Fasciolicides: efficacy, actions, resistance and its management.

The modes of action of fasciolicides are described. Closantel and other salicylanilides interfere with energy metabolism by uncoupling oxidative phosphorylation in the fluke. Other fasciolicides are believed to have a metabolic action-halogenated phenols (via uncoupling) and clorsulon (via inhibition of glycolysis)-but direct evidence is lacking. Benzimidazoles (in particular, triclabendazole) bind to fluke tubulin and disrupt microtubule-based processes. Diamphenethide inhibits protein synthesis in the fluke. Other potential drug actions may contribute to overall drug efficacy. In particular, a number of fasciolicides-salicylanilides, phenols, diamphenethide-induce a rapid paralysis of the fluke, so their action may have a neuromuscular basis, although the actions remain ill-defined. Resistance to salicylanilides and triclabendazole has been detected in the field, although drug resistance does not appear to be a major problem yet. Strategies to minimize the development of resistance include the use of synergistic drug combinations, together with the design of integrated management programmes and the search for alternatives to drugs, in particular, vaccines.

Fasciola hepatica: ultrastructural changes to the tegument of juvenile flukes following incubation in vitro with the deacetylated (amine) metabolite of diamphenethide.

Ultrastructural changes to the tegument of 5-week-old, 3-week-old and freshly-excysted Fasciola hepatica following in vitro incubation with the deacetylated (amine) metabolite of diamphenethide (DAMD, 10 microgramsml-1) were examined by transmission electron microscopy. A similar sequence of tegumental changes occurred in all three age groups of fluke, although, with increasing fluke age, the time before onset increased and the damage became more extensive. The 5-week-old flukes showed an initial stress response after 3 h, typified by blebbing of the apical plasma membrane, formation of microvilli and an accumulation and accelerated release of secretory bodies at the tegumental apex, as well as swelling of the basal infolds. The swelling increased in extent with progressively longer periods of incubation in DAMD, leading to extreme edema and sloughing of the tegument after 9 h. The 3-week-old flukes showed a stress response and swelling of the basal infolds after only 1.5 h, although sloughing of the tegument did not occur until after 9 h. In the freshly-excysted metacercaria, a stress response and some sloughing of the tegument were evident after only 0.5 h. At all stages of development, the ventral tegument was more severely affected than the dorsal. Changes also occurred to the tegumental cells which were indicative of a disruption in the synthesis and release of tegumental secretory bodies: the amount of GER became reduced, the cisternae became swollen and their ribosomal covering decreased, the Golgi complexes disappeared from the cells and the numbers of secretory bodies in the cells also decreased. The heterochromatin content of the nuclei increased and eventually the tegumental cells began to break down. Again, the changes became apparent more rapidly at the earlier stages of development. The ultrastructural changes to the tegument are linked to a possible mode of action for diamphenethide as an inhibitor of protein synthesis. In turn, the results may help to explain the drug's high efficacy against juvenile stages of F. hepatica.

The effect of diamphenethide on protein synthesis by the liver fluke, Fasciola hepatica.

The effect of the deacetylated (amine) metabolite of diamphenethide (DAMD, 10 micrograms ml-1) on the uptake and incorporation by adult Fasciola hepatica of radioactively labelled precursors of DNA, RNA and protein synthesis ([3H]thymidine, [3H]uridine and [3H]leucine, respectively) was measured by liquid scintillation counting. Comparison was made between the effects of DAMD and those of specific inhibitors of DNA, RNA and protein synthesis, namely, 5-fluorouracil, cordycepin and cycloheximide, respectively. DAMD caused a significant decrease in the overall uptake and incorporation of [3H]uridine by F. hepatica, decreased the incorporation of [3H]leucine and also caused a significant decrease in the overall protein content of the flukes. The effect of DAMD was similar to that of cycloheximide (1 x 10(-3) M), a potent inhibitor of protein synthesis, which also caused a significant decrease in the incorporation of [3H]leucine by the fluke and a decrease in the overall protein content of the fluke. Cordycepin (100 micrograms ml-1) caused a significant decrease in the protein content of the fluke, but had no effect on the uptake or incorporation of [3H]uridine. 5-Fluorouracil (1 x 10(-4) M) did not affect the uptake or incorporation of [3H]thymidine, nor did it decrease the protein content of the fluke. The results indicate that DAMD inhibits protein synthesis by F. hepatica, possibly by inhibiting RNA synthesis. The results are also consistent with previous morphological investigations involving DAMD.

The ionic response of the liver fluke, Fasciola hepatica to ouabain, monensin and the deacetylated (amine) metabolite of diamphenethide.

