Immunological investigations into naproxen-induced idiosyncratic hepatotoxicity

Background: Naproxen is a member of the aryl acetic acid class of non-steroidal anti-inflammatory drugs that has anti-inflammatory, antipyretic and analgesic properties. Naproxen is extensively metabolized in the liver by P450 enzymes to 6-O-desmethylnaproxen. Both parent drug and desmethyl metabolite undergo phase II biotransformation to their respective acyl glucuronide conjugated metabolites. Naproxen-induced idiosyncratic hepatotoxicity is a rare event. The mechanism of the tissue injury and the nature of the chemical entity involved in the adverse reaction have not been studied.
Aims: To explore and characterise naproxen (metabolite)-specific immune responses from patients with or without liver injury.
Methods: The chemical instability and protein reactivity of naproxen acyl glucuronide were investigated in vitro and an adduct with human serum albumin was generated. Peripheral blood mononuclear cells were isolated from patients and healthy volunteers. Lymphocyte proliferation and IFNγ secretion against naproxen, its major metabolites and the serum albumin adduct were examined. T-cell cloning by limiting dilution was undertaken in order to generate drug-specific T-cell clones and appropriate immune functional assays were used to characterize responses. Next generation sequencing was used in order to interrogate the transcriptional signature of peripheral blood mononuclear cells in response to naproxen and its major metabolites.
Results: Naproxen acyl glucuronide is chemically unstable with an identified degradation half-life of 2.72 hrs in 0.1 M phosphate buffer at 37°C (pH 7.4). Furthermore, irreversible binding was measured at 247.37 ± 57.64 nmol of drug per μmol serum albumin under 50 : 1 metabolite : protein molar ratio incubation at 37°C (pH 7.4). 6-O-desmethylnaproxen, but not naproxen or naproxen acyl glucuronide, exhibited lymphocyte stimulation in a patient with naproxen-induced liver injury. A CD4+CD8+ Th17 desmethylnaproxen – responsive T-cell clone was identified. The clone was found to be MHC-class II restricted and become activated via a pharmacological-interaction mechanism. 6-O-desmethylnaproxen exerts significant differential gene expression in peripheral blood mononuclear cells isolated from patients and healthy volunteers and an overall down-regulatory pattern was revealed.
Conclusion: Results show lymphocyte responses were directed to the primary desmethyl metabolite of naproxen, but not the parent drug or acyl glucuronide metabolite. In addition, the desmethyl metabolite shows consistent differential gene expression in human peripheral blood mononuclear cells. Taken together, this evidence suggests an overall immune-mediated basis for naproxen-induced idiosyncratic hepatotoxicity with a role for the inert oxidative metabolite. Whilst the FDA considers acyl glucuronide adduction of protein to be responsible for the liver reactions associated with carboxylic acid drugs such as naproxen, no evidence of this was found herein.