Chemotherapy for the treatment of alveolar echinococcosis: Where are we?

Alveolar echinococcosis (AE) is a severe liver disease due to infection with the Echinococcus multilocularis larval stage, called the metacestode. Management of AE is based on benzimidazole chemotherapy (albendazole or mebendazole), associated with surgery when possible. Benzimidazoles are the only compounds recommended for the treatment of AE; however, these are parasitostatic, which means that the parasite can resume growth when treatment is interrupted. Also, benzimidazoles can cause liver dysfunction which may prevent their use. Numerous drugs have been reported to have in vitro activity against E. multilocularis, but few had satisfactory in vivo activity, and none were clearly more effective than benzimidazoles. These drugs belong to various therapeutic categories including anti-infective agents (e.g. amphotericin B, mefloquine, pentamidine derivatives), anti-neoplastic compounds (e.g. imatinib, nilotinib, bortezomib), plant-extracted compounds (e.g. thymol, crocin, carvacrol) and others (e.g. metformin, verapamil, thiaclopride). These treatments are generally of limited interest due to their toxicity, their unfavorable pharmacokinetics, or the scarcity of studies involving humans. Apart from benzimidazoles, only amphotericin B, mefloquine and nitazoxanide have been reported to be used for human AE treatment, with unsatisfactory results. Few studies have aimed at developing innovative strategies for AE drug therapy, such as vectorization of drugs using nanoparticles. Altogether, this review emphasizes the urgent need for new therapeutic strategies in AE management, for which there is currently no curative chemotherapy.

Title: Chimiothérapie de l'échinococcose alvéolaire : où en sommes-nous ? Abstract: L'échinococcose alvéolaire (EA) est une maladie sévère du foie due à l'infection par la forme larvaire d'Echinococcus multilocularis, appelée métacestode. La prise en charge de l'EA repose sur la chimiothérapie par benzimidazolés (albendazole ou mébendazole), si possible associée à la chirurgie. Les benzimidazolés sont les seules molécules recommandées dans le traitement de l'EA, toutefois, ceux-ci sont parasitostatiques, ce qui signifie que le parasite peut reprendre sa croissance lors d'une interruption du traitement. Également, les benzimidazolés peuvent causer une dysfonction hépatique qui peut empêcher leur utilisation. De nombreux médicaments ont été rapportés comme ayant une activité in vitro contre E. multilocularis, mais peu d'entre eux avaient une activité in vivo satisfaisante et aucun n'était clairement plus efficace que les benzimidazolés. Ces médicaments appartiennent à diverses catégories, notamment les agents anti-infectieux (par exemple l'amphotéricine B, la méfloquine, des dérivés de la pentamidine), les composés antinéoplasiques (par exemple l'imatinib, le nilotinib, le bortézomib), les composés extraits de plantes (par exemple le thymol, la crocine, le carvacrol) et d'autres (par exemple metformine, vérapamil, thiaclopride). Ces traitements présentent généralement un intérêt limité en raison de leur toxicité, de leur pharmacocinétique défavorable ou de la rareté des études menées chez l'homme. Outre les benzimidazolés, seules l'amphotéricine B, la méfloquine et la nitazoxanide ont été utilisées dans le traitement de l'EA humaine, avec des résultats insatisfaisants. Peu d'études se sont intéressées à développer des stratégies médicamenteuses innovantes contre l'EA, comme la vectorisation de médicaments à l'aide de nanoparticules. Cette revue souligne le besoin urgent de nouvelles stratégies thérapeutiques dans la prise en charge de l'EA, pour lesquelles il n'existe pas de chimiothérapie curative.

Enhancing the therapeutic potential of P29 protein-targeted monoclonal antibodies in the management of alveolar echinococcosis through CDC-mediated mechanisms.

