Chemotherapy of schistosomiasis: present and future

Chemotherapy of schistosomiasis: present and future Conor R Caffrey Schistosomiasis is a chronic parasitic disease in tropical and subtropical regions and is associated with a variety of clinical syndromes that may lead to severe morbidity. Over the past 25 years, therapy and control of schistosomiasis has come to rely heavily on one drug, praziquantel (PZQ). This reliance is of concern should widespread treatment failure arise, particularly as measures are being undertaken to increase PZQ’s availability. This review summarizes the use, possible modes of action and limitations of PZQ, and recent attempts to derive synthetic analogs. Alternative artemisinin-based chemotherapies that have shown applicability in certain disease settings are also similarly examined. Looking forward, the review highlights some of the more experimental antischistosomals being evaluated (e.g. the trioxolanes), including those where knowledge of the parasite target (e.g. cysteine proteases and hemozoin formation) is more defined. Addresses Sandler Center for Basic Research in Parasitic Diseases, Byers Hall N508, University of California San Francisco, 1700 4th Street, San Francisco, CA 94158-2330, United States Corresponding author: Caffrey, Conor R ([email protected])

Current Opinion in Chemical Biology 2007, 11:433–439 This review comes from a themed issue on Orphan Diseases Edited by Kip Guy Available online 24th July 2007 1367-5931/$ – see front matter Published by Elsevier Ltd. DOI 10.1016/j.cbpa.2007.05.031

hepatic and intestinal schistosomiasis, is distributed in Africa, the Arabian peninsula, and South America; S. haematobium, causing the urinary form of the disease, is found in Africa and the Arabian peninsula; and S. japonicum, causing hepatosplenic and intestinal schistosomiasis, is found in parts of China and Indonesia. Water contamination with the parasite arises from eggs passed in the feces or urine that then hatch to release the free-swimming miracidium (pl. miracidia). This then invades the intermediate snail host, and after six to eight weeks of intense asexual multiplication (S. mansoni), the larval form (cercaria; pl. cercariae) escapes to await human contact. Cercariae actively invade skin, transform to schistosomula, and then migrate through the blood vasculature to the final maturation site (the hepatic portal and mesenteric venous systems for S. mansoni and S. japonicum, and the blood vessels of the urogenital system for S. haematobium) where the worms mate, mature, and commence egg-laying. Not all the eggs, however, escape, and with schistosomiasis mansoni and japonica, many are trapped in the liver to elicit an inflammatory reaction manifested by hepatomegaly—a condition seen primarily in children and adolescents [4]. Subsequently, the disease progresses to the chronic stage that involves massive and diffuse collagen deposition in the liver. This inexorable fibrosis, may, over the course of years, lead to occlusion of the hepatic portal vein, portal hypertension, gastrointestinal varices, and splenomegaly [4]. Apart from these severe manifestations of disease, schistosomiasis is more often associated with subtle, and perhaps, grossly underestimated [5] morbid effects such as physical and cognitive impairment, anemia, abdominal pain, and an intolerance of physical activity. Chronic schistosomiasis haematobia is a risk factor for squamous cell carcinoma of the bladder [6].

Introduction The parasite and disease

The mainstay chemotherapy: praziquantel

As a cause of morbidity, schistosomiasis (bilharzia) is second only to malaria among the parasitic diseases [1]. Considered as one of a number of ‘neglected tropical diseases’ (NTDs) [2], schistosomiasis afflicts populations living close to water (particularly lakes, ponds, and irrigation canals) that is contaminated with the parasite—the same water that is also required for everyday activities such as cooking, washing, and the generation of income through agriculture and/or fishing. The disease is endemic in approximately 70 countries, and estimates for 2003 suggest that 207 million people are infected and 779 million at risk with 85% of the latter in Africa alone [3]. The etiological agent of the disease is the Schistosoma bloodfluke, and three species account for most of the diseases occurring in humans: Schistosoma mansoni, causing

