Studies on the Metabolism of Tolmetin to the Chemically Reactive Acyl-Coenzyme A Thioester Intermediate in Rats

Abstract

ABSTRACT:Carboxylic acids may be metabolized to acyl glucuronides and acyl-coenzyme A thioesters (acyl-CoAs), which are reactive metabolites capable of reacting with proteins in vivo. In this study, the metabolic activation of tolmetin (Tol) to reactive metabolites and the subsequent formation of Tol-protein adducts in the liver were studied in rats. Two hours after dose administration (100 mg/kg i.p.), tolmetin acyl-CoA (Tol-CoA) was identified by liquid chromatography-tandem mass spectrometry in liver homogenates. Similarly, the acyl-CoA-dependent metabolites tolmetin-taurine conjugate (Tol-Tau) and tolmetin-acyl carnitine ester (Tol-Car) were identified in rat livers. In a rat bile study (100 mg/kg i.p.), the S-acyl glutathione thioester conjugate was identified, providing further evidence of the formation of reactive metabolites such as Tol-CoA or Tol-acyl glucuronide (Tol-O-G), capable of acylating nucleophilic functional groups. Three rats were treated with clofibric acid (150 mg/kg/day i.p. for 7 days) before dose administration of Tol. This resulted in an increase in covalent binding to liver proteins from 0.9 nmol/g liver in control rats to 4.2 nmol/g liver in clofibric acidtreated rats. Similarly, levels of Tol-CoA increased from 0.6 nmol/g to 4.4 nmol/g liver after pretreatment with clofibric acid, whereas the formation of Tol-O-G and Tol-Tau was unaffected by clofibric acid treatment. However, Tol-Car levels increased from 0.08 to 0.64 nmol/g after clofibric acid treatment. Collectively, these results confirm that Tol-CoA is formed in vivo in the rat and that this metabolite can have important consequences in terms of covalent binding to liver proteins.Drugs with a carboxylate functional group can be metabolized to chemically reactive metabolites that react with nucleophilic amino acids of proteins to form drug-protein adducts (Boelsterli, 2002;Bailey and Dickinson, 2003). Tolmetin (Tol; 5-[4Ј-methylbenzoyl]-1-methylpyrrole-2-acetic acid) is an example of a carboxylic acidcontaining drug which, in humans, is partly metabolized to the acyl glucuronide (Hyneck et al., 1987). In vitro studies have shown that the tolmetin-acyl glucuronide (Tol-O-G) is a reactive metabolite that reacts with the amino acid side chains of lysine, arginine, and serine present in human serum albumin (Ding et al., 1993(Ding et al., , 1995.Covalent binding of Tol has also been studied in humans, where Tol-protein adducts have been detected in plasma (Hyneck et al., 1988;Zia-Amirhosseini et al., 1994). Tol-protein adducts in plasma were believed to result from reaction of the acyl glucuronide with plasma proteins, since a good correlation between covalent plasmaprotein adducts and exposure to Tol-O-G was observed (Hyneck et al., 1988). In the liver, other metabolic pathways may contribute to Tol-protein adduct formation. In a recent study, it has been shown that the pyrrole moiety of Tol may undergo bioactivation to a reactive arene oxide (Chen et al., 2006). Other reports have shown that acyl-coenzyme A thioesters (acyl-CoAs) a...

Covalent Binding of Phenylacetic Acid to Protein in Incubations with Freshly Isolated Rat Hepatocytes

“…These results are consistent with an increasing number of reports showing that xenobiotic acyl-CoA thioesters are reactive electrophiles that contribute to covalent binding of carboxylic acid-containing compounds to liver proteins (Sallustio et al, 2000;Li et al, 2002a;Sidenius et al, 2004;Olsen et al, 2005Olsen et al, , 2007.…”

“…However, when we analyzed PAA rat hepatocyte incubation extracts for PA-SG, none of this conjugate could be detected, even with the use of a highly sensitive LC/MS/MS MRM detection method (subnanomolar sensitivity). We did not expect this result because we and others have been able to detect S-acyl-GSH conjugates for a number of carboxylic acid-containing drugs such as clofibric acid (Shore et al, 1995), zomepirac Olsen et al, 2005), tolmetin (Olsen et al, 2007), and ibuprofen (Grillo and Hua, 2008), which are known to form reactive S-acyl-CoA thioester intermediates. The lack of detection of PA-SG in rat hepatocyte incubation extracts may be in part caused by its rapid clearance from hepatocyte incubations, where PA-SG (1 M) was shown to be completely degraded after 6 min of incubation (degradation t 1/2 of ϳ1.7 min; Supplemental Data Fig.…”

Metabolic Activation of Mefenamic Acid Leading to Mefenamyl-S-Acyl-Glutathione Adduct Formation In Vitro and In Vivo in Rat

“…Thioester-linked GSH adducts are known metabolites of a range of carboxylic acid-containing drugs, e.g., clofibric acid , zomepirac (Grillo and Hua, 2003), diclofenac (Grillo et al, 2003a), tolmetin (Olsen et al, 2007), flunoxaprofen , and ibuprofen (Grillo and Hua, 2008). Mechanistic in vitro studies performed to investigate the relative contribution of acyl glucuronidation and S-acyl-CoA formation pathways leading to Sacyl-GSH thioester formation have indicated the S-acyl-CoA formation pathway to predominate in this regard, and therefore it may also be the bioactivation pathway predominating in vivo, leading to the acylation of protein nucleophiles by varied carboxylic acid-containing drugs.…”

Computer-Assisted HPLC Method Development for Determination of Tolmetin and Possible Kinetic Modulators of Its Oxidative Metabolism in Vivo

“…The non-steroidal anti-inflammatory drug tolmetin (TOL; 5-[4 0 -methylbenzolyl]-1-methylpyrrole-2-acetic acid) commonly used in the treatment of rheumatoid arthritis, osteoarthritis, and gout is subject to acyl glucuronidation [1], as well as oxidative transformation, where a thiol GSH adduct, namely 5-[4-methylbenzoyl]-1-methyl-3-glutathionylpyrrole-2-acetic acid (TOL-SG) has been identified in rat tissues treated with TOL [2]. Oxidative metabolism of TOL ( Fig.…”