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.

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.

Ivermectin-loaded lipid nanocapsules: toward the development of a new antiparasitic delivery system for veterinary applications.

Ivermectin (IVM) is probably one of the most widely used antiparasitic drugs worldwide, and its efficacy is well established. However, slight differences in formulation may change the plasma kinetics, the biodistribution, and in consequence, the efficacy of this compound. The present study focuses on the development of a novel nanocarrier for the delivery of lipophilic drugs such as IVM and its potential application in antiparasitic control. Lipid nanocapsules (LNC) were prepared by a new phase inversion procedure and characterized in terms of size, surface potential, encapsulation efficiency, and physical stability. A complement activation assay (CH50) and uptake experiments by THP-1 macrophage cells were used to assess the stealth properties of this nanocarrier in vitro. Finally, a pharmacokinetics and biodistribution study was carried out as a proof of concept after subcutaneous (SC) injection in a rat model. The final IVM-LNC suspension displayed a narrow size distribution and an encapsulation rate higher than 90 % constant over the evaluated time (60 days). Through flow cytometry and blood permanence measurements, it was possible to confirm the ability of these particles to avoid the macrophage uptake. Moreover, the systemic disposition of IVM in the LNC administered by the SC route was higher (p < 0.05) (1367 ng h/ml) compared to treatment with a commercial formulation (CF) (1193 ng.h/ml), but no significant differences in the biodistribution pattern were found. In conclusion, this new carrier seems to be a promising therapeutic approach in antiparasitic control and to delay the appearance of resistance.

Intestinal drug transport: ex vivo evaluation of the interactions between ABC transporters and anthelmintic molecules.

The family of ATP-binding cassette (ABC) transporters is composed of several transmembrane proteins that are involved in the efflux of a large number of drugs including ivermectin, a macrocyclic lactone (ML) endectocide, widely used in human and livestock antiparasitic therapy. The aim of the work reported here was to assess the interaction between three different anthelmintic drugs with substrates of the P-glycoprotein (P-gp) and the breast cancer resistance protein (BCRP). The ability of ivermectin (IVM), moxidectin (MOX) and closantel (CST) to modulate the intestinal transport of both rhodamine 123 (Rho 123), a P-gp substrate, and danofloxacin (DFX), a BCRP substrate, across rat ileum was studied by performing the Ussing chamber technique. Compared to the controls, Rho 123 efflux was significantly reduced by IVM (69%), CST (51%) and the positive control PSC833 (65%), whereas no significant differences were observed in the presence of MOX (30%). In addition, DFX efflux was reduced between 59% and 72% by all the assayed drug molecules, showing a higher potency than that observed in the presence of the specific BCRP inhibitor pantoprazole (PTZ) (52%). An ex vivo intestinal transport approach based on the diffusion chambers technique may offer a complementary tool to study potential drug interactions with efflux transporters such as P-gp and BCRP.

A pharmacology-based comparison of the activity of albendazole and flubendazole against Echinococcus granulosus metacestode in sheep.

Cyst echinococcosis (CE) is a zoonotic disease caused by the larval stage of the Echinococcus granulosus helminth parasite. The work reported here aimed to compare the efficacy of albendazole (ABZ) and flubendazole (FLBZ) against CE in naturally infected sheep. Additionally, their comparative pharmacokinetic behaviour and the assessment of serum liver enzymes activities were studied. Twelve (12) naturally infected sheep were allocated to the following experimental groups: unmedicated control group, FLBZ-treated and ABZ-treated. Treatments were orally performed every 48 h, over 55 days at dose rate of 10 (FLBZ) and 8.5 (ABZ) mg/kg (equimolar dose rates). The efficacy of the drug treatments was based on protoscoleces' vitality/viability. The kinetic disposition assessment included the Initial and Final Kinetic Studies which implicated the collection of blood samples after both the first and the last drug administration. Blood samples were processed to measure drug concentrations by HPLC. The protoscoleces' vitality observed in the untreated control group (98%) was significantly reduced in the presence of both ABZ and FLBZ. 90% of mice inoculated with protoscoleces in the control group developed hydatid cysts in their peritoneal cavity (viability study). However, only 25% (FLBZ) and 33% (ABZ) of mice inoculated with protoscoleces recovered from treated sheep, developed hydatid cysts in their abdominal cavity. Reduced FLBZ (R-FLBZ) was the main metabolite recovered in the bloodstream after oral administration of FLBZ to sheep. Low plasma concentrations of FLBZ parent drug were measured up to 48 h post-administration. ABZ was not detected in plasma at any time post-treatment, being its metabolites ABZ sulphoxide (ABZSO) and ABZ sulphone (ABZSO2) recovered in plasma. Hepatotoxicity due to the continued treatment with either ABZ or FLBZ was not observed. A 3-fold increase ethoxyresorufin O-deethylase activity, a cytochrome P450 1A (CYP1A)-dependent enzyme reaction, was observed in liver microsomes obtained from sheep receiving ABZ, compared to those of the unmedicated and FLBZ-treated animals. In conclusion, FLBZ is an available anthelmintic which may be developed into an effective and safe drug for the human CE treatment. Despite the low plasma concentrations measured by FLBZ/R-FLBZ, an important reduction in protoscoleces' vitality was observed in cysts located in sheep liver. Modern pharmaceutical technology may help to greatly improve FLBZ systemic exposure improving its efficacy against CE.

