Pediatr Blood Cancer 2014;61:297–301
Extended Duration of Prehydration Does Not Prevent Nephrotoxicity or
Delayed Drug Elimination in High-Dose Methotrexate Infusions:
A Prospectively Randomized Cross-Over Study
Torben Stamm Mikkelsen, MD, PhD,1* Aissata Diop Mamoudou, MD,1 Ruta Tuckuviene,
Peder Skov Wehner, MD, PhD,3 and Henrik Schroeder, MD1
Background. Alkalized hydration is used as supportive care to
prevent renal toxicity during infusions with high-dose methotrexate
(HDMTX). In children with acute lymphoblastic leukemia (ALL), the
hydration is commonly initiated 4 hours before start of the
methotrexate (MTX) infusion. To test if longer duration of prehydra-
tion would prevent MTX-induced renal toxicity, we preformed a
randomized cross-over study comparing 12–4 hours of hydration
before the infusion of HDMTX. Procedures. Children with ALL and
non-Hodgkin lymphoma that were treated with infusions of HDMTX
5 or 8 g/m2 were randomized to receive intravenous prehydration 12
or 4 hours before the first HDMTX infusion. Patients alternated
between 12 and 4 hours of prehydration in the subsequent HDMTX
infusions. Renal toxicity was defined as 50% increase in plasma
Key words: drug clearance; hydration; methotrexate; nephrotoxicity; pharmacokinetics
INTRODUCTION
Intravenous infusions with high-dose methotrexate (HDMTX)
are used in the treatment of childhood acute lymphoblastic
leukemia (ALL), non-Hodgkin lymphoma, and osteosarcoma
[1–3]. Typically, HDMTX (1–12 g/m2) is infused over 4–24 hours
and the methotrexate (MTX) clearance is subsequently monitored
by serial blood sampling until plasma concentrations are at non-
toxic levels (<0.2 mM). Delayed elimination of MTX is associated
with an increased risk of mucositis and hematopoietic toxicity [4,5].
Folinic acid is co-administered to mitigate this toxicity but at the
risk of rescuing the cancer cells when given in large amounts to
patients with very delayed elimination of MTX [6].
Intravenous hydration and urine alkalinization has long been
used to prevent MTX induced renal toxicity but only few
prospective studies have focused on optimization of this hydration
regimen [5,7–10]. Intravenous hydration ensures that patients have
an adequate urinary flow and alkalinization of the urine increases
the solubility of MTX in an effort to prevent crystallization within
the renal tubular system. When hydration was initially employed, it
significantly decreased the incidence of renal toxicity and increased
the clearance of MTX [5,9]. The ideal length of prehydration has
never been determined; as a consequence prehydration time varies
from a couple of hours to more than 24 hours between ALL
treatment protocols [5,11–13]. It has been suggested that extended
prehydration (i.e., 12–24 hours) can prevent renal toxicity in
patients who have had delayed MTX elimination in previous
HDMTX infusions [14]. Theoretically, longer prehydration time
could result in more stable urine pH and thereby prevent that MTX
precipitates as a consequence of short periods of acidic urine.
To determine whether extending prehydration beyond 4 hours is
beneficial in preventing delayed MTX clearance and renal toxicity,
we conducted a prospectively, randomized, cross-over study that
compared the effect of 4 hours versus 12 hours of prehydration in
patients with ALL or non-Hodgkin lymphoma who were treated
with HDMTX 5 or 8 g/m2.
C 2013 Wiley Periodicals, Inc.
DOI 10.1002/pbc.24623
Published online 3 September 2013 in Wiley Online Library
(wileyonlinelibrary.com).
MD, PhD,
2
creatinine after the HDMTX infusion. The plasma MTX concentration
was measured during and after the HDMTX infusion to determine if
the duration of prehydration would influence the systemic MTX
clearance. Results. A total of 47 patients (224 HDMTX infusions) with
a median age of 4.9 years were included in the study. The duration of
prehydration had no effect on MTX induced renal toxicity that
occurred in 18.5% of all HDMTX 5 g/m2 infusions and in 40.0% of all
HDMTX 8 g/m2 infusions. Similar the duration of prehydration had
no impact on the systemic clearance of MTX. Conclusion. Extending
prehydration beyond 4 hours does not reduce the risk of renal toxicity
or delayed MTX clearance after infusions with HDMTX 5–8 g/m2.
Pediatr Blood Cancer 2014;61:297–301. # 2013 Wiley Periodicals, Inc.
METHOD
Patients
Patients 1–21 years of age with ALL or non-Hodgkin-
lymphoma were eligible if treated with HDMTX 5 or 8 g/m2 on
the ALL treatment protocols: NOPHO-2000 or NOPHO-2008 from
the Nordic Association of Pediatric Hematology and Hematology
(NOPHO), or the non-Hodgkin-lymphoma treatment protocols
NHL-NOPHO 95 or European Inter-group Co-operation on
Childhood Non-Hodgkin-Lymphoma (EICNHL) protocol EURO-
LB 2002. Patients were treated in one of three pediatric oncology
departments in Denmark. Patients were excluded from the analysis
if they did not receive a full dose of HDMTX. Table SI shows the
HDMTX schedules used in the relevant treatment protocols. The
study was approved by the local ethics committee and conducted in
accordance with the Declaration of Helsinki [15]. Written informed
consent was obtained from all patients or parents before
randomization (ClinicalTrials.gov number, NCT00570817).
Additional Supporting Information may be found in the online version
of this article at the publisher’s web-site.
1
Department of Pediatric Oncology, Aarhus University Hospital,
Aarhus, Denmark; 2Department of Pediatric Oncology, Aalborg
University Hospital, Aalborg, Denmark; 3Department of Pediatric
Oncology, Odense University Hospital, Odense, Denmark
Conflict of interest: Nothing to declare.


