Population Pharmacokinetics of Intramuscular Artesunate in African Children With Severe Malaria: Implications for a Practical Dosing Regimen

Abstract

Parenteral artesunate (ARS) is the drug of choice for the treatment of severe malaria. Pharmacokinetics data on intramuscular ARS are limited with respect to the main treatment group that carries the highest mortality, namely, critically ill children with severe malaria. A population pharmacokinetic study of ARS and dihydroartemisinin (DHA) was conducted from sparse sampling in 70 Tanzanian children of ages 6 months to 11 years. All the children had been admitted with severe falciparum malaria and were treated with intramuscular ARS (2.4 mg/kg at 0, 12, and 24 h). Venous plasma concentration-time profiles were characterized using nonlinear mixed-effects modeling (NONMEM). A one-compartment disposition model accurately described first-dose population pharmacokinetics of ARS and DHA. Body weight significantly affected clearance and apparent volume of distribution (P < 0.001), resulting in lower ARS and DHA exposure levels in smaller children. An adapted dosing regimen including a practical dosing table per weight band is proposed for young children based on the pharmacokinetic model.Clinical Pharmacology & Therapeutics (2013); advance online publication 20 March 2013. doi:10.1038/clpt.2013.26.

Full text

Articles
nature publishing group
Severe falciparum malaria is one of the main causes of death
in African children, who account for >85% of the worldwide
malaria-related mortality.1 In children, the course of the disease
is more rapid than in adults, with most deaths occurring within
the rst 24 h aer admission despite parenteral antimalarial
treatment.2,3
Artesunate (ARS), administered parenterally, is currently
the drug of choice for the treatment of severe malaria in all
age groups and all malaria-endemic settings.4 A large trial
(SEAQUAMAT) carried out in Asia in a study population con-
sisting mostly of adult patients (n = 1,461) with severe falcipa-
rum malaria showed a 35% reduction in mortality with ARS
treatment as compared with quinine.
5
More recently, a larger
trial (AQUAMAT) performed in 5,425 African children with
severe malaria showed a 22.5% lower mortality in children
treated with parenteral ARS as compared with quinine.6
ARS has a broader stage-specicity and more potent para-
siticidal eect than quinine.
7,8
ARS is water-soluble and can be
administered either as an i.v. slow bolus or as an i.m. injection. e
latter route is more practical in the majority of African hospital
and clinic settings with limited facilities. e absorption of i.m.
ARS is rapid and reliable, with peak concentrations occurring
within 1 h.9,10 Aer being injected, ARS is rapidly and almost
completely converted into its active metabolite, dihydroarte-
misinin (DHA).
11
e elimination of ARS is very rapid, and anti-
malarial activity is determined mainly by DHA exposure levels.
DHA has a terminal elimination half-life of ~45 min
10,12,13
and is
~93% plasma protein–bound in patients with falciparum infec-
tion.
14
e current dosing recommendation for ARS in the treat-
ment of severe malaria is 2.4 mg/kg on admission, followed by
the same dose aer 12 h and then a daily dose until the patient is
able to take oral antimalarial therapy reliably. is recommenda-
tion, for the most part, was derived empirically from studies in
adults.15,16
In small children with severe disease, the pharmacokinetic
properties of antimalarials may dier from those reported in
Parenteral artesunate (ARS) is the drug of choice for the treatment of severe malaria. Pharmacokinetics data on
intramuscular ARS are limited with respect to the main treatment group that carries the highest mortality, namely,
critically ill children with severe malaria. A population pharmacokinetic study of ARS and dihydroartemisinin (DHA)
was conducted from sparse sampling in 70 Tanzanian children of ages 6 months to 11 years. All the children had been
admitted with severe falciparum malaria and were treated with intramuscular ARS (2.4 mg/kg at 0, 12, and 24 h). Venous
plasma concentration–time profiles were characterized using nonlinear mixed-effects modeling (NONMEM). A one-
compartment disposition model accurately described first-dose population pharmacokinetics of ARS and DHA. Body
weight significantly affected clearance and apparent volume of distribution (P < 0.001), resulting in lower ARS and DHA
exposure levels in smaller children. An adapted dosing regimen including a practical dosing table per weight band is
proposed for young children based on the pharmacokinetic model.
Received 8 August 2012; accepted 23 January 2013; advance online publication 20 March 2013. doi:10.1038/clpt.2013.26
Clinical Pharmacology & erapeutics
10.1038/clpt.2013.26
Articles
20March2013
93
5
8August2012
23January2013
Population Pharmacokinetics of Intramuscular
Artesunate in African Children With Severe
Malaria: Implications for a Practical Dosing
Regimen
ICE Hendriksen1,2, G Mtove3, A Kent4, S Gesase5, H Reyburn6, MM Lemnge5, N Lindegardh1,2,
NPJ Day1,2, L von Seidlein7, NJ White1,2, AM Dondorp1,2 and J Tarning1,2
1Mahidol-Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand; 2Centre for Tropical Medicine, Churchill
Hospital, University of Oxford, Oxford, UK; 3National Institute for Medical Research, Amani Centre, Tanga, Tanzania; 4Joint Malaria Programme, Moshi, United Republic
of Tanzania; 5National Institute for Medical Research, Tanga Medical Research Centre, Tanga, Tanzania; 6London School of Hygiene and Tropical Medicine, London, UK;
7Menzies School of Health Research, Casuarina, Australia. Correspondence: J Tarning ( joel@tropmedres.ac)
Open
CLINICAL PHARMACOLOGY & THERAPEUTICS | VOLUME 93 NUMBER 5 | MAY 2013 443

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adults.17–19 Only limited data are available on the pharmacoki-
netic properties of parenteral, rectal, and oral ARS in children
with moderate to severe malaria.
