Dissecting Parameters Contributing to the Underprediction of Aldehyde Oxidase-Mediated Metabolic Clearance of Drugs

Abstract

We investigated the effect of variability and instability in aldehyde oxidase (AO) content and activity on scaling of in vitro metabolism data. AO content and activity in human liver cytosol (HLC) and five recombinant human AO preparations (rAO) were determined using targeted proteomics and carbazeran oxidation assay, respectively. AO content was highly variable as indicated by the relative expression factor (REF, i.e., HLC to rAO content) ranging from 0.001-1.7 across different in vitro systems. The activity of AO in HLC degrades at a 10-fold higher rate in the presence of the substrate as compared to the activity performed after preincubation without substrate. To scale the metabolic activity from rAO to HLC, a protein-normalized activity factor (pnAF) was proposed wherein the activity was corrected by AO content, which revealed upto 6-fold higher AO activity in HLC versus rAO systems. A similar value of pnAF was observed for another substrate, ripasudil. Physiologically-based pharmacokinetic (PBPK) modeling revealed a significant additional clearance (CL; 66%), which allowed successful prediction of in vivo CL of four other substrates, i.e., O-benzyl guanine, BIBX1382, zaleplon and zoniporide. For carbazeran, the metabolite identification study showed that the direct glucuronidation may be contributing to around 12% elimination. Taken together, this study identified differential protein content, instability of in vitro activity, role of additional AO clearance and unaccounted metabolic pathways as plausible reasons for the underprediction of AO mediated drug metabolism. Consideration of these factors and integration of REF and pnAF in PBPK models will allow better prediction of AO metabolism.

Ontogeny of Human Liver Aldehyde Oxidase: Developmental Changes and Implications for Drug Metabolism

“…Therefore, an incubation time of 5 min was used for the activity assays in the HLC samples, whereas an incubation time of 10 min was used for the analysis of HH samples to allow equilibration of the substrate in the cell system. Consistent with our previous data, 34 the kinetics of 4-oxo carbazeran formation in HLC exhibited a Michaelis−Menten constant (K m ) of 7 μM (Figure S1). Accordingly, the activity assays in HLC and HH were carried out at 25 μM (∼5 times K m ).…”

“…The rate of formation of 4-oxo carbazeran showed a time-dependent decline in HLC incubation (Figure 1a), consistent with a previous observation. 34 Similarly, the rate of 4-oxo carbazeran formation in HH incubations was significantly higher at 10 min than at 30 min (Figure 1b). Therefore, an incubation time of 5 min was used for the activity assays in the HLC samples, whereas an incubation time of 10 min was used for the analysis of HH samples to allow equilibration of the substrate in the cell system.…”

“…There are several challenges associated with in vitro characterization of AO enzymes, including loss of activity during the processes of isolation, homogenization, and long-term storage. , The large interindividual variability in AO activity in both HLC and HH is an additional confounding factor. In this study, pediatric liver tissue for HLC preparation was procured from multiple sources, which may have caused a decline in AO activity due to unknown factors related to different handling methods, resulting in a poor correlation between AO content and activity.…”

“…An optimized trypsin digestion protocol and targeted LC-MS/MS assay were used to selectively quantify AO content in the pediatric HLC and HH samples. Briefly, ∼80 μg HLC protein or homogenate of 1 million HH in the Solubilization Buffer of the Mem-PER Plus Membrane Protein Extraction Kit was digested, as described previously . The purified AO protein (Pfizer, La Jolla, CA) was used as a calibrator, and the stable isotope labeled (SIL) peptides (two peptides, LILNEVSLLGSAPGGK and MIQVVSR) served as the internal standards.…”

“…Two sets of samples were analyzed, i.e., control (HH without carbazeran) and treatment (HH incubated with carbazeran) for statistical analysis using the metabolomics approach. The samples were analyzed by using Easy Spray 1200 series nanoLC coupled Q-Exactive HF MS (Thermo Fisher Scientific, Waltham, MA) with a previously 34 described method (Tables S4 and S5). The web-based XCMS-online platform was used to process the MS raw data (https://xcmsonline.scripps.edu).…”