Signaling-specific inhibition of the CB1 receptor for cannabis use disorder: phase 1 and phase 2a randomized trials

Abstract

Cannabis use disorder (CUD) is widespread, and there is no pharmacotherapy to facilitate its treatment. AEF0117, the first of a new pharmacological class, is a signaling-specific inhibitor of the cannabinoid receptor 1 (CB₁-SSi). AEF0117 selectively inhibits a subset of intracellular effects resulting from Δ⁹-tetrahydrocannabinol (THC) binding without modifying behavior per se. In mice and non-human primates, AEF0117 decreased cannabinoid self-administration and THC-related behavioral impairment without producing significant adverse effects. In single-ascending-dose (0.2 mg, 0.6 mg, 2 mg and 6 mg; n = 40) and multiple-ascending-dose (0.6 mg, 2 mg and 6 mg; n = 24) phase 1 trials, healthy volunteers were randomized to ascending-dose cohorts (n = 8 per cohort; 6:2 AEF0117 to placebo randomization). In both studies, AEF0117 was safe and well tolerated (primary outcome measurements). In a double-blind, placebo-controlled, crossover phase 2a trial, volunteers with CUD were randomized to two ascending-dose cohorts (0.06 mg, n = 14; 1 mg, n = 15). AEF0117 significantly reduced cannabis' positive subjective effects (primary outcome measurement, assessed by visual analog scales) by 19% (0.06 mg) and 38% (1 mg) compared to placebo (P < 0.04). AEF0117 (1 mg) also reduced cannabis self-administration (P < 0.05). In volunteers with CUD, AEF0117 was well tolerated and did not precipitate cannabis withdrawal. These data suggest that AEF0117 is a safe and potentially efficacious treatment for CUD.ClinicalTrials.gov identifiers: NCT03325595 , NCT03443895 and NCT03717272 .

Fig. 1. AEF0117 decreased the behavioral and physiological effects of cannabinoids.

a, In mice, after the acquisition phase (left panel, n = 26), AEF0117 (right panel, n = 13) decreased the number of infusions of the CB₁ agonist WIN55,212-2 compared to vehicle-treated mice (n = 13); P = 0.021: ANOVA treatment effects for AEF0117 (15 µg kg⁻¹). b, In monkeys (left panel, n = 4), AEF0117 dose-dependently decreased the number of THC infusions (4 µg kg⁻¹ per infusion). *P < 0.001, ^#P = 0.002 compared to vehicle (three sessions average), Tukey test. In monkeys (right panel, n = 4), after extinction of THC-reinforced responding, AEF0117 decreased reinstatement of drug seeking induced by a non-contingent THC injection (40 µg kg⁻¹, i.v.). ^$P = 0.0346, ^$$P = 0.0015 compared to non-contingent saline; ^##P = 0.0023, P = 0.0018 in AEF0117 5 µg kg⁻¹ and 15 µg kg⁻¹, respectively, ^###P = 0.0002 compared to THC + vehicle (AEF0117 0 µg kg⁻¹), Dunnett test, within-subjects design. AEF0117 inhibited the following effects of THC in mice. c, Increase in food intake, ^###P = 0.0003, ^***P = 0.0007 (Dunnett test, n = 16 for vehicle and AEF0117 15 µg kg⁻¹, n = 17 for THC, n = 8 for the other conditions). d, Impairment of long-term memory, ***P < 0.001: familiar versus novel object (P = 0.0002 for vehicle + AEF0117 0 µg kg⁻¹ and P < 0.0001 for THC + AEF0117 5 µg kg⁻¹); ^##P < 0.01: novel object after THC + AEF0117 0 µg kg⁻¹ versus novel object in the other two conditions (P = 0.0082 versus vehicle + AEF0117 0 µg kg⁻¹ and P = 0.0041 versus THC + AEF0117 5 µg kg⁻¹), Sidak test, n = 8 for vehicle and THC, n = 9 for AEF0117. e, Decrease in social interaction, ^**P = 0.0001: THC versus vehicle; ^#P = 0.0068, ^###P < 0.0001: AEF0117 + THC versus THC (Dunnett test, n = 24, 6, 15 and 14 for AEF0117 0 μg kg⁻¹, 5 μg kg⁻¹, 15 μg kg⁻¹ and 50 μg kg⁻¹ + vehicle; n = 27, 10, 19 and 18 for AEF0117 0 μg kg⁻¹, 5 μg kg⁻¹, 15 μg kg⁻¹ and 50 μg kg⁻¹ + THC, respectively). f, Increase in locomotor activity, P < 0.0001: treatment effect ANOVA (n = 18 for vehicle and THC, n = 19 for AEF0117 1.5 μg kg⁻¹, n = 10 for the other conditions). g, Impairment of sensory gating, P = 0.001: treatment effect ANOVA (n = 18 for vehicle and THC, n = 9 for AEF0117 50 μg kg⁻¹, n = 10 for the other conditions). h, Impairment in reality testing, ^*P = 0.0377: THC versus vehicle (unpaired t-test, one-tailed, n = 20 for AEF0117 0 μg kg⁻¹ vehicle, n = 19 for AEF0117 0 μg kg⁻¹ THC, n = 12 and 10 for AEF0117 15 μg kg⁻¹ and 50 μg kg⁻¹ per treatment dose). i, Catalepsy, ^***P = 0.0002: THC versus vehicle; ^#P = 0.037: AEF0117 + THC versus THC (Mann–Whitney test, n = 8 per condition). j, In rats, left: percentage increase in extracellular DA concentration from baseline over time; right: AUC of extracellular DA concentrations, ^**P = 0.0017, ^***P = 0.0008: AEF0117 + THC versus THC (Dunnett test, n = 7 for vehicle and n = 5, 6 and 7 for AEF0117 5 μg kg⁻¹, 15 μg kg⁻¹ and 50 μg kg⁻¹, respectively). Data are represented as mean ± s.e.m. inj., injection.

