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

Abstract

Atomoxetine (ATX) is a second-line nonstimulant medication used to control symptoms of attention deficit hyperactivity disorder (ADHD). Inconsistent therapeutic efficacy has been reported with ATX, which may be related to variable CYP2D6-mediated drug clearance. We characterized ATX metabolism in a panel of human liver samples as a basis for a bottom-up PBPK model to aid in ATX exposure prediction and control. Km, Vmax, and Clint values in pooled human liver microsomes (HLMs) were 2.4 µM, 479 pmol/min/mg protein, and 202 µl/min/mg protein, respectively. Mean population values of kinetic parameters are not adequate to describe variability in a population, given that Km, Vmax, and Clint values from single-donor HLMs ranged from 0.93 to 79.2 µM, 20.0 to 1600 pmol/min/mg protein, and 0.3 to 936 µl/min/mg protein. All kinetic parameters were calculated from 4-hydroxyatomoxetine (4-OH-ATX) formation. CYP2E1 and CYP3A contributed to 4-OH-ATX formation in livers with CYP2D6 intermediate and poor metabolizer status. In HLMs with lower CYP2D6 activity levels, 2-hydroxymethylatomoxetine (2-CH2OH-ATX) formation became a more predominant pathway of metabolism, which appeared to be catalyzed by CYP2B6. ATX biotransformation at clinically relevant plasma concentrations was characterized in a panel of pediatric HLM (n = 116) samples by evaluating primary metabolites. Competing pathways of ATX metabolism [N-desmethylatomoxetine (NDM-ATX) and 2-CH2OH-ATX formation] had increasing importance in livers with lesser CYP2D6 activity, but, overall ATX clearance was still compromised. Modeling ATX exposure to individualize therapy would require comprehensive knowledge of factors that affect CYP2D6 activity as well as an understanding of competing pathways, particularly for individuals with lower CYP2D6 activity.

Fig. 1.

Scheme of ATX metabolism in vivo. Figure reproduced and modified from Mattiuz et al. (2003) with permission from Drug Metabolism and Disposition.

Fig. 2.

Michaelis-Menten plots of 4-OH-ATX formation in 21 single-donor HLMs of varying CYP2D6 phenotype. Extensive and ultrarapid metabolizers are plotted in (A), whereas intermediate and poor metabolizers are plotted in (B). Extensive metabolizers were individuals with a CYP2D6 AS = 1–2. Extensive metabolizers were subcategorized as EM1 (green plots) or EM2 (blue plots), depending on CYP2D6 AS. Ultra-rapid metabolizers (UMs) were individuals with an AS > 2 (purple plots). Intermediate metabolizers (IMs) are individuals with a CYP2D6 AS = 0.5, which indicates one copy of a CYP2D6 allele with compromised activity (yellow plot). Poor metabolizers (PMs) have two no function CYP2D6 alleles (red plots). (PM, n = 2; IM, n = 1; EM1, n = 8, EM2 = 8, UM, n = 2).

Fig. 3.

Linear regression analysis evaluating ATX intrinsic clearance (C_lint) and CYP2D6 activity measured by dextromethorphan (DM) O-demethylation activity. Cl_int was calculated by taking the ratio of V_max/K_m from data obtained from 4-OH-ATX formation. Data from CYP2D6 phenotypic activity was provided by Xenotech, LLC. Data from 20 HLMs was included in this analysis, because one PM HLM preparation did not produce a reliable estimate of K_m (r² = 0.74, ***p < 0.0001). UM = purple dots, EM2 = blue dots, EM1 = green dots, IM = yellow dot, PM = red dot.

Fig. 4.

Formation rate of 4-OH-ATX (A), NDM-ATX (B), and 2-CH₂OH-ATX (expressed as NDM-ATX equivalents) (C) in heterologously expressed CYP enzymes incubated with ATX (1, 3, and 10 μM). Only those CYPs isoforms that displayed a concentration-dependent increase of metabolite formation that was greater than the limit of quantitation or the rate of formation by the negative control (vector) are shown. All experiments were conducted in duplicate.

Fig. 5.

Relative percentages of 4-OH-ATX and 2-CH₂OH-ATX formed when ATX coincubated with chemical inhibitors of specific CYP isoforms. Relative formation of 4-OH-ATX are reported with pooled HLMs (A) as well as adult single-donor HLMs with CYP2D6 AS = 0.5 (B) and 0 (C). Relative formation of 2-CH₂OH-ATX are reported with adult single donor HLMs with CYP2D6 AS = 0.5 (D) and 0 (E). All HLM preparations were obtained from Xenotech, LLC. All experiments were conducted in triplicate.

Fig. 6.

ATX metabolite formation stratified by CYP2D6, CYP2C19, and CYP2B6 genotype. These three CYPs are the most important contributors to 4-OH-ATX formation. Data from the 10 μM ATX pediatric HLM screen were used for these analyses. 4-OH-ATX formation rate is stratified by CYP2D6 activity score (A), NDM-ATX formation rate by CYP2C19 genotype (B), and 2-CH₂OH-ATX formation rate (expressed as NDM-ATX equivalents) by CYP2B6 genotype (C). Livers genotyped as CYP2C19 *2, *3, or *4 were collated as nonfunctional alleles (i.e., *0) because all have been reported to result in loss of activity. Because of the numerous number of allelic variants detected for CYP2B6, genotype groups were categorized as homozygous wild-type (*1/*1), heterozygous (*1/*X), and homozygous variant (*X/*X). All experiments were conducted in duplicate.

Fig. 7.

Formation of 4-OH-ATX (A), NDM-ATX (B), and 2-CH2OH-ATX (expressed as NDM-ATX equivalents) (C) as a function of age. This figure was generated using data from the pediatric HLM screen with 10 µM screen. A total of 116 individual livers was evaluated in duplicate 4-OH-ATX.