Testing Albendazole Resistance in Fasciola hepatica.

The egg development test is a useful in vitro tool to detect albendazole (ABZ) resistance in Fasciola hepatica. ABZ is the only flukicidal compound with ovicidal activity. The described test is based on the ABZ capacity to affect parasite egg development and hatching in susceptible parasites, while this effect is lost in ABZ-resistant liver fluke isolates. Among many advantages, it is noted that the diagnostic test can be performed on eggs isolated from fecal samples (sheep and cattle), avoiding the sacrifice of animals necessary in controlled efficacy trials. The egg development test described here is a simple, inexpensive, and accessible method, previously employed for diagnosis of ABZ resistance in F. hepatica.

In vitro and in vivo effects of chlorpyrifos and cypermethrin on blood cholinesterases in sheep.

The combination of the organophosphate (OP) chlorpyrifos (CPF) and the pyrethroid cypermethrin (CPM) is commonly marketed as pour-on formulations for the control of sheep lice, ked, and blowflies. CPF irreversibly inhibits acetylcholinesterases (AChE), while pyrethroids are not AChE inhibitors. However, combinations of pyrethroids with OPs showed a highly synergistic effect on AChE inhibition. Thus, the aim of the current work was to evaluate in vitro and in vivo the inhibitory potency of both pesticides, alone and in combination with AChE and butyrylcholinesterase (BChE) activities in sheep blood. In vitro, IC50 values were similar after CPF or CPF plus CPM incubations. The pour-on coadministration of recommended doses of CPF and CPM did not cause a significant inhibition of AChE and BChE in sheep blood. Only slight percentages of inhibition of their catalytic activities were observed when both drugs were given at 4-fold higher dose rates. The lower systemic availability of topical administration of OPs in sheep may help to explain the lower degree of inhibition of blood AChE and BChE in vivo. The results emerged from this research are a further contribution to the knowledge of the risks of implementing higher dosage regimens of OPs-containing antiparasitic formulations.

Assessment of serum pharmacokinetics and urinary excretion of albendazole and its metabolites in human volunteers.

BACKGROUND: Soil Transmitted Helminth (STH) infections negatively impact physical and mental development in human populations. Current WHO guidelines recommend morbidity control of these infections through mass drug administration (MDA) using albendazole (ABZ) or mebendazole. Despite major reductions in STH associated morbidity globally, not all programs have demonstrated the expected impact on prevalence of parasite infections. These therapeutic failures may be related to poor programmatic coverage, suboptimal adherence or the exposure of parasites to sub-therapeutic drug concentrations. As part of the DeWorm3 project, we sought to characterize the serum disposition kinetics and pattern of urinary excretion of ABZ and its main metabolites ABZ sulphoxide (ABZSO) and ABZ sulphone (ABZSO2) in humans, and the assessment of the duration and optimal time point where ABZ and/or its metabolites can be measured in urine as an indirect assessment of an individual's adherence to treatment. METHODOLOGY/PRINCIPAL FINDINGS: Consecutive venous blood and urine samples were collected from eight (8) human volunteers up to 72 h post-ABZ oral administration. ABZ/metabolites were quantified by HPLC. The ABZSO metabolite was the main analyte recovered both in serum and urine. ABZSO Cmax in serum was 1.20 ± 0.44 µg/mL, reached at 4.75 h post-treatment. In urine, ABZSO Cmax was 3.24 ± 1.51 µg/mL reached at 6.50 h post-ABZ administration. CONCLUSION/SIGNIFICANCE: Pharmacokinetic data obtained for ABZ metabolites in serum and urine, including the recovery of the ABZ sulphoxide derivative up to 72 h in both matrixes and the recovery of the amino-ABZ sulphone metabolite in urine samples, are suggesting the possibility of developing a urine based method to assess compliance to ABZ treatment. Such an assay may be useful to optimize ABZ use in human patients. TRIAL REGISTRATION: ClinicalTrials.gov NCT03192449.

Assessment of liver slices for research on metabolic drug-drug interactions in cattle.

