Identification and Characterization of Human UDP-Glucuronosyltransferases Responsible for the In Vitro Glucuronidation of Daphnetin

Liang, Sisi; Ge, Guangbo; Liu, Hui‐Xin; Zhang, Yanyan; Wang, Liming; Zhang, Jiangwei; Lu, Yin; Li, Wei; Fang, Zhong‐Ze; Wu, Jingjing; Li, Guohui; Yang, Ling

doi:10.1124/dmd.109.030734

Cited by 62 publications

(46 citation statements)

References 36 publications

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“…All analytical methods were shown to be precise and accurate. The intra-and inter-day precisions were less than 15%, with accuracy in the range of 86.7%-112.5% [19,[21][22][23] .…”

Section: Chemicals and Reagentsmentioning

confidence: 94%

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Substrate-dependent modulation of the catalytic activity of CYP3A by erlotinib

et al. 2011

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Aim: To ascertain the effects of erlotinib on CYP3A, to investigate the amplitude and kinetics of erlotinib-mediated inhibition of seven major CYP isoforms in human liver microsomes (HLMs) for evaluating the magnitude of erlotinib in drug-drug interaction in vivo. Methods: The activities of 7 major CYP isoforms (CYP1A2, CYP2A6, CYP3A, CYP2C9, CYP2D6, CYP2C8, and CYP2E1) were assessed in HLMs using HPLC or UFLC analysis. A two-step incubation method was used to examine the time-dependent inhibition of erlotinib on CYP3A.Results: The activity of CYP2C8 was inhibited with an IC 50 value of 6.17±2.0 μmol/L. Erlotinib stimulated the midazolam 1′-hydroxy reaction, but inhibited the formation of 6β-hydroxytestosterone and oxidized nifedipine. Inhibition of CYP3A by erlotinib was substratedependent: the IC 50 values for inhibiting testosterone 6β-hydroxylation and nifedipine metabolism were 31.3±8.0 and 20.5±5.3 μmol/L, respectively. Erlotinib also exhibited the time-dependent inhibition on CYP3A, regardless of the probe substrate used: the value of K I and k inact were 6.3 μmol/L and 0.035 min -1 for midazolam; 9.0 μmol/L and 0.045 min -1 for testosterone; and 10.1 μmol/L and 0.058 min -1 for nifedipine. Conclusion: The inhibition of CYP3A by erlotinib was substrate-dependent, while its time-dependent inhibition on CYP3A was substrateindependent. The time-dependent inhibition of CYP3A may be a possible cause of drug-drug interaction, suggesting that attention should be paid to the evaluation of erlotinib's safety, especially in the context of combination therapy.

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“…All analytical methods were shown to be precise and accurate. The intra-and inter-day precisions were less than 15%, with accuracy in the range of 86.7%-112.5% [19,[21][22][23] .…”

Section: Chemicals and Reagentsmentioning

confidence: 94%

“…Coumarin, diclofenac, dextromethorphan and ketoconazole were from ICN Biomedicals (Aurora, OH, USA). Human liver microsomes (HLMs) were prepared according to the method described by Guengerich (1989) and other previous reports [18,19] . Protein concentrations were determined using bovine serum albumin as a standard [20] .…”

Section: Chemicals and Reagentsmentioning

confidence: 99%

Substrate-dependent modulation of the catalytic activity of CYP3A by erlotinib

et al. 2011

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“…These findings suggest that the C-7 phenol group should receive more attention in further structure modifications aimed toward increasing the metabolic stability of 6,7-DHCs. To date, UGT1A6 and 1A9 have been identified as the major enzymes catalyzing C-7 glucuronidation of coumarins, including 4-MU, daphnetin, and fraxetin (Tukey and Strassburg, 2000;Liang et al, 2010;Xia et al, 2014). In our study, UGT1A6 and UGT1A9 were also effective in 7-O-glucuronidation of 6,7-DHCs.…”

Section: Discussionmentioning

confidence: 49%

Structural Modifications at the C-4 Position Strongly Affect the Glucuronidation of 6,7-Dihydroxycoumarins

