A Comprehensive Narrative Review of Protonitazene: Pharmacological Characteristics, Detection Techniques, and Toxicology

ABSTRACT

Protonitazene (PNZ) is a synthetic opioid emerging in Europe, Australia, North America and South America with a rapidly increasing number of intoxications. To describe PNZ's pharmacological characteristics, detection methods and the clinical presentation and management of PNZ intoxications, the PubMed database was searched for original articles in English concerning PNZ in any way. All articles were read and analysed completely for their suitability for inclusion, based on the article type, integrity and its description of PNZ pharmacology, toxicology and PNZ intoxications. Of the 21 articles resulting from the search, 16 articles were included. PNZ is a μ‐opioid receptor agonist that induces opioid‐like effects at subnanomolar concentrations at a much higher potency than morphine and fentanyl. 4′‐Hydroxy‐nitazene is a shared metabolite of most nitazenes and can be detected in urine for longer than most nitazenes, providing a way to detect nitazenes without knowing the parent nitazene. PNZ is detectable in whole blood, urine, bile, gastric contents and hair using several forms of mass spectrometry at subnanomolar concentrations but is not detectable in urine using traditional opioid test strips. More reports about monointoxications of PNZ and an appropriate public health response are necessary.

Summary

• The present paper concerns protonitazene, a new synthetic opioid that has been causing an increased number of deaths and injuries across the Western world.

• This paper is a comprehensive narrative review concerning every published article about protonitazene that was deemed suitable for inclusion.

• It draws conclusions about the pharmacology and the toxicology of protonitazene in a concise manner and makes suggestions for clinical implications (including the efficacy of naloxone on protonitazene intoxications) and future research.

1. Introduction

2‐Benzylbenzimidazole opioids (nitazenes) are a subclass of non‐fentanyl novel synthetic opioids. Nitazenes are characterized by their chemical structure (featuring a benzimidazole ring with an ethylamine and a benzyl group) and their great potency, which can be up to 1000 times that of morphine and up to 20 times that of fentanyl [1, 2, 3].

They were first synthesized in 1957 (2 years before the first synthesis of fentanyl) at the Swiss pharmaceutical company CIBA Aktiengesellschaft [2] by a team that consistently published about them until 1961 [2, 4, 5, 6, 7, 8, 9]. They were intended to be a more potent morphine alternative with a better side effect profile, but they never made it to market due to the high risk of addiction and overdoses due to their extreme potency, and all development of them was halted [10, 11].

Following the core scheduling of fentanyl analogues (first temporarily in 2018 and then permanently in 2023), the illicit recreational drug market started switching to alternatives that were not registered as Schedule I Substances [12]. Interestingly, researchers predicted the misuse of nitazenes as early as 1975 due to the relatively easy manufacturing process and extreme potency [13]. According to the United Nations Office on Drugs and Crime (UNODC), the number of nitazene analogues is now quickly rising, with the European Union Drugs Agency (EUDA) now monitoring 81 new opioids, 16 of which are nitazenes [14, 15]. As of 2024, the UNODC has listed a total of seven of these nitazenes as Schedule I Substances: etonitazene and clonitazene (added in 1961) and butonitazene, protonitazene, etonitazepyne, metonitazene and isotonitazene (all five added between 2021 and 2024) [16]. These last five nitazenes were added in recent years due to the sudden increase in sales and usage of these substances, mostly in North America (United States and Canada), Australia and Europe (United Kingdom, Slovenia, Estonia, Latvia, Belgium and the Netherlands) [14, 17].

Due to the potency of nitazenes in combination with often unknowing buyers and users of the substances, the UNODC, the World Health Organization (WHO) and the EUDA have warned and alerted agencies and health professionals of the emergence of these substances [14, 17, 18, 19]. The UNODC reports that synthetic opioids account for the largest burden of disease attributed to drug use disorders, as the high potency of these substances, including fentanyl and nitazenes, can cause overdoses with only minimal quantities of drug ingested [14].

Since 2019, 179 cases involving nitazenes have been reported to the UNODC early warning system, most of which were post‐mortem and about 85% of which were multidrug intoxications [18]. Importantly, the UNODC does not mandate reporting nitazene detections as of 2024. The total number of worldwide deaths related to nitazenes is currently controversial, as both the UNODC and the WHO have not yet published any such data. In Europe, at least 150 deaths have been confirmed to have involved nitazenes, and in Estonia, almost half the drug‐induced deaths in 2023 involved nitazenes [17]. The EUDA warns that all these numbers are probably underestimations due to the lack of routine testing for and detection of these nitazenes.

