Elimination of Schistosoma mansoni in infected mice by slow release of artemisone.

The current treatment of schistosomiasis is based on the anti-helminthic drug praziquantel (PZQ). PZQ affects only the adult stages of schistosomes. In addition, resistance to PZQ is emerging. We suggest a drug, which could serve as a potential alternative or complement to PZQ, and as a means of treating infections at earlier, pre-granuloma stage. Derivatives of the peroxidic antimalarial drug artemisinin have been indicated as alternatives, because both plasmodia and schistosomes are blood-feeding parasites. The mechanism of action of artemisinins is related to oxidative effects of the artemisinins on intracellular reductants leading to formation of cytotoxic reactive oxygen species. We used artemisone, which has improved pharmacokinetics and anti-plasmodial activity, and reduced toxicity compared to other artemisinins in clinical use against malaria. We infected adult mice by subcutaneous injection of S. mansoni cercariae (about 200) and treated them at various times post infection by the following methods: i. artemisone suspension administered by gavage (400-450 mg/kg); ii. subcutaneous injection of a gel containing a known concentration of artemisone (115-120 mg/kg); iii. subcutaneous insertion of the drug incorporated in a solid polymer (56-60 mg/kg); iv. intraperitoneal injection of the drug solubilized in DMSO (115-120 mg/kg). Drug administration in polymers was performed to enable slow release of the artemisone that was verified in vivo and in vitro bioassays using drug-sensitive malaria parasites. We found superior strong anti-schistosome effects up to a total reduction of worm number, mainly following repetitive treatments with the drug absorbed in the polymers (73.1% and 95.9% reduction in mice treated with artemisone in gel 7 and 14, and 21, 28 and 35 days post infection, respectively). The results indicate that artemisone has a potent anti-schistosome activity. Its main importance in this context is its effectiveness in treating hosts harboring juvenile schistosomes, before egg-deposition and induction of deleterious immune responses.

Praziquantel analogs with activity against juvenile Schistosoma mansoni.

Six amide and four urea derivatives of praziquantel were synthesized and tested for antischistosomal activity against juvenile and adults stages of Schistosoma mansoni in infected mice. Only one of these had significant activity against adult worms, but, unlike praziquantel, six of these had low to modest activity against juvenile worms. A praziquantel ketone derivative had the best combination of activity against juveniles and adults, but it had no effect on the motility of adult S. mansoni in ex vivo culture. Cytochrome P450 metabolic stability data support the hypothesis that the major trans-cyclohexanol metabolite of praziquantel plays an important role in the antischistosomal activity of this drug.

Spiroadamantyl 1,2,4-trioxolane, 1,2,4-trioxane, and 1,2,4-trioxepane pairs: relationship between peroxide bond iron(II) reactivity, heme alkylation efficiency, and antimalarial activity.

These data suggest that iron(II) reactivity for a set of homologous spiroadamantyl 1,2,4-trioxolane, 1,2,4-trioxane, and 1,2,4-trioxepane peroxide heterocycles is a necessary, but insufficient, property of animalarial peroxides. Heme alkylation efficiency appears to give a more accurate prediction of antimalarial activity than FeSO(4)-mediated reaction rates, suggesting that antimalarial activity is not merely dependent on peroxide bond cleavage, but also on the ability of reactive intermediates to alkylate heme or other proximal targets.

Histopathological changes in adult Schistosoma japonicum harbored in mice treated with a single dose of mefloquine.

New research has shown that mefloquine, an arylaminoalcohol used against malaria, is active against Schistosoma japonicum and Schistosoma mansoni in vivo. To enhance our understanding of the potential mechanism of action of mefloquine against schistosomiasis, we examined the dynamics of histopathological changes in adult S. japonicum. Mice infected with S. japonicum for 35 days were treated intragastrically with a single dose of mefloquine (400 mg/kg). One to 35 days after mefloquine administration, drug-induced histopathological alterations were studied. Twenty-four hours after treatment, S. japonicum showed signs of degeneration, including focal roughing and swelling of the tegument and/or muscles, dilatation of the gut, focal desquamation of gut epithelial cells, and a decrease in pigment particles. There was extensive degeneration of vitelline cells and appearance of pigment particles visible in the cytoplasm in female worms. The extent and severity of histopathological changes increased over time; 48 h posttreatment, two thirds of female worms and a quarter of male worms were classified as dead. Three to 14 days posttreatment, typical histological changes observed in surviving male worms were vesiculation, swelling of parenchymal tissues, and dilatation of gut. In females, there was disintegration and infiltration of inflammatory cells, forming dead worm abscesses and early stage of dead worm granuloma. Finally, 35 days posttreatment, only dead male and female worm granuloma were found. Our results provide further evidence of in vivo activity of mefloquine against adult schistosomes.