Xia

Wang

et al. 2015

Drug Metab Dispos

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Esculetin (6,7-dihydroxycoumarin) and its C-4 derivatives have multiple pharmacologic activities, but the poor metabolic stability of these catechols has severely restricted their application in the clinic. Glucuronidation plays important roles in catechols elimination, although thus far the effects of structural modifications on the metabolic selectivity and the catalytic efficacy of the human UDPglucuronosyltransferase (UGT) enzymes remain unclear. This study was aimed at exploring the structure-glucuronidation relationship of esculetin and its C-4 derivatives, including 4-methyl esculetin, 4-phenyl esculetin, and 4-hydroxymethyl esculetin as well as 4-acetic acid esculetin. It was achieved by identifying the main human UGTs responsible for the different reactions and by careful characterization of the reactions kinetics. These catechols, with the exception of 4-acetic acid esculetin, are selectively metabolized to the corresponding 7-O-glucuronides. UGT1A6 and UGT1A9 are the two major UGTs involved in the 7-O-glucuronidation of 4-methyl esculetin and esculetin. UGT1A6 was the major contributor for 7-O-glucuronidation of 4-hydroxymethyl esculetin, and UGT1A9 played a significant role in the 7-O-glucuronidation of 4-phenyl esculetin. The results of the kinetic analyses revealed that the K m values of the compounds, in both UGT1A9 and human liver microsomes, decreased with increasing hydrophobicity of the C-4 substitutions. The outcome of this was that C-4 hydrophobic and hydrophilic groups on 6,7-dihydroxycoumarin exhibited contrasting effects on UGT affinity. All of these findings provide helpful guidance for further structural modification of 6,7-dihydroxycoumarins with improved metabolic stability.

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“…The binding of ornidazole to microsomes was estimated according to eq. 3 (Austin et al, 2002;Liang et al, 2010): …”

Section: ) Michaelis-menten Modelmentioning

confidence: 99%

Stereoselective Glucuronidation of Ornidazole in Humans: Predominant Contribution of UDP-Glucuronosyltransferases 1A9 and 2B7

You

Chen

et al. 2013

Drug Metab Dispos

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Ornidazole [R,S-1-chloro-3-(2-methyl-5-nitro-1H-imidazol-1-yl)propan-2-ol] is a chiral 5-nitroimidazole class antimicrobial agent. This study aimed to investigate the principal metabolic pathway of ornidazole in humans and identify the major enzymes involved. A total of 19 metabolites were identified in human urine collected from patients with hepatobiliary diseases after an intravenous drip infusion of 500 mg of racemic ornidazole. Stereoselective glucuronidation, followed by renal excretion, was the principal metabolic pathway of ornidazole in humans, accounting for 37.3% of the administered dose. Screening assays with 12 available human recombinant UDP-glucuronosyltransferases (UGTs) demonstrated that UGT1A9 was the predominant UGT isoform involved in Rornidazole glucuronidation, whereas S-ornidazole glucuronidation was almost exclusively catalyzed by UGT2B7. Chemical inhibition study with niflumic acid and flurbiprofen supported these findings. Enzyme kinetic parameters were then determined in human liver microsomes (HLMs), human kidney microsomes (HKMs), UGT1A9, and 2B7. The K m values for UGT1A9 (15.6 6 1.6 mM for R-ornidazole) and 2B7 (3.8 6 0.9 mM for S-ornidazole) were quite similar to those determined in HLMs and HKMs (20.1 6 1.4 and 17.7 6 4.0 mM for R-ornidazole; 6.6 6 1.3 and 3.2 6 0.4 mM for S-ornidazole). The in vitro intrinsic clearance (CL int ) ratios of S-to R-ornidazole were approximately 4.3 in HLMs and 6.5 in HKMs, respectively. The hepatic and renal clearances were estimated based on the wellstirred model. Overall, stereoselective glucuronidation was the principal metabolic pathway of ornidazole in humans. Furthermore, UGT1A9 and 2B7 were the predominant UGT isoforms responsible for R-and S-ornidazole glucuronidation in humans, respectively.

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Identification and Characterization of Human UDP-Glucuronosyltransferases Responsible for the In Vitro Glucuronidation of Daphnetin

Cited by 62 publications

References 36 publications

Substrate-dependent modulation of the catalytic activity of CYP3A by erlotinib

Substrate-dependent modulation of the catalytic activity of CYP3A by erlotinib

Structural Modifications at the C-4 Position Strongly Affect the Glucuronidation of 6,7-Dihydroxycoumarins

Stereoselective Glucuronidation of Ornidazole in Humans: Predominant Contribution of UDP-Glucuronosyltransferases 1A9 and 2B7

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