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.     

Albendazole sulphoxide concentrations in plasma of endemic normals from a lymphatic filariasis endemic region using liquid chromatography

A simple and sensitive reversed-phase isocratic HPLC method for the determination of albendazole and its metabolites has been developed. The mobile phase consisting of acetonitrile-water-perchloric acid (70%) (30:110:0.06 (v/v/v)) was pumped at a flow rate of 0.80 ml/min on a 5 microm, reverse phase, Discovery RPamide C16 column with UV detection at 290 nm. The calibration graphs were linear in the range of 0.05- 1 microg/ml for albendazole, albendazole sulphoxide and albendazole sulphone. The limit of quantification was 50 ng/ml for albendazole, 25 ng/ml for albendazole sulphoxide and 30 ng/ml for albendazole sulphone. The within-day and day-to-day coefficient of variation averaged 4.98 and 6.95% for albendazole, 3.83 and 6.83% for albendazole sulphoxide and 3.44 and 5.51% for albendazole sulphone, respectively. The mean extraction recoveries of albendazole, albendazole sulphoxide and albendazole sulphone were 79.25, 93.03 and 88.78%, respectively. The method was applied to determine the plasma levels of albendazole sulphoxide in endemic normals administered with albendazole during pharmacokinetic studies.     

Drug transport mechanisms in helminth parasites: passive diffusion of benzimidazole anthelmintics

Anthelmintic molecules must reach their receptors inside target parasites to exert the pharmacological effect. Available data suggest that the main route of entry of antiparasitic drugs into helminth parasites would be through their external surface. However, it is unclear if trans-tegumental/cuticular penetration is the most important way of entry of benzimidazole (BZD) anthelmintics into their target parasites compared to oral ingestion. The relative involvement of active and passive transport mechanisms has not been defined. The goal of the work reported here was to determine the main processes involved in the entry of BZD anthelmintic molecules into the three main classes of helminth parasites. Adult specimens of Moniezia benedeni (cestode), Fasciola hepatica (trematode) and Ascaris suum (nematode) were incubated in Kreb's Ringer Tris buffer (pH 7.4, 37 degrees C) (1g parasite/10 ml incubation medium) for 15, 45, and 90 min, respectively, in the presence of a concentration gradient of either fenbendazole (FBZ), oxfendazole or triclabendazole sulphoxide (TCBZSO) (1-30 mol/ml, n=4). Dead helminth specimens were also incubated with the same drug concentration gradient. Specimens of F. hepatica with the oral route closed off by ligation were incubated with TCBZSO in the presence or absence of bovine serum albumin. After the incubation time elapsed, samples of parasite material were chemically extracted and prepared for high performance liquid chromatography analysis to measure drug/metabolite concentrations. Equivalent drug concentrations were measured within ligated and non-ligated liver flukes, demonstrating that BZD do mainly penetrate by trans-tegumental diffusion. The higher the concentration of BZD molecules in the incubation medium, the greater their concentration recovered within the helminth parasites. High correlation coefficients (>0.98) were obtained between initial drug concentration in the incubation medium and those measured inside the nematode, cestode, and trematode parasites. FBZ concentrations recovered from tissues of dead cestodes/nematodes over time were significantly greater compared to those measured in living parasites. These differences in drug diffusion may be related to the morphological/functional properties of the parasite's external surfaces. The outcome of the work reported here indicates that passive drug transfer through the external helminth surface is the main transport mechanism accounting for BZD accumulation into target parasites.     

Development of a liquid chromatography-tandem mass spectrometry with pressurized liquid extraction method for the determination of benzimidazole residues in edible tissues

A confirmatory and quantitative method of liquid chromatography-tandem mass spectrometry (LC-MS/MS) combined with a pressure liquid extraction (PLE) was developed for the determination of 11 benzimidazole and 10 metabolites of albendazole, fenbendazole and mebendazole in the muscles and livers of swine, cattle, sheep and chicken. For sample preparation, we used an automated technique of PLE method. The optimum extraction conditions were obtained using an 11 ml Accelerated Solvent Extraction (ASE) cells, acetonitrile/hexane as the extraction solvent. HPLC analysis was performed on a C18 column with gradient elution using acetonitrile and 5 mmol l(-1) formic ammonium as mobile phase. The analytes were detected in the positive ion multiple reaction monitoring (MRM) mode by the LC-ESI-MS/MS analysis. The recoveries of benzimidazole (BZDs) spiked at the levels of 0.5 μg kg(-1) ranged from 70.1% to 92.7%; the between-day relative standard deviations were no more than 10%. The limits of quantification were 0.02-0.5 μg kg(-1). The optimized method was successfully applied to monitor real samples containing BZDs, demonstrating the method to be simple, fast, robust and suitable for identification and quantification of BZDs residues in animal products.     

