Characterization of moclobemide N-oxidation in human liver microsomes

Abstract

1. Moclobemide undergoes morpholine ring N-oxidation to form a major metabolite 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 catalysing this reaction have been identified using inhibition, enzyme correlation, altered pH and heat pretreatment experiments. 3. N-oxidation followed single enzyme Michealis-Menten kinetics (0.02-4.0 mM). Km app and Vmax 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-oxidation, a probe reaction for flavin-containing monooxygenase (FMO) activity, (0.1 mM moclobemide, rS = 0:81; p < 0:005; 4 mM moclobemide, rS = 0:94; p = 0:0001). Correlationswere observed between moclobemide N-oxidation and specific cytochrome P450 (CYP) activities at both moclobemide concentrations (0.1 mM moclobemide, CYP2C19 rS = 0:66; p < 0:05; 4 mM moclobemide, CYP2E1 rS = 0:56; p < 0:05).5. The general P450 inhibitor, N-benzylimidazole, did not affect the rate of Ro12-5637 formation (0% inhibition versus control) at 1.3 mM moclobemide. Furthermore, the rate of Ro12-5637 formation in HLM was unaffected by inhibitors or substrates of specific P450s (<10% inhibition versus control). 6. Heat pretreatment of HLM in the absence of NADPH (inactivating FMOs) resulted in 97% inhibition of Ro12-5637 formation. N-oxidation activity was greatest when incubated at pH 8.5. These results are consistent with the reaction 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 potentially small contribution from cytochrome P450 isoforms.