Evaluation of rewarding effects of nitazene analogs: results from conditioned place preference tests and in vivo microdialysis experiments in mice

Abstract

In illicit drug markets, the most recently expanding new synthetic opioid subclass is benzimidazoles, also known as nitazenes, which were originally developed as analgesics in the 1950s. The emergence of this classical, potent drug family has attracted extensive research interest in the field of forensic toxicology; however, information on their psychological and physical dependence is very limited. Herein, we evaluated the rewarding effects of four nitazene analogs using a battery of in vivo experiments, with a positive control drug (isotonitazene). The four test materials, metonitazene, etodesnitazene, metodesnitazene, and flunitazene, were administered to male C57BL/6J mice by i.p. administration at 0.5, 2, 20, and 20 mg/kg, respectively. In comprehensive behavioral observation tests, representative opioid-related physiological and behavioral states, including analgesia, stereotypic circling behavior, hyperlocomotion, and Straub tail response, were observed. A set of conditioned place preference tests revealed that all the four analogs induced palatability in mice. Furthermore, measurements of dopamine levels in the nucleus accumbens shell by in vivo microdialysis resulted in significant elevations in all test material-treated groups, suggesting that the nitazenes elicit the rewarding effect through a neural circuit originating from the μ-opioid receptor activation at the ventral tegmental area. Our findings add important data regarding the psychological dependence of nitazenes and highlight the abuse potential of these four materials and other prevailing nitazene analogs.

INTRODUCTION

New psychoactive substances (NPS), which initially emerged in response to the international ban on classic drugs of abuse, continue to be popular, and the cumulative number of identified NPSs reached 1,184 in 2022 (Lee et al., 2022; Chaturvedi et al., 2023; Tirri et al., 2023) (World Drug Report 2023). Synthetic cathinones and synthetic cannabinoids account for more than 50% of the NPSs, but new synthetic opioids (NSOs) have become a growing category of NPS (Vandeputte et al., 2021a) (World Drug Report 2023). The most recently expanding NSO subclass is benzimidazoles (2-benzylbenzimidazoles), also known as “nitazenes”.

Nitazenes began to emerge on the illicit drug market in 2019 in response to the demand for alternatives to heroin following control measures to reduce the availability of fentanyl derivatives. Illegal markets in Europe, where most of the heroin originates from Afghanistan, also face a surging demand for NSOs as a reaction to the Taliban banning opium cultivation in 2022. The U.K. has already designated more than a dozen nitazene analogs as Class A (Siczek et al., 2021; Walton et al., 2022; Solimini et al., 2018) (European Drug Report 2024 (EU Early Warning System)). The nitazene analogs were first synthesized as potent analgesics by the Swiss pharmaceutical company, Chemische Industrie Basel (CIBA) in 1957 (Gross and Turrian, 1957; Hunger et al., 1957). Etonitazene was reported as the most potent nitazene analog with a 1,000-fold higher antinociceptive potency than that of morphine (Gross and Turrian, 1957; Hunger et al., 1960a; Ujváry et al., 2021). Starting with isotonitazene, the second most potent analog, these “classic” psychoactive substances began to reappear in the drug scene in Europe and North America in 2019, and several poisoning cases of a variety of nitazene analogs have been reported (Vandeputte et al., 2021b; Hasegawa et al., 2022; Walton et al., 2022). An autopsy case from metonitazene intoxication has also been reported in Japan (Morioka et al., 2023).

Nitazene analogs exhibit their antinociceptive activity by activating the μ-opioid receptor (MOR) pathway, like other opioids such as morphine and fentanyl (Walton et al., 2022). Vandeputte et al. pharmacologically evaluated a total of 29 nitazenes and their metabolites using β-arrestin 2 and mini-Gi recruitment (or GloSensor^® cAMP) assays for monitoring MOR activation. These results revealed that the potencies of two-thirds of the compounds examined were higher than fentanyl, with the highest potency displayed by N-desethyl isotonitazene (its EC₅₀ was two orders of magnitude lower than that of fentanyl) (Vandeputte et al., 2021b; De Vrieze et al., 2024). Antinociceptive activity and acute toxicity, such as respiratory inhibition, caused by nitazene analogs has been well studied using mice, rats, and rabbits by the CIBA group (Ujváry et al., 2021). In addition to these early in vivo studies, some recent studies have shown behavioral and physiological effects of isotonitazene on rats: e.g., antinociception (ED₅₀ = 4.22 μg/kg), catalepsy (ED₅₀ = 8.68 μg/kg), and hypothermia (at a dose of 30 μg/kg) (De Luca et al., 2022; Walton et al., 2023).

