Albendazole metabolites excretion in human saliva as a biomarker to assess treatment compliance in mass drug administration (MDA) anthelmintic programs.

Soil-transmitted-helminth (STH) infections continue to be a persistent global public health problem. Control strategies for STH have been based on the use of mass drug administration (MDA). Coverage and compliance assessment is critical to understanding the true effectiveness of albendazole (ABZ) in those MDA programs. The aims of this work were to characterize the pattern of albendazole and metabolites excretion in human saliva, and to develop a saliva-based biomarker (HPLC drug/metabolite detection) useful to accurately estimate the coverage/compliance in MDA campaigns. The study subjects were 12 healthy volunteers treated with a single oral dose of ABZ (400 mg). Saliva and blood (dried blood spot, DBS) samples were taken previously and between 2 and 72 h post-treatment. The samples were analyzed by HPLC with UV detection, C18 reversed-phase column. ABZ sulphoxide was the main analyte recovered up to 72 h p.t. in blood and saliva. The concentration profiles measured in the blood (DBS samples) were higher (P < 0.05) than those in saliva, however, this ABZ-metabolite was recovered longer in saliva. The in vivo measurement of drugs/metabolites in saliva samples from ABZ-treated volunteers offers strong scientific evidence to support the use of saliva as a valid biological sample for assessing compliance in MDA programs.

Ivermectin systemic availability in adult volunteers treated with different oral pharmaceutical formulations.

Ivermectin (IVM) is currently approved as an antiparasitic agent for human use in the treatment of onchocerciasis, lymphatic filariasis, strongyloidiasis, scabies, and pediculosis. Recent findings indicate that IVM may reach other pharmacological targets, which accounts for its proven anti-inflammatory/immunomodulatory, cytostatic, and antiviral effects. However, little is known about the assessment of alternative drug formulations for human use. OBJECTIVE: To compare the systemic availability and disposition kinetics of IVM orally administered as different pharmaceutical formulations (tablet, solution, or capsule) to healthy adults. EXPERIMENTAL DESIGN/MAIN FINDINGS: Volunteers were randomly assigned to 1 of 3 experimental groups and orally treated with IVM as either, a tablet, solution, or capsules at 0.4 mg/kg in a three-phase crossover design. Blood samples were taken as dried blood spots (DBS) between 2 and 48 h post-treatment and IVM was analyzed by HPLC with fluorescence detection. IVM Cmax value was higher (P < 0.05) after the administration of the oral solution compared to treatments with both solid preparations. The oral solution resulted in a significantly higher IVM systemic exposure (AUC: 1653 ng h/mL) compared to the tablet (1056 ng h/mL) and capsule (996 ng h/mL) formulations. The simulation of a 5-day repeated administration for each formulation did not show a significant systemic accumulation. CONCLUSION: Beneficial effects against systemically located parasitic infections as well as in any other potential therapeutic field of IVM application would be expected from its use in the form of oral solution. This pharmacokinetic-based therapeutic advantage without the risk of excessive accumulation needs to be corroborated in clinical trials specifically designed for each purpose.

Assessment of Diet-Related Changes on Albendazole Absorption, Systemic Exposure, and Pattern of Urinary Excretion in Treated Human Volunteers.

Soil-transmitted-helminth (STH) infections are a persistent global public health problem. Control strategies for STH have been based on the use of mass drug administration (MDA) mainly targeting preschool- and school-aged-children, although there is increasing interest in expanding treatment to include adults and others through community-wide MDA. Coverage assessment is critical to understanding the real effectiveness of albendazole (ALB) treatment in those MDA programs. The work described here aims to (i) evaluate the effect of type of diet (a heavy or light meal) and fasting before ALB treatment on the systemic disposition of ALB and its metabolites in treated human volunteers and (ii) evaluate the potential feasibility of detecting albendazole metabolites in urine. The data reported here demonstrate that the systemic availability of the active ALB-sulfoxide (ALBSO) metabolite was enhanced more than 2-fold after food ingestion (a heavy or light meal). ALB dissolution improvement related to the ingestion of food may modify the amount of drug/metabolites reaching the parasite, affecting drug efficacy and the overall success of MDA strategies. The measurement in urine samples of the amino-ALB-sulfone (NHALBSO2) derivative and ALBSO for up to 96 h suggests that it may be feasible to develop a noninvasive tool to evaluate compliance/adherence to ALB treatment.

