Metabolism and pharmacokinetics of mebendazole in Japanese pufferfish (Takifugu rubripes).

As a typical and broad-spectrum benzimidazole, mebendazole (MBZ) has long been used in human and veterinary medicine to treat parasitic infestations, and is widely employed in the aquaculture of Japanese pufferfish (Takifugu rubripes). However, there have been no studies examining the pharmacokinetic characteristics of MBZ in Japanese pufferfish. Furthermore, the presence of MBZ and its metabolites in animal-derived raw food represents a notable safety concern. Here, we investigated the metabolism of MBZ using a UPLC-Q-TOF system. Additionally, we evaluated the pharmacokinetics of MBZ and two metabolites, 2-amino-5(6)-benzoylbenzimidazole (MBZ-NH2) and 5-hydroxymebendazole (MBZ-OH), in Japanese pufferfish following intramuscular injection of 20 mg/kg MBZ. We detected three metabolites of MBZ (M1-M3), among which, 2-amino-5(6)-(a-hydroxybenzyl) benzimidazole (M3) was detected in an aquatic animal for the first time. The plasma dispositions of MBZ, MBZ-NH2, and MBZ-OH were characterized by low plasma clearance, medium distribution volume, and long terminal half-life. Moreover, these compounds were widely distributed in the muscle, from which they were rapidly cleared. The pharmacokinetics and metabolism of mebendazole in Japanese pufferfish are described for the first time in this study. Our findings provide a basis for the rational application of MBZ in Japanese pufferfish farming and contribute to our understanding of the metabolism of MBZ in cultured fish.

Mebendazole-induced M1 polarisation of THP-1 macrophages may involve DYRK1B inhibition.

OBJECTIVE: We recently showed that the anti-helminthic compound mebendazole (MBZ) has immunomodulating activity by inducing a M2 to M1 phenotype switch in monocyte/macrophage models. In the present study we investigated the potential role of protein kinases in mediating this effect. RESULTS: MBZ potently binds and inhibits Dual specificity tyrosine-phosphorylation-regulated kinase 1B (DYRK1B) with a Kd and an IC50 of 7 and 360 nM, respectively. The specific DYRK1B inhibitor AZ191 did not mimic the cytokine release profile of MBZ in untreated THP-1 monocytes. However, in THP-1 cells differentiated into macrophages, AZ191 strongly induced a pro-inflammatory cytokine release pattern similar to MBZ and LPS/IFNγ. Furthermore, like MBZ, AZ191 increased the expression of the M1 marker CD80 and decreased the M2 marker CD163 in THP-1 macrophages. In this model, AZ191 also increased phospho-ERK activity although to a lesser extent compared to MBZ. Taken together, the results demonstrate that DYRK1B inhibition could, at least partly, recapitulate immune responses induced by MBZ. Hence, DYRK1B inhibition induced by MBZ may be part of the mechanism of action to switch M2 to M1 macrophages.

N-Substituted Prodrugs of Mebendazole Provide Improved Aqueous Solubility and Oral Bioavailability in Mice and Dogs.

Mebendazole (MBZ) was developed as a broad-spectrum anthelmintic but has recently shown efficacy as an anticancer agent. The use of MBZ for cancer, however, is challenging due to its poor solubility leading to poor bioavailability. Herein, we developed a prodrug approach with various N-linked promoieties including acyloxymethyl, aminoacyloxymethyl, and substituted phosphonooxymethyl in attempt to improve these characteristics. Compound 12, containing an (((((isopropoxycarbonyl)oxy)methoxy)phosphoryl)oxy)methyl promoiety, showed a >10 000-fold improvement in aqueous solubility. When evaluated in mice, 12 displayed a 2.2-fold higher plasma AUC0- t and a 1.7-fold improvement in brain AUC0- t with a calculated oral bioavailability of 52%, as compared to 24% for MBZ-polymorph C (MBZ-C), the most bioavailable polymorph. In dogs, 12 showed a 3.8-fold higher plasma AUC0- t with oral bioavailability of 41% compared to 11% for MBZ-C. In summary, we have identified a prodrug of MBZ with better physicochemical properties and enhanced bioavailability in both mice and dog.

