Evidence That CYP2C19 is the Major (S)-Mephenytoin 4'-Hydroxylase in Humans

Goldstein, Joyce A.; Faletto, Michael B.; Romkes-Sparks, M; Sullivan, Theodore; Kitareewan, Sutisak; Raucy, Judy L.; Lasker, Jerome M.; Ghanayem, Burhan I.

doi:10.1021/bi00173a017

Cited by 319 publications

(154 citation statements)

References 42 publications

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“…mg protein-' (in the case of CYP 2C8), despite the use of identical expression systems (Table 1). Since these cytochromes P-450 have been described to hydroxylate tolbutamide (Brian et al, 1989;Relling et al, 1990;Goldstein, 1994b), this activity was chosen as a control for the metabolic competence of our microsomal fractions. All the expressed cytochromes P-450 2C were able to hydroxylate tolbutamide under standard incubation conditions (Table l), while no activity was detected in microsomes from control yeast.…”

Section: Resultsmentioning

confidence: 99%

“…Saccharornyces cerevisiae strain 334 was used for expression. Details of the protocols for construction of vectors and yeast transformation have been described elsewhere (Kaminsky et al, 1992;Goldstein et al, 1994b).…”

Section: Methodsmentioning

confidence: 99%

“…Very recently, it has been shown that cytochrome P-450 2C19 (CUP 2C19) is the isozyme responsible for S-mephenytoin oxidation (Goldstein et al, 1994b), but only limited knowledge exists about cytochrome P-450 2C8 (CUP 2C8) and cytochrome (Goldstein et al, 1994a;Guengerich, 199.5;Correia, 1995).…”

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confidence: 99%

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Oxidation of Tienilic Acid by Human Yeast‐Expressed Cytochromes P‐450 2C8, 2C9, 2C18 and 2C19

Jean

Lopez-Garcia

Dansette

et al. 1996

European Journal of Biochemistry

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Oxidation of tienilic acid by human cytochromes P-450 (CUP) 2C9,2C18, 2C8 and 2C19 was studied using recombinant enzymes expressed in yeast. CYP 2C9 was the best catalyst for 5-hydroxylation of tienilic acid (K, = 5 2 1 pM, k,,, = 1.7 +-0.2 min-I), 30-fold more potent in terms of k,,,/K,,, than CYP 2C18 (K, = 150?15 pM, k,, = 1.850.2min-I) and 300-fold more potent than CYP 2C8 ( K , = 145 +-15 pM, k,,, = 0.2 +-0.1 min-I). CYP 2C19 was unable to catalyze this hydroxylation under our experimental conditions. During this study, a marked effect of the ionic strength on the activities (hydroxylations of tienilic acid and tolbutamide) of these cytochromes P-450 expressed in the yeast strain 334 was observed. The effect was particularly great in the case of CYP 2C18, with a tenfold decrease of activity upon increasing ionic strength from 0.02 to 0.1. Specific-covalent binding of tienilic acid metabolites to cytochrome P-450 (incubations in the presence of 5 mM glutathione) was markedly higher upon tienilic acid oxidation by CYP 2C9 than by CYP 2C18 and CYP 2C8. Mechanism-based inactivation of cytochrome P-450 during tienilic acid oxidation was observed in the case of CYP 2C9 but was not detectable with CYP 2C18 and CYP 2C8. Tienilic acid thus appears to be a mechanism-based inhibitor specific for CYP 2C9 in human liver. Experiments performed with human liver microsomes confirmed that tienilic acid 5-hydroxylase underwent a time-dependent inactivation (apparent tllZ = 10 +-5 min) during 5-hydroxylation of tienilic acid.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Oxidation of Tienilic Acid by Human Yeast‐Expressed Cytochromes P‐450 2C8, 2C9, 2C18 and 2C19

Jean

Lopez-Garcia

Dansette

et al. 1996

European Journal of Biochemistry

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“…Only extensive metabolizers (EM), expressing functional CYP2C19, are able to hydroxylate (S)-mephenytoin to (S)-4´-hydroxymephenytoin, which is subsequently conjugated almost completely to the (S)-4´-hydroxymephenytoin glucuronide and excreted renally. 16,17 In CYP2C19 deficient poor metabolizers (PM), (S)-mephenytoin is biotransformed via slow N-demethylation, which in vitro is preferentially mediated by CYP2B6 (with minor contribution of CYP2C9), resulting in the formation of (S)-nirvanol. [18][19][20] (R)-mephenytoin is preferably demethylated to (R)-nirvanol and hydroxylated only to a small extent.…”

Section: Introductionmentioning

confidence: 99%

Assessment of urinary mephenytoin metrics to phenotype for CYP2C19 and CYP2B6 activity