Reversed-phase liquid chromatographic method with fluorescence detection for the simultaneous determination of albendazole sulphoxide, albendazole sulphone and albendazole 2-aminosulphone in sheep plasma

A rapid and sensitive HPLC method for the simultaneous quantification of albendazole sulphoxide (ABZ-SO), albendazole sulphone (ABZ-SO2) and albendazole 2-aminosulphone (ABZ-SO2NH2) in sheep blood plasma has been developed. Plasma samples were extracted with ethyl acetate under alkaline conditions. Separation was achieved on a C18 reversed-phase analytical column, in the presence of positively- (tetra-n-butylammonium hydrogen sulphate) and negatively-charged (octanesulphonate sodium) pairing ions, while detection was performed fluorometrically. Excitation and emission wavelengths were 290 and 320 nm, respectively. Limits of quantification were defined at 39 ng/ml for ABZ-SO, 4.95 ng/ml for ABZ-SO2 and 4 ng/ml for ABZ-SO2NH2. Accuracy data, in terms of recovery efficiency showed overall values (+/- S.E.M.) of 85.6 +/- 1.0% for ABZ-SO, 100.0 +/- 1.0% for ABZ-SO2 and 89.1 +/- 0.6% for ABZ-SO2NH2. The method was successfully applied to quantitatively determine the three albendazole metabolites in plasma samples collected from sheep that had been orally administered albendazole.     

Development of a high-performance liquid chromatography method to monitor the residues of benzimidazoles in bovine milk

A reversed-phase high-performance liquid chromatography with ultraviolet (UV) detection was developed that can determine 11 benzimidazole (BZDs) and 10 metabolites of albendazole, fenbendazole and mebendazole in bovine milk. Samples were extracted with acetonitrile and purified by mixed-mode cation exchange (MCX) solid phase extraction cartridges. LC separations were performed on a C₁₈ column with gradient elution using acetonitrile and ammonium acetate solution. The UV-detection was set at 292 nm. The method is very sensitive to each analyte with limits of quantification (LOQs) of lower than 10 μg kg⁻¹. The recoveries of the BZDs and their metabolites spiked in milk were more than 78% with between-day relative standard deviation values less than 16% at the concentration of 10, 50 and 100 μg kg⁻¹. The method developed has been successfully applied to monitoring real samples containing BZDs, which demonstrated that it is a simple, fast and robust method, and could be used as a regulatory toll to determine the residues of BZDs in milk.     

Speckle interferometry applied to pharmacodynamic studies: evaluation of parasite motility

The work reported here describes the application of the optical technique known as dynamic speckle interferometry to evaluate the motility of nematode parasites exposed to different anthelmintic drugs. This technique, a well proven tool for assessing the time evolution of different phenomena, is here successfully used to quantify parasite motility in pharmacodynamic assays. The characterization of the pharmacological properties of anthelmintic drugs is critical to optimize their use in parasite control. Besides, the evaluation of nematode motility is a relevant indicator of the pharmacodynamic effect of anthelmintic drugs. The application of this approach to study the motility of Haemonchus contortus (used as a model of nematode parasites) larvae exposed to different drugs is presented, showing its usefulness.Abbreviations ABZ albendazole - BZD benzimidazole - IVM ivermectin - LVM levamisole     

Determination of benzimidazole residues using liquid chromatography and tandem mass spectrometry

The determination of residues of benzimidazole using liquid chromatography and tandem mass spectrometry (LC–MS–MS) with ion spray ionization is described. Swine muscle tissue was spiked with a mixture of fifteen benzimidazoles, including metabolites of fenbendazole and albendazole. As clean-up procedure, an ethyl acetate extraction followed by solid-phase extraction on styrol-divinyl-benzene cartridge was used. The evaluation was performed by selecting the characteristic product ions for the benzimidazoles and using multiple reaction mode. 2-n-Butylmercaptobenzimidazole was used as internal standard. Blank muscle samples were fortified in the concentration range of 1–22 μg/kg. The limits of detection were below 6 μg/kg and the limits of quantification for most benzimidazoles were below 10 μg/kg. The matrix effect was checked using spiked muscle tissues of cattle and sheep as well as liver of cattle. Practical application will be shown by incurred egg material from laying hens treated with flubendazole. The recovery of the clean-up was mostly above 50% in muscle tissue and 70% in egg yolk.     