Metabolism of albendazole, ricobendazole and flubendazole in Haemonchus contortus adults: Sex differences, resistance-related differences and the identification of new metabolites.

Haemonchus contortus (family Trichostrongylidae, Nematoda), a hematophagous gastrointestinal parasite found in small ruminants, has a great ability to develop resistance to anthelmintic drugs. We studied the biotransformation of the three benzimidazole anthelmintics: albendazole (ABZ), ricobendazole (albendazole S-oxide; RCB) and flubendazole (FLU) in females and males of H. contortus in both a susceptible ISE strain and resistant IRE strain. The ex vivo cultivation of living nematodes in culture medium with or without the anthelmintics was used. Ultrasensitive UHPLC/MS/MS analysis revealed 9, 7 and 12 metabolites of ABZ, RCB and FLU, respectively, with most of these metabolites now described in the present study for the first time in H. contortus. The structure of certain metabolites shows the presence of biotransformation reactions not previously reported in nematodes. There were significant qualitative and semi-quantitative differences in the metabolites formed by male and female worms. In most cases, females metabolized drugs more extensively than males. Adults of the IRE strain were able to form many more metabolites of all the drugs than adults of the ISE strain. Some metabolites were even found only in adults of the IRE strain. These findings suggest that increased drug metabolism may play a role in resistance to benzimidazole drugs in H. contortus.

Biotransformation of flubendazole and fenbendazole and their effects in the ribwort plantain (Plantago lanceolata).

Although veterinary anthelmintics represent an important source of environmental pollution, the fate of anthelmintics and their effects in plants has not yet been studied sufficiently. The aim of our work was to identify metabolic pathways of the two benzimidazole anthelmintics fenbendazole (FBZ) and flubendazole (FLU) in the ribwort plantain (Plantago lanceolata L.). Plants cultivated as in vitro regenerants were used for this purpose. The effects of anthelmintics and their biotransformation products on plant oxidative stress parameters were also studied. The obtained results showed that the enzymatic system of the ribwort plantain was able to uptake FLU and FBZ, translocate them in leaves and transform them into several metabolites, particularly glycosides. Overall, 12 FLU and 22 FBZ metabolites were identified in the root, leaf base and leaf top of the plant. Concerning the effects of FLU and FBZ, both anthelmintics in the ribwort plantain cells caused significant increase of proline concentration (up to twice), a well-known stress marker, and significant decrease of superoxide dismutase activity (by 50%). In addition, the activities of four other antioxidant enzymes were significantly changed after either FLU or FBZ exposition. This could indicate a certain risk of oxidative damage in plants influenced by anthelmintics, particularly when they are under other stress conditions.

A Liquid Chromatography - Tandem Mass Spectrometry Approach for the Identification of Mebendazole Residue in Pork, Chicken, and Horse.

A confirmatory and quantitative method of liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the determination of mebendazole and its hydrolyzed and reduced metabolites in pork, chicken, and horse muscles was developed and validated in this study. Anthelmintic compounds were extracted with ethyl acetate after sample mixture was made alkaline followed by liquid chromatographic separation using a reversed phase C18 column. Gradient elution was performed with a mobile phase consisting of water containing 10 mM ammonium formate and methanol. This confirmatory method was validated according to EU requirements. Evaluated validation parameters included specificity, accuracy, precision (repeatability and within-laboratory reproducibility), analytical limits (decision limit and detection limit), and applicability. Most parameters were proved to be conforming to the EU requirements. The decision limit (CCα) and detection capability (CCß) for all analytes ranged from 15.84 to 17.96 µgkg-1. The limit of detection (LOD) and the limit of quantification (LOQ) for all analytes were 0.07 µgkg-1 and 0.2 µgkg-1, respectively. The developed method was successfully applied to monitoring samples collected from the markets in major cities and proven great potential to be used as a regulatory tool to determine mebendazole residues in animal based foods.

