Structural elucidation of rat biliary metabolites of corynoxeine and their quantification using LC‐MSn
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ABSTRACT Corynoxeine (COR) is one of 4 bioactive oxindole alkaloids in Uncaria species. In this work two phase I metabolites, namely 11‐hydroxycorynoxeine (M1) and 10‐hydroxycorynoxeine (M2), and two phase II metabolites, namely 11‐hydroxycorynoxeine 11‐ O‐β ‐ d ‐glucuronide (M3) and 10‐hydroxycorynoxeine 10‐ O‐β ‐ d ‐glucuronide (M4), were detected in rat bile after oral dose of COR (0.105 mmol/kg), by optimized high‐performance liquid chromatography–tandem mass spectrometry (LC‐MS n ) with electrospray ionization in positive ion mode. Structures of M1–4 were determined by LC‐MS n , nuclear magnetic resonance, circular dichroism and high‐resolution MS spectra. COR and its metabolites in rat bile were quantified by LC‐MS n . The LC‐MS n quantification methods for COR and its metabolites yielded a linearity with coefficient of determination ≥0.995 from 5.0 × 10 −10 to 5.0 × 10 −7 m . The recoveries of stability tests varied from 96.80 to 103.10%. Accuracy ranged from 91.00 to 105.20%. Relative standard deviation for intra‐day and inter‐day assay was <5.0%. After the oral dose 0.14% of COR was detected in rat bile from 0 to 8 h, in which in total 97.8% COR biotransformed into M1–4. M1 and M2 yielded 48.1 and 49.7%, which successively glucuronidated to M3 and M4 at 47.2 and 43.8%, respectively. Copyright © 2014 John Wiley & Sons, Ltd.Keywords:
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While N-glucuronidation is an important pathway for metabolism of aromatic amines, it has not been demonstrated for N-acetylbenzidine. A glucuronide of N-acetylbenzidine was synthesized and identified by mass spectrometry as N-acetylbenzidine-N'-glucuronide. This N'-glucuronide is acid labile with a t½ of 4 min at pH 5.3. A similar acid lability was also observed with benzidine-N-glucuronide. The formation of N-acetylbenzidine-N'-glucuronide was assessed with liver slices and microsomes prepared from human, dog and rat. When 0.014 mM [3H]N-acetylbenzidine was incubated with human liver slices a significant amount of N-acetylbenzidine-N'-glucuronide was produced (8–26% of the total radioactivity recovered). With higher concentrations of [3H]N-acetylbenzidine (1 mM) rat slices also produced N-acetylbenzidine-N'-glucuronide. However, N'-glucuronide formation was not detected with dog liver slices incubated with either 0.014 or 1 mM [3H]N-acetylbenzidine. N-Acetylbenzidine-N'-glucuronide formation was observed with microsomes prepared from human, dog and rat. To assess maximum activity four detergents were used at two concentrations. With or without detergent activation the relative amount of glucuronidation was human, dog> rat. The rate of benzidine N-glucuronide formation was 4.3- and 1.6- fold greater than N-acetylbenzidine-N'-glucuronide in dog and rat respectively, while in human both rates were similar (1.1-fold). With or without detergent activation the relative amount of benzidine-N-glucuronide formation was human > dog > rat. N-Glucuronidation of [3H]N, N'-diacetylbenzidine was not observed. Thus N-acetylbenzidine-N'-glucuronide formation appears to be an important pathway for metabolism of N-acetylbenzidine, especially in humans. Due to their acid lability, formation of the N-glucuronides of N-acetylbenzidine and benzidine provides a mechanism for hepatic detoxification and accumulation of these carcinogens in the bladder. A new model is described illustrating the effect of N-glucuronidation and the influence of N-acetylation on arylmono- and aryldiamine-induced bladder carcinogenesis.
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Benzidine
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Flurbiprofen is a nonsteroidal anti-inflammatory drug used as a racemic mixture. Although glucuronidation is one of its elimination pathways, the role of UDP-glucuronosyltransferase (UGT) in this process remains to be investigated. Thus, the kinetics of the stereoselective glucuronidation of racemic (R,S)-flurbiprofen by recombinant UGT isozymes and human liver microsomes (HLMs) were investigated, and the major human UGT isozymes involved were identified. UGT1A1, 1A3, 1A9, 2B4, and 2B7 showed glucuronidation activity for both (R)- and (S)-glucuronide, with UGT2B7 possessing the highest activity. UGT2B7 formed the (R)-glucuronide at a rate 2.8-fold higher than that for (S)-glucuronide, whereas the other UGTs had similar formation rates. The glucuronidation of racemic flurbiprofen by HLMs also resulted in the formation of (R)-glucuronide as the dominant form, which occurred to a degree similar to that by recombinant UGT2B7 (2.1 versus 2.8). The formation of (R)-glucuronide correlated significantly with morphine 3-OH glucuronidation (r = 0.96, p < 0.0001), morphine 6-OH glucuronidation (r = 0.91, p < 0.0001), and 3′-azido-3′-deoxythymidine glucuronidation (r = 0.85, p < 0.0001), a reaction catalyzed mainly by UGT2B7, in individual HLMs. In addition, the formation of both glucuronides correlated significantly (r = 0.99, p < 0.0001). Mefenamic acid inhibited the formation of both (R)- and (S)-glucuronide in HLMs with similar IC50 values (2.0 and 1.7 μM, respectively), which are close to those in recombinant UGT2B7. In conclusion, these findings suggest that the formation of (R)- and (S)-glucuronide from racemic flurbiprofen is catalyzed by the same UGT isozyme, namely UGT2B7.
