METABOLISM OF H/C-LABELED 4(-DEOXY-4(-EPIMETHYLAMINOAVERMECTIN B1A BENZOATE IN CHICKENS Identification of Novel Fatty Acid Conjugates of 4(-Deoxy-4(-epimethylaminoavermectin B1a

The metabolism of H/C-labeled 4(-deoxy-4(-epimethylaminoavermectin B1a (MAB1a) benzoate, the major homologue (>90%) of the avermectin insecticide emamectin benzoate, was studied in laying chickens. Ten Leghorn hens (Gallus domesticus) were orally dosed once daily for 7 days (1 mg/kg of body weight/day). Eggs and excreta were collected daily, and eggs were subsequently separated into whites and yolks. Chickens were euthanized within 20 hr after the last dose, and liver, kidney, heart, muscle, fat, ovaries, gizzard, gastrointestinal tract and contents, and carcass were collected. Approximately 70 and 6% of the total administered dose were recovered in the excreta plus gastrointestinal tract and contents and in the tissues plus eggs, respectively. Two novel metabolites, i.e. the 24-hydroxymethyl derivative of the parent compound (24-hydroxymethyl-4(-deoxy-4(-epimethylaminoavermectin B1a) and the N-demethylated derivative of 24-hydroxymethyl-4(-deoxy-4(-epimethylaminoavermectin B1a (24-hydroxymethyl4(-deoxy-4(-epiaminoavermectin B1a), were identified. In addition, eight fatty acid conjugates of each of these two metabolites, comprising 8–75% of total radioactive residues in tissues and eggs, were isolated and identified. Although this represents some of the most extensive in vivo fatty acid conjugation to a xenobiotic reported to date, potential human exposure to MAB1a residues from consumption of chicken would be extremely low, because the dosage level in this study was ;1000-fold greater than the MAB1a residue levels seen in crops and because the majority of the applied dose was recovered in the excreta. Based on these findings, the avian biotransformation of MAB1a differs substantially from the mammalian biotransformation. Emamectin benzoate (MK-0244) is the benzoate salt of the 40deoxy-40-epimethylamino derivative of abamectin, a macrocyclic lactone produced by the soil actinomycete Streptomyces avermitilis. It is currently being developed as a wide-spectrum Lepidopteran larvicide for use on leafy and fruiting vegetables, on cole crops, and on cotton. Emamectin benzoate is effective at extremely low use rates (,0.015 lb of active ingredient/acre). Emamectin benzoate is specified as a mixture of two homologous compounds, i.e. a minimum of .90% MAB1a and a maximum of ,10% MAB1b; these differ only by a methylene group on the C25 side chain (fig. 1). Because the structural difference between the two homologues is small and because comparative metabolism studies of MAB1a and MAB1b using rat liver slices have demonstrated homologous metabolism (Mushtaq M, unpublished results), it is likely that the metabolism of MAB1b would also be homologous to that of MAB1a in chickens. In previous emamectin benzoate metabolism studies in rats and goats, .99% of the administered dose was recovered in the excreta, with correspondingly low tissue residue levels (Mushtaq et al., 1996, 1997). In those studies, the parent compound was the major residue (85–95% of TRR) in tissues and feces, and the only identified metabolite was the N-demethylated derivative of the parent, AB1a (5– 15% of TRR). The purpose of this study was to determine the egg and tissue distribution, excretion, and metabolism of emamectin benzoate and its metabolites in laying hens after oral administration of the major homologue, MAB1a. Such results can be used to evaluate the potential for human exposure to emamectin benzoate residues from the consumption of eggs and tissue from chickens whose diet contained treated crops. The present results indicate that the metabolism of emamectin benzoate in chickens is remarkably different from that in mammals. Materials and Methods Test and Reference Chemicals. Ethanolic solutions of 5-[H]MAB1a benzoate (16.80 mCi/mg, 16.94 mCi/mmol) and 25-[C]MAB1a benzoate (20.86 mCi/mg, 20.69 mCi/mmol) were supplied by the Labeled Compound Synthesis Group of Merck Research Laboratories. The radiochemical purity of 1 Current address: Drug Safety & Metabolism-Animal Health, Schering-Plough Research Institute, P.O. Box 32, Lafayette, NJ 07848. 2 Abbreviations used are: MAB1a, 40-deoxy-40-epimethylaminoavermectin B1a; MAB1b, 40-deoxy-40-epimethylaminoavermectin B1b; 24-OH-AB1a, 24-hydroxymethyl-40-deoxy-40-epiaminoavermectin B1a; 24-OH-MAB1a, 24-hydroxymethyl-40-deoxy-40-epimethylaminoavermectin B1a; AB1, 40-deoxy-40-epiaminoavermectin B1 (a mixture of B1a and B1b homologues); AB1a, 40-deoxy-40epiaminoavermectin B1a; GIT, gastrointestinal tract and contents; APCI, atmospheric pressure chemical ionization; LSC, liquid scintillation counting; SPE, solid-phase extraction; EtOAc, ethyl acetate; TRR, total radioactive residue(s). Send reprint requests to: Christopher L. Wrzesinski, Schering-Plough Research Institute, P.O. Box 32, Lafayette, NJ 07848. 