Development and validation of an LC-MS/MS method for the novel PROTAC KT-474 in rat and mouse plasma: Application to dose-dependent pharmacokinetics and metabolite identification in mice.

Abstract

KT-474 is a novel orally bioavailable proteolysis-targeting chimera (PROTAC) that selectively degrades interleukin-1 receptor-associated kinase 4 (IRAK4) and is currently in phase 2 clinical trials for inflammatory diseases. In this study, we developed and validated a sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of KT-474 in rat and mouse plasma and successfully applied it to pharmacokinetic studies in mice. In addition, the metabolic stability of KT-474 was evaluated in plasma and liver microsomes, and its metabolites were identified in vitro. The developed method was validated in accordance with the U.S. Food and Drug Administration and the International Council for Harmonisation guidelines. KT-474 showed marked instability in human and rat plasma but moderate stability in mouse plasma. In contrast, microsomal studies indicated that the hepatic metabolic profiles of KT-474 were comparable between humans and mice, but they differed from those observed in rats, suggesting that mice may serve as a suitable surrogate species for predicting human hepatic metabolism. Metabolite identification revealed nine metabolites in the microsomes but only one in the mouse plasma. In the mouse microsomes, M3, formed through O-dealkylation, has been proposed as the major metabolite, whereas M10 has been identified as the sole metabolite in the mouse plasma, likely formed via non-enzymatic degradation. Pharmacokinetic studies in mice demonstrated that KT-474 exhibited a nonlinear pharmacokinetic profile up to an oral dose of 10 mg/kg, with oral bioavailability ranging from 16.93% to 25.08%. Collectively, these findings provide a comprehensive characterization of the pharmacokinetic and metabolic properties of KT-474 and support its further clinical development.