9,13,20–22
e primary aim of
this study was to characterize the population pharmacokinetic
properties of ARS and its active metabolite DHA in the treat-
ment of severe malaria in African children and to determine a
practical dosing regimen.
RESULTS
Clinical details
Seventy patients of ages 7 months to 11 years were included, of
whom 59 (84%) were <5 years of age. During the study, nine of
the children died (case fatality 13%). Of these, one died within
15 min of admission, and two died within the rst 24 h aer
admission. One patient of age 2.5 years had severe neurologic
sequelae 28 days aer discharge, comprising spastic hemiparesis,
blindness, and hearing impairment. At admission this child had
a count of 880,205 parasites/µl and plasma Plasmodium falcipa-
rum histidine-rich protein-2 concentration of 1,875 ng/ml, both
indicating an extremely heavy parasite burden. At the 3-month
follow-up, the neurologic sequelae had resolved completely.
Demographic, clinical, and laboratory characteristics of the
study population are described in Tab l e 1. Severe prostration,
severe acidosis, convulsions, and severe anemia were the most
common severity criteria. Eleven patients (16%) presented
with decompensated or compensated shock. In addition,
three patients (4.3%) had blood culture–proven septicemia
(unspeciated Gram-negative rods, Klebsiella pneumoniae, or
Staphylococcus aureus); none of these presented with shock. HIV
coinfection was detected in 3/70 (4.3%) of the patients. None of
these patients were receiving antiretroviral treatment.
Pretreatment with an oral antimalarial was reported with
respect to 31 patients (6 with quinine, 3 with amodiaquine, 13
with sulfadoxine-pyrimethamine, 8 with artemether-lumefan-
trine, and 1 with amodiaquine followed by artemether-lumefan-
trine). In addition, 12 patients had received pretreatment with
i.m. quinine within the 24 h preceding admission, the median
(range) total dose being 16.1 mg/kg (10.1–53.7 mg/kg).
Each of the patients received an i.m. ARS injection of 2.4 mg/
kg shortly aer admission. Supportive treatments included blood
transfusions and uid resuscitation. Hypoglycemia was corrected
with a 10% dextrose bolus at the time of admission and in those
who developed hypoglycemia aer admission (eight patients).
Peripheral blood asexual parasite counts aer 24 h were nega-
tive in 12/66 (18%) patients (24-h slide was not available for four
patients, three of them as a result of death). e geometric mean
(95% condence interval (CI)) parasite count aer 24 h was 1,128
(537–2,368) parasites/µl in the rest of the study population (n =
54). e overall geometric mean (95% CI) fractional reduction in
parasite counts at 24 h was 96% (94–98%).
Population pharmacokinetic-pharmacodynamic analysis
A total of 274 ARS and DHA postdose samples, randomly distrib-
uted over the rst 12 h of the study, were included in the model. A
one-compartment disposition model for both ARS and DHA was
adequate to describe the observed plasma concentration–time
data (Table 2). All combinations of two-compartment disposi-
tion models displayed signicant model misspecication despite
signicantly lower objective function values. A similar nding
has been reported previously for DHA.
23
Zero-order distribution/
absorption from the injection site(s) to the central compartment
provided the best description of the data, but too few samples
were collected during the absorption phase and the absorption
rate was therefore xed for an accurate estimation. Interindividual
variability could be estimated reliably for ARS and DHA clearance
and ARS volume of distribution, showing correlation between
ARS clearance and volume.
Table 1 Demographic, clinical, and laboratory characteristics of
children admitted with severe malaria
Variable Value
Total number of patients 70
Age (y) (median, range) 2.5 (0.6 to 11)
Weight (kg) 10.8 (9 to 13.5)
Weight-for-age Z-scorea−1.2 (1.0)
Coma (based on GCS/BCS) 19 (27%)
Prostration 46 (66%)
Convulsions 26 (37%)
Shockb11 (16%)
Respiratory distress 1 (1%)
Acidosis (base excess <−8 mmol/l) 28 (43%)
Hypoglycemia (glucose <3 mmol/l) 11 (16%)
Anemia (hemoglobin <5 g/dl) 21 (30%)
Hemoglobinuria 1 (1%)
Axillary temperature (°C) 38.3 (1.0)
Heart rate (beats/min) 158 (141 to 176)
Respiratory rate (breaths/min) 49 (40 to 58)
Glucose (mg/dl) 102 (88 to 127)
Blood urea nitrogen (mg/dl)c12 (8 to 16)
Hemoglobin (g/dl) 7.1 (5.1 to 9.2)
pHc7.39 (7.33 to 7.43)
HCO3 (mmol/l)c17.8 (13.2 to 21.5)
Base excess (mmol/l)c−7 (−13 to −3)
Aspartate transaminase (U/l)d71 (49 to 116)
Alanine aminotransferase (U/l)d25 (14 to 42)
Total bilirubin (mol/l)d31 (24 to 49)
HIV-positive 3/70 (4.3%)
Parasitemia (parasites/µl); (geometric
mean, 95% CI)
88,391 (53,547 to 145,909)
Plasma PfHRP2 (ng/ml);e (geometric
mean, 95% CI)
1,893 (1,387 to 2,584)
Data are median (interquartile range), mean (SD), or n (%), unless otherwise stated.
