Characterization of moclobemide N-oxidation in human liver microsomes.
1. Moclobemide underdergoes morpholine ring N-oxidation to form a major metabolite in plasma Rol2-5637. 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) respectively. 4. The N-oxidation of moclobemide strongly correlated with benzydamine N-oxidation a probe reaction for flavin-containing monoxygenase (FMO) activity (0.1 mM moclobemide, rs = 0.81, p < 0.005; 4 mM moclobemide, rs = 0.94, p = 0.0001). Correlations were observed between moclobemide N-oxidation and specific cytochromre P450 (CYP) activities at both moclobemide concentrations (0.1 mM moclobemide, CYP2C19 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 Rol2-5637 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 ilre consistent with the reaction being FMO medialtetd . 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.
Hoskins
J
J
Department of Clinical Pharmacology, Royal North Shore Hospital, St Leonards, NSW, Australia. janelle@med.usyd.edu.au
Shenfield
G
G
Murray
M
M
Gross
A
A
D001549:Benzamides / Q000494:pharmacology; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D006358:Hot Temperature; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D008862:Microsomes, Liver / Q000378:metabolism*; D020912:Moclobemide / Q000378:metabolism / Q000493:pharmacokinetics*; D008956:Models, Chemical; D008996:Monoamine Oxidase Inhibitors / Q000378:metabolism / Q000493:pharmacokinetics*; D009025:Morpholines / Q000494:pharmacology; D010100:Oxygen / Q000378:metabolism*; D020033:Protein Isoforms; D013696:Temperature; D013997:Time Factors
D001549:Benzamides; D008996:Monoamine Oxidase Inhibitors; D009025:Morpholines; D020033:Protein Isoforms; C062936:Ro 12-5637; D003577:Cytochrome P-450 Enzyme System; D020912:Moclobemide; D010100:Oxygen