Depletion and bioavailability of imidocarb residues in sheep and goat tissues.

The residual depletion of a commercial product containing imidocarb dipropionate in sheep and goat tissues was investigated. Additionally, the oral bioavailability of residues was determined in rats to evaluate the extent to which tissue imidocarb residues could be reabsorbed by consumers. Ten ewes and 5 goats were administered im with a commercial formulation containing imidocarb dipropionate (Carbesia cavalli, Shering-Ploug 121.15 mg/ml) at the single dose of 3 mg/kg bw corresponding to 2.1 mg/kg bw imidocarb base. Two sheep and 1 goat were slaughtered 15, 30, 60, 90 or 120 d after dosing and samples of muscle, injection site muscle, liver, omental and subcutaneous fat, and kidneys were collected. Samples of cerebral hemisphere, cerebellum, olfactory bulb, pineal and pituitaryglands were dissected. For the residue bioavailability study 7 groups of3 Wistar rats each, were dosed by gavage with imidocarb dipropionate standard in water (group 2, 3 and 4) or with imidocarb as a liver residue collected from prior dosed animals (group 5, 6 and 7) at 8.4. 16.8 or 33.6 microg/kg of imidocarb base respectively, for 5 d. Group I was control. All animals were sacrificed the day after the last drug administration and livers were collected. The highest drug levels in sheep and goats occurred in liver and kidney, suggesting that these tissues are targets for residues; muscle had negligible importance as storage tissue. Goats had a lower storage capability than sheep. The residue profile in sheep liver and omental fat showed a 30-d storage period to reach maximum concentrations, and suggested that imidocarb is redistributed. The high and long-lasting concentrations in brain showed its capacity to cross the blood-brain barrier and caused concern for potential neurotoxic effects. Detectable concentrations of imidocarb were not found in rat liver.

A cellular mechanism for imidocarb retention in edible bovine tissues.

Imidocarb dipropionate, formulated as Imizol, is used for the treatment and prophylaxis of bovine babesiosis. Several studies have shown that imidocarb remains detectable in edible ovine and bovine tissues for several months after dosing but the mechanism of retention remains unknown. In this study, the mechanism of imidocarb retention was investigated by measuring the binding of [14C]imidocarb to bovine hepatocytes, erythrocytes, sub-cellular fractions and isolated bovine macromolecules. The proportion of [14C]imidocarb (10 microM) bound to cells in suspension culture (1 x 10(7) cells.ml-1) was found to be substantially greater to hepatocytes (56.5%) than to erythrocytes (4.6%). Studies with washed erythrocytes reconstituted in plasma indicated that approximately 70% of the [14C]imidocarb was bound to plasma proteins, 10% to erythrocytes, and 20% remained free. Measurement of [14C]imidocarb binding to sub-cellular fractions prepared from bovine liver revealed preferential accumulation in the nuclear, rather than in the mitochondrial, microsomal or cytosolic fractions. Binding capacities of selected bovine macromolecules for [14C]imidocarb were in the order deoxy-ribonucleic acid (DNA) = ribonucleic acid (RNA) > > alpha 1-acid glycoprotein (AGP) > serum albumin (BSA) > haemoglobin (Hb). DNA binding sites for imidocarb remained unsaturated over the concentration range 0-100 microM [14C]imidocarb. Competitive binding studies between imidocarb and pentamidine or spermidine provided evidence for common DNA binding sites. These studies indicated that preferential binding of [14C]imidocarb to hepatocytes compared with erythrocytes observed in vitro was a result of substantial reversible binding to nucleic acids and that the same cellular mechanism may be implicated in the slow elimination of imidocarb from edible tissues in vivo.

Imidocarb residues in edible bovine tissues and in vitro assessment of imidocarb metabolism and cytotoxicity.

Imidocarb residues in liver and muscle were measured by HPLC after a single therapeutic dose to cattle (3 mg imidocarb dipropionate kg-1). Imidocarb and 7-ethoxycoumarin metabolism were compared in three different in vitro systems prepared from bovine liver: cultures of hepatocyte monolayers, precision-cut liver slices, and microsomes. The potential hepatotoxicity of imidocarb residues was tested on hepatocyte monolayers and assessed using the neutral red and lactate dehydrogenase leakage assays. The concentration of imidocarb (mean +/- SD) decreased between days 14 and 224 after treatment from 5.40 +/- 0.61 to 0.12 +/- 0.01 and from 1.05 +/- 0.31 to 0.06 +/- 0.02 microgram g-1 in liver and muscle, respectively. The depletion kinetics of imidocarb fitted a two-compartment model with alpha- and beta-phase half-lives of 31.7 and 48.5 days in liver and 34.9 and 120.7 days in muscle, respectively. Imidocarb metabolites were not detected in any in vitro system. 7-Ethoxycoumarin metabolism was found in all in vitro systems; the predominant metabolite produced by hepatocyte and liver slice cultures was umbelliferone glucuronide. Cytotoxicity of imidocarb (100 microM) to hepatocyte monolayers was maximal after 72 hr treatment and dose-dependent above 10 microM imidocarb. It is most likely that the hepatotoxicity of imidocarb is caused by the parent compound, because no evidence for imidocarb metabolism was found.

Imidocarb depletion from cattle liver and mechanism of retention in isolated bovine hepatocytes.

