Immune-mediated adverse drug reactions are difficult to predict and can be severe in nature. Recently observed genetic associations highlight the importance of specific human leukocyte antigen alleles in the development of certain reactions. The mechanisms underlying antigen formation and subsequent T-cell activation require further investigation. The drugs abacavir (HLA-B*57:01) and ximelagatran (HLA-DRB*07:01 and HLA-DQA*02:01) represent compounds associated with skin and liver reactions respectively, for which a HLA association has been reported. In order to investigate the mechanism of HLA-restricted T-cell activation a cohort of 400 healthy volunteers was established. Both functional lymphocytes and DNA were isolated and stored. Following sequence-based HLA-typing twenty-six individuals expressing HLA-B*57:01 were identified (1 homozygote, 25 heterozygotes) and 101 individuals (10 homozygotes, 91 heterozygotes) expressing HLA-DRB*07:01 were identified. T-cells from these volunteers were utilised in in vitro assays. The first assays employed had low sensitivity and were unable to detect any drug-specific T-cells either by proliferation or cytokine secretion. Seventy-four CD8+ abacavir-specific T-cell clones however were generated from 3/3 volunteers expressing HLA-B*57:01. These clones secreted an array of cytokines and cytotoxic mediators (IFN-γ, Granzyme B, perforin, Fas ligand) in response to drug incubation. Chemically reactive metabolites are frequently associated with adverse drug reactions. The metabolism of abacavir in both liver and immune cell preparations was therefore assessed. In human liver cytosol, abacavir was metabolised to three isomeric carboxylic acids (48±15% turnover at 20h). This reaction proceeded via a reactive aldehyde metabolite that could be trapped with methoxylamine. Metabolism was blocked by the addition of 4-methylpyrazole, an alcohol dehydrogenase inhibitor (1000µM = 91.7±3.9% inhibition; p<0.05). Low-level carboxylic acid formation could be detected in an S9 fraction (2%) and cytosol (3.6%) generated from antigen-presenting cells suggesting that T-cell clones are exposed to a small amount of the aldehyde metabolite during cellular assays. The parent drug however is the predominant chemical entity present. Cross-reactivity at the MHC-TCR interface was determined with a number of abacavir analogues. The interaction was found to be highly specific with only deuterated abacavir stimulating T-cell clones at a similar level to abacavir. Dihydro abacavir only stimulated clones when used at high concentrations. The alternative enantiomer of abacavir (1S,4R) and carbovir did not stimulate any clones at any concentration investigated. This work further highlights the involvement of T-cells in drug hypersensitivity reactions and the importance of previously described HLA associations. In addition, chemical restriction exists at the HLA-TCR interface and is key to the activation of abacavir-specific T-cell clones. The metabolism of abacavir to a protein-reactive metabolite can occur in antigen-presenting cells therefore the potential of reactive intermediates to activate T-cells in a HLA-restricted manner should be investigated.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:579332 |
| Date | January 2012 |
| Creators | Bell, Catherine |
| Contributors | Park, Kevin; Naisbitt, Dean |
| Publisher | University of Liverpool |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://livrepository.liverpool.ac.uk/10595/ |
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