Population pharmacokinetic

and pharmacogenetic
analysis of 6-mercaptopurine
in paediatric patients with
acute lymphoblastic
leukaemia
Ahmed F. Hawwa, Paul S. Collier, Jeff S. Millership,
Anthony McCarthy,
1
Sid Dempsey,
1
Carole Cairns
1
&
James C. McElnay
Clinical and Practice Research Group, School of Pharmacy, Medical Biology Centre, Queens University
Belfast and
1
Haematology and Oncology Outpatient Department, Royal Belfast Hospital for Sick
Children, The Royal Hospitals, Belfast Health and Social Care Trust, Belfast, UK
Correspondence
Professor Paul S. Collier, Clinical and
Practice Research Group, School of
Pharmacy, Medical Biology Centre,
Queens University Belfast, 97 Lisburn
Road, Belfast BT9 7BL, UK.
Tel: + 44 28 9097 2009
Fax: + 44 28 9024 7794
E-mail: p.collier@qub.ac.uk
----------------------------------------------------------------------
Keywords
6-mercaptopurine, acute lymphoblastic
leukaemia, NONMEM, pharmacogenetics,
population pharmacokinetics
----------------------------------------------------------------------
Received
10 July 2008
Accepted
5 August 2008
Published OnlineEarly
24 September 2008
WHAT IS ALREADY KNOWN ABOUT
THIS SUBJECT
The cytotoxic effects of 6-mercaptopurine (6-MP)
were found to be due to drug-derived intracellular
metabolites (mainly 6-thioguanine nucleotides and to
some extent 6-methylmercaptopurine nucleotides)
rather than the drug itself.
Current empirical dosing methods for oral 6-MP result
in highly variable drug and metabolite concentrations
and hence variability in treatment outcome.
WHAT THIS STUDY ADDS
The rst population pharmacokinetic model has been
developed for 6-MP active metabolites in paediatric
patients with acute lymphoblastic leukaemia and the
potential demographic and genetically controlled
factors that could lead to interpatient
pharmacokinetic variability among this population
have been assessed.
The model shows a large reduction in interindividual
variability of pharmacokinetic parameters when body
surface area and thiopurine methyltransferase
polymorphism are incorporated into the model as
covariates.
The developed model offers a more rational dosing
approach for 6-MP than the traditional empirical
method (based on body surface area) through
combining it with pharmacogenetically guided
dosing based on thiopurine methyltransferase
genotype.
AIMS
To investigate the population pharmacokinetics of 6-mercaptopurine (6-MP) active
metabolites in paediatric patients with acute lymphoblastic leukaemia (ALL) and
examine the effects of various genetic polymorphisms on the disposition of these
metabolites.
METHODS
Data were collected prospectively from 19 paediatric patients with ALL (n = 75
samples, 150 concentrations) who received 6-MP maintenance chemotherapy
(titrated to a target dose of 75 mg m
-2
day
-1
). All patients were genotyped for
polymorphisms in three enzymes involved in 6-MP metabolism. Population
pharmacokinetic analysis was performed with the nonlinear mixed effects
modelling program (NONMEM) to determine the population mean parameter
estimate of clearance for the active metabolites.
RESULTS
The developed model revealed considerable interindividual variability (IIV) in the
clearance of 6-MP active metabolites [6-thioguanine nucleotides (6-TGNs) and
6-methylmercaptopurine nucleotides (6-mMPNs)]. Body surface area explained a
signicant part of 6-TGNs clearance IIV when incorporated in the model (IIV
reduced from 69.9 to 29.3%). The most inuential covariate examined, however,
was thiopurine methyltransferase (TPMT) genotype, which resulted in the greatest
reduction in the models objective function (P < 0.005) when incorporated as a
covariate affecting the fractional metabolic transformation of 6-MP into 6-TGNs.
The other genetic covariates tested were not statistically signicant and therefore
were not included in the nal model.
CONCLUSIONS
The developed pharmacokinetic model (if successful at external validation) would
offer a more rational dosing approach for 6-MP than the traditional empirical
method since it combines the current practice of using body surface area in 6-MP
dosing with a pharmacogenetically guided dosing based on TPMT genotype.
British Journal of Clinical
Pharmacology
DOI:10.1111/j.1365-2125.2008.03281.x
826 / Br J Clin Pharmacol / 66:6 / 826837 2008 The Authors
Journal compilation 2008 The British Pharmacological Society
Introduction
6-Mercaptopurine (6-MP) is a purine antimetabolite widely
used in the maintenance chemotherapy of childhood
acute lymphoblastic leukaemia (ALL), the most common
cancer in children. 6-MP has been a key component of
almost every successful therapeutic regimen for low- to
mild-risk ALL. However, it was not until the early 1980s that
6-MP cytotoxic effects were found to be due to drug-
derived intracellular metabolite concentrations rather
than the plasma level of 6-MP itself [1].
Following oral administration, 6-MP undergoes exten-
sive biotransformation by three enzymes, two of which are
catabolic, xanthine oxidase (XO) and thiopurine S-methyl
transferase (TPMT) and one anabolic, hypoxanthine phos-
phoribosyl transferase (HPRT). XO metabolises 6-MP
to 6-thiouric acid (6-TU), whereas TPMT methylates 6-MP to
6-mMP. HPRT carries out the rst anabolic step to produce
6-thioinosine monophosphate (6-TIMP) and subsequently
the active 6-thioguanine nucleotides (6-TGNs). 6-TIMP
can alternatively be methylated by TPMT, yielding
6-methylmercaptopurine nucleotides (6-mMPNs) [2].
Finally, it is hypothesized that 6-TIMP is converted succes-
sively into 6-thioinosine diphosphate (6-TIDP) and triphos-
phate (6-TITP) to form 6-TIMP once again by the action of
the enzyme inosine triphosphatase (ITPA) [3].
Cytotoxic effects of 6-MP are achieved primarily
through the incorporation of 6-TGNs into the DNA of leu-
cocytes, due to their structural similarity to the endog-
enous purine based guanine [2, 4]. Moreover, 6-mMPNs are
strong inhibitors of purine de novo synthesis, which is a
well-established protocol to achieve immunosuppression
[5, 6]. Despite these facts, the pharmacokinetics of the
active metabolites of 6-MP remain poorly explored. A
better understanding of the disposition of 6-TGNs and
6-mMPNs would therefore be immensely helpful in
improving the design of dosing regimens for 6-MP.
In this study, the pharmacokinetics of 6-TGNs and
6-mMPNs in paediatric patients with ALL under 6-MP
maintenance chemotherapy were examined prospectively