Species differences in hepatic biotransformation of the anthelmintic drug flubendazole.

Flubendazole (FLBZ) is a broad-spectrum benzimidazole anthelmintic used in pigs, poultry, and humans. It has been proposed as a candidate for development for use in elimination programmes for lymphatic filariasis and onchocerciasis in humans. Moreover, FLBZ has shown promise in cancer chemotherapy, particularly for neuroblastoma. This work investigated the hepatic carbonyl-reducing pathway of FLBZ in different species, including humans. Microsomal and cytosolic fractions were obtained from sheep, cattle, pig, hen, rat, and human liver. Both subcellular fractions of each species converted FLBZ into a reduced metabolite (red-FLBZ). The rate of microsomal red-FLBZ production was highest in sheep (1.92 ± 0.13 nmol/min.mg) and lowest in pigs (0.04 ± 0.02 nmol/min.mg); cytosolic red-FLBZ production ranged from 0.02 ± 0.01 (pig) to 1.86 ± 0.61 nmol/min.mg (sheep). Only subcellular fractions from sheep liver oxidized red-FLBZ to FLBZ in a NADP+ -dependent oxidative reaction. Liver microsomes from both pigs and humans transformed FLBZ to red-FLBZ and a hydrolyzed metabolite. Very significant differences in the pattern of FLBZ metabolism were observed among the tested species and humans. These results reinforce the need for caution in extrapolating data on metabolism, efficacy, and safety of drugs derived from studies performed in different species.

Metabolic pathways of benzimidazole anthelmintics in harebell (Campanula rotundifolia).

Benzimidazoles anthelmintics, which enter into environment primarily through excretion in the feces or urine of treated animals, can affect various organisms and disrupt ecosystem balance. The present study was designed to test the phytotoxicity and biotransformation of the three benzimidazole anthelmintics albendazole (ABZ), fenbendazole (FBZ) and flubendazole (FLU) in the harebell (Campanula rotundifolia). This meadow plant commonly grows in pastures and comes into contact with anthelmintics through the excrements of treated animals. Suspensions of harebell cells in culture medium were used as an in vitro model system. ABZ, FLU and FBZ were not found to be toxic for harebell cells, which were able to metabolize ABZ, FLU and FBZ via the formation of a wide scale of metabolites. Ultrahigh-performance liquid chromatography coupled with high mass accuracy tandem mass spectrometry (UHPLC-MS/MS) led to the identification of 24, 18 and 29 metabolites of ABZ, FLU and FBZ, respectively. Several novel metabolites were identified for the first time. Based on the obtained results, the schemes of the metabolic pathways of these anthelmintics were proposed. Most of these metabolites can be considered deactivation products, but a substantial portion of them may readily be decomposed to biologically active substances which could negatively affect ecosystems.

Analysis of mebendazole binding to its target biomolecule by laser flash photolysis.

Mebendazole (MBZ) and related anticancer benzimidazoles act binding the ß-subunit of Tubulin (TU) before dimerization with α-TU with subsequent blocking microtubule formation. Laser flash photolysis (LFP) is a new tool to investigate drug-albumin interactions and to determine binding parameters such as affinity constant or population of binding sites. The aim of this study was to evaluate the interactions between the nonfluorescent mebendazole (MBZ) and its target biomolecule TU using this technique. Before analyzing the MBZ@TU complex it was needed to determine the photophysical properties of MBZ triplet excited state ((3)MBZ(⁎)) in different media. Hence, (3)MBZ(⁎) showed a transient absorption spectrum with maxima at 520 and 375 nm and a lifetime much longer in acetonitrile (12.5 µs) than in water (260 ns). The binding of MBZ to TU produces a greater increase of the lifetime of (3)MBZ(⁎) (25 µs). This fact and the strong electron acceptor capability observed for (3)MBZ* evidence that MBZ must not be located close to any electron donor amino acid of TU such as its tryptophan or cysteine residues. Adding increasing amounts of MBZ to aqueous TU was determined the MBZ-TU binding constant (2.0 ± 0.5 × 10(5)M(-1) at 298K) which decreased with increasing temperature. The LFP technique has proven to be a powerful tool to analyze the binding of drug-TU systems when the drug has a detectable triplet excited state. Results indicate that LFP could be the technique of choice to study the interactions of non-fluorescent drugs with their target biomolecules.