Alveolar echinococcosis (AE) is a highly lethal helminth infection. Current chemotherapeutic strategies for AE primarily involve the use of benzimidazoles (BZs) such as mebendazole (MDZ) and albendazole (ABZ), which exhibit limited efficacy. In a previous study, the vaccine of recombinant Echinococcus granulosus P29 (rEgP29) showed significant immunoprotection against E. granulosus in both mice and sheep. In the current study, we utilized hybridoma technology to generate five monoclonal antibodies (mAbs) against P29, among which 4G10F4 mAb exhibited the highest antigen-specific binding capacity. This mAb was selected for further investigation of anti-AE therapy, both in vivo and in vitro. In vitro, 4G10F4 inhibited a noteworthy inhibition of E. multilocularis protoscoleces and primary cells viability through complement-dependent cytotoxicity (CDC) mechanism. In vivo, two experiments were conducted. In the first experiment, mice were intraperitoneally injected with Em protoscoleces, and subsequently treated with 4G10F4 mAb (2.5/5/10 mg/kg) at 12 weeks postinfection once per week for 8 times via tail vein injection. Mice that were treated with 4G10F4 mAb only in dosage of 5mg/kg exhibited a significant lower mean parasite burden (0.89±0.97 g) compared to isotype mAb treated control mice (2.21±1.30 g). In the second experiment, mice were infected through hepatic portal vein and treated with 4G10F4 mAb (5mg/kg) at one week after surgery once per week for 8 times. The numbers of hepatic metacestode lesions of the 4G10F4 treatment group were significantly lower in comparison to the isotype control group. Pathological analysis revealed severe disruption of the inner structure of the metacestode in both experiments, particularly affecting the germinal and laminated layers, resulting in the transformation into infertile vesicles after treatment with 4G10F4. In addition, the safety of 4G10F4 for AE treatment was confirmed through assessment of mouse weight and evaluation of liver and kidney function. This study presents antigen-specific monoclonal antibody immunotherapy as a promising therapeutic approach against E. multilocularis induced AE.

PLGA-PEG-COOH nanoparticles are efficient systems for delivery of mefloquine to Echinococcus multilocularis metacestodes.

Alveolar echinococcosis (AE) is a severe disease caused by the infection with the larval stage of Echinococcus multilocularis, the metacestode. As there is no actual curative drug therapy, recommendations to manage AE patients are based on radical surgery and prophylactic administration of albendazole or mebendazole during 2 years to prevent relapses. There is an urgent need for new therapeutic strategies for the management of AE, as the drugs in use are only parasitostatic, and can induce toxicity. This study aimed at developing a drug delivery system for mefloquine, an antiparasitic compound which is highly active against E. multilocularis in vitro and in experimentally infected mice. We formulated mefloquine-loaded PLGA-PEG-COOH (poly-(lactic-co-glycolic acid)) nanoparticles that exhibit stable physical properties and mefloquine content. These nanoparticles crossed the outer acellular laminated layer of metacestodes in vitro and delivered their content to the inner germinal layer within less than 5 min. The in vitro anti-echinococcal activity of mefloquine was not altered during the formulation process. However, toxicity against hepatocytes was not reduced when compared to free mefloquine. Altogether, this study shows that mefloquine-loaded PLGA-PEG-COOH nanoparticles are promising candidates for drug delivery during AE treatment. However, strategies for direct parasite-specific targeting of these particles should be developed.

In vitro and in vivo efficacy of thiacloprid against Echinococcus multilocularis.

BACKGROUND: Alveolar echinococcosis (AE) is a chronic zoonosis caused by the larval form of Echinococcus multilocularis (E. multilocularis). Current chemotherapy against AE has relied on albendazole and mebendazole, which only exhibit parasitostatic and not parasiticidal efficacy. Therefore, novel compounds for the treatment of this disease are needed. METHODS: Phosphoglucose isomerase (PGI) assays were used for compound screening of seven neonicotinoids. The anti-parasitic effects of thiacloprid were then evaluated on E. multilocularis metacestode vesicles, germinal cells and protoscoleces in vitro. Human foreskin fibroblasts (HFF) and Reuber rat hepatoma (RH) cells were used to assess cytotoxicity. Glucose consumption in E. multilocularis protoscoleces and germinal cells was assessed by measuring uptake of 2-deoxyglucose (2-DG). Molecular docking was used to evaluate the potential binding sites of thiacloprid to acetylcholine receptors. In vivo efficacy of thiacloprid was evaluated in mice by secondary infection with E. multilocularis. In addition, ELISA and flow cytometry were used to evaluate the effects of cytokines and T lymphocyte subsets after thiacloprid treatment. Furthermore, collagen deposition and degradation in the host lesion microenvironment were evaluated. RESULTS: We found that thiacloprid is the most promising compound, with an IC50 of 4.54 ± 1.10 µM and 2.89 ± 0.34 µM, respectively, against in vitro-cultured E. multilocularis metacestodes and germinal cells. Thiacloprid was less toxic for HFF and RH mammalian cell lines than for metacestodes. In addition, thiacloprid inhibited the acetylcholinesterase activity in protoscoleces, metacestodes and germinal cells. Thiacloprid inhibited glucose consumption by protoscoleces and germinal cells. Subsequently, transmission electron microscopy revealed that treatment with thiacloprid damaged the germinal layer. In vivo, metacestode weight was significantly reduced following oral administration of thiacloprid at 15 and 30 mg/kg. The level of CD4+ T lymphocytes in metacestodes and spleen increased after thiacloprid treatment. Anti-echinococcosis-related cytokines (IL-2, IL-4, IL-10) were significantly increased. Furthermore, thiacloprid inhibited the expression of matrix metalloproteinases (MMPs 1, 3, 9, 13) and promoted collagen deposition in the host lesion microenvironment. CONCLUSIONS: The results demonstrated that thiacloprid had parasiticidal activity against E. multilocularis in vitro and in vivo, and could be used as a novel lead compound for the treatment of AE.

