Oral treatments of Echinococcus multilocularis-infected mice with the antimalarial drug mefloquine that potentially interacts with parasite ferritin and cystatin

doi:10.1016/j.ijantimicag.2015.07.016

International Journal of Antimicrobial Agents

Volume 46, Issue 5, November 2015, Pages 546-551

https://doi.org/10.1016/j.ijantimicag.2015.07.016 Get rights and content

Highlights

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Oral application of mefloquine (MEF) in mice acts against Echinococcus multilocularis similar to standard albendazole treatment.
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By affinity chromatography, MEF binds to Echinococcus ferritin and cystatin.
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Epoxy-Sepharose-immobilised MEF binds to human nicotinamide phosphoribosyltransferase.

Abstract

This study investigated the effects of oral treatments of Echinococcus multilocularis-infected mice with the antimalarial drug mefloquine (MEF) and identified proteins that bind to MEF in parasite extracts and human cells by affinity chromatography. In a pilot experiment, MEF treatment was applied 5 days per week and was intensified by increasing the dosage stepwise from 12.5 mg/kg to 200 mg/kg during 4 weeks followed by treatments of 100 mg/kg during the last 7 weeks. This resulted in a highly significant reduction of parasite weight in MEF-treated mice compared with mock-treated mice, but the reduction was significantly less efficacious compared with the standard treatment regimen of albendazole (ABZ). In a second experiment, MEF was applied orally in three different treatment groups at dosages of 25, 50 or 100 mg/kg, but only twice a week, for a period of 12 weeks. Treatment at 100 mg/kg had a profound impact on the parasite, similar to ABZ treatment at 200 mg/kg/day (5 days/week for 12 weeks). No adverse side effects were noted. To identify proteins in E. multilocularis metacestodes that physically interact with MEF, affinity chromatography of metacestode extracts was performed on MEF coupled to epoxy-activated Sepharose^®, followed by SDS-PAGE and in-gel digestion LC–MS/MS. This resulted in the identification of E. multilocularis ferritin and cystatin as MEF-binding proteins. In contrast, when human cells were exposed to MEF affinity chromatography, nicotinamide phosphoribosyltransferase was identified as a MEF-binding protein. This indicates that MEF could potentially interact with different proteins in parasites and human cells.

Graphical abstract

Introduction

The parasite Echinococcus multilocularis is an endoparasitic flatworm of the family Taeniidae. The life cycle of E. multilocularis is based on a predator–prey relationship. The definitive hosts are wild carnivores such as the red fox (Vulpes vulpes) and the arctic fox (Alopex lagopus), but the tapeworm also infects, and develops within the intestine of, domestic dogs and cats, increasing the infection pressure for humans [1], [2]. The definitive host sheds eggs that contain a first larval stage, the oncosphere. When taken up orally, oncospheres hatch as they reach the intestine, penetrate the intestinal wall and use the blood and lymphatic system for dissemination. They typically invade the liver, where they develop into the metacestode stage, which causes human alveolar echinococcosis (AE). AE is distributed in the Northern hemisphere, with endemic areas stretching from Northern America through Central and Eastern Europe to Central and East Asia including Northern parts of Japan [1]. The increase in the urban fox populations in Central Europe, together with the high prevalence rate of E. multilocularis in foxes, has resulted in increased environmental contamination with Echinococcus eggs and, as a consequence, has led to an increased risk of transmission to humans [2].

Human AE manifests itself by tumour-like infiltrative growth of metacestodes mainly in the liver, but other organs may also be affected. AE is often compared with a slow-growing liver cancer and, if untreated, the disease is usually lethal. The current strategy for treatment consists of surgical measures complemented by chemotherapy with mebendazole or albendazole (ABZ). In inoperable cases, chemotherapy has been shown to inhibit parasite proliferation with a parasitostatic effect, but benzimidazoles are rarely curative, resulting in a life-long duration of treatment, high costs and an elevated risk of side effects [3]. However, benzimidazoles have prolonged the average life expectancy of European patients at diagnosis from 3 years to 20 years [3]. Nevertheless, alternative options for chemotherapy with parasiticidal activity are needed [4].

Mefloquine (MEF) is a synthetic analogue of quinine commonly used in the treatment and prophylaxis of chloroquine-resistant Plasmodium falciparum malaria [5], [6]. The mechanism of action of MEF against Plasmodium spp. has not been completely elucidated, but several investigations indicated a disturbance of haemoglobin metabolism. Degradation of haemoglobin is usually followed by the formation of an insoluble polymer, hemozoin. However, MEF binds to haem, inhibits hemozoin formation and the oxidative and glutathione-dependent degradation of haem. The resulting MEF–haem complexes are toxic for the parasite, just like free haem is, causing parasite death [5]. It is not known whether a haem-related mode of action is relevant for the anti-echinococcal activity of MEF in vitro.

