Effect of artemether against Schistosoma haematobium in experimentally infected hamsters

The drug, artemether, has been shown to be active against the juvenile stages of Schistosoma japonicum and Schistosoma mansoni in experimentally infected animals, while it is less effective on adult worms. These findings have been confirmed in randomised controlled trials in humans. Consequently, it could be expected that artemether is also active against Schistosoma haematobium. We present here the first results from experiments assessing the effect of artemether on S. haematobium. Hamsters with a single infection received intra-gastrically an initial dose of 300 mg/kg artemether on day 14, 21 or 28, followed by further doses at varying treatment regimens. In all the treatment groups, the total and female worm reduction rates were highly significant, and ranged from 78 to 100% in hamsters harbouring juvenile schistosomes. Hamsters infected three times with S. haematobium, on days 0, 4 and 9, and repeatedly treated with artemether at the same dose as above, showed highly significant total and female worm reduction rates of between 94 and 99%. Artemether was also active against 77-day-old adult S. haematobium, since its administration on two consecutive days resulted in highly significant total and female worm reduction rates of 76-89%. Our findings confirm that artemether is also active against S. haematobium, especially the schistosomules. These results provide a basis for clinical trials in humans, for further assessment of the potential of artemether for schistosomiasis control.     

Study on the mechanism of action of artemether against schistosomes: the identification of cysteine adducts of both carbon-centred free radicals derived from artemether

Reaction of the antimalarial and anti-schistosome drug artemether (1) and catalytic amount of ferrous ion in the presence of excess cysteine gave two adducts of cysteine and previous postulated primary and secondary carbon-centred free radicals besides their other rearrangement products. This piece of further evidence for the presence of carbon-centred radicals, especially the secondary carbon-centred free radical for the first time by the isolation of its coupling adduct, will be help to understand the mechanism of action of artemether and other qinghaosu derivatives against parasites.     

Preventive effect of artemether in experimental animals infected with Schistosoma mansoni

The effect of artemether, an antimalarial drug developed from the plant Artemisia annua, has been tested against the larval stages of Schistosoma mansoni covering the time from skin penetration to the early adult liver-stage. The results show that the experimental animals used (hamster and mice) do not develop schistosomiasis mansoni if treated with artemether during the first month after infection. The parasite was found to be especially susceptible between the 3rd and 4th week after infection, resulting in worm reductions of 75.3-82.0% compared to non-treated controls. This level was boosted to 97.2-100% when the animals were subjected to various schedules of repeated treatment. Almost complete protection was also reached in parallel experiments with repeated infections carried out to mirror more closely the real situation of trickle infection.     

Evaluation of the anthelmintic effects of artesunate against experimental Schistosoma mansoni infection in mice using different treatment protocols

The therapeutic effects of artesunate against experimental Schistosoma mansoni infection in mice were analyzed. Previous studies showed that artesunate is highly effective against S. japonicum infection, but the action of this drug against S. mansoni remained uncovered. The present study examines the optical conditions for artesunate against S. mansoni and evaluates the effects of inhibiting the sexual maturation of adult worms. Mice infected with S. mansoni were orally administered with artesunate according to different schedules. Four consecutive administrations of 300 mg/kg of artesunate at 2-week intervals conferred almost total protection without the development of pathological lesions in the liver. The significant reduction in the number of eggs produced by surviving worms and the status of egg maturation suggested that artesunate inhibits sexual maturation. Electron microscopy revealed that artesunate caused morphological damage, especially on the worm tegument. Artesunate was also very effective in iron-deficient mice. Furthermore, the efficacy of artesunate was equal to or better than that of artemether against S. japonicum infection. Considering that artemether is more toxic, artesunate is currently one of the most efficient drugs against immature S. mansoni.     

Efficacy and safety of artemether in the treatment of chronic fascioliasis in Egypt: exploratory phase-2 trials

Background

Fascioliasis is an emerging zoonotic disease of considerable veterinary and public health importance. Triclabendazole is the only available drug for treatment. Laboratory studies have documented promising fasciocidal properties of the artemisinins (e.g., artemether).

Methodology

We carried out two exploratory phase-2 trials to assess the efficacy and safety of oral artemether administered at (i) 6×80 mg over 3 consecutive days, and (ii) 3×200 mg within 24 h in 36 Fasciola-infected individuals in Egypt. Efficacy was determined by cure rate (CR) and egg reduction rate (ERR) based on multiple Kato-Katz thick smears before and after drug administration. Patients who remained Fasciola-positive following artemether dosing were treated with single 10 mg/kg oral triclabendazole. In case of treatment failure, triclabendazole was re-administered at 20 mg/kg in two divided doses.