and, for the rst time, a population pharmacokinetic model
for 6-MP active metabolites, 6-TGNs and 6-mMPNs, in pae-
diatric patients with ALL was developed. In developing this
model, potential factors that could lead to variability in
6-MP cytotoxic metabolites that would be particularly
helpful in improving the dosing guidelines for 6-MP were
assessed.
Methods
Patients and data collection
The study was approved by the National Health Service
Ofce for Research Ethics Committees in Northern Ireland.
Informed parental consent was obtained for each child
before enrolment in the study. In addition, verbal assent
was obtained from older children (>10 years) after provi-
sion of a verbal description of the study and what it
involved.
Data were collected from19 paediatric patients attend-
ing the Haematology and Oncology Outpatient Depart-
ment at the Royal Belfast Hospital for Sick Children and
who had been diagnosed to be suffering from ALL. Blood
samples were taken from children who had been on
continuous/maintenance 6-MP therapy for at least 1
month and who had received constant daily doses for at
least 1 week. Patients who satised the above criteria but
who had received intensication therapy or red blood cell
(RBC) transfusion within the previous 2 months were
excluded.
Blood samples were obtained at a phase of treatment
when children had an indwelling cannula for vincristine
therapy and at least 12 h after the preceding 6-MP dose.
(Note, the blood sample was taken prior to the administra-
tion of vincristine.)
Maintenance chemotherapy for ALL patients consisted
of daily oral 6-MP and weekly methotrexate.The 6-MP dose
was titrated to the target protocol dose of 75 mg m
-2
day
-1
,
adjusted for each child according to leucocyte count and
the presence of clinically relevant infections. Additionally, a
monthly dose of intravenous vincristine was given to all
children irrespective of blood count. Chemotherapy was
administered usually for 2 or 3 years. The children had their
full blood counts assessed at each clinic visit (every 2
weeks) for bone marrow toxicity.
The blood samples (1.5 ml) were collected in ethylene-
diamine tetraaceticacid (EDTA) tubes and kept on ice until
centrifuged at 1000 g for 10 min to separate plasma from
RBC. Separated plasma was frozen at -20C in Eppendorf
tubes while RBC were washed twice with a balanced salt
solution, then suspended at a density of 8 10
8
RBC per
200 ml and kept frozen at -20C until required for further
processing. These latter samples for the determination of
metabolite content were taken on a maximumof ve occa-
sions (one sample per occasion), at monthly intervals, over
the study period.
A sample of blood (1 ml) from each patient, taken on
one occasion only, was collected in an EDTA tube and kept
at -20C without centrifugation for genotyping the
enzymes of interest (200 ml of whole blood was sufcient
for this purpose).
In addition to information on dosing and times of sam-
pling, the following data were collected for each child: age,
weight, surface area, gender, ongoing pathology (e.g. renal
and/or hepatic impairment), concomitant drug therapy,
lab test results and records of any side-effects experienced.
The demographic and clinical characteristics of the study
participants are shown in Table 1.
Assay of 6-MP metabolites
RBC concentrations of 6-MP active metabolites, 6-TGNs
and 6-mMPNs, were measured by a reversed high-
6-Mercaptopurine pharmacokinetics and pharmacogenetics
Br J Clin Pharmacol / 66:6 / 827
performance liquid chromatography methodology that
was developed earlier [7]. Intraday and interday coef-
cients of variation (CV) were 3.1 and 4.3%, and the limits
of quantication were 13 and 95 pmol per 8 10
8
RBC,
respectively.
Genotyping of TMPT, ITPA and XO
All patients were screened for seven common polymor-
phisms in three enzymes involved in 6-MP metabolism
(XO, TPMT and ITPA) that are potentially linked to the phar-
macodynamics, toxicity and treatment outcome of 6-MP;
two polymorphisms in XO (A1936G and A2107G) and
ITPA(C94Aand IVS2+21AC) and three polymorphisms
in TPMT (TPMT*3A, TPMT*3B and TPMT*3C).
Detection of the various single nucleotide polymor-
phisms (SNPs) in the enzymes genetic loci was based on
TaqMan genotyping assays (ABI, Foster City, CA, USA)
using somatic cell DNA extracted from patient blood
samples (QIAmp DNA Blood Mini kit; Qiagen, Hilden,
Germany). The conditions used for polymerase chain reac-
tion and subsequent detection of the genotypes were as
described in the manufacturers instructions.
Population pharmacokinetic modelling
The pharmacokinetics of 6-MP active metabolites, 6-TGNs
and 6-mMPNs, were determined using a population
approach in which concentrations from ALL patients were
analysed simultaneously to produce estimates of the phar-
macokinetic parameters. RBC concentrationtime proles
of 6-TGNs and 6-mMPNs were used for nonlinear mixed
effect modelling by extended least squares regression
using NONMEM (version VI, level 1.1 with double precision)
[8] installed on a personal computer in conjunction with
DIGITAL Visual Fortran compiler (version 5.0.A). Patients
were assigned randomly to either an index group (n = 15)
for the development of the pharmacokinetic model, or to
the validation group (n = 4) for the purpose of assessing
the predictive performance of the derived model. The rst-
order conditional estimation (FOCE) method with interac-
tion was used to estimate population mean parameters,
interindividual variability (IIV) in these parameters and
residual variability between measured and predicted
metabolite concentrations.
The concentrationtime courses of 6-MP metabolites
were described by using a one-compartment model with
rst-order absorption and elimination. The absorption rate
constant (k
a
) and the bioavailability factor (F) of the model
were xed at 1.3 h
-1
and 22%, respectively, according to the
literature [1, 9].The pharmacokinetic parameters estimated
from this model (implemented using PREDPP subroutine
ADVAN6) were clearance (CL) for 6-TGNs and 6-mMPNs
and the fractional metabolic transformation of 6-MP into
6-TGNs.
The relationship between the parent drug and its
metabolites was dened according to the following differ-
ential equations:
d A 6-MP
dt
F A 6-MP
Gut
a Gut
( )
= ( ) k
d A 6-MP
dt
F A 6-MP A 6-MP
Central
a Gut Central
( )
= ( ) ( ) k k
20
d A 6-TGNs
dt
FM A 6-MP
CL C 6-TG
Central
Central
-TGNs
( )
= ( )

