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In Vitro and In Vivo Efficacies of the EGFR/MEK/ERK Signaling Inhibitors in the Treatment of Alveolar Echinococcosis
research-article
Author(s):
Zhe Cheng
a
,
b ,
Zhijian Xu
a
,
b ,
Huimin Tian
c ,
Fan Liu
c ,
Xiu Li
a
,
b ,
Damin Luo
a
,
b ,
Yanhai Wang
a
,
b
,
Publication date (Electronic):
22 July 2020
Publisher:
American Society for Microbiology
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Abstract
Alveolar echinococcosis (AE), caused by the larval stage of the cestode Echinococcus multilocularis, is a lethal disease in humans. Novel therapeutic options are urgently needed since the current chemotherapy displays limited efficiency in AE treatment. In this study, we assessed the in vitro and in vivo effects of the epidermal growth factor receptor (EGFR)/MEK/extracellular signal-regulated kinase (ERK) signaling inhibitors, including BIBW2992, CI-1033, and U0126, on E. multilocularis.
ABSTRACT
Alveolar echinococcosis (AE), caused by the larval stage of the cestode Echinococcus multilocularis, is a lethal disease in humans. Novel therapeutic options are urgently needed since the current chemotherapy displays limited efficiency in AE treatment. In this study, we assessed the in vitro and in vivo effects of the epidermal growth factor receptor (EGFR)/MEK/extracellular signal-regulated kinase (ERK) signaling inhibitors, including BIBW2992, CI-1033, and U0126, on E. multilocularis. Our data showed that BIBW2992, CI-1033, and U0126 all displayed in vitro effects on the viability of the E. multilocularis metacestode. These inhibitors also showed protoscolicidal activities and caused severe ultrastructural alterations in the parasite. Moreover, BIBW2992 and CI-1033 exhibited potent proapoptotic effects on E. multilocularis metacestodes. Strikingly, a large portion of the apoptotic cells were found to be the germinative cells. In vivo studies showed that BIBW2992 and U0126 significantly reduced parasite burden, and the parasite obtained from BIBW2992-treated mice displayed impaired structural integrity of the germinal layer. In conclusion, these findings demonstrate the potential of EGFR-mediated signaling as a target for the development of novel anti-AE agents. The EGFR inhibitor BIBW2992 represents a promising drug candidate and/or a lead compound for anti-AE chemotherapy.
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Most cited references32
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Echinococcosis: Advances in the 21st Century
Hao Wen, Lucine Vuitton, Tuerhongjiang Tuxun … (2019)
SUMMARY Echinococcosis is a zoonosis caused by cestodes of the genus Echinococcus (family Taeniidae). This serious and near-cosmopolitan disease continues to be a significant public health issue, with western China being the area of highest endemicity for both the cystic (CE) and alveolar (AE) forms of echinococcosis. Considerable advances have been made in the 21st century on the genetics, genomics, and molecular epidemiology of the causative parasites, on diagnostic tools, and on treatment techniques and control strategies, including the development and deployment of vaccines. In terms of surgery, new procedures have superseded traditional techniques, and total cystectomy in CE, ex vivo resection with autotransplantation in AE, and percutaneous and perendoscopic procedures in both diseases have improved treatment efficacy and the quality of life of patients. In this review, we summarize recent progress on the biology, epidemiology, diagnosis, management, control, and prevention of CE and AE. Currently there is no alternative drug to albendazole to treat echinococcosis, and new compounds are required urgently. Recently acquired genomic and proteomic information can provide a platform for improving diagnosis and for finding new drug and vaccine targets, with direct impact in the future on the control of echinococcosis, which continues to be a global challenge.
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Clinical features and treatment of alveolar echinococcosis.
Peter Kern (2010)
Human alveolar echinococcosis is caused by the larval stage of Echinococcus multilocularis, occurring in at least 42 countries of the northern hemisphere. Recent studies in Europe and Asia have shown that the endemic area of E. multilocularis is larger than previously known and the parasite has regionally expanded from rural to urban areas. Diagnosis of alveolar echinococcosis is supported by results from imaging studies, histopathology and/or nucleic acid detection, and serology. The present review summarizes current understanding of clinical features, knowledge on appropriate treatment, and discusses ways to improve standards of care. High prevalences of this deadly disease have been discovered in surveys in parts of China. Clinical manifestations, diagnostic tools and the burden of disease were described, and are based on high case numbers. In Europe, excellent tools have been introduced, which improve disease management. Long-term observations in Switzerland provide an optimistic view, as the infection can be well controlled, if patients are cared for in specialized centres. An expert consensus summarizes the current recommendation for diagnosis and treatment of alveolar echinococcosis by the Informal Working Group on Echinococcosis of the WHO. Diagnosis and treatment of alveolar echinococcosis remains a challenge for clinicians. The updated WHO-recommendations aim to support decisions on diagnosis and treatment of alveolar echinococcosis. Anti-infective therapy is the backbone of treatment; surgery should be restricted to patients at an early stage of the disease. For the majority of cases continuous chemoprophylaxis with benzimidazoles is cost-effective and leads to a good quality of life for patients with this chronic disease.
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Repurposing drugs for the treatment and control of helminth infections
Gordana Panic, Urs Duthaler, Benjamin Speich … (2014)
1 Introduction Helminth infections caused by roundworms (nematodes) and flatworms (platyhelminths) comprise the greatest group of the neglected tropical diseases (NTDs) (Hotez et al., 2006). An estimated 11.5 million disability adjusted life years (DALYs) are attributed to intestinal nematode infections, schistosomiasis, lymphatic filariasis, onchocerciasis, food-borne trematodiasis, cysticercosis and echinococcosis (Murray et al., 2012). Most of the burden of these diseases results from disability (rather than premature death), influencing school attendance, child development and overall economic productivity, thus resulting in disease driven poverty traps (Hotez et al., 2006). In a recent special issue of the Disease Clinics of North America (Zumla and Keiser, 2012), cestode infestations (Brunetti and White, 2012), schistosomiasis (Gryseels, 2012), food-borne trematodiases (Fürst et al., 2012), filariases (Knopp et al., 2012a) and soil-transmitted helminthiases (Knopp et al., 2012b) were presented in great detail and hence for background information on these diseases, the reader is referred to these excellent publications. Preventive chemotherapy is the strategy of choice to control schistosomiasis, soil-transmitted helminthiases, lymphatic filariasis, onchocerciasis and food-borne trematodiases yet as emphasized in the following sections of this review, limited tools are available to treat these infections. In addition, for many of the available drugs, resistance is a threat since it has already developed in veterinary medicine and many of the drugs have an imperfect activity profile. Yet clear targets have been set to eliminate and control several of these diseases (http://www.unitingtocombatntds.org/downloads/press/london_declaration_on_ntds.pdf). Hence research and development (R&D) to find the next generation of anthelmintics is indispensable. However, a recent systematic assessment of databases of drug regulatory authorities and the World Health Organization (WHO) as well as clinical trial registries revealed a dry drug pipeline for NTDs, supported by the fact that no new chemical entity had been approved for these diseases in the past decade (Pedrique et al., 2013). Drug repurposing (also termed re-profiling, re-tasking, therapeutic switching or drug repositioning) is the process of developing new indications for existing, failed or abandoned drugs or advanced clinical candidates (Sekhon, 2013). Drug repurposing is a useful strategy to accelerate the drug development process due to lower costs, reduced risk and decreased time to market due to availability of preclinical data (Padhy and Gupta, 2011). This enables not only pharmaceutical companies but also public-sector researchers to engage in drug discovery and development efforts (O’Connor and Roth, 2005), and hence might result in treatment options for diseases almost exclusively addressed by public sector researchers, such as the neglected tropical diseases. Over the past years, a variety of drug-repurposing initiatives have been launched with particular attention to neglected tropical and rare diseases (Allarakhia, 2013), hence it is likely that these efforts will bear fruit in the next years. The aim of the present article is to highlight the status of drug repurposing for neglected helminth diseases. The focus is on the state-of-the-art treatments and how drug repurposing has been supplying the drug development pipeline for schistosomiasis, infections with major food-borne trematodes, Fasciola spp., Opisthorchis spp. and Clonorchis sinensis, soil-transmitted helminthiasis, Strongyloides stercoralis, the major human filariasis, lymphatic filariasis and onchocerciasis, taeniasis, neurocysticercosis and echinococcosis. Our review complements a recent article by Andrews and colleagues which summarized antiprotozoal drug repurposing for major parasitic protozoal diseases including malaria, trypanosomiasis, and leishmaniasis (Andrews et al., 2014). 