Absolute Quantitation of Drug-Metabolizing Cytochrome P450 Enzymes and Accessory Proteins in Dog Liver Microsomes Using Label-Free Standard-Free Analysis Reveals Interbreed Variability

Abstract Background Cornin is a commonly used herb in cardiology for its cardioprotective effect. The effect of herbs on the activity of cytochrome P450 enzymes (CYP450s) can induce adverse drug-drug interaction even treatment failure. Therefore, it is necessary to investigate the effect of cornin on the activity of CYP450s, which can provide more guidance for the clinical application of cornin. Methods Cornin (100 μM) was incubated with eight isoforms of CYP450s, including CYP1A2, 2A6, 3A4, 2C8, 2C9, 2C19, 2D6, and 2E1, in pooled human liver microsomes. The inhibition model and corresponding parameters were also investigated. Results Cornin exerted significant inhibitory effect on the activity of CYP3A4, 2C9, and 2E1 in a dose-dependent manner with the IC50 values of 9.20, 22.91, and 14.28 μM, respectively (p < 0.05). Cornin inhibited the activity of CYP3A4 non-competitively with the Ki value of 4.69 μM, while the inhibition of CYP2C9 and 2E1 by cornin was competitive with the Ki value of 11.31 and 6.54 μM, respectively. Additionally, the inhibition of CYP3A4 by cornin was found to be time-dependent with the KI/Kinact value of 6.40/0.055 min− 1·μM− 1. Conclusions The inhibitory effect of cornin on the activity of CYP3A4, 2C9, and 2E1 indicated the potential drug-drug interaction between cornin and drugs metabolized by these CYP450s, which needs further investigation and validation.

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Trapping of cis-2-Butene-1,4-dial to Measure Furan Metabolism in Human Liver Microsomes by Cytochrome P450 Enzymes

Drug Metabolism and Disposition ◽

10.1124/dmd.111.043679 ◽

2011 ◽

Vol 40 (3) ◽

pp. 596-601 ◽

Cited By ~ 23

Author(s):

Leah A. Gates ◽

Ding Lu ◽

Lisa A. Peterson

Keyword(s):

Cytochrome P450 ◽

Human Liver ◽

Liver Microsomes ◽

Human Liver Microsomes ◽

Cytochrome P450 Enzymes

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Enzymatic activity in turkey, duck, quail and chicken liver microsomes against four human cytochrome P450 prototype substrates and aflatoxin B1

Journal of Xenobiotics ◽

10.4081/xeno.2011.e4 ◽

2011 ◽

Vol 1 (1) ◽

pp. 4 ◽

Cited By ~ 10

Author(s):

Hansen W. Murcia ◽

Gonzalo J. Díaz ◽

Sandra Milena Cepeda

Keyword(s):

Cytochrome P450 ◽

Aflatoxin B1 ◽

Enzyme Activities ◽

High Performance ◽

Biochemical Characterization ◽

Liver Microsomes ◽

Cytochrome P450 Enzymes ◽

Catalytic Rate

Cytochrome P450 enzymes (CYP) are a group of monooxygenases able to biotransform several kinds of xenobiotics including aflatoxin B1 (AFB1), a highly toxic mycotoxin. These enzymes have been widely studied in humans and others mammals, but there is not enough information in commercial poultry species about their biochemical characteristics or substrate specificity. The aim of the present study was to identify CYPs from avian liver microsomes with the use of prototype substrates specific for human CYP enzymes and AFB1. Biochemical characterization was carried out in vitro and biotransformation products were detected by high-performance liquid chromatography (HPLC). Enzymatic constants were calculated and comparisons between turkey, duck, quail and chicken activities were done. The results demonstrate the presence of four avian ortholog enzyme activities possibly related with a CYP1A1, CYP1A2, CYP2A6 (activity not previously identified) and CYP3A4 poultry orthologs, respectively. Large differences in enzyme kinetics specific for prototype substrates were found among the poultry species studied. Turkey liver microsomes had the highest affinity and catalytic rate for AFB1 whereas chicken enzymes had the lowest affinity and catalytic rate for the same substrate. Quail and duck microsomes showed intermediate values. These results correlate well with the known in vivo sensitivity for AFB1 except for the duck. A high correlation coefficient between 7-ethoxyresorufin-Odeethylase (EROD) and 7-methoxyresorufin- O-deethylase (MROD) activities was found in the four poultry species, suggesting that these two enzymatic activities might be carried out by the same enzyme. The results of the present study indicate that four prototype enzyme activities are present in poultry liver microsomes, possibly related with the presence of three CYP avian orthologs. More studies are needed in order to further characterize these enzymes.

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Cytochrome P450 Enzymes Involved in the Metabolic Pathway of the Histamine 2 (H2)-Receptor Antagonist Roxatidine Acetate by Human Liver Microsomes

Arzneimittelforschung ◽

10.1055/s-0031-1300096 ◽

2011 ◽

Vol 51 (08) ◽

pp. 651-658

Author(s):

Makoto Sasaki ◽

Mitsuya Nakayama ◽

Satoshi Numazawa ◽

Takiko Oguro ◽

Seijiro Honma ◽

...