Given that the analgesic activity and euphoric effect of opioids are associated with abuse potential, investigations of the psychological and physical dependence of nitazene analogs should be accelerated. Currently, only a few early studies are available. A self-administration experiment using rhesus monkeys concluded that etonitazene was a positive reinforcer (Carroll and Meisch, 1978). A series of studies in nontolerant former morphine addicts at a U.S. Public Health Service Hospital revealed addictive potentials of clonitazene and etonitazene (Anonymous, 1959). Literature reporting experimental evidence about the rewarding effect on rodents is also limited. Evaluation of the effects of isotonitazene and metonitazene on dopamine (DA) transmission in the nucleus accumbens (NAc) shell (De Luca et al., 2022) and etonitazene-induced conditioned place preference (CPP) (Sala et al., 1992) are a few examples. Isotonitazene was recently found to induce CPP in mice at 0.025 and 0.05 mg/kg, although the details were not described (Tomiyama et al., a conference abstract in annual report of National Institute of Mental Health, National Center of Neurology and Psychiatry (fiscal year 2023)).

The purpose of the present study was to obtain further knowledge of the rewarding effects of nitazene analogs. To assess substances with a wide range of potency, we selected four analogs, metonitazene, etodesnitazene, flunitazene, and metodesnitazene, amongst various analogs, whose MOR binding activities were demonstrated in a previous study (EC₅₀ in the β-arrestin 2 assay was 8.14, 54.9, 377, and 548 nM, respectively). (Vandeputte et al., 2021b) (Fig. 1a-d). We evaluated their effects on mice by the CPP test and in vivo microdialysis experiment using isotonitazene as a reference compound (Fig. 1e). Our results demonstrated that all four test materials have the potential to elicit psychological dependence.

Fig. 1

Chemical structures of metonitazene (a), etodesnitazene (b), metodesnitazene (c), flunitazene (d), isotonitazene (e), and generic structure of 2-benzylbenzimidazole ‘nitazene’ opioids (f).

MATERIALS AND METHODS

Materials

All chemicals used in this study were of analytical grade or higher and purchased from the following sources: isotonitazene, metonitazene citrate, and etodesnitazene citrate, Cayman Chemical Company (Ann Arbor, MI, USA); metodesnitazene hydrochloride and flunitazene hydrochloride, Cayman Chemical Company or Chiron (Trondheim, Norway); atipamezole hydrochloride, butorphanol tartrate, and medetomidine hydrochloride, Meiji Seika Pharma (Tokyo, Japan); midazolam, Sandoz (Tokyo, Japan); and Kolliphor ELP, Merck (Darmstadt, Germany).

Isotonitazene was dissolved in a mixture of Kolliphor ELP and 0.9% saline in a ratio of 1:3, and metonitazene, etodesnitazene, metodesnitazene, and flunitazene were dissolved in 0.9% saline.

Animals

Six-week-old male C57BL/6J mice were obtained from Jackson Laboratories Japan (Kanagawa, Japan). The mice were housed in a plastic cage (3 mice per cage) with autoclaved paper bedding (Alpha-Dri, Shepherd Specialty Papers, Watertown, TN, USA). The mice were maintained in a temperature- and humidity-controlled room (24.5 ± 0.3°C and 49.0 ± 4.7% (mean ± SD)) on a 12-hr light-dark cycle with free access to food (CE-2, CLEA Japan, Tokyo, Japan) and water. The experiments were begun after a 2-week acclimation period. After the termination of each experiment, all animals were sacrificed by exsanguination through the abdominal aorta under 3% isoflurane anesthesia.