Genotoxicity of flubendazole and its metabolites in vitro and the impact of a new formulation on in vivo aneugenicity.

The anti-parasitic benzimidazole flubendazole has been used for many years to treat intestinal infections in humans and animals. Previous genotoxicity studies have shown that the compound is not a bacterial mutagen and a bone marrow micronucleus test, using a formulation that limited systemic absorption, was negative. The purpose of this study is to explore the genotoxicity of flubendazole and its main metabolites in in vitro micronucleus studies and to test a new oral formulation that improves systemic absorption in an in vivo micronucleus test. The isolated metabolites were also screened using the Ames test for bacterial mutagenicity. It was found that flubendazole, like other chemically related benzimidazoles used in anti-parasitic therapies, is a potent aneugen in vitro The hydrolysed metabolite of flubendazole is negative in these tests, but the reduced metabolite (R- and S-forms) shows both aneugenic and clastogenic activity. However, in vitro micronucleus tests of flubendazole in the presence of rat liver S9 gave almost identical signals for aneugenicity as they did in the absence of S9, suggesting that any clastogenicity from the reduced metabolite is not sufficient to change the overall profile. Like flubendazole itself, both metabolites are negative in the Ames test. Analysis of dose-response curves from the in vitro tests, using recently developed point of departure approaches, demonstrate that the aneugenic potency of flubendazole is very similar to related anti-parasitic benzimidazoles, including albendazole, which is used in mass drug administration programmes to combat endemic filarial diseases. The in vivo micronucleus test of the new formulation of flubendazole also showed evidence of induced aneugenicity. Analysis of the in vivo data allowed a reference dose for aneugenicity to be established which can be compared with therapeutic exposures of flubendazole when this has been established. Analysis of the plasma from the animals used in the in vivo micronucleus test showed that there is increased exposure to flubendazole compared with previously tested formulations, as well as significant formation of the non-genotoxic hydrolysed metabolite of flubendazole and small levels of the reduced metabolite. In conclusion, this study shows that flubendazole is a potent aneugen in vitro with similar potency to chemically related benzimidazoles currently used as anti-parasitic therapies. The reduced metabolite also has aneugenic properties as well as clastogenic properties. Treatment with a new formulation of flubendazole that allows increased systemic exposure, compared with previously used formulations, also results in detectable aneugenicity in vivo. Based on the lack of carcinogenicity of this class of benzimidazoles and the intended short-term dosing, it is unlikely that flubendazole treatment will pose a carcinogenic risk to patients.

Metabolism of drugs and other xenobiotics in giant liver fluke (Fascioloides magna).

1. Giant liver fluke Fascioloides magna is a dangerous parasite, which infects herbivores. It was imported to Europe from North America and started to spread. Benzimidazoles like albendazole, mebendazole, triclabendazole and salicylanilides closantel and rafoxanide are the most used anthelmintics to control fascioloidosis. However their effect might be altered via drug-metabolizing enzymes of this parasite. 2. The aim of our study was to determine the activities of drug-metabolizing enzymes in F. magna and the metabolism of above mentioned anthelmintics. 3. Activities of several oxidative, reductive and conjugative enzymes towards various model xenobiotic substrates were found in F. magna subcellular fractions. 4. Subcellular fractions from F. magna oxidized albendazole to its sulphoxide metabolite and reduced mebendazole to hydroxyl-mebendazole. Under ex vivo conditions, only very-low concentrations of these compounds were detected using high-performance liquid chromatography/mass spectrometry. 5. The results indicate that the giant liver fluke possesses the active xenobiotic-metabolizing system. The overexpression of this system may play an important role in parasite resistance against these anthelmintics.