3
6
k
me
NNs
Central
( )
d A 6-mMPNs
dt
FM A 6-MP
CL C 6-
Central
Central
-mMPNs
( )
= ( )

4
6
k
me
mmMPNs
Central
( )
where k
a
is the absorption rate constant of the parent drug,
k
20
is the elimination rate constant of 6-MP (k
20
= 0.53 h
-1
based on literature [1, 9]), k
me
is the metabolic transforma-
tion rate constant of the parent drug into either 6-TGNs or
6-MPNs. FMi is the fractional metabolic transformation into
the metabolite i (6-TGNs are designated by the number
3 and 6-mMPNs are designated by the number 4, FM
4
=
1 FM
3
), and A, C are the amount/concentration of the
drug or metabolite at the time t. k
20
= k
me
+ k
other
(k
other
is
the elimination rate constant of bioavailable 6-MP trans-
formed by other metabolic processes, k
other
= 0.22 k
20
=
0.1166 h
-1
based on the literature [9]).
Table 1
The demographic and clinical characteristics of the ALL population
Parameter n = 19
Gender (F : M) 6 : 13 (32% : 68%)
Age [median (range) years] 10 (317)
Weight [median (range) kg] 33.4 (13.277.5)
Body surface area [median (range) m
2
] 1.14 (0.592)
6-MP daily dose [median (range) mg] 50 (10100)
6-MP daily dose [median (range) mg kg
-1
] 1.42 (0.163.46)
6-MP daily dose [median (range) mg m
-2
] 40 (5.8876.47)
Co-medication during 6-MP chemotherapy
Methotrexate weekly dose [median (range) mg] 15 (525)
Cotrimoxazole (b.d. twice weekly) [median
(range) mg]
360 (120480)
Haematological parameters
Hb [median (range) g dl
-1
] 12.9 (10.916.5)
WBC [median (range) 10
9
l
-1
] 3.3 (1.29.1)
PLT [median (range) 10
9
l
-1
] 282 (66648)
ANC [median (range) 10
9
l
-1
] 1.68 (0.38.3)
XO, TPMT, and ITPA genotypes
XO A1936,G (heterozygotes/homozygotes) 1/0
XO A2107,G 1/0
TPMT*3A 1/0
TPMT*3B
TPMT*3C 2/0
ITPA C94,A 3/0
ITPA IVS2+21A,C 2/1
ALL, acute lymphoblastic leukaemia; 6-MP, 6-mercaptopurine; WBC, white blood
cells; PLT, platelets; ANC, absolute neutrophil count; XO, xanthine oxidase; TPMT,
thiopurine methyltransferase; ITPA, inosine triphosphatase.
A. F. Hawwa et al.
828 / 66:6 / Br J Clin Pharmacol
An exponential error model was used to describe the
deviations of the individuals clearance from the true (but
unknown) population mean values:
CL TVCL e
i
i CL
=
,
where CL
i
is the CL of the ith individual, TVCL is the typical
population estimate for CL and h
i,CL
is a random variable
that distinguish the i
th
individuals parameter from the
population mean values as predicted by the regression
model and is assumed to be normally distributed in the log
domain with a geometric mean of zero and a variance of

CL
2
. An exponential model was chosen for the IIV, since
pharmacokinetic parameters are usually log-normally dis-
tributed. In modelling this variability, the square root of the
variance was interpreted as the CV.
Residual variability, which describes the residual error
between the measured and the predicted metabolite con-
centrations, was modelled using additive, proportional and
combined error structures. The additive error model,
however, best described the residual variability. This vari-
ability could arise from intra-individual variability in the
pharmacokinetic parameters, inaccuracy in the timing of
sample collection and dosage administration, assay error,
model misspecication, or non-adherence to therapy.
C C
ij pred ij ij
= +
,

