Pharmacologic interaction between oxfendazole and triclabendazole: In vitro biotransformation and systemic exposure in sheep.

The aim of the current work was to evaluate a potential pharmacokinetic interaction between the flukicide triclabendazole (TCBZ) and the broad-spectrum benzimidazole (BZD) anthelmintic oxfendazole (OFZ) in sheep. To this end, both an in vitro assay in microsomal fractions and an in vivo trial in lambs parasitized with Haemonchus contortus resistant to OFZ and its reduced derivative fenbendazole (FBZ) were carried out. Sheep microsomal fractions were incubated together with OFZ, FBZ, TCBZ, or a combination of either FBZ and TCBZ or OFZ and TCBZ. OFZ production was significantly diminished upon coincubation of FBZ and TCBZ, whereas neither FBZ nor OFZ affected the S-oxidation of TCBZ towards its sulfoxide and sulfone metabolites. For the in vivo trial, lambs were treated with OFZ (Vermox® oral drench at a single dose of 5 mg/kg PO), TCBZ (Fasinex® oral drench at a single dose of 12 mg/kg PO) or both compounds at a single dose of 5 (Vermox®) and 12 mg/kg (Fasinex®) PO. Blood samples were taken to quantify drug and metabolite concentrations, and pharmacokinetic parameters were calculated by means of non-compartmental analysis. Results showed that the pharmacokinetic parameters of active molecules and metabolites were not significantly altered upon coadministration. The sole exception was the increase in the mean residence time (MRT) of OFZ and FBZ sulfone upon coadministration, with no significant changes in the remaining pharmacokinetic parameters. This research is a further contribution to the study of metabolic drug-drug interactions that may affect anthelmintic efficacies in ruminants.

Assessment of the pharmacological interactions between the nematodicidal fenbendazole and the flukicidal triclabendazole: In vitro studies with bovine liver microsomes and slices.

Parasitic diseases have a significant impact on livestock production. Nematodicidal drugs, such as fenbendazole (FBZ) or its oxidized metabolite oxfendazole (OFZ), can be used along with the trematodicidal triclabendazole (TCBZ), to broaden the spectrum of anthelmintic activity. However, co-exposure to these compounds could lead to drug-drug (D-D) interactions and eventually alter the clinical profile of each active principle. The aim of this study was to assess the presence of such interactions by means of two in vitro models, namely bovine liver microsomal fractions and bovine precision-cut liver slices (PCLSs). To this end, an in vitro assessment involving incubation of FBZ and TCBZ or a combination of FBZ and TCBZ was carried out. Results with microsomal fractions showed a 78.4% reduction (p = .002) in the rate of OFZ production upon co-incubation, whereas the sulfoxide metabolite of TCBZ (TCBZSO) exhibited a decreasing tendency. With PCLS, OFZ accumulation in the incubation medium increased 1.8-fold upon co-incubation, whereas TCBZSO accumulation decreased by 28%. The accumulation of FBZ and OFZ in the liver tissue increased upon 2-hr co-incubation, from 2.1 ± 1.5 to 18.2 ± 6.1 (p = .0009) and from 0.4 ± 0.1 to 1.3 ± 0.3 nmol (p = .0005), respectively. These results confirm the presence of D-D interactions between FBZ and TCBZ. Further studies are needed to determine the extent of involvement of drug-metabolizing enzymes and membrane transporters in interactions between compounds largely used in livestock production systems.

Gaining Insights Into the Pharmacology of Anthelmintics Using Haemonchus contortus as a Model Nematode.

