Flubendazole in cystic echinococcosis therapy: Pharmaco-parasitological evaluation in mice

Cystic echinococcosis (CE) caused by the parasite Echinococcus granulosus is an important public health problem worldwide. Flubendazole has shown poor in vivo efficacy against CE in humans and mice. However, flubendazole causes marked in vitro damage on E. granulosus protoscoleces. The goals of the current work were: a) to compare the plasma pharmacokinetic behaviour of flubendazole formulated as a hydroxipropyl-β-cyclodextrin aqueous solution or as a carboxymethyl celullose suspension, both given by the oral route to mice, b) to compare flubendazole clinical efficacy in secondary CE in mice after its administration as both formulations, c) to evaluate the flubendazole-induced morphological changes in hydatid cysts recovered from infected mice treated with both drug formulations. Flubendazole administration as a solution resulted in significantly higher plasma maximum concentration (C_max) and area under the concentration–time curve (AUC) values compared to those obtained after the flubendazole-suspension treatment. This enhanced drug availability correlated with an increased efficacy against secondary CE in mice observed for the flubendazole-solution formulation, while the suspension formulation did not reach differences with the untreated control group. Similar ultrastructural changes were observed in cysts recovered from flubendazole (both formulations) treated mice after 3, 6 and 9 months of infection, although the damage extension was greater after treatment with the flubendazole-solution formulation.     

Quantitative chromatographic determination of several benzimidazole anthelmintic molecules in parasite material

A specific, precise and accurate high-performance liquid chromatographic (HPLC) analytical method has been developed for the quantitative determination of different benzimidazole (BZD) anthelmintics in parasite material (Moniezia benedeni). Mebendazole (MBZ), oxibendazole (OBZ), flubendazole (FLBZ), albendazole (ABZ) ricobendazole (RBZ), albendazole sulphone (ABZSO(2)), fenbendazole (FBZ), oxfendazole (OFZ) and fenbendazole sulphone (FBZSO(2)) were measured simultaneously in M. benedeni, a sheep and cattle cestode parasite used as a model of the biological matrix. The recovery, linearity, precision, accuracy, limits of detection and quantification of the method were determined. Drug extraction from the parasite's tissue homogenate was performed using methanol (liquid phase extraction), and after solvent evaporation, the residual material was cleaned up by solid phase extraction prior to analysis by reversed-phase HPLC. The resolution of all the BZD molecules assayed was obtained on a C(18) reversed-phase (5 microm, 250 mm x 4.6 mm) column using acetonitrile and ammonium acetate as the mobile phase and ultraviolet (UV) detection at 292 nm. Regression analyses for all the BZD compounds assayed were linear at concentrations ranging from 1.61 to 64.21 nmol/100mg protein (triplicate determinations) showing correlation coefficients greater than 0.9922. The developed method is efficient for the simultaneous determination of several benzimidazole anthelmintic molecules in parasite material and useful for the ex vivo and in vivo characterisation of the kinetics of drug uptake/diffusion in target parasites, which seems to be relevant to optimise parasite control both in human and veterinary medicine.     

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.     

Food Security Monitoring via Mobile Data Collection and Remote Sensing: Results from the Central African Republic

The Central African Republic is one of the world’s most vulnerable countries, suffering from chronic poverty, violent conflicts and weak disaster resilience. In collaboration with Doctors without Borders/Médecins Sans Frontières (MSF), this study presents a novel approach to collect information about socio-economic vulnerabilities related to malnutrition, access to resources and coping capacities. The first technical test was carried out in the North of the country (sub-prefecture Kabo) in May 2015. All activities were aimed at the investigation of technical feasibility, not at operational data collection, which requires a random sampling strategy. At the core of the study is an open-source Android application named SATIDA COLLECT that facilitates rapid and simple data collection. All assessments were carried out by local MSF staff after they had been trained for one day. Once a mobile network is available, all assessments can easily be uploaded to a database for further processing and trend analysis via MSF in-house software. On one hand, regularly updated food security assessments can complement traditional large-scale surveys, whose completion can take up to eight months. Ideally, this leads to a gain in time for disaster logistics. On the other hand, recording the location of every assessment via the smart phones’ GPS receiver helps to analyze and display the coupling between drought risk and impacts over many years. Although the current situation in the Central African Republic is mostly related to violent conflict it is necessary to consider information about drought risk, because climatic shocks can further disrupt the already vulnerable system. SATIDA COLLECT can easily be adapted to local conditions or other applications, such as the evaluation of vaccination campaigns. Most importantly, it facilitates the standardized collection of information without pen and paper, as well as straightforward sharing of collected data with the MSF headquarters or other aid organizations.     

