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.
Introduction
Opioid overdose represents an escalating burden in the United States and around the globe (Skolnick, 2022). According to the Centers for Disease Control and Prevention, more than 105,000 drug overdose deaths occurred in 2022 in the United States (Ahmad et al., 2024). From 2020 to 2021, the rate of drug overdose fatalities involving synthetic opioids other than methadone increased 22%, whereas the rate of mortality involving heroin declined 32% (Spencer et al., 2022). Recently, the majority of synthetic opioid deaths have been due to fentanyl and fentanyl analogs. Subsequently, the US Drug Enforcement Administration designated fentanyl analogs as Schedule 1 compounds (Drug Enforcement Administration, 2018) as did China in 2019.
Also in 2019, following the scheduling of fentanyl analogs, nonfentanyl nitazenes (2-benzyl benzimidazole opioids) emerged in the illicit drug market (Papsun et al., 2022). This family of compounds, including etonitazene (ETZ), isotonitazene (ITZ), and metonitazene (MTZ), were first developed in the 1950s and 1960s as potential analgesics (Hunger et al., 1957). Nitazene analogs consist of a benzimidazole core with varying substitutions or deletions of the nitrite (R1), the N-ethylamine side chain (R2), or phenylalkyl chain (R3) (Fig. 1). Abused nitazenes are opioid agonists, with similar signs of opioid toxicity, including respiratory distress, confusion, nausea, and vomiting, which can precede coma or fatality (Montanari et al., 2022). Intravenous, sublingual, vaping, and intranasal routes of administration have been reported (Shover et al., 2021).
Fig. 1.
Structures of tested substituted nitazenes and standards. Structures of test compounds fentanyl, morphine, and DAMGO are shown. R1–R3 substitutions are shown.
Concern over the potential abuse of nitazenes was reported as early as 2012 (Lutz, 2012). Hospitalizations and deaths from nitazene analogs including ITZ and N-piperidinyl etonitazene (PIPETZ) have been reported (Krotulski et al., 2020; Shover et al., 2021; Calello et al., 2022). In 2020, ETZ and ITZ were emergency scheduled (Schedule I) by the US Drug Enforcement Administration, and continued reports of nitazene-mediated fatalities and hospitalizations (Papsun et al., 2022) triggered the following nitazenes to be emergency scheduled (Schedule I) in 2021 and 2022: butonitazene (BTZ), etodesnitazene (ETD), flunitazene (FTZ), metodesnitazene (MTD), MTZ, N-pyrrolidino etonitazene (PYRETZ), and protonitazene (PTZ) (Drug Enforcement Administration, 2021, 2022).
Despite their high potential to cause severe toxicities and death, only limited pharmacology of many nitazenes has been published. In early studies, using C6 rat glioma cells expressing the recombinant rat μ opioid receptor, ETZ had very high affinity (0.017 nM) for the [3H]sufentanil binding site, and high potency (1.12 nM) at stimulating [35S]GTPγS binding; this is more than 10-fold higher affinity and potency than fentanyl (Emmerson et al., 1996). ITZ stimulated the release of dopamine in rat nucleus accumbens shell to a greater extent than an equivalent dose of fentanyl (De Luca et al., 2022). Using HEK-293T cells, an array of substituted nitazenes showed no bias toward either βarrestin or Gi signaling pathways; ITZ was twice as potent as fentanyl at stimulating both pathways (Vandeputte et al., 2020, 2021). When evaluated in rats, PYRETZ, an ETZ analog (confirmed present in 21 overdose fatalities), induced opioid-like antinociceptive, cataleptic, and thermic effects and was equipotent to fentanyl in the hot plate test and at inducing catalepsy (Vandeputte et al., 2022). Subsequently, Vandeputte et al. (2023) evaluated ITZ and N-desethyl isotonitazene (DEI) in rats for hot plate antinociception, catalepsy score, and body temperature and found these nitazenes to be more potent than morphine in all assays and had faster onset and decline of in vivo effects. Taken together, there is strong evidence that structures based on a benzimidazole core have high potential for toxicity and are an emerging threat for raising mortalities, above and beyond that observed in the ongoing fentanyl crisis.
Therefore, to better understand the pharmacology of nitazenes, we set out to perform a comprehensive examination of 19 nitazene analogs at opioid receptors. We measured affinity, potency, and efficacy at μ opiod receptor (MOR), Δ opiod receptor (DOR), and κ opiod receptor (KOR) alongside morphine, fentanyl, Tyr-D-Ala-Gly-N-methyl-Phe-Gly-ol (DAMGO), U50,488H, and DPDPE. We then compared the pharmacologic measurements of the opioid agonists examined.
Materials and Methods
Materials.
