Conceived and designed the experiments: LT AS JK. Performed the experiments: LT AS. Analyzed the data: LT AS JK. Wrote the paper: LT JK.
The authors have declared that no competing interests exist.
Few effective drugs are available for soil-transmitted helminthiases and drug resistance is of concern. In the present work, we tested the efficacy of the veterinary drug monepantel, a potential drug development candidate compared to standard drugs
A motility assay was used to assess the efficacy of monepantel, albendazole, levamisole, and pyrantel pamoate
Monepantel showed excellent activity on
Monepantel reveals low or no activities against
Soil-transmitted helminthiases affect more than one billion people among the most vulnerable populations in developing countries. Currently, control of these infections primarily relies on chemotherapy. Only five drugs are available, all of which have been in use for decades. None of the drugs are efficacious using single doses against all soil-transmitted helminths (STH) species and show low efficacy observed against
The hookworm species
Helminth control relies primarily on the regular administration of anthelmintics, typically carried out within the framework of school-based deworming programs, once or twice a year
Since drug resistance to nematodes of veterinary importance is widely spread and increasing in frequency, most of the anthelmintic drug research and development efforts are motivated by veterinary needs
Monepantel (AAD1566) belongs to a new class of veterinary anthelmintics, the amino-acetonitrile derivatives. It has been proposed that monepantel interferes with nematode-specific acetylcholine receptor subunits, leading to body wall muscle paralysis and subsequent death of worms. Due to its unique mode of action, the drug has proven efficacy against nematodes infecting livestock which are resistant to current anthelmintic drugs
The aim of the present investigation was to study the activity of monepantel, compared with the reference drugs, albendazole, levamisole, and pyrantel pamoate, in five parasite-rodent models, that correspond to important human STH and
Monepantel was kindly provided by Novartis Animal Health, St-Aubin, Switzerland. Albendazole and pyrantel pamoate were purchased from Sigma-Aldrich (Buchs, Switzerland), and levamisole-hydrochloride from Fluka (Buchs, Switzerland).
For the
Three-week-old male Syrian Golden hamsters were purchased from Charles River (Sulzfeld, Germany). Four-week-old female NMRI mice and 3-week-old female C57Bl/6J mice were purchased from Harlan (Horst, The Netherlands). Three-week-old female Wistar rats were purchased from Harlan (Horst, The Netherlands).
All animals were kept in macrolon cages under environmentally-controlled conditions (temperature: 25°C, humidity: 70%, light/dark cycle 12 h/12 h) and had free access to water and rodent food (Rodent Blox from Eberle NAFAG, Gossau, Switzerland). They were allowed to acclimatize in the animal facility of the Swiss Tropical and Public Health Institute (Swiss TPH) for 1 week before infection. The current study was approved by the local veterinary agency based on Swiss cantonal and national regulations (permission no. 2070).
Infective
Embryonated
The
Infective
The larval or adult motility assay is currently the method of choice to evaluate drug sensitivity of different nematode species
Thirty L3 in 100 µl deionized water, supplemented with antibiotics, were placed in each well of a 96-well plate (Costar). The L3 had been harvested less than 1 month before the studies and were stored in deionized water containing 25 µg/ml amphotericin B (Sigma-Aldrich) and 1% (v/v) penicillin-streptomycin solution (10,000 U/ml penicillin and 10 mg/ml streptomycin, Sigma-Aldrich). Drugs were serially-diluted in HBSS (Gibco) supplemented with 25 µg/ml amphotericin B (Sigma Aldrich) and 1% (v/v) penicillin-streptomycin solution (10,000 U/ml penicillin and 10 mg/ml streptomycin, Sigma-Aldrich). Diluted drug (100 µl; 100–0.01 µg/ml, final concentration) were added to the wells and the plate was incubated for 72 h at room-temperature in a dark and humid box. Larval motility was evaluated under the microscope (magnification 20×) following addition of 100 µl hot water (∼90°C) and exposure to microscope light. A minimum of 2 wells served as controls, which were L3 incubated with the highest concentration of DMSO used in the test (2% v/v).
Drug susceptibility on adults was tested in 48-well plates (Costar). Three to 4 worms were added to wells containing 500 µl supplemented HBSS medium, containing 10% v/v fetal calf serum (Gibco). Drug dilutions (500 µl) were added and the plate was incubated for 72 h at 37°C and 5% CO2. The motility of adult worms was evaluated under the microscope (magnification 20×), after adding 500 µl hot water (∼90°C), using a viability scale (2: good motility, 1: lowered motility, and 0: no motility, death). A minimum of 3 worms served as controls which were incubated in the presence of the highest concentration of DMSO used in the test (2% v/v).
