Developmental changes in human liver CYP2D6 expression.

Within the human cytochrome P450 family, specific forms show developmental expression patterns that can affect drug clearance, efficacy, and safety. The objective of this study was to use dextromethorphan O-demethylase activity and quantitative Western blotting to identify CYP2D6 developmental expression patterns in a large (n = 222) and developmentally diverse set of pediatric liver samples. Immunodetectable levels of CYP2D6 protein determined for selected samples across all age categories showed a significant correlation with the corresponding dextromethorphan O-demethylase activity. Of gender, ethnicity, postmortem interval, and genotype, only increasing gestational age was associated with CYP2D6 activity and protein content in prenatal samples. In contrast, both age and genotype were associated with CYP2D6 expression in postnatal samples. CYP2D6 expression in liver samples from neonates less than 7 days of age was higher than that observed in first and second trimester samples, but not significantly higher than third trimester fetal samples. In contrast, expression in postnatal samples greater than 7 days of age was substantially higher than that for any earlier age category. Higher CYP2D6 expression also was observed in liver samples from Caucasians versus African Americans. Finally, using phenotype categories inferred from genotype, CYP2D6 activity was higher in postnatal samples predicted to be extensive or intermediate metabolizers versus poor metabolizers. These results suggest that age and genetic determinants of CYP2D6 expression constitute significant determinants of interindividual variability in CYP2D6-dependent metabolism during ontogeny.

Mechanism-based inactivation of cytochrome P450 2B6 by a novel terminal acetylene inhibitor.

N-(3,5-Dichloro-4-pyridyl)-3-(cyclopentyloxy)-4-methoxybenzamide (DCMB) is a known marker substrate for cytochrome p450 2B6. Based on the chemical template of DCMB, a novel terminal acetylene compound, N-(3,5-dichloro-4-pyridyl)-4-methoxy-3-(prop-2-ynyloxy)benzamide (TA) was synthesized and evaluated as a mechanism-based inactivator of p450 2B6. The pseudo first-order inactivation of expressed p450 2B6 by TA was both substrate and time-dependent. The kinetics of inhibition resulted in a maximal rate constant (k(inactivation)) of 0.09 min(-1) and an apparent K(I) of 5.1 microM. Incubation of expressed p450 2B6 with TA and NADPH resulted in a 68% loss in enzyme activity and a concurrent 62% loss in the formation of a reduced carbon monoxide complex, suggesting that heme destruction is the primary mode of enzyme inactivation. Enzyme inactivation of p450 2B6 was not reduced by the presence of 10 mM glutathione and was protected by incubation of excess DCMB with TA. The production of the carboxylic acid metabolite, N-(3,5-Dichloro-4-pyridyl)-3-(2-carboxyethoxy)-4-methoxybenzamide (TA-COOH), during the incubation of TA with 2B6 suggests that inactivation proceeds through a ketene intermediate. For 2B6 inactivation, the partition ratio was approximately 1.5 nmol TA-COOH formed/nmol P450 inactivated. Finally, TA was evaluated for mechanism-based inactivation of p450 3A4, 2C9, 2C19, 2D6, and 2E1 using human liver microsomes. In addition to 2B6, p450 2C forms were also found to be sensitive to TA-mediated inactivation, suggesting that subtle changes in the O-alkyl chain of the parent may be critical for the selectivity of enzyme inactivation.

Developmental expression of human hepatic CYP2C9 and CYP2C19.

The CYP2C subfamily is responsible for metabolizing many important drugs and accounts for about 20% of the cytochrome p450 in adult liver. To determine developmental expression patterns, liver microsomal CYP2C9 and -2C19 were measured (n = 237; ages, 8 weeks gestation-18 years) by Western blotting and with diclofenac or mephenytoin, respectively, as probe substrates. CYP2C9-specific content and catalytic activity were consistent with expression at 1 to 2% of mature values (i.e., specific content, 18.3 pmol/mg protein and n = 79; specific activity, 549.5 pmol/mg/min and n = 72) during the first trimester, with progressive increases during the second and third trimesters to levels approximately 30% of mature values. From birth to 5 months, CYP2C9 protein values varied 35-fold and were significantly higher than those observed during the late fetal period, with 51% of samples exhibiting values commensurate with mature levels. Less variable CYP2C9 protein and activity values were observed between 5 months and 18 years. CYP2C19 protein and catalytic activities that were 12 to 15% of mature values (i.e., specific content, 14.6 pmol/mg and n = 20; specific activity, 18.5 pmol/mg/min and n = 19) were observed as early as 8 weeks of gestation and were similar throughout the prenatal period. CYP2C19 expression did not change at birth, increased linearly over the first 5 postnatal months, and varied 21-fold from 5 months to 10 years. Adult CYP2C19 protein and activity values were observed in samples older than 10 years. The ontogeny of CYP2C9 and -2C19 were dissimilar among both fetal and 0- to 5-months postnatal samples, implying different developmental regulatory mechanisms.