Enfoque diagnóstico y terapéutico de la eosinofilia. A propósito de un caso / Diagnosis and treatment of eosinophilia. A case report

La eosinofilia es el aumento del número total de eosinófilos por encima de 500/μl. En la edad pediátrica la causa más frecuente es la parasitosis por helmintos; otras causas frecuentes son la ingesta de algunos fármacos y las enfermedades alérgicas. Presentamos el caso de una niña de cinco años con dolor abdominal y analítica con eosinofilia moderada en repetidas ocasiones, sin causa secundaria aparente y con normalidad en el resto de las pruebas complementarias realizadas en el centro de salud y en el hospital de referencia. Ante una eosinofilia persistente sin causa secundaria que la explique y con normalidad de las pruebas complementarias indicadas, se recomienda tratamiento empírico con antiparasitarios (AU)

Eosinophilia is the increased number of eosinophils above 500/μl. The most common cause in pediatric patients is parasitic worm disease; other common causes are ingestion of some drugs and allergic diseases. We present the case of a five year old girl with abdominal pain and moderate eosinophilia in repeated ocasions, without apparent secondary cause, and with normality in other complementary tests in the health center and in the reference hospital. In case of persistent eosinophilia without secondary causes and normal complementary tests, empiric antiparasitic therapy is recommended (AU)

Biotransformation of benzimidazole anthelmintics in reed (Phragmites australis) as a potential tool for their detoxification in environment.

Benzimidazole anthelmintics, the drugs against parasitic worms, are widely used in human as well as veterinary medicine. Following excretion, these substances may persist in the environment and impact non-target organisms. In order to test phytoremediation as a possible tool for detoxification of anthelmintics in environment, the biotransformation pathways of albendazole (ABZ) and flubendazole (FLU) were studied in reed (Phragmites australis) in vitro. Reed cells were able to uptake and biotransform both anthelmintics. Ten ABZ metabolites and five FLU metabolites were found. Some atypical biotransformation reactions (formation of glucosylglucosides, acetylglucosides and xylosylglucosides), which have not been described previously, were identified. Based on the obtained results, the schemes of metabolic pathways of ABZ and FLU in reed were proposed. Most of ABZ and FLU metabolites can be considered as anthelmintically less active; therefore uptake and biotransformation of these anthelmintics by reed could be useful for decrease of their toxicity in environment.

A pharmacology-based comparison of the activity of albendazole and flubendazole against Echinococcus granulosus metacestode in sheep.

Cyst echinococcosis (CE) is a zoonotic disease caused by the larval stage of the Echinococcus granulosus helminth parasite. The work reported here aimed to compare the efficacy of albendazole (ABZ) and flubendazole (FLBZ) against CE in naturally infected sheep. Additionally, their comparative pharmacokinetic behaviour and the assessment of serum liver enzymes activities were studied. Twelve (12) naturally infected sheep were allocated to the following experimental groups: unmedicated control group, FLBZ-treated and ABZ-treated. Treatments were orally performed every 48 h, over 55 days at dose rate of 10 (FLBZ) and 8.5 (ABZ) mg/kg (equimolar dose rates). The efficacy of the drug treatments was based on protoscoleces' vitality/viability. The kinetic disposition assessment included the Initial and Final Kinetic Studies which implicated the collection of blood samples after both the first and the last drug administration. Blood samples were processed to measure drug concentrations by HPLC. The protoscoleces' vitality observed in the untreated control group (98%) was significantly reduced in the presence of both ABZ and FLBZ. 90% of mice inoculated with protoscoleces in the control group developed hydatid cysts in their peritoneal cavity (viability study). However, only 25% (FLBZ) and 33% (ABZ) of mice inoculated with protoscoleces recovered from treated sheep, developed hydatid cysts in their abdominal cavity. Reduced FLBZ (R-FLBZ) was the main metabolite recovered in the bloodstream after oral administration of FLBZ to sheep. Low plasma concentrations of FLBZ parent drug were measured up to 48 h post-administration. ABZ was not detected in plasma at any time post-treatment, being its metabolites ABZ sulphoxide (ABZSO) and ABZ sulphone (ABZSO2) recovered in plasma. Hepatotoxicity due to the continued treatment with either ABZ or FLBZ was not observed. A 3-fold increase ethoxyresorufin O-deethylase activity, a cytochrome P450 1A (CYP1A)-dependent enzyme reaction, was observed in liver microsomes obtained from sheep receiving ABZ, compared to those of the unmedicated and FLBZ-treated animals. In conclusion, FLBZ is an available anthelmintic which may be developed into an effective and safe drug for the human CE treatment. Despite the low plasma concentrations measured by FLBZ/R-FLBZ, an important reduction in protoscoleces' vitality was observed in cysts located in sheep liver. Modern pharmaceutical technology may help to greatly improve FLBZ systemic exposure improving its efficacy against CE.

