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Molecular & Biochemical Parasitology 195 (2014) 23–29

Contents lists available at ScienceDirect

Molecular & Biochemical Parasitology

eview

chistosomiasis control: praziquantel forever?

onato Cioli ∗, Livia Pica-Mattoccia, Annalisa Basso, Alessandra Guidinstitute of Cell Biology and Neurobiology, National Research Council, Rome, Italy

r t i c l e i n f o

rticle history:eceived 8 April 2014eceived in revised form 7 June 2014ccepted 13 June 2014vailable online 21 June 2014

eywords:

a b s t r a c t

Since no vaccine exists against schistosomiasis and the molluscs acting as intermediate hosts are not easyto attack, chemotherapy is the main approach for schistosomiasis control. Praziquantel is currently theonly available antischistosomal drug and it is distributed mainly through mass administration programsto millions of people every year. A number of positive features make praziquantel an excellent drug,especially with regard to safety, efficacy, cost and ease of distribution. A major flaw is its lack of efficacyagainst the immature stages of the parasite. In view of its massive and repeated use on large numbers

chistosomiasisraziquantelxamniquinerugsesistanceechanism of action

of individuals, the development of drug resistance is a much feared possibility. The mechanism of actionof praziquantel is still unclear, a fact that does not favor the development of derivatives or alternatives.A large number of compounds have been tested as potential antischistosomal agents. Some of them arepromising, but none so far represents a suitable substitute or adjunct to praziquantel. The research ofnew antischistosomal compounds is an imperative and urgent matter.

© 2014 Elsevier B.V. All rights reserved.

ontents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242. Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243. Molluscicides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 244. Enter praziquantel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

4.1. Efficacy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 244.2. Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 254.3. Operational convenience . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 254.4. Cost . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 254.5. PZQ resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 254.6. Mechanism of action of PZQ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 264.7. Summary considerations on PZQ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

5. Other drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265.1. PZQ derivatives. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265.2. Oxamniquine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265.3. Antimalarial drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275.4. Furoxan. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

6. Bioinformatics and high throughput screenings . . . . . . . . . . . . . . . . . . . . . . . . . . .7. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

∗ Corresponding author at: IBCN-CNR, Via Ramarini 32, 00015 Monterotondo (RM), ItalE-mail addresses: dcioli@ibc.cnr.it (D. Cioli), lpica@ibc.cnr.it (L. Pica-Mattoccia), a.bass

ttp://dx.doi.org/10.1016/j.molbiopara.2014.06.002166-6851/© 2014 Elsevier B.V. All rights reserved.

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

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y. Tel.: +39 9009 1355; fax: +39 06 9009 1288.o@ibc.cnr.it (A. Basso), alessandra.guidi@ibc.cnr.it (A. Guidi).

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. Introduction

For countless centuries, schistosomiasis has been, and still is, aerious scourge for people living in tropical and sub-tropical areasf the world [1]. Estimates of the total number of currently infectedeople are usually around 200 million, ranging from 193 [2] to 2073] million, while the number of people at risk of infection has beenalculated to be between 600 and 779 million [2,3]. The develop-ent of water resources in several tropical countries has probably

ontributed to maintain these figures at relatively constant – if notncreasing – levels in recent years [3]. Mortality has been estimatedt 280,000 deaths/year in Sub-Saharan Africa [4], while the over-ll level of disability caused by schistosomiasis has been recentlye-evaluated and extended to include previously neglected effectsf chronic infection like anemia, growth stunting and diminishedhysical and mental fitness [5]. It is customary to summarize theituation by saying that, among parasitic diseases, schistosomiasisanks second after malaria for the number of people infected andor its health impact.

Such being the general picture of the disease, the immediateonnection that comes to mind of anyone considering possibleools for its control, is undoubtedly the word “praziquantel” (PZQ).ndeed, this drug is used today so extensively and so exclusivelyhat alternative options appear as something to which lip service,ather than real investment, is usually paid. Yet, we must avoidhe trap of an excessive ‘medicalization’ of the problem and we

ust first of all remind ourselves that schistosomiasis is a diseasef poverty, so that its full control could be achieved, in princi-le, just by removing the socio-economic causes that lay at itsasis [6]. We should not forget that the eradication of schistoso-iasis from Japan was hardly dependent on drugs for its success

7]. The often-recommended ‘integrated approach’ to control schis-osomiasis should comprise, among other measures, sanitation,ater supply, ecological interventions and health education. In the

ransmission of schistosomiasis, snails are the intermediate hosts,ut the real vector is man: it is a baffling truism that if peoplevoided urinating or defecating in or near water bodies, transmis-ion would be automatically interrupted, at least in places whereon-human hosts are absent. However, the rapid spread – even

n the most deprived settings – of electronic communication toolseems to remain a largely underused opportunity to raise aware-ess of health problems.