2 Schistosomiasis and food-borne trematodiases 2.1 Current treatment Since its discovery in the 1970s, praziquantel has replaced many other drugs as the sole treatment for a range of helmintic infections. Praziquantel is effective against all three major species of Schistosoma (S. mansoni, S. haematobium and S. japonicum), and is the standard treatment against C. sinensis, Opisthorchis viverrini and Opisthorchis felineus, and intestinal flukes (Keiser and Utzinger, 2004; Utzinger and Keiser, 2004). It is administered orally, is safe, and highly effective. As mentioned above, preventive chemotherapy is the strategy of choice to control schistosomiasis (WHO, 2006). This program is expected to expand ten-fold to include the treatment of 235 million people by 2018 (Knopp et al., 2013) which, on the one hand may significantly drive down morbidity and transmission but, on the other hand, would exacerbate drug pressure, likely resulting in resistance to praziquantel (Caffrey, 2007). Furthermore, praziquantel still has deficiencies: it is inactive against the juvenile stage of Schistosoma spp. and its (S)-enantiomer is inactive, a problem because at the moment, it is not separated from the (R)-enantiomer in production and hence the tablets are large and bitter (Stothard et al., 2013). Therefore, there is great motivation to seek alternative medications. In the case of fascioliasis, triclabendazole is the treatment of choice (Keiser et al., 2005). Triclabendazole is safe and effective against both the human and veterinary forms of the disease. However, there are two major setbacks: in veterinary medicine, triclabendazole resistance has already been documented (Brennan et al., 2007). Additionally, triclabendazole is not registered for use in many nations, and is therefore not always available for the treatment of human fascioliasis (Keiser et al., 2005). 2.2 Repurposed drugs 2.2.1 Antimalarials and their derivatives The largest anthelmintic drug repurposing success story by far has been the application of antimalarials against a wide variety of trematode infections, as well as other broader applications. Investigations of the use of antimalarials against Schistosoma spp. and the liver flukes are based on the rationale that these flukes have the same blood-feeding characteristic as Plasmodium spp., and therefore share heme degradation mechanisms (Toh et al., 2010). If antimalarials target this pathway, they may also be effective against blood-digesting flukes. However, antimalarials have shown activity also against the non-blood feeding intestinal flukes (Ingram et al., 2012b), not only indicating their broad-spectrum activity, but also hinting at other mechanisms of action. As emphasized earlier, the use of antimalarials in de-worming programs should be eyed with caution, as this could accelerate resistance to these drugs. Nevertheless, since malaria and schistosome infections largely overlap geographically, the wide use of antimalarials might be beneficial in co-infected patients (Keiser and Utzinger, 2012). 2.2.1.1 Artemisinins Artemisia annua (quinghao) has been used for thousands of years in Chinese medicine for a wide range of ailments, yet its antimalarial properties did not become known to the wider world until the 1970s, despite its prevalence as a common herb (Tu, 2011). Artemisinin, the plant’s active ingredient, is a sesquiterpene lactone, with a characteristic peroxide bridge that is widely believed to be the active pharmacophore. Though potent, its short half-life, poor bioavailability and the reliance on plant cultivation spurred a search for semi-synthetic and completely synthetic alternatives (Ho et al., 2014). The most widely used artemisinin-derivatives are the first-generation semi-synthetics artesunate and artemether. Artemisinin-based therapies have shown great efficacy against Schistosoma spp., Fasciola spp., Opisthorchis spp. and C. sinensis, as summarized in previous reviews (Keiser and Utzinger, 2007b, 2012; Utzinger et al., 2007). Briefly, artemether, artesunate and dihydroartemisinin (the active metabolite of all artemisinin compounds) share common antischistosomal therapeutic characteristics. In vivo studies have demonstrated that these artemisinins are highly effective against juvenile infections and only moderately effective against adult infections (Keiser and Utzinger, 2012; Li et al., 2014). This observation suggests that these drugs particularly show clinical benefit when used as prophylactic treatments. Indeed, as calculated by a recent meta-analysis by Liu and colleagues, administration of multiple low doses of artemether or artesunate over a 1–2 week period achieved a protection rate of 65–97% against schistosomiasis japonicum (Liu et al., 2011). In addition, a decreased risk was observed using artemether for the prevention of S. mansoni and S. haematobium infections (Utzinger et al. 2000; N’Goran et al. 2003). Contradictory findings were obtained when these agents were used in the treatment of chronic infections. While some studies reported high efficacy, low to moderate cure rates (CRs) were observed in other trials (Keiser and Utzinger, 2007a). However, overall the therapeutic effect of artesunate, artemether or artemisinin based combination therapy (ACTs) against schistosomiasis was inferior to praziquantel in clinical trials (Keiser and Utzinger, 2007a; Utzinger et al., 2010). Finally, the artemisinins often showed synergistic effects with praziquantel in clinical trials, as elucidated by two meta-analyses (Liu et al., 2011; Perez del Villar et al., 2012). The artemisinins have also been thoroughly characterized against food-borne trematodes in in vivo studies. Artemether and artesunate were both found to be very effective against the liver flukes Fasciola hepatica and C. sinensis, as well as the intestinal fluke Echinostoma caproni in the rodent infection models, achieving complete worm clearance (Keiser et al., 2006a,c,d), though lower worm burden reductions (WBRs) were achieved in the O. viverrini hamster infection model (66% and 78% for artemether and artesunate, respectively, administered as a single oral dose of 400 mg/kg) (Keiser et al., 2006d). Artemether and artesunate administered to sheep naturally infected with F. hepatica also achieved significant WBRs (91.3% at a dose of 140 mg/kg intramuscular (i.m.) and 91.9% at a dose of 40 mg/kg i.m. respectively), though embryotoxicity and toxicity were of potential concern (Keiser et al., 2008a, 2010e). Studies with the artemisinins have progressed to clinical trials in Fasciola-infected patients: in central Vietnam, patients subjected to a 10-day treatment course with artesunate, administered daily at a dose of 4 mg/kg, were less likely to report abdominal pain at hospital discharge than patients treated with a standard oral dose of 10 mg/kg triclabendazole. However, 3 months post-treatment, clinical and serological response rates were lower among artesunate recipients compared to triclabendazole-treated patients (Hien et al., 2008). This result mirrors an exploratory Phase 2 trial conducted in Egypt, where chronically infected patients treated with 6 × 80 mg and 3 × 200 mg artemether only achieved CRs of 35 and 6%, respectively (Keiser et al., 2011). Finally, low CR and egg reduction rate (ERR) of 4% and 32% were also observed after administration of artesunate (10 mg/kg in 3 split doses) to school-aged children infected with O. viverrini (Soukhathammavong et al., 2011). 