In vitro and in vivo pharmacology of nine novel synthetic cannabinoid receptor agonists

Abstract

Synthetic cannabinoid receptor agonists (SCRAs) are novel psychoactive substances that bind to and activate CB₁ receptors in the brain. The structural manipulations observed in newer SCRAs suggest that manufacturers have incorporated modern drug development techniques into their repertoire, often producing higher CB₁ receptor affinity than Δ⁹-tetrahydrocannabinol (Δ⁹-THC). This study examined nine SCRAs recently detected by forensic surveillance, some of which caused fatalities: 5F-MDMB-PICA, FUB-144, 5F-MMB-PICA, MMB-4en-PICA, MMB-FUBICA, 5F-EDMB-PINACA, APP-BINACA, MDMB-4en-PINACA, and FUB-AKB48. Compounds were evaluated for CB₁ and CB₂ receptor binding affinity and functional activation and for their effects on body temperature, time course, and pharmacological equivalence with Δ⁹-THC in Δ⁹-THC drug discrimination in mice. All SCRAs bound to and activated CB₁ and CB₂ receptors with high affinity, with similar or greater affinity for CB₂ than CB₁ receptors and stimulated [³⁵S]GTPγS binding in CB₁ and CB₂ expressing cell membranes. All compounds produced hypothermia, with shorter latency to peak effects for SCRAs than Δ⁹-THC. All SCRAs fully substituted for Δ⁹-THC in drug discrimination at one or more doses. Rank order potency in producing in vivo effects mostly aligned with rank order CB₁ receptor affinities. Potencies for Δ⁹-THC-like discriminative stimulus effects were similar across sex except Δ⁹-THC was more potent in females and 5F-MMB-PICA was more potent in males. In summary, 5F-EMDB-PINACA, 5F-MDMB-PICA, MDMB-4en-PINACA, FUB-144, FUB-AKB48, 5F-MMB-PICA, MMB-4en-PICA, and MMB-FUBICA are potent and efficacious SCRAs with pharmacology like that of past SCRAs that have been abused in humans. In contrast, APP-BINACA was efficacious, but had lower potency than most past SCRAs.

1.0. Introduction

In the U.S., forensic surveillance has detected the following indole- and indazole-derived synthetic cannabinoid receptor agonists (SCRAs) in recent years: 5F-MDMB-PICA, FUB-144, 5F-MMB-PICA (also known as MMB2201), MMB-4en-PICA (also known as MMB022), MMB-FUBICA, 5F-EDMB-PINACA, APP-BINACA, MDMB-4en-PINACA, and FUB-AKB48 (Figure 1) (Barceló et al., 2017; Drug Enforcement Administration, 2022; Institóris et al., 2022; Krotulski et al., 2020; Truver et al., 2020; Vaccaro et al., 2022). Several of these SCRAs have been associated with fatalities (e.g., 5F-EDMB-PINACA, FUB-AKB48) or other adverse health effects (e.g., FUB-144, MDMB-4en-PINACA) in human users (Drug Enforcement Administration, 2019, 2020; Food and Drug Administration, 2020; United Nations Office on Drugs and Crime, 2020). Therefore, the present study sought to examine the pharmacological effects of these SCRAs.

Figure 1.

Chemical structures of SCRA test compounds. Template regions of SCRAs are comprised of core, tail, linker and head, as labeled using 5F-EDMB-PINACA as an example.

SCRAs are novel psychoactive substances that bind to and activate CB₁ cannabinoid receptors in the brain. While these compounds produce an intoxication resembling that seen with Δ⁹-tetrahydrocannabinol (Δ⁹-THC), the primary psychoactive constituent of cannabis, their chemical structures differ significantly from the phytocannabinoids and preclude their coverage under regulatory controls governing marijuana. Hence, each new chemical remains “legal” until the responsible agency in a country has completed necessary procedures to regulate it under extant drug control policies. Once a structure or structural class is widely regulated, illicit manufacturers move on to novel structures that are not covered by the regulations. These structures still retain cannabinoid psychoactivity due to the remarkable diversity of cannabinoid structural templates that bind to and activate the CB₁ cannabinoid receptor (Pertwee, 1997). While early SRCAs were based primarily upon chemical structures covered in academic and patent literature, the variety of structural manipulations observed in subsequent generations of SCRAs provides evidence that manufacturers of these compounds have incorporated modern drug development techniques into their repertoire (for an excellent review, see Banister and Connor, 2018a).

As in legitimate drug development efforts, these techniques often result in refinement of the parent molecule and, in the case of SCRAs, in compounds with a higher CB₁ receptor affinity that is often accompanied by the same Δ⁹-THC-like intoxication, but with greater potency and/or efficacy than Δ⁹-THC, which acts as a partial agonist at the CB₁ receptor (Banister and Connor, 2018a, b). Unfortunately, intensification of cannabinoid-related toxicity and/or novel off-target actions also have produced newer SCRAs with a greater propensity for significant adverse effects as compared to Δ⁹-THC and first generation SCRAs (Alves et al., 2020; Cooper, 2016; Fantegrossi et al., 2014). Unlike drugs under development for therapeutic uses, novel SCRAs have only rarely been tested in preclinical animal models for safety (including abuse potential) before being “marketed” to human users (Howlett et al., 2021).

As a foundation for designation of appropriate regulatory controls, empirical data on the in vitro and in vivo activity of these new SCRAs are necessary. In addition, these data allow for probing of structure-activity relationships with novel structures, an activity that is fundamental for continued characterization of the pharmacophores of the two known cannabinoid receptors. In this study, nine SCRAs (Figure 1) were evaluated in vitro for CB₁ and CB₂ receptor binding affinity and functional activation and in vivo for their effects on body temperature, time course, and interoceptive effects in drug discrimination. This combination of pharmacological assays has been used previously to evaluate the cannabinoid abuse liability of SCRAs (Thomas et al., 2017; Wiley et al., 2015; Wiley et al., 2013).

2.0. Materials and Methods

2.1. Subjects

Adult male ICR mice (N=52) (30–44 g; Envigo, Frederick, MD, USA) were used in the temperature study, and adult male and female C57BL/6 mice (N=28 males; N=20 females) [20–25 g for males and 15–20 g for females at the beginning of the study; Jackson Laboratories, Bar Harbor, ME (n=12 males; n=12 females) and Envigo, Frederick, MD, USA (n=16 males; n=8 females)] were used in drug discrimination. ICR and C57BL/6 mice were chosen for temperature and drug discrimination studies, respectively, due to past use of these strains in these assays (Gamage et al., 2020; Gamage et al., 2017; Wiley et al., 2015; Wiley et al., 2013). Mice were maintained in a temperature- (20–26°C) and humidity-controlled (30–70%) environment with a 12-h light-dark cycle (lights on at 0700). All mice had free access to water in the home cage, and temperature mice had free access to food, whereas drug discrimination mice were lightly food restricted (i.e., fed about 2–3 g daily). Experiments complied with the Institutional Animal Care and Use Committee for RTI and with the ARRIVE guidelines. All research was conducted as humanely as possible, and followed the principles of laboratory animal care (National Research Council, 2011).

2.2. Apparatus

Temperature readings were taken using a BAT-12 Microprobe Thermometer with RET-3 Rectal Probe (PhysiTemp Instruments Inc., Clifton, NJ, USA). Experimental sessions for drug discrimination were conducted in mouse operant chambers (Coulbourn Instruments, Whitehall, PA), housed within light- and sound-attenuating cubicles. Each chamber contained two nose poke apertures (one at each side of the front panel), with stimulus lights located over each aperture, and a separate house light. A food dispenser was used to deliver 20-mg food pellets (Bioserv Inc., Frenchtown, NJ) into a food cup (with a light) centered between the two apertures. Illumination of lights, delivery of food pellets, and recording of nose pokes was controlled by a computer-based system (Coulbourn Instruments, Graphic State Software, Whitehall, PA).

