Effectiveness of synthetic trioxolane OZ78 against Schistosoma japonicum in mice and rabbits.

Antischistosomal activities of a synthetic peroxide OZ78 (an ozonide carboxylic acid) against Schistosoma japonicum have been studied in mice and rabbits. Among 132 mice used, 30 of them were infected with 80-100 S. japonicum cercariae for collection of juvenile and adult schistosomes applied in in vitro tests. The remaining 102 mice were infected with 40 schistosome cercariae used for experimental treatment. Other 13 rabbits infected each with 200 schistosome cercariae were treated orally with OZ78 42 days post-infection. Most treated mice and rabbits were sacrificed 4 weeks post-treatment to collect residual schistosomes for evaluation of the drug efficacy. OZ78 and its sodium salt (OZ78-Na salt) 10-60 µg/mL alone exhibited no in vitro effect against day 14, day 21 schistosomula, and day 35 adult schistosomes. But OZ78 and OZ78-Na salt 10 and 20 µg/mL together with hemin 80 µg/mL showed decrease in worm motor activity and severe damage to the worm tegument and intestine, and all worms died within 3 days post-incubation. After infected mice were treated orally with OZ78 at a single dose of 400 mg/kg for 1 day, 34.9% of the worms shifted to the liver. Three and 7 days post-treatment, 100% of the worms were recovered from the liver. Fourteen days post-treatment, 92.3% of the worms still remained in the liver and 7.7% of the worms returned back to the mesenteric veins. Male and female worms shifted to the liver revealed in apparent shrinkage, degeneration of worm body, depigmentation in gut, and disappearance of ova in the uterus of some female worms. Meanwhile, dead worm and dead worm fragments were found in the liver tissues. In mice infected with various stages of schistosomes and treated orally with single OZ78 400 mg/kg, moderate or potential effect of the drug against day 0 (3-h-old worm), day 7, day 14, and day 21 juvenile worms and day 28, day 35 as well as day 42 adult worms were observed, the differences of total or female worm burdens between each treated group and control group were statistically significant (P < 0.01 or P < 0.05). Among the various stages of juveniles, day 7 worms were more susceptible to OZ78 with worm reduction of 83.8%, while the effect of OZ78 against day 28 to day 42 adult worms were similar. Finally, rabbits infected with adult schistosomes and treated with OZ78 at a single dose of 45 mg/kg or a daily dose of 35 mg/kg for three consecutive days resulted in significantly lower total and female worm burdens in comparison with that of control (P < 0.05) with total and female worm reductions of 84.1% and 84.7% as well as 74.3% and 77.4%, respectively. The results demonstrate that OZ78 possesses effect against both juvenile and adult S. japonicum in mouse model, and also shows effect against adult schistosomes in rabbits.

Schistosoma japonicum-infected hamsters (Mesocricetus auratus) used as a model in experimental chemotherapy with praziquantel, artemether, and OZ compounds.

The purpose of the study is to better understand the antischistosomal properties of artemether, praziquantel, and ozonide (OZ) compounds (synthetic trioxolanes, secondary ozonides) in hamster (Mesocricetus auratus) model. A total of 230 male hamsters infected each with 100 Schistosoma japonicum cercariae were used in the study. Groups of five to ten hamsters were treated orally with artemether, praziquantel, and OZ78 or OZ277 7-35 days post-infection at single doses of 50, 100, 150, or 200 mg/kg. Untreated but infected hamsters in each batch of test served as the control. All treated hamsters were sacrificed 4 weeks post-treatment for collection of residual worms using perfusion technique. Nonparametric method (Mann-Whitney test) was used to analyze the data. In groups of five hamsters treated with artemether 7, 14, 21, 28, and 35 days post-infection at single doses of 150 and 200 mg/kg, the difference of mean worm burden between each treated group and control group was statistically significant (P<0.01). Apart from individual group, no difference in mean worm burden between each two groups of them was seen (P>0.05). Further test with various single doses of 50-200 mg/kg confirmed the similar susceptibility of 7-day-old juvenile and 35-day-old adult schistosomes to artemether. After administration of praziquantel 100 mg/kg to groups of five hamsters 7, 21, and 35 days post-infection, higher worm burden reduction of 95.5% was seen in the group with 35-day-old adult schistosomes while in the groups with 7- and 21-day-old juvenile schistosomes, poor efficacy was seen with mean worm burden reductions of 36.6% and 35.6%. In the same batch of hamster treated with praziquantel 200 mg/kg, the moderate effect of the drug against 7- and 21-day-old worms was seen, but their mean worm burden was significantly higher than that of the group with adult schistosomes. In comparison of artemether and praziquantel against various stages of schistosomes, the results further demonstrated that artemether possessed similar effect against juvenile and adult schistosomes in hamsters, while praziquantel was more effective against adult schistosomes than the juvenile ones in the same host. Finally, after administration of OZ78 and OZ277 to the groups of four to six hamsters with 14- and 35-day-old schistosomes at a single dose of 200 mg/kg, promising effect against juvenile and adult schistosome was observed with the mean worm burden and female worm burden reductions of 69.6-94.2% and 64.2-100% as well as 73.3-80.7% and 68.3-81.1%, respectively. The results indicate that in hamster model, praziquantel exhibits higher effect against adult schistosomes than the juvenile ones, while artemether and OZ compound display similar effect against both juvenile and adult schistosomes.