In the absence of a vaccine, efficient vector control, and water sanitation, the treatment and control of schistosomiasis relies heavily on a single drug, praziquantel (PZQ; 2(cyclohexylcarbonyl)-1,2,3,6,7,11b-hexahydro-4H-pyrazino(2,1-alpha)isoquinolin-4-one) [7]. A more expensive alternative, oxamniquine (6-hydroxymethyl-2-isopropylaminomethyl-7-nitro-1,2,3,4-tetrahydroquinoline), is also available, but its bioactivity is restricted to S. mansoni, and the drug has largely been replaced in favor of the more costeffective PZQ [8]. PZQ, on the contrary, is active against all schistosome species, and over 25 years of use in the field has established the drug as safe and effective—a single oral dose of 40–60 mg/kg being sufficient to achieve cure rates of 60–90% [7,9]. Today, PZQ is the recommended drug for disease treatment at either the community or individual

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level [10]—a recommendation underpinned by PZQ’s low cost of between 7 and 19 US cents per 600 mg tablet, depending on supplier [11]. The precise molecular target(s) of PZQ remains elusive; however, exposure of worms to PZQ causes a massive influx of calcium [12], contraction of the musculature [13], and disruption of the tegument [14]. The accumulated evidence, therefore, points to the involvement of calcium channels, and recent data with calcium-channel blockers and the actin depolymerization agent, cytochalasin D, support this notion [15]. Further, an unusual variant form of a calcium-channel beta subunit found only in schistosomes and some other flatworms may contribute to resistance to PZQ (reviewed in Reference [16]). Expression of this variant calcium-channel beta subunit in Xenopus oocytes conferred PZQ sensitivity to co-expressed but otherwise PZQ-insensitive, mammalian calcium-channel alpha1 subunits. Sensitivity could not be induced with the conventional S. mansoni beta subunit. It was determined that two consensus protein kinase C phosphorylation sites in the variant beta subunit had been altered and restoration of either of these by site-directed mutagenesis generated PZQ insensitivity [16]. However, tests on schistosome isolates less sensitive to PZQ did not identify sequence changes to the PKC sites [17], indicating that the decreased sensitivity to PZQ in these isolates must be due to some alternative mechanism(s). Most recently, PZQ has also been shown to inhibit nucleoside uptake by S. mansoni in vitro and that the calcium influx generated by PZQ might involve an activation of adenosine receptors that, in vertebrates, are known to modulate calcium channels [18]. Finally, the identification of actin as a receptor for PZQ by emplying PZQ bound to cellulose acetate membranes [19] could not be repeated [20]. The latter study used PZQ bound to Affigels 10 and 15 in a similar effort to trap parasite proteins, but found only non-specific binding to the Affigel matrix by both actin and myosin. One notable failing of PZQ is its decreased efficacy against immature parasites relative to adult worms. In fact, sensitivity of the parasite to the drug is biphasic: in mice, very young worms up to 7 days old are sensitive to PZQ, after which a period of decreasing responsiveness ensues to a low point at 28 days post-infection. Thereafter, sensitivity to PZQ is slowly restored being fully attained at about 40 days post-infection [21,22]. Similar data have also been reported for S. japonicum [23]. Therefore, in the context of its clinical use in areas of high transmission, a second treatment of PZQ, four to six weeks after the initial dose, is recommended to remove any parasites that have matured in the intervening period [24]. Commercially produced PZQ is a racemic mixture of levo ( ) and dextro (+) enantiomers due to the presence of an Current Opinion in Chemical Biology 2007, 11:433–439

