Pharmacometric evaluation of pre-referral rectal artesunate in children with severe malaria

Pharmacometric evaluation of pre-referral rectal artesunate in children with severe malaria | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 7 February 2026 V1 Latest version Share on Pharmacometric evaluation of pre-referral rectal artesunate in children with severe malaria Authors : Ayorinde Adehin 0000-0003-1351-6367 , Marie Onyamboko , Caterina Fanello , Nicholas White J , Nicholas Day , Richard Hoglund , and Joel Tarning 0000-0003-4566-4030 [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.177048556.61309462/v1 149 views 97 downloads Contents Abstract 1 Introduction 4 Discussion Author contributions Acknowledgements Additional information Supplementary Material References Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract not-yet-known not-yet-known not-yet-known unknown Aim Parenteral artesunate is the preferred first-line treatment for severe malaria. Pre-referral rectal artesunate suppositories are recommended where parenteral treatment is inaccessible. In this study, we compared dihydroartemisinin exposure and model-predicted early parasite clearance following rectal artesunate (10 mg/kg) and intravenous artesunate (2.4 mg/kg) in African children with severe malaria. Methods A total of 82 African children with severe malaria participated in a randomized crossover study in Democratic Republic of Congo. Forty children received rectal artesunate (10 mg/kg) while the other 42 received intravenous artesunate (2.4 mg/kg) as the first intervention, and then the other route of administration for the second dose. Blood samples were drawn for drug quantification, and nonlinear mixed-effects modelling was used to evaluate the pharmacokinetic properties of intravenous and rectal artesunate, and to simulate expected parasite clearance associated with these routes of administration. Results The estimated average bioavailability of rectal artesunate was 21% but was highly variable (IQR: 8 – 35%). Plasma exposure to dihydroartemisinin—the principal and bioactive metabolite of artesunate—did not differ significantly between rectal and intravenous administration. Predicted parasite reduction over the first 12 hours was similar for both routes, consistent with previously reported clinical benefits of rectal artesunate. Conclusions These findings support the 10mg/kg dose of rectal artesunate as a pre-referral intervention which should be more widely deployed in malaria endemic areas. not-yet-known not-yet-known not-yet-known unknown Formatted for British Journal of Clinical Pharmacology not-yet-known not-yet-known not-yet-known unknown Title Pharmacometric evaluation of pre-referral rectal artesunate in children with severe malaria Authors Ayorinde Adehin 1,2 , Marie A. Onyamboko 3 , Caterina Fanello 1,2 , Nicholas J. White 1,2 , Nicholas P. Day 1,2 , Richard M. Hoglund 1,2 , Joel Tarning 1,2* Affiliations 1 Mahidol Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand 2 Centre for Tropical Medicine and Global Health, Nuffield Department of Medicine, University of Oxford, UK 3 Kinshasa School of Public Health, University of Kinshasa, Kinshasa, Democratic Republic of Congo *Corresponding Author Joel Tarning (Email address: [email protected] ) not-yet-known not-yet-known not-yet-known unknown not-yet-known not-yet-known not-yet-known unknown 1 Abstract Aim Parenteral artesunate is the preferred first-line treatment for severe malaria. Pre-referral rectal artesunate suppositories are recommended where parenteral treatment is inaccessible. In this study, we compared dihydroartemisinin exposure and model-predicted early parasite clearance following rectal artesunate (10 mg/kg) and intravenous artesunate (2.4 mg/kg) in African children with severe malaria. Methods A total of 82 African children with severe malaria participated in a randomized crossover study in Democratic Republic of Congo. Forty children received rectal artesunate (10 mg/kg) while the other 42 received intravenous artesunate (2.4 mg/kg) as the first intervention, and then the other route of administration for the second dose. Blood samples were drawn for drug quantification, and nonlinear mixed-effects modelling was used to evaluate the pharmacokinetic properties of intravenous and rectal artesunate, and to simulate expected parasite clearance associated with these routes of administration. Results The estimated average bioavailability of rectal artesunate was 21% but was highly variable (IQR: 8 – 35%). Plasma exposure to dihydroartemisinin—the principal and bioactive metabolite of artesunate—did not differ significantly between rectal and intravenous administration. Predicted parasite reduction over the first 12 hours was similar for both routes, consistent with previously reported clinical benefits of rectal artesunate. Conclusions These findings support the 10mg/kg dose of rectal artesunate as a pre-referral intervention which should be more widely deployed in malaria endemic areas. What is already known about this subject? Severe malaria is a medical emergency; parenteral artesunate is the first-line treatment at facilities able to provide it. Pre-referral rectal artesunate (RAS) can bridge care, reducing risk of death or progression during transfer. WHO urges caution: without reliable referral completion and definitive treatment, RAS may not benefit and can be associated with worse outcomes. What this study adds? Dihydroartemisinin exposure was similar after rectal 10 mg/kg and intravenous 2.4 mg/kg artesunate Model-predicted early parasite clearance was similar for both routes Our findings support the use of 10 mg/kg RAS as a pre-referral dose, consistent with its intended use in the management of severe malaria 1 Abstract 1 Introduction Malaria continues to be a major mortality risk in tropical regions, accounting for an estimated 610,000 deaths in 2024 [1]. Untreated infections caused by Plasmodium falciparum can progress to severe