Spiro- and dispiro-1,2-dioxolanes: contribution of iron(II)-mediated one-electron vs two-electron reduction to the activity of antimalarial peroxides.

Fourteen spiro- and dispiro-1,2-dioxolanes were synthesized by peroxycarbenium ion annulations with alkenes in yields ranging from 30% to 94%. Peroxycarbenium ion precursors included triethylsilyldiperoxyketals and -acetals derived from geminal dihydroperoxides and from a new method employing triethylsilylperoxyketals and -acetals derived from ozonolysis of alkenes. The 1,2-dioxolanes were either inactive or orders of magnitude less potent than the corresponding 1,2,4-trioxolanes or artemisinin against P. falciparum in vitro and P. berghei in vivo. In reactions with iron(II), the predominant reaction course for 1,2-dioxolane 3a was two-electron reduction. In contrast, the corresponding 1,2,4-trioxolane 1 and the 1,2,4-trioxane artemisinin undergo primarily one-electron iron(II)-mediated reductions. The key structural element in the latter peroxides appears to be an oxygen atom attached to one or both of the peroxide-bearing carbon atoms that permits rapid beta-scission reactions (or H shifts) to form primary or secondary carbon-centered radicals rather than further reduction of the initially formed Fe(III) complexed oxy radicals.

Triclabendazole and its two main metabolites lack activity against Schistosoma mansoni in the mouse model.

Some have claimed that triclabendazole, a safe and efficacious drug for the treatment of fascioliasis, also exhibits antischistosomal properties, but results are conflicting. We assessed the effect of triclabendazole and its two main metabolites against two different strains of Schistosoma mansoni harbored in mice. Low worm burden reductions (18.6-35.9%) were observed in mice infected with an Egyptian strain of S. mansoni and treated with a single dose of 120 mg/kg 3 days before infection or single/double doses of 120-200 mg/kg 7 weeks after infection. Triclabendazole failed to significantly reduce hepatic and intestinal tissue egg loads, and eggs of all developmental stages were observed. Administration of 400 mg/kg of either triclabendazole, triclabendazole sulphone, or triclabendazole sulfphoxide to mice infected with a Liberian strain of S. mansoni resulted in worm burden reductions < 10%. In comparison, high worm burden reductions (82-100%) were observed in S. mansoni-infected mice treated with single oral doses of 400, 500, or 500 mg/kg twice a day praziquantel, regardless of the S. mansoni strain. We conclude that triclabendazole and its main metabolites display weak and inconsistent schistosomicidal activities.

Effect of artemether administered alone or in combination with praziquantel to mice infected with Plasmodium berghei or Schistosoma mansoni or both.

The artemisinins have become key drugs for the treatment and control of malaria, particularly within artemisinin-based combination therapies. Since the artemisinins also exhibit antischistosomal properties, their use in areas where malaria and schistosomiasis are co-endemic may have an effect on both diseases and co-infection might alter drug efficacy. We assessed the antimalarial and antischistosomal efficacies of artemether in mice infected with Plasmodium berghei or Schistosoma mansoni or both parasites concurrently. Three oral doses of 400 mg/kg artemether at 14-day intervals reduced total and female S. mansoni worm burdens by 98.7-100%, regardless of a concurrent P. berghei infection. When four daily doses of 55 mg/kg artemether were administered, which is a standard treatment schedule to cure P. berghei-infected mice, significantly lower total and female S. mansoni worm burden reductions were observed (73.1-89.2%). Artemether, administered at both of the above-mentioned treatment schemes, showed excellent antimalarial efficacy with no indications of delayed clearance of P. berghei or recrudescence, also in mice co-infected with S. mansoni. Co-infection with P. berghei had no effect on S. mansoni worm burden reductions following artemether-praziquantel combinations. Our findings point to the need for epidemiological studies in areas where malaria and schistosomiasis co-exist and where artemisinin-based combination therapies are introduced, since artemisinin-based combination therapies as part of a malaria control package may have ancillary benefits against schistosomiasis.

