Characterization of moclobemide N-oxidation in human liver microsomes

1. Moclobemide undergoes morpholine ring N-oxidation to form a major metabo lite in plasma, Ro12-5637. 2. The kinetics of moclobemide N-oxidation in human liver microsomes (HLM). (n = 6) have been investigated and the mixed-function oxidase enzymes cata lysing this reaction have been identified using inhibition, enzyme correlat ion, altered pH and heat pretreatment experiments. 3. N-oxidation followed single enzyme Michealis-Menten kinetics (0.02-4.0 m M). K-m (app) and V-max ranged from 0.48 to 1.35 mM (mean +/- SD 0.77 +/- 0 .34 mM) and 0.22 to 2.15 nmol mg(-1) min(-1) (1.39 +/- 0.80 nmol mg(-1) min (-1)), respectively. 4. The N-oxidation of moclobemide strongly correlated with benzydamine N-ox idation, a probe reaction for flavin-containing monooxygenase (FMO) activit y, (0.1 mM moclobemide, r(S) = 0.81; p < 0.005; 4 mM moclobemide, r(S) = 0. 94; p = 0.0001). Correlations were observed between moclobemide N-oxidation and specific cytochrome P450 (CYP) activities at both moclobemide concentr ations (0.1 mM moclobemide, CYP2C19 r(S) = 0.66; p < 0.05; 4 mM moclobemide , CYP2E1 r(S) = 0.56; p < 0.05). 5. The general P450 inhibitor, N-benzylimidazole, did not affect the rate o f Ro12-5637 formation (0% inhibition versus control) at 1.3 mM moclobemide. Furthermore, the rate of Ro12-5637 formation in HLM was unaffected by inhi bitors or substrates of specific P450s (< 10% inhibition versus control). 6. Heat pretreatment of HLM in the absence of NADPH (inactivating FMOs) res ulted in 97% inhibition of Ro12-5637 formation. N-oxidation activity was gr eatest when incubated at pH 8.5. These results are consistent with the reac tion being FMO mediated. 7. In conclusion, moclobemide N-oxidation activity has been observed in HLM in vitro and the reaction is predominantly catalysed by FMOs with a potent ially small contribution from cytochrome P450 isoforms.