check was performed
as followed: the final pharmacokinetic-pharmacodynamic model
was used to simulate original data sets at 1000 times. The 5th, 50th,
and 95th percentiles of simulated data were compared with the
observed data.
0.02 and 5.07
0.05 g/dL in NAR and control groups,
respectively).
The time-course of total or free MPA and MPAG concentrations
after intravenous infusion of MPA (5 mg/kg) to the control and NAR
groups are shown in Fig. 1, and the pharmacokinetic and pharma-
codynamic parameters by the non-compartmental analysis were
shown in Table 1. Although the maximum concentration (Cmax) of
total MPA in the NAR group was approximately 6% of that in the
control rats, the Cmax of free MPA in the NAR group was approxi-
mately 75% of that in the control rats (Fig. 1A, B and Table 1). The
AUC0e4h of total or free MPA was significantly lower in the NARs
compared with that in the controls (Table 1). Although the Cmax of
total MPAG was similar in the control and NAR groups, the Cmax of
free MPAG in the NAR group was 2.5-fold higher compared with
that in control rats (Fig. 1C, D and Table 1). The times corresponding
to Cmax for the total and free MPAG were shorter in NAR group than
in the controls (Fig. 1C, D).
2.8. Simulation for effects of protein binding
The effects of protein binding on the pharmacokinetics and
pharmacodynamics of MPA were evaluated using the simulation
from the final pharmacokinetic-pharmacodynamic model and final
parameters in control rats. One thousand data sets were simulated
under the condition of the free fraction at 1, 2, 4, and 8%. In order to
target the AUC0e4h of MPA at 15 mg h/mL, the MPA doses were set at
0.675,1.35, 2.7, and 5.4 mg/kg for the free MPA fraction at 1, 2, 4, and
8%, respectively. The AUC0e4h of the total and free MPA, and the
AUEC0e4h of IMPDH from the simulated data were calculated using
the linear trapezoidal method.
The time-course of IMPDH activity after an intravenous infusion
of MPA (5 mg/kg) in the control and NAR groups are shown in Fig. 2.
The IMPDH activity sharply decreased during the MPA intravenous
administration for 1 h, and gradually recovered after the infusion.
The IMPDH0 and the AUEC0e4h of IMPDH activity were not signif-
icantly different between both groups of rats (Table 1).
2.9. Statistical analysis
3.2. Relationship between total or free MPA concentration and
IMPDH activity
All data were expressed as mean standard error of the mean
(SEM). The statistical significance of the difference in mean values
between the two groups was analyzed using an unpaired t test if
the variance of the two groups were similar. Otherwise, the Man-
neWhitney's U test was used for the analysis. A value of P < 0.05
was considered to be statistically significant.
The relationships between the MPA concentration and IMPDH
activity in the control and NAR groups are shown in Fig. 3. The
IMPDH activity was inhibited by total and free MPA, concentration-
dependently. Although the inhibition curve of the NAR group was
Table 1
Pharmacokinetic and pharmacodynamic parameters of MPA after intravenous
infusion (5 mg/kg) in controls and NARs by the non-compartmental analysis.
Parameters
Total MPA:
Controls
NARs
Cmax
(
mg/mL)
28.5 2.5
46.0 4.3
1.80 0.10***
1.58 0.07***
AUC0e4h (mg$h/mL)
Free MPA:
Cmax
AUC0e4h
Total MPAG:
Cmax g/mL)
AUC0e4h g$h/mL)
Free MPAG:
Cmax g/mL)
AUC0e4h g$h/mL)
(
mg/mL)
0.597 0.052
0.961 0.089
0.447 0.024*
(
m
g$h/mL)
0.393 0.018***
(
m
5.54 1.13
13.2 4.1
5.64 0.37
7.08 0.68
(
m
(
m
1.33 0.27
3.16 0.97
3.34 0.22***
4.20 0.40
(
m
IMPDH activity:
IMPDH0
97.9 19.9
153 21
89.1 25.4
298 68
(m )
mol$secꢁ1$mol AMPꢁ1
AUEC0e4h
(m
mol$h$secꢁ1$mol AMPꢁ1
)
Cmax: maximum concentration of drug after administration, AUC: area under the
concentration time curve, IMPDH0: baseline IMPDH activity, AUEC: area under the
effect time curve. *P < 0.05, ***p < 0.001, significantly different from controls by
unpaired t test or ManneWhitney's U test. Mean SEM, n ¼ 5.
