Interpretation of anthelmintic activity using in vitro screens has, until now, relied on the detection of drug-induced effects on nematode development, viability and motility. A novel biochemical parameter dependent upon the spectrophotometric assay of acetylcholinesterase (AChE), an enzyme secreted in large quantities by certain trichostrongylid nematodes, has been developed to replace these often subjective indices of activity. Using Nippostrongylus brasiliensis, a worm frequently employed for primary screening, the secretion of this enzyme in the presence or absence of a large number of drugs in vitro was determined. During a 4-day incubation period in a complex undefined medium without serum. AChE was secreted by normal 4th larval and immature adult stages of the worm in a linear fashion. All modern broad-spectrum veterinary anthelmintics, regardless of their mode of action, dramatically reduced the amount of enzyme secreted. Correlation between the biochemical and observational parameters was excellent and the selectivity of the assay when based solely on enzyme secretion was not lost. Other advantages were that the time required for the activity of certain slow-acting compounds to be detected was reduced from 7 to 4 days and that close microscopical examination of the worms was not necessary.

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The cidal properties of some phenolic, halogenated diphenyl, salicylanilide, benzimidazole and diaminophenoxyalkane anthelmintics, against 6-week-old worms of Fasciola hepatica were assessed in vitro. In a conventional fluke culture medium containing RPMI 1640, supplemented with serum with or without rabbit erythrocytes or pink-ghosts, only the halogenated diphenyl and salicylanilide compounds showed activity at concentrations equal to or less than 100 microM. However, when basal, serum and cell-free RPMI 1640 was used, all compounds other than diamphenethide were highly active, their minimum lethal concentrations being some 25-125 times lower under these conditions. The inclusion of rabbit liver microsomes in the basal culture medium resulted in diamphenethide exhibiting cidal activity equivalent to that seen when its free-amine active metabolite was assayed. The possibility that the activity of many of these compounds was masked in vitro because of their serum binding properties is discussed. Recommendations are made that in vitro screens for new fasciolicides should be carried out in serum-free medium and that additional replicates containing mammalian liver microsomes and liver cytosolic extracts be included as means for the metabolic activation of certain otherwise undetectable prodrugs.

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