1982 Volume 30 Issue 10 Pages 3748-3756
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)-pyridazinone, 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 of O-C or N-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 nicotinamide adenine dinucleotide phosphate (NADPH) ; metabolism involving reduced nicotinamide adenine dinucleotide (NADH) was slight. In microsomes from non-pretreated, PB-and MC-pretreated animals, respectively, the Km values for the oxygenation of the carbon atom adjacent to the N atom were 1.1×10-2, 3.9×10-3 and 1.2×10-2M ; Vmax values were 0.483, 0.419 and 0.225μmol/4 nmol cyt. P-450/20 min ; these values for the oxygenation of the carbon atom adjacent to the O atom were 1.5×10-5, 1.9×10-4 and 1.8×10-4M and 0.065, 0.075 and 0.338μmol/4 nmol cyt. P-450/20 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 inducerpretreated microsomes was different, i.e. the proportions of type I and reverse type I spectrum were increased in PB-and MC-pretreated microsomes, respectively. Based on these results, it may be said that the two kinds of carbon atoms of the morpholino moiety are oxidized by two species of microsomal cytochrome P-450-dependent monooxygenation systems with different affinity and capacity, and that these oxygenation mechanisms are correlated with the substrate-binding differene spectra.