Combined moxidectin-levamisole treatment against multidrug-resistant gastrointestinal nematodes: A four-year efficacy monitoring in lambs.

Nematicide combinations may be a valid strategy to achieve effective nematode control in the presence of drug resistance. The goal of the current trial was to evaluate the pharmaco-parasitological performance of the moxidectin (MOX) and levamisole (LEV) combination after four years of continuous use in lambs naturally parasitized with multi-resistant gastrointestinal nematodes. At the beginning of the trial, 40 lambs were divided into four groups (n = 10), which were untreated (control) or subcutaneously treated with MOX (0.2 mg/kg), LEV (8 mg/kg) or with the combination MOX + LEV (administered separately at 0.2 and 8 mg/kg, respectively). Blood samples were collected at different times post-treatment and LEV and MOX plasma concentrations were measured by HPLC. The clinical efficacy of the continuous use of MOX + LEV combination was assessed with the controlled efficacy test (CET), performed at the beginning and end of the study, and with the faecal egg count reduction (FECR) test, performed over the four-year study period. No significant adverse pharmacokinetic changes were observed either for MOX or LEV after their co-administration to infected lambs. The CET (first year) showed efficacies of 84.3 % (Haemonchus contortus), 100 % (Teladorsagia circumcincta and Trichostrongylus axei), and 97.4 % (T. colubriformis). After the repetitive use of the combined treatment for four years, those efficacies remained high (100 %) and only decreased to 58 % against T. colubriformis. The evaluation of the FECR over the study period showed fluctuations in the performance of the combined administration. The initial FECR (2014) was 99 % (MOX), 85 % (LEV) and 100 % (MOX + LEV). The co-administration of MOX + LEV during the four-year experimental period resulted in a significantly higher anthelmintic effect (87 %) than that of MOX (42 %) or LEV (69 %) given alone. The combined use of MOX + LEV to control resistant gastrointestinal nematodes appears to be a valid strategy under specific management conditions. A high initial therapeutic response to the combination would be a relevant feature for the success of this tool.

Doramectin efficacy against Psoroptes ovis in sheep: Evaluation of pharmacological strategies.

The aims of this study were to evaluate the efficacy of two injectable formulations of doramectin (DRM) against Psoroptes ovis in sheep infested under controlled experimental conditions and to characterize the DRM plasma disposition kinetics in the infested animals. To this end, sheep were experimentally infested with a P. ovis strain from a farm with a history of treatment failure, and then treated either with DRM 1% (traditional preparation) on days 0 and 7 or with DRM 3.15% (long-acting formulation) on day 0. The efficacy of each treatment was calculated by counting live mites in skin scrapings. Plasma samples were obtained from each animal and DRM concentrations were measured by HPLC. After the two doses of DRM 1%, the maximum efficacy (98.8%) was reached on day 28, whereas after the single dose of DRM 3.15%, the maximum efficacy (100%) was reached on day 35 and ratified on day 42. The long-acting formulation allowed obtaining higher exposure and more sustained concentrations of DRM than the traditional preparation. Although both DRM formulations studied were effective according to international protocols, they did not reach 100% effectiveness in the time required for approved pharmaceutical products against sheep scab, according to Argentine regulations.

Pharmacokinetic-pharmacodynamic assessment of the ivermectin and abamectin nematodicidal interaction in cattle.

In a context of nematodicidal resistance, anthelmintic combinations have emerged as a reliable pharmacological strategy to control gastrointestinal nematodes in grazing systems of livestock production. The current work evaluated the potential drug-drug interactions following the coadministration of two macrocyclic lactones (ML) ivermectin (IVM) and abamectin (ABM) to parasitized cattle using a pharmacokinetic/pharmacodynamic (PK/PD) approach. The kinetic behavior of both compounds administered either separately or coadministered was assessed and the therapeutic response of the combination was evaluated under different resistance scenarios. In the pharmacological trial, calves received a single subcutaneous (s.c.) injection of IVM (100 µg/Kg); a single s.c. injection of ABM (100 µg/Kg) or IVM + ABM (50 µg/Kg each) administered in different injection sites to reach a final ML dose of 100 µg/Kg (Farm 1). Plasma samples were taken from those animals up to 20 days post-treatment. IVM and ABM plasma concentrations were quantified by HPLC. A parasitological trial was carried out in three farms with different status of nematodes resistance to IVM. Experimental animals received IVM (200 µg/Kg), ABM (200 µg/Kg) or IVM + ABM (100 µg/Kg each) in Farm 2, and IVM + ABM (200 µg/Kg each) in Farms 3 and 4. The anthelmintic efficacy was determined by fecal egg count reduction test (FECRT). PK analysis showed similar trends for IVM kinetic behavior after coadministration with ABM. Conversely, the ABM elimination half-life was prolonged and the systemic exposure during the elimination phase was increased in the presence of IVM. Although IVM alone failed to control Cooperia spp., the combination IVM + ABM was the only treatment that achieved an efficacy higher than 95% against resistant Cooperia spp. in all farms. In fact, when Cooperia spp. was the main genus within the nematode population and Haemonchus spp. was susceptible or slightly resistant to ML (Farms 2 and 4), the total FECR for the combination IVM + ABM was higher than 90%. Instead, when the predominant nematode genus was a highly resistant Haemonchus spp. (Farm 3), the total FECR after the combined treatment was as low as the single treatments. Therefore, the rational use of these pharmacological tools should be mainly based on the knowledge of the epidemiology and the nematode susceptibility status in each cattle farm.

