Lumefantrine

Cytotoxicity of sulphadiazine and lumefantrine (Sigma, USA) to Vero cells was evaluated by the methyl thiazolyl tetrazolium (MTT) assay (Chen et al., 2008 (link); Kavitha et al., 2010 ). Vero cells (2 × 10⁵) were seeded in 96-well plates and cultured in 10% FBS-DMEM for 12 h to obtain a monolayer. Vero cell monolayers were washed and directly subjected to lumefantrine (dilution from 50 to 1.563 μg L⁻¹) or sulphadiazine (dilution from 500 to 15.625 mg L⁻¹, from 100 to 3.125 mg L⁻¹ and from 30 to 0.9375 mg L⁻¹, respectively), which were diluted with 10% FBS-DMEM. The Vero cells were subsequently cultured for 24 and 48 h. As a control, Vero cells were treated with 200 μL 10% FBS-DMEM (blank control/DMEM group) and 20 μL dimethyl sulphoxide (DMSO) (1 μL mL⁻¹) (Sigma, USA) together with 180 μL 10% FBS-DMEM (solvent control/DMSO group). Supernatants were removed after culturing for 24 or 48 h, and the plates were washed twice by using phosphate-buffered saline (PBS) and pulsed by adding 10 μL of MTT (Solarbio, China) together with 90 μL 10% FBS-DMEM for 4 h under the same culture conditions. The supernatants were removed gently with pipettes and 110 μL formazan was added to each well. The plates were vibrated on a low-speed oscillator, and optical density (OD) was measured at 490 nm by using a microplate reader after 30 min (Tecan, Switzerland).

Primary analytical reference standards of purity ≥98% artemether and lumefantrine (Sigma-Aldrich, USA), deionized water (gifted by Tradewinds Chemist Limited, Ghana), analytical grade solvents including HPLC grade acetonitrile of purity ≥99.9% (LiChrosolv® Reag. Ph Eur. Supelco, Germany), and analytical grade orthophosphoric acid 85% (Supelco, Germany) were used.

Lumefantrine (Lu), artemether (Ar), and hypromellose were purchased from Sigma Aldrich (Taufkirchen, Germany). Model fake tablets were manufactured, containing the APIs Lumefantrine and artemether in different concentration ratios by direct compression. The total weight for each model tablet was 200 mg and the pharmaceutical excipient hypromellose was used to fill up the formulation. The composition of the analyzed tablets is visualized in Figure 6. Riamet^® tablets (Novartis) were purchased from a local pharmacy (Jena, Germany) and investigated. The coating of this tablet was removed for better conformity with the model tablets.

Chloroquine, quinine, mefloquine, desethylamodiaquine, lumefantrine, piperaquine, and pyronaridine were obtained from Sigma-Aldrich (St Quentin Fallavier, France). For the ex vivo chemosusceptibility assay, each isolate was aliquoted in 96-well plates previously treated with a gradient of antimalarial drug concentrations without culture adaptation, as previously described [37 (link)]. The plates were then incubated for 72 h at 37 °C in a controlled atmosphere with 10% O₂, 5% CO₂, and 85% N₂. An ELISA assay was then performed by targeting the HRP2 protein using the Malaria Ag Celisa kit (ref KM2159, Cellabs PTY LDT, Brookvale, Australia) to estimate parasite growth, as previously described [38 (link)].
Each batch of plates was controlled by assessing the Chloroquine-resistant P. falciparum strain W2 (MR4, Charlottesville, VA, USA) in between three and six independent experiments.