Abstract
Cryptosporidiosis is an enteric infection caused by the protozoan parasite, Cryptosporidium spp. and can be life threatening for the immunocompromised. There are many species of Cryptosporidium which infect humans and animals, with most human infections due to C. parvum and C. hominis. Cryptosporidium hominis is one of four pathogens most responsible for the global burden of severe diarrhoea in infants and toddlers, accounting for approximately 11% of the 8 million annual deaths of children under five. In New Zealand, cryptosporidiosis is continually listed as one of the top five notifiable human diseases. Crucially, there is neither an effective treatment nor a vaccine against cryptosporidiosis. Only one approved broad spectrum antiparasitic drug is available for humans, Nitazoxanide, which has moderate efficacy in children but no significant efficacy toward immunocompromised individuals. The aminoglycoside Paromomycin, which has similar efficacy to Nitazoxanide, is used as the ‘gold’ standard in research and as the last resort to treat cryptosporidiosis. A repurposed cancer drug, halofuginone lactate, is the only approved drug to treat neonatal livestock cryptosporidiosis.
In previous years, an experimental drug KDU731 has been identified as a more effective anti-cryptosporidial compound than Nitazoxanide and halofuginone lactate. KDU731 is a potent selective inhibitor of the Cryptosporidium phosphatidylinositol-4-OH kinase ATP binding site. For Nitazoxanide, halofuginone lactate and KDU731, the stage-specific anti-cryptosporidial mechanism is unknown. This project aimed to elucidate their anti-cryptosporidial effects on the asexual stages through an in vitro suite of phenotypic assays and create a baseline comparison for novel compounds in future drug discovery.
Using fluorescence microscopy and qPCR to detect parasitic growth, we calculated IC50 and IC90 values for each compound. Using the IC90 values from qPCR, the anti-cryptosporidial effects of our compounds were tested against the parasitic invasion of the cryptosporidial asexual life cycle, parasitic DNA replication, and the expression of genes specific to the sexual life cycle. Fluorescence microscopic counts of C. parvum, showed that our four compounds had no significant effects on C. parvum invasion of host cells when compared to pre-PFA fixed cells as a positive inhibitory control. Although halofuginone lactate (IC90 of 178 ± 8.39 nM) and KDU731 (IC90 of 146 ± 2.92 nM) showed significant efficacy toward inhibiting C. parvum growth compared to Nitazoxanide (IC90 of 19,860 ± 3.22 nM) and Paromomycin (IC90 of 52,250 ± 4.28 nM), the resazurin assay showed that all four drugs affected host mitochondrial health. At IC90 values, clinical dosing of Nitazoxanide and halofuginone lactate showed a significant decrease of <50 % in mean cell viability when compared to a single dose over a 48-hour period. Paromomycin and KDU731 treatment also resulted in reduced mean cell viability but was not significant and remained ≈ 80 %. Off target drug effects on host cells was further tested using cell cycle analysis using PI staining for flow cytometry. Our anti-cryptosporidial compounds significantly reduced the population of cells in the G2/M phase when compared to the cells-only control.
Summarily, the result of this project suggests that Paromomycin, Nitazoxanide, halofuginone lactate and KDU731 should not be used as baseline comparators for effective and safe anti-cryptosporidial drugs. Against the stage-specific effects of our drugs on the asexual cycle of C. parvum, invasion is unaffected, and future work needs to be done to optimise the DNA replication assay and qPCR assay for DMC1 expression for sexual characterisation. Cryptosporidium’s excystation and merozoite-egress in HCT-8 culture under drug conditions should also be explored, to determine whether the anti-cryptosporidial compounds mentioned, may affect cellular process required for infection and during merogony.