In vitro activation of µ opioid receptor and cardiac voltage-gated potassium channels hERG and KV7.1/KCNE1 of orphine analogs and their detections in forensic casework

<title>Abstract</title> <p> The new synthetic opioids market is rapidly evolving. The control of nitazene analogs in China has led to the emergence of new benzimidazolone (orphine) analogs since 2024. In addition to respiratory depression largely from µ opioid receptor (MOR) agonism, opioids can also lead to adverse cardiac effects, including arrhythmias and QT interval prolongation, in part through inhibition of the hERG channel (cardiac voltage-gated potassium channel K _V 11.1). In this study, the detection of orphines in Sweden is reported in seized samples and post-mortem casework. Also, 11 orphine analogs, including chlorphine and spirobrorphine, were examined for their <italic>in vitro</italic> MOR activity using an intracellular calcium release assay and <italic>in vitro</italic> hERG and K _V 7.1/KCNE1 activities using an automated patch clamp system. Brorphine, cychlorphine ( <italic>N</italic> -propionitrile chlorphine), and spirochlorphine were detected in eight seizures from 2020–2026, including in a nasal spray bottle, e-cigarette, and glass pipe. Cychlorphine was detected in six post-mortem cases from 2024–2025 with femoral blood concentrations of 0.5–16 ng/g. The MOR EC ₅₀ values for orphines ranged from 0.188–87.6 nM, where only cychlorphine was more potent than fentanyl. hERG channel conductance (G/G _max ) was completely inhibited by eight orphines (IC ₅₀  = 0.100–5.47 µM) likely through occupation of the channel’s canonical inner cavity as determined through <italic>in silico</italic> docking. Four orphines also significantly inhibited K _V 7.1/KCNE1 G/G _max . This demonstrates that most of the orphine analogs are potent MOR agonists and hERG inhibitors, suggesting not only high risk of respiratory depression, but also adverse cardiac effects, such as arrhythmia, Torsades de Pointes, and sudden cardiac death. </p>