In vitro and in vivo pharmacological evaluation of the synthetic cannabinoid receptor agonist EG-018

Abstract

Synthetic cannabinoid receptor agonists (SCRAs) possess high abuse liability and complex toxicological profiles, making them serious threats to public health. EG-018 is a SCRA that has been detected in both illicit products and human samples, but it has received little attention to date. The current studies investigated EG-018 at human CB₁ and CB₂ receptors expressed in HEK293 cells in [³H]CP55,940 competition binding, [³⁵S]GTPγS binding and forskolin-stimulated cAMP production. EG-018 was also tested in vivo for its ability to produce cannabimimetic and abuse-related effects in the cannabinoid tetrad and THC drug discrimination, respectively. EG-018 exhibited high affinity at CB₁ (21 nM) and at CB₂ (7 nM), but in contrast to typical SCRAs, behaved as a weak partial agonist in [³⁵S]GTPγS binding, exhibiting lower efficacy but greater potency, than that of THC at CB₁ and similar potency and efficacy at CB₂. EG-018 inhibited forskolin-stimulated cAMP with similar efficacy but lower potency, compared to THC, which was likely due to high receptor density facilitating saturation of this signaling pathway. In mice, EG-018 (100 mg/kg, 30 min) administered intraperitoneally (i.p.) did not produce effects in the tetrad or drug discrimination nor did it shift THC's ED₅₀ value in drug discrimination when administered before THC, suggesting EG-018 has negligible occupancy of brain CB₁ receptors following i.p. administration. Following intravenous (i.v.) administration, EG-018 (56 mg/kg) produced hypomotility, catalepsy, and hypothermia, but only catalepsy was blocked by the selective CB₁ antagonist rimonabant (3 mg/kg, i.v.). Additional studies of EG-018 and its structural analogues could provide further insight into how cannabinoids exert efficacy through the cannabinoid receptors.

Keywords: Behavior; Binding; CB(1); CB(2); Cannabinoid; Novel psychoactive substance; Signaling.

Figure 1.

Structures of EG-018, JWH-018, delta-9-tetrahydrocannabinol, CP55,940, and anandamide.

Figure 2.

[³H]CP55,940 (~1 nM) competition binding curves for CP55,940 (filled black circles), THC (filled green squares) and EG-018 (filled red triangles) using P2 membrane preparations from HEK293 cells stably expressing the (A) hCB₁ or (B) hCB₂ receptors. Compounds were incubated for 90 min at 30°C. Each data point represents the mean ± standard error of at least n=3 experiments performed in duplicate.

Figure 3.

Top Panel: Concentration response curves for CP55,940 (filled black circles), THC (filled green squares), EG-018 (filled red triangles), and anandamide (AEA; filled orange downward triangles) in [³⁵S]GTPγS binding using P2 membrane preparations from HEK293 cells expressing the (A) hCB₁ or (B) hCB₂ receptors. [³⁵S]GTPγS reactions were incubated for 1 h at 30°C. Bottom Panel: Concentration response curves inhibiting forskolin-stimulated cAMP production using intact HEK293 cells expressing the (C) hCB₁ or (D) hCB₂ receptors. and cAMP assay was incubated for 22 min at 37°C. Each data point represents the mean ± standard error of at least n=3 experiments performed in duplicate.

Figure 4.

Top Panel: Effects of THC (filled green squares) and EG-018 (filled red triangles) in mice trained to discriminate THC (5.6 mg/kg, i.p.) from vehicle using nose-poke response. Bottom Panel: Effects of EG-018 pretreatment (10 – 100 mg/kg, i.p.) on the discriminative stimulus effects of THC in mice trained to discriminate THC (5.6 mg/kg, i.p.) from vehicle using nose-poke response. Left Panels: percent THC-paired aperture responding. Right Panels: response-rates. Vehicle and THC training dose control tests are depicted on the left half of the X-axis. Values represent the mean ± standard error of data from 7 male C57/BL6J mice. The number of symbols indicates the increasing level of significance, i.e. 1 = p < 0.05, 2 = p < 0.01, 4 = p < 0.0001; * vs. Vehcontrol test (left x-axis), # vs. Veh+THC (PanelD)

Figure 5.

Top Panel: Effects of THC (filled green squares) and EG-018 (filled red triangles) on (A) locomotor activity (beam breaks during 10 min session), (B) catalepsy (ring immobility), (C) antinociception (latency to tail-flick), and (D) hypothermia (rectal temperature) in mice administered drugs i.p. 30 min prior to start of the experiment. Bottom Panel: Effects of EG-018 (56 mg/kg, i.v., 5 min) in mice given a 10 min pretreatment of vehicle (white bars) or rimonabant (3 mg/kg i.v.) on (E) locomotor activity (beam breaks during 10 min session), (F) catalepsy (ring immobility), (G) antinociception (latency to tail-flick), and (H) hypothermia. Values represent the mean±standard error of 6 male ICR mice. The number of symbols indicates the increasing level of significance, i.e. 1 = p < 0.05, 2 = p < 0.01, 3 = p < 0.001, 4 = p < 0.0001; * vs. Veh (A-D) or Veh+Veh (E-H), ^ vs. vehicle+rimonabant, # vs. EG-018+Veh, $ main effect of EG-018 treatment. V = Veh