C
ij
is the measured and C
pred,ij
is the model predicted
metabolite concentration of the i
th
individual at the j
th
sam-
pling time and e
ij
is the residual error term, which is a
random variable with zero mean and variance of s
2
. The
magnitude of this residual error or variability was
expressed as a standard deviation (SD).
Regression model
The initial analysis for the population pharmacokinetics of
6-MP metabolites, 6-TGNs and 6-mMPNs, was conducted
without including any patient covariates in the model
(BASE model). The conditional estimates of h
i,CL
were
obtained from this BASE model and then plotted against
the following covariates: age, gender, weight, body surface
area (BSA), TPMT, ITPA and XO genotypes in order to iden-
tify any potential relationship between CL and the covari-
ates. The inuence of the identied covariates on CL was
individually assessed by incorporating them in the BASE
model (univariate analysis). The regression relationship for
CL was modelled in a linear or nonlinear way for continu-
ous covariates:
Proportional: CL = q
CL
(1 + q
COV
COV)
Power: CL = q
CL
COV
qCOV
Exponential: q
CL
e
qCOV COV
where COV is a general continuous covariate and q
CL
and
q
COV
are the regression coefcients to be estimated by
NONMEM.
Categorical covariates (1 or 0) were examined using a
multiplicative model:

CL
CL COV
COV
=
where q
CL
is the population value in the absence of the
covariate (COV = 0) and q
COV
is the fractional change when
the covariate is present (COV = 1). Similar models were
used to investigate the effect of covariates on FM
3
.
For each model, the improvement in the t obtained on
addition of a xed effect variable (covariate) into the
model was assessed using a likelihood ratio test. The
change in the objective function value (DOBJF) produced
by the inclusion of a covariate represents a statistic that is
proportional to minus twice the log-likelihood of the data
and approximates a c
2
distribution with degrees of
freedom equal to the difference in the number of struc-
tural parameters (qs) between two models. A decrease in
the OBJF 6.63 was considered statistically signicant
(P < 0.01, 1 degree of freedom) for the addition of one xed
effect.
The goodness of t for each model was also assessed
by examining the precision of parameter estimates (i.e.
standard errors of the mean), the decrease in interindi-
vidual and residual variability, and graphs of residuals
(RES), weighted residuals (WRES) and measured metabo-
lite concentrations plotted separately against predicted
concentrations.
As a result of the univariate analysis, each model with
signicant effect was ranked according to its DOBJF com-
pared with the BASE model. The model with the largest
DOBJF was designated as the INTERMEDIATE model and
multiple regression analysis with forward selection was
performed where covariates were incorporated into the
INTERMEDIATE model one by one along the rank order
established by univariate analysis until all signicant cova-
riates were included and no further statistically signicant
reduction in OBJF was obtained (FULL model). The FULL
model was then subjected to stepwise, backward elimina-
tion to obtain the FINAL model. An increase in the OBJF
6.63 (P < 0.01, 1 degree of freedom) was required to
retain the covariate in the FINAL model.
Model evaluation
Since external validation using a newdataset fromanother
study is extremely difcult in paediatric studies, internal
validation using data splitting or resampling techniques is
an appropriate alternative [10]. In the present study, inter-
nal validation using the data-splitting technique was per-
formed to verify the predictive value of the population
model.
The predictive performance of the model was assessed
in terms of bias (mean prediction error) and precision (root
mean square prediction error) by comparing the measured
concentrations in the validation group (n = 4) with the cor-
responding predicted values by the population model
6-Mercaptopurine pharmacokinetics and pharmacogenetics
Br J Clin Pharmacol / 66:6 / 829
using post hoc Bayesian forecasting. This was achieved by
xing the structural and variance model parameters to the
values estimated in the nal population model.
The mean prediction error (ME) and root mean square
prediction error (RMSE) were determined according to the
following formulae [11]:
ME
N
pe
i
i
N
=
=

1
1
MSE
N
pe
i
i
N
=
=

1
2
1
RMSE MSE =
The prediction error (pe
i
) was calculated as:
pe
i
= C C
obs pred
where C
obs
and C
pred
represent the observed and predicted
concentrations, respectively.
In addition, WRES and measured metabolite concentra-
tions were plotted separately against the predicted con-
centrations to assess visually the deviations of model
predicted from observed metabolite concentrations in the
validation group.
In order to evaluate the performance of the nal model,
obtained by tting the full dataset (comprising index and
validation groups combined together), a posterior visual
predictive check was performed by simulating from
the nal estimates and comparing the distribution of
the observations with the simulated distribution. The
adequacy of the model was demonstrated by plotting the
time course of the observations along with the prediction
interval for the simulated values.
Results
On oral administration of 6-MP, large interindividual differ-
ences were observed as regards metabolite concentra-
tions and the course of elimination as shown in Figure 1. It
is apparent from the graph that patients with TPMT muta-
tions had higher 6-TGNconcentrations but compared with
other patients they had relatively low 6-mMPN concentra-
tions in erythrocytes.
At certain time points after treatment with the drug,
6-TU could also be identied in addition to the parent
drug, 6-MP. However, their low levels (due to the sampling
time chosen in this study as stated above) did not qualify
them for incorporation into NONMEM analysis. NONMEM
analysis in this pharmacokinetic study, therefore, was per-
formed using the measured erythrocyte levels of 6-TGNs
and 6-mMPNs in all samples (n = 75 samples, 150 concen-
trations) obtained from 19 paediatric patients with ALL
receiving 6-MP maintenance chemotherapy (a maximum
of ve samples was obtained per patient, one sample per
occasion).
Pharmacokinetic modelling
In the initial model, the data were described with a one-
compartment model with rst-order absorption and elimi-
nation since there were no points to enable the accurate
evaluation of the distribution phase. In addition, since most
of the kinetic data were collected in the post-absorption
phase, k
a
and F (the bioavailability factor) could not be
reliably estimated. Hence, their values were xed according
to the literature (1.3 h
-1
and 22%, respectively) throughout
the analysis [1, 9]. An additive error model best described
the residual variability and was used in the basic pharma-
cokinetic model (BASE) in order to be used for further
analysis.
The population estimates fromthe BASE model for FM
3
(the fractional metabolic transformation of 6-MP into
6-TGNs), CL
6-TGNs
(6-TGN clearance) and CL
6-mMPNs
(6-mMPN
0
5000
10000
15000
20000
25000
30000
4 3 2 1
Time (in months)
6
-
m
M
P
N
s