Progress made in understanding pharmacokinetic behaviour and pharmacodynamic mechanisms of drug action/resistance has allowed deep insights into the pharmacology of the main chemical classes, including some of the few recently discovered anthelmintics. The integration of pharmaco-parasitological research approaches has contributed considerably to the optimization of drug activity, which is relevant to preserve existing and novel active compounds for parasite control in livestock. A remarkable amount of pharmacology-based knowledge has been generated using the sheep abomasal nematode Haemonchus contortus as a model. Relevant fundamental information on the relationship among drug influx/efflux balance (accumulation), biotransformation/detoxification and pharmacological effects in parasitic nematodes for the most traditional anthelmintic chemical families has been obtained by exploiting the advantages of working with H. contortus under in vitro, ex vivo and in vivo experimental conditions. The scientific contributions to the pharmacology of anthelmintic drugs based on the use of H. contortus as a model nematode are summarized in the present chapter.

Comparative in vitro characterization of moxidectin and doramectin percutaneous absorption through bovine skin.

Topical formulations have achieved worldwide acceptance in veterinary medicine because their administration is an easy, less labor-intensive and nonstressing form. Any chemical compound that comes in contact with the skin has the potential to be locally and/or systemically absorbed. However, many factors related to the features of animal skin, composition of the topical formulation and to the drug itself can determine marked differences in the percutaneous absorption process. The aim of the current work was to characterize the pattern of in vitro percutaneous absorption for moxidectin (MXD) and doramectin (DRM), two of the most worldwide used topical macrocyclic lactone antiparasitic compounds in cattle. The work included the development of a simple and inexpensive in vitro assay useful to predict in vivo drug percutaneous absorption in cattle. Both drugs were administered as the commercial formulations intended for their topical administration to cattle. The in vitro studies were carried out using modified Franz-type vertical diffusion cells. Cattle skin slices of 500 µm thickness were prepared using a dermatome to separate the stratum corneum and upper epidermis from dermis and subcutaneous tissue. The receptor medium was sampled up to 72 h postadministration and drug concentrations were measured by HPLC. The parameters used to estimate the comparative in vitro skin permeation showed marked differences between DRM and MXD. A 5.29-fold longer lag time (T(lag)) was observed for DRM. Similarly, the flux (J) (2.93-fold) and the permeation coefficients (K(p) ) (2.95-fold) in cattle skin were significantly higher (P < 0.05) for DRM compared to those obtained for MXD. Additionally, the data obtained from the in vitro permeation studies was correlated with the plasma concentrations of both compounds achieved in vivo in cattle treated with the same topical formulations. Correlation coefficients between percentage of drug permeated in vitro vs. percentage of drug absorbed in vivo (up to 48 h post-treatment) were 0.856-0.887 (MXD) and 0.976-0.990 (DRM). However, the highest in vitro-in vivo correlations for both molecules were observed up to 24 h post-treatment A rapid screening method for testing different topical formulations can be achieved with the simple in vitro cattle skin permeation technique described here, which has been successfully adapted to test the comparative percutaneous absorption of MXD and DRM.

Potentiation of triclabendazole action in vivo against a triclabendazole-resistant isolate of Fasciola hepatica following its co-administration with the metabolic inhibitor, ketoconazole.

An in vivo study in the laboratory rat model has been carried out to monitor morphological changes in adult Fasciola hepatica over a 4-day period resulting from co-treatment with triclabendazole (TCBZ) and ketoconazole (KTZ), a cytochrome P450 inhibitor. Rats were infected with the triclabendazole-resistant Oberon isolate of F. hepatica, dosed orally with triclabendazole at a dosage of 10mg/kg live weight and ketoconazole at a dosage of 10mg/kg live weight. Flukes were recovered at 24, 48, 72 and 96 h post-treatment (p.t.) and changes to fluke ultrastructure were assessed using transmission electron microscopy (TEM). Results showed an increase in the severity of changes to the fluke ultrastructure with time p.t. Swelling of the basal infolds and the associated mucopolysaccharide masses became more severe with time. Golgi complexes, if present, were greatly reduced in size and number by 96 h p.t., and sub-tegumental flooding was seen from the 72 h time-period onwards. Some sloughing of the tegumental covering over the spines was observed at 96 h p.t. The results demonstrated that the Oberon isolate is more sensitive to TCBZ action in the presence of KTZ than to TCBZ alone, reinforcing the idea that altered drug metabolism is involved in the resistance mechanism. Moreover, they support the concept that TCBZ+inhibitor combinations (aimed at altering drug pharmacokinetics and potentiating the action of TCBZ) could be used in the treatment of TCBZ-R populations of F. hepatica.

