Repurposing of antiparasitic drugs: the hydroxy-naphthoquinone buparvaquone inhibits vertical transmission in the pregnant neosporosis mouse model
© Müller et al. 2016
Received: 11 December 2015
Accepted: 2 February 2016
Published: 17 February 2016
The three anti-malarial drugs artemiside, artemisone, and mefloquine, and the naphthoquinone buparvaquone known to be active against theileriosis in cattle and Leishmania infections in rodents, were assessed for activity against Neospora caninum infection. All four compounds inhibited the proliferation of N. caninum tachyzoites in vitro with IC50 in the sub-micromolar range, but artemisone and buparvaquone were most effective (IC50 = 3 and 4.9 nM, respectively). However, in a neosporosis mouse model for cerebral infection comprising Balb/c mice experimentally infected with the virulent isolate Nc-Spain7, the three anti-malarial compounds failed to exhibit any activity, since treatment did not reduce the parasite burden in brains and lungs compared to untreated controls. Thus, these compounds were not further evaluated in pregnant mice. On the other hand, buparvaquone, shown earlier to be effective in reducing the parasite load in the lungs in an acute neosporosis disease model, was further assessed in the pregnant mouse model. Buparvaquone efficiently inhibited vertical transmission in Balb/c mice experimentally infected at day 7 of pregnancy, reduced clinical signs in the pups, but had no effect on cerebral infection in the dams. This demonstrates proof-of-concept that drug repurposing may lead to the discovery of an effective compound against neosporosis that can protect offspring from vertical transmission and disease.
Neospora caninum is a cyst-forming apicomplexan parasite closely related to Toxoplasma gondii. N. caninum is one of the most important infectious causes of bovine abortion, stillbirth, and the birth of weak calves, with an economic impact of over 1.3 billion US dollars [1–3]. N. caninum infection can also result in birth of clinically healthy, but persistently infected calves transmitting the parasite to the next generation. In addition, N. caninum causes neuromuscular disease in dogs, and neosporosis has also been detected in a wide range of other species of livestock and wild animals worldwide.
Possible strategies to limit the economic impact of neosporosis include testing and culling of seropositive animals, discontinued breeding with offspring from seropositive cows, vaccination of susceptible and infected animals, and chemotherapeutical treatment of calves from seropositive cows [4, 5]. However, the most effective option is not always the most economic one  and the suitability of any of these options has to be assessed [6, 7].
As is the case for neglected diseases, one option for chemotherapy of neosporosis in cattle is the repurposing of drugs with well-documented antiparasitic activities [8–10]. The most prominent compounds in this context are certainly antimalarial drugs, for which enormous screening campaigns have been initiated, such as the high-throughput screening efforts of St. Jude Children’s Research Hospital (TN, USA), Novartis and GlaxoSmithKline [11–13]. While such large screening campaigns cannot be carried out for N. caninum, simply due to the lack of resources, one possibility is to use a piggy-back approach and perform efficacy testing of selected drugs that are already either marketed or in clinical trials for human and/or veterinary use. By screening a larger panel of such compounds, we here report on the results obtained with the three antimalarials artemisone, artemiside, and mefloquine, and the naphthoquinone buparvaquone.
Artemisone and artemiside are artemisinin derivatives  that have shown promising efficacy against experimentally induced toxoplasmosis in mice . Moreover, artemisone is highly active against N. caninum tachyzoites in vitro  and was shown to protect gerbils against cerebral infection . Artemiside exhibits excellent efficacy against malaria in a murine model , but has not been tested against neosporosis so far. Mefloquine, one of the most common antimalarials , is active in a mouse model of Schistosoma mansoni infection  and shows promising results in Echinococcus multilocularis infected mice , but its effect against N. caninum has never been assessed. Buparvaquone was also originally developed as an anti-malarial compound  and is now commercially available (Butalex ® ), for use in endemic regions against theileriosis in cattle. In other regions of the world, e.g., the EU, it is, however, not registered. In most cases only a single dose injection is required to cure Theileria-infected animals within a few days, with cure rates near to 100% when administered at the early stages of clinical disease . The mode of action of buparvaquone has not been clarified, but there are indications that it blocks the parasite respiratory chain , and resistance against buparvaquone has been associated with mutations in the cytochrome b gene of the parasite [24, 25]. Besides exhibiting an outstanding activity against Theileria spp. [26, 27], buparvaquone is also active against other protozoan parasites including Leishmania spp.  and Babesia spp. . Moreover, buparvaquone is highly active against N. caninum in vitro, and in a non-pregnant mouse model for acute disease we have recently shown that buparvaquone treatment protects mice against infection in the lungs, but not the brain, and prevents clinical signs of acute disease .
