5 DISTINCT HUMAN CYTOCHROMES MEDIATE AMITRIPTYLINE N-DEMETHYLATION IN-VITRO - DOMINANCE OF CYP 2C19 AND 3A4

The human cytochromes P450 (CYPs) mediating amitriptyline N-demethylat ion have been identified using a combination of enzyme kinetic and che mical inhibition studies. Amitriptyline was N-demethylated to nortript yline by microsomes from cDNA transfected human lymphoblastoid cells e xpressing human CYPs 1A2, 2C9, 2C19, 2D6, and 3A4. CYP 2E1 showed no d etectable activity. While CYP 2C19 and CYP 2D6 showed high affinity, C YP 3A4 showed low affinity; CYP 2C9 and 1A2 showed intermediate affini ties. Based on these kinetic parameters and estimated relative abundan ce of the different CYPs in human liver, CYP 2C19 was identified as th e major amitriptyline N-demethylase at low (therapeutically relevant) amitriptyline concentrations, whereas CYP 3A4 may be more important at higher amitriptyline concentrations. Chemical inhibition studies with ketoconazole and omeprazole indicate that CYP 3A4 is the major amitri ptyline N-demethylase at 100 mu mol/L amitriptyline, while CYP 2C19 is equally important at a substrate concentration of 5 mu mol/L. The CYP 1A2 inhibitor alpha-naphthoflavone and the CYP 2C9 inhibitor sulfaphe nazole produced much less inhibition of amitriptyline N-demethylation at both substrate concentrations. Quinidine produced no detectable inh ibition. The kinetics of amitriptyline N-demethylation by human liver microsome were consistent with a two enzyme model, with the high affin ity component exhibiting Michaelis Menten kinetics and the low affinit y component exhibiting Hill enzyme kinetics. No difference was apparen t in the kinetics of amitriptyline N-demethylation in two liver sample s with low levels of CYP 2C19 activity compared with two other samples with relatively normal 2C19 activity. This may reflect the importance of higher substrate concentration values in estimation of kinetic par ameters in vitro.