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Treatment with praziquantel (PZQ) has become virtually the sole basis of schistosomiasis control in sub-Saharan Africa and elsewhere, and the drug is reviewed here in the context of the increasing rate that it is being used for this purpose. Attention is drawn to our relative lack of knowledge about the mechanisms of action of PZQ at the molecular level, the need for more work to be done on schistosome isolates that have been collected recently from endemic areas rather than those maintained in laboratory conditions for long periods, and our reliance for experimental work mainly on Schistosoma mansoni, little work having been done on S. haematobium. There is no evidence that resistance to PZQ has been induced in African schistosomes as a result of its large-scale use on that continent to date, but there is also no assurance that PZQ and/or schistosomes are in any way unique and that resistant organisms will not be selected as a result of widespread drug usage. The failure of PZQ to produce complete cures in populations given a routine treatment should therefore solicit considerable concern. With few alternatives to PZQ currently available and/or on the horizon, methods to monitor drug-susceptibility in African schistosomes need to be devised and used to help ensure that this drug remains effective for as long a time as possible.

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The benzodiazepine Ro 11-3128 (methyl-clonazepam) presents several similarities with praziquantel with regard to its anti-schistosomal mode of action, since both drugs cause spastic paralysis, calcium influx and tegumental disruption in the parasites. In order to know whether the two compounds share the same binding sites in the schistosomes, we performed in vivo and in vitro competition experiments. We took advantage of the fact that Ro 11-3128 is active against immature Schistosoma mansoni (whereas praziquantel is inactive), and praziquantel is active against S. japonicum (which is insensitive to Ro 11-3128). An excess of praziquantel did not inhibit the activity of Ro 11-3128 against immature S. mansoni and an excess of Ro 11-3128 did not inhibit the activity of praziquantel against S. japonicum, suggesting that the schistosome binding sites of the two drugs are different. On the other hand, cytochalasin D, an agent known to perturb--among other things--calcium channel function, was capable of inhibiting the schistosomicidal activity of both praziquantel and Ro 11-3128, thus adding another element of similarity between the two anti-schistosomal agents. A similar, albeit partial, inhibition of the schistosomicidal activity of the two drugs was exerted by some of the classical calcium channel blockers. Taken together, these results suggest that praziquantel and Ro 11-3128, although binding to different schistosome receptor sites, may use the same basic anti-schistosomal effector mechanisms.

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The mechanism of action of praziquantel (PZQ), the drug of choice against schistosomiasis, is still unclear. Since exposure of schistosomes to the drug is associated with calcium influx and muscular contraction, calcium channels have been suggested as the target, although direct combination of PZQ with their subunits was never demonstrated. We report a hitherto unknown effect of PZQ, namely the inhibition of nucleoside uptake, as observed in living worms using radio-isotope labelled adenosine and uridine. This effect is clearly seen in schistosomes but is absent in mammalian cells in culture. Moreover it is a specific pharmacological effect seen exclusively with the active levo-R(-)stereo isomer of the drug, and is shared by at least one benzodiazepine having antischistosomal activity. This novel effect acquires significance given that schistosomes cannot synthesize purine nucleosides de novo. A possible relationship between this novel effect and the known action of PZQ on calcium channels is discussed, since adenosine is known to bind to specific receptors and to behave as an indirect antagonist of calcium release in mammalian cells. If calcium channels were correlated with adenosine receptors also in schistosomes, as they are in mammals, this would support the hypothesis that PZQ-induced calcium influx may be correlated to adenosine receptor blockade.

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Male and female schistosomes are generally assumed to form stable monogamous pairs for the whole span of their long existence in the mammalian host. Recent evidence from mixed infections has shown that Schistosoma mansoni males can displace S. intercalatum males from their homologous partners, but no information exists about the existence of similar phenomena within a single schistosome species. Here, we determine whether male S. mansoni can displace males of the same species from pre-formed pairs in vivo. The availability of clear-cut genetic markers of drug resistance in schistosomes was exploited to show that hycanthone sensitive S. mansoni males can displace homospecific hycanthone resistant males from pre-formed pairs and vice versa. The frequency of changes is dependent on the magnitude of the excess single males competing with paired worms. The possible mechanics and the biological significance of mate changing are discussed.

