Alveolar echinococcosis (AE) is a helminthic zoonotic disease caused by infection with the larval stage of the cestode parasite Echinococcus multilocularis . The prevalence of AE is highest in China, Central Asia, Russia, parts of Europe, and Japan . Adult-stage E. multilocularis release their eggs into the feces of the definitive host. The accidental ingestion of the eggs by intermediate hosts, such as small rodents, leads to the release of infective larvae (oncospheres) in the intestinal lumen. The oncospheres mainly reach the liver via the circulatory system, in which they grow into metacestodes (larval cysts) containing protoscoleces. After predation of the infected rodent by Canidae such as a fox, the protoscoleces transform and develop into adult tapeworms in the small intestine. Humans are infected as an aberrant intermediate host upon accidentally swallowing the parasite eggs. The growth of larval cysts in the liver leads to life-threatening conditions, such as organ dysfunction, several years after infection.
Radical surgical resection of the parasitic mass is the basis of treatment for AE and is usually accompanied by chemotherapy [1, 2]. In cases with inoperable advanced cysts of AE, the chemotherapeutic treatment involves albendazole (ABZ), which is not adequately efficacious . The mechanism of action of ABZ is the inhibition of tubulin polymerization in the intestinal cells of the parasite . During the clinical course, chemotherapy plays an important role in the process of treatment as an increase in the number of larval cysts in the liver is associated with a poor prognosis. However, the development of novel drugs for AE treatment has not advanced since the discovery of ABZ in the 1970s. Hence, many in vivo experiments have been performed to assess the efficacy of combination therapy with ABZ and other compounds . However, no successful result has been reported. In this study, we focused on the mitochondrial respiratory systems of the parasite as the drug target.
E. multilocularis employs an aerobic respiratory pathway, oxidative phosphorylation, or an anaerobic pathway, fumarate respiration, for its survival in various oxygen conditions [6–8]. The eggs of E. multilocularis excreted from the definitive host are exposed to oxygen during the long-term. The oncosphere and metacestodes (larval cysts) are reside in the artery and liver of the intermediate host, respectively. These stages, which are the target of treatment, can access the portal vein and hepatic vein that exhibit high arterial oxygen tension. However, it difficult for these stages to access oxygen owing to their size and site of infection. Adult worms live in the intestine of the definitive host, under low oxygen conditions. A similar change of the respiratory system is also found in other parasites [9–12].
Briefly, oxidative phosphorylation generally involves complexes I to IV, and V. Complex I oxidizes NADH and passes electrons to ubiquinone. Complex II receives electrons from succinate and functions as succinate-quinone reductase. Ubiquinone transfers the reducing equivalent to complex III, which passes it to complex IV via cytochrome c. Proton-motive force is generated across the inner mitochondrial membrane at complexes I, III, and IV, which drive ATP synthesis by complex V. In contrast, fumarate respiration involves complex I, II, and rhodoquinone. Complex I acts as a proton pump that is driven by the oxidation of NADH and contributes to the reduction of rhodoquinone. Complex II accepts electrons from rhodoquinol and catalyzes the reduction of fumarate to succinate as a terminal oxidase. Quinol-fumarate reductase activity is the reverse reaction of the succinate-quinone reductase activity of complex II under anaerobic conditions. As the advantage of fumarate respiration, ATP can be synthesized by the proton-pump activity of complex I and ATP synthase (complex V) even under low oxygen conditions.
Atovaquone (ATV) has long been used to treat infections by Plasmodium falciparum, Pneumocystis jirovecii, Babesia divergens, and Toxoplasma gondii as a specific inhibitor of the parasite mitochondrial complex III (cytochrome bc1 complex) [13–15]. We have previously reported that ATV inhibits mitochondrial complex III of E. multilocularis at extremely low concentrations and decreases the development of larval cysts in in vivo experiments . However, in the case of E. multilocularis, the effect of ATV alone is limited compared with that of ABZ in treated mice, because ATV inhibits only aerobic respiration . Therefore, in this study, the in vivo effects of combination therapy with ABZ and ATV were compared to those of standard oral ABZ treatments in mice with primary hydatid cysts after being orally infected with eggs of E. multilocularis as a natural infection model.