BCS, Blantyre Coma Score; CI, confidence interval; GCS, Glasgow Coma Score; PfHRP2,
Plasmodium falciparum histidine-rich protein-2.
aWeight-for-age Z-scores for children ≤10 years43; data missing for n = 1 child of
age >10 years. bChildren with compensated shock (n = 4) and decompensated
shock (n = 7) combined. cMissing data for n = 5 children because of missing i-STAT
measurements. dMissing data for n = 5, n = 6, and n = 4 children with respect to
aspartate transaminase, alanine aminotransferase, and total bilirubin, respectively.
eMissing data because of missing samples in n = 3 children with undetectable
PfHRP2 concentrations.
444 VOLUME 93 NU MBER 5 | MAY 2013 | www.nature.com/cpt

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When body weight was used as a xed allometric function on
all elimination clearance (power of 0.75) and apparent volume of
distribution (power of 1) parameters, a signicantly better model
t was observed (Δobjective function value = −14.6). DHA clear-
ance increased 10.2% per unit (g/dl) of decrease of hemoglobin.
Interindividual variability in clearance decreased from 63.6%
coecient of variation to 55.3% coecient of variation when this
covariate was included, suggesting that it accounts for a limited
but signicant part of the observed variability.
e nal model described the observed data well, with ade-
quate goodness-of-t diagnostics (Figure 1; Eta-shrinkage:
elimination clearance for ARS 10.6%, central volume of dis-
tribution for ARS 12.2%, elimination clearance for DHA 6.81;
Epsilon-shrinkage: 49.4 and 22.5% for ARS and DHA, respec-
tively). A prediction-corrected visual predictive check of the
nal model resulted in no model misspecication and good
simulation properties (Figure 2). e numerical predictive
check (n = 2,000) for ARS resulted in 4.35% (95% CI 1.45–9.42)
and 5.07% (95% CI 1.45–9.42) of observations above and below
the 90% prediction interval, respectively; for DHA, these values
were 2.97% (95% CI 1.79–9.52) and 5.95% (95% CI 1.79–8.92),
respectively.
ARS and DHA exposures aer the standard 2.4 mg/kg dose
were simulated at each body weight level (1,000 simulations
each at 1-kg intervals from 6 to 25 kg) using a uniform distri-
bution of hemoglobin concentrations within the observed range
(2.72–13.6 g/dl) to account for the observed covariate relationship
(Figure 3a,b). Children with body weights between 6 and 10 kg
showed a mean reduction of 20.4% (P < 0.0001) in DHA exposure
as compared with children with body weights between 21 and
25 kg (median (25th to 75th percentile) exposure: 3,380 (2,130–
5,470) ng × h/ml in the 6–10 kg patients, 3,780 (2,430–6,060) ng
Figure 1 Goodness-of-fit diagnostics of the final population pharmacokinetic model of (a,b,c) artesunate and (d,e,f) dihydroartemisinin in children with severe
malaria. The broken line represents a locally weighted least-squares regression; the solid line is the line of identity. The observed concentrations, population
predictions, and individual predictions were transformed into their logarithms (base 10).
10,000
1,000
100
Observations (nmol/l)
10
1
110 100
Population predictions (nmol/l)
1,000 10,000
10,000
1,000
100
Observations (nmol/l)
10
1
10
−4
−2
0
Conditional weighted residuals
2
4
123
Time (hours)
456
10 100
Individual predictions (nmol/l)
1,000 10,000
10,000
1,000
100
Observations (nmol/l)
10
1
110 100
Population predictions (nmol/l)
1,000 10,000
10,000
1,000
100
Observations (nmol/l)
10
1
10
−4
−2
0
Conditional weighted residuals
2
4
2
Time (hours)
46810
10 100
Individual predictions (nmol/l)
1,000 10,000
ab c
de f
Figure 2 Simulation-based diagnostics of the final model describing the
population pharmacokinetics of (a,c) artesunate and (b,d) dihydroartemisinin
in children with severe malaria. Graphs in a and b display a prediction-corrected
visual predictive check with venous plasma concentrations transformed into
their logarithms (base 10). Open circles represent observed data points; solid
lines represent the 5th, 50th, and 95th percentiles of the observed data; the
shaded area represents the 95% confidence interval (CI) of simulated (n = 2,000)
5th, 50th, and 95th percentiles. Graphs in c and d display the observed fraction
of data points below the limit of quantification (solid lines) and the 95% CI of the
simulated (n = 2,000) fraction below the limit of quantification (shaded area).