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Adult pairs of Schistosoma mansoni were kept in culture in the presence or absence of various bile salts and the numbers of parasite eggs deposited in vitro were monitored for 2 weeks. The hydrophilic bile salt tauroursodeoxycholic acid (TUDCA) was found to produce a highly significant increase in the number of eggs deposited during the 1st week of culture. The hydrophobic bile salt taurochenodeoxycholic acid (TCDCA) and the intermediately hydrophobic salt taurocholate (TCA) produced more moderate increases. These results expand previous data showing that schistosomes kept in the presence of portal blood have higher oviposition rates than schistosomes kept in systemic blood.

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The immunosuppressive fungal products cyclosporin A (CsA) and FK506 bind with high affinity to intracellular receptor proteins: cyclophilin (CYP) is one of the receptors for CsA and FK506-binding protein (FKBP) is one of the receptors for FK506. These proteins catalyze the in vitro isomerization from a cis to a trans conformation of peptidyl-prolyl bonds in oligopeptides. The relative importance of the peptidyl-prolyl cis-trans isomerase (PPI ase) activity of CYP compared to FKBP in schistosomes is not known. Here, we examine the effects of CsA and FK506 and show that the former inhibits PPIase activity in schistosome extracts, whereas the latter does not. Since CsA is specific for the CYP protein, this result is indicative of the fact that the PPIase activity in the parasite is mostly attributable to CYP. The observation that CsA was significantly more effective than FK506 as an antischistosomal agent, both in vivo and in vitro raises the possibility that killing of schistosomes is caused by the inhibition of schistosome CYP PPIase. We compared a number of Cs analogs for their antischistosomal effects and for the inhibition of CYP PPIase, but were unable to find a correlation between the two properties. We therefore conclude that the lethal effect of CsA is not directly linked to the inhibition of the enzymatic activity of schistosome CYPs.

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The notion that oxamniquine is active against Schistosoma mansoni but inactive against S. haematobium was confirmed using in vitro cultures of adult worms. Since oxamniquine and hycanthone have been shown to become effective upon activation by a schistosome enzyme, enzymatic tests were carried out to detect possible differences between the enzyme of S. mansoni and that of S. haematobium. It was found that the S. mansoni enzyme could activate hycanthone and, to a lesser extent, oxamniquine. The S. haematobium enzyme, on the other hand, was capable of activating hycanthone but virtually incapable of activating oxamniquine. It is concluded that the different activity of oxamniquine in the two species is due to differences in the drug-activating enzyme.

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The major antischistosomal drugs that have been or still are in use against infections with schistosomes are considered here together with some compounds that have not been in clinical use, but show interesting characteristics. Each individual compound presents aspects that may be enlightening about parasite biochemistry, parasite biology, and host-parasite relationships. Special attention is given to the mechanisms of action, an understanding of which is seen here as a major factor of progress in chemotherapy. Three compounds are currently in use, i.e., metrifonate, oxamniquine, and praziquantel, and all three are included in the World Health Organization list of essential drugs. They are analyzed in some detail, as each one presents advantages and disadvantages in antischistosomal therapy. The reported occurrence of drug-resistant schistosomes after treatment with oxamniquine and praziquantel suggests strict monitoring of such phenomena and encourages renewed efforts toward the development of multiple drugs against this human parasite.

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Two drug-resistant strains of Schistosoma mansoni were compared in this study in order to decide whether they are both mutated in the same gene with respect to drug-sensitive schistosomes. One of the two strains was isolated in the laboratory, while the other one originated from a treated uncured patient and was subsequently drug selected in the laboratory. The approach consisted in a genetic complementation test performed essentially by crossing the two strains and assessing resistance in the progeny. Since no reappearance of drug sensitivity was detected in the progeny, it was concluded that the two strains failed to complement and were therefore mutated in the same gene. This finding suggests that a single step of drug activation operates in sensitive schistosomes and is ineffective in resistant worms.

