Identification of carbadox metabolites formed by liver microsomes from rats, pigs and chickens using high-performance liquid chromatography combined with hybrid ion trap/time-of-flight mass spectrometry.

Carbadox (methyl-3-(2-quinoxalinylmethylene)-carbazate-N(1),N(4)-dioxide) is a chemotherapeutic growth promoter added to feed for starter pigs. In this work, the metabolism of carbadox in rat, pig and chicken liver microsomes has been studied firstly. The incubation mixtures were then processed and analyzed for metabolites with a sensitive and reliable method based on high-performance liquid chromatography combined with hybrid ion trap/time-of-flight mass spectrometry (LC/MS-IT-TOF). With the help of chromatographic behavior and accurate mass measurements, it is possible to rapidly and reliably characterize the metabolites of carbadox. The structural elucidations of these metabolites were performed by comparing the changes in the accurate molecular masses and fragment ions generated from precursor ions with those of parent drug. The present results showed that the metabolism of carbadox in liver microsomes had qualitative species-difference. A total of seven metabolites were identified in rat liver microsomes. Five metabolites (Cb1-Cb3, Cb5, Cb7) were observed in pig and chicken liver microsomes. In addition, metabolite Cb6 was also detected in chicken liver microsomes. The peak areas of the metabolites in the three species are different. For the formations of Cb1, Cb2, Cb5 and Cb6, the rank order was rat>chicken>pig; Cb3; pig~chicken>rat. Cb1, Cb2 and Cb3 have been previously reported, whereas the other four metabolites were novel. The N→O group reduction and hydroxylation followed by N→O group reduction were the main metabolic pathways for carbadox in the three species.

Application of electrospray ionization hybrid ion trap/time-of-flight mass spectrometry in the rapid characterization of quinocetone metabolites formed in vitro.

The application of electrospray ionization hybrid ion trap/time-of-flight mass spectrometry coupled with high-performance liquid chromatography (LC/MS-IT-TOF) in the rapid characterization of in vitro metabolites of quinocetone was developed. Metabolites formed in rat liver microsomes were separated using a VP-ODS column with gradient elution. Multiple scans of metabolites in MS and MS(2) modes and accurate mass measurements were automatically performed simultaneously through data-dependent acquisition in only a 30-min analysis. Most measured mass errors were less than 10 ppm for both protonated molecules and fragment ions using external mass calibration. The elemental compositions of all fragment ions of quinocetone and its metabolites could be rapidly assigned based upon the known compositional elements of protonated molecules. The structure of metabolites were elucidated based on the combination of three techniques: agreement between their proposed structure, the accurate masses, and the elemental composition of ions in their mass spectra; comparison of their changes in accurate molecular masses and fragment ions with those of parent drug or metabolite; and the elemental compositions of lost mass numbers in proposed fragmentation pathways. Twenty-seven phase I metabolites were identified as 11 reduction metabolites, three direct hydroxylation metabolites, and 13 metabolites with a combination of reduction and hydroxylation. All metabolites except the N-oxide reduction metabolite M6 are new metabolites of quinocetone, which were not previously reported. The ability to conduct expected biotransformation profiling via tandem mass spectrometry coupled with accurate mass measurement, all in a single experimental run, is one of the most attractive features of this methodology. The results demonstrate the use of LC/MS-IT-TOF approach appears to be rapid, efficient, and reliable in structural characterization of drug metabolites.

Population pharmacokinetics of enrofloxacin and its metabolite ciprofloxacin in chicken based on retrospective data, incorporating first-pass metabolism.

A population pharmacokinetic (PPK) model for enrofloxacin and its metabolite ciprofloxacin in chicken based on retrospective data was developed. Plasma concentrations of enrofloxacin and its metabolite ciprofloxacin were determined in blood samples from chicken administered either enrofloxacin via oral and intravenous routes or ciprofloxacin via intravenous injection. The disposition of enrofloxacin and ciprofloxacin was described simultaneously by an integrated mathematic model. Two compartments were used to describe the enrofloxacin and ciprofloxacin disposition profiles. The formation of ciprofloxacin was through the central compartment of enrofloxacin. The integrated model was estimated with nonlinear mixed effects model (NONMEM). The total clearance of enrofloxacin (CLEN) and ciprofloxacin (CLCP) was 0.613 L/h and 1.15 L/h, respectively. Correlation between CLEN, the central compartment volume of distribution for enrofloxacin (V2) and CLCP was estimated. After intravenous administration of enrofloxacin, the transformation rate of enrofloxacin to ciprofloxacin was 0.429 L/h. The bioavailability factor after oral administration was 0.926, and 12.6% of enrofloxacin after oral administration was transformed to ciprofloxacin via first-pass effect. Pharmacodynamic (PD) evaluation was performed using area under concentration time curve of active moiety from 0 to 24 h and MIC collected from literature. This study is the first one to use PPK method to investigate parent drug and its metabolite disposition and PDs using an integrated model in veterinary medicine.