0090-9556/98/2608-0786–794$02.00/0 DRUG METABOLISM AND DISPOSITION Vol. 26, No. 8 Copyright © 1998 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. 786 at A PE T Jornals on Jne 3, 2017 dm d.aspurnals.org D ow nladed from both chemicals was .97%. The ethanolic solutions of [H]MAB1a benzoate and [C]MAB1a benzoate were combined to yield 8 ml of a 15.2 mg/ml [H/C]MAB1a benzoate test solution. Unlabeled emamectin benzoate (MK0244) and AB1 (including both the AB1a and AB1b homologues) were used as reference standards and were supplied by Merck Research Laboratories. Preparation of the Dosing Capsules. Gelatin capsules (size 3 white ELC/ELC; Shionogi Corp., Whitsett, NC) were half-filled with cellulose powder, and a 100-ml aliquot of either ethanol (control) or the ethanolic test chemical solution (treated) was added to each capsule. The capsules were kept at room temperature in a hood, to allow the ethanol to evaporate, and were then completely filled with cellulose powder and sealed. Approximately 100 mg of cellulose was added to each capsule. The sealed capsules were then placed inside a larger capsule (size 2 clear AA/AA) for administration. Handling and Dosing of Chickens. Twenty top-quality, laying, Leghorn hens (Gallus domesticus) were procured from Avian Services (Frenchtown, NJ). They were each approximately 61 weeks of age, weighed between 1 and 2 kg, and were uniquely identified by wing band. Chickens were individually housed in galvanized metal cages (multi-stacking poultry laying cages) with ad libitum access to food (Purina Layena) and water. Daily food consumption was monitored by feed weight for each chicken. The chickens were allowed to acclimate to the metabolism cages, diet, and handling procedures for 14 days before the start of dosing and were maintained in a temperature-controlled room at 72°F, with 16 hr of light provided daily with fluorescent lights. After approximately 1 week of acclimation, healthy and actively laying chickens were randomly assigned to test (10 chickens) and control (5 chickens) groups. Chickens were dosed once daily (at approximately 1 mg/kg of body weight) for 7 consecutive days with capsules and were euthanized by carbon dioxide inhalation approximately 20 hr after administration of the last dose. The daily dose corresponded to approximately 10 ppm of emamectin benzoate in the diet, as calculated from food consumption. Sample Collection. Excreta were collected and composited once daily from all treated chickens. Eggs were collected twice daily from the treated chickens and were separated into whites and yolks. Excreta and eggs were collected from the control chickens before euthanasia only. Liver, kidney, heart, gizzard, GIT, ovaries (immature eggs), muscle (thigh and breast), fat (abdominal and muscle fat, with adhering skin), and carcass (minus head, feet, wings, and feathers) specimens were collected from each chicken. The interiors of the gizzards were flushed with water, and the contents were combined with the GIT specimen. After removal of the chickens, the cages housing the treated chickens were washed with soap and water and an aliquot of the wash was analyzed for radioactivity. All specimens were frozen after collection and were kept frozen except when aliquots were removed for analysis. Sample Homogenization and TRR Determination. Immediately after collection, the composite excreta samples were homogenized with water (1:1, w/v) in a Waring blender. GIT were composited into three samples (two treated and one control, each containing the GIT from five chickens) and likewise homogenized with water (1:1, w/v) in a Waring blender. In addition, water (1:1–3, w/v) was added to individual liver, kidney, heart, gizzard, egg yolk, and ovary specimens, and these were then homogenized with a Brinkman Polytron homogenizer (Brinkman Instruments, Westbury, NY). Egg white specimens were homogenized using a Brinkman Polytron homogenizer without the addition of water. Muscle and carcass specimens were chopped into small pieces (while still partially frozen) using a meat cleaver and were then passed three times through a Fleetwood meat grinder (W. W. Lowenstein, Inc., Newark, NJ). Fat specimens were homogenized by thorough chopping and mixing with a meat cleaver. Aliquots of tissue and