Pharmacologic characterization of substituted nitazenes at mu, kappa and delta opioid receptors suggests high potential for toxicity.

The benzimidazole opioids (BO, substituted nitazenes) are highly potent MOR agonists with heroin- or fentanyl-like effects. These compounds have caused hospitalizations and fatal overdoses. We characterized the in vitro pharmacology and structure-activity relationships of 19 nitazenes with substitutions at three positions of the benzimidazole core. Affinities were assessed using agonist radioligand binding assays at human mu, kappa, and delta opioid receptors (MOR, KOR, and DOR, respectively) heterologously expressed in Chinese hamster ovary (CHO) cells. Notably, for MOR binding, nine substituted nitazenes had significantly higher affinities than fentanyl including N-pyrrolidino etonitazene, N-pyrrilidino isonitazene, and N-desethyl isotonitazene; thirteen had sub-nanomolar affinities. Only metodesnitazene and flunitazene had significantly lower affinities than fentanyl. Affinities for the substituted nitazenes at KOR and DOR relative to MOR were 46-2580- and 180-1280-fold lower, respectively. Functional activities were assessed using [ 35 S]GTPγS binding assays. Four nitazenes had sub-nanomolar potencies at MOR: N-pyrrolidino etonitazene, N-pyrrilidino isonitazene, N-pyrrilidino protonitazene and N-desethyl isotonitazene. Ten substituted nitazenes had significantly higher potencies than fentanyl. All tested nitazenes were full MOR agonists. Potencies at KOR and DOR relative to MOR were 7.3-7920- and 24-9400-fold lower, respectively. Thus, many of these compounds are high affinity/high potency MOR agonists with elevated potential to elicit toxicity and overdose at low doses. Significance Statement The substituted nitazenes are a growing public health threat. The Although the 19 nitazenes tested vary in affinity, potency, and efficacy, a number are very high affinity, high potency, and high efficacy compounds - higher than fentanyl. Their pharmacology suggests high potential for harm.