Trichuris suis and Oesophagostomum dentatum Show Different Sensitivity and Accumulation of Fenbendazole,Albendazole and Levamisole In Vitro

Background

The single-dose benzimidazoles used against Trichuris trichiura infections in humans are not satisfactory. Likewise, the benzimidazole, fenbendazole, has varied efficacy against Trichuris suis whereas Oesophagostomum dentatum is highly sensitive to the drug. The reasons for low treatment efficacy of Trichuris spp. infections are not known.

Methodology

We studied the effect of fenbendazole, albendazole and levamisole on the motility of T. suis and O. dentatum and measured concentrations of the parent drug compounds and metabolites of the benzimidazoles within worms in vitro. The motility and concentrations of drug compounds within worms were compared between species and the maximum specific binding capacity (B_max) of T. suis and O. dentatum towards the benzimidazoles was estimated. Comparisons of drug uptake in living and killed worms were made for both species.

Principal findings

The motility of T. suis was generally less decreased than the motility of O. dentatum when incubated in benzimidazoles, but was more decreased when incubated in levamisole. The B_max were significantly lower for T. suis (106.6, and 612.7 pmol/mg dry worm tissue) than O. dentatum (395.2, 958.1 pmol/mg dry worm tissue) when incubated for 72 hours in fenbendazole and albendazole respectively. The total drug concentrations (pmol/mg dry worm tissue) were significantly lower within T. suis than O. dentatum whether killed or alive when incubated in all tested drugs (except in living worms exposed to fenbendazole). Relatively high proportions of the anthelmintic inactive metabolite fenbendazole sulphone was measured within T. suis (6–17.2%) as compared to O. dentatum (0.8–0.9%).

Conclusion/Significance

The general lower sensitivity of T. suis towards BZs in vitro seems to be related to a lower drug uptake. Furthermore, the relatively high occurrence of fenbendazole sulphone suggests a higher detoxifying capacity of T. suis as compared to O. dentatum.     

Treatment of Toxocara canis infections in mice with liposome-incorporated benzimidazole carbamates and immunomodulator glucan

Benzimidazole carbamates (mebendazole, albendazole and fenbendazole) are the most commonly used anthelmintic drugs for the treatment of larval toxocariasis (Toxocara canis) in paratenic hosts. However, the bioavailability of these drugs for tissues is very low due to their extremely low solubility, resulting in the administration of relatively high doses over a long period. To overcome this problem, neutral, negatively or positively charged and stabilized liposome drug carriers were examined in the chronic phase of T. canis infections in mice each orally inoculated with 1000 eggs. Moreover, liposomized albendazole and fenbendazole were co-administered with liposomized immunomodulator glucan. The highest efficacy of both drugs, evaluated 4 weeks after treatment, was recorded after their subcutaneous administration (ten doses of 25 mg kg(-1)) in stabilized liposomes and intramuscular co-administration of liposomized glucan (two doses of 5 mg kg(-1)). Fenbendazole was more effective in muscles (91.5%) whereas albendazole was more effective in the brain (92.2%). Liposomes with incorporated benzimidazole carbamate anthelmintics provide sustained drug-release reservoirs and can considerably enhance drug efficacy. Moreover, despite suppression by T. canis antigens, stimulation of the immune system by the immunomodulator glucan potentiates the effects of these antiparasitic drugs.     

Further investigation of the primary mechanism of benzimidazole resistance in Haemonchus contortus

The binding of the [³H] benzimidazole carbamates (BZCs)—albendazole (ABZ), oxibendazole (OBZ), parbendazole (PBZ), mebendazole (MBZ), fenbendazole (FBZ) and oxfendazole (OFZ)—to tubulin from three ecologically-related isolates of adult Haemonchus contortus has been examined. The extent of binding of each BZC was inversely proportional to the known resistance status of the isolate. Biochemically, the change in the formation of the BZC-tubulin complex was due to a reduction in the amount of drug bound to resistant tubulin, with no significant change in the association constant of the complex. The resistance factors derived from the binding data support the hypothesis that the complex is ligand-dependent, with the aryl-substituted BZCs—MBZ, OFZ and FBZ—demonstrating lower resistance factors than those of the alkyl-substituted BZCs—ABZ, OBZ and PBZ. Examination of the slope derived from plots of binding against protein concentration demonstrated that the failure of resistant or partially resistant isolates to bind was due to either a decrease in the number of binding sites or, more likely, to reduced stability of the BZC-tubulin complex rendering it unstable to charcoal extraction.     