(
p
m
o
l
/
8

1
0
8

R
B
C
s
)
0
200
400
600
800
1000
1200
4 3 2 1
Time (in months)
6
-
T
G
N
s

(
p
m
o
l
/
8

1
0
8

R
B
C
s
)
A
B
Figure 1
Individual 6-thioguanine nucleotide (6-TGN) (A) and
6-methylmercaptopurine nucleotide (6-mMPN) (B) concentration plots.
Patients having any mutation are highlighted and their corresponding
types are displayed. The therapeutic lower and upper limits suggested in
literature are indicated by the dashed lines.TPMT mutant (); ITPAmutant
(); TPMT and ITPA mutant ()
A. F. Hawwa et al.
830 / 66:6 / Br J Clin Pharmacol
clearance) were 0.028, 0.0125 l h
-1
and 0.0231 l h
-1
with
an interindividual variability (%CV) of 69.9 and 38.1% for
CL
6-TGNs
and CL
6-mMPNs
, respectively. The residual variability
corresponded to a SD of 0.18 and 9.04 mg l
-1
of packed
RBC for 6-TGNand 6-mMPNmetabolite levels, respectively.
Regression models
Plotting of the conditional estimates of h
i,CL
for the
metabolites 6-TGNs and 6-mMPNs in paediatric patients
with ALL fromthe BASE model vs. covariates showed some
potential relationships. An example is shown in Figure 2.
Univariate analyses showing covariates with signicant
effects on FM
3
, CL
6-TGNs
and CL
6-mMPNs
are presented in
Table 2. The addition of several covariates resulted in a
reduction of the OBJF of >6.63 (P < 0.01). These covariates
were TPMT mutations affecting FM
3
, CL
6-TGNs
and CL
6-mMPNs
parameters, along with weight (WT) and BSA, which
affected CL
6-TGNs
.
In the model that incorporated weight as a covariate
affecting CL
6-TGNs
, clearance was standardized using an allo-
metric model that is based on theoretical and empirical
evidence that CL is proportional to the
3
/
4
power of weight
[12].
The covariate that caused the largest reduction in the
OBJF was the dichotomous covariate model incorporating
the effect of TPMT mutations on FM
3
. Therefore, it was
declared as an INTERMEDIATE model, subsequent addition
to which of the signicant covariates was analysed. Multi-
variate analysis incorporating both BSA and TPMT muta-
tions as covariates showed that in the presence of the
effect of TPMT mutations on FM
3
, the effect of BSA on
CL
6-TGNs
remained signicant. The results of multivariate
analysis together with the various steps in building the
FULL model are presented in Table 3. Backward elimination
of any covariate from the FULL model increased the OBJF
by >6.63. Therefore, both factors TPMT mutations and BSA
were retained in the FINAL model.
Adequate correlations between predicted and
observed RBC concentrations of 6-TGNs and 6-mMPNs
were observed in the FINAL model, as shown in Figures 3
and 4. The scatterplots showthat the population predicted
and individual predicted concentrations of 6-TGNs and
6-mMPNs were in reasonable agreement with the mea-
sured concentrations around the line of identity, although
some underprediction could be observed, particularly at
higher concentrations.
Model evaluation
Bias (ME) and precision (RMSE) of the predictive perfor-
mance of the FINAL model were tested in the validation
group (n = 4) between the measured and predicted
metabolite concentrations. ME computed values for
6-TGNs and 6-mMPNs were -0.047 and 1.67 mg l
-1
, respec-
tively. The corresponding RMSE values were 0.16 and
4.84 mg l
-1
, respectively. Both were less than the residual
unexplained variability of the index group, which corre-
sponded to a SD of 0.188 and 9.04 mg l
-1
for 6-TGNs and
6-mMPNs, respectively.
The scatter plots of weighted residuals vs. model
predicted RBC concentrations of 6-TGNs and 6-mMPNs
(Figure 5) showed that both were randomly distributed
and the weighted residuals lay within 2 units of the null
ordinate of perfect agreement. Specic examples of the
predictive capability of the nal model are shown for two
representative individual patients in the validation group
in Figure 6, which shows the time course of measured and
post hoc predicted RBC concentrations of 6-TGNs and
6-mMPNs.
Final population pharmacokinetic model
The computed population structural parameter estimates,
the interpatient variability (CV%) and the residual variabil-
ity obtained by tting the full dataset to FINAL model are
presented in Table 4.
The nal population model for 6-TGNs and 6-mMPNs in
RBC was:
FM
TPMT
3
0 019 2 56 = ( ) . .
CL h BSA
6-TGNs
1.16
1 0 00914
1
( ) = ( ) .
CL h
6-mMPNs
1 0 0228
1
( ) = .
where BSA is body surface area in m
2
and TPMT = 1 in case
of mutation, otherwise = 0.
1.50
1.00
0.50
0.00
0.50
1.00
1.50
2.00
1.00 1.50
Surface area (m
2
)
h
i
,

