Altered drug influx/efflux and enhanced metabolic activity in triclabendazole-resistant liver flukes

Abstract

Triclabendazole (TCBZ) is a halogenated benzimidazole compound that possesses high activity against immature and adult stages of the liver fluke, Fasciola hepatica. The intensive use of TCBZ in endemic areas of fascioliasis has resulted in the development of liver flukes resistant to this compound. TCBZ sulphoxide (TCBZSO) and TCBZ sulphone (TCBZSO2) are the main molecules recovered in the bloodstream of TCBZ-treated animals. In order to gain some insight into the possible mechanisms of resistance to TCBZ, the goals of the work described here were: to compare the ex vivo transtegumental diffusion of TCBZ parent drug and its sulpho-metabolites (TCBZSO and TCBZSO2) into TCBZ-susceptible and -resistant liver flukes; and to assess the comparative pattern of TCBZ biotransformation by TCBZ-susceptible and -resistant F. hepatica. For the tegumental diffusion studies, TCBZ-susceptible (Cullompton) and -resistant (Sligo) adult flukes collected from untreated infected sheep were incubated (15-180 min) in KRT buffer containing either TCBZ, TCBZSO or TCBZSO2 (5 nmol.ml-1). For the metabolism studies, microsomal fractions obtained from TCBZ-susceptible and -resistant flukes were incubated for 60 min with TCBZ (40 microM), and the amount of the formed metabolic product (TCBZSO) was measured. Drug/metabolite concentrations were quantified by HPLC. All the assayed TCBZ-related molecules penetrated through the tegument of both TCBZ-susceptible and -resistant flukes. However, significantly lower (approximately 50%) concentrations of TCBZ and TCBZSO were recovered within the TCBZ-resistant flukes compared to the TCBZ-susceptible ones over the 180 min incubation period. The rate of TCBZ sulphoxidative metabolism into TCBZSO was significantly higher (39%) in TCBZ-resistant flukes. The flavin-monooxigenase (FMO) enzyme system appears to be the main metabolic pathway involved in the formation of TCBZSO in both TCBZ-susceptible and -resistant flukes. The altered drug influx/efflux and enhanced metabolic capacity identified in TCBZ-resistant liver flukes may account for the development of resistance to TCBZ.

Immature triclabendazole-resistant Fasciola hepatica: tegumental responses to in vitro treatment with the sulphoxide metabolite of the experimental fasciolicide compound alpha

“…The Sligo isolate has been shown to be resistant to the action of TCBZ in vivo Coles and Stafford 2001;McCoy et al 2005) and has been used in a number of in vitro studies (Robinson et al 2002(Robinson et al , 2004Alvarez et al 2005). Four-week-old juvenile flukes were removed from the livers of the rats under sterile conditions in a laminar flow cabinet.…”

Potentiation of triclabendazole sulphoxide-induced tegumental disruption by methimazole in a triclabendazole-resistant isolate of Fasciola hepatica

“…Moreover, TCBZ-resistant isolates have been shown to carry out the metabolism of TCBZ at a significantly higher rate than that by TCBZ-susceptible isolates (Robinson et al 2004;Alvarez et al 2005). It is this enhancement of oxidative metabolism that has been suggested as a possible mechanism of resistance within the fluke.…”

“…It is this enhancement of oxidative metabolism that has been suggested as a possible mechanism of resistance within the fluke. The FMO system represents the main metabolic pathway for the metabolism of TCBZ to triclabendazole sulphoxide (TCBZ.SO) by F. hepatica (Alvarez et al 2005). A previous scanning electron microscopical study showed that inhibition of this system by methimazole (MTZ) leads to more severe surface changes in a TCBZresistant isolate when incubated with TCBZ and TCBZ.SO than when incubated in the drug alone (Devine et al 2009).…”

Erratum to: Inhibition of triclabendazole metabolism in vitro by ketoconazole increases disruption to the tegument of a triclabendazole-resistant isolate of Fasciola hepatica

“…The FMO system probably represents the main pathway for the metabolism of TCBZ to TCBZ.SO, while the CYP 450 system probably plays a greater role in the conversion of TCBZ.SO to TCBZ. SO 2 (Alvarez et al 2005).…”