Efficacy of mebendazole and albendazole against Trichinella spiralis in mice

Changes in the sensitivity of Trichinella spiralis to anthelmintic treatment during the first 3 days of infection in mice were studied. Oral administration of either mebendazole or albendazole at 6.25 mh/kg 2 hr after exposure to infection eliminated 95-100% of the worms as determined at necropsy on day 7 postinoculation. Beyond the first day of infection the sensitivity of the parasite to benzimidazole therapy was much reduced and an oral dose of 50 mg/kg was only partially but significantly active against the adult worms. Despite decline in drug sensitivity during the enteral phase, gavage administration of either mebendazole or albendazole at 50 mg/kg for 5 consecutive days during the invasive phase of infection significantly reduced (96 and 67%, respectively) the number of larvae subsequently recovered from host musculature on day 45 postinoculation.     

The effect of an oil formulation on the systemic availability of oxfendazole.

Pure oxfendazole (OFZ) suspended in peanut oil and the commercial formulation 'Synanthic' were each intraruminally administered to Merino weaners at 5 mg kg-1. Plasma was collected and concentrations of OFZ, fenbendazole (FBZ) and FBZ sulphone (FBZ.SO2) were determined by HPLC. The maximum concentrations and area under the plasma concentration curve (AUC) of OFZ and FBZ were significantly higher (P less than 0.05) when OFZ was suspended in peanut oil than when administered as the commercial formulation.     

Dose-dependent activity of albendazole against benzimidazole-resistant nematodes in sheep: relationship between pharmacokinetics and efficacy

The relationship between the pharmacokinetic behaviour and the anthelmintic efficacy of albendazole (ABZ) against benzimidazole (BZD)-resistant nematodes was studied in sheep. A micronized ABZ suspension was orally administered at two different dose levels to sheep naturally infected with BZD-resistant gastrointestinal (GI) nematodes. The experimental animals were allocated into the following groups (n = 8): (a) untreated control; (b) orally treated with ABZ at 3.8 mg/kg b.w.; and (c) orally treated with ABZ at 7.5 mg/kg b.w. Plasma samples were obtained serially over 72 h post-treatment from both treated groups and analysed by HPLC to measure the concentrations of ABZ and its sulphoxide (ABZSO) and sulphone (ABZSO(2)) metabolites. Faecal egg counts were performed prior to treatment and at the necropsy day. All experimental animals were sacrificed 10 days after treatment to perform GI worm counts. While ABZ parent drug was not recovered in the bloodstream, ABZSO and ABZSO(2) were the molecules found in plasma. ABZSO was the metabolite measured at the highest concentrations in the bloodstream for up to 36 (treatment at 3.8 mg/kg) or 60 h (treatment at 7.5 mg/kg) post-administration. There was a proportional relationship between the administered ABZ dose and the measured plasma concentrations of both ABZ metabolites. Over a 100% increment on the plasma AUC values for the anthelmintically active ABZSO metabolite was observed at the 7.5 mg/kg compared to the 3.8 mg/kg treatment. The low efficacy patterns (< 24%) observed against the GI nematodes investigated indicate a high level of resistance to ABZ given at 3.8 mg/kg an efficacious therapeutic dose rate recommended in some countries. However, the higher and prolonged plasma drug concentration measured after the 7.5 mg/kg treatment resulted in an improved efficacy pattern (estimated by both faecal egg and adult worm counts) against most of the GI nematodes studied compared to that obtained at the lower dose rate. A direct relationship between drug pharmacokinetic behaviour and anthelmintic efficacy against BZD-resistant nematodes in sheep was shown in the current work, although individual variation precluded the observation of statistically significant differences in worm counts.     

Further investigations of the primary mechanism of benzimidazole resistance in Haemonchus contortus

The binding of the [³H] benzimidazole carbamates (BZCs)—albendazole (ABZ), oxibendazole (OBZ), parbendazole (PBZ), mebendazole (MBZ), fenbendazole (FBZ) and oxfendazole (OFZ)—to tubulin from three ecologically-related isolates of adult Haemonchus contortus has been examined. The extent of binding of each BZC was inversely proportional to the known resistance status of the isolate. Biochemically, the change in the formation of the BZC-tubulin complex was due to a reduction in the amount of drug bound to resistant tubulin, with no significant change in the association constant of the complex. The resistance factors derived from the binding data support the hypothesis that the complex is ligand-dependent, with the aryl-substituted BZCs—MBZ, OFZ and FBZ—demonstrating lower resistance factors than those of the alkyl-substituted BZCs—ABZ, OBZ and PBZ. Examination of the slope derived from plots of binding against protein concentration demonstrated that the failure of resistant or partially resistant isolates to bind was due to either a decrease in the number of binding sites or, more likely, to reduced stability of the BZC-tubulin complex rendering it unstable to charcoal extraction.     