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

A study has been carried out to investigate whether the action of triclabendazole (TCBZ) against Fasciola hepatica is altered by inhibition of drug metabolism. The cytochrome P450 (CYP 450) enzyme pathway was inhibited using ketoconazole (KTZ) to see whether a TCBZ-resistant isolate could be made more sensitive to TCBZ action. The Oberon TCBZ-resistant and Cullompton TCBZ-susceptible isolates were used for these experiments. The CYP 450 system was inhibited by a 2-h pre-incubation in ketoconazole (40 µM), then incubated for a further 22 h in NCTC medium containing either KTZ, KTZ + nicotinamide adenine dinucleotide phosphate (NADPH) (1 nM), KTZ + NADPH + TCBZ (15 µg/ml), or KTZ + NADPH + triclabendazole sulphoxide (TCBZ.SO; 15 µg/ml). Changes to fluke ultrastructure following drug treatment and metabolic inhibition were assessed using transmission electron microscopy. After treatment with either TCBZ or TCBZ.SO on their own, there was greater disruption to the TCBZ-susceptible than TCBZ-resistant isolate. However, co-incubation with KTZ + TCBZ, but more particularly KTZ + TCBZ.SO, led to more severe changes to the TCBZ-resistant isolate than with each drug on its own: for example, there was severe swelling of the basal infolds and their associated mucopolysaccharide masses, accompanied by an accumulation of secretory bodies just below the apex. Golgi complexes were greatly reduced or absent in the tegumental cells and the synthesis, production, and transport of secretory bodies were badly disrupted. With the TCBZ-susceptible Cullompton isolate, there was limited potentiation of drug action. The results support the concept of altered drug metabolism in TCBZ-resistant flukes and this process may play a role in the development of drug resistance.

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

A study has been carried out to investigate whether the action of triclabendazole (TCBZ) against Fasciola hepatica is altered by inhibition of drug metabolism. The cytochrome P450 (CYP 450) enzyme pathway was inhibited using ketoconazole (KTZ) to see whether a TCBZ-resistant isolate could be made more sensitive to TCBZ action. The Oberon TCBZ-resistant and Cullompton TCBZ-susceptible isolates were used for these experiments. The CYP 450 system was inhibited by a 2-h pre-incubation in ketoconazole (40 µM), then incubated for a further 22 h in NCTC medium containing either KTZ, KTZ + nicotinamide adenine dinucleotide phosphate (NADPH) (1 nM), KTZ + NADPH + TCBZ (15 µg/ml), or KTZ + NADPH + triclabendazole sulphoxide (TCBZ.SO; 15 µg/ml). Changes to fluke ultrastructure following drug treatment and metabolic inhibition were assessed using transmission electron microscopy. After treatment with either TCBZ or TCBZ.SO on their own, there was greater disruption to the TCBZ-susceptible than TCBZ-resistant isolate. However, co-incubation with KTZ + TCBZ, but more particularly KTZ + TCBZ.SO, led to more severe changes to the TCBZ-resistant isolate than with each drug on its own: in the syncytium, for example, there was severe swelling of the basal infolds and their associated mucopolysaccharide masses, accompanied by an accumulation of secretory bodies just below the apex. Golgi complexes were greatly reduced or absent in the tegumental cells and the synthesis, production, and transport of secretory bodies were badly disrupted. With the TCBZ-susceptible Cullompton isolate, there was limited potentiation of drug action. The results support the concept of altered drug metabolism in TCBZ-resistant flukes and this process may play a role in the development of drug resistance.

Enhancement of triclabendazole action in vivo against a triclabendazole-resistant isolate of Fasciola hepatica by co-treatment with ketoconazole.