[35S]GTPγS (1250 Ci/mmol), [3H]DAMGO (44–48 Ci/mmol), and [3H]U69,583 (44 Ci/mmol) were purchased from Perkin Elmer Life and Analytical Sciences (Boston, MA). [3H]DPDPE (48–52 Ci/mmol), DAMGO (Tyr-D-Ala-Gly-N-methyl-Phe-Gly-ol), DPDPE ([D-Pen2,D-Pen5]Enkephalin), (±)U50,488H, norbinaltorphimine, (nor-BNI (HCl)2), naltrexone HCl, morphine sulfate pentahydrate, and fentanyl HCl were obtained from the NIDA Drug Supply Program (Bethesda, MD). BTZ, clonitazene [CTZ], MTZ, PYRETZ, N-pyrrolidino protonitazene (PYRPTZ), the hydrochloride salts of DEI, FTZ, MTD, MTZ, PTZ, and the citrate salts of ETD, ETZ, 5-methyl etodesnitazene (5METD), isotodesnitazene (IDZ), ITZ, PIPETZ, N-piperidinyl isotonitazene (PIPITZ), propylnitazene (PPZ), N-pyrrolidino isotonitazene (PYRITZ), and N-pyrrolidino metonitazene (PYRMTZ) were purchased from Cayman Chemical (Ann Arbor, MI). Most other reagents were purchased from Sigma-Aldrich (St. Louis, MO).
Cell Culture.
CHO cells expressing human MOR (CHO-MOR) were purchased from Charles River Laboratories (Wilmington, MA, catalog no. CT6605). CHO-MOR cells were cultured in Hams F12 media supplemented with nonessential amino acids, 10% FBS (Atlas Biologicals, Fort Collins, CO), penicillin/streptomycin, and 200 µg/ml G418. CHO cells expressing human KOR (CHO-KOR) and human DOR (CHO-DOR) were obtained from Stanford Research Institute (Menlo Park, CA) and cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% FetalClone (Fisher, CHO-DOR) or 10% FBS (CHO-KOR), penicillin/streptomycin, and 200 µg/ml G418.
Membrane Preparation.
Cell membranes for receptor binding and [35S]GTPγS assays were prepared as described previously (Eshleman et al., 2020). Briefly, cells were grown to confluence, harvested, prepared in 50 mM Tris buffer (pH 7.5 at 4°C), scraped into calcium- and magnesium-free PBS, and centrifuged for 15 minutes at 500 × g. The resulting pellet was then homogenized in 2 ml of buffer using a polytron, diluted with an additional 11 ml of buffer, centrifuged for 15 minutes at 40,000 × g, then washed and recentrifuged using the same procedure. After covering with 3 ml of buffer, pellets were stored at –80°C until use.
Receptor Binding.
The receptor binding assays have been previously described (Eshleman et al., 2020). Radioligands were [3H]DAMGO (0.2–1.0 nM), [3H]U69,593 (0.2–0.8 nM), or [3H]DPDPE (0.1–0.8 nM) for competition binding at the MOR, KOR, or DOR, respectively, in a final volume of 1 ml. For each test compound, three assays were performed (biologic replicates) in duplicate (technical replicates) in 96-well plates using 50 mM Tris buffer (pH 7.4 at 25°C). Nonspecific binding was measured using 1 µM of the nonradiolabeled version of each radioligand. Cell membranes were incubated with radioligand and test compound at 25°C for 60 minutes. Incubations were terminated by rapid filtration through Perkin Elmer Filtermat A filters presoaked in 0.05% polyethylenimine using a Tomtec cell harvester (Hamden, CT). The filters were dried and spotted with scintillation cocktail. Radioactivity was measured using a Perkin Elmer MicroBeta plate 1405 scintillation counter.
[35S]GTPγS Assays.
Assay conditions were optimized for each receptor as previously described (Eshleman et al., 2020). Briefly, MOR cell membranes were prepared in 20 mM HEPES, 10 mM MgCl2, 100 mM NaCl, 1 mM EDTA, and 0.2 mM DTT (pH 7.4). KOR and DOR cell membranes were prepared in 20 mM HEPES, 10 mM MgCl2, 100 mM NaCl, and 0.2 mM DTT (pH 7.4). Cell membranes were incubated with [35S]GTPγS (50 pM), GDP (1 μM for MOR, 3 μM for KOR, and 10 μM for DOR), and the test opioid compound with buffer (20 mM HEPES, 10 mM MgCl2, 100 mM NaCl, 1 mM EDTA [MOR only], and 0.2 mM DTT, pH 7.4) in a final volume of 1 ml for 60 minutes at 25°C. Nonspecific [35S]GTPγS binding was measured in the presence of excess GTP (10 µM), and basal binding was measured in the absence of any drug. Samples were filtered and counted as described above. Three or more dose–response curves along with a reference full agonist (DAMGO, U50,488H, and DPDPE, for MOR, KOR, and DOR, respectively) were conducted in duplicate (technical replicates) in each experiment (biologic replicates) to identify full and partial agonist compounds.
