title: Carbamazepine Hypersensitivity: Linking Metabolism to the Immune Response
Creators: name: Yip, VLM
Publisher: name: University of Liverpool Repository
Year: 2016

description: Carbamazepine (CBZ) is an effective antiepileptic drug but has been associated with hypersensitivity reactions in up to 10% of patients. These reactions range from mild maculopapular exanthema to life-threatening conditions such as Stevens-Johnson syndrome and toxic epidermal necrolysis. The identification of CBZ-specific T cells and strong associations with specific human leukocyte antigen alleles provide evidence for immunological involvement. CBZ is extensively metabolised and forms several reactive metabolites. The aim of this thesis was to investigate the complex relationships between CBZ, its metabolism, the immune system, and genomics. Direct and microsomal incubations demonstrated that carbamazepine 10,11-epoxide (CBZE), the major metabolite of CBZ, formed a protein conjugate with human serum albumin (HSA) at His146. The same CBZE-modified HSA was also detected in patients tolerant of CBZ therapy. A second His146 CBZ-modified HSA adduct was identified in microsomal incubations, formed as a product of arene oxide providing the first chemical evidence that reactive metabolites of CBZ can modify soluble proteins. Healthy volunteers (n=8) and patients prescribed CBZ therapy (n=72) were recruited to investigate the influence of genetic variation on CBZ metabolism. Patient demographics and a mixture of rich and sparse pharmacokinetic (PK) samples were collected. Plasma levels of CBZ and four major metabolites were measured using a novel high performance liquid chromatography tandem mass spectrometric assay. There was significant variation in observed plasma concentrations of CBZ (14-fold) and its metabolites (approximately 30-fold). A population PK model was developed with nonlinear mixed effects modelling using the PK and clinical data collected from patients. Completion of autoinduction, total daily dosage and concomitant therapy with phenytoin were significant covariates that influenced the CBZ PK. Analysis of variance demonstrated that two single nucleotide polymorphisms (SNPs) in the gene ABCB1 and a single SNP in EPHX1 were significantly associated with altered plasma concentrations of CBZE. T cell clones (TCCs) were generated to CBZ and CBZE from two patients with a history of hypersensitivity to CBZ. All TCCs were CD4+ and secreted the cytokines IFN-γ, IL-13, granzyme B and perforin. TCC activation was MHC class II restricted and all TCCs were stimulated when CBZ was freshly added into the incubation mixture indicative of direct activation. A single TCC was activated when antigen presenting cells (APCs) were pulsed with CBZ indicative of a hapten mechanism. Transcriptomic analysis of peripheral blood mononuclear cells (PBMCs) from patients with a known history of CBZ hypersensitivity identified 9 CBZ/CBZE-specific mRNA and 39 CBZ/CBZE- miRNA transcripts. Pathway analysis mRNA and miRNA changes showed that antiviral response, psoriasis and inflammation were the most significant functions associated with exposure of cells from cases to CBZ and CBZE. In conclusion, these studies show that CBZ is transformed to stable and reactive metabolites, and these metabolites together with the parent drug, lead in susceptible individuals to an orchestrated response which involves transcriptional and immunological activation.