Impact of Echinococcus
Echinococcosis, caused by larval stages of Echinococcus (Cestoda, Plathelminthes), is a life-threatening disease affecting humans and livestock. Four distinct species within the genus Echinococcus have been identified, i.e. Echinococcus multilocularis, E. granulosus, E. vogeli and E. oligarthrus (Thompson, Reference Thompson and Thompson1986). At present, seven to nine species are described (Nakao et al., Reference Nakao, McManus, Schantz, Craig and Ito2007; Varcasia et al., Reference Varcasia, Garippa, Pipia, Scala, Brianti, Giannetto, Battelli, Poglayen and Micagni2008). All species are potentially zoonotic, two being of significant medical importance, namely E. multilocularis (small fox tapeworm) as the most pathogenic, and E. granulosus (dog tapeworm) as the most common (Rausch, Reference Rausch, Thompson and Lymbery1995; McManus et al., Reference McManus, Wenbao, Jun and Bartley2003). Echinococcus multilocularis infection causes alveolar echinococcosis (AE) in intermediate hosts and humans, and is restricted to the northern hemisphere. The current incidence rate for Germany, 0.07/100,000 persons, is probably underestimated by a factor of 3–5 (Jorgensen et al., Reference Jorgensen, an der Heiden, Kern, Schöneberg, Krause and Alpers2008). In contrast, E. granulosus, the causative agent of cystic echinococcosis (CE), occurs worldwide (Schantz et al., Reference Schantz, Chai, Craig, Eckert, Jenkins, Macpherson, Thakur, Thompson and Lymbery1995) with hyperendemic areas in South America, North and East Africa, southern Europe and Central Asia (McManus et al., Reference McManus, Wenbao, Jun and Bartley2003). Both, AE and CE are neglected diseases, and emergence (or re-emergence), especially in developing countries, is likely (McManus et al., Reference McManus, Wenbao, Jun and Bartley2003; Eckert & Deplazes, Reference Eckert and Deplazes2004), with an increasing economic impact due to the necessity of life-long treatments (Torgerson, Reference Torgerson2003).
Biology of Echinococcus
The habitat of the adult worms is the intestine of their respective final host (dogs for E. granulosus, foxes, dogs and cats for E. multilocularis), where sexual reproduction and subsequent egg production take place. Faecal shedding spreads the eggs into the environment, where they are accidentally taken up by suitable intermediate hosts, such as small rodents for E. multilocularis, and cattle and sheep for E. granulosus. Humans represent an aberrant intermediate host that acquires disease through the accidental ingestion of eggs, with serious consequences. Eggs contain the first larval stage, the oncosphere, which actively penetrates the intestinal lining, and migrates via blood and lymphatic vessels to the target sites. Most affected organs in humans are the liver for E. multilocularis, and the liver, lung and other sites in the case of E. granulosus. There, these oncospheres develop into metacestodes representing the second larval stage and the disease-causing stage. Within these metacestodes, protoscolex development takes place in natural intermediate hosts. If an infected intermediate host is ingested by a suitable definitive host, the life cycle is concluded. Protoscolex development in humans has only rarely been described (Eckert et al., Reference Eckert, Thompson and Mehlhorn1983).
Metacestodes are fluid-filled vesicles that can be separated into two distinct structural entities, namely a cellular and an acellular compartment (see fig. 1). The outer acellular surface of the metacestode is formed by the laminated layer, a carbohydrate-rich structure synthesized by the parasite, which, in terms of thickness, is much more prominent in E. granulosus metacestodes (Gottstein & Hemphill, Reference Gottstein and Hemphill1997). In addition, E. granulosus metacestodes are surrounded by a very prominent host-derived fibrous capsule, the adventitial layer composed of host connective tissue. The laminated layer plays a crucial role in the survival strategy of the parasite by modulating immunological and physiological reactions on the part of the host (Gottstein et al., Reference Gottstein, Dai, Walker, Stettler, Müller and Hemphill2002; reviewed in Siles-Lucas & Hemphill, Reference Siles-Lucas and Hemphill2002; Walker et al., Reference Walker, Baz, Dematteis, Stettler, Gottstein, Schaller and Hemphill2004a; Gottstein et al., Reference Gottstein, Haag, Walker, Matsumoto, Mejri and Hemphill2006). The actual larval tissue is formed by the germinal layer, its distal part, the tegument, being directly associated with the inner surface of the laminated layer. The tegument is characterized by microvilli-like extensions termed microtriches, which protrude well into the matrix of the laminated layer, thus increasing the resorbing surface of the parasite. In addition, the germinal layer contains highly differentiated cell types, including connective tissue, muscle cells and glycogen storage cells, as well as many undifferentiated cells (Eckert et al., Reference Eckert, Thompson and Mehlhorn1983).
Morphology and ultrastructure of Echinococcus metacestodes. (A) A lower magnification view of an opened metacestode exposing the inner germinal layer (GL)-associated tissue, and the acellular laminated layer representing the parasite surface (LL). Scale bar = 2.5 mm. (B) A higher magnification of the GL. Scale bar = 380 μm. (C) TEM through the metacestode wall. The parasite tissue is composed of the tegument (Te), a syncytial layer adjacent to the LL, with numerous microtriches (arrows) protruding into the LL. The germinal layer is composed of different cell types such as connective tissue cells, muscle cells (mus), glycogen storage cells (glyc), and undifferentiated cells with a large nucleus and nucleolus (uc). Scale bar = 2.5 μm.