When the costs of interventions are taken into account, there iso doubt that PZQ chemotherapy is today a very good buy, espe-ially when combined with the distribution of drugs against otherarasites. PZQ is unquestionably providing enormous benefits tondemic populations, since, among other things, it helps break theicious circle whereby poverty is a cause of disease and disease is aause of poverty. However, a more farsighted approach should con-emplate a substantial redressing of the balance from the presentverwhelming preponderance of mass drug distribution in favor ofther non-medical measures that may turn out to be more reward-ng in the long run.

. Vaccines

The major shortcoming of chemotherapy is that it does notrevent re-infection, thus requiring repeated treatments of peo-le living in endemic areas. Preventive vaccination would clearlyvercome this problem and the quest for a schistosomiasis vaccinectually represents a sizeable portion in the records of schistoso-

iasis research. Toward the end of the 1970s, optimism about the

easibility of a vaccine was encouraged by the finding that micexposed to irradiated cercariae exhibited over 80% resistance to

subsequent challenge with normal cercariae [8]. A number of

l Parasitology 195 (2014) 23–29

natural and recombinant antigens in various formulations weretested in an effort to identify the immunogen(s) active in irradi-ated cercariae, but none gave the expected high protection whentested in the mouse. WHO sponsored an independent trial to testsix antigens proposed by various research groups, but the resultswere flatly negative, since none of them reached the minimum goalof 40% protection in the mouse [9]. This may be construed as aturning point, since in subsequent years vaccine research main-tained a rather soft profile. Recent progress in the analysis of theschistosome genome, transcriptome and proteome, especially withregard to tegument proteins, has revived the hopes for a vaccine[10]. Undeniably though, the road to a safe, effective, long-lastingand cheap vaccine is still very long and frightfully crowded withuncertainties.

3. Molluscicides

Until the 1970s, molluscicides were at the forefront of schistoso-miasis control, to be later displaced by the newly available drugs forhuman use [11]. In spite of the adoption of a reasonably good chem-ical, niclosamide, the practice of mollusciciding has always facedserious problems. Local communities are understandably reluc-tant to accept that their water bodies turn yellowish while fishand other aquatic organisms undergo death and putrefaction [12].The molluscicidal effects are short-lived and a few surviving snailsare sufficient to subsequently re-populate treated sites. In addi-tion, the cost of chemicals is far from negligible, especially for largewater bodies. Today, the consensus seems to be that only underspecial circumstances focal mollusciciding may be recommendedas an adjunct to chemotherapy and other measures.

In spite of a substantial standstill in the practice of chemicalsnail control, a flourishing of reports has appeared over the years inthe literature, regarding plant-derived molluscicides that could bepotentially developed at the local level [13]. None of the proposedproducts, however, has been able, so far, to overcome the challengesof high efficacy and mass production.

On a related topic, snail control has been attempted using preda-tory or competing organisms like fish, prawns or different snailspecies [14], but practical applications of this interesting approachare as yet unavailable.

4. Enter praziquantel

The early events in the development of PZQ have been repeat-edly reviewed [15–17]. A series of compounds synthesized atMerck, Germany, in a project designed to find new tranquillizers,were passed on to Bayer to be screened for anthelmintic activity.The astonishing fact is that the screening for antischistosomal activ-ity of the initial compounds and of over 400 subsequently testedderivatives was carried out using mice infected with S. mansoni,complemented with in vitro observation of whole parasites [18].Yet, the selected product, PZQ, is such a highly optimized com-pound that it is still unsurpassed for safety and antiparasitic efficacyamong countless chemicals (analogs and otherwise) that have beentested up to this day.

The reasons for PZQ success can be classified under four mainheadings: efficacy, safety, operational convenience, price.

