Drug Recovery: Effect of Astemizole - Methylene Blue Combination Therapy against Plasmodium Strains in vitro

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Joyce Nyirongo
Lucy Kamau
Jemimah Simbauni
Victor Mwangi
Esther Kagasi
Faith Onditi
Hastings Ozwara


Plasmodium species are protozoa from the Apicomplexa phylum which cause malaria. In the tropics and sub-tropics, approximately 3.3 billion people are threatened by this disease. Artemisinin Combination Therapy, has been reported to have a possible emergence of resistance. Therefore, there is an urgent need for new drug formulations. 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 efficacy when used in combination has not been studied. Five concentrations ranging from 7.81 µg/ml to 125 µg/ml were combined in various ratios and assessed against two Plasmodium strains in vitro. Parasite load (per µl of blood) was determined by microscopy. The results were represented as mean ± standard error. ANOVA analysis was used to determine differences in the treatment groups at p<0.05. Antiplasmodial activity was observed in all drugs that were cultured with P. falciparum chloroquine sensitive (3D7) and chloroquine resistant (W2) strains. Strain dependent differences were observed in the efficacy scores of the tested drugs.  Astemizole-methylene blue combinations of ratios 1:1, 3:1 and 1:3 interacted antagonistically. The least antagonistic interactions were 3:1 and 1:3 ratios at 31.25 µg/ml against the Plasmodium strains (FIC of 2.2 and 2.6 respectively). Astemizole antagonized methylene blue in the combinations. This study provided information on the importance of astemizole-methylene blue combination therapy against malaria and emphasized on the relevance of drug repurposing in malaria. This study shows that the drugs work better as monotherapies and that combinations in these ratios have insignificant antiplasmodial activity.

Drug repurposing, malaria, astemizole, methylene blue, Plasmodium falciparum

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Nyirongo, J., Kamau, L., Simbauni, J., Mwangi, V., Kagasi, E., Onditi, F., & Ozwara, H. (2020). Drug Recovery: Effect of Astemizole - Methylene Blue Combination Therapy against Plasmodium Strains in vitro. South Asian Journal of Parasitology, 4(3), 9-17. Retrieved from https://journalsajp.com/index.php/SAJP/article/view/30118
Original Research Article


WHO | World malaria report 2019. WHO; 2020. [Online].


Accessed: Jul. 12, 2020.

Schneider P, Chan BH, Reece SE, Read AF. Does the drug sensitivity of malaria parasites depend on their virulence? Malar. J. 2008;7(1):257.

DOI: 10.1186/1475-2875-7-257

Matthews H, Usman-Idris M, Khan F, Read M, Nirmalan N. Drug repositioning as a route to anti-malarial drug discovery: Preliminary investigation of the in vitro anti- malarial efficacy of emetine dihydrochloride hydrate. Malar. J. 2013;12(1):359.

DOI: 10.1186/1475-2875-12-359

Wellems TE, Plowe CV. Chloroquine‐ Resistant Malaria. J. Infect. Dis. 2001; 184(6):770–776.

DOI: 10.1086/322858

Suwanarusk R, Russell B, Ong A, Sriprawat K, Chu C S, PyaePhyo A Renia L. Methylene blue inhibits the asexual development of vivax malaria parasites from a region of increasing chloroquine resistance. J. Antimicrob. Chemother. 2015; 70 (1): 124-129.

DOI: 10.1093/jac/dku326

WHO | World Malaria Report 2010. WHO; 2014.

Becker K, Tilley L, Vennerstrom JL, Roberts D, Rogerson S, Ginsburg H. Oxidative stress in malaria parasite-infected erythrocytes: Host-parasite interactions. Int. J. Parasitol. 2004;34(2): 163–189.

DOI: 10.1016/j.ijpara.2003.09.011

Zhou Z, Vorperian VR, Gong Q, Zhang S, January CT. Block of HERG potassium channels by the antihistamine astemizole and its metabolites desmethylastemizole and norastemizole. J. Cardiovasc. Electrophysiol. 1999;10(6):836–843.

DOI: 10.1111/j.1540-8167.1999.tb00264.x

Karapetyan YE, Sferrazza GF, Zhou M, Ottenberg G, Spicer T, Lasmézas CI. Unique drug screening approach for prion diseases identifies tacrolimus and astemizole as antiprion agents. Proc. Natl. Acad. Sci. U. S. A. 2013;110(17):7044–7049.

DOI: 10.1073/pnas.1303510110

Musonda CC, Whitlock GA, Witty MJ, Brun R, Kaiser M. Chloroquine- astemizole hybrids with potent in vitro and in vivo antiplasmodial activity. Bioorganic Med. Chem. Lett. 2009;19(2):481–484.

