In the current analysis, a novel parent-metabolite model to describe the population pharmacokinetics of AS and DHA in healthy subjects was developed. This is the first population pharmacokinetic analysis of AS and DHA conducted using data derived from a large number of healthy subjects following oral administration of AS. The model developed was stable and was able to predict AS and DHA data arising from single- and multiple-dosing of oral AS equally well.
AS was rapidly absorbed into the systemic circulation, with an absorption half-life of 10.8 minutes estimated in this analysis. The conversion of AS to DHA was very rapid and the concentration of DHA was measurable as early as 20 minutes post-dose for all subjects. The sensitivity of the assay used in this study enabled the measurement of AS concentrations up to eight hours post-dose and the measurement of DHA concentrations up to 12 hours post-dose, in 65% and 95% out of the total 1416 available samples, respectively. Therefore, we were able to characterize the distribution of lipophilic DHA to the peripheral tissue. The pharmacokinetic parameter estimates obtained using a non-linear mixed-effects modelling approach employed in this analysis are comparable with those obtained using non-compartmental analysis (unpublished data).
The pharmacokinetics of AS and DHA following orally administered AS in healthy subjects have been previously reported in different settings [23–26]. However, AS pharmacokinetics was not described in the reports by Benakis et al  and Na-Bangchang et a l . Navaratnam et al  compared the pharmacokinetics of AS and DHA after administration of oral and rectal AS in 12 healthy male Malaysian volunteers using non-compartmental approach. Following a single oral dose of 200 mg AS, the mean area under the plasma concentration-time curve (AUC) to time infinity for AS was reported to be 119 ng.h/mL, corresponding to a CL/F of 1680 L/h. A lower CL/F for AS (1190 L/h) was reported in the current analysis using data from a much larger sample size. The bioanalytical method used to measure AS plasma concentrations was also more sensitive (limit of quantification = 1 ng/mL) in current analysis. Teja-Isavadharm and colleagues  studied the single-dose pharmacokinetics of AS following the administration of 100 mg of oral AS in six healthy subjects and six patients with uncomplicated falciparum malaria. In their study, the range of apparent clearance and apparent volume of distribution in healthy subjects were 4.69-29 L/h/kg and 4.2-49.6 L/kg for AS, 1.66-3.26 L/h/kg and 1.99-4.45 L/kg for DHA, respectively. The weight normalized apparent clearance and apparent volume of distribution obtained in this analysis were 19.3 L/h/kg and 19.7 L/kg for AS, 1.52 L/h/kg and 1.88 L/kg for DHA, respectively. The estimates obtained in this present analysis are within the similar magnitude compared to their findings.
Several other studies have described the pharmacokinetics of DHA following the administration of oral AS in malarial patients [27–29]. Newton et al studied the disposition of DHA in 19 adult patients with acute uncomplicated falciparum malaria who were treated with 2 mg/kg of oral AS . DHA data was modelled using an open one-compartmental model with first order absorption and elimination assuming complete conversion of AS to DHA. The reported CL/F and V/F values were 73.1 L/h and 70.5 L, respectively. In another study, the pharmacokinetic characteristics of DHA were described in 26 malarial patients who received 100 mg of oral AS, using non-compartmental analysis . The reported AUC and t1/2 of DHA were 4.53 μmols.h/L and 0.66 h, respectively. These values correspond to a CL/F of 51.7 L/h and a V/F of 48.9 L. Similarly, Bethell et al reported the AUC and t1/2 of DHA of 1286 ng.h/mL and 1 h, respectively, in 10 children with moderately severe falciparum malaria who received 3 mg/kg of oral AS . These values correspond to a CL/F of 45.7 L/h and a V/F of 65.9 L. All these values for CL/F and V/F of DHA were lower than the value reported in this analysis. These differences could be resulted from the use of different assays, the effect of disease state (malaria), differences in the dose formulations, and/or the age-related difference in DHA metabolism capacity.
Population pharmacokinetics of AS and/or DHA in malaria patients have been described in four other papers [14–17]. Karunajeewa et al  proposed a three-compartment model (a rectal absorption compartment, a central compartment for AS and a central compartment for DHA) to describe the population pharmacokinetics of AS and DHA simultaneously in paediatric patients following administration of AS suppositories. Simpson et al  modelled only the DHA data pooled from five Phase II and III studies conducted in adult and paediatric malaria patients. Both the weight-normalized CL/F and V/F for AS obtained in our analysis are much larger than the ones reported by Karunajeewa et al (5.9 L/h/kg and 2.1 L/kg, respectively) . The larger CL/F and V/F for AS seen in our study might be attributed to the fact that the AS bioavailability is reduced when oral AS is given compared to rectal AS . On the other hand, the weight-normalized CL/F and V/F for DHA obtained in this study are smaller than those reported by the two studies. Karunajeewa et al reported values of 2.2 L/h/kg and 2.1 L/kg for the CL/F and V/F of DHA . The typical CL/F values of DHA reported by Simpson et al were 3.17 L/h/kg for a male and 2.03 L/h/kg for a female. For an adult weighted 70 kg, the typical value for V/F of DHA was 6.34 L/kg . This finding is also consistent with the observations that the AUC for DHA following oral administration of AS was higher than that following rectal AS [24, 30], suggesting that the bioavailability of DHA was increased when oral AS was given.
