KAE609 [(19R,39S)-5,79-dichloro-69-fluoro-39-methyl-29,39,49,99-tetrahydrospiro[indoline-3,19-pyridol[3,4-b]indol]-2-one] is a potent, fast-acting, schizonticidal agent being developed for the treatment of malaria. After oral dosing of KAE609 to rats and dogs, the major radioactive component in plasma was KAE609. An oxidative metabolite, M18, was the prominent metabolite in rat and dog plasma. KAE609 was well absorbed and extensively metabolized such that low levels of parent compound (£11% of the dose) were detected in feces. The elimination of KAE609 and metabolites was primarily mediated via biliary pathways ( ‡93% of the dose) in the feces of rats and dogs. M37 and M23 were the major metabolites in rat and dog feces, respectively. Among the prominent metabolites of KAE609, the isobaric chemical species, M37, was observed, suggesting the involvement of an isomerization or rearrangement during biotransformation. Subsequent structural elucidation of M37 revealed that KAE609, a spiroindolone, undergoes an unusual C-C bond cleavage, followed by a 1,2-acyl shift to form a ring expansion metabolite M37. The in vitro metabolism of KAE609 in hepatocytes was investigated to understand this novel biotransformation. The metabolism of KAE609 was qualitatively similar across the species studied; thus, further investigation was conducted using human recombinant cytochrome P450 enzymes. The ring expansion reaction was found to be primarily catalyzed by cytochrome P450 (CYP) 3A4 yielding M37. M37 was subsequently oxidized to M18 by CYP3A4 and hydroxylated to M23 primarily by CYP1A2. Interestingly, M37 was colorless, whereas M18 and M23 showed orange yellow color. The source of the color of M18 and M23 was attributed to their extended conjugated system of double bonds in the structures.
Numerous assays have been developed to investigate the interactions between G-protein-coupled receptors (GPCRs) and their ligands since GPCRs are key therapeutic targets. Reporter-based assays using the cAMP response element (CRE) coupled with bioluminescence from a luciferase reporter have been used extensively in vitro with high-throughput screens (HTS) of large chemical compound libraries. We have generated a transgenic mouse model (CRE luc) with a luciferase reporter under the control of a synthetic promoter that contains several CREs, which supports real-time bioimaging of GPCR ligand activity in whole animals, tissues, or primary cells. In the CRE luc model, GPCR signaling through the cAMP pathway can be detected from the target GPCR that is in a native cellular environment with a full complement of associated receptors and membrane constituents. Multiple independent lines have been produced by random integration of the transgene, resulting in tissue expression profiles covering the major organs. The goal of the CRE luc model is to accelerate the transition from HTS to profiling of GPCR small-molecule leads in preclinical animal disease models, as well as define the mechanism of action of GPCR drugs in three experimental formats: primary cells, tissue homogenates, and whole animals.
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