Data Analysis.
For receptor binding results, data were normalized to specific binding in the absence of test compound. Three or more independent receptor binding and [35S]GTPγS experiments were conducted (three biologic replicates) with duplicate determinations (two technical replicates). Experimenters were not blinded to compound identity. Drug order was randomized from experiment to experiment so that samples were measured from different plate locations. GraphPad Prism10 (San Diego, CA) was used to analyze the resulting data, with IC50 values converted to Ki values using the Cheng-Prusoff equation (Cheng and Prusoff, 1973). The Kd values used in the equation were 0.273 nM for [3H]DAMGO at MOR, 0.65 nM for [3H]U69,593 at KOR, and 0.789 nM for [3H]DPDPE at DOR. One-way ANOVA using logarithms of Ki values of test compounds and standards was used to determine if there were any differences in affinity. Dunnett’s multiple comparison test was used to generate multiple comparisons adjusted P values to conduct pairwise comparisons between test compounds and fentanyl. The cutoff for significance was set at P< 0.05.
For [35S]GTPγS assays, data were normalized to the maximal stimulation of a reference full agonist (DAMGO, U50,488H, and DPDPE, for MOR, KOR, and DOR, respectively). Basal [35S]GTPγS binding was first subtracted from stimulated binding before normalizing to maximal stimulation. Subsequently, GraphPad Prism10 was used to determine EC50 values and efficacies. As with receptor binding, we used one-way ANOVA to determine if there were differences in potencies or efficacies of test compounds and then used Dunnett’s multiple comparisons test to identify significant pairwise differences between test compounds and fentanyl (P < 0.05). To determine the correlation between binding affinities and functional potencies, a nonparametric Spearman correlation was computed for the data for each receptor. To determine the amplification ratio for each nitazene and reference compound, the ratio of binding affinity (Ki) to the functional potency (EC50) for each compound was calculated (Strange, 2008; Kozell et al., 2023).
Results
Substituted Nitazenes: Radioligand Binding at MOR.
At the MOR, the 19 substituted nitazenes had high affinities, as measured by displacement of [3H]DAMGO binding, and the measured affinities ranged from 0.206 nM (ETZ) to 19.3 nM (MTD) (Fig. 2A, Table 1). Fentanyl, a selective and highly abused MOR agonist, with which all substituted nitazene affinities were compared, had a Ki value of 1.255 nM, similar to the other prototypical agonists at MOR, morphine, and DAMGO, and indicative of high affinity (Ki values, Table 1). Notably, nine substituted nitazenes had significantly higher affinities than fentanyl, and 13 had subnanomolar affinities (Table 1). Only two compounds, MTD and FTZ, had significantly lower affinity than fentanyl (Fig. 3A). Some of the first synthesized nitazenes had the lowest affinities for MOR, including BTZ, CTZ, and FTZ.
TABLE 1.
Opioid receptor binding Ki values, substituted nitazenes
For MOR, KOR, and DOR, Hill slopes ranged from –0.65 to –2.71, –0.61 to –1.98, and –0.58 to –1.31, respectively.
