Candela Canton

Integrated assessment of ivermectin pharmacokinetics, efficacy against resistant Haemonchus contortus and P-glycoprotein expression in lambs treated at three different dosage levels

The main goals of the current work were: (a) to assess the ivermectin (IVM) systemic exposure and plasma disposition kinetics after its administration at the recommended dose, x5 and x10 doses to lambs, (b) to compare the clinical efficacy of the same IVM dosages in lambs infected with an IVM-resistant isolate of Haemonchus contortus, and (c) to assess the expression of the transporter protein P-glycoprotein (P-gp) in H. contortus recovered at 14 days after administration of the IVM dose regimens. There were two separated trials where IVM was administered either subcutaneously (SC, Experiment I) or intraruminally (IR, Experiment II). Each experiment involved twenty-four (24) lambs artificially infected with a highly resistant H. contortus isolate. Animals were allocated into 4 groups (n=6) and treated with IVM at either 0.2 (IVM x1), 1 (IVM x5) or 2mg/kg (IVM x10). Plasma samples were collected up to 12 days post-treatment and analysed by HPLC. An untreated-control Group was included to assess the comparative anthelmintic efficacy of the different treatments. The level of expression of Pgp in H. contortus specimens obtained from lambs both untreated and IR treated with the different IVM doses was quantified by real time PCR. Parametric and non-parametric tests were used to compare the statistical significance of the results (P<0.05). After the SC treatment, the IVM plasma area under the concentration-time curve (AUC0-LOQ) increased from 41.9 (IVM SCx1) up to 221 (IVM SCx5) and 287 (IVM SCx10)ng.day/mL and after the IR treatment from 20.8 (IVM IRx1) up to 121 (IVM IRx5) and 323 (IVM IRx10)ng.day/mL. Dose-adjusted AUC0-LOQ and Cmax were similar among doses, demonstrating dose proportionality for IVM after both SC and IR administration at the three different doses. The efficacies against resistant H. contortus after the SC treatment were 42% (IVM SC1), 75% (IVM SCx5) and 75% (IVM SCx10). However, the IR IVM treatment reached clinical efficacies ranging from 48% (IVM IRx1) up to 96% (IVM IRx5) and 98% (IVM IRx10). None of the IR IVM treatments increased the expression of P-gp in adult H. contortus at 14 days post-treatment compared to samples collected from the untreated control group. An enhanced parasite exposure of the drug at the abomasum may explain the improved efficacy against this recalcitrant H. contortus isolate observed only after the IR administration at 5- and 10-fold the IVM therapeutic dosage.

Resistant nematodes in cattle: Pharmaco-therapeutic assessment of the ivermectin- ricobendazole combination.

Nematodicidal combinations have been proposed as a valid strategy to achieve effective nematode control in the presence of drug resistance. The goals of this study were: (1) to compare the clinical efficacy (therapeutic response) of ivermectin (IVM) and ricobendazole (RBZ) given subcutaneously either by separate or combined administration to calves naturally infected with gastrointestinal nematodes resistant to IVM, and (2) to evaluate the potential pharmacokinetic (PK) and/or pharmacodynamic (PD) interactions occurring after the co-administration of both anthelmintics. Sixty male calves naturally infected with gastrointestinal nematodes resistant to IVM were randomly allocated into four groups (n=15). Untreated control: animals not receiving anthelmintic treatment; IVM alone: animals treated with IVM by subcutaneous (SC) injection (0.2mg/kg); RBZ alone: animals received RBZ by the SC route (3.75mg/kg); IVM+RBZ: animals treated with IVM and RBZ (0.2 and 3.75mg/kg, respectively), by SC injection in two separates sites. Eight animals of each treated group were randomly selected to perform the PK study. Plasma samples were taken from those animals up to 28days post-treatment. IVM and RBZ plasma concentrations were quantified by HPLC. The therapeutic response was determined by faecal egg count reduction test (FECRT). The proportions of third-stage larvae (L3) recovered from coprocultures were used to calculate the efficacy against the main parasite genera. The daily total egg deposition for each experimental group was estimated. Similar pharmacokinetic trends were obtained for both IVM and RBZ allying the single-drug and the combined treatments, which indicates the absence of PK interactions between both anthelmintics. The observed overall clinical drug efficacies were 48% (IVM alone), 94% (RBZ alone) and 98% (IVM+RBZ). Haemonchus spp. and Cooperia spp. were recovered in the coproculture after IVM treatment, suggesting that resistance to IVM includes both genera. In fact, the efficacy against Cooperia spp. was 83% (IVM), 98% (RBZ) and 98% (IVM+RBZ), while the efficacy against Haemonchus spp. was 0% (IVM), 97% (RBZ) and 100% (IVM+RBZ). The combination was the only treatment that achieved 100% clinical efficacy against IVM-resistant Haemonchus spp. The total egg excretion was reduced to 49.9% (IVM alone group), 6.3% (RBZ alone group) and 1.8% (IVM+RBZ combined group) compared to the untreated control. Although the combined treatment did not significantly increase the overall clinical efficacy in the current natural field conditions, an additive effect was achieved against IVM-resistant nematodes. In fact, the combination obtained significantly higher efficacy against IVM-resistant Haemonchus spp. than RBZ alone. Additionally, the epidemiological relevance of the reduction in the number of eggs excreted following the combined treatment is not negligible and should be taken into account in future studies. Further work is required to understand the advantages of nematodicidal combinations in different natural anthelmintic resistance scenarios.