In vivo and in vitro efficacy of crocin against Echinococcus multilocularis.

BACKGROUND: Alveolar echinococcosis (AE) is a fatal zoonosis caused by the larvae of Echinococcus multilocularis. However, current chemotherapy treatment options are based on benzimidazoles [albendazole (ABZ) and mebendazole], which have limited efficacy. Therefore, novel drugs are necessary for the treatment of this disease. METHODS: The anthelmintic effects of crocin were tested on E. multilocularis metacestodes, germinal cells and protoscoleces in vitro. Human foreskin fibroblasts (HFFs) and Reuber rat hepatoma (RH) cells were used to assess cytotoxicity. The in vivo efficacy of crocin was investigated in mice following secondary infection with E. multilocularis. Furthermore, collagen deposition and degradation in host tissues around the metacestodes were evaluated. RESULTS: In vitro, crocin had a median effective concentration of 11.36 µM against cultured E. multilocularis metacestodes, while it reduced germinal cell viability at a median inhibitory concentration of 10.05 µM. Crocin was less toxic to HFFs and RH mammalian cell lines than to metacestodes. Transmission electron microscopy revealed that crocin treatment resulted in structural damage in the germinal layer. In addition, 60.33 ± 3.06% of protoscoleces were killed by treatment with 10 µM crocin for 7 days, indicating that crocin has a parasiticidal effect. In vivo, the metacestode weight was significantly reduced after the administration of crocin at 50 mg/kg and 100 mg/kg (55.1 and 68.1%, respectively). Metacestode pathology showed structural disruption of the germinal and laminated layers after crocin treatment. The crocin- and ABZ-treated groups presented significant increases in the levels of interleukin (IL)-2 and IL-4. Furthermore, crocin inhibited the expression of matrix metalloproteinases (MMPs) (MMP2 and MMP9) and promoted collagen deposition in the metacestode. CONCLUSIONS: Crocin was demonstrated to exert parasiticidal activity against E. multilocularis in vitro and in vivo, and can be developed as a novel drug for the treatment of AE.

Impact of Albendazole on Cytokine and Chemokine Response Profiles in Echinococcus multilocularis-Inoculated Mice.

OBJECTIVE: Alveolar echinococcosis (AE) is a zoonosis caused by the larval stage of the metacestode Echinococcosis multilocularis with a tumor-like behavior in the targeted organ, especially in the liver. Surgery with albendazole is first-line modality for AE. Drug discontinuation is usually based upon the parasitic viability shown by the positron emission tomography (PET) scan. However, as a demanding and expensive method, it is not widely practiced in majority of the endemic regions. Further understanding on the cytokine and chemokine response profiles in AE patients may provide an interesting insight for potential markers in viability assessment. METHODS: Mice were inoculated with Echinococcus multilocularis intrahepatically to develop the hepatic AE murine model. Oral albendazole administration was then applied for three months after the first inoculation, and peripheral and regional immune cells including type 1 T helper cells (Th), Th2, Th17, regulatory T (Treg) cells, related cytokines, and chemokines were examined. RESULTS: The hepatic AE lesion was confirmed by ultrasound examination resulting in a successful rate of 70%. Among the 17 cytokines and chemokines detected, plasma levels of IL-23 were significantly higher in E. multilocularis-infected mice when compared to the control group; furthermore, more obvious increasing levels were found after albendazole treatment (p < 0.05). All chemokine levels other than eotaxin and MCP-3 were slightly higher in E. multilocularis-infected mice compared to the control group (p > 0.05). Eotaxin levels were significantly decreased in mice with E. multilocularis infection followed by albendazole treatment (p < 0.05). Both IL-17A and IL-23 expressions in hepatic AE lesions were significantly higher and related with disease activity. CONCLUSION: Albendazole administration influenced the balance of immune response and promotes the secretion of proinflammatory factors which is beneficial to parasite clearance. IL-23 seems to be associated with the successful albendazole treatment in mice with E. multilocularis infection; such a change could be translated into clinical application in the near future.