Falla en la eficacia de dos formulaciones de ivermectina contra Psoroptes ovis (Hering, 1838) en ovinos / Failure of efficacy of two ivermectin formulations against Psoroptes ovis (Hering, 1838) in sheep

Resumen El presente trabajo evaluó la relación entre la eficacia y la farmacocinética de dos formulaciones comerciales inyectables de ivermectina (IVM) en ovinos merino adultos artificialmente infestados con Psoroptes ovis. Los animales fueron tratados por vía subcutánea con IVM 1 % en dos dosis con un intervalo de aplicación de 7 días, (0.2 mg/kg) o con una única dosis de IVM 3.15%, (1.05 mg/kg). Se realizaron conteos semanales de ácaros vivos mediante raspajes de piel entre el día 0 y 28 post-tratamiento para determinar la eficacia de los tratamientos, y se tomaron muestras de sangre para medir las concentraciones de IVM en plasma. Se observó una disminución significativa en los conteos de ácaros a partir del día 14 post-tratamiento, sin embargo, se encontraron ácaros vivos en todos los muestreos para ambos grupos. En el Grupo IVM 1%, la máxima eficacia se observó el día 28 post tratamiento (93.3%), mientras que en el Grupo IVM 3,15% este registro se obtuvo el día 21 (95.9%). Mayores concentraciones de IVM fueron observadas en los animales tratados con la formulación 3.15 %. La falla para obtener una cura parasitológica tras el tratamiento con ambas formulaciones de IVM puede ser indicativo de la presencia de ácaros resistentes a este principio activo.

Abstract The current work evaluated the relationship between efficacy and pharmacokinetics of two commercial injectable formulations of ivermectin (IVM) in adult merino sheep artificially infested with Psoroptes ovis. Animals were treated subcutaneously with IVM 1% formulation (two doses on days 0 and 7) at 0.2 mg / kg or with a single dose of IVM 3.15% preparation at 1.05 mg / kg. Live mites were counted weekly by performing skin scrapings between days 0 and 28 post-treatment to determine the efficacy of each IVM formulation. Blood samples were taken up to 35 days post-treatment to measure IVM plasma concentrations. A significant decrease in mite counts was observed from day 14 post-treatment. However, live mites were found in all samples for both groups throughout the entire trial. After IVM 1% administration, the highest effcacy was observed on day 28 (93.3% whereas in the IVM 3,15% group was obtained on day 21 post treatment (95.9%). Higher IVM plasma concentrations were observed in animals treated with the IVM 3.15% formulation. Failure to obtain a parasitological cure after treatment with both IVM formulations may reflect the presence of resistant mites to this drug.

Impact on beef cattle productivity of infection with anthelmintic-resistant nematodes.

