Metabolic activation of carboxylic acids

118

134

ABSTRACT:Acyl glucuronides (AGs) formed from carboxylic acid-containing drugs have been considered to be a cause of idiosyncratic drug toxicity (IDT). Chemical stability of AGs is supposed to relate to their reactivity. In this study, the half-lives of 21 AGs of carboxylic drugs in potassium phosphate buffer (KPB), human serum albumin (HSA) solution, and human fresh plasma were analyzed in relation to the IDT risk derived from these drugs. The carboxylic drugs were classified into three safety categories of "safe," "warning," and "withdrawn" in terms of their IDT risk. As for the results, the half-lives of AGs in KPB correlated with the IDT risk better than those in HSA solution or in human fresh plasma with regard to the separation of the safe drugs from the warning drugs or the withdrawn drugs. In KPB, whereas the half-lives in the safe category were 7.2 h or longer, those in the withdrawn category were 1.7 h or shorter. The classification value of the half-life in KPB, which separated the safe drugs from the withdrawn drugs was calculated to be 3.6 h by regression analysis. In conclusion, this is the first report that clearly shows the relationship between the IDT risk and chemical stability of AGs in several in vitro systems. The KPB system was considered to be the best for evaluating the stability of AGs, and the classification value of the half-life in KPB serves as a useful key predictor for the IDT risk.

Section: Introductionmentioning

confidence: 99%

Predictability of Idiosyncratic Drug Toxicity Risk for Carboxylic Acid-Containing Drugs Based on the Chemical Stability of Acyl Glucuronide

Sawamura¹,

Okudaira²,

Watanabe³

et al. 2010

118

134

“…For a number of carboxylic acid-containing drugs associated with adverse allergic reactions, the toxicity is proposed to be linked to the metabolic activation of the carboxylic acid moiety through the formation of chemically reactive acyl glucuronides and/or acyl-coenzyme A (CoA) thioesters (Boelsterli, 2002;Skonberg et al, 2008), leading to the formation of immunogenic drug-protein adducts (Uetrecht, 2008). In the current study, the role of the chemically reactive acyl-CoA thioester metabolite in the formation of covalent adducts to hepatocyte protein was addressed.…”

mentioning

confidence: 99%

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

Grillo

Lohr²

2009

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.

“…Three possible metabolic pathways that might lead to the covalent binding to liver proteins have been proposed for carboxylic acids (Skonberg et al, 2008), as exemplified by diclofenac, a popular model compound for studies of liver protein adducts (Hargus et al, 1994). The first pathway that involves the formation of reactive acyl glucuronide has been shown to be responsible for covalent binding of BNX, FLX, and IBP to serum albumin in vitro Dong et al, 2005) and to plasma and liver tissue proteins in vivo in rat (Dong et al, 2005).…”

Section: Discussionmentioning

confidence: 99%

“…A third pathway that is possible for the bioactivation of NSAIDs is the formation of reactive acyl-CoA intermediate (Boelsterli, 2002;Skonberg et al, 2008). Practically all the carboxylic acids are thought to covalently modify proteins by this mechanism (Hertz and BarTana, 1988).…”

Section: Discussionmentioning

confidence: 99%

Glucuronidation and Covalent Protein Binding of Benoxaprofen and Flunoxaprofen in Sandwich-Cultured Rat and Human Hepatocytes

Dong

Smith²

2009

ABSTRACT:Benoxaprofen (BNX), a nonsteroidal anti-inflammatory drug (NSAID) that was withdrawn because of hepatotoxicity, is more toxic than its structural analog flunoxaprofen (FLX) in humans and rats. Acyl glucuronides have been hypothesized to be reactive metabolites and may be associated with toxicity. Both time-and concentration-dependent glucuronidation and covalent binding of BNX, FLX, and ibuprofen (IBP) were determined by exposing sandwich-cultured rat hepatocytes to each NSAID. The levels of glucuronide and covalent protein adduct measured in cells followed the order BNX > FLX > IBP. These results indicate that 1) BNXglucuronide (G) is more reactive than FLX-G, and 2) IBP-G is the least reactive metabolite, which support previous in vivo studies in rats. The proportional increases of protein adduct formation for BNX, FLX, and IBP as acyl glucuronidation increased also support the hypothesis that part of the covalent binding of all three NSAIDs to hepatic proteins is acyl glucuronide-dependent. Moreover, theses studies confirmed the feasibility of using sandwich-cultured rat hepatocytes for studying glucuronidation and covalent binding to hepatocellular proteins. These studies also showed that these in vitro methods can be applied using human tissues for the study of acyl glucuronide reactivity. More BNX-protein adduct was formed in sandwich-cultured human hepatocytes than FLX-protein adduct, which not only agreed with its relative toxicity in humans but also was consistent with the in vitro findings using rat hepatocyte cultures. These data support the use of sandwich-cultured human hepatocytes as an in vitro screening model of acyl glucuronide exposure and reactivity.Many types of acidic drugs form acyl glucuronides, and other xenobiotics are metabolized to carboxylic acids (Phase I metabolites), which subsequently undergo Phase II conjugation to form acyl glucuronides. Often such a glucuronide conjugate constitutes the major metabolite. The major site of conjugation for most compounds in humans is believed to be the liver. Modification of critical hepatic proteins by covalent binding of acidic drugs through reactive acyl glucuronides may provide a basis for direct hepatocyte toxicity or immune-mediated adverse reactions (Gillette, 1974;Faed, 1984;Boelsterli, 2002;Bailey and Dickinson, 2003).Some pharmaceutical companies have been conducting in vitro experiments by incubating acyl glucuronides with model proteins or peptides (Wang et al., 2004) to determine their reactivity, and hence possibly predict the relative extent of covalent protein binding in vivo in humans. This method requires chemical synthesis or biosynthesis of individual glucuronides for each drug candidate, a process that can be tedious and costly. In addition, hepatic tissue proteins are not present in the incubation, which makes it less likely to correlate covalent protein binding with hepatotoxicity. In vitro models involving hepatic materials, including liver homogenates, microsomal subcellular preparation, and isolated hepatocyte s...