Here we applied a standardized Balb/c mouse model infected with the N. caninum NcSpain-7 isolate  to demonstrate that of these four compounds buparvaquone may have the potential to limit vertical transmission in N. caninum infected animals without inducing detrimental effects on pregnancy.
Materials and methods
Tissue culture media, biochemicals, and drugs
If not stated otherwise, all tissue culture media were purchased from Gibco-BRL (Zürich, Switzerland), and biochemical reagents were from Sigma (St. Louis, MO, USA). Kits for molecular biology were purchased from Qiagen (Hilden, Germany). Buparvaquone was provided by Cross Vetpharm Group Limited (Dublin, Ireland). Mefloquine was kindly supplied by Mepha Pharma AG (Aesch BL, Switzerland). Artemiside was synthesized according to the literature procedure  and artemisone was purified and supplied by Cipla Mumbai Ltd.
Host cell cultivation and parasite cultures
Tachyzoites of the N. caninum Nc-Spain7 isolate were cultured and prepared for infection as described .
In vitro efficacy
In vitro efficacies of the compounds were determined using a N. caninum beta-galactosidase reporter strain (Nc-beta-gal) and human foreskin fibroblasts (HFF) as host cells. Briefly, HFF were seeded into 96-well-plates, grown to confluence, and infected with 103 Nc-beta-gal-tachyzoites per well in the presence of the compound to be tested or DMSO as a solvent control. After 3 days, medium was removed, and after one wash with PBS, cells were overlaid with 0.1 mL PBS containing Triton-X-100 (0.05%) and chlorophenyl-red-beta-galactoside (Roche, Rotkreuz, Switzerland). Absorption was continuously read at 570 nm using a 96-well-plate spectrophotometer (Versamax, Molecular Devices, Sunnyvale CA) . Host cell toxicity was determined by Alamar blue as described .
All protocols involving animals were approved by the Animal Welfare Committee of the Canton of Bern under the license BE115/14. All animals used in this study were handled in strict accordance with practices made to minimize suffering. Female and male BALB/c mice, 8 weeks of age, were purchased from a commercial breeder (Charles River, Sulzberg, Germany), and were maintained in a common room under controlled temperature and a 14 h/10 h light cycle according to the guidelines set up by the animal welfare legislation of the Swiss Veterinary Office (approval No. BE 105/14).
Assessment of the effects of mefloquine, artemisone, and artemiside on male BALB/c mice infected with N. caninum Spain-7
Male Balb/c mice were randomly distributed in four groups of six mice each and subcutaneously infected with 105 tachyzoites of the Nc-Spain7 isolate. At day 2 post infection (pi), one group received corn oil alone (placebo control), the others received mefloquine, artemiside, or artemisone (50 mg/kg/day suspended in 100 µL corn oil) for a period of 6 days by gavage once a day. At day 21 pi, the mice were euthanized. Blood was recovered by cardiac puncture and sera were obtained to assess humoral immune responses. Brains and lungs were removed for subsequent determination of parasite load and stored at −20 °C until further processing.
Assessment of the effects of buparvaquone on non-pregnant and pregnant BALB/c mice infected with N. caninum Spain-7
Overview of animal experiments presented in this study
4, 6 mice per group
3, 16 mice per group
Nc-Spain 7 (105)
Nc-Spain 7 (105), 7 days post mating
ARI, ARO, MEF, placebo; days 2–7 pi
BPQ, placebo; days 2–7 pi
21 days pi
non-pregnant: 21 days pi dams: 30 days after birth, i.e., 44 days pi
N. caninum load in brains and lungs, serum titer
Number of pups, N. caninum brain load, serum titer
Analysis of biological samples from in vivo experiments
To quantify the parasite load in brains and lungs, DNA purification was performed employing the DNeasy Blood & Tissue Kit (Qiagen, Basel, Switzerland) according to the standard protocol suitable for animal tissues. The DNA concentrations in all samples were determined using the QuantiFluor dsDNA System (Promega, Madison, Wi., USA) according to the manufacturer’s instructions and adjusted with sterile DNAse free water to 5 ng/µL. Quantification of parasite loads in brains and lungs was performed as described [30, 32]. Serum titers for N. caninum were assessed by ELISA as described [33, 34].
Statistical analysis of the parasite burdens in brains was done using the Kruskal–Wallis test followed by Wilcoxon rank-sum-test. Survival analysis of the pups was performed on the corresponding Kaplan–Meier estimator using the log-rank-test. Nominal data were analyzed using the Chi square test. All analyses were performed using the software package R .