ABSTRACTAims: The main goal of the current study was to evaluate, on a commercial beef cattle farm, the impact of infection with gastrointestinal nematodes resistant to both ivermectin (IVM) and moxidectin (MXD) on the productivity of calves.Methods: Male Aberdeen Angus calves, aged 9-11 months, with faecal nematode egg counts (FEC) ≥200 epg and body weight ≥190 kg, were allocated to two herds. Herd A (n = 90) grazed a maize-winter forage crop rotation and Herd B (n = 90) grazed a 2-year-old Agropyrum pasture. On Day 0 in each herd, calves were randomly allocated into five groups (n = 18), which were treated with 0.2 mg/kg IVM; 0.2 mg/kg MXD; 3.75 mg/kg ricobendazole (RBZ), both IVM and RBZ, or remained untreated. Faecal samples collected on Days -1 and 19 were used to determine the percentage reduction in FEC, and genera of the nematodes were determined by the identification of the third-stage larvae recovered from faecal cultures. Total weight gain was determined from body weights recorded on Days -1 and 91.Results: Overall mean reduction in FEC was 42% for IVM, 67% for MXD, 97% for RBZ and 99% for IVM + RBZ. The reduction in FEC for Cooperia spp. was ≤78% for IVM and MXD, and for Haemonchus spp. was 0 and 36% for IVM and MXD, respectively, confirming the presence of parasites resistant to both anthelmintics. Only IVM + RBZ treatment resulted in 100% efficacy against Haemonchus spp. The overall estimated mean total weight gain for calves treated with IVM was 15.7 (95% CI = 11.9-19.7) kg and for calves treated with IVM + RBZ was 28.8 (95% CI = 25-32.5) kg (p < 0.001). Mean total weight gain for calves treated with MXD was 23.5 (95% CI = 19.7-27.2) kg.Conclusions and clinical relevance: In calves naturally infected with resistant nematodes, under the production system assessed here, weight gains were lower in calves treated with anthelmintics that were moderately or highly ineffective compared to those treated with highly effective anthelmintics. These results demonstrate to farmers and veterinarians the importance of a sustainable and effective nematode control under field conditions.

Pharmacologic interaction between oxfendazole and triclabendazole: In vitro biotransformation and systemic exposure in sheep.

The aim of the current work was to evaluate a potential pharmacokinetic interaction between the flukicide triclabendazole (TCBZ) and the broad-spectrum benzimidazole (BZD) anthelmintic oxfendazole (OFZ) in sheep. To this end, both an in vitro assay in microsomal fractions and an in vivo trial in lambs parasitized with Haemonchus contortus resistant to OFZ and its reduced derivative fenbendazole (FBZ) were carried out. Sheep microsomal fractions were incubated together with OFZ, FBZ, TCBZ, or a combination of either FBZ and TCBZ or OFZ and TCBZ. OFZ production was significantly diminished upon coincubation of FBZ and TCBZ, whereas neither FBZ nor OFZ affected the S-oxidation of TCBZ towards its sulfoxide and sulfone metabolites. For the in vivo trial, lambs were treated with OFZ (Vermox® oral drench at a single dose of 5 mg/kg PO), TCBZ (Fasinex® oral drench at a single dose of 12 mg/kg PO) or both compounds at a single dose of 5 (Vermox®) and 12 mg/kg (Fasinex®) PO. Blood samples were taken to quantify drug and metabolite concentrations, and pharmacokinetic parameters were calculated by means of non-compartmental analysis. Results showed that the pharmacokinetic parameters of active molecules and metabolites were not significantly altered upon coadministration. The sole exception was the increase in the mean residence time (MRT) of OFZ and FBZ sulfone upon coadministration, with no significant changes in the remaining pharmacokinetic parameters. This research is a further contribution to the study of metabolic drug-drug interactions that may affect anthelmintic efficacies in ruminants.

Effects of fenbendazole and triclabendazole on the expression of cytochrome P450 1A and flavin-monooxygenase isozymes in bovine precision-cut liver slices.

Combinations of the anthelmintics fenbendazole (FBZ) and triclabendazole (TCBZ) have shown enhanced efficacy against the liver fluke Fasciola hepatica. This study aimed to measuring the constitutive expression of CYP1A1, CYP1A2, FMO1 and FMO3, thought to be involved in the metabolism of those compounds, by using an absolute quantitative real time (RT)-PCR approach in bovine precision-cut liver slices (PCLS). It also aimed to characterize the effects of FBZ and TCBZ (alone and in combination) on the expression and activity of the aforementioned isozymes. Both FMO1 and FMO3 were equally represented in control PCLS, whereas CYP1A2 was expressed more than CYP1A1 (P<0.05). PCLS cultured in the presence of beta naphthoflavone (ß-NF; CYP1A inducer) had higher mRNA levels of CYP1A1, CYP1A2, FMO1 and FMO3 (P<0.05). No clear-cut evidence of transcriptional effects of the anthelmintics were recorded. After incubation of PCLS with FBZ, there was a significant increase (P<0.05) vs. controls and TBCZ was observed for CYP1A1. PCLS treated with FBZ showed a higher (P<0.05) expression of CYP1A2 compared to controls, TCBZ alone, and the combination FBZ+TCBZ. The gene expression profiles of FMO1 and FMO3 were not affected by the presence of the anthelmintics; the only exception was an upregulation of FMO3 by TCBZ alone. The observed transcriptional effects of the xenobiotics were not mirrored by increased enzyme activities using prototypical substrates of the isozymes under study. Although further confirmatory studies are needed, these results suggest that PCLS represent an alternative in vitro tool for studies on the expression, regulation and function of relevant xenobiotic-metabolizing enzymes in cattle.