C
L
6
-
T
G
N
s
2.00
Figure 2
Plots of the conditional estimates of h
i,CL6-TGNs
vs. body surface area. The
solid line indicates the Lowess smooth line
6-Mercaptopurine pharmacokinetics and pharmacogenetics
Br J Clin Pharmacol / 66:6 / 831
For a hypothetical individual with population median
value of BSA (i.e. 1.14 m
2
), the model-predicted FM
3
, CL
6-TGNs
and CL
6-mMPNs
would be 0.019, 0.011 l h
-1
and 0.0288 l h
-1
,
respectively. The FM
3
would increase by 256% to 0.049 if
the patient had a TPMT mutation. The model for CL
6-TGNs
found that CL
6-TGNs
was proportional to the 1.16 power of
BSA, resulting in an estimated range of 0.00490.02 l h
-1
across the BSA range of 0.592.0 m
2
among the study
group.
The median individual Bayesian estimates for FM
3
,
CL
6-TGNs
, CL
6-mMPNs
and BSA normalized CL
6-TGNs
values
obtained by tting the full dataset from the study popula-
tion to the FINAL model are presented in Table 5. These
estimates are determined as a postprocessing step using
the measured concentrations, in contrast to the population
parameter estimates, which are derived from covariate
information. Since the individual Bayesian estimates are
drawn from a distribution where the population estimates
reect the posterior mode of the marginal likelihood dis-
tribution for that parameter, a possibility for differences in
this summary parameter could be introduced. In order to
evaluate the predictive performance of the nal model
obtained fromthe full dataset, a posterior predictive check
was performed (Figure 7).
The interpatient variability (CV%) for the population
pharmacokinetic parameters of CL
6-TGNs
and CL
6-mMPNs
were
33.6 and 33.2%, respectively. The interpatient variability of
CL
6-TGNs
was reduced from 65.6 to 33.6% by the inclusion of
BSA covariate, which explained most of the variability in
CL
6-TGNs
between individuals. The residual unexplained vari-
ability (SD) was 0.177 and 8.42 mg l
-1
for 6-TGNs and
6-mMPNs, respectively, which translates to a CV% of 24.3
Table 2
Summary of univariate analysis showing covariate models with signicant effects on FM
3
, CL
6-TGNs
or CL
6-mMPNs
of 6-TGN and 6-mMPN metabolites
Effect on Model DOBJF* Parameter estimate P-value
FM3

FM TPMT 3

TPMT
-8.69

FM3
= 0 129 .
<0.005
TPMT = 1 in case of mutation, otherwise = 0
CL6-TGNs


CL TGNs
BSA
6-
( ) BSA -7.95

CL TGNs 6-
= 0 163 .
<0.005
CL6-TGNs

CL TPMT TGNs 6-

TPMT
-7.32

CL TGNs 6-
= 0 333 .
<0.01
TPMT = 1 in case of mutation, otherwise = 0
CL6-mMPNs

CL TPMT mMPNs 6-

TPMT
-7.15

CL mMPNs 6-
= 0 0196 .
<0.01
TPMT = 1 in case of mutation, otherwise = 0
CL6-TGNs

CL TGNs 6-
( ) WT 33 40
0 75
.
.
-6.92

CL TGNs 6-
= 0 185 .
<0.01
*Reduction in the OBJF in comparison with the basic model (BASE). A DOBJF 6.63 is statistically signicant at P < 0.01 for 1 degree of freedom. 6-TGN, 6-thioguanine nucleotide;
6-mMPN, 6-methylmercaptopurine nucleotide; TPMT, thiopurine methyltransferase.
Table 3
Summary of stepwise model building
Model Effect DOBJF* P-value
BASE No covariates included
INTERMEDIATE TPMT mutations on FM3 -8.69 <0.005
FULL TPMT mutations on FM3 -12.85 <0.0005
BSA on CL6-TGNs
*Change in OBJF in comparison to preceding model. A DOBJF 6.63 is statistically
signicant at P < 0.01 for 1 degree of freedom. TPMT, thiopurine methyltrans-
ferase; BSA, body surface area.
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
0
Population predicted 6-TGN
concentrations (mg/L)
O
b
s
e
r
v
e
d

6
-
T
G
N
c
o
n
c
e
n
t
r
a
t
i
o
n
s

(
m
g
/
L
)
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
Individual predicted 6-TGN
concentrations (mg/L)
O
b
s
e
r
v
e
d

6
-
T
G
N
c
o
n
c
e
n
t
r
a
t
i
o
n
s

(
m
g
/
L
)
A
B
4.5 4 3.5 3 2.5 2 1.5 1 0.5
0 4.5 4 3.5 3 2.5 2 1.5 1 0.5
Figure 3
Scatter plots of observed vs. population predicted (A) and individual pre-
dicted (B) red blood cell (RBC) concentrations of 6-thioguanine nucle-
otides (6-TGNs) in the index group of the FINAL model. The solid line
represents the line of identity
A. F. Hawwa et al.
832 / 66:6 / Br J Clin Pharmacol
and 57.6% at the mean RBC concentrations of 6-TGNs and
6-mMPNs measured in the full dataset (0.729 and
14.61 mg l
-1
, respectively).
Discussion
Current empirical dosing methods for oral 6-MP result in
highly variable drug and metabolite concentrations, as
demonstrated by the present study and other investiga-
tors [13, 14]. The variability presumably arises in part from
individual differences in bioavailability. Other factors,
however, that could contribute to this variability were
evaluated in the present study for paediatric patients with
ALL using a population pharmacokinetic modelling
approach. The different factors studied were age, gender,
WT and BSA, along with various genetic factors such as
polymorphisms in XO, TPMT and ITPA enzymes.
Quantitativeevaluationof pharmacokinetic parameters
of 6-MP seems especially attractive and is a potentially
important prognostic factor in cancer chemotherapy [15].
In addition, the study of drug distribution is of particular
relevance for paediatric patients, given the effect of matu-
rational changes on organ function and body composition
that can affect drug disposition [16]. For these reasons, we
developed in this study, for the rst time, a population
pharmacokinetic model for 6-MP and its metabolites for
paediatric patients with ALL.
The pharmacokinetic model developed revealed con-
siderable IIV in the clearance of both 6-MP metabolites
investigated, 6-TGNs and 6-mMPNs. This variability,
however, coincides with the highly variable RBC concen-
trations of 6-TGNs and 6-mMPNs reported previously in
children taking identical doses of 6-MP [13, 14]. The esti-
mated IIV (CV%) of clearance in the BASE model, tted to
the index group, was 69.9 and 38.1% for 6-TGNs and
6-mMPNs, respectively. The model, however, showed large
reduction in the IIV of 6-TGN clearance (69.929.3%) and
a signicant reduction in the OBJF (7.95, P < 0.005) when
BSA alone was incorporated into the model as a covariate
affecting 6-TGN clearance. This indicated that large part of
the IIV in 6-TGN clearance was explained by differences in
BSA.
0 70 60 50 40 30 20 10
0
10
20
30
40
50
60
70
Population predicted 6-mMPN
concentrations (mg/L)
0 70 60 50 40 30 20 10
Individual predicted 6-mMPN
concentrations (mg/L)
O
b
s
e
r
v
e
d