Fig. 2. Time-course of IMPDH activity. Each IMPDH activity was obtained after intra-
venous infusion of MPA at 5 mg/kg for 1 h in controls (open cycle) and NARs (closed
cycle). Data are expressed as mean SEM of five rats.

K. Yoshimura et al. / Drug Metabolism and Pharmacokinetics 30 (2015) 441e448
445
(Km: 57.9
0.9 versus 54.7
9.1
m
g/mL, Vmax: 187
44 versus
A
135 37 ng minꢁ1$mg protein ꢁ1, respectively).
200
150
100
50
3.4. Pharmacokinetic and pharmacodynamic modeling of MPA
The final estimates and its relative SEM for the pharmacokinetic
and pharmacodynamic parameters by the non-linear mixed effects
modeling are showed in Table 2. The CLf in the control group was
3.61 L hꢁ1$kgꢁ1, and this value was significantly higher by 3.15-fold
in the NAR group. In contrast, the Qf, Vc,f, Vp,f, and VfMPAG were
significantly lower in the NAR group compared with the control
group (0.304-, 0.782-, 0.421-, and 0.571-fold, respectively). The
CLfMPAG values were not significantly different between the control
and NAR groups. The IC50,f and IMPDH0 were estimated as
163 13 ng/mL and 84.4 13.7 m
mol secꢁ1$mol AMPꢁ1, respec-
0
tively, and were not significantly different between both groups. A
large interindividual variability was observed in the IC50,f at 96.4%.
Since the Hill coefficient was not significantly different from one,
the sigmoidal maximal inhibitory model was reduced to the
maximal inhibitory model. The interindividual variability for the Vc,f
was not included in the final model by the result of the chi-squared
test. Both the goodness-of-fit and visual predictive check plots for
MPA and MPAG as well as IMPDH activity demonstrated that the
final model reasonably described the observed pharmacokinetic
and pharmacodynamic data for both groups (Figs. 4 and 5).
0 0.01 0.1
1
10 100
Total MPA (μg/mL)
B
200
150
100
50
3.5. Simulation for effect of protein binding
The AUC of the total and free MPA as well as AUEC of the IMPDH
activity were simulated using the final population pharmacokinetic
Table 2
Final pharmacokinetic and pharmacodynamic parameters of MPA in controls and
NARs by the non-linear mixed effects modeling.
0
Parameters
Estimates
RSE (%)
1
10
00.001 0.01 0.1
Mean parameters:
CLf (L$hꢁ1$kgꢁ1
)
3.61
3.60
3.04
4.36
1.67
2.00
3.15
0.304
0.782
0.421
0.571
163
17.4
9.36
10.8
11.9
24.4
32.7
27.6
39.8
5.20
47.5
47.3
8.28
16.2
Free MPA (μg/mL)
Qf (L$hꢁ1$kgꢁ1
Vc,f (L/kg)
)
Vp,f (L/kg)
CLfMPAG (L$hꢁ1$kgꢁ1
)
Fig. 3. Relationship between IMPDH activity and total (A) or free MPA concentrations
(B). MPA was intravenously infusion for 1 h at 0.5 (cross) or 5 mg/kg (open cycle) in
controls and 5 (closed cycle) or 15 mg/kg (closed triangle) in NARs. Data are expressed
as mean SEM of five rats.
