Structurally diverse fentanyl analogs yield differential locomotor activities in mice

Abstract

Synthetic narcotics have been implicated as the single greatest contributor to increases in opioid-related fatalities in recent years. This study evaluated the effects of nine fentanyl-related substances that have emerged in the recreational drug marketplace, and for which there are no existing or only limited in vivo data. Adult male Swiss Webster mice were administered fentanyl-related substances and their effects on locomotion as compared to MOR agonist standards were recorded. In locomotor activity tests, morphine (100, 180 mg/kg), buprenorphine (1, 10 mg/kg), fentanyl (1, 10 mg/kg), cyclopropylfentanyl (1, 10 mg/kg), cyclopentylfentanyl (10 mg/kg), (±)-cis-3-methylbutyrylfentanyl (0.1, 1, 10 mg/kg), ortho-methylacetylfentanyl (10 mg/kg), para-chloroisobutyrylfentanyl (100 mg/kg), ocfentanil (1, 10 mg/kg), and ortho-fluoroacrylfentanyl (0.1, 1, 10 mg/kg) elicited significant (p ≤ 0.05) dose-dependent increases in locomotion. However, 2,2,3,3-tetramethylcyclopropylfentanyl did not have any effects on locomotion, even when tested up to 100 mg/kg, and 4′-methylacetylfentanyl (10, 100 mg/kg) significantly decreased locomotion. The rank order of efficacy for stimulating locomotion (maximum effect as a % of fentanyl’s maximum effect) for fentanyl-related substances relative to MOR agonist standards was cyclopropylfentanyl (108.84 ± 20.21) > fentanyl (100 ± 15.3) > ocfentanil (79.27 ± 16.92) > morphine (75.9 ± 14.5) > (±)-cis-3-methylbutyrylfentanyl (68.04 ± 10.08) > ortho-fluoroacrylfentanyl (63.56 ± 19.88) > cyclopentylfentanyl (56.46 ± 8.54) > para-chloroisobutyrylfentanyl (22.44 ± 8.51) > buprenorphine (11.26 ± 2.30) > ortho-methylacetylfentanyl (9.45 ± 2.92) > 2,2,3,3-tetramethylcyclopropylfentanyl (6.75 ± 1.43) > 4′-methylacetylfentanyl (3.47 ± 0.43). These findings extend in vivo results from previous reports documenting additional fentanyl related-related substances that stimulate locomotion similar to known abused opioids while also identifying some anomalies.

1. Introduction

Fentanyl is a synthetic drug that produces its effects primarily via activation of the mu-opioid receptor (MOR) and has become increasingly problematic for its role in accidental overdose in the United States (Al-Hasani and Bruchas, 2011; Drug Enforcement Administration, 2017a,b, 2020; Hedegaard et al., 2018, 2019; Pathan and Williams, 2012; Spencer et al., 2019). Fentanyl is administered to patients as an analgesic for acute and chronic pain and as an anesthetic in combination with other complementary drugs (Bailey et al., 1985; Mather, 1983; Peng and Sandler, 1999; Scholz et al., 1996). Fentanyl and related substances are also consumed by persons with opioid use disorder (OUD) for their euphoric effects and alleviation of opioid withdrawal symptoms and sometimes unintentionally consumed as adulterants of other drugs (Comer and Cahill, 2019; Drug Enforcement Administration, 2016; Kuczynska et al., 2018). Recently, these drugs were reported to complicate conventional treatments for OUD and may discourage patients from seeking treatment (Varshneya et al., 2021a, 2022b). Non-fatal intoxications in persons with OUD involving fentanyl and its analogs have steadily increased in prevalence (Arfken et al., 2017; Chhabra et al., 2021; Kenney et al., 2018; Martinez et al., 2021; Ochalek et al., 2019, 2020). Fatalities involving fentanyl and related substances have also surged in recent years (Kuhlman et al., 2003; Martin et al., 2006; Thompson et al., 2007). In 2013, there were 3105 deaths in the United States involving synthetic opioids that increased over 18-fold to 56,516 in 2020 (Centers for Disease Control and Prevention, 2021), representing a 1820 % change. Moreover, synthetic opioids other than methadone, but including fentanyl-related substances, were involved in 82.3 % of the 68,630 opioid-related deaths in the United States in 2020 (Centers for Disease Control and Prevention, 2021). Given the increasing prevalence of both fatalities and non-fatal intoxications involving fentanyl and its analogs, this study sought to characterize the locomotor effects of several fentanyl-related substances that have emerged or prospectively, are likely to emerge in the recreational drug marketplace for which there are no existing or only limited in vivo data.