1. Precision-cut liver slices (PCLS) from food-producing animals have not been extensively used to study xenobiotic metabolism, and thus information on this field of research is sparse. 2. The aims of the present work were to further validate the technique of production and culture of bovine PCLS and to characterize the metabolic interaction between the anthelmintic albendazole (ABZ) and the flavin-monooxygenase (FMO) inhibitor methimazole (MTZ). 3. Nine steers were used as donors. PCLS were produced and incubated under two methods: a dynamic organ culture (DOC) incubator and a well-plate (WP) system. 4. Tissue viability, assessed through both structural and functional markers, was preserved throughout 12 h of incubation. ABZ was metabolized to its (+) and (-) albendazole sulfoxide stereoisomers (ABZSO) in bovine PCLS. The interaction between ABZ and MTZ resulted in a reduction (p < 0.001) in the rates of appearance of (+) ABZSO. Conversely, in presence of MTZ, the rates of appearance of (-) ABZSO increased under both systems (p < 0.05). 5. Both culture systems were suitable for assessing the interaction between ABZ and MTZ. 6. Overall, the results presented herein show that PCLS are a useful and reliable tool for short-term studies on metabolic drug-drug interactions in the bovine species.

The herbicide glyphosate is a weak inhibitor of acetylcholinesterase in rats.

The current work evaluated the inhibitory potency of the herbicide glyphosate (GLP) on acetylcholinesterase (AChE) activity in male and female rat tissues. The AChE activity in brain was higher (p<0.05) than those observed in kidney (females: 2.2-fold; males: 1.9-fold), liver (females: 6-fold; males: 6.9-fold) and plasma (females: 14.7-fold; males: 25.3-fold). Enzyme activities were higher in presence of 10mM GLP compared to those measured at an equimolar concentration of the potent AChE inhibitor dichlorvos (DDVP). Moreover, IC50s for GLP resulted between 6×10(4)- and 6.8×10(5)-fold higher than those observed for DDVP. In conclusion, GLP is a weak inhibitor of AChE in rats.

Plasma and ocular prednisolone disposition after oral treatment in cats.

OBJECTIVE: To evaluate the plasma and aqueous humor disposition of prednisolone after oral administration in cats. METHODS: Six cats were administered with a single oral dose of prednisolone (10 mg). Blood and aqueous humor samples were serially collected after drug administration. Prednisolone concentrations in plasma and aqueous humor were measured at 0.25, 0.5, 1.0, 1.5, 2.0, 3.0, 4.0, and 5.0 h after administration by a high-performance liquid chromatographic analytical method developed and validated for this purpose. RESULTS: Mean ± standard error (SE) of maximum plasma prednisolone concentration (300.8 ± 67.3 ng/mL) was reached at 1 h after administration. Prednisolone was distributed to the aqueous humor reaching a mean peak concentration of 100.9 ± 25.5 ng/mL at 1.25 h after administration. The mean ± SE systemic and aqueous humor exposure (AUC) was 553.3 ± 120.0 ng h/mL and 378.8 ± 64.9 ng h/mL, respectively. A high AUC(aqueous humor)/AUC(plasma) ratio was observed (0.68 ± 0.13). The mean half-life time of elimination in plasma and aqueous humor was 0.87 ± 0.16 h and 2.25 ± 0.44 h, respectively. CLINICAL SIGNIFICANCE: The observed high ratio between aqueous humor and plasma prednisolone concentrations indicates that extensive penetration of prednisolone to the anterior segment of the eye may occur. This is the first step that contributes to the optimization of the pharmacological therapeutics for the clinical treatment of uveitis.

Enhanced dissolution and systemic availability of albendazole formulated as solid dispersions.

Solid dispersions (SDs) containing the anthelmintic compound albendazole (ABZ) and either Pluronic 188 (P 188) or polyethylene glycol 6000 (PEG 6000) as hydrophilic carriers were formulated. Drug-polymers interactions in solid state were investigated using different techniques. Only a 4% of total ABZ was dissolved at 5 min post-incubation, reaching dissolution rates of 32.8% (PEG 6000) and 69.4% (P 188) in SDs. In this way, P 188 was substantially more efficient as ABZ dissolution promoter in comparison to PEG 6000, especially at the initial stages of the dissolution processes (<30 min). An increased systemic availability (p < 0.001) was obtained when ABZ was administered as ABZ-P 188 SDs, with a 50% enhancement in systemic exposure (AUC values) compared to treatment with an ABZ suspension. Consistently, the Cmax increased 130% (p < 0.001) following treatment with P 188 based SD ABZ formulation. For the ABZ-PEG 6000 SD formulation, the favorable effect on ABZ systemic availability did not reached statistical significance compared to the control group. The study reported here showed the utility of pharmacokinetic assays performed on mice as a model for preliminary drug formulation screening studies.

Improved albendazole dissolution rate in pluronic 188 solid dispersions.

