Identification of the human cytochromes p450 responsible for in vitro formation of R- and S-norfhoxetine. Journal of pharmacology and experimental therapeutics

Abstract

ABSTRACT The formation of R-and S-norfluoxetine was analyzed in vitro in human liver microsomes. Low apparent K m values for R-norfluoxetine formation of Յ8 M and S-norfluoxetine of Ͻ0.2 M were determined. R-Norfluoxetine formation rates in a characterized microsomal bank correlated with the catalytic activities for cytochrome P450 (CYP) 2D6, CYP2C9, and CYP2C8. Expressed CYP2C9, CYP2C19, and CYP2D6 formed R-norfluoxetine following incubation with 1 M Rfluoxetine and exhibited apparent K m values of 9.7, 8.5, and 1.8 M, respectively. Multivariate correlation analysis identified CYP2C9 and CYP2D6 as significant regressors with R-norfluoxetine formation. Antibodies to the CYP2C subfamily and CYP2D6 each exhibited moderate inhibition of Rnorfluoxetine formation. Therefore, CYP2D6 and CYP2C9 contribute to this biotransformation. At pharmacological concentrations of S-fluoxetine, S-norfluoxetine formation rates in the bank of microsomes were found to correlate only with CYP2D6 catalytic activity and only expressed CYP2D6 was found to be capable of forming S-norfluoxetine. Thus, it would appear that both CYP2D6 and CYP2C9 contribute to the formation of R-norfluoxetine, whereas only CYP2D6 is responsible for the conversion to S-norfluoxetine. Since the enantiomers of fluoxetine and norfluoxetine are inhibitors of CYP2D6, upon chronic dosing, the CYP2D6-mediated metabolism of the fluoxetine enantiomers would likely be inhibited, resulting in R-norfluoxetine formation being mediated by CYP2C9 and S-norfluoxetine formation being mediated by multiple high K m enzymes. Fluoxetine, a racemic mixture of R-and S-fluoxetine, is a selective serotonin reuptake inhibitor currently marketed for the treatment of depression and other disorders. R-fluoxetine was a drug candidate in development for use in psychiatric illness. The major route of metabolism of the enantiomers of fluoxetine is N-demethylation It has long been recognized that the enantiomers of fluoxetine and norfluoxetine are inhibitors of CYP2D6-mediated reactions. S-Fluoxetine and S-norfluoxetine are approximately 5-fold more potent in their ability to inhibit CYP2D6-mediated reactions than R-fluoxetine and R-norfluoxetine (K i values of 0.22, 0.31, 1.38, and 1.48 M, respectively) There have been a few studies in humans that have examined the clearance of the enantiomers of fluoxetine following both single and multiple doses of racemic fluoxetine. In a study examining the pharmacokinetics of a single dose of racemic fluoxetine a major role for CYP2D6 in S-fluoxetine metabolism was proposed, for S-fluoxetine clearance was 12-fold slower in poor metabolizers (PMs) of CYP2D6-mediated reactions than that observed in extensive metabolizers (EMs