Drug-induced liver injury (DILI) is of concern to the pharmaceutical industry, and reliable preclinical screens are required. Previously, we established an in vitro bile acid–dependent hepatotoxicity assay that mimics cholestatic DILI in vivo. Here, we confirmed that this assay can predict cholestatic DILI in clinical situations by comparing in vitro cytotoxicity data with in vivo risk. For 38 drugs, the frequencies of abnormal increases in serum alkaline phosphatase (ALP), transaminases, gamma glutamyltranspeptidase (γGT), and bilirubin were collected from interview forms. Drugs with frequencies of serum marker increases higher than 1% were classified as high DILI risk compounds. In vitro cytotoxicity was assessed by monitoring lactate dehydrogenase release from rat and human sandwich-cultured hepatocytes (SCRHs and SCHHs) incubated with the test drugs (50 μM) for 24 hours in the absence or presence of a bile acids mixture. Receiver operating characteristic analyses gave optimal cutoff toxicity values of 19.5% and 9.2% for ALP and transaminases in SCRHs, respectively. Using this cutoff, high- and low-risk drugs were separated with 65.4–78.6% sensitivity and 66.7–79.2% specificity. Good separation was also achieved using SCHHs. In conclusion, cholestatic DILI risk can be successfully predicted using a sandwich-cultured hepatocyte–based assay.

Genome-wide association studies indicate that several idiosyncratic adverse drug reactions are highly associated with specific human leukocyte antigen (HLA) alleles. For instance, abacavir, a human immunodeficiency virus reverse transcriptase inhibitor, induces multi-organ toxicity exclusively in patients carrying the HLA-B*57:01 allele. However, the underlying mechanism is unclear due to a lack of appropriate animal models. Previously, we developed HLA-B*57:01 transgenic mice and found that topical application of abacavir to the ears induced proliferation of CD8⁺ lymphocytes in local lymph nodes. Here, we attempted to reproduce abacavir-induced liver injury in these mice. However, oral administration of abacavir alone to HLA-B*57:01 transgenic mice did not increase levels of the liver injury marker alanine aminotransferase (ALT). Considering the importance of innate immune activation in mouse liver, we treated mice with CpG oligodeoxynucleotide (CpG-ODN), a toll-like receptor 9 agonist, plus abacavir. This resulted in a marked increase in ALT, pathological changes in liver, increased numbers of activated CD8⁺ T cells, and tissue infiltration by immune cells exclusively in HLA-B*57:01 transgenic mice. These results indicate that CpG-ODN-induced inflammatory reactions and/or innate immune activation are necessary for abacavir-induced HLA-mediated liver injury characterized by infiltration of CD8⁺ T cells. Thus, we developed the first mouse model of HLA-mediated abacavir-induced idiosyncratic liver injury. Further investigation will show that the proposed HLA-mediated liver injury model can be applied to other combinations of drugs and HLA types, thereby improving drug development and contributing to the development of personalized medicine.

Idiosyncratic drug toxicity (IDT) associated with specific human leukocyte antigen (HLA) allotype is a rare and unpredictable life-threatening adverse drug reaction for which prospective mechanistic studies in humans are difficult. Here, we show the importance of immune tolerance for IDT onset and determine whether it is susceptible to a common IDT, HLA-B*57:01-mediated abacavir (ABC)-induced hypersensitivity (AHS), using CD4⁺ T cell-depleted programmed death-1 receptor (PD-1)-deficient HLA-B*57:01 transgenic mice (B*57:01-Tg/PD-1^−/−). Although AHS is not observed in B*57:01-Tg mice, ABC treatment increases the proportion of cytokine- and cytolytic granule-secreting effector memory CD8⁺ T cells in CD4⁺ T cell-depleted B*57:01-Tg/PD-1^−/− mice, thereby inducing skin toxicity with CD8⁺ T cell infiltration, mimicking AHS. Our results demonstrate that individual differences in the immune tolerance system, including PD-1^highCD8⁺ T cells and regulatory CD4⁺ T cells, may affect the susceptibility of humans to HLA-mediated IDT in humans.