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Drug resistance in schistosomes is confined essentially to compounds of the hyconthone/oxamniquine family, since no documented case of resistance has so far been reported for the widely used drug praziquantel. The availability of strains of Schistosoma mansoni that are resistant to hyconthone and oxomniquine has permitted a detailed genetic and biochemical study of the mechanism of action of these compounds. Drugs must be activated by enzymatic esterification and this ultimately results in the production of an electrophilic moiety capable of alkylating DNA and other parasite macromolecules. As reviewed here by Donato Cioli, Livia Pica-Mattoccia and Sydney Archer, resistance is due to the loss of a drug-activating enzyme that is present in sensitive schistosomes and absent in resistant worms and in the mammalian hosts. Further study of this enzyme may yield valuable clues for drug design and for a basic understanding of parasite metabolism.

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Individual schistosomes of an hycanthone/oxamniquine-sensitive strain were crossed with individual schistosomes of the opposite sex and belonging either to the same sensitive population or to a different strain which exhibited high resistance to the two drugs. Schistosome crosses were performed by transfer of single worm pairs into the mesenteric veins of mice and the drug sensitivity/resistance of individual progeny worms was assessed using an in vitro test. Drug resistance behaved as an autosomal recessive trait, as shown by the results of the F1 and F2 generation and of the backcrosses. Drug-resistant worms appeared to be slightly less viable than their sensitive counterpart at all stages of the life cycle. The results are relevant for an interpretation of drug resistance and drug mechanisms and the approach used in this study may be applicable to different genetic markers in schistosomes.

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Crude extracts of hycanthone sensitive Schistosoma mansoni incubated at 37 degrees C in the presence of ATP and Mg2+ induced the covalent binding of tritiated hycanthone (HC) to macromolecules. The same behavior was shown by the HC sensitive species, Schistosoma rodhaini, whereas two independently isolated HC resistant S. mansoni strains had no detectable activity. Sensitive male schistosomes had more activity than females or immature worms. Virtually no activity was present in mouse liver, in human liver, in HeLa cells or in the naturally resistant species Schistosoma japonicum. The activity was destroyed by boiling or by Proteinase K treatment. Covalent binding of tritiated HC to macromolecules could be inhibited by cold HC, oxamniquine or IA-4, while none of the in vitro ineffective analogs, like lucanthone, UK-3883 or 4-desmethyl lucanthone, were inhibitory. These results strongly support the previously advanced suggestion that HC is activated by enzymatic mechanisms which are present only in drug sensitive schistosomes.

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The objective of this study is to determine whether various hycanthone resistant strains of schistosomes which have been independently isolated are all affected in the same gene. A strain obtained from a Brazilian patient was compared with a strain of Puerto Rican origin selected in the laboratory. If the mutation conferring resistance involved two different genes, one would expect that progeny of a cross between the two strains would show complementation, i.e. it would be sensitive to the drug. We have performed such a cross and obtained F1 hybrid worms which were essentially all resistant, thus suggesting that the mutation conferring resistance in the two strains involves the same gene.

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Condensation of hycanthone N-methylcarbamate (HNMC) with deoxyguanosine (dG) furnished a mixture of the N-1 and N2 adducts which were purified and characterized as their acetates. Condensation of HNMC with thymidine (T) gave the N-3 adduct in poor yield. Adenosine (A) and cytidine (C) did not react with HNMC. Incubation of schistosomes with either [3H]hycanthone (HC) or [3H]HNMC furnished DNA to which [3H]HC was covalently bound. The alkylated DNA was degraded enzymically and the radiolabeled nucleosides were separated using HPLC. Two major peaks were observed which coincided in retention time with the synthetic N-1 and N2 alkylated dG. Alkylated T was absent. Thus, the site of alkylation of DNA by either HC or HNMC is dG.