Assessment of P-glycoprotein gene expression in adult stage of Haemonchus contortus in vivo exposed to ivermectin.

The efflux transporter P-glycoprotein (P-gp) has been implicated in multidrug resistance of different nematode parasites affecting livestock species. Increased expression of P-gp in nematodes after their in vitro as well as in vivo exposure to anthelmintics suggests a role of P-gp in drug resistance. The current study evaluated the P-gp gene expression in a highly-resistant isolate of the sheep nematode Haemonchus contortus, selected after exposure to ivermectin (IVM) treatments at 10-fold the therapeutic dose. Four lambs were artificially infected with L3 (7000 L3/animal) of a previously selected IVM highly resistant H. contortus isolate. Forty five (45) days after infection, adult worms were collected at 0 (untreated), 6, 12 and 24 h post-oral IVM (2 mg/kg) administration. The relative transcription levels of different H. contortus P-gp genes were studied by quantitative real-time PCR (qPCR) and confirmed by RNA-seq. P-gp1 and P-gp11 gene expressions did not change throughout the experimental sampling period. P-gp3 and P-gp9.1 transcripts decreased significantly at both 12 and 24 h post IVM exposure. P-gp2 expression was progressively increased in a time-dependent manner at 1.81 (6 h), 2.08 (12 h) and 2.49 (24 h)-fold compared to adult worms not exposed (control 0 h) to IVM, although without reaching statistically significant differences (P > 0.05). P-gp12 was neither detected by qPCR nor by RNA-seq analysis. These relatively modest changes in the P-gp gene expression could not be enough to explain the high level of IVM resistance displayed by the H. contortus isolate under assessment. Overexpression of membrane drug transporters including P-gp has been associated with IVM resistance in different nematode parasites. However, some evidences suggest that resistance to IVM and other macrocyclic lactones may develop by multiple mechanisms. Further studies are needed to improve the understanding of resistance mechanisms in adult stages of H. contortus.

Failure of ivermectin efficacy against Psoroptes ovis infestation in cattle: Integrated pharmacokinetic-pharmacodynamic evaluation of two commercial formulations.

Psoroptic mange is an important parasitic disease that mainly affects beef cattle producing marked economic losses. Ivermectin (IVM) is considered one of the most effective treatments against psoroptic mange and is used worldwide to control both endo and ectoparasites in different species. The current work assessed the relationship between pharmacokinetic behavior of IVM and its efficacy against Psoroptes ovis after the subcutaneous administration of two commercial formulations in a cattle feedlot. Aberdeen Angus and Hereford steers were selected based on the presence of active mite infestations. Animals were allocated into 4 experimental groups and treated with a single (day 0) or repeated subcutaneous injection (days 0 and 7) of one of two commercial formulations of IVM (1%) at 0.2 mg/kg. Blood and skin samples were taken from 8 randomly selected animals of each experimental group to measure IVM concentrations by HPLC. Skin scrapings were also collected from six different sites in each animal, mites were counted and ranked based on a density score. Equivalent plasma concentrations of IVM were measured after the administration of IVM formulations under study. The repeated administration of both IVM formulations at day 0 and 7 accounted for a greater plasma drug availability compared with the single administration (P < 0.05). IVM was well distributed from the plasma to the skin without significant differences between both IVM formulations. There was a positive correlation between IVM concentrations in skin and plasma (r: 0.73 P < 0.0001). The mean ratios between IVM availabililty (measured as AUC) in the skin and in plasma were between 1.2 and 2.1. The repeated administration of IVM increased significantly the IVM concentrations in the skin of areas affected by mange. IVM failed to obtain a parasitological cure in the different groups affected by mange. The failure was observed with both formulations administeredat single or repeated doses. Based on the number of animals cured, the range of efficacy was between 0% on day 7 and 60% on day 28 post-treatment. No significant differences in the P. ovis density scores were observed after the IVM treatment at single or repeated doses. Additional studies are needed to confirm the presence of resistant strains of P.ovis and to establish the appropriate measures to control these parasitic infestations in feedlot cattle.

