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The in vitro metabolism of the calmodulin antagonist DY-9760e was investigated using liver microsomes from humans and three other animal species and compared with the in vivo metabolism in rats after intravenous administration of DY-9760e. Seven major metabolites were produced by human liver microsomes by the following metabolic pathways: N-dealkylation, phenyl hydroxylation, O-demethylation and imidazole oxidation. These metabolites were also produced by liver microsomes from monkeys, dogs and rats; additionally, a hydroxylated derivative of the indazole moiety was produced only by rat microsomes. To identify the structures of two imidazole ring metabolites whose authentic compounds could not be obtained, Escherichia coli co-expressing human cytochrome P450 CYP3A4 and NADPH-P450 reductase was used to biosynthesize these metabolites. NMR spectra elucidated the precise structures; oxidation occurred at the imidazole ring, and the subsequent ring-opening resulted in the generation of amide and formylamine groups. Glucuronide conjugates of the hydroxylated and O-demethylated derivatives were major components in rat bile. Therefore, DY-9760e metabolites generated in vitro correspond to the aglycones of the major metabolites observed in rat bile.

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DY-9760e, 3-[2-[4-(3-chloro-2-methylphenyl)-1-piperazinyl]ethyl]-5,6-dimethoxy-1-(4-imidazolylmethyl)-1H-indazole dihydrochloride 3.5 hydrate (Fig. 1), is a novel calmodulin antagonist being developed to treat acute ischemic stroke. It inhibits calmodulin-dependent enzymes and in so doing exerts neuroprotective effects in animals with induced strokes (Sugimura et al., 1997;Sato et al., 1999Sato et al., , 2003Sato et al., , 2004Fukunaga et al., 2000;Takagi et al., 2001;Hashiguchi et al., 2003). In experimental model rats with transient focal ischemia, DY-9760e significantly reduces the infarct volume when infused for 6 h from 1 h after the middle cerebral artery occlusion and also suppresses the increase in brain water content. Results of a previous study show that DY-9760e is metabolized by human liver microsomes in vitro, and the major metabolites of DY-9760e are identified as N-dealkylated (DY-9836), 4-hydroxylated (M3), O-demethylated (M5 and M7), and imidazole-oxidized derivatives (M6 and M8) ( Fig. 1; Tachibana et al., 2005b). The results of pharmacokinetic studies in rats after intravenous administration of DY-9760e show that DY9760e is extensively metabolized in the liver, and most of the administered DY-9760e is excreted into bile as glucuronide conjugates of hydroxylated and O-demethylated metabolites (Tachibana et al., 2005a,b). Therefore, DY-9760e is eliminated mainly through hepatic metabolic clearance.Although the general outline of DY-9760e metabolism is known, more specific information is needed to understand its complex metabolism. Thus, this study had two purposes. One purpose of this study was to identify which human P450 isozyme(s) are involved in the formation of the primary metabolites of DY-9760e, and to determine the enzyme kinetics for the formation of metabolites using human liver microsomes and microsomes expressing recombinant human P450 enzymes. These studies provide essential information for determining the contribution of each P450 isozyme to DY-9760e metabolism. The second purpose of the present study was to estimate how