First report of eprinomectin-resistant isolates of Haemonchus contortus in 5 dairy sheep farms from the Pyrénées Atlantiques département in France.

Infection of sheep by gastrointestinal nematodes (GIN) in pastoral systems such as those found in the South Western area of France, the Pyrénées Atlantiques, is one of the main reasons for economic loss and degradation of their welfare. In the present study, the efficacy of eprinomectin (EPN) was monitored on farms from this area following suspicion of lack of anthelmintic efficacy. Suspicions were raised by veterinarians, based on clinical signs ranging from milk and body condition loss, to anaemia, and mortality. Resistance was evaluated according to the World Association for the Advancement for Veterinary Parasitology (WAAVP) guidelines using fecal egg count reduction tests reinforced by individual analysis of drug concentration in the serum of all treated ewes by high-performance liquid chromatography (HPLC). EPN was administered by subcutaneous (SC) and topical (T) route according to manufacturer's requirements, as well as by the oral route (O) with the topical solution according to off-labelled practices in the field. For the first time in France, the presence of resistant isolates of Haemonchus contortus to EPN was observed in 5 dairy sheep farms. The HPLC dosages showed exposure of worms to concentrations compatible with anthelmintic activity for animals treated by the SC and O routes. By contrast, they showed under exposure to the drug of most individuals treated by the T route. EPN is the only null milk withdrawal anthelmintic molecule currently available. The presence of resistant isolates of the pathogenic H. contortus to EPN in this important dairy region requires an urgent change in grazing, and sometimes production, systems.

Interaction of macrocyclic lactones with the multidrug transporters: the bases of the pharmacokinetics of lipid-like drugs.

Like most drugs, macrocyclic lactone endectocides (MLs) exert their antiparasitic effects within the defined target tissues where parasites are located, and whose drug concentrations correlate with those in the plasma compartment. The process of drug distribution to the active site constitutes the link in the pharmacokinetic/pharmacodynamic relationship. In the past few years it has become evident that transporter proteins play a major role in regulating the distribution, elimination and metabolism of the antiparasitic macrocyclic lactones. The efflux transporter P-glycoprotein (P-gp) has received the most attention with regards to its strong interaction with ivermectin and other MLs. P-gp has been reported to be involved in restricting the absorption of these drugs, in enhancing their intestinal elimination, in the protection against their neurotoxicity and in the ML resistance mechanisms in parasites. This review focuses on the interaction of MLs with P-glycoprotein and with other multidrug resistance transporters. Given the structural and physicochemical diversity of these drugs, they constitute models of interest to study the major molecular determinants for the interaction with transporters. We will discuss the consequences of such interactions on ML pharmacokinetics and the possibility of benefiting from of drug/drug interaction to reverse multidrug resistance in several therapeutic fights such as against parasites and tumors.

P-glycoprotein interfering agents potentiate ivermectin susceptibility in ivermectin sensitive and resistant isolates of Teladorsagia circumcincta and Haemonchus contortus.

P-glycoprotein (P-gp) homologues, belonging to the ATP Binding Cassette (ABC) transporter family, are thought to play an important role in the resistance of gastro-intestinal nematode parasites against macrocyclic lactones. The aim of this study was to investigate the influence of various P-gp interfering compounds on the efficacy of ivermectin (IVM) in sensitive and resistant nematode isolates. The feeding of IVM resistant and sensitive Teladorsagia circumcincta and Haemonchus contortus first-stage larvae (L1) was assessed using a range of IVM concentrations (0.08-40 nm) with or without P-gp inhibitors: valspodar, verapamil, quercetin, ketoconazole and pluronic P85. The P-gp inhibitors were selected on the basis of their ability to interfere with P-gp transport activity in an epithelial cell line over-expressing murine P-gp. In the presence of P-gp interfering agents, the in vitro susceptibility to IVM of both sensitive and resistant isolates of T. circumcincta and H. contortus was increased. These results show that compounds interfering with P-gp transport activity could enhance IVM efficacy in sensitive isolates, and also restore IVM sensitivity in resistant nematodes. These results support the view that ABC transporters can play an important role in resistance to IVM, at least in the free-living stages of these economically important gastro-intestinal nematodes.