We previously reported the effects of fourteen fentanyl-related substances on nociception and locomotion in mice (Varshneya et al., 2019, 2021b) and of seven on their respiratory depressant effects (Varshneya et al., 2022a). We demonstrated that these fentanyl-related substances elicit significant dose-dependent antinociception and respiratory depression and that most, but not all substances tested, also elicit significant dose-dependent hyperlocomotion. We found that these effects were, in part, mediated by the MOR as indicated by significant rightward shifts in their dose-effect curves following pretreatment with naltrexone or naloxone. Here we report that other, previously uncharacterized, structurally-related fentanyl analogs will elicit effects in mice that are likely mediated by the MOR. Given the relevance of these emerging substances to public health, their characterization will be useful for regulatory scientists and policy makers (e.g., drug scheduling), forensic toxicologists (e.g., post-mortem toxicological analysis), and clinicians (e.g., patient care following non-fatal intoxications).

This study evaluated whether a selected subset of fentanyl-related substances would elicit hyperlocomotion in mice as measured by increases in distance traveled with comparable potency and efficacy to prototypical opioids. The drugs evaluated were morphine, buprenorphine, and fentanyl as MOR agonist standards, and cyclopropylfentanyl, cyclopentylfentanyl, (±)-cis-3-methylbutyrylfentanyl, ortho-methylacetylfentanyl, 4′-methylacetylfentanyl, para-chloroisobutyrylfentanyl, ocfentanil, ortho-fluoracrylfentanyl, and 2,2,3,3-tetramethylcyclopropylfentanyl as representative fentanyl-related substances. These drugs were chosen in part because of their diversity in chemical structure, their appearance in the recreational drug marketplace, and because several of them and their isomers or metabolites had been identified in fatal and non-fatal intoxications (Drug Enforcement Administration, 2021a,b; European Monitoring Centre for Drugs and Drug Addiction, 2018). This study clarifies whether these fentanyl-related substances have MOR agonist-like hyperlocomotor effects in mice similar to known abused opioids.

