Studies on the mechanism of flucloxacillin-induced liver injury

Abstract

Drug induced liver injury (DILI) due to the isoxazolyl β-lactam antibiotic, flucloxacillin, is a rare idiosyncratic adverse drug reaction. The underlying mechanism remains unclear but a recent association with the human leukocyte antigen class I allele, HLA-B*57:01, indicated a possible T-cell mediated reaction. This study aimed to identify further genetic determinants conferring susceptibility to this form of DILI and to study metabolic and immune mechanisms of this toxicity. Flucloxacillin DILI cases (n=150) and matched population controls (n=282) were genotyped for HLA-B*57:01, confirming the previous association with disease (OR = 40.1, 95% CI 22.7 – 70.7). Cases negative for HLA-B*57:01 (n=26) were genotyped for HLA-B alleles and this analysis showed a borderline significant association with HLA- B*13:02 (p = 0.0376). Genotyping of all cases for additional immune-related candidate genes such as KIR3DL1/KIR3DS1 and for variants detected in limited exome sequencing studies, performed by others, resulted in confirmation of a significant difference in frequency compared with community controls (n=235) for a Caspase-5 polymorphism (rs45483102) (OR = 2.39 95% CI 1.22 – 4.68). Reporter gene studies were performed to further investigate the ability of flucloxacillin to act as a ligand for the xenobiotic-sensing nuclear receptors, pregnane X receptor (PXR) and constitutive androstane receptor (CAR). No flucloxacillin activation of CAR was observed but flucloxacillin was confirmed to be a PXR ligand and studies comparing PXR activation by the isoxazolyl penicillin’s cloxacillin and dicloxacillin showed dicloxacillin to be a stronger PXR agonist than flucloxacillin. DILI cases due to cloxacillin (n=3) and dicloxacillin (n=2) were found not to possess the HLA-B*57:01 allele suggesting that the mechanism for DILI due to these drugs is different. Flucloxacillin metabolism pathways were studied using human liver microsomes, recombinant cytochrome P450 isoforms and rat B13/H cells which differentiate to a hepatocyte-like phenotype, with flucloxacillin penicilloic acid the major metabolite detected. Despite previous reports, formation of the metabolite 5'-hydroxymethyl flucloxacillin which was believed to be CYP3A4-mediated, could not be confirmed in any of the systems studied. Preliminary studies on T-cell mediated responses to flucloxacillin, by exposure of peripheral blood mononuclear cells (PBMCs) from HLA-B*57:01 expressing flucloxacillin-DILI patients and from HLA-B*57:01 positive and negative flucloxacillin-naïve donors to flucloxacillin, indicated increased expression of interferon-γ at the RNA level in 2 out of 3 of the patient samples but not in controls. This finding was generally consistent with reported findings by others. In summary, a novel HLA-B association involving some flucloxacillin DILI cases has been detected, HLA-B*57:01 does not appear to be an important risk factor for DILI due to other isoxazolyl penicillin’s and the ability to act as a PXR agonist appears to be a general feature of these penicillin’s so may not be directly relevant to the mechanism for flucloxacillin DILI. The confirmed association with caspase 5 may represent a minor additional risk factor for flucloxacillin DILI.