In England, as of October 2024, published data suggest the nitazene that caused the most fatalities is protonitazene (PNZ) [20] with chemical formula N,N‐diethyl‐2‐[5‐nitro‐2‐[(4‐propoxyphenyl)methyl]benzimidazol‐1‐yl]ethanamine (Figure 1), which has been confiscated by police in capsules labelled ‘OC80’, a label used to indicate OxyContin [21, 22]. Data on the relative occurrence of each nitazene for Europe as a whole is lacking, but the EUDA considers PNZ one of the most dangerous nitazenes as of 2024 due to its potency, easy manufacturing process and the fact that it is being marketed and sold as other substances, making it a major threat to public health [15, 17].

FIGURE 1.

Chemical structure of protonitazene.

The first nitazenes synthesized in the 1950s were largely overshadowed by etonitazene due to its potency, which ranges from hundreds to thousands of times that of morphine in animals [4]. Although etonitazene has been thoroughly studied since its discovery in the 1950s due to its dominance in the industry at the time, the other six Schedule I Substances mentioned earlier have only garnered attention in the last several years. Due to their contribution to overdose fatality, there is a sudden need for research into these substances [23].

Given that data suggest PNZ is the most prevalent nitazene in Western Europe in 2024 [20] but so little is known about it, this review aims to provide clinicians and toxicologists with an overview of PNZ by describing PNZ's pharmacological characteristics, detection methods and the clinical presentation and management of PNZ intoxications.

2. Methods

To answer the research questions, the PubMed database was searched via the NCBI interface on 21 October 2024 using the following search terms: protonitazene[Title/Abstract] NOT ‘systematic review’[Publication Type] NOT ‘review’[Publication Type].

The ‘Title/Abstract’ search term was added to eliminate articles that mention PNZ but lack unique data, such as articles that used PNZ as a comparison. PNZ does not possess any synonyms or MeSH terms in the PubMed database. The database was searched, and all articles were accessed for screening on 24 October 2024. Given the broad scope of this review and the limited number of articles on PNZ, all articles were read completely by the first author of the present paper, and their suitability for inclusion was evaluated. No automation tools were used in these processes.

Selection of articles was done according to PRISMA guidelines [24]. The primary inclusion criterion was that articles must contain information on PNZ related to the research questions—about pharmacological characteristics, detection in human tissues, clinical presentation and/or intoxication management.

The following exclusion criteria were used: Information on PNZ isomers and analogues was excluded, and articles concerning PNZ that lacked information related to the research questions were excluded. Animal studies were not excluded. Articles were screened for biases regarding conflicts of interest (COI) of the publishing authors, but no COIs were found, and no articles were excluded for this reason. Other, more general exclusion criteria—such as for languages other than English or publication dates before 2020—were not necessary as all published articles were in English and were published after 2022.

The reference lists of all included articles were also screened on 24 October 2024 but this delivered no additional articles to include in the review.

Data from each article were collected by the first author. All measures, outcomes and other information regarding PNZ were included. Any data regarding other opioids were excluded, except when necessary for comparison to previous data or other (opioid) substances. For the sake of comparison, all liquid sample concentrations were converted to concentrations in ng/mL using the molecular weight of PNZ of 410.5093 g/mol [25].

3. Results

The search resulted in 21 results, all of which were published in 2022–2024 (Figure 2). A total of five articles were excluded (Table S1). One article was a correction to another article. Two articles were excluded for concerning protonitazepyne (N‐pyrrolidino protonitazene), a structural analogue of PNZ, or iso‐protonitazene, a positional isomer of PNZ. Two articles concerned the presence of PNZ in wastewater, which was not relevant to the research questions.

FIGURE 2.

PRISMA flow diagram.

Sixteen articles remained, and they were searched in full by the first author specifically for relevant information on PNZ, as many articles described several nitazenes at once. The articles can be roughly divided into six case reports and 10 experimental studies (which often also contained short descriptions of case reports regarding the human tissues they used in their experiments) (Table S2).

3.1. Protonitazene Induces Highly Potent Opioid‐Like Effects and Is Partially Hepatically Metabolized Into Shared Metabolite 4′‐Hydroxy‐Nitazene

Glatfelter et al. [26] used rat brain membranes to show that PNZ is an effective, selective μ‐opioid receptor agonist. Kanamori et al. [27] and Kozell et al. [28] also tested the μ‐opioid receptor activity of several nitazenes. In vitro, PNZ is highly potent with a subnanomolar potency: It has a half‐maximal effective concentration (EC₅₀) of 0.07–0.35 ng/mL—stronger than morphine (0.49–20.94 ng/mL) but half as potent as fentanyl (0.037–1.56 ng/mL) [26, 27, 28]. In mice, PNZ induced ‘opioid‐like effects’, such as antinociception, hyper‐locomotion and hypothermia. In humans, PNZ can induce an opioid toxidrome with respiratory depression [29, 30, 31, 32]. Across all measures combined, PNZ was more potent in mice than fentanyl, metonitazene, butonitazene and morphine, but not as potent as etonitazene or isotonitazene [26, 27, 28]. The differences between these substances are near‐linear, with regression coefficients ranging from 0.8697 (p = 0.011) to 0.9764 (p = 0.0002).