| Drug | MOR [3H]DAMGO Ki (nM) ± S.E.M. (n) |
KOR [3H]U69,583 Ki (nM) ± S.E.M. (n) |
hDOR [3H]DPDPE Ki (nM) ± S.E.M. (n) |
|---|---|---|---|
| Fentanyl | 1.255 ± 0.084 (12) | 163 ± 11 (25) | 235 ± 12 (25) |
| Morphine | 1.71 ± 0.22 (12) | 29.2 ± 2.2**** (22) | 172.8 ± 8.8* (24) |
| DAMGO | 0.99 ± 0.11 (13) | ||
| U50,488H | 0.350 ± 0.034 (35)**** | ||
| DPDPE | 2.35 ± 0.16 (29)**** | ||
| BTZ | 1.47 ± 0.36 (3) | 491 ± 13**** (3) | 390 ± 110 (3) |
| CTZ | 3.9 ± 1.5 (4) | 707 ± 26**** (4) | 1770 ± 130**** (3) |
| DEI | 0.252 ± 0.056**** (3) | 650 ± 140**** (4) | 113 ± 31* (4) |
| ETD | 1.024 ± 0.097 (3) | 283 ± 45 (3) | 1310 ± 110**** (4) |
| ETZ | 0.206 ± 0.040**** | 167 ± 14 (4) | 124.2 ± 8.6 (3) |
| FTZ | 12.49 ± 0.71**** (3) | 2680 ± 110**** (3) | >8400#**** (3) |
| ITZ | 0.219 ± 0.039**** | 109.6 ± 9.1 (3) | 143 ± 36 (3) |
| IDZ | 1.85 ± 0.23 (4) | 114 ± 12 (3) | 631 ± 67** (3) |
| 5METD | 0.69 ± 0.15 (3) | 69 ± 22**** (5) | 397 ± 87 (3) |
| MTD | 19.3 ± 5.8**** (5) | 900 ± 140**** (3) | 4720 ± 760**** (4) |
| MTZ | 0.49 ± 0.15* (3) | 154 ± 15 (3) | 373 ± 46 (3) |
| PIPETZ | 0.51 ± 0.10* (4) | 1290 ± 110**** (3) | 607 ± 63* (3) |
| PIPITZ | 0.266 ± 0.066*** (3) | 380 ± 110 (3) | 101 ± 31**** (8) |
| PPZ | 0.92 ± 0.2339 (3) | 249 ± 69 (4) | 285 ± 41 (3) |
| PTZ | 0.295 ± 0.098**** (4) | 301 ± 68 (3) | 142 ± 18 (3) |
| PYRETZ | 0.240 ± 0.041**** (4) | 240 ± 24 (3) | 110.6 ± 3.9 (3) |
| PYRITZ | 0.260 ± 0.058**** (5) | 37.1 ± 9.3**** (3) | 54.4 ± 8.4**** (6) |
| PYRMTZ | 0.929 ± 0.047 (3) | 770 ± 270 **** (3) | 308 ± 73 (5) |
| PYRPTZ | 0.74 ± 0.11 (3) | 342 ± 79** (4) | 133 ± 21 (4) |
n, number of biologic experiments, each conducted in duplicate for nitazenes and reference compounds including fentanyl and morphine.
*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; one-way ANOVA followed by Dunnett’s multiple comparison test compared with fentanyl for each receptor.
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).
Some nitazenes differ only by the number of carbons or by substitution of a halide at R3 (Fig. 1). ETZ (ethoxy), PTZ (propoxy), and ITZ (methylethoxy), with two or three carbon chains, had similar subnanomolar affinities at MOR. By comparison with nitazenes with two or three carbons, MTZ (methoxy), with one carbon, and BTZ (butoxy) with four carbons, had decreased affinities as did the two nitazenes with halides at this position, CTZ and FTZ. PPZ (propyl), with the oxygen removed from the chain, also had decreased affinity.
Compared with the parent compounds, removal of the NO2 group at R1 caused a 5- to 8-fold affinity decrease for ETD and IDZ and a 40-fold shift for MTD (Fig. 2A). Substitution of a methyl group at R1 caused a 3.5-fold affinity decrease for 5METD. Substitution of a five carbon- or six carbon-ring (pyrrolidino or piperidinyl) at R2 caused either no change or a slight decrease in affinity for PIPETZ, PIPITZ, PYRETZ, PYRITZ, PYRMTZ, or PYRPTZ. Removal of an ethyl group from R2 had little effect on the affinity of DEI.
Substituted Nitazenes: Radioligand Binding at KOR and DOR.
In contrast to the high affinities at MOR, the substituted nitazenes had much lower affinities for binding to KOR (inhibition of [3H]U69,583), ranging from 37.1 nM for PYRITZ to 2680 nM for FTZ (Fig. 2B, Table 1). Similarly, fentanyl had lower affinity at KOR (163 nM). At DOR, substituted nitazenes also had lower affinities for inhibition of [3H]DPDPE binding, ranging from 54.4 nM for PYRITZ to >8400 nM for FTZ (Fig. 2C, Table 1). Thus, many substituted nitazenes had very high affinity for MOR and very high selectivity for MOR relative to KOR and DOR. Figure 3A shows the affinities sorted according to the rank order of affinities at MOR. Selectivity for MOR over KOR ranged from 46-fold (MTD) to 2580-fold (DEI). PYRETZ, PTZ, PIPITZ, PIPETZ, and DEI had more than 1000-fold selectivity for MOR over KOR (Fig. 3B). All tested nitazenes had greater than 170-fold selectivity for MOR over DOR, but only ETD and PIPETZ had more than 1000-fold selectivity (Fig. 3C). The rank order of affinities of nitazenes at KOR and DOR is shown in Fig. 3, D and E, with all compounds having Ki values greater than 30 nM at KOR (Fig. 3D) and greater than 50 nM at DOR (Fig. 3E).
TABLE 2.
