A Novel Approach for Predicting Acyl Glucuronide Reactivity via Schiff Base Formation:  Development of Rapidly Formed Peptide Adducts for LC/MS/MS Measurements

Abstract

A novel technique to study the reactivity of acyl glucuronide metabolites to protein has been developed and is described herein. Considered here are acyl glucuronide metabolites, which have undergone the rearrangement of the glucuronic acid moiety at physiological temperature and pH. The investigation of the reactivity of these electrophilic metabolites was carried out by measuring the rate of reaction of rearranged AG metabolites in forming the corresponding acyl glucuronide-peptide adduct in the presence of Lys-Phe. This differs from the parallel technique used in forming AG adducts of proteins that have been previously reported. In the study described here, the Schiff base adduct, diclofenac acyl glucuronide-Lys-Phe product, was generated and structurally elucidated by liquid chromatography tandem mass spectrometry (LC/MS/MS) analysis. The product structure was proved to be a Schiff base adduct by chemical derivatization by nucleophilic addition of HCN and chemical reduction with NaCNBH(3), followed by LC/MS/MS analysis. It is proposed here that the degree of reactivity of acyl glucuronides as measured by covalent binding to protein is proportional to the amount of its peptide adduct generated with the peptide technique described. The application of this technique to the assessment of the degree of reactivity of acyl glucuronide metabolites was validated by developing a reactivity rank of seven carboxylic acid-containing drugs. Consistency was achieved between the ranking of reactivity in the peptide technique for these seven compounds and the rankings found in the literature. In addition, a correlation (R(2) = 0.95) was revealed between the formation of a peptide adduct and the rearrangement rate of the primary acyl glucuronide of seven tested compounds. A structure effect on the degree of reactivity has demonstrated the rate order: acetic acid > propionic acid > benzoic acid derivatives. A rational explanation of this order was proposed, based on the inherent electronic and steric properties of each specific aglycone. In addition, adaptation of this technique to automation in order to more rapidly assess the ranking of reactivity of acyl glucuronide covalent binding to proteins by new chemical entities is proposed.

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

“…It was hypothesized that the benzoic acid derivative demonstrates the lowest reactivity due to resonance stabilization provided by the aromatic moiety, and the isopropionic acid derivative displays a lower reactivity than that of acetic acid derivative, possibly due to the higher steric hindrance capacity of the isopropyl group over the acetyl group (Wang et al, 2004). In our study, most AGs from the warning and withdrawn drugs are acetic acid or isopropionic acid derivatives except for furosemide AG, mefenamic acid AG, and probenecid AG.…”

“…It was reported that a structure effect on the degree of AG reactivity demonstrated the rate order: acetic acid Ͼ isopropionic acid Ͼ benzoic acid derivatives in covalent binding study using a small peptide (Wang et al, 2004). It was hypothesized that the benzoic acid derivative demonstrates the lowest reactivity due to resonance stabilization provided by the aromatic moiety, and the isopropionic acid derivative displays a lower reactivity than that of acetic acid derivative, possibly due to the higher steric hindrance capacity of the isopropyl group over the acetyl group (Wang et al, 2004).…”

Crystal structure of bis(acetylacetone)-p-naphthalenediimine, C20H22N2O2

“…Much attention has been drawn to the Schiff bases because of their potential applications in areas such as drugs, catalysis and supramolecular functional materials [1][2][3][4]. Schiff bases usually act as multi-dentate ligands in the construction of intriguing frameworks driven by coordination interactions [5][6][7].…”

Zomepirac Acyl Glucuronide Is Responsible for Zomepirac-Induced Acute Kidney Injury in Mice

“…Because of their electrophilic nature and ability to cause substitution reactions with nucleophilic groups in proteins or other macromolecules, AGs can covalently modify endogenous proteins and have been postulated to cause the adverse toxicity associated with carboxylic acid-containing drugs (Faed, 1984;Boelsterli, 2002). To assess the toxicity of AGs, several in vitro assay systems, such as stability assay by measuring half-lives in potassium phosphate buffer, peptide adducts assay, and immunostimulation assay, have been proposed (Wang et al, 2004;Sawamura et al, 2010;Jinno et al, 2013;Miyashita et al, 2014;Iwamura et al, 2015). However, the toxicity of AGs has remained controversial because direct evidence of in vivo AG toxicity has not been provided.…”