Typically, the fully mature E. granulosus metacestode (i.e. hydatid cyst) is a single-chambered or septated, but unilocular, cyst that shows expansive growth and causes thereby compression of neighbouring tissue, resulting in organ dysfunction and disease (Kern, Reference Kern2003, Reference Kern2006). In E. multilocularis infection, the metacestode represents a multi-vesicular structure that reproduces asexually, by exogenous formation and budding of daughter vesicles, which resembles progressive tumour-like growth (Ohbayashi et al., Reference Ohbayashi, Rausch and Fay1971; Ali-Khan et al., Reference Ali-Khan, Siboo, Gomersall and Faucher1983). This leads to the formation of a large and heterogeneous parasitic mass consisting of mostly peripheral actively proliferating sites and, in many cases, centrally located necrotic tissue. Metastasis formation may occur in other organs due to release of germinal layer cells into the blood or lymph system (Ali-Khan et al., Reference Ali-Khan, Siboo, Gomersall and Faucher1983; Eckert et al., Reference Eckert, Thompson and Mehlhorn1983; Mehlhorn et al., Reference Mehlhorn, Eckert and Thompson1983). Spontaneous cure of AE leading to calcified lesions is possible, but it is not known how commonly this occurs (reviewed in Gottstein & Hemphill, Reference Gottstein and Hemphill1997; Vuitton et al., Reference Vuitton, Zhang, Yang, Godot, Beurton, Mantion and Bresson-Hadni2006). In fact, mass screenings in endemic areas have shown that the number of established AE infections in humans is far lower than the number of humans exposed to the eggs of the parasite and therefore sero-positive (Rausch et al., Reference Rausch, Wilson, Schantz and McMahon1987; Bresson-Hadni et al., Reference Bresson-Hadni, Laplante, Lenys, Rohmer, Gottstein, Jacquier, Mercet, Meyer, Miguet and Vuitton1994; Bartholomot et al., 2002). Thus, innate or acquired immunity is able to control the parasite after infection. This opens the door for the development of appropriate immunotherapeutical tools.
Current therapies
Traditionally, treatment of echinococcosis relies on surgery and/or chemotherapy, depending on different factors such as metacestode size and location, viability status, the interaction between the expanding parasite and the adjacent host tissue, bacterial and fungal infection, and potential complications related to cyst rupture and spillage of protoscoleces (Kern, Reference Kern2003, Reference Kern2006).
In CE, radical resection of the cyst mass represents the traditional treatment strategy and is, in many instances, accompanied by chemotherapy. Protoscolicidal substances are often applied since there is a risk of spilling of cyst fluid containing protoscoleces, which would be responsible for metastasis formation (Stey & Jost, Reference Stey and Jost1993; Pawlowski, Reference Pawlowski, Anderson, Ouhelli and Kachani1997; Kern, Reference Kern2003, Reference Kern2006). PAIR (puncture, aspiration, injection, re-aspiration) is a technique introduced in the mid-eighties. It includes percutaneous puncture of the cysts under ultrasonic guidance, aspiration of substantial amounts of cyst fluid, injection of protoscolicidal substance (e.g. 95% ethanol), and re-aspiration of the fluid cyst content after 15–20 min. PAIR has been used in several hundred patients. Nevertheless, the efficacy and potential risks have not been fully evaluated, and more long-term studies are needed (Morris & Richards, Reference Morris and Richards1992; Brunetti et al., Reference Brunetti, Troia, Garlaschelli, Gulizia and Filice2004; Eckert & Deplazes, Reference Eckert and Deplazes2004).
For inoperable cases, chemotherapy with the benzimidazoles albendazole (ABZ), a broad-spectrum anthihelminthic, and mebendazole (MBZ) and the heterocyclic pyrazinoisoquinoline derivative praziquantel (PZQ) remains the only option. Pawlowski et al. (Reference Pawlowski, Eckert, Vuitton, Ammann, Kern, Craig, Dar, De Rosa, Filice, Gottstein, Grimm, MacPhearson, Sato, Todorov, Uchino, von Sinner, Wen, Eckert, Gemmell, Meslin and Pawlowski2001) have evaluated over 2000 well-documented inoperable cases of CE treated with benzimidazoles. When evaluated up to 12 months after initiation of chemotherapy, 30% of patients showed cyst disappearance, 50–70% exhibited cyst degeneration indicating improvement, and in 20–30% of patients E. granulosus metacestodes did not respond to chemotherapy (Pawlowski et al., Reference Pawlowski, Eckert, Vuitton, Ammann, Kern, Craig, Dar, De Rosa, Filice, Gottstein, Grimm, MacPhearson, Sato, Todorov, Uchino, von Sinner, Wen, Eckert, Gemmell, Meslin and Pawlowski2001). PZQ was proposed to be used alongside with benzimidazoles in CE-patients. PZQ exhibited a high efficacy against protoscoleces and metacestodes in animal experiments (Urrea-Paris et al., Reference Urrea-Paris, Moreno, Casado and Rodriguez-Caabeiro1999, Reference Urrea-Paris, Casado, Moreno and Rodriguez-Caabeiro2001), and the combined treatment with ABZ and PZQ given during the month prior to surgery increased the number of human patients with non-viable protoscoleces, as compared to therapy with ABZ alone (Cobo et al., Reference Cobo, Yarnoz, Sesma, Fraile, Aizcorbe, Trujillo, Diaz-de-Liano and Ciga1998).
For the treatment of AE, surgery is always accompanied by chemotherapy using benzimidazoles, which should last for at least 2 years post-surgery, and monitoring of patients should be continued for 10 years (Pawlowski et al., Reference Pawlowski, Eckert, Vuitton, Ammann, Kern, Craig, Dar, De Rosa, Filice, Gottstein, Grimm, MacPhearson, Sato, Todorov, Uchino, von Sinner, Wen, Eckert, Gemmell, Meslin and Pawlowski2001). Inoperable AE cases must undergo long-term chemotherapy, often life-long, which is based on ABZ and/or MBZ (Reuter et al., Reference Reuter, Jensen, Buttenschoen, Kratzer and Kern2000, Reference Reuter, Buck, Manfras, Kratzer, Seitz, Darge, Reske and Kern2004). Extensive animal experimentation and observations in human patients sufferering from AE have demonstrated that ABZ and MBZ exhibit a parasitostatic rather than a parasitocidal effect (El-On, Reference El-On2002; Reuter et al., Reference Reuter, Buck, Manfras, Kratzer, Seitz, Darge, Reske and Kern2004). Therefore, recurrence rates after interruption of therapy are high. Nevertheless, clinical studies have shown that chemotherapy has significantly increased the 10-year survival rate of inoperable or non-radically operated AE patients from 6–25% to 80–83% (Ammann & Eckert, Reference Ammann, Eckert, Thompson and Lymbery1995; Eckert & Deplazes, Reference Eckert and Deplazes2004).