VfMPAG (L/kg)
FCLf
FQf
FVc,f
FVp,f
FVfMPAG
IC50,f (ng/mL)
shifted to the left compared with that of the control group for the
total MPA (Fig. 3A), the curves of both groups overlapped for the
free MPA concentration (Fig. 3B).
IMPDH0
(m
mol$secꢁ1$mol AMPꢁ1
)
84.4
Interindividual variability (IIV: CV %):
IIV for CLf
IIV for Qf
IIV for Vp,f
IIV for CLfMPAG
IIV for VfMPAG
IIV for IC50,f
IIV for IMPDH0
Residual variability (proportional error: CV %):
MPA
MPAG
IMPDH activity
16.0
22.8
34.5
41.7
23.5
96.4
29.9
36.5
40.9
52.0
47.0
40.3
42.1
33.5
3.3. mRNA expression of CYPs and UGTs and UGT activity in liver
microsomes
We measured the mRNA expressions of four CYPs and five UGTs
in the liver of both rats (Supplemental Fig. 2) and also determined
the UGT activity in the liver microsomes. The mRNA expressions of
CYP1A2 and 2B2 were significantly lower in the NAR group
compared with the control (0.525-fold and 0.330-fold, respec-
tively). Furthermore, the mRNA expression of UGT1A7 was signif-
icantly higher in the NAR group compared with the control (1.70-
fold). The mRNA expressions of CYP2C11 and 3A2, and UGT1A1,
1A2, 1A6, and 1A8 were not significantly different between the
control and NAR groups.
12.3
22.4
32.2
20.7
30.8
15.8
CLf, total body clearance of free MPA; Qf, inter-compartment clearance of free MPA;
c,f, central volume of distribution of free MPA; Vp,f, peripheral volume of distri-
V
bution of free MPA; CLfMPAG, total body clearance of free MPAG; VfMPAG, volume of
distribution of free MPAG; IC50,f, half-maximal inhibitory concentration of free MPA;
IMPDH0, baseline IMPDH activity; RSE, relative standard error; CV, coefficient of
variation; FX, fold increase of parameter “X” of NARs compared with that of controls
(CLf of NARs ¼ FCLf ꢂ CLf; Qf of NARs ¼ FQf ꢂ Qf; Vc,f of NARs ¼ FVc,f ꢂ Vc,f; Vp,f of
NARs ¼ FVp,f ꢂ Vp,f; VfMPAG of NARs ¼ FVfMPAG ꢂ VfMPAG; CLfMPAG of NARs ¼ CLfMPAG).
Kinetic parameters for MPA glucuronidation by liver micro-
somes were not significantly different between controls and NARs

446
K. Yoshimura et al. / Drug Metabolism and Pharmacokinetics 30 (2015) 441e448
A
C
B
40
250
30
200
150
100
50
30
20
10
0
20
10
0
0
0
10
20
30
40
0
50 100 150 200 250
Population Predictions
0
10
20
30
Population Predictions (μg/mL)
Population Predictions (μg/mL)
(μmol·sec -1·mol AMP -1
)
E
D
F
250
200
150
100
50
40
30
30
20
10
0
20
10
0
0
0
50 100 150 200 250
Individual Predictions
0
10
20
30
0
10
20
30
40
Individual Predictions (μg/mL)
Individual Predctions (μg/mL)
(μmol·sec -1·mol AMP -1
)
Fig. 4. Goodness-of-fit plots of observed versus population predictions (AeC) or individual predictions (DeF) using the final model. (A, D) MPA concentrations of controls and NARs.
(B, E) MPAG concentrations of controls and NARs. (C, F) IMPDH activities of controls and NARs. Each dotted line shows the line of identity.
and pharmacodynamic parameters of the control, to examine the
effect of the free fraction of MPA (Fig. 6). The AUC0e4h of the free
MPA increased linearly according to the increase in the free fraction
of MPA (Fig. 6A). However, the AUEC0e4h of the IMPDH activity
decreased non-linearly with a larger variability compared with that
in the AUC0e4h of the free MPA (Fig. 6B).
