ABSTRACT:Carboxylic acid-containing nonsteroidal anti-inflammatory drugs (NSAIDs) can be metabolized to chemically reactive acyl glucuronide and/or S-acyl-CoA thioester metabolites capable of transacylating GSH. We investigated the metabolism of the NSAID mefenamic acid (MFA) to metabolites that transacylate GSH, leading to MFA-S-acyl-GSH thioester (MFA-SG) formation in incubations with rat and human hepatocytes and in vivo in rat bile. Thus, incubation of MFA (1-500 M) with rat hepatocytes led to the detection of MFA-1-␤-O-acyl glucuronide (MFA-1-␤-O-G), MFA-S-acylCoA (MFA-SCoA), and MFA-SG by liquid chromatography-tandem mass spectrometric analysis. The C max of MFA-SG (330 nM; 10-min incubation with 100 M MFA) was 120-to 1400-fold higher than the C max of drug S-acyl-GSH adducts detected from studies with other carboxylic acid drugs to date. MFA-SG was also detected in incubations with human hepatocytes, but at much lower concentrations. Inhibition of MFA acyl glucuronidation in rat hepatocytes had no effect on MFA-SG formation, whereas a 58 ؎ 1.7% inhibition of MFA-SCoA formation led to a corresponding 66 ؎ 3.5% inhibition of MFA-SG production. Reactivity comparisons with GSH in buffer showed MFA-SCoA to be 80-fold more reactive than MFA-1-␤-O-G forming MFA-SG. MFA-SG was detected in MFAdosed (100 mg/kg) rat bile, where 17.4 g was excreted after administration. In summary, MFA exhibited bioactivation in rat and human hepatocytes and in vivo in rat, leading to reactive acylating derivatives that transacylate GSH. The formation of MFA-SG in hepatocytes was shown not to be mediated by reaction with MFA-1-␤-O-G, and not solely by MFA-SCoA, but perhaps also by intermediary MFA-acyladenylate formation, which is currently under investigation.

ABSTRACT:Phenylacetic acid (PAA) represents a substructure of a class of nonsteroidal anti-inflammatory carboxylic acid-containing drugs capable of undergoing metabolic activation in the liver to acylcoenzyme A (CoA)-and/or acyl glucuronide-linked metabolites that are proposed to be associated with the formation of immunogenic, and hence potentially hepatotoxic, drug-protein adducts. Herein, we investigated the ability of PAA to undergo phenylacetyl-S-acyl-CoA thioester (PA-CoA)-mediated covalent binding to protein in incubations with freshly isolated rat hepatocytes in suspension. Thus, when hepatocytes were incubated with phenylacetic acid carboxy-14 C (100 M) and analyzed for PA-CoA formation and covalent binding of PAA to protein and over a 3-h time period, both PA-CoA formation and covalent binding to protein increased rapidly, reaching 1.3 M and 291 pmol equivalents/mg protein after 4 and 6 min of incubation, respectively. However, the covalent binding of PAA to protein was reversible and decreased by 72% at the 3-h time point. After 3 h of incubation, PAA was shown to be metabolized primarily to phenylacetyl-glycine amide (84%). No PAA-acyl glucuronide was detected in the incubation extracts. PA-CoA reacted readily with glutathione in buffer, forming PA-Sacyl-glutathione; however, this glutathione conjugate was not detected in hepatocyte incubation extracts. Coincubation of hepatocytes with lauric acid led to a marked inhibition of PA-CoA formation and a corresponding inhibition of covalent binding to protein. SDS-polyacrylamide gel electrophoresis analysis showed the formation of two protein adducts having molecular masses of ϳ29 and ϳ33 kDa. In summary, PA-CoA formation in rat hepatocytes leads to the highly selective, but reversible, covalent binding to hepatocyte proteins, but not to the transacylation of glutathione.

