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.

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.

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.

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.

Effects of the Antiparasitic Drug Moxidectin in Cattle Dung on Zooplankton and Benthic Invertebrates and its Accumulation in a Water-Sediment System.

Two anthelmintic macrocyclic lactones-ivermectin and moxidectin-have revolutionized parasite control in cattle. These drugs are only partly metabolized by livestock, and the main route of excretion is via feces. In seasonally inundated floodplains, cattle feces come into direct contact with surface water. Important differences in pharmacokinetics and pharmacodynamics between these drugs may bear on their ecotoxicology in aquatic ecosystems. Moxidectin strongly binds to organic matter and thereby may be consumed in aquatic food webs, but there is a scarcity of data on toxicity to freshwater invertebrates. The objectives of this work were to determine the effect of moxidectin spiked in cattle dung on survival and growth of three representative aquatic invertebrates: the zooplankton Ceriodaphnia dubia, the amphipod Hyalella curvispina, and the snail Pomacea canaliculata. Moxidectin-laced dung was added in microcosms and concentrations were measured in water, sediment + dung, roots of the aquatic plant Salvinia biloba, and the aforementioned invertebrates. The influence of moxidectin on nutrient concentrations was also evaluated. Dung was spiked with moxidectin to attain concentrations of 750, 375 and 250 µg kg-1 dung fresh weight, approximating those found in cattle dung at days 2, 3, and 5 following subcutaneous injection. Concentrations of moxidectin in dung during the first week of excretion were lethally toxic for the tested invertebrate taxa. The persistence of moxidectin in the sediment + dung and the uptake of the drug in roots of S. biloba increase its potential exposure to aquatic food webs. Moxidectin also reduced the rate of release of soluble reactive phosphorus to the water.

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.

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 .

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.

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.

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.

Comparative pharmacokinetic and pharmacodynamic response of single and double intraruminal doses of ivermectin and moxidectin in nematode-infected lambs.

AIMS: To compare the pharmacokinetics, distribution and efficacy (pharmacodynamic response) of intraruminal ivermectin (IVM) and moxidectin (MXD) administered at 0.2 and 0.4 mg/kg to naturally nematode-infected lambs, and to determine the ex vivo accumulation of these anthelmintics by Haemonchus contortus. METHODS: Romney Marsh lambs, naturally infected with IVM-resistant H. contortus, were allocated to treatment groups based on faecal nematode egg counts. They received 0.2 or 0.4 mg/kg IVM or MXD (n=10 per group), or no treatment (Control; n=6), on Day 0. Samples from four animals from each treatment group, including abomasal parasites, were obtained on Day 1. Plasma samples were also collected from Day 0 to 14, and a faecal egg count reduction test (FECRT) and a controlled efficacy trial were carried out on Day 14. Concentrations of IVM and MXD in plasma, in abomasal and intestinal tissues and in H. contortus were evaluated by high-performance liquid chromatography. Additionally, the ex vivo drug accumulation of IVM and MXD by H. contortus was determined. RESULTS: Peak plasma concentrations and the area under the concentration vs. time curve for both IVM and MXD were higher for 0.4 than 0.2 mg/kg treatments (p<0.05), but there were no differences for other parameters. Concentrations of IVM and MXD in the gastrointestinal target tissues and in H. contortus were higher compared to those measured in plasma. Concentrations of both drugs in H. contortus were correlated with those observed in the abomasal content (r=0.86; p<0.0001). The exposure of H. contortus to IVM and MXD was related to the administered dose. Mean FECRT and efficacy for removal of adult H. contortus was 0% for IVM at 0.2 and 0.4 mg/kg. For MXD, FECRT were >95% for both treatments, and efficacy against H. contortus was 85.1% and 98.1% for 0.2 and 0.4 mg/kg, respectively. The ex vivo accumulation of IVM and MXD in H. contortus was directly related to the drug concentration present in the environment and was influenced by the duration of exposure. CONCLUSION: Administration of IVM and MXD at 0.4 compared with 0.2 mg/kg accounted for enhanced drug exposure in the target tissues, as well as higher drug concentrations within resistant nematodes. The current work is a further contribution to the evaluation of the relationship between drug efficacy and basic pharmacological issues in the presence of resistant parasite populations.

Prevalencia de patología cardíaca en la enfermedad de Chagas: años 2004-2012. Programa y Red de Chagas de CABA / Prevalence of cardiac pathology in Chagas disease: 2004 -2012. Program and Chagas CABA network

El Instituto Universitario de Ciencias de las Salud ha mostrado un particular compromiso con la formación de sus estudiantes en la estrategia de Atención Primaria de la Salud, con las prácticas asistenciales dedicadas al 1er nivel de atención ambulatoria y a las patologías prevalentes en ese ámbito. Del mismo modo se han desenvuelto las actividades de formación en investigación. Como exponente de esa orientación, la revista Ciencias de la Salud publicó en el Vol. 2, N°1, 2011:4-9, el artículo “Prevalencia de la Enfermedad de Chagas” de Érica G. Morais, que había obtenido el premio “Futuros Líderes”, otorgado por el Curso Anual Internacional de Investigación en Ciencias de la Salud (IUCS-AMA, Prof. Carlos Álvarez Bermúdez). Aquella investigación formaba parte de un proyecto más amplio realizado en el Hospital Teodoro Álvarez entre 2004 y 2012, en el que participaron un conjunto de investigadores, que compartieron la autoría de la actual publicación. El Dr. Jorge Mitelman, Prosecretario de Ciencia y Técnica del IUCS e integrante de ese equipo, preparó además una reseña sobre la jornada del INCOSUR, realizada en abril del presente año, describiendo asimismo el proceso de desarrollo de la Ciudad de Buenos Aires, como área no endémica, para encarar las consecuencias de la enfermedad de Chagas

Closantel plasma and milk disposition in dairy goats: assessment of drug residues in cheese and ricotta.