Solids dispersions (SDs) have been proposed as an alternative to improve the dissolution rate of low solubility drugs. SDs containing albendazole (ABZ; 5, 10, 25, and 50% w/w) and Pluronic 188 (P 188) as hydrophilic carrier were formulated. The obtained SDs were assessed in comparison to physical mixtures (PMs). Drug-polymer interactions in solid state were investigated using Fourier-transform infrared spectroscopy, scanning electron microscopy, and X-ray diffraction analysis. No chemical interaction was found between ABZ and poloxamer. The dissolution profiles indicated that ABZ incorporated in SDs and PMs was rapidly released, reaching rapidly the steady state. Increased dissolution rates are usually observed at the highest polymer proportions. However, an opposite effect for SDs as well as for PMs was observed in the assays described here. The systems with the lowest P 188 percentages (SD4, SD3; PM4, PM3) tended to be more effective in increasing the ABZ dissolution rate. Such a result can be attributed to the fact that concentrated aqueous solutions of Poloxamer may form thermo-reversible gels. The physical-mechanical properties indicated that SDs possess improved flow and compacting properties compared to PMs. Thus, ABZ SDs would be more convenient for solid dosage form design and manufacture.

Enhancement of the drug susceptibility of a triclabendazole-resistant isolate of Fasciola hepatica using the metabolic inhibitor ketoconazole.

A study has been carried out to investigate whether the action of triclabendazole (TCBZ) is altered by using the metabolic inhibitor, ketoconazole (KTZ) to inhibit the cytochrome P450 (CYP 450) system within Fasciola hepatica. The Oberon TCBZ-resistant and Cullompton TCBZ-susceptible isolates were used for these experiments. The CYP 450 enzyme system was inhibited by a 2 h pre-incubation in KTZ (40 microM). Flukes were 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 microg/ml); or KTZ + 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 disruption to the TCBZ-susceptible isolate than the TCBZ-resistant isolate. However, co-incubation with KTZ and TCBZ/TCBZ.SO led to more severe surface changes to the TCBZ-resistant isolate than with each drug on its own, with greater swelling and blebbing of the tegument and even the loss of the apical plasma membrane in places. With the Cullompton isolate, there was limited potentiation of drug action in combination with KTZ, and only with TCBZ.SO. The results support the concept of altered drug metabolism within TCBZ-resistant isolates and indicate that this process may play a role in the development of drug resistance.

Potentiation of triclabendazole sulphoxide-induced tegumental disruption by methimazole in 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 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-susceptible isolates were used for these experiments. The FMO system was inhibited by a 2-h pre-incubation in methimazole (100 microM), 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). 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 triclabedazole-resistant isolate. However, co-incubation with MTZ+TCBZ, but more particularly MTZ+TCBZ.SO, led to more severe changes to the TCBZ-resistant isolate than with each drug on its own, with severe swelling of the basal infolds and mucopolysaccharide masses in the syncytium, accompanied by a reduction in numbers of secretory bodies. The synthesis and production of secretory bodies in the tegumental cells was severely affected as well. 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.

Enantiomeric behaviour of albendazole and fenbendazole sulfoxides in domestic animals: pharmacological implications.

Albendazole and fenbendazole are methylcarbamate benzimidazole anthelmintics extensively used to control gastrointestinal parasites in domestic animals. These parent compounds are metabolised to albendazole sulfoxide and fenbendazole sulfoxide (oxfendazole), respectively. Both sulfoxide derivatives are anthelmintically active and are manufactured for use in animals. They metabolites have an asymmetric centre on their chemical structures and two enantiomeric forms of each sulfoxide have been identified in plasma, tissues of parasite location and within target helminths. Both the flavin-monooxygenase and cytochrome P450 systems are involved in the enantioselective biotransformation of these anthelmintic compounds in ruminant species. A relevant progress on the understanding of the relationship among enantioselective metabolism and systemic availability of each enantiomeric form has been achieved. This article reviews the current knowledge on the pharmacological implications of the enantiomeric behaviour of albendazole sulfoxide and oxfendazole in domestic animals.

Effects of faecal residues of moxidectin and doramectin on the activity of arthropods in cattle dung.

Dung invertebrate colonization and degradation levels of faeces from cattle treated with endectocides were studied. Faeces of control and doramectin (DRM) (subcutaneous) and moxidectin (MXD) (subcutaneous and topical) treated animals were deposited on the field from 3 to 21 days post-treatment (pt). Pats were recovered after 6 to 42 days post-deposition (pd). Faecal weight, dry matter, arthropods number, and drugs concentrations were determined. Total arthropods number was higher in control (P<0.0001) than in the other groups from days 3 to 21 pt. Total number of insects recovered on days 3, 11, and 21 pt from control pats was significantly (P<0.001) higher than in treated-animal pats during all the trial. At day 21 pt, the insects' number in dung voided by DRM-treated cattle was (P<0.05) lower than in the other groups. Comparisons of dung degradation among treatments were inconclusive. A lower adverse effect was observed for MXD compared with DRM. No significant degradation of MXD or DRM was observed during the present trial.

