An automated method for the determination of subnanogram concentrations of eprinomectin in bovine plasma.

Eprinomectin is a potent anthelmintic compound that kills certain parasitic nematodes and arthropods of cattle. A sensitive and automated bioanalytical assay was developed for quantitation of eprinomectin in bovine plasma in support of clinical development of eprinomectin for use in all classes of cattle. This assay determined the concentration of eprinomectin in plasma by reversed-phase high performance liquid chromatography (HPLC) with fluorometric detection. Plasma sample preparation included liquid extraction performed by the Packard MultiPROBE robotics workstation, followed by solid phase extraction performed by the Gilson ASPEC XL automated workstation. The HPLC assay included automated pre-column derivatization with a fluorogenic reagent system which included trifluoroacetic anhydride and N-methylimidazole as the catalyst. This reversed-phase chromatographic analysis was based on the fluorescence detection of derivatized eprinomectin and an internal standard, L-648 548, which was similarly derivatized by the fluorogenic reagents. The assay was automated and validated for two concentration ranges of 0.05-10 and 0.5-200 ng ml-1. The lower limit of quantitation of eprinomectin in plasma was 0.05 ng ml-1. The %RSD of the assay was 10% or better at all concentrations. This automated analysis of eprinomectin was used for high-throughput clinical assays with acceptable accuracy and precision.

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.

Drug residue formation from ronidazole, a 5-nitroimidazole. VIII. Identification of the 2-methylene position as a site of protein alkylation.

Ronidazole protein-bound adducts were generated by the in vitro anaerobic incubation of [2-methylene-14C]ronidazole with microsomes from the livers of male rats. Acid hydrolysis of the protein adducts yielded an imidazole ring fragment bearing the radiolabel and an amino acid residue derived from the proteins. This fragment has been identified as carboxymethylcysteine by co-chromatography of the amino acid and its dansyl derivative with known standards under a variety of conditions. The carboxymethylcysteine was estimated to represent at least 15% of the radioactivity derived from the protein-bound adducts and provides unequivocal evidence that nucleophilic attack by protein cysteine thiols occurred at the 2-methylene position of ronidazole.

Dermal penetration of avermectin B1a in the rhesus monkey.

Forearms of rhesus monkeys were treated with [3H]avermectin B1a in three different vehicles and concentrations so that the penetration of avermectin B1a through skin could be determined. In order to simulate exposure of farm workers, such as mixer-loaders, applicators, and harvesters, to this pesticide, avermectin B1a was applied to the forearms of the monkeys as an emulsifiable concentrate (300 micrograms/monkey), a diluted emulsifiable concentrate (4.5 micrograms/monkey), and as a suspension in water (216 micrograms/monkey). After 1 or 10 hr of exposure, the treatment area was washed. The levels of radioactivity were determined in the urine, feces, plasma, and wash. On the basis of the amounts of radioactivity excreted in the urine and feces and the levels of radioactivity in the plasma after dermal application compared to those found after intravenous administration of the compound, less than 1% of the doses was absorbed. These data indicate that avermectin B1a would not readily penetrate the skin of farm workers exposed to it. Therefore, the hazard to farm workers exposed to this compound would be substantially mitigated.

Structural alterations that differentially affect the mutagenic and antitrichomonal activities of 5-nitroimidazoles.

Two approaches have been used to develop nonmutagenic 5-nitroimidazoles. Both approaches are based on knowledge of the likely mechanisms by which this class of compounds cause mutagenicity. The first approach involved incorporating readily oxidizable gallate derivatives into the molecule. In one case, a very weakly mutagenic active antitrichomonal agent was obtained. The second approach involved incorporating a substituent at the C4 position of the ring. This generally resulted in a large reduction in mutagenicity and a lowering of antitrichomonal activity in vitro. In certain cases, however, mutagenicity was dramatically reduced while moderate antitrichomonal activity was retained. For example, 1,2-dimethyl-4-(2-hydroxyethyl)-5-nitroimidazole (5) showed good antitrichomonal activity in vitro (ED50 = 2 micrograms/kg) while possessing only 4% of the mutagenicity of metronidazole.