2.2.1.2 Synthetic peroxides The success of the semi-synthetic artemisinins along with their pharmacologic draw-backs motivated the creation of fully synthetic derivatives in antimalarial drug discovery, the most investigated of these being the synthetic ozonides. The antimalarial OZ439 is currently in Phase 2 clinical trials (Wells, 2013). Two synthetic trioxolanes in particular have been widely investigated as potential anthelmintics- OZ78 and OZ288- whose trematocidal activity has been reviewed by Keiser and Utzinger (2007b). The review highlights the major in vivo studies which reveal interesting properties of these two compounds. First, their activity is comparable to those of the artemisinins and they exhibit the same increased in vivo activity against juvenile S. mansoni as compared to adult worms in mice and in hamsters. Second, in S. mansoni in vivo models, the adult WBR is affected by the rodent model, achieving only moderate WBRs in the mouse but high WBRs in the hamster model. However follow-up studies indicated similarly significant activity of OZ78 against S. japonicum juvenile and adult infection in hamsters, mice and rabbits, and of OZ277 in hamsters (Xiao et al., 2011, 2012). Interestingly, the ozonide OZ418 was recently found to be active against both juvenile and adult worms in the mouse S. mansoni infection model, and the adult S. haematobium hamster infection model (Keiser et al., 2012a), as well as moderately active against 7 day-old and adult S. japonicum- infected mice (Xue et al. 2014)- a result which shows that further modifications of the aryl ozonides can yield promising compounds. With regard to the food-borne trematodes, in vivo activity of OZ78 against the liver flukes F. hepatica and C. sinensis and the intestinal fluke E. caproni is very high but studies with O. viverrini showed no activity at all (Keiser and Utzinger, 2007c). Importantly, in follow-up studies, OZ78 was found to be effective against triclabendazole-resistant F. hepatica (100% WBR at oral dose of 100 mg/kg) (Keiser et al., 2007a). Oddly, the high activity of the OZs against F. hepatica could not be repeated in naturally-infected sheep, regardless of the administration route (Keiser et al., 2010b; Meister et al., 2013). This discrepancy could potentially be explained by pharmacokinetic differences observed in rat and sheep, as discussed by Meister and colleagues (Meister et al., 2013). Where OZ78 failed to treat F. hepatica in sheep, MT04 succeeded. MT04 is another synthetic peroxide, identified in the search for more effective analogues, which was found to have a 92% WBR and a 99% ERR in naturally infected sheep (Zhao et al., 2010; Wang et al., 2011; Meister et al., 2013). 2.2.1.3 Mefloquine Mefloquine is a 4-quinolinemethanol whose discovery as an antimalarial dates to around the same time as the artemisinins. Its antischistosomal activity was revealed in 2008 and was thoroughly covered in a review by Xiao et al. (2013). This review highlights some excellent antischistosomal properties of mefloquine. First, it is active against all three major Schistosoma spp. and against both the juvenile and adult stages, a characteristic that neither praziquantel nor the artemisinins possess. Second, it causes severe morphological damage to the fluke and acts independently of the host immune response. Third, initial results from clinical trials were promising; mefloquine- artesunate achieved an ERR of 95% against S. haematobium in school-aged children (Keiser et al., 2010c). In addition, pregnant women who used mefloquine as intermittent preventive treatment for the prevention of malaria showed significantly higher CRs and ERRs against S. haematobium than women treated with sulfadoxine-pyrimethamine (Basra et al., 2013). Moreover, mefloquine-related arylmethanols also show antischistosomal activity in vivo (Ingram et al., 2012a). The effectiveness of mefloquine against food-borne trematodes was briefly reviewed by Keiser et al. (2010a). This review highlighted that mefloquine is not active against F. hepatica nor C. sinensis, but interestingly, mefloquine was active against juvenile and adult O. viverini in a hamster infection model, achieving a WBR of 89% and 96% respectively, following a single oral dose of 300 mg/kg (Keiser et al., 2009a). However, mefloquine failed to produce an effect against O. viverini in clinical trials (Soukhathammavong et al., 2011). In a later study, mefloquine yielded significant results against C. sinensis infection in a rat model (when given at multiple dosages) and Paragonimus westermani infection in a dog model, but the results were not better than a treatment with praziquantel (Xiao et al., 2010). 2.2.2 Tribendimidine Also originating from China, tribendimidine is a Chinese anthelmintic drug which has a similar activity profile against the soil-transmitted helminths as albendazole (high activity against Ascaris lumbricoides and hookworms but only moderate activity against Trichuris trichiura) (Xiao et al., 2013). Besides its broad range of antinematodal activity, tribendimidine has demonstrated trematocidal activity in vivo against C. sinensis and O. viverrini (Keiser et al., 2010a; Xiao et al., 2013). A recent randomized open-label trial in Guangxi, PR China presented moderate CRs (44% following a single dose of 400 mg/kg and 58% following 400 mg/kg administered once daily for 3 days) and high ERR (98–99%), similar to praziquantel. Tribendimidine compared favorably to praziquantel with regards to appearance of adverse events (Qian et al., 2013). Similarly, tribendimidine cured 19 of 24 patients infected with O. viverrini and achieved an ERR of 99% (Soukhathammavong et al., 2011). Further studies with tribendimidine are currently ongoing (e.g. dose-finding, pharmacokinetic studies) in O. viverrini-infected patients in Laos with the ultimate goal of developing an alternative opisthorchicidal drug. To offset eventual resistance to praziquantel, using tribendimidine in combination with praziquantel is an attractive option. However, a recent study showed that praziquantel-tribendimidine administered to O. viverrini-infected hamsters, though achieving synergistic effects in vitro, had antagonistic effects in the hamster murine model (Keiser et al., 2013a). Further studies are required to shed light on this phenomenon. In contrast, in the C. sinensis rat model, tribendimidine (12.5–50 mg/kg) showed synergistic interactions with 150 mg/kg praziquantel (Keiser et al., 2009b). 2.2.3 Other compounds The most recent in vivo antitrematodal candidates come from a range of sources and fields, including cancer research, though expanding veterinary drug applications continues to be popular. The discoveries are described below and summarized in Table 1. 2.2.3.1 S. mansoni A (well-funded) cancer drug pipeline provides a cornucopia of compounds. Dissous and Grevelding (2011) recently explored the potential of repurposing cancer drugs, specifically protein kinase inhibitors, towards schistosomiasis. The principal rationale was the identification of select protein kinases as being essential to schistosome development and the subsequent identification of certain cancer drugs as inhibitors of these kinases. However, most of the drugs discussed were only inhibitors of egg development and had little effect on the adult worms themselves. The one exception was imatinib (Gleevec), a successful anti-leukemia agent, which was shown to be lethal to adult worms in vitro at high concentrations (Beckman and Grevelding, 2010). However, imatinib was ineffective in an S. mansoni infection model (Table 1) (Katz et al., 2013) which is not surprising, as the drug is highly bound to alpha-1-acid glycoprotein (Soo et al. 2010). Nonetheless, two anticancer agents have shown promising in vivo activity. Miltefosine, an alkylphospholipid, was initially developed to combat cutaneous metastasis of mammary carcinomas (van Blitterswijk and Verheij, 2013). As discussed in a complementary review by Andrews et al. (2014), it has also recently been licensed to treat leishmaniasis- a protozoan that also has cutaneous manifestations, though this drug treats both the visceral and cutaneous forms (Dorlo et al., 2012). Recently, it has shown in vivo activity in the S. mansoni mouse model at various stages of infection. Administered at 20 mg/kg for five consecutive days, commencing either on the day of infection (schistosomula stage), 21 days p.i. or 42 days p.i., miltefosine showed WBRs of 91%, 76% and 95%, respectively (Eissa et al., 2011a). A recent study by Eissa and colleagues also demonstrated its ovicidal, larvicidal and molluscicidal activity and its in vitro activity against S. haematobium adult worms (Eissa et al., 2011b). Interestingly, our own studies have shown that miltefosine was only moderately active against newly transformed schistosomula (NTS) of S. mansoni (unpublished findings). The antiandrogen, nilutamide, blocks the binding of testosterone to the androgen receptor and was initially marketed for the treatment of metastatic prostate cancer (Akaza, 2011). Recently, we have tested it in the S. mansoni mouse model since related hydantoin derivatives studied at Hoffmann-La Roche had intriguing antischistosomal properties (Link and Stohler, 1984) – a single 400 mg/kg oral dose achieved a WBR of 85% and an even higher WBR was observed when given in combination with praziquantel (Keiser et al., 2010d). However, nilutamide did display a stage specific effect – it was only moderately active against the juvenile stage (Keiser et al., 2010d). The advantages and disadvantages of using cancer drugs in the treatment of helmintic diseases should be carefully weighed. On the one hand, there is