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Genetic crosses between phenotypically resistant and sensitive schistosomes demonstrated that resistance to hycanthone and oxamniquine behaves like a recessive trait, thus suggesting that resistance is due to the lack of some factor. We hypothesized that, in order to kill schistosomes, hycanthone and oxamniquine need to be converted into an active metabolite by some parasite enzyme which, if inactive, results in drug resistance. Esterification of the drugs seemed to be the most likely event as it would lead to the production of an alkylating agent upon dissociation of the ester. An artificial ester of hycanthone was indeed active even in resistant worms, thus indirectly supporting our hypothesis. In addition, several lines of evidence demonstrated that exposure to hycanthone and oxamniquine results in alkylation of worm macromolecules. Thus, radioactive drugs formed covalent bonds with the DNA of sensitive (but not of resistant) schistosomes; an antiserum raised against hycanthone detected the presence of the drug in the purified DNA fraction of sensitive (but not of resistant) schistosomes; a drug-DNA adduct was isolated from hycanthone-treated worms and fully characterized as hycanthone-deoxyguanosine.

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Hycanthone-sensitive and hycanthone-resistant schistosomes (which are also sensitive and resistant to oxamniquine) were exposed in vitro to tritium-labelled oxamniquine. The initial uptake of the drug into the schistosomes was essentially the same for the 2 strains. The homogenate of worms incubated with tritiated oxamniquine was fractionated and a purified DNA fraction was obtained by ethanol precipitation, RNAase and protease digestion, repeated phenolchloroform extractions, CsC1 gradient centrifugation and extensive dialysis. The DNA fraction from sensitive worms contained radioactive oxamniquine at a level corresponding to about 1 drug molecule per 50,000 base pairs, while the DNA from resistant worms contained essentially no drug. The results support the hypothesis that oxamniquine, like hycanthone, exerts its activity by alkylating macromolecules of sensitive schistosomes. The possibility is discussed that oxamniquine may lack the mutagenic properties of hycanthone because it is not an intercalating agent.

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Adult Schistosoma mansoni of the hycanthone-sensitive and of the hycanthone-resistant strain were exposed in vitro to tritium-labeled hycanthone. The drug was taken up in similar amounts by the two strains, a result which is not compatible with hypothetical mechanisms of resistance based on reduced drug entry into the schistosomes. Labeled hycanthone was found to bind irreversibly to macromolecules of sensitive schistosomes, whereas the binding was minimal in resistant worms. In particular, the DNA of sensitive schistosomes showed high levels of tightly bound hycanthone, while the corresponding fraction of resistant schistosomes failed to do so. Female schistosomes and immature worms, which are less sensitive to hycanthone, showed a diminished drug-DNA binding with respect to adult males. Tritiated hycanthone N-methylcarbamate, which is effective against sensitive and resistant schistosomes, bound in similar amounts to the DNA of both strains. These results strongly support a previously proposed mechanism of action of hycanthone, which is based essentially on the alkylation of worm macromolecules by a drug derivative produced in sensitive schistosomes.

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On the basis of the remarkable biological similarities between hycanthone and oxamniquine and as a sequel to our finding that some esters of hycanthone are active against hycanthone-resistant schistosomes, we prepared oxamniquine acetate, oxamniquine N-methylcarbamate, and four substituted phenylsulfonohydrazones of oxamniquine aldehyde. These compounds were tested for their effect on survival of and on [3H]uridine incorporation into hycanthone-sensitive and -resistant Schistosoma mansoni. All of these derivatives were effective to a greater or lesser degree in killing worms and in inhibiting [3H]uridine incorporation in the sensitive strain, but none was effective in the resistant strain.

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The synthesis of a series of esters of hycanthone (HC) and 7-hydroxyhycanthone, their antitumor activity, and their antischistosomal effects on HC-sensitive and HC-resistant schistosomes are reported. Binding studies using tritium-labeled HC and hycanthone N-methylcarbamate (HNMC) with calf thymus DNA provided evidence that HNMC but not HC alkylated the DNA. Tritiated HNMC also bound to the DNA of intact HeLa cells exposed to the drug while very little tritiated HC bound to DNA under the same conditions. The mechanism proposed previously to account for the antischistosomal action of HC, namely, drug esterification followed by alkylation of DNA, applies also to the antitumor action of the drug as shown in Scheme I.

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