asymmetric center at 11b (indicated by an asterisk in Figure 1). Only the levo enantiomer is schistosomicidal [25], and attention has been focused on the difficult chemical challenge to selectively and cost-effectively synthesize levo-PZQ in bulk. The World Health Organization has backed this goal as the pure levo form, at half the dose of the racemate, has a similar cure rate but fewer side effects [26]. A useful forum for discussion of this topic is hosted by The Synaptic Leap (www. thesynapticleap.org), an organization whose mission is to encourage open-source collaborative approaches to tackling infectious diseases, including schistosomiasis. For such a successful drug, or perhaps because of it, surprisingly little SAR has been reported for derivatives of PZQ against schistosomes, particularly as five positions are amenable to chemical modification (see Figure 1 [27]). Efforts are underway to redress this by testing R1 variants originally synthesized by Bayer and amination substituents at R4 (not covered in the original patent literature [27]). All variants were less active than the parent compound, but in some cases, not by much, and it is possible that with continued SAR, including examination of all the available positions, promising lead compounds may yet be identified. These attempts to produce novel PZQ analogs are set against the fortunate absence of clinically relevant resistance to PZQ in the field. Indeed, the current consensus is that the drug will continue to form the backbone of strategies to control disease morbidity [28]. That stated, schistosome isolates less sensitive to the drug have appeared in areas of high transmission in Egypt [29] (albeit a decade later no further drug failures were identified in the same areas [30]). Also, with the greater dissemination of PZQ through control programs such as the Schistosomiasis Control Initiative [31,32] and the future coordination of programs providing drugs for a number of worm diseases [2,33], it may be just a matter of time before resistance to PZQ emerges. Certainly, with other fluke diseases (e.g. clonorchiasis in South East Asia), PZQ has already become less effective [34,35]. Therefore, it would be ill-advised not to continue the search for alternative chemotherapies for use either alone or, perhaps, in combination with PZQ, while the going is good. Alternative chemotherapy: artemisinin derivatives

Among the potential alternatives to PZQ, most data have been generated on various semi-synthetic derivatives of the sesquiterpene lactone, artemisinin, but in particular, artemether and artesunate (Figure 1), which are active against all species of schistosomes [36]. Artemisinin is found in the leaves of the sweet (Chinese) wormwood shrub, Artemisia annua, and its derivatives are now a vital cornerstone in the treatment and control of malaria, including chloroquine-resistant disease [37]. www.sciencedirect.com

Chemotherapy of schistosomiasis: present and future Caffrey 435

Figure 1

Structures of current anti-schistosomal drugs and experimental therapeutics discussed in the text.

The precise mechanism of action of the artemisinins against either malaria or schistosomiasis is unknown though it is thought to involve an interaction with hemin (the by-product of hemoglobin degradation) and the ironinstigated reduction of the endoperoxide bridge to produce carbon-centered free radicals that then alkylate parasite proteins [38]. Morphological effects on adult schistosomes include swelling and vacuolization of the tegument and damage to the female vitelline cells [39]. The anti-schistosomal effects of artemether and artesunate were first described in China for S. japonicum in the early 1980s, and it is there that these compounds are now approved for chemoprophylaxis (i.e. to prevent or slow the onset of patent chronic disease) of schistosomiasis japonica [40]. Numerous clinical trials conducted in the late 1990s with either artemether or artesunate (6 mg/kg once a week or every 2 weeks for up to 26 weeks) showed that the relative risk of developing a patent infection was decreased between 85 and 96% compared to placebo (summarized in Reference [39]). Elsewhere, www.sciencedirect.com

the protective efficacies of the artemisinins against S. mansoni or S. haematobium infection have been more variable but, nonetheless, encouraging in terms of reducing disease morbidity. Thus, clinical trials in West Africa between 2000 and 2004 with either long courses of artemether (6 mg/kg once every 3 or 4 weeks for up to 24 weeks) or artesunate, administered according to the protocols then prescribed for malaria monotherapy (e.g. 8 mg/kg once to three times a day over five days), yielded efficacies between 20 and 50% [41]. Most recently, 86–100% cure of light S. haematobium infection was achieved after treatment of small groups of children for falciparum malaria infection with the artemisinin-based combination therapy (ACT) of either amodiaquine/artesunate or sulfadoxine/pyrimethamine/artesunate [42]. The case for thoroughly evaluating the clinical potential of combining artemisinins and PZQ to treat schistosomiasis has been presented for some years now by Utzinger and Xiao and co-workers [36,39]. The argument stems primarily from the precedent that artemisinins are not Current Opinion in Chemical Biology 2007, 11:433–439

