Therapeutic Effects of Atranorin towards the Proliferation of Babesia and Theileria Parasites

Abstract

Atranorin (ATR), is a compound with multidirectional biological activity under different in vitro and in vivo conditions and it is effective as an antibacterial, antiviral, antiprotozoal and anti-inflammatory agent. In the current study, the in vitro as well as in vivo chemotherapeutic effect of ATR as well as its combined efficacy with the existing antibabesial drugs (diminazene aceturate (DA), atovaquone (AV) and clofazimine (CF)) were investigated on six species of piroplasm parasites. ATR suppressed B. bovis, B. bigemina, B. divergens, B. caballi and T. equi multiplication in vitro with IC₅₀ values of 98.4 ± 4.2, 64.5 ± 3.9, 45.2 ± 5.9, 46.6 ± 2.5, and 71.3 ± 2.7 µM, respectively. The CCK test was used to examine ATR's cytotoxicity and adverse effects on different animal and human cell lines, the main hosts of piroplasm parasites and it showed that ATR affected human foreskin fibroblasts (HFF), mouse embryonic fibroblast (NIH/3T3) and Madin-Darby Bovine Kidney (MDBK) cell viability in a dose-related effect with a moderate selective index. The combined efficacy of ATR with DA, CF, and AV exhibited a synergistic and additive efficacy toward all tested species. In the in vivo experiment, ATR prohibited B. microti multiplication in mice by 68.17%. The ATR-DA and ATR-AV combination chemotherapies were more potent than ATR monotherapy. These results indicate the prospects of ATR as a drug candidate for piroplasmosis treatment.

Keywords: Babesia sp.; Theileria sp.; atranorin; chemoprophylactic agents; drug discovery; piroplasmosis control.

Figure 1

The chemical structure of atranorin.

Figure 2

The relationship between the inhibition percentage and the concentrations of ATR (µM) on T. equi, B. divergens, B. bigemina, B. caballi, and B. bovis. The non-linear regression (curve fit analysis) in the GraphPad Prism software used for IC₅₀ calculations. The percentage of parasite growth inhibitory efficacy is calculated as the percentage of parasites inhibited divided by that of the positive control wells and the result was subtracted from the negative control wells.

Figure 3

In vivo chemotherapeutical prospect of ATR on B. microti and the chemotherapeutic potential of DA-IP, AV-oral, ATR-IP, ATR-DA, and ATR-AV treatment when compared with the positive group. The arrow shows 5 successive days of drug administration starting from day 4 to 8 p.i. The asterisks (*) show the significant variation (p < 0.05) between drug-treated and positive groups. Parasitemia was detected using Giemsa-stained thin blood smears by counting infected RBCs (iRBCs) among 2000 RBCs.

Figure 4

Hematology parameter changes in treated mice in vivo. Graphs showing the (A) hematocrit (HCT), (B) hemoglobin (HGB), and (C) red blood cells (RBCs) changes in treated mice compared to the infected-untreated mice. Asterisks (*) show significant variation (p < 0.05) between drug-treated and positive groups. The arrow shows five successive days of drug administration starting from day 4 to 8 p.i.

Figure 5

The molecular examination of B. microti in the blood of all treated groups on day 45. M refers to the marker, NC refers to the negative control group (untreated-uninfected), and PC refers to the positive control group (untreated-infected). The arrow indicates the 154 bp band length for B. microti positive cases.