2. Methods

2.1. Drugs

(1) Morphine, (4R,4aR,7S,7aR,12bS)-3-methyl-2,3,4,4a,7,7a-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-7,9-diol sulfate pentahydrate, was provided by the National Institute on Drug Abuse (Bethesda, MD, USA) Drug Supply Program. (2) Buprenorphine, (4R,4aS,6R,7R,7aR,12bS)-3-(cyclopropylmethyl)-6-(2-hydroxy-3,3-di methylbutan-2-yl)-7-methoxy-1,2,3,4,5,6,7,7a-octahydro-4a,7-ethano-4,12-methanobenzofuro[3,2-e]isoquinolin-9-ol hydrochloride was obtained from Spectrum Chemical (New Brunswick, NJ, USA). (3) Fentanyl, N-(1-phenethylpiperidin-4-yl)-N-phenylpropionamide citrate, was obtained from Sigma-Aldrich (St. Louis, MO, USA). Fentanyl-related substances: (4) cyclopropylfentanyl; N-(1-phenethylpiperidin-4-yl)-N-phenylcyclopropanecarboxamide hydrochloride, (5) cyclopentylfen tanyl; N-(1-phenethylpiperidin-4-yl)-N-phenylcyclopentanecarboxa mide hydrochloride, (6) (±)-cis-3-methylbutyrylfentanyl; cis-N-(3-methyl-1-phenethylpiperidin-4-yl)-N-phenylbutyramide hydrochloride, (7) ortho-methylacetylfentanyl; N-(1-phenethylpiperidin-4-yl)-N-(o-tolyl)acetamide hydrochloride, (8) 4′-methylacetylfentanyl; N-(1-(4-methylphenethyl)piperidin-4-yl)-N-phenylacetamide hydrochloride, (9) para-chloroisobutyrylfentanyl; N-(4-chlorophenyl)-N-(1-phenethylpiperidin-4-yl)isobutyramide hydrochloride, (10) ocfentanil; N-(2-fluorophenyl)-2-methoxy-N-(1-phenethylpiperidin-4-yl)acetamide hydrochloride, (11) ortho-fluoracrylfentanyl; N-(2-fluorophenyl)-N-(1-phenethylpiperidin-4-yl)acrylamide hydrochloride, and (12) 2,2,3,3-tetramethylcyclopropylfentanyl; 2,2,3,3-tetramethyl-N-(1-phenethylpiperidin-4-yl)-N-phenylcyclopropane-1-carboxamide hydrochloride were obtained from the Cayman Chemical Company (Ann Arbor, MI, USA). Drugs were obtained as dry powders and either dissolved in sterile saline (Fisher Scientific, Hampton, NH, USA) or suspended in 0.5 % methylcellulose (Sigma-Aldrich, St. Louis, MO, USA) in deionized water and were administered subcutaneously (SC) in a volume equivalent to 10 ml/kg body weight. Chemical structures for drugs 1–12 are shown in Fig. 1.

Fig. 1.

Chemical structures of (1) morphine, (2) buprenorphine, (3) fentanyl, (4) cyclopropylfentanyl, (5) cyclopentylfentanyl, (6) (±)-cis-3-methylbutyrylfentanyl, (7) ortho-methylacetylfentanyl, (8) 4′-methylacetylfentanyl, (9) para-chloroisobutyrylfentanyl, (10) ocfentanil, (11) ortho-fluoracrylfentanyl, and (12) 2,2,3,3-tetramethylcyclopropylfentanyl.

2.2. Subjects

Adult male Swiss Webster mice (N = 512; Crl:CFW(SW), Charles River Laboratories, Raleigh, NC, USA) weighing ~25–50 g at the time of testing were housed four subjects per cage in Association for Assessment and Accreditation of Laboratory Animal Care-accredited facilities. Subjects had ad libitum access to food (Teklad 7012 Rodent Diet; Envigo, Madison, WI, USA) and tap water. The vivarium was maintained at 22 °C ± 2 °C and 50 % ± 5 % humidity, with lights set to a 12-h light/dark cycle (lights on at 0600) and testing occurred during the light phase. Subjects were typically tested on weekdays between the hours of 1000 and 1600. Subjects were acclimated to the vivarium for at least one week before commencing experiments and were drug-naive before testing. All mice were drug and experimentally naïve prior to testing and only tested once. All procedures were carried out in accordance with the Guide for Care and Use of Laboratory Animals as adopted and promulgated by the National Institutes of Health (Committee for the Update of the Guide for the Care and Use of Laboratory Animals, 2011). The experimental protocol was approved by the Institutional Animal Care and Use Committee at Virginia Commonwealth University.