malaria, increasing the risk of death by more than 50-fold [2, 3]. A recent report suggests that 74% of malaria deaths in moderate to high transmission regions of sub-Saharan Africa occurred in children under 5 years of age [1]. Hence, reducing childhood mortality from severe malaria in these endemic and often resource-constrained areas is a priority for policy makers. Timely administration of artemisinin-derivatives immediately after diagnosis is critical for survival in severe malaria [4, 5]. These drugs are well-tolerated and are accompanied by a near-immediate biotransformation to dihydroartemisinin, the main bioactive metabolite which has potent and rapid antimalarial activity. Artemisinin derivatives have a broad asexual parasite-stage specificity and result in rapid parasite clearance because of their cidal-effect on ring-stage malaria parasites [6-8]. The high mortality associated with severe malaria in patients, demands that it be treated as a medical emergency [2]. As such, the administration of parenteral artesunate, is recommended as this provides the quickest and most reliable route for drug delivery and a rapid onset of drug action in severe malaria patients. This first-line treatment, given 12-hourly for a minimum of 24 h, is then followed by a full course of oral artemisinin-based combination therapy once tolerable [9]. While the World Health Organization (WHO) strongly recommends the administration of parenteral artesunate in severe malaria [10], the challenging circumstances that often characterize resource-constrained endemic regions often make this impracticable. Adequately equipped healthcare facilities may not be readily accessible at the community level where many cases present. In addition, these under-resourced centres often care for patients who are too ill to receive oral medications reliably but urgently require interventions to prevent progression of malaria to severe disease. In these circumstances, the administration of pre-referral rectal artesunate suppository (RAS), which reduced malaria associated mortality by 25% in the largest community-based study of severe malaria ever conducted [11], can provide a lifesaving stop-gap. The slowly increasing deployment of RAS as a pre-referral treatment option at the community level was interrupted by a temporary WHO moratorium imposed in early 2022 [10]. This was provoked by the findings of an observational study (“CARAMAL” [12, 13]) which reported an increased mortality in severely ill children given RAS. However, the causal relationship inferred in the CARAMAL study was strongly challenged [2, 14, 15]. There were major concerns over the role of confounders such as the accuracy of diagnosis, referral patterns, the quality of healthcare provided (notably the likely bias in RAS administration to more severely ill children), the impact of the Covid-19 pandemic, the heterogeneity and accuracy of reported outcomes, and the plausibility of the findings. An external review commissioned by WHO concurred that the CARAMAL study findings could not be used to support a causal relationship between the use of RAS and increased mortality[16]. A later revision of the WHO position [10, 17] emphasized the need for a strengthening of the continuum of care and the completion of referral as pre-requisites for RAS deployment, a position that is inconsistent with the primary rationale for RAS i.e., to initiate emergency effective treatment in precisely those settings where the continuum of care is weakest. Here, we investigated the pharmacokinetic and predicted pharmacodynamic properties of RAS in severe malaria with modelling and simulation. Using data gathered in African children with severe malaria, the predicted effectiveness of artesunate in clearing circulating malaria parasites when administered as an intravenous dose and as a rectal suppository was compared. not-yet-known not-yet-known not-yet-known unknown 1 2 Methods 2.1 Ethics Ethical approval was obtained from the Ethics Committee of the Kinshasa School of Public Health, the Ministry of Public Health of the Democratic Republic of the Congo, and the Oxford University Tropical Research Ethics Committee (OXTREC), as previously reported[18]. The study was conducted in accordance with the Declaration of Helsinki. Study materials were developed in English and translated into French; the patient information sheet and informed consent forms were additionally translated into Lingala by a certified translator. Safety reporting complied with the International Council for Harmonisation (ICH) Harmonised Tripartite Guideline for Good Clinical Practice (1996). 2.2 Study design The data used in the present study came from a clinical trial in 82 African children admitted with severe falciparum malaria in the Democratic Republic of Congo [18]. This study was a randomized, open-label, 2-arm, individual crossover clinical trial which assigned participants to each of the study-arms based on three blocks of body-weight stratification, namely 6.0 to 12.9 kg, 13.0 to 23.9 kg, and 24.0 to 34.0 kg. Infection status was confirmed by a positive malaria Ag Pf/Pan SD BIOLINE rapid diagnostic test at the point of enrolment. 2.3 Drugs Intravenous artesunate was manufactured by Guilin Pharmaceuticals, China, and intravenous quinine was from Rotex, Germany. Artesunate suppositories, supplied as 100-mg doses, were produced by Catalent, Germany Eberbach, GmbH, and packed by Scanpharm, Copenhagen, Denmark. 