Metabolism of 4-ethoxy-2-methyl-5-morpholino-3(2H)-pyridazinone (emorfazone). V. Effect of inducer pretreatment on oxygenation of the morpholino moiety in guinea pigs.

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Abstract
The in vivo and in vitro metabolism and the cytochrome P-450 substrate-binding difference spectrum of emorfazone, 4-ethoxy-2-methyl-5-morpholino-3(2H)-pyridazionone [maximum velocity], were studied in non-pretreated and in phenobarbital (PB)- and 3-methylcholanthrene(MC)-pretreated male guinea pigs. In non-pretreated animals, emorfazone was primarily metabolized to 5-(N-carboxymethyl-N-2-hydroxyethylamino)-4-ethoxy-2-methyl-3(2H)-pyridazinone (M-8) and 5-[2-(carboxymethyloxy)ethylamino]-4-ethoxy-2-methyl-3(2H)-pyridazinone (M-9), produced by oxidative cleavage O.sbd.C or N.sbd.C bonds in the morpholino moiety. The M-8:M-9 ratios were 10.1, 2.0 and 0.3 at doses of 20,100 and 500 mg/kg emorfazone, respectively. In PB-pretreated animals, these ratios were 1.6, 0.7 and 0.1; in MC-pretreated animals they were > 100, 16.9 and 5.8 at doses of 20, 100 and 500 mg/kg, respectively, indicating that PB pretreatment increased the production of M-9, whereas MC-pretreatment lead to the production of greater amounts of M-8. In in vitro experiments, the morpholino moiety was oxidatively metabolized by microsomes in the presence of reduced NADPH; metabolism involving reduced NADH was slight. In microsomes from non-pretreated, PB- and MC-pretreated animals, respectively, the Km values for the oxygenation of the C atom adjacent to the N atom were 1.1 .times. 10-2, 3.9 .times. 10-3 and 1.2 .times. 10-2 M; Vmax [maximum velocity] values were 0.483, 0.419 and 0.225 .mu.mol/4 nmol cytochrome P-450 per 20 min; these values for the oxygenation of the C atom adjacent to the O atom were 1.5 .times. 10-5, 1.9 .times. 10-4 and 1.8 .times. 10-4 M and 0.065, 0.075 and 0.338 .mu.mol/4 nmol cytochrome P-450 per 20 min. With increasing substrate concentrations, the apparent substrate-binding difference spectrum in microsomes from the non-pretreated group gradually changed from a reverse type I to a type I spectrum. Each binding difference spectral pattern with inducer-pretreated microsomes was different, i.e., the proportions of type I and reverse type I spectrum were increased in PB- and MC-pretreated microsomes, respectively. Evidently the 2 kinds of C atoms of the morpholino moiety are oxidized by 2 species of microsomal cytochrome P-450-dependent monooxygenation systems with different affinity and capacity, and these oxygenation mechanisms are correlated with the substrate-binding difference spectra.