2.3. Drugs/Chemicals

Δ⁹-THC was obtained from the National Institute on Drug Abuse (NIDA, Bethesda, MD, USA) through the NIDA Drug Supply Program. 5F-EMDB-PINACA, 5F-MDMB-PICA, FUB-144, FUB-AKB48 were provided by the Drug Enforcement Administration (DEA, Dulles, VA, USA). MDMB-4en-PINACA and MMB-4en-PICA were obtained from Cayman Chemical (Ann Arbor, MI, USA). APP-BINACA, MMB-FUBICA, and 5F-MMB-PICA were obtained from Synthcon (Colorado Springs, CO, USA). Purity was > 97% for all compounds. For in vivo studies, compounds were dissolved in 7.8% Polysorbate 80 (Fisher Scientific, Fair Lawn, NJ, USA), and 92.2% saline (Patterson Veterinary Supply, Columbus, OH, USA). All doses of all compounds were injected intraperitoneally (i.p.) at a volume of 10 ml/kg, except that 20 mg/kg of FUB-144, 6 mg/kg MMB-FUBICA, and 60 mg/kg MMB-4en-PICA were administered at 20 ml/kg due to solubility problems at higher concentrations. For drug discrimination, all doses of all compounds were injected 30 min before the start of the session.

For the binding studies, [³H]CP55,940 (Perkin Elmer Life Sciences, Waltham, MA, USA), CP55,940 and MDMB-4en-PINACA were dissolved in 100% ethanol, and 5F-MDMB-PICA, FUB-144, FUB-AKB48, and 5F-EDMB-PINACA were dissolved in 100% acetonitrile. Compound stocks were stored at −20°C in silanized glass dram vials under a blanket of nitrogen and sealed with Teflon tape. MMB-FUBICA, MMB-4en-PICA, 5F-MMB-PICA and APP-BINACA were dissolved in 100% DMSO, aliquoted and stored at −20°C or −80°C ([³H]CP55,940). Guanosine 5’ diphosphate (GDP; Sigma Aldrich, St. Louis, MO, USA), unlabeled GTPγS (Sigma Aldrich, St. Louis, MO, USA), and [³⁵S]GTPγS (1213–1282 Ci/mmol; Perkin Elmer Life Sciences, Boston, MA, USA) were dissolved in distilled water, aliquoted and stored at −80°C. Scintillation fluid (MicroScint-20 and Ultima Gold) was obtained from Perkin Elmer Life Sciences.

2.4. Receptor Binding and Agonist-Stimulated [³⁵S]GTPγS Binding

Human embryonic kidney (HEK293) cells stably expressing either the hCB₁ or hCB₂ receptor were grown in Dulbecco’s Modified Eagle’s Media/F12 (10–092-CV; Corning, Manassas, VA, USA) with 10% fetal bovine serum (FBS-CBT; Rocky Mountain Biological Laboratory, CO, USA), 50 unit/ml penicillin/streptomycin (Thermo Fisher Scientific; Waltham, MA, USA), 500 μg/mL G418 (ant-gn-5; Invivogen, San Diego, CA), and 2–4 μg/mL puromycin (ant-pr-1; Invivogen) in 5-layer multi-flasks (353144, Corning Falcon, Corning, NY, USA) to approximately 90% confluence under 5% CO₂ at 37°C. Cells were detached using 1 mM EDTA in phosphate buffered saline (PBS; Sigma Aldrich, St. Louis, MO, USA), pelleted in PBS at 200 × g for 6 min, then suspended in ice-cold membrane buffer (50 mM Tris Base, 1 mM EGTA, 3mM MgCl₂, pH 7.4) containing protease inhibitor cocktail (Pierce Protease Inhibitor mini-tablet, Thermo Scientific, A32953) and homogenized by Brinkmann Polytron 3000 with Kinematica 95/ PT-DA 3012/2 S generator probe for 15 s at 22,000 RPM. Cell homogenates were centrifuged at 1600 × g for 10 min at 4°C, the supernatant was collected, and the pellet was homogenized again and centrifuged at 1600 × g for 10 min at 4°C. The supernatants from the two spins were pooled and spun at 40,000 × g for 1 h at 4°C resulting in a P2 pellet. The P2 pellet was resuspended in membrane buffer and the protein amount was quantified by the Bradford method (Thermo Scientific Pierce Coomassie Plus, PI23238) using absorbance mode in a Clariostar plate reader (BMG Labtech; Cary, NC, USA). The membrane preparations were diluted to 1 mg/mL, aliquoted at 1.1 mL, snap frozen in liquid nitrogen, and stored at −80°C until the day of the experiment at which point they were thawed slowly on ice. The membrane preparation was partially diluted, re-homogenized with 10 strokes of a glass Dounce homogenizer, and then further diluted to 5× the final assay concentration. Membrane preparations from these cell lines contain approximately 2.1 pmol/mg of hCB₁ receptors or 1.2 pmol/mg of hCB₂ receptors, respectively, as previously reported (Gamage et al., 2020).

For receptor binding, reactions were carried out in assay buffer containing 50 mM Tris Base, 100 mM NaCl, 3 mM MgCl₂, 0.2 mM EGTA and 5 mg/ml fatty acid free bovine serum albumin (BSA; A7030; Sigma Aldrich, St. Louis, MO, USA) BSA and 10 μg total protein per well. Reactions were carried out for 90 min at 30°C with ~0.8 nM [³H]CP55,940 and concentrations of compounds on a logarithmic scale. Amount of radioligand added for each experiment was determined by pipetting 50 μL of each nominal concentration stock and adding 20 mL of Ultima Gold scintillation cocktail and analysis on a Packard TriCarb 2300TR scintillation counter. Non-specific binding was determined by addition of excess unlabeled SR141716 ligand (10 μM). For receptor signaling, membranes (10 μg protein determined by Bradford method) were pre-equilibrated for 30 min at 30°C with 30 μM GDP and drugs followed by addition of 0.1 nM [³⁵S]GTPγS in a volume of 10 μL (2% final volume) to ensure compounds were pre-equilibrated close to the final concentration. Non-specific binding was determined by including 30 μM unlabeled GTPγS. Binding for all reactions was terminated by vacuum filtration through a Perkin Elmer GF/C filter plate using a Perkin Elmer FilterMate followed by rinsing with 4–6 mL of cold rinse buffer (50 mM Tris, 0.1% BSA, pH 7.4) per sample. After plates were dried, 35 μL of Microscint-20 (Perkin Elmer) was added to each well and the plates were sealed with TopSeal-A and counted on a Packard TopCount NXT.

2.5. Temperature

2.5.1. Temperature Dose-Effect.

Separate groups of mice were randomly assigned to each compound (Δ⁹-THC, 5F-MDMB-PICA, 5F-EMDB-PINACA, MDMB-4en-PINACA, FUB-144, FUB-AKB48, APP-BINACA, MMB-FUBICA, 5F-MMB-PICA, and MMB-4en-PICA; n=6/compound). Doses of a compound, including vehicle, were evaluated within-subject. Most mice were only exposed to one cannabinoid, whereas some of the mice (n=8) were re-used to evaluate a second cannabinoid. Temperature assessments occurred up to twice per week with a minimum of a three-day washout between assessments. Prior to injection, baseline rectal temperature values were obtained. After baseline measurements, mice were injected with vehicle or a cannabinoid. Rectal temperature was measured again at 30, 45, and 60 min after injection.

2.5.2. Temperature Time Course.

Following a minimum three-day washout after the dose-effect study, 48 of the mice that were used in the dose-effect study were given an additional drug and vehicle administration (n=6/group) to determine the time course of the hypothermic effects of the cannabinoids. Each mouse was exposed to vehicle, and 1–3 cannabinoids during this phase of the study. Only a single dose of each cannabinoid was examined for time course effects. Doses were chosen based on their ability to produce significant hypothermia in the absence of severe adverse effects in the temperature dose-effect curve (e.g., prolonged, or repeated seizures). Temperature data were collected 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, and 24 h after administration.

