In vivo and in vitro metabolism of the designer benzodiazepine, bretazenil: a comparison of pooled human hepatocytes and liver microsomes with postmortem urine and blood samples

TL;DR

This study identifies how the designer drug bretazenil is metabolized in the body and suggests key markers for detecting its use in clinical and forensic settings.

Contribution

The study discovers a new benzodiazepine metabolism pathway involving hydroxylation and cysteine conjugation.

Findings

A total of 26 bretazenil metabolites were identified using in vitro and postmortem samples.

Hydroxylation on the pyrrolidine ring was the predominant metabolic pathway.

Three metabolites are recommended as significant markers for bretazenil use.

Abstract

Benzodiazepines are often used with other drugs like opioids, potentially leading to severe intoxications. Bretazenil, an imidazo-tetrahydropyrrolo-1,4-benzodiazepine, developed in the 1980s but never marketed as a medicine, has recently appeared on the illicit drug market. Given its high potency, short elimination half-life, and potential for rapid metabolism, it is essential to identify markers for bretazenil consumption for clinical and forensic purposes. Our study aimed to thoroughly explore bretazenil’s metabolism using web-based in silico prediction tools, in vitro incubation with pooled human liver microsomes and hepatocytes, and to compare these results with authentic postmortem blood and urine samples. The in silico prediction revealed 16 metabolites, mainly formed by hydroxylation (phase I) and further O-glucuronidation, sulfation, and methylation (phase II) reactions. High-resolution mass spectrometry and software-aided data processing of in vitro and in vivo samples identified a total of 26 metabolites. Eight metabolites were detected in vitro, 15 in postmortem urine, and 11 in postmortem blood. Hydroxylation on the pyrrolidine ring was predominant. Other phase I reactions, including combinations of dihydroxylation, hydroxylation, reduction, and carboxylation as well as phase II glucuronidation and sulfation on the pyrrolidine ring, imidazole ring, or the tert-butyl chain, were also identified. Additionally, we discovered a new benzodiazepine biotransformation pathway via hydroxylation and cysteine conjugation in both human hepatocytes and blood. Due to bretazenil’s extensive metabolism, we recommend hydroxy-bretazenil (B14), reduced hydroxy-bretazenil (B6), and reduced dihydroxy-bretazenil (B1) as significant markers for detecting bretazenil use.

The online version contains supplementary material available at 10.1007/s00204-025-04213-x.

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