All experiments were conducted according to the Animal Research Reporting of In Vivo Experiments guideline. The protocol was approved by the Animal Experiment Committee of the Tokyo Metropolitan Institute of Public Health.

The dosage of each test material was determined primarily based on MOR activity assessment of nitazene analogs in vitro (Vandeputte et al., 2021b) and analgesic potency (Hunger et al., 1960a; Hunger et al., 1960b). Other available information was also assessed including reports on the CPP and/or in vivo microdialysis studies for morphine and fentanyl (Fadda et al., 2005; Hataoka et al., 2017) as well as our preliminary behavior observation test for isotonitazene in ICR mice. We have carefully set the dosage of the four analogs in the main CPP tests and in vivo microdialysis experiments after confirming the results of behavioral observation tests.

Behavioral observation test

The dose(s) of each test material for the behavioral observation test was determined as follows: 0.5 mg/kg for metonitazene, 2 mg/kg for etodesnitazene, and 10 and 20 mg/kg for both metodesnitazene and flunitazene. It is noted that we have applied low and high dosage levels to the last two chemicals due to their relatively low potency. In order to perform the CPP test appropriately (described below), in the behavioral observation tests a dose was used that elicited a clear biological behavior response in the drug-treated group but which had no effect on behavior at 6 hr after administration.

Twenty-one mice were divided into 7 groups of 3 animals each: vehicle (saline), metonitazene, etodesnitazene, metodesnitazene (low), metodesnitazene (high), flunitazene (low), and flunitazene (high). The mice were individually placed in a plastic cage (CL-0103-2; CLEA Japan) for 30 min prior to drug administration to allow them to become acclimated to the environment, and then i.p. administered with vehicle or test chemicals at the doses as described above at a volume of 10 mL/kg. The mice were observed at 15, 30, 60, 120, and 360 min after administration, using the general, neurological, and autonomic assessment tables modified from Irwin’s test (Irwin, 1968; Inomata et al., 2017; Takeda et al., 2020). All behavioral and physiological endpoints were evaluated by visual observation or palpation without special instruments (except forceps and sticks) according to the original Irwin’s test and RIKEN modified SHIRPA (https://ja.brc.riken.jp/lab/jmc/shirpa/). Each item was scored by two trained observers with a scale from –3 to +3 (suppressed behavior: −3, −2, and −1, normal behavior: 0, and excited behavior: +1, +2, and +3). The strength of biological effects was determined by the mean value for each item. When the mean value was from 0 to ±0.5, the result was deemed not to be a biological effect. When the mean value was from ±0.5 to ±1.0, a biological effect was suspected. When it was from ±1.0 to ±3.0, the result was deemed to be a biological effect.

Conditioned place preference (CPP) test

CPP tests were performed as previously described (Hataoka et al., 2017). The CPP apparatus consisted of a two-compartment polyvinyl chloride box with black and white colors, separated by a shutter (W 400 mm × D 200 mm × H 250 mm; Bioresearch Center, Aichi, Japan). The black box had a slippery floor and the white box had a rough floor. On day 1, the mice were free to acclimate to the two compartments for 15 min. Thereafter, the mice were allowed to explore again for 15 min, to measure the time spent in each box (pre-test). The measurement of the time a mouse spent in the box was visually assessed by observers with a stopwatch. The box where the mice stayed longer was designed as the vehicle treatment box, and the other box was the drug treatment box. On day 2, the mice were treated i.p. with vehicle or test materials at a volume of 10 mL/kg and were confined for 30 min in the relevant box. After 6 hr, the mice previously treated with the drug were administered vehicle and confined for 30 min in the other box, and vice versa. This training session was repeated for three days (day 2–4). On day 5 (24 hr after the last treatment), the mice were placed in the box, allowing mice to move freely, and the time spent in each of the boxes was recorded for 15 min (post-test). The CPP score was calculated by subtracting the pre-test time from the post-test time in the drug-paired compartment.

