Analysis of drug-protein adducts and their role in drug hypersensitivity

Abstract

Idiosyncratic drug reactions are extremely severe and are not attributed to the normal pharmacology of a drug. Elucidating their toxicological mechanisms have been challenging. In recent years, idiosyncratic drug-induced liver injury (iDILI) has been linked with the immune system proposing that tissue damage may be mediated by the cytotoxicity of T-cells. Furthermore, several associations have been found with iDILI and gene polymorphisms which code for human leukocyte antigen (HLA). The hapten model of drug hypersensitivity proposes that a drug-modified peptide presented by HLA may interact with T-cells. Therefore, drug-protein adducts may be uniquely placed to interact with risk HLA alleles. However, this link has not yet been defined experimentally. The aims of this thesis were to define the role of T-cells in several forms of iDILI, to define the chemistry of drug-protein adducts and ultimately to link these adducts to risk HLA alleles and T-cell activation. The antibiotic treatment co-amoxiclav is the most common cause of iDILI and it has been linked with several HLA alleles including the DR15 haplotype. Three patients with co-amoxiclav-induced liver injury were recruited and PBMCs were isolated to examine the presence of drug-reactive T-cells in the memory T-cell population. Amoxicillin-reactive T-cells were characterised from all three patients displaying a mixture of T-cell phenotypes. Furthermore, T-cells were activated in a processing dependent manner suggesting the potential for hapten mediated T-cell activation. However, T-cells were not restricted to any specific HLA allele. Amoxicillin-protein adducts were detected, using mass spectrometric and proteomics methods in the serum of drug tolerant patients where the covalent modification of several lysine residues was detected on HSA. When incorporated into T-cell assays, amoxicillin-modified HSA was not able to activate patient T-cells. These reactions were therefore investigated at the peptide level to elucidate the interaction of amoxicillin-protein adducts with specific HLA alleles. Drug-modified peptides were designed containing lysine to bind amoxicillin and anchor residues to bind DR15 risk HLA alleles to study haptens in co-amoxiclav iDILI. Peptides were purified to remove free drug and were fully characterised using mass spectrometry. Positional derivatives were generated where the amoxicillin-modified lysine was placed in different positions on the peptide backbone. Upon examining the memory T-cell population from a co-amoxiclav patient who was positive for DR15, amoxicillin-modified peptide-specific T-cells were detected. T-cells responded with specificity for the location of amoxicillin modification on the peptide backbone. Furthermore, amoxicillin-modified peptides were restricted to risk HLA alleles of the DR15 haplotype. The drugs terbinafine and ticlopidine are associated with the HLA-A*33 serotype in iDILI patients, despite their unique pharmacological mechanism and chemistry. We sought to define the mechanistic potential of these drugs to activate the immune system and elucidate whether they follow similar or different toxicological mechanisms. Terbinafine can be bioactivated to the chemically reactive aldehyde TBF-A. We were unable to isolate TBF-A or terbinafine reactive T-cells; therefore, we sought to define the chemistry of TBF-A to be able to eventually incorporate drug-protein adducts into future T-cell assays. TBF-A was shown to bind to small molecules, whole proteins and model peptides via adducts with several amino acids including cysteine, histidine and tyrosine. To investigate T-cell responses to ticlopidine, naive T-cells were primed from healthy donors positive for the risk HLA allele. T-cells isolated from positively primed donors demonstrated ticlopidine-specific T-cell activation. Furthermore, T-cells were activated in a direct, processing independent manner and were restricted to the risk HLA allele. Several drugs can cause severe hypersensitivity reactions without the need for presentation via specific HLA alleles. Elucidating the chemistry of adducts in these haptens may help define why compounds such as the nitroso metabolite of sulfamethoxazole, SMX-NO, are such potent immunogens. Here, we sought to detect the presence of SMX-NO adducts in drug-tolerant patients. An in vitro characterisation of SMX-NO adducts revealed promiscuous binding to cysteine, lysine and tyrosine in addition to the detection of oxidised lysine residues. Similar adducts were also detected in patient sera. Herein, these data have examined several reactive drugs or metabolites and their associated drug-protein adducts. We have demonstrated that adducts can be presented as drug-modified peptides to activate T-cells in a highly specific HLA-restricted manner. Furthermore, the formation of these adducts may drive several co-signals required for the priming and activation of T-cells. These studies lay the foundations for future studies to elucidate precise protein targets implicated in drug hypersensitivity.