Thermal stability of antiparasitic macrocyclic lactones milk residues during industrial processing.

The chemical stability of residues of different antiparasitic macrocyclic lactone compounds in milk subjected to thermal treatment was assessed. Concentrations of ivermectin (IVM), moxidectin (MXD) and eprinomectin (EPM) in sheep milk, equivalent to those measured in vivo in milk excretion studies, were subjected to 65 degrees C over 30 min or to 75 degrees C for 15 s. Residue concentrations of IVM, MXD and EPM in milk were measured by high-performance liquid chromatography (HPLC) (fluorescence detection) before and after heat treatment of the drug-fortified milk samples. No evidence of chemical loss was obtained in either of the thermal treatments under evaluation. The stability of the parent compounds in milk was evidenced by the lack of bioconversion products (metabolites) after both thermal treatments. Only very minor changes on drug concentrations were observed at the end of the treatments, which fell within the limits of the variation of the validated analytical method. In conclusion, residue concentrations of macrocyclic lactones are unaffected by industrial-simulated milk thermal procedures. Based on the reported findings, it can be postulated that residue concentrations of IVM, MXD and EPM measured in raw sheep milk may be used to estimate consumer exposure and dietary intake for these veterinary drugs.

Ivermectin (3.15%) long-acting formulations in cattle: absorption pattern and pharmacokinetic considerations.

Ivermectin (IVM) is a broad-spectrum antiparasitic drug extensively used in veterinary medicine. The composition of the pharmaceutical preparation affects IVM absorption and its systemic availability. After the introduction of the first approved IVM formulation (propylene glycol/glycerol formal 60:40) used at 200 microg/kg, different pharmaceutical modifications have been assayed to extend IVM persistent endectocide activity. Recently, IVM 3.15% long-acting (IVM-LA) preparations to be administered at 630 microg/kg to cattle were introduced into the veterinary pharmaceutical market. The work reported here was designed to evaluate the comparative IVM absorption pattern and plasma concentration profiles obtained after subcutaneous administration of the classic pioneer IVM formulation (1%) and two different commercially available IVM-LA preparations (3.15%) to cattle. Twenty-eight Holstein heifers were divided in four experimental groups (n=7) and treated subcutaneously as follows--Group A: IVM 1% given at 200 microg/kg, Group B: IVM 1% administered at 630 microg/kg, Group C: IVM-LA (A) injected at 630 microg/kg and Group D: IVM-LA (B) given at 630 microg/kg. Blood samples were taken between 0.5 and 90 days post-treatment and IVM plasma concentrations were determined by HPLC with fluorescence detection. There were no differences in the persistence of IVM plasma concentrations after the administration of IVM 1% formulation at the two used dose levels (200 and 630 microg/kg). Higher peak plasma concentration (C(max)) and shorter mean residence time (MRT) were obtained for IVM 1% given at 630 microg/kg (Group B) compared to the treatments with both IVM-LA preparations. The IVM-LA (A) formulation showed a more extended absorption process than IVM-LA (B) preparation, which accounted for a longer persistence of detectable IVM plasma concentrations. The parasitological implications of the observed differences in peak plasma concentrations (C(max) values) and in the IVM concentration levels measured from day 20, and afterwards until day 90 post-treatment, between the different preparations assayed need to be elucidated. The characterization of the absorption patterns and kinetic behaviour obtained after injection of these novel long-acting formulations used at three times the therapeutic dose recommended for the classic IVM preparation in cattle is a further contribution to the field.

Assessment of the main metabolism pathways for the flukicidal compound triclabendazole in sheep.