Klaassen

Jetter²,

Tomalik-Scharte³

et al. 2007

Eur J Clin Pharmacol

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OBJECTIVES: (S)-Mephenytoin is selectively metabolised to (S)-4'-hydroxymephenytoin by CYP2C19. The urinary excretion of 4'-hydroxymephenytoin reflects the activity of individual enzymes. We evaluated fractioned urinary collection and beta-glucuronidase pre-treatment in order to determine the optimal CYP2C19 metrics. We also assessed whether urinary excretion of N-desmethylmephenytoin (nirvanol) might be a useful CYP2B6 metric in in vivo studies. METHODS: A 50-mg dose of mephenytoin was administered to 52 volunteers as a component of phenotyping cocktails in four separate studies. Urine was collected up to 166 h post-dose. Urinary excretion of 4'-hydroxymephenytoin and nirvanol was quantified by liquid chromatography-tandem mass spectrometry, and common CYP2C19 and CYP2B6 genotypes were determined. RESULTS: Cumulative excretion of 4'-hydroxymephenytoin in urine with beta-glucuronidase treatment collected from before mephenytoin administration up to 12-16 h thereafter showed the greatest difference between CYP2C19 genotypes and the lowest intra-individual variability (7%). Renal elimination of nirvanol was highest for a *4/*4 individual and lowest for individuals carrying the *5/*5 and *1/*7 genotype, but lasted for several weeks, thus making its use in cross-over studies difficult. CONCLUSION: Cumulative urinary excretion of 4'-hydroxymephenytoin 0-12 h post-administration is a sensitive and reproducible metric of CYP2C19 activity, enabling the effect of a drug on CYP2C19 to be assessed in a small sample size of n=6 volunteers. While nirvanol excretion may reflect CYP2B6 activity in vivo, it is not useful for CYP2B6 phenotyping. We evaluated fractioned urinary collection and β-glucuronidase pre-treatment to assess the optimal CYP2C19 metric. Furthermore, we addressed whether urinary excretion of N-desmethylmephenytoin (nirvanol) might be a useful CYP2B6 metric in vivo.Methods: 50 mg of mephenytoin was administered to 52 volunteers as part of phenotyping cocktails in four separate studies. Urine was collected up to 166 hours postdose. Urinary excretion of 4'-hydroxymephenytoin and nirvanol was quantified by LC-MS/MS, and common CYP2C19 and CYP2B6 genotypes were determined.Results: Cumulative excretion of 4'-hydroxymephenytoin in urine with β-glucuronidase treatment collected from before mephenytoin administration up to 12-16 hours thereafter showed the greatest difference between CYP2C19 genotypes and the lowest intraindividual variability (7 %). Renal elimination of nirvanol was highest for a *4/*4 individual and lowest for individuals carrying the *5/*5 and *1/*7 genotype, but lasted for several weeks, thus making its use in cross-over studies difficult. Conclusion:Cumulative urinary excretion of 4'-hydroxymephenytoin 0-12 hours is a sensitive and reproducible metric of CYP2C19 activity, allowing a small sample size of n=6 volunteers to assess the effect of a drug on CYP2C19. While nirvanol excretion may reflect CYP2B6 activity in vivo, it is not useful for CYP2B6 phenotyping.3

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“…Extensive metabolizer (EM) and poor metabolizer (PM) polymorphism for mephenytoin [3,4] is well known and has been studied extensively. It has been shown that CYP2C19 is a candidate for mephenytoin 4'-hydroxylation [5,6]. However, the role of CYP2C18 and the relationships between the other CYP2C subfamily members and the CYP2C18 gene have not been clearly demonstrated.…”

Section: Introductionmentioning

confidence: 99%

Genetic analysis of the cytochrome P‐450IIC18 (CYP2C18) gene and a novel member of the CYP2C subfamily

et al. 1996

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The CYP2CI8 gene was investigated in order to characterize its molecular basis in the CYP2C subfamily. A mutation of the CYP2C18 gene was identified at the 5'-flanking region of the gene, which could be detected by digestion with DdeI. The allele frequency of the mutant CYP2C18 gene was 21.4%. Genotypes of the polymorphic DdeI site of the CYP2C18 gene were found to be completely consistent with that of the polymorphic CYP2C19 gene (the ml mutant). The CYP2CI8 and CYP2CI9 genes were suggested to be linked and located close together on the chromosome. Clones containing the 5'-flanking region of a member of the CYP2C subfamily were obtained from the PCR products from human genomic DNA. The nucleotide sequence of the clone proved to be 90.5% identical to the corresponding region of the CYP2C18 gene. This is very likely to be a novel member of the CYP2C subfamily.

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Evidence That CYP2C19 is the Major (S)-Mephenytoin 4'-Hydroxylase in Humans

Cited by 319 publications

References 42 publications

Oxidation of Tienilic Acid by Human Yeast‐Expressed Cytochromes P‐450 2C8, 2C9, 2C18 and 2C19

Oxidation of Tienilic Acid by Human Yeast‐Expressed Cytochromes P‐450 2C8, 2C9, 2C18 and 2C19

Assessment of urinary mephenytoin metrics to phenotype for CYP2C19 and CYP2B6 activity

Genetic analysis of the cytochrome P‐450IIC18 (CYP2C18) gene and a novel member of the CYP2C subfamily

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