title: Bioactivation of Trimethoprim to Protein-Reactive Metabolites in Human Liver Microsomes.
abstract: The formation of drug-protein adducts via metabolic activation and covalent binding may stimulate an immune response or may result in direct cell toxicity. Protein covalent binding is a potentially pivotal step in the development of idiosyncratic adverse drug reactions (IADRs). Trimethoprim (TMP)-sulfamethoxazole (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 previously demonstrated that TMP is bioactivated to chemically reactive intermediates that can be trapped in vitro by N-acetyl cysteine (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 covalent binding 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 covalent binding that was NADPH dependent and increased with incubation time and concentration of both protein and TMP. The estimated covalent binding was 0.8 nmol Eq TMP/mg protein, which is comparable to the level of covalent binding for several other drugs that have been associated with covalent binding-induced toxicity and/or IADRs. NAC and selective inhibitors of CYP2B6 and CYP3A4 significantly reduced TMP covalent binding. 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.
authors.affiliation: Divisions of Clinical Pharmacology, Toxicology, and Therapeutic Innovation, Children's Mercy Hospital, University of Missouri, Kansas City, Missouri (J.L.G., J.S.L.); Department of Medicinal Chemistry, University of Kansas School of Pharmacy, Lawrence, Kansas (Y.M.K., R.P.H.); Baker & McKenzie LLP, Dallas, Texas (S.A.R.); and Center for Integrative Chemical Biology and Drug Discovery, Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, North Carolina (K.L.) jlgoldman@cmh.edu.
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Goldman
authors.affiliation: Divisions of Clinical Pharmacology, Toxicology, and Therapeutic Innovation, Children's Mercy Hospital, University of Missouri, Kansas City, Missouri (J.L.G., J.S.L.); Department of Medicinal Chemistry, University of Kansas School of Pharmacy, Lawrence, Kansas (Y.M.K., R.P.H.); Baker & McKenzie LLP, Dallas, Texas (S.A.R.); and Center for Integrative Chemical Biology and Drug Discovery, Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, North Carolina (K.L.).
authors.forename: Yakov M
authors.initials: YM
authors.lastname: Koen
authors.affiliation: Divisions of Clinical Pharmacology, Toxicology, and Therapeutic Innovation, Children's Mercy Hospital, University of Missouri, Kansas City, Missouri (J.L.G., J.S.L.); Department of Medicinal Chemistry, University of Kansas School of Pharmacy, Lawrence, Kansas (Y.M.K., R.P.H.); Baker & McKenzie LLP, Dallas, Texas (S.A.R.); and Center for Integrative Chemical Biology and Drug Discovery, Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, North Carolina (K.L.).
authors.forename: Steven A
authors.initials: SA
authors.lastname: Rogers
authors.affiliation: Divisions of Clinical Pharmacology, Toxicology, and Therapeutic Innovation, Children's Mercy Hospital, University of Missouri, Kansas City, Missouri (J.L.G., J.S.L.); Department of Medicinal Chemistry, University of Kansas School of Pharmacy, Lawrence, Kansas (Y.M.K., R.P.H.); Baker & McKenzie LLP, Dallas, Texas (S.A.R.); and Center for Integrative Chemical Biology and Drug Discovery, Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, North Carolina (K.L.).
authors.forename: Kelin
authors.initials: K
authors.lastname: Li
authors.affiliation: Divisions of Clinical Pharmacology, Toxicology, and Therapeutic Innovation, Children's Mercy Hospital, University of Missouri, Kansas City, Missouri (J.L.G., J.S.L.); Department of Medicinal Chemistry, University of Kansas School of Pharmacy, Lawrence, Kansas (Y.M.K., R.P.H.); Baker & McKenzie LLP, Dallas, Texas (S.A.R.); and Center for Integrative Chemical Biology and Drug Discovery, Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, North Carolina (K.L.).
authors.forename: James S
authors.initials: JS
authors.lastname: Leeder
authors.affiliation: Divisions of Clinical Pharmacology, Toxicology, and Therapeutic Innovation, Children's Mercy Hospital, University of Missouri, Kansas City, Missouri (J.L.G., J.S.L.); Department of Medicinal Chemistry, University of Kansas School of Pharmacy, Lawrence, Kansas (Y.M.K., R.P.H.); Baker & McKenzie LLP, Dallas, Texas (S.A.R.); and Center for Integrative Chemical Biology and Drug Discovery, Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, North Carolina (K.L.).
authors.forename: Robert P
authors.initials: RP
authors.lastname: Hanzlik
mesh_terms: D000111:Acetylcysteine / Q000494:pharmacology; D000892:Anti-Infective Agents, Urinary / Q000009:adverse effects / Q000493:pharmacokinetics*; D001711:Biotransformation; D006801:Humans; D008862:Microsomes, Liver / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D014295:Trimethoprim / Q000009:adverse effects / Q000493:pharmacokinetics*
chemical_list: D000892:Anti-Infective Agents, Urinary; D011506:Proteins; D014295:Trimethoprim; D000111:Acetylcysteine