An in vivo study in the laboratory rat model was carried out to monitor morphological changes in adult Fasciola hepatica over a 4-day period resulting from combination treatment of triclabendazole (TCBZ) and the metabolic inhibitor, ketoconazole (KTZ). Rats were infected with the TCBZ-resistant Oberon isolate of F. hepatica and divided into 3 groups at 12 weeks post-infection. The first group was dosed orally with TCBZ at a dosage of 10mg/kg and KTZ at a dosage of 10mg/kg. Flukes were recovered at 24, 48, 72 and 96 h post-treatment (p.t.). A second group of rats was treated with TCBZ alone (10mg/kg) and sacrificed at 96 h p.t. The third group acted as untreated controls. Surface changes were monitored by scanning electron microscopy (SEM). In flukes from the TCBZ+KTZ-treated group, the results showed a progressive and time-dependent increase in the level of disruption to the tegumental syncytium. Swelling, furrowing, blebbing and sloughing of the syncytium increased with time p.t. Another feature seen was a thick layer of tegumental shedding in some fluke samples at different times p.t. By comparison, flukes treated with TCBZ alone remained unaffected. The results demonstrated that the Oberon isolate is only sensitive to drug action in the presence of ketoconazole, indicating that combining triclabendazole with a metabolic inhibitor could be used to preserve the effectiveness of the drug against TCBZ-resistant populations of F. hepatica.

Piperonyl butoxide enhances triclabendazole action against triclabendazole-resistant Fasciola hepatica.

A study has been carried out to determine whether the action of triclabendazole (TCBZ) against the liver fluke, Fasciola hepatica is altered by inhibition of the cytochrome P450 (CYP 450)-mediated drug metabolism pathway. The Oberon TCBZ-resistant and Cullompton TCBZ-susceptible fluke isolates were used for these experiments, the basic design of which is given in the paper by Devine et al. (2010a). Piperonyl butoxide (PB) was the CYP P450 inhibitor used. Morphological changes resulting from drug treatment and following metabolic inhibition were assessed by means of transmission electron microscopy. After treatment with either TCBZ or TCBZ.SO on their own, there was greater disruption to the TCBZ-susceptible than TCBZ-resistant isolate. However, co-incubation with PB+TCBZ, but more particularly PB+TCBZ.SO, led to greater changes to the TCBZ-resistant isolate than with each drug on its own, with blebbing of the apical plasma membrane, severe swelling of the basal infolds and their associated mucopolysaccharide masses in the syncytium and flooding in the internal tissues. Golgi complexes were greatly reduced or absent in the tegumental cells and the synthesis and production of secretory bodies were badly disrupted. The mitochondria were swollen throughout the tegumental system and the somatic muscle blocks were disrupted. With the TCBZ-susceptible Cullompton isolate, there was a limited increase in drug action following co-incubation with PB. The results provide evidence that the condition of a TCBZ-resistant fluke can be altered by inhibition of drug metabolism. Moreover, they support the concept that altered drug metabolism contributes to the mechanism of resistance to TCBZ.

Inhibition of cytochrome P450-mediated metabolism enhances ex vivo susceptibility of Fasciola hepatica to triclabendazole.

A study has been carried out to investigate whether the action of triclabendazole (TCBZ) against Fasciola hepatica is altered by inhibition of drug metabolism. The cytochrome P450 (CYP P450) system was inhibited using piperonyl butoxide (PB). The Oberon TCBZ-resistant and Cullompton TCBZ-susceptible isolates were used for these experiments. The CYP P450 system was inhibited by a 2 h pre-incubation in PB (100 mum). Flukes were then incubated for a further 22 h in NCTC medium containing either PB; PB+nicotinamide adenine dinucleotide phosphate (NADPH) (1 nm); PB+NADPH+TCBZ (15 microg/ml); or PB+NADPH+TCBZ.SO (15 microg/ml). Morphological changes resulting from drug treatment and following metabolic inhibition were assessed using scanning electron microscopy. After treatment with either TCBZ or TCBZ.SO alone, there was greater disruption to the TCBZ-susceptible than the resistant isolate. However, co-incubation with PB and TCBZ/TCBZ.SO lead to more severe surface changes to the TCBZ-resistant Oberon isolate than with each drug on its own. With the TCBZ-susceptible Cullompton isolate, there was limited potentiation of drug action, and only with TCBZ.SO. The results support the concept of altered drug metabolism in TCBZ-resistant flukes and this process may play a role in the development of drug resistance.