The first CPP test was conducted as a positive control experiment consisting of 3 groups: vehicle (mixture of Kolliphor ELP and saline), 0.05 mg/kg of isotonitazene, and 0.1 mg/kg of isotonitazene (6 mice in each group). The main experiment consisted of 5 groups: vehicle (saline), metonitazene (0.5 mg/kg), etodesnitazene (2 mg/kg), metodesnitazene (20 mg/kg), and flunitazene (20 mg/kg) (6 mice in each group).

In vivo microdialysis

Surgery: Mice, anesthetized i.p. with a combination of medetomidine (0.15 mg/kg), midazolam (2 mg/kg), and butorphanol (2.5 mg/kg), were held in a stereotaxic apparatus (SR-5M-NT; Narishige, Tokyo, Japan). A microdialysis probe (D-I-6-01: 0.22 mm outer diameter, 1 mm membrane length; Eicom, Kyoto, Japan) was implanted into the NAc shell of the mouse brain at the following coordinates; AP: +1.76 mm, ML: +0.5 mm relative to bregma and DV: −5.0 mm from the skull (Paxinos and Franklin, 2008; Hataoka et al., 2019). The probes were secured to the skull using dental acrylic. For the recovery from anesthesia, the mice were s.c. injected with atipamezole (0.45 mg/kg) and placed on a heating pad controlled at 38°C. The mice were allowed at least 20 hr for recovery from surgery before initiating the experiments.

Procedure: The probes were perfused with artificial cerebrospinal fluid (NaCl: 148.8 mM, CaCl₂ 2H₂O: 1.80 mM, and KCl: 4.02 mM with pH 7.2–7.4) at a perfusion rate of 1.5 μL/min, using a syringe pump (ESP-64; Eicom). One hour after the reflux, the dialysate samples were collected at 20-min intervals using an autoinjector (EAS-20S; Eicom). After confirmation of a stable level of the signal, 5 dialysate samples (100 min) were collected to establish a baseline level of extracellular DA. Subsequently, mice were treated i.p. with test materials at a volume of 10 mL/kg, then 9 dialysate samples (180 min) were collected for post-administration analysis. Samples were measured using an HTEC-500 HPLC system with an electrochemical detector (Eicom) and an ion-exchange column (CAX; Eicom) maintained at 35°C. The mobile phase was composed of 0.1 M ammonium acetate buffer (pH 6.0) and methanol (7:3 v/v), 0.05 M sodium sulfate, and 50 mg/L EDTA-2Na at a flow rate of 250 μL/min. The results were shown as relative values of the baseline data (mean values of the 5 samples before administration were defined as 100%). After the microdialysis, the mice were sacrificed and their brains were histologically examined to validate the probe placement.

Isotonitazene was first examined for a positive control with doses of 0 (vehicle; a mixture of Kolliphor ELP and saline) and 0.1 mg/kg (5 mice in each group). The four analogs were then evaluated with the same dose as the CPP tests: vehicle (saline), metonitazene (0.5 mg/kg), etodesnitazene (2 mg/kg), metodesnitazene (20 mg/kg), and flunitazene (20 mg/kg) (6 mice in the vehicle group and 8 mice in the metodesnitazene, flunitazene, metonitazene, and etodesnitazene treated group).

Statistical analysis

Comparisons of multigroup values were analyzed by Dunnett’s test in CPP tests and in vivo microdialysis experiments. Comparisons of values between 2 groups in the in vivo microdialysis experiments using isotonitazene were analyzed by Welch’s t-test. One-tailed tests were used for all statistical analyses. Differences in values were considered significant when p-values were less than 0.05. Statistical analyses were performed using StatLight (Yukms, Kanagawa, Japan).

RESULTS

Behavioral and physiologic state of the mice treated with the four nitazene analogs

To elucidate the behavioral and physiologic effects of metonitazene, etodesnitazene, metodesnitazene, and flunitazene, we observed mice i.p. administered with each drug for 6 hr, using the comprehensive observation protocol (behavioral observation test).

