NADPH-Dependent Covalent Binding of [³H]Paroxetine to Human Liver Microsomes and S-9 Fractions: Identification of an Electrophilic Quinone Metabolite of Paroxetine

Abstract

The primary pathway of clearance of the methylenedioxyphenyl-containing compound and selective serotonin reuptake inhibitor paroxetine in humans involves P450 2D6-mediated demethylenation to a catechol intermediate. The process of demethylenation also results in the mechanism-based inactivation of the P450 isozyme. While the link between P450 2D6 inactivation and pharmacokinetic interactions of paroxetine with P450 2D6 substrates has been firmly established, there is a disconnect in terms of paroxetine's excellent safety record despite the potential for bioactivation. In the present study, we have systematically assessed the NADPH-dependent covalent binding of [(3)H]paroxetine to human liver microsomes and S-9 preparations in the absence and presence of cofactors of the various phase II drug-metabolizing enzymes involved in the downstream metabolism/detoxification of the putative paroxetine-catechol intermediate. Incubation of [(3)H]paroxetine with human liver microsomes and S-9 preparations resulted in irreversible binding of radioactive material to macromolecules by a process that was NADPH-dependent. The addition of reduced glutathione (GSH) to the microsomal and S-9 incubations resulted in a dramatic reduction of covalent binding. Following incubations with NADPH- and GSH-supplemented human liver microsomes and S-9, three sulfydryl conjugates with MH(+) ions at 623 Da (GS1), 779 Da (GS2), and 928 Da (GS3), respectively, were detected by LC-MS/MS. The collision-induced dissociation spectra allowed an insight into the structure of the GSH conjugates, based on which, bioactivation pathways were proposed. The formation of GS 1 was consistent with Michael addition of GSH to the quinone derived from two-electron oxidation of paroxetine-catechol. GS 3 was formed by the addition of a second molecule of GSH to the quinone species obtained via the two-electron oxidation of GS 1. The mechanism of formation of GS 2 can be rationalized via (i) further two-electron oxidation of the catechol motif in GS 3 to the ortho-quinone, (ii) loss of a glutamic acid residue from one of the adducted GSH molecules, and (iii) condensation of a cysteine-NH 2 with an adjacent carbonyl of the ortho-quinone to yield an ortho-benzoquinoneimine structure. Inclusion of the catechol-O-methyltransferase cofactor S-adenosylmethionine (SAM) in S-9 incubations also dramatically reduced the covalent binding of [(3)H]paroxetine, a finding that was consistent with O-methylation of the paroxetine-catechol metabolite to the corresponding guaiacol regioisomers in S-9 incubations. While the NADPH-dependent covalent binding was attenuated by GSH and SAM, these reagents did not alter paroxetine's ability to inactivate P450 2D6, suggesting that the reactive intermediate responsible for P450 inactivation did not leave the active site to react with other proteins. The results of our studies indicate that in addition to the low once-a-day dosing regimen (20 mg) of paroxetine, efficient scavenging of the catechol and quinone metabolites by SAM and GSH, respective...

Identification of Cytochrome P450 Isoforms Involved in the Metabolism of Paroxetine and Estimation of Their Importance for Human Paroxetine Metabolism Using a Population-Based Simulator

“…This finding is supported by results from van der Lee et al (2007), who stated that combining phenytoin (a CYP3A4 inducer) with paroxetine decreased paroxetine levels. These inferences are also supported by the finding that paroxetine is a weak mechanismbased inhibitor of CYP3A , especially given that the reactive species most likely does not leave the active site before inactivation (Zhao et al, 2007), also suggesting involvement of CYP3A4 in paroxetine metabolism.…”

“…The mechanism-based inhibition (MBI) is most likely caused by an irreversible binding of a paroxetinereactive metabolite, one that does not leave the active site, to the heme complex in the P450 enzyme (Bertelsen et al, 2003;Zhao et al, 2007). The MBI kinetic constants (K inact and K I ) have been determined for CYP2D6 and CYP3A (Bertelsen et al, 2003;Obach et al, 2007).…”

Demonstration of the Innate Electrophilicity of 4-(3-(Benzyloxy)phenyl)-2-(ethylsulfinyl)-6-(trifluoromethyl)pyrimidine (BETP), a Small-Molecule Positive Allosteric Modulator of the Glucagon-Like Peptide-1 Receptor

“…Inclusion of NADPH and GSH in liver microsomal incubations led to an even more rapid decline of BETP and the quantitative conversion to GSH adduct M1, which is indicative of a detoxifying metabolic pathway that competes with protein covalent binding. The propensity of GSH to reduce microsomal covalent binding has been noted with several drugs that are bioactivated to electrophilic species (Zhao et al, 2007;Obach et al, 2008).…”

Base-catalyzed reactions of environmentally relevant N-chloro-piperidines. A quantum-chemical study

“…This antidepressant is an important representative of an emerging group of pharmacologically active 4-aryl piperidines (Scheme 2). Recent studies have shown that paroxetine and its metabolites [17][18][19][20] have the potential to accumulate in waste waters, 3, 21 but also in the tissue of fish 22 as a result of discharges of this antidepressant into surface waters from municipal wastewater treatment plants. Therefore, interest in conducting the environmental risk assessment of paroxetine continues unabated.…”