LKY-047: First Selective Inhibitor of Cytochrome P450 2J2

Phuc, Nguyen Minh; Wu, Zhexue; Yuseok, O; Lee, Jee-Hyun; Oh, Sangtaek; Song, Gyu Yong; Liu, Kwang-Hyeon

doi:10.1124/dmd.117.075036

Cited by 13 publications

(7 citation statements)

References 28 publications

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“…Moreover, the range of obtained IC 50 values was much narrower than those previously reported, such as 0.6–1.3 (Liu et al, ), 0.68–2.61 (Dahlinger et al, ), 0.87–1.82 (Li et al, ), 0.6–1.8 (Kozakai et al, ), 0.67–1.4 (Kim et al, ) and 0.79–1.29 (Peng et al, ), indicating that this method is more accurate than previous cocktail assays. In addition, the measured IC 50 values were consistent with previously reported values (Chen et al, ; Donato et al, ; Eagling, Tjia, & Back, ; Kim et al, ; Kozakai et al, ; Lafite et al, ; Lee et al, a, ; Liu et al, , 2015; Perloff et al, ; Phuc et al, ; Stresser et al, ; Walsky & Obach, ; Walsky et al, b; Wu et al, ), demonstrating that the IC 50 values of NCEs against P450 isoform‐mediated metabolism could be determined precisely using a cocktail assay instead of individual substrate incubations, which would save a tremendous amount of time in DDI studies for NCEs.…”

Section: Discussionsupporting

confidence: 88%

“…The structure of the 12 P450 probe substrates, their metabolites and internal standard used in the assays are shown in Figure . The substrates of the cocktail doses established for the incubation study (Table ) were selected based on the recommendations of the US FDA (2016) and our previously published data (Lee et al, a; Phuc et al, ). In the case of CYP3A, three substrates were selected due to the existence of multiple binding sites within the CYP3A active site (Kenworthy et al, ; Lee, Shon, & Liu, ).…”

Section: Resultsmentioning

confidence: 99%

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Screening of ten cytochrome P450 enzyme activities with 12 probe substrates in human liver microsomes using cocktail incubation and liquid chromatography–tandem mass spectrometry

Kim

Lee

et al. 2019

Biopharm & Drug Disp

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Testing for potential drug interactions of new chemical entities is essential when developing a novel drug. In this study, an assay was designed to evaluate drug interactions with 10 major human cytochrome P450 (P450) enzymes incubated in liver microsomes, involving 12 probe substrates with two cocktail incubation sets used in a single liquid chromatography-tandem mass spectrometry (LC-MS/MS) run. The P450 substrate composition in each cocktail set was optimized to minimize solvent effects and mutual drug interactions among substrates as follows: cocktail A was composed of phenacetin for CYP1A2, bupropion for CYP2B6, amodiaquine for CYP2C8, diclofenac for CYP2C9, S-mephenytoin for CYP2C19, and dextromethorphan for CYP2D6; cocktail B was composed of coumarin for CYP2A6, chlorzoxazone for CYP2E1, astemizole for CYP2J2, and midazolam, nifedipine, and testosterone for CYP3A. Multiple probe substrates were used for CYP3A owing to the multiple substrate-binding sites and substrate-dependent inhibition. After incubation in human liver microsomes, each incubation mixture was pooled and all probe metabolites were simultaneously analysed in a single LC-MS/MS run. Polarity switching was used to acquire the negative-ion mode for hydroxychlorzoxazone and positive-ion mode for the remaining analytes. The method was validated by comparing the inhibition data obtained from incubation of each individual probe substrate alone and with the substrate cocktails. The half-maximal inhibitory concentration values obtained from the cocktail and individual incubations were well correlated and in agreement with previously reported values. This new method will be useful in assessing the drug interaction potential of new chemical entities during new drug development. KEYWORDS drug interaction, high-throughput screening, IC 50 , liquid chromatography-tandem mass spectrometry, new chemical entities *These authors contributed equally to this work.

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Section: Discussionsupporting

confidence: 88%

Section: Resultsmentioning

confidence: 99%

Screening of ten cytochrome P450 enzyme activities with 12 probe substrates in human liver microsomes using cocktail incubation and liquid chromatography–tandem mass spectrometry

Kim

Lee

et al. 2019

Biopharm & Drug Disp

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“…Linezolid and cyp3cide were from the Pfizer sample bank. LKY-047 was prepared according to the method of Phuc, et al (2017). 1-(3-chlorophenyl)-3-phenylurea was prepared using the method of Mohd Siddique, et al, (2016).…”