Fig. 2. Distribution of participants.

Participant flow for the SAD (a), MAD (b) and phase 2a (c) studies. AEF, AEF0117; crit., criteria.

Fig. 3. AEF0117 decreased positive subjective ratings and cannabis self-administration in research volunteers with CUD in the phase 2a study.

AEF0117 (1 mg) significantly decreased positive subjective ratings of cannabis measured by VAS (0–100 mm). a,d, Intoxication subscale comprising the arithmetic mean of the ratings of the ‘I feel a Good Effect’ and ‘I feel High’ items. Rating of the ‘Felt Good Cannabis Effect’ (b,e) and of the ‘Cannabis Cigarette Liking’ (c,f) items. a–c, MMRM global crossover analysis (n = 13 per dose) shows a significant effect of treatment for (Treatment × Time, P = 0.0036 and Dose × Treatment × Time, P = 0.0017) (a) and (Treatment × Dose × Time, P = 0.0034) (b). Significant Treatment × Sequence interactions were found for all the outcomes, P = 0.0182 (a), P < 0.0001 (b) and P = 0.0318 (c). d–f, When the pre-planned MMRM parallel group analysis (first dosing period only, placebo n = 14, AEF0117 n = 6 per dose) was performed to eliminate the sequence effect, a significant decrease was observed for all the outcomes (Treatment × Time, P = 0.0381 (d), P = 0.0032 (e), P = 0.0126 (f); Treatment × Dose × Time, P = 0.0368 (e)). g,h, AEF0117 1 mg significantly decreased cannabis self-administration as measured by the number of cannabis puffs purchased by the participants. g, The MMRM global crossover analysis (n = 13 per dose) showed a significant Treatment effect (P = 0.0254), a Treatment × Dose × Session interaction (P = 0.0009) and a Treatment × Sequence interaction (P = 0.0085). h, The pre-planned MMRM parallel group analysis showed a significant decrease in self-administration (Dose × Treatment × Session, P = 0.0344). i, Exploratory analysis as a function of the dosing sequence (n = 6 for AEF0117 1 mg first or n = 7 for placebo first) for ‘Felt Good Cannabis Effect’. The ratings after AEF0117 were similar in the two sequences, whereas responding under placebo was lower if participants had previously received AEF0117, indicating a long-lasting effect of AEF0117. j,k, When AEF0117 was administered in the first study period (AEF0117 first, n = 6 for the 0.06-mg cohort; n = 7 and 8 for the 1-mg cohort for placebo and AEF0117, respectively), detectable levels of AEF0117 were observed in the second dosing period when participants received placebo ≥14 d after AEF0117 administration. This was not the case when AEF0117 was administered in the second dosing period (placebo first, n = 8 and 7 per dose for the 0.06-mg and 1-mg cohorts, respectively). Data are represented as mean ± s.e.m. over the different days of testing for each timepoint (a–h,j,k) or as overall rating over the 5 d of testing and the five timepoints (i). AEF, AEF0117; D, days; SA, self-administration; PD, pre-dose.

Fig. 4. Effects of AEF0117 on mood, food intake and body weight in the phase 2a study.