Correspondence to: Torben Stamm Mikkelsen, Aarhus University
Hospital, Skejby, Brendstrupgaardsvej 100, 8200 Aarhus N, Denmark.
E-mail: torben.mikkelsen@ki.au.dk
Received 27 March 2013; Accepted 9 May 2013
298 Mikkelsen et al.
Study Design and Treatment
We used a randomized, open-labeled, cross-over design to deter-
mine if duration of prehydration had an effect on MTX elimination
and MTX-induced renal toxicity. After stratification according to
HDMTX dosage and study-center, patients were randomly assigned
to receive 12- or 4-hour prehydration before the first HDMTX
course, and then to alternate between the two prehydration-
schedules through the following HDMTX courses (Fig. S1). The
inclusion of patients and obtainment of informed written consent
was performed by one physician at each of the three institutions.
Randomization was performed by a computer software program
(http://www.randomization.com) and concealed in envelops until
informed consent was obtained from the individual patients.
The prehydration fluid, consisting of 5% glucose with 40 mmol/L
sodium bicarbonate and 20 mmol/L potassium chloride, was infused
through a central venenous catheter at a rate of 150 ml/m2/hour.
Urine volume and pH was measured at every voiding and extra
bicarbonate was administered if needed to achieve a urine pH  7.0.
The IV hydration was continued at a rate of 125 ml/m2/hour through
the HDMTX course until the plasma MTX concentration was below
0.2 mM. The HDMTX infusions were only started if patients had
WBC 1.0  109/L or ANC 0.5  109/L, normal renal function,
and liver aminotransferases ASAT/ALAT not elevated more than 10
times upper limit of normal. The HDMTX was given as a 24-hour
intravenous infusion with 1/10 of the dose infused during the first
hour, and the remaining 9/10 as a continuous infusion over the
following 23 hours. Blood samples were drawn through a central
venous catheter at 23, 36, 42, 48, 54, and 66 hours following
initiation of the MTX infusion and at least twice daily thereafter,
until the plasma MTX concentration was below 0.2 mM. Plasma
MTX concentrations were measured using a fluorescence polariza-
tion immunoassay on a TDx instrument with reagents from Abbott
according to the manufacturer’s instructions. The first dose of folinic
acid (leucovorin or isovorin) was administered intravenously
36 hours after start of the 8 g/m2 HDMTX infusion or 42 hours
after start of the 5 g/m2 HDMTX infusion and then at intervals of
6 hours until MTX plasma concentrations were below 0.2 mM.
All patients received at least six doses of isovorin 7.5 mg/m2 or
leucovorin 15 mg/m2 and an increased dosage was administered to
patients with plasma MTX concentrations above 3 mM at 36 hours
or above 1 mM at 42 hours after start of the HDMTX infusion. To
prevent renal toxicity, intravenous hydration was increased to
4,500 ml/m2/day if the plasma creatinine concentration rose to 1.5
times the pre-treatment concentration, or if the 36-hour plasma
MTX concentration was 3 mM or 42-hour plasma MTX
concentration was 1 mM. MTX induced renal toxicity was
defined as a 1.5-fold change in plasma creatinine from baseline to
36 hours after start of the HDMTX infusion. Gastrointestinal
toxicity (mucositis) was registered daily by patients and parents
during the HDMTX infusion and until 2 weeks after. Ten to fourteen
days after the HDMTX infusion, MTX-related hematopoietic
toxicities were assessed as the concentration of platelets,
hemoglobin and leucocytes. Hematopoietic toxicities were graded
according to the version 2.0 National Cancer Institute Common