Effect of mefloquine administered orally at single, multiple, or combined with artemether, artesunate, or praziquantel in treatment of mice infected with Schistosoma japonicum.

The purpose of the study is to explore the efficacy of mefloquine administered orally at single, multiple doses, or in combination with artesuante, artemether, or praziquantel in mouse--Schistosoma japonicum model. A total of 205 mice were divided into 4 batches and each batch of mice was infected percutaneously with 40 S. japonicum cercariae for 35 days. The infected mice were treated orally with mefloquine at single doses, multiple daily doses, or combined with artesunate, artemether, or praziquantel, while infected but untreated mice served as control. All treated animals were killed 4 weeks post-treatment for assessment of effect. When infected mice were treated orally with mefloquine at single or multiple daily doses under the same total dose levels, the tendency to decrease the efficacy was seen. Particularly, when a lower single dose of 100 mg/kg was divided equally into five daily doses of 20 mg/kg, the efficacy decreased statistically significant (P<0.05), i.e., the total worm and female worm reductions of 67.9% and 73.4% decreased to 31.3% and 30.3%, respectively. In infected mice treated with mefloquine or artesuante at a single dose of 100 mg/kg, a moderate effect against schistosomes was observed. No further significant reduction of total and female worm burdens was seen, when the two drugs combined together at the same dose level. On the other hand, administration of mefloquine combined with artesunate at single dose of 50 mg/kg, which exhibited no effect against schistosomes, resulted in significant reduction of total and female worm burdens in comparison with the groups treated with mefloquine and artesunate alone at the same dose level. Similar results were observed in treatment of infected mice with mefloquine in combination with artemether at the smaller dose of 50 mg/kg. The total worm burden was significantly lower than that of control and the female worm burden was also significant lower than that of groups treated with mefloquine and artemether alone. Interestingly, in administration of mefloquine 100 mg/kg combined with artemether 100 mg/kg to the infected mice, all female worms were killed and the total worm burden was also statistically significant lower than that of groups treated with either drug alone. Finally, when infected mice were treated with mefloquine combined with prazqiuatel at single dose of 50 mg/kg, no apparent improvement in efficacy was seen. Administration of mefloquine 100 mg/kg combined with praziquantel 100 mg/kg, only the difference of female worm burdens between praziquantel group and combined treatment group was statistically significant. The results indicate that under the same dose level of mefloquine, the efficacy of single dose is superior to that of multiple daily doses; mefloquine combined with artesunate or artemether at an invalid or moderate effective dose may show synergistic effect, especially the effect against female worms; no prominent synergistic effect is observed, when the similar dose level of mefloquine in combination with praziquantel.

Further study on mefloquine concerning several aspects in experimental treatment of mice and hamsters infected with Schistosoma japonicum.

Antischistosomal properties of mefloquine against Schistosoma japonicum have been further studied. A total of 260 mice were divided into four batches, and three batches of them were infected percutaneously with 40 S. japonicum cercariae. In the remaining batch, mice were infected with 20, 40, or 80 S. japonicum cercariae. Other 45 hamster, divided into two batches, were each infected two or three times with 50 S. japonicum cercariae at days 0 and 7 or 0, 14, and 21. The infected mice and hamsters were treated orally with single doses of mefloquine or praziquantel at various intervals post-infection, while infected but untreated mice and hamsters served as control. All treated animals were killed 4 weeks post-treatment for assessment of effect. In hamsters concurrently infected with 14- and 21-day-old or 14-, 21-, and 35-day-old schistosomes and treated orally with mefloquine at a single dose of 100 and 200 mg/kg, the total worm burdens were significantly lower than that of control (P < 0.05 or P < 0.01) with worm burden reductions of 45.4% and 89.9% as well as 82.5% and 90.6%, respectively. In the first batch of mice treated with mefloquine and four structurally related amino alcohol antimalarials 5 weeks post-infection at a single dose of 400 mg/kg, mefloquine, quinine, and quinidine possessed similar potential effect with total worm burden reductions of 80.9-90.3%, while halofantrine and lumefantrine showed moderate and poor effect with total worm burden reductions of 67.5% and 38.4%, respectively. In the second batch of mice infected with 20, 40, and 80 S. japonicum cercariae and treated orally with mefloquine at a single dose of 200 and 400 mg/kg 5 weeks post-infection, similar effects were seen in groups of mice with various infection intensity, the total worm burden reductions were 59.9-73.0% (200 mg/kg) and 85.0-89.1% (400 mg/kg). In the other two batches of mice infected with various stages of schistosomes and treated orally with mefloquine and praziquantel at a single dose of 200 or 400 mg/kg, potential and moderate effects of praziquantel against d0 worms (3-h-old) and adult worms (28- and 35-day-old) with total worm burden reductions of 83.6-95.6% and 42.4-69.3% were observed, but no effect against various stages of juvenile schistosome was seen. Under the two single doses used, mefloquine exhibited no effect against d0 worms, but showed moderate or potential effect against various stages of juvenile and adult schistosomes with total worm burden reductions of 56.3-89.1% (200 mg/kg) and 81.1-100% (400 mg/kg). The results indicate that mefloquine shows potential effect on hamsters concurrently infected with various stages of juvenile and adult S. japonicum; among the four structurally related amino alcohol antimalarials tested, quinine and its isomer quinidine exhibit potential effect against adult S. japonicum similar to that of mefloquine, while halofantrine and lumefantrine posses moderate and poor effect; no impact of infection intensity on the effect of mefloquine against schistosomes was observed in mice; under the same dose level, the effect of mefloquine against development stages of juvenile and adult S. japonicum is superior to that of praziquantel.