DOI: 10.1016/j.bmcl.2008.11.047

Chong CR, Chen X, Shi L, Liu JO, Sullivan DJ. A clinical drug library screen identifies astemizole as an antimalarial agent. Nat. Chem. Biol. 2006;2(8):415–416.

DOI: 10. 1038/nchembio806

Corsello SM, Bittker JA, Liu Z, Gould J, McCarren P, Hirschman JE. The drug repurposing hub: A next-generation drug library and information resource. Nature Medicine, vol. Nature Publishing Group. 2017;23(4):405–408.

DOI: 10.1038/nm.4306

Meissner PE, Mandi G, Coulibaly B, Witte S, Tapsoba T, Mansmann U. Methylene blue for malaria in Africa: Results from a dose-finding study in combination with chloroquine. Malar. J. 2006;5.

DOI: 10.1186/1475-2875-5-84

Mwangi VI, Mumo RM, Kiboi DM, Omar S A, Ng’ang’a ZW, Ozwara HS. Methylene blue inhibits lumefantrine-resistant Plasmodium berghei. J. Infect. Dev. Ctries. 2016;10(6):635–642.

DOI: 10.3855/jidc.7556

Amantea D, Certo M, Bagetta G. Drug repurposing and beyond: The fundamental role of pharmacology. Functional Neurology. CIC Edizioni Internazionali s.r.l. 2015;30(1):79–81.

DOI: 10.11138/FNeur/2015.30.1.079

Nzila A, Ma Z, Chibale K. Drug repositioning in the treatment of malaria and TB. Future Medicinal Chemistry, Future Med Chem. 2011;3(11):1413–1426.

DOI: 10.4155/fmc.11.95

Fivelman QL, Adagu IS, Warhurst DC. Modified fixed-ratio isobologram method for studying in vitro interactions between atovaquone and proguanil or dihydroartemisinin against drug-resistant strains of Plasmodium falciparum. Antimicrob. Agents Chemother. 2004; 48(11):4097–4102.

DOI: 10.1128/AAC.48.11.4097-4102.2004

Kumar M, Okombo J, Mambwe D, Taylor D, Lawrence N, Reader J, Chibale K. Multistage antiplasmodium activity of astemizole analogues and inhibition of hemozoin formation as a contributor to their mode of action. ACS Infect. Dis. 2019;5(2):303–315.

DOI: 10.1021/acsinfecdis.8b00272

Akoachere M, Buchholz K, Fischer E, Burhenne J, Haefeli WE, Schirmer RH, Becker K. In vitro assessment of methylene blue on chloroquine-sensitive and -resistant Plasmodium falciparum strains reveals synergistic action with artemisinins. Antimicrob. Agents Chemother. 2005;49(11):4592–4597.

DOI: 10.1128/AAC.49.11.4592-4597.2005

Krettli AU, Andrade-Neto VF, Brandão MD GL, Ferrari WMS. The search for new antimalarial drugs from plants used to treat fever and malaria or plants randomly selected: A review. Mem. Inst. Oswaldo Cruz. 2001;96(8):1033–1042.

DOI: 10.1590/S0074-02762001000800002

Dormoi J, Pradines B. Dose responses of proveblue methylene blue in an experimental murine cerebral malaria model. Antimicrobial Agents and Chemotherapy. American Society for Microbiology (ASM). 2013;57(8):4080–4081.

DOI: 10.1128/AAC.00634-13

Lee JH, Kim JW, Jeon SY, Park BK, Han BG. Assessment of general and cardiac toxicities of astemizole in male cynomolgus monkeys: Serum biochemistry and action potential duration. Toxicol. Res. 2008;24(4):289–295.

DOI: 10.5487/TR.2008.24.4.289

Aykul S, Martinez-Hackert E. Determination of half-maximal inhibitory concentration using biosensor-based protein interaction analysis. Anal. Biochem. 2016;508:97–103.

DOI: 10.1016/j.ab.2016.06.025

Te Dorsthorst DTA, Verweij PE, Meis JFG M, Punt NC, Mouton JW. Comparison of fractional inhibitory concentration index with response surface modeling for characterization of in vitro interaction of antifungals against itraconazole- susceptible and -resistant Aspergillus fumigatus isolates. Antimicrob. Agents Chemother. 2002;46(3):702–707.

DOI: 10.1128/AAC.46.3.702-707.2002

Huang RY, et al. Isobologram analysis: A comprehensive review of methodology and current research. Frontiers in Pharmacology. Frontiers Media S.A., 2019;10:1222.

DOI: 10.3389/fphar.2019.01222

Wambaugh MA, Denham ST, Ayala M, Brammer B, Stonhill MA, Brown JCS. Synergistic and antagonistic drug interactions in the treatment of systemic fungal infections. Elife. 2020;9:1–26.

DOI: 10.7554/eLife.54160