P-glycoproteins play a role in ivermectin resistance in cyathostomins.

Anthelmintic resistance is a global problem that threatens sustainable control of the equine gastrointestinal cyathostomins (Phylum Nematoda; Superfamily Strongyloidea). Of the three novel anthelmintic classes that have reached the veterinary market in the last decade, none are currently licenced in horses, hence current control regimens focus on prolonging the useful lifespan of licenced anthelmintics. This approach would be facilitated by knowledge of the resistance mechanisms to the most widely used anthelmintics, the macrocyclic lactones (ML). There are no data regarding resistance mechanisms to MLs in cyathostomins, although in other parasitic nematodes, the ABC transporters, P-glycoproteins (P-gps), have been implicated in playing an important role. Here, we tested the hypothesis that P-gps are, at least in part, responsible for reduced sensitivity to the ML ivermectin (IVM) in cyathostomins; first, by measuring transcript levels of pgp-9 in IVM resistant versus IVM sensitive third stage larvae (L3) pre-and post-IVM exposure in vitro. We then tested the effect of a range of P-gp inhibitors on the effect of IVM against the same populations of L3 using the in vitro larval development test (LDT) and larval migration inhibition test (LMIT). We demonstrated that, not only was pgp-9 transcription significantly increased in IVM resistant compared to IVM sensitive L3 after anthelmintic exposure (p < 0.001), but inhibition of P-gp activity significantly increased sensitivity of the larvae to IVM in vitro, an effect only observed in the IVM resistant larvae in the LMIT. These data strongly implicate a role for P-gps in IVM resistance in cyathostomins. Importantly, this raises the possibility that P-gp inhibitor-IVM combination treatments might be used in vivo to increase the effectiveness of IVM against cyathostomins in Equidae.

Cross-resistance to moxidectin and ivermectin on a meat sheep farm in France.

Resistance to ivermectin and moxidectin was explored by a faecal egg count reduction test in two sheep flocks with suspected anthelmintic resistance. The FECRT confirmed one suspicion, with a mean percentage of reduction in egg excretion within the treated groups of 0% for ivermectin (CI 95%: -228 to 58) and 13% for moxidectin (CI 95%: -152 to 70). This was further explored by a controlled efficacy test. An experimental infection of 18 naïve lambs was set up using infective larvae isolated from this flock (5000 L3/lamb). Compared to the control group, abomasal worm burdens (Teladorsagia circumcincta) were reduced by 90% [CI 95%: 81.5-94.8] and 85% [CI 95%: 72.4-92.2] after ivermectin (p<0.05) and moxidectin (p<0.05) treatment respectively. Again, compared to the control group, there was a reduction for intestinal strongyles (Trichostrongylus colubriformis) of 100% and 99% [CI 95%: 97.5-99.7] for ivermectin and moxidectin respectively. No difference was found between the efficacy of moxidectin and ivermectin. Pharmacokinetic values indicated that the strongyles were submitted to anthelmintic concentrations usually lethal to them. This trial demonstrated the first multiple resistance of ovine strongyles in France.

Impaired secretion of heart lipoprotein lipase in cyclophosphamide-treated rabbit.

Cyclophosphamide administration into fasted rabbits induces a hypertriglyceridaemia and a defect in vascular lipoprotein lipase. Heart LPL activity was more than 50% decreased after antimitotic treatment in fasted animals. The tissue distribution of lipoprotein lipase activity was followed in heart using recycling perfusion. Cyclophosphamide administration resulted in a profound decline in the heparin-releasable lipoprotein lipase activity, concordant with a higher recovery in the residual heart tissue. The effects were more pronounced in fasted than in fed animals. In agreement, the proportion of neosynthesized [35S]methionine-labelled lipoprotein lipase released by heparin was decreased by 50% following antimitotic treatment. The lipolysis of very low density lipoprotein-labelled triacylglycerols was found 2.5-fold reduced in hearts from cyclophosphamide-treated rabbits as compared to controls. These results suggest that a defective secretion of lipoprotein lipase may contribute to the poor expression of lipolytic activity in the vascular bed and to the occurrence of hypertriglyceridaemia during cyclophosphamide treatment.

