High‐throughput screening of inhibitory potential of nine cytochrome P450 enzymes <i>in vitro</i> using liquid chromatography/tandem mass spectrometry

Kim, Minjung; Kim, Hyojin; Cha, In‐June; Park, Jang-Su; Shon, Ji‐Hong; Liu, Kwang-Hyeon; Shin, Jae‐Gook

doi:10.1002/rcm.2110

Cited by 152 publications

(149 citation statements)

References 37 publications

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“…In human liver microsomes in vitro, bupropion, caffeine and chlorzoxazone incubated with coumarin (a specific CYP2A6 substrate) show no inhibitory effects on coumarin 7-hydroxylation [35][36][37]. To the best of our knowledge, there are no human in vivo studies on the inhibitory properties of bupropion on CYP2E1 activity, and bupropion does not inhibit chlorzoxazone metabolism in vitro in human liver microsomes [37,38]. As caffeine was dosed Table 3 Effect of nicotine administration on disposition kinetics of cotinine-d4 …”

Section: Discussionmentioning

confidence: 99%

Effects of nicotine on cytochrome P450 2A6 and 2E1 activities

Hukkanen

Jacob

Peng

et al. 2010

Brit J Clinical Pharma

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WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT• Smoking slows the metabolism of nicotine and accelerates the metabolism of chlorzoxazone.• Nicotine is a useful probe for phenotyping cytochrome P450 2A6 activity and chlorzoxazone is a frequently used probe for CYP2E1 activity.• The tobacco smoke constituents responsible for the reduced CYP2A6 and increased CYP2E1 activities are unknown. WHAT THIS PAPER ADDS• This study demonstrates that CYP2A6 and CYP2E1 activities are not affected by nicotine dosing.• High-dose nicotine treatment has a low potential of interaction with CYP2A6 and CYP2E1 substrates.• The mechanisms of tobacco smoke-elicited changes in CYP2A6 and CYP2E1 activities are yet to be determined. AIMSSmoking slows the metabolism of nicotine and accelerates the metabolism of chlorzoxazone, which are probe reactions for cytochrome P450 2A6 (CYP2A6) and CYP2E1 activities, respectively. We aimed to determine the role of nicotine in these metabolic effects of cigarette smoking. METHODSThe study had a single-blind, randomized, crossover two-arm design. Twelve healthy smokers were given two transdermal patches with 42-mg nicotine a day or placebo patches, each for 10 days. The subjects abstained from smoking during the study arms. Oral chlorzoxazone was given on day 7 and deuterium-labelled nicotine-d2 and cotinine-d4 infusion on day 8. RESULTSThere was no significant influence of transdermal nicotine administration on pharmacokinetic parameters of nicotine-d2 or on the formation of cotinine-d2. Nicotine decreased the volume of distribution (62.6 vs. 67.7 l, 95% confidence interval of the difference -9.7, -0.6, P = 0.047) of infused cotinine-d4. There were no significant differences in disposition kinetics of chlorzoxazone between the treatments. CONCLUSIONSCYP2A6 and CYP2E1 activities are not affected by nicotine. The tobacco smoke constituents responsible for the reduced CYP2A6 and increased CYP2E1 activities remain unknown.

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Section: Discussionmentioning

confidence: 99%

Effects of nicotine on cytochrome P450 2A6 and 2E1 activities

Hukkanen

Jacob

Peng

et al. 2010

Brit J Clinical Pharma

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“…So-called cocktail assays, in which several enzyme activities are determined in parallel by liquid chromatography-tandem mass spectrometry (LC-MS/MS) are particularly useful in this regard. Although a number of such assays have been published in recent years (Dierks et al, 2001;Testino and Patonay, 2003;Weaver et al, 2003;Kim et al, 2005;Tolonen et al, 2007;Lahoz et al, 2008), there is still a need for optimizing the selection of probe substrates and their composition to get the most significant information.…”

Section: Introductionmentioning

confidence: 99%

Profiling Induction of Cytochrome P450 Enzyme Activity by Statins Using a New Liquid Chromatography-Tandem Mass Spectrometry Cocktail Assay in Human Hepatocytes