Keyword(s):

Cytochrome P450 ◽

Receptor Antagonist ◽

Metabolic Pathway ◽

Human Liver ◽

Liver Microsomes ◽

Human Liver Microsomes ◽

Cytochrome P450 Enzymes ◽

H2 Receptor Antagonist ◽

H2 Receptor ◽

Roxatidine Acetate

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Effect of testicular steroids on catalytic activities of cytochrome P450 enzymes in porcine liver microsomes

Food and Chemical Toxicology ◽

10.1016/j.fct.2006.10.023 ◽

2007 ◽

Vol 45 (4) ◽

pp. 676-681 ◽

Cited By ~ 40

Author(s):

G. Zamaratskaia ◽

W.J. Gilmore ◽

K. Lundström ◽

E.J. Squires

Keyword(s):

Cytochrome P450 ◽

Liver Microsomes ◽

Cytochrome P450 Enzymes ◽

Porcine Liver ◽

Catalytic Activities ◽

Testicular Steroids

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Grapefruit juice, lyophilized grapefruit juice, and powdered whole grapefruit inhibit cytochrome P450-mediated triazolam hydroxylation by beagle dog liver microsomes

Journal of Veterinary Pharmacology and Therapeutics ◽

10.1111/j.1365-2885.2009.01124.x ◽

2010 ◽

Vol 33 (2) ◽

pp. 189-195 ◽

Cited By ~ 6

Author(s):

M. J. HANLEY ◽

R. CERUNDOLO ◽

N. RADWANSKI ◽

M. H. COURT

Keyword(s):

Cytochrome P450 ◽

Grapefruit Juice ◽

Liver Microsomes ◽

Beagle Dog ◽

Dog Liver

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Characterization of O-demethylations and Aromatic Hydroxylations Mediated by Cytochromes P450 in the Metabolism of Flavonoid Aglycons

Croatica Chemica Acta ◽

10.5562/cca3528 ◽

2019 ◽

Vol 92 (1) ◽

pp. 115-123 ◽

Cited By ~ 4

Author(s):

Goran Benković ◽

Hrvoje Rimac ◽

Željan Maleš ◽

Siniša Tomić ◽

Zoran Lončar ◽

...

Keyword(s):

Cytochrome P450 ◽

Human Liver ◽

Cytochromes P450 ◽

Liver Microsomes ◽

Cytochrome P450 Enzymes ◽

Uv Detector ◽

Aromatic Hydroxylation ◽

Metabolism Of Xenobiotics ◽

Kinetics Of

One of the most important groups of metabolic enzymes is cytochrome P450 superfamily. These enzymes are important in terms of the catalytic diversity and the large number of xenobiotics that are detoxified or activated by converting to reactive metabolites. Flavonoids are xenobiotics to which humans are exposed through diet. Data on their oxidative metabolism mediated by cytochromes P450 are limited. The aim of this study was to determine the enzymatic kinetics of O-demethylation and aromatic hydroxylation of flavonoid aglycons on recombinant cytochrome P450 enzymes and human liver microsomes systems. The study was performed on ten flavonoids, namely 3,7-dihydroxyflavone, 7-hydroxyflavone, acacetin, apigenin, flavone, galangin, kaempferol, naringenin, sakuranetin, and tangeretin using liquid chromatography coupled with mass spectrometry and UV detector. Most relevant enzyme involved in metabolism of flavonoid aglycons is CYP1A2, and its catalytic effectiveness ranges from 0.5 to 2.9 × 106 M–1 min–1. Having in mind high expression and involvement of CYP1A2 in metabolism of xenobiotics including drugs, and its intraindividual differences in expression and activity, potential of drug-flavonoid competitive interactions/inhibitions should be considered when consuming dietary supplement and foods rich in flavonoids.

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Assessment of inhibitory effects on major human cytochrome P450 enzymes by spasmolytics used in the treatment of overactive bladder syndrome

Therapeutic Advances in Urology ◽

10.1177/1756287217708951 ◽

2017 ◽

Vol 9 (7) ◽

pp. 163-177

Author(s):

Dominik Dahlinger ◽

Sevinc Aslan ◽

Markus Pietsch ◽

Sebastian Frechen ◽

Uwe Fuhr

Keyword(s):

Cytochrome P450 ◽

Liver Microsomes ◽

Fold Increase ◽

Pharmacokinetic Data ◽

Cytochrome P450 Enzymes ◽

Trospium Chloride ◽

Screening Experiments ◽

Human Cytochrome

Background: The objective of this study was to examine the inhibitory potential of darifenacin, fesoterodine, oxybutynin, propiverine, solifenacin, tolterodine and trospium chloride on the seven major human cytochrome P450 enzymes (CYP) by using a standardized and validated seven-in-one cytochrome P450 cocktail inhibition assay. Methods: An in vitro cocktail of seven highly selective probe substrates was incubated with human liver microsomes and varying concentrations of the seven test compounds. The major metabolites of the probe substrates were simultaneously analysed using a validated liquid chromatography tandem mass spectrometry (LC-MS/MS) method. Enzyme kinetics were estimated by determining IC50 and Ki values via nonlinear regression. Obtained Ki values were used for predictions of potential clinical impact of the inhibition using a static mechanistic prediction model. Results: In this study, 49 IC50 experiments were conducted. In six cases, IC50 values lower than the calculated threshold for drug–drug interactions (DDIs) in the gut wall were observed. In these cases, no increase in inhibition was determined after a 30 min preincubation. Considering a typical dosing regimen and applying the obtained Ki values of 0.72 µM (darifenacin, 15 mg daily) and 7.2 µM [propiverine, 30 mg daily, immediate release (IR)] for the inhibition of CYP2D6 yielded a predicted 1.9-fold and 1.4-fold increase in the area under the curve (AUC) of debrisoquine (CYP2D6 substrate), respectively. Due to the inhibition of the particular intestinal CYP3A4, the obtained Ki values of 14 µM of propiverine (30 mg daily, IR) resulted in a predicted doubling of the AUC for midazolam (CYP3A4 substrate). Conclusions: In vitro/ in vivo extrapolation based on pharmacokinetic data and the conducted screening experiments yielded similar effects of darifenacin on CYP2D6 and propiverine on CYP3A4 as obtained in separately conducted in vivo DDI studies. As a novel finding, propiverine was identified to potentially inhibit CYP2D6 at clinically occurring concentrations.

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