The drug effects on eggs were observed using a modified protocol of the egg hatch test
L3 or adults were collected from the intestines of infected mice and 3–4 worms were distributed to each well of 48 or 96-well plates (Costar), containing 100 µl (L3) or 500 µl (adults) RPMI medium supplemented with 5% (v/v) amphotericin B (Sigma Aldrich) and 1% (v/v) penicillin-streptomycin solution (10,000 U/ml penicillin and 10 mg/ml streptomycin, Sigma-Aldrich). Drug dilutions (100 µl for testing on L3 or 500 µl for adults) ranging from 200–50 µg/ml, final concentration, were added and the plate was incubated for 72 h at 37°C and 5% CO2. The larval and adult motilities were evaluated under the microscope (magnification 20–80×) using a viability scale (3: good motility, 2: low motility, 1: very low motility, and 0: death), as described by Stepek and colleagues
The immunosuppressive treatment of hamsters was stopped at least 2 days before treatment. Hamsters were housed individually from day 20 post-infection (p.i.) onwards. On days 21 and 22 (
Hamsters were treated on day 23 p.i. (
Treatment with dexamethasone was stopped at least 2 days before treatment. Mice were housed individually from day 40 onwards. A fecal sample was examined from each mouse and egg negative animals were excluded from the study. Groups of 4 infected animals were assigned to treatment (monepantel 600 mg/kg, albendazole 600 mg/kg, levamisole 200 mg/kg, or pyrantel pamoate 300 mg/kg) or control groups. Expelled worms were counted from the collected stools 24 h and 48 h after treatment. On day 7 post-treatment, mice were killed by the CO2 method and the remaining worms in the gut counted
Five days p.i, 1 group of 4 rats was treated orally with 32 mg/kg monepantel. Four rats were left untreated and served as controls. Seven days post-treatment, the rats were dissected. The intestine was removed, opened and incubated for 3 h at 37°C in PBS, as described before
One hour p.i, 2 groups of 4 mice were treated with single oral doses of 600 mg/kg monepantel or 600 mg/kg albendazole. A third group of 4 mice was left untreated and served as control. On day 7 post-treatment, the mice were killed by the CO2 method. The lungs and livers were removed, cut with fine scissors, and incubated in 0.9% NaCl for 24 h at 37°C to allow larvae to migrate out of the organs into the saline
The average of motility scores for one drug was calculated for each concentration and normalized into percentage, relative to control. IC50 values were expressed based on the median effect principle using CompuSyn (version 1.0). The r value represents the linear correlation coefficient of the median-effect plot, indicating the goodness of fit, hence the accuracy of the IC50
The effects of monepantel and reference drugs on L3 and adult worms of
Drugs |
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IC50 in µg/ml (r) | IC50 in µg/ml (r) | IC50 in µg/ml (r) | ||||
L3 | Adults | L3 | Adults | L3 | Adults | |
Monepantel | >100 (0.70) | 1.7 (0.93) | >100 (n.d.) | >100 (0.32) | 78.7 (1.0) | >200 (n.d.) |
Albendazole | 32.4 (0.87) | >100 (0.77) | >100 (n.d.) | >100 (0.57) | >200 (0.39) | >200 (0.40) |
Levamisole-HCl | 1.6 (0.95) | >100 (0.79) | 0.5 (0.97) | 13.4 (0.99) | 33.1 (1.0) | 16.5 (1.0) |
Pyrantel pamoate | 90.9 (0.84) | >100 (0.79) | 2.0 (0.94) | 7.6 (0.98) | 95.5 (0.99) | 34.1 (0.99) |
IC50s (µg/ml) were calculated for monepantel, albendazole, levamisole, and pyrantel pamoate after 72 h on L3 and adult stages of
The sensitivity of
As shown in
Group | % Embryonation at 24 h (SD) | % Hatching at 48 h (SD) |
Control | 100 (3.5) | 100 (9.8) |
Monepantel | 99.5 (9.3) | 68.6 (9.2) |
Albendazole | 9.5 (5.2) |
1.5 (3.0) |
Levamisole-HCl | 88.6 (8.2) |
58.5 (3.5) |
Pyrantel pamoate | 95.4 (7.0) | 95.3 (12.5) |
SD = standard deviation.
*P-value <0.001 (Fisher's exact test).