2.3. Measurement of locomotion

Locomotor activity tests were conducted in eight commercially obtained, automated activity monitoring devices each enclosed in sound- and light-attenuating chambers that recorded movement via computer-controlled circuitry (AccuScan Instruments, Columbus, OH, USA). The interior of each device was divided into separate 20 × 20 × 30 cm fields permitting the independent and simultaneous measurement of two subjects. Sixteen photobeam sensors per axis were spaced 2.5 cm apart along the walls of the chamber and detected movement. A fan mounted in each test chamber provided ventilation and masking noise. On a test day, subjects were transported to the laboratory where they acclimated for ~30 min. Subjects were injected (SC) with either vehicle or drug and immediately placed in the test chambers where their movement was recorded for 120 min. Subjects administered vehicle were tested concurrently with drug-administered subjects. Doses of fentanyl-related substances were selected to include a sufficient number of doses such that: (1) maximum mean effects between at least two doses significantly differed from one another (as defined by non-overlapping SEMs); and (2) at least at one time interval, the effects of at least one dose was significantly different (p ≤ 0.05) from vehicle; or (3) until 100 mg/kg, SC was tested. Based on these criteria, fentanyl-related substances were tested at doses of 0.1, 1, and 10 (and 100 if necessary) mg/kg, SC. Fentanyl (0.1, 1, and 10 mg/kg, SC), morphine (1, 10, 100, and 180 mg/kg, SC), and buprenorphine (0.1, 1, and 10 mg/kg, SC) were tested as comparators (Janssen, 1975). Results for fentanyl and morphine have been presented previously (Varshneya et al., 2019, 2021b). The total distance traveled (cm) within each 10-min bin during the experimental session was recorded for each mouse. All locomotor activity tests used a between-subjects, acute dose design.

2.4. Data analysis

Distance traveled (cm) was the primary dependent variable and is reported as mean values (±SEM) for groups of subjects at each drug dose. Statistical significance was assessed by either one-way or two-way analyses of variance (ANOVA; Gaussian distribution of residuals assumed; sphericity assumed). Fisher’s LSD post-hoc analyses were used for all pairwise comparisons. Dose-effect curves for locomotion were further analyzed to determine the estimated dose required for eliciting 100 m of travel using nonlinear regression. Data for morphine were fit to the following model: [Agonist] vs. response – Variable slope, Y = Bottom + (XĤillslope) * (Top-Bottom) / (XĤillSlope + EC₅₀HillSlope). Fentanyl, cyclopropylfentanyl, (6) (±)-cis-3-methylbutyrylfentanyl, ocfentanil, and ortho-fluoracrylfentanyl generated inverted U-shaped curves relating distance traveled to dose and were modeled with a lognormal distribution (Y = (A/X) * exp(−0.5 * (ln(X/GeoMean) / ln (GeoSD))^2) where parameters ‘A’ and ‘GeoMean’ were constrained to values > 0, and parameter ‘GeoSD’ was constrained to values > 1). Buprenorphine, cyclopentylfentanyl, ortho-methylacetylfentanyl, 4′-methylacetylfentanyl, para-chloroisobutyrylfentanyl, and 2,2,3,3-tetramethylcyclopropylfentanyl did not produce marked effects on locomotion and therefore were not appropriate for modeling. G*Power 3.1.9.7 was used to a priori calculate the sample sizes required to detect statistically significant differences for a large effect (F-test for ANOVA: 0.40) given alpha (0.05) and power (0.8) for all tests (Cohen, 1988; Faul et al., 2007, 2009). Comparisons were considered statistically significant if p ≤ 0.05. Analyses were performed with software (GraphPad Prism 9.4.1 (681) for Microsoft Windows 10 × 64; GraphPad Software, San Diego, CA, USA).