10,000
1.0
0.8
0.6
0.4
Fraction censored
0.2
0
0120
Time (hours)
12
1,000
100
Concentration (nmol/l)
10
1
1234 1
Time (hours)
234
ab
cd
CLINICAL PHARMACOLOGY & THERAPEUTICS | VOLUME 93 NUMBER 5 | MAY 2013 445

Articles
× h/ml in the 11–15 kg patients, 4,100 (2,570–6,590) ng × h/ml in
the 16–20 kg patients, and 4,240 (2,700–6,840) ng × h/ml in the
21–25 kg patients).
is suggests that smaller children need higher doses of ARS
to attain ARS exposures similar to those in children with higher
body weights. Using the nal model, we evaluated various dos-
ing regimens based on body weight bands. e proposed regi-
men (Tabl e 3 ) resulted in similar exposures in all weight bands
aer the rst dose of i.m. ARS (Figure 3c,d). e same simula-
tions were performed assuming a uniform distribution of body
weights at dierent levels of hemoglobin; these resulted in lower
DHA exposures with decreasing hemoglobin levels (Figure 4).
ere was no statistical dierence between survivors and non-
survivors with respect to total exposure (ARS P = 0.8060, DHA
P = 0.4828) or maximum concentration (ARS P = 0.7655, DHA
P = 0.6865) aer the rst dose. Similarly, an exposure–response
relationship could not be established using nonlinear mixed-
eects modeling (NONMEM) in a time-to-event approach.
However, this might be a consequence of the relatively low num-
ber of deaths and the high proportion of pretreatments with
dierent antimalarial drugs, doses, and administration routes.
DISCUSSION
Parenteral ARS is currently the drug of choice for the treatment of
severe malaria. Optimal treatment strategies depend on detailed
knowledge of the pharmacokinetic properties of drugs in the tar-
get population in which the drug is used. Age, disease status, and
severity are all factors that may aect drug absorption, distribu-
tion, metabolism, and elimination.
17
e importance of pharma-
cokinetics in determining the therapeutic response is illustrated
by the study of artemether, the first parenteral artemisinin
derivative that was compared with quinine for eectiveness in
the treatment of severe malaria in large clinical trials.24,25 In a
meta-analysis of randomized trials in severe malaria, artemether
signicantly reduced mortality in Southeast Asian adults but did
not do so in African children.26 Subsequent pharmacokinetic
studies showed that the oil-based artemether was released slowly
and erratically from the i.m. injection site, and that this likely
counterbalanced its pharmacodynamic advantages relative to qui-
nine.
10,27
Despite having pharmacodynamic properties similar to
those of artemether, ARS is superior to artemether as a treatment
because of its more favorable pharmacokinetic properties.28
Dosing regimens for children are oen derived from studies
in adults, and this practice has led to substantial underdosing
of several antimalarials in children. A pharmacokinetic study of
sulfadoxine- pyrimethamine in African children with uncom-
plicated falciparum malaria showed that, with the usual dose of
25/1.25 mg/kg, the area under the concentration–time curve in
children 2–5 years of age was half that in adults. is may have
caused failure of antimalarial treatment in small children, thereby
contributing to the spread of resistance. is information came
decades aer the introduction of sulfadoxine-pyrimethamine.
18
It has also been shown that piperaquine exposure levels are lower
in small children if a standard body weight-based dose regimen
is followed.19
Only limited data are available on the pharmacokinetics of
parenteral, rectal, and oral ARS in children. is is the rst
population pharmacokinetic study of i.m. ARS in African chil-
dren with severe malaria. A one-compartment model accurately
described the distribution of ARS and DHA. ARS was converted
rapidly into DHA, the ARS elimination half-life being ~26 min.
is is in agreement with ARS half-life values reported in other
Figure 3 Simulated total first-dose exposure levels (AUC0–12h) of (a) ARS and (b) DHA after the standard 2.4 mg/kg dosing in children at different body weights.
Simulated total first-dose exposure levels (AUC0–12h) of (c) ARS and (d) DHA after the suggested adjusted dose regimen (Table 3). Open circles represent median
values, and bars indicate the 25th to 75th percentiles of simulations (1,000 simulations at each body weight). The broken line represents the median exposure for
the largest weight group (i.e., 700 h × ng/ml and 1,230 h × ng/ml for ARS and DHA, respectively). ARS, artesunate; AUC0–12h, area under the concentration–time
curve from time point 0 to 12 h; DHA, dihydroartemisinin.
6
0
500
1,000
ARS AUC0−12 h (h × ng/ml)
1,500
810121416
Body weight (kg)
18 20 22 24 6810 12 14 16
Body weight (kg)
18 20 22 24
a
0
500
1,000
DHA AUC0−12 h (h × ng/ml)
2,500
2,000
1,500
b
6
0
500
1,000
ARS AUC0−12 h (h × ng/ml)
1,500
810121416
Body weight (kg)
18 20 22 24 6810 12 14 16
Body weight (kg)
18 20 22 24
c
0
500
1,000
DHA AUC0−12 h (h × ng/ml)
2,500
2,000
1,500
d
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pharmacokinetic studies of i.m. ARS
9,10,29
but is considerably
longer than that associated with the i.v. route.