Kinetic evidence for phosphatidylethanolamine and triacylglycerol as preferential substrates for hepatic lipase in HDL subfractions: modulation by changes in the particle surface, or in the lipid core.

Human HDL subfractions, HDL2 (d: 1.085-1.125) and HDL3 (d: 1.125-1.19) labelled with 2-[14C]linoleoylphosphatidylethanolamine and tri-[3H]oleoylglycerol, were incubated with partially purified hepatic triacylglycerol lipase, isolated from human post-heparin plasma. Kinetics of hydrolysis of these two HDL-lipid substrates were followed and were compared to those previously obtained on phosphatidylcholine (G. Simard et al (1989) Biochim. Biophys. Acta 1001, 225-233). (1) The apparent Km obtained for HDL-triacylglycerol was half that for HDL-phosphatidylethanolamine, but the estimated Vmax was higher for the latter. Hence, despite a lower affinity, more molecules of phosphatidylethanolamine than of triacylglycerol were found hydrolysed. A strong correlation was observed between the hepatic lipase activity added and the maximal degradation rates for phosphatidylethanolamine measured in HDL2 and HDL3. (2) A linear relationship was observed in both HDL2 and HDL3 between the respective degradations of the two substrates. The number of phosphatidylethanolamine molecules hydrolysed exceeded that of triacylglycerol by 30% in HDL2 and by 70% in HDL3. HDL2 were 2- and 4-times more reactive than HDL3 for the hydrolysis of phosphatidylethanolamine and triacylglycerol, respectively, taking the Vmax/Km ratio as an indicator of catalytic efficiency. In both HDL subfractions, the calculated Vmax/Km value was 30-50-fold higher for PE and TG than for PC. (3) HDL particles were modified either on their surface by selective enrichment in free cholesterol or in their inner-core by replacement of esterified cholesterol by triacylglycerol in presence of a source of neutral lipid transfer activity. A mild cholesterol enrichment stimulated the phosphatidylethanolamine and triacylglycerol reactivities by 30-60% towards hepatic lipase, whereas increasing the triacylglycerol concentration in HDL was followed by a proportional increase in the amounts of triacylglycerol hydrolysed with no effect on phospholipid degradation.

Kinetics and anthelmintic efficacy of topical eprinomectin when given orally to goats.

Preliminary data suggest that topical eprinomectin in goat shows an individual variation in anthelmintic efficacy when used off-license at a dose rate of 0.5 or 1.0mg/kg BW. As a result, the use of oral administration of topical formulation of eprinomectin tends to develop in dairy goat farms in France. The plasma levels and milk excretion as well as the anthelmintic efficacy of eprinomectin were determined in goats following oral administration of a topical formulation of the drug at dose rates of 0.5 and 1mg/kg BW. The area under the concentration-time curve (AUC) values were 17.62 ± 9.68 ng day/ml and 6.56 ± 4.00 ng day/ml for plasma and milk respectively after the administration of 0.5mg/kg BW and 45.32 ± 13.90 ng day/ml and 13.88 ± 1.77 ng day/ml for plasma and milk, respectively after the administration of 1mg/kg BW. The milk-to-plasma ratio ranged from 0.33 to 0.36 and the amount of drug recovered in the milk was 0.4% of the total administered dose. The maximum concentrations of eprinomectin residues determined in milk after oral treatment were < 20 µg/kg (Maximum Residue Limit in goat milk). The anthelmintic efficacy of the oral administration of topical eprinomectin was 100% through Faecal Egg Count Reduction Test in natural infection and ≥ 99.8% through Controlled Test in experimental infection (Haemonchus contortus and Trichostrongylus colubriformis). Additional information is needed about the fate of the vehicles used for topical formulation when given by oral route concerning food safety.

