Stadelmann Britta, Rufener Reto, Aeschbacher Denise, Spiliotis Markus, Gottstein Bruno, Hemphill Andrew
Institute of Parasitology, Vetsuisse Faculty, University of Berne, Berne, Switzerland.
PLoS Negl Trop Dis. 2016 Mar 11;10(3):e0004535. doi: 10.1371/journal.pntd.0004535. eCollection 2016 Mar.
The metacestode (larval) stage of the tapeworm Echinococcus multilocularis causes alveolar echinococcosis (AE), a very severe and in many cases incurable disease. To date, benzimidazoles such as albendazole and mebendazole are the only approved chemotherapeutical treatment options. Benzimidazoles inhibit metacestode proliferation, but do not act parasiticidal. Thus, benzimidazoles have to be taken a lifelong, can cause adverse side effects such as hepatotoxicity, and are ineffective in some patients. We here describe a newly developed screening cascade for the evaluation of the in vitro efficacy of new compounds that includes assessment of parasiticidal activity. The Malaria Box from Medicines for Malaria Venture (MMV), comprised of 400 commercially available chemicals that show in vitro activity against Plasmodium falciparum, was repurposed. Primary screening was carried out at 10 μM by employing the previously described PGI assay, and resulted in the identification of 24 compounds that caused physical damage in metacestodes. Seven out of these 24 drugs were also active at 1 μM. Dose-response assays revealed that only 2 compounds, namely MMV665807 and MMV665794, exhibited an EC50 value below 5 μM. Assessments using human foreskin fibroblasts and Reuber rat hepatoma cells showed that the salicylanilide MMV665807 was less toxic for these two mammalian cell lines than for metacestodes. The parasiticidal activity of MMV665807 was then confirmed using isolated germinal layer cell cultures as well as metacestode vesicles by employing viability assays, and its effect on metacestodes was morphologically evaluated by electron microscopy. However, both oral and intraperitoneal application of MMV665807 to mice experimentally infected with E. multilocularis metacestodes did not result in any reduction of the parasite load.
多房棘球绦虫的成虫期（幼虫期）会引发泡型包虫病（AE），这是一种非常严重且在很多情况下无法治愈的疾病。迄今为止，苯并咪唑类药物如阿苯达唑和甲苯达唑是唯一获批的化疗治疗选择。苯并咪唑类药物可抑制成虫期增殖，但不具有杀寄生虫作用。因此，苯并咪唑类药物必须终生服用，可能会引起如肝毒性等不良副作用，且对某些患者无效。我们在此描述一种新开发的筛选流程，用于评估新化合物的体外疗效，其中包括对杀寄生虫活性的评估。疟疾防治创新联盟（MMV）的疟疾药物盒由400种对恶性疟原虫具有体外活性的市售化学品组成，该药物盒被重新利用。通过使用先前描述的PGI测定法在10μM浓度下进行初步筛选，结果鉴定出24种对成虫期造成物理损伤的化合物。这24种药物中有7种在1μM浓度下也具有活性。剂量反应测定表明，只有2种化合物，即MMV665807和MMV665794，其半数有效浓度（EC50）值低于5μM。使用人包皮成纤维细胞和鲁伯大鼠肝癌细胞进行的评估表明，水杨酰苯胺MMV665807对这两种哺乳动物细胞系的毒性低于对成虫期的毒性。然后通过活力测定法，利用分离的生发层细胞培养物以及成虫期囊泡，证实了MMV665807的杀寄生虫活性，并通过电子显微镜对其对成虫期的影响进行了形态学评估。然而，对实验感染多房棘球绦虫成虫期的小鼠口服和腹腔注射MMV665807均未导致寄生虫负荷的任何降低。
