Investigations on potential drug candidates and metabonomics-based diagnostic biomarker discovery for human soil-transmitted helminthiases

Abstract

Soil-transmitted helminthiases (STHs) are diseases caused by nematode worms. The most common species affecting humans are Ascaris lumbricoides, Necator americanus Ancylostoma duodenale, Trichuris trichiura and Strongyloides stercoralis. More than 1 billion people are infected globally. Most at risk are the 3 billion poorest people of the world, particularly children. Heavy infections cause iron-deficiency anemia to growth stunting and intellectual retardation. STHs occur often concomitantly with other infections such as malaria. Currently, STHs morbidity control relies on only five drugs (albendazole, mebendazole, levamisole, pyrantel pamoate and ivermectin). These present limited efficacies, especially when administered in single dose against T. trichiura and hookworm infections. Although anthelmintic drug resistance has not yet appeared as a major public health problem, emergence of drug resistance may be inevitable. The situation is precarious and new drugs are urgently needed. In addition, existing tools for in vitro drug sensitivity testing are based on a viability assessment assay which lacks convenience and hinders high-throughput screening rates. Also, the lack of accurate diagnostic tools to detect STHs and malaria infections hampers an optimal management of these diseases. This work aimed first to set up nematode-rodent models at the Swiss Tropical and Public Health Institute (Swiss TPH), improve drug screening assays and evaluate potential new treatments for human STHs. Prior to this thesis, monepantel (AAD 1566), tribendimidine, nitazoxanide and oxantel pamoate had been identified as potential drug candidates for STHs. Secondly, we aimed to strengthen our understanding of the impact of a murine malaria and hookworm co-infection on the host’s metabolism and explore the potential of metabolic profiling as multiplexing diagnostic tool. Once the animal models corresponding to human helminthiases have been established, (Ancylostoma ceylanicum, N. americanus and Trichuris muris), the Alamar Blue, the MTT and the acid phosphatase assays, as well as the xCELLigence System, isothermal microcalorimetry, and the feeding-inhibition assay (A. ceylanicum only) were tested and compared to the current assay of choice, the motility assay. For T. muris, the Alamar Blue assay compared most favorably to the motility assay since it is precise and cost-effective. For A. ceylanicum, no alternative assay was found better than the motility assay for testing on L3, whereas the xCELLigence System was found accurate and convenient for adult worms. The potential of monepantel was assessed against A. ceylanicum, N. americanus, T. muris, Ascaris suum and Strongyloides ratti. In vivo, the veterinary drug showed good and moderate activities respectively, against A. ceylanicum (10 mg/kg: 100% worm burden reduction) and N. americanus (10 mg/kg: 58.3% worm burden reduction), but failed to show sufficient anthelmintic properties in the other three models. Tribendimidine, a Chinese anthelmintic, and its metabolites dADT and AdADT were tested using the hookworm models A. ceylanicum and Heligmosomoides bakeri. In A. ceylanicum-infected hamsters, a single oral dose of 10 mg/kg resulted in 74.8% worm burden reduction. In the H. bakeri model, a single oral dose of 2 mg/kg achieved a worm burden reduction of 100%. The metabolite AdADT showed moderate activity against both parasites. The combination tribendimidine-levamisole displayed an additive to synergistic behavior in the A. ceylanicum model in vivo. Nitazoxanide, an anti-protozoal drug was evaluated against A. ceylanicum and T. muris. In vitro, it had a marked effect on A. ceylanicum adult worms (IC50 = 0.74 µg/ml) and on T. muris L3 and adult worms (IC50s = 0.27 and 12.87 µg/ml, respectively). However, the drug lacked efficacy in both models in vivo. The “old” anthelmintic oxantel pamoate was studied in the A. ceylanicum, N. americanus and T. muris models. The drug lacked anti-hookworm activity in vivo (10 mg/kg), but showed promising trichuricidal properties in vitro and in vivo (ED50 = 4.7 mg/kg). Moreover, the combination oxantel pamoate-mebendazole revealed highly synergistic properties. Murine H. bakeri and Plasmodium berghei single and co-infection (delayed and simultaneous) models were established for metabolic analysis. Urine and plasma samples were subjected to 1H nuclear magnetic resonance (NMR) spectroscopy and subsequent multivariate analysis in order to identify infection-discriminating metabolic fingerprints. Characteristic metabolic fingerprints have been found for each of the infection scenarios. We detected two unknown metabolites and confirmed the accumulation of urinary pipecolic acid in P. berghei-infected mice. Pipecolic acid may therefore represent a candidate for human malaria diagnostics. 1H NMR spectroscopy was found powerful for detecting metabolic changes in the co-infection model, but still presents some drawbacks as diagnostic tool in its actual form.