2.6. Drug Discrimination

2.6.1. Acquisition.

Training for the Δ⁹-THC discrimination procedure was similar to that described previously (Marusich et al., 2018; Vann et al., 2009; Wiley et al., 2015). Briefly, each mouse was placed in a standard operant conditioning chamber with two nose poke apertures. Mice were trained to respond on one of the two apertures following administration of 5.6 mg/kg Δ⁹-THC and to respond on the other aperture following vehicle injection according to a fixed ratio 10 (FR10) schedule of food reinforcement, under which 10 consecutive responses on the correct (drug-appropriate) aperture resulted in delivery of a food pellet. Responses on the incorrect aperture reset the ratio requirement on the correct aperture. Daily injections were administered on a double alternation sequence of Δ⁹-THC and vehicle (e.g., drug, drug, vehicle, vehicle). Daily 15-min training sessions were held Monday-Friday until the mice met three criteria for 8 of 10 sessions: 1) the first completed FR10 was on the correct aperture, 2) ≥ 80% of the total responding occurred on the correct aperture, and 3) response rate was ≥ 0.2 responses/s for males and ≥ 0.1 responses/s for females. When these criteria were met, acquisition of the discrimination was established, and substitution testing began. One female mouse did not meet these criteria and was dropped from the study.

2.6.2. Substitution dose-effect curves.

Stimulus substitution tests were typically conducted on Tuesdays and Fridays during 15 min test sessions, with maintenance of training continuing on intervening days. During test sessions, responses on either aperture delivered reinforcement according to an FR10 schedule. Prior to each test, mice must have completed the first FR10 on the correct aperture, ≥ 80% of the total responding occurred on the correct aperture, and response rate was ≥ 0.2 responses/s for males or ≥ 0.1 responses/s for females during the prior day’s training session. In addition, the mouse must have met these same criteria during previous training sessions with the alternate training compound (training drug or vehicle).

A dose-effect curve for Δ⁹-THC was determined in all mice prior to testing with any SCRA. Two female and four male mice were removed from the study after completing the initial Δ⁹-THC dose-effect curve for failure to maintain reliable stimulus control on training days. Their data were excluded from all graphs and analyses. Following completion of the initial Δ⁹-THC dose-effect curve, 5F-MDMB-PICA, 5F-EMDB-PINACA, FUB-144, FUB-AKB48, APP-BINACA, MMB-FUBICA, 5F-MMB-PICA, and MMB-4en-PICA were evaluated in subsets of male and female mice (n=8/sex unless otherwise indicated in the figure legends). To ensure that discrimination was maintained, control tests with vehicle and 5.6 mg/kg Δ⁹-THC (i.e., the training dose) were conducted in between dose-effect curves.

2.7. Data Analysis

2.7.1. In Vitro Data.

All in vitro data were analyzed using GraphPad Prism 9.3. [³⁵S]GTPγS data were calculated as % stimulation over basal [(Treatment – Basal) / Basal * 100] and were fit using non-linear regression using the equation ‘log(agonist) vs. response (three parameters)’. For competition radioligand binding data, the radioligand [³H]CP55,940 K_d was constrained to 1.2 as determined from previous saturation binding experiments using a one-site model (Gamage et al., 2019). The free concentration was constrained to the average amount of radioligand from the experimental replications (0.79 ± 0.13 nM, N=10) as determined by daily scintillation counts of the radioligand preparation. K_i values were calculated using the equation ‘One site – fit K_i^’ in Prism. Each data point represents the mean of at least N=3 experiments performed in triplicate. Calculated pK_i and pEC₅₀ values were analyzed by two-way ANOVA (compound × receptor subtype) to determine differences in affinity and potency between compounds and whether these differed at hCB₁ and hCB₂ receptors.

2.7.2. Temperature.

All in vivo data were analyzed with Number Cruncher Statistical Systems software (NCSS Statistical Software, Kaysville, UT, USA), and figures were graphed using GraphPad Prism (GraphPad Software, San Diego, CA, USA). Rectal temperature values were expressed as the difference between control temperature (before injection) and temperature following drug or vehicle administration (Δ°C). Separate within-subject analyses of variance (ANOVAs) were used to analyze the Δ°C dose-effect curve for each compound at each timepoint (30, 45, 60 min), and determine which doses produced a significant difference in Δ°C compared to vehicle. Within-subject ANOVAs (time × dose) were used to analyze the time course of °C and determine the timepoints at which °C differed from baseline (time 0) and differed from vehicle. Significant effects of dose or time were further analyzed with Tukey post hoc tests (α = 0.05) as necessary.

2.7.3. Drug Discrimination.

For each discrimination session, percentage of responses on the drug aperture and response rate (responses/s) were calculated. ED₅₀ values were calculated on the linear part of the drug aperture selection dose-effect curve for each drug using least squares linear regression analysis, followed by calculation of 95% confidence intervals. Since mice that responded fewer than 10 times during a test session did not respond on either aperture enough times to earn a reinforcer, their data were excluded from analysis of drug aperture selection, but their response rate data were included. Response-rate data were analyzed using mixed model ANOVAs with dose as a within-subject factor and sex as a between-subject factor. A mean substitution procedure was used to maintain equal n’s across conditions in the case of missing data (i.e., 10 mg/kg FUB-AKB48 was only tested in 6 of 8 males and in 2 of 8 females). Data for 0.3 mg/kg FUB-144 were excluded from analyses because this dose was not tested in all subjects, but these data were retained in graphs. Significant ANOVAs were further analyzed with Tukey post hoc tests (α = 0.05) to specify differences between means.

3.0. Results

3.1. Cannabinoid Receptor Binding and Agonist-Stimulated [³⁵S]GTPγS Binding

All compounds completely displaced [³H]CP55,940, a synthetic cannabinoid agonist, at both hCB₁ (Figure 2 A–B) and hCB₂ (Figure 2 C–D) receptors expressed in HEK293 cell membrane preparations with high affinity (Table 1). Significant main effects of compound on the calculated affinity (pK_i) [F(9, 66) = 18.18, p < 0.0001] and receptor subtype [F(1, 66) = 73.35, p < 0.0001], as well as an interaction [F(9, 66) = 2.832, p < 0.01], were found. The rank order affinity for hCB₁ was 5F-EDMB-PINACA > CP55,940 > 5F-MDMB-PICA = FUB-AKB48 = MDMB-4en-PINACA > 5F-MMB-PICA > MMB-FUBICA > FUB-144 > MMB-4en-PICA > APP-BINACA. The rank order affinity for hCB₂ was MDMB-4en-PINACA > 5F-EDMB-PINACA > 5F-MDMB-PICA > CP,44930 > FUB-AKB48 > FUB-144 > 5F-MMB-PICA > APP-BINACA > MMB-4en-PICA > MMB-FUBICA. All compounds exhibited similar affinity for both receptors or greater affinity for CB₂ than CB₁, except that MMB-FUBICA exhibited slightly greater (approximately 2-fold) affinity for CB₁ (Table 1). FUB-144 [p < 0.0001], APP-BINACA [p < 0.001] and MDMB-4en-PINACA [p < 0.05] had 23-, 12-, and 7-fold greater affinity at hCB₂ receptors than at hCB₁, respectively.

Figure 2.

Displacement of [³H]CP55,940 (Panels A-B) and stimulation of [³⁵S]GTPγS turnover (Panels E-F) at the hCB₁ receptor (± SEM), expressed in HEK293 cell membrane. Displacement of [³H]CP55,940 (Panels C-D) and stimulation of [³⁵S]GTPγS turnover (Panels G-H) at the hCB₂ receptor (± SEM). Both receptors were expressed in HEK293 cell membrane. Each point reflects the mean (±SEM) of at least n=3 replicates performed in duplicate.

Table 1.

Binding affinity and potency and efficacy for stimulation of [³⁵S]GTPγS turnover at hCB₁ and hCB₂ receptors. EC₅₀ and K_i values in nM are provided in parenthesis below pEC₅₀ and pK_i values, respectively.

