Plasma Pharmacokinetics and Pharmacodynamic Effects of the 2-Benzylbenzimidazole Synthetic Opioid, Isotonitazene, in Male Rats

Abstract

Isotonitazene is an illicit synthetic opioid associated with many intoxications and fatalities. Recent studies show that isotonitazene is a potent μ-opioid receptor (MOR) agonist in vitro, but little information is available about its in vivo effects. The aims of the present study were to investigate pharmacokinetics of isotonitazene in rats, and relate pharmacokinetic parameters to pharmacodynamic effects. Isotonitazene and its metabolites were identified and quantified by liquid chromatography tandem quadrupole mass spectrometry (LC-QQQ-MS). Male Sprague-Dawley rats with jugular catheters and subcutaneous (s.c.) temperature transponders received isotonitazene (3, 10, 30 μg/kg, s.c.) or its vehicle. Blood samples were drawn at 15, 30, 60, 120, and 240 min post-injection, and plasma was assayed using LC-QQQ-MS. At each blood draw, body temperature, catalepsy scores, and hot plate latencies were recorded. Maximum plasma concentrations of isotonitazene rose in parallel with increasing dose (range 0.2–9.8 ng/mL) and half-life ranged from 23.4–63.3 min. The metabolites 4’-hydroxy nitazene and N-desethyl isotonitazene were detected, and plasma concentrations were below the limit of quantitation (0.5 ng/mL) but above the limit of detection (0.1 ng/mL). Isotonitazene produced antinociception (ED₅₀=4.22 μg/kg), catalepsy-like symptoms (ED₅₀=8.68 μg/kg), and hypothermia (only at 30 μg/kg) that were significantly correlated with concentrations of isotonitazene. Radioligand binding in rat brain tissue revealed that isotonitazene displays nM affinity for MOR (Ki=15.8 nM), while the N-desethyl metabolite shows even greater affinity (Ki=2.2 nM). In summary, isotonitazene is a potent MOR agonist whose pharmacodynamic effects are related to circulating concentrations of the parent drug. The high potency of isotonitazene portends substantial risk to users who are exposed to the drug.

1. Introduction

The constant evolution of new psychoactive substances (NPS) in non-medical (i.e., recreational) drug markets presents immense challenges for law enforcement personnel, healthcare providers, forensic toxicologists, analytical chemists, and research pharmacologists (Adamowicz and Nowak, 2022; Pardo et al., 2019; Rudin et al., 2021). When a specific NPS first appears on the market, there is usually little toxicological or pharmacological information about the substance. In the interest of public health and safety, it is imperative to carry out preclinical investigations to determine the biological effects of these drugs (Baumann et al., 2018). However, once such studies are finally completed and published, the drug of interest may no longer be prevalent, since the average lifetime of a particular drug may only be a few months (Pallavi Upadhyay, 2021). Novel synthetic opioids (NSOs) represent a class of NPS with increasing complexity, as various fentanyl analogues disappear from the marketplace and are replaced with non-fentanyl structural templates (Baumann et al., 2020; Morrow et al., 2019; Solimini et al., 2018). In general, all NSOs act as agonists at the μ-opioid receptor (MOR) to exert their effects, and most were first developed as potential analgesic agents but never approved for use in humans (Prekupec et al., 2017; Trescot et al., 2008). Unfortunately, as drug users continue to misuse synthetic opioids recreationally, the adverse effects of opioids pose serious public health and safety concerns. Synthetic opioid toxicity manifests in varying ways, from gastrointestinal effects, decreased heart rate, and decreased blood pressure, to life-threatening respiratory depression (Bowdle, 1998; Pergolizzi et al., 2017). The production of these effects may not only come from the synthetic opioid itself, but may involve active metabolites of the drug, some of which can be much more potent than the parent drug (Trescot et al., 2008).

At present, a predominant non-fentanyl subclass of NSOs is the 2-benzylbenzimidazoles (Montanari et al., 2022; NPS Discovery, 2021a; Walton et al., 2021). The 2-benzylbenzimidazole opioids were first synthesized in Switzerland during the late 1950’s and were tested as analgesic agents in preclinical studies (Hunger et al., 1960, 1957). However, these so-called “nitazene analogues” were never approved for clinical use (Ujváry et al., 2021). In 2019, N,N-diethyl-2-[[4-(1-methylethoxy)phenyl]methyl]-5-nitro-1H-benzimidazole-1-ethanamine (isotonitazene), a drug from the original patent of the 2-benzylbenzimidazoles (Hoffman et al., 1960), emerged on the recreational drug market in the United States of America (USA) (Krotulski et al., 2020). Since the initial identification of isotonitazene, other nitazenes have appeared, with eight new nitazene analogues found in toxicology samples from 2019 to 2021 [npsdiscovery.org]. Isotonitazene has been identified in hundreds of toxicology samples since its first appearance, including samples from postmortem and drugged driving (DUID) cases (Drug Enforcement Administration, 2020; Krotulski et al., 2020; Mueller et al., 2021; Shover et al., 2021; Walton et al., 2021). Due to the imminent danger to public health and safety, the Drug Enforcement Administration (DEA) of the USA, and other regulatory agencies, placed isotonitazene into schedule I control in 2020 (Drug Enforcement Administration, 2020; European Monitoring Centre for Drugs and Drug Addiction (EMCDDA), 2020). More recently, the DEA placed seven other nitazene analogues into schedule I control: butonitazene, etodesnitazene, flunitazene, metodesnitazene, metonitazene, N-pyrrolidino etonitazene, and protonitazene (Drug Enforcement Administration, 2021). Etonitazene and clonitazene, two analogues from the original patent, have both been schedule I drugs since 1961.

