Standardization of the physicochemical parameters to assess in vitro the beta-hematin inhibitory activity of antimalarial drugs

Intraerythrocytic plasmodia form hemozoin as a detoxification product of he moglobin-derived heme. An identical substance, beta -hematin (BH), can be o btained in vitro from hematin at acidic pH. Quinoline-antimalarials inhibit BH formation. Standardization of test conditions is essential for studying the interaction of compounds with this process and screening potential inh ibitors. A spectrophotometric microassay of heme polymerization inhibitory activity (HPIA) (Basilico et al, Journal of Antimicrobial Chemotherapy 42, 55-60, 1998) previously reported was used to investigate the effect of pH a nd salt concentration on BH formation. The yield of BH formation decreased with pH. Moreover, under conditions used in the above HPIA assay (18 h, 37 degreesC, pH = 2.7), several salts including chloride and phosphate inhibit ed the process. Aminoquinoline drugs formulated as salts (chloroquine-phosp hate, primaquine-diphosphate), but not chloroquine-base, also inhibited the reaction. Interference by salts was highest at low pH and decreased at hig her pH (pH 4). Here, we describe different assay conditions that eliminate these problems (BHIA, beta -hematin inhibitory activity). By replacing hema tin with hemin as the porphyrin and NaOH solution with DMSO as solvent, the formation of BH was independent of pH up to pH 5.1. No interference by sal ts was observed over the pH range 2.7-5.1. Dose-dependent inhibition of BH formation was obtained with chloroquine-base, chloroquine-phosphate, and ch loroquine-sulfate at pH 5.1, Primaquine was not inhibitory. The final produ ct, characterized by solubility in DMSO, consists of pure BH by FT-IR spect roscopy. The BHIA assay (hemin in DMSO, acetate buffer pH 5 +/- 0.1, 18 h a t 37 degreesC) is designed to screen for those molecules forming pi-pi inte ractions with hematin and thus inhibiting beta -hematin formation. (C) 2000 Academic Press.