3. Results

3.1. Results from locomotor activity tests

Fig. 2 shows total distance traveled (cm) in the 2-h test session as a function of dose for each drug in locomotor activity tests. Morphine [100 and 180 mg/kg; F(4, 59) = 17.06, p < 0.0001], buprenorphine [1 and 10 mg/kg; F(3, 44) = 3.388, p = 0.0262], fentanyl [1 and 10 mg/kg; F(3, 60) = 28.76, p < 0.0001], cyclopropylfentanyl [1 and 10 mg/kg; F(3, 28) = 17.12, p < 0.0001], cyclopentylfentanyl [10 mg/kg; F(4, 43) = 2.833, p = 0.0359], (±)-cis-3-methylbutyrylfentanyl [0.1, 1, and 10 mg/kg; F (3, 28) = 13.27, p < 0.0001], ortho-methylacetylfentanyl [10 mg/kg; F(4, 43) = 3.430, p = 0.0161], para-chloroisobutyrylfentanyl [100 mg/kg; F(4, 43) = 5.901, p = 0.0007], ocfentanil [1 and 10 mg/kg; F(3, 28) = 8.613, p = 0.0003], and ortho-fluoroacrylfentanyl [0.1, 1, 10 mg/kg; F(3, 27) = 5.495, p = 0.0044] produced significant increases (p ≤ 0.05) in distance traveled relative to vehicle at least at one dose tested. An inverted U-shaped dose-response function related distance traveled to increasing dose was observed for fentanyl, cyclopropylfentanyl, (±)-cis-3-methylbutyrylfentanyl, ocfentanil, and ortho-fluoroacrylfentanyl. Unlike other drugs, 2,2,3,3-tetramethylcyclopropylfentanyl did not have significant effects on locomotion and 4′-methylacetylfentanyl [10, 100 mg/kg; F(4, 43) = 8.684, p < 0.0001] significantly decreased locomotion relative to vehicle.

Fig. 2.

Dose effects in locomotor activity tests for (1) morphine, (2) buprenorphine, (3) fentanyl, (4) cyclopropylfentanyl, (5) cyclopentylfentanyl, (6) (±)-cis-3-methylbutyrylfentanyl, (7) ortho-methylacetylfentanyl, (8) 4′-methylacetylfentanyl, (9) para-chloroisobutyrylfentanyl, (10) ocfentanil, (11) ortho-fluoracrylfentanyl, and (12) 2,2,3,3-tetramethylcyclopropylfentanyl. Symbols represent the mean (±SEM) distance traveled (cm) during the 120-min experimental session for n = 8–16 mice per dose. Significant differences between drug and vehicle are indicated by asterisks: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

Fig. 3 shows distance traveled (cm) in 10-min bins as a function of time for each drug during locomotor activity tests. MOR agonist standards significantly increased distance traveled at several timepoints: morphine 10 mg/kg (40–90 min), 100 mg/kg (20–120 min), and 180 mg/kg (20–120 min) [F(44, 649) = 6.459, p < 0.0001]; buprenorphine 0.1 mg/kg (50 and 70 min), 1 mg/kg (10–70 min), and 10 mg/kg (10–70 min) [F(33, 308) = 2.745, p < 0.0001]; and fentanyl at 1 mg/kg (10–120 min), and 10 mg/kg (50–120 min) [F(33, 660) = 4.100, p < 0.0001]. Fentanyl-related substances significantly increased distance traveled at several timepoints: cyclopropylfentanyl [1 mg/kg (10–120 min) and 10 mg/kg (30–120 min); F(33, 308) = 3.364, p < 0.0001], cyclopentylfentanyl [10 mg/kg (10–80 min); F(33, 308) = 16.28, p < 0.0001], (±)-cis-3-methylbutyrylfentanyl [0.1 mg/kg (10–60 min), 1 mg/kg (10–120 min), and 10 mg/kg (50–120 min); F(33, 308) = 6.208, p < 0.0001], ortho-methylacetylfentanyl [10 mg/kg (10–70 min) and 100 mg/kg (10–20 min); F(44, 473) = 2.214, p < 0.0001], para-chloroisobutyrylfentanyl [100 mg/kg (10–60 min); F(44, 473) = 5.830, p < 0.0001], ocfentanil [0.1 mg/kg (20 min), 1 mg/kg (10–120 min), and 10 mg/kg (10, 30, 50–120 min); F(33, 308) = 2.937, p < 0.0001], ortho-fluoroacrylfentanyl [0.1 mg/kg (10–70 min), 1 mg/kg (10–120 min), and 10 mg/kg (80–120 min); F(33, 297) = 8.660, p < 0.0001]. In contrast, 4′-methylacetylfentanyl significantly decreased distance traveled at several timepoints [0.1 mg/kg (40 min), 1 mg/kg (10 min), 10 mg/kg (10–30 min), and 100 mg/kg (10–50 min); F(44, 473) = 3.256, p < 0.0001], and 2,2,3,3-tetramethylcyclopropylfentanyl [F(44, 473) = 0.9214, p = 0.6182] did not have a significant time × treatment interaction.