Toxicity Criteria.
Study End Points
The primary end point was incidence of high-risk plasma MTX
concentrations, defined as plasma MTX concentrations above 3 mM
Pediatr Blood Cancer DOI 10.1002/pbc
at 36 hours, or above 1 mM at 42 hours after start of the HDMTX
infusion; concentrations above these levels have previously been
associated with a high risk of toxicity and therefore requires
increased folinic acid dosages [5,16–19]. The key secondary end
point was MTX induced renal toxicity. Additional clinical end
points considered were median MTX plasma exposure from 23 to
66 hours after start of the HDMTX infusion (AUC23–66hours),
changes in plasma creatinine, total dosage of folinic acid, and
duration of hospitalization after initiation of the HDMTX infusion.
The exposure to methotrexate assessed as AUC23–66h was
calculated in STATA 11 using the trapezoid method.
Statistical Analysis
Sample-size estimation was based on MTX pharmacokinetic
data from the NOPHO-92 ALL treatment protocol, in which the
prevalence of delayed MTX elimination was 0.18 for patients
treated with 4 hours of prehydration [20]. Based on clinical
observations from institutions in Scandinavia which have used long
prehydration regimens, we assumed that the prevalence of delayed
MTX elimination would be less than 10% in HDMTX infusions
preceded by 12 hours of prehydration. When considering each
HDMTX infusion as an independent observation within patients,
we estimated that 75 infusions with each prehydration length
(HDMTX 5 g/m2) would be needed to provide 80% power of
detecting a 50% difference between the prehydration-regimens at a
two-sided significance level of 0.05.
Serial evaluations of HDMTX infusions within each patient
were considered non-independent observations and a mixed-effects
logistic regression was used to assess the relationship between the
primary endpoint and duration of prehydration. Delayed elimina-
tion was the primary outcome variable and was clustered by patient
with baseline creatinine included as a covariate. We also used the
first HDMTX infusion with 4-hour prehydration and the first with
12-hour prehydration from each patient to determine if duration of
prehydration had an effect on delayed MTX elimination and
incidence of MTX-related renal toxicity (increase in plasma
creatinine 50% from baseline, McNemar’s test). When comparing
the other endpoints including plasma MTX concentrations, total
folinic acid dose, hospitalization time, plasma MTX concentration
time above 1 mM and change in plasma creatinine, we calculated
the median values from the HDMTX courses with 4 hours and
12 hours of prehydration, respectively, from each patient. The
difference between prehydration regimens for these secondary
endpoints was tested with a Wilcoxon matched-pairs signed-rank
test. A P-value of <0.05 was considered statistically significant. All
statistical analyses were performed with STAT11 software.
RESULTS
Plasma Methotrexate Pharmacokinetics
A total of 47 patients with a median age of 4.9 years were
included in the study. Twelve patients received between two and
four infusions of HDMTX 8 g/m2 and 35 patients received between
two and nine infusions of HDMTX 5 g/m2. Patient characteristics
are summarized in Table I. The median duration of prehydration in
the 4-hour prehydration group was 4 hours (5th percentile 4 hour;
95th percentile 6 hour) and in the 12-hour prehydration group it was
12 hours (5th percentile 12 hour; 95th percentile 13.5 hour). The
MTX concentrations at end of the HDMTX infusion did not differ
 Prehydration for High-Dose Methotrexate 299