20,30
is is because
the elimination rate of ARS is limited by the rate of absorption
from the i.m. injection site (i.e., “ip-op” pharmacokinetics).9
e volume of distribution values, maximum concentration
values, and area under the concentration–time curves of ARS
and DHA are also comparable with the ndings from a previ-
ous small and densely sampled pharmacokinetic study of i.m.
ARS in children.
9
However, the area under the concentration–
time curve of DHA in this study was considerably lower than
that reported in adult patients and healthy volunteers aer i.v.
administration of ARS.30,31 Despite the lower predicted maxi-
mum concentration value aer i.m. injection as compared with
i.v. administration, this is still far greater than the in vitro–
dened DHA concentration value of 2.28 ng/ml that is required
for 99% inhibition (IC
99
). e excellent bioavailability aer i.m.
injection (~90%),
9,29
fast absorption, and comparable estimates
for ARS and DHA exposure support the use of i.m. ARS as a
suitable alternative to i.v. ARS.30
Our study had only a relatively small number of patients, thereby
limiting its power to detect covariate relationships. A parsimonious
Table 3 Proposed body weight–adjusted dosing regimen for i.m.
artesunate
Weight (kg) Dose i.m. (mg)
Prepared
solution (ml)aDose i.m. (mg/kg)
6–7 20 2 2.86–3.33
8–9 25 2.5b2.78–3.13
10–11 30 3b2.73–3.00
12–13 35 3.5b2.69–2.92
14–16 40 2 2.50–2.86
17–20 50 2.5 2.50–2.94
21–25 60 3 2.40–2.86
i.m., intramuscular.
aFor children of body weights <14 kg, dilute to 10 mg/ml; for children with body
weights ≥14 kg, dilute to 20 mg/ml. bDivide the dose equally and administer into both
thighs.
Figure 4 (a) Simulated total first-dose exposure levels (AUC0–12h) of DHA
after the standard 2.4 mg/kg dosing in children at different hemoglobin
levels. Open circles represent median values, and bars indicate the 25th
to 75th percentiles of simulations (1,000 simulations at each hemoglobin
level). (b) Simulated total first-dose exposure (AUC0–12h) of DHA in children at
different body weights at very low (open circles: 3 g/dl), low (open squares:
8 g/dl), and normal (open triangles: 13 g/dl) hemoglobin levels. The broken
line represents the median exposure for the largest weight group (i.e.,
1,230 h × ng/ml). AUC0–12h, area under the concentration–time curve from
time point 0 to 12 h; DHA, dihydroartemisinin.
3456789
Hemoglobin (g/dl)
10 11 12 13
6
0
1,000
2,000
3,000
4,000
0
1,000
2,000
3,000
4,000
810121416
Body weight (kg)
18 20 22 24
a
b
DHA AUC
0−12 h
(h × ng/ml)
DHA AUC0−12 h (h × ng/ml)
Table 2 Parameter estimates of the final model describing
the population pharmacokinetics of artesunate and
dihydroartemisinin in children (n = 70) with severe malaria
Variable
Population
estimatea (% RSEb)95% CIb
Fixed effects
CL/FARS (l/h) 45.8 (8.10) 38.8–53.7
V/FARS (l) 28.2 (11.4) 22.7–35.2
CL/FDHA (l/h) 22.4 (8.40) 19.2–26.5
V/FDHA (l) 13.5 (9.69) 11.2–16.3
DUR (min) 1.00 (fixed) —
Covariate effect
Negative effect of hemoglobin
on CL/F DHA (%)
10.2 (14.9) 6.84–12.8
Random effects
ηCL/F ARS 0.415 (45.3) 0.0890–0.755
ηV/F ARS 0.680 (54.6) 0.111–1.373
ηCL/F ARS ~ ηV/F ARS 0.497 (52.3) 0.0732–0.969
ηCL/F DHA 0.306 (37.9) 0.136–0.546
σ
ARS 0.0942 (29.2) 0.0266–0.249
σ
DHA 0.211 (12.5) 0.122–0.320
Post hoc estimatesc
CL/FARS (l/h/kg) 4.27 1.18–11.0
V/FARS (l/kg) 2.58 0.479–8.06
t1/2 ARS (h) 0.425 0.238–0.727
Cmax ARS (ng/ml) 943 329–5,090
AUC0–12h ARS (h × n/ml) 570 281–2,170
CL/FDHA (l/h/kg) 2.01 0.736–5.95
V/FDHA (l/kg) 1.24 —
t1/2 DHA (h) 0.427 0.145–1.18
Tmax DHA (h) 0.608 0.321–1.04
Cmax DHA (ng/ml) 547 284–890
AUC0–12h DHA (h × ng/ml) 890 297–2,510
ARS, artesunate; AUC0–12h, area under the concentration–time curve from time point
0 to 12 h; CL/F, elimination clearance; Cmax, predicted maximum concentration;
DHA, dihydroartemisinin; DUR, duration of zero-order absorption; F, intramuscular
bioavailability; t1/2, terminal elimination half-life; Tmax, time to maximum
concentration; V/F, central volume of distribution; η, interindividual variability;
ηCL/F ~ ηV/F , correlation of random effects on CL/F and V/F; σ, additive residual variance.