Spiro and dispiro-1,2,4-trioxolanes as antimalarial peroxides: charting a workable structure-activity relationship using simple prototypes.

This paper describes the discovery of synthetic 1,2,4-trioxolane antimalarials and how we established a workable structure-activity relationship in the context of physicochemical, biopharmaceutical, and toxicological profiling. An achiral dispiro-1,2,4-trioxolane (3) in which the trioxolane is flanked by a spiroadamantane and spirocyclohexane was rapidly identified as a lead compound. Nonperoxidic 1,3-dioxolane isosteres of 3 were inactive as were trioxolanes without the spiroadamantane. The trioxolanes were substantially less effective in a standard oral suspension formulation compared to a solubilizing formulation and were more active when administered subcutaneously than orally, both of which suggest substantial biopharmaceutical liabilities. Nonetheless, despite their limited oral bioavailability, the more lipophilic trioxolanes generally had better oral activity than their more polar counterparts. In pharmacokinetic experiments, four trioxolanes had high plasma clearance values, suggesting a potential metabolic instability. The toxicological profiles of two trioxolanes were comparable to that of artesunate.

Triggering of high-level resistance against Schistosoma mansoni reinfection by artemether in the mouse model.

Artemether, a methyl ether derivative of dihydroartemisinin, not only exhibits antimalarial properties, but also possesses strong activity against schistosomula, the immature stages of a parasitic worm that can cause schistosomiasis. To test if the effect would be similar to that of irradiation with respect to the induction of immunologic protective responses, groups of mice were infected with Schistosoma mansoni cercariae and treated with artemether at 1-3 weeks post-infection. Control mice were either infected with normal cercariae or with cercariae exposed to radiation that permitted early development but not maturation of the parasites. The mice were challenged six weeks after the initial infection, and the mean numbers of schistosomes recovered in the various groups were calculated upon dissection eight weeks post-challenge. The administration of artemether two weeks after the initial infection resulted in 58% protection, while giving the drug three weeks post-infection increased the level of protection to 81%. This level of protection is as high as that normally obtained by immunization with irradiated cercariae (84% in the present study) and is superior to the level of resistance obtained with any individual schistosome vaccine candidate antigen thus far reported.

Schistosoma japonicum: In vitro effects of artemether combined with haemin depend on cultivation media and appraisal of artemether products appearing in the media.

We recently found that the exposure of schistosomes in vitro to artemether plus haemin can lead to parasite death, while exposure to each compound singly had no effect. Since these observations might be relevant to understanding the mechanism of action of artemether against schistosomes, we conducted additional experiments. First, we performed a comparative appraisal of Schistosoma japonicum survival after incubation in two media, namely Hanks' balanced salt solution (HBSS) and RPMI 1640, supplemented with inactivated calf serum, antibiotics and different concentrations of artemether and/or haemin. Worm mortalities were consistently higher and occurred faster in HBSS when compared to RPMI 1640. Second, we investigated the behaviour of artemether in different chemical systems, including reduced glutathione or cysteine, in the presence of haemin or ferrous sulfate. Cleavage of the endoperoxide bridge of artemether occurred in all experiments, consistently forming five different products, of which one has not been described previously. Third, RPMI 1640 and HBSS media were supplemented with artemether and haemin in the presence of S. japonicum. The consumption of artemether in RPMI 1640 was much faster than in HBSS. Trace amounts of artemether and five free radical reaction products of artemether could be detected in RPMI 1640 after 24-48 h. In contrast, large amounts of artemether remained without the formation of free radical products in HBSS under the same conditions. These findings coincide with significantly higher schistosome mortalities employing HBSS instead of RPMI 1640. Our results suggest that it is artemether or an active metabolite thereof (most likely a carbon-centred free radical), rather than any free radical reaction product that is harmful for the worms. We speculate that schistosomes ingest artemether and cleave it in their gut. This cleavage is induced by haemin or another iron-containing molecule. These findings might be a further step forward in elucidating the mechanism of action of artemether against schistosomes.