Compound	CB1 pKia,b	[35S]GTPγS Turnover b		CB2 pKia,b	[35S]GTPγS Turnover b
		CB1 pEC50c	CB1 Emaxd		CB2 pEC50c	CB2 Emaxd
Δ9-THCe	8.09 ± 0.0182 (8.05) 3	7.05 ± 0.015 (89.9) 3	128 ± 3.82 3	7.5 ± 0.0691 (32) 3	7.69 ± 0.295 (20.3) 3	46.5 ± 7.94 3
CP55,940	9.10 ± 0.0518 (0.797) 17	8.38 ± 0.0717 † (4.19) 13	335 ± 23.5 ‡ 13	9.21 ± 0.0909 (0.618) 13	9.15 ± 0.0548 (0.715) 13	126 ± 11.9 13
5F-EDMB-PINACA	9.42 ± 0.0441 (0.378) 3	8.80 ± 0.0798 (1.60) 3	387 ± 69.0 † 3	9.51 ± 0.0217 (0.312) 3	9.36 ± 0.137 (0.442) 3	161 ± 8.36 3
5F-MDMB-PICA	8.90 ± 0.0772 (1.25) 3	8.29 ± 0.109 (5.07) 3	380 ± 27.4 ^ 3	9.36 ± 0.202 (0.437) 3	8.78 ± 0.211 (1.65) 3	189 ± 25.2 3
FUB-AKB48	8.89 ± 0.265 (1.30) 4	8.16 ± 0.162 ^ (6.94) 4	368 ± 55.5 ‡ 4	9.08 ± 0.0188 (0.823) 3	9.37 ± 0.318 (0.424) 4	131 ± 16.5 4
MDMB-4en-PINACA	8.85 ± 0.0252 * (1.40) 3	8.48 ± 0.0679 (3.30) 3	304 ± 42.8 ‡ 3	9.67 ± 0.0651 (0.213) 3	8.87 ± 0.510 ‡ (1.34) 3	63.6 ± 9.95 3
5F-MMB-PICA	8.19 ± 0.121 (6.45) 3	7.57 ± 0.0647 (27.0) 3	295 ± 21.2 † 3	8.47 ± 0.336 (3.40) 3	8.09 ± 0.200 † (8.19) 3	77.8 ± 13.3 3
MMB-FUBICA	7.92 ± 0.241 (12.0) 3	7.22 ± 0.210 (59.7) 3	278 ± 29.3 † 3	7.65 ± 0.0253 (22.5) 3	7.65 ± 0.162 † (22.1) 3	52.1 ± 6.73 3
FUB-144	7.57 ± 0.272 ‡ (27.2) 3	7.04 ± 0.106 ^ (90.6) 4	218 ± 31.5 4	8.93 ± 0.299 (1.19) 3	8.34 ± 0.111 (4.61) 3	194 ± 8.45 3
MMB-4en-PICA	7.41 ± 0.0865 (39.0) 3	7.22 ± 0.0470 (60.7) 4	298 ± 31.9 ‡ 4	7.81 ± 0.0388 (15.6) 3	7.91 ± 0.332 (12.4) 5	77.6 ± 7.17 5
APP-BINACA	6.97 ± 0.208 † (107) 3	6.46 ± 0.112 ‡ (344) 3	180 ± 25.1 3	8.03 ± 0.0874 (9.34) 3	8.65 ± 0.759 (2.24) 3	83.2 ± 2.72 3

^aValues represent pK_i ± SEM for [³H]CP55,940 displacement at specified receptor.

^bFor each measure, n is shown below the K_i/EC₅₀ (nM) or E_max value.

^cValues represent pEC₅₀ ± SEM for [³⁵S]GTPγS binding at specified receptor.

^dValues represent % stimulation over basal

^eAll values for Δ⁹-THC are from (Gamage et al., 2020).

^*p < 0.05

^{^}p < 0.01

^†p < 0.001

^‡p < 0.0001 compared to hCB₂

All compounds tested stimulated [³⁵S]GTPγS binding in membranes of HEK293 cells expressing either hCB₁ (Figure 2 E–F) or hCB₂ receptors (Figure 2 G–H). Main effects of compound [F(9, 66) = 18.18, p < 0.0001] and receptor subtype [F(1, 66) = 73.35, p < 0.0001], and a significant interaction [F(9, 66) = 2.832, p < 0.001], were found for potency. The rank order potency of compounds at hCB₁ were 5F-EDMB-PINACA > MDMB-4en-PINACA > CP55,940 > 5F-MDMB-PICA > FUB-AKB48 > 5F-MMB-PICA > MMB-FUBICA > MMB-4en-PICA > FUB-144 > APP-BINACA. The rank order potency of compounds at hCB₂ were FUB-AKB48 = 5F-EDMB-PINACA > CP55,940 > MDMB-4en-PINACA > 5F-MDMB-PICA > APP-BINACA > FUB-144 > 5F-MMB-PICA > MMB-4en-PICA > MMB-FUBICA. Overall, compounds exhibited greater potency at hCB₂ than hCB₁ receptors with EC₅₀ values in the nanomolar and sub-nanomolar ranges (Table 1). FUB-AKB48 [p < 0.01], FUB-144 [p < 0.01], CP55,940 [p < 0.001], and APP-BINACA [p < 0.0001] exhibited 16-, 20-, 7-, and 153-fold greater potency at hCB₂ receptors than at hCB₁ receptors, respectively. In addition to their marked potency, all SCRAs displayed high efficacy similar to that of CP55,940, which is consistent with other abused SCRAs previously tested in our lab (Gamage et al., 2018; Gamage et al., 2019). In HEK293 membranes expressing hCB₁, FUB-144 [p < 0.05] and APP-BINACA [p < 0.01] were significantly less efficacious than CP55,940, 5F-EDMB-PINACA, 5F-MDMB-PICA, and FUB-AKB48 and were also the only two compounds with efficacies that were not significantly different between receptor types (Table 1). No statistically significant differences in efficacy were observed between compounds in HEK293 membranes expressing hCB₂.

3.2. Temperature Dose-Effect

Figure 3 shows the effects of Δ⁹-THC and the nine SCRAs on change in temperature in male mice at three post-injection timepoints (i.e., 30, 45 and 60 min). Whereas all compounds produced dose-dependent hypothermia over the dose ranges tested, 5F-EDMB-PINACA, 5F-MDMB-PICA, and MDMB-4en-PINACA consistently produced significant decreases in temperatures (compared to vehicle) at lower doses than Δ⁹-THC and the other SCRAs across the three timepoints [30 min, Fig. 3, panel A: 5F-MDMB-PICA F(3,15) = 22.21, p < 0.0001; 5F-EMDB-PINACA F(3,15) = 13.33, p < 0.001; MDMB-4en-PINACA F(4, 20) = 57.49, p < 0.0001; 45 min, Fig. 3, panel B: 5F-MDMB-PICA F(3,15) = 30.17, p < 0.0001; 5F-EMDB-PINACA F(3,15) = 32.52, p < 0.0001; MDMB-4en-PINACA F(4,20) = 26.99, p < 0.0001; 60 min, Fig. 3, panel C: 5F-MDMB-PICA F(3,15) = 53.97, p < 0.0001; 5F-EMDB-PINACA F(3,15) = 68.78, p < 0.0001; MDMB-4en-PINACA F(4,20) = 19.17, p < 0.0001]. Maximal observed hypothermia for these three potent SCRAs occurred at 60 min post-injection (at the 10 mg/kg dose) and was substantial, with temperature decreases ranging from −8.1 to −9.4 °C as compared to pre-injection temperature.

Figure 3.

Effects of vehicle, Δ⁹-THC and nine SCRAs on rectal temperature (°C) in male ICR mice at 30 (panel A), 45 (panel B), and 60 min (panel C) after i.p. injection. Each value represents the mean (± SEM) of data from 6 mice. * indicate statistically significant differences compared to respective vehicle for the compound (p < 0.05).

