Identification of cytochrome P4503A as the major enzyme sub-family responsible for the metabolism of 22,23-dihydro-13-O-[(2-methoxyethoxy)methyl]-avermectin B1 aglycone by rat liver microsomes.

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.

Stability of ivermectin in rumen fluids.

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.

Fate of 4"-epiacetylamino-4"-deoxyavermectin B1 in rats.

Distribution, excretion, and metabolism of 4"-epiacetylamino-4"-deoxyavermectin B1 (AAB1), a new avermectin, were determined in Sprague-Dawley VAF rats. The rats were dosed orally for 7 consecutive days at approximately 6 mg/kg body weight with [5-3H]AAB1 as a 1.2 mg/ml aqueous suspension containing 0.5% methyl cellulose. Rats were killed at approximately 7 hours and 1, 2, and 5 days after the last dose. The major route of excretion of drug residues was via feces, with less than 1% of the dose found in urine. The radioactive residue levels in tissues and blood followed the order GI > liver approximately equal to fat approximately equal to kidney > muscle > plasma approximately equal to red blood cells and were comparable in male and female rats. HPLC-radiochromatographic profiles revealed that 4"-epiamino-4"-deoxyavermectin B1a was the major metabolite in all tissue samples and plasma samples, and was usually the major residue at later time points. The results indicate that N-deacetylation of AAB1 was the primary route of metabolism in rats. A distinct feature of the metabolism was a sex difference in the extent of metabolism. When metabolite profiles of male and female rats killed at the same time were compared, less parent drug and more of the N-deacetylated metabolite were found in the female rats, indicating that the drug was metabolized more extensively in female rats than in male rats. The sex difference in the extent of metabolism was also demonstrated in vitro.

Environmental effects of the usage of avermectins in livestock.

Abamectin (avermectin B1) and ivermectin (22,23-dihydroavermectin B1) are high molecular weight hydrophobic compounds, active against a variety of animal parasites and insects. Numerous environmental fate and effects studies have been carried out in the development of these two compounds as antiparasitic agents and for abamectin as a crop protection chemical. They were found to be immobile in soil (Koc > or = 4000), rapidly photodegraded in water (degradation half-life (t1/2) in the summer 0.5 days or less) and as thin films on surfaces (t1/2 < 1 day), and aerobically degraded in soil (ivermectin in soil/feces mixtures (t1/2) = 7-14 days; avermectin B1a in soils, t1/2 = 2-8 weeks) to less bioactive compounds. Abamectin is not taken up from the soil by plants, nor is it bioconcentrated by fish (calculated steady-state bioconcentration factor of 52, with rapid depuration). Daphnia magna is the fresh water species found to be most sensitive to ivermectin and abamectin (LC50 values of 0.025 and 0.34 ppb respectively); fish (e.g. rainbow trout) are much less sensitive to these compounds (LC50 values of 3.0 ppb and 3.2 ppb, respectively). In the presence of sediment, toxicity toward Daphnia is significantly reduced. The metabolism and degradation of ivermectin and abamectin result in reduced toxicity to Daphnia. Abamectin and ivermectin possess no significant antibacterial and antifungal activity. They display little toxicity to earthworms (LC50 values of 315 ppm and 28 ppm in soil for ivermectin and abamectin, respectively) or avians (abamectin dietary LC50 values for bobwhite quail and mallard duck of 3102 ppm and 383 ppm, respectively), and no phytotoxicity. Residues of the avermectins in feces of livestock affect some dung-associated insects, especially their larval forms. This does not delay degradation of naturally formed cattle pats under field conditions; however, in some cases, delays have been observed with artificially formed pats. Based on usage patterns, the availability of residue-free dung and insect mobility, overall effects on dung-associated insects will be limited. As abamectin and ivermectin undergo rapid degradation in light and soil, and bind tightly to soil and sediment, they will not accumulate and will not undergo translocation in the environment, minimizing any environmental impact on non-target organisms resulting from their use.

Proton chemical shifts in fluorocinnamate-chymotrypsin complexes.

Binding of cinnamate or fluorocinnamate anions to alpha-chymotrypsin is accompanied by chemical shift changes at each proton of the cinnamate structure. The direction and magnitude of these shifts are consistent with the expected binding of these inhibitors at the active site of the enzyme. The protein-induced fluorine chemical shift effects at each position in the aromatic ring are compared to the shift effects observed when a hydrogen occupies the same position. There is no discernible relation between the proton and fluorine chemical shifts, leading to the conclusion that those factors dominantly responsible for the shift effects are different for the two sets of data.

Infrared spectroscopy of retinoids.

The Fourier transform infrared spectra of 15 purified retinoids were compared. Retinoids with conjugated C = O groups revealed the presence of a band between 1730 and 1630 cm-1 X A characteristic of retinoids is the presence of a band between 1650 and 1620 cm-1 due to C = C stretching where three to four conjugated C = O and C = C bonds were present or between 1610 and 1555 cm-1 where more conjugated unsaturations were present. The presence of cis double bonds was confirmed by a band at 1380 cm-1 while unsubstituted trans double bonds gave absorbances at 990 to 955 cm-1. Epoxy rings, which are present in some retinoids, resulted in bands at 1250, 880, and 790 cm-1 while a furan structure was confirmed by bands at 1175, 1083, and 1065 cm-1.

Identification of a decarboxylation product of retinoic acid.

The oxidative decarboxylation of retinoic acid was investigated utilizing a model system consisting of all-trans-retinoic acid, H2O2 and horseradish peroxidase. The decarboxylation products were purified by high-performance liquid chromatography on bonded, octadecylsilane columns. Based on mass spectral, nuclear magnetic resonance, ultraviolet and Fourier transform infrared analyses, the major decarboxylation product was identified as a 4-oxo-C19 aldehyde with a hydroxyl group on the side chain at C9, specifically 8-(2,6,6-trimethyl-3-oxocyclohex-1-enyl)2,6-dimethyl-6-hydroxyoctatrienal.

Solvent effects on the time-dependent photoisomerization of methyl retinoate.

The photoisomerization of methyl all-trans-retinoate is dependent upon both solvent polarity and irradiation time. The all-trans-isomer isomerizes most rapidly in solvents of high polarity. Methyl 11-cis-retinoate is maximally produced after three hours in dimethyl sulfoxide, while it is not formed to an appreciable extent in heptane. Methyl 13-cis-retinoate forms maximally after two hours in heptane, then decrease in concentration by one-half to about the same level maximally formed in dimethyl sulfoxide. The solvent and time dependences of the other isomers are presented to allow selection of conditions for maximal production of desired isomers. The results for methyl retinoate appear applicable for retinoic acid.