Characterization of CYP2C19 and CYP2C9 from human liver: respective roles in microsomal tolbutamide, S-mephenytoin, and omeprazole hydroxylations.
Individuals with drug metabolism polymorphisms involving CYP2C enzymes exhibit deficient oxidation of important therapeutic agents, including S-mephenytoin, omeprazole, warfarin, tolbutamide, and nonsteroidal anti-inflammatory drugs. While recombinant CYP2C19 and CYP2C9 proteins expressed in yeast or Escherichia coli have been shown to oxidize these agents, the capacity of the corresponding native P450s isolated from human liver to do so is ill defined. To that end, we purified CYP2C19, CYP2C9, and CYP2C8 from human liver samples using conventional chromatographic techniques and examined their capacity to oxidize S-mephenytoin, omeprazole, and tolbutamide. Upon reconstitution, CYP2C19 metabolized S-mephenytoin and omeprazole at rates that were 11- and 8-fold higher, respectively, than those of intact liver microsomes, whereas neither CYP2C9 nor CYP2C8 displayed appreciable metabolic activity with these substrates. CYP2C19 also proved an efficient catalyst of tolbutamide metabolism, exhibiting a turnover rate similar to CYP2C9 preparations (2.0-6.4 vs 2.4-4.3 nmol hydroxytolbutamide formed/min/nmol P450). The kinetic parameters of CYP2C19-mediated tolbutamide hydroxylation (Km = 650 microM, Vmax = 3.71 min-1) somewhat resembled those of the CYP2C9-catalyzed reaction (Km = 178-407 microM, Vmax = 2.95-7.08 min-1). Polyclonal CYP2C19 antibodies markedly decreased S-mephenytoin 4'-hydroxylation (98% inhibition) and omeprazole 5-hydroxylation (85% inhibition) by human liver microsomes. CYP2C19 antibodies also potently inhibited (>90%) microsomal tolbutamide hydroxylation, which was similar to the inhibition (>85%) observed with antibodies to CYP2C9. Moreover, excellent correlations were found between immunoreactive CYP2C19 content, S-mephenytoin 4'-hydroxylase activity (r = 0.912; P < 0. 001), and omeprazole 5-hydroxylase activity (r = 0.906; P < 0.001) in liver samples from 13-17 different subjects. A significant relationship was likewise observed between microsomal tolbutamide hydroxylation and CYP2C9 content (r = 0.664; P < 0.02) but not with CYP2C19 content (r = 0.393; P = 0.184). Finally, immunoquantitation revealed that in these human liver samples, expression of CYP2C9 (88. 5 +/- 36 nmol/mg) was 5-fold higher than that of CYP2C19 (17.8 +/- 14 nmol/mg) and nearly 8-fold higher than that of CYP2C8 (11.5 +/- 12 nmol/mg). Our results, like those obtained with recombinant CYP2C enzymes, indicate that CYP2C19 is a primary determinant of S-mephenytoin 4'-hydroxylation and low-Km omeprazole 5-hydroxylation in human liver. Despite its tolbutamide hydroxylase activity, the low levels of hepatic CYP2C19 expression (relative to CYP2C9) may preclude an important role for this enzyme in hepatic tolbutamide metabolism and any polymorphisms thereof.
Lasker
J M
JM
Department of Biochemistry, Mount Sinai School of Medicine, New York, New York 10029, USA.
Wester
M R
MR
Aramsombatdee
E
E
Raucy
J L
JL
D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000378:metabolism; D001189:Aryl Hydrocarbon Hydroxylases; D002848:Chromatography, DEAE-Cellulose; D002852:Chromatography, Ion Exchange; D065731:Cytochrome P-450 CYP2C19; D065727:Cytochrome P-450 CYP2C8; D065729:Cytochrome P-450 CYP2C9; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006900:Hydroxylation; D007700:Kinetics; D008617:Mephenytoin / Q000493:pharmacokinetics*; D008862:Microsomes, Liver / Q000201:enzymology*; D006899:Mixed Function Oxygenases / Q000302:isolation & purification / Q000378:metabolism*; D009853:Omeprazole / Q000493:pharmacokinetics*; D039181:Steroid 16-alpha-Hydroxylase; D013250:Steroid Hydroxylases / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity; D014044:Tolbutamide / Q000493:pharmacokinetics*; D014462:Ultrafiltration
D000894:Anti-Inflammatory Agents, Non-Steroidal; D003577:Cytochrome P-450 Enzyme System; D014044:Tolbutamide; D006899:Mixed Function Oxygenases; D013250:Steroid Hydroxylases; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases; C045793:CYP2C19 protein, human; C450259:CYP2C8 protein, human; D065731:Cytochrome P-450 CYP2C19; D065727:Cytochrome P-450 CYP2C8; D039181:Steroid 16-alpha-Hydroxylase; D009853:Omeprazole; D008617:Mephenytoin