Triclabendazole (TCBZ) is an halogenated benzimidazole (BZD) compound worldwide used to control immature and adult stages of the liver fluke Fasciola hepatica. The purpose of this investigation was to characterize in vitro the patterns of hepatic and ruminal biotransformation of TCBZ and its metabolites in sheep. TCBZ parent drug was metabolized into its sulphoxide (TCBZSO), sulphone (TCBZSO2) and hydroxy derivatives by sheep liver microsomes. The same microsomal fraction was also able to oxidize TCBZSO into TCBZSO2 and hydroxy-TCBZSO (HO-TCBZSO). TCBZ sulphoxidation was significantly (P < 0.001) inhibited after inactivation of the flavin-monooxygenase (FMO) system (77% inhibition) as well as in the presence of the FMO substrate methimazole (MTZ) (71% inhibition). TCBZ sulphoxidative metabolism was also reduced (24% inhibition, P < 0.05) by the cytochrome P450 inhibitor piperonyl butoxide (PB). The rate of TCBZSO conversion into TCBZSO2 was also significantly inhibited by PB (55% inhibition), MTZ (52% inhibition) and also following FMO inactivation (58% inhibition). The data reported here indicate that the FMO is the main enzymatic pathway involved in TCBZ sulphoxidation (ratio FMO/P450 = 3.83 +/- 1.63), although both enzymatic systems participate in a similar proportion in the sulphonation of TCBZSO to form the sulphone metabolite (ratio FMO/P450 = 1.31 +/- 0.23). Additionally, ketoconazole (KTZ) did not affect TCBZ sulphoxidation but decreased (66% inhibition, P < 0.05) the formation of TCBZSO2. Similarly, inhibition of TCBZSO2 production was observed after incubation of TCBZSO in the presence of KTZ and erythromycin (ETM). Conversely, thiabendazole (TBZ) and fenbendazole (FBZ) did not affect the oxidative metabolism of both incubated substrates. The sheep ruminal microflora was able to reduce the sulphoxide (TCBZSO) into the parent thioether (TCBZ). The ruminal sulphoreduction of the HO-TCBZSO derivative into HO-TCBZ was also demonstrated. The rate of sulphoreduction of HO-TCBZSO was significantly (P < 0.05) higher than that observed for TCBZSO. The metabolic approach tested here contributes to the identification of the different pathways involved in drug biotransformation in ruminant species. These findings on the pattern of hepatic and ruminal biotransformation of TCBZ and its main metabolites are a further contribution to the understanding of the pharmacological properties of widely used anthelmintics in ruminants. Comprehension of TCBZ metabolism is critical to optimize its flukicidal activity.

Moxidectin and ivermectin metabolic stability in sheep ruminal and abomasal contents.

The oral administration of macrocyclic lactones to sheep leads to poorer efficacy and shorter persistence of the antiparasitic activity compared to the subcutaneous treatment. Gastrointestinal biotransformation occurring after oral treatment to ruminant species has been considered as a possible cause of the differences observed between routes of administration. The current work was addressed to evaluate on a comparative basis the in vitro metabolism of moxidectin (MXD) and ivermectin (IVM) in sheep ruminal and abomasal contents. Both compounds were incubated under anaerobic conditions during 2, 6 and 24 h in ruminal and abomasal contents collected from untreated adult sheep. Drug concentrations were measured by high-performance liquid chromatography with fluorescence detection after sample clean up and solid phase extraction. Neither MXD nor IVM suffered metabolic conversion and/or chemical degradation after 24-h incubation in ruminal and abomasal contents collected from adult sheep. Unchanged MXD and IVM parent compounds represented between 95.5 and 100% of the total drug recovered in the ruminal and abomasal incubation mixtures compared with those measured in inactive control incubations. The partition of both molecules between the solid and fluid phases of both sheep digestive contents was assessed. MXD and IVM were extensively bound (>90%) to the solid material of both ruminal and abomasal contents collected from sheep fed on lucerne hay. The results reported here confirm the extensive degree of association to the solid digestive material and demonstrates a high chemical stability without evident metabolism and/or degradation for both MXD and IVM in ruminal and abomasal contents.

Comparative hepatic and extrahepatic enantioselective sulfoxidation of albendazole and fenbendazole in sheep and cattle.