Subjective ratings before dosing with AEF0117 at 9:00 (a,c,e) and then 2.5 h after AEF0117 administration, before cannabis smoking (b,d,f) for the subscale of the 44-item VAS used to measure precipitated cannabis withdrawal Irritable (a,b), Anxious (c,d) and Miserable (e,f). In the MMRM analysis performed for all three subscales, a small but significant effect was found for the ‘Irritable’ subscale (Treatment × Day × Time interaction, P = 0.0373). No significant changes were found for the other two endpoints used to measure precipitated withdrawal: daily caloric intake (g) and daily body weight (h). For body weight, day 6 is the day of discharge 24 h after the last administration of AEF0117 at day 5 (n = 13 per dose cohort). Red circles indicate data from the same participant (included in the mean value calculations and statistical MMRM analysis). Data are represented as mean + s.e.m.

Extended Data Fig. 1. Structure and in vitro effects of AEF0117.

(a) Chemical structure of AEF0117. (b) AEF0117 reversed THC-induced decreases in cellular respiration (1 μM) in HEK293 cells transfected with the human CB₁ (hCB₁). When cells are transfected with a mutant CB₁ (hCB₁-Mut), which invalidates the putative pregnenolone binding site, AEF0117 does not reverse THC’s effects (n = 3 and 4 for 2 and 1 independent experiments for THC, Vehicle, AEF0117 100 nM; n = 4 for all other conditions). (c) AEF0117 did not modify THC-induced inhibition of cAMP levels (n = 3 per dose). (d) AEF0117 did not modify binding of the CB₁ agonist, CP55,940 (n = 3 per dose). (e) AEF0117 reduced the increase in p-ERK1/2 induced by THC (30 nM) in CHO cells stably transfected with the hCB₁ (CHO-hCB₁; n = 4 per dose). (f) AEF0117 reduced the increase in p-ERK1/2 induced by THC (10 µM) in STHdh^Q7/Q7 cells expressing the mouse CB₁ (4 independent experiments, n = 3 each). Data are represented as mean ± s.e.m.

Extended Data Fig. 2. Pharmacokinetics of AEF0117 and THC in mice, rats, healthy volunteers, and volunteers with cannabis use disorder (CUD).

Plasma and brain concentrations after a single per os administration of AEF0117 in male and female mice (n = 3 per sex and per time point, data for males and females were averaged) after different doses of AEF0117 (a) 0.3 mg/kg; (b) 4 mg/kg and (c) 10 mg/kg and (d) in male rats after 1.6 mg/kg of AEF0117 (n = 3 per time point). Plasma concentration of AEF0117 from the multiple ascending dose (MAD) study in healthy volunteers after: (e) the first dose (due to severe weather conditions, there was no sample available in the cohort of AEF0117 2 mg at 24 h); and (f) the seventh and last dose (n = 5 for 0.6 mg; n = 6 for the other dose cohorts). Data are represented as mean ± s.e.m.

Extended Data Fig. 3. Effects of AEF0117 and of the CB₁ orthosteric antagonist rimonabant in mice.

Compared to Vehicle-treated animals, Rimonabant (Rimo 10 mg/kg; ip) but not AEF0117 (5, 15, 50 µg/kg and 5 and 15 mg/kg per os) decreased: (a) body weight in diet-induced obesity (DIO) mice, Days 5 to 15: P < 0.05 (from Day 5 to 15: P = 0.026, 0.0133, 0.0162, 0.008, 0.0076, 0.0053, 0.0054, 0.0044, 0.0027, 0.0032, 0.0018, respectively), Rimo vs Vehicle (Dunnett test) and (b) food intake in DIO mice, Day 1 to 5: P < 0.0001, Rimo vs Vehicle (Tukey test). N = 18 for vehicle; n = 10 for AEF0117 5 µg/kg, n = 7 for AEF0117 50 µg/kg, n = 8 for all other conditions. The repeated administration (28 days, once a day) of rimonabant (10 mg/kg) but not AEF0117 (0, 0.05, 5, 15 mg/kg, per os) increased anxiety- and depression-related behavior as shown by: the decrease in the percentage of (c) time spent in, and (d) visits to, the open arms of the Elevated Plus Maze (EPM) (for AEF0117: n = 5 for Vehicle, n = 8 for all other conditions; for Rimo: n = 6 for Vehicle, n = 8 for 10 mg/kg), *P = 0.0209, **P = 0.0026, Rimo 10 vs 0 mg/kg (unpaired t-test) and (e) the decrease in sucrose intake (n = 6 for Vehicle, n = 8 for all other conditions), *P = 0.0279, Rimo 10 vs 0 mg/kg (unpaired t-test). Rimonabant (10 mg/kg) but not AEF0117 (0.15 mg/kg) precipitated withdrawal in THC-dependent animals as shown by an increase in the duration of (f) head shaking and (g) paw tremors [n = 19 for Vehicles (VEH) groups, n = 7 for Rimo and n = 6 for AEF0117 groups], *P = 0.0351, ***P < 0.0001, Rimo vs Vehicle + THC (Dunnett test). AEF0117 (0.3 or 10 mg/kg, per os) did not significantly increased plasma concentrations of corticosterone (n = 3 per sex) in (h) male mice or (i) female mice. Data in a through g are represented as mean + s.e.m. Horizontal and vertical red lines in h and i represent mean ± s.e.m. respectively.

