Fasciolagigantica: Parasitological and scanning electron microscopy study of the in vitro effects of ivermectin and/or artemether

Objective

To evaluate the in vitro effects of different concentrations of ivermectin and/or artemether on Fasciolagigantica worms and to study the parasitological changes and tegumental alterations using scanning electron microscopy (SEM).

Methods

Fasciola gigantica worms were incubated in vitro for 24 and 48 h with three concentrations of either ivermectin or artemether (10, 20 and 50 μg/ml) or both in half concentration of either (5, 10 and 25 μg/ml).

Results

Exposure of Fasciola worms to 25 + 25 μg/ml of combined drug regimens or to 50 μg/ml of either ivermectin or artemether for 48 h led to 100%, 41.7% and 75% worm killing which were accompanied by a significant reduction in egg laying capacity and significant increase in dead eggs maximally recorded in combined drug regimens. SEM of the flukes incubated for 48 h with combined drug regimens showed maximal tegumental disruption with swelling of the worm body, roughness, blebbing, sloughing and complete loss of spines. Disruption to the tegument of the flukes induced by artemether was more than that of ivermectin.

Conclusions

Artemether alone or combined with ivermectin in half doses had potent fasciocidal activities. Besides, half doses of combined drug regimens had higher ovicidal effects than each drug alone. In vivo studies are recommended to explore the efficacy of combined regimens against Fasciola infection.     

The potential of artemether for the control of schistosomiasis

Schistosomiasis continues to rank – following malaria – at the second position of the world's parasitic diseases in terms of the extent of endemic areas and the number of infected people. There is yet no vaccine available and the current mainstay of control is chemotherapy with praziquantel used as the drug of choice. In view of concern about the development of tolerance and/or resistance to praziquantel, there is a need for research and development of novel drugs for the prevention and cure of schistosomiasis. Interestingly, derivatives of artemisinin, which are already effectively used in the treatment of malaria, also exhibit antischistosomal properties. Significant advances have been made with artemether, the methyl ether derivative of artemisinin. We review the discovery of the antischistosomal activity of artemether by Chinese scientists two decades ago; the detailed laboratory studies of the susceptibility of, and effect on, the different developmental stages of Schistosoma japonicum, Schistosoma mansoni and Schistosoma haematobium to artemether; the possible mechanism of action and the potential long-term toxicity. Finally, we look at the effect of combined treatment with artemether and praziquantel; and clinical findings thus far obtained from randomised controlled trials with oral artemether for the prevention of patent infections and morbidity. The review intends to create a forum for strategic discussion of how these laboratory and clinical findings could be translated into public health actions. We conclude that artemether – as part of integrated current control measures and adapted to specific socio-ecological and epidemiological settings – has considerable potential to significantly reduce the current burden of schistosomiasis in many parts of the world.     

Electronically stabilized versions of the antimalarial acetal trioxanes artemether and artesunate

From 9-substituted DHA, several new artemisinin-derived C-10 acetal ethers and esters were prepared with either a 9-fluoro or a 9-sulfonyl substituent. The very strong inductive electron-withdrawing C-9 substituent is shown to retard considerably C-10 ionization (acid-promoted etherification) of 9-fluoro-DHA and 9-sulfonyl-DHA.     

In-vitro culture of Plasmodium falciparum: Utility of modified (RPNI) medium for drug-sensitivity studies using SYBR Green I assay

Studies were carried out to establish the potential of RPNI medium for drug-sensitivity studies using the MSF assay. The drug sensitivity of standard anti-malarials was compared using both the (³H) Hypoxanthine incorporation assay and the MSF assay. The media supplements used during the study have been human serum, FBS and ALBUMAX-II. Drug sensitivity of two parasite lines, adapted to grow separately in conventional as well as in RPNI medium was compared to observe the effect of RPNI medium on functional characteristics of the parasite. The results revealed identical IC₅₀ values of standard anti-malarials obtained by both the (³H) Hypoxanthine incorporation assay and the MSF assay and no untoward effect of FBS and ALBUMAX-II could be noticed on the chemo-sensitivity of standard anti-malarials. Apart from this the chemo-sensitive response of parasite line adapted to grow in RPNI medium was observed to be intact. These findings showed that RPNI medium has potential to be used for chemo-sensitivity studies and the MSF assay being more convenient was observed to be most suitable assay for bio evaluation of new molecules.     

Rapid characterization of artemether and its in vitro metabolites on incubation with bovine hemoglobin, rat blood and dog blood by capillary gas chromatography–chemical ionization mass spectrometry

A fast and sensitive analytical method was developed to characterize artemether and its metabolites in small amounts in body fluids. The extracts were derivatized with N-methyl-N-trimethylsilyltrifluoroacetamide, separated on an optimized capillary gas chromatographic system and identified by chemical ionization mass spectrometry by using ammonia as reagent gas. The analytical assay is demonstrated on samples extracted from bovine hemoglobin, rat blood and dog blood. Full mass spectra of artemether and three metabolites were obtained at a level of 1·10⁻⁶ g/ml.     