McGready et al  characterized the population pharmacokinetics of DHA in pregnant women with acute uncomplicated falciparum malaria following a three-day dosing of oral AS (4 mg/kg/day) and atovaquone plus proguanil. However, the pharmacokinetics of AS was not evaluated because AS was detectable only in about 6.5% of the total available samples. The pharmacokinetic parameter estimates for DHA were therefore derived using AS dose in DHA equivalents. The CL/F of DHA was reported to be 1.77 L/h/kg, which is similar in our analysis. However, the estimate for DHA apparent volume of distribution in healthy Korean subjects obtained in this analysis is about 60% lower compared to the pregnant Karen patients in their study (4.63 L/kg). The larger volume of distribution seen in their study might be attributed to the physiological changes during pregnancy and the effect of the disease state.
Stepniewska et al conducted a population pharmacokinetic study of AS in African children with acute malaria from six months to five years old . The subjects received either the fixed dose combination of AS and amodiaquine or the separate tablets of both drugs. The DHA data was modelled using nonlinear mixed-effects approach. The weight normalized CL/F of DHA reported in their study was 0.636 L/h/kg, which was almost 60% lower than the value analysis in healthy adults. The discrepancy could be related to the developmental changes of metabolizing enzymes that take place in the young children. It has been demonstrated that the glucuronidation of DHA was catalyzed by UDP-glucuronosyltransferases (UGTs), in particular UGT1A9 and UGT2B7 . The capacity of these metabolizing enzymes in young children could be much less than the full capacity in adults  and therefore resulted in lower CL/F of DHA. In a review on developmental patterns of UGT system, de Wildt et al suggested that the use of per-kg model for clearance is adequate to address developmental changes in young children and may lead the underestimation of clearance by up to 200% in chilren under 3.4 kg of body weight . The reported weight-normalized V/F of DHA by Stepniewska et al was 2.285 L/kg , which was quite similar to the value obtained in this analysis.
In the present analysis, food intake was found to significantly delay the absorption of AS. When AS dose was administered after the intake of high-fat and high-caloric meal, the population absorption half-life of AS increased from 10.8 minutes to 67.5 minutes. However, the extent of absorption was not altered significantly. Body weight affected CLM/F significantly and therefore was included as a covariate in the final model. The average CLM/F for a healthy subject with 61.5 kg of body weight was estimated to be 93.8 L/h. A unit deviation in body weight would result in 1.9 unit deviation in the CLM/F from the population estimate. None of the other covariates tested was significant determinants of the variability seen. Co-administration of PYR did not affect any of the pharmacokinetic parameters of AS and DHA.
Remarkable time-dependent pharmacokinetics of ARN has been observed in both healthy subjects and in malaria patients after single or repeated oral and rectal administration of ARN dose [32–35]. Auto-induction of CYP2B6 and CYP2C19 was proposed to be the main mechanism causing the decline of plasma ARN concentrations [36–38]. A semi-physiological pharmacokinetic model taking into account the autoinduction phenomenon has been developed for ARN in healthy subjects . Conflicting observations have been reported concerning the autoinduction phenomenon after the administration of AS dose. In their unconvincing report, Khanh et al  observed a decline in DHA concentrations in 6 malaria patients following repeated AS dosing. However, the decline in either AS or DHA concentration was not observed in two other studies [41, 42]. In this analysis, the type of dosing was tested in the covariate analysis to investigate any differences in the pharmacokinetics of AS and DHA following the administration of single- and multiple-dose of AS. None of the AS and DHA pharmacokinetic parameters was affected by the type of AS dosing received at a significance level of 0.05. Visual predictive check plots in Figure 4 shows similar distributions for AS and DHA observations following single- or multiple-dose of AS, indicating no sign of decline in AS and DHA concentrations after three repeated daily AS dosing. The model described the AS and DHA observations equally well regardless the type of dosing received by the healthy subjects. In addition, the mean AS/DHA AUC0-8 ratios were similar after single and multiple doses (0.10 and 0.11, respectively). This provides evidence that there is little or no induction of the metabolic enzymes involved in the metabolism of AS following once daily dosing over three days.