L. Maté

Hepatic and extra-hepatic metabolic pathways involved in flubendazole biotransformation in sheep.

Flubendazole (FLBZ) is a broad-spectrum benzimidazole anthelmintic compound used in pigs, poultry and humans. Its potential for parasite control in ruminant species is under investigation. The objective of the work described here was to identify the main enzymatic pathways involved in the hepatic and extra-hepatic biotransformation of FLBZ in sheep. Microsomal and cytosolic fractions obtained from sheep liver and duodenal mucosa metabolised FLBZ into a reduced FLBZ metabolite (red-FLBZ). The keto-reduction of FLBZ led to the prevalent (approximately 98%) stereospecific formation of one enantiomeric form of red-FLBZ. The amounts of red-FLBZ formed in liver subcellular fractions were 3-4-fold higher (P<0.05) compared to those observed in duodenal subcellular fractions. This observation correlates with the higher (P<0.05) carbonyl reductase (CBR) activities measured in the liver compared to the duodenal mucosa. No metabolic conversion was observed following FLBZ or red-FLBZ incubation with sheep ruminal fluid. Sheep liver microsomes failed to convert red-FLBZ into FLBZ. However, this metabolic reaction occurred in liver microsomes prepared from phenobarbital-induced rats, which may indicate a cytochrome P450-mediated oxidation of red-FLBZ. A NADPH-dependent CBR is proposed as the main enzymatic system involved in the keto-reduction of FLBZ in sheep. CBR substrates such as menadione and mebendazole (a non-fluoride analogue of FLBZ), inhibited this liver microsomal enzymatic reaction, which may confirm the involvement of a CBR enzyme in FLBZ metabolism in sheep. This research is a further contribution to the understanding of the metabolic fate of a promissory alternative compound for antiparasitic control in ruminant species.

Comparative tissue pharmacokinetics and efficacy of moxidectin, abamectin and ivermectin in lambs infected with resistant nematodes: Impact of drug treatments on parasite P-glycoprotein expression.

Graphical abstract Highlights ► Macrocyclic lactones share some structural and physico-chemical properties. ► The kinetic-dynamic differences of these compounds should be addressed under in vivo standardized conditions. ► Lower concentrations of moxidectin were obtained in resistant Haemonchus contortus. ► Moxidectin showed a higher efficacy against resistant H. contortus. ► The P-glycoprotein expression in H. contortus was increased after ivermectin treatment.

In vivo and ex vivo assessment of the interaction between ivermectin and danofloxacin in sheep.

The impact of an efflux pump-related interaction between ivermectin and danofloxacin on their intestinal transport (ex vivo) and disposition kinetics (in vivo) was assessed. Eighteen male Corriedale sheep were randomly assigned to one of three groups. Animals in Group A received 0.2mg/kg ivermectin by SC injection, those in Group B were given 6 mg/kg danofloxacin SC on two occasions 48 h apart and those in Group C were treated with both compounds at the same rates. Plasma concentrations of ivermectin and danofloxacin were measured by HPLC using fluorescence detection. Ex vivo intestinal drug transport activity was measured by the use of the Ussing chamber technique. Plasma concentrations of ivermectin in the first 6 days after injection tended to be higher in Group C than Group A. Contemporaneous treatment with ivermectin significantly increased systemic exposure to danofloxacin (AUC values were 32-35% higher) and prolonged the elimination half-life of danofloxacin (40-52% longer). Ex vivo, incubation with ivermectin significantly decreased the efflux transport of rhodamine 123, a P-glycoprotein substrate, in sheep intestine, but no significant effect of danofloxacin on transport activity was observed. Evaluation of the interaction of danofloxacin with the breast cancer resistance protein (BCRP) showed that pantoprazole and ivermectin significantly decreased danofloxacin secretion in the rat intestine. Thus, the ivermectin-induced reduction of danofloxacin efflux transport observed in this study may involve BCRP activity but the involvement of P-glycoprotein cannot be ruled out.

Pretreatment with the inducers rifampicin and phenobarbital alters ivermectin gastrointestinal disposition.

The goal of the study was to evaluate the effects of rifampicin (RFP) and phenobarbital (PBT) on the plasma and gastrointestinal disposition kinetics of ivermectin (IVM) subcutaneously administered to Wistar rats. Fifty seven rats were used. Animals in Group I were the noninduced (control) group. Those in Groups II and III received a treatment with RFP (160 mg/day) and PBT (35 mg/day), respectively, both given orally during eight consecutive days as induction regimen. The IVM pharmacokinetic study was started 24 h after the RFP and PBT last administration. Animals received IVM (200 microg/kg) by subcutaneous injection. Rats were sacrificed between 6 h and 3 days after IVM administration. Blood and samples of liver tissue, intestinal wall and luminal content of jejunum were collected from each animal. IVM concentrations were measured by high performance liquid chromatography. IVM disposition kinetics in plasma and tissues was significantly modified by the PBT treatment, but not by RFP. Despite the enhanced CYP3A activity observed after the pretreatment with RPF and PBT, there were no marked changes on the percentages of IVM metabolites recovered from the bloodstream in induced and noninduced animals. An enhanced P-glycoprotein-mediated intestinal transport activity in pretreated animals (particularly in PBT pretreated rats) may explain the drastic changes observed on IVM disposition.

