Human cystic and alveolar echinococcosis are helmintic zoonotic diseases caused by infections with the larval stages of the cestode parasites Echinococcus granulosus and E. multilocularis, respectively. Both diseases are progressive and chronic, and often fatal if left unattended for E. multilocularis. As a treatment approach, chemotherapy against these orphan and neglected diseases has been available for more than 40 years. However, drug options were limited to the benzimidazoles albendazole and mebendazole, the only chemical compounds currently licensed for treatment in humans. To compensate this therapeutic shortfall, new treatment alternatives are urgently needed, including the identification, development, and assessment of novel compound classes and drug targets. Here is presented a thorough overview of the range of compounds that have been tested against E. granulosus and E. multilocularis in recent years, including in vitro and in vivo data on their mode of action, dosage, administration regimen, therapeutic outcomes, and associated clinical symptoms. Drugs covered included albendazole, mebendazole, and other members of the benzimidazole family and their derivatives, including improved formulations and combined therapies with other biocidal agents. Chemically synthetized molecules previously known to be effective against other infectious and non-infectious conditions such as anti-virals, antibiotics, anti-parasites, anti-mycotics, and anti-neoplastics are addressed. In view of their increasing relevance, natural occurring compounds derived from plant and fungal extracts are also discussed. Special attention has been paid to the recent application of genomic science on drug discovery and clinical medicine, particularly through the identification of small inhibitor molecules tackling key metabolic enzymes or signalling pathways.
In human infections, established E. granulosus cysts (or metacestodes) can develop and reach the mature, fertile state. Protoscoleces are then produced from the germinative layer inside the cyst. Spillage of viable protoscoleces after spontaneous or traumatic cyst rupture or during surgical intervention can give rise to new cysts (recurrence). Drugs against CE have been tested both against the metacestode and against the protoscoleces. In AE patients the metacestode usually does not produce protoscoleces, and the majority of the studies carried out to assess the therapeutic efficacy of drugs against AE have been done in the larval stage of the parasite. However, some authors have also tested defined drugs against E. multilocularis protoscoleces isolated from in vivo metacestodes obtained in the murine model [10]. For both CE and AE, activity of the compounds can be assayed in vitro and in vivo, although the number of agents reaching human clinical trial has been very limited. Lately, drugs have also been tested in vitro against stem cells derived from the germinative layer of the metacestode [11,12], both for CE and AE. Measurement of drug activity against protoscoleces is mainly directed towards the identification of effective compounds to reduce the risk of CE recurrence after surgery. The parasitocidal effect of drugs against protoscoleces can be measured in vitro using simple procedures involving vital staining with eosin or other vital dyes [13]. Some authors combine this vital staining with the investigation of the ultra-structural changes originated after drug exposure as seen in electron microscopy [14], the measurement of indirect markers of parasite damage including nucleosomal fragmentation and apoptosis-related enzyme activities in treated protoscoleces [14], and, in few cases, the assessment of cyst formation capacity of in vitro treated protoscoleces after intraperitoneal injection into rodents, compared with non-treated parasites [15]. A novel movement-based assay has been recently developed for in vitro-drug screening using E. multilocularis protoscoleces cultured in microwell plates. Morphological effects caused by the active compounds tested are then directly measured and quantified by image analysis [10]. Assays against protoscoleces can also be done after the intra-cyst inoculation of the drug to test its scolicidal activity [16], and by the administration of the drug to rodent models shortly before or after intraperitoneal infection with viable protoscoleces to mimic accidental spillage in the peritoneal cavity during a surgical intervention [17]. In general, drug testing against protoscoleces in any of those modalities is of advantage to translate the results mainly to avoid secondary CE in patients. As we mention hereinafter, a number of drugs and compounds have shown good protoscolicidal activity. Thus, drugs against the metacestode are much more urgently needed. Drug activity can be measured in vitro against both E. granulosus s. l. and E. multilocularis metacestodes maintained in culture, although E. multilocularis is the preferred experimental model due to the relative simplicity in obtaining parasite material from experimentally infected mice and the feasibility of maintaining and multiplying in vitro cultured vesicles of the parasite compared to E. granulosus cysts (Fig 1) [5,18]. However, the in vitro vesicular model has some limitations regarding the extrapolation of the in vitro effects to the in vivo scenario. First, assessment of total loss of parasite viability after treatment (parasitocidal effect) is difficult. This has been most frequently approached by the study of macroscopic alterations (e. g. , shrinking of cysts or detachment of the germinative layer) [19], microscopical changes in the germinative layer (destruction of the cells in the layer) [20], or leaking of parasite-derived compounds to the culture medium and subsequent assessment of their associated enzyme (e. g. alkaline phosphatase, phosphoglucoisomerase) activity [5,21,22]. Although these assays correlate well with parasite damage, parasitocidal activity could only