Measurement of ivermectin concentrations in target worms and host gastrointestinal tissues: influence of the route of administration on the activity against resistant Haemonchus contortus in lambs

The influence of the administration route on the relationship between efficacy and ivermectin concentration profiles achieved in the bloodstream, the gastrointestinal mucosal tissues/fluid contents and within a target abomasal parasite (Haemonchus contortus) was evaluated in lambs. Twenty-six (26) parasitized lambs were assigned into three experimental groups: untreated (control) and ivermectin treated by the subcutaneous and intraruminal route at 0.2mg/kg. Blood samples were collected between 0 and 15 days post-treatment (plasma disposition study). Four animals from each group were sacrificed at day 3 post-treatment. Mucosa and content samples from abomasum and small intestine and adult specimens of H. contortus were collected. Drug concentrations were measured by HPLC. Individual fecal egg counts were evaluated at -1, 3 and 15 days post treatment. Post-mortem examination was done at day 15 post-treatment. Adult nematodes recovered from the digestive tract were counted and identified by species. Ivermectin plasma availability was higher (P<0.05) after the subcutaneous administration (129 ng.d/ml) compared to the intraruminal treatment (58.4 ng.d/ml). However, ivermectin concentrations measured in the gastrointestinal contents were higher in lambs treated by the intraruminal route. The mean ivermectin concentrations achieved (3 days post-treatment) in the abomasal content were 143 ng/g (intraruminal) and 2.53 ng/g (subcutaneous). Ivermectin concentrations were 15-fold higher in H. contortus recovered from intraruminally treated lambs. Whereas the subcutaneous administration reduced the number of adult nematodes from 4376 to 1300, the number of adult nematodes after the treatment with ivermectin given by the intraruminal route was 206 (P<0.05). The higher ivermectin concentrations achieved in the digestive tract shortly after the intraruminal treatment may account for the observed enhanced efficacy compared to the parenteral administration against parasites of reduced susceptibility.     

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.     

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.     

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     

Exploring flubendazole formulations for use in sheep. Pharmacokinetic evaluation of a cyclodextrin-based solution

ABSTRACT: BACKGROUND: Flubendazole (FLBZ) is a poor water solubility broad-spectrum BZD methylcarbamate. Anthelmintic compounds cyclodextrins (CDs) are usually used to increase aqueous solubility of poor hydrosoluble compounds. The comparative in vitro aqueous solubility of FLBZ and other BZD anthelmintics in the presence of hydroxypropyl-betacyclodextrin (HPbetaCD) was evaluated in the current work. Additionally, the comparative pharmacokinetic behaviour of FLBZ (and its metabolites) administered by the intraruminal (i.r.) or intraabomasal (i.a.) routes to sheep as either an aqueous CDs-based solution or a conventional carboximethylcellulose (CMC) suspension was assessed. Drug solubility studies involving albendazole, mebendazole, oxfendazole and FLBZ were performed in an aqueous solution (pH 1.2 or 7.4) with or without HPbetaCD (10 %, w/v). The pharmacokinetic study involved two experiments. Experiment 1: In a crossover study, sheep received either a FLBZ-CDs solution (n = 3) or a FLBZ-CMC suspension (n = 3) by the i.r. route (3.8 mg/kg). The treatment Groups were reversed after a 21-days washout period. Experiment 2: sheep (n = 4) were treated by the i.a. route with the FLBZ-CDs solution (3.8 mg/kg). Plasma and abomasal fluid samples were collected between 0 and 72 h posttreatment. Samples were analysed by HPLC. RESULTS: Improvement of FLBZ aqueous solubility due to CDs resulted markedly higher than that observed for mebendazole and albendazole. However, oppositely to what was expected, the absorption-related pharmacokinetic parameters did not show any marked formulation-dependant effect. After the i.a. administration of FLBZ, the AUC and the Tmax of the parent compound were significantly (P < 0.05) reduced, which is consistent with ruminal bypass. CONCLUSION: The administration of FLBZ as a CDs-based solution, does not seem to achieve great practical relevance for parasite control in sheep.