HLA-DR7 and HLA-DQ2: Transgenic mouse strains tested as a model system for ximelagatran hepatotoxicity

Abstract

The oral thrombin inhibitor ximelagatran was withdrawn in the late clinical trial phase because it adversely affected the liver. In approximately 8% of treated patients, drug-induced liver injury (DILI) was expressed as transient alanine transaminase (ALT) elevations. No evidence of DILI had been revealed in the pre-clinical in vivo studies. A whole genome scan study performed on the clinical study material identified a strong genetic association between the major histocompatibility complex alleles for human leucocyte antigens (HLA) (HLA-DR7 and HLA-DQ2) and elevated ALT levels in treated patients. An immune-mediated pathogenesis was suggested. Here, we evaluated whether HLA transgenic mice models could be used to investigate whether the expression of relevant HLA molecules was enough to reproduce the DILI effects in humans. In silico modelling performed in this study revealed association of both ximelagatran (pro-drug) and melagatran (active drug) to the antigen-presenting groove of the homology modelled HLA-DR7 molecule suggesting "altered repertoire" as a key initiating event driving development of DILI in humans. Transgenic mouse strains (tgms) expressing HLA of serotype HLA-DR7 (HLA-DRB1*0701, -DRA*0102), and HLA-DQ2 (HLA-DQB1*0202,-DQA1*0201) were created. These two lines were crossed with a human (h)CD4 transgenic line, generating the two tgms DR7xhCD4 and DQ2xhCD4. To investigate whether the DILI effects observed in humans could be reproduced in tgms, the mice were treated for 28 days with ximelagatran. Results revealed no signs of DILI when biomarkers for liver toxicity were measured and histopathology was evaluated. In the ximelagatran case, presence of relevant HLA-expression in a pre-clinical model did not fulfil the prerequisite for reproducing DILI observed in patients. Nonetheless, for the first time an HLA-transgenic mouse model has been investigated for use in HLA-associated DILI induced by a low molecular weight compound. This study shows that mimicking of genetic susceptibility, expressed as DILI-associated HLA-types in mice, is not sufficient for reproducing the complex pathogenesis leading to DILI in man.

Fig 1. Identified antigen-binding groove binding-site.

Antigen-binding groove binding-site identification of homology modelled HLA-DR7 using SiteMap (A–front view, B–top view). Blue–hydrogen bond donor region, red–hydrogen bond acceptor region, yellow–hydrophobic region, white–binding site grid.

Fig 2. Verification of normal and comparable profiles between wt and tg mice.

Representation of lymphoid cell subsets from PBMC (A) and spleen (B) to compare wt and tg mice using mouse specific tracer antibodies. *^/** significant difference, * p≤0.05 ** p≤0.01.

Fig 3. Representative surface-marker expression on cells from wt and tg mice.

A and B illustrate the expression surface markers hCD4 and HLA DR/DQ on PBMCs, respectively. C displays the T-cell population and shows that hCD4 in the tgms is almost exclusively expressed on T-cells also expressing mCD4.

Fig 4. ALT levels in animals treated with ximelagatran for 28 days.

ALT levels in mice compared before and after 28 days of ximelagatran treatment. A fold change of one equals no change between start of treatment and end of treatment; fold change of two equals two times higher ALT levels.

Fig 5. Structures of drug in HLA-DR7 model.

Docked structure of melagatran (A–brown) and ximelagatran (B–blue) in the antigen-binding groove of the homology modelled HLA-DR7.