Identification of a Novel Glutathione Adduct of Diclofenac, 4′-Hydroxy-2′-Glutathion-Deschloro-Diclofenac, Upon Incubation With Human Liver Microsomes

Abstract

ABSTRACT:Clinical use of the nonsteroidal anti-inflammatory drug diclofenac (DF) is associated with an incidence of idiosyncratic hepatoxicity. The formation of reactive metabolites of DF in vivo has been proposed to be responsible for such toxicity. One type of reactive metabolite, a benzoquinone imine of DF formed through oxidation by cytochromes P450, can be trapped by glutathione in vitro in liver microsomes to form glutathione (GS) adducts. Three GS adducts from DF were reported in the literature, namely, 5-hydroxy (OH)-4-glutathione-DF, 4-OH-3-glutathione-DF and 5-OH-6-glutathione-DF, and they all have the same molecular weight of 616. Recently, we developed a sensitive and high throughput method for the detection of GS adducts from liver microsome incubation. This method uses a constant neutral loss scan of m/z 129, a "structurecharacteristic" fragment for GS adduct, on an automated chipbased nanoelectrospray (Advion NanoMate 100) attached to a tandem mass spectrometer (Sciex API 3000). The analysis of GS adducts from human liver microsome incubation with DF by the NanoMate 100-API 3000 method unambiguously revealed a new adduct ion with m/z 583 (MH ؉ ), in addition to the known adduct peak with m/z 617 (MH ؉ ). This new adduct was further confirmed to be 4-OH-2-glutathion-deschloro-diclofenac by liquid chromatography (LC) tandem mass spectrometry (MS), LC/MS-NMR, and comparison to a synthetic standard.Diclofenac (DF) is a nonsteroidal anti-inflammatory drug that is widely used for the treatment of osteoarthritis and rheumatoid arthritis (Small, 1989). Chemically reactive metabolites of DF have been proposed to explain the idiosyncratic hepatoxicity associated with the clinical use of the drug (Banks et al., 1995;Miyamoto et al., 1997;Shen et al., 1999;Tang et al., 1999b;Tang, 2003). One type of reactive metabolite associated with the formation of protein adducts (Maggs et al., 1995) is a benzoquinone imine intermediate generated via oxidation by cytochromes P450 (Shen et al., 1999;Poon et al., 2001). Metabolism of DF has been studied extensively (Corcoran et al., 2000), and it was proposed that P450-catalyzed oxidation of DF resulted in the formation of DF-2,5-quinone imine and DF-1Ј,4Ј-quinone imine intermediates through initial formation of 5-OH-DF and 4Ј-OH-DF, respectively (Tang et al., 1999a). These reactive intermediates can be trapped by glutathione (GSH) to form GS adducts (Baillie and Slatter, 1991) in vitro (in liver microsomes or hepatocytes), as well as in vivo (in bile and urine; often detected as degradation products, N-acetylcysteine, of the initially formed GS adduct). Three GS adducts from DF were reported in the literature, namely, 5-OH-4-GS-DF, 4Ј-OH-3Ј-GS-DF, and 5-OH-6-GS-DF (Tang et al., 1999a), and they all have the same molecular weight of 616.A neutral loss (NL) scan of m/z 129, a "structure-characteristic" fragment (loss of glutamate) for glutathione adducts, by liquid chromatography-tandem mass spectrometry (LC/MS/MS) was developed to detect reactive metabolite formation (Baill...

Formation of the Accumulative Human Metabolite and Human-Specific Glutathione Conjugate of Diclofenac in TK-NOG Chimeric Mice with Humanized Livers

“…The chemical structure of M4 was proposed as a 29-hydroxy-39-glutathionyl analog based on the assumption that this monochlorinated metabolite was generated via diclofenac-29,39-oxide, because M4 had not been detected in the incubation mixture of II and NADPH-fortified human liver microsomes. On the other hand, 49-hydroxy-29-(glutathion-S-yl)monoclofenac was proposed by Yu et al (2005), and designated as M5 later (Boerma et al, 2014). Similar to M4, M5 was generated in an incubation mixture of diclofenac and human liver microsomes fortified with glutathione, and its chemical structural assignment was obtained via comparison of mass and 1 H NMR spectra with those of a chemically synthesized authentic standard.…”

“…Monochlorinated metabolites M4 and M5 have been previously detected in incubation mixtures of diclofenac with human liver microsomes fortified with glutathione (Yan et al, 2005;Yu et al, 2005). Following oral administration of diclofenac to a control TK-NOG mouse, no glutathione conjugates at m/z 583 were detected in the bile.…”

“…When the ion at m/z 583 was subjected to CID (Fig. 7A) Yan et al (2005) for M4 and Yu et al (2005) for M5. To determine that this biliary metabolite is M4 or M5, further studies including isolation of the metabolite from the bile sample and subsequent NMR analyses are inevitable.…”

“…Experiments using recombinant enzymes and specific inhibitors demonstrated that CYP2C9 was involved in the formation of M4, and this metabolite was not detected in incubations with either rat or monkey liver microsomes. Around the same time, Yu et al (2005) reported another monochlorinated glutathione conjugate with the same molecular weight, 49-hydroxy-29-(glutathion-S-yl)monoclofenac (M5), followed by the discovery of the latest glutathione conjugate, 29-(glutathion-S-yl) monoclofenac (M6) (Wen et al, 2008;Boerma et al, 2012). Although M4, M5, and M6 are generated in in vitro studies using recombinant enzymes or liver microsomes fortified with glutathione, in vivo formation of these metabolites has not been previously reported.…”

Rapid detection and identification of N-acetyl-L-cysteine thioethers using constant neutral loss and theoretical multiple reaction monitoring combined with enhanced product-ion scans on a linear ion trap mass spectrometer

“…This approach permits the preselection of an analyte group [9]. The preselection criterion may be a specific product ion such as m/z 113 in glucuronides, [10] a specific neutral loss such as 162 Da involving a glucose substructure as present in glycosides [9], or the neutral loss of 129 Da of glutathione metabolites [11]. The choice of transition determines the choice of the survey scan mode.…”