Adverse reactions against benzimidazoles under long-term chemotherapy include elevation of transaminases, proteinuria, loss of hair, gastrointestinal disturbances, neurological symptoms (vertigo/dizziness), leucopenia, headache, abnormal liver biopsy, abdominal pain, fever, urticaria, thrombocytopenia, allergic shock (due to cyst collapse and liberation of E. granulosus cyst fluid) and bone marrow toxicity. A study comprised of 3282 echinococcosis patients treated with ABZ showed that most side-effects were associated with the gastrointestinal tract but no fatal cases involving ABZ therapy were described. In 3.8% of these cases, permanent discontinuation of treatment had to be undertaken (reviewed by Pawlowski, Reference Pawlowski, Anderson, Ouhelli and Kachani1997; Kern, Reference Kern2003). As suggested by animal experimentation, MBZ and ABZ may induce embryotoxic or teratogenic effects, and it is recommended that these drugs are not used for the treatment of pregnant women (Horton, Reference Horton1989, Reference Horton1997). Constant monitoring of drug serum levels is suggested in order to avoid toxic reactions.
Taken together, besides benzimidazoles, novel drugs are required.
Methods for in vitro and in vivo evaluation of anti-echinococcal drugs
Both in vitro and in vivo laboratory models have been used for drug evaluation (reviewed in Siles-Lucas & Hemphill, Reference Siles-Lucas and Hemphill2002). Historically, the primary assessment of anti-echinococcal drug candidates has often been performed in mice or gerbils by evaluating parasite mass and/or health parameters of the host. This has led to the extensive use of animal experimentation, and has often yielded inconclusive results. In vitro culture of Echinococcus metacestodes has subsequently proven to be a suitable tool for the primary assessment of drug susceptibility by counting damaged metacestodes under the light microscope, followed by ultrastructural analysis by scanning and transmission electron microscopy (see, for example, Ingold et al., Reference Ingold, Bigler, Thormann, Cavaliero, Gottstein and Hemphill1999). A rapid test for viability is eosin exclusion (Lawton et al., Reference Lawton, Walchshofer and Sarciron2001). Another method consists in sampling culture supernatant and measuring enzymes released from damaged metacestodes (e.g. Stettler et al., Reference Stettler, Siles-Lucas, Sarciron, Lawton, Gottstein and Hemphill2001). Furthermore, viability and proliferative capacity of metacestodes can be analysed by quantification of 14-3-3 gene expression (Matsumoto et al., Reference Matsumoto, Müller, Hemphill, Oku, Kamiya and Gottstein2006; Spicher et al., 2008a). In vitro cultures also represent an ideal model system for studies on drug uptake and respective metabolic changes imposed upon the parasite (Hemphill et al., Reference Hemphill, Stettler, Walker, Siles-Lucas, Fink and Gottstein2002a). In vitro chemotherapy studies on CE have mostly, but not exclusively, focused on protoscoleces, since these are easily cultured. Conversely, their differentiation into metacestodes is a time-consuming process and can easily take 4–6 months (Walker et al. Reference Walker, Baz, Dematteis, Stettler, Gottstein, Schaller and Hemphill2004a, Reference Walker, Rossignol, Torgerson and Hemphillb). More recently, optimized in vitro culture conditions have been developed by Brehm and co-workers (Spiliotis et al., Reference Spiliotis, Tappe, Sesterhenn and Brehm2004; reviewed by Brehm & Spiliotis, Reference Brehm and Spiliotis2008). These in vitro methods reduce the number of animal experiments, but do not render them completely obsolete. In order to assess long-term viability and infectivity, metacestodes treated with the best drug candidates are still injected into rodents (Hemphill et al., Reference Hemphill, Stettler, Walker, Siles-Lucas, Fink and Gottstein2002).
In vitro and in vivo studies with benzimidazoles
Since benzimidazoles, especially ABZ, are the most relevant chemotherapeutics for the treatment of AE and CE to date, most preclinical in vitro and in vivo studies have been performed with these compounds, with a focus on comparing the activities of different benzimidazole derivatives, and on different formulations and modes of application. In rodents, efficacies of oral administration are dependent on the duration of treatment and the age of the parasite. Efficacy rises with prolongation of the treatment, but is distinctly lower for infections that have been persisting for extended periods of time (Wangoo et al., Reference Wangoo, Ganguly and Mahajan1987). Increased doses produce better results, although clear parasitocidal effects are never completely achieved (Taylor et al., Reference Taylor, Richards and Morris1989). Moreover, phenomena related to drug resistance have been described (Morris & Taylor, Reference Morris and Taylor1990). Conflicting reports exist on the most suitable mode of administration of benzimidazoles. It was postulated that parenteral administration of benzimidazoles resulted in a higher efficacy than other routes in animals experimentally infected with E. multilocularis (reviewed in Siles-Lucas & Hemphill, Reference Siles-Lucas and Hemphill2002). Combinations of ABZ with other compounds were tested in order to obtain better treatment efficacies. For instance, synergistic effects were reported for combinations of ABZ with the dipeptide methyl ester Phe-Phe-OMe (Sarciron et al., Reference Sarciron, Walchshofer, Walbaum, Arsac, Descotes, Petavy and Paris1997). In addition, novel formulations of benzimidazoles, either as pro-drugs (Walchshofer et al., Reference Walchshofer, Delabre-Defayolle, Paris and Petavy1990), liposome-entrapped compounds (Wen et al., Reference Wen, New, Muhmut, Wang, Wang, Zhang, Shao and Craig1996) or colloidal, intravenously injectable formulations (Rodrigues et al., Reference Rodriguez-Fonseca, Amils and Garret1995) were tested in rodents and showed enhanced efficacy at lower doses than the parental compounds. However, these studies have not really been translated into clinical applications, with one exception. Chai et al. (Reference Chai, Menghebat, Wei, Deyu, Bin, Jincao, Chen, Xiong, Yiding, Xiuling, Dolikun, Guliber, Yanchun, Fanghua and Shuhua2004) reported on improved efficacy of ABZ emulsion compared to ABZ tablets or capsules for the treatment of liver CE. Experimental prophylactic therapy of E. granulosus protoscoleces was carried out as a model that would mimic spillage during surgery, by treating protoscoleces with PZQ (Urrea-Paris et al., Reference Urrea-Paris, Casado, Moreno and Rodriguez-Caabeiro2001) or a combination of PZQ and ABZ (Casado et al., Reference Casado, Urrea-Paris, Moreno and Rodriguez-Caabeiro2001) prior to injection into mice. Against E. granulosus infection in rodents, a combination of fenbendazol and netobimin (Garcia-Llanazares et al., Reference Garcia-Llamazares, Alvarez-de-Felipe, Redondo-Cardena, Voces-Alonso and Prieto-Fernandez1997) showed synergistic effects, allowing the administration of lower drug dosage. Oxfendazole, like ABZ, is a benzimidazole, used in veterinary medicine for the control of nematode infections, and has a similar antimicrobial spectrum but a longer half-life. Experimental treatments of naturally E. granulosus-infected sheep and goats suggested that oxfendazole may be as efficacious as ABZ, but does not require daily uptake of the drug (Blanton et al., Reference Blanton, Wachira, Zeyhle, Njoroge, Magambo and Schantz1998; Dueger et al., Reference Dueger, Moro and Gilman1999). Another benzimidazole derivative effective againt E. multilocularis metacestodes in vitro is methiazole (Reuter et al., Reference Reuter, Manfras, Merkle, Harter and Kern2006).