Expression profiling of Echinococcus multilocularis miRNAs throughout metacestode development in vitro.

The neglected zoonotic disease alveolar echinococcosis (AE) is caused by the metacestode stage of the tapeworm parasite Echinococcus multilocularis. MicroRNAs (miRNAs) are small non-coding RNAs with a major role in regulating gene expression in key biological processes. We analyzed the expression profile of E. multilocularis miRNAs throughout metacestode development in vitro, determined the spatial expression of miR-71 in metacestodes cultured in vitro and predicted miRNA targets. Small cDNA libraries from different samples of E. multilocularis were sequenced. We confirmed the expression of 37 miRNAs in E. multilocularis being some of them absent in the host, such as miR-71. We found a few miRNAs highly expressed in all life cycle stages and conditions analyzed, whereas most miRNAs showed very low expression. The most expressed miRNAs were miR-71, miR-9, let-7, miR-10, miR-4989 and miR-1. The high expression of these miRNAs was conserved in other tapeworms, suggesting essential roles in development, survival, or host-parasite interaction. We found highly regulated miRNAs during the different transitions or cultured conditions analyzed, which might suggest a role in the regulation of developmental timing, host-parasite interaction, and/or in maintaining the unique developmental features of each developmental stage or condition. We determined that miR-71 is expressed in germinative cells and in other cell types of the germinal layer in E. multilocularis metacestodes cultured in vitro. MiRNA target prediction of the most highly expressed miRNAs and in silico functional analysis suggested conserved and essential roles for these miRNAs in parasite biology. We found relevant targets potentially involved in development, cell growth and death, lifespan regulation, transcription, signal transduction and cell motility. The evolutionary conservation and expression analyses of E. multilocularis miRNAs throughout metacestode development along with the in silico functional analyses of their predicted targets might help to identify selective therapeutic targets for treatment and control of AE.

In vivo efficacy of combination therapy with albendazole and atovaquone against primary hydatid cysts in mice.

Alveolar echinococcosis (AE) is caused by the larval stage of Echinococcus multilocularis. Chemotherapy for AE involves albendazole (ABZ), which has shown insufficient efficacy. More effective chemotherapy for AE is needed. Previously, we have demonstrated that atovaquone (ATV), an antimalarial, inhibits mitochondrial complex III of E. multilocularis and restricts the development of larval cysts in in vivo experiments. Therefore, in this study, we evaluated the efficacy of ABZ and ATV combination therapy on E. multilocularis in culture and in vivo experiments. Protoscoleces were treated with 50 µM ABZ and/or ATV in the medium; the duration of parasite elimination was determined under aerobic and anaerobic culture. In the in vivo experiment, the effects of ABZ and ATV combination treatment in BALB/c mice infected orally with eggs from the feces of an adult-stage E. multilocularis-infected dog were compared with those of standard oral ABZ therapy. In the culture assay, the duration of elimination associated with ABZ and ATV combination treatment was shorter than that associated with ATV alone under aerobic conditions. Protoscolex viability progressively reduced owing to the combination treatment under anaerobic conditions; however, either drug used singly did not exhibit antiparasitic effects under hypoxia. Furthermore, compared with ABZ alone, the combination treatment significantly reduced the growth of the primary cyst in the liver of mice infected orally with parasite eggs (P = .011). ATV enhances the effect of ABZ in the treatment of AE in mice.

Anti-echinococcal effect of verapamil involving the regulation of the calcium/calmodulin-dependent protein kinase II response in vitro and in a murine infection model.