As shown in Table 1, all four test materials evoked strikingly similar effects on mice. Both metodesnitazene and flunitazene resulted in a dose-dependent increase of the responses, and the strength of their effects was slightly lower in even the high-dose groups (20 mg/kg), compared with metonitazene and etodesnitazene groups. Amongst general behaviors, stereotype (circling behavior) was enhanced at early time points, and grooming and verticalness were suppressed in the treated groups. Notably, a strong suppression of pain response was observed in all treated groups despite the short duration in the low-dose metodesnitazene and flunitazene groups. In the metonitazene and etodesnitazene groups, the effect on pain response lasted for 120 min. Sound response and touch response were sporadically induced in mice treated with etodesnitazene, metodesnitazene, and flunitazene (Table 1).

Table 1. Scores in mice i.p. administreted with four nitazene analogs in the behavioral observation test.

Neurological behaviors such as spontaneous activity, abnormal gait, and Straub tail reaction were markedly enhanced by the nitazene analogs. The spontaneous activity of hyperlocomotor was frequently observed in the treated mice. In addition, a marked suppression of tendon reflex was observed in the metonitazene group for 60 min (Table 1).

Regarding the items of autonomical behaviors, palpebral opening was induced by all test materials although the clear effects of metodesnitazene and flunitazene were observed only at early time points. Exophthalmos was observed only in the metonitazene group. Of note, respiratory inhibition was never observed in any of the groups at least by visual inspection (Table 1).

Overall, metonitazene elicited the strongest effects among the four analogs, and metodesnitazene and flunitazene showed weaker effects (even in the high-dose groups) in our dose setting. However, the overtly enhanced or suppressed items among general, neurological, and autonomic behaviors were largely common in the four nitazene analogs, and all the effects disappeared 6 hr after administration. Consequently, it seemed reasonable to apply the doses of 0.5, 2, 20, and 20 mg/kg to the CPP test and in vivo microdialysis for metonitazene, etodesnitazene, metodesnitazene, and flunitazene, respectively.

Effects of the four nitazenes on CPP in mice

We next performed 2 sets of CPP tests. In the first experiment, we confirmed that isotonitazene showed a significant place preference at 0.05 and 0.1 mg/kg under the present conditions (Fig. 2a).

Fig. 2

Effects of the nitazene analogs on conditioned place preference in mice: Conditioning was preformed by i.p. administration of (a) isotonitazene; vehicle, 0.05 and 0.1 mg/kg (n = 6) and (b) four nitazene analogs; vehicle, metonitazene (0.5 mg/kg), etodesnitazene (2 mg/kg), metodesnitazene (20 mg/kg), and flunitazene (20 mg/kg) (n = 6 each). Data are expressed as CPP scores: post-test time minus pre-test time in the drug-paired compartment. Values represent the mean ± SEM. Statistical analysis was performed using Dunnett’s test. * p < 0.05, vs. vehicle.

In the main experiment, mice conditioned with metonitazene (0.5 mg/kg), etodesnitazene (2 mg/kg), metodesnitazene (20 mg/kg), or flunitazene (20 mg/kg) showed significantly high CPP scores compared with vehicle control mice (Fig. 2b), indicating that all these nitazene analogs induced palatabilities in mice.

Effects of the four nitazenes on DA levels in the NAc shell

To futher investigate the rewarding effects of metonitazene, etodesnitazene, metodesnitazene, and flunitazene, we next evaluated DA transmissions in the NAc shell induced by the four nitazene analogs using an in vivo microdialysis technique. In the first microdialysis experiment, we confirmed that a positive control material, isotonitazene, with a dose of 0.01 mg/kg induced a significant increase of the extracellular DA level in the NAc shell between 20 and 160 min after the treatment (Fig. 3a).

