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Simultaneous Assessment of Cytochrome P450 Activity in Human Hepatocytes for Ligand-Mediated Activation of the
Pregnane X Receptor (PXR), Constitutive Androstane Receptor (CAR), and Aryl Hydrocarbon Receptor (AhR)
Abstract # 197
Susan P. Rhodes*, Jennifer N. Otten, Scott D. Kragerud, Gary P. Hingorani, Dylan P. Hartley, and Ronald B. Franklin, Array BioPharma Inc, Boulder, CO 80301, USA Available at www.arraybiopharma.com
CYP2B6 Bupropion 7'-Hydroxylase Activity
Abstract
Table 1. LC-MS/MS Conditions for CYP450 Probe Substrates and Their
CYP1A2 mRNA Levels
CYP2B6 Bupropion Hydroxylase Activity
CYP2B6 Bupropion Hydroxylase Activity
Respective Metabolites
The human pregnane X receptor (PXR), constitutive androstane receptor (CAR), and aryl hydrocarbon receptor (AhR) are known to regulate gene expression of the cytochrome P450 (CYP) enzymes, CYP3A4, CYP2B6, and CYP1A2 mRNA Levels
CYP1A2 mRNA Levels
CYP1A2 respectively. The objective of this study was to assess, simultaneously, the induction of CYP3A4, MRM Transitions for Parent, Metabolite, and Internal
Liquid Chromatography
CYP2B6, and CYP1A2 activity in cultured human hepatocytes treated with various prototypical ligands of PXR, Standard
Gradient
AhR. To evaluate the xenobiotic-mediated induction of hepatocellular gene expression, rifampicin (RIF; 10 μM) and phenobarbital (PB; 1 mM) were used for CYP3A4, CITCO (1 μM) and artemisinin (ART; 50 μM) were Transition (m/z)
Time (min)
used for CYP2B6, and 3-methylcholanthrene (3-MC; 1 μM) and omeprazole (OME; 50 μM) were utilized for CYP1A2 induction. Hepatocyte cultures in 24-well plates were treated with each compound for two days, followed by a thirty-minute incubation of the hepatocyte culture along with the addition of three marker substrates for specific CYP activity: midazolam (CYP3A4; 5 μM), bupropion (CYP2B6; 50 μM), and phenacetin (CYP1A2; 100 μM). The assessment of CYP activity was performed with a novel liquid chromatography-mass spectrometry method which simultaneously assessed activity of CYP3A4, CYP2B6, and CYP1A2 in a single three minute method by examining the formation of the probe substrate metabolites, 1'-hydroxymidazolam, hydroxybupropion, CYP2B6 Bupropion Hydroxylase Activity
CYP2B6 Bupropion Hydroxylase Activity
and acetaminophen, respectively. The potential for inhibitory as well as activation effects of each probe substrate CYP1A2 mRNA Levels
CYP1A2 mRNA Levels
on the metabolism of the other probe substrates were evaluated, and it was shown that the inclusion of the three probe substrates showed no inhibitory or activating effects on the other CYP activities. In four separate preparations of donor human hepatocytes, the incorporation of all three probe substrates in a single incubation *See 'Materials & Methods' section
achieved similar results as compared with the conventional activity assay ( i.e. separate incubations for each probe substrate). In addition, the average fold-induction of CYP3A4, CYP2B6, and CYP1A2 activity was comparable Figure 1. LC-MS/MS Total Ion Chromatogram
between the three substrate assay and the conventional assay. Induction of CYP3A4, CYP2B6, and CYP1A2 activity was supported further by mRNA analysis for each of the genes. Interestingly, ART (50 μM) inhibited the activities of CYP2B6 and CYP3A4, which masked the observed induction of CYP2B6 as well as CYP3A4 mRNA levels. In conclusion, the use of this substrate cocktail method in monitoring cytochrome P450 induction in cultured human hepatocytes resulted in a higher throughput methodology as compared to the conventional CYP3A4 Midazolam 1'-Hydroxylase Activity
Fig. 3A. Comparison of the substrate cocktail and single substrate conventional assays: average fold induction of CYP2B6 bupropion 7'-hydroxylase activity
methodology, while conserving the inductive responses of prototypical inducers.