Ultrastructural alterations in adult Schistosoma mansoni caused by artemether

Progress has been made over the last decade with the development and clinical use of artemether as an agent against major human schistosome parasites. The tegument has been identified as a key target of artemether, implying detailed studies on ultrastructural damage induced by this compound. We performed a temporal examination, employing a transmission electron microscope to assess the pattern and extent of ultrastructural alterations in adult Schistosoma mansoni harboured in mice treated with a single dose of 400 mg/kg artemether. Eight hours post-treatment, damage to the tegument and subtegumental structures was seen. Tegumental alterations reached a peak 3 days after treatment and were characterized by swelling, fusion of distal cytoplasma, focal lysis of the tegumental matrix and vacuolisation. Tubercles and sensory organelles frequently degenerated or collapsed. Typical features of subtegumental alterations, including muscle fibres, syncytium and parenchyma tissues, were focal or extensive lysis, vacuolisation and degeneration of mitochondria. Severe alterations were also observed in gut epithelial cells and vitelline cells of female worms. Our findings of artemether-induced ultrastructural alterations in adult S. mansoni confirm previous results obtained with juvenile S. mansoni and S. japonicum of different ages

Recent investigations of artemether, a novel agent for the prevention of schistosomiasis japonica, mansoni and haematobia.

Two decades ago, a group of Chinese scientists discovered the antischistosomal properties of artemether, a derivative of the antimalarial drug artemisinin. However, it was only recently that the importance of this finding was recognized internationally, following a collaborative effort between Chinese, European and African scientists, who investigated the effects of artemether against the major human schistosome species. Laboratory studies revealed that artemether exhibits the highest activity against juvenile stages of the parasites, while adult worms are significantly less susceptible. There was no indication of neurotoxicity following repeated high doses of artemether given fortnightly for up to 5 months. Randomized controlled clinical trials confirmed that artemether, orally administered at a dose of 6 mg/kg once every 2-3 weeks, results in no drug-related adverse effects, and significantly reduces the incidence and intensity of schistosome infections. The risk that these treatment regimens might select for resistance, particularly for resistant-plasmodia, appears to be low. Combined treatment with artemether and praziquantel, given to animals harbouring juvenile and adult schistosome worms, resulted in significantly higher worm burden reductions than each drug administered singly. In conclusion, artemether-integrated with other control strategies-has considerable potential for reducing the current burden of schistosomiasis in different epidemiological settings.

Schistosoma japonicum: effect of artemether on glutathione S-transferase and superoxide dismutase.

Glutathione S-transferase (GST) and superoxide dismutase (SOD) are major antioxidant enzymes of schistosomes that are involved in detoxification processes. To study the effect of artemether on these enzymes, mice infected with adult Schistosoma japonicum, were treated with artemether either at a subcurative (100 mg/kg) or a curative dose (300 mg/kg). Schistosomes were recovered 24-72 h post-treatment separated by sex and used for GST and SOD activity measurements. Female worms showed consistently higher GST inhibitions than males. For instance, 24 h after administration of 100 mg/kg artemether, GST activities of female worms were inhibited by 23.3%, as compared to 12.7% in males. Both activities were significantly lower when compared to worms recovered from untreated mice. Slightly higher inhibitions were observed at the higher dose of artemether, which gradually increased to levels of 52.5-55.1%, 72 h post-treatment. GST inhibitions could be reversed by application of 1,4-dithiothreitol at a concentration of 10 mmol/L. Adding L-cysteine also reduced GST inhibitions, but in female worms, GST activities remained significantly higher than in worms from untreated animals. Administration of 300 mg/kg artemether resulted in significant reductions of SOD activities in both sexes. In conclusion, these results suggest that the inhibition of GST and, to a lesser extent also SOD enzymes, could lead to increased schistosome susceptibility to oxidant attacks and might be linked with the antischistosomal action of artemether.