Fig. 3.

Time course effects in locomotor activity tests for (1) morphine, (2) buprenorphine, (3) fentanyl, (4) cyclopropylfentanyl, (5) cyclopentylfentanyl, (6) (±)-cis-3-methylbutyrylfentanyl, (7) ortho-methylacetylfentanyl, (8) 4′-methylacetylfentanyl, (9) para-chloroisobutyrylfentanyl, (10) ocfentanil, (11) ortho-fluoracrylfentanyl, and (12) 2,2,3,3-tetramethylcyclopropylfentanyl. Symbols represent the mean (±SEM) distance traveled (cm) in 10-min bins for n = 8–16 mice per dose.

Table 1 shows the maximum total distance traveled, maximum total distance traveled as a percentage of fentanyl’s maximum effect, and estimated dose (mg/kg) required to elicit a level of effect equal to 100 m of travel. The highest level of effect for MOR agonist standards was observed for subjects administered fentanyl (1 mg/kg) which elicited 34,905 ± 5355 cm of travel during the 120-min test session. The rank order of efficacy (maximum effect as a % of fentanyl’s maximum effect) for the compounds evaluated was cyclopropylfentanyl (108.84 ± 20.21) > fentanyl (100 ± 15.3) > ocfentanil (79.27 ± 16.92) > morphine (75.9 ± 14.5) > (±)-cis-3-methylbutyrylfentanyl (68.04 ± 10.08) > ortho-fluoroacrylfentanyl (63.56 ± 19.88) > cyclopentylfentanyl (56.46 ± 8.54) > para-chloroisobutyrylfentanyl (22.44 ± 8.51) > buprenorphine (11.26 ± 2.30) > ortho-methylacetylfentanyl (9.45 ± 2.92) > 2,2,3,3-tetramethylcyclopropylfentanyl (6.75 ± 1.43) > 4′-methylacetylfentanyl (3.47 ± 0.43). Fig. 3 shows distance traveled as a function of dose and time for each drug. The rank order of potency (estimated dose [mg/kg] required to elicit 100 m of travel [95 % CI]) for fentanyl-related substances relative to MOR agonist standards was ortho-fluoroacrylfentanyl 0.0430 mg/kg [0.0150–0.1151], (±)-cis-3-methylbutyrylfentanyl 0.0760 mg/kg [0.0391–0.1448] > ocfentanil 0.1480 mg/kg [0.0611–0.3402] > cyclopropylfentanyl 0.2070 mg/kg [0.0891–0.4692] > fentanyl 0.23 mg/kg [0.1139–0.4555] > morphine 15.3 mg/kg [5.1165–46.3444]. The remaining substances tested did not produce a level of effect that was sufficient for modeling.

Table 1.

Results from locomotor activity tests.

#	Drug	Maximum effect (distance traveled in cm ± SEM)	Maximum effect as a % of fentanyl’s maximum effect	Estimated dose (mg/kg) required to elicit 100 m of travel
1	Morphine	26,491 ± 5066	75.9 ± 14.5	15.3
2	Buprenorphine	3931 ± 804	11.26 ± 2.30	N/A
3	Fentanyl	34,905 ± 5355	100 ± 15.3	0.23
4	Cyclopropylfentanyl	37,992 ± 7054	108.84 ± 20.21	0.2070
5	Cyclopentylfentanyl	19,706 ± 2981	56.46 ± 8.54	N/A
6	(±)-Cis-3-methylbutyrylfentanyl	23,750 ± 3520	68.04 ± 10.08	0.0760
7	Ortho-methylacetylfentanyl	3299 ± 1018	9.45 ± 2.92	N/A
8	4′-Methylacetylfentanyl	1212 ± 151	3.47 ± 0.43	N/A
9	Para-chloroisobutyrylfentanyl	7833 ± 2971	22.44 ± 8.51	N/A
10	Ocfentanil	27,670 ± 5907	79.27 ± 16.92	0.1480
11	Ortho-fluoroacrylfentanyl	22,187 ± 6939	63.56 ± 19.88	0.0430
12	2,2,3,3-Tetramethylcyclopropylfentanyl	2358 ± 498	6.75 ± 1.43	N/A

Efficacy estimates are expressed as maximum effect (total distance traveled in cm) and maximum effect as a % of fentanyl’s maximum effect.

