1. Ivermectin was extensively metabolized by human liver microsomes to at least 10 metabolites. The structure of many of them (mostly hydroxylated and demethylated) was determined by 1H-NMR and LC/MS. 2. To determine which human cytochrome P450 isoform(s) is responsible for the metabolism of ivermectin, chemical inhibitors including sulphaphenazole, quinidine, furafylline, troleandomycin (TAO) and diethyldithiocarbamate (DDC) were used to evaluate their effect on ivermectin metabolism. TAO, a specific inhibitor of cytochrome P4503A4, was the most potent inhibitor, inhibiting the total metabolism as well as formation of each metabolite. Metabolism was also inhibited by an anti-human cytochrome 3A4 antibody by 90%. 3. When ivermectin was incubated with microsomes from cells expressing CYP1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1 or 3A4 at 4 mg/ml protein concentrations, metabolic activity was only detected with the microsomes containing CYP3A4. The metabolic profile from cDNA-expressed CYP3A4 microsomes was qualitatively similar to that from human liver microsomes. 4. Thus, cytochrome P4503A4 is the predominant isoform responsible for the metabolism of ivermectin by human liver microsomes.

Abamectin (AVM) and ivermectin (IVM) are each metabolized by rat liver microsomes to 3‘‘-O-desmethyl (3‘‘-ODMe), 24-hydroxymethyl (24-OHMe), and 26-hydroxymethyl (26-OHMe) derivatives. Microsomes from rats pretreated with dexamethasone (Dex), but not 3-methylcholanthrene (3MC), increased the formation of 3‘‘-ODMe metabolites of both AVM and IVM. Troleandomycin inhibited formation of 3‘‘-ODMe metabolites by >80% by microsomes from Dex-induced rats. Therefore, cytochrome P450 3A plays a major role in this metabolic pathway. Formation of the 26-OHMe metabolites was markedly increased by microsomes from 3MC-treated but not Dex-treated rats. Formation of 24-OHMe from AVM, but not IVM, was slightly increased by microsomes from 3MC-treated rats. Consistent with this observation, anti-rat cytochrome P450 1A1 inhibited formation of 26-OHMe metabolites of AVM and IVM by 90 and 40%, respectively. This antibody also inhibited formation of the 24-OHMe metabolite from AVM by 60% but not from IVM. Thus, cytochrome P450 1A1 is involved in the hydroxylation of the 26-methyl group of both AVM and IVM as well as the 24-methyl group of AVM but not the 24-methyl group of IVM. Keywords: Avermectin; ivermectin; metabolism; rat; cytochrome P450; isoform

To determine whether ivermectin is metabolized in the rumen, in vitro studies were conducted with the tritium-labelled H2B1a component of ivermectin in rumen fluid from sheep and cattle. No detectable metabolism occurred over 24 h in in vitro incubations at 38 degrees C. The viability of the microbes in the rumen fluids was demonstrated by the conversion of 17% and 11% of [14C]cellulose to 14CO2 in 24 h in the incubations with sheep and steer rumen fluids respectively. The results indicate that ivermectin is not metabolized in the rumen. Based on the lack of in vitro metabolism of ivermectin in rumen fluid, the similarity of in vitro liver microsomal metabolism with in vivo metabolism of the avermectins and the physicochemical properties of the avermectins, any disappearance of ivermectin in vitro from rumen fluid is probably a result of binding to solids or surfaces. Apparent discrimination by dung beetles, where observed, between control faeces and faeces from cattle or sheep treated with ivermectin or abamectin therefore must be attributable to chance, to factors unrelated to treatment or to factors such as changes in amino acid composition rather than the production of volatile metabolites of ivermectin.

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1. Metabolism of 22,23-dihydro-13-O-[(2-methoxyethoxy)methyl]-avermectin B1 aglycone (MEM-H2B1), a new avermectin, by rat liver microsomes has been studied. Metabolites identified were formed by demethylation of the methoxyethoxymethoxy (MEM) side chain, loss of the MEM side chain, partial cleavage and further oxidation of the MEM side chain, and oxidation of the aglycone after cleavage of the MEM side chain. 2. The specific cytochrome P450 isoforms involved in the metabolism of MEM-H2B1 were identified through immunoinhibition studies. Among several antibodies prepared against various cytochrome P450s, only anti-rat P4503A IgG inhibited MEM-H2B1 metabolism by liver microsomes from the untreated rat. Moreover, troleandomycin, a selective suicide inhibitor for enzymes of the cytochrome P4503A family, inhibited the total metabolism by > 80%. These results clearly indicate that cytochrome P4503A is primarily responsible for the metabolism of MEM-H2B1. 3. Secondary metabolism was evident in the metabolism of MEM-H2B1 by dexamethasone and phenobarbital induced liver microsomes, where different isoform(s) of cytochrome P4503A could be involved in these multiple step reactions.