a great deal of effort to minimize toxic effects of cancer drugs due to their long treatment course. If a cancer drug is deemed safe when administered over a long period, there is a good chance it is also safe as an anthelmintic, where treatment is annual or twice annual (if part of regular preventive chemotherapy programs) but short (i.e. single dose). However, due to the severity and life threatening state of many cancers, the benefit of taking the drugs may outweigh potential severe adverse events. Thus, just because a drug is approved for cancer treatment, does not mean it is without significant adverse events. Yet a drug used in preventive chemotherapy programs must have an excellent tolerability profile. Extending the application of veterinary anthelmintics continues to be a source of human anthelmintic candidates. Abdulla et al. (2009) showed that rafoxanide, a salicylanilide derivative and a veterinary anthelmintic, caused a 50–56% WBR in mice at a single oral dose of 50 mg/kg. This compound was identified from a screen of a commercially available FDA-approved library. This same screen also identified anisomycin, lasalocid sodium, diffractic acid, gamboic acid and niclosamide as hits for in vivo testing in the S. mansoni mouse model. Unfortunately, these drugs failed to reduce worm burden in vivo, the exception being lasalocid sodium which caused a WBR of 41–44% when administered at 100 mg/kg orally. Rafoxanide was not originally in the library, but due to niclosamide’s excellent in vitro profile (but poor in vivo results), rafoxanide was tested as well, as it is structurally related to niclosamide. Clorsulon (a veterinary flukicidal drug) caused a WBR in S. mansoni-infected mice of 88%, 96% and 98% when treated with either a single, double or triple dose of 5 mg/kg one week apart starting from the 4th week postinfection (Mossallam et al., 2007). Yet in our own studies only moderate activity against NTS were observed in vitro when the NTS were incubated with 10 μM clorsulon for 72 h (unpublished observations). Another re-purposing strategy is to increase the anthelmintic spectrum of activity through slight modification of the molecule. For example, BTP-Iso, a novel benzimidazole (in the same family as triclabendazole) was recently investigated as an antischistosomal. At a dose of 300 mg/kg given to S. mansoni-infected mice, BTP-Iso showed statistically significant higher reductions (p < 0.01) in female (77%), male (35%), and total worms (55%) in comparison with the control group (El Bialy et al., 2013). 2.2.3.2 F. hepatica Two fasciocidal drugs were moved directly into clinical testing, without previous publications of in vivo studies. In Iran, 46 patients who were positive for Fasciola 3 months after triclabendazole treatment were given 1.5 g/day metronidazole orally for three weeks. Two months after end of therapy, stool exams became negative in 35 patients (81%), of which 31 patients (72%) presented both negative serology and stool exams. At 12 months, 28 patients were examined and all were negative (Mansour-Ghanaei et al., 2003). Nitazoxanide, a thiazolide derivative and a pyruvate ferredoxin oxireductase inhibitor, has a broad spectrum of activity. The US Food and Drug Administration (FDA) approved nitazoxanide in 2002 for the treatment of diarrhea caused by Cryptosporidium species and Giardia intestinalis in pediatric patients 1–11 years of age, and in 2004 for its use in adults (Hemphill et al., 2006). Two clinical trials elucidated nitazoxanide’s fasciocidal effects. In northern Peru, a double-blind placebo-controlled trial showed moderate effects in children and adults: a 7-day course of nitazoxanide resulted in CRs of 60% in adults and 40% in children (Favennec et al., 2003). In the Atlixco municipality in Mexico, a study carried out in 50 Mexican schoolchildren showed nitazoxanide to be effective against light F. hepatica infections. Children diagnosed with fascioliasis were administered nitazoxanide at 7.5 mg/kg body weight, every 12 hours over seven days. The efficacy against fascioliasis was 94% and 100% after first and second treatment courses, respectively. Its efficacy was also very high against protozoan and intestinal helminths (Zumaquero-Rios et al., 2013). However, this second study did not have a control group, and therefore could not be compared to a placebo or treatment with triclabendazole. Given these somewhat contradictory findings, larger randomized control trials should be conducted to elucidate the role of nitazoxanide in the treatment of fascioliasis. Myrrh has been marketed as an antischistosomal, though its actual effect is controversial (Abdul-Ghani et al., 2009; Yakoot, 2010). Despite a 2004 field trial in Egypt, showing a CR of 88% and 94% CR, 2 and 3 months after treatment (Abo-Madyan et al., 2004), the overall consensus based on bench and field publications is that it is ineffective as a fasciocidal agent (Botros et al., 2009; Keiser et al., 2010a). In the veterinary field, the benzimidazoles are often used as broad-spectrum anthelmintics, as discussed further in the soil-transmitted helminths section below. In livestock, the use of oxfendazole has previously been restricted to the treatment of roundworm, strongyloides and pinworm infection. However, very recently, it was demonstrated that it has potent anti-flukicidal activity: 100% WBR was achieved after a single oral dose of 30 mg/kg was given to F. hepatica infected pigs (Ortiz et al. 2014). 3 Soil-transmitted helminthiasis and strongyloides 3.1 Current treatment The two benzimidazoles, albendazole and mebendazole, as well as pyrantel pamoate and levamisole are the four available treatments against soil-transmitted helminths, marketed between 1966 (pyrantel pamoate) and 1980 (albendazole) (Keiser and Utzinger, 2010; WHO, 2013). These drugs are widely used (in 2010, 328 million children were treated with albendazole or mebendazole (Anonymous, 2012)) since, as mentioned, the current strategy to control morbidity due to the most common soil-transmitted helminths (A. lumbricoides, T. trichiura and the hookworm species Ancylostoma duodenale and Necator americanus) is by regular treatment of at risk populations (WHO, 2006, 2011). The limitations of these drugs are widely known. Briefly, using a single dose regimen they all are very efficacious against A. lumbricoides, yet for the treatment of hookworm infections only albendazole produces satisfying CRs, while all drugs achieve poor CRs against infections with T. trichiura (Keiser and Utzinger, 2008). As no new anthelmintic drugs are on the horizon, repurposing of existing drugs is an essential strategy to improve treatment of soil-transmitted helminths, particularly T. trichiura infections (Keiser and Utzinger, 2008; Olliaro et al., 2011). The current drug of choice against S. stercoralis infections is ivermectin, which is widely used in mass drug administration programs to combat lymphatic filariasis (Knopp et al., 2012a). A single dose of 200 μg/kg results in a CR of 88% against S. stercoralis (Keiser and Utzinger, 2010). The drug has the disadvantages that it is not licensed for treating S. stercoralis infections in several countries (e.g. Switzerland, Germany, Great Britain), that the ideal dosage schedule of ivermectin has yet to be evaluated, and that it can cause harmful and dangerous treatment effects in individuals co-infected with Loa loa which is an insect-borne filarial infection (Bisoffi et al., 2013; Greaves et al., 2013). People not tolerating ivermectin can alternatively use albendazole, which is inferior compared to ivermectin regarding ERRs and CRs; however, it still achieves satisfactory efficacy (Keiser and Utzinger, 2010; Knopp et al., 2012a). In addition, mebendazole achieves CRs and ERRs of 67–98% against S. stercoralis when given in multiple doses (Musgrave et al., 1979; Shikiya et al., 1990, 1991; Zaha et al., 2000). Of note, even though ivermectin is not listed as an essential drug against soil-transmitted helminths (WHO, 2013), several studies have shown that by combining ivermectin with the standard drug albendazole or mebendazole, the efficacy against soil-transmitted helminths (especially against T. trichiura) can be improved significantly (a summary of trials is presented in Keiser et al. (2012b)). 3.2 Repurposed drugs 3.2.1 Drugs evaluated in clinical testing 3.2.1.1 Nitazoxanide Nitazoxanide, mentioned above for its protozoan and fasciocidal properties, showed (in manufacturer-financed trials) very high CRs against A. lumbricoides, T. trichiura, S. stercoralis and even against A. duodenale when the drug was given in multiple doses (Romero Cabello et al., 1997; Abaza et al., 1998; Juan et al., 2002; Diaz et al., 2003). The drug also demonstrated superior activity than the standard drugs in in vitro screening against Trichuris muris (Tritten et al., 2012). However, a recent clinical trial using a single dose of 1000 mg nitazoxanide could not confirm the initial results obtained in earlier trials and in the laboratory. Nitazoxanide achieved low CRs and ERRs (7% and 13%, respectively) against T. trichiura in school-aged children on Pemba, Tanzania. Additionally the drug was slightly less well tolerated compared to standard treatment and hence nitazoxanide was not recommended as an alternative treatment against soil-transmitted helminthiasis (Speich et al., 2012). Of note, within the same trial, the efficacy of a single dose nitazoxanide (1000 mg) against intestinal protozoa was also only moderate (Speich et al., 2013). 