be totally proven after inoculation of the treated parasitic material into a rodent model, or, alternatively, re-culturing the material in vitro in order to prove the lack of parasite growth. Second, the development of the cysts in primary infections in natural hosts (after oral infection with parasite eggs) differs from the development of the cysts in vitro, both for CE and AE. For CE, cyst growth usually leads to the formation of the external laminated and adventitial layers of variable thickness [23]. In AE patients, cysts grow forming a stromatous mass [23]. In both cases, access of drugs to the living parasite cells is more limited than in the in vitro conditions. In addition, because cells growing in vitro are not the exact dissociated replicates of their in vivo counterparts, in vitro models do not take into consideration the potentially synergistic antiparasitic effect of the host' s immune system, which may, at least partially, explain some of the outcomes observed in in vivo models. Nevertheless, the in vitro vesicular model has shown to be very valuable for the high-throughput screening of compounds, specially applying defined assays that show to correlate well with the loss of parasite activity, e. g. the measurement of phosphoglucoisomerase (PGI) activity in the culture medium after drug exposure, avoiding the unnecessary use of experimental animals [24]. Nevertheless, concentration range of drugs tested in vitro should consider that bioavilability in vivo, both in plasma and intra-cyst, can be three to ten-fold lower than in the in vitro systems (e. g. , see “Albendazole” section). Additionally, toxicological and adverse effects should be taken into account when high-dose in vitro studies are to be translated to in vivo conditions. Similar limitations, although less pronounced, are found in the in vivo rodent models. These are usually based on the experimentally-induced development of the parasite after intraperitoneal injection of viable protoscoleces (for E. granulosus) or micro-cyst (for E. multilocularis) into rodents (secondary infection) [5]. Development of the parasite within the peritoneal cavity resembles more accurately the situation of a recurrence in CE natural hosts than that of a primary infection. However, this approach has some limitations when attempting to recreate a natural (usually primary liver) infection in the natural host of E. multilocularis. It should also be noted that intraperitoneal rodent models may be hampered by variations in the efficacy of systemic drug treatments. Differences in cyst location (e. g. , intraperitoneal vs. intraparenchymatous) may have important consequences in terms of parasite' s exposure and achievable tissue concentration of the drug. Additionally, the elected time point after infection to start the drug treatment is of importance for the outcome of the treatment [25]. In few cases, the treatment has been done directly in naturally infected animals for CE, such as sheep [26]. The in vivo models have been also used to assess parasite viability after in vitro treatment [27]. Assessment of parasite damage after treatment in in vivo models has been usually done by estimating the mean cyst number and, more frequently, the mean biomass weight of cysts developed in infected, treated rodents compared with those figures from infected, non-treated animals [13]. Occasionally, drug-induced cyst damage and scolicidal activity has been assessed macro- and microscopically in in vivo models [28]. When only cyst weight differences are found, cysticidal activity cannot be guaranteed and instead a parasitostatic effect could be attributed to the tested drug. Of special importance is the assessment of drug effectiveness against the stem cells of the parasite in AE, since the uncontrolled growth of the metacestode and the growing foci distant from the primary lesion are presumably propitiated by the stem cells present in the germinative layer of E. multilocularis [29]. The recent availability of the genome and transcriptome data for both E. granulosus and E. multilocularis is also of importance for the definition of new drugs that could target defined parasite-specific molecules [11,30,31]. Very few drugs have reached trials in patients. The best characterized drugs are BMZ, which have been use both against CE and AE for many years. Lately, recommendations on the use of BMZ for the stage-specific treatment of CE and AE patients have been produced [3]. Nevertheless, evidence-based data supporting specific dosage and duration of BMZ treatment, either for the prevention of recurrence in CE due to protoscoleces spillage or for the fully effective elimination of the parasite, are not available to date. For other drugs different from BMZ, assessment of treatment efficacy in patients has been usually done retrospectively (e. g. , for praziquantel) [32], and in a low number of patients with heterogeneous clinical pictures and management, precluding the extraction of sound conclusions and hampering the generalization of the obtained results [3]. A wide range of chemically synthetised molecules, including anti-virals, antibiotics, anti-parasites, anti-mycotics, immunomodulatory agents, and inhibitor compounds of key metabolic enzymes or signalling pathways have been evaluated in in vitro and in vivo experimental settings in order to assess their potential effect against human CE and AE. However, some of these synthetic compounds have been studied comparatively less thoroughly that their counterparts of the BZD family. Thus, their actual chemotherapeutic efficacy and safety of use remains unclear in many cases. A summary of these molecules and compounds, together with their therapeutic performance is presented in Table 3. Following, detailed information is given on the different compounds, their mode of action (when available), and effectivity against E. granulosus and E. multilocularis in vitro and in vivo at the different dosages and administration regimes