MEF also exhibits considerable efficacy against other helminths such as Schistosoma mansoni, Schistosoma japonicum, Opisthorchis viverrini, Brugia patei and Brugia malayi [6], [7], [8]. We have previously demonstrated the efficacy of MEF against AE in experimentally infected mice when the drug was applied intraperitoneally but not when applied orally [9]. In this study, we present two distinct oral MEF treatment protocols in E. multilocularis-infected mice and show that MEF treatment, when applied orally as a suspension in honey at a dosage of 100 mg/kg twice a week, exhibits anti-echinococcal activity comparable with ABZ applied orally at 200 mg/kg/day. We also show that (i) the iron-binding protein ferritin and (ii) cystatin, a potential immunomodulator in parasite infections, bind to MEF and thus possibly could be targeted by MEF in E. multilocularis metacestodes. In contrast, MEF affinity chromatography of the human cancer cell line Caco2 results in binding of the enzyme nicotinamide phosphoribosyltransferase (NAPRT).

Section snippets

Biochemicals and compounds

Unless stated otherwise, all culture media and reagents were purchased from Gibco BRL (Zürich, Switzerland) and biochemical reagents were from Sigma (St Louis, MO). MEF was kindly supplied by Mepha Pharma AG (Aesch, Switzerland).

In vitro culture

Culture of E. multilocularis isolate H95 was carried out as previously described [10]. The human colon carcinoma cell line Caco2 was maintained as previously described by Müller et al. [11].

Experimental infection of mice and treatments with mefloquine or albendazole

Female BALB/c mice (age 9 weeks; mean body weight 25 g) were housed in a

Oral application of mefloquine acts against Echinococcus multilocularis metacestodes in experimentally infected mice

Initial experiments carried out previously had shown that MEF treatment of E. multilocularis-infected mice at 25 mg/kg twice a week by the oral route did not lead to a reduction in parasite weight, whilst intraperitoneal application had a profound effect [9].

In a pilot experiment, an intensified MEF treatment protocol characterised by a stepwise increase of drug concentration (see Supplementary Fig. S1) was applied. During an initial period of 9 days, 12.5 mg/kg MEF was applied five times,

Discussion

As the pilot study showed, a stepwise increase of oral MEF input was significantly effective but had detrimental effects, resulting in a lower efficacy of MEF compared with ABZ as well as obvious adverse side effects once concentrations >100 mg/kg were applied.

The second study demonstrated that oral application of MEF at 100 mg/kg twice a week for 12 weeks in experimentally infected mice acts against E. multilocularis metacestodes in a similar manner as oral application of ABZ (200 mg/kg on 5 days

Acknowledgments

The authors thank Myriam Siffert (University of Bern, Bern, Switzerland) for responsibly taking care of the animals, and Sophie Lagache (Core Facility of Proteomics and Mass Spectrometry, University of Bern) for careful analysis of protein samples.

Funding: This study was supported by the Swiss National Science Foundation [grant no. 31003A_160108/1], the Fondation Sana and the Bangerter-Rhyner Foundation.

Competing interests: None declared.