MPA. According to the simultaneous population pharmacokinetic
and pharmacodynamic analysis, the CLf of the NARs was 3.15-fold
higher compared with that of the controls, indicating the intrinsic
clearance was dramatically higher in NARs.
It has been reported that the recombinant rat UGT1A1, 1A6, and
1A7 showed MPA-glucuronization activity, and UGT1A7 showed
the highest apparent activity followed by 1A6 and 1A1 [23]. In this
study, we measured the mRNA expression levels of five UGTs and
found that the mRNA expression of UGT1A7 was higher in the NAR
group than in the control group. However, kinetic parameters for
MPA glucuronidation by liver microsomes were not significantly
different between controls and NARs. Therefore, precise mecha-
nisms of increased intrinsic clearance in NARs were unknown and
remain to be clarified in the future study.
The plasma free fraction of MPA was significantly higher in the
NAR group by approximately 12-fold than in the controls (24.8
versus 2.1%), but not 100%. The plasma albumin level of the NAR
group was barely detected at 0.0042% [27], but the concentrations of
globulins were reported to be higher in the NARs than in the control
rats [10]. Indeed, we confirmed that there was no difference in the
total protein concentrations between two groups. Since approxi-
4. Discussion
The pharmacokinetics of MPA shows a large inter- and intra-
individual variability and monitoring of its AUC after administra-
tion has been used in individualized therapy for organ
transplantations [6,9]. The plasma protein binding of MPA is over
98% [7] and, therefore, its pharmacokinetics and pharmacody-
namics could change in patients with hypoalbuminemia. In this
study, the pharmacokinetics and pharmacodynamics of MPA were
examined in NARs, and the modeling and simulation approach
using these experimental data enables the quantitative assessment
of the effects of protein binding on the associated IMPDH activity, as
a biomarker of the pharmacodynamics of MPA.
The non-compartmental pharmacokinetic analysis following
the intravenous administration of MPA showed that the AUC0e4h of
the total MPA in the NAR group was approximately 3% compared
with that in the controls, and that of the free MPA was approxi-
mately 40% of the controls. Before the experiment, we speculated
that the AUC0e4h values of the free MPA in the NAR and control
groups were similar, and that the pharmacokinetic differences in
the NARs were mainly attributable to the higher free fraction of
mately 30e40% of bumetanide was bound to rat a- and b-globulins
in NARs [11], MPA also might be bound to globulins in the plasma of
NARs. The Vc,f, Vp,f and VfMPAG significantly decreased in the NARs
compared with the controls, indicating that the distribution as well
as the hepatic intrinsic metabolism were changed in the NARs.
Concerning the pharmacodynamics, the inhibition curve for
IMPDH activity against the free MPA concentrations was similar for

K. Yoshimura et al. / Drug Metabolism and Pharmacokinetics 30 (2015) 441e448
447
A
C
B
10
250
200
150
100
50
50
8
6
4
2
0
40
30
20
10
0
0
0
1
2
3
4
0
1
2
3
4
0
1
2
3
4
Time (h)
Time (h)
Time (h)
F
E
D
2.5
2.0
1.5
1.0
0.5
0.0
10
250
200
150
100
50
8
6
4
2
0
0
0
1
2
3
4
0
1
2
3
4
0
1
2
3
4
Time (h)
Time (h)
Time (h)
Fig. 5. Visual predictive check of MPA and MPAG concentrations and IMPDH activity in control rats (AeC) or NARs (DeF). (A, D) MPA concentrations. (B, E) MPAG concentrations. (C,
F) IMPDH activities. Open circles shows observed data after intravenous infusion of MPA in 10 control rats or in 10 NARs. Although the MPA doses were 0.5 and 5 mg/kg for control
and 5 and 15 mg/kg for NAR groups, each observed data was normalized to 5 mg/kg using the individual estimated pharmacokinetic and pharmacodynamic parameters. Dotted
lines shows 5th or 95th percentile, and gray lines shows 50th percentile of 1000 simulated concentrations after intravenous infusion of 5 mg/kg of MPA using the final phar-
macokinetic and pharmacodynamic parameters of the controls or NARs.
