Reactive Metabolites

One of the major driving forces behind the use of NMR in toxicology has no doubt been the regulatory pressure on pharmaceutical companies to fully characterize metabolites of novel drug candidates in animal species as part of drug evaluation submissions. An internal motivation for these companies is also to identify reactive or toxic metabolites of novel drug candidates at the earliest stage possible so as to minimize attrition in later, more expensive, stages of the development pipeline. Hyphenated LC-NMR-MS has developed into a powerful technique for analyzing metabolites of novel drugs from complex mixtures, and its applications are reviewed elsewhere [23, 24].

A key illustrative example of the use of NMR for identifying reactive metabolites is the characterization of certain reactive and volatile metabolites, which is impossible by LC-MS and only feasible by LC-NMR [33]. Ester glucuronide metabolites of many acidic drugs are unstable in aqueous solutions at pH 7.4, due to the susceptibility of the acyl groups to internal and external nucleophilic attack. The chemical reactivity is well established and has been implicated in adverse drug reactions due to protein binding to bile transporter proteins [33]. In brief, the initial drug P-1-O-acyl glucuronide is formed enzymatically in the body as a polar conjugate that is readily excreted in the urine or bile (depending on the molecular weight). However, the drug moiety can then internally acyl-migrate around the glucuronide ring from positions 1 to 4 to produce positional isomers which also mutarotate. Analysis of reactive drug P-1-O-acyl glucuronides in urine requires collecting samples over ice and adjusting to acidic pH to stabilize the parent conjugate. LC-NMR was used to identify the highly reactive a-1-O-acyl isomer of naproxen glucuronide, which was previously discounted in drug metabolism and pharmacokinetic studies. Dynamic stopped-flow LC-NMR provided key kinetic data that confirmed that the highly reactive a-1-O-acyl isomer is part of the overall kinetic scheme. As this is a general chemical reaction of carbohydrates, observed also for zomepirac, tolmetin, and difluni-

sal acyl glucuronides, the presence of this isomer in vivo is therefore possible. This finding may have toxicological implications in the reactivity of these metabolites toward important cellular proteins. Similarly, LC-NMR-MS may be used to identify other reactive conjugates, including glutathione pathway metabolites and acyl coenzyme A thioester conjugates.

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