Modified Hederagenin Derivatives Demonstrate Ex Vivo Anthelmintic Activity against Fasciola hepatica.

Infection with Fasciola hepatica (liver fluke) causes fasciolosis (or fascioliasis) and poses a considerable economic as well as welfare burden to both the agricultural and animal health sectors. Here, we explore the ex vivo anthelmintic potential of synthetic derivatives of hederagenin, isolated in bulk from Hedera helix. Thirty-six compounds were initially screened against F. hepatica newly excysted juveniles (NEJs) of the Italian strain. Eleven of these compounds were active against NEJs and were selected for further study, using adult F. hepatica derived from a local abattoir (provenance unknown). From these eleven compounds, six demonstrated activity and were further assessed against immature liver flukes of the Italian strain. Subsequently, the most active compounds (n = 5) were further evaluated in ex vivo dose response experiments against adult Italian strain liver flukes. Overall, MC042 was identified as the most active molecule and the EC50 obtained from immature and adult liver fluke assays (at 24 h post co-culture) are estimated as 1.07 µM and 13.02 µM, respectively. When compared to the in vitro cytotoxicity of MDBK bovine cell line, MC042 demonstrated the highest anthelmintic selectivity (44.37 for immature and 3.64 for adult flukes). These data indicate that modified hederagenins display properties suitable for further investigations as candidate flukicides.

Flukicidal effects of abietane diterpenoid derived analogues against the food borne pathogen Fasciola hepatica.

Control of liver fluke infections remains a significant challenge in the livestock sector due to widespread distribution of drug resistant parasite populations. In particular, increasing prevalence and economic losses due to infection with Fasciola hepatica is a direct result of drug resistance to the gold standard flukicide, triclabendazole. Sustainable control of this significant zoonotic pathogen, therefore, urgently requires the identification of new anthelmintics. Plants represent a source of molecules with potential flukicidal effects and, amongst their secondary metabolites, the diterpenoid abietic acids can be isolated in large quantities. In this study, nineteen (19) chemically modified abietic acid analogues (MC_X) were first evaluated for their anthelmintic activities against F. hepatica newly excysted juveniles (NEJs, from the laboratory-derived Italian strain); from this, 6 analogues were secondly evaluated for their anthelmintic activities against adult wild strain flukes. One analogue, MC010, was progressed further against 8-week immature- and 12-week mature Italian strain flukes. Here, MC010 demonstrated moderate activity against both of these intra-mammalian fluke stages (with an adult fluke EC50 = 12.97 µM at 72 h post culture). Overt mammalian cell toxicity of MC010 was inferred from the Madin-Darby bovine kidney (MDBK) cell line (CC50 = 17.52 µM at 24 h post culture) and demonstrated that medicinal chemistry improvements are necessary before abietic acid analogues could be considered as potential anthelmintics against liver fluke pathogens.

Fasciola hepatica Cathepsin L Zymogens: Immuno-Proteomic Evidence for Highly Immunogenic Zymogen-Specific Conformational Epitopes to Support Diagnostics Development.

Fasciola hepatica, the common liver fluke and causative agent of zoonotic fasciolosis, impacts on food security with global economic losses of over $3.2 BN per annum through deterioration of animal health, productivity losses, and livestock death and is also re-emerging as a foodborne human disease. Cathepsin proteases present a major vaccine and diagnostic target of the F. hepatica excretory/secretory (ES) proteome, but utilization in diagnostics of the highly antigenic zymogen stage of these proteins is surprisingly yet to be fully exploited. Following an immuno-proteomic investigation of recombinant and native procathepsins ((r)FhpCL1), including mass spectrometric analyses (DOI: 10.6019/PXD030293), and using counterpart polyclonal antibodies to a recombinant mutant procathepsin L (anti-rFhΔpCL1), we have confirmed recombinant and native cathepsin L zymogens contain conserved, highly antigenic epitopes that are conformationally dependent. Furthermore, using diagnostic platforms, including pilot serum and fecal antigen capture enzyme-linked immunosorbent assay (ELISA) tests, the diagnostic capacities of cathepsin L zymogens were assessed and validated, offering promising efficacy as markers of infection and for monitoring treatment efficacy.

An Abies procera-derived tetracyclic triterpene containing a steroid-like nucleus core and a lactone side chain attenuates in vitro survival of both Fasciola hepatica and Schistosoma mansoni.

Two economically and biomedically important platyhelminth species, Fasciola hepatica (liver fluke) and Schistosoma mansoni (blood fluke), are responsible for the neglected tropical diseases (NTDs) fasciolosis and schistosomiasis. Due to the absence of prophylactic vaccines, these NTDs are principally managed by the single class chemotherapies triclabendazole (F. hepatica) and praziquantel (S. mansoni). Unfortunately, liver fluke resistance to triclabendazole has been widely reported and blood fluke insensitivity/resistance to praziquantel has been observed in both laboratory settings as well as in endemic communities. Therefore, the identification of new anthelmintics is necessary for the sustainable control of these NTDs in both animal and human populations. Here, continuing our work with phytochemicals, we isolated ten triterpenoids from the mature bark of Abies species and assessed their anthelmintic activities against F. hepatica and S. mansoni larval and adult lifecycle stages. Full 1H and 13C NMR-mediated structural elucidation of the two most active triterpenoids revealed that a tetracyclic steroid-like nucleus core and a lactone side chain are associated with the observed anthelmintic effects. When compared to representative mammalian cell lines (MDBK and HepG2), the most potent triterpenoid (700015; anthelmintic EC50s range from 0.7 µM-15.6 µM) displayed anthelmintic selectivity (selectivity indices for F. hepatica: 13 for newly excysted juveniles, 46 for immature flukes, 2 for mature flukes; selectivity indices for S. mansoni: 14 for schistosomula, 9 for immature flukes, 4 for adult males and 3 for adult females) and induced severe disruption of surface membranes in both liver and blood flukes. S. mansoni egg production, a process responsible for pathology in schistosomiasis, was also severely inhibited by 700015. Together, our results describe the structural elucidation of a novel broad acting anthelmintic triterpenoid and support further investigations developing this compound into more potent analogues for the control of both fasciolosis and schistosomiasis.

Optimized conditions for the in vitro excystment of Calicophoron daubneyi metacercariae.

Paramphistomosis, caused by Calicophoron daubneyi, is an emerging infection of ruminants throughout Western Europe. Despite its prevalence, many questions remain regarding the basic biology of this parasite and how it interacts with its host. Consequently, there is a need to develop methods to study C. daubneyi in vitro to improve our understanding of rumen fluke biology. Towards this, we aimed to identify a suitable protocol for in vitro excystment of C. daubneyi metacercariae. Six methods that have been used to excyst metacercariae from a number of trematode species were tested with C. daubneyi metacercariae. Three of these achieved an average of >50% excystment whilst one method, which included an acid-pepsin treatment, incubation in reducing conditions and an alkaline/bile salt solution to activate the larvae, consistently gave >80% excystment. The latter protocol also showed no detrimental effect on the motility of newly excysted juvenile (NEJ) parasites when observed for up to 24 h in RPMI 1640 medium post-excystment. The successful production of C. daubneyi NEJs in vitro is a significant step forward, and will enable the discovery of infective stage-specific parasite antigens and facilitate drug screening trials, to aid the development of much needed diagnostic and therapeutic options for paramphistomosis.