Genotoxicity evaluation of Mequindox in different short-term tests.

Quinoxaline-1,4-dioxides (QdNOs) are the potent heterocyclic N-oxides with interesting biological properties such as antibacterial, anticandida, antitubercular, anticancer and antiprotozoal activities. Here, we tested and compared the mequindox (MEQ) for mutagenic abilities in a battery of different short term tests according to OECD guidelines. When compared with the controls, a strong mutagenicity of MEQ and carbadox (CBX) was observed with an approximate concentration-effect relationship in Salmonella reverse mutation test, chromosome aberration test, unscheduled DNA synthesis assay and HGPRT gene mutation test, in the absence and presence of S(9)-mix. In in vivo micronucleus test, CBX produced significant increase in the proportion of micronucleus formation than MEQ in mice bone marrow cells. From these results, we can conclude that MEQ had a strong genotoxic potential to mammalian cells in vitro as well as in vivo and its mutagenicity is slightly higher than CBX. Our results, for the 1st time, discuss the genotoxic potential of MEQ. These results not only confirm the earlier findings about CBX but also extend the knowledge and awareness about the genotoxic risk of QdNO derivatives.

Two generation reproduction and teratogenicity studies of feeding quinocetone fed to Wistar rats.

To investigate the reproductive toxicity and teratogenic potential of quinocetone, a growth promoting agent, Wistar rats were fed different diets containing 0, 50, 300 and 1800 mg/kg quinocetone or 300 mg/kg olaquindox. Groups of 15 males and 30 females (F(0)) were fed through a 10-week prebreed period as well as during mating, gestation, parturition and lactation. At weaning, 12 males and 24 females of F(1) generation weanlings per group were selected randomly as parents for F(2) generation. Selected F(1) weanlings were exposed to the same diet and treatment as their parents. At the highest quinocetone group, body weights in F(0) and F(1) rats, fetal body weight on day 21 after birth and number of viable fetuses in F(0) and F(1) generation significantly decreased. In teratogenicity study, groups of 12 males and 24 females were fed with the same diets through a 12-week prebreed period and matting periods. Pregnant rats were subjected to cesarean section on GD 20 for teratogenic examination. At the highest quinocetone group, body weights and feed efficiency, fetal body lengths, tail lengths, litter weights and number of viable fetuses significantly decreased. The NOAEL for reproduction/development of quinocetone for rats was estimated to be 300 mg/kg diet.

In vitro biotransformation and investigation of metabolic enzymes possibly responsible for the metabolism of bisdesoxyolaquindox in the liver fractions of rats, chicken, and pigs.

Bisdesoxyolaquindox is a reduced metabolite of olaquindox which is used as a medicinal feed additive in veterinary medicine. The relevant metabolism studies of bisdesoxyolaquindox have been carried out for the first time in rat, chicken, and pig liver subcellular fractions in order to understand the metabolic enzymes that are possibly responsible for the metabolism of olaquindox. The metabolites were characterized by high-performance liquid chromatography combined with hybrid ion trap/time-of-flight mass spectrometry. The major metabolic pathways of bisdesoxyolaquindox in the three species were the oxidation of hydroxyl to bisdesoxyolaquindox-2'-carboxyl acid (O10) and the N-dealkylation of the side chain to 3-methylquinoxaline 2-carboxamide (O12). Other metabolic pathways were also proposed which involved the direct methyl oxidation and N-oxide on the quinoxaline ring in the three species as well as N-hydroxylation only in rat. The intrinsic clearance values in the liver microsomes for O10 and O12 were ranked in the order of chicken>pig≫rat and rat>pig≫chicken, respectively. Inhibition studies indicated that 8-methoxypsoralen, 4-methylpyrazole and α-naphthoflavone could inhibit the formations of O10 and O12 in all species. Quinidine, troleandomycin, diethyldithiocarbamate, and disulfiram showed an interspecies difference in the inhibition of the formation of two metabolites. In rat and pig liver cytosol, 4-methylpyrazole, menadione and chlorpromazine strongly inhibited the formation of O10. Both diethyldithiocarbamate and disulfiram were found to inhibit O10 formation in rat cytosol but not in pig cytosol. These results indicated the following: In rat liver microsomes, CYP2A might be involved in the formation of O10, and CYP1A, CYP2A and CYP2E would be involved in the O12 formation. In pig liver microsomes, CYP1A and CYP2E might catalyze the formations of O10 and O12. In rat cytosol, alcohol dehydrogenase, aldehyde oxidase and aldehyde dehydrogenase should catalyze the O10 formation. In pig cytosol, alcohol dehydrogenase and aldehyde oxidase might be involved in the formation of O10. In chicken, it was found that various CYP isoenzymes were capable of catalyzing the two reactions; none of the inhibitors of cytosol enzymes inhibited O10 formation in chicken cytosol.