The enantioselective sulfoxidation of the prochiral anthelmintic compounds albendazole (ABZ) and fenbendazole (FBZ) was investigated in liver, lung and small intestinal microsomes obtained from healthy sheep and cattle. The microsomal fractions were incubated with a 40 microM concentration of either ABZ or FBZ. Inhibition of the flavin-containing monooxygenase (FMO) system was carried out by preincubation with 100 microM methimazole (MTZ) either with or without heat pretreatment (2 min at 50 degrees C). ABZ and FBZ were metabolized to the (+) and (-) enantiomers of their sulfoxide metabolites, named albendazole sulfoxide (ABZSO) and oxfendazole (OFZ), respectively. ABZ sulfoxidation rates were higher (p < 0.001) than those observed for FBZ. The FMO-mediated liver sulfoxidation of ABZ was enantioselective (100%) toward the (+) ABZSO production in both species. Liver sulfoxidation of FBZ by FMO was also enantioselective toward (+) OFZ (sheep = 65%; cattle = 79%). Cytochrome P450 was found to be mainly involved in the production of (-) ABZSO in the liver. MTZ did not affect the sulfoxidation of ABZ by lung microsomes, which may indicate that FMO is not involved in the production of ABZSO in this tissue. A significant (p < 0.05) inhibition of (-) ABZSO production by liver microsomes was observed after ABZ incubation in the presence of erythromycin (cattle = 21%) and ketoconazole (sheep = 36%). Both CYP3A substrates induced a reduction in the production of (-) ABZSO (sheep = 67-78%, cattle = 50-78%) by lung microsomes. Overall, the results reported here contribute to the identification of the metabolic pathways involved in the biotransformation of benzimidazole anthelmintics extensively used for parasite control in ruminants.

Pharmacokinetic evaluation of four ivermectin generic formulations in calves.

The plasma concentration profiles of four randomly chosen ivermectin (IVM) generic formulations (IVM G1-G4) were compared after their subcutaneous (SC) administration to healthy calves. The disposition of other avermectin-type endectocide compounds, doramectin (DRM) and abamectin (ABM), was also assessed in the same pharmacokinetic trial. Forty-two parasite-free Aberdeen Angus male calves were randomly allocated into six treatment groups. Animals in each group (n = 7) received SC treatment (200 microg/kg) with one of the commercially available endectocide formulation used in the trial. Blood samples were taken into heparinised vacutainer tubes from the jugular vein prior to and up to 35 days post-treatment. The recovered plasma was analysed by HPLC with fluorescence detection. Large kinetic differences were observed among the DRM, ABM and IVM formulations under evaluation. The DRM plasma concentration profiles were higher than those measured for ABM and all the IVM generic formulations. The higher and sustained plasma concentrations of DRM accounted for greater area under concentration-time curve (AUC) and longer mean residence time (MRT) values compared to those obtained for both ABM and the IVM generic preparations. The pattern of IVM absorption from the site of subcutaneous administration showed differences among the generic formulations under evaluation. The IVM G2 preparation showed higher peak plasma concentration and AUC values (P < 0.05) compared to those obtained after the administration of the IVM G1 formulation. Longer (P < 0.05) MRT values were obtained after the administration of the IVM G3 compared to other IVM generic preparations. The kinetic behaviour of ABM did not show significant differences with that described for most of the IVM formulations. This study demonstrates that major differences on drug kinetic behaviour may be observed when using different endectocide injectable formulations in cattle.

Breed differences on the plasma availability of moxidectin administered pour-on to calves.

The pharmacokinetic profile of avermectin and milbemycin compounds is affected by different drug- and host-related factors. This work reports the influence of cattle breeds on the plasma kinetics of moxidectin (MXD) after topical (pour-on) administration. Parasite-free Aberdeen Angus and Holstein calves were treated with a commercial MXD pour-on formulation at 500 microg/kg. Blood samples were collected over a period of 35 days post-treatment and the recovered plasma was analysed by high performance liquid chromatography using fluorescence detection. MXD was detected in plasma from two hours up to 35 days post-treatment in animals from both breeds. A slow MXD absorption and delayed peak plasma concentration were observed in Aberdeen Angus compared to Holstein calves. Significant lower systemic availability (expressed as AUC) (P<0.01) and peak plasma concentration (C(max)) (P<0.05) were also observed in Aberdeen Angus calves, although the plasma mean residence time (MRT) and elimination half-lives (T(1/2el)) of MXD in both breeds were similar. The pharmacokinetic differences observed between cattle breeds contribute to explain the variability in the pattern of clinical efficacy for pour-on administered endectocide compounds reported in different field trials.

Loperamide-induced enhancement of moxidectin availability in cattle.