High-performance liquid chromatography with UV detection and diode-array UV confirmation of isonicotinic acid hydrazide in cattle milk

A method for the determination of isonicotinic acid hydrazide (isoniazid) in milk was developed. Milk was deproteinized with trichloroacetic acid. Isoniazid was condensed with cinnamaldehyde and assayed on a reversed-phase HPLC system, with good sensitivity and accuracy (10 μg/l) with UV detection at 330 nm. Use of solid-phase extraction with a C₁₈ cartridge allows the detection limit to be lowered to 0.1 μg/l with UV detection and confirmation of isoniazid hydrazone from the diode-array UV spectrum.     

Simultaneous determination of moricizine and its sulphoxidation metabolites in biological fluids by high-performance liquid chromatography

A simultaneous assay for moricizine, its two sulphoxidation metabolites, morizine sulphoxide and moricizine sulphone, using high-performance liquid chromatography (HPLC) is described. The drug and metabolites and clozapine (internal standard) in biological fluids were extracted using pentanesulphonic acid into diethyl ether. The ethereal extract was evaporated to dryness and the residue was redissolved in the mobile phase (methanol-water-triethylamine, 65:35:0.5, v/v). The analyses were performed on a μBondapak reversed-phase C₁₈ column housed in a Waters Z-module, linked to a C₁₈ pre-column, with a run-time of 12 min. The retention times were 2.7, 3.5, 6.2 and 9.7 min for moricizine sulphone, moricizine sulphoxide, moricizine and clozapine, respectively. The recovery of the compounds from plasma ranged from 89.9% for the sulphoxide to 98.1% for clozapine. The limits of detection of the assay for moricizine, moricizine sulphoxide and moricizine sulphone were 20, 10 and 5 ng/ml, respectively.     

Modified high-performance liquid chromatographic method for the determination of ertapenem in human urine: enhanced selectivity and automation

A column-switching, reversed-phase high-performance liquid chromatographic (HPLC) assay for a new structurally unique carbapenem antibiotic, ertapenem, in urine has been improved for selectivity and automated using a Packard MultiPROBE II EX pipetting station. The method uses column-switching for on-line extraction of the urine sample. The extraction column, analytical column, mobile phase, and timing of the column-switching valve have been changed to enhance selectivity for the analyte over endogenous background material. Sample transfer and dilution prior to direct-injection into the HPLC system have been accomplished using a Packard MultiPROBE II EX robotic liquid handling system.     

Determination of esomeprazole and its two main metabolites in human, rat and dog plasma by liquid chromatography with tandem mass spectrometry

A LC-MS/MS method was developed for quantitative determination of esomeprazole, and its two main metabolites 5-hydroxyesomeprazole and omeprazole sulphone in 25 microL human, rat or dog plasma. The analytes and their internal standards were extracted from plasma into methyl tert-butyl ether - dichloromethane (3:2, v/v). After evaporation and reconstitution of the organic extract the analytes were separated on a reversed-phase LC column and measured by atmospheric-pressure positive ionisation MS. The linearity range was 20-20,000 nmol/L for esomeprazole and omeprazole sulphone, and 20-4000 nmol/L for 5-hydroxyesomeprazole. The extraction recoveries ranged between 80 and 105%. The intra- and inter-day imprecision were less than 9.5% with accuracy between 97.7% and 100.1% for all analytes.     

Determination of deracoxib in feline plasma samples using high performance liquid chromatography

A new HPLC procedure for the determination of deracoxib, a selective cyclooxygenase-2 inhibitor, has been developed and validated. Following a liquid-liquid extraction using isopropyl alcohol and chloroform, samples were separated by isocratic reversed-phase HPLC on an Atlantis C18 column and quantified using UV detection at 252 nm. The mobile phase was a mixture of 10 mM potassium phosphate (pH 4.5) and acetonitrile, with a flow-rate of 1.0 ml/min. The procedure produced a linear curve over the concentration range 10-1500 ng/ml. The development of the assay allowed the determination of pharmacokinetic parameters after oral administration of deracoxib in cats and would be suitable for other pharmacokinetic studies.