The metabolism of olaquindox in rats, chickens and pigs.

Olaquindox is a growth-promoting feed additive for food-producing animals. Its toxicities were reported to be closely related to the metabolism. To provide the interpretation of toxicities in animals, this study explored the metabolism of olaquindox in rats, chickens and pigs of different genders by qualitative metabolite profiling. Animals were fed olaquindox in an oral dose, and then their urine, plasma, feces, liver, kidney and muscle were collected. Liquid chromatography combined with hybrid ion trap/time-of-flight mass spectrometry was used for structural investigation and identification of metabolites. The structures of metabolites were elucidated based on the accurate MS² spectra and comparison of their changes in accurate molecular masses and fragment ions with those of parent drug or metabolite. A total of 18, 18 and 16 metabolites of rats, chickens and pigs were identified, respectively. Among the identified metabolites, 8 known metabolites were confirmed as an early study had stated, and 15 metabolites were found for the first time in vivo. The major metabolic pathways of olaquindox were proposed to be N-O reduction and oxidation of hydroxyl to carboxylic acid followed by N-O reduction. The qualitative species difference on the metabolite profiles of olaquindox among the three species was observed. However, metabolite profiles of olaquindox appeared to be qualitatively similar between female and male for the same species. The proposed metabolic pathways of olaquindox in animals will provide comprehensive data to clarify the metabolism of olaquindox among different species, and will give scientific explanation for toxicities and residues of olaquindox.

The metabolism and N-oxide reduction of olaquindox in liver preparations of rats, pigs and chicken.

Olaquindox, N-(2-hydroxyethyl)-3-methyl-2-quinoxalinecarboxamide-1,4-di-N-oxide, is one of the quinoxaline-dioxides used widely as an antimicrobial growth promoter in pig production. Its toxicities were reported to be closely related to the formation of N-oxide reductive metabolites. The present study presents the metabolism and N-oxide reduction of olaquindox incubated with liver microsomes and liver cytosol of rats, pigs and chicken. Metabolites were identified and characterized with a novel LC/MS-ITTOF. Thirteen metabolites were found in liver microsomes of rats, three of which were identified to be novel. Seven metabolites were found in liver microsomes of pigs and chicken. The N-oxide reduction was the major metabolic pathway of olaquindox in liver microsomes of the three species. The N1-reduction of olaquindox to metabolite O2 was found in not only liver microsomes but also cytosol of the three species in the presence of NAD(P)H under hypoxic conditions. The N1-reduction could be inhibited by air and carbon monoxide, and be significantly stimulated by riboflavin under various conditions. The N1-reduction in the liver cytosol of rats and pigs could be enhanced by menadione, but the reduction in liver cytosol of chicken could not be. The N1-reduction activities in all animals were not abolished when liver microsomes and cytosol were boiled. These findings suggested that the N1-reduction of olaquindox could be mediated by non-enzymatic and enzymatic conditions. This N1-reduction of olaquindox could also be catalyzed by a quinone-dependent reducing system in liver cytosol of rats and pigs. Moreover, liver cytosol of rats and pigs had an ability of N4-reduction that catalyzed olaquindox to metabolite O1 in the presence of benzaldehyde under hypoxic conditions, but the liver cytosol of chicken did not. The N4-reduction could be inhibited markedly in the cytosol rats and pigs by menadione, chlorpromazine and promethazine. In addition, 7-hydroxycoumarin was also found to inhibit the formation of O1 in the cytosol of rats. The inhibitory results suggested that the N4-reduction might be catalyzed by aldehyde oxidase in the cytosol of pigs, and by aldehyde oxidase and xanthine oxidase in the cytosol of rats. In conclusion, the N1-reduction and N4-reduction of olaquindox are mediated by multiple mechanisms and significant species differences are involved in both reductions. This work is a contribution to the understanding of toxicities and the relativities between toxicities and metabolism of olaquindox.