4.1. Efficacy

When measured by parasite egg excretion about four weeks

after treatment with 40 mg/kg, the effects of PZQ can be verybroadly summarized as 60–90% cure (no eggs in feces) and 80–95%average reduction in the number of excreted eggs in noncuredpatients. This can be regarded as a very good result, but it was

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ointed out [19] that 100% cure is seldom achieved and that thesegures are probably overestimated due to the relative insensitivityf diagnostic methods. The standard dose of 40 mg/kg may be a sub-urative one, but increasing the dose to 60 mg/kg does not seem tomprove results [20]. Alternative explanations are thus necessary.n important fact in the mode of action of PZQ is that schistosomesre susceptible for the first few days after infection, but then sus-eptibility decreases to a minimum around day 28, to resume againradually to a maximum after weeks 6-7 [21–23]. If an individualarbors immature parasites at the time of treatment – a situationost likely to occur in areas of intense transmission of infection –

ure will not be achieved. This is probably an explanation for a fewnstances where unusually low cure was obtained [24]. To obviatehe problem of low susceptibility of immature stages, it was pro-osed to administer a second dose of PZQ two weeks later [25],hen immature forms have progressed to maturity, a procedure

hat actually resulted in higher cure rates [26].As currently used, PZQ is a racemic mixture of two stereoiso-

ers, only one of which is endowed with antischistosomalroperties [18], while the other one contributes a portion of sideffects [27], is responsible for the unpleasant taste of the medica-ion [28] and represents 50% of the bulk of tablets that are oftenifficult to swallow for children. Current efforts to devise an eco-omically viable production of PZQ as a single enantiomer [29] willopefully result in a much improved drug.

In addition to its schistosomicidal activity, PZQ exerts remark-ble effects on a number of other trematodes (Opisthorchis,aragonimus, Fasciolopsis, Heterophyes, Metagonimus spp.), with theotable exception of Fasciola spp. [30]. PZQ is also effective againstost cestodes (Hymenolepis, Echinococcus, Diphyllobothrium, Tae-

ia spp.), with the exception of some larval cestode infections, likeydatid disease and sparganosis [30]. It may be mentioned that,ven before its introduction into human therapy, PZQ had beenarketed as a dog cestocide under the name Droncit®. The activity

f PZQ against these additional parasites clearly adds to its attrac-iveness in many areas where polyparasitism is often the rule.

.2. Safety

A massive amount of data has been collected over the yearsn the subject of PZQ safety, with regard to both immediate andelayed effects, and the overwhelming evidence points to the con-lusion that PZQ may be considered the safest of all anthelminticrugs. The same conclusion applies to different geographical sett-

ngs [31], different parasite species [32], different patient ages [33]nd conditions. Reversing previous practice, an informal WHO con-ultation concluded that pregnant and lactating women should alsoe treated, since the benefits of treatment clearly exceed hypothet-

cal risks [34].Short-term adverse reactions do occur in a significant number of

ases, but they are usually mild and of short duration. The frequencynd the severity of side effects is directly correlated with the pre-reatment intensity of infection, suggesting that a proportion ofhe reactions are likely to be due to dying schistosomes and to theelease of their products. Very rare instances of allergic reactionsave been reported, but only in one case allergy could be directlyttributed to PZQ on the basis of specific desensitization [35].

.3. Operational convenience

Over 42 million people were treated with PZQ in 2012, anmpressive figure, although it represents only 14.4% of the pop-

lation estimated to be in need of treatment [36]. Such a largecale distribution occurred largely through the school system andas made possible by the fact that PZQ is given as a single oralose, does not require direct medical supervision, does not produce

l Parasitology 195 (2014) 23–29 25

serious side effects and can be easily dosed on the basis of children’sheight [37]. On these premises, PZQ is generally administered bymass treatment without previous individual diagnosis. In high riskareas (50% prevalence of infection) all school-age children and alladults are targeted for treatment.

The current strategy of preventive chemotherapy envisages –where co-endemicity exists – the simultaneous administration ofmedication against lymphatic filariasis, onchocerciasis and soil-transmitted helminthiasis, a practice that represents a formidableboost to the cost efficiency of chemotherapy campaigns.

4.4. Cost

At the time of its introduction into human therapy, the costof PZQ represented a major obstacle to its mass distribution, butalready in 1983 the Korean company Shin Poong stepped into themarket with a new manufacturing process and brought about aconsiderable price reduction. Nowadays the average cost of PZQis around US$ 0.20 per treatment [38], while roughly the sameamount is spent for drug distribution. Merck KgaA has pledged tomake freely available up to 250 million tablets PZQ/year and othermanufacturers and partner organizations will make additional con-tributions, but, as stated in a recent WHO document, ‘the gap inavailability of praziquantel is huge and pledged amounts will notfill it in the near future’ [36].