Fig. 3

Time course of the changes of extracellular dopamine (DA) levels in NAc shell of mice after i.p. administrations of the nitazene analogs: Administered with (a) isotonitazene (vehicle and 0.1 mg/kg, n = 5) and (b) four nitazene analogs; vehicle (n = 6), metonitazene (0.5 mg/kg, n = 8), etodesnitazene (2 mg/kg, n = 8), metodesnitazene (20 mg/kg, n = 7), and flunitazene (20 mg/kg, n = 7). Note that one mouse in each metodesnitazene and flunitazene group was excluded from the analysis due to a dialysis solution leakage stemming from the vigorous circling behavior after the administration. Basal values of the DA levels were 1.27 ± 0.27 nM in (a) (n = 10) and 0.86 ± 0.08 nM (b) (n = 36). Values represent the percent changes from the basal levels (mean ± SEM). Statistical analysis was performed using Welch’s t-test (a) or Dunnett’s test (b). *p < 0.05, vs. vehicle.

In the second microdialysis experiment, mice were divided into 5 groups, and administered vehicle or the four nitazene analogs with the same dose setting as the CPP test. Significant increases in the DA levels were observed from 20 min to at least 80 min after the administration in all four nitazene analogs-treated groups (Fig. 3b). Elevated levels of DA in the NAc shell were comparable among the four groups with a peak range of 227–253%. Metonitazene and flunitazene significantly increased the extracellular DA levels in the NAc shell from 20 min to 180 min and 40 min to 160 min post-administration, respectively. Etodesnitazene and metodesnitazene also induced increases in DA levels but their levels decreased in the latter half of the post-administration observation period (Fig. 3b).

DISCUSSION

In the present study, we demonstrated that four nitazene analogs have rewarding effects on mice. Opioids induce rewarding effects by increasing DA release in the NAc shell by inhibiting GABAergic neurons, which is regulated by the activation of MOR in the ventral tegmental area (Funada, 2005; De Luca et al., 2022). The results of our CPP tests and in vivo microdialysis experiments strongly suggested that the nitazene analogs can elicit the rewarding effect through a similar mechanism. Our results from CPP tests and in vivo microdialysis experiments using isotonitazene reproduced previous findings reported by two studies: Tomiyama et al. using a combination of CPP tests and in vivo microdialysis experiments with doses of 0.025 and 0.05 mg/kg isotonitazene reported that isotonitazene induced drug dependence in mice (a conference abstract in annual report of National Institute of Mental Health, National Center of Neurology and Psychiatry, fiscal year 2023), and De Luca et al. using in vivo microdialysis with doses of 0.001–0.01 mg/kg isotonitazene reported that isotonitazene elicited an increase in DA the NAc shell in rats (De Luca et al., 2022). Of the four analogs, only metonitazene has previously been shown to induce a significant increase of DA in the NAc shell in rats (De Luca et al., 2022). Our findings add important data regarding the rewarding effects of the nitazenes in the light of the recent emergence of a large number of nitazene analogs in the drug circuit and highlight the possibility that other nitazene analogs may also have rewarding effects.

Our comprehensive behavioral observation test revealed that these the four compounds tested induced not only analgesic effects (e.g., decreases of pain response) but also stereotypic circling behavior, hyperlocomotor activity, and the Straub tail response, which were commonly observed in rodents administered opioids (Lowery et al., 2011; Huang et al., 2017). As morphine and fentanyl induced hyperlocomotor activity and the Straub tail response via MOR (Nath et al., 1994; Varshneya et al., 2021), the nitazene analogs are likely to evoke these behaviors via MOR.

We conducted CPP tests and in vivo microdialysis experiments using animals exposed to only one dose of each material (this is a limitation of the present study); however, our results may add to the knowledge of the relative strength of addictiveness of these nitazene analogs to some extent because we could obtain similar levels of responses by all four analogs at in the behavior tests. Thus, based on the range of the doses in our experiments, we depict a possible order of the addictive effects of the five compounds used in this study as follows: isotonitazene > metonitazene > etodesnitazene > flunitazene ≈ metodesnitazene (dosed at 0.05, 0.5, 2, 20, and 20 mg/kg, respectively). This order coincides well with the level of antinociceptive potency: relative potency to morphine was 500, 100, 70, 1, and 1, respectively (Ujváry et al., 2021), as well as the potency of MOR activation: EC₅₀ of MOR-β-arrestin 2 recruitment was 1.63, 8.14, 54.9, 377, and 548 nM, respectively (Vandeputte et al., 2021b). These comparisons further support the suggestive mechanism of the rewarding effects of nitazenes. Of course, further studies for the evaluation of the dose-response of the CPP and/or DA level in the NAc shell are required. In particular, since the rewarding effect of a drug can be elicited at lower doses than those at which other central nervous system effects are induced (Funada, 2005), whether the lower doses of the tested compounds can result in positive results in CPP tests and in vivo microdialysis experiments need to be explored.

