Membrane Permeability Drives the Extreme Potency of Fentanyl

Fentanyl is a leading cause of drug overdose deaths in the United States, yet the mechanism driving its extreme <i>in vivo</i> potency is poorly defined. Here we developed novel computational and experimental approaches to examine whether the membrane contributes to the <i>in vivo</i> potency of fentanyl. Using weighted-ensemble continuous constant pH molecular dynamics (WE-CpHMD) simulations, we estimated the permeability of ionized fentanyl to be approximately 10^-7 cm/s, about 100-fold higher than that of ionized morphine and several more orders of magnitude higher than those of ionized naloxone and isotonitazene. Simulations revealed that all opioids deprotonate when diffusing below the lipid headgroup region, with isotonitazene and naloxone deprotonating closer to the hydrophobic core. Mean first passage time calculation revealed fentanyl's rapid kinetics for bidirectional membrane transport, suggesting that it partitions into and permeates the membrane while also redistributing back into solution from both the membrane core and intracellular compartment. Consistently, cell washout experiments making use of a BRET senor demonstrated that fentanyl, but not morphine, is retained by cells and can repartition into solution to reactivate the mu-opioid receptor. The IAM-HPLC measurement confirmed fentanyl's superior phospholipophilicity. These findings support the hypothesis that membrane permeation is a major driver of fentanyl's extreme analgesic potency, rapid onset, and short duration of action, revealing a fundamental mechanism underlying opioid toxicity, with implication for developing more effective countermeasures. WE-CpHMD provides a valuable tool for mechanistic elucidation of membrane permeation of ionizable molecules, which remains poorly understood.