Disposition and metabolism of finasteride in dogs.

Finasteride (FIN) is a potent 5 alpha-reductase inhibitor that has shown clinical success in treating men with benign prostatic hyperplasia. In the study of biological effects and metabolism of FIN in animals, the dog serves as the primary modality. This study was conducted to determine the pharmacokinetics and fate of FIN after oral administration of single doses of [14C]FIN to dogs at 10 and 80 mg/kg (N = 2 and 3, respectively), and also after intravenous infusion at 5 mg/kg (N = 2). Plasma, urine, and feces were analyzed for total 14C content. Parent drug and metabolites in plasma and excreta were measured by HPLC/UV/radioassay and identified by NMR spectroscopy and MS, FIN was subject to extensive biotransformation before excretion. Structures were determined for the major metabolites in plasma, urine, and feces. The primary metabolic events for FIN were hydroxylation of the t-butyl side chain to give hydroxymethyl-FIN (metabolite I), which is oxidized further to form the carboxylic acid derivative (metabolite IV), and hydroxylation at positions B alpha and 15. Terminal half-life of FIN after the intravenous dose was 3.4 hr. Plasma clearance and volume of distribution at steady-state were 4.8 ml/min/kg and 1.1 liter/kg. Dogs showed rapid absorption after oral administration of the low dose, with Cmax reached in the 1-2 hr, bioavailability was estimated to be > 90%. After either dosing route, 45% of the plasma radioactivity (as represented by AUC) was parent drug, 43% was metabolite I, and 1% was metabolite IV. After oral administration, the 80 mg/kg dose was absorbed slowly, with the highest levels of radioactivity in plasma reached in 4-30 hr. Average Cmax value for FIN and metabolite I increased in a dose-related, but nonproportional, manner. Compared with the 10 mg/kg dose, it seems the higher dose was reasonably well-absorbed, as indicated by the nearly proportional increase of AUC values of total radioactivity and FIN. Composition of plasma metabolites observed at the 80 mg/kg dose level was similar to that observed previously for the low dose, suggesting that an increase in plasma exposure was effected in dogs receiving FIN at 80 mg/kg in toxicity studies. Most of the administered radioactivity was recovered in feces after all doses. Little of the intravenous and low oral doses, but > 50% of the 80 mg/kg oral dose, was excreted as intact FIN, suggesting that metabolism might have been saturated at the high dose.

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.

Comparative metabolic disposition of ivermectin in fat tissues of cattle, sheep, and rats.

In the study of the metabolic disposition of ivermectin in cattle, sheep, and rats, a group of nonpolar metabolites was detected in the fat tissues of these animals. Upon saponification or esterase treatment, these nonpolar metabolites gave rise to polar products that were similar to the ivermectin metabolites present in the liver. A hypothesis was thus proposed that the polar ivermectin metabolites produced in the liver were esterified and stored in the fat as nonpolar entities, which can be reconverted back to the polar metabolites chemically or enzymatically. To substantiate this hypothesis, the regeneration of polar metabolites from the nonpolar metabolites was studied and hydrolysis products were characterized to establish the basic structure of the alcohol portion of the metabolites. Furthermore, chromatographic comparisons were made between radiolabeled in vivo metabolites and synthetic fatty acid ester samples. These results established the general structural class of the ivermectin nonpolar metabolites and also confirmed the unusual metabolic pathway of ivermectin disposition in fat tissue.

High-performance liquid chromatographic determination of N-(2-methyl-2-propyl)-3-oxo-4-aza-5 alpha-androst-1-ene-17 beta-carboxamide, a 4-azasteroid, in human plasma from a phase I study.