both groups, although NARs showed higher sensitivity compared
with controls in the case of total MPA concentrations. According to
the constructed population pharmacokinetic and pharmacody-
namic model, the calculated IC50 value for free MPA was 163 ng/mL
in both rats. These results indicated that the free MPA concentra-
tions were better pharmacodynamic biomarker compared with the
total MPA concentrations. The IC50 of MPA for the proliferation of
rat PBMCs stimulated with T- or B-cell mitogens were reported as
241 nM (77 ng/mL) and 118 nM (38 ng/mL) for T- and B-cells,
respectively [28], corresponding with our IC50 value.
In this study, we successfully constructed a simultaneous
pharmacokinetic and pharmacodynamic model of MPA using the
experimental data obtained from the control and NARs by the
nonlinear mixed effects modeling approach. The predicted values
of the final model corresponded reasonably well with the observed
concentrations of MPA and MPAG, as well as the IMPDH activity in
both groups of rats. The obtained parameters indicated that
although the hepatic metabolism and distribution of free MPA were
changed in the NARs, the pharmacodynamic sensitivity to free MPA
was similar between NARs and control rats. The interindividual
variability of the IC50 for free MPA was revealed to be as high as
96.4%, although the interindividual variability of the CLf was as
small as 16.0%. These results suggest that there might be a larger
interindividual variability in the pharmacodynamics of MPA than in
its pharmacokinetics in humans.
parameters of the control rats. Since the consensus target AUC0e12h
of MPA has been proposed as 30e60 g h/mL in patients with renal
transplants [6], we set the target AUC0e4h of the total MPA to be
15 g h/mL in the simulation study. In this situation, the AUC0e4h of
the free MPA will increase linearly from 0.15 to 1.2 g h/mL based on
m
m
m
the change in the free fraction of MPA from 1 to 8%. In contrast, the
AUEC0e4h of the IMPDH activity will decrease non-linearly from
299 to 164 m
mol h$secꢁ1$mol AMPꢁ1 with an increase in the free
fraction of MPA from 1 to 8%. Patients with hepatic or renal
transplants may suffer from hepatic or renal failure due to a relapse
of the primary disease, rejection, or longitudinal adverse effects
caused by calcineurin inhibitors. Renal or hepatic dysfunction de-
creases serum albumin. Thus, it is possible that increasing the dose
of MPA based on the total AUC0e12h might cause extra-
immunosuppression, leading to an increase in adverse effects (for
example, leukopenia and risk of infection), especially in patients
with hypoalbuminemia.
In conclusion, the metabolism and disposition of free MPA were
altered in the NARs compared with the control rats, in addition to
the higher free fraction of MPA. We successfully constructed a
simultaneous pharmacokinetic-pharmacodynamic model of MPA
using a nonlinear mixed effects modeling approach, and the
following simulation study quantitatively clarified an important
issue concerning the free MPA concentration associated with the
immunosuppressive effect. In patients with decreased protein
binding of MPA, dosage adjustments that are based on the total AUC
might cause unnecessary overexposure to MPA.
We quantitatively evaluated the effect of protein binding on the
pharmacokinetics and pharmacodynamics of MPA using the final

448
K. Yoshimura et al. / Drug Metabolism and Pharmacokinetics 30 (2015) 441e448
[2] Kaufman DB, Shapiro R, Lucey MR, Cherikh WS, Bustami RT, Dyke DB.
A
Immunosuppression: practice and trends. Am J Transpl 2004;4:38e53.
[3] Minagawa K, Yamamori M, Katayama Y, Matsui T. Mycophenolate mofetil:
fully utilizing its benefits for GvHD prophylaxis. Int J Hematol 2012;96:10e25.
