Site-directed mutation studies of human liver cytochrome P-450 isoenzymes in the CYP2C subfamily

Abstract

Evidence from human studies in vivo and in vitro strongly suggests that the methylhydroxylation of tolbutamide and the 4-hydroxylation of phenytoin, the major pathways in the elimination of these two drugs, are catalysed by the same cytochrome P-450 isoenzyme(s). In the present study we used site-directed mutagenesis and cDNA expression in COS cells to characterize in detail the kinetics of tolbutamide and phenytoin hydroxylations by seven CYP2C proteins (2C8, 2C9 and variants, and 2C10) in order to define the effects of small changes in amino acid sequences and the likely proteins responsible in the metabolism of these two drugs in man. Tolbutamide was hydroxylated to varying extents by all expressed cytochrome P-450 isoenzymes, although activity was much lower for the expressed 2C8 protein. While the apparent Km values for the 2C9/10 isoenzymes (71.6-131.7 microM) were comparable with the range of apparent Km values previously observed in human liver microsomes, the apparent Km for 2C8 (650.5 microM) was appreciably higher. The 2C8 enzyme also showed quite different sulphaphenazole inhibition characteristics. The 4-hydroxylation of phenytoin was also more efficiently catalysed by the 2C9/10 enzymes. These enzymes showed similarities in kinetics of phenytoin hydroxylation and sulphaphenazole inhibition compared with human liver phenytoin hydroxylase. Also of interest was the observation that, among the 2C9 variants, small differences in amino acid composition could appreciably affect both tolbutamide and phenytoin hydroxylations. The amino acid substitution Cys-144-->Arg increased both the rates of tolbutamide and phenytoin hydroxylations, while the Leu-359-->Ile change had a greater effect on phenytoin hydroxylation. We conclude that: (1) although 2C8 and 2C9/10 proteins metabolize tolbutamide. only 2C9/10 proteins play a major role in human liver; (2) 2C9/10 proteins also appear to be chiefly responsible for phenytoin hydroxylation; and (3) subtle differences in the amino acid composition of these 2C9/10 proteins can affect the functional specificities towards both tolbutamide and phenytoin.

Role of Cytochrome P450 2C8 in Drug Metabolism and Interactions

“…Additionally, CYP2C8 is involved, to a minor extent, in the metabolism of the sulfonylureas gliclazide, glyburide, and tolbutamide, the dipeptidyl peptidase 4 inhibitor sitagliptin, and the PPARa agonist sipoglitazar (Table 1; Relling et al, 1990;Srivastava et al, 1991;Veronese et al, 1993;Elliot et al, 2007;Vincent et al, 2007;Zharikova et al, 2009;Nishihara et al, 2012). Tolbutamide p-methyl hydroxylation has been used as a marker reaction for CYP2C8 activity in several in vitro studies.…”

Optimization of yeast‐expressed human liver cytochrome P450 3A4 catalytic activities by coexpressing NADPH‐cytochrome P450 reductase and cytochrome b5

“…A fruitful alternative consists of the expression of cloned cDNA, coding for individual forms of P450 enzymes, in an adequate heterologous system. Several human liver P450 from the subfamilies involved in xenobiotic oxidation have already been expressed in different systems, including recombinant simian virus 40 in COS cells and references therein; Romkes et al, 1991;Veronese et al, 1991), recombinant vaccinia viruses in human hepatoma Hep G2 cells (Aoyama et al, 1990a and1990b, and references therein) and recombinant plasmids in the yeast Succharomyces cerevisiae (Yasumori et al, 1989;Brian et al, 1989;Brian et al, 1990;Renaud et al, 1990;Eugster et al, 1990;Ching et al, 1991; Truan, G., Cullin, C., Reisdorf, P., Urban, P. and Pompon, D., unpublished results). P450 NF25 (CYP3A4) is important in pharmacology and toxicology, not only because it is probably the major form of human liver (Guengerich and Turvy, 1991) but also because it is involved in the metabolism of numerous widely used drugs such as nifedipine (Guengerich et al, 1986a), erythromycin and troleandomycin (Renaud et al, 1990 and references therein), quinidine (Guengerich et al, 1986b), cyclosporin A (Kronbach et al, 1988;Aoyama et al, 1989;Combalbert et al, 1989), 1 7a-ethynylestradiol (Guengerich, 1988), midazolam (Kronbach et al, 1989), lidocaine (Bargetzi et al, 1989;Imaoka et al, 1990), and diltiazem (Pichard et al, 1990).…”

CYP2C9 Amino Acid Residues Influencing Phenytoin Turnover and Metabolite Regio- and Stereochemistry

“…Before the present study, the only information available regarding specific CYP2C9 amino acids that may influence the interaction between the enzyme and phenytoin came from in vitro studies that evaluated the effect of CYP2C9 genetic variants on phenytoin turnover (Veronese et al, 1993;Rettie et al, 1999;Takanashi et al, 2000). These early studies demonstrated that expression of the CYP2C9*2 (R144C) and CYP2C9*3 (I359L) alleles resulted in diminished metabolism of phenytoin because of a reduction in K m , K cat , or a combination of these two kinetic effects.…”

“…However, beyond a single report that the I359L (CYP2C9.3) variant increases the percentage of (R)-p-HPPH produced (Takanashi et al, 2000), nothing is known about phenytoin's binding determinants within the active site of CYP2C9. This is especially surprising because one of the first studies employing sitedirected mutagenesis to investigate the activity of CYP2C9 genetic variants involved phenytoin as the probe drug (Veronese et al, 1993).…”

“…Phenytoin's primary metabolic pathway is hydroxylation by CYP2C9 to 5-(4-hydroxyphenyl)-5-phenylhydantoin (p-HPPH) (Veronese et al, 1993). Approximately 95% of the p-HPPH produced by CYP2C9 has the (S) configuration (Scheme 1).…”