Antiplasmodial Activity Assay of 3-Chloro-4-(4-chlorophenoxy) Aniline Combinations with Artesunate or Chloroquine In Vitro and in a Mouse Model

Malaria is the eighth highest contributor to global disease burden with 212 million cases and 429,000 deaths reported in 2015. There is an urgent need to develop multiple target drug to curb growing resistance by Plasmodia due to use of single target drugs and lack of vaccines. Based on a previous study, 3-chloro-4-(4-chlorophenoxy) aniline (ANI) inhibits Plasmodia enoyl acyl carrier protein reductase. This study aimed at evaluating the antiplasmodial activity of ANI combinations with artesunate (AS) or chloroquine (CQ) against P. falciparum in vitro based on the semiautomated microdilution assay and P. berghei in vivo based on Peters' 4-day test. Data were analysed by linear regression using version 5.5 of Statistica, 2000. From the results, on the one hand, a combination of 1.1 ng/ml AS and 3.3 μg/ml of ANI inhibited 50% growth of W(2), while a combination of 0.8 ng/ml of AS and 2.6 μg/ml of ANI inhibited 50% growth of 3D(7). On the other hand, a combination of 22 ng/ml CQ and 3.7 μg/ml of ANI inhibited 50% growth of W(2), while a combination of 4.6 ng/ml CQ and 3.1 μg/ml of ANI inhibited 50% growth of 3D(7). In in vivo assays, a combination of ED(50) concentrations of AS and ANI cleared all parasites, while 1/2 and 1/4 ED(50) combinations inhibited 67.0% and 35.4% parasite growth, respectively. ED(50) combinations of CQ and ANI inhibited 81.0% growth of parasites, while 1/2 and 1/4 ED(50) combinations inhibited 27.3% and 10.2% parasite growth. Assuming a linear relationship between percentage chemosuppression and combination ratios, only 0.88 mg/kg of AS combined with 1.68 mg/kg of ANI or 1.78 mg/kg of CQ with 3.15 mg/kg of ANI inhibited 50% parasite growth in vivo. ANI combinations with AS or CQ are thus potential antimalarial drug combinations if their clinical efficacy and safety are ascertained.