Age‐dependent Expression Analysis of Major Drug Metabolizing Enzymes in Human Liver
Authors: Mehrotra Aanchal , Boberg Mikael , Vrana Marc , Gaedigk Andrea , Pearce Robin E , Leeder Steven , Prasad Bhagwat 
Published: November 2023

Empirical body weight or body surface area normalized approaches to determine pediatric dosing from adults can result in undesired pharmacokinetics due to differential expression of drug metabolizing enzymes (DMEs) in children vs. adults. This can lead to drug toxicity or lack of efficacy. Although there is limited expression data available for some DMEs in children, the data is mostly based on non‐selective methods. Therefore, the purpose of this study is to quantify protein expression of 65 phase I and phase II DMEs in a large cohort of human liver samples using LC‐MS/MS quantitative proteomics.

LC‐MS/MS quantifiable surrogate peptides for highly abundant human DMEs were selected and quantitative proteomic methods were developed and validated. Microsomal and cytosolic fractions were obtained from 129 pediatric and 45 adult human liver samples and processed for LC‐MS/MS analysis as previously described [1–3]. Utilizing 1, 2 or 3 unique surrogate peptides per protein, 13 DMEs have been quantified to date in 44 pediatric (13 neonates+infants (<1yr); 20 children (1 to <12yrs); 11 adolescents (12 to <18yrs)) and 7 adult (>18 yrs) liver samples under optimum conditions of reproducibility. Each sample was digested and independently analyzed on three different days to control for inter‐day analytical variability. Heavy peptide internal standards, an external protein internal standard and pooled adult microsomes or cytosol were used as quality control measures. Protein expression data of DMEs from various age groups were compared using Kruskal‐Wallis test with Dunn's multiple comparison test.

Of the 13 proteins analyzed, CYP2A6, CYP3A4, CYP2C9, CYP2E1, CYP reductase, UGT1A6, UGT2B7, UGT2B15, and CES1 showed a significant (p value < 0.05), 2 – 10 fold increase in protein expression from neonates to adults. CYP3A7 exhibited a sharp >60‐fold decrease in protein expression within the first year of life. EPHX2 protein expression increased ~50% when neonates were compared to adolescents and significantly decreased in adults. For EPHX1 and CYP2J2 no changes were noted. Associations were observed between protein levels of CYP2A6 and CYP3A4 (r2 = 0.74), UGT2B7 and UGT2B15 (r2 = 0.71), and UGT2B15/UGT2B7 and UGT1A6 (r2 = 0.37 and 0.33, respectively), suggesting that expression of these proteins are regulated by similar mechanisms in vivo. Complete data acquisition of 65 DMEs from 129 pediatric and 45 adult samples are in progress and will be presented. Whether genetic polymorphism is a confounding factor in determination of age‐dependent variability, we are also conduct genotyping using DMET chip and PGRN‐seq arrays on these samples.

Quantification of DME protein expression levels is crucial for the development of physiologically based pharmacokinetic (PBPK) models to predict drug disposition in children. The protein expression data obtained in this investigation will also be invaluable for future studies to determine fundamental genetic and/or epigenetic mechanisms that drive age‐dependent and interindividual variability in the expression of these proteins.

Support or Funding Information

Funded by NIH/NICHD1R01HD081299‐01A1.