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Chem Res Toxicol. 2007 Nov;20(11):1649-57. Epub 2007 Oct 2.

NADPH-dependent covalent binding of [3H]paroxetine to human liver microsomes and S-9 fractions: identification of an electrophilic quinone metabolite of paroxetine.

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  • 1Pharmacokinetics, Dynamics and Metabolism Department, Pfizer Global Research and Development, Groton, Connecticut, USA.

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, respectively, serves as an explanation for the excellent safety record of paroxetine despite the fact that it undergoes bioactivation.

PMID:
17907785
[PubMed - indexed for MEDLINE]
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