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 elsewhere.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 compensated shock.40
Children ≤14 years with a clinical diagnosis of severe malaria confirmed by Plasmodium
lactate dehydrogenase (pLDH)-based rapid diagnostic test (OptiMAL, Diamed, Cressier, Switzerland) were recruited, and written informed consent was obtained from the respective parents or caregivers. Severe malaria was defined 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 flaring, costal indrawing/recession or use of accessory muscles, severe tachypnea) or acidotic breathing (“deep” breathing), shock (capillary refill time ≥3
sec and/or temperature gradient and/or systolic blood pressure <70
mm Hg), severe symptomatic anemia (<5
g/dl with respiratory distress), 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 assessment of plasma Plasmodium falciparum
histidine-rich protein-2 (a measure 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 aminotransferase, alanine transaminase, γ-glutamyltransferase, total bilirubin, creatinine, and urea, by Reflotron, 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.
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 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.
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 empirically with i.v. antibiotics. Convulsions were treated with i.v. diazepam or phenobarbitone if persisting. Peripheral blood smears were repeated after 24
Blood samples (1.5
ml) were drawn from an indwelling catheter into prechilled fluoride oxalate tubes42
for ARS and DHA quantification before the first dose (at baseline). Four subsequent samples 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-generated randomization (STATA version 12 (Stata, College Station, TX).
Immediately after blood collection, the blood samples for drug measurements were centrifuged at 4 °C at 2,000g
min. Plasma samples (0.5
ml) were stored at −80 °C and shipped on dry ice to the MORU Department of Clinical Pharmacology, Bangkok, Thailand, for drug quantification. ARS drug content and quality were checked in vials taken randomly from the purchase lots (see Supplementary Data
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 precision 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.
Venous plasma concentrations were transformed 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 estimation 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 physiological 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 backward 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 final model with the observed variability were performed for different body weights to obtain representative population estimates of the exposure levels during the first day of dosing (area under the concentration–time curve from time point 0 to 12
h) after prospective dose regimens. No drug exposure target is defined for parenteral ARS; therefore, for the purpose of arriving at a practical parenteral dosing regimen, different body weight “bins” were evaluated to ensure similar target exposures in all weight bands in agreement with the exposure in children of body weight 25
kg. The same simulations were used to evaluate the effect of other significant covariates on drug exposure.
Peripheral blood smears were taken at admission and after 24
h. Reduction in parasite load over 24
h, survival, and severe neurologic sequelae were evaluated as part of the pharmacodynamic 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.