aComputed population mean values from nonlinear mixed-effects modeling are
calculated for a typical patient with a body weight of 10.9 kg and a hemoglobin
value of 7.1 g/dl. bAssessed by nonparametric bootstrap method (n = 974 successful
iterations out of 1,000) of the final pharmacokinetic model. Relative standard error
(% RSE) is calculated as 100 × (SD/mean value). 95% Confidence interval (95% CI) is
displayed as the 2.5–97.5 percentiles of bootstrap estimates. cPost hoc estimates are
displayed as median values with 2.5–97.5 percentiles of empirical Bayes estimates.
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approach (P < 0.001 for a covariate to be retained in the model) was
applied in order to avoid artifactual covariate relationships. e
most signicant covariate identied in the present study was body
weight. is has also been reported in other population pharma-
cokinetic studies of oral and rectal ARS in pediatric32,33 and mixed
adult–pediatric
13
populations. In general, physiological processes
do not scale linearly with body weight; consequently children
with lower body weights will have higher body weight–normal-
ized elimination clearance values. is has also been reported
previously for other antimalarials.
18,19
Dosing simulations using
an exact dose of 2.4 mg/kg resulted in lower ARS and DHA expo-
sures in small children as compared with those with body weights
of 25 kg, suggesting the need for higher dosing in small children. In
addition, hemoglobin concentration was also a signicant covari-
ate, resulting in lower DHA exposure in more anemic children.
Severe anemia is a common presenting feature of severe malaria
in young children. In severe malarial anemia there is usually sub-
stantial intravascular hemolysis, and the released heme may cause
iron-mediated degradation of the artemisinin peroxide bridge.
is is also the cause of the degradation of ARS in hemolyzed
plasma samples ex vivo.
34
Hemoglobin concentration was found
to be associated with reduced exposure levels of ARS and DHA,
independent of body weight. is supports the idea that young
children with severe malaria, who are generally more anemic than
older children, should receive adjusted higher doses.35,36
Underdosing in young children with severe malaria may have
immediate adverse consequences with respect to outcomes.
Parenteral and oral ARS are extremely well tolerated. e only
dose-dependent toxicity identied to date is neutropenia. A
recent study carried out in Cambodia has shown that oral ARS
at a dose of 6 mg/kg/day for 7 days resulted in a reduction in
neutrophil counts (to <1.0 × 10
3
/μl) and short-term neutropenia
in 19% of the patients.
37
Because oral ARS has a bioavailability of
~80%,
38,39
this corresponds to a total parenteral dose of 33.6 mg/
kg, which would be reached only if parenteral treatment were
continued for 14 doses. In the AQUAMAT trial, the median
(interquartile range) number of doses of parenteral treatment
in surviving children was 3 (2–4) doses.6
ARS is currently the rst-choice treatment for severe malaria,
and a product produced according to Good Manufacturing
Practices, which is WHO prequalied and available. To facilitate
implementation of an optimized dosing regimen in the treatment
of severe malaria in African children, we dened a simplied
weight-band–based dosing regimen based on the current popu-
lation pharmacokinetics model, taking into account accuracy and
practicality issues. We considered 0.5 ml to be the minimum vol-
ume of prepared ARS solution that can be measured accurately
and administered with commonly available types of syringes.
Because the weight bands are smaller for children with body
weights <14 kg, we propose an incremental ARS dose increase of
5 instead of 10 mg and a dilution of 10 mg/ml for i.m. administra-
tion in this group, similar to the dosage regimen in the current
study. In children with body weights of <14 kg, doses should be
split and administered in both thighs if injection volumes exceed
2 ml. In children of body weights ≥14 kg, larger injection volumes
can be avoided by using a dilution of 20 mg/ml. Binning of weight
bands was also chosen in accordance with the currently available
vial, which contains a dose of 60 mg, demarcating the upper limits
of weight bands at 25 kg.
The dosing recommendations we propose do not extend
beyond the weight ranges of the children included in this study.
No children with body weight <6.5 kg were included; therefore
more studies of ARS population pharmacokinetics are needed to
evaluate dosing in these very small children and to support the
current dosing recommendations. More extensive sampling in
the rst 15 min and during the rst 12 h aer the dose could give
more information than the current study provided.
In conclusion, ARS and DHA exposures were lower in small
children aer i.m. administration of ARS in severe malaria, war-
ranting dose adaptation in this group. Independently, hemolytic
anemia may aggravate the lower exposures in young children. We
propose a body weight–adjusted and convenient dosing regimen
for i.m. ARS in children with body weights between 6 and 25 kg.