Mode of action of benzimidazoles
When in vitro cultures of metacestodes were treated with ABZ, microtubular ultrastructure was affected (Rubino et al., Reference Rubino, Fiori, Lubinu, Monaco and Cappucinelli1983; Ingold et al., Reference Ingold, Bigler, Thormann, Cavaliero, Gottstein and Hemphill1999) suggesting that ABZ interfered with tubulin polymerization (Lacey, Reference Lacey1990). Molecular genetics revealed that sensitivity to benzimidazoles in evolutionary distant organisms such as fungi, nematodes, platyhelmithes and various protozoa was correlated with the presence of specific alleles of β-tubulin genes (Driscoll et al., Reference Driscoll, Dean, Reilly, Bergholz and Chalfie1989; Katiyar et al., Reference Katiyar, Gordon, Mclaughlin and Edlind1994; Kwa et al., Reference Kwa, Veenstra, Van Dijk and Roos1995; Henriquez et al., Reference Henriquez, Ingram, Muench, Rice and Roberts2008), the substitution of Phe to Tyr in position 200 being sufficient for switching from benomyl sensitivity to resistance in nematodes (Kwa et al., Reference Kwa, Veenstra, Van Dijk and Roos1995). Molecular modelling of the putative ABZ binding site in ABZ-resistant Acanthamoeba β–tubulin revealed 13 residues, four of which showed sequence variation as compared to tubulins of sensitive organisms. Resistance was conferred when, besides Phe200, Phe167 was also replaced (Henriquez et al., Reference Henriquez, Ingram, Muench, Rice and Roberts2008). In E. multilocularis, cDNAs of three β-tubulin genes were identified, two of them having a Phe in position 200 (Brehm et al., Reference Brehm, Kronthaler, Jura and Frosch2000). The IC50 values of ABZ in organisms regarded as sensitive to ABZ was between 30 nm in the protozoon Giardia lamblia (Katiyar et al., Reference Katiyar, Gordon, Mclaughlin and Edlind1994, confirmed by own observations) and more than 10 μm in nematode larvae (Satou et al., Reference Satou, Koga, Matsuhashi, Koike, Tada and Nikaido2002). Clear-cut IC50 values for E. multilocularis metacestodes are not found in the literature. Metacestodes treated with nearly 40 μm albendazolesulphoxide (ABZSO) showed symptoms with increasing severity over time (e.g. Ingold et al., Reference Ingold, Bigler, Thormann, Cavaliero, Gottstein and Hemphill1999) and totally disintegrated only after 21 days (Reuter et al., Reference Reuter, Manfras, Merkle, Harter and Kern2006). This indicates that cytotoxic effects of ABZ (or its metabolites) due to tubulin depolymerization may be hampered in Echinococcus by: (1) the presence of an ABZ-insensitive β-tubulin complementing the sensitive isoform; (2) rapid ABZ catabolism; (3) toxicity of ABZ catabolites rather than ABZSO itself; and/or (4) a good regenerative power due to insensitive stem cells. Sensitivity of mammalian cells (carrying the benzimidazole-resistant allele 200-Tyr) lies between an IC50 around 0.25 μm in HL60 cells and 20 μm in Vero cells (nocodazole, another benzimidazole, is, however, 10- to 20-fold more toxic; Katiyar et al., Reference Katiyar, Gordon, Mclaughlin and Edlind1994). This suggests that ABZ may also bind with low affinity to mammalian β-tubulin, and/or the existence of another, unknown, mode of action of ABZ that may be responsible for ABZ side-effects in patients, as mentioned above. In this respect, it is noteworthy to mention that Xiao et al. (Reference Xiao, Feng and Yao1995) found the downregulation of enzymes involved in carbohydrate metabolism upon benzimidazole treatment of rodents bearing E. granulosus metacestodes.
Novel chemotherapeutical treatment options
Novel and improved therapeutical tools are needed in order to optimize treatment of CE and AE. They should have a better selectivity, thus a much greater therapeutic window than ABZ, and be parasitocidal rather than parasitostatic. Unfortunately, the pharmaceutical industry is not developing novel treatment options besides benzimidazoles against these neglected diseases. Therefore, novel chemotherapeutics have to be identified issuing from existing drugs by one of the following strategies: (1) in vitro testing of broad-spectrum anti-infective drugs, either in parallel with, or followed by, small animal experimentation; (2) in vitro testing of drugs inhibiting proliferation of cancer cells for their effects on the viability of Echinococcus metacestodes and protoscoleces.