Recent pharmacological studies show that isotonitazene is a potent MOR agonist in vitro with an EC₅₀=3.72 nM for the recruitment of Gi in MOR-transfected cells, approximately 9-fold more potent than fentanyl (EC₅₀=34.6 nM) (Vandeputte et al., 2021a). Additionally, the primary metabolite of isotonitazene, N-desethyl isotonitazene, has an even higher MOR potency (EC₅₀=1.16 nM) (Vandeputte et al., 2021a). A number of nitazene analogues exhibit in vitro potency similar to isotonitazene, with the most potent compounds ranging from 0.614 to 8.14 nM (i.e., N-desethyl isotonitazene, etonitazene, N-desethyl etonitazene, protonitazene, and metonitazene) (Vandeputte et al., 2021a). With the exception of older literature from the 1960s, scant information is available about the in vivo pharmacology and toxicology of isotonitazene or other nitazene analogues (Ujváry et al., 2021). Recent publications highlight the in vivo actions of isotonitazene in mice and rats (De Luca et al., 2022; Lee et al., 2022), but no studies have investigated the pharmacokinetics of isotonitazene in laboratory rats. To this end, the purpose of the present study was to use current analytical methods to determine the plasma pharmacokinetics of isotonitazene in rats, then examine the relationship between pharmacokinetic parameters and pharmacodynamic effects. Our preclinical findings will provide vital information about the in vivo potency and efficacy of isotonitazene, which complement existing in vitro findings and provide further insights into the public health risks of the compound.

2. Materials & Methods

2.1. Chemicals and reagents

Isotonitazene, N-desethyl isotonitazene, 5-amino isotonitazene, 4’-hydroxy nitazene, and isotonitazene-D7 drug standards were purchased as powders from Cayman Chemical (Ann Arbor, MI, USA) and prepared at 1 mg/mL in methanol. Drug-naïve rat plasma and brain tissue were purchased from BioIVT (Westbury, NY, USA). Ethyl acetate, N-butyl chloride, and LC-MS grade solvents (e.g., water, methanol) were purchased from Honeywell Chemicals (Charlotte, NC, USA). Formic acid ampoules were purchased from ThermoFisher Scientific (Waltham, MA, USA). Sodium borate decahydrate and dimethylsulfoxide (DMSO) were purchased from Sigma-Aldrich, Millipore (St. Louis, MO, USA). Saline was purchased from Hospira (Lake Forest, IL, USA). Tris pH 7.4, disodium EDTA, MgCl₂ × 6 H₂O, bovine serum albumin (BSA, heat shock free, protease free, fatty acid free), and sodium metabisulfite were also obtained from Sigma-Aldrich, Millipore (St. Louis, MO, USA).

For the drug administration studies, isotonitazene (Cayman Chemical) was dissolved in a solution of 10% DMSO in saline, and this solution served as the vehicle for injection. Each milligram (mg) of powdered drug was dissolved in 100 μL of DMSO using sonication, followed by the addition of 900 μL of 0.9% sterile saline to yield a stock solution of 1 mg/mL isotonitazene in vehicle. Serial dilutions of the 1 mg/mL stock were prepared in 10% DMSO vehicle to yield 30, 10, and 3 μg/mL solutions for injection. Doses were administered at μg/kg body weight.

2.2. Analytical methods

Two spiking solutions (final concentrations 1.0 ng/μL and 0.1 ng/μL) containing isotonitazene, N-desethyl isotonitazene, 5-amino isotonitazene, and 4’-hydroxy nitazene were prepared by serial dilution in methanol from the 1 mg/mL stock standards for quantitative analysis (Figure 1). The matrix volume was 200 μL. The calibration range containing seven non-zero calibrators was evaluated from 0.5 to 50 ng/mL. Low, mid, and high controls were evaluated at 1.6, 8, and 40 ng/mL, respectively. The limit of quantitation was 0.5 ng/mL and the limit of detection was 0.1 ng/mL; both values were administratively set based on experiments performed during method development. Isotonitazene-D7 (20 μL, 0.1 ng/ μL) was used as the internal standard. The authentic rat plasma samples were stored at −80 °C prior to analysis.

Figure 1:

Isotonitazene and its metabolites: N-desethyl isotonitazene, 5-amino isotonitazene, and 4’-hydroxy nitazene

A basic liquid-liquid extraction (LLE) was used to prepare the samples for analysis (Krotulski et al., 2020). Plasma (200 μL) was aliquoted and spiked appropriately if calibrator or control. 20 μL of internal standard was added to each calibrator, control, and rat plasma sample at a final concentration of 10 ng/mL. Borax buffer (1 mL, pH 10.4, 10 mM) and extraction solvent (3 mL, 70:30 N-butyl chloride:ethyl acetate) was added to all samples. Samples were rotated for approximately 15 minutes and centrifuged for 10 minutes at 4600 rpm. The organic layer was separated from the aqueous layer and dried under nitrogen using a TurboVap at 35 °C for approximately 30 minutes to remove excess solvent. The samples were reconstituted in 200 μL of initial chromatographic conditions (i.e., 60A:40B) and transferred to autosampler vials for analysis.

Instrumental analysis was performed according to a validated analytical method developed for human blood by Walton et al., after matrix matching isotonitazene to plasma (Walton et al., 2021). To matrix match to plasma, each control level (i.e., 1.6, 8, 40 ng/mL) was run in quintuplicate against the calibration curve prepared in blood demonstrating a side-by-side comparison of the two biological matrices. In order for the results to be acceptable, the mean concentration of the five control samples for each control level prepared in plasma must be ±20% of the target value and the percent coefficient of variation must be less than 20% (AAFS Standards Board, 2019).

This methodology used a Waters Xevo TQ-S Micro liquid chromatograph tandem quadrupole mass spectrometer coupled with a Waters Acquity I-class ultra-performance liquid chromatograph (LC-QQQ-MS) (Milford, MA, USA). Chromatographic separation was achieved using linear reverse phase gradient elution with an Agilent InfinityLab Poroshell C-18 120 (2.7 μm, 3.0 × 100 mm) analytical column (Table 1), and a multiple reaction monitoring (MRM) method containing 2–3 abundant ions per drug was employed (Table 2) (Walton et al., 2021). The chromatographic separation of isotonitazene and metabolites is shown in Figure 2. Mobile phase A (MPA) consisted of 0.1% formic acid in water and mobile phase B (MPB) consisted of 0.1% formic acid in methanol. Positive electrospray ionization (ESI+) was used. The flow rate was 0.4 mL/min. The injection volume was 5 μL. The column temperature was 30 °C. Data was processed using Waters MassLynx^™ Software (Milford, MA, USA).