Closantel (CLS) is currently used in programs for the strategic control of gastrointestinal nematodes. CLS is extralabel used in different dairy goat production systems. From available data in dairy cows, it can be concluded that residues of CLS persist in milk. The current work evaluated the concentration profiles of CLS in plasma and milk from lactating orally treated dairy goats to assess the residues pattern in dairy products such as cheese and ricotta. Six (6) female Saanen dairy goats were treated orally with CLS administered at 10 mg/kg. Blood and milk samples were collected between 0 and 36 days post-treatment. The whole milk production was collected at 1, 4, 7, and 10 days post-treatment to produce soft cheese and ricotta. CLS concentrations in plasma, milk, cheese, whey, and ricotta were determined by HPLC. The concentrations of CLS measured in plasma were higher than those measured in milk at all sampling times. However, the calculated withdrawal time for CLS in milk was between 39 and 43 days postadministration to dairy goats. CLS residual concentrations in cheese (between 0.93 and 1.8 µg/g) were higher than those measured in the milk used for its production. CLS concentrations in ricotta were sixfold higher than those in the milk and 20-fold higher than those in the whey used for its production. The persistent and high residual concentrations of CLS in the milk and in the cheese and ricotta should be seriously considered before issuing any recommendation on the extralabel use of CLS in dairy goat farms.

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.

P-glycoprotein in sheep liver and small intestine: gene expression and transport efflux activity.

The role of the transporter P-glycoprotein (P-gp) in the disposition kinetics of different drugs therapeutically used in veterinary medicine has been demonstrated. Considering the anatomo-physiological features of the ruminant species, the constitutive expression of P-gp (ABCB1) along the sheep gastrointestinal tract was studied. Additionally, the effect of repeated dexamethasone (DEX) administrations on the ABCB1 gene expression in the liver and small intestine was also assessed. The ABCB1 mRNA expression was determined by real-time quantitative PCR. P-gp activity was evaluated in diffusion chambers to determine the efflux of rhodamine 123 (Rho 123) in the ileum from experimental sheep. The constitutive ABCB1 expression was 65-fold higher in the liver than in the intestine (ileum). The highest ABCB1 mRNA expression along the small intestine was observed in the ileum (between 6- and 120-fold higher). The treatment with DEX did not elicit a significant effect on the P-gp gene expression levels in any of the investigated gastrointestinal tissues. Consistently, no significant differences were observed in the intestinal secretion of Rho 123, between untreated control (Peff S-M = 3.99 × 10(-6)  ± 2.07 × 10(-6) ) and DEX-treated animals (Peff S-M = 6.00 × 10(-6)  ± 2.5 × 10(-6) ). The understanding of the efflux transporters expression and activity along the digestive tract may help to elucidate clinical implications emerging from drug interactions in livestock.

Effects of sub-lethal exposure of rats to the herbicide glyphosate in drinking water: glutathione transferase enzyme activities, levels of reduced glutathione and lipid peroxidation in liver, kidneys and small intestine.

Glyphosate (GLP), the active ingredient of many weed killing formulations, is a broad spectrum herbicide compound. Wistar rats were exposed during 30 or 90 days to the highest level (0.7 mg/L) of GLP allowed in water for human consumption (US EPA, 2011) and a 10-fold higher concentration (7 mg/L). The low levels of exposure to the herbicide did not produce histomorphological changes. The production of TBARS was similar or tended to be lower compared to control animals not exposed to the herbicide. In rats exposed to GLP, increased levels of reduced glutathione (GSH) and enhanced glutathione peroxidase (GPx) activity may act as a protective mechanism against possible detrimental effects of the herbicide. Overall, this work showed certain biochemical modifications, even at 3-20-fold lower doses of GLP than the oral reference dose of 2mg/kg/day (US EPA, 1993). The toxicological significance of these findings remains to be clarified.

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

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

Macrocyclic lactones and cellular transport-related drug interactions: a perspective from in vitro assays to nematode control in the field.

Macrocyclic lactones (MLs) are antiparasitic drugs used against endo-ectoparasites. Regarding the wide use of MLs in different species, it is likely that drug-drug interactions may occur after their co-administration with other compounds. A new paradigm was introduced in the study of the pharmacology of MLs during the last years since the interactions of MLs with ATP-binding cassete (ABC) transporters have been described. The current review article gives an update on the available information concerning drug-drug interactions involving the MLs. The basis of the methodological approaches used to evaluate transport interactions, and the impact of the pharmacology-based modulation of drug transport on the MLs disposition kinetics and clinical efficacy, are discussed in an integrated manner. A different number of in vitro and ex vivo methods have been reported to study the characterization of the interactions between MLs and ABC transporters. The production of the ABC transporters knockout mice has provided valuable in vivo tools to study this type of drug-drug interaction. In vivo trials performed in different species corroborated the effects of ABC transporter modulators on the pharmacokinetics behaviour of MLs. Important pharmacokinetic changes on plasma disposition of MLs have been observed when these compounds are co-administered with P-glycoprotein modulators. The modulation of the activity of P-glycoprotein was evaluated as a strategy not only to increase the systemic availability of MLs but also to improve their clinical efficacy. The understanding of the MLs interactions may supply relevant information to optimize their use in veterinary and human therapeutics.