Similarly, the other six SCRAs also produced significant decreases in temperature (compared to vehicle) at each of the three timepoints. With the exception of APP-BINACA, these SCRA-induced decreases first occurred at doses that were 3- to 10-fold lower than the lowest dose of Δ⁹-THC (30 mg/kg) that decreased temperature [30 min, Fig. 2, panel A: Δ⁹-THC F(4,20) = 73.83, p < 0.0001; FUB-144 F(4,20) = 34.93, p < 0.0001; FUB-AKB48 F(3,15) = 62.63, p < 0.0001; MMB-FUBICA F(3,15) = 109.08, p < 0.0001; 5F-MMB-PICA F(4,20) = 56.44, p < 0.0001; MMB-4en-PICA F(4,20) = 70.70, p < 0.0001; APP-BINACA F(3,15) = 56.63, p < 0.0001; 45 min, Fig. 2, panel B: Δ⁹-THC F(4,20) = 91.12, p < 0.0001; FUB-144 F(4,20) = 24.14, p < 0.0001; FUB-AKB48 F(3,15) = 114.24, p < 0.0001; MMB-FUBICA F(3,15) = 84.63, p < 0.0001; 5F-MMB-PICA F(4,20) = 49.70, p < 0.0001; MMB-4en-PICA F(4,20) = 69.36, p < 0.0001; APP-BINACA F(3,15) = 54.80, p < 0.0001; 60 min, Fig. 2, panel C: Δ⁹-THC F(4,20) = 63.45, p < 0.0001; FUB-144 F(4,20) = 25.19, p < 0.0001; FUB-AKB48 F(3,15) = 115.21, p < 0.0001; MMB-FUBICA F(3,15) = 57.87, p < 0.0001; 5F-MMB-PICA F(4,20) = 81.53, p < 0.0001; MMB-4en-PICA F(4,20) = 42.52, p < 0.0001; APP-BINACA F(3,15) = 15.17, p < 0.0001]. APP-BINACA was the only SCRA assessed that was less potent at decreasing temperature than Δ⁹-THC at all three timepoints. Further, up to a dose of 100 mg/kg, it was not as efficacious at decreasing temperature compared to Δ⁹-THC and the other SCRAs. By contrast, across the dose range tested for the five SCRAs with medium potencies (i.e., FUB-144, FUB-AKB48, MMB-FUBICA, 5F-MMB-PICA, and MMB-4en-PICA), maximal hypothermia produced was similar to that produced by Δ⁹-THC (within 1.5 °C).

3.3. Temperature Time Course

Figure 4 shows effects of Δ⁹-THC and the nine SCRAs on temperature over the course of 24 h in male mice. Data are organized into panels within this figure based on the dose that was examined during the time course analysis. In Figure 4, Panel A, Δ⁹-THC (100 mg/kg), the positive control for this study, significantly decreased temperature (compared to vehicle at the same timepoint and compared to pre-injection baseline temperature) across the entire 8-h initial period of measurement [dose × time interaction: F(11,55) = 13.58, p < 0.0001]; however, by 24 h post-injection, its hypothermic effect had disappeared. Peak effect occurred from 1 to 3 h post-injection. By contrast, the significant hypothermic effect of APP-BINACA (100 mg/kg) lasted only 4 h, as compared to vehicle [Fig. 4, panel A; dose × time interaction: F(11,55) = 43.53, p < 0.0001]. In addition, visual inspection of the graph showed that its maximum hypothermic effect was less than that of Δ⁹-THC at an identical dose.

Figure 4.

Effects of vehicle and single doses of Δ⁹-THC and nine SCRAs on rectal temperature (°C) in male ICR mice measured before injection (0 h) and post-injection at 0.5 h, 0.75 h, at hourly intervals from 1 to 8 h, and at 24 h. Test drug data illustrated in each panel is identified in the symbol legend below the figure. Filled symbols represent data for the compound whereas unfilled symbols show data for respective vehicle across the same time period. Each value represents the mean (± SEM) of data from 6 mice. * indicate statistically significant differences compared to pre-injection (0 h) measurement whereas # indicate statistically significant differences compared to the respective vehicle injection at the same timepoint (p < 0.05).

As shown in Figure 4, Panel B, a 3 mg/kg dose of each of the three most potent SCRAs (5F-MDMB-PICA, 5F-EMDB-PINACA, and MDMB-4en-PINACA) also produced significant hypothermia (compared to vehicle at the same timepoint) [dose × time interaction: 5F-MDMB-PICA F(11,55) = 16.57, p < 0.0001; 5F-EMDB-PINACA F(11,55) = 19.62, p < 0.0001; MDMB-4en-PINACA F(11,55) = 25.70, p < 0.0001], but the magnitude and duration of these effects varied across the three compounds. Onset of hypothermic effect was evident by the first measurement at 30 min post-injection for all three compounds, with the largest decreases occurring from 30 min to 1 h. Duration of action was shortest for 5F-EDMB-PINACA and MDMB-4en-PINACA, with their hypothermic effects dissipating over the first 3 to 4 h post-injection, respectively, as compared to vehicle at the same timepoint. 5F-MDMB-PICA’s hypothermic effect waned more slowly, with significant decreases in temperature (compared to vehicle at same time point) still evident up to 7 h post-injection. Mice had recovered to normal body temperature by 24 h.

A 10 mg/kg dose of FUB-AKB48 and 5F-MMB-PICA also significantly decreased temperature compared to vehicle at the same timepoint as shown in Figure 4, Panel C [dose × time interaction: FUB-AKB48 F(11,55) = 6.14, p < 0.0001; 5F-MMB-PICA F(11,55) = 55.80, p < 0.0001]. Whereas FUB-AKB48 decreased temperature only at measurements conducted 1 h or less post-injection, 5F-MMB-PICA’s hypothermic effect dissipated slowly over 3 h and was no longer present a 4 h. Figure 4, Panel D shows that various doses (6–60 mg/kg) of the remaining three SCRAs also significantly decreased temperature in a time-dependent manner [dose × time interaction: MMB-FUBICA F(11,55) = 97.50, p < 0.0001; MMB-4en-PICA F(11,55) = 50.18, p < 0.0001; FUB-144 F(11,55) = 12.23, p < 0.0001]. For each compound, onset of hypothermia occurred by the time of the first measurement at 30 min post-injection. The effects of FUB-144 (20 mg/kg) and MMB-FUBICA (6 mg/kg) were no longer evident after 1 and 2 h, respectively; whereas the hypothermic effect of MMB-4en-PICA (60 mg/kg) more gradually dissipated with decreased temperature still noted at 5 h post-injection.

3.4. Drug Discrimination

Figure 5 shows dose-effect curves for Δ⁹-THC in male (left panels) and female (right panels) mice, with percentage of Δ⁹-THC-associated aperture responding (top panels) and response rate (bottom panels) as dependent variables. As expected, mice of both sexes showed full, dose-dependent substitution for Δ⁹-THC (Figure 5; males, top left panel and females, top right panel). Further, during control tests with vehicle and the 5.6 mg/kg training dose of Δ⁹-THC, mice of both sexes responded predominantly on the vehicle- and Δ⁹-THC-associated apertures, respectively, across the course of the study (left side of x-axis in Figure 5, top panels). Although maximal substitution was similar across sex, Δ⁹-THC was 1.5-fold more potent in females than males, as indicated by its lower ED₅₀ in this sex (with non-overlapping 95% confidence intervals; Table 2). As shown in Figure 5, Δ⁹-THC also altered response rates (compared to respective response rates after vehicle administration), with a significant decrease in rates observed in male mice at 10 mg/kg (bottom left panel) and significant increases seen at doses of 3 and 5.6 mg/kg in female mice (bottom right panel) [sex × dose interaction: F(5,190) = 2.39, p < 0.05]. In addition, males exhibited significantly greater response rates under vehicle conditions than females.

Figure 5.

