Xylazine exacerbates fentanyl-induced respiratory depression and prevents rescue by naloxone in mice

Xylazine is a veterinary sedative and widespread adulterant of illicit opioids, where it is commonly detected in combination with the potent synthetic µ opioid receptor (MOR) agonist fentanyl in fatal overdose. We used whole body plethysmography to evaluate the respiratory effects of xylazine in the presence and absence of fentanyl in awake C57BL/6 mice of both sexes. We asked whether suppression of breathing by xylazine-adulterated fentanyl resisted reversal with naloxone. We identified receptor mechanisms necessary to reverse effects of xylazine-adulterated fentanyl on breathing. Xylazine rapidly and dose-dependently suppressed minute ventilation, respiratory frequency, and tidal volume, producing profound respiratory depression at low doses. These effects were dependent on α-2 adrenergic receptors (α2ARs) and were blocked by the α2AR antagonist atipamezole. Xylazine, combined with a dose of fentanyl with modest respiratory effects, suppressed breathing with greater efficacy than when administered alone. A dose of naloxone sufficient to reverse fentanyl-induced respiratory depression was ineffective in reversing the respiratory suppression induced by xylazine-adulterated fentanyl. By contrast, combinations of naloxone with atipamezole rapidly and fully reversed respiratory suppression induced by xylazine-adulterated fentanyl. The same dose of atipamezole, administered alone, produced significant, but markedly delayed reversal. Xylazine suppresses breathing via activation of α2ARs, an effect enhanced by coadministration with the MOR agonist fentanyl. Respiratory suppression inflicted by the mixture of xylazine and fentanyl resisted reversal by naloxone but was fully reversible by subsequent coadministration of both naloxone and atipamezole.