3.2.1.2 Oxantel pamoate Oxantel pamoate, a veterinary drug which was introduced on the market in 1974, revealed high efficacy against T. trichiura in a number of exploratory trials (Garcia, 1976; Lee et al., 1976; Lee and Lim, 1978). The drug was only revisited recently, more than three decades later. Laboratory studies demonstrated a significantly higher activity of oxantel pamoate compared to the benzimidazoles, levamisole or pyrantel pamoate against T. muris in vitro and in vivo (Keiser et al., 2013b). A randomized controlled double blind trial recently confirmed the high efficacy of oxantel pamoate (20 mg/kg) against T. trichiura (26% CR and 93% ERR). Hence oxantel pamoate-albendazole, due to its broad spectrum of activity could be useful in the control of soil-transmitted helminthiasis (Speich et al., 2014). Further studies have been planned with the ultimate goal of elucidating the potential of adding oxantel pamoate to the current drug armamentarium. 3.2.1.3 Tribendimidine A large phase 2 trial in Côte d’Ivoire confirmed the excellent activity of tribendimidine against hookworm and A. lumbricoides (N’Goran et al. under review). Following promising results with tribendimidine against Strongyloides ratti in the rat model (Keiser et al., 2008b) an exploratory trial in China documented a CR of 55% in patients infected with S. stercoralis (Steinmann et al., 2008). 3.2.1.4 The benzimidazoles The benzimidazoles are a large family, widely used in veterinary medicine, and not only include albendazole and mebendazole (and the fasciocidal drug triclabendazole mentioned earlier) but also flubendazole, fenbendazole, oxfendazole, thiabendazole and oxibendazole (Olliaro et al., 2011). In addition, oxfendazole and cambendazole are promising broad spectrum benzimidazoles used in veterinary medicine, which have not yet been tested in humans. The benzimidazoles have a broad spectrum of activity including whipworms, roundworms and hookworms. For example, multiple doses of thiabendazole have similarly high efficacy against S. stercoralis as compared to ivermectin, however the drug was less tolerated (Gann et al., 1994; Zaha et al., 2000; Bisoffi et al., 2011). Clinical trials conducted with flubendazole, fenbendazole, oxibendazole and thiabendazole against soil-transmitted helminths and S. stercoralis are summarized in Table 2. 3.2.2 Compounds in early clinical testing and potential veterinary drugs An excellent summary of potential drug development candidates for human soil-transmitted helminthiasis has recently been provided by Olliaro and colleagues (Olliaro et al., 2011). Four promising compound classes are worth highlighting. First, emodepside is a cyclic depsipeptide and a broad-spectrum veterinary anthelmintic used for companion animal gastrointestinal nematode infections (Harder et al., 2003; Geary and Mackenzie, 2011). It showed high activity against Trichuris spp., Ancylostoma spp. and other nematodes in in vitro and in vivo studies (Harder and von Samson-Himmelstjerna, 2002; Altreuther et al., 2009). Moreover, the closely related PF1022A was potent in clearing T. muris infections in mice at various stages of infection (Kulke et al. 2014). In vivo studies on Haemonchus contortus have shown that it has a different mode of action than the benzimidazoles and ivermectin (von Samson-Himmelstjerna et al., 2005) hence the drug will likely be active against parasites resistant to the benzimidazoles and ivermectin. Second, doramectin which belongs to the avermectins, showed high efficacy against S. ransomi, S. papillosus, T. suis and A. suum in pigs (Stewart et al., 1996; Yawzinski et al., 1997) as well as against Trichuris spp. in cattle (Jones et al., 1993; Saeki et al., 1995). Doramectin showed even more favorable results against nematodes compared to ivermectin, which could be explained by its lower clearance and higher bioavailability (Lumaret et al., 2012). The closely related milbemycin family is also a potent drug class. Milbemycin oxime has proven to be efficacious against Ascaris spp., hookworm and Trichuris spp. in cats and dogs (Blagburn et al., 1992; Catton and Van Schalkwyk, 2003). On the other hand only low ERRs were found against T. trichiura in baboons (Reichard et al., 2007). Third, moxidectin is another commercially available broad-spectrum antiparasitic from the veterinary field (Reinemeyer and Cleale, 2002). It is a semisynthetic methoxime derivative of naturally occurring nemadectin with a novel mode of action (Awasthi et al., 2013). Moxidectin showed high efficacy in several in vivo studies (i.e. in cattle, swine, sheep) against Ascaris spp., Trichuris spp. and Strongyloides spp. (Bauer and Conraths, 1994; Coles et al., 1994; Stewart et al., 1999; Reinemeyer and Cleale, 2002; Ranjan and DeLay, 2004; Lyons et al., 2006). Finally, from a completely different medical arena, cyclosporine A is an immunosuppressive agent which is commonly used for organ transplantation (Cohen et al., 1984). Its antischistosomal properties were first described in 1981 (Bueding et al., 1981) followed by studies against S. stercoralis in dogs and S. ratti in rats (Armson et al., 1995). Treatment with cyclosporine A seems to be of benefit in cases of opportunistic, possibly life threatening infections of S. stercoralis activated by immunosuppression (Schad, 1986). 4 Filariasis 4.1 Current treatment Though filariasis is a collection of diseases, the three major control programs target lymphatic filariasis (Global Program to Eliminate Lymphatic Filariasis) or onchocerciasis (African Program for Onchocerciasis Control and the Onchocerciasis Elimination Program for the Americas), and a major cornerstone of all three programs is mass drug administration. Regardless of the disease, the recommended treatments are limited: ivermectin, albendazole and diethylcarbamazine (DEC), ivermectin being the most widely recommended drug. The use and status of these drugs, including their limitations, have recently been reviewed by Katiyar and Singh (2011). Briefly, resistance to ivermectin has already been documented and its administration to onchocerciasis patients co-infected with loaiasis results in severe adverse events (Kamgno et al., 2007). Albendazole’s efficacy is heavily debated and DEC is contraindicated in patients with onchocerciasis (Katiyar and Singh, 2011). Additionally, the treatment regimens required for these drugs in order to fully stop transmission are long: according to current control guidelines, ivermectin or DEC-albendazole need to be administered once a year for 5 years to interrupt LF transmission (Anonymous, 2009, 2014). The long treatment schedule is partly because all three drugs predominantly act on microfilaria only (the larvae shed by the adult worms), with DEC showing some adult stage activity (Dreyer et al., 2006). This latter point is problematic, as the adult stage still survives in the host, ready to shed more microfilaria once the drugs are absent from the host, continuing to cause pathology in onchocerciasis patients and also continuing the transmission cycle. Thus, new drug candidates should necessarily target adult worms as well. Additionally, as all filariasis-causing nematodes (except L. loa) carry with them the endosymbiotic Wolbachia bacteria necessary for the parasite’s development, efforts have been made to target these bacteria. 