2.4 Intervention and sample collection Participants in study-arm one received approximately 10 mg/kg rectal artesunate suppository at the beginning of the study, and then 2.4 mg/kg intravenous artesunate after 12 h. A reverse sequence of drug administration was applied in the second study-arm. All participants were administered quinine doses throughout the entire period of the study. This was given as a 20-mg-salt/kg loading dose followed by two 10-mg-salt/kg doses at 8-h interval. Blood samples were collected just before each drug administration, and then at 5, 15, 30, and 45 min and 1, 2, 3, 4, 6, and 8 h post-dose. Pathogen decline was checked in blood film at 0 (pre-dose), 6, and 12 h post-dose, and every 12 h thereafter until 2 consecutive blood films were negative for malaria parasites. Artesunate and dihydroartemisinin contents of blood samples were quantified using a liquid chromatography-tandem mass spectrometry technique [19]. Assay precision was evaluated at 2.9, 51.7 and 546 ng/mL for artesunate, and at 5.87, 117 and 1880 ng/mL for dihydroartemisinin. Imprecision, expressed as coefficient of variation, was less than 7% for both analytes, and the lower limits of quantification were 1.19 ng/mL and 1.96 ng/mL for artesunate and dihydroartemisinin, respectively. 2.5 Pharmacometric Analyses Plasma levels of artesunate and dihydroartemisinin were modelled in NONMEM (version 7.5.1) using a nonlinear mixed-effects approach. Pirana (version 21.11.1 [20]) and Perl-Speaks-NONMEM (PsN version 5.3.0 [21]) were used in the management of modelling workflow. Data post-processing and generation of relevant plots were done in R (version 4.3.0). Model parameters were assumed to be log-normally distributed and between-subject variabilities in these parameters were described by (Eq. 1), \[\theta_{i}=\ \theta\times exp(\eta_{i})\ \ \ \ \ \ \ \ \ (Eq.\ 1)\] where \(\theta\) is the population estimate of a pharmacokinetic parameter and \(\theta_{i}\) is the value of the pharmacokinetic parameter in the \(i^{\text{th}}\) subject. \(\eta_{i}\) is the \(i^{\text{th}}\) subject’s deviation from the population value. Model parameters were MU-referenced, and a 15% between-subject variability (variance of 0.0225) was assumed and fixed on model parameters for which random effects were not estimated, as recommended [22]. Entries below the assay limits of quantification were handled with the M3 method [23], and residual unexplained variability was captured by an additive error model in the log-domain (which is essentially an exponential error model on the arithmetic scale). The data setup also accounted for pre-dose drug levels where applicable, and instances where doses were re-administered after an initial expulsion. Parameter estimates and relevant metrics were derived from a three-stage approach comprising an initial iterative two-stage (ITS) method to provide good initial eta estimates. This was followed by a stochastic approximation expectation maximization (SAEM) step for the estimation of model parameters, and a Monte Carlo importance sampling (IMP) step for the computation of the objective function value (OFV) for hypothesis testing. Competing models were evaluated using the likelihood-ratio test, setting the type 1 error rate at 5%. Goodness of fit plots and biological plausibility were also considered during model selection. A joint parent-metabolite model that assumed a complete conversion of artesunate in the central compartment to dihydroartemisinin was developed in a sequential step, fitting the parent data first and then including the metabolite data. One-, two- and three-compartment structural models were tested for parent and metabolite. In addition, first order, zero order and n -transit compartment models were considered for the absorption of rectal artesunate. Having established the structural model, allometric scaling of individual body weight, based on a 70 kg reference body weight, was applied to all clearance (with an exponent of 0.75) and volume (with an exponent of 1.0) parameters [24]. Further noting that dihydroartemisinin is eliminated by glucuronidation [25], a maturation function that captures the age-dependence of the uridine 5’-diphospho-glucuronosyltransferase (UGT) pathway was fixed in the model as earlier described by Jamsen et al [26, 27]. Physiologically plausible relationships between model parameters and covariates comprising sex, axillary temperature and haemoglobin/haematocrit were tested in a stepwise manner. In addition, the sequence of rectal administration of artesunate was tested as a categorical covariate on rectal absorption parameters. The p -value for forward addition was set at 0.01 while that of the backward elimination step was set at 0.001. Parameter estimates of the final model were assessed for robustness by relative standard errors derived from 1000 nonparametric bootstraps. In addition, relevant plots and simulation-based diagnostics were assessed to determine adequacy of fit. The Wilcoxon sign-ranked test was applied to model-derived AUC0-12h for dihydroartemisinin in the study subjects to assess the presence, or not, of a significant difference in exposure when artesunate was administered through the intravenous and rectal routes. Given that all subjects were administered quinine alongside intravenous or rectal artesunate, the use of study participants’ parasite-count-over-time data as pharmacodynamic endpoints could be problematic. Moreover, only blood-film readings of parasite biomass for three occasions within 12 h were available, limiting the reliability of such data for the estimation of pharmacodynamic parameters of parasite clearance in severe malaria where portions of parasitized red cells are sequestered. Hence, published pharmacodynamic parameters [28] estimated in a comparable disease condition was added to the final pharmacokinetic model. These authors modelled the conversion of artesunate to dihydroartemisinin which subsequently drove parasite killing dynamics using an E max model implemented through an indirect effect compartment. The model assumed that artesunate’s contribution to parasite killing was conserved, as complete conversion to dihydroartemisinin was implemented in the pharmacokinetic model. In addition, the model accounted for differential killing of artemisinin-resistance and artemisinin-susceptible parasites. Hence, our joint parent-metabolite model was used for the simulation of dynamic dihydroartemisinin levels given the standard 2.4 mg/kg intravenous dose, the 10 mg/kg body-weight-band doses for rectal artesunate i.e., 100 mg for ≤ 12.9 kg, 200 mg for 13 kg – 23.9 kg, and 300 mg for ≥ 24 kg), and the 10 mg/kg exact body-weight-based doses. In our simulation of drug effect, the parasite compartment was initialized with the median total parasite biomass derived from the plasma Plasmodium falciparum HRP2 (Pf HRP2) levels recorded on admission. Estimation of total parasite biomass, shown in (Eq. 2), capturing circulating and sequestered parasites was according to a relation earlier published by Dondorp et al [29]. \[Total\ parasite\ biomass=12\times\ plasma\ PfHRP2\ \times\ \left(1-\text{hematocrit}\right)\ \times\ body\ weight\ \times\ 10^{13}\ (Eq.