Effect of the metabolic inhibitor, methimazole on the drug susceptibility of a triclabendazole-resistant isolate of Fasciola hepatica.

SUMMARY: A study has been carried out to investigate whether the action of triclabendazole (TCBZ) is altered in the presence of a metabolic inhibitor. The flavin monooxygenase system (FMO) was inhibited using methimazole (MTZ) to see whether a TCBZ-resistant isolate could be made more sensitive to TCBZ action. The Oberon TCBZ-resistant and Cullompton TCBZ-sensitive isolates were used for these experiments. The FMO system was inhibited by a 2-h pre-incubation in methimazole (100 microM). Flukes were then incubated for a further 22 h in NCTC medium containing either MTZ; MTZ+nicotinamide adenine dinucleotide phosphate (NADPH) (1 nM); MTZ+NADPH+TCBZ (15 microg/ml); or MTZ+NADPH+triclabendazole sulphoxide (TCBZ.SO) (15 microg/ml). Morphological changes resulting from drug treatment and following metabolic inhibition were assessed using scanning electron microscopy. After treatment with either TCBZ or TCBZ.SO alone, there was greater surface disruption to the triclabendazole-susceptible than -resistant isolate. However, co-incubation with MTZ and TCBZ/TCBZ.SO lead to more severe surface changes to the TCBZ-resistant isolate than with each drug on its own; this was not seen for the TCBZ-susceptible Cullompton isolate. Results of this study support the concept of altered drug metabolism in TCBZ-resistant flukes and this process may play a role in the development of drug resistance.

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

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.

Changes to oxfendazole chiral kinetics and anthelmintic efficacy induced by piperonyl butoxide in horses.

REASONS FOR PERFORMING THE STUDY: The study of novel pharmacological strategies to control parasitism in horses is required since many parasite species have developed resistance to anthelmintic drugs. OBJECTIVES: To evaluate the effects of piperonyl butoxide (PB) (a metabolic inhibitor) on the plasma availability and enantiomeric behaviour of oxfendazole (OFZ) given orally to horses, and to compare the clinical efficacy of OFZ given either alone or co-administered with PB in naturally parasitised horses. METHODS: Fifteen naturally parasitised crossbred male ponies were allocated into 3 groups (n = 5) and treated orally as follows: Group I (control) received distilled water as placebo; Group II was dosed with OFZ (10 mg/kg bwt); and Group III was treated with OFZ (10 mg/kg bwt) co-administered with PB (63 mg/kg bwt). Jugular blood samples were obtained over 120 h post treatment. Three weeks after treatments, all experimental horses were subjected to euthanasia. RESULTS: The observed maximum plasma concentration (Cmax) and area under the concentration vs. time curve (AUC) values for OFZ increased 3- and 5-fold, respectively, in the presence of PB. The plasma concentration profiles of fenbendazole (FBZ), a metabolite generated from OFZ, were significantly lower after the treatment with OFZ alone (AUC = 0.8 microg x h/ml) compared to those obtained after the OFZ + PB treatment (AUC = 2.7 microg x h/ml). The enhanced pharmacokinetic profiles correlated with increased anthelmintic efficacy. The combination OFZ + PB showed 100% efficacy against mature nematode parasites. The efficacy against cyathostome L3 larvae increased from 94% (Group II) to 98.7% (Group III). Consistently, the number of L4 larvae recovered from OFZ + PB treated horses (Group III) (n = 146) was significantly lower (P<0.05) than that recovered from Group II (n = 1397). CONCLUSIONS: The use of PB as a metabolic inhibitor may be useful to enhance OFZ activity against mature and migrating larvae of different parasite species in horses. POTENTIAL RELEVANCE: Metabolic inhibitors may be used to enhance the activity of benzimidazole anthelmintics and extend the effective lifespan of benzimidazole drugs in the face of increasing resistance.

