Antimalarial activity of methylene blue against selected drug resistant Plasmodium parasites

Abstract

Malaria continues to be a major global health problem mainly due to emergence of parasites that are resistant to well-established antimalarials. This persistent threat of resistance has continually pushed for the search for new molecules with novel mechanisms of action and in some cases reversion to earlier phased out drugs. We aimed at developing Pyrimethamine (PYR) resistant P. berghei by gene manipulation using the transfection technique and determine in vivo activity of Methylene Blue (MB) on P. berghei parasites resistant to the standard antimalarials chloroquine, lumefantrine and pyrimethamine. In addition, the study set out to determine MB’s activity against P. falciparum in vitro. PYR resistant P. berghei lines were developed through introducing plasmid DNA that conferred PYR resistance in wild parasites via transfection. The antimalarial activity of methylene blue was investigated in vivo on groups of Balb/C mice infected with either CQ, LM or PYR resistant rodent malaria parasite, P. berghei. Negative control groups were also included. Data collected was subjected to paired t-test for the comparison of means of the different experimental groups. Parasitaemia data generated from the in vitro experiments was subjected non linear regression analysis of log/dose response curves to determine the MB IC50. Successful genetic transformation of wild parasites was confirmed by PCR detection of TgDHFR gene in the genomic DNA of transfected P. berghei. 45 mg/kg daily dose of MB was found to inhibit over 99% of the erythrocytic parasite growth of LM and PYR resistant P. berghei for up to 6 days post treatment (DPT) and 49.2% suppression in CQ resistant parasites to day 4 post treatment. MB treated mice survived longer than mice in the other treatment groups. At the same time, antiplasmodial activity against CQ resistant P. falciparum was observed and the IC50 determined at 27.33nM. Glutathione (GSH) plays a critical role in parasite detoxification by degrading heme and protection against oxidative stress. Methylene Blue, a glutathione reductase inhibitor prevents reduction of oxidized glutathione thus increasing levels of toxic cellular hydrogen peroxide. This mode of action provides a new avenue in the production of antimalarials against resistant parasites. The minimal suppression against CQ resistant P. berghei could be linked to higher levels of GSH these parasites have in relation to their sensitive counterparts. Transfection provides a powerful tool in malaria research. The antimalarial activities displayed suggest that the phenothiazine structure could serve as a lead compound for further drug development and combination therapy. This may be the new approach to tackle increased prevalence of resistant malaria especially in Africa. In conclusion, the results obtained highlight that MB is effective in inhibiting P. falciparum growth and that it can be integrated in a new low cost antimalarial therapy against lumefantrine and pyrimethamine resistant parasites based on its activity on the drug resistant P. berghei and the P. yoelii 17X used. The suppressive activity demonstrated on the P. yoelii 17X shows it has potential activity across species. These findings suggest that MB provides a suitable candidate for first line drug in the management of resistant falciparum malaria as well as a potential candidate in the production of more effective and resilient combination-therapy drugs for malaria control.