UGT2B7
Glucuronide
Glucuronosyltransferase
Flurbiprofen
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Glucuronide
UGT2B7
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1. The disposition of diflunisal (DF) was investigated in bile-exteriorized and intact homozygous Gunn rats given 10 and 50 mg/kg doses i.v. and in Wistar rats given 10mg/kg doses i.v.2. In Gunn rats, DF sulphate, DF acyl glucuronide, and a hitherto unidentified metabolite of DF, a conjugate of 3-hydroxy-DF, were identified as the major metabolites, accounting for ∼ 37%, 16% and 11% respectively of 10mg/kg doses and 35%, 24% and 15% respectively of 50 mg/kg doses in bile-exteriorized animals. There was no evidence for formation of DF phenolic glucuronide.3. Total plasma clearance of DF and formation clearances of DF to DF sulphate and 3-hydroxy-DF were little affected by increase of dose from 10 to 50mg DF/kg, whereas formation clearance of DF to DF acyl glucuronide was increased, but not significantly.4. In Gunn rats with undisturbed bile flow into the gut, recoveries of DF sulphate and total 3-hydroxy-DF in urine increased to ∼48% and 25% dose respectively at the expense of DF acyl glucuronide through enterohepatic recirculation.5. In bile-exteriorized Wistar rats, DF phenolic glucuronide, DF acyl glucuronide, DF sulphate and 3-hydroxy-DF accounted for 16%, 27%, 14% and 2%, respectively, of 10mg/kg doses. In intact Wistar rats, urinary recoveries of the metabolites were 15%, 13%, 23% and 5%, respectively.6. Thus in comparison to Wistar rats, phenolic glucuronidation of DF was absent or negligible in homozygous Gunn rats, acyl glucuronidation was significantly decreased, sulphation was unchanged, and the 3-hydroxylation of DF was significantly enhanced.
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Diflunisal
Enterohepatic circulation
Glucuronosyltransferase
Hydroxylation
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Substrates that are specific for certain UDP-glucuronosyltransferase (UGT) isoforms are usually used as specific inhibitors to identify UGT isoforms responsible for the glucuronidation of drugs. 1-Naphthol and 4-nitrophenol are probe substrates for human UGT1A6. In the present study, we found that UGT1A1-catalyzed estradiol 3-O-glucuronide formation and UGT1A4-catalyzed imipramine N-glucuronide formation in human liver microsomes were prominently decreased in the presence of 1-naphthol, but those by recombinant human UGT1A1 and UGT1A4, respectively, were not. Interestingly, when recombinant UGT1A6 was added in the reaction mixture, these activities by recombinant UGT1A1 and UGT1A4 were diminished in the presence of 1-naphthol. To interpret this phenomenon, the inhibitory effects of 1-naphthol O-glucuronide and UDP, products of the glucuronidation of 1-naphthol, were investigated. We found that UDP strongly inhibited the UGT1A1 (Ki = 7 μM) and UGT1A4 (Ki = 47 μM) activities in a competitive manner for the 5′-diphosphoglucuronic acid binding. These results suggest that UDP produced by UGT1A6-catalyzed 1-naphthol glucuronidation, but not 1-naphthol O-glucuronide and 1-naphthol per se, is the actual inhibition substance. Next, we examined the inhibitory effects of 15 compounds that are substrates of UGTs on estradiol 3-O-glucuronide formation in human liver microsomes compared with those by recombinant UGT1A1. Among them, 4 compounds (1-naphthol, 2-naphthol, 4-nitrophenol, and 4-methylumbelliferone) with high turnover rates (Vmax/Km value >200 μl/min/mg) showed more potent inhibition of the activity in human liver microsomes compared with that by the recombinant UGT1A1. Thus, we should pay attention to the inhibitory effects of UDP on UGT, which may cause erroneous evaluations in inhibition studies using human liver microsomes.