4.2 Drugs targeting Wolbachia Amongst the new antifilarial drugs, doxycycline is the most studied. It is a broad-spectrum antibacterial and antiprotozoal whose effects against Wolbachia are considered a breakthrough in antifilarial treatment (Katiyar and Singh, 2011). In onchocerciasis patients, a treatment regimen of 100 mg of doxycycline per day for 6 weeks slowly eliminated Wolbachia and resulted in long-term embryogenesis block of the adult worms (Hoerauf et al., 2003). The death of Wolbachia also leads to macrofilaricidal effects, as adults worms extracted from LF patients treated with 200 mg of doxycycline per day for 4 weeks were found to be either sterile or dead (Debrah et al., 2007). Findings by Taylor and colleagues also showed macrofilaricidal effects in LF patients, though the same level of potency could not be observed in onchocerciasis patients (Taylor et al., 2005; Hoerauf et al., 2008b). In addition, doxycycline is potentially microfilaricidal as well, as shown in LF patients treated with 100–200 mg per day for 6 weeks (Supali et al., 2008). Doxycycline, given prior to antifilarial treatment, appears to be more effective in suppressing microfilaremia in bancroftian filariasis over the long term than the standard treatments alone, while also resulting in fewer severe adverse events (Turner et al., 2006). LF symptoms could also be reduced by doxycycline, regardless of active filarial infection as demonstrated in a trial in Ghana (Mand et al., 2012). This symptomatic alleviation also extends to LF patients who presented with hydrocele (Debrah et al., 2009). The effects of doxycycline administered in combination with the standard drugs have also been characterized. For example, a combination of doxycycline and albendazole was far more effective at suppressing circulating microfilaria than doxycycline or albendazole alone (nearly 100% suppression vs. 69% and 89%, respectively when checked at day 365 post-treatment) and even succeeded in completely clearing 42% of patients of microfilaremia (Gayen et al., 2013). Different trials also assessed doxycycline/ivermectin and found that the combination was more effective than ivermectin or doxycycline alone (Masud et al., 2009; Turner et al., 2010). Similarly, a 21-day course of doxycycline at 200 mg administered orally to LF patients in Orissa, India, followed by a single 6 mg/kg dose of DEC was effective at completely clearing microfiladermia. Interestingly, the co-administration of doxycycline followed by DEC was also effective at clearing adult worm nests, and less effective when the doxycycline treatment was shortened to 10 days (Mand et al., 2009). Doxycycline was even found to be effective against Mansonella perstans, one of two agents that cause serous cavity filariasis. In a randomized open-label trial in Mali, 97% and 75% of M. perstans patients treated with 200 mg doxycycline for 6 weeks were found to be amicrofilaremic at 12 and 36 months post-treatment, respectively. This finding is very significant as the current standard treatments are not effective against M. perstans (Coulibaly et al., 2009). Last but not least, given that L. loa is the only filarial nematode that does not possess Wolbachia endosymbionts, the use of Wolbachia-targeting antibiotics is a possible safe alternative for the treatment of this disease. Doxycycline was already shown to be safe in onchocerciasis patients co-infected with loaisis (Wanji et al., 2009; Turner et al., 2010), but its long treatment course leaves room for short-course therapy innovations. Considering the importance of a potent anti-Wolbachia treatment, there is ongoing research to also elucidate the effects of other potent broad-spectrum antibiotics on Wolbachia. Rifampicin was found to be effective in reducing Wolbachia bacteria, as well as embryogenesis and microfilaria production in a small number of onchocerciasis patients, though not as efficiently as doxycycline (Specht et al., 2008). However a short-course therapy of 5 days rifampicin was documented to be completely ineffective (Richards et al., 2007). Meanwhile azithromycin was observed to be only marginally effective against Wolbachia and showed no effect on the worms in onchocerciasis patients in Ghana (Hoerauf et al., 2008a). In another study, oxytetracycline (the parent compound of doxycycline) had macrofilaricidal properties in cattle infected with Onchocerca ochengi, however this success has yet to be translated to human treatment (Nfon et al., 2007). 4.3 Drugs targeting the worms As mentioned above, drugs effective against the filarial adult stage should be investigated with high priority. The potential of the benzimidazole flubendazole, a veterinary drug used to treat gastrointestinal nematode infection, as a pre-clinical macrofilaricide candidate for preventive treatment of onchocerciasis and lymphatic filariasis is currently being studied in a project lead by the Drugs for Neglected Diseases initiative (DNDi). These investigations have been reviewed by Mackenzie and Geary (2011). In the late 1980s and early 1990s, flubendazole was shown to be macrofilaricidal against a variety of filariases-causing agents in vivo and even in a clinical trial for onchocerciasis (Dominguez-Vazquez et al., 1983; Van Kerckhoven and Kumar, 1988; Franz et al., 1990). Interestingly, it has no microfilaricidal effects (Van Kerckhoven and Kumar, 1988), which indicates that it could be safe for L. loa co-infected patients as well. The drug is, however, very poorly orally absorbed and hence the current focus of the project lead by DNDi is to improve the drug’s bioavailability. A new cyclodextrin formulation is currently being investigated with mixed results: while it achieved higher plasma concentrations when administered orally to Echinococcus granulosus-infected mice, no significant pharmacokinetic effects were observed when it was administered to sheep via the intraruminal nor the intraabomasal route (Ceballos et al., 2009, 2012). Nonetheless, other drugs on the horizon continue to target microfilaria while also having some effects on the adult stage. Unsurprisingly, moxidectin has shown potent microfilaricidal effects and long term sterilization of females. Furthermore, early-stage clinical trials have shown that it is safe and well tolerated in humans in doses of 3–36 mg and has a longer half-life than ivermectin (Cotreau et al., 2003; Awadzi et al. 2014). It is being evaluated in ongoing Phase 2 and 3 clinical trials via a partnership between APOC, TDR and Wyeth (Taylor et al., 2009; Babalola, 2011). Nonetheless, tolerability studies are needed to confirm that moxidectin is safe in patients co-infected with L. loa and whether the drug is efficacious against ivermectin-resistant filaria. Other anthelmintics tested include the above mentioned cyclic depsipeptides, emodepside and PF1022A, and levamisole. Emodepside showed potent microfilaricidal effects and even some macrofilaricidal effects (but not against Brugia malayi) in vivo (Zahner et al., 2001), hence there is great interest in the potential of emodepside as a novel antifilarial (http://r4d.dfid.gov.uk/PDF/Outputs/DNDI/evolutions_of_dndi_portfolio.pdf). With regards to levamisole, though previous studies from the 1980s showed it had no significant treatment effect for LF or onchocerciasis patients (Awadzi et al., 1982; McMahon, 1981), the drug was revisited in a clinical trial in 2004 where levamisole was given alone (2.5 mg/kg) or co-administered with ivermectin or albendazole. Here as well, it proved to be ineffective, alone or in combination (Awadzi et al., 2004). Nitazoxanide, mentioned earlier in this review, and its metabolite tizoxanide were tested against B. malayi worms in vitro and in vivo. The drugs were lethal to adult worms at 20 μg/ml and significantly decreased microfilaria release at 5 μg/ml. However, they were ineffective against Wolbachia and eventually were shown to be ineffective in clearing the worms in vivo (Rao et al., 2009). Considering the success of antimalarials against trematodes, as well as the co-endemicity of malaria and many helmintic diseases, it is always tempting to test antimalarials on other worms as well. Recently, an open randomized control trial investigated the effects of the antimalarials quinine, chloroquine, amodiaquine and artesunate, based on previous in vivo successes or case studies, on patients infected with L. loa. However, none of the treatments were found to be effective (Kamgno et al., 2010). 5 Cestode infections 5.1 Current treatment Niclosamide, originally developed as a molluscicide, was introduced in 1959 as the first synthetic drug against taeniasis, characterized by an excellent therapeutic index and high efficacy (WHO/FAO/OIE, 2005). It is a poorly water soluble halogenated salicylanilide with a low bioavailability, which is an advantage since a systemic action is not desired (Pearson and Hewlett, 1985; WHO/FAO/OIE, 2005). Approximately one decade later, in 1972, the taenicidal activity of praziquantel, an acylated isoquinoline-pyranzine, was discovered jointly by Bayer AG and E. Merck (Andrews et al., 1983). Both drugs are still the recommended first line treatments against intestinal cestodes including Taenia solium, Taenia saginata, Diphyllobothrium spp. and Hymenolepis nana (WHO/FAO/OIE, 2005). Praziquantel, which is well absorbed from the intestine, presents additional activity against T. solium cysticerci located in the host’s tissues (i.e. central nervous system, subcutaneous tissue, and the eye). A single oral dose of praziquantel is highly effective against taeniasis; nevertheless multiple treatment courses over 2 weeks are recommended for uncomplicated cysticercosis. Albendazole, is also recommended for the treatment of (neuro)-cysticercosis and is generally more active than praziquantel (Sotelo et al., 1988, 1990; Takayanagui and Jardim, 1992; WHO/FAO/OIE, 2005). Regardless, therapy for neurocysticercosis needs to be individualized depending on the patient’s state of disease. Anthelmintics, surgery, and symptomatic treatment with analgesics, corticosteroid drugs, and antiepileptic drugs, to relieve headache, perilesional inflammation and seizures, are the main pillars of the therapeutic approach (Carpio, 2002; Garcia et al., 2011). Surgical resection of the entire parasitic lesion is indicated in all operable cases of cystic and alveolar echinococcosis. Puncture-aspiration-injection-re-aspiration (PAIR) treatment is applied in inoperable cases of cystic echinococcosis: cysts are punctured and exposed to protoscolicidal substances (i.e. ethanol 95%) for at least a quarter of an hour (WHO, 1996). Chemotherapy is key to preventing secondary echinococcosis and to reducing the risk of re-occurrence following surgery (Brunetti et al., 2010). Mebendazole and albendazole are both recommended for the treatment of echinococcosis. The albendazole dose regimen is more convenient because its phase-1 metabolite, albendazole sulfoxide, also exhibits metacestodicidal properties and thus the drug disposition is greater than for mebendazole (Ingold et al., 1999). Praziquantel itself possesses less pronounced activity against echinococcosis than albendazole. Combination treatment of praziquantel and albendazole seems sensible, because both drugs display a different mechanism of action and praziquantel increases the bioavailability of albendazole sulfoxide when the two are given together (Cobo et al., 1998; Kern, 2006; Garcia et al., 2011). Nonetheless, insufficient clinical data are available to provide treatment recommendations for this combination (Pawłowski, 2001; Bygott and Chiodini, 2009). 