The DA levels in the NAc shell in the etodesnitazene and metodesnitazene groups declined slightly faster than those of isotonitazene, metonitazene, and flunitazene groups in the 180 min-monitoring of the microdialysis tests. The difference in the DA releasing property may partially be attributed to differences in the clearance process of bioactive compounds from the blood and brain. Etodesnitazene and metodesnitazene have a structural similarity in the absence of a nitro group in R1 position of the backbone (they are called “desnitro-analogs”), thus, may undergo a different metabolic pathway from the other three analogs (Fig. 1f). Taoussi et al. performed a comparative analysis of the metabolism of four nitazene analogs, isotonitazene, metonitazene, etodesnitazene, and metodesnitazene, using forensic cases and in vitro metabolite profiling technique with human hepatocyte. In their proposed metabolic pathway, the main first transformation of the parent compounds is N-deethylation at the N,N-diethylethanamine side chain or O-dealkylation at R2 position (Fig. 1f). If the first step is N-deethylation, an N-desethyl metabolite with comparable potency to the parent compound (Vandeputte et al., 2021b; De Vrieze et al., 2024) is produced before the detoxification (O-glucuronidation). By contrast, if the first step is O-dealkylation, the resultant metabolites seem more quickly to be glucuronidated. Interestingly, etodesnitazene and metodesnitazene were more frequently involved in the “O-dealkylation-first” route in their in vitro and postmortem samples (Taoussi et al., 2024). Another study evaluating etodesnitazene metabolites in rat serum also revealed the most abundant metabolite was O-dealkylated one (Grigoryev et al., 2023). Therefore, it is possible that etodesnitazene and metodesnitazene were received faster detoxification compared to other three analogs in our microdialysis experiments. Further studies are necessary to explore the types of nitazene metabolites, their binding capacity at MOR, and their biological effects.

In a meeting held in Tokyo on August 2023, the Ministry of Health, Labour and Welfare of Japan recently announced the reinforcement of surveillance and control of NPSs as the number of designer drug retailers, which had almost disappeared in 2015, are rapidly reviving with more than 300 shops currently in operation in Japan. Nitazene analogs are one of the main targets of the regulatory bodies. Apart from the five analogs evaluated here, more than 10 nitazene analogs are controlled as narcotics or designated substance (“Shitei-Yakubutsu”) as of year-end 2024. With rampant global circulation, nitazenes are suspected of contributing to the new wave of North America “opioid crisis” and a dramatic increase in the incidence of opioid-related acute intoxications and fatalities (Di Trana et al., 2022). The increase in nitazene-related fatal cases may stem from some factors such as their very high potency as agonists of MOR and the risks of accidental intake due to being mixed into heroin, fake diazepam, and fake codeine. However, it is also noted that information on their pharmacological and toxicological is very limited since they were never approved for medical use, unlike other opioids. In this respect, new evidence of the psychological dependence of nitazenes from the current study may contribute to increased awareness and suppression of this newly prevailing, dangerous subclass of NSOs.

ACKNOWLEDGMENTS

We would like to thank Dr. Motomu Shimizu for the technical support for in vivo microdialysis experiments. We would also like to thank the members of the Pharmaceutical Affairs Section, Health and Safety Division, Bureau of Public Health, Tokyo Metropolitan Government for their financial and administrative support.

Conflict of interest

The authors declare that there is no conflict of interest.

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