Opioid receptor functional values, substituted nitazenes
| Drug | MOR EC50 (nM) ± S.E.M. % Max Stimulation (n) |
KOR EC50 (nM) ± S.E.M. % Max Stimulation (n) |
DOR EC50 (nM) ± S.E.M. % Max Stimulation (n) |
|---|---|---|---|
| Fentanyl | 22.7 ± 2.7 96.2% ± 2.4% (13) |
379 ± 29 79.8% ± 2.7% (31) |
1120 ± 120 67.5% ± 2.8% (24) |
| Morphine | 26.6 ± 4.2 86.7% ± 2.6% (11) |
79.6 ± 7.4**** 88.1% ± 2.2% (29) |
792 ± 77 80.2% ± 2.0%** (26) |
| DAMGO | 18.9 ± 2.0 96.1% ± 1.5% (12) |
||
| U50,488H | 0.87 ± 0.10**** 97.26% ± 0.93% (40)**** |
||
| DPDPE | 7.17 ± 0.62**** 102.0% ± 1.2% (29)**** |
||
| BTZ | 12.3 ± 3.1 104.4% ± 4.2% (6) |
3430 ± 390**** 69% ± 10% (3) |
1280 ± 210 85% ± 12% (3) |
| CTZ | 78 ± 14** 104.2% ± 3.7% (3) |
2360 ± 330**** 37.4% ± 8.3%**** (4) |
2260 ± 750 43.0% ± 9.6%* (3) |
| DEI | 0.053 ± 0.014**** 111.2% ± 4.8% (3) |
420 ± 100 93.5% ± 4.0% (5) |
501 ± 22 110.0% ± 5.4%**** (3) |
| ETD | 26.7 ± 8.5 92.8% ± 9.5% (3) |
850 ± 190 104.2% ± 7.4%** (5) |
3330 ± 790*** 40.1% ± 3.5%** (4) |
| ETZ | 1.14 ± 0.33**** 99.4% ± 5.9% (4) |
510 ± 160 85.3% ± 6.9% (4) |
311 ± 29**** 92.0% ± 4.7%** (4) |
| FTZ | 168.5 ± 4.4**** 95.4% ± 6.7% (3) |
6200 ± 2200*** 63% ± 21% (4) |
>8000#**** 15.0% ± 7.6%**** (3) |
| IDZ | 43.3 ± 3.7 96.2% ± 6.9% (3) |
405 ± 38 92.2% ± 4.1% (4) |
3160 ± 590*** 62.2% ± 4.3% (3) |
| ITZ | 1.45 ± 0.28**** 103.5% ± 6.5% (3) |
344 ± 99 87.7% ± 3.5% (6) |
548.6 ± 8.1*** 114.6% ± 7.5%** (4) |
| 5METD | 10.5 ± 1.6 99.3% ± 3.5% (4) |
273 ± 59 86% ± 13% (3) |
1240 ± 110 75.4% ± 7.2% (3) |
| MTD | 294.4 ± 8.7**** 90.2% ± 6.0% (3) |
2150 ± 460**** 103% ± 10% (4) |
>7000**** 24.7% ± 8.0%**** (3) |
| MTZ | 7.27 ± 0.94* 95.3% ± 8.3% (3) |
860 ± 250 85% ± 11% (5) |
2470 ± 320** 79% ± 13% (4) |
| PIPETZ | 8.47 ± 0.81 98.4% ± 6.7% (3) |
1610 ± 370*** 38.3% ± 5.8%**** (4) |
2370 ± 330* 71.0% ± 3.1% (3) |
| PIPITZ | 2.12 ± 0.27**** 88% ± 10% (3) |
740 ± 160 79.0% ± 7.3% (5) |
353.8 ± 5.7** 103.7% ± 8.1%**** (3) |
| PPZ | 4.13 ± 0.37**** 98.5% ± 1.0% (4) |
1740 ± 440*** 67.9% ± 2.1% (4) |
1220 ± 280 83.6% ± 9.6% (4) |
| PTZ | 2.47 ± 0.77**** 93.0% ± 3.3% (6) |
1150 ± 210** 105.6% ± 3.8%** (5) |
2230 ± 420* 109.5% ± 7.1%**** (7) |
| PYRETZ | 0.73 ± 0.16**** 106.1% ± 2.1% (3) |
430 ± 140 56.4% ± 7.2%* (4) |
828 ± 95 106.6% ± 4.5%**** (3) |
| PYRITZ | 0.574 ± 0.012**** 97.8% ± 2.1% (3) |
105 ± 20* 90.5% ± 7.0% (3) |
60.4 ± 2.0**** 101.3% ± 1.9%*** (3) |
| PYRMTZ | 18.2 ± 3.6 95.3% ± 5.5% (4) |
1000 ± 330 42.9% ± 7.9%**** (5) |
1590 ± 130 94.8% ± 6.2%** (3) |
| PYRPTZ | 0.30 ± 0.10**** 93.8% ± 8.7% (4) |
293 ± 79 91% ± 15% (3) |
399 ± 49** 109.6% ± 4.5%**** (3) |
n, number of biologic experiments, each conducted in duplicate for nitazenes and reference compounds including fentanyl and morphine.
