Opioids are effective analgesics but are addictive and negatively impact multiple physiologic functions. Opioid abuse has reached epidemic levels and led to an exponential increase in deaths and societal costs, with a disproportionate impact on women. Opioid-related mortality has been attributed primarily to extreme acute inhibitory effects on CNS centers controlling breathing {opioid-induced respiratory depression; OIRD). Synthetic opioids such as fentanyl also impair alveolar-capillary gas exchange {A-a 02 gradient), cause upper airway dysfunction, and increase chest wall rigidity {Wooden Chest Syndrome) via synchronous and tonic activation of normally reciprocal respiratory muscles. The opioid receptor antagonist naloxone reverses opioid overdoses but has unwanted side-effects, highlighting an urgent need for developing and testing novel therapeutic reversing agents. We have developed several potent, thiol-based compounds including our lead compound D-Cysteine ethyl ester {D-CYSee) that in rodents reverse the opioid-induced triad of deleterious respiratory effects without affecting analgesia. Here we will test the dose-dependent effects of fentanyl {a major cause of the current opioid crisis), D-CYSee and naloxone in adult female goats which show respiratory responses to opioids similar to humans. Our preliminary data in goats indicate moderate to high doses of IV fentanyl acutely depressed total ventilation and respiratory rate while increasing tidal volume as seen in humans. High dose fentanyl also acutely increased the A-a oxygen gradient and led to increased tonic activity of intercostal and abdominal { expiratory) muscles (consistent with "wooden chest") while also prolonging post-inspiratory muscle activation and causing obstructive apnea. Like naloxone, D-CYSee injections immediately following high doses of fentanyl rapidly reversed ventilatory suppression and restored blood gases, but unlike naloxone sedation and analgesia was maintained. To date the multiple negative opioid-induced effects have not been simultaneously studied, nor has the novel reversing agent D-CYSee been tested in a species with a human-like physiologic response to opioids. We will address these and other critical knowledge gaps by completing three Specific AIMS: AIM 1 will test the hypothesis that our lead compound D-CYSee will be as effective as naloxone in reversing the acute {<15 min) fentanyl-induced compromise of vital physiological functions while preserving analgesia; AIM 2 will test the hypothesis that D-CYSee will prevent the acute and delayed fentanyl-induced attenuation of vital hypoxic and/or hypercapnic ventilatory responses; AIM 3 will characterize the pharmacokinetics of free and protein-bound plasma D-CYSee (and fentanyl) using established liquid chromatography-mass spectrometry (LC-MS), based on the premise that knowledge of the temporal distribution D-CYSee and potential metabolites will inform the mechanism(s) of this novel drug. Completing these studies will provide insights into the multiple negative effects of opioids beyond OIRD in a human-like pre-clinical model, validate a novel reversal agent which could aid in the fight against the opioid crisis, and provide critical pharmacokinetics data on this promising new compound.

Opioids cause fatalities by lowering oxygen in the blood by: 1) inhibiting cells in the brain that determine the rate and depth of breathing, 2) decreasing oxygen transfer from the lung to the blood, and 3) abnormal contraction of respiratory muscles in the chest wall and airways. Although naloxone (Narcan) can reverse the immediate harmful effects of opioids, it also has negative side-effects and limited effect times. Here, we aim to characterize the negative physiological effects of fentanyl (a major cause of the current opioid crisis) and the effectiveness of a known (naloxone) and novel, potent and long-lasting reversing agent (D-CYSee) in a large pre-clinical animal model (adult female goats) with physiologic responses to opioids that are more human-like than rodents.