Effect of the anti-parasitic compounds pyrvinium pamoate and artemisinin in enzymatic and culture assays: Data on the search for new anti-echinococcal drugs

The dataset presented herein is related to a previous research article titled “Mitochondrial Complex III in Larval Stage of Echinococcus multilocularis as a Potential Chemotherapeutic Target and in vivo Efficacy of Atovaquone Against Primary Hydatid Cysts” [1]. In this report, data were collected by screening drugs for echinococcosis. We investigated the inhibitory activities of artemisinin and pyrvinium pamoate against the mitochondrial respiratory enzymes in E. multilocularis protoscoleces. Artemisinin did not inhibit mitochondrial complexes I, II, and III. However, pyrvinium pamoate inhibited complex I at 11 μM, although complexes II and III were not inhibited. In the culture assay, E. multilocularis protoscoleces were treated with atovaquone (ATV), rotenone, praziquantel, artemisinin, and pyrvinium pamoate at a final concentration of 50 µM in different culture media. The viability of protoscoleces was compared under aerobic and anaerobic conditions via culture experiments. The survival days of E. multilocularis protoscoleces were evaluated in the drug-treated group compared with those in the non-treated group. The results of these culture assays revealed that praziquantel and artemisinin did not eliminate the protoscoleces under both aerobic and anaerobic conditions. However, a stronger elimination ability was observed with the co-administration of praziquantel or artemisinin with ATV than with ATV alone under aerobic conditions. Pyrvinium pamoate completely killed protoscoleces at 5 and 7 days under aerobic and anaerobic conditions, respectively. Pyrvinium pamoate behaved identically to rotenone, the complex I inhibitor, in the culture treatment assay. The data serve as a reference for the development of novel anti-echinococcal drugs.

praziquantel, artemisinin, and pyrvinium pamoate at a final concentration of 50 μM in different culture media. The viability of protoscoleces was compared under aerobic and anaerobic conditions via culture experiments. The survival days of E. multilocularis protoscoleces were evaluated in the drug-treated group compared with those in the non-treated group. The results of these culture assays revealed that praziquantel and artemisinin did not eliminate the protoscoleces under both aerobic and anaerobic conditions. However, a stronger elimination ability was observed with the coadministration of praziquantel or artemisinin with ATV than with ATV alone under aerobic conditions. Pyrvinium pamoate completely killed protoscoleces at 5 and 7 days under aerobic and anaerobic conditions, respectively. Pyrvinium pamoate behaved identically to rotenone, the complex I inhibitor, in the culture treatment assay. The data serve as a reference for the development of novel anti-echinococcal drugs. ©

Value of the Data
• The data provide the inhibitory activity of artemisinin and pyrvinium pamoate against the mitochondrial respiratory chain of E. multilocularis . Artemisinin did not inhibit mitochondrial complexes I, II, and III in E. multilocularis protoscoleces. Pyrvinium pamoate inhibited the mitochondrial complex I activity of E. multilocularis protoscoleces at IC 50 11.3 μM.
• The data describe the efficacy of the combination of atovaquone with praziquantel or artemisinin against E. multilocularis protoscoleces in culture assays. The combined administration shortened the duration of parasite elimination compared with atovaquone alone under the aerobic conditions, but had no effect under anaerobic conditions. • In culture experiments, pyrvinium pamoate, which inhibited mitochondrial complex I, completely eliminated E. multilocularis protoscoleces at 6 and 7 days under aerobic and anaerobic condition, respectively.

Data Description
The data in Table 1 show that pyrvinium pamoate inhibited mitochondrial complex I at IC 50 11.3 μM but did not inhibit complexes II and III (IC 50 > 30 μM). Artemisinin did not block mitochondrial complexes I, II, and III (IC 50 > 40 μM). Fig. 1 shows the anti-parasitic effect of 50 μM atovaquone (ATV), rotenone, praziquantel, artemisinin, pyrvinium pamoate, and combination chemotherapy of ATV and other drugs in a culture experiment using E. multilocularis protoscoleces. Praziquantel and artemisinin did not eliminate E. multilocularis protoscoleces under both aerobic and anaerobic conditions. Interestingly, under aerobic conditions, a stronger elimination ability was observed with the co-administration of praziquantel or artemisinin with  ATV than with ATV alone. Pyrvinium pamoate killed all protoscoleces at 6 days under aerobic conditions and 95 % at 7 days under anaerobic conditions.