1. The ionic response of the liver fluke, Fasciola hepatica to perturbation of Na,K-pump activity has been determined by atomic absorption spectrophotometry. 2. The Na+/K(+)-ATPase inhibitor ouabain (1 x 10(-4) M) induced a marked reduction in K+ levels; Na+ and Ca2+ levels also fell. 3. The sodium ionophore monensin (1 x 10(-4) M) also caused a decrease in K+ levels, to below that of Na+. 4. Brief pretreatment with ouabain (1 x 10(-4) M, 15 min) followed by monensin treatment did not affect the decline in K+ levels, but did prevent the short-lived Na+ decline observed with monensin alone. 5. The deacetylated (amine) metabolite of the fasciolicide diamphenethide caused a short-lived drop in Na+ levels, but otherwise produced little change in ion levels within the fluke.

[Manifestation of the mutagenic effect of acemidophen depending on its metabolism in various mammalian species (acemidophen--a promutagen)]. / Proiavlenie mutagennogo éffekta atsemidofena v zavisimosti ot ego metabolizma u raznykh vidov mlekopitaiushchikh (atsemidofen--promutagen).

While studying the specific variability of metabolism of the fasciolacide anthelmintic acemidophen, it is found that it possesses no mutagenic activity. This activity is peculiar to its metabolite-free amine L2, formed in mice organism by deacetylation. Thus, acemidophen is shown to be promutagen.

Diamphenethide--a reassessment of its pharmacological action.

At a concentration of 1 x 10(-4) M (28.84 micrograms/ml), with a solvent concentration of 1.0% (v/v) ethanol, the deacetylated (amine) metabolite of diamphenethide (DAMD) causes an initial stimulation of activity, followed by suppression, leading to a paralysis within 3 h. These changes are accompanied by an increase in muscle tone of more than 200 mg. However, ethanol alone at a concentration of 1.0% (v/v) causes an initial stimulation of activity and increase in muscle tone (approximately 550 mg). If the concentration of DAMD is kept at 1 x 10(-4) M (28.84 micrograms/ml) but the solvent concentration reduced [e.g., 0.05% (v/v) dimethyl sulphoxide], then only a suppression of motility and flaccid paralysis are observed. This response is also seen at the lower concentration of 10 micrograms/ml, which corresponds to the maximum blood levels of DAMD in vivo. The sodium ionophore monensin induces a suppression of motility, leading to a rapid flaccid paralysis (in approximately 1.5 h at 1 x 10(-7) M, and within a few minutes at higher concentrations). Ouabain, an inhibitor of Na+/K+-ATPase activity, also causes a suppression of motility, but this is accompanied by an increase in muscle tone, leading to a spastic paralysis (in approximately 2.5 h at 1 x 10(-3) M, and 3.5 h at 1 x 10(-4) M). Pretreatment with ouabain (1 x 10(-3) M for 15 min) followed by monensin (1 x 10(-5) M) reverses the original effect of monensin by inducing a rapid spastic paralysis (in approximately 50 min).(ABSTRACT TRUNCATED AT 250 WORDS)

Fasciola hepatica in vitro: increased susceptibility to fasciolicides in a defined serum-free medium.

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.

The interaction between the deacetylated (amine) metabolite of diamphenethide (DAMD) and cytochemically demonstrable Na+/K+-ATPase activity in the tegument of Fasciola hepatica.

The relative effects on tegumental Na+/K+-ATPase activity in Fasciola hepatica of the deacetylated (amine) metabolite of diamphenethide (DAMD) (10 micrograms/ml, 18 h) and the Na+/K+-ATPase inhibitor ouabain (0.1 mM, 0.5 h) have been assessed cytochemically. In the normal tegument, Na+/K+-ATPase activity is particularly concentrated along the invaginations of the apical plasma membrane and the infoldings of the basal plasma membrane. Ouabain pretreatment significantly reduces the overall level of Na+/K+-ATPase activity, but does not induce swelling of the basal infolds. In contrast, DAMD does not significantly reduce either the level or distribution of Na+/K+-ATPase activity, but does cause a pronounced swelling of the basal infolds. The results are discussed in relation to the postulated action of diamphenethide as an inhibitor of Na+/K+-ATPase activity.

Fasciola hepatica: tegumental surface alterations following treatment in vitro with the deacetylated (amine) metabolite of diamphenethide.