Imizol injection (imidocarb) is used for the prevention and treatment of babesiosis in cattle. Studies in sheep indicate that imidocarb is retained in edible tissues (Aliu et al.). In the present study we have set up and validated a high-performance liquid chromatography based method to investigate the retention of imidocarb in cattle liver. Imidocarb was still detectable 224 d after a single therapeutic dose with a half-life of 42.7 d. The mechanism of imidocarb retention by bovine liver was modelled using isolated bovine hepatocytes. Incubations with isolated hepatocytes indicated that [14C]imidocarb binding was dependent on hepatocyte number and showed signs of saturation. Bound [14C]imidocarb could be eluted from hepatocytes with buffer and extracted with solvents. Equilibrium dialysis under denaturing conditions (Sun and Dent) indicated that 3% of the [14C]imidocarb was covalently bound to macromolecules. Although the hepatocyte preparations demonstrated the capacity for phase I and II 7-ethoxycoumarin metabolism no metabolites of [14C]imidocarb were found. Further in vitro binding studies involving sub-cellular fractionation indicated that [14C]imidocarb is partitioned largely in the nuclear fraction of bovine liver homogenates and that it binds to deoxyribonucleic acid.

Efficacy, toxicity and metabolism of imidocarb dipropionate in the treatment of Babesia ovis infection in sheep.

An intramuscular dose of 1.2 mg kg-1 of imidocarb dipropionate (IMDP) was effective in controlling fatal infections with Babesia ovis in sheep. The sheep were infected by the intravenous injection of sheep blood containing B ovis. A severe recrudescence of infection occurred in most sheep 10 to 14 days after therapy but this could be controlled by a second dose of 1.2 mg kg-1 IMDP. Studies on the toxicology, residues and metabolism of IMDP showed this to be a safe dosage regimen. Transient or mild signs of toxicity were seen at 2.4 and 4.8 mg kg-1 IMDP. Severe toxicity was observed in sheep treated with 9.6 mg kg-1.

Influence of induced disease states on the disposition kinetics of imidocarb in goats.

The influence of fever, induced by different agents, on the disposition kinetics of imidocarb was determined in goats. Escherichia coli endotoxin (0.2 microgram/kg), Trypanosoma evansi (10(7) in 1 ml sterile glucose citrate), and Infectious Bovine Rhinotracheitis virus (10(6.5)TCID50) were the agents administered to induce the febrile state. In control and febrile animals the two-compartment model was used to describe the disposition kinetics of the drug. Fever caused significant changes to occur in the apparent volume of distribution and the body (systemic) clearance of imidocarb, but the half-life remained unchanged. The statistical significance of the changes in these pharmacokinetic parameters varied with the etiology of the febrile state. E. coli endotoxin and IBR virus caused corresponding decreases in apparent volume of distribution and clearance of imidocarb, while fever induced with T. evansi caused highly significant increases in both pharmacokinetic parameters. It was concluded that the alterations in the disposition kinetics of imidocarb that occurred in the febrile goats were related not only to the febrile reaction per se but also to the pathophysiology of the disease condition.

Influence of Escherichia coli endotoxin-induced fever on pharmacokinetics of imidocarb in dogs and goats.

The pharmacokinetics of imidocarb, administered as an IV bolus dose (4 mg/kg), was studied in normal and Escherichia coli endotoxin-induced febrile dogs and goats. In the febrile group, the drug was administered 1 hour after injection of the endotoxin. The plasma and urine concentrations of imidocarb were measured by spectrophotometry. The decline in plasma drug concentrations in both species was analyzed, using a 2-compartment open model. With the exception of the coefficient A and the volume of central compartment, E coli endotoxin-induced fever produced the same changes in kinetic determinants in both species. Fever significantly decreased the distribution rate constant in both dogs (P less than 0.05) and goats (P less than 0.01). The elimination rate constant and, in turn, the half-life were not altered by the endotoxin-induced fever in either species. The volume of distribution at steady-state was significantly lower (P less than 0.01) in the febrile dogs and goats. The body clearance of imidocarb was also significantly lower in the febrile dogs (P less than 0.05) and goats (P less than 0.01). The decreased apparent volume of distribution and lower body clearance of imidocarb could explain the higher plasma values of the drug in the febrile, compared with normal, animals.

Absorption, distribution, and excretion of imidocarb dipropionate in sheep.

Spectrophotometric and thin-layer chromatographic methods for determination of imidocarb in biological specimens are described. Following intravenous injection of imidocarb (2.0 mg/kg) into 3 sheep, plasma concentrations, initially averaging 10.8 microgram/ml, decreased to an average of 1.9 microgram/ml within 1 hour and then to less than 1 microgram/ml within the next 4 hours. When imidocarb (4.5 mg/kg) was injected intramuscularly (IM) into 7 sheep, peak plasma concentrations averaging 7.9 microgram/ml were achieved within 4 hours and then rapidly decreased to 4.6 microgram/ml within the next 2 hours. Plasma values then decayed very slowly by first-order kinetics and trace amounts were still present 4 weeks after treatment. Imidocarb was bound to plasma proteins and the apparent volume of distribution was estimated to be slightly higher than the total body water. The concentrations of the drug in the plasma and in the erythrocytes were approximately equal. Detectable amounts were present in all examined tissues 4 weeks after IM administration Twenty-four hours after IM administration, the highest concentrations were in kidney, liver, and brain. The 14C-labeled imidocarb could be detected in all regions of the central nervous system examined, in the hypophysis, and in the pineal body. Metabolic or biotransformation products were not detected by the methods used. Of the administered IM dose, 11 to 17% was excreted in the urine within 24 hours; thereafter, the excretion rate was low, and detectable amounts were still present in the urine for 4 weeks. Renal clearance of imidocarb was less than glomerular filtration rate, indicating net tubular reabsorption. The relatively high concentration of imidocarb in the bile suggests that the bile is an important route of excretion. High concentrations were also found in the mild of lactating ewes, but the drug could not be detected in the plasma of lambs fed milk from these ewes.