Assessment of the pharmacological interactions between the nematodicidal fenbendazole and the flukicidal triclabendazole: In vitro studies with bovine liver microsomes and slices.

Parasitic diseases have a significant impact on livestock production. Nematodicidal drugs, such as fenbendazole (FBZ) or its oxidized metabolite oxfendazole (OFZ), can be used along with the trematodicidal triclabendazole (TCBZ), to broaden the spectrum of anthelmintic activity. However, co-exposure to these compounds could lead to drug-drug (D-D) interactions and eventually alter the clinical profile of each active principle. The aim of this study was to assess the presence of such interactions by means of two in vitro models, namely bovine liver microsomal fractions and bovine precision-cut liver slices (PCLSs). To this end, an in vitro assessment involving incubation of FBZ and TCBZ or a combination of FBZ and TCBZ was carried out. Results with microsomal fractions showed a 78.4% reduction (p = .002) in the rate of OFZ production upon co-incubation, whereas the sulfoxide metabolite of TCBZ (TCBZSO) exhibited a decreasing tendency. With PCLS, OFZ accumulation in the incubation medium increased 1.8-fold upon co-incubation, whereas TCBZSO accumulation decreased by 28%. The accumulation of FBZ and OFZ in the liver tissue increased upon 2-hr co-incubation, from 2.1 ± 1.5 to 18.2 ± 6.1 (p = .0009) and from 0.4 ± 0.1 to 1.3 ± 0.3 nmol (p = .0005), respectively. These results confirm the presence of D-D interactions between FBZ and TCBZ. Further studies are needed to determine the extent of involvement of drug-metabolizing enzymes and membrane transporters in interactions between compounds largely used in livestock production systems.

Pharmaco-parasitological evaluation of the ricobendazole plus levamisole nematodicidal combination in cattle.

The goals of the current study were to evaluate the potential pharmacokinetic (PK) interactions and the clinical efficacy occurring after the subcutaneous (s.c.) administration of ricobendazole (RBZ) and levamisole (LEV) given both separately and co-administered to calves naturally infected with susceptible gastrointestinal nematodes. The clinical efficacy was shown in two seasons, winter and spring, with predominance of different nematode populations. Groups of 15 calves were treated with RBZ alone, LEV alone and RBZ + LEV combination, and an untreated group was kept as a Control. RBZ and LEV plasma concentrations were quantified by HPLC. The clinical efficacy was determined by the faecal egg count reduction test. RBZ and LEV have similar plasma persistence, being detected in plasma over 24 hr post-treatment. No PK interactions were observed after the combined treatment, with similar PK parameters (p > .05) obtained for the single-drug and the combination-based strategy. In winter, the observed clinical efficacies were 96%, 99% and 100% for groups treated with RBZ, LEV and RBZ + LEV, respectively; however, in spring, the efficacies were 95%, 93% and 96% for the same groups. Remarkably, the combination was the only treatment that achieved 100% clinical efficacy against both Haemonchus spp and Ostertagia spp in winter; but the increased presence of Ostertagia spp. in spring (28% in untreated group) determined a tendency to reduced efficacies compared to winter time (only 10% of Ostertagia spp. in untreated group), even for the combined treatment. Overall, in a scenario where the nematode population is susceptible, the RBZ + LEV treatment may be a valid combination in cattle to delay the development of resistance, especially in winter when this combination achieved 100% of efficacy. Thus, selection of anthelmintic resistance will never occur. In fact, this is one of the greatest challenges for the whole cattle production system: to be one step ahead of anthelmintic resistance.

Pharmacokinetic assessment of the monepantel plus oxfendazole combined administration in dairy cows.