6
-
m
M
P
N
c
o
n
c
e
n
t
r
a
t
i
o
n
s

(
m
g
/
L
)
s
0
10
20
30
40
50
60
70
O
b
s
e
r
v
e
d

6
-
m
M
P
N
c
o
n
c
e
n
t
r
a
t
i
o
n
s

(
m
g
/
L
)
A
B
Figure 4
Scatter plots of observed vs. population predicted (A) and individual pre-
dicted (B) red blood cell (RBC) concentrations of 6-methylmercaptopurine
nucleotides (6-mMPNs) in the index group of the FINAL model. The solid
line represents the line of identity
0 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1
0 25 20 15 10 5
1.5
1
0.5
0
0.5
1
1.5
Model predicted 6-TGN
concentrations (mg/L)
W
e
i
g
h
t
e
d

r
e
s
i
d
u
a
l
s
0.6
0.4
0.2
0
0.2
0.4
0.6
0.8
1
Model predicted 6-mMPN
concentrations (mg/L)
W
e
i
g
h
t
e
d

r
e
s
i
d
u
a
l
s
A
B
Figure 5
Scatter plots of weighted residuals vs. predicted concentrations of
6-thioguanine nucleotides (6-TGNs) (A) and 6-methylmercaptopurine
nucleotides (6-mMPNs) (B) in red blood cells (RBC)
6-Mercaptopurine pharmacokinetics and pharmacogenetics
Br J Clin Pharmacol / 66:6 / 833
Both the WT and BSA had signicant effects on 6-TGN
clearance in this study. BSA, however, was found to be a
better predictor of clearance than WT. The nal model
obtained by tting the full dataset indicated that CL
6-TGNs
increased by 116% per 1-m
2
increase in the BSA resulting
in an estimated range of 0.00490.02 l h
-1
across the BSA
range of 0.592.0 m
2
among the study group.
Clinical experience also conrms the effect of impaired
renal function on the concentration of 6-MP metabolites
and its clinical outcome. It has been shown that patients
with impaired renal function have increased susceptibility
to 6-MP side-effects. In the present study, none of the
patients had clinically signicant renal impairment and
hence renal function was mainly proportional to patients
BSA.
The rationale behind the use of BSA as a criterion for
dosing in anticancer chemotherapy was outlined about
50 years ago [17], giving rise to the practice of using BSA
for dosing anticancer therapy. The current approach for
dosing anticancer drugs is to administer a standard dose,
which is normalized to BSA, and then to adjust or individu-
alize subsequent doses based on the severity of drug tox-
icity. Even though this clearly has practical and economic
implications, its clinical value has, in recent years, been
questioned [18]. One objection to the use of BSA in mea-
suring drug dosage was the difculty in measuring BSA.
Apart from the inaccuracies inherent in methods for BSA
calculation [19], they are dependent on the accuracy of
weight and height measurements used in BSA calculation.
Another alternative was the use of normograms to avoid
inaccurate determination of BSA. However, the reliability of
these normograms tends to differ [20].
Another objection was that pharmacokinetic studies of
anticancer drugs revealed substantial interpatient variabil-
ity in plasma drug concentrations when the dose was
based on BSA [13, 21]. The potential consequences of this
variability in systemic drug exposure are life-threatening
toxic effects in patients who are exposed to excessive drug
concentrations and tumour progression in patients who
achieve subtherapeutic drug concentrations. The identi-
cation of specic factors that account for variability in drug
disposition among patients can, therefore, lead to more
rational dosing methods.
In the present study, the most inuential covariate
examined was TPMT genotype on the fraction of meta-
bolic transformation, FM
3
. Its inclusion in the model
resulted in the greatest reduction in OBJF (DOBJF = -8.69,
P < 0.005). Hence, it explained part of the variability in FM
3
between individuals. In addition, the nal model predicted
an increase in FM
3
of 256%if the patient had a TPMT muta-
tion. This would mean preferential 6-TGN production and
6-mMPNunderproduction if the patient had a TPMT muta-
tion. This is in perfect agreement with previous studies,
which demonstrated an inverse correlation betweenTPMT
activity and the levels of 6-TGNs in RBC, suggesting that
an inherited decrease in the methylation step (due to the
TPMT mutation) results in shunting 6-MP metabolism
away from 6-mMPNs towards overproduction of 6-TGNs
[22, 23]. More importantly, the optimal dose of 6-MP, which
was determined in the standard empirical fashion, was also
related to TPMT genotype [22, 24].
No prior studies, however, have provided specic 6-MP
dosing guidelines based onTPMT genotype. In the present
study, for the rst time, the potential utility of TPMT geno-
type in prospectively dening the percentage of 6-MP con-
verted to 6-TGNs or 6-mMPNs as predicted by FM
3
was
illustrated. This would identify patients who are less toler-
ant of 6-MP standard doses based on their capacity to
metabolize the drug. One limitation of the developed
model, however, was its inability to account for variation in
TPMT activity among patients having the wild-type. This
4500 4000 3500 3000 2000 1500 1000 500 2500
0.0
0.2
0.4
0.6
0.8
10
15
20
25
C
o
n
c
e
n
t
r
a
t
i
o
n