\ 2)\] The resulting pharmacokinetic (PK) / pharmacodynamic (PD) model was used for simulation tasks assessing the extent of fractional parasite reduction in children administered intravenous or rectal artesunate. Where applicable, virtual populations of children were simulated using the WHO child growth chart [30]. Simulations, done in R using the RsSimulx package (https://simulx.lixoft.com/rssimulx/), explored pathogen population decline in artesunate-resistant and artesunate-susceptible parasite populations. The EC50 value of 8.63 ng/mL reported by Lohy Das et al[28] was utilized for the estimation of time-above drug-level required for parasite clearance. 1 2 Methods not-yet-known not-yet-known not-yet-known unknown 1 3 Results 3.1 Clinical descriptions A total of 82 African children with severe malaria participated in a randomized individual crossover study conducted in 2015 in Kinshasa, Democratic Republic of Congo. This study has been reported previously[18]. Forty children started in the 10 mg/kg rectal artesunate suppository (RAS) arm while the other 42 received intravenous artesunate (2.4 mg/kg) as the first intervention. They then received the other route of administration for the second dose. All children also received intravenous quinine (20 mg salt/kg infusion followed by 10 mg/kg, 8 hourly). Two of the children in the RAS arm did not proceed to the intravenous artesunate stage of the study because of death (n = 1) and the critical state of illness (n = 1). RAS was re-administered in 5 children after an initial expulsion of the earlier dose. Hypoglycaemic children at admission, defined by a blood glucose level of <3 mmol/L, received 5 mL/kg transfusions of 10% dextrose-saline. In addition, children with haemoglobin levels of participants developed complications that were not present at admission or reported by caregivers during the trial. These comprised blackwater fever, convulsions, posturing, coma, deterioration of the coma score, severe anaemia, and respiratory distress. Additional data on the demographics, clinical and laboratory characteristics of participants are provided in Table 1. At the end of the study, a total of 2,500 artesunate and dihydroartemisinin plasma samples were available for further pharmacokinetic analyses. 3.2 Pharmacokinetic and pharmacodynamic analysis Artesunate and dihydroartemisinin concentration-time profiles in plasma was modelled using a nonlinear mixed-effect technique implemented in NONMEM version 7.5.1. Initially, dihydroartemisinin data was ignored and artesunate data fitted separately for all data in the rectal and intravenous administration arms. Separate fits of intravenous and rectal artesunate data were adequately described by two-compartment structural models, both showing objective function value (OFV) drops of 267.2 and 58.5 units, respectively. Replacing the initially assumed first-order absorption model for rectal artesunate with a zero-order absorption model did not significantly improve the fit to the data. The introduction of a third disposition compartment led to no significant drop in OFV (0.5 units) with rectal artesunate data. In contrast, the addition of a third compartment to the intravenous artesunate model led to an OFV drop of 29.4 units. This numerical drop in OFV provided no visible improvement in goodness of fit plots and resulted in an unusually long biological half-life of 3 h. As such, a simpler two-compartment model was carried forward. Our choice of a two-compartment structural model for artesunate from the earlier step was consistent with findings from a pooled fit of data from the two routes of administration where an OFV drop of 319.9 units was recorded. However, parameters associated with the peripheral compartment were considered unreliable because of tailing observed concentration values at time-points beyond 6 h post-dose in some individuals and the influence of the erratic absorption of RAS. To address this challenge, pharmacokinetic parameters of a two-compartment structural model were estimated from IV-data in patients whose first dose was intravenous artesunate. Allometric scaling for body weight was applied on all clearance and volume parameters in this model, and the individual parameter estimates associated with the peripheral compartment (peripheral volume of distribution and intercompartmental clearance) were subsequently Table 1. Demographic, clinical features and laboratory characteristics of 82 children with severe falciparum malaria at enrolment 1 3 Results Total number of patients 82 Age (years) 4 (2 – 8) Body weight in kg (median, IQR) 15 (12 – 25) Height in cm (median, IQR) 101 (89 – 129) Sex (M/F) 41/82 (50%) Coma (Glasgow coma score) 14/82 (17%) Prostration 65/82 (79%) Convulsion 5/82 (6%) Respiratory distress 64/82 (78%) Hypoglycaemia (blood glucose < 40 mg/dL; 2.2 mmol/L) 4/82 (5%) Anaemia (Haemoglobin < 5 g/dL) 25/82 (31%) Haematocrit (median, IQR) 0.19 (0.15 – 0.26) Haemoglobin in g/dL (median, IQR) 6.45 (4.93 – 6.98) Axillary temperature in °C 38.0 (37.1 – 38.9) Parasite density; parasites/µL (median, IQR) 72,534 (3,862 – 242,471) Plasma P f HRP2 ng/mL (median, IQR) 1,469 (565 – 3,664) Data are presented as median [IQR: Interquartile range] or as number of patients (%) not-yet-known not-yet-known not-yet-known unknown fixed in