4.5. PZQ resistance

The massive and exclusive use for many decades of a single drughas obviously raised legitimate fears that PZQ-resistant schisto-somes may sooner or later appear. While the experience with otheranti-infective agents justifies such fears on theoretical grounds,another theoretical consideration points to the opposite direction.As previously mentioned, only a minor proportion of people at riskactually receive treatment, thereby leaving ample ‘refugia’ [39] forsensitive parasites. Thus, it is sadly ironic that the very inability toprovide complete drug coverage may prevent further disasters.

Leaving aside theoretical considerations, one should askwhether any evidence for the development of PZQ resistance hasappeared so far in the field or in the laboratory.

Extremely low cure rates (18%) were reported in Senegal [24],but this occurred in a special focus of very intense transmission,suggesting that low cure may have been largely due to the presenceof many immature parasites (see Section 4.1).

Eggs obtained from treated and uncured Egyptian patients gaverise to schistosomes that showed decreased susceptibility whentested in the laboratory [40]. However, such insensitivity was onlyof moderate degree, was often unstable and investigations carriedout ten years later in the same area failed to show any hint of PZQresistance [41].

A number of travelers returning with schistosomiasis fromendemic areas had to be repeatedly treated (sometimes unsuccess-fully) to clear the infection. However, most of these were infectionscaused by S. haematobium (see [42] for a list) whose eggs areretained for a long time in tissues and diagnosis was rarely obtainedon the basis of egg excretion. In any event, no highly resistantschistosome isolate was obtained from these patients. Also, it ispossible that people coming from non-endemic areas may lack animmunological component that has been shown to contribute toPZQ activity in experimental animals [43].

Different geographical S. mansoni isolates were shown to differin their sensitivity to PZQ [44], with somewhat lower susceptibility

when coming from areas of previous PZQ usage, but differenceswere relatively modest and only detectable at low doses.

A laboratory strain of S. mansoni was repeatedly subjected tosub-lethal PZQ doses in subsequent generations and drug-selected

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arasites were shown to exhibit a decreased susceptibility withespect to unselected schistosomes [45]. This partial resistance wasonfirmed in other laboratories, but, again, it was of rather limitedagnitude. Genetic crosses between selected and unselected schis-

osomes indicated a co-dominant type of inheritance [46].More recently, PZQ selection was carried out at the infected snail

tage and the schistosomes thus obtained were reported to be lessensitive to PZQ at low and intermediate doses [47].

When all these pieces of information are taken together, it seemsafe to conclude that no overt occurrence of PZQ resistance hasppeared so far in the field and that reported sporadic cases ofecreased drug sensitivity may often lend themselves to alternativexplanations and are not of sufficient magnitude to undermine theublic health value of PZQ. Likewise, laboratory data are based onelatively minor differences in PZQ sensitivity, at least when com-ared with the solid resistance to another antischistosomal drug,xamniquine (see later).

There is obviously no guarantee that serious PZQ resistance willever appear; the worry has solid rational justifications and theuest for alternative drugs is becoming more urgent every day.

.6. Mechanism of action of PZQ

It is remarkable that after so many years of use and so manyillion people treated, the mechanism of action of PZQ is still

nsettled. However, the early effects exerted by the drug on thechistosome have been quite well described and can be summa-ized under three main headings: (i) calcium influx into wholearasites, (ii) muscle contraction and (iii) surface modifications15]. It is tempting to link these three phenomena into a singlehread, assuming that the key event is calcium influx, which in turnauses muscle contraction and tegument alterations. Evidence col-ected in recent years gives strong but not definitive support to thisypothesis [48].

It was initially observed that schistosomes possess two regu-atory � subunits of voltage-activated calcium channels, one of

hich can be defined ‘variant’ since it has an unusual structurend lacks two serine residues that constitute putative phosphory-ation sites in the ‘conventional’ subunit. When the variant subunit

as co-expressed in Xenopus oocytes together with a mammalian1 subunit, the resulting channel exhibited a novel PZQ sensitiv-

ty, consisting in increased Ca2+ currents in the presence of therug. A mutagenized variant subunit where the two candidatehosphorylation sites had been reconstituted, no longer exhibitedhis functional peculiarity. Conversely, a conventional mammalian

subunit mutagenized to lose the two phosphorylation sitesehaved functionally like the variant schistosome subunit. The ideahat Ca2+ channels containing the variant � subunit could be the tar-et of PZQ action was reinforced by the finding that other organismshat are susceptible to PZQ (Taenia solium, Clonorchis sinensis) alsoossess the variant � subunit.