*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, one-way ANOVA followed by Dunnett’s multiple comparison test compared with fentanyl for each receptor.
Substituted Nitazenes: Agonist Activity at MOR.
In the [35S[GTPγS binding assay, which measures the interaction of MOR with Gi/o following agonist stimulation, all tested nitazenes were full or near full agonists as compared with maximal stimulation by DAMGO (Fig. 4A, Table 2). There were no significant differences among nitazenes with respect to efficacy (Fig. 6B). The 19 substituted nitazenes had more variation in potencies as MOR agonists, ranging from 0.053 nM (DEI) to 294 nM (MTD), than was observed for their radioligand binding affinities for the MOR (Fig. 6A, Table 2). Fentanyl had an EC50 value of 22.7 nM, similar to morphine (Table 2). To better compare the relative efficacies of nitazenes and standards, which all exhibit maximal/near maximal efficacy at MOR, we calculated and sorted the agonist affinity/EC50 ratios, or amplification ratios, of all the drugs tested (Fig. 7A) (Strange, 2008). One-way ANOVA indicated that drugs differed with respect to their amplification ratios (F = 37.67, P < 0.0001), with post-hoc testing indicating that DEI and PYRPTZ had significantly higher amplification ratios than fentanyl.
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.
Eleven substituted nitazenes had significantly higher potencies than fentanyl, and four had subnanomolar potencies at the MOR (Fig. 6A, Table 2). Differences in potencies paralleled differences in affinities as discussed above. ITZ (Fig. 4A), ETZ, and PTZ, with two or three carbon chains at R3, had similar low nanomolar potencies (1.14–2.47 nM) (Table 2). MTZ and BTZ, with one or four carbons, were less potent (7.27–12.3 nM). All of these compounds had higher potencies than fentanyl. CTZ and FTZ, with halide substitutions, were less potent than fentanyl. Removal of the NO2 group at R1 caused 20- to 40-fold potency decreases for ETD and IDZ (Fig. 4A) and substitution of a methyl group at R1 caused a 10-fold potency decrease for 5METD. Most compounds with five carbon ring (pyrrolidino) substitution at R2 (PYRETZ, PYRITZ, and PYRPTZ) had subnanomolar affinity. PYRMTZ had a 2-fold decreased potency. Substitution of a six carbon ring at R2 had minimal effect on potency for PIPITZ, whereas potency decreased for PIPETZ (∼8-fold). There was an excellent correlation between MOR binding affinities and functional EC50 values (Fig. 5A). We observed two outliers to this generality, because both DEI and PYRPTZ, which have very high amplification ratios at MOR, have much higher potencies than radioligand binding affinities.
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.
Substituted Nitazenes: Agonist Activity at KOR and DOR.
In the functional assays for KOR, agonist efficacy varied, with all being partial to full agonists as defined by maximal stimulation of [35S[GTPγS binding by U50,488H (Figs. 4B and 6B, Table 2). Unlike MOR, a number of nitazenes were partial agonists, with some being weak partial agonists. Four compounds had significantly lower efficacies than fentanyl: CTZ (37.4%), PIPETZ (38.3%), PYRMTZ (42.9%), and PYRETZ (56.4%). PTZ and ETZ had higher efficacies than fentanyl. One-way ANOVA indicated that drugs differed with respect to their KOR amplification ratios (F = 21.39, P < 0.0001), with post-hoc testing indicating that DEI, PIPETZ, PYRETZ, PYRMTZ, and PYRPTZ had significantly higher amplification ratios than fentanyl (Fig. 7B). Fentanyl had a mid-nanomolar potency, lower than that of morphine. Only PYRITZ had higher potency (105 nM) than fentanyl, whereas seven compounds had significantly lower potencies. Potencies at KOR ranged from 105 nM to 6200 nM (FTZ) (Figs. 4B and 6E). KOR potencies relative to MOR are shown in Fig. 6C. Selectivity for MOR over KOR ranged from 7- to 9-fold (IDZ and MTD) to greater than 7900-fold (DEI). PYRPTZ, PYRETZ, PTZ, ETZ, PPZ, and PIPITZ had more than 300-fold selectivity for MOR over KOR (Fig. 6C). There was an excellent correlation between KOR binding affinities and functional EC50 values (Fig. 5B). The rank order of potencies of nitazenes at KOR is shown in Fig. 6E, with all compounds having EC50 values greater than 100 nM at KOR.
