Preclinical pharmacology of N-pyrrolidino "nitazene" opioids appearing in nonmedical drug markets.

N-pyrrolidino (PYR) nitazenes are a subset of 2-benzylbenzimidazole opioids appearing in illicit drug markets, but there is limited information about the pharmacology of these compounds. Here, we examined the opioid-like effects of PYR nitazenes with varying alkoxy chain lengths (ie, methoxy, ethoxy, propoxy, and isopropoxy) compared with fentanyl. Opioid receptor binding and functional assays were employed to determine affinities and potencies of the compounds in vitro. Acute opioid-like effects were examined in mice, using hot plate antinociception, motor activity, and body temperature measures. PYR nitazenes displayed high affinity at the μ-opioid receptor (Ki = 1-12 nM), with weaker affinities at δ and κ sites. The compounds were potent and efficacious agonists in assays measuring μ-opioid receptor-mediated inhibition of cAMP formation (EC50 = 0.03-0.91 nM, Emax = 104%-106%) compared with fentanyl (EC50 = 0.23 nM, Emax = 100%). The nitazenes also induced β-arrestin 2 recruitment, but with weaker potencies. In mouse studies, the compounds induced dose-dependent antinociception, hyperactivity, and hypothermia. Across in vivo measures, the ethoxy, propoxy, and isopropoxy compounds (ED50 = 0.002-0.033 mg/kg) were more potent than fentanyl (ED50 = 0.081-0.178 mg/kg). The acute in vivo effects of ethoxy and isopropoxy analogs were fully or partially reversed by naloxone (0.3 mg/kg). PYR nitazenes with intermediate alkoxy chain lengths are most potent for eliciting opioid-like effects in vitro and in mice. In particular, the ethoxy, propoxy, and isopropoxy analogs are more potent than fentanyl, highlighting an elevated risk for overdose with these compounds. SIGNIFICANCE STATEMENT: The present study characterized the pharmacology of N-pyrrolidino nitazene opioids that vary by alkoxy chain length and are associated with overdose cases in humans. The ethoxy, isopropoxy, and propoxy N-pyrrolidino nitazenes were found to be more potent than fentanyl in both in vitro and in vivo assays, suggesting that these compounds pose a serious overdose risk to humans who are unknowingly exposed to the compounds in the illicit opioid supply.