Principal Findings

CRs achieved with 6×80 mg and 3×200 mg artemether were 35% and 6%, respectively. The corresponding ERRs were 63% and nil, respectively. Artemether was well tolerated. A high efficacy was observed with triclabendazole administered at 10 mg/kg (16 patients; CR: 67%, ERR: 94%) and 20 mg/kg (4 patients; CR: 75%, ERR: 96%).

Conclusions/Significance

Artemether, administered at malaria treatment regimens, shows no or only little effect against fascioliasis, and hence does not represent an alternative to triclabendazole. The role of artemether and other artemisinin derivatives as partner drug in combination chemotherapy remains to be elucidated.     

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.     

Effect of praziquantel together with artemether on Schistosoma japonicum parasites of different ages in rabbits

The efficacy of combined treatment with praziquantel and artemether against infection with Schistosoma japonicum was tested on infected rabbits, in which 7-to 14-day-old schistosomules and 42-day-old adult schistosomes were simultaneously present. Rabbits were treated orally with praziquantel and artemether using various dosages and schedules. The therapeutic effects were evaluated by estimating the mean total worm burden (TWB) and female worm burden (FWB) and comparing them with the worm burdens in control animals treated with praziquantel or artemether alone. When the rabbits received praziquantel in a single dose (50 mg/kg), or daily for 2-6 days (30-60 mg/kg), the TWB was reduced by 28-66% and the FWB by 26-65%. In rabbits treated with artemether the reductions were 44-56% and 35-54%, respectively. Treatment with praziquantel in combination with artemether resulted in a significantly greater reduction of worm burden than was found for the groups treated with praziquantel or artemether alone, using the same dosages and schedules. TWB was reduced by 79-92%, and FWB by 80-93%. The results demonstrated that when rabbits infected simultaneously with schistosomules and adult schistosomes were treated with praziquantel in combination with artemether, the effects of the individual drugs could be increased significantly.     

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.     

Population pharmacokinetics of Artemether and dihydroartemisinin in pregnant women with uncomplicated Plasmodium falciparum malaria in Uganda

ABSTRACT: BACKGROUND: Malaria in pregnancy increases the risk of maternal anemia, abortion and low birth weight. Approximately 85.3 million pregnancies occur annually in areas with Plasmodium falciparum transmission. Pregnancy has been reported to alter the pharmacokinetic properties of many anti-malarial drugs. Reduced drug exposure increases the risk of treatment failure. The objective of this study was to evaluate the population pharmacokinetic properties of artemether and its active metabolite dihydroartemisinin in pregnant women with uncomplicated P. falciparum malaria in Uganda. METHODS: Twenty-one women with uncomplicated P. falciparum malaria in the second and third trimesters of pregnancy received the fixed oral combination of 80 mg artemether and 480 mg lumefantrine twice daily for three days. Artemether and dihydroartemisinin plasma concentrations after the last dose administration were quantified using liquid chromatography coupled to tandem mass-spectroscopy. A simultaneous drug-metabolite population pharmacokinetic model for artemether and dihydroartemisinin was developed taking into account different disposition, absorption, error and covariate models. A separate modeling approach and a non-compartmental analysis (NCA) were also performed to enable a comparison with literature values and different modeling strategies. RESULTS: The treatment was well tolerated and there were no cases of recurrent malaria. A flexible absorption model with sequential zero-order and transit-compartment absorption followed by a simultaneous one-compartment disposition model for both artemether and dihydroartemisinin provided the best fit to the data. Artemether and dihydroartemisinin exposure was lower than that reported in non-pregnant populations. An approximately fourfold higher apparent volume of distribution for dihydroartemisinin was obtained by noncompartmental analysis and separate modeling compared to that from simultaneous modeling of the drug and metabolite. This highlights a potential pitfall when analyzing drug/metabolite data with traditional approaches. CONCLUSION: The population pharmacokinetic properties of artemether and dihydroartemisinin, in pregnant women with uncomplicated P. falciparum malaria in Uganda, were described satisfactorily by a simultaneous drug-metabolite model without covariates. Concentrations of artemether and its metabolite dihydroartemisinin were relatively low in pregnancy compared to literature data. However, this should be interpreted with caution considered the limited literature available. Further studies in larger series are urgently needed for this vulnerable group.     

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.     