[4] Premaud A, Le Meur Y, Debord J, Szelag JC, Rousseau A, Hoizey G, et al.
Maximum A posteriori Bayesian estimation of mycophenolic acid pharma-
cokinetics in renal transplant recipients at different postgrafting periods. Ther
Drug Monit 2005;27:354e61.
[5] Staatz CE, Tett SE. Clinical pharmacokinetics and pharmacodynamics of
mycophenolate in solid organ transplant recipients. Clin Pharmacokinet
2007;46:13e58.
[6] van Gelder T, Hilbrands LB, Vanrenterghem Y, Weimar W, de Fijter JW,
Squifflet JP, et al. A randomized double-blind, multicenter plasma concen-
tration controlled study of the safety and efficacy of oral mycophenolate
mofetil for the prevention of acute rejection after kidney transplantation.
Transplantation 1999;68:261e6.
[7] Nowak I, Shaw LM. Mycophenolic-Acid binding to human serum-albumin e
characterizationandrelationtopharmacodynamics. ClinChem1995;41:1011e7.
[8] Glander P, Hambach P, Braun K-P, Fritsche L, Giessing M, Mai I, et al. Pre-
transplant inosine monophosphate dehydrogenase activity is associated with
clinical outcome after renal transplantation. Am J Transpl 2004;4:2045e51.
[9] Li H, Mager DE, Sandmaier BM, Storer BE, Boeckh MJ, Bemer MJ, et al. Phar-
macokinetic and pharmacodynamic analysis of inosine monophosphate de-
hydrogenase activity in hematopoietic cell transplantation recipients treated
with mycophenolate mofetil. Biol Blood Marrow Transpl 2014;20:1121e9.
[10] Nagase S, Shimamune K. Albumin-deficient rat mutant. Science 1979;205:
590e1.
30
1.5
1.0
0.5
0.0
20
10
0
1 2
4
8
Free Fraction (%)
B
[11] Kim EJ, Lee MG. Pharmacokinetics and pharmacodynamics of intravenous
bumetanide in mutant Nagase analbuminemic rats: importance of globulin
binding for the pharmacodynamic effects. Biopharm Drug Dispos 2001;22:
147e56.
500
[12] Kim EJ, Lee AK, Kim SH, Kim SG, Lee MG. Pharmacokinetics and pharmaco-
dynamics of intravenous azosemide in mutant Nagase analbuminemic rats.
Drug Metab Dispos 2003;31:194e201.
[13] Choi YH, Bae SK, Oh JM, Kim S-O, Lee MG. Pharmacokinetics of intravenous
methotrexate in mutant Nagase analbuminemic rats. Biopharm Drug Dispos
2007;28:385e92.
[14] Miida H, Arakawa S, Shibaya Y, Honda K, Kiyosawa N, Watanabe K, et al.
Toxicokinetic and toxicodynamic analysis of clofibrate based on free drug
concentrations in Nagase analbuminemia rats (NAR). J Toxicol Sci 2008;33:
349e61.
[15] Hebbar V, Shen GX, Hu R, Kim BR, Chen C, Korytko PJ, et al. Toxicogenomics of
resveratrol in rat liver. Life Sci 2005;76:2299e314.
[16] Tian H, Ou J, Strom SC, Venkataramanan R. Activity and expression of various
isoforms of uridine diphosphate glucuronosyltransferase are differentially
regulated during hepatic regeneration in rats. Pharm Res 2005;22:2007e15.
[17] Omiecinski CJ, Hassett C, Costa P. Developmental expression and in situ
localization of the phenobarbital-inducible rat hepatic mRNAs for cyto-
chromes CYP2B1, CYP2B2, CYP2C6, and CYP3A1. Mol Pharmacol 1990;38:
462e70.
400
300
200
100
0
1 2
4
8
Free Fraction (%)
Fig. 6. Effect of free fraction on pharmacokinetics and pharmacodynamics of MPA.