A sensitive and selective high-performance liquid chromatographic method has been developed for the quantitative determination of N-(2-methyl-2-propyl)-3-oxo-4-aza-5 alpha-androst-1-ene-17 beta-carboxamide (I) in human plasma. I, a 5 alpha-reductase inhibitor and a potential therapeutic agent for benign prostatic hyperplasia, is a member of the family of compounds referred to as the 4-azasteroids. The 4-N-methyl analogue of the drug was used as the internal standard and calibration curves were developed at two levels of sensitivity to cover a large dynamic range of plasma concentrations. Drug was isolated from biological fluids with a solid-phase C18 extraction column; the analyte was further purified by adsorption and desorption from a second extraction column (CN cartridge). Evaluation of the isolation method revealed that it was reproducible and drug recoveries equalled ca. 90%. Chromatography was carried out on a C8 column (5 micron) with ultraviolet detection at 210 nm. The detection limit was ca. 10 ng/ml for I. Human plasma levels are reported for I following single-dose oral administration of 50,200 and 400 mg of drug; urinary excretion data are reported for a single volunteer given 400 mg of I.

Confirmation of clorsulon residues in cattle kidney by capillary gas chromatography-negative-ion chemical-ionization mass spectrometry.

A confirmatory assay for residues of the anthelmintic agent clorsulon [4-amino-6-(trichloroethenyl)-1,3-benzenedisulfonamide] in cattle kidney tissue has been developed. The assay involves isolation of a drug-containing fraction by solvent extraction, methylation of the analyte, and fused-silica capillary column gas chromatography-negative-ion chemical-ionization mass spectrometry of the pentamethyl derivative of clorsulon. The intensities of four negative ions [m/z 406 and 408 (trichloro species) and m/z 413 and 415 (dichloro species)] are monitored. Confirmation of the presence of drug in an analyte requires that all four ions appear at the appropriate retention time with their intensity ratios within 10-15% of those arising from analysis of the reference standard, methylated clorsulon; the lower limit of detection is 3 ppb. Quantification of the drug is based on the intensity of the m/z 406 ion. Identification and quantification of residues by the gas chromatographic-mass spectrometric assay gave results in good agreement with those obtained with an electron-capture gas chromatographic assay.

Use of stable isotopes in the elucidation of drug biotransformation processes.

This article surveys the role of stable isotope labelling and mass spectrometric detection in the recognition and characterization of drug metabolites. Applications of the so-called isotope cluster technique, useful for both recognition of drug-related compounds and elucidation of their structures, are presented. The use of deuterium-labelled g.l.c./mass spectrometric derivatization reagents to facilitate the determination of drug metabolite structures via 'peak shifts' is illustrated.

Oxidative and defluorinative metabolism of fludalanine, 2-2H-3-fluoro-D-alanine.

The antibacterial agent fludalanine [2-2H-3-fluoro-D-alanine (DFA)] is a potent inhibitor of bacterial alanine racemase, an enzyme required for the generation of D-alanine, an essential component of the bacterial cell wall. Primary metabolism of DFA involves its oxidation to fluoropyruvate (FP); this organic fluoride is then rapidly reduced to fluorolactate (FL) which is the major organic metabolite in laboratory animals. Gas-liquid chromatographic chemical ionization mass spectrometric assays were developed for these two metabolites. FL is the predominant organofluoride metabolite of DFA in the circulation. FP was detected in the urine although recovery was very low. The rapid conversion of FP to FL precludes assay of the former in serum. Maximum serum FL concentrations in the rat appear about 1 hr after the dose of DFA and are relatively constant for several hours thereafter. The peak FL concentration is proportional to the dose of DFA; repeated daily dosing of DFA appears to cause neither saturation nor induction of metabolic pathways. Comparison of FL concentrations determined using the GC/MS assay with those based on an enzymic method specific for L-(+)-FL demonstrated that only the latter isomer is found in the plasma of monkeys dosed with DFA. In vivo exchange studies involving the alpha-proton of FL indicate that a small FP pool exists and is in equilibrium with FL. A crude pyruvate dehydrogenase complex isolated from beef heart mitochondria was shown to produce equimolar quantities of acetate, CO2, and fluoride from FP.

