Regulation of cytochrome P450 2C9 expression in primary cultures of human hepatocytes

Abstract

Cytochrome P450 2C9 (CYP2C9) expression is regulated by multiple nuclear receptors including the constitutive androstane receptor (CAR) and pregnane X receptor (PXR). We compared coregulation of CYP2C9 with CYP2B6 and CYP3A4, prototypical target genes for human CAR and PXR using human hepatocyte cultures treated for three days with the PXR activators clotrimazole, rifampin, and ritonavir; the CAR/PXR activator phenobarbital (PB); and the CAR-selective agonists CITCO, (6-(4-chlorophenyl)imidazo[2,1-beta][1,3]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl)oxime) and phenytoin. Clotrimazole, rifampin, ritonavir, phenytoin, and phenobarbital induced CYP2C9 consistent with previous findings for CYP3A4. We observed EC(50) values of 519 microM (phenobarbital), 11 microM (phenytoin), and 0.75 microM (rifampin), similar to those for CYP3A4 induction. Avasimibe, a potent PXR activator, produced nearly identical concentration-dependent CYP2C9 and CYP3A4 activity profiles and EC(50) values. In 17 donors, rifampin increased mean basal CYP2C9 activity from 59 +/- 43 to 143 +/- 68 pmol/mg protein/min; fold induction ranged from 1.4- to 6.4-fold. Enzyme activity and mRNA measurements after rifampin, CITCO and PB treatment demonstrated potency and efficacy consistent with CYP2C9 regulation being analogous to CYP3A4 rather than CYP2B6. We demonstrate that hepatic CYP2C9 is differentially regulated by agonists of CAR and PXR, and despite sharing common regulatory mechanisms with CYP3A4 and CYP2B6; this enzyme exhibits an induction profile more closely aligned with that of CYP3A4.

Application of Micropatterned Cocultured Hepatocytes to Evaluate the Inductive Potential and Degradation Rate of Major Xenobiotic Metabolizing Enzymes

“…The HepatoPac cultures show a higher and consistent response for both of the CYP2C genes compared with the monocultures. In the HepatoPac model we observed that a 3.5-fold change in mRNA and up to a 7-fold change in activity with phenobarbital, a dual PXR/CAR transactivator (Sahi et al, 2009). Phenobarbital showed the highest inductive response for CYP2C9 expression and activity in the HepatoPac model, perhaps owing to its dual activation of PXR and CAR.…”

Complex Drug Interactions of HIV Protease Inhibitors 2: In Vivo Induction and In Vitro to In Vivo Correlation of Induction of Cytochrome P450 1A2, 2B6, and 2C9 by Ritonavir or Nelfinavir

“…Note the log scale for control CYP2B6 activity, whereas CYP1A2 and CYP2C9 are displayed on a linear scale. CYP2C9 expression is regulated, at least in part, by PXR (Sahi et al, 2009). A correlation analysis of fold induction of CYP1A2, CYP2C9, CYP2B6, and CYP3A showed no significant correlations (R 2 Յ 0.1; data not shown), implying minimal coregulation of these enzymes or an insufficient dynamic range to discern a correlation.…”

CYP2C9*1BPromoter Polymorphisms, in Linkage withCYP2C19*2, Affect Phenytoin Autoinduction of Clearance and Maintenance Dose

“…Thus, it is possible that the extent of drug interactions after cotreatment with known PXR/CAR activators and CYP2C9 inducers such as rifampin or phenytoin or barbiturates might differ between individuals with the CYP2C9*1B versus other CYP2C9*1 genotypes. Although CYP2C9 is not highly induced in human hepatocytes by phenytoin (Sahi et al, 2009) (Fig. 7), hepatic CYP2C9 is also induced by rifampin, phenobarbital, and avasimibe (Chen et al, 2004;Sahi et al, 2009).…”

“…Although CYP2C9 is not highly induced in human hepatocytes by phenytoin (Sahi et al, 2009) (Fig. 7), hepatic CYP2C9 is also induced by rifampin, phenobarbital, and avasimibe (Chen et al, 2004;Sahi et al, 2009). Moreover, CYP2C9 is among the three most highly induced CYPs in human duodenum in patients treated with clinical doses of oral rifampin (E. Schuetz, unpublished observation).…”