Pharmacologic Characterization of Substituted Nitazenes at μ, κ, and Δ Opioid Receptors Suggests High Potential for Toxicity

Abstract

The benzimidazole opioids (substituted nitazenes) are highly potent μ opiod receptor (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 μ, κ, and Δ opioid receptors (MOR, KOR, and DOR, respectively) heterologously expressed in 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; 13 had subnanomolar 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- to 2580-fold and 180- to 1280-fold lower, respectively. Functional activities were assessed using [³⁵S]GTPγS binding assays. Four nitazenes had subnanomolar 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- to 7920-fold and 24- to 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: Substituted nitazenes are a growing public health threat. Although the 19 nitazenes tested vary in their opioid receptor pharmacology, a number are very high affinity, high potency, and high efficacy compounds- higher than fentanyl. Their pharmacology suggests high potential for harm.

Fig. 1.

Structures of tested substituted nitazenes and standards. Structures of test compounds fentanyl, morphine, and DAMGO are shown. R1–R3 substitutions are shown.

Fig. 2.

Concentration–response curves of substituted nitazenes and fentanyl in radioligand binding assays at MOR, KOR, and DOR. Data are the means ± S.E.M. of (A) three to five biologic experiments (MOR), (B) three to four biologic experiments (KOR), and (C) three to six biologic experiments (DOR) conducted in duplicate. Note the differences in the x axis range among MOR (–12 to –6) and KOR and DOR (–9 to –5).

Fig. 3.

Substituted nitazenes sorted by pKis and affinity ratios illustrate relative affinities. (A) pKis sorted by affinity for MOR, indicating the high affinities for MOR and the lower affinities for KOR and DOR. In the row labeled DAMGO/U50/DPDPE, DAMGO, U50,488H, and DPDPE correspond to MOR, KOR, and DOR, respectively. The ratios of the affinities for MOR compared with KOR (B) and compared with DOR (C), emphasizing the selectivity of affinities for MOR. Substituted nitazenes sorted by pKis for KOR (D) and DOR (E).

Fig. 4.

Concentration–response curves of agonist activity in [35S]GTPγS functional assays. All biologic experiments were conducted in duplicate and normalized to the maximal effect of (A) DAMGO (MOR), (B) U50,488H (KOR), or (C) DPDPE (DOR) that was tested in each experiment. Data are the means ± S.E.M. of (A) three to seven biologic experiments (MOR), (B) four to six biologic experiments (KOR), and (C) three to seven biologic experiments (DOR) conducted in duplicate.

Fig. 5.

Correlation of binding affinities and functional potencies of nitazenes at MOR, KOR, and DOR. As a group, binding affinities and functional potencies of nitazenes at (A) MOR, (B) KOR, and (C) DOR are highly correlated. Spearman’s correlations are listed on each panel. P values are <0.001.

Fig. 6.

Heat map of potencies (pEC₅₀) and efficacies (% E_max) of nitazenes and standards to stimulate [³⁵S]GTPγS binding for MOR, KOR, and DOR. (A and B) Data are sorted according to hMOR potencies and efficacies. (C) Relative potencies, MOR compared with KOR. Note that DEI is above the range of the legend, at 7920. (D) Relative potencies, MOR compared with DOR. Note that DEI is above the range of the legend, at 9450. Heat map of sorted potencies (pEC₅₀ values) of nitazenes and standards to stimulate [³⁵S]GTPγS binding for (E) KOR and (F) DOR.

Fig. 7.

Amplification ratios of nitazenes and standards at MOR, KOR, and DOR. To determine the amplification ratio for each nitazene and reference compound, the ratio of the binding affinity (K_i) to the functional potency (EC₅₀) for each nitazene was calculated (Strange, 2008).