Tumorigenicity of nitrated derivatives of pyrene, benz[a]anthracene, chrysene and benzo[a]pyrene in the newborn mouse assay.

Eight nitropolycyclic aromatic hydrocarbons (PAHs), including 1- and 4-nitropyrene, 1,3-, 1,6- and 1,8-dinitropyrene, 7-nitrobenz[a]anthracene, 6-nitrochrysene and 6-nitrobenzo-[a]pyrene and their parent PAHs were tested fro tumorigenicity in the newborn mouse model by i.p. administration at 1, 8, and 15 days after birth. Both pyrene and 1-nitropyrene induced similar incidences of hepatic tumors in males, yielding a 12-15% and a 21-28% tumor incidence at total doses of 700 and 2800 nmol per mouse, respectively. Liver tumors did not occur in females and the 3-10% lung tumor yield in both sexes was similar to that found in solvent-treated controls. The presumed proximate carcinogen, 1-nitrosopyrene, administered at 700 nmol per mouse, caused liver tumors in 45% of the males and in 9% of the females. 4-Nitropyrene was more tumorigenic than pyrene or 1-nitropyrene; at a dose of 2800 nmol, it induced liver tumors in 83% of the males and 7% of the females, with a lung tumor yield of 38 and 31%, respectively. Female mice treated with 200 nmol of 1,3-, 1,6- or 1,8-dinitropyrene did not develop liver tumors but the hepatic tumor incidence in males was 20, 32 and 16%, respectively, which was significantly greater than that found in mice treated with pyrene. In male mice administered 2800 nmol of benz[a]anthracene, the hepatic tumor incidence was 79%, while treatment with 7-nitrobenz[a]anthracene showed an incidence of only 28%. Similarly, 560 nmol of benzo[a]pyrene caused a 49% liver tumor yield in males while those given 6-nitrobenzo[a]pyrene had a 28% incidence. Treatment with benzo[a]pyrene also induced a 35 and 48% lung tumor incidence in males and females while the comparable values in 6-nitrobenzo[a]pyrene-treated mice were 14 and 2%. Chrysene administered at 2800 nmol per mouse induced hepatic and lung tumors in 41% and 21% of the males, respectively; at the 700-nmol dose, it induced only liver tumors in 29% of the males and in none of the females. In contrast, treatment with 6-nitrochrysene at 700 nmol per mouse resulted in a 76 and 23% hepatic tumor incidence in males and females, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

Mutagenicity of benzylic acetates, sulfates and bromides of polycyclic aromatic hydrocarbons.