Furthermore, employing the currently achieved genomic sequencing efforts, the conventional strategies (1) and (2) will be complemented by two molecular genetic and in silico approaches, namely (3) the exploitation of the similarities between the parasite and mammalian signalling machineries, with a special focus on targeting specific signalling receptors; and (4) the search for suitable molecular targets for compounds of known modes of action.
(1) Chemotherapeutical activities of anti-infective drugs
Besides benzimidazoles, promising compounds with in vitro protoscolicidal action are cetrimide (Frayha et al., Reference Frayha, Bikhazi and Kachachi1981), and the ionophore monensin (Rogan & Richards, Reference Rogan and Richards1986), but these drugs are rather ineffective against metacestodes. The imidazothiazole levamisole, an acetylcholine agonist, and the macrolide antibioticum ivermectin, a chloride-channel activator classically used against nematode infections, were shown to exhibit in vitro activities similar to benzimidazoles (Casado et al., Reference Casado, Rodriguez-Caabeiro, Jiménez, Criado and de Armas1989; Martinez et al., Reference Martinez, Perez-Serrano, Bernadina and Rodriguez-Caabeiro1999; but see also Reuter et al., Reference Reuter, Manfras, Merkle, Harter and Kern2006). In rodents infected with E. multilocularis metacestodes, mytomicin C, piperazine and quinolone derivates, alkylaminoethers, and propargylic alcohols exhibited parasitostatic effects, at either lower or comparable levels as benzimidazoles (reviewed in Siles-Lucas & Hemphill, Reference Siles-Lucas and Hemphill2002). PZQ has been used for the treatment of AE, but experimental data in animals have shown that the efficacy of PZQ against E. multilocularis metacestodes was not satisfactory (Marchiondo et al., Reference Marchiondo, Ming, Andersen, Slusser and Conder1994). Also, treatment of E. multilocularis-infected mice with alpha-difluoromethylornithine was not successful (Miyaji et al., Reference Miyaji, Katakura, Matsufuji, Murakami, Hayashi, Oku, Okamoto and Kamiya1993).
When E. multilocularis metacestodes were treated with the thiazolide nitazoxanide (NTZ), a broad-spectrum anthelminthic also effective against enteric bacteria, Giardia and Cryptosporidium (Hemphill et al., Reference Hemphill, Müller and Esposito2006), 0.3 μm caused a total disintegration after 21 days, 3 μm after 14 days and 30 μm after 7 days at most (Reuter et al., Reference Reuter, Manfras, Merkle, Harter and Kern2006). NTZ was thus more efficient in vitro than ABZ (see above). Ultrastructural analysis revealed that NTZ induced significant distortion of the germinal layer in vitro (Stettler et al., Reference Stettler, Fink, Walker, Gottstein, Geary, Rossignol and Hemphill2003) and severe damage in E. granulosus protoscoleces and the germinal layer of in vitro cultured E. granulosus metacestodes (Walker et al., Reference Walker, Rossignol, Torgerson and Hemphill2004b). In vitro studies on E. multilocularis and E. granulosus employing NTZ derivatives (see Esposito et al., Reference Esposito, Mueller and Hemphill2007; Hemphill et al., Reference Hemphill, Müller and Müller2007) showed that metacestodicidal and protoscolicidal activity of this class of drugs depends largely on the presence of the nitro-thiazole moiety (Stadelmann & Hemphill, in preparation). Echinococcus multilocularis metacestodes treated with 32 μm NTZ for 14 days were non-viable when introduced into susceptible mice for 5 months (Stettler et al., Reference Stettler, Fink, Walker, Gottstein, Geary, Rossignol and Hemphill2003). In another study, metacestodes treated with the same amount of NTZ were viable in gerbils (Reuter et al., Reference Reuter, Manfras, Merkle, Harter and Kern2006). In contrast to ABZ, in vivo studies in rodents showed little or no effects of thiazolides, most likely due to their rapid metabolism. In this respect, Stettler et al. (Reference Stettler, Rossignol, Fink, Walker, Gottstein, Merli, Theurillat, Thormann, Dricot, Segers and Hemphill2004) showed that NTZ, orally applied to E. multilocularis-infected mice, either alone or in combination with ABZ, exhibited a profound antiparasitic efficacy, with the ABZ/NTZ combination yielding the most promising results. Analysis of pharmacokinetics showed that the half-life of ABZ-sulphoxide, a metabolite of ABZ, increased upon application of ABZ in combination with NTZ (Stettler et al., Reference Stettler, Rossignol, Fink, Walker, Gottstein, Merli, Theurillat, Thormann, Dricot, Segers and Hemphill2004). Therefore, the increased efficacy observed in mice could be a result of both the direct effects of NTZ and ABZ on the parasite and the inhibition of ABZ catabolism by NTZ. Combination treatment with ABZ/NTZ at concentrations as low as 3 and 4 μm, respectively, for 3 weeks inhibited, however, re-growth of parasites during 8 months of drug discontinuation and, also, bioassay in gerbils did not result in viable parasite infections (Reuter et al., Reference Reuter, Manfras, Merkle, Harter and Kern2006). Thus, combined ABZ/NTZ treatment exhibited a parasitocidal effect. The mode of action and especially targets of NTZ in Echinococcus are unknown. In G. lamblia, a nitroreductase was identified by affinity chromatography and subsequent enzymological studies as a potential target (Müller et al., Reference Müller, Wastling, Sanderson, Müller and Hemphill2007b). Furthermore, recombinant protein disulphide isomerases from G. lamblia and N. caninum were inhibited by NTZ and other thiazolides (Müller et al., Reference Müller, Sterck, Hemphill and Müller2007a, Reference Müller, Naguleswaran, Müller and Hemphill2008a).