Metabolic disposition of ivermectin in tissues of cattle, sheep, and rats.

The metabolic disposition of ivermectin, a new antiparasitic drug, has been studied in cattle, sheep, and also in rats dosed with the drug labeled with tritium in the C-22,23 positions. In the edible tissues of these animals, the unaltered drug was the major tissue residue component and was quantitated by HPLC-reverse isotope dilution assay. The depletion half-lives of the drug ranged between 1 and 6 days, similar to those of the total tissue residue in these species. Most metabolites present in the liver tissues were more polar than the parent drug. Based on spectral (NMR, mass spectrometric) analysis and chromatographic comparison with authentic compounds prepared by in vitro rat or steer microsomal incubations, three of these metabolites have been isolated and identified as the hydroxylation derivatives of ivermectin, i.e. 24-hydroxymethyl-H2B1a, its monosaccharide, and 24-hydroxymethyl-H2B1b.

Drug metabolite identification: stable isotope methods.

This paper focuses on stable isotope labeling of drugs, in combination with mass spectrometry (MS)-based methods, to facilitate the recognition and identification of metabolites and the employment of stable isotope-labeled derivatization reagents (e.g., bis-trimethylsilylacetamide-d18) in the structure elucidation of metabolites from unlabeled drugs via gas-liquid chromatography-MS techniques. In both cases, it is the so-called isotope peak shift that permits generation of data useful for metabolite identification. Furthermore, judicious labeling of a drug permits characterization of drug-related species (metabolites) by MS-based recognition of isotope cluster signatures. Studies using stable isotope-labeled drugs are exemplified by work on aminopyrine and isopropylantipyrine metabolism; examples of the derivatization peak shift approach include those from studies of timolol and cyproheptadine.

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.

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.

Capillary column gas-liquid chromatography selected ion monitoring assay for [13C, 15N]N-methyltryptamine in human urine: failure to detect conversion of [13C,15N]tryptamine in schizophrenia patients.

A capillary column gas-liquid chromatography selected ion monitoring-based method was developed for the measurement of [13C,15N]N-methyltryptamine ( NMT ) in human urine. The method was employed to establish the extent of conversion of [13C,15N]tryptamine to the correspondingly labeled NMT in schizophrenic patients in an attempt to demonstrate whether methylation of tryptamine plays a role in schizophrenia. Mass spectrometric detection in the assay procedure is via chemical ionization (Isobutane) with monitoring of the MH+ ions of the trimethylsilyl derivatives of [13C,15N] NMT and the internal standard, [2H3,13C,15N] NMT . The assay possesses a sensitivity limit (using 200 ml of urine) of ca. 0.1 ng/ml, corresponding to substrate conversion of ca. 0.00005% with a 75 mg dose (i.v.) of labeled tryptamine. Evidence for methylation was found with only one of seven patients studied; the extent of substrate conversion for the one individual was only 0.0001%. These results do not support the indoleamine--methylation hypothesis of schizophrenia.

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.

Biochemical and biomedical applications of capillary column GLC using SIM.

This report focuses on recent applications of capillary column GLC to the analysis of drugs and metabolites, other xenobiotics, natural products, and environmental contaminants in samples of biological origin. The increasing use of selected ion monitoring, combined with stable isotope methods as a means of GLC detection and quantification, is emphasized. Specific topics covered include the use of capillary column GLC for 1) determining the pharmacokinetics of timolol (BLOCADREN) in human volunteers by simultaneous oral and intravenous administration of this beta-blocker and its 13C3-labeled analog; 2) measuring residue levels of the forced molting agent, xylonidine, in yolk and albumen of chicken eggs; 3) monitoring animal plasma and tissues for the depletion of 5-hydroxy-1,3-dioxane and 4-hydroxymethyl-1,3-dioxolane (isomeric components of glycerol formal, an animal drug formulation component); 4) detecting 13C, 15N-methyltryptamine (a biosynthesis product from 13C, 15N-tryptamine) in human urine; and 5) measuring estradiol levels in biological samples.