TABLE I. Demographics and Characteristics of Patients

Diagnosis Age (years) BSA (m2) Gender Number of infusions

HDMTX dosage ALL/lymphoma Median (range) Median (range) Female/male Total Median (range)
5 g/m (n ¼ 35)
2
32/3 4.9 (2.0–19.6) 0.78 (0.54–2.23) 15/20 189 5 (2–9)
8 g/m2 (n ¼ 12) 11/1 5.2 (2.5–19.0) 0.78 (0.60–1.85) 6/6 35 3 (2–4)
Total (n ¼ 47) 43/4 4.9 (2.0–19.6) 0.78 (0.54–2.23) 21/26 224 4 (2–9)
HDMTX, high-dose methotrexate; ALL, acute lymphoblastic leukemia; BSA, body surface areas.

between the 4- and 12-hour prehydration groups for patients who
received HDMTX 5 g/m2 (median: 85.0 mM vs. 86.9 mM; P ¼ 0.56)
or patients who received HDMTX 8 g/m2 (median: 129 mM vs.
126 mM; P ¼ 0.65). Furthermore, there was no difference between
the 4- and 12-hour prehydration regimens when plasma MTX
concentrations were compared at 36 and 42 hours after start of the
HDMTX infusion, regardless of HDMTX dose (Table II). Duration
of prehydration did not have any impact on MTX exposure assessed
as AUC (Table II). In an explorative analysis we compared plasma
MTX concentrations between the 12- and 4-hour prehydration
within patient subgroups defined by age (1–12 and 12–21 years);
sex (male and female); patients with ALL; and baseline plasma
creatinine (4–26, 27–41, and 41–88 mM); there was no significant
differences between the two prehydration regimens within any of
these specified patient subgroups (data not shown).
Delayed Elimination
In this study, delayed elimination of MTX was observed in 47%
(95% CI: 40–54%) of the infusions with HDMTX 5 g/m2 and in
74% (95% CI: 57–88%) of the infusions with HDMTX 8 g/m2.
There was no significant association between delayed MTX
elimination and duration of prehydration when adjusted for
baseline plasma creatinine in a mixed-effects logistic regression
model OR 0.97 (95% CI, 0.89; 1.05; P ¼ 0.41). Furthermore, there
was no difference in median folinic acid dosage during the first
66 hours following initiation of the HDMTX infusion between the
4- and 12-hour prehydration (median: 82.5 mg/m2 vs. 75.0 mg/m2;
P ¼ 0.31). The frequency of gastrointestinal toxicity was low and
grade III to IV mucositis occurred only in 9% of the infusions.
Grade III–IV anemia was only seen after 1% of the HDMTX
infusions but blood transfusions were needed after 51/131 (39%) of
the infusions. Duration of prehydration had no impact on risk of
grade III or VI mucositis or hematopoietic toxicities. Prehydration
did not have any impact on duration of hospitalization regardless of
HDMTX dosage.
Renal Toxicity
All patients had baseline plasma creatinine concentrations
below the upper limit of normal (Table III). Plasma creatinine
concentrations increased significantly from baseline to 36 hours
after start of the HDMTX infusions (P < 0.001), but this change did
not differ between the 4-hour prehydration (median: 15%; range
33% to 690%) and the 12-hour prehydration (median: 23%;
range: 42% to 452%) (P ¼ 0.38). MTX induced renal toxicity
occurred in 18.5% of all HDMTX 5 g/m2 infusions and in 40.0% of
all HDMTX 8 g/m2 infusions. The 12-hour prehydration did not
reduce the risk of developing MTX induced renal toxicity in the
HDMTX 5 g/m2 group nor the HDMTX 8 g/m2 group.
We further explored whether the 12-hour prehydration had any
preventive effect on renal toxicity in the subgroup of patients who
had at least one event of severe delayed MTX elimination. Eleven
patients with a mean age of 12.7 years (range: 4–20 years) had
plasma MTX concentrations above 5 mM at the 42-hour time point
and all of these infusions were accompanied by a 1.5-fold increase
in plasma creatinine (n ¼ 15 infusions). Of these 15 HDMTX
infusions, 10 were preceded by a 12-hour prehydration and only 5
by a 4-hour prehydration. There were no significant differences in
median plasma MTX concentrations at 36, 42, or 48 hours after start
of the MTX infusions when the two prehydration regimens were
compared within these patients (Table IV). Likewise, there was no