Hepatic hyper-vitaminosis A: importance of retinyl ester level determination.

We report the case of a 32-year-old man with portal hypertension without cirrhosis due to chronic vitamin A intoxication. Portal hypertension revealed by oesophageal varice rupture progressively worsened and ascites occurred 5 years after the patient stopped vitamin A intake. Initially, serum retinyl palmitate concentration was increased whereas serum retinol concentration was normal. There was no hepatic fibrosis on light microscopic examination of liver biopsy specimens. Five years after the patient stopped excessive vitamin A intake, serum retinol and retinol-binding protein concentrations were below the normal range even though there was an increased hepatic retinyl ester content. This was attributed to the late development of peri-sinusoidal fibrosis. This case mainly shows the importance of retinyl ester level determination: serum retinyl palmitate should be measured immediately after intoxication and hepatic retinyl esters should be measured initially and particularly later. Indeed, later serum and hepatic retinol levels in chronic hyper-vitaminosis A may be normal and lead to under-estimation of liver vitamin A overload.

Total parenteral nutrition decreases liver oxidative metabolism and antioxidant defenses in healthy rats: comparative effect of dietary olive and soybean oil.

BACKGROUND: Total parenteral nutrition (TPN) is used for critically ill patients undergoing surgery, after trauma, or during disease conditions that favor oxidative stress. We studied the effect of TPN on liver oxidative metabolism and antioxidant defenses in rats, and we compared the effect of soybean oil- and olive oil-based diets. METHODS: Seven-week-old rats (n = 28) were divided into four groups. Two experimental groups received a TPN solution containing soybean oil (TPN-S) or a mixture of olive/soybean oil, 80/20 (TPN-O), IV for 6 days. Orally fed animals received a solid diet including soybean oil (Oral-S) or olive/soybean oil, 80/20 (Oral-O). The following parameters were measured: DL-alpha-tocopherol, vitamin A, malondialdehyde and thiobarbituric acid reactive substances (MDA-TBARS), and total radical-trapping antioxidant parameter (TRAP) in serum; DL-alpha-tocopherol, vitamin A, glutathione (GSH), and catalase (Cat) activity in liver homogenate; fatty acids from phospholipid, cytochrome P-450 content, NADPH-cytochrome c2 reductase activity in liver microsomes; superoxide dismutase (SOD), glutathione peroxidase (Gpx), glutathione reductase (GR), glutathione transferase (GST), and glucose-6-phosphate dehydrogenase (G6PD) in liver cytosol. RESULTS: The soybean or olive oil diets modified the liver microsomal fatty acid phospholipid composition, but the unsaturation index remained unchanged. TPN specifically increased the saturation of the membrane. The cytochrome P-450 level and the NADPH-cytochrome c2 reductase, SOD, Gpx, Cat, and GST activities were unchanged by soybean oil or olive oil diet but decreased receiving TPN. CONCLUSIONS: In rats, TPN decreased the liver oxidative metabolism and enzymatic antioxidant defenses. This may be related to saturation of the liver microsomal fatty acids.

Enhancement of moxidectin bioavailability in lamb by a natural flavonoid: quercetin.

Moxidectin is an antiparasitic drug widely used in cattle, sheep and companion animals. Due to the involvement of P-glycoprotein (P-gp) and cytochrome P450 3A in the metabolism of moxidectin, we studied the influence of various P-gp interfering agents (ivermectin, quercetin and ketoconazole) on the metabolism of 14C moxidectin in cultured rat hepatocytes over 72 h. This in vitro study allowed selection of compounds which are able to increase the moxidectin bioavailability in lambs. From this, the modulation of moxidectin pharmacokinetics in plasma of lambs was studied after co-administration of 0.2 mg kg(-1) moxidectin (subcutaneously (SC)) and 0.2 mg kg(-1) ivermectin (SC), or 10 mg kg(-1) quercetin (SC), or 10 mg kg(-1) ketoconazole (orally). Ivermectin and quercetin increased significantly the quantity of 14C moxidectin in the rat hepatocytes. Ketoconazole co-administration led to a higher concentration of moxidectin in the rat hepatocytes. In vivo, only quercetin was able to modify the pharmacokinetics of moxidectin in plasma of lambs by increasing significantly the area under the plasma concentration-time curve. This study allowed the use of a natural agent, quercetin, to improve the bioavailability of moxidectin.