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only safe but are also fortuitously most active against those juvenile schistosomes least susceptible to PZQ [36,43]. Indeed, in a number of animal models, a combination of artemether or artesunate with PZQ is more effective in reducing worm [36] and egg burdens [36,44] than either compound alone. However, the data thus far accumulated from human trials are less clear on the benefits of an artemisinin/PZQ combination therapy over the standard use of PZQ alone. Thus, in Gabon (with S. haematobium), no significant additional cure rate was observed after PZQ/ artesunate therapy (81%) over PZQ (73%) alone (placebo was 20%), although egg production was decreased slightly more in the combination therapy [45]. In Senegal (with S. mansoni), although after 5 weeks the artesunate/PZQ therapy was more effective (69% cure rate) than PZQ alone (44%), by 12 and 24 weeks post-treatment this difference had been lost. Also, no difference in egg counts was measured at any time after treatment [46]. It seems, therefore, that more clinical trials are needed to determine whether artemisinin/PZQ combination therapy offers any significant advantages and even if so determined, whether the inevitable higher cost of such a combination therapy makes it practicable. Also, an important concern regarding the advancement of the artemisinins, either alone or in combination with PZQ, to specifically treat schistosomiasis, is the risk of driving artemisinin resistance in malaria in areas where both diseases are endemic [37,47]. That stated, artemisinin-based therapies for malaria are already widely available, and it is unknown how they might influence transmission and morbidity of schistosomiasis unless adequate surveillance is undertaken. Experimental chemotherapies: trioxolanes and alkyl-amino-alkane-thiosulfates

The artemisinins have proven fundamental in providing rapid treatment of uncomplicated and chloroquine-resistant malaria [37]. However, as a drug class they have drawbacks: a semi-synthetic manufacturing pathway, poor bioavailability, and short plasma half-lives [48,49]. It was logical, therefore, to search for and identify a class of fully synthetic endoperoxide alternatives that are relatively cheap, easy to synthesize, and offer better pharmacokinetics. Such is the case for the 1,2,4-trioxolanes (secondary ozonides or OZs [49,50]) one or more of which were, or continue to be, part of the Medicines for Malaria drug discovery portfolio; www.mmv.org). OZs are also bioactive against trematode flatworm parasites [51] and have been tested in animal models of schistosomiasis with promising results [52]. Single doses of 200 mg/kg of two compounds, OZ78 and OZ288 (Figure 1), decreased juvenile (21-day old) and adult (49-day old) worm burdens by between 72 and 95% in mice or hamsters infected with S. mansoni or S. japonicum. Encouragingly, the study identified that the OZ structural requirements necessary for trematocidal and anti-malarial activity are different, thereby offering the promise of mitigating the development of drug resistance to malaria [52]. Current Opinion in Chemical Biology 2007, 11:433–439

The alkylamino alkane thiosulfates (Figure 1) have a long history of research in Brazil [53] and are effective against mature S. mansoni infections (>50 days) in mice at single oral doses of between 400 and 800 mg/kg [54]. Preferential killing of female worms (64–100%) is a hallmark of these compounds with the consequence that fewer eggs are laid to induce organ pathology or to contribute to transmission. Though toxicity is a problem, it seems, nevertheless, worthwhile to further explore these chemically facile compounds to attempt to resolve this issue, as well as understand their pharmacokinetics and whether they are active against juvenile stages (for prophylaxis) [54]. Target-based drug discovery

To date, anti-schistosomal drugs have originated from industrial and academic phenotypic (whole organism) discovery programs, in the absence of firm data as to either the molecular target or mechanism of action. The approach has worked well, yielding effective and safe drugs in PZQ and the artemisinins. To multiply the opportunities for lead identification, however, targetbased drug discovery, based on a thorough understanding of the biochemistry of the parasite and its exploitable (and perhaps unique) molecular pathways, will be a powerful approach—an approach that is already bearing fruit [55,56]. Example of a molecular target: cysteine proteases

A primary research focus at this author’s institute is the application of cysteine protease inhibitors (CPIs) as therapy of parasitic protozoan diseases, including Chagas’ disease and African Sleeping Sickness [57] (http:// www.ucsf.edu/mckerrow/fruit.html). This has been recently extended to include helminths with the demonstration of schistosomicidal activity by the peptidomimetic, vinyl sulfone CPI known as K11777 (Figure 1). Upon intraperitoneal (IP) administration at 25 mg/kg twice daily for 14 days to mice harboring pre-patent S. mansoni infections, K11777 eliminated parasite eggs and decreased worm burdens by >88% [55]. Against mature infections, at 50 mg/kg twice daily for eight days, K11777 also had a pronounced affect on worm and egg burdens. Though the dosing regimens were longer than would be considered practicable for field use, the study is significant in illustrating how a CPI drug candidate, already in late-stage preclinical tests against one parasitic disease (Chagas’ disease) [58], might find an application in another disease setting based on the unifying concept that cysteine protease activity is vital to the survival of many parasites. Example of a mechanistic target: formation of hemozoin

