Brorphine analogues as new psychoactive substances: in vitro and in vivo pharmacological characterization
Background: The emergence of new synthetic opioids (NSOs) has added complexity to recreational opioid markets worldwide. While NSOs with diverse chemical structures have emerged, brorphine – which was first identified in 2019 - currently remains the only NSO with a piperidine benzimidazolone scaffold. However, the emergence of new generations of NSOs, including brorphine analogues, can be anticipated. This study explores the pharmaco-toxicological effect profile of brorphine and four newly synthesized analogues with differing para-ring substituents (orphine, fluorphine, chlorphine, iodorphine) as potential NSOs.
Methods: In vitro characterization focused on the µ-opioid receptor (MOR), the primary molecular target for opioids. [3H]DAMGO competition binding assays in rat brain tissue were performed to evaluate the MOR binding affinity of the analogues. In addition, two cell-based NanoBiT® (Promega) second messenger (β-arrestin2 and mini-Gαi) recruitment assays were used to evaluate their intrinsic MOR activation potential. To aid in the interpretation of in vitro results, we further investigated the effects of intraperitoneal (IP) administration of the drugs (0.01-15 mg/kg) in male CD-1 mice. Antinociceptive effects were evaluated using tail flick and tail pinch assays. Respiratory depression, the primary cause of opioid-related fatalities, was studied using non-invasive plethysmography.
Results: Binding assays indicated that all analogues bind to MOR with nanomolar affinity. While analogues with smaller-sized substituents (i.e., orphine, fluorphine) showed the highest MOR affinity, chlorphine, brorphine and iodorphine were generally the most active in the MOR activation assays. This was corroborated by in vivo findings, as all analogues generally increased the threshold to acute mechanical and thermal pain stimuli, but only brorphine and chlorphine induced maximum antinociception in both assays. Furthermore, most of the analogues negatively impacted respiration (respiratory rate, breath length, and tidal volume), with brorphine- and chlorphine-induced respiratory depression being the most pronounced. Pretreatment with naloxone (6 mg/kg) indicated non-opioid mechanisms of action for some compounds, which may contribute to their eventual toxicity.
Conclusions: Brorphine-like piperidine benzimidazolones are MOR agonists inducing varying degrees of antinociception and respiratory depression in vivo. While the current study is limited to male CD-1 mice, similar effects can be anticipated in humans. Notably, with the exception of brorphine, all studied analogues are currently non-controlled in most countries. Hence, the dynamic nature of the NSO market bolsters the possibility of brorphine analogues to appear in the future, filling the void left by (newly banned) NSOs. This situation requires close monitoring, as our study shows that brorphine-like opioids may potentially cause substantial harm in users.
