# Insights into the human metabolism and in silico receptor activity of gidazepam and desalkylgidazepam

## TL;DR

This study explores how the body processes gidazepam and its metabolite desalkylgidazepam, and how they interact with brain receptors, offering insights into their effects and detection.

## Contribution

The study identifies specific blood markers for gidazepam and provides in silico evidence for its prodrug behavior and TSPO agonism.

## Key findings

- Gidazepam is metabolized into desalkylgidazepam, N-acetyl gidazepam, and N-glucuronide forms.
- Desalkylgidazepam undergoes hydroxylation and O-glucuronidation, but does not bind to TSPO.
- In silico modeling shows gidazepam has lower GABAAR affinity but interacts with α1β2 subunit transmembrane domains.

## Abstract

Desalkylgidazepam, an active gidazepam metabolite, first appeared on the illicit drug market in 2022 and has been detected in polydrug intoxication cases. Since both benzodiazepines and their metabolites can result from gidazepam metabolism, it is important to identify markers that specifically indicate consumption of each compound. We therefore investigated the human metabolism of gidazepam and desalkylgidazepam by incubating them with human hepatocytes and analyzing the resulting samples, along with human blood from a confirmed desalkylgidazepam-positive case, using liquid chromatography-high-resolution mass spectrometry. To further assess their pharmacological profile, we examined the activity of gidazepam, desalkylgidazepam, and their potential (3R)- and (3S)-hydroxy metabolites at γ-aminobutyric acid A (GABAAR) and 18 kDa translocator protein (TSPO) receptors in silico, using AutoDock Tools and UCSF Chimera. Gidazepam was metabolized through N-desalkylation (yielding desalkylgidazepam), N-acetylation, and N-glucuronidation. Conversely, desalkylgidazepam was subjected to hydroxylation and subsequent O-glucuronidation reactions. Notably, gidazepam demonstrated a lower affinity at GABAAR’s prominent α1/γ2 site compared to desalkylgidazepam and its (3R)- and (3S)-hydroxy metabolites. However, its interaction with the transmembrane domains of the α1β2 subunit may account for its anxiolytic effects. For the TSPO receptor, gidazepam and 3-hydroxy desalkylgidazepam metabolites showed higher binding affinity, whereas desalkylgidazepam did not bind to TSPO. Our findings suggest blood markers specific to gidazepam, namely gidazepam-N-glucuronide and N-acetyl gidazepam, are essential for confirming gidazepam consumption. In addition, in silico modelling supports the hypothesis that gidazepam functions as a prodrug via GABAAR and as an agonist at TSPO. Further research is necessary to clarify designer benzodiazepine activity at TSPO.

The online version contains supplementary material available at 10.1007/s00204-025-04249-z.