A high oxfendazole dose to control porcine cysticercosis: pharmacokinetics and tissue residue profiles.

Oxfendazole (OFZ) is efficacious for porcine cysticercosis at 30 mg/kg. OFZ is not registered to be used at this dose. The assessment of the OFZ and metabolites [(fenbendazole sulphone (FBZSO2), fenbendazole (FBZ)] plasma pharmacokinetic and tissue residue profiles after its oral administration to pigs and the withdrawal period for human consumption were reported. Forty-eight pigs allocated into two groups received OFZ (30 mg/kg) orally as a commercial (CF) or as experimental formulation (SMF). Samples (blood, muscle, liver, kidney and fat) were collected over 30 days post-treatment and analyzed by HPLC. OFZ was the main compound recovered in plasma, followed by FBZSO2 and low FBZ concentrations. OFZ AUC0-LOQ (209.9±33.9 µg·h/ml) and Cmax (5.40±0.65 µg/ml) parameters for the CF tended to be higher than those for the SMF (AUC0-LOQ: 159.4±18.3 µg h/ml, Cmax: 3.80±0.35 µg/ml). The highest total residue (OFZ+FBZSO2+FBZ) concentrations were quantified in liver, followed by kidney, muscle and fat tissue. FBZSO2 residue levels were the highest found in muscle (0.68±0.39 µg/g) and fat (0.69±0.39 µg/g). In liver and kidney the highest residues corresponded to FBZ (5.29±4.36 µg/g) and OFZ (2.86±0.75 µg/g), respectively. A withdrawal time of 17 days post-treatment was established before tissues are delivered for human consumption.

Flubendazole and ivermectin in vitro combination therapy produces a marked effect on Echinococcus granulosus protoscoleces and metacestodes.

The aim of the present work was to evaluate the in vitro efficacy of the flubendazole (FLBZ) and ivermectin (IVM) combination against Echinococcus granulosus protoscoleces and metacestodes. Protoscoleces and groups of ten peritoneal cysts obtained from BALB/c mice were incubated with the two drugs, either separately or in combination, at the following final concentrations: 10 microg/mL FLBZ, 1 microg/mL FLBZ, 1 microg/mL IVM, 10 microg/mL FLBZ + 1 microg/mL IVM, and 1 microg/mL FLBZ + 1 microg/mL IVM. The maximum protoscolicidal effect was found with the combination 10 microg/mL FLBZ + 1 microg/mL IMV. After 1 day of incubation, the presence of numerous blebs in the tegument of protoscoleces was observed. Ultrastructural studies revealed that the primary site of damage was the tegument of the parasite. The effect of the two drugs on hydatid cysts obtained from mice was more rapidly detected in cysts treated with the combination of FLBZ + IVM than when drugs were used separately. Ultrastructural studies revealed that the germinal layer of treated cysts lost the multicellular structure feature and underwent considerable degenerative changes after in vitro treatment. The outcomes obtained demonstrated the favorable effect of the combination of FLBZ and IVM against E. granulosus.

In vitro and in vivo effects of flubendazole on Echinococcus granulosus metacestodes.