Fig. 6.
Heat map of potencies (pEC50) and efficacies (% Emax) 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 (Ki) to the functional potency (EC50) for each nitazene was calculated (Strange, 2008).
In functional assays with DOR, agonist efficacy varied, with most being partial to full agonists as defined by maximal stimulation by DPDPE (Fig. 4C, Table 2). Two compounds, MTD and FTZ, had little agonist activity, and ETN and CTZ had significantly less stimulation of [35S]GTPγS binding compared with fentanyl. One-way ANOVA indicated that drugs differed with respect to their DOR amplification ratios (F = 19.12, P < 0.0001) (Fig. 7C), with post-hoc testing indicating that CTZ, FTZ, MTD, and PYRITZ had significantly higher amplification ratios than fentanyl. Fentanyl had low micromolar potency—lower than morphine. PYRITZ (60.4 nM), ETZ (311 nM), PIPITZ (353.8 nM), PYRPTZ (399 nM), and ITZ (548.6 nM) were more potent than fentanyl, whereas seven compounds were less potent (Fig. 6F). Potencies ranged from 60.4 nM to >8000 nM (FTZ) (Figs. 5C and 6F). Selectivity for MOR over DOR ranged from 24-fold (MTN) to greater than 9400-fold (DEI) (Fig. 6D. There was an excellent correlation between DOR binding affinities and functional EC50 values (Fig. 5C).
Discussion
Overdoses and deaths due to substituted nitazenes have been increasing in the United States since 2019 (Papsun et al., 2022). Of the 19 nitazenes studied here, all were high potency full agonists at MOR with high selectivity for MOR relative to DOR and KOR, consistent with other reports (Vandeputte et al., 2020, 2021, 2022, 2023). Most exhibited higher affinity at MOR than fentanyl, in some cases by a factor of close to 10. Most were also more potent than fentanyl by up to a factor of 430. Efficacies at MOR were almost uniformly consistent with full or near full agonism. At DOR and KOR, a number of nitazenes were partial agonists, including some that were weak partial agonists or showed no agonist activity at all. We calculated amplification ratios to more fully characterize agonist efficacy, which is useful for making relative comparisons between full/near full agonists (Strange, 2008). We found that most nitazenes had higher amplification ratios than fentanyl—some by a factor >10—consistent with higher efficacy. DEI and PYRPTZ both exhibited particularly high amplification ratios. Interestingly, DEI was recently identified as a superagonist relative to the full agonist DAMGO when measuring arrestin recruitment (Malcolm et al., 2023). As a class, the nitazenes appear to exceed fentanyl and fentanyl analogs with respect to their activities at MOR. Consistent with these results, ETZ, ITZ, and DEI are more potent than morphine and fentanyl in antinociceptive and catalepsy assays in rats (Vandeputte et al., 2023). ITZ pharmacokinetics have been examined in rats, with dose-dependent half-lives ranging from approximately 20–60 minutes (Walton et al., 2023), very similar to the terminal half-life of fentanyl in rats (Bjorkman et al., 1994), which suggests comparable withdrawal and risk of re-narcotization for ITZ and fentanyl. The pharmacokinetics for the vast majority of other nitazenes has not been reported.
Examination of structure activity relationships highlighted that two or three carbon chain substitutions at R3, relative to one or four carbon chain, exhibited higher (subnanomolar) affinities and higher potencies at MOR—up to a 10-fold difference. Removal of the nitrite group at R1 significantly decreased affinity and potency by up to 40-fold. Ring substitutions or removal of an ethyl group at R2 had limited effect on affinity and decreased potency by up to 8-fold. DEI and PYRPTZ had much higher potencies than radioligand binding affinities. Nitazene selectivity for MOR relative to KOR and DOR was 10s to 1000s fold higher when considering affinity or potency. Therefore, specific structural changes to the parent molecule can lead to differences in selectivity to the MOR and thus makes these analogs extremely likely to cause toxicity and/or overdose.
In the limited cases where comparison is possible, our affinity and potency results are highly correlated with previous reports (Toll et al., 1998; Vandeputte et al., 2021, 2023). Recent work has also described the importance of alkyl chain length at R1, highlighting higher affinities and potencies of two or three carbon chain nitazenes (Glatfelter et al., 2023). We have confirmed and extended this finding to additional nitazenes with R2 and R3 substitutions. A metabolite of ITZ, DEI, had similar affinity and higher potency at MOR than the parent compound ITZ, in agreement with earlier work (Vandeputte et al., 2022; Walton et al., 2023).