Section: Methodsmentioning

confidence: 99%

Linezolid Metabolism Is Catalyzed by Cytochrome P450 2J2, 4F2, and 1B1

Obach

2022

Drug Metab Dispos

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The oxazolidinone antibacterial linezolid has been in clinical use for over twenty years, yet knowledge of the contributions of specific cytochrome P450 enzymes to the metabolic clearance of this drug were mostly unknown. In this investigation it was revealed that three P450 enzymes that had not been previously explored in linezolid metabolism, CYP2J2, CYP4F1, and CYP1B1, catalyzed the 2-hydroxylation and deethyleneation of the morpholine moiety of linezolid. The intrinsic clearance for linezolid metabolism in pooled human liver microsomes was low at 0.51 µL/min/mg protein, consistent with its in vivo clearance in humans, and the K M was high (>200 µM). In recombinant human P450 enzymes, a rank order of intrinsic clearance values for linezolid 2-hydroxylation were CYP2J2>>CYP4F2>CYP2C8>CYP1B1≈CYP2D6≈CYP3A4>CYP1A1>CYP3A5, with nine other P450 enzymesshowing no linezolid metabolism The effect of selective inhibitors for these eight P450 enzymes on linezolid metabolism in pooled human liver microsomes was evaluated to provide estimates of the relative fractional contributions of these enzymes to linezolid metabolism. These experiments suggest that CYP2J2 and CYP4F2 contribute about 50% each to linezolid hepatic metabolism. It is proposed that the oxidative metabolic clearance of linezolid is primarily catalyzed by these two unusual P450 enzymes and that this explains the lack of observation of meaningful effects of common perpetrators of drug interactions on linezolid pharmacokinetics. SIGNIFICANCE STATEMENTLinezolid is an important antibacterial drug, but the enzymes involved in its oxidative metabolism were unknown. In this paper, evidence is shown that supports an important role for two enzymes not frequently associated with the metabolism of drugs: CYP2J2 and CYP4F2. These observations offer insight to understand the results of clinical drug-drug interaction studies conducted on linezolid.

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“…The IC 50 values were calculated using WinNonlin software (Pharsight, Mountain View, CA). The apparent kinetic parameters for inhibitory activity (K i ) were estimated by graphical methods, such as Dixon and Lineweaver-Burk plots, and were more accurately determined by nonlinear least-squares regression analysis, on the basis of the best enzyme inhibition model using the WinNonlin software (Miners et al, 2011;Phuc et al, 2017). The models…”

Section: Methodsmentioning

confidence: 99%

Ginsenoside Rc Is a New Selective UGT1A9 Inhibitor in Human Liver Microsomes and Recombinant Human UGT Isoforms

et al. 2019

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Ginseng is known to have inhibitory effects on UGT1A9 activity. However, little is known about the inhibitory effects of ginsenosides, the major active compounds in ginseng, on UGT1A9 activity. In vitro investigation of UGT1A9 inhibition by ginsenosides was carried out using human liver microsomes (HLMs). Among 10 ginsenosides, ginsenoside Rc was the strongest inhibitor of UGT1A9-mediated mycophenolic acid glucuronidase activity. Further inhibition kinetic studies using HLMs suggested that ginsenoside Rc competitively and noncompetitively inhibited UGT1A9-mediated propofol and mycophenolic acid glucuronidation activities, with K i values of 2.83 and 3.31 mM, respectively. Next, to investigate whether the inhibitory effect of ginsenoside Rc is specific to the UGT1A9 isoform, we studied the inhibitory potency of ginsenoside Rc on nine human uridine diphospho-glucuronosyltransferase (UGT) activities using recombinant human UGT isoforms. Ginsenoside Rc exhibited a 12.9-fold selectivity (which was similar to niflumic acid at 12.5-fold) for UGT1A9 inhibition. Ginsenoside Rc at 50 mM also inhibited none of the other UGT isoform-specific activities above 12.0%, except for UGT1A9 (>91.5%) in HLMs, indicating that ginsenoside Rc might be used as a selective UGT1A9 inhibitor in reaction phenotyping studies of new chemical entities. Considering lower plasma concentrations (0.01 mM) of ginsenoside Rc in healthy subjects and no induction potential on UGT isoforms, ginsenoside Rc does not cause pharmacokinetic drug interactions with other coadministered drugs metabolized by UGT1A9. SIGNIFICANCE STATEMENT Ginsenoside Rc selectively inhibited UGT1A9-mediated propofol and mycophenolic acid glucuronidation activities in human liver microsomes and recombinant uridine diphospho-glucuronosyltransferase (UGT) isoforms. It exhibited a 12.9-fold selectivity for UGT1A9 inhibition. Therefore, ginsenoside Rc might be used as a selective UGT1A9 inhibitor in reaction phenotyping studies of new chemical entities, such as niflumic acid.

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LKY-047: First Selective Inhibitor of Cytochrome P450 2J2

Cited by 13 publications

References 28 publications

Screening of ten cytochrome P450 enzyme activities with 12 probe substrates in human liver microsomes using cocktail incubation and liquid chromatography–tandem mass spectrometry

Screening of ten cytochrome P450 enzyme activities with 12 probe substrates in human liver microsomes using cocktail incubation and liquid chromatography–tandem mass spectrometry

Linezolid Metabolism Is Catalyzed by Cytochrome P450 2J2, 4F2, and 1B1

Ginsenoside Rc Is a New Selective UGT1A9 Inhibitor in Human Liver Microsomes and Recombinant Human UGT Isoforms

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