Background:Butaselen is an ebselen analog that is under clinical trials for treating hepatic and pulmonary
fibrosis. Our previous studies showed that butaselen is mainly present in human plasma in the form of M2, a
free Se-methylated metabolite.Objective:This study aimed to investigate the metabolic mechanisms of butaselen.Methods and Results:Butaselen was incubated with human plasma. Butaselen immediately disappeared, and the
butaselen-HSA (human serum albumin) adduct was detected by HPLC-HRMS, showing that butaselen covalently
binds to HSA. The butaselen-HSA adduct was precipitated using acetonitrile and then incubated with PBS, Cys, and
GSH for 1 hour. The product was M1, a reduced form of butaselen. The results indicated that HSA, Cys, and GSH
can reduce the butaselen-HSA covalent bond. The binding site for butaselen could be the cysteine-34 residue of HSA
through pronase and trypsin hydrolysis. Incubating butaselen with cysteine, butaselen-Cys, butaselen-2Cys, and M1
were generated, indicating the covalent binding and reduction of butaselen by cysteine. We incubated liver microsomes
and cytosol with butaselen, 6.22 and 246 nM M2 were generated, respectively. The results demonstrated that
cytosolic enzymes are mainly involved in M2 production. The amount of M2 in the liver cytosol decreased from 246
nM to 2.21 nM when 10 mM m-anisic acid (a specific TPMT enzyme inhibitor) was added, showing that TPMT is
responsible for M2 formation.Conclusion:Butaselen was covalently bound to HSA, and the binding site was the cysteine-34 residue of HSA. The
butaselen-HSA adduct was reduced by free thiol compounds to generate M1. M1 was further metabolized to M2 by
cytosolic TPMT. This study provides a basis for studying the pharmacokinetics of selenium-containing drugs.