Carbamazepine Hypersensitivity: Linking Metabolism to the Immune Response

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

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

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 hypothesised, 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 the subjects. The relative amount of adduct was moderately positively correlated with plasma concentrations of CBZ (r2 = 0.44, p = 0.002) and CBZE (r2 = 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 merits further immunological investigation in hypersensitive patients

The HLA-A*31:01 allele: influence on carbamazepine treatment

Vincent Lai Ming Yip,1,2 Munir Pirmohamed1,2 1MRC Centre for Drug Safety Science, Institute of Translational Medicine, Department of Molecular and Clinical Pharmacology, University of Liverpool, 2Department of Clinical Pharmacology,The Royal Liverpool and Broadgreen University Hospital NHS Trust, Liverpool, UK Abstract: Carbamazepine (CBZ) is an effective anticonvulsant that can sometimes cause hypersensitivity reactions that vary in frequency and severity. Strong associations have been reported between specific human leukocyte antigen (HLA) alleles and susceptibility to CBZ hypersensitivity reactions. Screening for HLA-B*15:02 is mandated in patients from South East Asia because of a strong association with Stevens–Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN). HLA-A*31:01 predisposes to multiple phenotypes of CBZ hypersensitivity including maculopapular exanthema, hypersensitivity syndrome, and SJS/TEN in a range of populations including Europeans, Japanese, South Koreans and Han Chinese, although the effect size varies between the different phenotypes and populations. Between 47 Caucasians and 67 Japanese patients would need to be tested for HLA-A*31:01 in order to avoid a single case of CBZ hypersensitivity. A cost-effectiveness study has demonstrated that HLA-A*31:01 screening would be cost-effective. Patient preference assessment has also revealed that patients prefer pharmacogenetic screening and prescription of alternative anticonvulsants compared to current standard of practice without pharmacogenetic testing. For patients who test positive for HLA-A*31:01, alternative treatments are available. When alternatives have failed or are unavailable, HLA-A*31:01 testing can alert clinicians to 1) patients who are at increased risk of CBZ hypersensitivity who can then be targeted for more intensive monitoring and 2) increase diagnostic certainty in cases where hypersensitivity has already occurred, so patients can be advised to avoid structurally related drugs in the future. On the basis of the current evidence, we would favor screening all patients for HLA-A*31:01 and HLA-B*15:02 prior to starting CBZ therapy. Keywords: carbamazepine, oxcarbazepine, hypersensitivity, adverse drug reaction, pharmacogenetics, HL