Table 1:

Chromatographic separation

Time (min)	Flow (mL/min)	%MPA	%MPB
Initial	0.4	60.0	40.0
1.00	0.4	60.0	40.0
2.00	0.4	70.0	30.0
5.50	0.4	40.0	60.0
6.00	0.4	60.0	40.0
7.00	0.4	60.0	40.0

Table 2:

Mass spectrometer parameters for isotonitazene and metabolites

Analyte	RT (min)	Precursor Ion (m/z)	Product Ions (m/z)	Dwell (s)	Cone (V)	Collision (V)
Isotonitazene	6.34	411.2	100.0*	0.011	46	20
			106.9			52
			72.0			42
N-Desethyl Isotonitazene	6.45	383.2	72.0*	0.011	48	20
			311.9			18
			130.0			56
4’-Hydroxy Nitazene	1.79	369.2	100.0*	0.011	46	24
			72.0			32
5-Amino Isotonitazene	1.21	381.2	100.0*	0.011	48	22
			72.0			42
Isotonitazene-D7	6.34	418.2	100.0*	0.011	50	24
			72.0			46
			107.9			52

^*Quantification ion.

Figure 2:

Chromatographic separation of 5-amino isotonitazene (1.21 min), 4’-hydroxy nitazene (1.79 min), isotonitazene (6.34 min), and N-desethyl isotonitazene (6.45 min). The other peaks in the chromatogram are various nitazene analogues from the original method (Chromatography from Walton et al., 2021).

2.4. Animals and surgery

Male Sprague-Dawley rats (250–300 g) purchased from Envigo (Frederick, MD, USA) were group housed (2 – 3 per cage) under conditions of controlled temperature (22 ± 2 °C) and humidity (45% ± 5%), with ad libitum access to food and water. Lights were on between 7:00 a.m. and 7:00 p.m. The Institutional Animal Care and Use Committee of the National Institute on Drug Abuse (NIDA), Intramural Research Program (IRP), approved the animal experiments, and all procedures were carried out in accordance with the National Institutes of Health, Guide for the Care and Use of Laboratory Animals. The approved NIDA IRP Animal Study Proposal was 20-OSD-35. Vivarium facilities are fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care. Experiments were designed to minimize the number of animals included in the study.

Rats were anesthetized with intraperitoneal (i.p.) ketamine and xylazine (75 and 5 mg/kg) (Covetrus, Dublin, OH, USA), and venous catheters were surgically implanted into the right jugular vein. Catheters were constructed of Silastic medical grade tubing (Dow Corning, Midland, MI, USA) linked to vinyl tubing (SCI, Lake Havasu City, AZ, USA) using a 23 g stainless steel connector. In brief, the proximal Silastic end of the catheter was advanced to the atrium, while the distal vinyl end was exteriorized on the nape of the neck and plugged with a metal stylet. Immediately after catheter implantation, while rats were still under anesthesia, temperature transponders (model IPTT-300; Bio Medic Data Systems, Seaford, DE, USA) were surgically implanted to allow for noninvasive measurement of body temperature (Truver et al., 2020). The temperature transponder emits radio frequency signals received by a compatible handheld reader system (DAS-7006/7r; Bio Medic Data Systems). The transponders are cylindrical in shape (14×2 mm) and were implanted s.c. along the midline of the back, posterior to the shoulder blades, via a prepackaged sterile guide needle delivery system. Rats were single housed postoperatively and given four to seven days to recover from surgery.

2.5. Animal experiments

Rats were brought into the laboratory in their home cages on the day of an experiment and allowed 1 h to acclimate to the surroundings. Polyethylene extension tubes were attached to 1 mL tuberculin syringes and filled with sterile saline before being connected to the vinyl end of the catheters. Blood sampling, performed by an investigator remote from the animal, was facilitated by threading the extension tubes outside the cage. Catheters were flushed with 0.3 mL of 48 IU/mL heparin saline (Thomas Scientific, Swedesboro, NJ, USA) to facilitate blood withdrawal. To prepare the isotonitazene for injection, a 100 μL aliquot of 1 mg/mL isotonitazene was diluted in 10%DMSO:saline vehicle to yield concentrations of 3, 10, and 30 μg/mL. Groups of rats received s.c. injections of either vehicle (control) or 3, 10, or 30 μg/kg isotonitazene on the lower back between the hips. The specific doses administered were chosen based on pilot studies which examined the effects of various doses on catalepsy and antinociception in rats. Rats were randomly assigned to each dose group. Blood samples (400 μL) were withdrawn via catheters immediately before and at 15, 30, 60, 120, and 240 min after injection.

Samples were collected into 1 mL tuberculin syringes and transferred to 1.5 mL plastic tubes containing 5 μL of 1000 IU/mL heparin (Thomas Scientific) as an anticoagulant and 5 μL of 250 mM sodium metabisulfite as a preservative. Blood was centrifuged at 1000 g for 10 min at 4 °C. Plasma was decanted into cryovials and stored at −80 °C until analysis. To maintain volume and osmotic homeostasis, rats received an equal volume of saline solution via the intravenous catheter after each blood withdrawal.

Body temperature, catalepsy scores, and hot plate latency were determined prior to each blood withdrawal as described previously (Bergh et al., 2021). Catalepsy was operationally defined by the presence of three specific symptoms: 1] immobility, 2] flattened body posture, and 3] splayed limbs, as described below. The rats that received isotonitazene in our experiments displayed varying degrees of catalepsy-like rigid immobility. We chose to gauge catalepsy by observing the rats, rather than using the more traditional bar test. Rat behaviors were observed by an experienced rater for 1 min just prior to the measurement of body temperature via the handheld reader system. The behavioral rater was blind to treatment conditions. On each test day, one investigator prepared isotonitazene solutions and administered the drug to rats, whereas another investigator performed the behavioral scoring without knowing the dose being administered to the animal. During the 1 min observation period, catalepsy behaviors were scored based on the presence of 1] immobility (i.e., frozen in place), 2] flattened body posture (i.e., belly pressed to the cage floor), and 3] splayed limbs (i.e., forelimbs and/or hindlimbs extended laterally away from the body) (Truver et al., 2020). At each time point, each symptom was scored as either 1=absent or 2=present. For each animal, catalepsy scores at each time point were summed, yielding a minimum score of 3 and a maximum possible score of 6. To assess hot plate latency, rats were placed on a hot plate analgesia meter (IITC Life Sciences, Woodland Hills, CA, USA) set at 52 °C after the blood draw. The rats remained on the hot plate until they exhibited hind paw licking in response to the heat stimulus (Truver et al., 2020). The maximum time spent on the hot plate was 45 seconds to prevent tissue damage, and the time spent on the hot plate was recorded using a timer triggered by a foot pedal.

