In vitro and in vivo pharmacological characterization of fentanyl analogs

Opioid overdose is the leading cause of unintentional drug-related deaths in the United States (US), largely due to the use of illicitly manufactured synthetic opioids like fentanyl and its analogs. The pharmacological properties of novel fentanyl analogs are largely unknown. Thus, this study characterized opioid-like activities of six novel fentanyl analogs (acryl fentanyl, β-hydroxythio fentanyl, cyclohexyl fentanyl, 4-fluoroisobutyrly fentanyl, furanyl fentanyl, and tetrahydrofuranyl fentanyl) in vitro and in vivo, in comparison to morphine and fentanyl. In radioligand binding assays using [³H](D-Ala², N-MePhe⁴, Gly-ol)-enkephalin (DAMGO) and the human mu opioid receptor (MOR) stably expressed in Chinese hamster ovary cells, all test substances except cyclohexyl fentanyl exhibited sub-nanomolar affinity for MOR. Furthermore, assessments of stimulation of [³⁵S]guanosine 5′-O-[γ-thio]triphosphate (GTPγS) binding at the MOR indicated that all test substances functioned as MOR agonists (E_max in % of DAMGO: 69.8–105), except for cyclohexyl fentanyl (13.3 %). In the warm water tail withdrawal procedure (55 °C) using male CD1 mice, cumulative injections of each test substance (S.C., N = 8 per group) produced dose-dependent increases in tail-flick latency (ED₅₀s: 0.158–3.18 mg/kg), which were blocked by the opioid receptor antagonist naltrexone. The antinociceptive potencies of each test substance were similar to or less than that of fentanyl (ED₅₀: 0.122 mg/kg). Additional in vivo studies using morphine discrimination, locomotor activity, and assessments of precipitated withdrawal further corroborated the MOR effects of these fentanyl analogs. Importantly, all the analogs produced morphine-like subjective effects suggesting that these compounds may have abuse liabilities similar to morphine.