Idelalisib 8-oxidation as an enzyme-selective catalytic marker of human aldehyde oxidase in HepG2 cells: Application to the investigation of enzyme inactivation

Aldehyde oxidase is a cytosolic drug-metabolizing enzyme. Idelalisib is a phosphoinositide-3-kinase-delta inhibitor that undergoes extensive biotransformation, mainly via 8-oxidation, which is catalyzed predominantly by human aldehyde oxidase (AOX1) and, to a lesser extent, cytochromes P450 (P450). In the present study, the primary objective was to test the hypothesis that idelalisib 8-oxidation is an enzyme-selective catalytic marker of AOX1 in HepG2 human hepatocellular carcinoma cells that express AOX1 but has little or no CYP3A functionality, and this approach is effective in identifying AOX1 inactivators. Enzyme kinetic analysis indicated comparable values of k cat , K m , and ratio of k cat /K m (catalytic efficiency) for idelalisib 8-oxidation catalyzed by HepG2 cells and human liver cytosol, whereas V max was greater for liver cytosol-catalyzed idelalisib 8-oxidation. Human recombinant AOX1, CYP3A4, and CYP3A5 were major catalysts of idelalisib 8-oxidation. However, experiments with a panel of enzyme-selective chemical inhibitors indicated that AOX1, but not P450 or xanthine oxidase, was responsible for idelalisib 8-oxidation in HepG2 cells and human liver cytosol. The HepG2 cell-catalyzed idelalisib 8-oxidation assay correctly identified time-dependent AOX1 inactivators (erlotinib, O -desmethylerlotinib, O -didesmethylerlotinib, hydralazine) and non-inactivators (gefitinib, O -desmethylgefitinib, O -desmorpholinopropylgefitinib, raloxifene). The data from the experiment to address our secondary objective indicated that erlotinib dosing increased the accumulation of idelalisib and 8-oxo-idelalisib in mouse plasma, liver, and kidney. In conclusion, 1) idelalisib 8-oxidation is an AOX1-selective catalytic marker in HepG2 cells that can be utilized to study AOX1 catalytic function; and 2) the in vivo mouse pharmacokinetic approach is not effective in identifying erlotinib as an inhibitor of idelalisib 8-oxidation.