excreta homogenates (0.2–1 g) were then assayed in replicate (three or more determinations) for TRR by radiocombustion analysis using a Packard model 307 sample oxidizer, followed by LSC. The TRR values for egg whites, egg yolks, and ovaries were determined by direct LSC of triplicate aliquots (0.2–0.4 g) of each specimen. The accuracy of direct LSC of these specimens was demonstrated by a comparison of results obtained by radiocombustion analysis followed by LSC with those obtained by direct LSC of composite samples (data not shown). Sample Extraction. Tissue homogenates (liver, muscle, fat, and ovaries) were composited by combining equal amounts of each individual homogenized specimen. Aliquots of egg yolk homogenates from all eggs collected in the study were similarly composited and were additionally composited according to treatment day, as for excreta. Aliquots of composited tissue, excreta, and egg yolk homogenates (1–2 g) were extracted either with acetone/EtOAc (1:1, v/v) or with acetone/5% aqueous sodium chloride (1:1, v/v) (fat only), the extracts were partitioned with EtOAc, and the organic layers from this partitioning were fractionated by cation exchange SPE, as described in detail elsewhere (Mushtaq et al., 1996, 1997). The EtOAc/NH3 residue fractions from the SPE fractionation contained 80–95% of the total radioactivity in the original samples (data not shown). Initial attempts were made to analyze the EtOAc/ NH3 SPE fractions by reverse-phase HPLC using a C18 column. However, for all samples except excreta, recoveries were generally unacceptably low, ranging from 30 to 90% (data not shown). Column recoveries were improved to .95% using normal-phase HPLC (method 1; table 1). Therefore, suitable aliquots from the EtOAc/NH3 SPE fractions were mixed with unlabeled AB1 and MK-0244 standards, dried under N2, and reconstituted in approximately 100 ml of methanol. Triplicate 10-ml aliquots were counted directly for recovery calculations, and the remaining solution (;70 ml) was analyzed by normal-phase HPLC (method 1). Metabolite Isolation. Approximately 50 g of composite excreta homogenate was extracted with acetone/EtOAc, and the organic extract was fractionated by cation exchange SPE. After concentration, the EtOAc/NH3 SPE fraction was fractionated by semipreparative HPLC (method 2; table 1), and selected fractions were pooled into four crude residue fractions. The crude 24-OH-MAB1a and 24-OH-AB1a fractions were combined and refractionated by HPLC method 4. The collected fractions containing the 24-OH-MAB1a and 24-OH-AB1a metabolites from this second HPLC fractionation were then individually purified by HPLC method 1. The two purified residues were then analyzed by NMR spectrometry and/or MS. Similarly, the crude MAB1a and AB1a fractions from semipreparative HPLC were subjected to additional HPLC separation. Nonlabeled MK-0244 and AB1 standards were added as appropriate, and the crude MAB1a and AB1a residue fractions were refractionated by HPLC method 3. The individual residues coeluting with the respective added standards were then analyzed by HPLC method 1. The identities of MAB1a and AB1a were thus confirmed by cochromatography with reference standards, using two HPLC methods with different selectivities. Approximately 40 g of the homogenate from the liver specimen with the highest TRR level was extracted as described above. After concentration, the EtOAc/NH3 SPE fraction was fractionated by HPLC method 1 (table 1), and selected fractions were pooled into five crude residue fractions. Two of these crude residue fractions contained fatty acid conjugates of 24-OH-MAB1a and of 24-OH-AB1a. The fatty acid conjugates of both of these metabolites eluted as a single peak with this HPLC method. These two crude residue fractions were fractionated into seven residue peaks by HPLC method 5 (table 1). All resolved fatty acid conjugates were then identified by MS analysis. One of the seven residue peaks from each of the two crude residue fractions was shown to contain two separate conjugates. The purified 24-OH-MAB1a oleate residue was also analyzed by NMR spectrometry. Lipase Hydrolysis. To each of two 20-ml glass vials was added an aliquot of the EtOAc/NH3 SPE fraction from the liver with the highest TRR level, containing approximately 0.7 mg of parent equivalent. The aliquots were concentrated to dryness under N2 and resuspended in 5 ml of phosphate buffer FIG. 1. Structure of emamectin benzoate (MK-0244) and positions of the radiolabels. 787 METABOLISM OF H/C-EMAMECTIN BENZOATE IN CHICKENS at A PE T Jornals on Jne 3, 2017 dm d.aspurnals.org D ow nladed from