ABSTRACT For the first time, over 100,000 deaths were caused by drug overdose in a 12-month period (April 2020 - April 2021) in the United States. Synthetic opioids, primarily fentanyl, accounted for over 60% of all overdose deaths during this span (87% of opioid overdose deaths). Over the past decade, the number of overdose deaths involving synthetic opioids has risen 20-fold, approaching 60,000 in 2020. The emergence of fentanyl analogs, some of which are much more potent than fentanyl (e.g., carfentanil, reported to be ~100 times more potent than fentanyl), pose serious risk to public health. Another dangerous characteristic of exposure to these drugs is that they are predominantly taken unknowingly which, combined with their potency, increases the risk of overdose. The opioid receptor antagonist naloxone is the only FDA-approved treatment for opioid overdose. While naloxone has saved countless lives, its effectiveness is limited by its short duration of action and that its antagonism is competitive – that is, that the effects of naloxone can be surmounted by taking more of an opioid agonist. Clinical reports suggest that larger or more frequent doses of naloxone are required to reverse opioid overdose involving carfentanil or other fentanyl analogs, and preclinical studies show reduced effectiveness of opioid antagonists to antagonize the effects of carfentanil relative to other opioid agonists. The novel opioid receptor antagonist methocinnamox (MCAM) binds non-competitively at the mu opioid receptor and has extremely long-lasting effects. A recent study demonstrated that some effects of MCAM are mediated through binding an allosteric site on the mu opioid receptor. This finding warrants further study and provides rationale for evaluating the potential of using mixtures of antagonists to reverse opioid-induced ventilatory depression. The proposed studies use whole-body plethysmography in rats to address current trends in opioid overdose death and the need for development of new treatment options for opioid overdose, testing the hypotheses that the effects of mixtures of opioid agonists will be greater than each drug when given alone, and that mixtures of the opioid antagonists naloxone and MCAM will be more potent than naloxone alone at reversing the effects of opioid agonists on ventilation. Aim 1 will determine the nature of the interaction between the effects of heroin, fentanyl, and carfentanil on ventilation. Aim 2 will determine the nature of the interactions between naloxone, MCAM, and diprenorphine for reversing the ventilatory depressive effects of heroin, fentanyl, and carfentanil, and begin to assess the mechanism of this interaction. The proposed studies will determine the nature of interactions between opioid agonists commonly involved in opioid overdose and evaluate whether mixtures of opioid antagonists might be more effective alternatives to naloxone for reversing opioid overdose. Results from these studies will provide valua...