In common with some other blood-feeding organisms, for example, Plasmodium and the reduvid bug, Rhodnius, schistosomes sequester hemoglobin-derived heme as hemozoin (beta-hematin) [59,60] in order to avoid the toxicity associated with free heme [61]. The anti-malarial, chloroquine www.sciencedirect.com

Chemotherapy of schistosomiasis: present and future Caffrey 437

Table 1 Desired product profile for new anti-schistosomal drugs (adapted from Reference [65])  Different chemical class than PZQ to offset development of resistance  Activity against all major species infecting humans  Bioactivity against all developmental stages in human host (PZQ has limited activity against immature parasites) including parasite eggs  Oral use and short course, preferably one dose  Safety equal to or better than PZQ  Safety in the absence of diagnosis in children and during pregnancy  Inexpensive (PZQ currently costs between 7 and 19 US cents)  A long shelf life under tropical conditions

and reasonably tractable procedure with schistosomes [56,72,73]. On the chemistry front, the establishment of drug development portfolios for other NTDs by organizations such as the MMV and the Drugs for Neglected Diseases initiative (www.dndi.org), together with the concomitant accumulation of target and scaffold-focused small-molecule libraries at academic, governmental, and industrial screening centers [65], should provide a repository of chemical jump-starts for novel schistosomicides. In this context, and as is ongoing at this institute, a standardized whole-organism screen will be central to generating useful lead compounds and optimizing structure–activity relationships.

Acknowledgements (CQ), is thought to prevent the accumulation of heme as hemozoin in malaria eventually leading to parasite death (reviewed in References [62,63]), and the hypothesis was, therefore, that disruption of the schistosome’s ability to do the same would be detrimental to parasite survival [64]. CQ, when administered daily at 50 mg/kg IP to patent S. mansoni infections in mice (42–49 days old) had little effect on worm or egg burdens. However, when administered every other day to mice with pre-patent infections (7–28 days post-infection) at 60 mg/kg IP, CQ was significantly prophylactic. Also, the reduction in worm and egg burdens was associated with a decreased accumulation of hemozoin in the parasite gut [64].

Conclusions In the 28 years since the first clinical trials, PZQ has been a tremendous success—a single dose therapy that is effective, non-toxic, and relatively cheap. The bar is, therefore, set very high for any new anti-schistosomal compound to compete with PZQ as a cornerstone of schistosomiasis control (Table 1) [65]. Nonetheless, PZQ has several short-comings, and there is an increased awareness that now is the time to identify new drugs, either to complement PZQ or replace it should it fail. This endeavor will advance on a number of fronts with improved biological and chemical tools for target identification and chemical lead prosecution. First is the availability of comprehensive EST datasets for both S. mansoni and S. japonicum [66–68] (http://verjo18.iq.usp.br/schisto/; http://verjo18.iq.usp.br/thiago/Agilent-schisto/cgi-bin/ agilentSearch.pl; http://compbio.dfci.harvard.edu/tgi/cgibin/tgi/gimain.pl?gudb=s_mansoni) and substantial (and improving) genome sequence information (http://www. sanger.ac.uk/Projects/S_mansoni/; http://www.genedb. org/genedb/smansoni/). These data together with information detailing the stage and sex-specificity of gene transcription will be vital when choosing appropriate drug targets given the complexity of the schistosome life-cycle (summarized in Reference [69]) [70,71]. Target validation in some cases may be facilitated by RNA interference, which has gained a foothold as a cost-effective www.sciencedirect.com

My sincere thanks to Matthew Todd, School of Chemistry, University of Sydney, for access to his unpublished data regarding synthesis of PZQ analogs, James H McKerrow of this institute, and Daniel G Colley of the Center for Tropical and Emerging Global Diseases and the Department of Microbiology, University of Georgia, for critically reading the manuscript.

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