Effects of the benzimidazole anthelmintic drug flubendazole on rat embryos in vitro.

Filarial diseases affect millions of people in poverty-stricken areas. In 2011, an investigation of the potential of flubendazole as a safe, highly efficacious, and field-usable macrofilaricidal drug was begun by Drug for Neglected Diseases initiative. As part of the preclinical development program, whole embryo culture was used to investigate the potential embryotoxicity of flubendazole and its metabolites, reduced and hydrolyzed flubendazole. Albendazole was included as a comparator. Flubendazole and albendazole showed similar potency in affecting rat embryonic development in vitro, inducing retardation of growth and dysmorphogenic effects at concentrations ≥0.5 µg/mL. The head, optic and otic systems, branchial arches and posterior body portion were affected. Diffuse areas of cell death were seen in various embryonic districts. The No Observed Effect Level (NOEL) was 0.25 µg/mL for both drugs. Reduced and hydrolyzed flubendazole were less embryotoxic than the parent compound, with NOELs 4-fold and >40-fold higher than that of flubendazole, respectively.

Nematodicidal activity of flubendazole and its reduced metabolite on a murine model of Trichinella spiralis infection.

BACKGROUND: Flubendazole (FLBZ) is a broad-spectrum benzimidazole anthelmintic compound. The parent FLBZ is metabolized to its reduced (R-FLBZ) and hydrolyzed (H-FLBZ) metabolites. There are no data on the potential nematodicidal activity of R-FLBZ, the main plasma metabolite found in sheep and mice. The goal of the current work was to assess the efficacy of FLBZ and R-FLBZ against Trichinella spiralis in a mouse model. METHODS: Both compounds were administered to Balb/c mice infected with T. spiralis as either a cyclodextrin aqueous solution or as a carboxymethylcellulose suspension. Treatments were performed orally (5 mg/kg) at 1 day after infection with T. spiralis. The efficacy of the treatments was assessed at day 6 after infection. RESULTS: While the efficacy obtained for FLBZ and R-FLBZ administered as a solution was 94 and 98%, respectively, the efficacies obtained after the treatment with FLBZ suspensions were 38% (FLBZ) and 64% (R-FLBZ). CONCLUSION: Under the current experimental conditions, a high nematodicidal efficacy of both FLBZ and R-FLBZ administered as solution preparations was observed.

The metabolism of flubendazole and the activities of selected biotransformation enzymes in Haemonchus contortus strains susceptible and resistant to anthelmintics.

Haemonchus contortus is one of the most pathogenic parasites of small ruminants (e.g. sheep and goat). The treatment of haemonchosis is complicated because of recurrent resistance of H. contortus to common anthelmintics. The aim of this study was to compare the metabolism of the anthelmintic drug flubendazole (FLU) and the activities of selected biotransformation enzymes towards model xenobiotics in 4 different strains of H. contortus: the ISE strain (susceptible to common anthelmintics), ISE-S (resistant to ivermectin), the BR strain (resistant to benzimidazole anthelmintics) and the WR strain (resistant to all common anthelmintics). H. contortus adults were collected from the abomasums from experimentally infected lambs. The in vitro as well as ex vivo experiments were performed and analysed using HPLC with spectrofluorimetric and mass-spectrometric detection. In all H. contortus strains, 4 different FLU metabolites were detected: FLU with a reduced carbonyl group (FLU-R), glucose conjugate of FLU-R and 2 glucose conjugates of FLU. In the resistant strains, the ex vivo formation of all FLU metabolites was significantly higher than in the susceptible ISE strain. The multi-resistant WR strain formed approximately 5 times more conjugates of FLU than the susceptible ISE strain. The in vitro data also showed significant differences in FLU metabolism, in the activities of UDP-glucosyltransferase and several carbonyl-reducing enzymes between the susceptible and resistant H. contortus strains. The altered activities of certain detoxifying enzymes might protect the parasites against the toxic effect of the drugs as well as contribute to drug-resistance in these parasites.