METHODS
Study design. This pharmacokinetic assessment of ARS was part of
the AQUAMAT trial (registration number ISRCTN 50258054), a large
multinational trial for which the results have been published else-
where.6 This substudy was conducted at Teule Hospital in Muheza,
Tanzania, from May 2009 to July 2010. Except for the additional blood
sampling, the procedures for this substudy were part of the AQUAMAT
study protocol.6 Ethical approval was obtained from the Tanzania
Medical Research Coordinating Committee and the Oxford Tropical
Research Ethics Committee. A total of 18 patients were co-enrolled in
the “FEAST” trial evaluating fluid bolus therapy in children with com-
pensated shock.40
Children ≤14 years with a clinical diagnosis of severe malaria con-
rmed by Plasmodium lactate dehydrogenase (pLDH)-based rapid diag-
nostic test (OptiMAL, Diamed, Cressier, Switzerland) were recruited,
and written informed consent was obtained from the respective parents
or caregivers. Severe malaria was dened as the presence of at least one
of the following: coma (Glasgow Coma Score ≤10 or Blantyre Coma
Score ≤2 in preverbal children), convulsions (duration >30 min or ≥2
episodes in the 24 h preceding admission), respiratory distress (nasal alar
aring, costal indrawing/recession or use of accessory muscles, severe
tachypnea) or acidotic breathing (“deep” breathing), shock (capillary rell
time ≥3 sec and/or temperature gradient and/or systolic blood pressure
<70 mm Hg), severe symptomatic anemia (<5 g/dl with respiratory dis-
tress), hypoglycemia (<3 mmol/l), hemoglobinuria, severe jaundice, or,
in older children, a convincing history of anuria or oliguria. Patients
who had received full treatment with parenteral quinine or a parenteral
artemisinin derivative >24 h before admission were excluded.
Physical examination was carried out at admission, and a venous blood
sample was taken for peripheral blood parasite count, quantitative assess-
ment of plasma Plasmodium falciparum histidine-rich protein-2 (a meas-
ure of total body parasite burden),41 HIV serology (SD Bio-Line HIV
1/2 3.0; Standard Diagnostics, Kyonggi-do, Korea/Determine HIV-1/2,
Abbott Laboratories, IL), blood culture, liver function tests (aspartate ami-
notransferase, alanine transaminase, γ-glutamyltransferase, total bilirubin,
creatinine, and urea, by Reotron, Roche Diagnostics, Basel, Switzerland),
hematocrit, biochemistry, and acid–base parameters (EC8+ cartridge for
i-STAT handheld blood analyzer, Abbott Laboratories, Abbott Park, IL).
Hematocrit was reported from the i-STAT reading or, when not available,
measured by HemoCue (HemoCue AB, Ängelholm, Sweden) (n = 5). A
neurologic examination was conducted at discharge, and repeated at day
28 in children who had not made a full neurologic recovery at discharge.
Antimalarial treatment. ARS (Guilin Pharmaceutical Factory,
Guangxi, China) was administered as an i.m. injection (2.4 mg/kg)
shortly after admission, again at 12 h and 24 h, and daily thereafter. The
448 VOLUME 93 NU MBER 5 | MAY 2013 | www.nature.com/cpt

Articles
contents of each 60 mg vial of ARS were dissolved in 1 ml 5% sodium
bicarbonate (provided with the drug) and further diluted with 5 ml
5% dextrose (final concentration of 10 mg/ml) before administration
as a deep i.m. injection into the anterolateral thigh. Dosing was based
on the measured body weight of the patient, and injection volumes of
>2–3 ml were split and divided into two injections, one in each thigh.
When the patient was well enough to take oral medication, but after
a minimum of 24 h (two doses of i.m. ARS), a full 3-day course of oral
artemether-lumefantrine (Co-artem; Novartis, Basel, Switzerland) was
given to complete the treatment.
Patient management. Vital signs and glucose were monitored at least
every 6 h and also at any sign of deterioration in clinical condition. A
majority of the patients (i.e., other than those who were able to be orally
fed) received an infusion with dextrose 5%. Hypoglycemia (defined
here as blood glucose <3 mmol/l) was treated with an i.v. bolus of 5 ml/
kg of 10% dextrose. Blood transfusion (20 ml/kg) was given to children
with hemoglobin concentrations of <5 g/dl. Fluid bolus was given to
children with signs of shock.40 All the children were treated empiri-
cally with i.v. antibiotics. Convulsions were treated with i.v. diazepam
or phenobarbitone if persisting. Peripheral blood smears were repeated
after 24 h.
Blood sampling. Blood samples (1.5 ml) were drawn from an indwell-
ing catheter into prechilled fluoride oxalate tubes42 for ARS and DHA
quantification before the first dose (at baseline). Four subsequent sam-
ples were taken from each patient at preset random time points within
the following time windows: 0–1, 1–4, 4–12, and 12–24 h after the first
dose. Randomization of sampling times was done by computer-gener-
ated randomization (STATA version 12 (Stata, College Station, TX).
