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.

Fig. 1.

Proposed pathway of TMP bioactivation and protein covalent binding.

Fig. 2.

Structure, synthesis, and labeling of [¹⁴C]-TMP. The black dot indicates the position of the ¹⁴C label.

Fig. 3.

Effects of TMP concentration, protein concentration, incubation time, and NAC on the covalent binding of [¹⁴C]-TMP to HLMs. Pooled HLMs from 16 donors were incubated in the presence of NADPH at 37 ± 1°C. (A) TMP concentration ranged from 50 to 250 µM, whereas protein and time were constant at 1500 µg/ml protein and 120 minutes, respectively. (B) Protein concentrations ranged from 250 to 1500 µg/ml, whereas incubation time (120 minutes) and TMP (100 µM) were constant. (C) Incubation times ranged from 45 to 120 minutes, while protein (1500 µg/ml) and TMP (100 µM) concentrations were constant. (D) NAC (5 mM) was included in the incubation (100 µM TMP, 1500 µg protein/ml, 120 minutes). An aliquot (100 µl) of the incubation mixture was applied to absorbent filter paper strips and the strips were processed as outlined in the Materials and Methods. Radioactivity associated with the precipitated protein was estimated by liquid scintillation counting. CVB, covalent binding.

Fig. 4.

Effect of NADPH on the covalent binding of [¹⁴C]-TMP to HLMs. Pooled HLMs were incubated under standard conditions in the absence or presence of NADPH, with [¹⁴C]-TMP (50–250 µM) at 37 ± 1°C for 120 minutes. An aliquot (100 µl) of the incubation mixture was applied to absorbent filter paper strips as outlined in the Materials and Methods. Radioactivity associated with the precipitated protein was estimated by liquid scintillation counting. A low level of apparent covalent binding was noted in those incubations without NADPH and this amount was considered background and subtracted. CVB, covalent binding.

Fig. 5.

Effects of various P450 isoform-selective inhibitors on the covalent binding of [¹⁴C]-TMP to pooled HLMs were analyzed as described in the Materials and Methods. Each bar represents the mean and standard deviation. CVB, covalent binding.