SHORT COMMUNICATION Bioactivation of trimethoprim to protein-reactive metabolites in human liver microsomes Running Title Page: Covalent protein binding of trimethoprim in vitro Corresponding Author: Idiosyncratic adverse drug reactions: IADRs Trimethopri

Abstract

ABSTRACT: The formation of drug-protein adducts via metabolic activation and covalent binding (CVB) may stimulate an immune response or result in direct cell toxicity. Protein CVB is a potentially pivotal step in the development of idiosyncratic adverse drug reactions (IADRs). Trimethoprimsulfamethoxazole (TMP-SMX) is a combination antibiotic that commonly causes IADRs. Recent data suggest that the contribution of the TMP component of TMP-SMX to IADRs may be underappreciated. We have previously demonstrated that TMP is bioactivated to chemically reactive intermediates that can be trapped in vitro by N-acetylcysteine (NAC), and we have detected TMP-NAC adducts (i.e., mercapturic acids) in the urine of patients taking TMP-SMX. However the occurrence and extent of TMP CVB to proteins was unknown. To determine the ability of TMP to form protein adducts, we incubated [ 14 C]TMP with human liver microsomes in the presence and absence of NADPH. We observed protein CVB that was NADPH-dependent and increased with incubation time and concentration of both protein and TMP. The estimated CVB was 0.8 nmol-equivalent TMP/mg protein, which is comparable to the level of CVB for several other drugs that have been associated with CVB induced toxicity and/or IADRs. Selective inhibitors of CYPs 2B6 and 3A4, as well as NAC, significantly reduced TMP CVB. These results demonstrate for the first time that TMP bioactivation can lead directly to protein adduct formation suggesting that TMP has been overlooked as a potential contributor of TMP-SMX IADRs