The polyene macrolide amphotericin B (AMB) is an antifungal compound binding to ergosterol in the cell membrane, thus causing membrane depolarization and cell damage by oxidative stress (Blum et al., Reference Blum, Perkhofer, Haas, Schrettl, Würzner, Dierich and Lass-Flörl2008). In a formulation as desoxycholate, it was shown to inhibit E. multilocularis larval growth in vitro and in human patients in vivo (Reuter et al., Reference Reuter, Merkle, Brehm, Kern and Manfras2003a, Reference Reuter, Buck, Grebe, Nüssle-Kügele, Kern and Manfrasb). Major drawbacks of AMB are its intravenous application mode and its nephrotoxic side-effects. Moreover, AMB is only parasitostatic (Reuter et al., Reference Reuter, Buck, Grebe, Nüssle-Kügele, Kern and Manfras2003b). Nevertheless, a few cases of progressive human AE have been treated with AMB, and prolonged application of AMB over months to years appears to be feasible, as side-effects have been milder than expected (Reuter et al., Reference Reuter, Buck, Grebe, Nüssle-Kügele, Kern and Manfras2003b). Itraconazole, an antimycotic with a similar mode of action as AMB, was also slightly effective against metacestodes, but only parasitostatic (Reuter et al., Reference Reuter, Manfras, Merkle, Harter and Kern2006).
Isoprinosine, an inosine derivative commercialized as an immunostimulant, affected viability of protoscoleces in vitro, thus not mediated by the immune system of the host (Lawton et al., Reference Lawton, Walchshofer and Sarciron2001).
Artemisinin and artemisinin-derivatives are widely used in malaria chemotherapy. In vitro testing on a number of artemisinin-derivatives showed that artesunate (AS) and dihydroartemisinin (DHA) at 40 μm caused considerable damage to metacestodes in vitro. However, in vivo treatment in mice was ineffective. Again, combination therapies of ABZ with DHA and AS, respectively, were slightly more efficacous than ABZ alone, but results closely missed statistical significance (Spicher et al., Reference Spicher, Roethlisberger, Lany, Stadelmann, Keiser, Ortega-Mora, Gottstein and Hemphill2008b).
(2) Studies on antiproliferative drugs
There are a number of links between growth of cancer cells and Echinococcus and other parasites (reviewed in Klinkert & Heussler, Reference Klinkert and Heussler2006), namely their proliferative capacity, the potential to modulate the immune response, the secretion of proteolytic enzymes in order to reach their target sites or organs, and the capacitiy of metastasis formation. Echinococcus multilocularis metacestodes behave like malignant tumours, and there is an association between the uncontrolled proliferation and growth and the overexpression in metacestodes of a family of proteins named 14-3-3 (Siles-Lucas et al., Reference Siles-Lucas, Felleisen, Hemphill, Wilson and Gottstein1998, Reference Siles-Lucas, Nunes and Zaha2001). 14-3-3 proteins are found in all eukaryotic cells and participate in protein kinase signalling pathways. They function as phosphoserine/phosphothreonine-binding modules and have an effect on phosphorylation-dependent events such as DNA-damage checkpoints and prevention of apoptosis (reviewed in Siles-Lucas & Gottstein, Reference Siles-Lucas and Gottstein2003). Some 14-3-3 proteins have been found to be aberrantly expressed in tumour cells, being either pro- or antitumorigenic. In fact, when Echinococcus 14-3-3 sequences were aligned with other 14-3-3 isoforms of other organisms, those overexpressed in metacestodes were grouped with the tumour-growth-related zeta-isoforms (Siles-Lucas et al., Reference Siles-Lucas, Nunes and Zaha2001). This indicates that antitumour agents interacting with 14-3-3-triggered pathways could have the potential to interfere in growth of Echinococcus metacestodes.
Doxorubicin, or hydroxyldaunorubicin, is a DNA-interacting drug widely used in chemotherapy, and is commonly used in the treatment of a wide range of cancers. The parasiticidal properties of doxorubicin against the metacestode of E. multilocularis were investigated after binding of that drug to polyisohexylcyanoacrylate nanoparticles, a colloidal biodegradable drug carrier. A reduction of the hepatic parasite development and a reduced viability of the metacestode were observed in mice injected with 5 mg/kg body weight, but 7.5 mg/kg body weight did not appear to be more efficient. Free doxorubicin or unbound nanoparticles had no antiparasitic activity (Liance et al., Reference Liance, Nemati, Bories and Couvreur1993).
Another class of antitumour agents with proven antiparasitic activities are isoflavonoids. Isoflavonoids are substances formed by plant tissue in response to physiological stimuli such as infectious agents, with reported anti-oxidant, antibacterial, antiviral and antifungal activity (Dakora & Phillips, Reference Dakora and Phillips1996). They are composed of a characteristic 15-carbon backbone ring structure connected by a heterocyclic pyrane (3-C) bridge (C6–C3–C6) (Reynaud et al., Reference Reynaud, Guilet, Terreux, Lussignol and Walchshofer2005), with the two aromatic rings generally containing a number of phenolic hydroxyl groups. Genistein, a major component of soya, is the most prominent isoflavonoid, and inhibits growth and metastasis of a number of cancer cell lines (breast, prostate, skin, colon). Genistein also stimulates the synthesis of transforming growth factor-β (TGF-β), which itself inhibits cancer cell proliferation (Messina, Reference Messina1999). Besides other targets, genistein acts on a number of signalling pathways, by functioning as a kinase inhibitor (tyrosine kinase, mitogen activated protein (MAP) kinase, ribosomal S6 kinase). Our own studies showed that genistein was highly effective against E. multilocularis metacestodes in vitro (Naguleswaran et al., Reference Naguleswaran, Spicher, Vonlaufen, Ortega-Mora, Torgerson, Gottstein and Hemphill2006). However, genistein has a disadvantage, in that it also exerts oestrogenic effects by binding to oestrogen receptor-β (Pike et al., Reference Pike, Brzozowski, Hubbard, Bonn, Thorsell, Engström, Ljunggren, Gustafsson and Carlquist1999), and this renders genistein unfavourable for long-term treatment applications. Binding to the oestrogen receptor-β has been proven to take place through the hydroxyl-group associated with the B-ring of the molecule. We have therefore tested a number of isoflavonoids in vitro, which do not carry this hydroxyl-group and therefore do not meet the steric requirements to bind to the oestrogen receptor-β. One of these compounds, Rm6423, exhibits pronounced antiparasitic activity against E. multilocularis metacestodes, as well as against E. granulosus metacestodes and protoscoleces (Naguleswaran et al., Reference Naguleswaran, Spicher, Vonlaufen, Ortega-Mora, Torgerson, Gottstein and Hemphill2006). Further, examination of culture medium revealed increased leakage of parasite proteins into the medium during treatment, and zymography demonstrated a loss in the activity of metalloproteases. The molecular basis of the efficacy of genistein and its derivative Rm6423 have not been elucidated, but these compounds could interfere in signalling, for instance through inhibiting the tyrosine kinase activity associated with, for example, the epidermal growth factor receptor identified in E. multilocularis (Spiliotis et al., Reference Spiliotis, Konrad, Gelmedin, Tappe, Bruckner, Mosch and Brehm2006).