Ethical approval: Animal studies were performed in strict

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2023, International Journal for Parasitology: Drugs and Drug Resistance
Alveolar echinococcosis (AE) is caused by infection with the fox tapeworm E. multilocularis. The disease affects humans, dogs, captive monkeys, and other mammals, and it is caused by the metacestode stage of the parasite growing invasively in the liver. The current drug treatment is based on non-parasiticidal benzimidazoles. Thus, they are only limitedly curative and can cause severe side effects. Therefore, novel and improved treatment options for AE are needed.
Mefloquine (MEF), an antimalarial agent, was previously shown to be effective against E. multilocularis in vitro and in experimentally infected mice. However, MEF is not parasiticidal and needs improvement for successful treatment of patients, and it can induce strong neuropsychiatric side-effects. In this study, the structure-activity relationship and mode of action of MEF was investigated by comparative analysis of 14 MEF derivatives. None of them showed higher activity against E. multilocularis metacestodes compared to MEF, but four compounds caused limited damage. In order to identify molecular targets of MEF and effective derivatives, differential affinity chromatography combined with mass spectrometry was performed with two effective compounds (MEF, MEF-3) and two ineffective compounds (MEF-13, MEF-22). 1′681 proteins were identified that bound specifically to MEF or derivatives. 216 proteins were identified as binding only to MEF and MEF-3. GO term enrichment analysis of these proteins and functional grouping of the 25 most abundant MEF and MEF-3 specific binding proteins revealed the key processes energy metabolism and cellular transport and structure, as well as stress responses and nucleic acid binding to be involved. The previously described ferritin was confirmed as an exclusively MEF-binding protein that could be relevant for its efficacy against E. multilocularis. The here identified potential targets of MEF will be further investigated in the future for a clear understanding of the pleiotropic effects of MEF, and improved therapeutic options against AE.
Status and prospect of novel treatment options toward alveolar and cystic echinococcosis
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Cystic echinococcosis (CE) and alveolar echinococcosis (AE) are the two most important global parasitic infectious diseases caused by species of Echinococcus granulosus and E. multilocularis, respectively. Although numerous trials have been performed in search of novel therapeutic options to curb the neglected zoonosis, no other nonsurgical options are currently available to replace the licensed anti echinococcal drugs albendazole (ABZ) and mebendazole (MBZ). A safer and more effective treatment plan for echinococcosis is therefore urgently needed to compensate for this therapeutic shortfall. Here, we present a review of the literature for state-of-the-art valuable anti-parasitic compounds and novel strategies that have proved effective against CE and AE, which includes details about the pharmaceutical type, practical approach, experimental plan, model application and protoscolecidal effects in vivo and in vitro. The content includes the current application of traditional clinical chemicals, the preparation of new compounds with various drug loadings, repurposing findings, combined programs, the prospects for Chinese herbal medicines, non-drug administrations and the exploration of target inhibitors based on open-source information for parasitic genes. Next the conventional experimental projects and pharmacodynamic evaluation methods are systematically summarized and evaluated. The demands to optimize the construction of the echinococcosis model and improve the dynamic monitoring method in vivo are also discussed given the shortcomings of in vivo models and monitoring methods.
Laparoscopic or open treatment for hepatic alveolar echinococcosis: A single-institution experience
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Citation Excerpt :
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Between January 2018 and December 2019, 213 hepatic AE patients were admitted to the authors’ institution. Among them, 165 patients (77.46%, 165/213) underwent surgery. Of 13 patients who underwent laparoscopic treatment, two required conversion to open surgery. The remaining 11 patients (group 1) were analyzed. During the same period, 154 patients underwent open surgery, but only 14 records were compatible with the criteria for the laparoscopic approach and were reviewed retrospectively (group 2).
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Laparoscopic surgery provides a safe and efficacious approach for hepatic AE in selected patients. Large, prospective, randomized trials are needed to confirm its superiority.
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Conventional anthelmintics such as albendazole could not achieve complete cure of trichinellosis till now. The antimalarial mefloquine mediates oxidative stress and disrupts lysosomal functions leading to cell death. Therefore, the aim of this work was to investigate the effect of mefloquine on experimental acute and chronic trichinellosis and to clarify the possible mechanisms of such effects. Mice were divided into four groups; Group I: Uninfected untreated control (20 mice); Group II: Infected untreated control (40 mice); Group III: infected and treated with albendazole (400 mg/kg) (40 mice); Group IV: infected and treated with mefloquine (300 mg/kg) (40 mice). All infected treated groups were equally subdivided into 2 subgroups; (a) treated on the 2^nd day post infection (dpi) for 3 days, (b) treated on the 35^th dpi for 5 days. Parasitological adults and larvae counting besides immunohistopathological examination of intestines and muscles were done. Biochemical assay of oxidant/antioxidant status, apoptotic, cytoprotective and inflammatory biomarkers in intestinal and muscle homogenates were achieved. Results showed that both albendazole and mefloquine significantly reduced adults and larvae counts with higher efficacy of albendazole in the intestinal phase and superiority of mefloquine in the muscle phase. The superiority of mefloquine was indicated by increased inflammatory immune infiltration and decreased anti-apoptotic immunohistochemical markers expression in both jejunal and muscle tissues. Biochemically, mefloquine treatment showed highly significant oxidative, apoptotic and inflammatory effects. So, our results suggest that mefloquine might be a superior treatment for chronic trichinellosis.
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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.

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¹: These two authors contributed equally to this work.

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Oral treatments of Echinococcus multilocularis-infected mice with the antimalarial drug mefloquine that potentially interacts with parasite ferritin and cystatin

Highlights

Abstract

Graphical abstract

Introduction

Section snippets

Biochemicals and compounds

In vitro culture

Experimental infection of mice and treatments with mefloquine or albendazole

Oral application of mefloquine acts against Echinococcus multilocularis metacestodes in experimentally infected mice

Discussion

Acknowledgments

Trends Parasitol

J Biochem Biophys Methods

Biochem Biophys Res Commun

Travel Med Infect Dis

Biochim Biophys Acta

Mol Biochem Parasitol

Trends Cell Biol

Parasitol Today

Biological, epidemiological, and clinical aspects of echinococcosis, a zoonosis of increasing concern

Clin Microbiol Rev

The global burden of alveolar echinococcosis

PLoS Negl Trop Dis