As most nitazenes are known to be metabolized hepatically, Jadhav et al. [33] used human liver microsomes (HLM) and human liver S9 fractions (HS9) to study the metabolism of PNZ in vitro. The study found PNZ to be primarily metabolized by cytochrome P450 2D6 and 2B6 and primarily excreted through urine. This, combined with the knowledge of its short half‐life of 6–9 min in HLMs and HS9, respectively [33], necessitates a testing method for a metabolite that is more stable and shared among parent nitazenes to facilitate a single detection method for all current (and possible future) nitazene analogues.

Although several articles [27, 29, 34, 35, 36, 37] describe methods of testing for several nitazenes at once, a shared metabolite among all nitazenes that have a 5‐nitro group (N,N‐diethylamine) and an associated phenyl ether (this includes PNZ) was detected, namely, 4′‐hydroxy‐nitazene, produced by O‐dealkylation (Figure 3) [38, 39, 40]. This metabolite has been detected in urine at ratios relative to PNZ of 15.9–38.3 [37, 39], indicating it is a good target molecule for detection of nitazenes in urine and whole blood, as nitazenes currently are underreported due to a lack of routine detection [41]. Being able to detect a shared metabolite will facilitate the process of including nitazenes in routine drug tests [39]. Whether this metabolite itself is active has not been sufficiently studied, but as O‐dealkylation is a common activation pathway for more traditional opioids, it is possible that 4′‐hydroxy‐nitazene is an active metabolite. Aside from the first experimental test of this metabolite in urine by Ameline et al. [39] and Maruejouls et al. [37], who tested for 4′‐hydroxy‐nitazene experimentally as an ‘additional’ test, there is no literature yet regarding actual applications of detection and quantification of this metabolite to test for PNZ in human intoxications.

FIGURE 3.

O‐dealkylation of protonitazene to 4′‐hydroxy‐nitazene.

3.2. Protonitazene Is Detectable in Whole Blood, Urine and Hair Using Mass Spectrometry

PNZ is detectable in many tissues, including whole blood (central and peripheral), urine, bile, gastric contents and hair. Of these, whole blood and urine are the most tested [31, 34, 35, 37, 38, 39]. Importantly, traditional routine opioid test strips used on urine that can detect morphine, heroin and sometimes fentanyl are not able to detect any nitazenes due to their chemical structure. In the existing literature, PNZ concentrations in whole blood and urine have been mostly analysed using liquid chromatography–tandem mass spectrometry (LC‐MS/MS), ultra‐high performance liquid chromatography–tandem mass spectrometry (UHPLC‐MS/MS) and liquid chromatography‐triple quadrupole mass spectrometry (LC‐QQQ‐MS) [31, 34, 35, 37, 38, 39]. These methods of mass spectrometry (MS), however, always require comparison to an authentic standard. Reference‐free detection in human tissues would make the identification of non‐fentanyl novel synthetic opioids including PNZ much faster. Hollerbach et al. [36] devised a method of detecting PNZ in whole blood using 33 m cyclic structures for lossless ion manipulations‐ion mobility spectrometry/mass spectrometry (SLIM‐IMS/MS) to bypass this need for comparison with authentic standards.

Using traditional methods of MS alone, isotonitazene and PNZ cannot be separated, as they are positional isomers (m/z 411) and can only be separated by their retention times [27, 38, 41]. PNZ has the longest retention time of all Schedule I nitazenes, but this retention time is only marginally longer than that of isotonitazene, and they usually only differ by mere seconds, making it hard to separate these two substances [35, 41]. The new method devised by Hollerbach et al. [36] is more accurate, allowing for the separation of PNZ and isotonitazene into two partially separated peaks for the identification of both substances simultaneously. They also found that most nitazenes, including PNZ, may exhibit up to three gas‐phase isomers, further requiring this need for a more accurate and thorough detection method such as 33 m cyclic SLIM‐IMS/MS.