Studies were performed to determine the direct mutagenicity of the acetates and some bromides and sulfates of hydroxymethyl polycyclic aromatic hydrocarbons in S. typhimurium strains TA98 and TA100. Benzylic acetates, bromides and sulfates were synthesized and characterized. The compounds tested were benzyl alcohol, 5-hydroxymethylchrysene, 1-hydroxymethylpyrene, 6-hydroxymethylbenzo[a]pyrene, 6-(2-hydroxyethyl)benzo[a]pyrene, 6-hydroxymethylanthanthrene, 9-hydroxymethylanthracene, 9-hydroxymethyl-10-methylanthracene, 7-hydroxymethylbenz[a]anthracene, 7-(2-hydroxyethyl)benz[a]anthracene, 12-hydroxymethylbenz[a]anthracene, 7-hydroxymethyl-12-methylbenz[a]anthracene, 12-hydroxymethyl-7-methylbenz[a]anthracene, 1-hydroxy-3-methylcholanthrene, 2-hydroxy-3-methylcholanthrene, 3-hydroxy-3, 4-dihydrocyclopental[cd]pyrene and 4-hydroxy-3, 4-dihydrocyclopental[cd]pyrene. The benzylic sulfate esters of 6-hydroxymethylbenzo[a]pyrene and 7-hydroxymethylbenz[a]anthracene were the most mutagenic compounds, whereas the aliphatic sulfate ester of 7-hydroxyethylbenz[a]anthracene did not cause an increase in mutations above background. All meso-anthracenic benzylic acetate esters were mutagenic in both strains with various degrees of activity, whereas the corresponding non-benzylic esters were inactive, as expected. Of the non-meso-benzylic acetate esters, only the 3-acetoxy-3, 4-dihydrocyclopenta[cd]pyrene was mutagenic. In the benzylic bromide series, only the eight mesoanthracenic were mutagenic, whereas benzyl bromide and 5-bromomethylchrysene were inactive. The aliphatic bromides, 6-(2-bromoethyl)benzo[a]pyrene and 7-(2-bromoethyl)benz[a]anthracene did not display significant activity. The potencies of the acetate esters more accurately reflect the mutagenicity because the rate of solvolysis did not compete with the reactivity of the esters with bacterial DNA. In the case of benzylic sulfates and bromides, the rate of solvolysis was very rapid and could have diminished the level of mutagenicity, depending on the assay conditions. These results demonstrate that meso-anthracenic benzylic acetates, sulfates and bromides are mutagenic, whereas benzylic acetate esters attached to other carbon atoms are inactive.

Studies on the mechanism of activation and the mutagenicity of ronidazole, a 5-nitroimidazole.

Substantial evidence implicates the obligatory nucleophilic attack by water at C4 for the elimination of the carbamate and subsequent immobilization by electrophilic attack on protein thiols. Consequently, the strong correlation between the structural requirements for protein alkylation and for mutagenicity in TA100 suggests a possible role of nucleophilic addition at C4 or at the 2-methylene carbon for the expression of mutagenicity. Further studies directed at evaluating this possibility are currently in progress.

Urinary metabolites of the antiprotozoal agent cis-3a,4,5,6,7,7a- hexahydro-3-(1-methyl-5-nitro-1H-imidazol-2-yl)-1,2-benzisoxazole in the rat.

1H-NMR and MS were employed to identify 13 rat urinary metabolites of 14C-labeled cis-3a,4,5,6,7,7a- hexahydro-3-(1-methyl-5-nitro-1H-imidazol-2-yl)-1,2-benzisoxazole (MK-0436). The major free (unconjugated) metabolite was cis-3a,4,5,6,7, 7a-hexahydro-3-carboxamido-1,2-benzisoxazole; it was also the second most abundant metabolite released during hydrolysis of the conjugated fraction. All other identified metabolites were hydroxylated analogues substituted at C(4)-C(7a) of the cyclohexane ring. the 4-equatorial,5-axial,7a-triol was the second most abundant metabolite excreted in an unconjugated form. Four monohydroxy (5-axial, 6-axial, 6-equatorial, 7-equatorial) metabolites of the drug were identified; they were found in the conjugated fraction only and were released by hydrolysis. The 5-axial hydroxy compound is the major conjugated metabolite and is overall the most abundant of all the metabolites. Six dihydroxy metabolites were identified: one was found exclusively in the free state, three as conjugates only (including the 7-axial,7a-diol, which is the major dihydroxy species), and two both free and conjugated. A second triol was found both free and conjugated.

Drug residue formation from ronidazole, a 5-nitroimidazole. V. Cysteine adducts formed upon reduction of ronidazole by dithionite or rat liver enzymes in the presence of cysteine.