Development of a Specific Monoclonal Antibody-Based ELISA to Measure the Artemether Content of Antimalarial Drugs

Artemether is one of the artemisinin derivatives that are active ingredients in antimalarial drugs. Counterfeit and substandard antimalarial drugs have become a serious problem, which demands reliable analytical tools and implementation of strict regulation of drug quality. Structural similarity among artemisinin analogs is a challenge to develop immunoassays that are specific to artemisinin derivatives. To produce specific antibodies to artemether, we used microbial fermentation of artemether to obtain 9-hydroxyartemether, which was subsequently used to prepare a 9-O-succinylartemether hapten for conjugation with ovalbumin as the immunogen. A monoclonal antibody (mAb), designated as 2G12E1, was produced with high specificity to artemether. 2G12E1 showed low cross reactivities to dihydroartemisinin, artemisinin, artesunate and other major antimalarial drugs. An indirect competitive enzyme linked immunosorbent assay (icELISA) developed showed a concentration causing 50% of inhibition for artemether as 3.7 ng mL⁻¹ and a working range of 0.7–19 ng mL⁻¹. The icELISA was applied for determination of artemether content in different commercial drugs and the results were comparable to those determined by high-performance liquid chromatography analysis. In comparison with reported broad cross activity of anti-artemisinin mAbs, the most notable advantage of the 2G12E1-based ELISA is its high specificity to artemether only.     

Evidence of an increased incidence of day 3 parasitaemia in Suriname: an indicator of the emerging resistance of Plasmodium falciparum to artemether

The emerging resistance to artemisinin derivatives that has been reported in South-East Asia led us to assess the efficacy of artemether-lumefantrine as the first line therapy for uncomplicated Plasmodium falciparum infections in Suriname. This drug assessment was performed according to the recommendations of the World Health Organization in 2011. The decreasing number of malaria cases in Suriname, which are currently limited to migrating populations and gold miners, precludes any conclusions on artemether efficacy because adequate numbers of patients with 28-day follow-up data are difficult to obtain. Therefore, a comparison of day 3 parasitaemia in a 2011 study and in a 2005/2006 study was used to detect the emergence of resistance to artemether. The prevalence of day 3 parasitaemia was assessed in a study in 2011 and was compared to that in a study in 2005/2006. The same protocol was used in both studies and artemether-lumefantrine was the study drug. Of 48 evaluable patients in 2011, 15 (31%) still had parasitaemia on day 3 compared to one (2%) out of 45 evaluable patients in 2005/2006. Overall, 11 evaluable patients in the 2011 study who were followed up until day 28 had negative slides and similar findings were obtained in all 38 evaluable patients in the 2005/2006 study. The significantly increased incidence of parasite persistence on day 3 may be an indication of emerging resistance to artemether.     

Effect of artemether,artesunate, OZ78, praziquantel,and tribendimidine alone or in combination chemotherapy on the tegument of Clonorchis sinensis

We investigated the morphological effects of half-strength treatments with praziquantel, artemether, artesunate, OZ78 and tribendimidine as well as combinations of praziquantel with artemether, artesunate, OZ78 and tribendimidine and an artesunate–tribendimidine combination in rats harboring adult Clonorchis sinensis. Rats were infected with C. sinensis, dosed orally with single agents or combination treatments and flukes recovered at 3 or 5 days post-treatment. The number of flukes was counted, the viability recorded and surface changes monitored by scanning electron microscopy. Drug effects induced by the individual drugs at sub-curative doses 3 days post-treatment were minor with the exception of flukes recovered from rats treated with artemether and tribendimidine. Treatment with the praziquantel combinations of artesunate, OZ78 and tribendimidine did not produce a greater disruption of the tegument than the individual drugs 3 days post-treatment. On the other hand, at this time point many worms treated with artemether–praziquantel had died and eruptions, roughening or blebbing were observed on all worms examined. Five days post-treatment flukes exposed to any of the praziquantel combinations in rats had died. Rats treated with an artesunate–tribendimidine combination resulted in a rapid death of flukes, 3 days post-treatment all worms had been expelled.In conclusion, we have confirmed the promising clonorchicidal properties of different drug combinations in rats. Differences in the extent and time-scale of tegumental disruption have been observed. The effect of drug combinations against C. sinensis requires further scientific inquiry, e.g. in transmission electron microscopy studies and in the C. sinensis-rabbit model.     