To assist in the diagnosis of a PNZ intoxication, being able to detect trace amounts of PNZ in blood and urine is crucial. Specific levels of detection or levels of quantification for 33 m cyclic SLIM‐IMS/MS were not mentioned. The lowest level of detection and level of quantification were achieved by Maruejouls et al. [37] using LC‐MS/MS, with a level of detection of 0.05 ng/mL and a level of quantification of 0.1 ng/mL.

Kintz et al. [41] found that PNZ is also detectable and quantifiable in human hair in vitro using LC‐MS/MS (level of detection = 0.1 pg/mg; level of quantification = 1 pg/mg), although this has not yet been done in vivo or on any post‐mortem human subjects. Gas chromatography MS has not been tested in this regard. Detection of nitazenes in human hair is advantageous due to its longer detection window, less invasive collection methods and the ability to store the human hair samples at room temperature. The method is more effective on darker hair, as N‐containing drugs bind more readily to melanin, which is more prevalent in darker hair [41]. All samples tested by Kintz et al. were dark in colour, meaning it is possible this method of detection has a higher level of detection and level of quantification for lighter‐coloured hair than was reported for the darker hair. Kintz et al. did not test any metabolite of PNZ, including 4′‐hydroxy‐nitazene.

3.3. Protonitazene Intoxications in Humans at Subnanomolar Concentrations Can Be Managed With Naloxone

In the 16 selected articles, there are a total of 24 descriptions of human intoxications with PNZ, 18 of which were fatal or post‐mortem [29, 30, 31, 34, 37, 38, 39]. All intoxications were mixed drug intoxications, often with several opioids including several nitazenes. The reported intoxications mostly presented themselves as severe opioid intoxications, with non‐responsive patients suffering respiratory depression and hypothermia, further indicating that PNZ can induce an opioid toxidrome. There is one record of a living patient in a complete coma (Glasgow coma score 3) due to PNZ intoxication, who recovered to a Glasgow coma score of 12 before arrival to the ED through oxygen and ventilation via bag‐valve‐mask alone [30]. This patient did not receive naloxone. Naloxone was administered in six of the non‐fatal cases, all with good results [29, 30, 31, 32]. Partridge et al. [29] postulated that nitazene intoxications, including those involving PNZ, may require higher doses of naloxone for full recovery as compared to intoxications with more traditional opioids.

Although PNZ is available in tablets, powders, crystalline solids and liquids, it is currently mostly used to ‘cut’ other substances (most notably heroin) and, as such, is mostly injected intravenously [29, 32, 42, 43, 44, 45, 46]. Because it is used to cut other substances, it is highly prevalent in prisons [37, 41, 46, 47, 48, 49]. Syrjanen et al. [31] reported the first documented case of PNZ added to vaping e‐liquid without the users' knowledge (both patients reported vaping tetrahydrocannabinol, which was present as well). In a different paper, Syrjanen et al. [32] reported a cluster of unrelated patients with PNZ intoxications who all reported ‘snorting’ ketamine and were all unaware of the presence of PNZ in the powder. None of these patients reported an intention to ingest opioids. Schumann et al. [30] reported a woman who had been using PNZ for months without serious side effects who suddenly lost consciousness and stopped breathing, requiring cardiopulmonary resuscitation and naloxone before eventually recovering, demonstrating the unpredictability of an individual's response to nitazenes even in the setting of tolerance according to the authors of the paper.

Of the 25 cases, 22 [29, 31, 34, 37, 38, 39] reported urine and/or whole blood concentrations of PNZ, all measured using various MS methods compared with authentic standards. One post‐mortem case reported by Papsun et al. [34] involved a whole blood concentration of 1400 ng/mL, which was considered an outlier and was not included in the following numbers. Of the fatal cases, the mean (range) urine concentration was 1.7 ng/mL (0.4–2.9; n = 7), and the mean (range) whole blood concentration was 5.8 ng/mL (0.1–25; n = 15). Of the non‐fatal cases, no urine concentrations were reported, but the mean (range) whole blood concentration was 2.2 ng/mL (0.7–3.0; n = 4).

4. Discussion

PNZ is a highly potent synthetic opioid emerging in Western countries and South America that acts as a μ‐receptor agonist and is fatal at subnanomolar concentrations. It can be detected individually using LC‐MS/MS, UHPLC‐MS/MS or LC‐QQQ‐MS in urine, whole blood, bile, gastric contents and hair with very low levels of detection and levels of quantification, but this always requires comparison with authentic samples. Other techniques such as gas chromatography MS, high‐resolution MS or immunoassays have not yet been researched regarding detection of PNZ. Hair testing is unlikely to be useful in acute settings but can assist in surveillance through detection of nitazenes in post‐mortem cases [41]. Naloxone seems to be an adequate treatment for PNZ intoxications and overdoses, but according to Partridge et al. [29], higher doses may be required compared to intoxications with more traditional opioids.