In vivo effect of single oral dose of artemether against early juvenile stages of Schistosoma mansoni Egyptian strain

The current treatment and control of schistosomiasis, rely on a single drug, praziquantel, although, it has minor activity against juvenile stages of the parasite. Studies have shown that artemether (ART) exhibits effects against juveniles of Schistosoma mansoni Liberian and Puerto Rican strains, Schistosoma japonicum and Schistosoma haematobium. Aiming to assess the in vivo activity of single oral dose of ART against early juvenile stages of S. mansoni Egyptian strain, this study was established. Mice were treated with ART (400 mg/kg) at two time points evenly spaced over the period of larval development (7 and 21 days post-infection; pi), and a third treatment point (day 49 pi) was included to elucidate when susceptibility decreases. Administration of ART on day 7 pi reduced the total worm burden by 85.94%. The greatest reductions were seen when treatment was given on day 21 pi, with total and female worm burden reductions of 91.52% and 90.57%, respectively, and cessation of oviposition. Similar dose given on day 49 pi reduced total worm burden by 55.17% and female worm burden by 66.51%. Moreover, it induced significant reduction in the tissue egg load and significant alterations in the oogram pattern with decreased immature eggs and increased dead eggs. Antipathological activities were evident in significant reductions in granulomata count and diameter. In conclusion, ART exhibits major in vivo schistosomicidal effects against the early larval migratory stages of S. mansoni Egyptian strain, mainly the 21-day old schistosomula, hence preventing disease progression and morbidity.     

Schistosoma mansoni: N-acetylcysteine downregulates oxidative stress and enhances the antischistosomal activity of artemether in mice

Artemether (Art), a derivative of the antimalarial artemisinin, also exhibit antischistosomal properties. N-acetylcysteine (NAC) has a diversity of applications, largely because of the chemical properties of the thiol moiety present in its structure. The ability of this moiety to sweep reactive oxygen species is well-established with NAC. This study investigates the ability of NAC to enhance the therapeutic potential of Art against adult Schistosoma mansoni infection and evaluates the protective role of this antioxidant on S. mansoni-induced oxidative stress. Mice were divided into five groups; normal (i), infected control (ii), infected treated with NAC, 300 mg/kg 5 days a week/4weeks (iii), infected treated with Art (300 mg/kg) 7 weeks post infection (iv) and infected treated with both NAC and Art (v). Results showed that Art produced a significant reduction in total number of worms when used alone. Also, it decreased hepatic ova count significantly accompanied with an increase in the percentage of dead ova. Treatment with NAC alone increased the percentage of dead ova; meanwhile, it enhanced the decrease in total number of worms and hepatic ova count when used with Art. Infection with S. mansoni significantly increased tissue GSH, GR, SOD and serum ALT and GGT, while decreased the activities of GST, GPx and the levels of proteins and albumin compared to normal control. Treatment with NAC alone approximately recovered the contents of GSH, activities of GPx and levels of serum albumin, ALT and GGT relative to normal control. A tendency for normalization in activities of the antioxidant enzymes mentioned above and serum levels of liver function tests was observed in the groups treated with Art alone or Art + NAC. Conclusion: NAC downregulates oxidative stress induced by S. mansoni infection and enhances the therapeutic potential of artemether against adult schistosomes.     

Fasciola hepatica: tegumental alterations in adult flukes following in vitro and in vivo administration of artesunate and artemether

The tegumental changes in adult Fasciola hepatica induced by artemether and artesunate were assessed utilizing scanning electron microscopy (SEM). F. hepatica were incubated with artemether and artesunate for 48h at a concentration of 10microg/ml in the absence or presence of haemin. For the latter experiment both, a triclabendazole-resistant and sensitive F. hepatica isolate were used. For the in vivo studies rats were treated with single 200mg/kg oral doses of artemether and artesunate and flukes recovered from the bile ducts after 24-96h. SEM analysis of the flukes incubated in the presence of the drugs without haemin showed only minor and localized damage of the tegument. In the presence of haemin extensive tegumental damage, including sloughing, blebbing and eruptions, particularly in the ventral and dorsal mid-body and tail region, was evident. No difference in the extent of damage could be observed between artemether and artesunate and between the triclabendazole-resistant and non-resistant flukes. After 24h in vivo disruption of the tegument was evident in the artemether-treated flukes, and the damage increased in severity 48-72h post-treatment. Sloughing, swelling and extensive furrowing of the tegument was observed in several flukes, in particular in the tail region and the ventral apical cone region. In the artesunate treatment, tegumental damage was evident after 72h, but seemed slightly less pronounced when compared to the artemether-treated specimens examined at the same time point. Concluding our experiments confirm that artemether and artesunate are potent fasciocidal drugs and the tegument of adult F. hepatica appears to be a target for the action of these drugs.     

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.     

Artemether/hydroxypropyl-β-cyclodextrin host–guest system: Characterization, phase-solubility and inclusion mode

An inclusion complex of the antimalarial artemether (ATM) in hydroxypropyl-β-cyclodextrin (HPβCD) was prepared and characterized. The phase-solubility diagram for the drug showed an increase in water solubility and gave an apparent binding constant of 220 M⁻¹. According to ¹H NMR and 2D NMR spectroscopy (ROESY), the inclusion mode involves two CH₃ from the drug orientated in the HPβCD cavity. The complex was characterized by Powder X-ray diffraction and thermal analysis. In addition, the complex produces a 1.81-fold enhancement in apparent bioavailability compared to artemether.