Hydrolytic Metabolism of Withangulatin A Mediated by Serum Albumin Instead of Common Esterases in Plasma.

Background and objectives: The oral bioavailability of withangulatin A (WA) is low and may undergo first-pass metabolism because of the presence of two esters bonds. This study aimed to identify the hydrolysis behavior and mechanism of WA, thus enriching its structure-pharmacokinetic relationship.

Methods: The in vivo pharmacokinetic studies of WA in rats were first investigated, followed by in vitro assays including metabolic stability, phenotyping identification and metabolic kinetics assays. After screening out the responsible enzymes with higher catalytic capacity, molecular docking study was performed to demonstrate the interaction mode between WA and metabolic enzymes. Then, metabolites in human serum albumin (HSA) were identified by LC-TOF-MS/MS.

Results: In rats, the oral bioavailability of WA was only 2.83%. In vitro, WA was hydrolyzed in both rat and human plasma and could not be inhibited by selective esterase inhibitors. Physiologic concentration of HSA not recombinant human carboxylesterases (rhCES) could significantly hydrolyze WA, and it had a similar hydrolytic capacity with human plasma to WA. Furthermore, WA could stably bind to HSA by forming hydrogen bonds with Lys199 and Arg410, accompanied by the metabolic reaction of the lactone ring opening.

Conclusion: The study showed that WA underwent obvious hydrolysis in rat and human plasma, which implied a strong first-pass effect. Serum albumin rather than common esterases primarily contributed to the hydrolytic metabolism of WA in plasma.