Ceballos et al. BMC Veterinary Research 2010, 6:8 http://www.biomedcentral.com/1746-6148/6/8 RESEARCH ARTICLE Open Access Unchanged triclabendazole kinetics after co- administration with ivermectin and methimazole: failure of its therapeutic activity against triclabendazole-resistant liver flukes Laura Ceballos1,2†, Laura Moreno1,2†, Luis Alvarez1,2, Laura Shaw3, Ian Fairweather3, Carlos Lanusse1,2* Abstract Background: The reduced drug accumulation based on enhanced drug efflux and metabolic capacity, identified in triclabendazole (TCBZ)-resistant Fasciola hepatica may contribute to the development of resistance to TCBZ. The aim of this work was to evaluate the pharmacokinetics and clinical efficacy of TCBZ administered alone or co- administered with ivermectin (IVM, efflux modulator) and methimazole (MTZ, metabolic inhibitor) in TCBZ-resistant F. hepatica-parasitized sheep. Sheep infected with TCBZ-resistant F. hepatica (Sligo isolate) were divided into three groups (n = 4): untreated control, TCBZ-treated (i.r. at 10 mg/kg) and TCBZ+IVM+MTZ treated sheep (10 i.r., 0.2 s.c. and 1.5 i.m. mg/kg, respectively). Plasma samples were collected and analysed by HPLC. In the clinical efficacy study, the animals were sacrificed at 15 days post-treatment to evaluate the comparative efficacy against TCBZ- resistant F. hepatica. Results: The presence of IVM and MTZ did not affect the plasma disposition kinetics of TCBZ metabolites after the i.r. administration of TCBZ. The AUC value of TCBZ.SO obtained after TCBZ administration (653.9 ± 140.6 μg.h/ml) was similar to that obtained after TCBZ co-administered with IVM and MTZ (650.7 ± 122.8 μg.h/ml). Efficacy values of 56 and 38% were observed for TCBZ alone and for the combined treatment, respectively. No statistical differences (P > 0.05) were observed in fluke counts between treated groups and untreated control, which confirm the resistant status of the Sligo isolate. Conclusions: The presence of IVM and MTZ did not affect the disposition kinetics of TCBZ and its metabolites. Thus, the combined drug treatment did not reverse the poor efficacy of TCBZ against TCBZ-resistant F. hepatica. Background resistant populations, which is now a worrying problem Triclabendazole (TCBZ, 6-chloro-5(2-3 dichlorophe- in several areas of the world [2,3]. noxy)-2-methyl thio-benzimidazole), an halogenated Parasites have several possible strategies to achieve benzimidazole (BZD) thiol derivative, shows high effi- drug resistance, including changes in the target mole- cacy against both the immature and mature stages of cule, in drug uptake/efflux mechanisms and in drug Fasciola hepatica in sheep and cattle, which is a differ- metabolism [4]. At least two mechanisms appear to be ential feature compared to other available trematodicidal implicated in TCBZ resistance in F. hepatica: increased drugs [1]. As a consequence of its excellent activity drug efflux and enhanced oxidative metabolism [5-7]. against the liver fluke, it has been extensively used and TCBZ and its sulphoxide metabolite (TCBZ.SO) are this has inevitably promoted the selection of TCBZ- both substrates of P-glycoprotein (Pgp) [8]. Over-expres- sion of Pgp has been implicated in the resistance to * Correspondence: [email protected] macrocyclic lactones (ivermectin (IVM), moxidectin † Contributed equally (MXD)) [9,10], closantel and BZDs in nematodes [11]), 1 Laboratorio de Farmacología, Facultad de Ciencias Veterinarias, Universidad although the exact nature of the role has yet to be Nacional del Centro de la Provincia de Buenos Aires (UNCPBA), Campus ex vivo Universitario, 7000, Tandil, Argentina established [12]. Different experiments support © 2010 Ceballos et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Ceballos et al. BMC Veterinary Research 2010, 6:8 Page 2 of 8 http://www.biomedcentral.com/1746-6148/6/8 the hypothesis of the involvement of Pgp over-expres- Chemical Company (St Louis, USA). The different sol- sion in the resistance of F. hepatica to TCBZ. Higher vents (HPLC grade) and buffer salt used for sample levels of TCBZ and TCBZ.SO were observed within extraction or chromatographic methods were purchased TCBZ-resistant flukes when drug efflux from the para- from Baker Ind. (Phillipsburg, USA). site was decreased by IVM [7], a well recognized Pgp substrate/inhibitor [9,13]. It has been demonstrated that Animals and Experimental design TCBZ and its main metabolites, TCBZ.SO and TCBZ- Twelve (12) healthy intact male Corriedale sheep (53.8 ± sulphone (TCBZ.SO2) may induce tegumental damage 2.6 kg) aged 14-16 months and obtained from a farm in liver flukes [14]. Additionally, an increased oxidative locatedinanareafreeofF. hepatica were involved in metabolic capacity has been described as complementary this trial. Additionally, the absence of liver fluke infec- TCBZ resistance mechanism in F. hepatica [5,6]. In fact, tion