Mass Spectrometric Characterization of Circulating Covalent Protein Adducts Derived from Epoxide Metabolites of Carbamazepine in Patients

Abstract

Carbamazepine (CBZ) is an effective antiepileptic drug that has been associated with hypersensitivity reactions. The pathogenesis of those reactions is incompletely understood but is postulated to involve a complex interplay between the drug's metabolism, genetic variation in human leukocyte antigens, and adverse activation of the immune system. Multiple T-cell activation mechanisms have been hypothesized including activation by drug-peptide conjugates derived from proteins haptenated by reactive metabolites. However, definitive evidence of the drug-protein adducts in patients has been lacking. In this study, mass spectrometry was used to characterize protein modifications by microsomally-generated metabolites of CBZ and in patients taking CBZ therapy. CBZ 10,11-epoxide (CBZE), a major electrophilic plasma metabolite of CBZ, formed adducts with glutathione-S-transferase pi (GSTP; Cys47) and human serum albumin (HSA; His146 and His338, but not Cys34) in vitro via notably divergent side-chain selectivity. Both proteins were adducted at the same residues by undefined monoxygenated metabolites ([O]CBZ) when they were incubated with human liver microsomes, NADPH and CBZ. There was also evidence for formation of a CBZ adduct at His146 and His338 of HSA derived via dehydration from an intermediate arene oxide adduct. Glutathione trapping of reactive metabolites confirmed microsomal production of CBZE and indicated simultaneous production of arene oxides. In 15 patients prescribed CBZ therapy, [O]CBZ-modified HSA (His146) was detected in all subjects. The relative amount of adduct was moderately positively correlated with plasma concentrations of CBZ (r = 0.44, p = 0.002) and CBZE (r = 0.35, p = 0.006). Our results have provided the first chemical evidence for microsomal production of [O]CBZ species that are able to escape the microsomal domain to react covalently with soluble proteins. This study has also demonstrated the presence of circulating [O]CBZ-modified HSA in patients without hypersensitivity reactions who were receiving standard CBZ therapy. The implications of those circulating adducts for susceptibility to CBZ hypersensitivity merit further immunological investigation in hypersensitive patients.

Mechanisms leading to T-cell activation in drug hypersensitivity

“…For example, the cytochrome P450 enzymes are targeted by many short-lived reactive metabolites, and haptenation of these proteins can lead to both drug-specific and auto-immune responses [10]. Stable metabolites that are able to escape from the liver can also haptenate circulating proteins, for example, carbamazepine 10-11 epoxide was found to bind to albumin in patients receiving carbamazepine therapy [11]. Both haptenic structures and carrier proteins are vital for T-cell and antibody recognition, however, haptenic structures have a dominant role in both events.…”

Evaluation of clinical and genetic factors in the population pharmacokinetics of carbamazepine

“…CBZ and metabolites in plasma samples were assayed by liquid chromatography-tandem mass spectrometry using the method reported in Yip et al 24 The calibration ranges for the analytes were as follows: CBZ, 0. This article is protected by copyright.…”

“…1), which is pharmacologically active 2 and electrophilic. 5 Other metabolites include 2-hydroxycarbamazepine (2OH-CBZ), 3-hydroxycarbamazepine (3OH-CBZ), 4 2,3-dihydroxycarbamazepine, and the o-quinone of the catechol. 2 Multiple cytochrome P450 (CYP) isoforms are involved in the formation of these metabolites.…”

“…Those metabolites were chosen because they are chemically reactive (CBZE) or their generation is via reactive arene oxide intermediates (2OH-CBZ and 3OH-CBZ). 24 The model was used to investigate the influence of physiological factors, concomitant medications, and genetic variation in enzymes and transporters associated with the metabolism and disposition of CBZ.…”

The Mechanistic Differences in HLA-Associated Carbamazepine Hypersensitivity

“…The clinical symptoms of patients suffering from CBZ-mediated ADRs range from milder skin exanthemas to severe and life-threatening syndromes [33]. They comprise maculopapular exanthema (MPE) where patients exhibit a rash with macules or erythematous and maculopapular lesions as well as cutaneous ADRs involving multiple organs.…”