Developmental Expression of CYP2B6: A Comprehensive Analysis of mRNA Expression, Protein Content and Bupropion Hydroxylase Activity and the Impact of Genetic Variation

Abstract

Although CYP2B6 catalyzes the biotransformation of many drugs used clinically for children and adults, information regarding the effects of development on CYP2B6 expression and activity are scarce. Utilizing a large panel of human liver samples (201 donors: 24 fetal, 141 pediatric, and 36 adult), we quantified CYP2B6 mRNA and protein expression levels, characterized CYP2B6 (bupropion hydroxylase) activity in human liver microsomes (HLMs), and performed an extensive genotype analysis to differentiate CYP2B6 haplotypes such that the impact of genetic variation on these parameters could be assessed. Fetal livers contained extremely low levels of CYP2B6 mRNA relative to postnatal samples and fetal HLMs did not appear to catalyze bupropion hydroxylation; however, fetal CYP2B6 protein levels were not significantly different from postnatal levels. Considerable interindividual variation in CYP2B6 mRNA expression, protein levels, and activity was observed in postnatal HLMs (mRNA, ∼40,000-fold; protein, ∼300-fold; activity, ∼600-fold). The extremely wide range of interindividual variability in CYP2B6 expression and activity was significantly associated with age (P < 0.01) following log transformation of the data. Our data suggest that CYP2B6 activity appears as early as the first day of life, increases through infancy, and by 1 year of age, CYP2B6 levels and activity may approach those of adults. Surprisingly, CYP2B6 interindividual variability was not significantly associated with genetic variation in CYP2B6, nor was it associated with differences in gender or ethnicity, suggesting that factors other than these are largely responsible for the wide range of variability in CYP2B6 expression and activity observed among a large group of individuals/samples.

Age-Dependent Absolute Abundance of Hepatic Carboxylesterases (CES1 and CES2) by LC-MS/MS Proteomics: Application to PBPK Modeling of Oseltamivir In Vivo Pharmacokinetics in Infants

“…The samples were stratified based on the following age categories: neonatal (0-27 days; n = 4), infancy (28-364 days; n = 17), early childhood (1 to ,6 years; n = 30), middle childhood (6 to ,12 years; n = 37), adolescence (12-18 years; n = 48), and adulthood (.18 years; n = 35) (Williams et al, 2012). The microsomal and cytosolic fractions from all samples were prepared using established protocols (Gibbs et al, 1996;Shirasaka et al, 2015;Pearce et al, 2016). Detailed donor demographic information is provided in Supplemental Table 2.…”

Ontogeny of Phase I Metabolism of Drugs

“…When assessing the consecutive steps from CYP gene to protein activity, mRNA expression, protein expression, and protein activity are the consecutive potential sources of information on ontogeny. Similar to mRNA, protein quantification (either by immunoblot or proteomics) does not necessary correlates with protein activity, as has nicely been illustrated for CYP2B6 ontogeny . While the fetal liver samples expressed protein concentrations (pmol CYP2B6/mg protein) to a similar extent as neonatal hepatic samples, no related protein activity (marker: bupropion hydroxylation) was observed in the fetal samples …”

Characterization of Atomoxetine Biotransformation and Implications for Development of PBPK Models for Dose Individualization in Children

“…Heterologously expressed cytochrome P450 enzymes (Cypex Bactosomes Dundee, United Kingdom), adult and pediatric single-donor HLMs, and pooled human HLMs (n = 200 donors) were purchased from XenoTech LLC (Lenexa, KS). HLMs prepared from liver samples from pediatric donors were obtained through the Liver Tissue Cell Distribution System (Minneapolis, MN; Pittsburgh, PA), National Institutes of Health #N01-DK-7-0004/HHSN267200700004C, #HHSN276201200017C, the University of Maryland Brain and Tissue Bank for Developmental Disorders, and XenoTech LLC, and have been described elsewhere (Pearce et al, 2015).…”