Ketone reduction drives major circulating metabolites of GDC-8264 in humans

GDC-8264 (flizasertib) is a small molecule inhibitor of receptor-interacting serine/threonine-protein kinase 1 currently in clinical development. From a phase I dose escalation study in healthy subjects, 2 major circulating metabolites, M5 and M3, were identified and M5 was estimated to be >10% of total drug-related material. Mass spectrometric analysis, characterized by a prominent neutral loss of water, indicated that M5 was a ketone reduction product (+2 Da), whereas M3 was identified as its glucuronide conjugate. Comparison with synthetic standards confirmed M5 as the (S)-alcohol, resulting from stereospecific reduction. The formation of M5 was found to be NADPH dependent and mainly produced from human liver cytosolic but not microsomal fractions. Using chemical inhibitors and recombinant enzymes, the reaction was attributed primarily to carbonyl reductase 1 (CBR1), with minimal contributions from aldo-keto reductases (AKR1Cs) and negligible involvement of 11β-hydroxysteroid dehydrogenase 1. Assessment of extrahepatic metabolism revealed a distinct rank order of specific activity: intestine ≫ kidney ≈ liver > lung. Furthermore, M5 underwent efficient reoxidation to the parent GDC-8264, mediated mainly by CYP2C19 and CYP1A2, with potential contribution from CBR1. Collectively, CBR1-mediated stereoselective ketone reduction is the primary determinant of GDC-8264's circulating metabolite profile, whereas the dynamic interconversion (reduction to M5 and reoxidation back to the parent) represents a critical factor for GDC-8264 pharmacokinetics. SIGNIFICANCE STATEMENT: This study demonstrated that a clinical drug candidate, GDC-8264, has a major human circulating metabolite M5 that was formed via stereoselective ketone reduction primarily mediated by carbonyl reductase 1. This highlights (1) the critical role of carbonyl reductase 1 for ketone-containing molecules as a potential major drug metabolizing enzyme that warrants attention in new chemical entity development and that (2) reversible reduction-reoxidation as well as enterohepatic recycling could play an important role to drive parent drug exposures.