Immediately aer blood collection, the blood samples for drug mea-
surements were centrifuged at 4 °C at 2,000g for 7 min. Plasma samples
(0.5 ml) were stored at −80 °C and shipped on dry ice to the MORU
Department of Clinical Pharmacology, Bangkok, ailand, for drug
quantication. ARS drug content and quality were checked in vials taken
randomly from the purchase lots (see Supplementary Data online).6
Drug analysis. ARS and DHA plasma concentrations were measured
using liquid chromatography–tandem mass-spectrometry.43 Quality
control samples at low, middle, and high concentrations were analyzed
in triplicate within each analytical batch to ensure accuracy and preci-
sion during the analysis. The total coefficients of variation were <8%
for all quality control samples. The lower limit of quantification was set
at 1.2 ng/ml for ARS and 2.0 ng/ml for DHA.
Pharmacokinetic modeling. Venous plasma concentrations were trans-
formed into molar units and modeled as natural logarithms, using
NONMEM v.7 (ICON Development Solutions, Ellicott City, MD).
ARS and DHA were modeled simultaneously, using a drug–metabolite
model with complete in vivo conversion of ARS into DHA (for details,
see Supplementary Data online). The first-order conditional esti-
mation method with interaction was used throughout the modeling.
Model selection was based on the objective function values computed
by NONMEM, goodness-of-fit graphical analysis, and physiologi-
cal plausibility. Potential covariates were investigated using a stepwise
forward addition and backward elimination approach. A P value of
0.05 was used in the forward step and a P value of 0.001 in the back-
ward step to compensate for the relatively small population studied.
Simulation-based diagnostics (visual and numerical predictive checks)
and bootstrap diagnostics were used to evaluate the performance of the
final model.44
Monte Carlo simulations using the nal model with the observed vari-
ability were performed for dierent body weights to obtain representative
population estimates of the exposure levels during the rst day of dosing
(area under the concentration–time curve from time point 0 to 12 h) aer
prospective dose regimens. No drug exposure target is dened for paren-
teral ARS; therefore, for the purpose of arriving at a practical parenteral
dosing regimen, dierent body weight “bins” were evaluated to ensure
similar target exposures in all weight bands in agreement with the expo-
sure in children of body weight 25 kg. e same simulations were used
to evaluate the eect of other signicant covariates on drug exposure.
Pharmacodynamics. Peripheral blood smears were taken at admis-
sion and after 24 h. Reduction in parasite load over 24 h, survival, and
severe neurologic sequelae were evaluated as part of the pharmacody-
namic analysis. The effects of ARS and DHA exposures on outcomes
were investigated using a time-to-event analysis in NONMEM, with
predicted drug concentrations being used to modulate the hazard
function in a traditional maximum effect (Emax) relationship. Group
comparisons were performed using the nonparametric Mann–
Whitney U-test in STATA.
SUPPLEMENTARY MATERIAL is linked to the online version of the paper at
http://www.nature.com/cpt
ACKNOWLEDGMENTS
We are grateful to the patients and their caregivers. We thank Ben Amos from
Teule Hospital in Muheza for microbiology and laboratory management.
Permission to publish this work was given by the director general, National
Institute for Medical Research, Tanzania. This work was supported by
The Wellcome Trust of Great Britain (grants 076908 and 082541) and was
coordinated as part of the Mahidol-Oxford Tropical Medicine Research
Programme funded by the Wellcome Trust of Great Britain.
AUTHOR CONTRIBUTIONS
J.T., I.C.E.H., and A.M.D. wrote the manuscript. J.T., I.C.E.H., N.L., N.P.J.D., L.v.S.,
N.J.W., and A.M.D. designed research. I.C.E.H., G.M., and A.K. performed
research. J.T., I.C.E.H., and N.L. analyzed data. S.G., H.R., and M.M.L.
contributed new reagents/analytical tools.
CONFLICT OF INTEREST
The authors declared no conflict of interest.
Study Highlights
WHAT IS THE CURRENT KNOWLEDGE ON THIS TOPIC?
3 Parenteral ARS is currently the drug of choice for the
treatment of severe malaria in all age groups. e cur-
rent recommended dosing regimen was, for the most
part, derived empirically from the results of studies
in adults. Most deaths from severe malaria occur in
children, but pharmacokinetic data on i.m. ARS in this
age group are scarce.
WHAT QUESTION DID THIS STUDY ADDRESS?
3 We studied the pharmacokinetic properties of i.m.
ARS and its active metabolite, DHA, in the treatment
of severe malaria in African children. From our nd-
ings, we derived a practical dosing regimen that avoids
underdosing in smaller children.
WHAT THIS STUDY ADDS TO OUR KNOWLEDGE
3 Body weight signicantly aected elimination clearance
and apparent volume of distribution, resulting in lower
exposure levels of ARS and DHA in smaller children.
HOW THIS MIGHT CHANGE CLINICAL PHARMACOLOGY
AND THERAPEUTICS
3 We propose an adapted ARS dosing regimen for small
children, including a practical dosing table per weight
band.
CLINICAL PHARMACOLOGY & THERAPEUTICS | VOLUME 93 NUMBER 5 | MAY 2013 449

Articles
© 2013 American Society for Clinical Pharmacology and Therapeutics
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