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2.3. Human Leukocyte Antigen (HLA) Class II Binding Assay and Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) Detection of Ximelagatran Bound to HLA-DR Molecules

To reconfirm the inhibitory effect of ximelagatran on the binding of the ligand peptide to HLA-DRB1*07:01 [], a DELFIA-based binding assay was conducted. As previously reported [], ximelagatran inhibited the binding of the ligand peptide to HLA-DRB1*07:01, whereas no inhibitory effect was observed for HLA-DRB1*01:01 and DRB1*15:01 (). LC-MS/MS analysis of the same incubation samples revealed that the presence of HLA-DRB1*01:01 significantly increased the concentration of ximelagatran extracted from HLA class II binding assay samples (). Although the differences were not significant for HLA-DRB1*07:01 and DRB1*15:01, they appeared to be marginally significant (p = 0.087 and p = 0.084, respectively). Taken together, ximelagatran bound to all three HLA-DR molecules was detected in this setting. In contrast, the comparison between ximelagatran concentrations in (1) the presence of HLA-DR molecules and the ligand peptides; and (2) the presence of HLA-DR molecules and absence of the ligand peptides showed that the extent of the decrease of ximelagatran bound to the HLA-DR molecules by the addition of the ligand peptides was different between the three HLA-DR alleles (). The difference was significant (p < 0.05) for HLA-DRB1*01:01 and was marginally significant (p = 0.076) for DRB1*15:01, but just the overall trend for DRB1*07:01.

3. Discussion

In order to evaluate the possible direct interaction of ximelagatran with the peptide binding groove of HLA-DRB1*07:01, a series of in silico simulations and in vitro measurement of ximelagatran bound to HLA-DR molecules were performed.

3. Discussion

Taken together, these results indicate that ximelagatran directly interacts with the peptide binding groove of HLA-DRB1*07:01 and competes with ligand peptides for this binding site. In the cases of abacavir for HLA-B*57:01 [,], and lapatinib for HLA-DRB1*07:01 [], in both of which drugs increase the loading of ligand peptides, it is easy to imagine the immune stimulation by the enhanced antigen presentation and/or the presentation of neo-antigens. Unlike these cases, it may be somewhat difficult to associate the inhibited antigen presentation by ximelagatran with the immune stimulation. However, considering the simulated binding mode of ximelagatran at the peptide binding groove of HLA-DRB1*07:01, there is a possibility that there are some kinds of peptides that are loaded onto HLA-DRB1*07:01 de novo and/or in a different manner in the presence of ximelagatran. Although further investigations are needed to reveal the conclusive mechanism, the present study strongly supports the interaction between ximelagatran and the peptide binding groove of HLA-DRB1*07:01, which could alter the immune response in some way and lead to the idiosyncratic ximelagatran-induced hepatotoxicity. In vitro studies using hepatocyte co-cultured with macrophages or lymphocytes carrying various HLA alleles will support our hypothesis and give us additional information to understand the detailed mechanism of ximelagatran-induced hepatotoxicity.

2.1. Docking Simulations

The interaction modes of ximelagatran or melagatran with HLA-DRB1*07:01 and control alleles DRB1*01:01 and DRB1*15:01, both of which are associated with IDTs caused by other drugs, were predicted by in silico docking stimulations. The binding affinities were judged by a scoring function of generalized-Born volume integral/weighted surface area (GBVI/WSA_dG), which is considered to express protein-ligand binding free energy []. The lowest GBVI/WSA_dG values (kcal/mol) of the complexes between ximelagatran or melagatran and the three HLA-DR molecules are shown in . These values are much lower than those of the complexes between HLA-B*14:02 and nevirapine-related compounds [], and ones between HLA-B*58:01 and allopurinol-related compounds [] simulated by the same software system, indicating ximelagatran and melagatran have high potential to interact with these HLA molecules. The binding modes of ximelagatran and melagatran at the peptide binding grooves of HLA-DRB1*01:01, DRB1*07:01, and DRB1*15:01 with the lowest GBVI/WSA_dG values in each complex are shown in and , respectively. In the complex with HLA-DRB1*07:01, the oxime form of ximelagatran showed a higher binding affinity than the hydroxylamine form. Ximelagatran showed higher simulated binding affinities than melagatran for all three HLA-DR molecules.

1. Introduction

Ximelagatran (Exanta®/Exarta®) is an orally-available anticoagulant agent that directly inhibits thrombin and was developed for the prevention and treatment of thromboembolism. Although ximelagatran was generally well tolerated in short-term use (<12 days), an elevated serum alanine aminotransferase (ALT) level of >3× upper limit of normal was found to develop during long-term treatment (>35 days) in 7.9% of patients []. Due to the concerns for its potential liver toxicity, the development of ximelagatran was terminated and it was withdrawn from the world market in 2006. A retrospective case-control pharmacogenetic study of ALT elevation during long-term treatment of ximelagatran revealed a strong genetic association between elevated ALT and the HLA alleles DRB1*07 and DQA1*02, suggesting a possible immune-mediated pathogenesis []. Furthermore, a competitive binding assay revealed that ximelagatran was able to inhibit the binding of the ligand peptide to HLA-DRB1*07:01, supporting the specific involvement of HLA-DRB1*07:01 in the idiosyncratic ximelagatran-induced hepatotoxicity [].