Characterization of glutathione conjugates of duloxetine by mass spectrometry and evaluation of in silico approaches to rationalize the site of conjugation for thiophene containing drugs.

The in vitro bioactivation of the selective serotonin and norepinephrine reuptake inhibitor duloxetine was investigated using liver microsomes and cytosol, expressed glutathione transferase, and recombinant P450 2D6 and 1A2. In the presence of glutathione, several conjugates were identified and characterized using a combination of direct infusion nanoelectrospray mass spectrometry on an LTQ/Orbitrap and liquid-chromatography mass spectrometry on a triple quadrupole. Structural characterization of these conjugates revealed that glutathione conjugation occurred on naphthalene rather than on thiophene and likely proceeded via a reactive epoxide intermediate. Experiments with recombinant P450s and the isoform specific inhibitors quinidine and furafylline suggested that both P450 2D6 and 1A2 were involved in the bioactivation of duloxetine. To explore the utility of in silico approaches to address bioactivation issues, MetaSite and two docking approaches (rigid and induced-fit docking) utilizing publicly available human P450 crystal structures or a homology model for P450 2C19 were used to predict the sites of bioactivation for duloxetine as well as the thiophene containing compounds tienilic acid, suprofen, ticlopidine, methapyrilene, and OSI-930 for which glutathione conjugates on the thiophene moiety have been reported. MetaSite and induced fit docking but not rigid docking correctly predicted that naphthalene rather than thiophene was the preferred site of bioactivation for duloxetine by P450 2D6. MetaSite predictions were also consistent with literature reports that thiophene was the site of glutathione conjugation for tienilic acid, suprofen, and OSI-930 but not for ticlopidine or methapyrilene. Of the two docking approaches investigated, induced fit docking results were consistent with thiophene as the site of bioactivation for all compounds to which it was applied. In conclusion, our investigation identified the likely bioactivation pathway for duloxetine and demonstrated the utility of in silico approaches MetaSite and induced fit docking to address potential bioactivation liabilities.