5.2 Repurposed drugs 5.2.1 Taeniasis and neurocysticercosis The food and drug administration (FDA) approved nitazoxanide, mentioned several times earlier in this review, as an antiprotozoal agent against Cryptosporidium species and Giardia intestinalis in 2002 (Rossignol, 2009). Its chemical structure stems from the taenicide niclosamide and therefore, unsurprisingly, activity was reported against T. saginata and H. nana infections in the 1980s (Table 3) (Rossignol and Maisonneuve, 1984). Hence, nitazoxanide is not a genuine example of a repurposed drug for taeniasis; still its strong taenicidal activity makes it attractive for off-label use in cases where patients do not respond to praziquantel or niclosamide (Vermund et al., 1986; Lateef et al., 2008). Similarly, quinacrine (mepacrine), which is an ancient antimalarial agent and antigiardial drug, can be used to treat niclosamide-tolerant T. saginata infections (Gardner et al., 1996; Koul et al., 2000). The recent promising results achieved with tribendimidine against O. viverrini and C. sinensis liver fluke infections (see above) inspired further exploration of its anthelmintic activity profile against cestodes. In an open-label randomized clinical trial, including 15 patients infected with Taenia spp., a single oral dose of 200 mg (5–14 year old children) or 400 mg (⩾15 years) tribendimidine resulted in a CR of 67%. Due to diagnostic challenges and the relatively small numbers of patient enrolled in the study, the authors pointed out that the observed cestocidal effect of tribendimidine should rather be recognized as an indication of possible activity rather than as a proof-of-concept (Steinmann et al., 2008). A distinct anticestodal potential of tribendimidine was demonstrated in mice infected with Hymenolepis microstoma, where a triple dose of 50 mg/kg tribendimidine, given over three consecutive days, achieved a WBR of more than 95% (Kulke et al., 2012). No cestocidal effect was observed however, when mice were treated with deacetylated amidantel. This result is somehow unexpected as deacetylated amidantel is supposed to be the major active metabolite of tribendimidine, hence further studies are required (Xiao et al., 2009; Yuan et al., 2010). Paromomycin (Humatin®) is an aminoglycoside antibiotic with activity against gram-negative and various gram-positive bacteria. It is marketed to decrease bacterial load in the gut in hepatic coma, and for the treatment of amoebiasis and giardiasis. Recently, it has been licensed in India for leishmaniasis therapy (Davidson et al., 2009). In the 1960s the taenicidal activity of paromomycin was discovered by chance in the course of amoebiasis treatments, where co-infected patients shed segments of T. saginata (Salem and el-Allaf, 1969). In a follow-up clinical study it was demonstrated that daily treatments for 1–3 days with 30–50 mg/kg paromomycin resulted in a success rate of 89–100% against T. saginata. In addition, an efficacy of approximately 90% was observed against H. nana following a 30 mg/kg single or double dose of paromomycin (Salem and el-Allaf, 1969). Tamoxifen is a first generation selective estrogen receptor modulator (SERM), which acts as an antagonist on estrogen receptors in breast tissue, while possessing an agonistic effect in other tissues. It is used as an adjuvant for breast cancer and the palliative treatment of metastatic or locally advanced mamma carcinoma (den Hollander et al., 2013). It has been demonstrated that estradiol plays an essential role in regulating the asexual reproduction of Taenia crassiceps cysticerci in mice, possibly by interfering with the host’s cellular immune response (Terrazas et al., 1994), and as a result, tamoxifen was tested against T. crassiceps cysticerci. Tamoxifen reduced the parasite load by 80% and 50% in female and male mice, respectively. In addition, loss of motility and reduced reproduction could be demonstrated in vitro (Vargas-Villavicencio et al., 2007). A follow-up study showed that tamoxifen decreased the intestinal establishment of T. solium in hamsters by approximately 70%. Moreover, tamoxifen induced morphological changes: the recovered tapeworms appeared like scolices without strobilar development and consequently showed an 80% reduction in length (Escobedo et al., 2013). Yet the taenicidal and cysticidal potential of tamoxifen has not been evaluated in humans and livestock. Metrifonate is an organophosphorous compound applied as an insecticide and veterinary anthelmintic. In humans it has been, for example, for the treatment of schistosomiasis (Holmstedt et al., 1978). A case report published in 1981 about a man with a heavy cutaneous cysticercosis infection, described that two treatment courses of metrifonate at 10 mg/kg/day for six days were able to clear about 75% of the nodules and reduced the size of the remaining ones. Nevertheless, the observed adverse drug events caused by metrifonate’s unspecific cholinesterase inhibition were so strong that atropine had to be given to relieve the symptoms (Tschen et al., 1981). 5.2.2 Echinococcosis In vitro and in vivo studies indicate that Echinococcus multilocularis and E. granulosus can be included in the broad activity spectrum of nitazoxanide. In vitro treatment of E. multilocularis metacestodes with nitazoxanide lead to morphological and ultrastructure alterations and inhibited larval growth, while E. granulosus protoscoleces and metacestodes suffered deleterious effects as well (Stettler et al., 2003; Walker et al., 2004; Reuter et al., 2006). Subsequent in vivo experiments showed that a combination of nitazoxanide and albendazole reduced parasite weight by about 4 times in the murine model (Stettler et al., 2004). A good effect was observed in patients with disseminated cystic echinococcosis on affected soft tissue, muscles, or viscera, who received nitazoxanide additionally to the standard albendazole chemotherapy (with or without praziquantel). Nevertheless, nitazoxanide shows no effect on chronic and extensive body lesions (Perez-Molina et al., 2011). A 3-month treatment course with the anticancer drugs daunorubicin, idarubicin, cytarabine, and fludarabine did not affect the size and contents of the echinococcus cysts in a patient suffering from acute leukemia and cystic echinococcosis of the liver. The antifungal agent amphotericin B was additionally applied within the chemotherapy schedule, but did not show parasitocidal activity against E. granulosus cysts either (Ali et al., 2005). However, in vitro data suggest that amphotericin B effectively inhibits the growth of E. multilocularis metacestodes (Reuter et al., 2003b). Amphotericin B is also able to halt parasite growth in E. multilocularis-infected patients and might therefore play a role as salvage treatment, when resistance or intolerance to benzimidazoles occurs (Reuter et al., 2003a). The anthelmintic agent ivermectin was tested against E. multilocularis in hamsters with intraperitoneal inoculation of protoscolices, and in rats with transportal inoculation of protoscolices. The drug showed no effect in either case (Inaoka et al., 1987). In addition, in vitro results confirmed that ivermectin is also ineffective against E. multilocularis larvae (Reuter et al., 2006). The spectrum of activity of antimalarials, such as mefloquine or the artemisinins, was also assessed against echinococcosis. Mefloquine presented a dose dependent activity against metacestodes in vitro, although a reduction of the parasite weight in E. multilocularis-infected mice was only achieved by intraperitoneal mefloquine administration and not with oral treatments (Kuster et al., 2011). Artesunate and dihydroartemisinin, however, had no effect