Albendazole failure to control resistant nematodes in lambs: lack of effect of fasting-induced improvement on drug absorption

Enhanced plasma availability of albendazole sulphoxide (ABZSO), the active metabolite of albendazole (ABZ), has been described in feed-restricted sheep. The aim of the present work was to determine if the absorption-related pharmacokinetic changes derived from fasting animals prior to drug treatment would modify the clinical efficacy of ABZ against resistant gastrointestinal nematodes in lambs. Forty Corriedale lambs, naturally infected with resistant gastrointestinal nematodes, were divided into 4 groups. Controls were fed ad libitum and did not receive any drug treatment. Treated animals were fed ad libitum up to 30 min prior to treatment with ABZ (3.8 mg/kg) by the intraruminal route. The control (fasted) animals were not fed during the 24-hr period prior to the start of the experiment and did not receive any drug treatment. A second treated group of animals were fasted 24 hr prior to the treatment with ABZ, as previously described for the fed-treated group. Blood samples were collected over a period of 72 hr post-treatment from 6 animals in each treated group. Plasma samples were analyzed by high performance liquid chromatography. The pharmacokinetic parameters were statistically compared using parametric statistical tests. The estimation of the efficacy of the different treatments was performed by the fecal egg count reduction test (FECRT). Additionally, 4 animals randomly chosen from the control-fed and treated groups were killed 13 days post-treatment to evaluate the efficacy against different adult nematode parasites. The results were statistically compared by parametric and non-parametric tests. Significantly (P < 0.05) higher Cmax and AUC values were observed for both the ABZSO and ABZ-sulphone (ABZSO(2)) metabolites in the fasted compared to the fed animals. These kinetic results may be due to a fasting-induced delay in the GI transit time which increases ABZ dissolution and GI absorption. However, a poor ABZ efficacy (measured as FECRT), compatible with a high degree of nematode resistance, was obtained in both fed (48%) and fasted (49%) animals. Haemonchus contortus and Trichostrongylus colubriformis appeared as the more reluctant species with respect to ABZ treatment. The efficacy against H. contortus ranged between 37 (fed) and 54% (fasted) and against T. colubriformis between 0% (fed) and 16% (fasted). Under these experimental conditions, the fasting-induced improvement on ABZ systemic availability (>60%) did not improve its activity against nematodes with a high degree of resistance. However, the data described here for a highly resistant nematode population should not discourage the use of fasting as a practical and well-proven management tool for parasite control in ruminants.     

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.     

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.     

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.     

Exploring the Potential of Flubendazole in Filariasis Control: Evaluation of the Systemic Exposure for Different Pharmaceutical Preparations

The goal of elimination of the human filariases would benefit greatly from the use of a macrofilaricidal agent. In vivo trials in humans and many experimental animal models suggest that flubendazole (FLBZ) is a highly efficacious macrofilaricide. However, since serious injection site reactions were reported in humans after parenteral FLBZ administration, the search for alternative pharmaceutical strategies to improve the systemic availability of FLBZ and its metabolites has acquired urgency in both human and veterinary medicine. The goal of the current work was to compare the systemic exposure of FLBZ formulated as either an aqueous hydroxypropyl-β-cyclodextrin (CD) or aqueous carboxymethyl cellulose (CMC) suspension or a Tween 80-based formulation (TWEEN) in rats and jirds (Meriones unguiculatus). Healthy animals of both species were allocated into four experimental groups of 44 animals each: FLBZ-CD_oral and FLBZ-CD_sc, treated with the FLBZ-CD formulation by the oral or subcutaneous routes, respectively; FLBZ-TWEEN_sc, dosed subcutaneously with the FLBZ-TWEEN formulation; and FLBZ-CMC_oral, treated orally with the FLBZ suspension. The FLBZ dose was 5 mg/kg. FLBZ and its hydrolyzed (H-FLBZ) and reduced (R-FLBZ) metabolites were recovered in plasma samples collected from rats and jirds treated with the different FLBZ formulations. In both species, FLBZ parent drug was the main analyte recovered in the bloodstream. In rats, FLBZ systemic exposure (AUC_0-LOQ) was significantly (P<0.05) higher after the FLBZ-CD treatments, both oral (4.8±0.9 µg.h/mL) and subcutaneous (7.3±0.6 µg.h/mL), compared to that observed after oral administration of FLBZ-CMC suspension (0.93±0.2 µg.h/mL). The same differences were observed in jirds. In both species, parenteral administration of FLBZ-TWEEN did not improve the systemic availability of FLBZ compared to FLBZ-CD_oral treatment. In conclusion, formulation approaches that enhance the availability of flubendazole in the rat and jird may have therapeutic implications for a drug with poor or erratic bioavailability.     