TABLE II. Plasma Methotrexate After 4- and 12-Hour Prehydration (Median and Range)

HDMTX 5 g/m2 HDMTX 8 g/m2

Plasma PK variable 4-Hour 12-Hour n P
4-Hour 12-Hour n P

23-Hour (mM) 85.0 86.9 35 0.56 123 132 12 0.93
47–209 44–217 57–206 49–226
36-Hour (mM) 2.0 2.2 35 0.64 3.8 3.3 12 0.27
0.8–11.7 0.8–16.4 1.0–7.5 1.2–23.4
42-Hour (mM) 0.8 0.9 35 0.17 1.71 1.2 12 0.40
0.3–6.2 0.3–5.7 0.4–6.0 0.5–25.5
AUC 17.8 17.5 35 0.34 33.0 25.4 12 0.39
4.5–132 6.3–105 8.6–162 10–691
HDMTX, high-dose methotrexate; AUC, area under the time concentration curve; PK, pharmacokinetics. Median of each patient’s HDMTX
infusions was used to calculate the median and range in the two infusions. Area under the time concentration curve (AUC) (mmol hour/L) was
calculated as AUC23–48 for HDMTX 5 g/m2 and AUC23–66 for HDMTX 8 g/m2.
Wilcoxon matched-pairs signed-rank test.
Pediatr Blood Cancer DOI 10.1002/pbc
300 Mikkelsen et al.

TABLE III. MTX Induced Renal Toxicity Assessed as Change in Serum Creatinine After the 4- and 12-Hour Prehydration and 5 g/m2
Methotrexate (Median and Quartiles)

HDMTX 5 g/m2 HDMTX 8 g/m2

Unit 4-Hour 12-Hour P
4-Hour 12-Hour P

Baseline creatinine mM 36 34 0.43 22 28 0.75
26–42 26–43 18–56 19–50
Change in creatininea % 22 19 0.48 41 50 0.63
5–31 5–30 22–80 15–98
HDMTX, high-dose methotrexate. Median of each patient’s HDMTX infusions was used to calculate the median and range in the two infusions.