against E. multilocularis in vivo (Spicher et al., 2008), despite excellent in vitro activity. Furthermore, in vitro incubation of E. multilocularis larvae with artemether did not induce destruction of parasite vesicles (Reuter et al., 2006). Ethanol (70–95%), hypertonic saline (15–30%) and cetrimide solution (0.5%) are considered to be scolicidal agents for PAIR treatment with relatively low risk according to the WHO (1996). Efforts were made to discover more potent scolicidal agents to reduce the recurrence rate following PAIR and surgical interventions. For instance, chlorhexidine gluconate, which is a commonly available, inexpensive, and non-toxic antiseptic agent, proved to be highly active at a concentration of 0.04% following intracystic injection (Topcu et al., 2009). Moreover, in vitro data confirm that intracystic albendazole sulfoxide injections at concentrations of 50–100 μg/ml are highly scolicidal and additional studies in rabbits indicate that this treatment lacks biliary and systemic toxicity (Erzurumlu et al., 1998; Adas et al., 2009). Recently, a water-soluble albendazole sulfoxide salt was developed (Bayverm®-PI, Bayer HealthCare), which enabled the preparation of a liquid dosing form and henceforth systemic and even intracystic injections. Importantly, systemic application circumvents the low oral absorption rate of albendazole and significantly elevates drug disposition (Lanusse et al., 1998). A proof of concept trial in sheep infected with E. granulosus (n = 7) was performed, which demonstrated that intracystic instillation without reaspiration of albendazole sulfoxide significantly decreased cyst size and lead to death of all protoscoleces and daughter cysts 6 months post-injection (Deger et al., 2000). Further studies in animals and human subjects are warranted to evaluate if this therapeutic approach could substitute the current lengthy oral albendazole therapy. Albendazole and mebendazole are currently the recommended drugs for chemotherapy of echinococcosis; hence it is reasonable that other benzimidazole derivatives are investigated. For instance flubendazole induced 90% reduction in cyst weight compared to untreated mice infected with E. granulosus. This is particularly remarkable, as lower quantities of flubendazole were detected in plasma and within the cysts compared to albendazole treatments, suggesting a higher potency of flubendazole (Ceballos et al., 2011). Finally, it could be demonstrated that oxfendazole (60 mg/kg) alone and in combination with praziquantel was able to decrease protoscolex viability and reduce the diameter of liver and lung cysts in sheep naturally infected with E. granulosus following short-term treatment regimens (⩽6 weeks) (Gavidia et al., 2010). Notably, triclabendazole has shown potency in vitro and it would be interesting to see if it is as successful as the other benzimidazoles in vivo (Richter et al., 2013). 6 Conclusion We have summarized drug repurposing efforts against a wide array of helminth infections. Drug repurposing is an important strategy, in particular for these neglected tropical diseases since drug discovery and development has languished in this field. Moreover, the time, effort and resources saved renders drug repurposing a smart and ethical choice, as it can result in faster and more affordable access to new medicines. Several dozen compounds were identified by systematically searching the literature and presented in our review. This rather low number mirrors the challenges helminth drug discovery efforts still face. In vitro screens are often low throughput and cumbersome, as described for the T. muris assay (Wimmersberger et al., 2013). Furthermore, we lack adequate animal models; prominent examples are the two most important human filarials O. volvulus and Wuchereria bancrofti, and the problematic L. loa. As such, it is currently not possible to test drugs on these species directly in vivo and most testing is conducted with infection models from analogous animal filariasis infections, such as O. ochengi in cattle (Geary and Mackenzie, 2011). Interestingly, only a few compound classes have been investigated and several compounds have been studied against different helminth species. Antimalarial and anticancer compounds were most widely studied, though most of the successful anthelmintic compounds still come from the antimalarial and veterinary fields. Though most repurposed drugs still come from veterinary anthelmintic R & D, gradually compounds from fields such as antiprotozoal and anticancer research are being increasingly investigated (Fig. 1). As mentioned in this review these drug classes were not randomly selected but rather based on biological features (flukes have the same blood-feeding characteristic as Plasmodium spp.) and possible drug targets (identification of select protein kinases as being essential to schistosome development) of these parasites. With the availability of genomes, e.g. B. malayi or the L. loa genome, it is the hope of many that compound libraries containing drugs’ known modes of action can be matched in silico against helminth-specific gene targets, therefore providing potential drug candidates. In addition, unsurprisingly the benzimidazole class of compounds has been studied extensively. However, given that there is known cross-resistance between the various benzimidazoles in animal health, it is worth highlighting that a good clinical candidate arising from this group would rather replace albendazole and mebendazole than serve as backup. Overall, disappointingly few compounds have been pursued into clinical testing, which does little to alleviate the dry anthelmintic pipelines. A notable exception however is the trichuricidal drug oxantel pamoate. As public–private partnerships continue to expand, new compounds and compound libraries have been made openly accessible. For example, recently the Medicines for Malaria Venture made available an open-source library consisting of 200 drug-like and 200 probe-like compounds, which was screened against S. mansoni in vitro and in vivo. Though the in vivo results were moderate at best, it did provide two potential new scaffolds to of interest: the N,N′-diarylurea and 2,3-dianilinoquinoxaline derivatives (Ingram-Sieber et al., 2014). This is an example illustrating that closer collaborations between different research fields would be of great benefit in the field of drug discovery and development for helminth diseases. More of such collaborations are needed to take bench findings to the field and to turn field successes into affordable and useable medicines. Conflict of interest The authors state that they present no conflict of interest in writing this review.
Author and article information
Journal
Journal ID (nlm-ta): Antimicrob Agents Chemother
Journal ID (iso-abbrev): Antimicrob. Agents Chemother
Journal ID (hwp): aac
Journal ID (pmc): aac
Journal ID (publisher-id): AAC
Title:
Antimicrobial Agents and Chemotherapy
Publisher:
American Society for Microbiology
(1752 N St., N.W., Washington, DC
)
ISSN
(Print):
0066-4804
ISSN
(Electronic):
1098-6596
Publication date
(Electronic preprint):
1
June
2020
Publication date
(Electronic):
22
July
2020
Publication date Collection:
August
2020
Publication date PMC-release: 22
July
2020
Volume: 64
Issue: 8
Electronic Location Identifier: e00341-20
Affiliations
[a
]State Key Laboratory of Cellular Stress Biology, School of Life Sciences, Xiamen University,
Xiamen, Fujian, China
Author notes
Address correspondence to Yanhai Wang,
wangyh@
123456xmu.edu.cn
.
Citation Cheng Z, Xu Z, Tian H, Liu F, Li X, Luo D, Wang Y. 2020. In vitro and in vivo efficacies of the EGFR/MEK/ERK signaling inhibitors in the treatment of alveolar echinococcosis. Antimicrob Agents Chemother 64:e00341-20. https://doi.org/10.1128/AAC.00341-20.
Author information
Yanhai Wang
https://orcid.org/0000-0002-0002-2467
Article
Publisher ID: 00341-20DOI: 10.1128/AAC.00341-20
PMC ID: 7526812
PubMed ID: 32482675
SO-VID: 03d61124-11c7-4ba3-98b8-013331f7430c
Copyright © Copyright © 2020 Cheng et al.
License:
This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.
History
Date
received
: 21
February
2020
Date
revision requested
: 30
April
2020
Date
accepted
: 25
May
2020
Page count
supplementary-material: 1,
Figures: 5,
Tables: 0,
Equations: 0,
References: 37,
Pages: 10,
Words: 5834
Funding
Funded by: National Parasitic Resources Center;
Award ID: NPRC-2019-194-30
Award Recipient
:
Zhe Cheng
Funded by: National Natural Science Foundation of China (NSFC), https://doi.org/10.13039/501100001809;
Award ID: 81772211
Award Recipient
:
Zhe Cheng
Funded by: National Natural Science Foundation of China (NSFC), https://doi.org/10.13039/501100001809;
Award ID: 81572018
Award Recipient
:
Yanhai Wang
Funded by: National Natural Science Foundation of China (NSFC), https://doi.org/10.13039/501100001809;
Award ID: 81871679
Award Recipient
:
Fan Liu
Categories
Subject:
Experimental Therapeutics
Custom metadata
cover-date August 2020
ScienceOpen disciplines: Infectious disease & Microbiology