further model development steps (further details can be found in the supplementary material, Table S1 ). With a two-compartment structural model established for artesunate in a combined fit of intravenous- and rectal-dose data, different absorption models were tested. A single transit absorption compartment model significantly improved data fit, leading to an OFV drop of 108.3 units. No further improvements were seen with more transit compartments nor were there benefits with the use of a zero-order absorption model. Having established the structural model for artesunate, dihydroartemisinin data were introduced into the model, allometric scaling for the effect of body weight on clearance and volume parameters of dihydroartemisinin was assumed, and multiple structural model compartments were tested. Dihydroartemisinin concentration-time data were best described by a two-compartment structural model in the joint parent-metabolite model (ΔOFV = -480.5). The structural model describing the disposition of dihydroartemisinin was consistent with the findings from an alternative approach of fixing all model parameters for artesunate in a model of dihydroartemisinin. This approach resulted in an OFV drop of 400.7 units with the introduction of a second compartment. The addition of a third compartment led to no further improvement. Age-dependence of the dihydroartemisinin elimination pathway was incorporated into the model as earlier described [27], leading to an increase in OFV (1.8 units). Thereafter, a stepwise covariate search was carried out with covariates comprising sex, haemoglobin/haematocrit and axillary temperature. However, none of the tested covariate relationships significantly improved the model fit (supplementary Table S2 ). The final model described the observed data adequately, and parameter estimates are provided in Table 2. Goodness-of-fit plots further supporting model adequacy is shown in Figure 1. Additional pharmacokinetic parameters from the posthoc step are shown in Table 3. The rectal bioavailability of artesunate was variable, with an estimated coefficient of variation of about 90%. Despite this, there was no significant difference in paired dihydroartemisinin exposure between intravenous and rectal doses (Wilcoxon signed-rank test, p = 0.22, α = 0.05 [31]). Model estimates in the children showed that RAS provided about 19% (IQR: 8 – 36%) of the systemic dihydroartemisinin derivable from an intravenous dose of artesunate. Epsilon-shrinkage for artesunate and dihydroartemisinin were 4.6% and 8.3%, respectively. A numerical predictive check (n = 2000) for dihydroartemisinin showed that 1.7 % (95% CI: 1.2% – 4.1%) of observations were below the prediction interval while 1.7% (95% CI: 1.5% – 3.8%) of observations were above the prediction interval. Visual predictive checks for artesunate and dihydroartemisinin are shown in Figure 2 and additional visual predictive checks for censored observations are provided in the supplementary file (Figure S1 & S2 ). 3.3 Simulations A published pharmacodynamic model (PD) [28] was implemented and combined with the final joint parent-metabolite pharmacokinetic model. The resulting PK/PD model assumed a dihydroartemisinin-dependent clearance of malaria parasites in infected patients (Figure 3). This model also accounted for the differential elimination of parasites (particularly in the early ring state) by artemisinin derivatives, a phenomenon that results from mutations in the kelch protein (PfK13) of Plasmodium falciparum [32, 33]. To assess the predicted effectiveness of RAS and parenteral artesunate, a virtual population of 21,800 children, comprising equal numbers of males and females and a uniform distribution of age (i.e., 100 children per month of age-group; 12 - 120 months) was created. The creation of this virtual population relied on the use of percentiles provided by the WHO Child Growth Standards for the creation of realistic age-for-body-weight data (supplementary Figure S3 ). Table 2. Parameter estimates of the final model describing the population pharmacokinetics of artesunate and dihydroartemisinin in 82 children with severe malaria not-yet-known not-yet-known not-yet-known unknown Artesunate FRC (%) 19.5 (16.4%) 11.7 – 23.2 2.82 (26.0%) 1.65 – 4.51 MTT (h) 1.1 (6.5%) 0.9 – 1.2 0.35 (21.1%) 0.22 – 0.50 CLARS (L/h) 270.4 (6.9%) 232.9 – 302.4 0.25 (38.0%) 0.09 – 0.46 VCentral (ARS) (L) 39.7 (11.3%) 30.7 – 47.5 0.0225 fixed - QARS (L/h) 14.9a fixed - 0.0225 fixed - VPeripheral (ARS) (L) 14.0a fixed - 0.0225 fixed - Dihydroartemisinin CLDHA (L/h) 85.6 (5.5%) 76.1 – 94.4 0.135 (23.0%) 0.08 – 0.20 VCentral (DHA) (L) 71.5 (6.1%) 63.9 – 80.5 0.0225 fixed - QDHA (L/h) 4.1 (16.3%) 2.9 – 5.2 0.0225 fixed - VPeripheral (DHA) (L) 13.3 (27.9%) 10.1 – 27.9 0.679 (66.5%) 0.22 – 2.66 Residual unexplained variability expressed as standard deviation σARS 1.4 (8.6%) 1.2 – 1.6 σDHA 0.6 (6.8%) 0.5 – 0.7 Between-subject variability (BSV) is presented as VarianceRSE: Relative standard errora: Values estimated from intravenous artesunate data in subjects whose first dose was intravenous artesunate and then fixed for the combined modelFRC: Rectal bioavailability; MTT: Mean transit time; CL: Clearance; Q: Intercompartmental clearance; V: Volume of distribution; ARS: Artesunate; DHA: Dihydroartemisinin; IV: Intravenous; RC: Rectal; CI: Confidence interval Figure 1. Goodness of fit plots for the final model describing the population pharmacokinetics of artesunate (A, B, C, D) and dihydroartemisinin (E, F, G, H) in children with severe malaria. Solid lines represent line of identity. Broken lines show locally weighted least-square regressions. Table 3. A summary of posthoc estimates from the joint parent-metabolite model of artesunate in 82 African children with severe malaria Artesunate F ARS (%) 21.05 8.10 – 39.47 t 1/2 ARS (h) 0.51 0.45 – 0.59 T max, ARS (RC) (h) 0.63 0.51 – 0.78 C O, ARS (IV) (ng/mL) 3,875 3,777 – 3,975 C max, ARS (RC) (ng/mL) 247 127 – 445 AUC 0-12 h, ARS (IV) (ng/mL x h) 407 338 – 497 AUC 0-12 h, ARS (RC) (ng/mL x h) 400 147 – 817 Dihydroartemisinin F REL, DHA (%) 18.97 8.23 – 36.16 t 1/2 DHA (h) 1.69 1.11 – 2.53 T max, DHA (IV) (h) 0.22 0.18 – 0.24 T max, DHA (RC) (h) 1.18 0.97 – 1.44 C max, DHA (IV) (ng/mL) 1,146 1,053 – 1,214 C max, DHA (RC) (ng/mL) 445 195 – 799 AUC 0-12 h, DHA (IV) (ng/mL x h) 1,148 879 – 1,365 AUC 0-12 h, DHA (RC) (ng/mL x h) 958 332 – 2,118 Artesunate + Dihydroartemisinin AUC 0-12 h, ARS + DHA, IV dose (nM) 5,047 4,127 – 5,942 AUC 0-12 h, ARS + DHA, Rectal dose (nM) 4,693 1,600 – 9,110 F ARS : Rectal bioavailability of artesunate; F REL, DHA : Relative bioavailability of DHA; AUC: Area under the plasma concentration vs time curve; t 1/2 : Terminal elimination half-life; T max : Time to reach maximum plasma concentration; C 0 : Initial concentration; C max : Maximum plasma concentration. ARS: Artesunate; DHA: Dihydroartemisinin; IV: Intravenous; RC: Rectal; CI: Confidence interval; IV: Intravenous dose; ARS: Artesunate; DHA: Dihydroartemisinin. Relative bioavailability of DHA calculated as: (AUC DHA, rectal / Dose ARS, rectal ) / (AUC DHA, IV / Dose ARS, IV ). All parameters are summaries of model-predicted values Figure 2. Simulation-based diagnostics of the final model describing the population pharmacokinetics of artesunate in children with severe malaria, after receiving intravenous (A) & (C) and rectal (B) & (D) artesunate. Grey shades represent the 95% confidence intervals of simulations around the 5 th , 50 th and 95 th percentiles of observed data. Broken straight lines represent assay limit of quantification. Figure 3. The final pharmacokinetic-pharmacodynamic model of artesunate and dihydroartemisinin in African children and with severe malaria. V: volume of distribution; CL: drug clearance; Q: intercompartmental clearance; k tr : transfer rate constant; k growth : growth rate constant of the malaria parasite; k e0 : equilibration rate constant between plasma and effect site. Dosing was applied according to body-weight bands to reflect the recommended dosing regimen of RAS. A 100-mg RAS dose was applied when body weight was ≤ 12.9 kg, a 200-mg RAS dose was applied at 13 to 23.9 kg body weight, and a 300-mg RAS dose was applied at body weights of ≥ 24 kg. Each virtual subject was assumed to have an initial parasite biomass estimated[29] using the median haematocrit value (0.19) and the median plasma PfHRP2 value (1469 ng/mL) recorded at admission in study subjects. DHA was available in plasma above reported the EC 50 value of 8.63 ng/mL[28] for a median (5 th to 95 th percentile) duration of 5.91 h (2.81 h – 11.77 h). The simulated median (5 th to 95 th percentile) malaria parasite reduction during the first 12 h after intravenous artesunate dose was 88.7 % (82.7% – 92.3%) for an artemisinin-susceptible infection, and 64.0% (53.3% – 71.3%) for artemisinin-resistant infection (Figure 4). At 10 mg/kg dose of RAS, the median parasite reduction was 86.4% (48.0% – 92.2%) and 59.8% (19.5% – 70.9%) in susceptible and resistant infections, respectively. For comparison, Figure 5 shows the parasite reduction in blood films of study participants in 12 h. Overall, confidence intervals derived by simulation of fractional parasite reduction with intravenous ARS or RAS overlapped, suggesting comparability of both routes of administration in delivering parasite reduction in severe malaria. The dynamic pathogen model from Lohy Das et al [28] assumed that parasite clearance was driven solely by DHA, omitting the contribution of artesunate—which in contrast to oral administration persists in the system for more than six hours following RAS administration. To address this potential underestimation, we also simulated the parasite reduction in the first 12 h post dose using a modified model that incorporated the combined effect of artesunate and dihydroartemisinin (supplementary Figure S4). Predicted values for artemisinin-resistant and artemisinin-susceptible parasites were 62.5% (26.3% – 72.1%) and 88.1% (58.4% – 92.8%), respectively, for RAS. The corresponding values were 66.2% (58.1% – 72.7%) and 89.9% (85.5% – 93.0%) for artemisinin-resistant and artemisinin-susceptible parasites, respectively, after intravenous administration. These values were comparable to those predicted by the original dynamic pathogen and support the benefits from the administration of RAS. Simulated dihydroartemisinin exposure (AUC) was comparable between RAS dosing based on exact body weight (10 mg/kg dose) and a practical weight-band RAS dosing (Figure 6). 4 Discussion Variability in the pharmacokinetics of rectally administered artesunate in children has long been recognized[34], raising concerns whether RAS provides adequate exposure [35]. However, once absorbed, the pharmacodynamic effects of RAS and parenteral artesunate are the same. Here, we used data from an individual cross over study conducted in African children admitted to hospital with a diagnosis of severe malaria to describe the pharmacokinetic properties of ARS and DHA following intravenous and rectal administration, with a population-based modelling technique. Combining this joint parent-metabolite pharmacokinetic model with published parasite clearance parameters allowed us to predict the effectiveness of rectal and intravenous artesunate in the elimination of malaria parasites. ARS and DHA were each described by a two-compartment structural model with large between-subject variability in the volume of distribution of the peripheral compartment of DHA (variance of 0.68, equivalent to a CV of 99%). While previous ARS/DHA models based on sparse data commonly used one-compartment disposition models [34, 36, 37], the multi-compartment model in the present study is consistent with findings in other data-rich studies [38, 39]. The retention of a random effect on the volume of distribution of the peripheral compartment for DHA was necessary for model stability despite its low precision (RSE ~ not-yet-known