BACKGROUND: Echinococcosis, which is caused by the larvae of cestodes of the genus Echinococcus, is a parasitic zoonosis that poses a serious threat to the health of humans and animals globally. Albendazole is the drug of choice for the treatment of echinococcosis, but it is difficult to meet clinical goals with this chemotherapy due to its low cure rate and associated side effects after its long-term use. Hence, novel anti-parasitic targets and effective treatment alternatives are urgently needed. A previous study showed that verapamil (Vepm) can suppress the growth of Echinococcus granulosus larvae; however, the mechanism of this effect remains unclear. The aim of the present study was to gain insight into the anti-echinococcal effect of Vepm on Echinococcus with a particular focus on the regulatory effect of Vepm on calcium/calmodulin-dependent protein kinase II (Ca2+/CaM-CaMKII) in infected mice. METHODS: The anti-echinococcal effects of Vepm on Echinococcus granulosus protoscoleces (PSC) in vitro and Echinococcus multilocularis metacestodes in infected mice were assessed. The morphological alterations in Echinococcus spp. induced by Vepm were observed by scanning electron microscopy (SEM), and the changes in calcium content in both the parasite and mouse serum and liver were measured by SEM-energy dispersive spectrometry, inductively coupled plasma mass spectrometry and alizarin red staining. Additionally, the changes in the protein and mRNA levels of CaM and CaMKII in infected mice, and in the mRNA levels of CaMKII in E. granulosus PSC, were evaluated after treatment with Vepm by immunohistochemistry and/or real-time quantitative polymerase chain reaction. RESULTS: In vitro, E. granulosus PSC could be killed by Vepm at a concentration of 0.5 µg/ml or higher within 8 days. Under these conditions, the ultrastructure of PSC was damaged, and this damage was accompanied by obvious calcium loss and downregulation of CaMKII mRNA expression. In vivo, the weight and the calcium content of E. multilocularis metacestodes from mice were reduced after treatment with 40 mg/kg Vepm, and an elevation of the calcium content in the sera and livers of infected mice was observed. In addition, downregulation of CaM and CaMKII protein and mRNA expression in the livers of mice infected with E. multilocularis metacestodes was found after treatment with Vepm. CONCLUSIONS: Vepm exerted a parasiticidal effect against Echinococcus both in vitro and in vivo through downregulating the expression of Ca2+/CaM-CaMKII, which was over-activated by parasitic infection. The results suggest that Ca2+/CaM-CaMKII may be a novel drug target, and that Vepm is a potential anti-echinococcal drug for the future control of echinococcosis.

Serotonin stimulates Echinococcus multilocularis larval development.

BACKGROUND: Serotonin is a phylogenetically ancient molecule that is widely distributed in most metazoans, including flatworms. In addition to its role as a neurotransmitter, serotonin acts as a morphogen and regulates developmental processes. Although several studies have focused on the serotonergic nervous system in parasitic flatworms, little is known on the role of serotonin in flatworm development. METHODS: To study the effects of serotonin on proliferation and development of the cestode Echinococcus multilocularis, we cloned the genes encoding the E. multilocularis serotonin transporter (SERT) and tryptophan hydroxylase (TPH), analyzed gene expression by transcriptome analysis and whole mount in situ hybridization (WMISH) and performed cell culture experiments. RESULTS: We first characterized orthologues encoding the SERT and TPH, the rate-limiting enzyme in serotonin biosynthesis. WMISH and transcriptomic analyses indicated that the genes for both SERT and TPH are expressed in the parasite nervous system. Long-term treatment of parasite stem cell cultures with serotonin stimulated development towards the parasite metacestode stage. Mature metacestode vesicles treated with serotonin showed increased rates of incorporation of the thymidine analogue 5-ethynyl-2'-deoxyuridine (EdU), indicating stimulated cell proliferation. In contrast, treatment with the selective serotonin reuptake inhibitor paroxetine strongly affected the viability of parasite cells. Paroxetine also caused structural damage in metacestode vesicles, suggesting that serotonin transport is crucial for the integrity of parasite vesicles. CONCLUSIONS: Our results indicate that serotonin plays an important role in E. multilocularis development and proliferation, providing evidence that the E. multilocularis SERT and TPH are expressed in the nervous system of the protoscolex. Our results further suggest that the E. multilocularis SERT has a secondary role outside the nervous system that is essential for parasite integrity and survival. Since serotonin stimulated E. multilocularis metacestode development and proliferation, serotonin might also contribute to the formation and growth of the parasite in the liver.

Digest the Sugar, Kill the Parasite: A New Experimental Concept in Treating Alveolar Echinococcosis.

