Phenacetin had been used as a prodrug of acetaminophen, but it was withdrawn from the market due to methemoglobinemia and renal failure. We found that phenacetin was hydrolyzed to p-phenetidine by AADAC, and subsequently metabolized to N-hydroxyl p-phenetidine by CYP1A2 and CYP2E1. N-Hydroxy p-phenetidine had been suggested to cause methemoglobinemia, and this proposal was proven by our in vitro experiment using red blood cells and recombinant human AADAC, CYP1A2, and CYP2E1. Administration of phenacetin to mice caused methemoglobinemia, and pre-treatment of tri-o-tolylphosphate, an AADAC inhibitor, attenuated the plasma methemoglobin level, indicating that the AADAC-dependent hydrolysis triggers off methemoglobinemia. Ketoconazole, another substrate of AADAC, rarely causes severe liver injury when it was orally administered. Hydrolysis and subsequent N-hydroxylation have been considered to be associated with ketoconazole-induced hepatotoxicity. Overexpression of AADAC in HepaRG cells with an adenovirus expression system elicited the cytotoxicity of ketoconazole, and the cytotoxicity in human primary hepatocytes was attenuated by diisopropylfluorophosphate, an AADAC inhibitor. Thus, the involvement of AADAC in drug-induced toxicity was demonstrated.
Flupirtine had been used as an analgesic, but the European Medicines Agency restricted its use due to liver toxicity. We found that flupirtine was efficiently hydrolyzed by CES2, followed by N-acetylation to produce an acetylamino form by NAT2. The hydrolyzed metabolite has been supposed to be non-enzymatically converted to quinone diimine. This quinone diimine could be detected by trapping with N-acetylcystein (NAC). Liver S9 samples from NAT2 slow acetylators showed higher NAC conjugate formation than those from rapid acetylators, being affected by CES2-dependent flupirtine hydrolase activity. The cytotoxicity by flupirtine in HepG2 cells was increased by overexpression of CES2, indicating that the hydrolysis of flupirtine would be a key pathway to cause hepatotoxicity.
In summary, we demonstrated several esterases including well-characterized esterase CES and orphan esterase AADAC are relevant to drug-induced toxicity, in addition to pharmacological activation of prodrugs. It should be noted that hydrolyzed metabolites may lead to toxicity.