Neither monepantel nor albendazole had an effect on the viability of larvae or adult
A reduction in viability of
The worm expulsion rates and worm burden reductions determined for monepantel, albendazole, levamisole, and pyrantel pamoate administered to
Group | Dose(mg/kg) | Mean number of worms (SD) | Mean number of expelled worms (SD) | Worm expulsion rate (%) | Worm burden reduction (%) | |
Control 1 | – | 29.5 (21.2) | 0 | 0 | – | – |
Control 2 | – | 24.5 (8.8) | 0 | 0 | – | – |
Control 3 | – | 29.7 (4.2) | 0.3 (0.6) | 1.1 | – | – |
Control 4 | – | 27.0 (4.0) | 0.3 (0.6) | 1.2 | – | – |
Monepantel | 51 | 22.3 (10.1) | 9.5 (4.4) | 42.7 | 56.8 | 0.046 |
101 | 16.0 (9.7) | 16.0 (9.7) | 100 | 100 | ||
Albendazole | 1.254 | 11.0 (6.9) | 7.8 (3.8) | 70.5 | 87.8 | <0.001 |
2.53 | 6.3 (7.1) | 6.3 (7.1) | 100 | 100 | ||
52 | 6.3 (7.1) | 6.3 (7.1) | 100 | 100 | ||
Levamisole-HCl | 102 | 17.5 (7.2) | 7.8 (2.5) | 44.3 | 60.2 | 0.057 |
Pyrantel pamoate | 103 | 10.3 (9.3) | 6.5 (6.2) | 63.4 | 87.2 | 0.057 |
SD = standard deviation. The numbers in superscript refer to the corresponding control group.
*Kruskal Wallis test comparing the median of the worm burdens of control and treated hamsters (all doses versus control),
Mann-Whitney U-test comparing the median of the worm burdens of control and treated hamsters (one dose versus control).
As presented in
Group | Dose(mg/kg) | Mean number of worms (SD) | Mean number of expelled worms (SD) | Worm expulsion rate (%) | Worm burden reduction (%) | |
Control | – | 8.0 (7.5) | 0 (0) | 0 | – | – |
Monepantel | 20 | 19.0 (12.0) | 7.3 (4.5) | 38.6 | 0 | 0.830 |
10 | 8.0 (4.4) | 4.7 (3.1) | 58.3 | 58.3 | ||
Albendazole | 10 | 6.7 (2.5) | 6.7 (2.5) | 100 | 100 | 0.028 |
5 | 7.7 (3.1) | 5.3 (3.8) | 69.6 | 70.8 |
SD = standard deviation.
*Kruskal Wallis test comparing the median of the worm burdens of control and treated hamsters (all doses versus control).
The worm expulsion rates and worm burden reductions determined for monepantel, albendazole, levamisole, and pyrantel pamoate administered to
Group | Dose(mg/kg) | Mean number of worms (SD) | Mean number of expelled worms (SD) | Worm expulsion rate (%) | Worm burden reduction (%) | |
Control | – | 90.0 (41.5) | 1.0 (1.2) | 0 | – | – |
Monepantel | 600 | 106.6 (159.1) | 1.7 (0.6) | 1.6 | 0 | 0.536 |
Albendazole | 600 | 140.3 (116.6) | 69.3 (48.1) | 49.4 | 20.2 | 0.536 |
Levamisole-HCl | 200 | 29.0 (60.9) | 26.3 (54.6) | 90.5 | 95.9 | 0.036 |
Pyrantel pamoate | 300 | 282.3 (246.6) | 26.7 (20.6) | 9.4 | 0 | 0.095 |
SD = standard deviation.
Mann-Whitney U test comparing the median of the worm burdens of control and treated mice.
Monepantel displayed negligible effect on the worm burden in the mice's lungs and liver, 7 days after treatment with 600 mg/kg (worm burden reduction = 3.3%,
Group | Dose(mg/kg) | Mean number of worms following treatment (SD) | Worm burden reduction (%) | |
Control | – | 48.4 (37.33) | – | – |
Monepantel | 600 | 46.8 (21.33) | 3.3 | 0.886 |
Albendazole | 600 | 11.35 (13.26) | 76.6 | 0.171 |
SD = standard deviation.
Mann-Whitney U test comparing the median of the worm burdens of control and treated mice.
To date, only five drugs are included in the WHO model list of essential medicines to treat infections with human STH. Most of these anthelmintics were discovered before the 1980s. Though there is no evidence yet for emerging resistance to any of these drugs in human helminth populations, there are worrying signs that anthelminthic efficacy may be declining
Monepantel activates signaling
In addition, monepantel lacked activity in
Finally, although only one high dosage was tested, our data indicate that
Like
In the present investigation, albendazole, levamisole, and pyrantel pamoate have been extensively studied
Interestingly, contradictory results were obtained with albendazole, levamisole, and pyrantel pamoate against adult
In conclusion, to our knowledge, we have for the first time analyzed the efficacy of monepantel in animal models corresponding to human intestinal helminthiases. A recently developed target product profile suggested that a drug development candidate for the treatment of infections with STH should ideally target all stages (at least adult and ova) and species of the major geohelminths such as
We thank R. Kaminsky from Novartis Animal Health, St-Aubin, Switzerland, for supplying us with monepantel (AAD1566) and for his helpful comments on the manuscript. We acknowledge Mireille Vargas and Augustine Corfu for excellent technical assistance.