Monepantel (MNP) is a novel anthelmintic compound launched into the veterinary pharmaceutical market. MNP is not licenced for use in dairy animals due to the prolonged elimination of its metabolite monepantel sulphone (MNPSO2 ) into milk. The goal of this study was to evaluate the presence of potential in vivo drug-drug interactions affecting the pattern of milk excretion after the coadministration of the anthelmintics MNP and oxfendazole (OFZ) to lactating dairy cows. The concentrations of both parent drugs and their metabolites were measured in plasma and milk samples by HPLC. MNPSO2 was the main metabolite recovered from plasma and milk after oral administration of MNP. A high distribution of MNPSO2 into milk was observed. The milk-to-plasma ratio (M/P ratio) for this metabolite was equal to 6.75. Conversely, the M/P ratio of OFZ was 1.26. Plasma concentration profiles of MNP and MNPSO2 were not modified in the presence of OFZ. The pattern of MNPSO2 excretion into milk was also unchanged in animals receiving MNP plus OFZ. The percentage of the total administered dose recovered from milk was 0.09 ± 0.04% (MNP) and 2.79 ± 1.54% (MNPSO2 ) after the administration of MNP alone and 0.06 ± 0.04% (MNP) and 2.34 ± 1.38% (MNPSO2 ) after the combined treatment. The presence of MNP did not alter the plasma and milk disposition kinetics of OFZ. The concentrations of the metabolite fenbendazole sulphone tended to be slightly higher in the coadministered group. Although from a pharmacodynamic point of view the coadministration of MNP and OFZ may be a useful tool, the presence of OFZ did not modify the in vivo pharmacokinetic behaviour of MNP and therefore did not result in reduced milk concentrations of MNPSO2 .

Understanding the main route of drug entry in adult Fasciola hepatica: Further insights into closantel pharmacological activity.

Closantel (CLS) is highly effective against adult liver flukes after its oral or subcutaneous (sc) administration in ruminants. Trans-tegumental diffusion and oral ingestion are the two potential routes available for the entry of drugs into Fasciola hepatica. The work reported here contributes to improve the understanding of CLS pharmacology. The main goals of were: I) to determine the pattern of in vivo CLS accumulation into adult F. hepatica and relevant tissues in CLS-treated sheep; II) to investigate the influence of the physicochemical composition of the incubation medium on the CLS diffusion process into adult F. hepatica; III) to assess the ovicidal activity of CLS against F. hepatica eggs; and IV) to investigate the in vivo effect of CLS treatment on glutathione S-transferases activity in adult liver flukes exposed to CLS. Fourteen healthy sheep were each orally infected with 75 F. hepatica metacercariae. Sixteen (16) weeks after infection, animals were treated with CLS by oral (n = 6, 10 mg/kg) or sub-cutaneous (sc) (n = 6, 5 mg/kg) route. At 12, 24 and 36 h post-treatment, animals were sacrificed (n = 2) and samples of blood, bile and adult F. hepatica were collected. In addition, flukes recovered from non-treated sheep (n = 2) were ex vivo incubated (60 min) in the presence of CLS in either RPMI or bile as incubation medium. CLS concentration was measured by HPLC. The ovicidal activity of CLS was investigated using eggs obtained from the bile of untreated sheep. Finally, glutathione S-transferase activity in F. hepatica recovered from untreated and CLS-treated sheep was assessed. In the in vivo studies, the highest CLS concentrations were measured in plasma and adult liver flukes. A positive correlation was observed between CLS concentration in plasma and in F. hepatica. Results obtained in the current work indicate that the in vivo accumulation of CLS into adult liver flukes occurs mainly by the oral route. After ex vivo incubation, the uptake of CLS by the parasite was markedly diminished in the presence of bile compared with that observed in the presence of RPMI as incubation medium. CLS lacks ovicidal activity at therapeutically relevant concentrations. Lastly, CLS significantly increased glutathione S-transferase activity in flukes recovered at 12 h (oral treatment) and 24 h (sc treatment), compared to the control liver flukes.

Host pharmacokinetics and drug accumulation of anthelmintics within target helminth parasites of ruminants.

