Carvalho et al. Parasites Vectors (2020) 13:362 https://doi.org/10.1186/s13071-020-04235-7 Parasites & Vectors RESEARCH Open Access Activities of artesunate-based combinations and tafenoquine against Babesia bovis in vitro and Babesia microti in vivo Leonardo J. M. Carvalho1,2,3* , Bunduurem Tuvshintulga1, Arifn B. Nugraha1, Thillaiampalam Sivakumar1 and Naoaki Yokoyama1,2,4 Abstract Background: Babesiosis represents a veterinary and medical threat, with a need for novel drugs. Artemisinin-based combination therapies (ACT) have been successfully implemented for malaria, a human disease caused by related parasites, Plasmodium spp. The aim of this study was to investigate whether ACT is active against Babesia in vitro and in vivo. Methods: Mefoquine, tafenoquine, primaquine, methylene blue and lumefantrine, alone or in combination with artesunate, were tested in vitro against Babesia bovis. Parasite growth was verifed using a SYBR green I-based fuores- cence assay. Mice infected with Babesia microti were treated with mefoquine or tafenoquine, alone or in combination with artesunate, and parasitemia was verifed by microscopy and PCR. Results: All drugs, except lumefantrine, showed in vitro activity against B. bovis, with methylene blue showing the most potent activity (concentration 0.2 μM). Combination with artesunate led to improved activity, with mefoquine showing a striking 20-fold increase in activity. Tafenoquine (10 mg/kg, base), combined or not with artesunate, but not mefoquine, induced rapid clearance of B. microti in vivo by microscopy, but mice remained PCR-positive. Blood from mice treated with tafenoquine alone, but not with tafenoquine-artesunate, was infective for naive mice upon sub-inoculation. Conclusions: Tafenoquine, and most likely other 8-aminoquinoline compounds, are promising compounds for the development of ACT for babesiosis. Keywords: Babesia, Artesunate, Tafenoquine Background side efects associated with the administration of these Te genus Babesia contains a very diverse group of drugs and reports of resistance indicate the need for the piroplasmid organisms, such as Babesia bovis, Babesia discovery and development of new drugs for treating ani- bigemina, Babesia microti and Babesia caballi [1]. Babe- mal babesiosis. Human babesiosis is caused largely by siosis caused by these Babesia species represents a veteri- a rodent parasite B. microti and can lead to severe and nary and medical threat. In the case of animal babesiosis, even fatal infections especially in immunocompromised diminazene aceturate and imidocarb have been estab- patients [3]. However, the currently established treat- lished as efective therapeutic agents [2]. However, the ments, the combinations of quinine plus clindamycin or atovaquone plus azithromycin, are limited because *Correspondence: [email protected] 3 Laboratory of Malaria Research, Oswaldo Cruz Institute, Fiocruz, Rio de of substantial adverse efects and lack of efcacy due to Janeiro, Brazil drug resistance [4–6]. Terefore, there is an active search Full list of author information is available at the end of the article for new drugs to treat babesiosis. © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://crea- tivecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdo- main/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Carvalho et al. Parasites Vectors (2020) 13:362 Page 2 of 9 A number of compounds have been screened in the potential combinations (primaquine, tafenoquine and past years, some of them showing promising results. methylene blue). Among the classes of compounds that have been screened, antimalarial drugs are prominent [7], because Methods Plasmodium spp. and Babesia spp. are closely related Aim, design and setting of the study organisms, often sharing same drug targets. As men- Tis study was designed to investigate the activity of tioned, the antimalarial drugs quinine and atovaquone selected antimalarial drugs, alone or in combination with are currently used to treat human babesiosis, and radical artesunate, against B. bovis in vitro and the efcacy of cure has been demonstrated in experimental B. microti the most active combinations in vivo against B. microti. infection using a novel atovaquone-based drug combi- For the in vitro studies, each drug was added to B. bovis nation with an endochin-like quinolone [8]. Atovaquone cultures at serial dilutions, alone or in combination with has also been shown to be highly active against B. bovis artesunate. Drugs that showed activity in vitro alone and and B. divergens [9]. Methylene blue, a ‘rediscovered’ with an improved profle when combined with artesunate antimalarial drug, has been