Differentiation between low- and high-efficacy CB1 receptor agonists using a drug discrimination protocol for rats

Abstract

Rationale: The "subjective high" from marijuana ingestion is likely due to Δ(9)-tetrahydrocannabinol (THC) activating the central cannabinoid receptor type 1 (CB1R) of the endocannabinoid signaling system. THC is a weak partial agonist according to in vitro assays, yet THC mimics the behavioral effects induced by more efficacious cannabinergics. This distinction may be important for understanding similarities and differences in the dose-effect spectra produced by marijuana/THC and designer cannabimimetics ("synthetic marijuana").

Objective: We evaluated if drug discrimination is able to functionally detect/differentiate between a full, high-efficacy CB1R agonist [(±)AM5983] and the low-efficacy agonist THC in vivo.

Materials and methods: Rats were trained to discriminate between four different doses of AM5983 (0.10 to 0.56 mg/kg), and vehicle and dose generalization curves were determined for both ligands at all four training doses of AM5983. The high-efficacy WIN55,212-2 and the lower-efficacy (R)-(+)-methanandamide were examined at some AM5983 training conditions. Antagonism tests involved rimonabant and WIN55,212-2 and AM5983. The separate (S)- and (R)-isomers of (±)AM5983 were tested at one AM5983 training dose (0.30 mg/kg). The in vitro cyclic adenosine monophosphate (cAMP) assay examined AM5983 and the known CB1R agonist CP55,940.

Results: Dose generalization ed50 values increased as a function of the training dose of AM5983, but more so for the partial agonists. The order of potency was (R)-isomer > (±)AM5983 > (S)-isomer and AM5983 > WIN55,212-2 ≥ THC > (R)-(+)-methanandamide. Surmountable antagonism of AM5983 and WIN55,212-2 occurred with rimonabant. The cAMP assay confirmed the cannabinergic nature of AM5983 and CP55,940.

Conclusions: Drug discrimination using different training doses of a high-efficacy, full CB1R agonist differentiated between low- and high-efficacy CB1R agonists.

Fig. 1

Chemical structures of cannabinergics used in the drug discrimination studies

Fig. 2

Effects of the cannabinergics (±)-AM5983 and (−)-CP55,940 on cAMP levels in HEK-293 cells expressing rat CB₁R. Cells were stimulated in the presence of 2 μM forskolin (FSK) for 30 min. The data are the average of five experiments performed in triplicates for each ligand

Fig. 3

Generalization test data for AM5983 and Δ⁹-THC (top) and the corresponding response rate data (bottom) for rats trained to discriminate between vehicle and 0.10 mg/kg AM5983 (group 1a). Data points are based on one to two observations for each rat (n =12–18, AM5983, and n =11–22, Δ⁹-THC) and were obtained on separate test days. Vehicle response rate (mean ± SEM) was 0.42±0.05 responses per s (horizontal lines), based on the initial six reinforcement cycles of the nondrug maintenance sessions immediately preceding the above tests. Asterisk indicates a significant difference from the vehicle rate at P ≤0.05 (Holm–Sidak post hoc multiple comparison procedure involving a control mean)

Fig. 4

Generalization test data for four cannabinergics (top) and the corresponding response rate data (bottom) for rats trained to discriminate between vehicle and 0.18 mg/kg AM5983 (group 1b). Data points are based on one observation for each rat (n =11–12, AM5983, and n =9–12, Δ⁹-THC; mAEA; and WIN55,212-2) and were obtained on separate test days. Vehicle response rate (mean ± SEM) was 0.61±0.04 responses per s (horizontal lines), based on the initial six reinforcement cycles of the nondrug maintenance sessions immediately preceding the above tests. Asterisk indicates significant difference from the vehicle rate at P ≤0.05 (Holm–Sidak post hoc multiple comparison procedure involving a control mean)

Fig. 5

Antagonism test data for AM5983 and WIN55,212-2 (top) and the corresponding response rate data (bottom) for rats trained to discriminate between vehicle and 0.18 mg/kg AM5983 (group 1b). Tests were conducted 20 min after concurrent i.p. administrations of 1 mg/kg rimonabant (SR) and AM5983 or WIN55,212-2. Data points are based on one observation for each rat (n =8–10 for both drug combinations) and were obtained on separate test days. Vehicle response rate (mean ± SEM) was 0.50±0.03 responses per s (horizontal lines), based on the initial six reinforcement cycles of the nondrug maintenance sessions immediately preceding the above tests. Asterisk indicates a significant difference from the vehicle rate at P ≤0.05 (Holm–Sidak post hoc multiple comparison procedure involving a control mean)

Fig. 6

Generalization test data for five cannabinergics (top) and the corresponding response rate data (bottom) for rats trained to discriminate between vehicle and 0.30 mg/kg AM5983 (group 2a). Data points are based on one observation for each rat (n =11–12, AM5983 and AM4971; n =9–11, Δ⁹-THC; n =10–12, mAEA; and n =10–11, AM5760) and were obtained on separate test days. Vehicle response rate (mean ± SEM) was 0.46±0.03 responses per s (horizontal lines), based on the initial six reinforcement cycles of the nondrug maintenance sessions immediately preceding the above tests. Asterisk indicates a significant difference from the vehicle rate at P ≤0.05 (Holm–Sidak post hoc multiple comparison procedure involving a control mean)

Fig. 7

Generalization test data for four cannabinergics (top) and the corresponding response rate data (bottom) for rats trained to discriminate between vehicle and 0.56 mg/kg AM5983 (group 2b). Data points are based on one observation for each rat (n =8–12, AM5983; n =8–9, Δ⁹-THC; n =8–10, mAEA; and n =8–9, WIN55,212-2) and were obtained on separate test days. Vehicle response rate (mean ± SEM) was 0.84±0.18 responses per s (horizontal lines), based on the initial six reinforcement cycles of the nondrug maintenance sessions immediately preceding the above tests. Asterisk indicates a significant difference from the vehicle rate at P ≤0.05 (Holm–Sidak post hoc multiple comparison procedure involving a control mean)

Fig. 8

Antagonism test data for AM5983 and WIN55,212-2 (top) and the corresponding response rate data (bottom) for rats trained to discriminate between vehicle and 0.56 mg/kg AM5983 (group 2b). Tests were conducted 20 min after concurrent i.p. administrations of 1 mg/kg rimonabant (SR) and AM5983 or WIN55,212-2. Data points are based on one observation for each rat (n =8–9 for both drug combinations) and were obtained on separate test days. Vehicle response rate (mean ± SEM) was 0.57±0.04 responses per s (horizontal lines), based on the initial six reinforcement cycles of the nondrug maintenance sessions immediately preceding the above tests. Asterisk indicates a significant difference from the vehicle rate at P ≤0.05 (Holm–Sidak post hoc multiple comparison procedure involving a control mean)

Fig. 9

Graphical display of the changes in milligram per kilogram of the ED₅₀ values (Y-axis) for the four study cannabinergics as a function of the training dose (range, 0.10 to 0.56 mg/kg) of AM5983 (X-axis)