The effect of the deacetylated (amine) metabolite of diamphenethide (10 micrograms/ml) on the tegumental surface of Fasciola hepatica over a 24 h period in vitro has been determined by scanning electron microscopy. Blebbing begins around the oral sucker after 3 h and then passes backwards along the body, reaching the ventral sucker and midbody by 6 h, and finally the posterior end of the body (by 12 h). Initially, the blebs are small, the tegument surrounding the spines is swollen and the tegument generally has a smooth, swollen appearance. This submerges the spines below the body surface. At higher magnification the surface is seen to bear microvillous-like projections in addition to the blebs and surface pitting is deeper than normal. Later on, the blebs increase in size and burst, causing lesions and loss of spines. Lesions begin to appear on the oral cone and ventral sucker after 6 h, in the midbody by 12 h and on the dorsal surface of the posterior region after 24 h. By this time the damage is extensive: around the oral and ventral suckers, and over large areas of the oral cone and midbody region the tegument has been stripped off to expose the basal lamina beneath. The dorsal surface of the fluke is consistently more severely affected than the ventral surface.

Fasciola hepatica: tegumental changes induced in vitro by the deacetylated (amine) metabolite of diamphenethide.

The effect of the deacetylated (amine) metabolite of diamphenethide (10 mugm/ml) on the tegument of Fasciola hepatica over a 24 hr period in vitro has been determined by means of transmission electron microscopy. In the tegumental syncytium, there is an initial accumulation of T2 secretory bodies at the apical surface (after 6 hr), together with increased exocytosis of secretory bodies and blebbing of the surface membrane. After 9 hr, the two surfaces of the fluke show different tegumental responses to drug treatment with a marked swelling of the basal infolds in the dorsal tegument, while the ventral tegument remains normal. By 18 hr, the swelling in the dorsal tegument is very severe, the entire basal region becoming edematous. In some areas, the tegument becomes detached to expose the basal lamina. The ventral tegument retains a fairly normal morphology, although there is a slight swelling of the basal infolds. The edema spreads internally to the cell bodies, beginning after 9 hr on the dorsal side of the fluke and 18 hr on the ventral side. By 18 hr, the flooding on the dorsal side is very severe and the cells attenuated, retaining few contacts with the surrounding parenchyma. From 9 hr onwards, there are progressive changes in cell structure, including a decrease in amount of granular endoplasmic reticulum and extent of its ribosomal covering, a decrease in numbers of secretory bodies, a swelling of the trans-most Golgi cisternae and disruption of the release of secretory bodies, and a swelling and disorganization of the mitochondria. The results are discussed in relation to the postulated activity of the deacetylated (amine) metabolite of diamphenethide as a Na+ ionophore.

The ability of diamphenethide to control immature Fasciola hepatica in sheep at a lower than standard dose level.

Groups of eight Welsh Mountain sheep were dosed with diamphenethide at the rate of 70 mg/kg bodyweight at either one, four, six or eight weeks after artificial infection with approximately 300 Fasciola hepatica metacercariae. Comparisons were made with similarly infected but undosed sheep and with sheep which were neither infected nor dosed. The good clearance of flukes up to six weeks of age (above 97 per cent on pooled data) was reflected in the plasma concentrations of the accepted liver damage marker enzymes glutamate dehydrogenase and gamma-glutamyl transpeptidase. Highly significant correlations were demonstrated between the numbers of flukes recovered, the plasma levels of these enzymes and haemoglobin and plasma albumin values. At 70 mg/kg, diamphenethide was shown to be able to control F hepatica populations of up to six weeks of age. The systematic use of diamphenethide at this dose level at intervals of up to six weeks during the period of metacercarial challenge should prevent ovine fascioliasis.

Productivity in sheep treated with diamphenethide at different times after infection with Fasciola hepatica.

Sheep with potentially fatal Fasciola hepatica burdens were treated with diamphenethide 4, 8 or 12 weeks after infection. Specific effects of fluke on growth rate and wool growth over and above any anorectic effects were measured by including a pair fed group to each infected group. After drenching, feed intakes returned to that of the uninfected control group. With the exception of sheep drenched in Week 4 after infection, all sheep had significantly lower production after drenching than the control sheep. No compensatory growth was observed and productivity remained depressed for the duration of the trial (26 weeks).

Fasciola hepatica: motility response to fasciolicides in vitro.