shown to be active in vitro were selected for in vivo efcacy testing using BALB/c against Babesia and Teileria parasites, but the activity mice infected with B. microti. All studies were performed in vivo was disappointing [10]. Out of several compounds at the National Research Center for Protozoan Diseases, with antimalarial activity, including mefoquine, halo- Obihiro, Japan. fantrine, artesunate, artelenic acid and the combination quinine plus clindamycin, only two 8-aminoquinolines, In vitro Babesia growth inhibition assay WR006026 and WR238605 (“tafenoquine”), were able Babesia bovis (Texas strain) was cultivated in purifed to cause a 100% suppression of B. microti infection in bovine red blood cells (RBCs) using a microaerophilic, the hamster model [11]. Te 8-aminoquinolines, indeed, stationary-phase culture system, as previously described show consistent activity against Babesia. A recent study [23]. Babesia bovis-infected RBCs (iRBCs) were culti- confrmed the potent activity of tafenoquine against vated at 1% parasitemia and 2.5% hematocrit in 96-well B. microti infection [12], and similar results have been plates using M199 media containing 40% bovine serum obtained with primaquine and 4-methyl-primaquine [13, with or without the following drugs in serial dilutions: 14]. More recently, extensive screening of the malaria box lumefantrine (LUM: 0.1–200 μM), mefoquine hydro- compound library led to the identifcation of novel leads chloride (MEF: 0.1–100 μM), primaquine bisphosphate with promising antibabesial activity in vitro and in vivo (PRI: 0.1–100 μM), tafenoquine succinate (TAF: 0.1–100 [7, 9, 15]. Artemisinin derivatives have shown substan- μM), and methylene blue (MB: 0.01–1 μM), alone or in tial inhibitory activity against diferent species of Babe- combination with sodium artesunate (ARS: 0.1–100 μM) sia in vitro, but only limited activity in vivo [11, 16–21]. (all drugs were purchased from Sigma-Aldrich, Tokyo, In the case of malaria, one successful strategy to improve Japan). Stock solutions for all drugs were prepared using treatment efcacy has been the use of drug combination DMSO (Sigma-Aldrich) as a solvent, except for methyl- therapies, that is, the use of two or more drugs with dif- ene blue (MilliQ water as solvent). Te cultures, in trip- ferent mechanisms of action to overcome low efcacy, licate wells for each concentration of the drugs, were resistance and pharmacokinetic limitations. Today, the incubated in an atmosphere of 5% O 2 and 5% CO 2 at 37 use of the so-called ACTs (artemisinin combination °C for 4 days without replacement of the medium. Maxi- therapies) has become the frst line of treatment against mum fnal concentration of DMSO in the wells ranged non-complicated malaria in most endemic countries, in from 0.1% to 0.5%, and control wells (no drug) were pre- two- or three-drug combinations [22]. Tis strategy has pared using culture medium containing DMSO at these not been properly explored in babesiosis. One of the few concentrations. Assays were run in triplicate for each studies indicated that the combination of artemisinin drug, and at least three independent assays were run for and lumefantrine in vitro had a synergistic efect against each drug. At the end of the 4-day incubation period, B. gibsoni [20]. Te activity and the efcacy of diferent parasite growth was measured using a fuorescent-based artemisinin-based combinations have not been explored assay, as previously described [24, 25]. Briefy, SYBR in greater detail with other Babesia species of veterinary green (Lonza Rockland Inc., Rockland, ME, USA) diluted and medical importance, such as B. bovis and B. microti. 1:10,000 in lysing bufer was added to the wells and incu- Terefore, we propose to address the suitability of difer- bated at room temperature in the dark for 4 h. Ten, ent ACTs as potential antibabesial drugs in in vitro and plates were read in a fuorescence spectrophotometer in vivo studies, using established drug partners of arte- (Fluoroskan Ascent; Termo Fisher Scientifc, Waltham, misinins (lumefantrine and mefoquine) as well as new MA, USA) (emission wavelength: 450nm; absorbance Carvalho et al. Parasites Vectors (2020) 13:362 Page 3 of 9 wavelength: 518 nm) and the half maximal inhibitory GTT T-3’)/inner reverse primer Bab3 (5’-AAG CCA concentrations (IC50s) were calculated [26]. TGC GAT TCG CTA AT-3’). Te expected size of the fnal PCR product was 154 bp. In vivo tests: chemotherapeutic evaluation in mice All protocols were approved by the Obihiro University Parasite sub‑inoculation ethical committee on animal experimentation (approval In the TAF-treated and ARS-TAF-treated groups, 30 days number: 19-105).