INTRODUCTION: The E. multilocularis laminated layer (LL) is a heavily glycosylated parasitic structure that plays an important role in protecting the larval stage (metacestode) of this parasite from physiological and immunological host reactions. We elaborated an experimental design with the idea to modify the (glycan) surface of the LL by a targeted digestion. This should allow the host defense to more easily recognize and attack (or kill) the parasite by immune-mediated effects. METHODS: Experimentally, E. multilocularis (clone H95) metacestodes were cultured in vitro with or without addition of α1-3,4,6-galactosidase or ß1-3-galactosidase in the medium. Morphological changes were subsequently measured by microscopy at different time points. Parasites were then recovered at day 5 and reinjected into mice for assessing their viability and infectious status. For finally recovered parasites, the respective load was assessed ex vivo by wet weight measurement, and host-related PD1 and IL-10 levels were determined as the key immunoregulators by using flow cytometry. RESULTS: Our experiments demonstrated that the parasite vesicular structure can be directly destroyed by adding galactosidases into the in vitro culture system, resulting in the fact that the parasite metacestode vesicles could not anymore infect and develop in mice after this glycan digestion. Moreover, when compared to the mice inoculated with E. multilocularis metacestode without galactosidases, PD1 expression was upregulated in CD4+ Teffs from mice inoculated with E. multilocularis metacestode pretreated with ß1-3-galactosidase, with a lower IL-10 secretion from CD4+ Teffs; there was no difference of PD1 and IL-10 expression levels regarding CD4+ Teff from mice inoculated with E. multilocularis metacestode pretreated with α1-3,4,6-galac-tosidase. DISCUSSION: We raised our hypothesis that this "aborting" effect may be linked to an altered PD1 and IL-10 response fine-tuning between immunopathology and immune protection. These findings justify a continuation of these experiments upon therapeutical in vivo administration of the enzymes.

Application of target repositioning and in silico screening to exploit fatty acid binding proteins (FABPs) from Echinococcus multilocularis as possible drug targets.

Fatty acid binding proteins (FABPs) are small intracellular proteins that reversibly bind fatty acids and other hydrophobic ligands. In cestodes, due to their inability to synthesise fatty acids and cholesterol de novo, FABPs, together with other lipid binding proteins, have been proposed as essential, involved in the trafficking and delivery of such lipophilic metabolites. Pharmacological agents that modify specific parasite FABP function may provide control of lipid signalling pathways, inflammatory responses and metabolic regulation that could be of crucial importance for the parasite development and survival. Echinococcus multilocularis and Echinococcus granulosus are, respectively, the causative agents of alveolar and cystic echinococcosis (or hydatidosis). These diseases are included in the World Health Organization's list of priority neglected tropical diseases. Here, we explore the potential of FABPs from cestodes as drug targets. To this end, we have applied a target repurposing approach to identify novel inhibitors of Echinococcus spp. FABPs. An ensemble of computational models was developed and applied in a virtual screening campaign of DrugBank library. 21 hits belonging to the applicability domain of the ensemble models were identified, and 3 of the hits were assayed against purified E. multilocularis FABP, experimentally confirming the model's predictions. Noteworthy, this is to our best knowledge the first report on isolation and purification of such four FABP, for which initial structural and functional characterization is reported here.

Short communication: Efficacy of albendazole in Echinococcus multilocularis-infected mice depends on the functional immunity of the host.

Alveolar echinococcosis (AE) is a deadly parasitic disease that requires lifelong treatment with albendazole. Development of host immunity is pivotal with regard to the clinical outcome of AE, but its influence on conventional albendazole treatment is unknown. Using T-cell deficient athymic nude mice, we demonstrated that functional immunity is required for albendazole to be efficacious against murine AE. These results call for attention given the increasing number of immunocompromised patients with AE.

Drug repurposing applied: Activity of the anti-malarial mefloquine against Echinococcus multilocularis.

The current chemotherapeutical treatment against alveolar echinococcosis relies exclusively on benzimidazoles, which are not parasiticidal and can induce severe toxicity. There are no alternative treatment options. To identify novel drugs with activity against Echinococcus multilocularis metacestodes, researchers have studied potentially interesting drug targets (e.g. the parasite's energy metabolism), and/or adopted drug repurposing approaches by undertaking whole organism screenings. We here focus on drug screening approaches, which utilize an in vitro screening cascade that includes assessment of the drug-induced physical damage of metacestodes, the impact on metacestode viability and the viability of isolated parasite stem cells, structure-activity relationship (SAR) analysis of compound derivatives, and the mode of action. Finally, once in vitro data are indicative for a therapeutic window, the efficacy of selected compounds is assessed in experimentally infected mice. Using this screening cascade, we found that the anti-malarial mefloquine was active against E. multilocularis metacestodes in vitro and in vivo. To shed more light into the mode of action of mefloquine, SAR analysis on mefloquine analogues was performed. E. multilocularis ferritin was identified as a mefloquine-binding protein, but its precise role as a drug target remains to be elucidated. In mice that were infected either intraperitoneally with metacestodes or orally with eggs, oral treatment with mefloquine led to a significant reduction of parasite growth compared to the standard treatment with albendazole. However, mefloquine was not acting parasiticidally. Assessment of mefloquine plasma concentrations in treated mice showed that levels were reached which are close to serum concentrations that are achieved in humans during long-term malaria prophylaxis. Mefloquine might be applied in human AE patients as a salvage treatment. Future studies should focus on other repurposed anti-infective compounds (MMV665807, niclosamide, atovaquone), which showed stronger in vitro activity against E. multilocularis than mefloquine.