assayed by different authors. While information on the use of BMZ against AE and CE has been compiled only from 2008, drugs and compounds different from BMZ are presented historically, allowing the reader to identify most of the agents that have been assayed to seek for their potential use in the treatment of human AE and CE. In the last few years, a growing number of plant- and fungus-derived products have been tested against CE and AE, seeking for alternative natural compounds for the effective treatment of both diseases. Nevertheless, the vast majority of these molecules have been exclusively assayed in vitro against E. granulosus protoscoleces, and only three of them have been evaluated for its activity in the murine in vivo model (Table 4). In general, these compounds are naturally occurring biocides with low toxicity, acting synergistically with synthetic compounds against pathogen resistant strains, and as such they can be used as general purpose disinfectants [189]. Although some of them have shown effects potentially similar to ABZ, none of them have been tested, alone or in combination, in CE or AE patients. Drugs assayed against CE and AE can be classified in scolicidal and/or cysticidal. Application of scolicidal compounds either intra-cyst or peri-interventionally is used to minimise the risk of recurrence in CE patients during surgical or percutaneous procedures. To be effective, scolicidal agents must deliver their effects in the shortest therapeutic time period without eliciting undesirable adverse events. In recent years several compounds have been proposed as candidate drugs to substitute the recommended 20% saline solution as scolicidal agent. From these, glucose 50%, cetrimide, and H2O2 have a rapid effect on protoscoleces, but should be further evaluated for potential cytotoxic effects before being safely recommended for human use. Biogenic selenium particles, thymol, and several plant extracts have also shown promising scolicidal properties, and are regarded as non-toxic compounds at the concentrations used to exert their chemotherapeutic activity. Nevertheless, the main drawback of most of those compounds lies in the highly variable procedures involved in their in-house preparation, an inherent feature that makes difficult the assessment of the results obtained in different studies. In this regard, thymol is a commercially available compound and as such could be easily used in standardized trials to prove its therapeutic performance, particularly in those clinical cases in which biliary communication of cysts could advise against intra-cyst injection of 20% saline or alcohol as scolicidal agents. The efficacy of scolicidal compounds at the systemic level (usually peri-operatively) has been far lesser investigated. In this specific clinical setting, candidate drugs should ideally combine high solubility and intra-cyst bioavailability with elevated scolicidal efficiency, easy administration regimen (preferably at low dose), and absence of side effects. Monensin, praziquantel, imanitib and 2-methoxyestradiol may well comply with most of these requirements, but further in vivo assays are needed to clearly demonstrate the adjunctive activity of these drugs at different regimes and also to prove their safety of use. The advantages of defining a good scolicidal agent rely in its use to avoid secondary CE in patients. Disadvantages of testing only the scolicidal activity of drugs is that a good scolicidal agent can show no activity against the metacestode, and that scolicidal drugs are of no use in AE patients. In spite of this, majority of drug testing studies against Echinococcus are still performed in vitro against protoscoleces. Drugs directed against the larval stages of E. granulosus and E. multilocularis should also share the same characteristics already mentioned for systemic scolicidal compounds. Unfortunately, most of the drugs assayed to date against CE and AE cysts do not fulfil these criteria. Many other factors may influence the parasitocidal activity of a given drug treatment against CE and AE. Particularly relevant is the time during the dosing interval of the administered drug, for which there is currently no consensus, not even for the best known compounds used until now, the members of the BMZ family. Other variables that could affect the effectivity of the treatment include the number, size, location, developmental stage and condition of cysts. Most of these variables are usually not considered during the chemotherapeutic evaluation process of a given compound. An additional and important variable affecting the outcome of drug treatment in CE and AE is the time post-infection in which drugs are used. In this respect, it has been shown by several authors that the in vivo treatment of CE and AE is more effective when applied at early post-infection times regardless of the drug used. This fact may explain, at least partially, the failure or limited success of therapies in CE and AE patients frequently reported in the literature, where treatments usually starts at a late, chronic stage of the disease. Similarly, the route of administration seems to be crucial for some compounds to exert their activity, since some drugs demonstrate parasitocidal properties in intraperitoneal administration but show little or no effect in more convenient regimes, e. g. , oral administration. Both CE and AE are still very much neglected diseases for which the current drug of choice is ABZ. Accessibility to ABZ is impaired by limited distribution and elevated cost not only in socioeconomically disadvantages areas, but also in a number of developed countries. Additionally, this compound seems to exert a parasitostatic, rather than a parasitocidal, effect against both parasites and no alternative drug is available for patients with AE who experienced severe side effects and cannot be treated with ABZ (or MBZ). This also seems to be the case for the vast majority of alternative drugs