IDENTIFICATION OF 3 CYTOCHROME-P450 ISOZYMES INVOLVED IN N-DEMETHYLATION OF CITALOPRAM ENANTIOMERS IN HUMAN LIVER-MICROSOMES

Using in vitro techniques, the present study demonstrates that CYP2D6, 2C19 and 3A4 are involved in N-demethylation of citalopram (CIT) enan tiomers. Human liver microsome incubations performed with specific inh ibitors of these three CYP isozymes have shown up to 60%, inhibition o f demethylcitalopram production. cDNA expressed human cytochrome P-450 3A4, 2C19 and 2D6 isozymes, but not CYP1A2, were identified to be inv olved in N-demethylation of CIT enantiomers. Kinetics using cDNA expre ssed CYP2C19 and CYP3A4 show K-m values in the same range: 198 mu M, 2 11 mu M for CYP2C19 and 169 mu M, 163 mu M for CYP3A4 for S- and R-CIT demethylation, respectively. In contrast, kinetics using cDNA express ed CYP 2D6 show a K-m of 18 mu M and 22 mu M for S- and R-CIT demethyl ation, respectively. Nevertheless, kinetics using cDNA expressed CYP2C 19 and 3A4 have a range of V-max values ten times higher than that of CYP2D6. For this reason, intrinsic clearance values (V-max/K-m) for S- and R-CIT were within a small range for these three isozymes: 0.25 to 0.39 mu lh(-1) x pmol(-1) of CYP. CYP2D6 has an opposite stereoselect ivity in the biotransformation of CIT enantiomers than CYP2C19 and 3A4 ; the SIR ratios of the intrinsic clearance were 0.71, 1.57 and 1.37, respectively. Taking into account that CYP isozymes are expressed at v arious levels, CYP2D6, which is expressed at lower levels than CYP2C19 and CYP3A4, plays a minor role in the biotransformation of CIT enanti omers. These results confirm that the use of cDNA expressed CYP isozym es is a potent tool for the measurement of kinetic constants and help to predict clearance modifications of CIT enantiomers, especially in p oor metabolizers of mephenytoin (with a CYP2C19 deficiency) or patient s comedicated with potent CYP2C19 or 3A4 inhibitor(s). For instance, f luvoxamine (100 mu M) inhibits CIT N-demethylation by 64% in microsome s.