Drug transfer into target helminth parasites.

The pharmacokinetics of an anthelmintic drug includes the time course of drug absorption, distribution, metabolism and elimination from the host and determines the concentration of the active drug that reaches the location of the parasite. However, the action of the anthelmintic also depends on the ability of the active drug to reach its specific receptor within the target parasite. Thus, drug entry and accumulation in target helminths are important issues when considering how best to achieve optimal efficacy. Passive drug transfer through the external helminth surface is the predominant entry mechanism for most widely used anthelmintics and is discussed in this article. Despite the structural differences between the external surface of nematodes (the cuticle) and the external surface of cestodes and trematodes (the tegument), the mechanism of drug entrance into both types of helminth depends on the lipophilicity of the anthelmintic and this is the major physicochemical determinant for the drug to reach a therapeutic concentration in the target parasite. Understanding the processes that regulate drug transfer into helminth parasites is an important aspect in improving the control of parasites in human and veterinary medicine.

Milk excretion of ivermectin and moxidectin in dairy sheep: assessment of drug residues during cheese elaboration and ripening period.

Ivermectin (IVM) and moxidectin (MXD) are broad-spectrum endectocide antiparasitic drugs extensively used in food-producing animals. The patterns of IVM and MXD excretion in milk were comparatively characterized following their subcutaneous administration (200 microg.kg(-1) of body weight) to lactating dairy sheep. The relationship between milk excretion and plasma disposition kinetics of both compounds was characterized. A pool of milk collected from all of the animals in each experimental group was used for cheese elaboration. IVM and MXD residual concentrations were assessed during the cheese-making process and ripening period. IVM and MXD concentrations were measured in plasma, milk, and milk product (whey, curd, and cheese) samples using an HPLC-based methodology with fluorescence detection. IVM and MXD were extensively distributed from the bloodstream to the mammary gland, and large quantities, particularly of MXD, were excreted in milk. Residual concentrations of both compounds were recovered in milk up to 30 (IVM) and 35 (MXD) days post-treatment. The total fraction of the administered dose excreted in milk for MXD was significantly higher than that of IVM. During cheese production, the highest residual concentrations of both molecules were measured in the curd. Thirty-four percent of the total drug residue measured in the pooled milk collected from treated sheep was lost during the cheese-making process. The lowest residual concentrations were measured in the whey. IVM and MXD concentrations in the elaborated cheese tended to increase during the ripening period, reaching the highest residual level at 40 days of cheese maturation. The long persistence of milk residual concentrations of MXD and IVM in lactating dairy sheep and the high concentrations found in cheese and other milk-related products should be seriously considered before recommendation of the extralabel use of these antiparasitic drugs in dairy animals.

Loperamide modifies the tissue disposition kinetics of ivermectin in rats.

Ivermectin (IVM) is a broad-spectrum antiparasitic drug extensively used in human and veterinary medicine that is largely excreted in bile and faeces. Loperamide (LPM) is an opioid derivative that reduces gastrointestinal secretions and motility. Both IVM and LPM have been reported to act as P-glycoprotein substrates (P-GP). The goal of the present work was to study the LPM-induced modifications to the pattern of tissue distribution for IVM. Thirty-six Wistar male rats were randomly allocated to two groups (n = 18) and treated subcutaneously with IVM alone or co-administered with LPM. Rats were killed at different times post-treatment and samples (blood and tissues) were collected and analyzed by HPLC. The presence of LPM induced a marked enhancement in the IVM plasma concentrations, resulting in a significantly higher area under concentration time curve (AUC) value (P < 0.01) than that obtained after the administration of IVM alone. Significantly higher IVM availabilities in the liver tissue and small intestine wall (P < 0.05) were obtained in the presence of LPM. There were no statistically significant differences in drug availability in the large intestinal wall after both treatments. However, LPM induced a marked decrease in the amount of IVM recovered in the large intestinal lumen content. The ratio between IVM concentrations in the large intestinal luminal content and plasma at day 1 post-treatment was 4.64-fold higher in the absence of LPM. The delayed intestinal transit time caused by LPM accounting for an extended plasma-intestine recycling time, and a potential competition between IVM and LPM for the P-GP-mediated bile-intestinal secretion processes, may account for the enhanced IVM systemic availability reported in the current study.

