The pharmacokinetics and drug-drug interactions of ivermectin in Aedes aegypti mosquitoes

Abstract

Mosquitoes are vectors of major diseases such as dengue fever and malaria. Mass drug administration of endectocides to humans and livestock is a promising complementary approach to current insecticide-based vector control measures. The aim of this study was to establish an insect model for pharmacokinetic and drug-drug interaction studies to develop sustainable endectocides for vector control. Female Aedes aegypti mosquitoes were fed with human blood containing either ivermectin alone or ivermectin in combination with ketoconazole, rifampicin, ritonavir, or piperonyl butoxide. Drug concentrations were quantified by LC-MS/MS at selected time points post-feeding. Primary pharmacokinetic parameters and extent of drug-drug interactions were calculated by pharmacometric modelling. Lastly, the drug effect of the treatments was examined. The mosquitoes could be dosed with a high precision (%CV: ≤13.4%) over a range of 0.01-1 μg/ml ivermectin without showing saturation (R2: 0.99). The kinetics of ivermectin were characterised by an initial lag phase of 18.5 h (CI90%: 17.0-19.8 h) followed by a slow zero-order elimination rate of 5.5 pg/h (CI90%: 5.1-5.9 pg/h). By contrast, ketoconazole, ritonavir, and piperonyl butoxide were immediately excreted following first order elimination, whereas rifampicin accumulated over days in the mosquitoes. Ritonavir increased the lag phase of ivermectin by 11.4 h (CI90%: 8.7-14.2 h) resulting in an increased exposure (+29%) and an enhanced mosquitocidal effect. In summary, this study shows that the pharmacokinetics of drugs can be investigated and modulated in an Ae. aegypti animal model. This may help in the development of novel vector-control interventions and further our understanding of toxicology in arthropods.

Fig 1. Ivermectin dosing precision and linearity of Aedes aegypti mosquitoes.

Mosquitoes (n = 30) were treated with blood containing 0.01, 0.05, 0.1, 0.5, and 1 μg/ml ivermectin. The amount of ivermectin shows a linear relationship with the applied concentration in blood (regression line R²: 0.99, black dashed line), while the coefficient of variation (CV%) was ≤13.4% (numbers above the regression line). The grey circles correspond to ivermectin amounts recovered from single extracted mosquitoes. The red cross depicts the median amount of ivermectin recovered from the mosquitoes.

Fig 2. Goodness-of-fit plots for the final model (dashed lines: Local weighted smooth).

Observed data vs. population predictions (top left), observed data vs. individual predictions (top right), absolute conditional weighted residuals (CWRES) vs. individual predictions (bottom left), and CWRES over time (bottom right).

Fig 3. Concentration-time course of ivermectin single and combination treatments in Aedes aegypti mosquitoes.

Aedes aegypti mosquitoes were treated with ivermectin (IVM: 0.1 μg/ml) alone or in combination with ketoconazole (KCZ: 5 μg/ml), rifampicin (RIF:10 μg/ml), ritonavir (RTV: 10 μg/ml), or piperonyl butoxide (PBO: 10 μg/ml). At 0, 6, 12, 24, 48, and 72 h post treatment, the drug concentration in up to 20 live mosquitoes was analysed. Concentration-time profiles of ivermectin single and combination treatments are illustrated in the left column and the profile of the respective CYP modulator in the right column. Large circles depict the mean concentration and the small circles (single treatment: white, combination treatment: red) represent the drug amount extracted per individual mosquito.

Fig 4. Drug effect of mono- and combination treatments on mosquito mortality, fecundity, and fertility.

Aedes aegypti mosquitoes were treated with blank human blood, blood containing ivermectin (IVM: 0.1 μg/ml), ketoconazole (KCZ: 5 μg/ml), rifampicin (RIF: 10 μg/ml), ritonavir (RTV: 10 μg/ml) or piperonyl butoxide (PBO: 10 μg/ml), or a drug combination of ivermectin (0.1 μg/ml) plus ketoconazole (5 μg/ml), rifampicin (10 μg/ml), ritonavir (RTV: 10 μg/ml) or piperonyl butoxide (10 μg/ml). 3 batches of 50 mosquitoes were investigated per treatment group. Median percentage survival of the mosquitoes was assessed after 1, 2, 3, and 4 days. The error bars correspond to the range. The fecundity, the amount of eggs excreted, and fertility, the proportion of hatched larvae, were measured after 4 days. The floating-bars display the range and the line in the middle the median value.