2.6. Binding experiments

Opioid receptor binding assays were carried out as described previously (Vandeputte et al., 2022), with minor modifications. Briefly, whole rat brains minus cerebellum (BioIVT) were thawed on ice, homogenized in 50 mM Tris HCl at pH 7.5 using a Kinematica Polytron (setting 6 for 20 s), and centrifuged at 30,000 g for 15 min at 4 °C. The supernatant was discarded, and the pellet was resuspended in fresh buffer and spun again at 30,000 g for 15 min. The pellet was resuspended to yield 100 mg/mL wet weight. Ligand binding experiments were conducted in polypropylene tubes containing 300 μL Tris buffer and 100 μL of tissue suspension for 1 h at room temperature. Radioligands were used at 1 nM final concentration. Specifically, [³H]DAMGO, [³H]DADLE, and [³H]U69,593 (all from Perkin Elmer Life Sciences, Waltham, MA, USA) were used to label MOR, the delta opioid receptor (DOR), and the kappa opioid receptor (KOR), respectively. Non-specific binding was determined in the presence of 10 μM naloxone in all cases. Stock solutions of 10 mM morphine, isotonitazene, and its metabolites were prepared in 100% DMSO and stored at −80 °C. On the day of an experiment, aliquots of stock solution were diluted in 50 mM Tris buffer to yield the appropriate concentrations for binding assays. Incubations were terminated by rapid vacuum filtration over Whatman GF/B filters using a cell harvester (Brandel Instruments, Gaithersburg, MD, U.S.). Filters were washed three times with ice cold buffer, transferred to 24-well plates, and Cytoscint (MP Biomedicals, Irvine, CA, USA) was added. Radioactivity was counted the following day using a Perkin Elmer MicroBeta2 liquid scintillation counter. Raw cpm data were normalized to percent of radioligand bound, and Ki values were determined using nonlinear regression analysis (GraphPad Prism, San Diego, CA, USA).

2.7. Data analysis and statistics

Pharmacokinetic and pharmacodynamic data were statistically evaluated using GraphPad Prism version 8.02 (GraphPad Software, San Diego, CA, USA). Time-concentration profiles for plasma isotonitazene were evaluated by two-way analysis of variance (dose × time) followed by Dunnett’s multiple comparisons test to compare dose effects at each time point. Time course data were further analyzed using Kinetica software (ThermoFisher) to calculate pharmacokinetic constants including maximal concentration (C_max), time of maximal concentration (T_max), area-under-the-curve at 4 h (AUC_4h), elimination constant (K_e) and plasma half-life (t_1/2). Pharmacokinetic constants for each analyte were compared by one-way analysis of variance (dose) followed by Tukey’s tests, or by unpaired t-tests, to determine differences between dose groups. Time course data for hot plate latencies, catalepsy scores, and body temperature were evaluated by two-way analysis of variance (dose × time) followed by Dunnett’s multiple comparisons test. ED₅₀ potency estimates for analgesic and cataleptic effects were calculated by nonlinear regression, using the mean hot plate latencies and mean catalepsy scores over the first 60 min post-injection. The relationships between plasma concentrations of analytes and hot plate latencies, catalepsy scores, or body temperature were assessed using a Pearson’s correlation analysis. Specifically, for each subject, the mean value for isotonitazene concentration across the first 60 min was plotted with respect to the mean hot latency, catalepsy score, and temperature over the same time period. For the radioligand binding studies, Ki values were calculated by nonlinear regression analysis using GraphPad Prism. p < 0.05 was used as the minimum threshold for statistical significance for all comparisons.

3. Results

3.1. Method adaptation

The analytical method from Walton et al., which was previously validated in human blood, was successfully matrix matched to plasma for the detection and quantitation of isotonitazene and its metabolites (Table 3) (Walton et al., 2021). Mean concentrations for each control level (i.e., low, mid, and high) were within ±20% of the target values of 1.6, 8, and 40 ng/mL. During this study, isotonitazene was evaluated quantitatively while the metabolites, N-desethyl isotonitazene, 4’-hydroxy nitazene, and 5-amino isotonitazene, were evaluated only qualitatively due to their concentrations less than the limit of quantitation (0.5 ng/mL). No other adaptations to the existing method were needed to identify isotonitazene and metabolites in plasma.

Table 3:

Results of matrix matching isotonitazene to plasma

	Mean Concentration (± Std Dev)	% Deviation	% Coefficient of Variation
Low Control (1.6 ng/mL)	1.47 ± 0.06	8.12	3.94
Mid Control (8 ng/mL)	7.46 ± 0.27	6.75	3.60
High Control (40 ng/mL)	38.7 ± 1.25	3.37	3.24

3.2. Pharmacokinetics

Isotonitazene was detected in all plasma samples after the administration of 3, 10, and 30 μg/kg of isotonitazene to rats. No drug was detected in the vehicle control rats. Figure 3A shows the time-concentration profiles for isotonitazene in plasma, and Table 4 shows the pharmacokinetic constants that were calculated from the time-course data. The plasma concentrations of isotonitazene were significantly affected by dose administered (F[2, 84]=126.60, p<0.0001) and time (F[5, 84]=71.99, p<0.0001), with a significant dose × time interaction (F[10, 84]=23.79, p<0.0001). Concentrations of isotonitazene rose linearly with the dose administered, and the drug was eliminated rapidly. Dunnett’s post hoc tests revealed that concentrations of isotonitazene after the 10 μg/kg dose were significantly greater than those after the 3 μg/kg dose at 15 and 30 min post-injection. Likewise, concentrations of isotonitazene after the 30 μg/kg dose were significantly greater than those after the 3 and 10 μg/kg doses for the first 60 min post-injection.

