Safety and efficacy of hydroxyurea and eflornithine against most blood parasites Babesia and Theileria

Abstract

Background: The plenteous resistance to and undesirable consequences of the existing antipiroplasmic therapies have emphasized the urgent need for new chemotherapeutics and drug targets for both prophylaxis and chemotherapy. Hydroxyurea (HYD) is an antineoplastic agent with antitrypanosomal activity. Eflornithine (α-difluoro-methyl ornithine, DFMO) is the best choice therapy for the treatment of late-stage Gambian human African trypanosomiasis.

Methods: In this study, the inhibitory and combination efficacy of HYD and DFMO with existing babesicidal drugs (diminazene aceturate (DA), atovaquone (ATV), and clofazimine (CLF)) deoxyribonucleotide in vitro against the multiplication of Babesia and Theileria. As well as, their chemotherapeutic effects were assessed on B. microti strain that infects rodents. The Cell Counting Kits-8 (CCK-8) test was used to examine their cytotoxicity on human foreskin fibroblast (HFF), mouse embryonic fibroblast (NIH/3T3), and Madin-Darby bovine kidney (MDBK) cells.

Findings: HYD and DFMO suppressed the multiplication of all tested species (B. bigemina, B. bovis, B. caballi, B. divergens, and T. equi) in a dose-related manner. HFF, NIH/3T3, or MDBK cell viability was not influenced by DFMO at 1000 μM, while HYD affected the MDBK cell viability at EC50 value of 887.5±14.4 μM. The in vitro combination treatments of DFMO and HYD with CLF, DA, and ATV exhibited synergistic and additive efficacy toward all tested species. The in vivo experiment revealed that HYD and DFMO oral administration at 100 and 50 mg/kg inhibited B. microti multiplication in mice by 60.1% and 78.2%, respectively. HYD-DA and DFMO-DA combined treatments showed higher chemotherapeutic efficacy than their monotherapies.

Conclusion: These results indicate the prospects of HYD and DFMO as drug candidates for piroplasmosis treatment, when combined mainly with DA, ATV, and CLF. Therefore, further studies are needed to combine HYD or DFMO with either ATV or CLF and examine their impact on B. microti infection in mice.

Fig 1. The relationship between the relative fluorescence units (RFUs) and the log concentrations of HYD (nM) (A) and DFMO (nM) (B) on B. bovis, B. bigemina, B. divergens, B. caballi and T. equi.

The non-linear regression (curve fit analysis) in GraphPad Prism software (GraphPad Software Inc. USA) was used to calculate the IC₅₀’s. 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.

Fig 2. In vivo chemotherapeutic potential of HYD and DFMO on B. microti.

Graph reveals the chemotherapeutic potential of DA-IP, HYD-IP, and DFMO-IP compared to the infected-untreated 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 iRBCs among 5000 RBCs.

Fig 3. In vivo growth inhibition of HYD and DFMO against B. microti.

Graph reveals the chemotherapeutic potential of HYD–DA and DFMO–DA when compared to the positive group. The asterisks (*) show significant variation (P < 0.05) between drug-treated and positive groups. The arrow shows 5 successive days of drug administration starting from day 4 to 8 p.i.

Fig 4. Hematology parameter changes in DFMO- and HYD-treated groups in vivo.

Graphs showing the hematocrit (HCT) (A), red blood cells (RBCs) (B), and hemoglobin (HGB) (C) 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 5 successive days of drug administration starting from day 4 to 8 p.i.