Moxidectin (MXD) is a milbemycin endectocide compound active at extremely low dosages against a wide variety of nematode and arthropod parasites. Different pharmacological approaches are currently being tested to delay the bile-faecal elimination and to obtain increased systemic availability for endectocide molecules in ruminants. Loperamide (LPM) is an opioid derivative, whose main pharmacological action is to abolish intestinal propulsive peristaltic waves. The influence of LPM on the pattern of faecal excretion of MXD and on its plasma disposition following intravenous (i.v.) and subcutaneous (s.c.) administrations to cattle was evaluated in the current work. Parasite-free calves were treated with MXD given either alone at 200 microg/kg by i.v. (Experiment 1) and s.c. (Experiment 2) administrations or coadministered with LPM subcutaneously injected at 0.4 mg/kg. Blood and faecal samples were collected over a period of 20 (Experiment 1) and 40 (Experiment 2) days post-treatment. The recovered plasma and faecal samples were extracted and analysed by high-performance liquid chromatography (HPLC) using fluorescence detection. Significantly higher MXD plasma concentrations were obtained after the coadministration of MXD + LPM compared with treatments with MXD alone by both routes. The higher MXD plasma concentration profiles measured after the coadministration with LPM accounted for the significantly higher AUC values obtained following the i.v. (> 46%) and s.c. (> 38%) treatments. A reduced MXD body clearance was observed in the presence of LPM. The appearance of MXD in faeces was significantly delayed after the i.v. and s.c. coadministrations of MXD with LPM (T(1/2app)=5.87 and 10.6 h, respectively) than that observed after the treatment with MXD alone (T(1/2app)=3.48 and 5.12 h). A delayed MXD peak concentration in faeces collected from MXD + LPM-treated animals compared with those receiving MXD alone, was observed. The delayed intestinal transit time caused by LPM and a potential competition between MXD and LPM for the P-glycoprotein-mediated bile/intestinal secretion processes, may account for the enhanced MXD systemic availability measured in cattle in the current work.

In vitro ruminal biotransformation of benzimidazole sulphoxide anthelmintics: enantioselective sulphoreduction in sheep and cattle.

The comparative in vitro sulphoreduction of the (+) and (-) enantiomers of albendazole sulphoxide (ABZSO) and oxfendazole (OFZ) by ruminal fluid obtained from sheep and cattle, was investigated, under anaerobic conditions, in this study. Ruminal fluid samples were obtained from Holstein steers fitted with a permanent rumen fistula and from Corriedale lambs via an oesophageal tube. Albendazole sulphoxide, incubated as either the racemic (rac) mixture or as each individual enantiomeric form, was extensively sulphoreduced to form albendazole (ABZ) by ruminal fluid from both species. The concentrations of ABZ formed at different incubation times were between 55 and 158% greater after the incubation of cattle ruminal fluid with (+) ABZSO, compared with that produced when (-) ABZSO was the incubated substrate. Similarly, the concentrations of ABZ were 1.3--3.0-fold higher when (+) ABZSO was incubated with sheep ruminal fluid. Significantly higher rates of depletion were observed for the (+) enantiomeric form when ABZSO was incubated with ruminal fluid from both species. The rates of ABZ formation from both ABZSO enantiomeric forms were significantly higher in sheep compared with cattle ruminal fluid. Fenbendazole (FBZ) was the metabolite formed after the incubation of the racemic form of OFZ with ruminal fluid obtained from both species. The metabolic profile of both OFZ enantiomers followed a similar pattern to that observed for ABZSO enantiomers. A bi-directional chiral inversion of one enantiomer into its antipode was observed. The (+) enantiomer appeared in the incubation medium when (-) ABZSO was the incubated substrate, and also the (-) antipode was detected after (+) ABZSO incubation with ruminal fluid obtained from both species. The results reported here demonstrate an enantioselective ruminal sulphoreduction of ABZSO and OFZ (substrate enantioselectivity). These findings contribute to interpret the chiral behaviour of benzimidazole-sulphoxide anthelmintics.

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.

Influence of diet on the pattern of gastrointestinal biotransformation of netobimin and albendazole sulphoxide in sheep.

The in vitro biotransformation of the anthelmintic compounds, netobimin (NTB) pro-drug and albendazole sulphoxide (ABZSO), by ruminal fluid obtained from sheep fed either hay or concentrate-based diets was investigated. No metabolic activity was observed in boiled samples of ruminal fluid, which confirms the importance of ruminal microflora in the metabolism of the xenobiotics under investigation. NTB pro-drug was efficiently biotransformed by ruminal fluid in vitro. Albendazole (ABZ) and its sulphoxide derivative were the metabolic products recovered. The thioether ABZ was formed by sulphoreduction of ABZSO by ruminal fluid in vitro. A more efficient nitroreduction of NTB and sulphoreduction of ABZSO were observed for ruminal fluid collected from sheep fed the concentrate diet. The type of diet determines the composition and distribution of the microbial population in the rumen; this affects the pattern of drug biotransformation in the gastrointestinal tract, which may impact on drug therapy.

