Analysis of the efficacy of aminophenylamidines and cyclooctadepsipeptides and their mode of action (2014)

Parasitic helminths including cestodes, trematodes and nematodes, have an enormous impact on human and animal health and cause tremendous financial losses. Therefore, the urgent demand for modern and safe treatment options against helminthoses is obvious. In addition to the optimisation of existing drug classes, two major objectives are in the focus of research to increase treatment options against helminth infections: (i) the evaluation of combinations, to increase the anthelmintic spectrum, to improve the efficacy of the respective drugs and to decelerate the development of resistance and (ii) the identification and development of safe and highly efficacious new drug classes with novel, resistance-breaking modes of action. The present cumulative thesis, consisting of five publications, describes investigations addressing both of the above mentioned aspects by comparing the potential of the aminophenylamidines amidantel, tribendimidine and deacylated amidantel (dAMD) and of the cyclooctadepsipeptides emodepside and PF1022A for the treatment against helminth infections of humans and animals in monotherapy as well as in several combinations. Since activitiy of the nematicidal aminophenylamidines against cestodes has already been published, but respective reports were somewhat informal, publication 1 aimed on the evaluation of the efficacy of tribendimidine and dAMD against the tapeworm Hymenolepis microstoma in a controlled laboratory trial. Whereas treatments with three dosages of up to 100 mg/kg dAMD did not result in any significant reduction of the worm burdens, in marked contrast, treatments with 3× 50 mg/kg tribendimidine, which is the parent compound of dAMD, resulted in high parasite clearance. In publication 2, substantial concentration-dependent anthelmintic potencies of aminophenylamidines and cyclooctadepsipeptides as well as of some drug combinations were detected against larval and adult stages of the rat hookworm Nippostrongylus brasiliensis and against first larval stages of Trichinella spiralis using three in vitro assays. Consequently, in publication 3 the same derivatives were found to be highly efficacious using the N. brasiliensis in vivo model in rats. Moreover, tested drug combinations of two cholinergic drugs or an aminophenylamidine plus a cyclooctadepsipeptide resulted in additive effects, making not only the individual drugs but also combinations interesting for further evaluation. Furthermore, a pharmacokinetic analysis revealed that after oral administration of tribendimidine, only its metabolite dAMD was detectable in rat blood samples. Finally, electron microscopic analysis of hookworms treated with the test compounds revealed information about their mode of action. Worms treated with PF1022A showed signs of flaccid paralysis, whereas individuals treated with cholinergic drugs including aminophenylamidines and levamisole, showed strong contraction along the whole body of the worm suggesting spastic paralysis. Following the establishment and parasitological characterisation of a Trichuris muris mouse model, publication 4 investigated the in vivo potency of the aminophenylamidines and the cyclooctadepsipeptides against patent whipworm infections using three consecutive dosages by different routes of administration. Only treatments with relatively high dosages of tribendimidine or dAMD resulted in complete elimination of the worm burden whereas treatments with fairly low dosages of the cyclooctadepsipeptides emodepside and PF1022A, especially after oral administration, resulted in full parasite clearance. Due to the high efficacy, PF1022A was further evaluated in single and multiple oral treatment schemes targeting developmental stages including histotropic larvae. Again, dose-dependent efficacies were observed. However, using a single dose regimen, only high dosages cured the larval whipworm infections completely. But since only a very basic formulation was tested, reduction of the required dosages can likely be achieved by optimisation of the formulation. The cyclooctadepsipeptides showed high efficacy against all nematode species tested. However, their mode of action is not fully understood. The calcium-gated and voltage-dependent potassium channel SLO-1 is generally accepted to be crucial for the nematicidal effects of cyclooctadepsipeptides in Caenorhabditis elegans but direct activation of SLO-1 channels by any cyclooctadepsipeptide has not been demonstrated. Therefore, manuscript 5 reports the identification and characterisation of SLO-1 channels from various nematodes. After identification and cloning of slo-1 orthologs from a wide phylogenetic range of parasitic nematodes, SLO-1 and its potential activation by cyclooctadepsipeptides were analysed by heterologous expression in Xenopus laevis oocytes. Accordingly, it was demonstrated for the first time, that emodepside directly induces currents via C. elegans SLO-1. Due to the obtained insights regarding their mode of action and their potential to cure helminthoses in a single-treatment regimen, the cyclooctadepsipeptides might currently be the most interesting new class for the treatment against helminthoses of human and livestock. In addition to monotherapies, the use of combinations containing cyclooctadepsipeptides and cholinergic drugs such as tribendimidine should also be further investigated. The combinations were shown to have increased efficacy against the rat hookworm and presumably the risk of selection for resistance would be decreased, since two completely independent targets are affected.