Placental and fetal toxicity of albendazole sulphoxide in Wistar rats.

This work characterized the effects of albendazole sulphoxide (ABZSO) on placental and fetal parameters in Wistar rats on day 20 of gestation. ABZSO was fed in laboratory chow at 0, 2.5, 5, 10, 20 or 30 mg/kg/d from day 6 to 15 of gestation to pregnant rats. Data of resorptions, placental and fetal characteristics and fetal skeletal malformations were recorded. Resorption percentages in the 20 and 30 mg/kg/d groups were significantly higher compared to the control group. Placentas of ABZSO-treated rats had lower weight and smaller size than untreated rats. The fetal weight and size were lower in the 5 mg/kg/d dose compared to no treatment. In the 5, 10 and 20 mg/kg/d groups, reductions in ossification process were observed. ABZSO induced malformations and/or fetal death when orally administered to pregnant rats. This data contributes to characterization of the reproductive toxicity of ABZSO, the main active metabolite of albendazole.

Disposition of doramectin milk residues in lactating dairy sheep.

Doramectin (DRM) is a broad spectrum macrocyclic lactone antiparasitic drug not approved for use in dairy animals. However, DRM and other endectocide compounds are widely used extra-label to control endo- and ectoparasites in dairy sheep. The plasma disposition kinetics and the pattern of DRM excretion in milk were characterized following its subcutaneous administration to lactating dairy sheep. DRM concentration profiles were measured in plasma and milk samples after validation of a specific HPLC-based methodology. DRM was detected between 1 h and 30 days post-treatment. DRM concentrations of 0.48 ng.mL(-1) (plasma) and 1.03 ng.mL(-1) (milk) were measured at 30 days post-treatment. DRM was extensively distributed from the bloodstream to the mammary gland, and large concentrations were excreted in milk. The peak concentrations and total amount of DRM recovered in milk (expressed as area under the concentration versus time curve) were 3-fold higher than those measured in plasma; 2.44% of the total DRM dose was excreted in milk. The long persistence of DRM milk residues should be seriously considered before its extra-label use in dairy animals is recommended.

Farmacocinética y toxicología de neomicina y paromomicina en ovinos

Los objetivos del presente plan experimental fueron: Estudiar todas las variables farmacocinéticas de neomicina y paromomicina tras su administración por vías intravenosa, intramuscular y subcutánea a ovinos. Determinar la biodisponibilidad de neomicina y paromomicina tras su administración por las vías intramuscular y subcutánea a ovinos. Calcular la dosis de ataque y dosis de mantenimiento. Corroborar prácticamente la eficacia de los planes diseñados. Determinar el margen de seguridad y caracterizar el cuadro tóxico agudo en ovinos, tras la administración de neomicina y paromomicina por infusión intravenosa cada 15 minutos; correlacionando los niveles plasmáticos de estos antibióticos con la sintomatología tóxica

Farmacocinética y toxicología de neomicina y paromomicina en ovinos

Los objetivos del presente plan experimental fueron: Estudiar todas las variables farmacocinéticas de neomicina y paromomicina tras su administración por vías intravenosa, intramuscular y subcutánea a ovinos. Determinar la biodisponibilidad de neomicina y paromomicina tras su administración por las vías intramuscular y subcutánea a ovinos. Calcular la dosis de ataque y dosis de mantenimiento. Corroborar prácticamente la eficacia de los planes diseñados. Determinar el margen de seguridad y caracterizar el cuadro tóxico agudo en ovinos, tras la administración de neomicina y paromomicina por infusión intravenosa cada 15 minutos; correlacionando los niveles plasmáticos de estos antibióticos con la sintomatología tóxica

Farmacocinética y toxicología de neomicina y paromomicina en ovinos

Los objetivos del presente plan experimental fueron: Estudiar todas las variables farmacocinéticas de neomicina y paromomicina tras su administración por vías intravenosa, intramuscular y subcutánea a ovinos. Determinar la biodisponibilidad de neomicina y paromomicina tras su administración por las vías intramuscular y subcutánea a ovinos. Calcular la dosis de ataque y dosis de mantenimiento. Corroborar prácticamente la eficacia de los planes diseñados. Determinar el margen de seguridad y caracterizar el cuadro tóxico agudo en ovinos, tras la administración de neomicina y paromomicina por infusión intravenosa cada 15 minutos; correlacionando los niveles plasmáticos de estos antibióticos con la sintomatología tóxica