In vitro efficacies of artemisone, artemiside, mefloquine and buparvaquone against N. caninum tachyzoites
In vivo efficacies in a Balb/c mouse model for cerebral N. caninum infection
Treatment of pregnant mice with buparvaquone protects pups from vertical transmission of N. caninum
Effects of buparvaquone treatment on clinical signs, mortality, fertility and cerebral N. caninum Nc-Spain7 infection in non-pregnant mice, dams and pups
Dams and pups
Number of dams
Nc positive dams
Total number of pups
Litter size average
Nc positive pups
In order to investigate whether drug repurposing may be a suitable strategy for obtaining suitable chemotherapeutic agents against neosporosis, we have tested four compounds, namely artemiside, artemisone, mefloquine, and buparvaquone, with proven efficacies against apicomplexan parasites in vitro and in mouse models. Three of these four compounds, artemiside, artemisone, and mefloquine did not affect the cerebral parasite load when assessed in a chronic infection model for N. caninum infection. This demonstrates that a low IC50 value against N. caninum tachyzoites obtained in vitro (in the case of artemisone; see also ) is not a predictive parameter for good in vivo effects. Moreover, our results are not in line with previous findings concerning effects of artemisone against neosporosis in gerbils . However, in that study artemisone was administered by intraperitoneal injection as a formulation solubilized in DMSO, and the authors used another isolate (NcIS491) from Israel , which most likely differs in virulence from the Nc-Spain7 isolate used in this study. In a previous study on experimentally induced murine toxoplasmosis, DMSO solutions of artemisone and artemiside had been applied s.c. , but we refrained from this application mode due to potential side effects that is induced by this treatment based on own observations and on previously published results . Oral application of mefloquine, shown to be effective against schistosomiasis  and more recently against Echinococcus infections in mice  did not exhibit any effects against N. caninum in this study.
Buparvaquone drastically limits the proliferation of N. caninum tachyzoites in vitro. In addition, we have previously shown that administration of buparvaquone protected mice that were infected with a high dosage (2 × 106) of N. caninum tachyzoites of the Nc-Liverpool isolate against acute neosporosis . In the present study, by using a 20 times reduced infection dose (105 tachyzoites), which does not lead to acute symptoms but to chronic infection followed by vertical transmission in dams , we demonstrate that in the short term, buparvaquone reduces the cerebral parasite load– as seen in the non-pregnant mice that were euthanized at 3 weeks pi. However, in the longer term, as seen in the dams that were euthanized 30 days after birth, that is, 44 days pi, no reduction in the cerebral parasite load was evident. This confirms earlier observations that buparvaquone is parasiticidal against N. caninum only after long term treatment . Thus, 6 days of treatment is too short to eradicate the parasite from the dams, but the action of the drug during this short time span is sufficient to consistently impair vertical transmission to the offspring as evidenced by a significantly lower cerebral parasite load and a lower mortality in the offspring. Under the conditions in which we run our in vivo experiments (SPF animals, controlled access, qualified personnel), in uninfected, untreated animals, postnatal mortality does not occur. In the vertical transmission model , postnatal mortality occurs only upon dose dependent infection of the dams. Pups of infected dams die within 3 weeks; pups of not infected dams survive. The model is reproducible as shown in a more recent study .
In conclusion, this study shows that repurposing of established drugs facilitates the identification of compounds that may be applied for treatment of neosporosis, thus avoiding a long and costly drug development process. Other examples of potentially interesting compounds identified earlier through this avenue, and providing interesting results both in vitro and in vivo are miltefosine  and compounds belonging to the class of bumped kinase inhibitors (BKIs) that act against calcium-dependent protein kinase 1, a target that is found in most apicomplexans [32, 39]. BKIs were originally developed to combat malaria . Buparvaquone is already in use against Theileria infections in cattle in endemic countries. Thus, it would be of interest to determine the incidence of neosporosis in buparvaquone treated cattle in those regions as compared to a control population that has not been treated. Moreover, studies in cattle will show whether buparvaquone treatments are able to eradicate N. caninum from the mother cows or whether they reduce the parasite to a low level chronic infection which may reactivate as soon as drug treatment is withdrawn. After establishment of a suitable treatment scheme, the effects on vertical transmission in cattle could be determined.
The authors declare that they have no competing interests.
RKH and NWH synthesized and provided the drugs used in this experiment. JM performed cell culture and in vitro studies. JM, AAM, AH and VM performed the animal experiments. JM, VM and AAM did the euthanasia, isolation of tissues and PCR assays, JM, AH, AAM and RKH wrote the paper. All authors read and approved the final manuscript.
We acknowledge the financial support by the Swiss National Science Foundation (Grant No. 310030 146162). RKH acknowledges funding from the South African Medical Research Council (MRC) with funds from National Treasury under its Economic Competitiveness and Support Package. Cipla Mumbai Pty Ltd. is thanked for the supply of purified artemisone, and we also thank Prof. Luis Ortega-Mora, Universidad Complutense Madrid, for providing the N. caninum Spain-7 isolate.
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