not-yet-known not-yet-known unknown Figure 4. Box plots showing the simulated relative reduction of malaria parasites at 12 h post-dose in children infected with Plasmodium falciparum . Boxes and Whiskers represent the median, interquartile range and 90% prediction interval of the simulated parasite reduction associated with intravenous administration of 2.4 mg/kg of artesunate (A) and weight-based rectal administration of 10 mg/kg of artesunate (B). Figure 5. Plots of parasite reduction over a 12-h period in 82 children with severe malaria who were administered intravenous or rectal artesunate. (A) A box plot showing parasite reduction following the administration of rectal and intravenous artesunate. Boxes and Whiskers represent median, interquartile range, 5 th and 95 th percentiles. (B) A plot of the median decline in parasite count following the administration of intravenous artesunate. (C) A plot of the median decline in parasite count (expressed as % baseline) following the administration of rectal artesunate suppository . Bars are the 5 th to 95 th percentiles. IV: Intravenous. Figure 6. Box plots showing the simulated area under the plasma concentration vs time curve (AUC) and the maximum plasma concentration of dihydroartemisinin in children. Boxes and Whiskers represent median, interquartile range and 90 % prediction interval of simulated profiles. (A) A boxplot showing the simulated AUC of dihydroartemisinin for 0 to 12 h in children administered intravenous and rectal doses of artesunate. (B) A boxplot showing the simulated C max of dihydroartemisinin in children administered intravenous and rectal artesunate. 67%). This wide between-subject variability is consistent with previous reports in children with severe malaria [34, 40]. While the source of this wide variability could not be identified in the present study, we note that children enrolled in the study presented with a range of haemoglobin concentrations 25 , comorbidities, and received additional interventions (i.e., standard of care) that might have impacted the disposition kinetics of ARS and DHA. A first-order absorption model with one transit compartment described the rectal uptake of artesunate better than a zero-order absorption process despite the limited surface area provided by the rectum [41] and the tendency for this to be rate-limiting for drug absorption. Further, the rectal absorption of artesunate was rapid but erratic as reflected by the large between-subject variability in the mean transit time (variance of 0.35, equivalent to a CV of 65%) and the rectal bioavailability of RAS (variance of 2.82, estimated[42] CV of about 90%). This marked variability in rectal drug absorption is not unexpected as it likely reflects the interplay between anatomical differences along the upper, middle, and lower rectum; the local physiological conditions at the time of administration (such as rectal fluid volume or the presence of faecal matter); and patient-related factors, particularly in children, who may have involuntary bowel movements after RAS administration [43]. Notwithstanding, the DHA exposure derived from 10 mg/kg doses of RAS in the study children were not significantly different from those seen in children who took 2.4 mg/kg IV doses ( p = 0.22). Our posthoc analysis also showed that, on the average, RAS provided about 19% of DHA derivable from ARS administered via the IV route. Thus, with the much higher dose of 10 mg/kg RAS, severely ill children were still being exposed to sufficient bioactive DHA needed for the clearance of malaria parasite. We also considered the possibility that a low rectal bioavailability of RAS may not necessarily indicate significant drug loss. Rather, it may have resulted from the suppository moving from the lower rectum to the upper rectum after administration. At this point, the drug would enter the portal circulation and undergo extensive first-pass metabolism to DHA. An attempt to account for this alternative first-pass pathway in the joint parent-metabolite model was unsuccessful due to stability issues and was subsequently dropped. A few covariates have been reported to explain between-subject variabilities in some pharmacokinetic parameters of ARS and DHA. For example, a faster clearance of DHA in males than in females in a study of adults and children [34], and an effect of haemoglobin levels 25, [36] on the clearance of DHA have been observed. However, in the present study, sex, axillary temperature, haemoglobin/haematocrit were not significant covariates for any of the pharmacokinetic parameters of ARS and DHA. The absence of adults in our study population could potentially explain the lack of an effect of sex on the clearance of DHA. In addition, 53/82 patients in the present study were transfused with blood and all patients got dextrose-saline infusion prior-to and during drug administration and sample collection, likely suppressing the ability to clearly identify an effect of haemoglobin on any disposition parameter as previously reported. The joint pharmacokinetic parent-metabolite model showed good predictive performance in the simulation-based diagnostics, supporting its suitability for the simulation of different dosing regimens. Hence, we simulated drug levels derivable for a 2.4 mg/kg intravenous dose, 10 mg/kg RAS dose according to body-weight bands (i.e., 100 mg for ≤ 12.9 kg, 200 mg for 13 kg – 23.9 kg, and 300 mg for ≥ 24 kg), and the 10 mg/kg dose according to exact body weights in a virtual population of children. Population-based simulations of RAS administration on the basis of body-weight bands showed that intravenous ARS and RAS provided comparable DHA exposures as reflected by median AUC 0-12h , and the 5 th and 95 th percentiles 1078 ng/mL × h [573 – 2,005] vs 936 ng/mL × h [107 – 4,442]). Dosing of RAS according to exact body weight provided no advantage when compared with dosing according to realistic body weight bands. Artemisinin and its derivatives are short-acting