Wilcoxon matched-pairs signed-rank test. aChange from baseline (pre-treatment) to 36 hours after start of the HDMTX infusion.

difference between prehydration regimens when the change in
plasma creatinine was compared between prehydration regimens in
this patient subgroup (median: 20.2% vs. 24.5%; P ¼ 0.32).
DISCUSSION
Using a cross-over study design, we tested the hypothesis that
12 hours of prehydration prior to infusions with HDMTX (5 or 8 g/m2)
would reduce the risk of renal toxicity and delayed MTX
elimination as compared with 4 hours of prehydration. We found
no statistically significant difference between the two hydration
regimens.
Prehydration is employed because typically 10% of the
HDMTX dosage is infused during the first hour and readily
excreted through the kidneys resulting in very high MTX
concentrations in the renal tubule. There is substantial evidence
that the glomerular filtration rate decreases in the early hours
following initiation of a HDMTX infusion and that MTX-induced
renal toxicity is related to impaired clearance of MTX [21]. In this
study, we found that renal function decreased significantly within
the first 36 hours after start of the HDMTX infusion but extended
duration of prehydration was not able to prevent the incidence
of renal toxicity. A 50% increase in plasma creatinine was
observed in 18.5% (MTX 5 g/m2) and 40% (MTX 8 g/m2) of all
HDMTX infusions. This relatively high frequency of renal toxicity
is similar to what we have previously observed and we have no
obvious explanation to why others experience much less renal
toxicity [21].
A weakness of our study was the heterogeneity of the study
population (i.e., age, gender, cancer treatment protocol) as this
potentially masked an effect of prehydration duration in one of
these subgroups of patients. Age, gender, treatment protocol, and
pre-treatment creatinine concentration have been shown to impact
MTX clearance [16,22]. However, in the current study, we found no
significant differences in MTX concentrations or incidence of renal
toxicity between prehydration regimens when patients were
grouped by gender, age, baseline plasma creatinine, or cancer
protocol. Furthermore, there was no difference in MTX pharmaco-
kinetics when compared between prehydration regimens in patients
who had at least one HDMTX infusion with severely delayed MTX
elimination (42-hour MTX  5 mM). This suggests that prehydra-
tion beyond 4 hours does not have any effect even in patients who
are predisposed to develop renal toxicity or impaired MTX
clearance.
The extensive hydration used together with HDMTX infusions
increases the urinary diuresis at least threefold compared to normal.
In future studies, it would be interesting to test if a further increase
in diuresis, for example, by using prehydration 200 ml/m2/hour
would prevent MTX induced renal toxicity. Increased diuresis or
urine alkalinization could theoretically prevent MTX precipitation
in the early phase of the HDMTX infusion where the urine MTX
concentration is high [10]. However, it is also plausible that factors
other than tubular crystallization of MTX are responsible for the
delayed MTX elimination. Recent studies have shown that genetic
variations in genes encoding liver and kidney transporter proteins
account for some of the variability in MTX clearance [22–24].

TABLE IV. Plasma Methotrexate Pharmacokinetics in 11 Patients With One or More Infusions With 42 Hours MTX > 5 mM (Median
[Range])

Plasma PK variable 4-Hour 12-Hour P

23-Hour (mM) 104 134 0.32
70–127 80–183
36-Hour (mM) 3.7 11.9 0.21
3.0–7.1 2.2–23.4
42-Hour (mM) 1.2 5.7 0.32
1.1–6.0 0.9–25.5
Plasma creatinine pre-treatment 42 61 0.40
10–55 19–81
Change in plasma creatinine (%) 20.2 24.5 0.32
0.0–690 12.3; 452.6
HDMTX, high-dose methotrexate; MTX, methotrexate.
Wilcoxon matched-pairs signed-rank test.
Pediatr Blood Cancer DOI 10.1002/pbc

In conclusion, the results from this study provide no evidence
that extending prehydration beyond 4 hours can reduce the risk of
delayed MTX elimination or renal toxicity after infusions with
HDMTX 5 and 8 g/m2. Interestingly, the frequency of very-delayed
MTX elimination combined with renal toxicity did not differ
between prehydration-groups, indicating that even in patients at
high risk of experiencing delayed MTX elimination there would be
no benefit from long prehydration time. Overall our findings suggest
that it is sufficient to start prehydration the same day as the
HDMTX-infusion is scheduled, thus keeping the hospitalization
time at a minimum.
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