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Glucuronosyltransferase
UGT2B7
Lipophilicity
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This study aimed to characterize the glucuronidation pathway of licochalcone A (LCA) in human liver microsomes (HLM). HLM incubation systems were employed to catalyze the formation of LCA glucuronide. The glucuronidation activity of commercially recombinant UDP-glucuronosyltransferase (UGT) isoforms toward LCA was screened. Kinetic analysis was used to identify the UGT isoforms involved in the glucuronidation of LCA in HLM. LCA could be metabolized to two monoglucuronides in HLM, including a major monoglucuronide, namely, 4-O-glucuronide, and a minor monoglucuronide, namely, 4'-O-glucuronide. Species-dependent differences were observed among the glucuronidation profiles of LCA in liver microsomes from different species. UGT1A1, UGT1A3, UGT1A7, UGT1A8, UGT1A9, UGT1A10 and UGT2B7 participated in the formation of 4-O-glucuronide, with UGT1A9 exhibiting the highest catalytic activity in this biotransformation. Only UGT1A1 and UGT1A3 were involved in the formation of 4'-O-glucuronide, exhibiting similar reaction rates. Kinetic analysis demonstrated that UGT1A9 was the major contributor to LCA-4-O-glucuronidation, while UGT1A1 played important roles in the formation of both LCA-4-O- and 4'-O-glucuronide. UGT1A9 was the major contributor to the formation of LCA-4-O-glucuronide, while UGT1A1 played important roles in both LCA-4-O- and 4'-O-glucuronidation.
UGT2B7
Glucuronide
Glucuronosyltransferase
Biotransformation
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The metabolism of benzo(a)pyrene (20 ..mu..M) to phenolic metabolites was examined in the non-recirculating perfused rat liver. The majority of free and conjugated phenols remained in the liver. Glucuronide conjugates were the primary metabolites released into bile and perfusate. Glucuronide and sulfate conjugates were concentrated approximately 3-fold in bile, suggesting transport by an active process. Release of glucuronide conjugates and free phenols into the perfusate was facilitated by albumin. Inhibition of glucuronidation by pretreatment with galactosamine (600 mg/kg, i.p.) decreased the intracellular concentration and release of glucuronide conjugates by >90%, without affecting the formation or release of sulfate conjugates or free phenols. Perfusion with sulfate-free buffer decreased the intracellular concentration of sulfate conjugates by 60%, but did not affect the rate of sulfate conjugate release from the liver. Intracellular concentrations of glucuronides and unconjugated phenols were unaffected by inhibition of sulfation; however, release of glucuronides into perfusate was enhanced approximately 2.5-fold. These data suggest that glucuronidation can regulate rates of production and release of benzo(a)pyrene phenols from liver.
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Renal physiology
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Although conjugation with glucuronic acid is a major process for converting many xenobiotics into hydrophilic, excretable metabolites, relatively little has been reported concerning interindividual variability of glucuronidation in human populations. Oxazepam, a therapeutically active metabolite of diazepam, is one of a number of C3-hydroxylated benzodiazepines for which glucuronide conjugation is the predominant pathway of biotransformation. The drug is normally formulated as a racemic mixture of inactive (R) and active (S) enantiomers. In the present study we have investigated the use of oxazepam as a potential probe drug for studying the variability of glucuronide conjugation, and for demonstrating the extent to which genetic factors may be responsible. In preliminary studies we determined oxazepam pharmacokinetics metabolite profiles after administration of racemic (R,S) oxazepam to eleven human volunteers. The (S) glucuronide was preferentially formed and excreted in nine of the eleven subjects. The ratios of (S) to (R) glucuronide metabolites (S/R ratios) were 3.87 ± 0.79 (mean ± SD) and 3.52 ± 0.60 in urine and plasma, respectively. However, both ratios were significantly lower in two subjects (p < 0.01). In these two atypical subjects, the half-life of (R,S) oxazepam was also markedly longer (14.7 and 15.9 h) than in the other subjects (8.1 ± 3.2 h). A good correlation (rs = 0.90) between the S/R-glucuronide ratio in urine and the plasma clearance of (R,S) oxazepam suggested that a low S/R ratio may be a marker of poor elimination of oxazepam. In further investigations, the drug was administered to 66 additional subjects. The S/R-glucuronide ratio in 8 h pooled urine was bimodally distributed, with 10% of all subjects possessing ratios below an apparent antimode of 1.9. A survey of the in vitro formation of oxazepam glucuronides by microsomes from 37 human livers also showed that 10% of the livers displayed an abnormally high apparent Michaelis constant (Km) for the formation of the (S) glucuronide, but not of the (R) glucuronide. These results suggest that the glucuronidation of the pharmacologically active (S) enantiomer of oxazepam is decreased in a significant percentage (10%) of Caucasian individuals. The observed in vitro differences in apparent kinetics of the S-glucuronidation reaction may reflect defects at the genetic level, leading to structural changes in the isozyme(s) of UDP-glucuronyltransferase that catalyse this reaction.
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Glucuronide
Biotransformation
Active metabolite
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