Figure 3:

Time-concentration profiles for plasma isotonitazene in rats (Panel A), the structure of isotonitazene (Panel B), chromatographic peak of isotonitazene (Panel C). Male Sprague Dawley rats received s.c. isotonitazene (3, 10, or 30 μg/kg), and serial blood samples (0.4 mL) were withdrawn immediately before and at 15, 30, 60, 120, and 240 min post-injection. Plasma was assayed for isotonitazene and metabolites as described in Materials and Methods. Data are ng/mL expressed as mean±SEM for N=5–6 rats per group. Vertical lines through symbols represent SEM; when no vertical line is visible, the SEM is within the symbol. Solid symbols indicate significant effects compared to the 3 μg/kg dose group at a given time point (Dunnett’s p<0.05).

Table 4:

Pharmacokinetic constants for plasma isotonitazene after systemic administration of the drug to male rats

Dose (μg/kg, s.c.)	Cmax (ng/mL)	Tmax (min)	AUC4h (min * ng/mL)	Ke (1/min)	T1/2 (min)
3 μg/kg	0.46±0.09	15	n.d.*	n.d.	n.d.
10 μg/kg	2.20±0.23	15	4031±1241	0.0222±0.0031	34.1±5.2
30 μg/kg	6.84±1.10	15	24729±3237	0.0125±0.0007	56.4±3.1

Data are expressed as mean ±SEM for N=6 rats per group.

^*n.d. = could not be determined.

The dose of isotonitazene significantly influenced C_max value (F[2,14]=36.95, p<0.0001), whereby the C_max after 30 μg/kg was significantly greater than C_max after 10 μg/kg and 3 μg/kg doses. T_max was found at the earliest time point (15 min) in all dose groups. AUC, K_e and t_1/2 values could not be calculated for the 3 μg/kg isotonitazene dose group because of a limited number of samples with quantifiable drug amounts, so statistical tests for AUC, K_e and t_1/2 were evaluated using unpaired t-tests. The AUC after 30 μg/kg isotonitazene was significantly greater than AUC after 10 μg/kg (t=6.412, df=9, p<0.001), and the dose administered significantly affected values for K_e (t=2.783, df=9, p<0.01) and t_1/2 (t=3.488, df=9, p<0.01). In the latter case, t_1/2 after 30 μg/kg was significantly greater than that after 10 μg/kg, indicating the possibility of delayed drug clearance after isotonitazene doses >10 μg/kg (See Table 4).

In the present study, 4’-hydroxy nitazene and N-desethyl isotonitazene were detected in rat plasma samples, while 5-amino isotonitazene was not. Importantly, the concentrations of the 4’-hydroxy and N-desethyl metabolites were below the limit of quantitation. 4’-Hydroxy nitazene was qualitatively identified at low concentrations in some samples from all three dose groups, while N-desethyl isotonitazene was qualitatively identified at low concentrations only after the 10 and 30 μg/kg doses. Because the results for isotonitazene metabolites could not be quantified, time-concentration profiles for 4’-hydroxy nitazene and N-desethyl isotonitazene are not presented. Even though metabolite concentrations were not quantitated, the detection of N-desethyl isotonitazene is toxicologically significant because of the reported high potency of this metabolite when compared to isotonitazene (Vandeputte et al., 2021a).

3.3. Pharmacodynamic effects

The time-course effects of s.c. isotonitazene administration on hot plate latency, catalepsy score, and body temperature are depicted in Figure 4. Hot plate latency was defined as the time interval from placement of the rat on the hot plate until the animal responded with hind paw licking, measured in sec. Hot plate latency was significantly affected by isotonitazene dose (F (F[3,114]=155.0, p<0.0001) and time after drug administration (F[5,114]=104.7, p<0.0001), with a significant dose × time interaction (F[15,114]=22.67, p<0.0001) (Figure 4A). The maximum cutoff latency was 45 sec, and both the 10 and 30 μg/kg doses produced maximal responses. Dunnett’s post hoc tests showed that 3 μg/kg isotonitazene produced a significant increase in hot plate latency compared to vehicle control only at the 15-min time point, whereas 10 μg/kg isotonitazene significantly increased latencies from 15 to 60 min post-injection. The 30 μg/kg dose produced increases in latency compared to vehicle control for 120 min post-injection. Nonlinear regression analysis of the mean hot plate latency over the first 60 min post-injection revealed an ED₅₀=4.22±0.23 μg/kg for the induction of antinociceptive effects.

Figure 4:

Time course effects of isotonitazene on hot plate latency (Panel A), catalepsy score (Panel B), and body temperature (Panel C). Male Sprague Dawley rats received s.c. isotonitazene (3, 10, or 30 μg/kg) or its 10% DMSO:saline vehicle, and pharmacodynamic measures were determined at 0 (pre-injection), 15, 30, 60, 120, and 240 min post-injection. Hot plate latency (sec) was measured using an analgesia meter, catalepsy score was determined using a numerical scale, and body temperature (°C) was measured non-invasively. Data are expressed as mean±SEM for N=6 rats per group. Vertical lines through symbols represent SEM; when no vertical line is visible, the SEM is within the symbol. Solid symbols indicate significant effects compared to the vehicle control group at a given time point (Dunnett’s p<0.05)

Catalepsy score was determined by rating the absence (1) or presence (2) of immobility, flattened body posture, and splayed limbs at each time point. Catalepsy score was significantly influenced by isotonitazene dose (F[3,114]=134.3, p>0.0001) and time after drug administration (F[5,114]=54.90, p<0.0001), with a dose × time interaction (F[15,114]=15.08, p<0.0001) (Figure 4B). The maximum possible catalepsy score was 6, and 30 μg/kg isotonitazene produced maximal catalepsy responses. Dunnett’s post hoc tests showed that 3 μg/kg isotonitazene did not alter catalepsy score with respect to vehicle control at any time point, while 10 μg/kg isotonitazene significantly increased catalepsy scores at 15 and 30 min post-injection. The 30 μg/kg dose of isotonitazene produced maximal catalepsy scores for 60 min post-injection, and scores remained significantly elevated above control for 120 min post-injection. Nonlinear regression analysis of the mean catalepsy score over the first 60 min post-injection revealed an ED₅₀=8.68±0.43 μg/kg for the induction of cataleptic effects.