Given the tremendous and escalating health and societal impacts of fentanyl, a high affinity and high potency synthetic opioid, these results highlight the possibility that many nitazenes may have similar or higher potential for overdose and toxicity. In fact, treatment of a metonitazene overdose group, which was associated with a need for cardiopulmonary resuscitation, required a significantly higher number of naloxone boluses compared with treatment of a fentanyl overdose group (Amaducci et al., 2023). In another recent study, the in vitro potencies of MTZ, ETZ, ITZ, PTZ, and BTZ at MOR were highly correlated with their in vivo potencies for hypothermia, increased locomotion, and antinociception, with some exhibiting higher potencies in vitro and in vivo than fentanyl (Glatfelter et al., 2023). Similarly, ITZ and MTZ exhibited very high in vitro affinity and potency at MOR, high in vivo antinociceptive potency, as well as high potency to increase dopamine in the nucleus accumbens (De Luca et al., 2022). DEI led to more potent anti-nociception and greater and longer lasting apnea and respiratory depression (Malcolm et al., 2023).
The first strength of this study is the large number of nitazenes examined at the three main classes of opioid receptors in parallel in binding and functional assays, with comparisons made to important reference opioids like morphine and fentanyl. A second strength is its high potential public health relevance considering the appearance of nitazenes in the illicit market and tremendous public health impact of synthetic opioids. There are a few limitations. The first is that we did not perform characterization of nitazene activity at nonopioid receptors, including targets that mediate fentanyl-related toxicities that may contribute to fatal overdoses (Torralva et al., 2020). The second is that experimenters were not blinded to the drugs tested. Risk of bias is mitigated by drug order randomization within experiments and by concurrent comparisons to standards such as fentanyl and morphine within in each experiment. A final limitation is that we have not extended any of the findings to in vivo models.
Nitazenes may exhibit fentanyl-like (or higher) potential for acute toxicities that result from excessive MOR activation. Notably, a number of nitazenes exhibit higher affinity, higher potency, and higher efficacy than fentanyl at opioid receptors. What is less clear is whether they are associated with potentially fatal effects such as vocal cord closure, which has been associated with fentanyl and may be mediated by off-target activities (Torralva et al., 2020). This possibility is yet to be characterized for nitazenes. Further work characterizing the toxicity potential of nitazenes, and a more complete pharmacological characterization, will be key to understanding nitazene-induced fatal overdoses.
Data Availability
With permission of the US Drug Enforcement Administration, the raw data and GraphPad files that support this research article’s findings are available from the corresponding author on request.
Abbreviations
- BTZ
butonitazene
- CTZ
clonitazene
- DAMGO
Tyr-D-Ala-Gly-N-methyl-Phe-Gly-ol
- DEI
N-desethyl isotonitazene
- DPDPE
[D-Pen2, D-Pen5]enkephalin
- DOR
Δ opiod receptor
- ETZ
etonitazene
- ETD
etodesnitazene
- 5METD
5-methyl etodesnitazene
- FTZ
flunitazene
- IDZ
isotodesnitazene
- ITZ
isotonitazene
- KOR
κ opiod receptor
- MOR
μ opiod receptor
- MTZ
metonitazene
- MTD
metodesnitazene
- PIPETZ
N-piperydinyl etonitazene
- PIPITZ
N-piperidinyl isotonitazene
- PPZ
propylnitazene
- PTZ
protonitazene
- PYRETZ
N-pyrrolidino etonitazene
- PYRITZ
N-pyrrolidino isotonitazene
- PYRMTZ
N-pyrrolidino metonitazene
- PYRPTZ
N-pyrrolidino protonitazene
Authorship Contributions
Participated in research design: Kozell, Eshleman, W. Schutzer, Janowsky, Abbas.
Conducted experiments: Kozell, Eshleman, Wolfrum, Swanson, Bloom, Benware, Schmachtenberg, K. Schutzer.
Performed data analysis: Kozell, Eshleman, Wolfrum, Swanson, Bloom, Benware, Schmachtenberg, Abbas.
Wrote or contributed to the writing of the manuscript: Kozell, Eshleman, K. Schutzer, W. Schutzer, Janowsky, Abbas.
Footnotes
Author and/or study funding was provided by the United States Department of Justice, Drug Enforcement Administration [Grant D-22-OD-0001], Department of Veterans Affairs Career Scientist Program [Grant 14S-RCS-006], National Institutes of Health National Institute on Drug Abuse [Grant ADA12013], Food and Drug Administration [Grant CDER-20-I-0546], the OHSU Physician Scientist Award [Grant 60678300], and the Portland VA Research Foundation [Grant 429999].
The contents do not represent the views of the US Department of Veterans Affairs, US Department of Justice, Drug Enforcement Administration, Food and Drug Administration, or the United States Government.
No author has an actual or perceived conflict of interest with the contents of this article.
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