The aim of the present work was to determine the efficacy of flubendazole (FLBZ) against Echinococcus granulosus metacestodes by using in vitro and in vivo models. Groups of 50 microcysts developed in vitro, and groups of 10 peritoneal cysts were obtained from Balb C mice with experimental secondary infections of 8 months. The cysts were placed in Leighton tubes containing 10 ml of culture medium. FLBZ was added to the medium resulting in final concentrations of 5 and 1 microg/ml for mycrocysts treatment and 10, 5, and 1 microg/ml for murine cysts treatment. In vivo treatment was performed on 20 mice that developed an experimental secondary hydatid disease over a period of 11 months. FLBZ was given (1.5 mg/kg) by the oral route once a day for 50 days. A loss of turgidity was detected in all in vitro drug treated cysts irrespective of the drug concentration or parasite origin. Inspection of treated cysts by scanning electron microscopy (SEM) revealed that the germinal layer lost it characteristic multicelular structure. These results were confirmed on the ultrastructural level by transmission electron microscopy (TEM), treated metacestodes had undergone considerable degenerative changes after the in vitro treatment. The results obtained after the in vivo treatment with FLBZ showed no significant difference between the control and treated groups related to the weight of cyst masses. However, the ultrastructural study at TEM of cysts that developed in mice from the treated group revealed alterations in the germinal layer with the presence of numerous vacuoles. With regard to the ultrastructural study at SEM, only cellular debris of the germinal layer could be seen. In conclusion, the data obtained clearly demonstrate that in vitro and in vivo treatment with FLBZ is effective against E. granulosus metacestodes.

Ivermectin disposition kinetics after subcutaneous and intramuscular administration of an oil-based formulation to cattle.

Slight differences in formulation may change the plasma kinetics and ecto-endoparasiticide activity of endectocide compounds. This work reports on the disposition kinetics and plasma availability of ivermectin (IVM) after subcutaneous (SC) and intramuscular (IM) administration as an oil-based formulation to cattle. Parasite-free Aberdeen Angus calves (n = 24; 240-280 kg) were divided into three groups (n = 8) and treated (200 microg/kg) with either an IVM oil-based pharmaceutical preparation (IVM-TEST formulation) (Bayer Argentina S.A.) given by subcutaneous (Group A) and intramuscular (Group B) injections or the IVM-CONTROL (non-aqueous formulation) (Ivomec, MSD Agvet) subcutaneously administered (Group C). Blood samples were taken over 35 days post-treatment and the recovered plasma was extracted and analyzed by HPLC using fluorescence detection. IVM was detected in plasma between 12 h and 35 days post-administration of IVM-TEST (SC and IM injections) and IVM-CONTROL formulations. Prolonged IVM absorption half-life (p < 0.05) and delayed peak plasma concentration (p < 0.001) were obtained following the SC administration of the IVM-TEST compared to the IVM-CONTROL formulation. No differences in total plasma availability were observed among treatments. However, the plasma residence time and elimination half-life of IVM were significantly longer after injection of the IVM-TEST formulation. IVM plasma concentrations were above 0.5 ng/ml for 20.6 (CONTROL) and 27.5 days (IVM-TEST SC), respectively (p < 0.05). The modified kinetic behaviour of IVM obtained after the administration of the novel oil-based formulation examined in this trial, compared to the standard preparation, may positively impact on its strategic use in cattle.

Impact of liposomes as delivery systems in veterinary medicine.

The development of drug resistance and the inability of the drug to reach the location of the etiologic agents are major challenges for anti-infective and cancer therapies. As the development of new drugs with improved pharmacodynamic and pharmacokinetic properties is a slow and difficult process, drug delivery systems appear as promising alternatives. Liposomes are lipid vesicles formed when phospholipids are exposed to an aqueous environment. They arrange themselves in bilayers and close up, forming a vesicle. During this process they capture the aqueous phase of the dispersion, and any substance dissolved in it, within the vesicle. Liposomes have remarkable features that make them an almost ideal delivery system. They are biodegradable, with few side effects, can deliver drugs with different physico-chemical properties together and can be formulated for different routes of administration. The potential to modify the pharmacokinetic behaviour of encapsulated drugs to deliver them selectively to the site of action is the most important feature of liposomes as drug delivery systems. Liposomes are already used in human medicine for he treatment of bacterial, viral and parasitic diseases, and cancer. They have also been proven useful as immunoadjuvants and vaccines. Liposomes are used in certain avian vaccines. The possible uses of liposomes and their impact in veterinary medicine in the treatment of infectious diseases and cancer as well as in the prevention of diseases are discussed in the present article.

Pharmacokinetics of ticardillin after intravenous administration in sheep

Se midieron las concentraciones séricas de ticarcilina entre las 0.042 y las 12 h en seis ovinos adultos y sanos, luego de la administración de 40 mg/Kg por la vía intravenosa. La curva semilogarítmica concentración versus tiempo fue desripta monoexponencialmente. El análisis farmacocinético dio como resultados una constante de tasa de eliminación de 2,0214 ñ 0,657 1h, una vida media de eliminación de 0,37 ñ 0,097 h, un volumen de distribución de 398 ñ 111 ml/Kg y un aclaramiento corporal de 744,43 ñ 199,24 ml/h.Kg. La conclusión fué que la ticarcilina es una droga moderadamente distribuida y rápidamente eliminada, cuando se la administra en forma endovenosa a ovinos