Simultaneous assessment of cytochrome P450 activity in cultured human hepatocytes for compound-mediated induction of CYP3A4, CYP2B6, and CYP1A2.
BACKGROUND
The human nuclear receptors 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, 3A4, 2B6, and 1A2, respectively. In conventional CYP induction studies, the activity of each CYP enzyme is assessed in a separate incubation with the appropriate marker substrate. The objective of this study was to assess, simultaneously, the induction of CYP3A4, CYP2B6, and CYP1A2 activity in cultured human hepatocytes treated with various prototypical ligands of PXR, CAR, and AhR by utilizing an optimized substrate cocktail, as well as a rapid, sensitive liquid chromatography-mass spectrometry method.


METHODS
To evaluate the xenobiotic-mediated induction of hepatocellular gene expression, the prototypical inducers rifampicin (10 μM) and phenobarbital (1 mM) were used for CYP3A4, CITCO (1 μM) and artemisinin (50 μM) were used for CYP2B6, and 3-methylcholanthrene (1 μM) and omeprazole (50 μM) were utilized for induction of CYP1A2. Primary human hepatocytes were treated with each compound for 48h, followed by a 30-min 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; 100μM). The assessment of CYP activity was performed with a rapid, sensitive liquid chromatography-tandem mass spectrometry method which simultaneously assessed activity of CYP3A4, CYP2B6, and CYP1A2 in a single 3-min method by examining the formation of the probe substrate metabolites, 1'-hydroxymidazolam, hydroxybupropion, and acetaminophen, respectively.


RESULTS
The average fold-induction of CYP3A4, CYP2B6, and CYP1A2 activity was comparable between the cocktail and the conventional assay.


CONCLUSIONS
The combination of three marker substrates in a single 30-min incubation, in addition to a rapid, sensitive LC-MS/MS method, resulted in an efficient and robust method for assessing cytochrome P450 induction as compared to the conventional methodology.
Array BioPharma Inc., 3200 Walnut Street, Boulder, Colorado 80301, USA. Susan.Rhodes@arraybiopharma.com
Susan P
SP
Rhodes
Jennifer N
JN
Otten
Gary P
GP
Hingorani
Dylan P
DP
Hartley
Ronald B
RB
Franklin
D001189:Aryl Hydrocarbon Hydroxylases / Q000096:biosynthesis*; D018929:Cell Culture Techniques; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D000090702:Constitutive Androstane Receptor; D019388:Cytochrome P-450 CYP1A2 / Q000096:biosynthesis*; D065702:Cytochrome P-450 CYP2B6; D051544:Cytochrome P-450 CYP3A / Q000096:biosynthesis*; D055808:Drug Discovery / Q000379:methods*; D004790:Enzyme Induction; D022781:Hepatocytes / Q000187:drug effects / Q000201:enzymology*; D006801:Humans; D008024:Ligands; D010089:Oxidoreductases, N-Demethylating / Q000096:biosynthesis*; D000077297:Pregnane X Receptor; D018336:Receptors, Aryl Hydrocarbon / Q000378:metabolism; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D011987:Receptors, Steroid / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry; D015262:Xenobiotics / Q000494:pharmacology
D000090702:Constitutive Androstane Receptor; D008024:Ligands; D000077297:Pregnane X Receptor; D018336:Receptors, Aryl Hydrocarbon; D018160:Receptors, Cytoplasmic and Nuclear; D011987:Receptors, Steroid; D015262:Xenobiotics; D001189:Aryl Hydrocarbon Hydroxylases; C504573:CYP1A2 protein, human; C585599:CYP2B6 protein, human; D019388:Cytochrome P-450 CYP1A2; D065702:Cytochrome P-450 CYP2B6; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D010089:Oxidoreductases, N-Demethylating