Comparative assessment of the access of albendazole, fenbendazole and triclabendazole to Fasciola hepatica: effect of bile in the incubation medium.

The work reported here describes the comparative ability of albendazole (ABZ), fenbendazole (FBZ) and triclabendazole (TCBZ) to penetrate through the tegument of mature Fasciola hepatica, and the influence of the physicochemical composition of the incubation medium on the drug diffusion process. The data obtained from the trans-tegumental diffusion kinetic studies were complemented with the determination of lipid-to-water partition coefficients (octanol-water) for the benzimidazole (BZD) anthelmintic drugs assayed. Sixteen-week-old F. hepatica were obtained from untreated artificially infected sheep. The flukes were incubated (37 degrees C) over 60 and 90 min in incubation media (pH 7.4) prepared with different proportions of ovine bile and Krebs' Ringer Tris (KRT) buffer (100, 75, 50, 25 and 0% of bile) containing either ABZ, FBZ or TCBZ at a final concentration of 5 nmol/ml. After the incubation time expired, the liver fluke material was chemically processed and analysed by high performance liquid chromatography (HPLC) to measure drug concentrations within the parasite. Additionally, the octanol-water partition coefficients (PC) for each molecule were calculated (as an indicator of drug lipophilicity) using reversed phase HPLC. The 3 BZD molecules were recovered from F. hepatica at both incubation times in all incubation media assayed. The trans-tegumental diffusion of the most lipophilic molecules ABZ and FBZ (higher PC values) tended to be greater than that observed for TCBZ. Interestingly, the uptake of ABZ by the liver flukes was significantly greater than that measured for TCBZ, the most widely used flukicidal BZD compound. This differential uptake pattern may be a relevant issue to be considered to deal with TCBZ-resistant flukes. Drug concentrations measured within the parasite were lower in the incubations containing the highest bile proportions. The highest total availabilities of the 3 compounds were obtained in liver flukes incubated in the absence of bile. Altogether, these findings demonstrated that the entry of the drug into a target parasite may not only depend on a concentration gradient, the lipophilicity of the molecule and absorption surface, but also on the physicochemical composition of the parasite's surrounding environment.

Transtegumental diffusion of benzimidazole anthelmintics into Moniezia benedeni: correlation with their octanol-water partition coefficients.

The experiments described here report on the correlation between the ex vivo diffusion of different benzimidazole (BZD) anthelmintics into the cestode parasite Moniezia benedeni, and their octanol-water partition coefficients (P.C.). The characterisation of the drug diffusion process into target parasites is relevant to understand the mechanism of drug penetration and the pharmacological activity of anthelmintic drugs. Specimens of the tapeworm M. benedeni, used as a helminth parasite model, were obtained from untreated cattle killed at the local abattoir. The collected parasites were incubated (5-210 min) with either fenbendazole (FBZ), albendazole (ABZ), ricobendazole (RBZ), oxfendazole (OFZ), mebendazole (MBZ), oxibendazole (OBZ), or thiabendazole (TBZ), in a Kreb's Ringer Tris buffer medium at a final concentration of 5 nmol/ml. After the incubation time elapsed, samples of parasite material were chemically extracted and prepared for high performance liquid chromatography (HPLC) analysis to measure drug/metabolite concentrations. Additionally, the octanol-water P.C. for each molecule was estimated as an indicator of drug lipophilicity, using reversed phase HPLC analysis. All the incubated drugs were recovered from the tapeworms as early as 5 min post incubation. There was a high correlation (r=0.87) between drug lipophilicity, expressed as octanol-water P.C. (Log P), and drug availability within the parasite. The most lipophilic BZD compounds (FBZ, ABZ, and MBZ), with P.C. values higher than 3.7, were measured at significative higher concentrations within the tapeworm compared to those drugs with the lowest P.C. values. Considering the results from the current and previous studies, it is clear that passive diffusion is a major mechanism of BZD penetration into cestode parasites, where lipid solubility is a determinant factor influencing the diffusion of these anthelmintic molecules through the parasite tegument.