Pharmacokinetics of ivermectin in the yak (Bos grunniens).

The yak (Bos grunniens) belongs to the cattle family Bovidae and lives in the mountains of China and adjacent areas. Due to the physiological adaptations of yak to its environment and the lack of data, the ivermectin pharmacokinetic was studied following a single subcutaneous dose at the recommended dose for cattle (0.2 mg kg(-1)). The observed peak plasma concentration (Cmax) was 48.93 ng ml(-1) and the time to reach Cmax (Tmax) was 0.73 day. These results show a faster rate of absorption than in cattle. The values for the absorption half-life (t(1/2a)), the distribution half-life (t(1/2alpha)) and the terminal half-life (t(1/2beta)) were 0.31, 0.74 and 4.82 days, respectively. The calculated area under the concentration-time curve (AUC) was 146.2 ng day ml(-1) and the mean residence time (MRT) was 3.57 days. The availability of ivermectin appears low in yaks in comparison to cattle but equivalent to that reported in horses and is likely to be due to physiological characteristics of this species.

Efficacy of eprinomectin pour-on against gastrointestinal nematodes and the nasal bot fly (Oestrus ovis) in sheep.

The efficacy of the pour-on formulation of eprinomectin, at a dose rate of 0.5 mg/kg bodyweight, was assessed in sheep against three main species of gastrointestinal nematodes and against the nasal bot fly, Oestrus ovis, and some pharmacokinetic parameters were determined for 21 days after the treatment. By comparison with untreated control sheep, infected experimentally with Haemonchus contortus, Teladorsagia circumcincta and Trichostrongylus colubriformis, eprinomectin was 100 per cent effective against the two abomasal species and 99.5 per cent effective against T. colubriformis. In ewes naturally infected with the nasal bot fly, the efficacy of the drug against O. ovis was 97.7 per cent. The mean (se) systemic area under the curve (AUC) was 56.0 (26.2) ng/day/ml and the mean residence time was 5.3 (1.0) days, but there were wide variations between individual sheep.

Characterisation of macrocyclic lactone resistance in two field-derived isolates of Cooperia oncophora.

The anthelmintic sensitivity of two field-derived isolates (designated FI001 and FI004) of cattle nematodes from beef farms in Scotland were investigated in a controlled efficacy test (CET). Efficacies of ivermectin pour-on (IVM-PO), IVM injectable (IVM-INJ) and moxidectin pour-on (MOX-PO) formulations were assessed. In each group, five helminth-naïve calves were infected experimentally with 50,000 third stage larvae from either isolate and administered with anthelmintic at the manufacturers' recommended dose rate 28 days later. For each isolate, nematode burdens were compared between treatment and control groups to determine efficacy. Nematode species composition, based on data derived from the untreated control groups' burden estimations, were 39 and 14% Cooperia oncophora and 61 and 86% Ostertagia ostertagi for isolates FI001 and FI004, respectively. Macrocyclic lactone resistance in C. oncophora was confirmed for both FI001 and FI004 isolates. Efficacies (as determined by nematode burden analysis) of 4, 21 and 31% for FI001, and 10, 1 and 74% for FI004, were obtained for IVM-INJ, IVM-PO and MOX-PO, respectively. Efficacy based on faecal egg count reduction at seven days post anthelmintic administration were 8, 99 and 100% for FI001, and 37, 20 and 100% for FI004 for IVM-INJ, IVM-PO and MOX-PO, respectively. In summary, this study details two macrocyclic lactone resistant isolates of C. oncophora obtained from cattle from two distinct geographical locales in the UK.

Influence of Pluronic 85 and ketoconazole on disposition and efficacy of ivermectin in sheep infected with a multiple resistant Haemonchus contortus isolate.

