Anti-Plasmodial Activity Of Astemizole-Methylene Blue Combination Therapy Against Chloroquine Sensitive And Resistant Plasmodium Falciparum And Their Safety In Balb/C Mice

ABSTRACT

Plasmodium is protozoa from the Apicomplexa phylum which causes malaria. In the tropics and sub-tropics, approximately 3.3 billion people are at risk. Artemisinin Combination Therapy (ACT), the current prime treatment, has been reported to have a possible emergence of resistance. This is a major obstacle that contributes to high mortality. Drug repurposing offers an appealing alternative to de novo drug development. Although astemizole and methylene blue have been reported to have anti-malarial properties, their safety when used in combination has not been explored. This study aimed at evaluating the optimum growth of Plasmodium falciparum 3D7 and W2, efficacy of astemizole-methylene blue combination therapy these strains and the safety of the combination therapy.To establish this, the growth potential of Plasmodium falciparum 3D7 and W2 was assessed by maintaining a continuous culture. Afterwhich, eight concentrations of astemizole-methyelene (1:1,3:1 and 3:1) using drug concentrations of range 7.81 μg/ml to 1 mg/ml, were assessed in triplicate against the Plasmodium strains. The parasites were cultured in complete media containing human erythrocytes in 96 well plates at 36.80C. Parasitemia was determined by microscopy and non-linear regression was used to determine the interactions of the drugs. Combinations that had high efficacy AST-MB 3:1 and 1:3 were administered in Balb/c mice (N=25) intraperitoneally. Clinical symptoms, hematology, biochemistry, and gross pathology were assessed and results presented as mean ± standard error of mean. ANOVA was used to analyze the results. Differences were considered significant if P values were less than 0.05(p< 0.05), F values were more than 2.78. Astemizole-methylene blue 3:1 (31.25 μg/ml and IC50 of 22.28±0.24 μg/ml) was the most efficacious drug combinations against P. falciparum 3D7 (F= 8.439, p=0.017). Whereas astemizole-methylene blue 1:3 (31.25 μg/ml and IC50 of 15.07±0.60 μg/ml was the most efficacious drug combinations against P. falciparum W2 (F= 5.428, p=0.0035). In spite of this, all astemizole-methylene blue drug combinations showed antagonism (FIC˃2). Also, astemizole was found to be less efficacious against both parasite strains in comparison to methylene blue. Frome the toxicity study, astemizole-methylene blue 3:1 drug combinations was associated with lower weight of the heart (F= 8.967, df=4, p=0.007) and liver (F= 4.339, p=0.0001) compared to the negative controls. This indicates abnormalities in these organs. Astemizole-methylene blue 3:1 reduced the platelet levels to undetectable amounts (F= 27.40, df=4, p=0.005). Both Plasmodium falciparum 3D7 and W2 had good and similar growth potentials, astemizole-methylene blue combinations were efficacious against both parasite strains and astemizole-methyelene blue 3:1 adversely affected Balb/c mice. This study recommends evaluating methylene blue and astemizole combinations with lower concentrations of astemizole to counter the effects.