Any form of MS detection takes longer than a urine test strip, but, as mentioned earlier, routine opioid test strips currently in rotation cannot detect any nitazenes. Opioid test strips for nitazenes are currently in development. BTNX Inc. announced their Rapid Response Nitazene Test Strips in early 2024, priced at USD $2.25 per test [50]. This specific test—a rapid visual immunoassay for qualitative detection of nitazenes—however is dedicated for forensic use only and has a cut‐off sensitivity concentration of 2000 ng/mL, which is multitudes higher than any of the concentrations reported in the literature so far, including all fatalities.

A comprehensive narrative review of PNZ like the present paper has not yet been published. These findings are important for clinicians and toxicologists to understand, as PNZ intoxications often go undetected and untreated, causing unnecessary suffering and fatality.

There are three main shortcomings in the literature represented in this review. The first is that there is very little literature on PNZ. More research on PNZ in the forms of case reports and experimental studies is necessary, specifically regarding monointoxications and research on a wider geographical plane.

The second shortcoming concerns the 25 case reports in the current literature. All these case reports were multidrug intoxications, with two to nine drugs present in every case, most of which were other opioids including other nitazenes. This limits many aspects of research done on these case reports, including the fatal concentrations and the efficacy of naloxone on PNZ intoxications. As for the fatal concentrations, it is noteworthy that although the reported fatal cases have a higher mean whole blood concentration than the non‐fatal cases (5.8 ng/mL vs. 2.2 ng/mL, respectively), the ranges overlap, with a fatality reporting a PNZ concentration as low as 0.1 ng/mL [37], whereas a person with a concentration of 3 ng/mL recovered successfully [29]. This does not provide much insight generally due to the many limitations of these cases including the multidrug nature and the post‐mortem aspect. It is possible that this is related to the receptor sensitivity tolerance of the individual patients, but it is more likely that this is due to the co‐intoxications in each of these cases. Crucially, there is selection and reporting bias due to an overrepresentation of lethal overdoses in current literature, possibly skewing these interpretations to represent the most severe cases.

Finally, the rapid pace at which papers on this topic are being published limits the scope of the present review. It is possible that relevant articles regarding PNZ were published between the conduction of the literature search and the publication of this article that were not included in this review.

Naloxone appears to be effective in treating PNZ intoxications, with most patients who received naloxone recovering completely, but the exact efficacy of naloxone and the effective dosage amounts are unknown due to the complicated nature of the currently reported intoxications. In the reviewed case reports, naloxone was administered through intranasal, intramuscular and intravenous routes [29, 30, 32]. The efficacy of naloxone in these cases cannot be properly assessed due to the coingestants, on which naloxone has no effect, such as alcohol or benzodiazepines. As the in vivo half‐life of PNZ is not known, there are insufficient data to determine whether repeat naloxone dosing is required to combat PNZ intoxications, as it is with several other opioids with long half‐life times. If they present themselves, cases concerning monointoxications with only PNZ ought to be reported to learn more about these aspects of the substance. Furthermore, the statement made by Partridge et al. [29] regarding the requirement of higher doses of naloxone for nitazene intoxications must be nuanced by noting that case reports can only retrospectively document how much naloxone was given and, as such, cannot state that the naloxone ‘was required’. To do this, further study is necessary.

5. Conclusion

PNZ infiltrating the illicit drug market in Western countries (specifically North America, Australia and Western and Northern Europe) and South America, often sold under the guise of another substance [29, 32, 42, 43, 44, 45, 51, 52], is highly threatening to public health. Aside from legal action that has already been taken such as the recent registration of PNZ as a Schedule I Substance, more enforcement and control of these dangerous nitazenes is necessary.

An appropriate public health response may also aid in controlling new synthetic opioids like PNZ. Clinicians ought to be educated about the presence of these drugs in the market and their hallmarks such as the symptoms and methods for treatment and detection. More research into PNZ specifically is necessary, as its comparison to other nitazenes is only limited [36, 38, 41].

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Table S1 List of excluded articles.

Table S2 Methods and findings of the selected articles.

Verbeek J. and Brinkman D., “A Comprehensive Narrative Review of Protonitazene: Pharmacological Characteristics, Detection Techniques, and Toxicology,” Basic & Clinical Pharmacology & Toxicology 137, no. 2 (2025): e70078, 10.1111/bcpt.70078.

Data Availability Statement

Data sharing is not applicable to this article as no new data were created or analyzed in this study.