Non-specific mechanisms involving ATP-binding cassette drug efflux transporters may play an important role in xenobiotic clearance in ovine gastro-intestinal nematodes. By using transporter inhibitors, the aim of this trial was to assess the possibility of increasing drug bioavailability in the host in an attempt to improve treatment efficacy. Thirty-six lambs were infected with 5000 multiple-drug resistant Haemonchus contortus third stage larvae and separated into six groups (n=6): ivermectin alone (IVM; 0.2 mg/kg body-weight, BW), ketoconazole alone (KET; 10 mg/kg BW), Pluronic 85 alone (P85; 4 mg/kg BW), IVM+KET, IVM+P85 or untreated control. Ivermectin was administered once on day 28 post-infection for all appropriate groups, whereas KET and P85 were administered as five separate doses on day 26-30 post-infection inclusive. The resultant data showed that concomitant administration of KET or P85 with IVM induced increases in plasma and tissue concentrations of IVM in treated animals, resulting in a two-fold increase in the area under the time-concentration curve (p<0.05). Faecal egg counts and worm burdens of the IVM+KET and IVM+P85 groups were lower than in the untreated, KET and P85 alone control animals. Worm burdens were reduced by between 16% and 51% with IVM+KET and IVM+P85 respectively compared to untreated control animals. The co-administration of P85 with IVM increased the efficacy by 34%, compared with IVM alone, in terms of worm count reduction of the multi-resistant isolate of H. contortus.

Estimation of absolute oral bioavailability of moxidectin in dogs using a semi-simultaneous method: influence of lipid co-administration.

Moxidectin is a long-acting anthelmintic drug for which little is known about its kinetic behaviour in dogs and its oral absolute bioavailability has never been reported. We studied the pharmacokinetics of moxidectin in dogs, with a special emphasis on oral bioavailability and the influence of lipid co-administration, by using a semi-simultaneous method of administration. Ten Beagle dogs were dosed orally and then intravenously (i.v.) with 0.2 mg/kg moxidectin. The oral application was conducted with or without corn oil co-administration. Moxidectin concentration-time profiles in plasma were analysed using a compartmental modelling approach, designed to fit the oral and i.v. kinetic disposition curves simultaneously. In contrast to what happens in other species, our study indicates that the bioavailability of orally given moxidectin in dogs is nearly total (90.2 +/- 7.4%), and is not enhanced by lipid co-administration. The clearance, the volume of distribution, the mean residence time and the terminal half-life were similar to what was already described for other species. Finally our trial suggests that the body condition (degree of obesity) is likely to be a major determinant of moxidectin kinetics in dogs because of its modulation of the volume of distribution that indirectly controls the terminal half-life of the drug.

The interaction between moxidectin and MDR transporters in primary cultures of rat hepatocytes.

The interaction of moxidectin (a macrocyclic lactone, ML) with P-glycoprotein (P-gp), multidrug resistance associated proteins (MRPs) and breast cancer resistance protein (BCRP) was studied in primary cultures of rat hepatocytes by measuring the intracellular accumulation of [14C]-moxidectin over 72 h in the presence of specific inhibitors: for P-gp, verapamil (10 microM); for MRPs, MK571 (100 microM), indomethacin (10 microM) and probenecid (3.8 mM); and for BCRP, fumitremorgin C (5 microM). The P-gp and MRP inhibitors increased significantly (P < 0.01) by 48.7%, 49.8%, 49.9% and 57.2% the area under the time-intracellular concentration curve (AUC) of moxidectin in rat hepatocytes, while the BCRP inhibitor, fumitremorgin C, had no effect on the AUC compared with the control. In addition, the mRNAs of all the drug transporters studied were detected in rat hepatocytes from 0 to 72 h. Using this cellular model it has been shown that MRP inhibitors increase moxidectin intracellular concentrations to a similar extent as the P-gp inhibitor. The identification of all the transporters that interact with MLs remains a challenge, which currently concerns several important therapeutic fields.