An apparently unrelated observation was made in the planarianugesia japonica, which is able to regenerate both its head and

ts tail when amputated at the two ends. If these planarians werexposed to PZQ soon after a double truncation, the resulting regen-rated worms invariably presented two heads instead of a head and

tail [49]. Suppression of planarian calcium channel � subunits byNAi inhibited the double head phenomenon, although – contraryo expectation – inhibition was more pronounced when the con-entional subunit, rather than the variant subunit, was suppressed.hus, in spite of some conflicting details, even in this system theiological activity exerted by PZQ appears to be broadly depend-

nt on the activity of calcium channels. The analogy between thechistosome and planarian systems has been recently extended tohow that compounds inducing regenerative bipolarity are oftenndowed with antischistosomal properties and vice versa, implying

l Parasitology 195 (2014) 23–29

possible new research avenues to uncover antischistosomal drugs[50].

The schistosomicidal activity of PZQ can be partially inhibitedby some classical calcium channel inhibitors (nicardipine, nifedip-ine) and is completely abolished if schistosomes are pre-incubatedwith the actin depolymerizing agent cytochalasin D [51]. This wasinitially interpreted as an effect of cytochalasin D on calcium chan-nels (as documented in other mammalian systems), but it was latershown that PZQ-mediated calcium influx into the schistosomes isnot at all inhibited by cytochalasin D, rather it is largely increased[52]. This presents us with the puzzling situation in which schisto-somes inundated with a large amount of calcium fail to exhibit theexpected sequence of events leading to tegument disruption anddeath. A completely analogous coexistence of high calcium levelsand of undisturbed survival is presented by immature stages ofS. mansoni exposed to PZQ, to which they are largely insensitive.These phenomena seem to contradict the basic assumption thatcalcium is the key agent of PZQ schistosomicidal effects, but it mustbe admitted that our knowledge of the detailed molecular eventsconnected with PZQ activity are still rather crude [52].

A number of alternative hypotheses on PZQ mechanism of actionhave been put forward and are detailed in previous reviews [15,53].

4.7. Summary considerations on PZQ

PZQ is not a perfect drug. Its major fault is the lack of activityagainst immature schistosomes, a potential source of unsatisfac-tory results upon mass administration. Its racemic compositioncontributes undue amounts of side effects and complicates prac-tical administration. Its still unclear mechanism of action preventsthe rational design of improved analogs. Finally, even if PZQ werean intrinsically perfect drug, its being the only medication availableagainst schistosomiasis would urge the development of alternativedrugs. The fact that no clinically relevant resistance has appearedover thirty years after its introduction is another fantastic testi-mony to the qualities of PZQ, but cannot be taken as a guaranteefor the future. The combined high standards of safety and efficacymake PZQ a drug that is very hard to beat or even to match, but thechallenge cannot be shirked.

5. Other drugs

The number of compounds that have been tested as possibleantischistosomal agents is so large that it would be difficult toacknowledge them all. What follows is an incomplete mention ofthose compounds that, as of now, appear to hold some promise forthe development of new antischistosomal drugs.

5.1. PZQ derivatives

A relatively small number of PZQ derivatives have been syn-thesized and tested after the introduction of the parent drug intohuman use. No compound promised better performance than PZQand scanty information could be derived from structure–activityrelationships. Modifications of the aromatic ring generally led todecreased activity [54]; moderate activity against juvenile wormswas found in some compounds, but was not accompanied bysatisfactory performance against adults [55]; substitutions in thecyclohexyl group gave compounds with decreased activity [56].

5.2. Oxamniquine

Certainly not a new drug, oxamniquine (OXA) was used longbefore the introduction of PZQ, to treat many millions of peopleinfected with S. mansoni. The main limitation of OXA is that it is not

D. Cioli et al. / Molecular & Biochemical Parasitology 195 (2014) 23–29 27

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ctive against S. haematobium or S. japonicum, a fact that discour-ged its use outside of South America, where only S. mansoni exists.he restricted market of OXA prevented its competitive productionnd the expected price reduction, so that today PZQ is cheaper thanXA and has replaced it even in countries, like Brazil, where OXAas been for many years the successful cornerstone of control pro-rams. With respect to both safety and efficacy against S. mansoni,XA has proved to be at least as good as PZQ, sharing its advantagesf single oral administration and mild side effects [15].