Enantioselective binding of albendazole sulphoxide to cytosolic proteins from helminth parasites.

The pharmacological effect of the active albendazole metabolite, albendazole sulphoxide (ABZSO), depends on its sustained presence at the site of parasite location and its binding to helmith beta-tubulin. ABZSO is found in the plasma and tissues of albendazole-treated animals in two enantiomeric forms: (+)ABZSO and (-)ABZSO. Knowledge of enantioselectivity in drug action is necessary, since any difference in target proteins affinity between enantiomers may have implications on the pharmacological effect of this anthelmintic molecule. The binding of ABZSO to mammalian and helminth parasites cytosolic proteins, as well as the differential binding of both enantiomers, were studied. Cytosolic proteins from Moniezia expansa (cestode), Ascaris suum (nematode), Fasciola hepatica (trematode), rat liver and brain as well as purified porcine brain tubulin were used. Drug analysis was performed by HPLC using both C18 and chiral columns. ABZSO protein binding was quantitatively different between parasite species (4.17, 2.5 and 1.07 ng/mg for cestode, nematode and trematode, respectively); this binding to helminth cytosolic proteins was enantioselective. Enantiomeric ratios of (-)ABZSO/(+)ABZSO as a percentage were: 43/57 (Ascaris), 36/64 (Moniezia) and 91/9 (Fasciola). Conversely, the binding of ABZSO to mammalian cytosolic proteins showed no enantioselectivity. The overall binding affinity of ABZSO for mammalian cytosolic proteins was lower than that observed in helminth proteins. The characterization of the comparative binding pattern of ABZSO enantiomers to cytosolic proteins from helminth parasites and mammalian tissues may contribute to understanding the pharmacological properties of this chiral anthelmintic molecule.

Ex vivo diffusion of albendazole and its sulfoxide metabolite into ascaris suum and Fasciola hepatica.

The current experiments compare the pattern of ex vivo uptake (diffusion) of albendazole (ABZ) and albendazole sulfoxide (ABZSO) by Ascaris suum and Fasciola hepatica. Specimens of A. suum and F. hepatica were collected from untreated animals (pigs and sheep, respectively) and incubated with either ABZ or ABZSO for different time periods (5-180 min). After incubation. the parasite material was analysed by HPLC to quantify the amount of ABZ and/or ABZSO. The parent drug and its active ABZSO metabolite were recovered from the parasites after ex vivo incubation for different time periods throughout the assay. Total drug availability in A. suum, expressed as area under the concentration versus time curve (AUC) over 180 min of incubation, was significantly greater (P<0.05) for ABZ parent drug (AUC = 4.19 +/- 0.59 microg x h xg(-1)) compared with the more polar ABZSO metabolite (AUC = 0.25 +/- 0.01 micro x h x g(-1)). Similar results were observed after the incubation of both molecules with F. hepatica, where the AUC values obtained were 10.6 +/- 0.28 microg x h x g(-1) and 2.04 +/- 0.33 microg x h x g(-1) for ABZ and ABZSO, respectively. The greater diffusion and availability of ABZ in both helminths correlate with the higher lipophilicity of the parent drug, compared with its sulfoxide metabolite. The amount of both molecules measured in A. suum was significantly lower (P<0.05) than that recovered in F. hepatica. The complexity of the histological structure of the nematode cuticle compared with the external tegument of the trematode may account for such a difference in drug diffusion between the species. These findings complement previous observations on the patterns of in vivo uptake of ABZ by different helminth parasites, contributing to the understanding of the pharmacological anthelmintic action of these moieties.

