Praziquantel: its use in control of schistosomiasis in sub-Saharan Africa and current research needs.

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.

Antischistosomal drugs: past, present ... and future?

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.

Hycanthone resistance in schistosomes correlates with the lack of an enzymatic activity which produces the covalent binding of hycanthone to parasite macromolecules.

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.

Binding of tritiated hycanthone and hycanthone N-methylcarbamate to macromolecules of drug-sensitive and drug-resistant schistosomes.

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.

Mode of action of the schistosomicide hycanthone: site of DNA alkylation.

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.

Effect of hycanthone administered in vivo upon the incorporation of radioactive precursors into macromolecules of Schistosoma mansoni.

Mice infected with Schistosoma mansoni were treated with hycanthone or with 8-chloro-2[2-(diethylamino)ethyl]-2H-[1]benzothiopirano-[4,3, 2-cd]-indazole-5-methanesulphonate (IA-4). Schistosomes were obtained by perfusion at various times after drug administration and tested for their ability to incorporate radioactive precursors of DNA, RNA and protein. In adult worms, male or female, the incorporation of radioactive thymidine was severely and irreversibly inhibited after treatment with either drug. Uridine and leucine incorporations were also inhibited, though to a lesser extent. On the contrary, the synthetic activities of immature worms were unaffected by hycanthone and only partially or temporarily depressed by IA-4. Hycanthone-resistant schistosomes, when tested between 1 and 7 days after treatment, showed a pattern of precursor incorporation which was virtually identical to that of untreated worms. These results are consistent with the hypothesis that hycanthone and IA-4 may kill schistosomes by interfering with their nucleic acid synthesis.

Synthesis and biological properties of some 6H-pyrido[4,3-b]carbazoles.

The effect of methyl substitution on the biological properties of the ellipticines was reexamined. 9-Hydroxy-6H-pyrido[4,3-b]carbazole was synthesized and shown to be devoid of antitumor activity in murine P388 lymphocytic leukemia in mice. 5-(Hydroxymethyl)-11-methyl-6H-pyrido[4,3-b]carbazole (46) and its N-methylcarbamate (48) were synthesized and their effect on macromolecular synthesis in HeLa cells and their antitumor properties were compared with those of ellipticine. In contrast to the alkaloid 1 and the hydroxymethyl derivative 46, which produced partially reversible inhibition of [3H]thymidine incorporation, the carbamate ester irreversibly blocked incorporation of the tritiated pyrimidine. The ester was also a more potent antitumor agent in P388 lymphocytic leukemia than 1 or 46.

Preparation and antischistosomal and antitumor activity of hycanthone and some of its congeners. Evidence for the mode of action of hycanthone.

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.

Studies on some derivatives of oxamniquine.

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.

Studies on the mode of action of oxamniquine and related schistosomicidal drugs.

Adult Schistosoma mansoni were incubated for 1 hour in vitro with various drugs and then returned into the mesenteric veins of permissive animal hosts. Survival of schistosomes was assessed 3-4 weeks later by portal perfusion. Under these conditions, oxamniquine and hycanthone proved effective in killing S. mansoni, whereas UK-3883, lucanthone and lucanthone-4-desmethyl had no lethal activity. The same drugs which were schistosomicidal in vitro also persistently inhibited DNA, RNA, and protein synthesis in S. mansoni, whereas they were only transiently inhibitory against Schistosoma japonicum, against hycanthone-resistant S. mansoni and against immature worms. When drugs were administered in vivo to infected mice and the synthesis of macromolecules was assayed in vitro on worms obtained 1 or 3 days after treatment, not only oxamniquine and hycanthone, but also UK-3883 and lucanthone, proved effective in inhibiting the synthesis of macromolecules in sensitive--but not in resistant--S. mansoni. It is suggested that oxamniquine, like hycanthone, may exert its schistosomicidal activity by inhibiting nucleic acid synthesis in the parasite.

Genetic analysis of hycanthone resistance in Schistosoma mansoni.

Interbreeding between hycanthone-resistant and hycanthone-sensitive schistosomes was achieved using a worm transfer technique which considerably reduced the length and the complexity of the operations generally involved in performing schistosome genetic crosses. A mouse was considered to harbor resistant schistosomes if, three weeks or more after a single intrasmuscular injection of 80 mg/kg hycanthone schistosome eggs were still excreted in the feces, at least one normal worm pair was obtained by perfusion, or miracidia could be seen hatching from the liver. The F1 hybrid progeny from crosses between sensitive and resistant schistosomes proved to be sensitive to hycanthone, irrespective of whether the resistant parent was the male or the female. The resistant phenotype reappeared in back-crosses and in the F2 progeny. These results could be confirmed using the traditional technique of single sex infections. It can thus be concluded that hycanthone resistance behaves like an autosomal recessive trait. These results suggest that hycanthone-resistant schistosomes are deficient in some factor, possibly an enzymatic activity which transforms hycanthone into a biologically active molecule, as suggested in a recent hypothesis on the mode of action of hycanthone.

Binding of oxamniquine to the DNA of schistosomes.

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.

Evidence for the mode of antischistosomal action of hycanthone.

Evidence is presented which supports the hypothesis that the mode of action, or a slight variant thereof, suggested by Hartman and Hulbert (11) to account for the mutagenic effects of hycanthone (HC) is the mechanism whereby HC exerts its antischistosomal activity. HC is metabolically activated to a reactive ester which, upon dissociation, alkylates DNA. If resistant schistosomes are unaffected because they cannot convert HC to a reactive ester they should be killed upon direct exposure to an appropriately esterified drug. Hycanthone N-methylcarbamate (HNMC) was synthesized and shown to bind to DNA and also alkylate 4-(p-nitrobenzyl)-pyridine. When tested with schistosomes kept in vitro, HNMC caused an irreversible inhibition of 3H-uridine incorporation not only in sensitive S. mansoni (as HC does) but also in HC-resistant and immature S. mansoni worms and S. japonicum worms which are only transiently inhibited by HC. After in vitro contact with HNMC for 1 h both sensitive and resistant schistosomes died in three weeks if either kept in culture or re-transplanted into the host animal. Mice infected with HC-resistant schistosomes showed a drastic worm reduction after in vivo HNMC administration.

Lack of correlation between schistosomicidal and anticholinergic properties of hycanthone and related drugs.

Visual observation of the motor activity of Schistosoma mansoni kept in vitro showed an increase of activity in the presence of hycanthone (HC). In addition, HC caused a delay in the paralytic effects of carbachol. Similar results were observed in the presence of oxamniquine (OXA). The same pattern of motor activity, however, was shown by HC-resistant worms, by Schistosoma japonicum, and by worms exposed to drug precursors (lucanthone and UK-3883), which are not schistosomicidal in vitro. Other analogs with in vitro killing activity (IA-4 and IA-4 N-oxide) showed minimal anticholinergic effects. The anticholinergic effects of HC and OXA were quickly reversible in vitro and in vivo, whereas their antischistosomal effects are irreversible and delayed. Incubation of schistosomes with high concentrations of carbachol or with anticholinergic drugs failed to compete with the schistosomicidal effects of HC. These results are viewed as contradictory to the hypothesis that HC kills schistosomes by blocking their acetylcholine receptors.

Praziquantel and the benzodiazepine Ro 11-3128 do not compete for the same binding sites in schistosomes.

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.

The anti-schistosomal drug praziquantel is an adenosine antagonist.

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.

Genetic complementation analysis of two independently isolated hycanthone-resistant strains of Schistosoma mansoni.

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.