TY - JOUR T1 - Role of epoxide hydrolases and cytochrome P450s on metabolism of KZR-616, a first-in-class selective inhibitor of the immunoproteasome JF - Drug Metabolism and Disposition JO - Drug Metab Dispos DO - 10.1124/dmd.120.000307 SP - DMD-AR-2020-000307 AU - Ying Fang AU - Henry Johnson AU - Janet L. Anderl AU - Tony Muchamuel AU - Dustin McMinn AU - Christophe Morisseau AU - Bruce D. Hammock AU - Christopher Kirk AU - Jinhai Wang Y1 - 2021/01/01 UR - http://dmd.aspetjournals.org/content/early/2021/07/03/dmd.120.000307.abstract N2 - KZR-616 is an irreversible tripeptide epoxyketone-based selective inhibitor of the human immunoproteasome. Inhibition of the immunoproteasome results in anti-inflammatory activity in vitro and, based on promising therapeutic activity in animal models of rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE), KZR-616 is being developed for potential treatment of multiple autoimmune and inflammatory diseases. The presence of a ketoepoxide pharmacophore presents unique challenges in the study of drug metabolism during lead optimization and clinical candidate profiling. This study presents a thorough and systematic in vitro and cell-based enzymatic metabolism and kinetic investigation to identify the major enzymes involved in the metabolism and elimination of KZR-616. Upon exposure to liver microsomes in the absence of NADPH, KZR-616 and its analogs were converted to their inactive diol derivatives with varying degrees of stability. Diol formation was also shown to be the major metabolite in pharmacokinetic studies in monkeys and correlated with in vitro stability results for individual compounds. Further study in intact hepatocytes and a hepatocellular carcinoma cell line revealed that KZR-616 metabolism was sensitive to an inhibitor of microsomal epoxide hydrolase (mEH) but not inhibitors of cytochrome P450 (CYP) or soluble epoxide hydrolase (sEH). Primary human hepatocytes were determined to be the most robust source of mEH activity for study in vitro. These findings also suggest that the exposure of KZR-616 in vivo is unlikely to be affected by co-administration of inhibitors or inducers of CYP and sEH. Significance Statement This work presents a thorough and systematic investigation of metabolism and kinetic of KZR-616 and other peptide epoxyketones in in vitro and cell-based enzymatic systems. Gained information could be useful in assessing novel covalent proteasome inhibitors during lead compound optimization. The study also demonstrates a robust source of in vitro metabolism identification that correlated very well with in vivo PK metabolism for peptide epoxyketones. ER -