The metabolism of avermectins B1a, H2B1a, and H2B1b by liver microsomes.

The avermectins area a new class of structurally related antiparasitic agents isolated from Streptomyces avermitilis. The major polar metabolites isolated from in vitro incubations of [3H]avermectins B1a, H2B1a, and H2B1b with either rat or steer liver microsomes have been isolated and identified as the C24-methyl alcohols of the parent compounds. A smaller quantity of a more polar metabolite has also been identified as the monosaccharide of the C24-methyl alcohols of avermectin H2H1b from rat liver microsomal incubation and avermectin H2B1a from steer liver microsomal incubation. The mass spectra and 300-MHz 1H-NMR spectra permitted assignment of structures to these metabolites. Together these two metabolites represent 50-80% of the total radioactivity more polar than the parent compounds. The metabolite profiles on reverse-phase HPLC demonstrate that the rat and steer are qualitatively similar in the production of these two polar metabolites.

Oxidation reactions by prostaglandin cyclooxygenase-hydroperoxidase.

oxidations of organic sulfides, amines, and even enzymes catalyzed by purified and microsomal forms of prostaglandin cyclooxygenase-hydroperoxidase have been studied using O2 incorporation into arachidonic acid to monitor oxygenase and [14C]15-hydroperoxyprostaglandin E2 reduction to prostaglandin E2 to measure hydroperoxidase. The oxygenase was protected by phenol against the irreversible deactivation induced by low levels of hydroperoxides. Furthermore, the EPR signal noted during reactions with the microsomal enzyme probably reflected the adventitious oxidation of endogenous materials. As described previously for phenol and other reducing cosubstrates, methyl phenyl sulfide (MPS) increased hydroperoxidase activity at all concentrations studied, while stimulating oxygenase at low levels and inhibiting it at 5-10 mM. In stoichiometric equivalence with 15-hydroperoxyprostaglandin E2 reduction, MPS was enzymatically oxidized to its analogous sulfoxide, methylphenyl sulfoxide, acquiring an oxygen atom exclusively from the hydroperoxide and demonstrating some chiral character. In contrast, other oxidizable compounds such as N,N-dimethylphenylenediamine and aminopyrine reacted via radical intermediates. Phenylbutazone, which is oxidized using dissolved molecular oxygen, did not compete with MPS oxidation. Hence, MPS was oxidized while bound to the enzyme, whereas the amine oxidation occurred in solution via an enzyme-formed oxidant. The Soret peak noted with cyclooxygenase-hydroperoxidase was examined as a possible measure of this binding, but was also noted in denatured and deactivated enzyme, suggesting that its relevance should be reconsidered. Despite the similarities in their drug-metabolizing profiles, cyclooxygenase-hydroperoxidase is clearly distinct from cytochrome P-450. The mechanism of this hydroperoxidase is considered in the context of other more extensively studied peroxidases.

Urinary metabolites of timolol from humans and laboratory animals. Syntheses and beta-adrenergic blocking activities.

Syntheses are reported for three metabolites (2-4) of timolol (1) formed by oxidative metabolism of the morpholine ring. GLC-MS comparisons are presented which establish that the two metabolites whose structures were previously in question are identical with their synthetic counterparts 2 and 3. In 2, metabolic oxidation of the 4-morpholinyl group of 1 had occurred at the carbon next to oxygen to give the 2-hydroxy-4-morpholinyl moiety, whereas in 3, the morpholine of 1 has been oxidized one step further and then ring opened to produce the N-(2-hydroxyethyl)glycine substituent. Biological testing of synthetic samples of the three major metabolites from human urine (3, 4, and 6) indicated that only 4, in which the morpholine moiety has been degraded to a 2-hydroxyethylamino group, had significant beta-adrenergic blocking activity (one-seventh that of timolol in anesthetized dogs).