2-Methoxyestradiol (2-ME), an endogenous metabolite of oestrogen with both anti-angiogenic and antitumour effects (reviewed in Schumacher & Neuhaus, Reference Schumacher and Neuhaus2001), was shown to downregulate the pro-tumorigenic 14-3-3-ζ-isoform in a number of cancer cell types (Kumar et al., Reference Kumar, Garcia, Orsborn, Levin and Slaga2003), an isoform also overexpressed in Echinococcus metacestodes (Siles-Lucas & Gottstein, Reference Siles-Lucas and Gottstein2003). Application of 2-ME (2–10 μm) to E. multilocularis metacestodes in vitro caused severe damage and downscaled 14-3-3 transcription levels. In vivo however, 2-ME had no significant effects, although a combination therapy comprised of ABZ and 2-ME produced slightly better results than ABZ alone (Spicher et al., Reference Spicher, Naguleswaran, Ortega-Mora, Müller, Gottstein and Hemphill2008a). The mechanism of action of 2-ME in cancer cells has been attributed to interference in microtubule stability and dysregulation of hypoxia-inducible factor (Attalla et al., Reference Attalla, Makela, Adlercreutz and Andersson1996; Klauber et al., Reference Klauber, Parangi, Flynn, Hamel and D'Amato1997; Mabjeesh et al., Reference Mabjeesh, Escuin, LaVallee, Pribluda, Swartz, Johnson, Willard, Zhong, Simons and Giannakakou2003), inducing cancer cells to undergo apoptosis via extrinsic and intrinsic pathways. It is not known how 2-ME exerts its effects on Echinococcus.
NTZ, previously introduced as an anti-infective drug, also inhibits the proliferation of colon cancer cells in vitro, probably by interfering with glutathione-S-transferase (GST) class π, an isoform overexpressed in many proliferating cells (Müller et al., Reference Müller, Sidler, Nachbur, Wastling, Brunner and Hemphill2008b). In E. granulosus and E. multilocularis, the only GSTs characterized so far have some sequence homologies to the mammalian class μ (Liebau et al., Reference Liebau, Müller, Lucius, Walter and Henkle-Dührsen1996; Fernandez et al., Reference Fernandez, Chalar, Martinez, Musto, Zaha and Fernandez2000). The catalytic properties of recombinant GST of E. multilocularis had, however, higher similarities to mammalian classes α and π, with, especially, a high conjugating activity on ethacrynic acid, an anticancer drug (Liebau et al., Reference Liebau, Müller, Lucius, Walter and Henkle-Dührsen1996). In principle, GSTs may have two opposite effects on drugs, namely by inactivating drugs or by activating ineffective pro-drugs. The latter effects have been employed as an anticancer drug strategy (Rooseboom et al., Reference Rooseboom, Commandeur and Vermeulen2004) and may be further developed as an anti-Echinococcus strategy.
Cyclosporin A (CA) is an inhibitor of the protein phosphatase calcineurin and acts as an antiproliferative for lymphocytes, employed mainly as an immunosuppressant after organ transplantation (Ozbay et al., Reference Ozbay, Karamperis and Jørgensen2007). CA also exhibits anti-echinococcal activities in mice. While administration of CA in five consecutive daily doses, beginning 2 days prior to infection with E. granulosus protsocoleces, resulted in significant reduction in cyst numbers and cyst masses measured at 20 weeks post-infection, no changes in cyst mass and numbers were noted when the drug was administered 18 weeks post-infection. Ultrastructural examination of the germinal membrane and laminated layer of late-treated E. granulosus revealed abnormalities in all cysts studied, whereas control and early-treated hydatids were normal (Hurd et al., Reference Hurd, Mackenzie and Chappell1993). In contrast to E. granulosus, CA did not have any antiparasitic activity against E. multilocularis infection in mice, and its immunosuppressive activity was shown to be more effective than its parasitostatic effect (Liance et al., Reference Liance, Bresson-Hadni, Vuitton, Lenys, Carbillet and Houin1992). Potential targets for CA in Echinococcus are unknown.