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.

Influence of 25-hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3 on expression of P-glycoprotein and cytochrome P450 3A in sheep ☆

In order to improve calcium and phosphorus balance, beef cattle and dairy cows can be supplemented with vitamin D. However, different vitamin D metabolites have been shown to increase expression of P-glycoprotein (P-gp, MDR1, ABCB1) and cytochrome P450 3A (CYP3A) in rodents as well as in cell culture systems. As such interferences might have an impact on pharmacokinetics of some drugs widely-used in veterinary medicine, we investigated the expression of P-gp, CYP3A, vitamin D receptor (VDR), pregnane X receptor (PXR) and retinoid X receptor α (RXRα) in sheep either treated orally with 6μg/kg body weight (BW) 25-hydroxyvitamin D3 (OHD3) for ten days before sacrifice or 12h after intravenous injection of 0.5μg/kg BW 1,25-dihydroxyvitamin D3 (1,25- (OH)2D3). Down-regulation of ruminal, jejunal and hepatic, but not renal P-gp could be found with 25-OHD3 supplementation. Interestingly, this effect on P-gp was not observed in tissues from 1,25-(OH)2D3-treated sheep. In contrast, 1,25-(OH)2D3 induced a significant up-regulation of renal and jejunal CYP3A expression, while 25-OHD3 had no impact. Renal expression of VDR and PXR was also increased by treatment with 1,25-(OH)2D3, while jejunal PXR expression was only stimulated in sheep supplemented with 25-OHD3. Either treatments increased renal, but not ruminal, jejunal or hepatic expression of RXRα. These results demonstrate that the impact of large doses of vitamin D metabolites on different target organs and potential interactions with other medications should be further investigated in vitro and in vivo to understand the effects of vitamin D metabolites on metabolism and excretion pathways in livestock.

Ivermectin Pharmacokinetics, Metabolism, and Tissue/Egg Residue Profiles in Laying Hens

The goals were to determine the ivermectin (IVM) plasma pharmacokinetics, tissue and egg residue profiles, and in vitro metabolism in laying hens. Experiments conducted were (1) 8 hens were intravenously treated with IVM and blood samples taken; (2) 88 hens were treated with IVM administered daily in water (5 days) (40 were kept and their daily eggs collected; 48 were sacrificed in groups (n = 8) at different times and tissue samples taken and analyzed); (3) IVM biotransformation was studied in liver microsomes. Pharmacokinetic parameters were AUC = 85.1 ng·day/mL, Vdss = 4.43 L/kg, and T1/2el = 1.73 days. Low IVM tissue residues were quantified with the highest measured in liver and skin+fat. IVM residues were not found in egg white, but significant amounts were quantified in yolk. Residues measured in eggs were greater than some MRL values, suggesting that a withdrawal period would be necessary for eggs after IVM use in laying hens.

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.

Phase 1 and phase 2 metabolic activities along the small intestine in adult male sheep.

Metabolic activities of several xenobiotic metabolizing enzymes were evaluated in both hepatic and enteric subcellular fractions obtained from Corriedale × Merino crossbreed rams by using a biochemical approach. Microsomes obtained from the different segments of sheep small intestinal mucosa displayed cytochrome P450 (CYP)-dependent N-demethylations but not O-deethylase activities apparently occurred. CYP-mediated N-demethylations neither decreased nor increased along the small intestinal mucosa. Percentages of activity for erythromycin N-demethylase in the small intestine were between 29% (duodenum) and 45% (ileum) from that measured in the liver, whereas those determined for triacetyl-oleandomycin N-demethylation ranged between 10% (duodenum) and 15% (jejunum) of the same hepatic activity. Conversely, metabolic rates for aminopyrine and chlorfeniramine N-demethylations in the gut mucosa ranged between 3% and 7% compared to their respective hepatic enzyme activities. Sheep enteric mucosa also displayed metabolic reactions typically mediated by flavin-containing monooxygenases (FMOs), carbonyl reductases (CBRs), carboxylesterases (CES), glutathione S-transferases (GSTs) and uridine diphosphoglucuronyltransferases (UGTs). The FMO-mediated sulfoxidation of methimazole was 2.6-fold higher (P < 0.01) in the ileal compared to the duodenal mucosa. Percentages of activity for the microsomal CBR-dependent biotransformation of menadione were between 12% (ileum) and 19% (duodenum-jejunum) of the total activity measured in the liver; metabolic rates measured in duodenum and jejunum were ∼1.7-fold higher (P < 0.05) than that observed in the ileum. The microsomal CES activity (using p-nitrophenyl acetate as substrate) was around twofold higher in duodenum (P < 0.05) and jejunum (P < 0.01) in comparison to the ileum. Cytosolic GST-dependent activities (toward 1-chloro, 2,4-dinitrobenzene) were similar in the mucosa of duodenum, jejunum and ileum. Microsomal UGT activities (toward 1-naphthol) in duodenum and jejunum were three- and fourfold higher, respectively, compared to that measured in the ileum. The small intestinal mucosa may play a critical defensive role due to its involvement in the detoxification of toxic compounds prior to absorption. In addition, gut metabolic reactions may contribute to the presystemic metabolism of orally administered drugs. These results are a further contribution to the understanding of the relevance of the extra-hepatic metabolism of xenobiotics in ruminant species.

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.