The effects of a wide range of fasciolicides on the in vitro motility of Fasciola hepatica have been determined by means of an isometric transducer system. Carbon tetrachloride and diamphenethide do not affect movement at concentrations up to 500 and 100 micrograms/ml, respectively; at 1000 micrograms/ml, however, carbon tetrachloride induces a rapid tonic paralysis. Brotianide and the deacetylated metabolite of diamphenethide cause a rapid flaccid paralysis of the fluke at concentrations of 1.0 micrograms/ml and above. In contrast, the effect of MK-401 is a long-term one, a flaccid paralysis occurring after 20 hr only at 200 micrograms/ml. Praziquantel also produces a flaccid paralysis of the fluke, but this follows an initial increase, then decrease in muscle tone. The effect is rapid at 500 micrograms/ml, but long-term at 100 and 200 micrograms/ml; at these lower concentrations there is also a stimulation of activity. Oxyclozanide , rafoxanide, niclofolan , bithionol, and hexacholorophene induce a rapid spastic paralysis of the fluke at concentrations of 1.0 micrograms/ml and above. Both phasic and tonic components are evident in the response at concentrations of 1.0 micrograms/ml and below; the phasic component disappears at higher concentrations. Nitroxynil produces a similar effect, evident at higher concentrations. Among the benzimidazoles, mebendazole, oxfendazole, and albendazole sulphoxide cause a suppression of motility, whilst thiabendazole and albendazole produce a stimulation of movement. The effects are not rapid, however, for only mebendazole at 500 micrograms/ml causes total inactivity of the fluke within a 12-hr period. Possible explanations for these effects on fluke motility are discussed.

Effects of diamfenetide on metabolic and excretory functions of Fasciola hepatica in vitro.

The effect of diamfenetide (DFT) on the time course of production of end-products of glucose metabolism, tissue ATP levels and NH3 production by adult Fasciola hepatica in vitro was determined. Products of glucose metabolism are increased between 6 and 24 hr incubation in 10(-4) M DFT. Tissue ATP levels and NH3 production are decreased during this time period. The observed metabolic effects of DFT are manifested at a much later time after drug exposure than previously described membrane-disruptive events indicating that metabolic effects of DFT on F. hepatica may be secondary to the initial effects on surface membranes.

Fasciola hepatica: effects of diamfenetide free amine on in vitro physiology, biochemistry, and morphology.

Short-term (1-3 hr) incubations in vitro of immature and adult Fasciola hepatica with 10(-4) to 10(-5) M free amine of diamfenetide (DPT-FA) demonstrated a time/dose-dependent, irreversible paralysis that involved an increase in muscular tension and decrease in contraction amplitude. The following events occurred preceding or concomitant with the paralysis: influx of Na+, decrease in surface membrane potential, increase in wet weight, swellings on the ventral surface, and inhibition of 3-O-methyl glucose transport. These events were all consistent with a disturbance in surface membrane functions. The effects of DPT-FA were more severe in immature flukes (3-5 weeks postinfection) than adults which agrees with observed in vivo efficacy.

The effect of five fasciolicides on malate dehydrogenase activity and mortality of Fasciola gigantica, Fasciolopsis buski and Paramphistomum explanatum.

The effect of oxyclozanide, hexachlorophene, nitroxynil, rafoxanide and diamphenethide on malate dehydrogenase activity of homogenates of Fasciola gigantica, Fasciolopsis buski and Paramphistomum explanatum was investigated. The ratio of oxaloacetate reduction to malate oxidation in homogenates of Fasciola gigantica, Fasciolopsis buski and P. explanatum was 4.5:1, 3.6:1 and 5.2:1 respectively. Oxyclozanide and rafoxanide at 10(-3) M inhibited enzyme activity by 100% in homogenates from all three species while hexachlorophene at 10(-3) M also caused 100% inhibition in homogenates from Fasciola gagantica and P. explanatum but only 65% of malate oxidation in Fasciolopsis buski homogenates. Nitroxynil at 10(-3) M produced 60% inhibition in F. buski homogenates yet had little effect at this concentration on preparations from the other species. Little inhibition was seen with diamphenethide, even at high concentrations. Rapid death of Fasicola gigantica and P. explanatum resulted in vitro when 10(-3) M oxyclozanide, hexachlorophene, nitroxynil or rafoxanide, were added to the incubation medium. Fasciolopsis buski was killed by 10(-3) M oxyclozanide but at this concentration the remaining compounds only caused reduced activity. Assay of malate dehydrogenase following drug treatment in vitro failed to show any appreciable reduction in enzyme activity in Fasciola gigantica and P. explanatum but oxyclozanide and hexachlorophene produced inhibition in Fasciolopsis buski. The mode of action of these compounds is discussed.

Studies of the effect of diamphenethide and oxyclozanide on the metabolism of Fasciola hepatica.

Studies were made of the effects of diamphenethide-amine on glucose transport, glycogen breakdown, adenine nucleotides, metabolites and excretory products in both parenchymal and bile duct flukes in vitro and in bile duct flukes in vivo. The most consistent and pronounced effect observed was an elevation of malate levels. There appeared to be no differences between responses of parenchymal and of bile duct flukes to diamphenethide-amine. For comparative purposes the effect of oxydozanide was assessed on most of these parameters in bile duct flukes in vitro. Not all the changes caused by oxyclozanide were characteristic of an uncoupler; however, the pattern of changes in the metabolites was markedly different from that caused by diamphenethide-amine.