AUC0e4h of total MPA was targeted at 15 g h/mL, and MPA dose was set as 0.675, 1.35,
[18] Iida J, Kudo T, Shimada K, Yatsuno Y, Yamagishi S, Hasegawa S, et al. Inves-
m
tigation of the safety of topical metronidazole from
a pharmacokinetic
2.7, and 5.4 mg/kg for free fraction at 1, 2, 4, and 8%, respectively. (A) AUC0e4h of total
(white box) and free (grey box) MPA obtained from model simulations. (B) AUEC0e4h of
IMPDH activity obtained from model simulations. Box plots represent the 5th
percentile, lower quartile, median, upper quartile, and 95th percentile values obtained
from 1000 simulated data sets.
perspective. Biol Pharm Bull 2013;36:89e95.
[19] Elbarbry FA, McNamara PJ, Alcorn J. Ontogeny of hepatic CYP1A2 and CYP2E1
expression in rat. J Biochem Mol Toxic 2007;21:41e50.
[20] Myers AL, Hassan HE, Lee IJ, Eddington ND. Repeated administration of oxy-
codone modifies the gene expression of several drug metabolising enzymes in
the hepatic tissue of male sprague-dawley rats, including glutathione S-
transferase A-5 (rGSTA5) and CYP3A2. J Pharm Pharmacol 2010;62:189e96.
[21] Ma J, Zheng L, Deng T, Li CL, He YS, Li HJ, et al. Stilbene glucoside inhibits the
glucuronidation of emodin in rats through the down-regulation of UDP-
glucuronosyltransferases 1A8: application to a drug-drug interaction study
in radix polygoni multiflori. J Ethnopharmacol 2013;147:335e40.
Conflict of interest
[22] Kessler FK, Ritter JK. Induction of
a rat liver benzo[a]pyrene-trans-7,8-
None of the authors has any conflict of interest related to this
study.
dihydrodiol glucuronidating activity by oltipraz and beta-naphthoflavone.
Carcinogenesis 1997;18:107e14.
[23] Miles KK, Stern ST, Smith PC, Kessler FK, Ali S, Ritter JK. An investigation of
human and rat liver microsomal mycophenolic acid glucuronidation: evi-
dence for a principal role of UGT1A enzymes and species differences in UGT1A
specificity. Drug Metab Dispos 2005;33:1513e20.
Acknowledgments
[24] Kawanishi M, Yano I, Yoshimura K, Yamamoto T, Hashi S, Masuda S, et al.
Sensitive and validated LC-MS/MS methods to evaluate mycophenolic acid
pharmacokinetics and pharmacodynamics in hematopoietic stem cell trans-
plant patients. Biomed Chromatogr 2015;29:1309e16.
This study was supported in part by JSPS KAKENHI number
25460210.
[25] Bullingham R, Monroe S, Nicholls A, Hale M. Pharmacokinetics and bioavail-
ability of mycophenolate mofetil in healthy subjects after single-dose oral and
intravenous administration. J Clin Pharmacol 1996;36:315e24.
[26] Beal S, Boeckmann A, Sheiner L. NONMEM user's guides. NONMEM project
group. San Francisco: University of California at San Francisco; 1992.
[27] Nagase S, Shimamune K, Shumiya S. Albumin-deficient rat mutant: an animal
model for analbuminemia. Jikken Dobutsu 1980;29:33e8.
[28] Jain J, Almquist SJ, Heiser AD, Shlyakhter D, Leon E, Memmott C, et al. Char-
acterization of pharmacological efficacy of VX-148, a new, potent immuno-
suppressive inosine 5ʹ-monophosphate dehydrogenase inhibitor. J Pharmacol
Exp Ther 2002;302:1272e7.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.dmpk.2015.10.004.
References
[1] Allison AC, Eugui EM. Mycophenolate mofetil and its mechanisms of action.
Immunopharmacology 2000;47:85e118.