Body temperature was measured non-invasively using a handheld reader that was sensitive to the signals emitted from the implanted temperature transponder. Body temperature was significantly affected by isotonitazene dose (F[3,114]=18.53, p<0.0001) and time after drug administration (F[5,114]=5.386, p<0.0002), with a dose × time interaction (F[15,114]=4.127, p<0.0001) (Figure 4C). Isotonitazene produced hypothermia at the highest dose administered (i.e., 30 μg/kg, s.c.), which reached ~3°C below vehicle control levels. Post hoc tests showed that 3 and 10 μg/kg doses of isotonitazene had no effect on temperature, whereas 30 μg/kg isotonitazene produced significant hypothermic responses compared to vehicle control from 15 to 120 min post-injection.

3.4. Correlation analysis

Since we collected pharmacokinetic and pharmacodynamic data from the same individual rats, we were able to examine relationships among the variables measured using Pearson’s correlation analysis. Data from individual rats were used to create a correlation matrix. The correlation between mean circulating concentrations of isotonitazene and mean hot plate latencies over the first 60 min post-injection are shown in Figure 5A. Hot plate latencies were positively correlated with isotonitazene concentrations (r = 0.6946, p<0.0020). Correlation between mean circulating concentrations of isotonitazene and mean catalepsy scores over the first 60 min post-injection are shown in Figure 5B. Catalepsy scores were positively correlated with isotonitazene concentrations (r = 0.8747, p<0.0001). Correlation between mean circulating concentrations of isotonitazene and mean temperature measures over the first 60 min post-injection are shown in Figure 5C. Body temperature was negatively correlated with isotonitazene plasma concentrations (r = −0.8120, p<0.0001).

Figure 5:

Correlations between mean plasma isotonitazene concentrations and hot plate latency (Panel A), catalepsy score (Panel B), and body temperature (Panel C). Mean plasma concentration of isotonitazene, hot plate latency, catalepsy score, and body temperature over the first 60 min post-injection were determined for each rat (N=17), and values were subjected to Pearson’s correlation analyses. Isotonitazene was positively correlated with hot plate latency (p<0.0020), positively correlated with catalepsy score (p<0.0001), and negatively correlated with body temperature (p<0.001).

3.5. Radioligand binding

Dose-response curves depicting the affinity of isotonitazene and its metabolites for MOR, DOR, and KOR in rat brain membranes are shown in Figure 6. [³H]DAMGO, [³H]DADLE, and [³H]U69593 were used to label MOR, DOR and KOR, respectively. Ki affinity values for isotonitazene, its metabolites, and the opioid standard compounds morphine and fentanyl are shown in Table 5. Isotonitazene displayed high affinity for MOR (Ki=15.8 ± 3.1 nM) when compared to DOR (Ki=745.8 ± 265 nM) and KOR (Ki=691.0 ± 220.0 nM). However, the affinity of isotonitazene for MOR was less than that of the comparator compounds morphine (Ki=2.1±0.4) and fentanyl (Ki=4.4±1.0), which had similar affinities. With regard to the metabolites, N-desethyl isotonitazene displayed ~7-fold higher affinity for MOR than the parent compound (Ki=2.2±0.4 nM). While N-desethyl isotonitazene displayed slightly higher affinity than isotonitazene at DOR (Ki=610.2±108.0 nM), it displayed weaker affinity at KOR (Ki=838.9±120 nM). 5-amino isotonitazene displayed lower binding affinity at MOR (Ki=41.9±8.0 nM) when compared to isotonitazene, and this metabolite displayed weak affinity for DOR (>10,000 nM) and KOR (Ki=949.3±323.0 nM.) The findings with 5-amino isotonitazene confirm the importance of the nitro group for high-affinity MOR binding for the nitazene analogues.

Figure 6:

Radioligand binding of isotonitazene and its metabolites at the μ-opioid receptor (MOR) labeled with [³H]DAMGO (Panel A), the δ-opioid receptor (DOR) labeled with [³H]DADLE (Panel B), and the κ-opioid receptor (KOR) labeled with [³H]U69593 (Panel C) in rat brain membranes. Ki values for inhibition of binding were calculated using nonlinear regression analyses and summarized in Table 4. Binding data are expressed as mean±SD from N=3 separate experiments performed in triplicate. Vertical lines through the symbols represent SD; when no vertical line is visible, the SD is within the symbol.

Table 5:

Effects of isotonitazene and its metabolites on opioid receptor binding in comparison to fentanyl and morphine

Drug	MOR Binding (Ki, nM)	DOR Binding (Ki, nM)	KOR Binding (Ki, nM)
Isotonitazene	15.8 ± 3.1	745.8 ± 265	691.0 ± 220
5-Amino Isotonitazene	41.9 ± 8.0	>10,000	949.3 ± 323.0
N-Desethyl Isotonitazene	2.2 ± 0.4	610.2 ± 108.0	838.9 ± 120.0
-	-	-	-
Fentanyl	4.4 ± 1.0	932.1 ± 292.0	365.0 ± 113.0
Morphine	2.1 ± 0.4	442.1 ± 150.0	146.1 ± 61.9

[³H]DAMGO, [³H]DADLE, and [³H]U69593 were used to label MOR, DOR, and KOR respectively. Data are expressed as mean±SD for N=3 experiments performed in triplicate.

4. Discussion

Isotonitazene was first synthesized in the late 1950s, as one of a series of 2-benzylbenzimidazole compounds that were investigated as novel analgesic agents (Hoffman et al., 1960; Hunger et al., 1960, 1957). Isotonitazene and related analogues were never approved for clinical use, and the compounds remained in relative obscurity until their emergence in the recreational drug market in the USA during 2019 (Krotulski et al., 2020; Vandeputte et al., 2021b; Walton et al., 2021). Isotonitazene was first reported in a confiscated drug powder, and soon after it began appearing in biological samples from human case work (Blanckaert et al., 2020; Krotulski et al., 2020). Vandeputte et al. (2021a) demonstrated that isotonitazene is a potent MOR agonist in vitro (EC₅₀=3.72 nM), but information about the in vivo effects of isotonitazene is limited (De Luca et al., 2022; Hunger et al., 1960; Lee et al., 2022), especially in rodent models used to study emerging opioids. Here we report the characterization of isotonitazene pharmacokinetics and pharmacodynamics in male rats. Our study reveals three main findings: 1) isotonitazene displays dose-proportional plasma pharmacokinetics, and its metabolites are present at much lower concentrations than the parent drug; 2) isotonitazene induces dose-dependent antinociception, catalepsy, and temperature effects that are consistent with MOR agonist actions in rats; 3) isotonitazene exhibits nM binding affinity at MOR in rat brain tissue (Ki=15.8±3.1 nM), while its metabolite, N-desethyl isotonitazene, exhibits even higher MOR affinity (Ki=2.2±0.4 nM). Despite the high affinity of N-desethyl isotonitazene for MOR, the extraordinarily low concentrations of this metabolite in plasma suggest it might not contribute to the in vivo effects of isotonitazene, at least in rats.