Effects of vehicle, Δ⁹-THC and nine SCRAs on percentage of responses that occurred on the Δ⁹-THC-associated aperture (top panels) and response rate (bottom panels) in male (left panels) and female (right panels) C57 mice trained to discriminate 5.6 mg/kg Δ⁹-THC from vehicle. Control tests with vehicle (Veh) and 5.6 mg/kg Δ⁹-THC (THC) were conducted prior to each dose-effect curve, with results shown at the left side of the panels. For Δ⁹-THC, each point represents the mean (± SEM) of data for 24 male or 16 female mice. For the SCRAs, each point represents the mean (± SEM) of data for 7–9 male or 7–8 female mice, except for % responding on the Δ⁹-THC-associated aperture for 0.1 mg/kg 5F-MDMB-PICA (n=1 male and 4 females), 100 mg/kg APP-BINACA (n=3 males and 1 female), 10 mg/kg MMB-4en-PICA (n=1 male and 1 female), 3 mg/kg 5F-MMB-PICA (n=6 males), 0.1 mg/kg 5F-EDMB-PINACA (n=2 males), and 10 mg/kg FUB-AKB48 (n=3 males and 2 females). In addition, only 5 males and 2 females were tested with 0.3 mg/kg FUB-144. Except for FUB-144, the n for the higher doses of the indicated compounds was reduced because data for % Δ⁹-THC aperture responding were excluded due to fewer than 10 overall responses. * indicate sex × dose interactions, with a significant post hoc difference for the indicated sex and dose (p < 0.05) compared to the respective vehicle (at left side of each panel). # indicate significant main effects for dose across both sexes (p < 0.05).

Table 2.

Potencies for substitution in Δ⁹-THC discrimination. Molecular weights are provided in parentheses below the compound names. ED₅₀s (± 95% confidence limits) are expressed in μmol/kg in the columns on the left and mg/kg in the columns on the right.

Compound	ED50 (μmol/kg)*		ED50 (mg/kg)*
	Males	Females	Males	Females
Δ9-THC (314.45)	6.878 (5.94–7.95)	4.488 (3.45–5.85)	2.163 (1.87–2.50)	1.411 (1.08–1.84)
5F-MDMB-PICA (376.46)	0.046 (0.04–0.06)	0.068 (0.05–0.09)	0.017 (0.01–0.02)	0.025 (0.02–0.03)
5F-EDMB-PINACA (391.48)	0.057 (0.04–0.09)	0.059 (0.05–0.07)	0.022 (0.01–0.03)	0.023 (0.02–0.03)
MDMB-4en-PINACA (357.50)	0.071 (0.05–0.11)	0.143 (0.11–0.19)	0.025 (0.02–0.04)	0.051 (0.04–0.07)
5F-MMB-PICA (362.45)	0.891 (0.67–1.19)	4.950 (3.21–7.63)	0.323 (0.24–0.43)	1.794 (1.16–2.76)
MMB-FUBICA (382.44)	2.613 (1.82–3.75)	3.022 (2.28–4.01)	0.999 (0.70–1.43)	1.156 (0.87–1.53)
FUB-AKB48 (403.49)	4.181 (3.31–5.28)	4.115 (2.78–6.10)	1.687 (1.34–2.13)	1.660 (1.12–2.46)
MMB-4en-PICA (342.43)	4.217 (3.37–5.28)	5.436 (2.85–10.37)	1.444 (1.15–1.81)	1.861 (0.98–3.55)
FUB-144 (349.44)	8.625 (5.69–13.07)	8.921 (6.92–11.50)	3.014 (1.99–4.57)	3.117 (2.42–4.02)
APP-BINACA (364.45)	148.453 (106.54–206.86)	ND	54.104 (38.83–75.39)	ND

^*N=7–9 mice/curve, with exception of Δ⁹-THC where n=24 male and 16 female mice.

ND = not determined. The ED₅₀ for females for APP-BINACA could not be determined because only one mouse responded at the highest dose, and this mouse had a response rate of 0.01 responses/s. Therefore, an accurate ED₅₀ could not be determined.

The effects of SCRAs evaluated in the discrimination procedure are also shown in Figure 5. In male mice, each of the nine SCRAs tested showed full, dose-dependent substitution for Δ⁹-THC (top left panel), with potencies ranging from 0.046 μmol/kg for 5F-MDMB-PICA to 148 μmol/kg for APP-BINACA (Table 2). Based upon their potencies for producing Δ⁹-THC-like effects, the SCRAs can be separated into three general groups, as is apparent visually in Figure 5 (top left panel). 5F-MDMB-PICA, 5F-EDMB-PINACA, and MDMB-4en-PINACA were approximately equipotent and comprised the group with the greatest potencies, ranging from 0.046 to 0.071 μmol/kg (Table 2). The second group was comprised of MMB-FUBICA, FUB-AKB48, MMB-4en-PICA, and FUB-144 (potencies comparable with that of Δ⁹-THC and ranging from 2.613 to 8.625 μmol/kg). In males, 5F-MMB-PICA did not fit neatly into this categorical scheme, as it was 2.9- to 9.7-fold more potent than the 4 compounds in the second group and 13- to 19-fold less potent than the 3 compounds in the higher potency group. The relatively low potency of APP-BINACA (148 μmol/kg) placed it in a (third) category of its own. Although APP-BINACA fully substituted for Δ⁹-THC, it did so only at the relatively high dose of 100 mg/kg and substitution was accompanied by significant response rate suppression [dose main effect: F(3,42) = 29.81, p < 0.0001]. In male mice, some, but not all, of the other SCRAs also significantly decreased response rates (compared to vehicle) within the dose range tested [5F-EDMB-PINACA sex × dose interaction: F(5,70) = 17.3, p < 0.0001; 5F-MDMB-PICA sex × dose interaction: F(4,56) = 4.44, p < 0.001; FUB-AKB48 sex × dose interaction: F(3,42) = 14.71, p < 0.0001; MMB-4en-PICA sex × dose interaction: F(3,42) = 7.41, p < 0.001; 5F-MMB-PICA sex × dose interaction: F(4,60) = 2.91, p < 0.05; MMB-FUBICA dose main effect: F(3,42) = 31.34, p < 0.0001]; however, in most instances, full substitution for Δ⁹-THC also was observed at one or more doses that were lower than the dose(s) that decreased responding. 5F-EDMB-PINACA, MMB-FUBICA, and APP-BINACA are the only compounds for which the single dose that fully substituted (> 80% responding on the Δ⁹-THC-associated aperture) in males concomitantly significantly suppressed response rate.

As shown in Figure 5 (top right panel), SCRAs produced a pattern of substitution in female mice that was similar to that observed in male mice, with two exceptions (APP-BINACA and 5F-MMB-PICA). In females, eight of the nine SCRAs fully and dose-dependently substituted for Δ⁹-THC. Although the level of substitution for APP-BINACA did not reach 80% (designated “full substitution”), the 100 mg/kg dose significantly increased responding on the Δ⁹-THC-associated aperture, as it did in males. In addition, as with males, potency could be used as a basis for separation of the SCRAs into the same three groups. The most potent group included 5F-MDMB-PICA, 5F-EDMB-PINACA, and MDMB-4en-PINACA, which were approximately equipotent to each other and across sex (Table 2). Five of the remaining SCRAs (MMB-FUBICA, FUB-AKB48, MMB-4en-PICA, FUB-144, and 5F-MMB-PICA) comprised the second group of compounds, with ED₅₀s ranging from 3.02 to 8.92 μmol/kg and comparable to that of Δ⁹-THC (Table 2). Dissimilar to the grouping pattern for males, 5F-MMB-PICA potency was clustered with the other mid-potency compounds in females. Hence, it was less potent in females than males (i.e., higher ED₅₀ with non-overlapping 95% confidence intervals; Table 2). As with males, APP-BINACA was in a category of its own in females. Substantial substitution occurred only at 100 mg/kg, a dose that also significantly suppressed overall responding [dose main effect: F(3,42) = 29.81, p < 0.0001]. In females, only three of the compounds produced changes in response rates compared to vehicle conditions. In addition to APP-BINACA, MMB-4en-PICA and MMB-FUBICA significantly altered response rates [sex × dose interaction for MMB-4en-PICA: F(3,42) = 7.41, p < 0.001; dose main effect for MMB-FUBICA: F(3,42) = 31.34, p < 0.0001]; however, females also had lower overall response rates than males across several of the compounds [5F-EDMB-PINACA sex main effect: F(1,70) = 45.19, p < 0.0001; 5F-MDMB-PICA sex main effect: F(1,56) = 11.42, p < 0.01; FUB-144 sex main effect: F(1,42) = 18.02, p < 0.001; MDMB-4en-PINACA sex main effect: F(1,39) = 11.63, p < 0.01; 5F-MMB-PICA sex main effect: F(1,60) = 8.09, p < 0.05].