by the positive controls, CITCO (1 μM) and ART (50 μM) in primary cultures of human hepatocytes. Note the inhibitory effect of ART on bupropion hydroxylase activity. Data are expressed as fold induction over VC (0.2% DMSO). Percent difference in the average fold induction as measured by activity CYP3A4 Midazolam 1'Hydroxylase Activity
CYP3A4 Midazolam 1'Hydroxylase Activity
between the substrate cocktail and conventional method is 16% and 10% for CITCO and ART respectively.
Fig. 4B. Comparison of the substrate cocktail and single substrate conventional assays: average fold induction of 1A2 mRNA levels by the positive
controls, 3-MC (1 μM) and OME (50 μM) in primary cultures of human hepatocytes. Data are expressed as fold induction over VC (0.2% DMSO). Introduction
Percent difference in the average fold induction as measured by mRNA levels between the substrate cocktail and conventional method is 21% and 0.2% CYP2B6 mRNA Levels
ƒ Cytochrome P450 induction of CYP3A4, CYP2B6, or CYP1A2 is important to consider in the selection of clinical CYP2B6 mRNA Levels
CYP2B6 mRNA Levels
candidate compounds in order to avoid the potential for adverse drug-drug interactions during clinical ƒ The human pregnane X receptor (PXR), constitutive androstane receptor (CAR), and aryl hydrocarbon receptor (AhR) are known to regulate the gene expression of CYP3A4, CYP2B6, and CYP1A2 respectively ƒ Emerging evidence that CAR plays an important role in regulating xenobiotic metabolism provides the rationale for Results & Discussion
ƒ Conventionally, enzyme activity determinations are carried out as single probe substrate incubations and require CYP3A4 Midazolam 1'Hydroxylase Activity
CYP3A4 Midazolam 1'Hydroxylase Activity
We have successfully developed a marker substrate cocktail and a novel LC-MS/MS separate LC-MS/MS methods for each metabolite method to simultaneously assess ligand-mediated activation of PXR, CAR, and AhR via ƒ Here, we have developed a probe substrate incubation cocktail along with a novel LC-MS/MS method to determine cytochrome P450 induction activities of CYP3A4, CYP2B6, and CYP1A2 in human hepatocytes that gene expression of CYP3A4, CYP2B6, and CYP1A2, respectively, in cultures of fresh allow for simultaneous assessment of enzyme activity in a single, three-minute method CYP2B6 mRNA Levels
CYP2B6 mRNA Levels
The incorporation of all three probe substrates in a single thirty-minute incubation among Materials and Methods
four donor livers achieved similar results as compared with the conventional activity assay The average fold-induction of CYP3A4, CYP2B6, and CYP1A2 activity was comparable ƒ Materials
between the substrate cocktail assay and the conventional assay ƒ Four individual donors of fresh plated human hepatocytes were purchased from CellzDirect (Pittsboro, NC) ƒ Rifampicin, phenobarbital, CITCO, artemisinin, 3-methylcholanthrene, omeprazole, midazolam, bupropion, Induction of CYP3A4, CYP2B6, and CYP1A2 activity was supported further by mRNA Fig. 2A. Comparison of the substrate cocktail and single substrate conventional assays: average fold induction of CYP3A4 midazolam 1'-hydroxylase
phenacetin, and all other chemicals were purchased through Sigma-Aldrich (St. Louis, MO) activity by the positive controls, RIF (10 μM) and PB (1 mM) in primary cultures of human hepatocytes. Data are expressed as fold induction over VC (0.2% DMSO). Percent difference in average fold induction as measured by activity between the substrate cocktail and conventional method is 11% and 13% for Assay conditions
Fig. 3B. Comparison of the substrate cocktail and single substrate conventional assays: average fold induction of CYP2B6 mRNA levels by the positive