Anthelmintic drugs require effective concentrations to be attained at the site of parasite location for a certain period to assure their efficacy. The processes of absorption, distribution, metabolism and excretion (pharmacokinetic phase) directly influence drug concentrations attained at the site of action and the resultant pharmacological effect. The aim of the current review article was to provide an overview of the relationship between the pharmacokinetic features of different anthelmintic drugs, their availability in host tissues, accumulation within target helminths and resulting therapeutic efficacy. It focuses on the anthelmintics used in cattle and sheep for which published information on the overall topic is available; benzimidazoles, macrocyclic lactones and monepantel. Physicochemical properties, such as water solubility and dissolution rate, determine the ability of anthelmintic compounds to accumulate in the target parasites and consequently final clinical efficacy. The transcuticular absorption process is the main route of penetration for different drugs in nematodes and cestodes. However, oral ingestion is a main route of drug entry into adult liver flukes. Among other factors, the route of administration may substantially affect the pharmacokinetic behaviour of anthelmintic molecules and modify their efficacy. Oral administration improves drug efficacy against nematodes located in the gastroinestinal tract especially if parasites have a reduced susceptibility. Partitioning of the drug between gastrointestinal contents, mucosal tissue and the target parasite is important to enhance the drug exposure of the nematodes located in the lumen of the abomasum and/or small intestine. On the other hand, large inter-animal variability in drug exposure and subsequent high variability in efficacy is observed after topical administration of anthelmintic compounds. As it has been extensively demonstrated under experimental and field conditions, understanding pharmacokinetic behaviour and identification of different factors affecting drug activity is important for achieving optimal parasite control and avoiding selection for drug resistance. The search for novel alternatives to deliver enhanced drug concentrations within target helminth parasites may contribute to avoiding misuse, and prolong the lifespan of existing and novel anthelmintic compounds in the veterinary pharmaceutical market.

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.

Gaining Insights Into the Pharmacology of Anthelmintics Using Haemonchus contortus as a Model Nematode.

Progress made in understanding pharmacokinetic behaviour and pharmacodynamic mechanisms of drug action/resistance has allowed deep insights into the pharmacology of the main chemical classes, including some of the few recently discovered anthelmintics. The integration of pharmaco-parasitological research approaches has contributed considerably to the optimization of drug activity, which is relevant to preserve existing and novel active compounds for parasite control in livestock. A remarkable amount of pharmacology-based knowledge has been generated using the sheep abomasal nematode Haemonchus contortus as a model. Relevant fundamental information on the relationship among drug influx/efflux balance (accumulation), biotransformation/detoxification and pharmacological effects in parasitic nematodes for the most traditional anthelmintic chemical families has been obtained by exploiting the advantages of working with H. contortus under in vitro, ex vivo and in vivo experimental conditions. The scientific contributions to the pharmacology of anthelmintic drugs based on the use of H. contortus as a model nematode are summarized in the present chapter.

Hepatic biotransformation pathways and ruminal metabolic stability of the novel anthelmintic monepantel in sheep and cattle.

Monepantel (MNP) is a new amino-acetonitrile derivative anthelmintic drug used for the treatment of gastrointestinal (GI) nematodes in sheep. The present work investigated the main enzymatic pathways involved in the hepatic biotransformation of MNP in sheep and cattle. The metabolic stability in ruminal fluid of both the parent drug and its main metabolite (monepantel sulphone, MNPSO2 ) was characterized as well. Additionally, the relative distribution of both anthelmintic molecules between the fluid and particulate phases of the ruminal content was studied. Liver microsomal fractions from six (6) rams and five (5) steers were incubated with a 40 µm of MNP. Heat pretreatment (50 °C for 2 min) of liver microsomes was performed for inactivation of the flavin-monooxygenase (FMO) system. Additionally, MNP was incubated in the presence of 4, 40, and 80 µm of methimazole (MTZ), a FMO inhibitor, or equimolar concentrations of piperonyl butoxide (PBx), a well-known general cytochrome P450 (CYP) inhibitor. In both ruminant species, MNPSO2 was the main metabolite detected after MNP incubation with liver microsomes. The conversion rate of MNP into MNPSO2 was fivefold higher (P < 0.05) in sheep (0.15 ± 0.08 nmol/min·mg) compared to cattle. In sheep, the relative involvement of both FMO and CYP systems (FMO/CYP) was 36/64. Virtually, only the CYP system appeared to be involved in the production of MNPSO2 in cattle liver. Methimazole significantly reduced (41 to 79%) the rate of MNPSO2 production in sheep liver microsomes whereas it did not inhibit MNP oxidation in cattle liver microsomes. On the other hand, PBx inhibited the production of MNPSO2 in liver microsomes of both sheep (58 to 98%, in a dose-dependent manner) and cattle (almost 100%, independently of the PBx concentration added). The incubation of MNP and MNPSO2 with ruminal contents of both species showed a high chemical stability without evident metabolism and/or degradation as well as an extensive degree of adsorption (83% to 90%) to the solid phase of the ruminal content. Overall, these results are a further contribution to the understanding of the metabolic fate of this anthelmintic drug in ruminants.