Mouse Red Blood Cell–Mediated Rare Xenobiotic Phosphorylation of a Drug Molecule Not Intended to Be a Kinase Substrate

Abstract

Phosphorylation of xenobiotics is rare, probably owing to a strong evolutionary pressure against it. This rarity may have attracted more attention recently as a result of intentionally designed kinase-substrate analogs that depend on kinase-catalyzed activation to form phosphorylated active drugs. We report a rare phosphorylated metabolite observed unexpectedly in mouse plasma samples after an oral dose of a Tankyrase inhibitor that was not intended to be a kinase substrate, i.e., ()-2-(4-(6-(3,4-dimethylpiperazin-1-yl)-4-methylpyridin-3-yl)phenyl)-8-(hydroxymethyl)quinazolin-4(3)-one (AZ2381). The phosphorylated metabolite was not generated in mouse hepatocytes. In vitro experiments showed that the phosphorylation of AZ2381 occurred in mouse whole blood with heparin as anticoagulant but not in mouse plasma. The phosphorylated metabolite was also produced in rat, dog, and human blood, albeit at lower yields than in mouse. Divalent metal ions are required for the phosphorylation since the reaction is inhibited by the metal chelator EDTA. Further investigations with different cellular fractions of mouse blood revealed that the phosphorylation of AZ2381 was mediated by erythrocytes but did not occur with leukocytes. The levels of O incorporation into the phosphorylated metabolite when inorganicO-phosphate and -O-ATP were added to the mouse blood incubations separately suggested that the phosphoryl transfer was from inorganic phosphate rather than ATP. It remains unclear which enzyme present in red blood cells is responsible for this rare phosphorylation.

The Importance of Tracking “Missing” Metabolites: How and Why?

“…The metabolites falling under this category can thus be easily missed without the relevant experiments, such as the aforementioned siponimod cholesterol ester metabolite M17 (Figure 6B) and a rare erythrocyte-mediated phosphorylation metabolite of AZ2381 not formed in cryopreserved hepatocytes. 121 Sometimes, metabolites from non-host origin, i.e., gut microbiome-derived drug metabolites, can also be efficiently re-absorbed into circulation and appear as a major circulating metabolite, as in the case of the ozanimod oxadiazole ring scission metabolite RP101124 representing 15% of DRM in circulation (Figure 2C). 28 From a human 14 C mass balance study of pimasertib, a novel phosphoethanolamine conjugate metabolite M554 was observed as the major metabolite in circulation (30% of AUC) and in excreta (17% of dose) (Figure 8B).…”

“…When the responsible enzymes are unknown or the metabolism is solely from extrahepatic origin, typical in vitro metabolism studies using liver-derived cells and subcellular fractions will not be the appropriate system to capture these metabolites. The metabolites falling under this category can thus be easily missed without the relevant experiments, such as the aforementioned siponimod cholesterol ester metabolite M17 (Figure B) and a rare erythrocyte-mediated phosphorylation metabolite of AZ2381 not formed in cryopreserved hepatocytes . Sometimes, metabolites from non-host origin, i.e., gut microbiome-derived drug metabolites, can also be efficiently re-absorbed into circulation and appear as a major circulating metabolite, as in the case of the ozanimod oxadiazole ring scission metabolite RP101124 representing 15% of DRM in circulation (Figure C) .…”