Mass Spectrometric Characterization of Circulating Covalent Protein Adducts Derived from a Drug Acyl Glucuronide Metabolite: Multiple Albumin Adductions in Diclofenac Patients

Abstract

Covalent protein modifications by electrophilic acyl glucuronide (AG) metabolites are hypothetical causes of hypersensitivity reactions associated with certain carboxylate drugs. The complex rearrangements and reactivities of drug AG have been defined in great detail, and protein adducts of carboxylate drugs, such as diclofenac, have been found in liver and plasma of experimental animals and humans. However, in the absence of definitive molecular characterization, and specifically, identification of signature glycation conjugates retaining the glucuronyl and carboxyl residues, it cannot be assumed any of these adducts is derived uniquely or even fractionally from AG metabolites. We have therefore undertaken targeted mass spectrometric analyses of human serum albumin (HSA) isolated from diclofenac patients to characterize drug-derived structures and, thereby, for the first time, have deconstructed conclusively the pathways of adduct formation from a drug AG and its isomeric rearrangement products in vivo. These analyses were informed by a thorough understanding of the reactions of HSA with diclofenac AG in vitro. HSA from six patients without drug-related hypersensitivities had either a single drug-derived adduct or one of five combinations of 2-8 adducts from among seven diclofenac N-acylations and three AG glycations on seven of the protein's 59 lysines. Only acylations were found in every patient. We present evidence that HSA modifications by diclofenac in vivo are complicated and variable, that at least a fraction of these modifications are derived from the drug's AG metabolite, and that albumin adduction is not inevitably a causation of hypersensitivity to carboxylate drugs or a coincidental association.

Human mass balance study of TAS-102 using 14C analyzed by accelerator mass spectrometry

“…Therefore, the un-extractable radioactivity is not a human-specific observation. Covalent protein adduct formation of drug-related material is not uncommon, especially with the nonsteroidal anti-inflammatory drugs (NSAIDs) [30–37]. The non-extractable nature of drug-related radioactivity in plasma indicates that it is no longer available for free diffusion into tissues, which has been interpreted as a detoxification reaction, though this may not be completely innocuous because the haptenated proteins may be antigenic [38].…”

Approaches to the diagnosis and management of patients with a history of nonsteroidal anti-inflammatory drug–related urticaria and angioedema

“…Moreover, a glucuronide acyl modification of different lysine residues of albumin was reported for diclofenac, ketoprofen, and acetaminophen. 54,55 In vitro prediction of an SNIUAA reaction on this basis is not yet feasible. …”

Diclofenac and Its Acyl Glucuronide: Determination of In Vivo Exposure in Human Subjects and Characterization as Human Drug Transporter Substrates In Vitro

“…Therefore, one of the aims of the present study was to characterize both as substrates of liver and kidney transporters in vitro. In contrast to the rodent (Lagas et al, 2010), only limited data are available for DF-AG in human plasma (Hammond et al, 2014), with earlier studies largely focused on dose recovery and profiling of human excreta (John, 1979;. Therefore, a second aim of the study was to determine DF-AG exposure in normal healthy human volunteers following a single oral DF dose.…”