In vitro evaluation of cytotoxicity caused by carbamazepine and its metabolites in association to carbamazepine-induced hypersensitivity reactions

Carbamazepine (CBZ), an anticonvulsant and mood-stabilising drug, is known to cause delayed type hypersensitivity reactions. These reactions occur only in a minority of patients treated with the drug, but often result in severe clinical outcomes. Although an association between CBZ-induced hypersensitivity reactions and HLA alleles has been demonstrated, the underlying mechanism(s) of toxicity are poorly understood. Cell death caused by CBZ and one of its metabolites, 9-acridinecarboxaldehyde (9-AC) was investigated. CBZ did not show cytotoxic effects in concentrations ranging from sub-therapeutic to supra-therapeutic. By contrast, 9-AC caused apoptosis in the lymphoblastoid cell line (50µM and 24 hours of exposure) and primary PBMCs (50 µM and 2 hours of exposure). PBMCs from 20 CBZ-naïve individuals showed significant inter-individual variability in the susceptibility to the cytotoxic effect of 9-AC. To further investigate the observed inter-individual variability, 331 immortalised lymphoblast cell lines of unrelated individuals from 4 populations were exposed to CBZ, CBZ-10,11 epoxide or 9-AC and cell viability was measured after 24 hour exposure. Considerable inter-individual variability in the cytotoxic response was observed for all three compounds. The genome wide association study (GWAS) revealed two genetic polymorphisms in dual oxidase 1 (DUOX1) and RP11-354|13.2 that were linked to cell toxicity at low concentrations of all three compounds. A SNP in DUOX1 was investigated further because of its biological plausibility. Genotyping of 153 patients did not show an association between this SNP and CBZ-induced hypersensitivity in Caucasians. Due to the higher than normal frequency of the DUOX1 variant in non-Caucasian patients (20%), the involvement of DUOX1 in the predisposition to CBZ-induced hypersensitivity reactions in non-Caucasians could not be excluded. To elucidate how T cell activation occurs in CBZ-induced hypersensitivity reactions, the protein binding capability of CBZ and two of its metabolites, CBZ-10,11 epoxide (CBZE) and 9-AC, to human serum albumin and glutathione S-transferase π was assessed. Only CBZE was found to bind covalently to these proteins. For 9-AC, no covalent products were observed but an indication of reversible binding was detected. Finally, newly developed genotyping methods for HLA-A*31:01 were investigated in comparison to sequence based typing. The methods were based on SSP-PCR. One of the methods showed exact accordance with the current gold standard but PCR failed to amplify the gene of interest and the control gene in considerable amount of samples (13.1%), while the second SSP-PCR typing method showed less reliability. In conclusion, the mechanisms of CBZ hypersensitivity has been investigated using a number of approaches designed to elucidate bioactivation of the drug, and how cytotoxicity links with genetic factors. The genomic approach may have the potential to identify novel biomarkers, but needs further studies with larger sample size