Lumefantrine

The WWARN QA/QC proficiency testing program for pharmacology laboratories assesses the ability of pharmacology laboratories to assay blood or plasma samples for concentrations of antimalarial compounds and their metabolites. Participation in the proficiency testing program is open to all laboratories doing either therapeutic efficacy studies or other research on antimalarial drug exposure. The program currently offers plasma-based samples for eight antimalarial drug compounds and metabolites: chloroquine/desethylchloroquine, mefloquine/carboxymefloquine, primaquine/carboxyprimaquine, amodiaquine/desethylamodiaquine, piperaquine, lumefantrine/desbutyl-lumefantrine, dihydroartemisinin, and artesunate. Commercially obtained and controlled plasma is spiked with accurately weighed certified reference materials. All active ingredients and the plasma are controlled by the manufacturer and reflected in certificates of analysis. Each analyte is sent in a range of concentrations, including the highest and lowest concentrations expected to be found in clinical samples (Table 1), which allows each laboratory to test the limits of its assay.

After obtaining community support and written parental consent, 190 children aged 1–3 yrs from Ilaita 1–7 and Sunuhu 1 and 2 were enrolled in March 2006. Demographic information was collected from all participating children and the location of each child's home was recorded using a hand-held GPS receiver (Garmin 12XL). Each child was clinically examined: axillary temperature was measured using digital thermometers, spleen palpated and a standard questionnaire of common signs and symptoms of malarial illness was administered. Hemoglobin (Hb) was measured using a portable device (HemoCue®, Ångholm, Sweden). A 5 ml venous blood sample was collected using Heparin-Vacutainer® tubes (Becton Dickinson, NJ, USA) and 2 blood slides (thick and thin films) made for determination of malarial infection. Bed net usage of both mother and child was queried. All children with parasitologically confirmed malaria (see below) were treated with Coartem® (arthemeter-lumefantrine); those with moderate to severe anaemia (i.e. Hb <7.5 g/dl) received an antimalarial treatment (Coartem®) plus 4 weeks of iron and folate supplementation according to national treatment guidelines.
As the initial enrolment did not yield the required sample size, the study area was extended to include Kamanokor and Ingamblis villages and 74 further children were enrolled into the study from the 1^st to the 3^rd regular bleed time points (May to September 2006).Enrolment procedures were identical with the exception of the 5 ml venous blood sample, which was not collected from these later enrolments. A 250 µl finger prick blood sample was collected instead.

The PK/PD modelling now allows for artemisinin absorption and conversion (described above), so the ability to track more than two drug concentrations simultaneously and convert them into a drug-killing rate is crucial. This feature is absent from previous pharmacological models of malaria, which track only a single drug [1] (link) although we previously extended the methodology to track up to two drugs [13] . Existing pharmacological models typically use a standard differential equation [1] (link) to find a mathematical description for the rate of change in total parasite growth and death rates where P is the number of parasites in the infection, t is time after treatment (days), a is the parasite growth rate (per day), f(C) represents the drug-dependent rate of parasite killing which depends on the drug concentration C, and f(I) the killing resulting from the hosts background immunity.
As antimalarial drugs are now typically deployed as combination therapies and as each drug may affect parasites in its unconverted and/or converted forms, predicting the changing numbers of parasites requires an expansion of Equation 9 where r is the number of drugs, the drug effect f(C_d) is the effect of each drug, d. Note that we regard each active entity as a distinct “drug”. For example artemether-lumefantrine (AR-LF) includes three drug forms lumefantrine (LF), artemether (AR) (unconverted) and its active metabolite DHA (dihydroartemisinin). Note that Equation 10 assumes drugs kill independently; this is discussed further below.
Integrating Equation 10 allows us to predict the number of parasites at any time, t, after treatment with any number of drugs. This was done by first integrating Equation 9 using the separation-of-variables technique
Integrating both sides of Equation 11 gives so
Taking the exponential of both sides (and noting that a times 0 = 0) gives so
The problem is now to integrate f(C). Assuming there are r separate drugs/metabolites with antimalarial activity. In this case, f(C) becomes
So for each drug/metabolite d we need to calculate its concentration over time C_d using the compartment model Equations (7 and 8) and the substitute C_d into the killing rate equation
Note in Equation 14, is the maximum drug killing V_max for drug d.
Substituting Equation 13 into 12 gives or, equivalently,
Note that C_d may be a complicated expression (including Equations 7 and 8) and so has to be integrated numerically. As before [13] , if the predicted parasite number (P_t) falls below 1 we assume the infection has been cleared and the patient cured, immunity is currently ignored (see Winter & Hastings [13] for further discussion).

This was a longitudinal, single-arm, prospective study to evaluate P. falciparum tolerance to ART and its derivatives in children with uncomplicated malaria aged 6 months to 14 years in 3 health facilities in the Greater Accra region of Ghana. The study focused mainly on day 3 post artemether-lumefantrine (AL) treatment parasitaemia, 72-h ex vivo RSA after dihydroartemisinin (DHA) exposure, 72-h parasite clearance in vitro against a panel of 6 drugs (ART, AS, artemether [AM], DHA, amodiaquine [AQ], lumefantrine [LUM], and the following molecular markers of drug tolerance / resistance: Single Nucleotide Polymorphisms (SNPs), Multiple Nucleotide Polymorphisms (MNPs), Insertions & Deletions (INDEL) in Pfk13, Pfcoronin, P. falciparum multidrug resistance protein 1 (Pfmdr1), multidrug resistance protein 2 (Pfmdr2), dihydrofolate reductase (Pfdhfr), dihydropteroate synthetase (Pfdhps), signal peptide peptidase (Pfspp), and multidrug resistance-associated protein 2 (Pfmrp2) genes. It sought to set up correlates of ART tolerance.

Poly(acrylic acid) (PAA, Carbomer, M_W = 1.8, 450, 4000 kg/mol) was purchased from
Sigma-Aldrich (St. Louis, MO), lumefantrine (LMF) from Nanjing Bilatchem
Industrial Co. (Nanjing, China), dichloromethane (ChromAR grade) from
Thermo Fisher Scientific (Fair Lawn, NJ), and ethanol from Decon Laboratories
(King of Prussia, PA). All materials were used as received.