Characterization of a highly sensitive and selective novel trapping reagent, stable isotope labeled glutathione ethyl ester, for the detection of reactive metabolites

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• 作者：Toshikazu Yamaoka ; ^{toshikazu.yamaoka@mb.kyorin-pharm.co.jp" class="auth_mail" title="E-mail the corresponding author} ; Yoshiaki Kitamura ^{yoshiaki.kitamura@mb.kyorin-pharm.co.jp" class="auth_mail" title="E-mail the corresponding author}
• 关键词：Bioactivation ; Glutathione trapping assay ; Idiosyncratic toxicity ; Reactive intermediate ; Stable isotope
• 刊名：Journal of Pharmacological and Toxicological Methods
• 出版年：2015
• 出版时间：November-December 2015
• 年：2015
• 卷：76
• 期：Complete
• 页码：83-95
• 全文大小：1758 K

文摘

Glutathione (GSH) trapping assays are widely used to predict the post-marketing risk for idiosyncratic drug reactions (IDRs) in the pharmaceutical industry. Although several GSH derivatives have been introduced as trapping reagents for reactive intermediates, more sensitive and selective reagents are desired to prevent the generation of erroneous results. In this study, stable isotope labeled GSH ethyl ester (GSHEE-d₅) was designed and its detection capability was evaluated.

Methods

GSHEE-d₅ was synthesized and its detection potential was compared with stable isotope labeled GSH ([¹³C₂,¹⁵N]GSH) as a reference trapping reagent. The trapping reagents were added to human liver microsomes as a 1:1 mixture with GSHEE or GSH, respectively, and incubated with seven IDR positive drugs and three IDR negative drugs. The adducts formed between the reagents and reactive metabolites were analyzed by unit resolution mass spectrometer (MS) using isotope pattern-dependent scan with neutral loss filtering.

Results

A single-step reaction of GSH and ethanol-d₆ produced GSHEE-d₅ with a yield of 85%. The GSHEE-d₅ assay detected adducts with all seven IDR positive drugs, and no adducts were detected with the three IDR negative drugs. In contrast, the [¹³C₂,¹⁵N]GSH assay failed to detect adducts with three of the IDR positive drugs. In the case of diclofenac, the GSHEE-d₅ assay showed a 4-times greater signal intensity than the [¹³C₂,¹⁵N]GSH assay.

Discussion

GSHEE-d₅ enabled the detection of reactive metabolites with greater sensitivity and selectivity than [¹³C₂,¹⁵N]GSH. These results demonstrate that GSHEE-d₅ would be a useful trapping reagent for evaluating the risk of IDRs with unit resolution MS.