Extended Data Fig. 4. Effects of AEF0117 on endocannabinoids (AEA and 2-AG) and pregnenolone’s downstream steroids (allopregnanolone, DHEA and testosterone) in male and female rats.

(a) N-arachidonoylethanolamine (Anandamide, AEA) and (b) 2-Arachidonylglycerol (2-AG) data were averaged for males and females. (c) Allopregnanolone in females. (d) Allopregnanolone in males. (e) Dehydroepiandrosterone (DHEA) in females. (f) DHEA in males. (g) Testosterone in males. For all parameters, the timepoints were: 14 days of treatment at 5 h (D14-5h) and 24 h (D14-24h) after dosing with AEF0117, and 24 h after the last administration on day 28 (D28-24h). For (a) and (b) n = 6 per time point for vehicle (AEF0117 0 mg/kg), n = 12 per time point for all the other doses of AEF0117. For (c-g), n = 3 per time point for vehicle (AEF0117 0 mg/kg), n = 6 per time point for the other doses of AEF0117 (2, 9, 36, mg/kg) except n = 5 for AEF0117 36 mg/kg at D14-5h time point in (d, f, g) and n = 5 for AEF0117 36 mg/kg at D28-24h in (g). Data are represented as mean + s.e.m.

Extended Data Fig. 5. Effects of AEF0117 on endocannabinoids (AEA and 2-AG) and pregnenolone’s downstream steroids (allopregnanolone, DHEA and testosterone) in male and female dogs.

(a) N-arachidonoylethanolamine (Anandamide, AEA) and (b) 2-Arachidonylglycerol (2-AG) data were averaged in males and females. (c) Allopregnanolone in females. (d) Allopregnanolone in males. (e) Dehydroepiandrosterone (DHEA) in females. (f) DHEA in males. (g) Testosterone in males. PD= Pre-dose levels. n = 3 per sex per time point. Data are represented as mean ± s.e.m.

Extended Data Fig. 6. Effects of single doses of AEF0117 on endocannabinoids (AEA and 2-AG), pregnenolone, and pregnenolone’s downstream steroids in male and female healthy volunteers.

(a) N-arachidonoylethanolamine (Anandamide, AEA). (b) 2- Arachidonoylglycerol (2-AG). (c) Pregnenolone. (d) Dehydroepiandrosterone (DHEA). (e) Allopregnanolone. (f) Testosterone (only males). (g) Cortisol. (h) Estradiol. (i) Progesterone. Dunnett’s multiple comparisons test after ANOVA were performed. There were statistically significantly higher levels of pregnenolone (c, # P = 0.0306; ## P = 0.0051; ### P < 0.0001) in the AEF0117 6 mg group compared to placebo, but this difference was already observed at pre-dose, suggesting an effect independent of AEF0117 dosing. Higher pre-dose levels in the 6 mg group compared to placebo were also observed for allopregnanolone (e, ### P = 0.0006). In the 0.2 mg group compared to placebo there were lower concentrations of 2-AG 4 h after dosing (b, * P = 0.0223) and lower concentrations of allopregnanolone at 0.5 h after dosing (e, * P = 0.0467). Data are represented as mean ± s.e.m. Single ascending dose (SAD) and day 1 results of the multiple ascending dose (MAD) studies were averaged, n = 16 for Placebo per time point n = 6 per 0.2 mg/kg per time point, n = 12 [except n = 10 in (f)] per 0.6 mg/kg per time point, n = 18 per 2 mg/kg per time point and n = 12 per 6 mg/kg per time point. PD = Pre-dose levels.