(3) The Echinococcus signalling machinery as a novel drug target
More recently, the excellent work of Brehm et al. has shed light on a number of developmental factors that E. multilocularis metacestodes share with other metazoans (for a review refer to Brehm et al., Reference Brehm, Spiliotis, Zavala-Gongora, Konrad and Frosch2006). These include signalling systems that employ receptor tyrosine kinases of the epidermal growth factor (EGF) (Spiliotis et al., Reference Spiliotis, Kroner and Brehm2003, Reference Spiliotis, Konrad, Gelmedin, Tappe, Bruckner, Mosch and Brehm2006), the insulin/insulin-like growth factor (Ins/IGF)-receptor families (Konrad et al., Reference Konrad, Kroner, Spiliotis, Zavala-Gongora and Brehm2003), and the surface serine/threonine kinases of the closely related transforming growth factor-β (TGF-β) and bone morphogenetic protein (BMP)-receptor families (Zavala-Gongora et al., Reference Zavala-Gongora, Kroner, Bernthaler, Knaus and Brehm2006). A cytokine that has significant homologies to mammalian EGF has been shown to be tenfold upregulated in E. multilocularis metacestodes cultured under conditions that promote growth and differentiation (Spiliotis et al., Reference Spiliotis, Kroner and Brehm2003). EmSkip, a novel member of the SNW/SKIP family of transcriptional co-regulators was found to be expressed in the Echinococcus metacestodes and protoscoleces during an infection of the intermediate host (Gelmedin et al., Reference Gelmedin, Zavala-Gongora, Fernandez and Brehm2005). EmSkip interacts with EmSmadA and EmSmadB, two TGF-β/BMP signal transducers of E. multilocularis (Zavala-Gongora et al., Reference Zavala-Gongora, Kroner, Wittek, Knaus and Brehm2003), indicating a role of this protein in TGF-β signalling processes in the parasite. In addition, downstream signalling elements of the MAP kinase cascade have been identified and characterized (Spiliotis & Brehm, Reference Spiliotis and Brehm2004; Spiliotis et al., Reference Spiliotis, Tappe, Bruckner, Mosch and Brehm2005, Reference Spiliotis, Konrad, Gelmedin, Tappe, Bruckner, Mosch and Brehm2006). The Echinococcus MAP kinase cascade factors share similarities, but also have differences, compared to their mammalian counterparts. The differences would represent prime candidate targets for the development of novel anthelminthic drugs. For instance, analysis of receptor activation has shown that the E. multilocularis insulin receptor EmIR interacts readily with insulin from the host. Moreover, TGF-β receptor EmTR1, and possibly also the EGF-receptor EmER, interact with their corresponding host ligands (reviewed in Brehm et al., Reference Brehm, Spiliotis, Zavala-Gongora, Konrad and Frosch2006). Thus, parasite and host have evolved means of communication that would largely influence the developmental biology of both parasite and host. These receptor–ligand systems certainly play a central role in host–parasite interaction processes, and thus represent interesting drug targets (Brehm et al., Reference Brehm, Spiliotis, Zavala-Gongora, Konrad and Frosch2006). Cancer research has generated an enormous number of compounds that interfere in the functional activities of homologous receptors or respective downstream kinases (for review see Sioud & Leirdal, Reference Sioud and Leirdal2007), and the challenge will be to identify those drugs, or respective derivatives, that inhibit these receptors, or the corresponding downstream enzymes, in a parasite-specific manner.
(4) In silico approaches
Mathis et al. (Reference Mathis, Wild, Boettger, Kapel and Deplazes2005) have been the first to exploit the current genomic sequence information to define a drug target in Echinococcus in silico, and subsequently confirmed their hypothesis experimentally. In bacteria, the ribosomes are important antibiotic targets, and macrolides such as erythromycin and clarithromycin are agents that bind to the nascent peptide exit tunnel near the peptidyltransferase centre of large subunit rRNA (Rodriguez-Fonseca et al., Reference Rodriguez-Fonseca, Amils and Garret1995). Higher eukaryotes carry a guanine at position 2058 of both cytoplasmic and mitochondrial rRNAs, and this modification at this position has been demonstrated to confer the resistance of eukaryotic cells to macrolide antibiotics. In contrast, the mitochondrial rRNA of E. multilocularis carries an adenine at sequence position 2058, which would be predictive for susceptibility (Sander et al., Reference Sander, Prammananan, Meier, Frischkorn and Bottger1997), while the nucleus-encoded rRNA is characterized by a guanine at 2058 (Mathis et al., Reference Mathis, Wild, Boettger, Kapel and Deplazes2005). Upon in vitro culture of E. multilocularis metacestodes with clarithromycin, parasites, as expected, exhibited severely impaired growth characteristics, presented morphologically altered mitochondria and displayed a lack of microtriches, all in a dose-dependent manner. Adult worms were also severely affected, lost their motility and displayed morphological alterations such as shortening and constriction of proglottids and increased vacuolization. This study (Mathis et al., Reference Mathis, Wild, Boettger, Kapel and Deplazes2005) is the first in Echinococcus to encourage the use of sequence-based in silico approaches for the exploitation of drugs, the mode of action of which is well studied at the molecular level and the corresponding target is precisely defined. However, a prerequisite for this is the availability of more comprehensive Echinococcus genome sequence information. In 2008, shotgun sequencing of the E. multilocularis genome (c. 270 MB) was completed (http://www.sanger.ac.uk/Projects/Echinococcus/). This opens the door for the identification of drug targets by affinity chromatography followed by mass spectrometry (MS)-based sequencing and for reverse genetics by overexpressing or silencing genes of interest. Transient transfections of primary cells of E. multilocularis have already been performed (Spiliotis et al., Reference Spiliotis, Lechner, Tappe, Scheller, Krohne and Brehm2008).
Concluding remarks
As outlined in this review, considerable efforts have been undertaken in order to improve the therapeutical options for the treatment of CE and AE. Benzimidazole-based treatments have considerably improved the prognosis of patients, but new developments are wanted since the current treatments are only parasitostatic and have numerous side-effects. Parasitocidal compounds in vivo have not been identified to date, but drugs with different modes of action, such as NTZ, AMB and oestradiol- and artemisinin-derivatives, having shown promising results in preclinical studies, may be tested in combination with ABZ or related benzimidazoles in order to develop a suitable parasiticidal therapy. So far, academic institutions provide a scientific basis for novel treatment options, but financial constraints constantly limit the further development of promising therapies. Therefore, considerably more input is needed by the pharmaceutical industry and governmental agencies, in order to provide solutions for these neglected diseases. Clearly, a medium- to high-throughput metacestode screening system, allowing preliminary in vitro assessments of novel drug classes and respective conclusions on structure–activity relationships, needs to be developed. Besides traditional screening methods, genomic approaches, focused on the discovery of the receptor–ligand interactions and associated signalling pathways that influence the parasite–host interactions, will provide new opportunities and promising targets for follow-up studies on novel therapeutical options.
Acknowledgements
The authors want to acknowledge the financial support by the Foundation der Schweizerischen Mobiliar Versicherungen, the Swiss Life Foundation, the Novartis Research Foundation, and the Swiss National Science Foundation (31-111780). J.M. is recipient of a Novartis Research Fellowship. We also thank Bruno Gottstein, Britta Stadelmann and Norbert Müller for helpful discussions and support.