Comparative metabolism of albendazole and albendazole sulphoxide by different helminth parasites.

Albendazole (ABZ) is a broad-spectrum benzimidazole anthelmintic widely used in human and veterinary medicine. The aim of the current work was to characterise the sulphoxidative metabolism of ABZ, and the sulphoreduction of ABZ sulphoxide (ABZSO), by microsomal (Ms) and cytosolic (Cyt) fractions of three different helminth species: Fasciola hepatica, Moniezia expansa and Ascaris suum. After the incubation assays, parasite material was analysed by HPLC to characterise the metabolic product formed. Both the Ms and Cyt fractions of the three parasites studied were able to oxidise ABZ into ABZSO in a non-enantioselective fashion. Oxidation of ABZ was greater in the Ms fraction of the trematode (50%) than in both cestode (19%) and nematode (14%) parasites. Only the incubation of ABZSO with both subcellular fractions of M. expansa generated ABZ as a metabolic product. The results obtained here indicate that helminths have the capacity to biotransform benzimidazole compounds; however, this metabolic activity differs qualitatively and quantitatively among helminth species.

Uptake of albendazole and albendazole sulphoxide by Haemonchus contortus and Fasciola hepatica in sheep.

The pattern of in vivo uptake of albendazole (ABZ) and its major metabolite, ABZ-sulphoxide (ABZSO), by Haemonchus contortus and Fasciola hepatica recovered from ABZ-treated sheep, was investigated. Concentration profiles of both compounds were simultaneously measured in target tissues/fluids from the same infected sheep. In addition, the proportion of the (+) and (-) ABZSO enantiomers was determined in plasma, bile and F. hepatica recovered from treated sheep. Sheep naturally infected with H. contortus were intraruminally (i.r.) treated with ABZ (micronized suspension, 7. 5mg/kg) and the plasma concentrations of ABZSO and ABZ-sulphone (ABZSO(2)) determined in addition to the concentration of ABZ and ABZSO in H. contortus, abomasal mucosa and fluid content samples. In addition, F. hepatica artificially infected sheep were treated i.r. with the same ABZ suspension (7.5mg/kg), and samples of blood, bile, liver tissue and adult flukes were collected and analysed by HPLC to determine the concentrations of ABZ and both enantiomers of ABZSO. ABZSO and ABZSO(2) were the analytes recovered in plasma with ABZ and ABZSO present in H. contortus. ABZ was the analyte recovered at the highest concentration in H. contortus and abomasal mucosa, whereas higher concentrations of ABZSO were measured in abomasal fluid content. Only low concentrations of ABZ were detected in F. hepatica and bile, but markedly higher concentrations of ABZ were measured in liver tissue. ABZSO was the main molecule recovered in F. hepatica, plasma and bile samples collected from ABZ-treated sheep. The (+) enantiomer of ABZSO was recovered at a higher proportion in plasma (75%), bile (78%) and F. hepatica (74%) after ABZ administration to infected sheep.

Liver sulphoxidative metabolism of albendazole in rat: enantioselectivity and effect of methimazole.

The aim of the present work was to evaluate the effects of methimazole (MTZ) on the enantioselective sulphoxidation of albendazole (ABZ) by rat liver microsomes and tissue slices. Albendazole sulphoxide (ABZSO) was the metabolite recovered after the incubation with ABZ in both liver preparations. MTZ significantly reduced ABZSO production both in microsomes and slices. ABZSO production decreased as a function of MTZ concentration. The sulphoxidation reaction performed by rat liver explants in the presence of MTZ was 65% lower than that observed in controls. The reduction in the production of ABZSO in the presence of MTZ was mainly due to a lower production of (+) ABZSO. The results reported further contribute to the understanding of the enantioselective metabolism of ABZ. In addition, the work presented provides information on the comparison of two different liver tissue preparations for the evaluation of xenobiotic metabolism.