was checked by analysis of F. hepatica eggs in co-incubation of TCBZ or TCBZ.SO with methimazole faeces, following routine procedures [20]. Animals were (MTZ), a flavin monooxygenase (FMO) enzymatic sys- housed individually during the experiment and for 20 tem inhibitor, lead to more severe surface morphological days before the start of the study. Animals were fed on changes in TCBZ-resistant F. hepatica, compared to that a commercial balanced concentrate diet. Water was pro- observed after incubation with TCBZ or TCBZ.SO alone vided ad libitum. Animal procedures and management [15]. protocols were carried out in accordance with the Ani- The interaction between co-administered drugs may mal Welfare Policy (Act 087/02) of the Faculty of Veter- induce changes in the pharmacokinetic behaviour of inary Medicine, Universidad Nacional del Centro de la either molecule. Increased albendazole sulphoxide Provincia de Buenos Aires (UNCPBA), Tandil, Argen- plasma concentrations in lambs after co-administration tina http://www.vet.unicen.edu.ar and internationally of albendazole (intraruminally, i.r.) with IVM (subcuta- accepted animal welfare guidelines [21]. neously, s.c.), was previously reported [16]. Similarly, Animals were each orally infected with eighty (80) after the co-administration to sheep of IVM and TCBZ metacercariae of a TCBZ-resistant F. hepatica isolate, by the intravenous (i.v.) route, an enhanced TCBZ.SO named Sligo. For details of the history of the Sligo iso- plasma concentration was achieved [17]. On the other late, see previous works [5,22,23]. Sixteen weeks after hand, MTZ inhibition of TCBZ oxidative metabolism by infection, animals were randomly distributed into three sheep liver microsomes has been reported [18]. How- experimental groups (n = 4 each): Group I, which repre- ever, MTZ did not affect TCBZ disposition kinetics in sented the untreated control group; Group II, in which sheep after the administration of both compounds by animals were treated with TCBZ (Fasinex®, Novartis) by the i.v. route [19]. the i.r. route at 10 mg/kg dose rate; and Group III, in Both modified influx/efflux and enhanced metabolism which animals were simultaneously treated with TCBZ may account for the development of resistance to TCBZ (Fasinex®, Novartis) by the i.r. route (10 mg/kg) and in F. hepatica. As a consequence, it opens up the possi- IVM (Ivosint®, Biogénesis) by the s.c. route (0.2 mg/kg, bility of modulating drug efflux and metabolism in the internal face of the thigh). Additionally, animals in TCBZ-resistant fluke, by co-administering TCBZ with Group III were treated by the intramuscular (i.m.) route MTZ and IVM, with the aim of reversing anthelmintic (Semitendinosus muscle) with MTZ (2.5% aqueous solu- resistance. Furthermore, in vivo drug-drug interaction tion) at a dose rate of 1.5 mg/kg [24]. MTZ administra- between these drugs may modify the overall disposition tion was performed 30 min after TCBZ/IVM treatment. kinetics and pattern of drug distribution of TCBZ to the Blood samples (5 ml) were taken by jugular venipunc- liver fluke. The aims of the current work were: a) to tures into heparinised Vacutainers® tubes (Becton Dick- investigate the potential effect of MTZ and IVM on the inson, USA) before administration (time 0) and at 1, 3, plasma concentrations profiles of TCBZ and its metabo- 6, 9, 12, 18, 24, 30, 36, 48, 72, 96, 120 and 144 h post- lites in sheep and b) to study the clinical efficacy of treatment. Plasma was separated by centrifugation at TCBZ alone or when co-administered with MTZ and 3000 g for 15 min, placed into plastic tubes and frozen IVM against TCBZ-resistant F. hepatica. at -20°C until analyzed by high performance liquid chro- matography (HPLC). Methods Chemicals Clinical efficacy study Pure reference standards (97-99%) of TCBZ and its Fifteen days after treatment all animals were stunned TCBZ.SO and TCBZ.SO2 metabolites, were provided by and exsanguinated immediately. Adult F. hepatica speci- Novartis Animal Health (Basel, Switzerland) (Batch # mens were recovered from the common bile ducts and AMS 215/102, HI-1025/1 and JG-5161/6, respectively). the gall bladder of each sheep and counted according to MTZ and IVM were purchased from Sigma-Aldrich the World Association for the Advancement of Ceballos et al. BMC Veterinary Research 2010, 6:8 Page 3 of 8 http://www.biomedcentral.com/1746-6148/6/8 Veterinary Parasitology (W.A.A.V.P) guidelines [25]. The 6.74%) (TCBZ), 87.1% (CV: 6.03%) (TCBZ.SO) and efficacy of each anthelmintic treatment was determined 90.1% (CV: 4.75%) (TCBZ.SO2). Precision (intra- and by the comparison of F. hepatica burdens in treated ver- inter-assay) was determined by analysing replicates of sus untreated animals. The following equation expresses fortified plasma samples (n = 5) with each compound the percent efficacy (%E) of a drug treatment against F.