Resistance-induced changes in triclabendazole transport in Fasciola hepatica: ivermectin reversal effect

Triclabendazole (TCBZ) and albendazole (ABZ) are flukicidal benzimidazole compounds extensively used in veterinary medicine. Although TCBZ has excellent activity against mature and immature stages of the liver fluke, Fasciola hepatica, ABZ action is restricted to flukes older than 12 wk. The intensive use of TCBZ has resulted in the development of resistance. To gain insight into the mechanisms of resistance to TCBZ, the ex vivo diffusion of TCBZ, TCBZ sulfoxide (TCBZSO, the active metabolite of TCBZ), and ABZ into TCBZ-susceptible and -resistant adult flukes was compared. TCBZ-susceptible (Cullompton) and -resistant (Sligo) flukes were incubated in Krebs-Ringer Tris buffer with either TCBZ, TCBZSO, or ABZ (5 nmol/ ml) for 90 min. Drug/metabolite concentrations were quantified by high-performance liquid chromatography. All the assayed molecules penetrated through the tegument of both susceptible and resistant flukes. However, significantly lower concentrations of TCBZ and TCBZSO were recovered within the TCBZ-resistant flukes. In contrast, ABZ entrance into the susceptible and resistant flukes was equivalent. The influx/efflux balance for TCBZ, TCBZSO, and ABZ in susceptible and resistant flukes in the presence or absence of a substrate (ivermectin) of the drug transporter P-glycoprotein was assessed. The ivermectin-induced modulation of P-glycoprotein activity decreased TCBZ efflux from the resistant flukes. Higher concentrations of TCBZ and TCBZSO were recovered from the resistant liver flukes in the presence of ivermectin. Thus, an altered influx/efflux mechanism may account for the development of resistance to TCBZ in F. hepatica.     

Effects of ivermectin and doramectin faecal residues on the invertebrate colonization of cattle dung

Abstract: The effects of avermectin [ivermectin (IVM) and doramectin (DRM)] faecal residues on dung colonization and degradation by invertebrates were evaluated during late spring in the east of La Pampa province, Argentina. The study was conducted after collection of faecal material from animals (10 steers per group) allocated to the following groups: untreated control group (CG) and groups treated subcutaneously (200 μg/kg) with either DRM (DG) or a long‐acting formulation of IVM (IG). Fifty pats (550 g each) per group were collected, prepared and deposited on the field on days 3, 7, 16 and 29 post‐treatment (pt). Eight pats per group were recovered after 7, 14, 21, 42, 100 and 180 days post‐deposition (pd) on the field. The weight, percentage of dry matter, number of arthropods and nematodes from faeces were determined. The faecal concentrations of IVM and DRM were measured by high performance liquid chromatography (HPLC) throughout the trial period to correlate the pattern of drug degradation in dung with pd time. The total number of arthropods in dungs from CG was higher (P < 0.05) than those counted between days 3 and 29 pt in IG and DG. A decrease in the number of Coleoptera larvae (P < 0.05) between days 21 and 42 days pd was observed in both treated groups. Diptera larvae counts in CG pats were significantly higher (P < 0.05) than those obtained in treated groups in the 7‐ and 14‐day‐old pats. A lower number (P < 0.05) of Collembola, compared with pats from CG, was recovered from IG and DG pats deposited at days 3 and 7 pt and exposed from day 42. The counts of Acari in pats from treated animals were lower (P < 0.05) than those observed in CG pats at 3, 8 and 16 days pt. There were no differences neither in adult Scarabaeidae recovered nor in the proportions of dung buried and destroyed by great dung beetles. Dung specific nematodes were reduced (P < 0.05) in IG and DG pats from 3 and 7 days pt compared with those of CG pats. The comparative results shown here demonstrate that the negative effects of both IVM and DRM on dung colonization are similar. The pattern of drug degradation in the environment was very slow. High residual concentrations of both active parent compounds were recovered in dungs exposed in the field for up to 180 days pd. Concentrations as high as 13 ng/g (IVM) and 101 ng/g (DRM) were measured in faeces obtained from pats deposited on day 27 pt and exposed to the environment during 180 days. The results show a decrease in invertebrate colonization of dung recovered from IVM‐ and DRM‐treated cattle, which is in agreement with the large drug residual concentrations measured in faeces.     