antimalarial drugs, and their activity is thought to be dependent on drug exposure above a minimum inhibitory concentration ( MIC ). However, the values for these MICs are poorly characterized in vivo [44]. As such, we relied on information from a published model that described the dynamic relationship between ARS/DHA plasma levels and parasite clearance. Our choice model was based on measured pharmacokinetic and parasite-clearance data in patients with uncomplicated falciparum malaria in southeast Asia[28]. The resulting pharmacometric model used for the simulation of parasite clearance considered ARS as a prodrug and relied on the concentration of DHA for the killing (i.e., an indirect sigmoidal E max model) of artemisinin-resistant and artemisinin-susceptible parasites. This PK/PD model is even more appropriate as it captures the now-emerging differential response of malaria parasites to artemisinin-containing drugs in parts of Africa[45] and Asia[46]. The predicted median parasite reduction was similar with intravenous ARS compared to RAS (about 89% vs 86% for susceptible parasites and 64% vs 60% for resistant parasites) during the first 12 hours after drug administration. This model prediction is also consistent with empirical median parasite reduction after 12 h of 84% (IQR: 50 – 95%) following RAS dose and 69% (IQR: 46 – 94%) following intravenous artesunate dose (Figure 6) in the study population. We, however, observed a wider variability in the predicted parasite clearance with RAS compared with intravenous ARS. A broad variability in predicted response to RAS appears to be largely related to its variable rectal bioavailability. This could make it unsuitable for children with diarrhoea which may sometime occur in severe malaria. Diarrhoea alters the viscosity of the rectal content and bowel movement [47], and as such, could compromise an effective uptake of RAS. Despite this interplay between rectal bioavailability and parasite clearance, the significance of the use of pre-referral RAS in reducing mortality from severe malaria is undiminished. Simulations also show that, on the average, plasma levels of DHA remained above its reported EC 50 for a about 6 h within a 12-hour period. It is important to note that RAS is not intended as treatment, but a pre-referral intervention in critically ill children who are being transported to adequately resourced medical facilities where proper care would be administered. There is no alternative. Thus, any drug exposure that can be achieved through RAS is undoubtably better than no treatment, and the drug exposure resulting from an initial administration of RAS during the expansion of the parasite biomass in the blood stream could be lifesaving. The interpretation of our findings have some limitations. This was a hospital-based study and therefore does not reflect the typical context of RAS use. Only one child died, indicating markedly lower disease severity compared with other prospective severe malaria studies in African children. This was largely because prostration and severe anaemia were the predominant clinical features in this cohort. These—carry a low risk of death in the absence of other severe manifestations, and the definition of respiratory distress used here was broad. Although participants also received parenteral quinine, pharmacokinetic interactions between RAS and quinine are highly unlikely. This study was also conducted before the WHO review of artesunate dosing for children weighing < 20 kg, which increased the recommended dose from 2.4 mg/kg to 3 mg/kg. In conclusion, this study demonstrated that rectal administrations of ARS to children with severe falciparum malaria at a dose of 10mg/kg resulted in comparable antimalarial exposures to intravenous artesunate at a dose of 2.4mg/kg This supports the current RAS dose, and initiatives to increase its deployment in malaria endemic areas. Author contributions JT, MAO, CF, NJW, NPD contributed to conceptualization, investigation, and data collection. JT carried out bioanalysis AA, RMH, JT contributed to formal data analysis AA, RMH, JT contributed to manuscript preparation, editing and review All authors reviewed and approved the final manuscript. Acknowledgements This work was supported by the Wellcome Trust–Mahidol University–Oxford Tropical Medicine Research Programme, with additional support from the Li Ka Shing Foundation. Pharmacokinetic data analysis was supported in part by the Bill & Melinda Gates Foundation (INV-006052). This research was funded wholly or in part by the Wellcome Trust (grant numbers 106698/Z/14/Z and 220211). not-yet-known not-yet-known not-yet-known unknown 1 Competing interests None declared. 1 Competing interests Additional information Supplementary materials are available online. Supplementary Material File (image1.emf) Download 604.39 KB File (image2.emf) Download 91.55 KB File (image4.emf) Download 11.99 KB File (image5.emf) Download 9.28 KB File (image6.emf) Download 19.35 KB References 1. 1. 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Authors Affiliations Ayorinde Adehin 0000-0003-1351-6367 Mahidol Oxford Tropical Medicine Research Unit View all articles by this author Marie Onyamboko Universite de Kinshasa View all articles by this author Caterina Fanello Mahidol Oxford Tropical Medicine Research Unit View all articles by this author Nicholas White J Mahidol Oxford Tropical Medicine Research Unit View all articles by this author Nicholas Day Mahidol Oxford Tropical Medicine Research Unit View all articles by this author Richard Hoglund Mahidol Oxford Tropical Medicine Research Unit View all articles by this author Joel Tarning 0000-0003-4566-4030 [email protected] Mahidol Oxford Tropical Medicine Research Unit View all articles by this author Metrics & Citations Metrics Article Usage 149 views 97 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Ayorinde Adehin, Marie Onyamboko, Caterina Fanello, et al. 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