|Budget Amount *help
¥2,200,000 (Direct Cost : ¥2,200,000)
Fiscal Year 1995 : ¥800,000 (Direct Cost : ¥800,000)
Fiscal Year 1994 : ¥1,400,000 (Direct Cost : ¥1,400,000)
Propranolol (PL), a beta-blocker, was oxidized to 4-hydroxylated (4-OH-PL), 5-hydroxylated (5-OH-PL) and N-desisopropylated (NDP) metabolites in human liver microsomes. The PL aromatic hydroxylations were suppressed by anti-rat cytochrome P450-2D2 (P450-2D2) antibody and its specific inhibitor (quinidine) concentration dependently. The PL N-desalkylation was decreased by alpha-naphthoflavone (a P450-1A inhibitor) and highly correlated with phenacetin O-deethylation, an index of P450-1A2 activity. These results suggest that the naphthalene ring hydroxylations and the side chain N-desalkylation were mediated by P450-2D6 and P450-1A2, respectively.
Purified rat P450-2D2 was incubated with [3H]-PL in the presence of NADPH,and relationship between radioactivity binding and decrease in enzyme activity was examined. Radioactivity bound to P450-2D2 was suppressed by anti-P450-2D2 antibody in a concentration dependent manner, and the enzyme activity was decereased time-dependently, which showed
first-order kinetics. Radioactivity was localized at P450-2D2 apoprotein, and corresponded well to the decreased enzyme activity. From these results, it is thought that PL is biotransformed by P450-2D2 to a chemically reactive metabolite, which covalently binds to the enzyme itself, resulting in its inhibition.
4-OH-PL strongly suppressed P450-2D enzyme activity after preincubation with rat liver microsomes in the presence of NADPH.Further metabolism of 4-OH-PL was examined in rat liver microsomes, and the possibilityarose that superoxide was involved in the elimination of 4-OH-PL in the microsomes. It was found that 4-OH-PL was converted to 1,4-naphthoquinone in xanthine-xanthine oxidase system, a well known superoxide-generating system. The quinone was identified as an metabolite of 4-OH-PL also in the system containing rat liver microsomes. Taking these results and findings into account, it seems likely that PL is converted to 4-OH-PL by P450-2D enzymes in human and rat liver microsomes. 4-OH-PL thus obtained is converted by superoxide supplied from microsomal electron transfer system to 1,4-naphthoquinone, which covalently binds to P450-2D enzymes, resulting in the inhibition. Less