Oxfendazole flukicidal activity in pigs.

Although oxfendazole (OFZ) is a well know broad-spectrum benzimidazole anthelmintic, the assessment of its potential trematodicidal activity remains unexplored. OFZ administration at single high doses has been recommended to control Taenia solium cysticercus in pigs. The current study investigated the flukicidal activity obtained after a single high (30mg/kg) oral dose of OFZ in pigs harbouring a natural Fasciola hepatica infection. Sixteen (16) local ecotype pigs were randomly allocated into two (2) experimental groups of 8 animals each named as follow: Untreated control and OFZ treated, in which animals received OFZ (Synanthic(®), Merial Ltd., 9.06% suspension) orally at 30mg/kg. At seven (7) days post-treatment, all the animals were sacrificed and direct adult liver fluke counts were performed following the WAAVP guidelines. None of the animals involved in this experiment showed any adverse event during the study. OFZ treatment as a single 30mg/kg oral dose showed a 100% efficacy against F. hepatica. In conclusion, the trial described here demonstrated an excellent OFZ activity against F. hepatica in naturally infected pigs, after its administration at a single oral dose of 30mg/kg.

Field trial assessment of ivermectin pharmacokinetics and efficacy against susceptible and resistant nematode populations in cattle

The study compared the pharmacokinetic (PK) behaviour and anthelmintic efficacy against susceptible and resistant nematodes following subcutaneous (SC) and oral administration of ivermectin (IVM) to cattle. Six commercial farms were involved: Farms 1 and 2 (IVM-susceptible nematode population) and Farms 3, 4, 5 and 6 (IVM-resistant nematode population). On each farm, forty-five calves naturally infected with gastrointestinal (GI) nematodes were randomly allocated into three groups (n = 15): untreated control, IVM SC administration, and IVM oral administration (both at 0.2 mg/kg). PK assessment (plasma and faeces) was performed on Farm 1. Efficacy was determined by Faecal Egg Count Reduction Test. IVM systemic availability upon SC administration (421 ± 70.3 ng·d/mL) was higher (P < 0.05) compared to the oral treatment (132 ± 31.3 ng·d/mL). However, higher (P < 0.05) faecal IVM concentrations were observed following oral treatment (9896 ± 1931 ng·d/mL) compared to SC administration (4760 ± 924 ng·d/mL). Similar (91-93%) IVM efficacy was observed on Farms 1 and 2 by both routes. Efficacy against resistant nematodes was slightly higher on Farms 3 and 4 after the oral (63 and 82%, respectively) compared to the SC (36 and 68%, respectively) treatment. However, there was complete therapeutic failure (0% efficacy) on Farm 5 and a very low response on Farm 6 (40 and 41% for SC and oral administration, respectively). Although larger faecal concentrations following IVM oral administration may increase drug exposure of GI adult worms, this does not always improve efficacy against resistant nematodes. The potential therapeutic advantages of oral treatments should be cautiously assessed, especially in presence of anthelmintic resistance.

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.

Strategies to Optimize the Efficacy of Anthelmintic Drugs in Ruminants

Anthelmintic resistance in human and animal pathogenic helminths has been spreading in prevalence and severity. Multidrug resistance is a widespread problem in livestock animals. The use of available pharmacology-based information is critical to the design of successful future approaches for parasite control. Relevant scientific work supporting the main strategies to optimize anthelmintic therapy in ruminants under the current drug-resistance scenario is described here. We emphasize the need for further integrated pharmaco-parasitological knowledge to extend the lifespan of both traditional and novel anthelmintic compounds, and to progress in the identification of complementary/alternative measures of parasite control in livestock animals.