Efficacy of cyclooctadepsipeptides and aminophenylamidines against larval, immature and mature adult stages of a parasitologically characterized trichurosis model in mice

Abstract

Background: The genus Trichuris includes parasites of major relevance in veterinary and human medicine. Despite serious economic losses and enormous impact on public health, treatment options against whipworms are very limited. Additionally, there is an obvious lack of appropriately characterized experimental infection models. Therefore, a detailed parasitological characterization of a Trichuris muris isolate was performed in C57BL/10 mice. Subsequently, the in vivo efficacies of the aminophenylamidines amidantel, deacylated amidantel (dAMD) and tribendimidine as well as the cyclooctadepsipeptides emodepside and in particular PF1022A were analyzed. This was performed using various administration routes and treatment schemes targeting histotropic and further developed larval as well as immature and mature adult stages.

Methodology/principal findings: Duration of prepatent period, time-dependent localization of larvae during period of prepatency as well as the duration of patency of the infection were determined before drugs were tested in the characterized trichurosis model. Amidantel showed no effect against mature adult T. muris. Tribendimidine showed significantly higher potency than dAMD after oral treatments (ED50 values of 6.5 vs. 15.1 mg/kg). However, the opposite was found for intraperitoneal treatments (ED50 values of 15.3 vs. 8.3 mg/kg). When emodepside and PF1022A were compared, the latter was significantly less effective against mature adults following intraperitoneal (ED50 values of 6.1 vs. 55.7 mg/kg) or subcutaneous (ED50 values of 15.2 vs. 225.7 mg/kg) administration. Only minimal differences were observed following oral administration (ED50 values of 2.7 vs. 5.2 mg/kg). Triple and most single oral doses with moderate to high dosages of PF1022A showed complete efficacy against histotropic second stage larvae (3 × 100 mg/kg or 1 × 250 mg/kg), further developed larvae (3 × 10 mg/kg or 1 × 100 mg/kg) and immature adults (3 × 10 mg/kg or 1×100 mg/kg). Histotropic first stage larvae were only eliminated after three doses of PF1022A (3 × 100 mg/kg) but not after a single dose.

Conclusions/significance: These results indicate that the cyclooctadepsipeptides are a drug class with promising candidates for further evaluation for the treatment of trichurosis of humans and livestock animals in single dose regimens.

Figure 1. Analysis on egg shedding in the course of patency of the infection.

The graph shows the arithmetic mean values with standard deviations of the absolute numbers of eggs per gram feces between days 35 and 105 p.i. with a group size of nine animals. Due to the death of one mouse, group size was reduced to eight starting from day 70 p.i.

Figure 2. Analysis on the occurrence of specific stages of T. muris in the course of the period of prepatency.

Presented is the occurrence of first stage larvae in the luminal content of the guts (free stages), of histotropic first, second and third stage larvae (histotropic stages) and of third and fourth stage larvae as well as immature and mature adults attached to the epithelium while extruding their posterior parts into the lumen of the guts (extruding stages) between days 1 and 31 p.i. Based on dissection of three mice per time point, the graph shows three data points for each stage and time point. If the count was found to be zero for a specific stage in each of the three independent counts, data points are not shown.

Figure 3. Analysis on the occurrence of T. muris in the course of patency of the infection.

(A) Absolute worm counts in the course of patency of the infection. The graph shows the arithmetic mean values and standard deviations of the absolute number of recovered worms during time with a group size of six animals per time point. Mean worm counts were compared to day 35 p.i. using One-Way-ANOVA followed by Dunnet's post hoc test. **, p<0.01 vs. day 35. (B) Sex ratio of T. muris in the course of patency of the infection. Graph shows the arithmetic means with standard deviations of the recovered male worms expressed as percentage of total recovered worms with a group size of six animals per time point. ***, p<0.001 vs. day 35.

Figure 4. In vivo dose-response curves of dAMD (blue) and tribendimidine (red) after oral (A) and intraperitoneal (B) treatments against mature adults of T. muris.

Dose-response curves show the arithmetic mean values and standard errors of the mean with a group size of five animals per drug and dose. Efficacy was calculated as relative number of recovered worms compared to the no-drug control in percentage. Dosages were log₁₀ transformed and logistic regressions were calculated with top values constrained between 0 and 100%. Efficacies were set to zero if mean of the worm counts was higher than the mean of the corresponding control group. Furthermore, the corresponding SEM values of the affected groups start from zero. The no-drug controls were set to 10⁻⁴ mg/kg to allow log₁₀ transformation of dosages.

Figure 5. In vivo dose-response curves of emodepside (red) and PF1022A (blue) after oral (A), intraperitoneal (B) or subcutaneous (C) treatments against mature adults of T. muris.

Dose-response curves show the arithmetic mean values with standard errors of the mean with a group size of five animals per drug and dose. Efficacy was calculated as relative number of recovered worms compared to the no-drug control in percentage. Dosages were log₁₀ transformed and logistic regressions were calculated with top values constrained between 0 and 100%. Triangles indicate a single dose of PF1022A (light blue), circles three doses of PF1022A (dark blue) and squares three doses of emodepside (red). Efficacies were set to zero if mean of the worm counts was higher than the mean of the corresponding control group. Furthermore, the corresponding SEM values of the affected groups start from zero. The no-drug controls were set to 10⁻⁴ mg/kg to allow log₁₀ transformation of dosages.

Figure 6. In vivo efficacy of PF1022A against L3, L4 and immature adults (A, B), histotropic L2 (C, D) and histotropic L1 (E, F) of T. muris using both, single (A, C, E) and triple (B, D, F) dose regimens.

Box plots show the median numbers and quartiles of recovered adult T. muris after treatment against immature stages with wiskers representing minimal and maximal values. Group sizes were 5 mice per drug and dose. +, arithmetic mean; *, p<0.01 vs. control; **, p<0.001 vs. control.