Transfer of flubendazole and tylosin at cross contamination levels in the feed to egg matrices and distribution between egg yolk and egg white.

Chemical residues may be present in eggs from laying hens' exposure to drugs or contaminants. These residues may pose risks to human health. In this study, laying hens received experimental feed containing flubendazole or tylosin at cross contamination levels of 2.5, 5, and 10% of the therapeutic dose. Eggs were collected daily and analysis of the whole egg, egg white, and egg yolk was performed using liquid chromatography tandem mass spectrometry. Highest concentrations of the parent molecule flubendazole, as well as the hydrolyzed and the reduced metabolite, were detected in egg yolk. Residue concentrations of the parent molecule were higher compared with those of the metabolites in all egg matrices. No tylosin residue concentrations were detected above the limit of quantification for all concentration groups and in all egg matrices. Neither molecule exceeded the set maximum residue limits.

Import and efflux of flubendazole in Haemonchus contortus strains susceptible and resistant to anthelmintics.

Drug entry into the body of a helminth is a key factor in the efficacy of anthelmintics. The present project was designed to study the ex vivo uptake and efflux of the benzimidazole anthelmintic flubendazole (FLU) in four strains of H. contortus: the ISE strain (fully susceptible to anthelmintics), the ISE-S strain (resistant to ivermectin), the BR strain (resistant to benzimidazoles) and the WR strain (multi-resistant). The transport of FLU between dead and living nematodes was also compared as well as the effect of verapamil, an inhibitor of the main efflux ABCB1 transporter (P-glycoprotein), on FLU accumulation in nematodes. The obtained results showed that FLU is able to effectively enter H. contortus adults due to high FLU lipophilicity. Passive diffusion is probably the only mechanism in both FLU import and efflux from nematodes. No differences in FLU transport were found among four H. contortus strains with different sensitivity to anthelmintics. No active FLU efflux from H. contortus and no effect of verapamil were observed, indicating that H. contortus cannot protect itself against FLU by the active removal of this anthelmintic from its body.

The activity of drug-metabolizing enzymes and the biotransformation of selected anthelmintics in the model tapeworm Hymenolepis diminuta.

The drug-metabolizing enzymes of some helminths can deactivate anthelmintics and therefore partially protect helminths against these drugs' toxic effect. The aim of our study was to assess the activity of the main drug-metabolizing enzymes and evaluate the metabolism of selected anthelmintics (albendazole, flubendazole, mebendazole) in the rat tapeworm Hymenolepis diminuta, a species often used as a model tapeworm. In vitro and ex vivo experiments were performed. Metabolites of the anthelmintics were detected and identified by HPLC with spectrofluorometric or mass-spectrometric detection. The enzymes of H. diminuta are able to reduce the carbonyl group of flubendazole, mebendazole and several other xenobiotics. Although the activity of a number of oxidation enzymes was determined, no oxidative metabolites of albendazole were detected. Regarding conjugation enzymes, a high activity of glutathione S-transferase was observed. A methyl derivative of reduced flubendazole was the only conjugation metabolite identified in ex vivo incubations of H. diminuta with anthelmintics. The results revealed that H. diminuta metabolized flubendazole and mebendazole, but not albendazole. The biotransformation pathways found in H. diminuta differ from those described in Moniezia expanza and suggest the interspecies differences in drug metabolism not only among classes of helminths, but even among tapeworms.