Advancing drug discovery and treatment evaluation for schistosomiasis : improved screening, drug repurposing and rescuing, and the metabonomics of praziquantel treatment

Abstract

Schistosomiasis is a widespread chronic inflammatory disease caused by a parasitic blood fluke of the Schistosoma genus, primarily by S. mansoni, S. haematobium and S. japonicum. More than 200 million people in low-resource rural dwellings of the tropics and sub-tropics are affected, 90% of which are in Africa and many of which are children. The chronic disease is characterized by anemia, abdominal pain, bloody stool or urine, portal hypertension, swelling and degradation of the liver, spleen, intestines or the urogenital organs, caused by hypersensitivity reactions to eggs that become lodged in visceral organs. The main strategy for control endorsed by the World Health Organization is morbidity reduction by mass administration of the only drug available, praziquantel. The treatment is safe and effective but carries important drawbacks such as its lack of efficacy against juvenile worm stages and its child-unfriendly bitter taste. Importantly, the reliance on a single drug to treat millions is unwise and treatment alternatives are required. Yet schistosomiasis falls into the category of neglected tropical diseases, so called because their treatment and drug candidate arsenal is marginal compared to their public health impact. This PhD thesis had two major aims. The first was to mitigate this need for novel antischistosomal drug candidates by exploring automated in vitro drug screening systems and by invoking drug repurposing, rescuing and re-designing strategies to fill the pipeline. The second was to enhance our understanding of schistosomiasis and its treatment with praziquantel in pre-school and school-aged children with a clinical dose-finding trial and complementary metabonomic investigations. In testing 11 fluorescence/luminescence-based viability/cytotoxicity markers, resazurin, Vybrant® and CellTiter-Glo® presented as markers that correlated with S. mansoni larval stage viability. Of these, CellTiter-Glo® could determine IC50 values for some standard drugs in the range of microscopically assessed values and was 100% accurate in identifying hits from a small sub-set of a screen. An evaluation of a new calorimetry device, the calScreener™, showed that heat flow and heat flow fluctuation signals from single adult stage worms of two nematodes could be used to monitor worm viability over time. However, this did not extend to S. mansoni adult stage nor larval stage worms. A literature review of drug repurposing anthelmintic in vivo and clinical trial candidates detailed the continued reliance on veterinary drug discovery as a source for new compounds, largely for nematodes. Antimalarials were found to be an important contributor of antischistosomal drug candidates, though additional sources, such as anticancer drugs are increasingly in the foreground. To expand repurposing sources, we screened an open-source library of 1600 FDA approved compounds against S. mansoni in vitro and in vivo. Our screen identified 121 hits against Newly Transformed Schistosomula (NTS, the larval stage) at a concentration of 10 µM and 36 active compounds on the adult stage at a concentration of 33 µM. Despite needing to discard many compounds for pharmacokinetic or toxicity reasons, 11 compounds were tested further in vivo, with doramectin (10 mg/kg) and clofazimine (400 mg/kg) showing significant worm burden reductions of 60.1% and 82.7%, respectively. In seeking to revive a pre-praziquantel era antischistosomal candidate, Ro 13-3978 was characterized for its activity against S. mansoni. In vitro, it displayed only minor activity against adult S. mansoni. In vivo it was more potent than praziquantel with an ED50 of 14.6 and 138.9 mg/kg against adult and juvenile stage worms respectively. Hepatic shift and SEM studies of ex-vivo worms showed minimal activity within the first 24 h but by 48 h were worms are shunted to the liver and the tegument thoroughly damaged. Further investigations with immunohistochemistry on histological sections of treated versus non-treated mice indicated significant recruitment of macrophages and B cells to the worm at 24 h post-treatment and additional recruitment of T cells and neutrophils at 48 h post treatment. Oxamniquine was re-designed by synthesizing organometallic derivatives. Three compounds- a ferrocenyl, a ruthenocenyl and a benzyl derivative- presented potent in vitro activity against adult S. mansoni worms, reducing their viability by > 75% within 4 to 7 h after exposure. This contrasted with oxamniquine, which presents no activity in vitro. In S. mansoni-infected mice, a 100 mg/kg dose of the derivatives reduced worm burden by 76 to 93%, comparable to the worm burden reduction of oxamniquine at the same dose. Our randomized controlled dose-finding clinical trial of praziquantel in school-aged and pre-school aged children infected with S. mansoni in southern Cote d’Ivoire showed slightly lower efficacy of praziquantel in pre-school versus school-aged children. When administered either a placebo or 20, 40, 60 mg/kg praziquantel, cure rates for pre-schoolers were 37.1, 62.2, 72.2 and 71.4%, respectively, as compared to those for school-aged children which were 11.9, 30.4, 68.8 and 82.9%, respectively. An Emax model predicted an egg reduction rate of 99% with 65 mg/kg praziquantel for school-aged children while for pre-schoolers, this was not in range. Metabonomic analyses of urine samples from this same clinical trial revealed that heavily infected children could be differentiated from non-infected children prior to treatment, indicating potential urinary biomarkers of morbidity. Treatment with praziquantel produced a plethora of metabolic responses 24h after treatment, which differed between pre-school and school aged children, which may be linked to the diferring treatment success rates between the two groups. Most metabolites were correlated to energy, liver and gut microbial metabolisms. In conclusion, we present CellTiter Glo® as a possible hit/no hit pre-screening tool for in vitro assays. Moreover, our FDA library screen proposes additional sources of drug indications from which antischistosomal compounds and scaffolds could be sourced. Meanwhile Ro 13-3978 is an efficacious antischistosomal, active against multiple stages of infection, and presents as an excellent pre-clinical candidate worthy of further investigations. The oxamniquine derivatives described elucidate a successful drug re-design strategy using organometallic derivatization. Finally, praziquantel treatment itself has been better characterized for an increasingly important demographic population, pre-school aged children, and illustrated through a systemic metabolic lens.