Potency estimates are expressed as dose (mg/kg) required to produce a level of effect equal to 100 m of travel.

Data are mean ± SEM for n = 8–16 mice per group.

N/A: impossible to estimate based upon the slope of the dose-effect curve.

4. Discussion

This study represents the first reported assessments of cyclopropylfentanyl, cyclopentylfentanyl, (±)-cis-3-methylbutyrylfentanyl, ortho-methylacetylfentanyl, 4′-methylacetylfentanyl, para-chloroisobutyrylfentanyl, ocfentanil, ortho-fluoroacrylfentanyl, and 2,2,3,3-tetramethylcyclopropylfentanyl on locomotion in mice and adds to our knowledge of fentanyl-related substances. In locomotor activity tests, all drugs elicited significant dose-dependent hyperlocomotion in mice except for 2,2,3,3-tetramethylcyclopropylfentanyl and 4′-methylacetylfentanyl, the latter of which significantly decreased locomotion. The onset of hyperlocomotion for buprenorphine, fentanyl and the behaviorally active fentanyl-related substances was rapid following SC administration, with significant changes in locomotion occurring within the first 10-min bin at one or more of the tested doses. In contrast, morphine significantly altered locomotion beginning at the second 10-min bin.

Duration of activity varied as a function of drug and dose, but notably several fentanyl-related substances produced levels of activity similar to the most efficacious fentanyl dose (1 mg/kg) during the final bin of the 2-h session, including cyclopropylfentanyl (1 mg/kg), (±)-cis-3-methylbutyrylfentanyl (1 mg/kg), ocfentanil (1 and 10 mg/kg), and ortho-fluoroacrylfentanyl (1 mg/kg). These four compounds were also more potent than fentanyl as determined by their estimated dose required to produce 100 m of travel. In so far as the potency and duration of action for inducing hyperlocomotion of this subset of compounds applies to other behavioral effects, these data suggest that they may pose a similar or greater risk than fentanyl with respect to overdose, reversibility by naloxone, and potential renarcotization following initial overdose rescue (Rzasa Lynn and Galinkin, 2018). Currently, all of the fentanyl-related substances tested have a Schedule I designation in the United States per the Controlled Substances Act (CSA). Our data provide preliminary evidence that these drugs warrant further evaluation for their abuse potential and toxicity.