Plasmodium vivax: in vitro susceptibility of blood stages to synthetic trioxolane compounds and the diamidine DB75

Plasmodium vivax is an important human pathogen causing malaria in more temperate climates of the world. Similar to Plasmodium falciparum, the causative agent for malaria tropica, drug resistance is beginning to emerge for this parasite species and this hampers adequate treatment of infection. We have used a short-term ex vivo drug assay to monitor activity of OZ277 (RBx-11160), a fully synthetic anti-malarial peroxide, and the diamidine DB75 against P. vivax. For both compounds as well as the anti-malarial reference compounds artesunate, artemether, and chloroquine, the in vitro IC(50) values were determined in one-cycle hypoxanthine incorporation assays. Results from such assays were found to be very similar compared to IC(50) values obtained from one-cycle P. falciparum hypoxanthine assays. We demonstrate the anti-parasite activity of OZ277 and the reference compounds to be faster than that of DB75. These data warrant clinical testing of OZ277 against P. vivax malaria and support recent data on clinical activity against P. vivax for DB75.     

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.     

Artemether Combined with shRNA Interference of Vascular Cell Adhesion Molecule-1 Significantly Inhibited the Malignant Biological Behavior of Human Glioma Cells

Artemether is the derivative extracted from Chinese traditional herb and originally used for malaria. Artemether also has potential therapeutic effects against tumors. Vascular cell adhesion molecule-1 (VCAM-1) is an important cell surface adhesion molecule associated with malignancy of gliomas. In this work, we investigated the role and mechanism of artemether combined with shRNA interference of VCAM-1 (shRNA-VCAM-1) on the migration, invasion and apoptosis of glioma cells. U87 human glioma cells were treated with artemether at various concentrations and shRNA interfering technology was employed to silence the expression of VCAM-1. Cell viability, migration, invasiveness and apoptosis were assessed with MTT, wound healing, Transwell and Annexin V-FITC/PI staining. The expression of matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9) and phosphorylated Akt (p-Akt) was checked by Western blot assay. Results showed that artemether and shRNA-VCAM-1 not only significantly inhibited the migration, invasiveness and expression of MMP-2/9 and p-Akt, but also promoted the apoptosis of U87 cells. Combined treatment of both displayed the maximum inhibitory effects on the malignant biological behavior of glioma cells. Our work revealed the potential therapeutic effects of artemether and antiVCAM-1 in the treatments of gliomas.     

Plasmodium vinckei: selection of a strain exhibiting stable resistance to arteether

A strain of rodent malaria parasite Plasmodium vinckei showing >12-fold resistance to arteether has been selected after exposure to sub-curative doses of drug in 44 sequential passages over a period of 700 days. Experimentally induced resistance was found to be stable after drug free maintenance of parasites for 11 serial passages over a period of 100 days. Cross-sensitivity studies have shown that apart form resistance to related derivatives like artemether and artesunic acid, the derived parasites also show resistance to quinine and mefloquine.     

Evaluation of Influence of Single Nucleotide Polymorphisms in Cytochrome P450 2B6 on Substrate Recognition Using Computational Docking and Molecular Dynamics Simulation

In this study, we investigated the influence of single nucleotide polymorphisms on the conformation of mutated cytochrome P450 (CYP) 2B6 proteins using molecular dynamics (MD) simulation. Some of these mutations influence drug metabolism activities, leading to individual variations in drug efficacy and pharmacokinetics. Using computational docking, we predicted the structure of the complex between the antimalarial agent artemether and CYP2B6 whose conformations were obtained by MD simulation. The simulation demonstrated that the entire structure of the protein changes even when a single residue is mutated. Moreover, the structural flexibility is affected by the mutations and it may influence the enzyme activity. The results suggest that some of the inactive mutants cannot recognize artemether due to structural changes caused by the mutation.     

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.     

Tegumental alterations in juvenile Schistosoma haematobium harboured in hamsters following artemether treatment

We report the findings of a detailed temporal study on tegumental alterations in juvenile Schistosoma haematobium, induced by artemether, using scanning electron microscopy. Hamsters infected with S. haematobium cercariae for 28 days were treated intragastrically with a single dose of 300 mg/kg artemether. Groups of two hamsters were killed 24 h, 72 h and 7 days after treatment, and schistosomula were recovered from livers by perfusion and subsequent systematic examination of the tissue, before routinely processing for scanning electron microscopic examination. Most schistosomula collected 24 h after artemether administration showed severe tegumental damage, usually including swelling, fusion, vesiculation, peeling and collapse of enlarged sensory structures. After 72 h, tegumental damage had increased and schistosomula generally showed contraction with extensive swelling, erosion and peeling of the tegument. Seven days post-treatment, severe tegumental damage was only seen in a single male specimen with swelling of the worm body and destruction of the oral sucker. The other schistosomula showed only light to moderate damage, suggesting that schistosomula surviving the treatment began to recover. Our findings of tegumental damage following artemether treatment correlate with the efficacy of this novel antischistosomal drug in killing the juvenile stages of S. haematobium and complement recent findings with S. japonicum and S. mansoni.