The use of this substrate cocktail method in monitoring cytochrome P450 induction ƒ Hepatocytes were cultured in 24-well collagen-coated tissue culture plates with Matrigel® overlay controls, CITCO (1 μM) and ART (50 μM) in primary cultures of human hepatocytes. Note the increase of CYP2B6 mRNA levels by ART despite its lack of ƒ Hepatocytes from each of four donors were treated for two consecutive days with rifampicin (10 μM), CYP3A4 mRNA Levels
effect on bupropion hydroxylase activity. Data are expressed as fold induction over VC (0.2% DMSO). Percent difference in the average fold induction as resulted in a higher throughput methodology as compared to the conventional measured by mRNA levels between the substrate cocktail and conventional method is 15% and 38% for CITCO and ART respectively. methodology, while conserving the inductive responses of prototypical inducers (1 mM), CITCO (1 μM), artemisinin (50 μM), 3-methylcholanthrene (1 μM), or omeprazole (50 μM); negative CYP3A4 mRNA Levels
CYP3A4 mRNA Levels
control cultures were treated with solvent alone (0.2% DMSO) CYP1A2 Phenacetin O-Deethylase Activity
ƒ The culture medium was exchanged every 24 hours with fresh supplemented culture medium containing vehicle or inducer over the two-day (48-hour) experiment CYP1A2 Phe nace tin O-De e thylase Activ ity
CYP1A2 Phe nacetin O-Dee thylase Activ ity
ƒ After two days of treatment, the media were aspirated and replaced with 10 mM Hepes-HBSS buffer supplemented either as a cocktail or as a single agent with 5 μM midazolam (CYP3A4), 50 μM bupropion References
(CYP2B6), and 100 μM phenacetin (CYP1A2) ƒ Cells were incubated at 37°C/5% CO for 30 minutes ƒ The reaction was stopped by the addition of 100 μL medium into 100 μL 25% acetonitrile and 75% water with 1 1. Faucette, S. et. al. JPET 320: 72-80 (2007)
μM labetalol included as internal standard 2. Faucette, S. et. al. JPET 317: 1200-1209 (2006)
ƒ LC-MS/MS conditions
ƒ LC-MS/MS system:
CYP3A4 mRNA Levels
CYP3A4 mRNA Levels
3. Unadkat, D. et. al. Biopharm. Drug Dispos. 28: 257-262 (2007)
CYP1A2 Phenacetin O-Deethylase Activity
CYP1A2 Phe nace tin O-De ethylase Activity
ƒ API4000 triple quadrupole mass spectrometer Analyte detection and separation:
ƒ Supelco Ascentis RP-Amide HPLC column (5 cm x 2.1 mm, 5 μm) ƒ Mobile Phase A: HPLC-grade water + 0.1% acetic acid Please e-mail inquires to Susan.Rhodes@arraybiopharma.com
ƒ Mobile Phase B: 25:75 (v/v) methanol:acetonitrile ƒ Total ion abundance of 6 specific multiple reaction monitoring (MRM) transitions in electrospray positive ionization (ESI+) mode was monitored Fig. 2B. Comparison of the substrate cocktail and single substrate conventional assays: average fold induction of CYP3A4 mRNA levels by the positive
controls, RIF (10 μM) and PB (1 mM) in primary cultures of human hepatocytes. Data are expressed as fold induction over VC (0.2% DMSO). Percent ƒ Total run time for a single injection was three minutes difference in the average fold induction as measured by mRNA levels between the substrate cocktail and conventional method is 33% and 30% for RIF and PB respectively. Fig. 4A. Comparison of the substrate cocktail and single substrate conventional assays: average fold induction of CYP1A2 phenacetin O-deethylase
activity by the positive controls, 3-MC (1 μM) and OME (50 μM) in primary cultures of human hepatocytes. Data are expressed as fold induction over VC
(0.2% DMSO). Percent difference in the average fold induction as measured by activity between the substrate cocktail and conventional method is 2% and
4% for 3-MC and OME respectively.

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