Though little to no in vivo data have been previously published for the fentanyl-related substances tested in this study, several of these compounds have been investigated elsewhere in vitro. Eshleman et al. characterized 22 fentanyl analogs including cyclopropylfentanyl, cyclopentylfentanyl, and 2,2,3,3-tetramethylcyclopropylfentanyl (Eshleman et al., 2020). These three compounds exhibited selectivity for MOR over KOR and DOR, with 2,2,3,3-tetramethylcyclopropylfentanyl being the least selective of the three. Cyclopropylfentanyl and cyclopentylfentanyl had higher binding affinities at MOR compared to fentanyl and morphine, whereas 2,2,3,3-tetramethylcyclopropylfentanyl had approximately 763- and 408-fold lower binding affinity at MOR compared to fentanyl and morphine, respectively. In the [³⁵S]GTPγS functional binding assay, cyclopropylfentanyl and cyclopentylfentanyl had comparable efficacy to fentanyl, with cyclopropylfentanyl being nearly twice as potent as fentanyl and four-fold more potent than cyclopentylfentanyl. Conversely 2,2,3,3-tetramethylcyclopropylfentanyl had a mean E_max of 18.3 % and was over 30-fold less potent than fentanyl. Collectively, these in vitro findings generally corroborate the behavioral effects observed in the present study, particularly the divergence in locomotor activating effects for cyclopropylfentanyl and cyclopentylfentanyl vs. 2,2,3,3-tetramethylcyclopropylfentanyl and the potency separation between cyclopropylfentanyl and cyclopentylfentanyl. Furthermore, these data are consistent with the hypothesis that the locomotor activating effects of cyclopropylfentanyl and cyclopentylfentanyl are likely mediated by MOR agonism. A report from Hassanien et al. (2020) included in vitro evaluation of binding affinity, efficacy, and potency of 33 fentanyl-related substances at MOR, including cyclopropylfentanyl, cyclopentylfentanyl, ortho-methylacetylfentanyl, ortho-fluoroacrylfentanyl, and para-chloroisobutyrylfentanyl. Rank order of efficacy in the [³⁵S]GTPγS binding assay was similar, but not identical to our reported rank order of efficacy in the locomotor activity assay. Potency comparisons are more limited due to the number of compounds tested that did not produce sufficient locomotor activity to estimate the dose required to produce 100 m traveled, but notably, ortho-fluoroacrylfentanyl was the most potent compound tested in this study both in vivo and in vitro (approximately 5.3- and 2.3-fold more potent than fentanyl, respectively).

Several of these fentanyl-related substances (most commonly cyclopropylfentanyl and ocfentanil) have been identified in published post-mortem toxicology reports (Allibe et al., 2018; Brunetti et al., 2021; Coopman et al., 2016). Cyclopropylfentanyl is particularly notable in this regard as it is one of the highest efficacy fentanyl-related substances in the locomotor assay tested in our laboratory to date. Moreover, and consistent with the results of others, this study confirms that cyclopropylfentanyl is greater in potency than morphine—and even fentanyl itself—which may suggest that it poses an increased overdose risk for unsuspecting users (Bergh et al., 2021). We have also reported that ocfentanil produces oxycodone-like discriminative stimulus effects in mice, suggesting that they elicit similar subjective effects in humans, and ocfentanil was twice as potent as fentanyl in substituting for oxycodone (Walentiny et al., 2019). Interestingly, valerylfentanyl, a drug which failed to stimulate locomotor activity in mice at doses up to 100 mg/kg produced antinociception in a warm-water tail-withdrawal assay (Varshneya et al., 2019). Valerylfentanyl also substituted for oxycodone in the drug discrimination procedure with valerylfentanyl being less potent than morphine (Walentiny et al., 2018). This finding suggests that drugs with even lower efficacy relative to those in this study such as ortho-methylacetylfentanyl, 2,2,3,3-tetramethylcyclopropylfentanyl, and 4′-methylacetylfentanyl could produce analgesic or abuse-related effects comparable to opioids used clinically and recreationally, but this will need to be empirically determined (Santos et al., 2022).

Taken together, the results of this study demonstrate that most of the fentanyl-related substances tested can produce significant increases in locomotor activity like the MOR-selective agonist standards morphine, buprenorphine, and fentanyl, suggesting that their effects are likely mediated by the MOR. Converging evidence from other preclinical in vivo and in vitro studies further implicate MOR in mediating the locomotor effects of a subset of these compounds. Future studies should confirm these findings with additional tests using MOR-selective antagonists, as well as assays measuring other MOR-mediated endpoints including antinociception and respiratory depression. Overall, this research demonstrates that fentanyl-related substances can elicit hyperlocomotion in mice. This procedure offers a high throughput approach to compare structurally-related drugs to known in-class standards to generate information regarding onset and duration of action, potency, and efficacy at the whole animal level. Such information can guide future research to better understand the spectrum of behavioral and physiological effects of these and other fentanyl-related substances in order to promote public health and safety and guide regulatory actions.

Data availability

Data will be made available on request.