Sporadic instances of OXA resistance observed in Brazil and theuplication of the phenomenon in the laboratory permitted the

solation of S. mansoni strains that were highly refractory to therug, surviving doses ∼500-fold higher than those that are lethalo sensitive parasites. Genetic crosses between sensitive and resis-ant schistosomes led to the conclusion that OXA resistance is aecessive trait controlled by a single autosomal gene [57]. This sug-ested the existence of a schistosome ‘factor’ that is essential toonvert the prodrug OXA into the active compound. A series of fur-her biochemical data (summarized in [58]) narrowed down theypothesis and predicted that a parasite sulfotransferase is thectivating enzyme and that a loss of its function is at the basis ofXA resistance [59]. This prediction was recently confirmed using

linkage mapping approach that unambiguously identified the S.ansoni sulfotransferase gene and permitted the crystallographic

nalysis of the enzyme and of its interaction with the drug [60].his represents the first complete elucidation of an anthelminticrug’s mechanism of action, and – most importantly – opens theay to a structure-based redesign of OXA to extend its activity to

he S. haematobium sulfotransferase analog. Thus, it is now a real-stic hope that a new broad-spectrum OXA may represent the longought-after partner/substitute of PZQ.

.3. Antimalarial drugs

Derivatives of artemisinin are known for their antimalarialctivity, but have also been found to possess antischistosomalroperties. In general, these types of compounds have the notable

stages of S. mansoni life cycle. Yellow boxes indicate different approaches that caniquine are not active on immature worms, whereas artemisinins and antimalarials

characteristic of being more active against the immature schisto-some stages than against the adults – just the opposite of PZQ –a feature suggesting combined treatments as their ideal utilization(Fig. 1). Results from clinical trials show that artesunate alone giveslower cure rates than PZQ, while a combination of an artemisininderivatives plus praziquantel is more effective than PZQ alone [61].Taking advantage of the activity of artemisinins on early stages ofinfection, a prophylactic approach consisting of the administrationof repeated doses of these drugs proved to confer significant pro-tection when compared to placebo [61]. A limitation to the use ofartemisinins against schistosomiasis consists in the risk that thismay favor the development of drug resistant plasmodia in areas ofcoendemicity.

Artemisinins contain an endoperoxide bridge that is implicatedin their mechanism of action. Synthetic endoperoxide-containingcompounds are currently the object of active research and somepromising leads have been identified that are effective against bothadult and immature schistosomes [62].

Another antimalarial drug that was found to possess antischis-tosomal activity is mefloquine [63]. As with artemisinins, activityagainst immature schistosomes is higher than against adults.Mefloquine derivatives are currently under investigation.

5.4. Furoxan

While the antioxidant defenses of vertebrates are largelydependent on two enzymes, glutathione reductase and thiore-doxin reductase, schistosomes rely on a single multifunc-tional selenocysteine-containing enzyme, thioredoxin–glutathionereductase (TGR) [64,65]. This enzyme is essential for parasite sur-vival and has been the target of extensive high throughput screensleading to the identification of oxadiazole 2-oxides as a class ofpotential antischistosomal agents [66]. One of these compounds,

furoxan, showed in vitro activity against adult and juvenile wormsat �M concentrations, was highly effective in vivo when adminis-tered once daily for 5 days by intraperitoneal injections and had atoxicity slightly higher than PZQ for mammalian cells.

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. Bioinformatics and high throughput screenings

Recent advances in genome sequencing and the availability ofunctional databases have become essential prerequisites and com-lements for any large scale investigation of parasite targets andotential drugs. The creation of a ‘TDR targets database’ well exem-lifies this trend [67].

Along similar lines, the differential analysis of schistosomeranscripts before and after exposure to PZQ has been used as aool to identify drug targets [68].

The successful exploitation of high throughput screening ofompound libraries using a defined molecular target (typically annzyme) has been exemplified above with regard to furoxan [64].uch defined targets, however, are not commonly available andesort is made to whole organisms in vitro. In this case, larval stagesre preferred to adult parasites because they are more easily avail-ble in large numbers, but one has to take into account the differentrug susceptibility of different life cycle stages. Screening can beased on various methods of parasite labeling or even on the auto-atic detection of morphological changes [69].

. Concluding remarks

It is possible that PZQ may remain the antischistosomal drugf choice for many additional years. However, since the loomingevelopment of resistant parasites would represent an enormousisaster for millions of people, it is imperative that alternative inter-ention tools be actively researched and promptly developed.

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