(
m
g
/
L
)
Time (hrs)
4500 4000 3500 3000 2000 1500 1000 500 2500
Time (hrs)
0.0
0.1
0.2
0.3
0.4
0.5
0.6
6
8
10
12
14
16
18
20
C
o
n
c
e
n
t
r
a
t
i
o
n

(
m
g
/
L
)
A
B
Figure 6
Longitudinal assessment of the predictive performance of the FINAL
model in two representative patients from the validation dataset: (A)
12-year-old boy and (B) 6-year-old girl. () Observed and () model-
predicted 6-thioguanine nucleotide (6-TGN) concentrations. ()
Observed and () predicted 6-methylmercaptopurine nucleotide
(6-mMPN) concentrations
A. F. Hawwa et al.
834 / 66:6 / Br J Clin Pharmacol
could be the reason for underprediction observed in the
model for 6-mMPN concentrations at higher levels. Higher
concentrations of 6-mMPNs are probably due to high
TPMT activity that could not be accounted for by the
model. Phenotyping patients, however, by measuring
TPMT enzymatic activity could resolve this limitation and
allowfor individualization of drug doses among the whole
population.
The other genetic covariates tested in this study (ITPA,
XO mutations) were not statistically signicant and there-
fore not included in the FINAL model. It should be noted,
however, that the IIV not explained by the model might
have resulted, in part, from these covariates. It is also pos-
sible that the small number of patients studied (n = 19)
was not enough to identify more potential sources of IIV.
Therefore, the developed population model will need to
be tested and rened by larger prospective studies that
have the potential to encounter and quantify more
covariates.
The reasonably large residual (unexplained) variability
in the model might be due to large IIV in the pharmaco-
kinetic parameters, interoccasional variability, assay errors,
or model misspecication. Since the data in most patients
were collected for a period of several months, this might
have added to the variability within the same patient.
Moreover, the signicant IIV in drug absorption may
explain some of the variability obtained with the orally
administered 6-MP [25]. Non-adherence to therapy could
also have contributed to this variability. Children with ALL
who have reached remission always remain under a pro-
longed and complex course of chemotherapy in spite of
being practically asymptomatic. These factors are likely
to contribute to missing some doses given the home
therapy of 6-MP and the absence of obvious conse-
quences at the time. Therefore, non-adherence is
somehow expected in this group of patients and should
be considered.
Although the robustness of the pharmacokinetic
model developed in this study was validated internally
using the data-splitting technique, it is highly recom-
mended that the model be subjected to external valida-
tion before using it to guide dosage adjustments. If
successful, this would offer a more rational dosing
approach than the traditional empirical method, since it
would combine the current practice of using BSA in 6-MP
dosing with a pharmacogenetically guided dosing based
on TPMT genotype.
Table 4
Population pharmacokintics of 6-TGNs and 6-mMPNs, obtained by tting the full dataset (19 children) to the BASE and FINAL models
Parameter Symbol
Base model Final model
Estimate w (CV%) Estimate w (CV%) SE (%)
FM3

FM3 0.0295 0.0191 64.1
CL6-TGNs (l h
-1
)

CL6-TGNs 0.0138 65.7 0.00914 33.6 56.8
CL6-mMPNs (l h
-1
)

CL6-mMPNs 0.0227 33.9 0.0228 33.1 14.2
TPMT qTPMT 2.56 35.9
BSA qBSA 1.16 49.0
wCL6-TGNs 0.431 0.113 81.2
wCL6-mMPNs 0.115 0.11 82.6
s6-TGNs (mg l
-1
) 0.171 0.177 17.5
s6-mMPNs (mg l
-1
) 9.04 8.42 19.3
Structural models:
TVFM
FM
TPMT
3 3
=
TPMT
TVCL BSA
-TGNs -TGNs 6 6
= ( )

CL
BSA
TVCL
-mMPNs -mMPNs 6 6
=
CL
Random effects models:
CL TVCL e
-TGNsi 6-TGNs
i CL6-TGNs
6
=
,
CL TVCL e
-mMPNsi 6-mMPNs
i CL6-mMPNs
6
=
,
where FM3 is the fractional transformation of 6-MP into 6-TGNs; TPMT, 1 if the patient had TPMT mutation and 0 otherwise; CL6-TGNs is 6-TGN clearance; BSA is body surface area
in m
2
; CL6-mMPNs is 6-mMPN clearance; w is the interindividual variability; h is squared w; s6-TGNs and s6-mMPNs are the residual variabilities of 6-TGNs and 6-mMPNs. 6-TGN,
6-thioguanine nucleotide; 6-mMPN, 6-methylmercaptopurine nucleotide; TPMT, thiopurine methyltransferase.
Table 5
Individual Bayesian estimates obtained fromthe FINAL population model
Parameter Median (P5, P95)
FM3 (no TPMT mutation) 0.0191
FM3 (with TPMT mutation) 0.0491
CL6-TGNs (l h
-1
) 0.0112 (0.0046, 0.0235)
CL6-mMPNs (l h
-1
) 0.0226 (0.0153, 0.0310)
CL6-TGNs (l h
-1
m
-2
)* 0.0082 (0.0059, 0.0151)
P5, 5th percentile; P95, 95th percentile. *Calculated using the body surface area
of each individual. 6-TGN, 6-thioguanine nucleotide; 6-mMPN, 6-
methylmercaptopurine nucleotide; TPMT, thiopurine methyltransferase.
6-Mercaptopurine pharmacokinetics and pharmacogenetics
Br J Clin Pharmacol / 66:6 / 835
Competing interests
None declared.
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