Albendazole solid dispersions against alveolar echinococcosis: a pharmacotechnical strategy to improve the efficacy of the drug.

Alveolar echinococcosis is a neglected parasitic zoonosis caused by Echinococcus multilocularis. The pharmacological treatment is based on albendazole (ABZ). However, the low water solubility of the drug produces a limited dissolution rate, with the consequent failure in the treatment of the disease. Solid dispersions are a successful pharmacotechnical strategy to improve the dissolution profile of poorly water-soluble drugs. The aim of this work was to determine the in vivo efficacy of ABZ solid dispersions using poloxamer 407 as a carrier (ABZ:P407 solid dispersions (SDs)) in the murine intraperitoneal infection model for secondary alveolar echinococcosis. In the chemoprophylactic efficacy study, the ABZ suspension, the ABZ:P407 SDs and the physical mixture of ABZ and poloxamer 407 showed a tendency to decrease the development of murine cysts, causing damage to the germinal layer. In the clinical efficacy study, the ABZ:P407 SDs produced a significant decrease in the weight of murine cysts. In addition, the SDs produced extensive damage to the germinal layer. The increase in the efficacy of ABZ could be due to the improvement of water solubility and wettability of the drug due to the surfactant nature of poloxamer 407. In conclusion, this study is the basis for further research. This pharmacotechnical strategy might in the future offer novel treatment alternatives for human alveolar echinococcosis.

The importance of uninterrupted albendazole treatment in patients with unresectable alveolar echinococcosis undergoing liver transplantation.

BACKGROUND: This study focused on the monitoring of patients who had undergone liver transplantation (LT) because of unresectable alveolar echinococcosis (AE). The role of long-term administration of albendazole (ABZ) in patients with residual/recurrent AE lesions and without AE lesions was evaluated. METHODS: Albendazole therapy was prescribed to patients diagnosed with AE 4-6 weeks after LT on the background of continuous suppressive therapy while following the protocol for managing patients after LT. Clinical data (general condition, blood counts, and level of hepatic transaminases), ultrasound scans (USs), magnetic resonance imaging (MRI) or multispiral computed tomography (MSCT), and serological data were collected from four patients with residual/recurrent AE lesions and without AE lesions. The results of the USs, MRI, and MSCT examinations at diagnosis and at the end of follow-up were retrospectively reviewed for all patients. RESULTS: Observation of patients over a long period (up to 10 years) showed that the continuous (without interruption) use of ABZ restrained the development of metacestodes. Interruptions in taking the drug, associated with the manifestation of hepatotoxicity in some patients, led to the development of lesions in other organs in which the parasite had not previously been detected. No new foci were found in the transplanted livers of the patients. CONCLUSION: Liver transplantation, together with continuous anti-relapse chemotherapy, prolongs the patient's life, both in the absence and in the presence of metastases in other organs.

In vitro effects of lonidamine and 6-aminonicotinamide against Echinococcus granulosussensu stricto and Echinococcus multilocularis.

Echinococcosis is a zoonotic disease caused by cestode species of the genus Echinococcus, which demonstrates considerable medical and veterinary concerns. The development of novel drugs for echinococcosis treatment is urgently needed. In this study, we demonstrated that lonidamine (LND) and 6-aminonicotinamide (6-AN) exhibited considerable in vitro effects against both larval- and adult-stage of E. granulosussensu stricto (s. s.) and E. multilocularis. The combination of LND and 6-AN exhibited a significantly higher activity than the single drug treatment. These results highlight the therapeutic potential of LND, 6-AN and the combination of LND and 6-AN for the treatment of echinococcosis.

Anti-echinococcal activity of menthol and a novel prodrug, menthol-pentanol, against Echinococcus multilocularis.