When ronidazole (1-methyl-5-nitroimidazole-2-methanol carbamate) is reduced by either dithionite or rat liver microsomal enzymes in the presence of cysteine, ronidazole-cysteine adducts can be isolated. Upon reduction with dithionite ronidazole can react with either one or two molecules of cysteine to yield either a monosubstituted ronidazole-cysteine adduct substituted at the 4-position or a disubstituted ronidazole-cysteine adduct substituted at both the 4-position and the 2-methylene position. In both products the carbamoyl group of ronidazole has been lost. The use of rat liver microsomes to reduce ronidazole led to the formation of the disubstituted ronidazole-cysteine adduct. These data indicate that upon the reduction of ronidazole one or more reactive species can be formed which can bind covalently to cysteine. The proposed reactive intermediates formed under these conditions may account for the observed binding of ronidazole to microsomal protein and the presence of intractable drug residues in the tissues of animals treated with this compound. They may also account for the mutagenicity of this compound in bacteria.

Drug residue formation from ronidazole, a 5-nitroimidazole. VI. Lack of mutagenic activity of reduced metabolites and derivatives of ronidazole.

The potential toxicity of ronidazole residues present in the tissues of food-producing animals was assessed using the Ames mutagenicity test. Since ronidazole is activated by reduction, reduced derivatives of ronidazole and metabolites formed by enzymatic reduction of ronidazole were tested for mutagenicity. When tested at levels several orders of magnitude higher than that at which ronidazole was mutagenic, 5-amino-4-S-cysteinyl-1,2- dimethylimidazole , a product of the dithionite reduction of ronidazole in the presence of cysteine, the 5-N-acetylamino derivative of ronidazole and 5-amino-1,2- dimethylimidazole all lacked mutagenic activity in Ames strain TA100. The metabolites of ronidazole formed by the incubation of ronidazole with microsomes under anaerobic conditions were also not mutagenic. These data demonstrate that although ronidazole is a potent mutagen, residues from it which may be present in the tissues of food-producing animals lack any mutagenic activity.

Effect of liver enzymes on the mutagenicity of nitroheterocyclic compounds: activation of 3a,4,5,6,7,7a-hexahydro-3-(1-methyl-5-nitro-1H-imidazol-2-yl)- 1,2-benzisoxazole and deactivation of nitrofurans and nitroimidazoles in the Ames test.

The effect of liver enzymes (S9) on the mutagenic response of nitroimidazoles and nitrofurans in the Ames test was evaluated with strain TA100. A diminished response was observed with a 5-nitroimidazole and 5-nitrofurans when the S9 preparation was incorporated in the agar layer. Preincubation with S9 under anaerobic conditions prior to adding the bacteria resulted in a greater and sometimes complete loss of the mutagenic effect. The loss of mutagenic potency was dependent on both incubation time and quantity of the S9 preparation. These results suggest that metabolites formed after reductive metabolism are neither mutagenic (presumably due to the loss of the nitro group) nor capable of activation to mutagenic metabolites. One 5-nitroimidazole, 3a,4,5,6,7,7a-hexahydro-3-(1-methyl-5-nitro -1H-imidazol-2-yl)-1,2-benzisoxazole (MK-0436), gave an increased response in the presence of S9 in both the plate test and when preincubated under aerobic conditions. 7 metabolites were produced by the incubation. 4 monooxygenated metabolites were isolated and found to possess significant mutagenic activity. 2 synthetic dihydroxy analogs were more mutagenic than MK-0436. Similar results were obtained with S9 preparations from human liver and the livers of control, phenobarbital and Aroclor-1254 pretreated rats.

Drug residue formation from ronidazole, a 5-nitro-imidazole. IV. The role of the microflora.

When radioactive 1-methyl-5-nitroimidazole-2-methanol carbamate, ronidazole, labeled at the 4,5-ring positions was administered orally to germ-free and conventional rats, a much larger fraction of the radioactivity was excreted in the feces of the conventional animals. Determination of the total radioactive residues present in the carcass, blood, plasma, liver, fat and kidney 5 days after dosing indicated that the carcass of the germ-free animals contained a greater quantity of residue than that of conventional rats. On the other hand, the blood of the conventional animals contained a much higher level of radioactivity than that of the germ-free animals. These results show that while the microflora influence the distribution of the drug their presence is not obligating for the formation of persistent tissue residues in rats dosed with ronidazole.