Our pharmacokinetic findings in rats demonstrate that isotonitazene C_max and AUC increase dose-proportionally over the dose range tested, which is indicative of simple linear kinetics. On the other hand, we observed that isotonitazene t_1/2 after 10 μg/kg (34.1±5.2 min) is significantly shorter than the t_1/2 after 30 μg/kg (56.4±3.1 min), suggesting the possibility of delayed clearance at high drug doses. In support of this notion, Lee et al. (2022) recently showed that isotonitazene has a t_1/2 of 1.64 h (i.e., 98.4 min) in mice receiving a dose of 200 μg/kg via i.p. injection (Lee et al., 2022). The experiments of Lee et al. purposefully employed a high dose of isotonitazene to induce respiratory depression in mice, as a means to test the ability of their newly developed vaccine to ameliorate adverse effects of the drug. Although it is difficult to extrapolate pharmacokinetic data across species, the available findings from rodents indicate that isotonitazene doses above 10 μg/kg might engender nonlinear kinetics, resulting in drug accumulation due to impaired clearance. Future studies are warranted to address this hypothesis. It is noteworthy that isotonitazene C_max values that we measured in rats are in the low ng/mL range, in agreement with findings from the mouse study of Lee et al. (2022) and from human toxicology case work (Mueller et al., 2021; Walton et al., 2021). The present animal findings supplement forensic cases which show that isotonitazene, and perhaps other potent nitazene analogues, are found at very low concentrations in biological samples, which presents challenges for detecting and quantitating these substances.

During this study, our confirmatory analysis detected two of the known metabolites for isotonitazene (N-desethyl isotonitazene and 4’-hydroxy nitazene), and these metabolites were found at concentrations below the limit of quantitation. The presence of these two metabolites is consistent with information available about the in vivo metabolism of isotonitazene (Krotulski et al., 2020; Vandeputte et al., 2021a). The presence of N-desethyl isotonitazene is potentially significant as it has a higher MOR binding affinity (i.e., Figure 6 herein) and agonist potency (EC₅₀=1.16 nM) (Vandeputte et al., 2021a) when compared to the parent compound. 5-Amino isotonitazene was absent in the rat samples, though this may be attributed to concentrations below the limit of our assay detection or poor stability of the metabolite (Walton et al., 2021). It is important to consider that in vivo pharmacokinetic data from rat plasma may not be directly comparable to data from in vitro metabolism studies or human sample analysis. In typical metabolism studies, saturating drug concentrations are incubated with liver microsomes or hepatocytes under static conditions to induce biotransformation of the parent drug, which can produce metabolites that are not seen in authentic human cases. Krotulski et al. (2020) previously determined using an in vitro analysis that isotonitazene can produce four metabolites. In the human case study, the authors discovered 5-amino isotonitazene in the majority of the blood samples analyzed, while N-desethyl isotonitazene was primarily found in the urine samples (Krotulski et al., 2020). In contrast to the human findings, the current studies only detected 4’-hydroxy nitazene and N-desethyl isotonitazene in rat plasma. The summed findings suggest that there are differences with regard to metabolism and disposition of isotonitazene across various species. In any case, the low concentrations of isotonitazene metabolites that we observed in plasma suggest these analytes may not contribute to the in vivo effects of isotonitazene in rats.

We found that isotonitazene induces dose-dependent opioid-like pharmacodynamic effects in rats, including antinociception, catalepsy, and temperature changes. The profile of pharmacodynamic effects produced by isotonitazene in rats mirrors that of other recently encountered NSOs like U-47700 and cyclopropylfentanyl (Bergh et al., 2021; Truver et al., 2020), but isotonitazene is more potent. In particular, present results support that isotonitazene is an ultrapotent antinociceptive agent, with an ED₅₀=4.22 μg/kg, s.c., in the rat hot plate test. Our data are consistent with the original findings of Hunger et al. (1960) who reported that isotonitazene is 500-fold more potent than morphine in the mouse warm water tail flick test. Although the exact ED₅₀ for isotonitazene is not given in the original investigations of Hunger et al.(1960), an extrapolated ED₅₀ value of 10 μg/kg, s.c., can be calculated based on the morphine ED₅₀ of 5 mg/kg, s.c., in the tail flick test. Our hot plate latency data with isotonitazene are similar to those recently reported by De Luca et al. (2022) who showed that i.v. isotonitazene produces antinociception with an ED₅₀ of 1.56 μg/kg in male rats. Lee et al. (2022) found that isotonitazene has an ED₅₀=20 μg/kg, i.p., in the mouse hot plate assay, which is somewhat less potent than our results. However, Lee et al. (2022) used a different species, route of administration, and dosing paradigm to measure antinociceptive effects, when compared to our experiments. The present studies did not examine the pharmacodynamic effects of standard opioid compounds for comparison. However, Vandeputte et al. (2022), recently reported potency values for morphine (ED₅₀=3.94 mg/kg, s.c.) and fentanyl (ED₅₀=20.9 μg/kg, s.c.) in the rat hot plate assay, under conditions identical to the present work. Thus, the overall findings indicate that isotonitazene is ~1000-fold more potent than morphine, and ~5-fold more potent than fentanyl, as an antinociceptive agent in the rat hot plate test.

