Covalent adduction of carbamazepine 10, 11-epoxide with human serum albumin and glutathione S-transferase pi: implications for carbamazepine hypersensitivity

Abstract

BackgroundCarbamazepine (CBZ) is an effective antiepileptic drug. However, it can cause cutaneous adverse reactions in up to 10% of patients. These reactions include life-threatening conditions such as Stevens-Johnson syndrome and toxic epidermal necrolysis. It has been hypothesised that CBZ is metabolised to reactive metabolites that form haptens with cellular proteins, which are then presented by specific HLA alleles, HLA-B*1502 and HLA-A*3101, activating the immune system and triggering hypersensitivity in susceptible individuals. CBZ 10, 11-epoxide (CBZ-E) is a major circulating metabolite of CBZ and retains therapeutic activity. It is also sufficiently electrophilic to react with glutathione and proteins in vitro. However, the structures of the protein adducts were unknown. Our aim was to determine the structure of these CBZ-E protein adducts using high sensitivity proteomics tools. MethodsInitially, synthetic CBZ-E was incubated with human glutathione S-transferase pi (hGSTP) or human serum albumin (HSA). Both proteins form adducts with various reactive chemicals. Subsequently, to determine whether metabolically generated CBZ-E could also form protein adducts, CBZ was incubated with human supersomes (CYP3A4, CYP2E1, CYP2B6) and hGSTP. Tryptic peptide analysis by advanced liquid chromatography-tandem mass spectrometry was used to characterise CBZ-E adduct formation. FindingsAn adduct was identified at cys-47 of hGSTP, and adducts were identified at his146 and his338 of HSA after incubation with CBZ-E. After incubation of CBZ with human supersomes and hGSTP the dihydrohydroxy thioether adduct at cys47 of hGSTP was again identified, indicating that metabolic activation of CBZ to CBZ-E in vivo might also yield stable protein adducts. InterpretationWe have shown for the first time, to our knowledge, that CBZ-E, an abundant CBZ metabolite in man, can form covalent adducts with hGSTP and HSA in vitro. These adducts and similar adducts of other proteins could be responsible for triggering hypersensitivity reactions. Further work will focus on confirming the presence of HSA-CBZ-E adducts in patients and determining the immunogenicity of these adducts in vitro. FundingUK Medical Research Council, ICON, GlaxoSmithKline, AstraZeneca, the Medical Evaluation Unit.