ABSTRACT:Flunoxaprofen (FLX) is a chiral nonsteroidal anti-inflammatory drug that was withdrawn from clinical use because of concerns of potential hepatotoxicity. FLX undergoes highly stereoselective chiral inversion mediated through the FLX-S-acyl-CoA thioester (FLX-CoA) in favor of the (R)-(؊)-isomer. Acyl-CoA thioester derivatives of acidic drugs are chemically reactive species that are known to transacylate protein nucleophiles and glutathione (GSH). In this study, we investigated the relationship between the stereoselective metabolism of (R)-(؊)-and (S)-(؉)-FLX to FLX-CoA and the subsequent transacylation of GSH forming FLX-S-acyl-glutathione (FLX-SG) in incubations with rat hepatocytes in suspension. Thus, when hepatocytes (2 million cells/ml) were treated with (R)-(؊)-or (S)-(؉)-FLX (100

ABSTRACT:The 2-methyl substituted indole, 2MI [2-(4-(4-(2,4-dichlorophenylsulfonamido)-2-methyl-1H-indol-5-yloxy)-3-methoxyphenyl)acetic acid] is a potent dual inhibitor of 1) chemoattractant receptorhomologous molecule expressed on T-helper type-2 cells and 2) D-prostanoid receptor. During evaluation as a potential treatment for asthma and allergic rhinitis, 2MI was identified as a mechanism-based inactivator of CYP3A4 in vitro. The inactivation was shown to be irreversible by dialysis and accompanied by an NADPH-dependent increase in 2MI covalent binding to a 55-to 60-kDa microsomal protein, consistent with irreversible binding to CYP3A4. Two glutathione (GSH) adducts, G1 and G2, were identified in vitro, and the more abundant adduct (G1) was unambiguously determined via NMR to be GSH adducted to the 3-position of the 2-methylindole moiety. The potential for a clinical drug-drug interaction arising from mechanism-based inactivation of CYP3A4 by 2MI was predicted using a steady-state model, and a 4.3-to 7.5-fold increase in the exposure of midazolam was predicted at anticipated therapeutic concentrations. To better assess the potential for in vivo drug-drug interactions, the Sprague-Dawley rat was used as an in vivo model. An excellent in vitro-in vivo correlation was observed for the reduction in enzyme steady-state concentration (E ss/Ess ) as well as the change in the exposure of a prototypical CYP3A substrate, indinavir (area under the curve (AUC) for indinavir/AUC). In summary, 2MI was identified as a potent mechanism-based inactivator of CYP3A and was predicted to elicit a clinically relevant drug-drug interaction in humans at an anticipated therapeutic concentration.Prostaglandin D 2 , the major cyclooxygenase product formed in activated mast cells, is an important mediator of the inflammatory response associated with asthma. PGD 2 activity is mediated through activation of chemoattractant receptor-homologous molecule expressed on T-helper type-2 cells (CRTH2) and D-prostanoid (DP) receptor, and antagonism of CRTH2 and DP has been proposed as a potential therapeutic target for the treatment of asthma and allergic rhinitis (Pettipher, 2008). The 2-substituted methyl indole, 2MI [2-(4-(4-(2,4-dichlorophenylsulfonamido)-2-methyl-1H-indol-5-yloxy)-3-methoxyphenyl)acetic acid] (Fig. 1) is a potent dual DP and CRTH2 antagonist and a potential treatment for asthma and allergic rhinitis. Preliminary absorption, distribution, metabolism, and excretion studies revealed that it was also a potent mechanism-based inhibitor of CYP3A4.Mechanism-based inactivation (MBI) of CYP3A4 has been associated with clinically relevant drug-drug interactions (DDIs) (Zhou et al., 2005;Kalgutkar et al., 2007;Riley et al., 2007). Unlike competitive inhibition, metabolic activity after MBI is restored only after de novo enzyme synthesis (Silverman, 1995;Hollenberg, 2002), resulting in a prolonged reduction of enzyme activity. This irreversible inactivation occurs as the result of P450-mediated bioactivation of the parent compound to re...

Ibuprofen is metabolized to chemically reactive acyl glucuronide and S-acyl-CoA metabolites that are proposed to transacylate glutathione (GSH) forming ibuprofen-S-acyl-GSH (I-SG) in vivo. Herein, we report the detection of novel metabolites of ibuprofen, namely ibuprofen-N-acyl-cysteinylglycine