Opioids are known to cause catalepsy in rats that is characterized by rigid immobility (Ling and Pasternak, 1982; Pasternak et al., 1983). Rat behavioral assays showed that isotonitazene produces catalepsy-like symptoms, but the potency to induce these effects is slightly right-shifted (ED₅₀=8.68 μg/kg, s.c.) when compared to its antinociceptive actions. The fact that higher opioid doses are required to recruit catalepsy, as compared to antinociception, has been observed previously by us (Bergh et al., 2021; Truver et al., 2020) and other labs (Pöyhiä and Kalso, 1992; Taracha et al., 2009). It is noteworthy that catalepsy was evaluated using a behavioral scoring method based on three observable symptoms - immobility, flattened body posture, and splayed limbs- rather than using the more familiar bar test. Nevertheless, our findings demonstrate that isotonitazene is capable of producing sustained immobility and motor impairment in rats. Our catalepsy results agree with those of De Luca et al. (2022) who demonstrated that 10 μg/kg, i.v., isotonitazene is accompanied by profound sedation in rats. Isotonitazene also produced robust hypothermia at the highest dose administered (30 μg/kg, s.c.), and this effect is similar to that produced by other opioids including morphine and fentanyl (Adler et al., 1988; Rawls and Benamar, 2011). Hypothermia was utilized as a proxy for adverse opioid effects, such as bradycardia and respiratory depression, since these changes emerge in parallel with hypothermia after high-dose opioid administration in rodents [e.g., (Wong et al., 2017)]. In an early study by Geller et al., the threshold doses of s.c. morphine and fentanyl which produced significant hypothermia in rats were 30 mg/kg and 200 μg/kg, respectively (Geller et al., 1983). Based on the data from Geller et al. (1983), our findings suggest that isotonitazene is ~1000-fold more potent than morphine, and ~6-fold more potent than fentanyl, in its ability to produce hypothermia. More research is needed to establish the relationship between therapeutic (i.e., antinociception) and adverse effects (i.e., respiratory depression) for nitazene analogues and other NSOs emerging in recreational drug markets.

Because we measured plasma drug concentrations and pharmacodynamic effects in the same subjects, we were able to assess correlations between the various endpoints using data from individual animals. In short, mean plasma concentrations of isotonitazene over the first 60 min post-injection were positively correlated with hot plate latencies and catalepsy scores, but negatively correlated with body temperatures. We have observed similar significant correlations between circulating drug levels and pharmacodynamic endpoints when examining the opioid-like effects of other NSOs, such as U-47700 and cyclopropylfentanyl (Bergh et al., 2021; Truver et al., 2020). In the present study, there appeared to be threshold amounts of plasma isotonitazene that were associated with the recruitment of specific pharmacodynamic effects. For example, maximal hot plate latencies were always observed when mean isotonitazene concentrations exceeded 1 ng/mL, whereas cataleptic and hypothermic effects were not observed until drug concentrations were substantially higher. It is difficult to discern the clinical relevance of our correlational data from rats, but the findings suggest that once a certain threshold amount of isotonitazene is achieved in plasma (e.g., 4 ng/mL), adverse effects of the drug are imminent.

We found that isotonitazene exhibits nM affinity for MOR (Ki=15.8±3.1 nM), with much less affinity for DOR (Ki=745±265 nM) and KOR (Ki=691±220). The binding results confirm that isotonitazene is a MOR-selective compound in rat brain tissue, but the drug displays weaker MOR binding affinity when compared to morphine (Ki=2.1±0.4 nM) and fentanyl (Ki=4.4±1.1 nM). Furthermore, the N-desethyl metabolite displays even higher MOR higher affinity (Ki=2.2±0.4 nM) than isotonitazene itself. The binding affinity of 4’-hydroxy nitazene was not determined in this study, but the 5-amino metabolite displayed somewhat lower activity at MOR (Ki=41.9±8.0 nM) when compared to isotonitazene. The present isotonitazene binding data in rat brain tissue revealed key differences when compared to functional data measuring G protein recruitment in MOR-transfected cells (i.e., see Vandeputte et al., 2021). Specifically, the MOR binding affinity of isotonitazene is weaker than that of morphine and fentanyl, whereas the MOR functional potency of isotonitazene for G protein recruitment (EC₅₀=3.72 nM) is much greater than that of morphine (EC₅₀=385 nM) and fentanyl (EC₅₀=34.6 nM) (Vandeputte et al., 2021b). The precise mechanism(s) responsible for the discrepancies between MOR binding and functional assays are not well understood, but the available evidence suggests that in vitro functional assays in MOR-transfected cells might provide a more accurate estimate of in vivo opioid effects, at least for the compounds investigated here.

In summary, we provide the first characterization of plasma pharmacokinetics for the non-fentanyl synthetic opioid isotonitazene in male rats. We adapted a sensitive analytical method from human case work for the detection of isotonitazene and three of its metabolites for use in this study. Isotonitazene displayed dose-proportional pharmacokinetics in rats and has a relatively short half-life. N-Desethyl isotonitazene and 4’-hydroxy nitazene are present in rat plasma, but these were often below the limit of quantitation. The metabolite identification is consistent with what is often seen in authentic human samples after ingestion of isotonitazene (Krotulski et al., 2020; Walton et al., 2021). Importantly, the data show that isotonitazene exerts opioid-like effects in rats, and the drug is approximately 1000-fold more potent than morphine as an antinociceptive agent. It was found that isotonitazene exhibits nM affinity for MOR in rat brain tissue, which agrees with recent findings from MOR-transfected cells. N-Desethyl isotonitazene exhibited higher MOR affinity than isotonitazene, but the low circulating concentrations of this metabolite suggest that it may not contribute to the effects of systemically administered isotonitazene. Although the popularity of isotonitazene has waned in recent months, similar analogues, such as metonitazene and N-pyrrolidino etonitazene, are increasingly detected in toxicological samples (Krotulski et al., 2021; NPS Discovery, 2021b). In the case of N-pyrrolidino etonitazene, this drug is reportedly even more potent than isotonitazene as an analgesic agent in rats (NPS Discovery, 2021c; Vandeputte et al., 2022). Continued vigilance is needed to assess the potential risks posed by nitazene analogues and other NSOs as they continue to appear on recreational drug markets worldwide.

Highlights.

• Isotonitazene displays nM affinity (Ki=15.8 nM) for the rat μ-opioid receptor.

• Isotonitazene induces antinociception (ED₅₀=4.22 μg/kg) and catalepsy (ED₅₀=8.68 μg/kg).

• Plasma concentrations of isotonitazene increase in parallel with the dose administered.

• Two isotonitazene metabolites were detectable in plasma but could not be quantified.

• Opioid effects of isotonitazene are related to circulating levels of the parent drug.