Unchanged triclabendazole kinetics after co-administration with ivermectin and methimazole: failure of its therapeutic activity against triclabendazole-resistant liver flukes

Background  

The reduced drug accumulation based on enhanced drug efflux and metabolic capacity, identified in triclabendazole (TCBZ)-resistant Fasciola hepatica may contribute to the development of resistance to TCBZ. The aim of this work was to evaluate the pharmacokinetics and clinical efficacy of TCBZ administered alone or co-administered with ivermectin (IVM, efflux modulator) and methimazole (MTZ, metabolic inhibitor) in TCBZ-resistant F. hepatica-parasitized sheep. Sheep infected with TCBZ-resistant F. hepatica (Sligo isolate) were divided into three groups (n = 4): untreated control, TCBZ-treated (i.r. at 10 mg/kg) and TCBZ+IVM+MTZ treated sheep (10 i.r., 0.2 s.c. and 1.5 i.m. mg/kg, respectively). Plasma samples were collected and analysed by HPLC. In the clinical efficacy study, the animals were sacrificed at 15 days post-treatment to evaluate the comparative efficacy against TCBZ-resistant F. hepatica.     

Cattle nematodes resistant to macrocyclic lactones: Comparative effects of P-glycoprotein modulation on the efficacy and disposition kinetics of ivermectin and moxidectin

The role of the drug efflux pump, known as P-glycoprotein, in the pharmacokinetic disposition (host) and resistance mechanisms (target parasites) of the macrocyclic lactone (ML) antiparasitic compounds has been demonstrated. To achieve a deeper comprehension on the relationship between their pharmacokinetic and pharmacodynamic behaviors, the aim of the current work was to assess the comparative effect of loperamide, a well-established P-glycoprotein modulator, on the ivermectin and moxidectin disposition kinetics and efficacy against resistant nematodes in cattle. Fifty (50) Aberdeen Angus male calves were divided into five (5) experimental groups. Group A remained as an untreated control. Animals in the other experimental Groups received ivermectin (Group B) and moxidectin (Group C) (200 μg/kg, subcutaneuosly) given alone or co-administered with loperamide (0.4 mg/kg, three times every 24 h) (Groups D and E). Blood samples were collected over 30 days post-treatment and drug plasma concentrations were measured by HPLC with fluorescence detection. Estimation of the anthelmintic efficacy for the different drug treatments was performed by the faecal egg count reduction test (FECRT). Nematode larvae were identified by pooled faecal cultures for each experimental group. Cooperia spp. and Ostertagia spp. were the largely predominant nematode larvae in pre-treatment cultures. A low nematodicidal efficacy (measured by the FECRT) was observed for both ivermectin (23%) and moxidectin (69%) in cattle, which agrees with a high degree of resistance to both molecules. Cooperia spp. was the most abundant nematode species recovered after the different drug treatments. The egg output reduction values increased from 23% to 50% (ivermectin) and from 69% to 87% (moxidectin) following their co-administration with loperamide. Enhanced systemic concentrations and an altered disposition of both ML in cattle, which correlates with a tendency to increased anthelmintic efficacy, were observed in the presence of loperamide. Overall, the in vivo modulation of P-glycoprotein activity modified the kinetic behavior and improved the efficacy of the ML against resistant nematodes in cattle. The work provides further evidence on the high degree of resistance to ML in cattle nematodes and, shows for the first time under field conditions, that modulation of P-glycoprotein may be a valid pharmacological approach to improve the activity and extend the lifespan of these antiparasitic molecules.