Alveolar echinococcosis is one of the most dangerous parasitic zoonoses. This disease, widely distributed in the northern hemisphere, is caused by the metacestode stage of the tapeworm Echinococcus multilocularis. All surgical and non-surgical patients should perform chemotherapy with benzimidazoles, mainly with albendazole. However, the efficacy of albendazole is variable due to its deficient pharmacokinetic properties. Therefore, the need to find new therapeutic alternatives for the treatment of alveolar echinococcosis is evident. Menthol is a natural compound of low toxicity, used in industries such as cosmetics and gastronomy and generally recognized as safe by the Food and Drug Administration. In addition, menthol has important pharmacological effects and is effective against a wide variety of organisms. The development of prodrugs allows improving the pharmacokinetic properties of the parental drug. To improve lipophilicity and therefore the bioavailability of menthol, a novel prodrug called menthol-pentanol was developed by masking the functional polar group of menthol by linking n-pentanol by a carbonate bond. The aim of the current work was to evaluate the in vitro and in vivo efficacy of menthol and menthol-pentanol against E. multilocularis. Menthol-pentanol had a greater protoscolicidal effect than menthol. In addition, the prodrug demonstrated a similar clinical efficacy to albendazole. The increase in lipophilicity of the prodrug with respect to menthol was reflected in an increase in its antiparasitic activity against E. multilocularis. Thus, menthol-pentanol appears as a promising candidate for further evaluation as a potential alternative for the treatment of alveolar echinococcosis.

Successful surgical management of hepatic alveolar echinococcosis by inductive therapy with albendazole - a case report.

We report the case of a 65-year-old female patient with hepatic alveolar echinococcosis (AE) caused by Echinococcus multilocularis. This infrequent zoonosis has a considerable morbidity and mortality. The malignant appearing hepatic mass was initially misdiagnosed as cholangiocarcinoma of the right hepatic lobe (segments VII, VIII, and IVa, sized 10.9 cm × 7.6 cm) involving the right and middle hepatic vein and extending close to the left hepatic vein. During exploratory laparotomy, the frozen-section biopsy was indicative of AE (World Health Organization [WHO] classification: stage P3N0M0). Due to the high operative risk, it was decided to pretreat the patient with albendazole as inductive therapy in order to remove the AE secondarily in accordance with the patient's request. After year-long treatment with albendazole (under strict control of the maximum blood levels), a right hemihepatectomy was successfully performed. Postoperative treatment with albendazole had to be stopped prematurely after 11 months due to considerable subjective intolerance and a more-than-tenfold elevation of transaminases despite normal therapeutic albendazole blood levels. A 18F-FDG-PET/CT scan revealed no evidence of AE residues. Conducting follow-up examinations by 18F-FDG-PET/CT scans every 2 years is planned in order to recognize possible recurrence at an early stage.

Mitochondrial complex III in larval stage of Echinococcus multilocularis as a potential chemotherapeutic target and in vivo efficacy of atovaquone against primary hydatid cysts.

Echinococcus multilocularis employs aerobic and anaerobic respiration pathways for its survival in the specialized environment of the host. Under anaerobic conditions, fumarate respiration has been identified as a promising target for drug development against E. multilocularis larvae, although the relevance of oxidative phosphorylation in its survival remains unclear. Here, we focused on the inhibition of mitochondrial cytochrome bc1 complex (complex III) and evaluated aerobic respiratory activity using mitochondrial fractions from E. multilocularis protoscoleces. An enzymatic assay revealed that the mitochondrial fractions possessed NADH-cytochrome c reductase (mitochondrial complexes I and III) and succinate-cytochrome c reductase (mitochondrial complexes II and III) activities in the aerobic pathway. Enzymatic analysis showed that atovaquone, a commercially available anti-malarial drug, inhibited mitochondrial complex III at 1.5 nM (IC50). In addition, culture experiments revealed the ability of atovaquone to kill protoscoleces under aerobic conditions, but not under anaerobic conditions, indicating that protoscoleces altered their respiration system to oxidative phosphorylation or fumarate respiration depending on the oxygen supply. Furthermore, combined administration of atovaquone with atpenin A5, a quinone binding site inhibitor of complex II, completely killed protoscoleces in the culture. Thus, inhibition of both complex II and complex III was essential for strong antiparasitic effect on E. multilocularis. Additionally, we demonstrated that oral administration of atovaquone significantly reduced primary alveolar hydatid cyst development in the mouse liver, compared with the untreated control, indicating that complex III is a promising target for development of anti-echinococcal drug.