Preparation of the mitochondrial fraction from E. multilocularis protoscoleces
All experiments were performed as described previously [1] . The infected cotton rats ( Sigmodon hispidus) were sacrificed by isoflurane overdose to obtain the cyst tissues containing E. multilocularis (Nemuro strain) protoscoleces. The tissues were passed through a metal mesh and shredded completely. Thereafter, the tissues were repeatedly suspended and washed in a tall beaker to isolate protoscoleces using the difference in buoyancy between the protoscoleces and other tissue [1,2] . The parasite materials were homogenized with a motor-driven homogenizer to prepare the mitochondrial fraction of E. multilocularis protoscoleces, while icing them. The homogenate was diluted with the mitochondrial preparation buffer (210 mM mannitol, 10 mM sucrose, 1 mM disodium EDTA, and 50 mM Tris-HCl; pH 7.5) supplemented with 10 mM sodium malonate to five times the volume of the original protoscolex sediment, and then centrifuged at 800 × g for 10 min (4 °C) to remove cell debris and nuclei. The supernatant was then centrifuged at 8,0 0 0 × g for 10 min (4 °C) to obtain the mitochondrial pellet. The pellet was re-suspended in mitochondrial preparation buffer (without malonate) and centrifuged at 8,0 0 0 × g for 10 min (4 °C). The enriched mitochondrial fraction was suspended in mitochondrial preparation buffer without malonate.

Enzyme assays and 50% inhibitory concentration (IC50) determination
All enzyme assays using mitochondrial fractions were performed in 0.5 or 1 mL reaction mixtures at 25 °C using an absorption spectrophotometer. Before the assay, the mitochondrial suspension was thawed at room temperature and refrozen in a deep freezer. The refrozen sample was thawed again after 1 h. This freezing and thawing process was performed before the assay to ensure that the mitochondrial membrane was permeable to the solutes. Without this process, NADH cannot pass through the mitochondrial membrane as a substrate. The reagents used in each assay were mixed with the reaction buffer (30 mM potassium phosphate and 1 mM MgCl 2 , pH 7.5). The final mitochondrial protein concentration was 50 μg/mL. NADH-decyl rhodoquinone reductase activity (complex I), succinate quinone reductase activity (complex II), and succinate-cytochrome c reductase activity (complexes II and III) were measured using a SHI-MADZU spectrophotometer UV-30 0 0 (Kyoto, Japan) as described previously [1,2] . The IC 50 values of pyrvinium pamoate and artemisinin were determined by calculating approximation lines from three or more points of anteroposterior concentration to the IC 50 values.

Culture treatment of E. multilocularis protoscoleces
The metacestodes from one cotton rat at 4 months after infection with 50 eggs were full of mature protoscoleces and were enough to perform the culture experiment. Praziquantel alone cannot inhibit the mitochondrial respiratory system [3] . However, praziquantel can exert its effects in synergy with albendazole, although synergy with ATV has not been demonstrated [4] . The complementary culture experiment based on our previous report was performed to determine the efficacy of the combination therapy of ATV with praziquantel or artemisinin. Additionally, the efficacy of pyrvinium pamoate, which inhibited complex I of E. multilocularis , was ascertained in a culture assay under aerobic and anaerobic conditions. In this experiment, rotenone was used as the control to compare with previous report findings [1,2] .
The obtained protoscoleces were cultured in Connaught Medical Research Laboratories 10 6 6 medium (Gibco, Grand Island, NY, USA) containing 23 mM 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid, 0.5% (w/v) D ( + )-glucose, 0.4 mM sodium taurocholate (Wako Pure Chemical Industries), 0.5% (w/v) yeast extract (Difco Laboratories, Detroit, MI, USA), 57 mM sodium hydrogen carbonate, 2 mM L-glutamine (Gibco), 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco). Half of the medium was replaced on day 3. For anaerobic cultures, six-well plates were sealed in plastic containers with oxygen-detecting agents and oxygen scavengers (Aneromeito®, Nissui Pharmaceutical, Tokyo, Japan) to maintain the oxygen concentration under 0.3% at 37 °C. To assess the effect of chemical compounds against E. multilocularis protoscoleces, the parasites were treated with rotenone, praziquantel, artemisinin, pyrvinium pamoate, and ATV at a final concentration of 50 μM in the culture medium. The control group was supplemented with 0.5% (v/v) dimethyl sulfoxide (DMSO), and all conditions were assayed in triplicate. The viability of protoscoleces was determined by microscopic observation of more than 170 protoscoleces per well using the trypan blue exclusion test. The protoscoleces were observed daily for 7 consequent days, and the efficacy of the treatment was evaluated by the duration of parasite elimination based on previous reports [1,2,5,6] .

Ethics Statement
The procedures in this manuscript are in strict accordance with the National Institutes of Health guide for the Care and Use of Laboratory animals. Furthermore, the protocol for animal experiments was approved by the ethics committee of the Hokkaido Institute of Public Health (permit numbers: K25-2 and K29-4).

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that have, or could be perceived to have, influenced the work reported in this article.