4.0. Discussion

A large number of SCRAs have been identified and characterized for their affinity and/or potency in radioligand binding to or functional activity at CB₁ receptor (Ametovski et al., 2020; Cannaert et al., 2020; Gamage et al., 2018; Glatfelter et al., 2022; Patel et al., 2020; Pike et al., 2021; Potts et al., 2020; Tai and Fantegrossi, 2017). Characterization of the chemical structures of these SCRAs typically focuses on four manipulatable regions of the molecule: core, tail, linker and head (Figure 1; Banister and Connor, 2018b; Potts et al., 2020). Although the limited number and structural diversity of the SCRAs tested herein prevents thorough investigation of structure-activity relationships of this cannabinoid class vis-à-vis CB₁ receptor affinity and activation, several observations are pertinent. First, the core of each SCRA tested herein was comprised of an indole or indazole, which is consistent with the core structures of numerous psychoactive SCRAs that have appeared previously in illicit markets (Banister and Connor, 2018b; Wiley et al., 2014; Wiley et al., 2017b). Systematic comparisons of CB₁ receptor activation of SCRAs has revealed that compounds with indazole cores tend to show higher CB₁ receptor affinity and to be of equal or slightly greater potency in activating β-arrestin-2 recruitment as compared to those with indole cores (Cannaert et al., 2020; Pike et al., 2021). The results of the present study are consistent with these findings, as observed in the greater affinity and the slightly greater potency (EC₅₀) of the indazole-based 5F-EDMB-PINACA as compared to its close analog, the indole-based 5F-MDMB-PICA (Figure 1 and Table 1). Similarly, indazole-based MDMB-4en-PINACA displayed greater affinity and potency than indole-based MMB-4en-PICA.

Second, in the head region of the molecules, greater CB₁ receptor affinity and activation was conferred by incorporation of a tert-leucinate than a valinate constituent, as seen most notably in differences between 5F-MDMB-PICA and 5F-MMB-PICA, differing structurally only by one structural moiety in the head region (tert-butyl vs. iso-propyl). These results are consistent with a recent report investigating the structure-activity relationships of 5F-MDMB-PICA and its 5F-pentylindole analogs (Glatfelter et al., 2022). This difference is also evident between MDMB-4en-PINACA and MMB-4en-PICA, although they also have different cores. Previous reports also have shown that tert-leucinate confers an advantage over valinate in CB₁ receptor binding and activation of β-arrestin-2 recruitment (Cannaert et al., 2020; Pike et al., 2021). In addition, a benzyl group is often present in many SCRAs in the head region, such as APP-BINACA (Figure 1). While a benzyl group was shown to be more potent than the corresponding isopropyl when 5F-MPP-PICA was compared to 5F-MMB-PICA (Cannaert et al., 2020), benzyl generally affords lower affinity or potency than the corresponding tert-butyl or iso-propyl in other SCRAs (Ametovski et al., 2020; Cannaert et al., 2020; Pike et al., 2021). Consistently, APP-BINACA showed significantly lower affinity and potency than 5F-EDMB-PINACA (tert-butyl) and 5F-MMB-PICA (iso-propyl).

The tail region of the SRCAs generally prefers hydrophobic substituents of an appropriate size. Our results showed that both the fluoropentyl group and the pentenyl group (i.e., 5F-MDMB-PICA and MDMB-4en-PINACA) provided good CB₁ affinity and potency, although the fluoropentyl group conferred slightly greater affinity than the pentenyl (5F-MMB-PICA vs. MMB-4en-PICA), consistent with previous reports (Cannaert et al., 2020). This tail region is hypothesized to correspond with the C-3 constituent of the prototypic phytocannabinoid Δ⁹-THC (Huffman et al., 1994). Although based on a completely different tricycle ring skeleton, structure-activity relationship investigation of THC analogs with halogen or double bond-bearing C-3 constituents has revealed similar trends in potency and efficacy (Griffin et al., 2001; Keimowitz et al., 2000; Martin et al., 1999). These results reinforce the hypothesis of cross-scaffold correspondence of the tail region of SCRAs and the C-3 constituent of the THCs. In addition, in both SCRAs and Δ⁹-THC analogs, affinity and potency generally increase and then decrease with the elongation of the alkyl constituents: propyl < butyl ≤ pentyl ≈ hexyl > heptyl (Aung et al., 2000; Huffman et al., 2003; Martin et al., 1999; Wiley et al., 1998). However, the significantly lower CB₁ affinity and potency of the butyl analog APP-BINACA likely resulted from the presence of benzyl group in the head group, instead of the butyl tail group.

Finally, the carboxamide of the linker region is common to all tested compounds with exception of FUB-144, which has a carbonyl group, a group that is also present in some previously reported SCRAs. FUB-144 also contains a unique tetramethylcyclopropyl head constituent; hence, conclusions regarding structural diversity in the linker region are not possible. In addition to differences in CB₁ receptor affinity and potency for G-protein activation, eight of the SCRAs show enhanced efficacy for activation of the CB₁ receptor (E_max range = 218–387% of basal) compared to the less efficacious Δ⁹-THC (E_max = 128%), as has been reported for other SCRAs (Sachdev et al., 2019; Thomas et al., 2017). Interestingly, APP-BINACA, the compound with the least affinity for the CB₁ receptor, also exhibited efficacy that was closest to that of Δ⁹-THC (E_max = 180%).

While CB₁ receptor affinity and activation is most relevant for determination of the psychoactivity of SCRAs, many of these compounds also have considerable affinity for the CB₂ receptor, as reported herein and elsewhere (Huffman, 2005). In the present study, CB₂ receptor affinities ranged from 0.213 – 22.1 nM (pK_i: 7.65 – 9.67), as compared to a larger range of CB₁ receptor affinities (0.378 – 107 nM, or pK_i: 6.97 – 9.42). Interestingly, one goal of early research with SCRAs was to maximize CB₂ receptor affinity and its presumed conferral of potential therapeutic effects (Atwood et al., 2012; Riether, 2012), an effort that initially proved difficult without concomitant increase of CB₁ receptor affinity. While cannabinoids selective for the CB₂ receptor have since been developed, structure-activity relationship studies are scarce (Marriott and Huffman, 2008). In the present study, the highest CB₂ receptor affinities were for compounds that also showed the highest CB₁ receptor affinities (Table 1); however, reasonable separation (with CB₂ receptor preference) was seen with FUB-144 (23-fold) and APP-BINACA (12-fold).