Extended Data Fig. 7. Objective sleep measures with the Actiwatch®.

(a) Sleep onset. (b) Sleep efficiency. (c) Sleep Percentage. (d) Snooze time. (e) Wake bouts. (f) Wakefulness after sleep onset. The performed MMRM analysis for all parameters showed a statistically significant overall Treatment effect (no significant interaction with Dose or Days) for Snooze time (Treatment, P = 0.0071) and number of Wake bouts (Treatment, P = 0.0237). Relative to placebo, AEF0117 increased Snooze time and decreased the number of Wake bouts. Day 1 is the morning before the first AEF0117 dose and Day 6 is 24 h after the last AEF0117 dose on Day 5. n = 13 per dose cohort. Data are represented as mean + s.e.m.

Extended Data Fig. 8. Subjective sleep ratings measured by items of the sleep questionnaire using Visual Analog Scales (VAS) in the Phase 2a study.

(a) I slept well last night. (b) I fell asleep easily last night. (c) I woke up early this morning. (d) I feel clear-headed this morning. (e) I woke often last night. (f) I was satisfied with my sleep last night. (g) I had a lot of dreams last night. (h) How many hours did you sleep last night? The only statistically significant effect was a Dose*Treatment interaction for the ‘I woke often last night’ item. The two doses of AEF0117 had opposite effects on this parameter: 0.06 mg decreasing and 1 mg increasing ratings of waking often; neither AEF0117 dose differed significantly from placebo for this rating. Day 1 is the morning before the first AEF0117 dose and Day 6 is 24 h after the last dosing on Day 5. n = 13 per dose cohort. Data are represented as mean + s.e.m.

Extended Data Fig. 9. Effects of AEF0117 on mood after cannabis smoking in the Phase 2a study.

The subscales of the 44-item VAS used to measure precipitated cannabis withdrawal: (a) Irritable, (b) Anxious, and (c) Miserable were also used to measure mood alterations after smoking cannabis. In this context, in the MMRM analysis performed for all 3 subscales a small but significant effect was found for the ‘Irritable’ subscale (Treatment*Day interaction, P = 0.0418). Small but statistically significant changes were also found in the MMRM analysis for some of the other subscales used to measure mood effects of AEF0117 during cannabis smoking: (d) a decrease for the ‘Social’ subscale (Treatment*Day interaction, P = 0.0215) and (e) an increase for the ‘Bad effect’ subscale (Dose*Treatment*Day interaction P = 0.0388). n = 13 per dose cohort. Data are represented as mean + s.e.m. Red-encircled dots represent data from one participant, the same individual highlighted by red encircled dots in Fig. 4.

Extended Data Fig. 10. Effects of AEF0117 on endocannabinoids (AEA and 2-AG), pregnenolone, pregnenolone’s downstream steroids, THC and its metabolites in volunteers with cannabis use disorder (CUD) in the Phase 2a study.

(a) N-arachidonoylethanolamine (Anandamide, AEA). (b) 2-Arachidonoylglicerol (2-AG). (c) Pregnenolone. (d) Dehydroepiandrosterone (DHEA). (e) Allopregnanolone. (f) Testosterone. (g) Cortisol (h) Estradiol (only males). (i) Progesterone (only males). (j) Δ⁹-tetrahydrocannabinol (THC). (k) 11-OH THC. (l) 11-COOH THC. A MMRM analysis for each dose cohort (n = 13) was performed. (a) For AEA the MMRM overall crossover analysis identified a significant Dose*Treatment*Time interaction (P = 0.0015). When the analysis was performed for each dose cohort, a significant Treatment effect was found for the 0.06 dose only (P = 0.0063). This effect seemed to be due to higher basal AEA levels (PD) before AEF0117 dosing (P = 0.0079) that remained high 3 h post dosing (P = 0.0397), suggesting the effect was not caused by AEF0117 administration. The MMRM global crossover analysis also identified significant Dose*Treatment*Time interactions for: (d) Testosterone, P = 0.0012 and (e) Allopregnanolone, P = 0.0009. However, when these analyses were performed for each dose cohort independently, no significant effect of Treatment or Treatment*Time interaction was found. Other MMRM analysis did not revealed any significant effects of Treatment or Treatment*Time. Data are represented as mean ± s.e.m. of plasma concentrations. PD = pre-dose levels obtained 30 min before AEF0117 administration. Black arrows indicate cannabis smoking at 3.5 and 5.5 h after AEF0117 dosing. * P = 0.0397 and ** P = 0.0079 AEF0117 0.06 mg compared to placebo.