PROJECT SUMMARY: According to the Centers for Disease Control (CDC), synthetic opioids are currently the most common cause of overdose death in the U.S., and have increased opioid deaths by >500% from 2015- 2020. In 2020 alone, over 80% of the 69,700 opioid deaths were attributed to these synthetics (fentanyl). Despite the widespread availability of naloxone, the only FDA approved treatment for opioid overdose, deaths from fentanyl and fentanyl analogues (F/FA) continue to rise in parallel with increasing reports and pharmacological evidence of F/FA resistance to naloxone. High doses of rapidly injected F/FA cause airway obstruction from vocal cord closure (VCC) and severe chest wall rigidity (CWR) within 60-90 seconds, lethal effects that can persist for up to 10 minutes and appear to be resistant to naloxone. In contrast, morphine derived opiates (e.g. heroin) cause respiratory depression and mild muscle rigidity that is responsive to naloxone, but are not known to cause VCC in humans. The rapidly lethal phenomenon of VCC suggests unique pharmacological mechanisms underlying F/FA effects and may be a factor driving the dramatic increase in community overdose deaths. In support of this hypothesis, our published pharmacological data demonstrate F/FA, but not morphine or naloxone, have affinity for off-site targets that regulate these F/FA-induced effects. These in vitro data include F/FA concentrations that may be physiologically relevant to humans, based on available models of brain lipid concentrations for F/FA and by comparison with plasma levels in decedents of fentanyl overdose. Additionally, we have demonstrated in our published animal model that intravenous fentanyl induces VCC in <10 seconds, is not reversed by high dose naloxone and involves these off-site receptor targets. These data suggest that the development of effective therapies for overdose requires a biological model that replicates clinical effects of F/FA toxicity and a re-conceptualization of the underlying causes of F/FA overdose deaths to include VCC, in addition to respiratory depression. Therefore, the goal of this Fast-track proposal is to use an animal model of fentanyl toxicity, to identify specific drug classes that address F/FA induced VCC and respiratory depression. There are currently no Federal Drug Administration approved treatments that target these F/FA toxicity effects, and this project directly addresses the need for the development of a new class of therapeutics, specific to F/FA overdose.