Feidt¹,

Klein²,

Hofmann³

et al. 2010

Drug Metab Dispos

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ABSTRACT:Human hepatocytes in primary culture are a very useful model to directly assess induction of gene expression by xenobiotics. We developed a cytochrome P450 (P450) activity cocktail assay using model substrates for the seven important P450s 1A2 (phenacetin), 2B6 (bupropion), 2C8 (amodiaquine), 2C9 (tolbutamide), 2C19 (Smephenytoin), 2D6 (propafenone), and 3A4 (atorvastatin). Metabolite formation was determined by liquid chromatography-tandem mass spectrometry in hepatocyte culture supernatants. Atorvastatin has not been previously assessed as a CYP3A probe drug. We demonstrate highly selective atorvastatin ortho-hydroxylation by CYP3A4 by recombinant P450s. In human liver microsomes orthohydroxyatorvastatin formation was highly correlated with CYP3A4 protein content (r s ‫؍‬ 0.78, p < 0.0001, n ‫؍‬ 150). We profiled induction of these P450 activities in primary human hepatocytes after treatment with 30 M atorvastatin, lovastatin, pravastatin, rosuvastatin, and simvastatin for 24 to 72 h. Except for pravastatin, all statins induced P450 activities to various degrees, approximately in the order atorvastatin > simvastatin > lovastatin > rosuvastatin. Inducibility of P450s followed the order CYP2C8 > CYP3A4 > CYP2C9 > CYP2B6 > CYP2C19 ϳ CYP2D6 > CYP1A2. The strongest induction was observed for amodiaquine N-desalkylation, which was induced approximately 20-fold. Quantitative reverse transcription-polymerase chain reaction confirmed corresponding changes on the mRNA level with even more dramatic induction up to almost 100-fold. These data suggest a broader inducing effect of statins on cytochrome P450s and possibly other absorption, distribution, metabolism, and excretion genes than previously known, thus further emphasizing their drug-drug interaction potential. Our cocktail assay should be helpful for economical use of human hepatocytes in the assessment of P450 induction by drugs and drug candidates.

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“…Such P450-substrate cocktails are increasingly used for the determination of basal and induced P450 activities (Zhou et al, 2004). Indeed, the use of a variety of substrate combinations, numbers of substrates, or analytical methods has revealed no significant differences between the results obtained from individual or cocktail analyses (Dierks et al, 2001;Kim et al, 2005;Lahoz et al, 2008a). In addition, expression of various other genes related to xenobiotic metabolism and transport was also measured for comparison purposes.…”

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confidence: 99%

Stable Expression, Activity, and Inducibility of Cytochromes P450 in Differentiated HepaRG Cells

Anthérieu

Chesné²,

Li³

et al. 2009

Drug Metab Dispos

230

143

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ABSTRACT:HepaRG cells possess the unique property to differentiate in vitro and to express various functions of mature hepatocytes, including the major cytochromes P450 (P450s). In the present study, we carefully analyzed mRNA expression and activity of the major P450s and their responsiveness to three prototypical inducers, phenobarbital, rifampicin, and omeprazole, in differentiated HepaRG cell cultures over a 4-week period after low and high seeding. Only minor differences were observed in P450 activities when measured by two cocktails of probe substrates, probably related to the choice and/or concentration of substrates. Similar results were obtained from the two cell seeding conditions. Expression and activities of several P450s were dimethyl sulfoxidedependent. However, basal P450 expression and activities as well as their responsiveness to the prototypical inducers were well maintained over the 4-week period, and a good correlation was observed between transcript levels and corresponding activities. Thus, CYP1A2, CYP2B6, and CYP3A4 were found to accurately respond to their respective prototypical inducers, i.e., omeprazole, phenobarbital, and rifampicin. Likewise, basal expression of several phase II enzymes, transporters, and nuclear receptors, and response to inducers were also well preserved. More genes were found to be induced in HepaRG cells than in primary human hepatocytes, and no marked variation was noticed between the different passages. Taken together, these data support the conclusion that HepaRG cells represent a promising surrogate to primary human hepatocytes for xenobiotic metabolism and toxicity studies.

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High‐throughput screening of inhibitory potential of nine cytochrome P450 enzymes in vitro using liquid chromatography/tandem mass spectrometry

Cited by 152 publications

References 37 publications

Effects of nicotine on cytochrome P450 2A6 and 2E1 activities

Effects of nicotine on cytochrome P450 2A6 and 2E1 activities

Profiling Induction of Cytochrome P450 Enzyme Activity by Statins Using a New Liquid Chromatography-Tandem Mass Spectrometry Cocktail Assay in Human Hepatocytes

Stable Expression, Activity, and Inducibility of Cytochromes P450 in Differentiated HepaRG Cells

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