In vitro anti-trypanosomal effect of ivermectin on Trypanosoma evansi by targeting multiple metabolic pathways

Abstract

High cytotoxicity and increasing resistance reports of existing chemotherapeutic agents against T. evansi have raised the demand for novel, potent, and high therapeutic index molecules for the treatment of surra in animals. In this regard, repurposing approach of drug discovery has provided an opportunity to explore the therapeutic potential of existing drugs against new organism. With this objective, the macrocyclic lactone representative, ivermectin, has been investigated for the efficacy against T. evansi in the axenic culture medium. To elucidate the potential target of ivermectin in T. evansi, mRNA expression profile of 13 important drug target genes has been studied at 12, 24, and 48 h interval. In the in vitro growth inhibition assay, ivermectin inhibited T. evansi growth and multiplication significantly (p < 0.001) with IC₅₀ values of 13.82 μM, indicating potent trypanocidal activity. Cytotoxicity assays on equine peripheral blood mononuclear cells (PBMCs) and Vero cell line showed that ivermectin affected the viability of cells with a half-maximal cytotoxic concentration (CC₅₀) at 17.48 and 22.05 μM, respectively. Data generated showed there was significant down-regulation of hexokinase (p < 0.001), ESAG8 (p < 0.001), aurora kinase (p < 0.001), casein kinase 1 (p < 0.001), topoisomerase II (p < 0.001), calcium ATPase 1 (p < 0.001), ribonucleotide reductase I (p < 0.05), and ornithine decarboxylase (p < 0.01). The mRNA expression of oligopeptidase B remains refractory to the exposure of the ivermectin. The arginine kinase 1 and ribonucleotide reductase II showed up-regulation on treatment with ivermectin. The ivermectin was found to affect glycolytic pathways, ATP-dependent calcium ATPase, cellular kinases, and other pathway involved in proliferation and maintenance of internal homeostasis of T. evansi. These data imply that intervention with alternate strategies like nano-formulation, nano-carriers, and nano-delivery or identification of ivermectin homologs with low cytotoxicity and high bioavailability can be explored in the future as an alternate treatment for surra in animals.

Keywords: HMI-9 medium; Ivermectin; Surra; Trypanosoma evansi.

Fig. 1

Dose-dependent effect of ivermectin on the proliferation of Trypanosoma evansi. The trypanosomes were cultured in HMI-9 medium and exposed to ivermectin for 72 h. The 24 h data was analyzed by curve fitting technique to compute minimal 50% inhibitory concentration (IC₅₀) of ivermectin against T. evansi

Fig. 2

Cumulative growth analysis of trypanosomes in HMI-9 medium in the absence or presence of different concentrations of ivermectin. Significance was determined using two-way ANOVA comparing treated and control, p < 0.05

Fig. 3

Dose-dependent effect of ivermectin on mammalian cells. Equine PBMCs and Vero cell line were exposed with ivermectin for 48 h and cell viability was determined by luminescent cell viability assay

Fig. 4

qPCR analysis of mRNA expression kinetics of a hexokinase; b trans-sialidase; c trypanothine reductase; d ESAG8; e aurora kinase; f oligopeptidase B; g casein kinase 1; h aurora kinase; i topoisomerase II; j calcium ATPase 1; k ribonucleotide reductase I; l ribonucleotide reductase II; and m ornithine decarboxylase after treatment with IC₅₀ of ivermectin for 48 h. The values are expressed as relative quantity with respect to the control