One of the prominent CB₁ receptor-mediated effects of Δ⁹-THC in rodents is hypothermia (Compton et al., 1996), an effect that is shared by SCRAs (Wiley et al., 2017b). The nine SCRAs tested herein produced significant decreases in body temperature. Although their rank order potencies for inducing hypothermia (5F-EDMB-PINACA = 5F-MDMB-PICA = MDMB-4en-PINACA > 5F-MMB-PICA = MMB-FUBICA > FUB-AKB48 = FUB-144 = MMB-4en-PICA > Δ⁹-THC > APP-BINACA) mostly corresponded with their rank order CB₁ receptor affinities (listed in order of high to low affinity in Table 1), one exception was FUB-AKB48, which showed less relative potency than would have been predicted by its CB₁ receptor affinity, possibly because of differences in pharmacokinetic properties with the presence of the large and lipophilic adamantyl head group. Previous studies have shown that SCRAs detected in illicit markets typically are more potent than Δ⁹-THC at producing CB₁ receptor-mediated effects (Wiley et al., 2015; Wiley et al., 2013) and, with exception of APP-BINACA, the results reported here support this finding, as dose-effect curves for each of the other SCRAs were to the left of Δ⁹-THC’s dose-effect curve. Notably, a previous study using radiotelemetry to measure body temperature reported that APP-BINACA failed to produce any hypothermia during a 6-h period (Sparkes et al., 2022). Together, these results are consistent with the compound’s relatively low affinity for the CB₁ receptor [> 100 nM herein and 25 nM in (Sparkes et al., 2022)] and lower efficacy (180% vs. >300%) as compared to many other abused SCRAs. Δ⁹-THC itself is comparatively less potent than would have been predicted by the CB₁ receptor binding affinity recently reported by our lab (Gamage et al., 2020); however, greater alignment between rank order potency and affinity for Δ⁹-THC vis-à-vis the SCRAs is seen when a previously reported value (~41 nM) is used (Martin et al., 1995; Showalter et al., 1996). In addition to the enhanced hypothermic potency observed with eight SCRAs as compared to Δ⁹-THC, it is also noteworthy that the maximal degree of hypothermia produced by each of these compounds exceeded that of Δ⁹-THC at one or more timepoints, as previously reported with other SCRAs (Grim et al., 2016a; Wiley et al., 2015).

Analysis of the temperature time course data showed that peak hypothermic effects occurred at 0.5–1 h for the SCRAs and at 2 h for Δ⁹-THC. Duration of hypothermia varied across the compounds, with 5F-MDMB-PICA and MMB-4en-PICA showing the most extended periods of substantial hypothermia. The long half-life of 5F-MDMB-PICA for producing hypothermia has been demonstrated previously in male rats (Krotulski et al., 2021). This compound has also been shown to produce hypothermia, analgesia and catalepsy in male mice, effects that were reversed by administration of the CB₁ receptor antagonist rimonabant (Glatfelter et al., 2022). In the present study, determination of equivalence of doses across the compounds, or dose-dependence of the duration of hypothermic effect, was difficult because only one dose of each cannabinoid was examined in the time course analysis. Although some mice were tested with multiple compounds for hypothermic effects, there was no evidence that multiple testing affected sensitivity to the test compounds.

In addition to their hypothermic effects, each of the SCRAs produced dose-dependent increases in responding on the Δ⁹-THC-associated aperture in male and female mice trained to discriminate Δ⁹-THC from vehicle, similar to what has been found in past studies with other SCRAs examined in rats and primates (Gatch and Forster, 2019; Gatch et al., 2022; Hruba and McMahon, 2017; Jarbe and Raghav, 2017). Δ⁹-THC discrimination as an animal model of the subjective effects of psychoactive cannabinoids in humans is well established (Balster and Prescott, 1992). Further, because substitution for Δ⁹-THC in this model is predictive of Δ⁹-THC-like subjective effects in humans, the FDA has recommended its use as a core component of preclinical abuse liability profiling for cannabinoids (Food and Drug Administration, 2010), especially since self-administration of cannabinoids has proved difficult (Lefever et al., 2014). Previous research has shown that Δ⁹-THC’s discriminative stimulus effects are CB₁ receptor-mediated (Wiley et al., 1995); hence, not unexpectedly, the SCRAs with the highest affinities for the CB₁ receptor (with exception of FUB-AKB48, as noted above) were those with the greatest potencies for Δ⁹-THC-like responding in both sexes. Further, with exception of 5F-EDMB-PINACA, MMB-4en-PICA, MMB-FUBICA, and APP-BINACA, each of the other five SCRAs fully substituted for Δ⁹-THC at one or more doses that did not significantly decrease overall responding in both sexes, suggesting that these five SCRAs would share Δ⁹-THC’s higher abuse potential in humans. Indeed, human use of each of these compounds has been reported (Barceló et al., 2017; Drug Enforcement Administration, 2022; Institóris et al., 2022; Krotulski et al., 2020; Truver et al., 2020; Vaccaro et al., 2022). Potencies for the Δ⁹-THC-like compounds were similar across sex with two exceptions: Δ⁹-THC was more potent in females than males and 5F-MMB-PICA was more potent in males than females. While the greater potency of Δ⁹-THC’s discriminative stimulus effects in female rats has been previously reported (Wiley et al., 2017a), species and strain also affect its potency across the sexes in this assay (Wiley et al., 2021). Sex differences in the in vivo effects of some, but not all, SCRAs have been reported; however, research is sparse (Wiley et al., 2021; Wiley et al., 2019; Wiley et al., 2017a). Additional research will be needed to determine the extent of sex differences in cannabinoid effects and the degree to which these differences are related to pharmacodynamic versus pharmacokinetic effects.

Interestingly, 5F-MDMB-PICA, 5F-MMB-PICA, MDMB-4en-PINACA, and FUB-AKB48 all fully substituted for Δ⁹-THC in drug discrimination at one or more doses in male mice that did not produce hypothermic effects at any timepoint, indicating that there are potency differences between these two assays for some SCRAs. The few past studies examining hypothermic and discriminative stimulus effects of SCRAs via i.p. dosing in rodents show mixed results regarding potency differences across assays. JWH-018, JWH-073, and CP47,497 were more potent in their Δ⁹-THC-like discriminative stimulus effects than hypothermic effects. The lowest dose that fully substituted for Δ⁹-THC did not significantly alter temperature compared to vehicle (Brents et al., 2013; Grim et al., 2016b; Marshell et al., 2014). In contrast, doses of AB-CHMINACA and AB-PINACA (two other SCRAs) that fully substituted for Δ⁹-THC produced significant hypothermia (Wiley et al., 2015). Additional research is needed to determine how common it is for SCRAs to show large potency differences in these two assays, and the SCRA characteristics for which this potency difference occurs.

5.0. Conclusion

In summary, eight of the compounds tested herein (5F-EMDB-PINACA, 5F-MDMB-PICA, MDMB-4en-PINACA, FUB-144, FUB-AKB48, 5F-MMB-PICA, MMB-4en-PICA, and MMB-FUBICA) are potent and efficacious SCRAs in vitro and in vivo. Their high CB₁ receptor binding affinity and potent activation of the receptor, coupled with their potency in producing long-lasting hypothermia and Δ⁹-THC-like discriminative stimulus effects, are like those of past SCRAs that have been abused in humans (Castaneto et al., 2014). In contrast, APP-BINACA shows low CB₁ receptor affinity and produces Δ⁹-THC-like in vivo effects only at a high (100 mg/kg) dose, suggesting that its cannabimimetic effects are low in potency.

Abbreviations:

SCRA

synthetic CB₁ receptor agonist

Δ⁹-THC

Δ⁹-tetrahydrocannabinol

5F-EDMB-PINACA

ethyl 2-(1-(5-fluoropentyl)-1H-indazole-3-carboxamido)-3,3-dimethylbutanoate

5F-MDMB-PICA

methyl (2S)-2-{[1-(5-fluoropentyl)-1H-indole-3-carbonyl]amino}-3,3-dimethylbutanoate

APP-BINACA

N-[(2S)-1-amino-1-oxo-3-phenylpropan-2-yl]-1-butylindazole-3-carboxamide

FUB-144

(1-(4-fluorobenzyl)-1H-indol-3-yl)(2,2,3,3-tetramethylcyclopropyl)methanone

FUB-AKB48

N-((3s,5s,7s)-adamantan-1-yl)-1-(4-fluorobenzyl)-1H-indazole-3-carboxamide

MDMB-4en-PINACA

methyl 3,3-dimethyl-2-{[1-(pent-4-en-1-yl)-1H-indazole-3-carbonyl]amino}butanoate

MMB-4en-PICA

methyl 3-methyl-2-[(1-pent-4-enylindole-3- carbonyl)amino]butanoate

5F-MMB-PICA

methyl (2S)-2-[[1-(5-fluoropentyl)indole-3-carbonyl]amino]-3-methylbutanoate

MMB-FUBICA

methyl 2-(1-(4-fluorobenzyl)-1H-indole-3-carboxamido)-3-methylbutanoate.