Metabolism of albendazole in cattle, sheep, rats and mice.

Nine metabolites of albendazole were identified in the urine of cattle, sheep, rats, and mice which had been given the labeled drug orally. Metabolic conversions included oxidation at sulfur, alkyl and aromatic hydroxylation, methylation at both nitrogen and sulfur, and carbamate hydrolysis. Unchanged albendazole was present in urine in only minor amounts. Albendazole (ABZ), methyl [5-(propyltbio)1H-benzimidazol2-yl]carbamate, is the first broad-spectrum antheLmintic to have useful efficacy at low dose levels against all major economically important helniinth parasites infecting domestic animals (1). ABZ is widely used in sheep and cattle for control of gastrointestinal nematodes and tapeworms, lungworms, and liver flukes (2). It is also effective against metacestodes of Taenia saginaw in calves (3) and more exotic but difficult infections of Paragonimus kellicotti in cats (4) and Filaroides hirthi in dogs (5). The present study identifies the urinary metabolites of ABZ in cattle, sheep, rats, and mice. Toxicity, pharmacokinetics, distribution and metabolism of drug in tissues, and methods for determination ofdrug in biological samples will be reported elsewhere. Materials and Methods Animal Studies. Four steers (average weight 145 kg), housed in metabolism cages, were dosed orally by gelatine capsule with 20 mg of ‘4C-ABZ per kg (0.760 zCi/mg). Urine collected in the period 0-72 hr after administration was pooled and used for the isolation and identification of most of the metabolites in this study. In a second study, two steers were similarly treated with 15 mg of ‘4C-ABZ per kg (3.26 iCi/mg). A quantitatively similar mixture of urine metabolites was produced and is reported herein. Two male sheep (average weight 24 kg), housed in metabolism cages, were dosed orally by gelatine capsule with 10 mg of ‘4C-ABZ per kg (7.81 zCi/mg). Urine was collected and analyzed in a manner similar to that used in the cattle study. Cattle and sheep urine was kept frozen until use. Six virgin female Sprague-Dawley rats (Blue Spruce Farms, Altamont, N.Y.), each weighing approximately 200 g, were individually housed in metabolism cages. In separate studies, rats were dosed with 13.25 mg of ‘4C-ABZ per kg (0.5 10 sCi/mg) or 14.9 mg of ‘4C-labeled metabolite A per kg (0.178 zCi/mg). Doses were suspended in 1 ml of water containing 1% carboxymethylcellulose and 0.2% Tween-80 and administered to each rat by stomach tube. Urine was separated from feces by a secondary openweave cage floor. Urine was collected in 24-hr intervals and kept frozen until use. In a separate study for isolation of metabolites E and G, two rats were dosed with 100 mg of ‘4C-A per kg (0.788 iCi/mg) and urine collected in the period 0-24 hr. Twelve Charles River CD male albino mice, each weighing approximately 25 g, were dosed orally with 13.25 mg of ‘4C-ABZ per kg (1.09 MCi/mg). Dosing and urine collection was carried out in a manner similar to that used in the rat study. Instrumental Analysis. Radiometric assays were performed on Packard Tri-Carb model 577 or 3255, or Beckman model LS-355 liquid-scintillation spectrometers with a modified Bray’s scintillation fluid or Instagel (Packard lnstrumcnt Co., Downers Grove, Ill.). Modified Bray’s solution was prepared from naphthalene (250 g), 2,5-diphenyloxazole (16 g), p-bis(2-(5phenyloxazolyl)]benzene (0 8 g), toluene (1600 ml), p-dioxane (1600 ml), and ethanol (475 ml). Quenching was corrected by use of an external standard. Melting points were determined in capillary tubes with a Thomas-Hoover or Mel-Temp apparatus and are uncorrected. Melting points of benzimidazole carbamates are dependent upon rate of heating and the temperature at which the samples are inserted; they melt with decomposition. IR spectra were determined in Nujol-mull with a PerkinElmer model 727B spectrophotometer. NMR spectra were obtained in a Varian model T-60 spectrometer equipped with an Ortex model S47 digital signal averager. Electron-impact mass spectra were obtained with a Hitachi/Perkin-Elmer model RMU-6E spectrometer. Field-desorption mass spectra were obtained in a Varian model MAT-CH5-DF mass spectrometer. Chemical-ionization mass spectra were obtained in a Finnegan model 3200 GC/MS instrument with an INCOS data system. Thin-layer Chromatography. Glass plates coated with silica gel (2SO-p thickness) containing an inorganic phosphor(GF; Analtech, Newark, Del.) were used. Compounds were visualized by UV light or autoradiography on medical X-ray film (Type NS-5T, Kodak), developed for highest contrast. In metabolite isolation studies, radioactive zones from developed plates were scraped, eluted with isopropanol or methanol, and evaporated under N2. In metabolite-quantitation studies, plate scrapings of discrete zones corresponding to the metabolites were assayed by scintillation counting as described above. Synthetic Methods. All synthetic metabolites and intermediates gave elemental analyses (C, H, N) which agreed with theoretical values to within 0.4%. Melting points and molecular formulae are given in the text for all intermediates not previously reported. Chemical names and molecular formulae of metabolites are given in table 1. The structure of each synthesized standard was confirmed by IR, NMR, and mass spectrometry. Synthetic samples were used for comparison with isolated metabolites. ‘4C-A BZ. Several syntheses of ‘4CABZ were carried out. In each case, the chemical of high specific activity was demonstrated to have radiochemical purity >99% by TLC and autoradiography or radioscanning. It was diluted with nonradioactive ABZ to an appropriate specific activity before use. ABZ, uniformly ‘4C-labeled in the benzene ring, was prepared from ‘4C-benzene in overall 45% radiochemical yield by the following sequence of reactions: 4C-benzene nitrated with nitronium trifluoromethanesulfonate gave ‘4C-nitrobenzene; catalytic reduction over 5% Pd/C in methanolic HC1 gave ‘4C-aniline hydrochloride and thiocyanation with NH4SCN and Br2 in acetic acid/ether followed by reaction with acetic anhydride gave ‘4C-4-thiocyanatoacetanilide; nitration with fuming nitric acid in methanesulfonic acid gave ‘4C-2-nitro-4-thiocyanatoacetanilide (6); reduction with NaBH4 in methanol at 0#{176}C gave ‘4C-4-acetamido-3-nitrothiophenol at A PE T Jornals on A ril 8, 2017 dm d.aspurnals.org D ow nladed from