Differences in plasma and abomasal kinetics of albendazole and its metabolites in calves grazed on pasture or fed a grain-based diet.

We evaluated the comparative plasma and abomasal fluid disposition kinetics of albendazole (ABZ) and its metabolites in calves either grazing on pasture or fed a grain-based concentrate diet. Six male Holstein calves (weight 180 to 200 kg) were allowed to graze on lush pasture for three weeks before intraruminal administration of ABZ at 10 mg kg-1(pasture group). After a three-week wash-out period, the same animals were housed and fed on a grain-based concentrate diet for three weeks prior to receiving the same ABZ treatment (concentrate group). Jugular blood and abomasal fluid samples were collected over 120 hours post-treatment. Plasma and abomasal fluid samples were analysed by high performance liquid chromatography (HPLC). The digesta transit time was measured using cobalt (Co) as a fluid marker; abomasal fluid and faecal samples were collected and Co concentrations measured by atomic absorption spectrophotometry. Complementary studies of the in vitro dissolution of ABZ particles at different pH values were also conducted. The pH of abomasal fluid collected from animals kept under both feeding conditions was registered. Increased concentrations of ABZ sulphoxide (ABZSO) and sulphone (ABZSO2) in plasma, resulting in significantly higher Cmax and area under the curve (AUC) values for both metabolites, were obtained in calves fed on the concentrate diet compared to those grazing on pasture. Enhanced abomasal fluid levels of ABZ and ABZSO were observed in concentrate-fed calves. The mean retention time of the digestive fluid marker in the gastrointestinal (GI) tract was significantly longer in the animals fed the grain-based diet. The in vitro dissolution of ABZ at a pH value equivalent to that obtained in the abomasum of the concentrate-fed calves (1.75) was significantly greater than that obtained at the pH registered in pasture-fed animals (2.00). The characterisation of the kinetic/metabolic behaviours and the resultant efficacy of antiparasitic drugs in animals reared under different management conditions may be relevant in increasing parasite control in livestock.

Bioequivalence of ivermectin formulations in pigs and cattle.

The vehicle in which endectocide compounds are formulated plays a relevant role in their absorption kinetics and resultant systemic availability. The pharmaceutical bioequivalence and comparative plasma disposition kinetics of ivermectin (IVM), following the subcutaneous administration of two injectable formulations to pigs and cattle were investigated using parallel experimental designs. Sixteen parasite-free male Duroc Jersey-Yorkshire crossbred pigs (90-110 kg) (Expt 1) and 16 parasite-free male Holstein calves (100-120 kg) (Expt 2) were divided into two groups and treated subcutaneously at either 300 (pigs) or 200 (calves) microg/kg with two different propylene glycol/glycerol formal (60: 40) based IVM formulations; in both experiments pigs or calves in Group A received the test (IVM-TEST) formulation and those in Group B were treated with the reference formulation (IVM-CONTROL). Heparinized blood samples were taken from 0 h up to either 20 (pigs) or 30 (calves) days post-treatment and plasma was extracted, derivatized and analysed by high performance liquid chromatography (HPLC) using fluorescence detection. Early detection of IVM (12 h) with a peak plasma concentration (C(max)) between 33 and 39 ng/mL was observed in pigs. The drug was detected in plasma up to 20 days post-administration of either formulation, resulting in elimination half-lives between 3.47 and 3.80 days. There were no differences between the IVM-TEST and IVM-CONTROL formulations in the kinetic parameters (except t(max)) obtained in pigs. IVM was detected in plasma between 12 h and 30 days post-administration of both formulations under investigation in cattle. The plasma disposition kinetics of IVM in calves was similar following treatment with both formulations. C(max) values (between 40.5 and 46.4 ng/mL) were achieved at 2 days post-administration of both formulations. None of the estimated kinetic parameters were statistically different between drug formulations. The injectable IVM formulations investigated were bioequivalent after their subcutaneous administration to both pigs and calves at recommended dose rates.