Journal of Pharmacological and Toxicological Methods
Original articleA simple in vitro model to study the stability of acylglucuronides
Introduction
UDP-glucuronosyltransferase (UGTs) catalyzes the transfer of glucuronic acid from UDP-glucuronic acid to a nucleophilic center of a substrate, resulting in a substrate conjugate that is joined by a 1-O-β-glycosidic bond. These phase II metabolites are generally accepted to be inactive metabolites that are readily excreted from the body. Acylglucuronide conjugates appear to be an exception to this general rule as they are chemically unstable in vitro and in vivo. The reactivity of these metabolites is due to the nucleophilic substitution at the ester group linking the aglycone and the glucuronic acid (Faed, 1984, Spahn-Langguth and Benet, 1992). The resultant ester bond undergoes an intramolecular rearrangement, in which the substrate moiety migrates away from the 1-O-β position, re-exposing the hemiacetal function (Spahn-Langguth & Benet, 1992). Further, acylglucuronides have been shown to form covalent adducts with proteins. Spahn-Langguth, Dahms, and Hermening (1996) and Bailey and Dickenson (1996) demonstrated that several carboxylate drugs formed protein adducts through an acylglucuronidation pathway causing potential toxicity. Thus, models for predicting the glucuronide-mediated toxicity of new chemical entities are desirable for drug discovery support programs. An excellent correlation was shown between the extent of covalent binding to albumin in vitro and the apparent first order degradation rates of the acylglucuronide with six drugs (Benet et al., 1993). This implies that unstable acylglucuronides have tendency to covalently bind to protein. However, studies assessing the stability of acylglucuronides require authentic β-1-O-acylglucuronide prepared synthetically or isolated from bile of animals following dosing of aglycone or isolated from microsomal incubation of aglycone. The preparation of authentic acylglucuronide is often the limiting factor for an early detection of highly reactive acylglucuronides of new chemical entities. Bolze et al. (2002) developed an in vitro screening model to determine the reactivity of acylglucuronide metabolites from carboxylic drugs. We developed an in vitro model, which is more reliable and simpler, since no β-glucuronidase is required.
Section snippets
Chemicals
Zomepirac, ibuprofen, gemfibrozil (Fig. 1), and UDPGA were purchased from Sigma Chemical Co. (St. Louis, MO). Zomepirac acyglucurode, ibuprofen acyglucurode, gemfibrozil acyglucurode, compounds A, B, C, and D, and their corresponding acyglucurodes were synthesized in Merck and Co., Inc. All other reagents were of analytical grade.
Rat liver microsomes
Rats were purchased from Harlan (Indianapolis, IN). Hepatic microsomes from male Sprague–Dawley rats were prepared by standard differential centrifugation methods (
Results and discussion
Assessing the stability of acylglucuronides requires authentic acylglucuronide prepared synthetically or isolated from bile of animals following dosing of aglycone or isolated from microsomal incubation of aglycone. This step is often the limiting factor for early detection of highly reactive acylglucuronides of new chemical entities. We developed a simple in vitro model to assess the stability of acylglucuronide that does not require preparation and purification of authentic β-1-O
References (8)
- et al.
Chemical and immunochemical comparison of protein adduct formation of four carboxylate drugs in rat liver and plasma
Chemical Research in Toxicology
(1996) - et al.
Predictability of the covalent binding of acidic drugs in man
Life Sciences
(1993) - et al.
Development of an in vitro screening model for the biosynthesis of acyl glucuronide metabolites and the assessment of their reactivity toward human serum albumin
Drug Metabolism & Disposition
(2002) - et al.
In vitro and in vivo trans-esterification of 1-[2(R)-(2-amino-2-methylpropionylamino)-3-(1H-indol-3-yl)propionyl]-3(S)-benzyl-piperidine-3-carboxylic acid ethyl ester and the effects of ethanol on its pharmacokinetics in rats
Pharmaceutical Research
(2004)
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Current address: Department of Drug Metabolism, Cardiovascular Therapeutics, Palo Alto, CA 94303, USA.