The in vitro effect of mefloquine and praziquantel against juvenile and adult Schistosoma japonicum.

Mefloquine, an antimalarial drug, has been found to be effective against various stages of schistosomes in vivo. The purpose of the study is to explore the in vitro effect of mefloquine against adult and juvenile Schistosoma japonicum and to compare its efficacy with praziquantel. Three-hour-old schistosomula were prepared by penetrating the mouse skin with schistosome cercariae, while schistosomes 7-, 14-, and 35-day-old were collected from mice infected with S. japonicum cercariae for 7, 14, and 35 days by perfusion. Schistosomes were placed to each of 24 wells of a Falcon plate and maintained in Hanks' balanced salt solution-20% calf serum. Besides observation on the direct in vitro effect of mefloquine and praziquantel, adult worms exposed to mefloquine and praziquantel for 1 and 4 h were transferred to the medium without the drugs and incubated continuously for another 72 h. The reversible effect of mefloquine and praziquantel was assessed by the recovery of the worm motor activity and parasite survival. The minimal effective concentration of mefloquine against adult schistosomes in vitro was 10 microg/mL, which revealed that the worm motor activity was first stimulated, then decreased significantly, followed by bleb formation, focal swelling and elongation of the worm body, cessation of gut peristalsis, and death of 56.3% (18/32) worms within 24-72 h. Similar appearance was seen in the adult worms exposed to higher mefloquine concentration of 20 and 30 microg/mL, but all worms died within 4-24 h. The adult schistosomes exposed to praziquantel 1-30 microg/mL showed fast spasmodic contraction of the worm body, followed by bleb formation along the tegument, feeble movement of oral sucker, and death of a part of males and females 72 h after incubation. When male and female schistosomes exposed to mefloquine 10 and 20 microg/mL for 1 and 4 h were transferred to the medium without the drug, no apparent recovery of worm motor activity and survival was seen. In case of worms exposed to praziquantel at the same concentration for 1 and 4 h before replacement of drug-free medium, a well recovery of worm motor activity, looseness of worm body, and reduction or disappearance of blebs along the tegument were observed. Mefloquine also exhibited in vitro effect against 3-h-old and 7- and 14-day-old schistosomula which was similar to that seen in adult worms, but all or parts of worms showed decrease in motor activity or even death (3-h-old and 7-day-old schistosomula) at a lower mefloquine concentration of 5 microg/mL. In 14 day-old schistosomula exposed to praziquantel 1-30 microg/mL, spasmodic contraction and significant decrease in motor activity of the worm body with movement of oral and ventral suckers were observed, but no death of worm was seen during a 3-day incubation period. The results indicate that in vitro mefloquine exhibits a direct killing effect against adult and juvenile S. japonicum which is different from that of praziquantel. Meanwhile, the juvenile schistosomes are more susceptible to mefloquine than the adult ones. Furthermore, the in vitro effect of mefloquine against adult schistosomes is irreversible, while that of praziquantel is reversible.

Glutathione inhibits the antischistosomal activity of artemether.

OBJECTIVE: To observe the effect of reduced glutathione (GSH) and glutathione depleting agent 1-chloro-2,4-dinitrobenzene (CDNB) on the susceptibility of adult Schistosoma japonicum to artemether (Art) in combination with hemin in vitro. METHODS: In vitro, malondialdehyde(MDA) levels were determined in five-week-old worms incubated without or with Art, hemin, GSH, and CDNB, either alone or in combination, for 24 h, and the remaining worms were continuously incubated up to 96 h for worm survival assessment. In vivo, GSH levels were determined in worms freshly recovered from mice 6, 12 and 24 h after treatment with Art 300 mg/kg. RESULTS: In vitro, GSH decreased the proportion of worms killed by Art plus hemin, but CDNB rendered the worms susceptible to the killing. The above-mentioned distinguishing features of GSH and CDNB were associated with their reverse effect on worm lipid peroxidation induced by Art-hemin system. In vivo, Art led to a slight decrease followed by a significant increase in the parasite GSH levels. CONCLUSION: GSH might play an important role in the defense of the worms against Art-generated toxic peroxides and free radicals.