Identification of in vitro (rat liver postmitochondrial S9 fraction) metabolites of the antiprotozoal agent 3a,4,5,6,7,7a-hexahydro-3-(1-methyl-5-nitro-1H-imidazol-2-yl)-1,2-benzisoxazole .

Twelve in vitro oxygenated metabolites of 3a,4,5,6,7,7a-hexahydro-3-(1-methyl-5-nitro-1H-imidazol-2-yl)-1,2-benzisoxazole (MK-0436) were produced by incubation of this antiprotozoal agent with the postmitochondrial supernatant (S9) fraction isolated from the livers of rats treated with phenobarbital. Metabolite structure elucidation was achieved using NMR and mass spectrometry. Seven monohydroxy and two dihydroxy metabolites were fully characterized; two other metabolites were partially characterized as dihydroxy derivatives of the drug. The major in vitro metabolite is the 5 axial hydroxy compound, and a minor metabolite is the corresponding ketone. In all cases metabolite formation involved biotransformation on the hexahydrobenzisoxazole ring.

Epoxidation reactions catalyzed by rat liver cytochromes P-450 and P-448 occur at different faces of the 8,9-double bond of 8-methylbenz[a]anthracene.

8-Methylbenz[a]anthracene (8-MeBaA) transdihydrodiol metabolites were isolated by reversed-phase and normal-phase HPLCs from incubations of 8-MeBaA with liver microsomes or a reconstituted system containing purified cytochrome P-448 and epoxide hydrolase. Regardless of the enzyme source, the metabolically formed 8-MeBaA trans-3,4- and -5,6-dihydrodiols were found to be enriched in one enantiomeric isomer and differed only in the degree of optical purity. The 8-MeBaA trans-8,9-dihydrodiol formed by liver microsomes from either untreated or phenobarbital-treated rats was enriched with the (+)-enantiomer. In contrast, the 8-MeBaA trans-8,9-dihydrodiol formed either by liver microsomes from 3-methylcholanthrene-treated rats or by the reconstituted rat liver enzyme system containing cytochrome P-448 and epoxide hydrolase was enriched with the (-)enantiomer. These results indicate that, in catalyzing the formation of 3,4- and 5,6-epoxide intermediates, the interaction with the unsubstituted 3,4- and 5,6-double bonds of 8-MeBaA by the different forms of cytochrome P-450 occur preferentially on the same face of the aromatic plane and they differ only in the degree of stereoselectivity. However, different forms of cytochrome P-450 may interact with different faces of the aromatic plane at the methyl-substituted 8,9-double bond of 8-MeBaA, resulting in the formation of trans-8,9-dihydrodiols enriched in different enantiomeric forms. This demonstrates that different forms of cytochrome P-450 may catalyze the epoxidation reaction preferentially at different sides of the methyl-substituted double bond of a planar polycyclic hydrocarbon molecule. These properties may be used to further classify and to understand the enzyme-substrate interactions of the different forms of cytochrome P-450 in the drug-metabolizing enzyme systems.

Drug residue formation from ronidazole, a 5-nitroimidazole. I. Characterization of in vitro protein alkylation.

The metabolic activation of [14C]ronidazole by rat liver enzymes to metabolite(s) bound to macromolecules was investigated. The alkylation of protein by [14C]ronidazole metabolite(s) was catalyzed most efficiently by rat liver microsomes, in the absence of oxygen utilizing NADPH as a source of reducing equivalents. Based on a comparison of total ronidazole metabolized versus the amount bound to microsomal protein, approximately one molecule alkylates microsomal protein for every 20 molecules of ronidazole metabolized. Protein alkylation was strongly inhibited by sulfhydryl-containing compounds such as cysteine and glutathione whereas methionine had no effect. Based on HPLC analysis of ronidazole, cysteine was found not to inhibit microsomal metabolism of ronidazole ruling out a decrease in the rate of production of the reactive metabolite(s) as the mechanism of cysteine inhibition.