assayed to date, since in vivo assays have evidenced changes in cyst weight, but reduction in cyst number are rarely observed. Could ABZ or other drug from those that have already shown parasitostatic effects be parasitocidal under specific conditions? In an attempt to improve the intra-cyst bioavailability of ABZ, different formulations have been assessed aiming to increase the solubility, absorption, and stability of the drug. These improvements have translated into enhanced drug effectivity and should be further explored, e. g. by using nanoparticles that lead to increased intra-cyst drug levels, or novel drug enantiomers with higher bioavailability and activity. An additional strategy to overcome the problem of drugs’ solubility is represented by the salification of sulfynil-benzimidazoles with sodium hydroxide solution. The proposed two-step production of the enantiomers of RBZNa and TCBZSONa (i. e. enantioseparation of racemic sulfoxides by HPLC on chiral stationary phase and successive transformation of the benzimidazole scaffold into a sodium salt form) is simple and it seems to be suitable for implementation on a semi-industrial or industrial scale. The main benefit of using water-soluble RBZNa and TCBZSONa salts is the possibility to prepare novel anthelmintic formulations with higher levels of bioavailability, downscaling currently recommended human dosage, thus possibly decreasing side adverse events than those currently reported for RBZ, ABZ and TCBZ. A second line of investigation that seems promising is the use of therapies based on the combination of two or more agents, since some components have shown to act synergistically, e. g. together with ABZ, against the parasite, and importantly some of them have shown activity specifically against the stem cells of the parasite. Development of synergistic combinations of drugs can overcome toxicity and other side effects associated with high doses and/or long time dosage of single drugs. From those molecules assayed in in vivo models as an alternative for BMZ compounds, very few have reached clinical use. One example is nitazoxanide, which showed high activity in preliminary in vitro and in vivo studies, but when used in patients obtained results were discouraging. Clinical translation of drugs assayed in vivo has not been tackled systematically, and number of treated patients have been usually low and their clinical status too variable to extract robust evidence-based conclusions. Thus, there is still an urgent need for defining new compounds or improved formulations of those already assayed, and also for a careful design of clinical protocols that could lead to the draw of a broad international consensus on the use of a defined drug, or a combination of drugs, for the effective treatment of CE and AE both in complicated and non-complicated cases. Interestingly, data on the genome of both E. granulosus and E. multilocularis have been recently released [30,31]. These data have shown that the parasites display several sets of family molecules related both with the activity of already known drugs (e. g. , praziquantel) and also with the activity of potentially new drugs that could find their targets in specific parasite enzymes (e. g. , protein kinases). Reasons for the reduced efficacy reported for some drugs against larval stages and potential new targets could be extracted from these genomic data. An orphan drug is defined as a compound that has been developed specifically to treat a rare medical condition. It is easier to gain marketing approval for an orphan drug, and there may be other financial incentives to encourage the development of compounds which might otherwise lack a sufficient profit motive. Despite the fact that both CE and AE are classified as orphan diseases (http: //www. orpha. net/consor/cgi-bin/index. php? lng=EN), none of the drugs tested so far against these diseases have been specifically developed against them, but have previously been licensed based on the activity demonstrated against other infectious and non-infectious conditions.
Human cystic and alveolar echinococcosis (CE and AE), caused by the larval stages of the helminths Echinococcus granulosus and E. multilocularis, respectively, are progressive and chronic diseases affecting more than 1 million people worldwide. Both are considered orphan and neglected diseases by the World Health Organization. As a treatment approach, chemotherapy is limited to the use of benzimidazoles, drugs that stop parasite growth but do not kill the parasite. To compensate this therapeutic shortfall, new treatment alternatives are urgently needed. Here, we present the state-of-the-art regarding the alternative compounds and new formulations of benzimidazoles assayed against these diseases until now. Some of these new and modified compounds, either alone or in combination, could represent a step forward in the treatment of CE and AE. Unfortunately, few compounds have reached clinical trials stage in humans and, when assayed, the design of these studies has not allowed evidence-based conclusions. Thus, there is still an urgent need for defining new compounds or improved formulations of those already assayed, and also for a careful design of clinical protocols that could lead to the draw of a broad international consensus on the use of a defined drug, or a combination of drugs, for the effective treatment of CE and AE.
medicine and health sciences
vesicles
tropical diseases
parasitic diseases
animal models
routes of administration
model organisms
pharmaceutics
experimental organism systems
drug administration
neglected tropical diseases
pharmacology
oral administration
cellular structures and organelles
drug metabolism
research and analysis methods
echinococcosis
mouse models
pharmacokinetics
helminth infections
cell biology
biology and life sciences
drug therapy
Progress in the pharmacological treatment of human cystic and alveolar echinococcosis: Compounds and therapeutic targets