The selective reversible FAAH inhibitor, SSR411298, restores the development of maladaptive behaviors to acute and chronic stress in rodents

Abstract

Enhancing endogenous cannabinoid (eCB) signaling has been considered as a potential strategy for the treatment of stress-related conditions. Fatty acid amide hydrolase (FAAH) represents the primary degradation enzyme of the eCB anandamide (AEA), oleoylethanolamide (OEA) and palmitoylethanolamide (PEA). This study describes a potent reversible FAAH inhibitor, SSR411298. The drug acts as a selective inhibitor of FAAH, which potently increases hippocampal levels of AEA, OEA and PEA in mice. Despite elevating eCB levels, SSR411298 did not mimic the interoceptive state or produce the behavioral side-effects (memory deficit and motor impairment) evoked by direct-acting cannabinoids. When SSR411298 was tested in models of anxiety, it only exerted clear anxiolytic-like effects under highly aversive conditions following exposure to a traumatic event, such as in the mouse defense test battery and social defeat procedure. Results from experiments in models of depression showed that SSR411298 produced robust antidepressant-like activity in the rat forced-swimming test and in the mouse chronic mild stress model, restoring notably the development of inadequate coping responses to chronic stress. This preclinical profile positions SSR411298 as a promising drug candidate to treat diseases such as post-traumatic stress disorder, which involves the development of maladaptive behaviors.

Figure 1

Synthesis of SSR411298. The compound was prepared in 24% yield by carbamation of (trans)-3-[-5-(6-methoxynaphtalen-1-yl)-1,3-dioxan-2-yl]propanamine (6) with chloroacetamide. Compound (6) was obtained via the dioxane formation of 2-(6-methoxynaphtalen-1-yl)propan-1,3-diol (5) with 4,4-diethoxybutanamine, in 93% yield. Compound (5) was isolated in 42% yield from the reduction of diethyl-2-(6-methoxynaphtalen-1-yl)propanedioate (4). Compound (4) was synthesized by alkylation of ethyl-(6-methoxynaphtalen-1-yl)-acetate (3) with ethylcarbonate in 81% yield. Compound (3) was prepared in two steps, in 57% overall yield, consisting in a substitution-deshydratation sequence of 6-methoxy-1,2,3,4-tetrahydronaphtalen-1-one (1) with ethyl bromoacetate, followed by an oxydation of the naphtalene ring by DDQ.

Figure 2

(A) Inhibitory effect of SSR411298 on mouse brain FAAH. Brain homogenates were incubated with 10 μM of [³H]anandamide in the absence (controls) or presence of increasing concentrations of SSR411298 (3 × 10⁻¹¹ M to 10⁻⁵ M). Data are the mean ± SEM of 4 independent experiments; (B) Reversibility of the effects of SSR411298 and PMSF on mouse brain FAAH activity by dialysis. For each concentration, data are the mean ± SEM of mouse brain FAAH activity estimated by measuring [³H]ethanolamine production, expressed in percent of mean of controls after 16 to 18 hours at 25 °C without (black bars) or with dialysis (hatched bars). *P < 0.05 versus non dialyzed (Student’s t-test); (C) Ex vivo effect of SSR411298 on mouse brain FAAH activity 2 hours after intraperitoneal or oral administration; (D) Time-course of p.o. treatment with SSR411298 on mouse brain FAAH activity. Each point represents the mean ± SEM of 2 to 5 independent experiments. [³H]ethanolamine production are expressed as percent of mean of controls (vehicle treated mice). ID₅₀ are calculated on specific [³H]ethanolamine production (equal to the total minus non-specific production).

Figure 3

Effects of SSR411298 on levels of (A) anandamide (AEA), (B) palmitoylethanolamide (PEA), (C) 2-Arachidonoyl glycerol (2-AG) and (D) oleoylethanolamine (OEA) in the hippocampus of mice. Data represent mean ± SEM. *P < 0.05; **P < 0.01;***P < 0.001 (Kruskal-Wallis multiple comparison test versus vehicle treated group). N = 8 mice per group.

Figure 4

Potentiation by SSR411298 of turning behavior induced by intra-striatal infusion of anandamide (AEA) in mice. (A) Dose-response of AEA; (B) Potentiation by SSR411298 of the effects of AEA. Data represent the mean ± SEM. **P < 0.01 (Dunnett's t-test) vs. control or AEA-treated group. N = 12 to 24 mice per group.

Figure 5

Effect of SSR411298 and Δ⁹-THC during generalization sessions in a drug discrimination procedure. The ratio represents the percentage of responses on the WIN 55,212-2-associated hole (●). Response rate is the number of responses per second (○). The number alongside each (●) symbol indicates the number of animals with complete generalization to WIN 55,212-2. SSR411298 at 10, 30, 100 and 500 mg/kg produced no generalization to WIN 55,212-2 whereas Δ⁹-THC produced complete generalization to WIN 55,212-2 in 10/12 rats. Data represent the mean ± SEM. N = 12.

Figure 6

Study of the potential cannabimimetic activity of SSR411298. (A) PBQ: number of stretches; (B), Ambulation: number of beam interruptions on the horizontal sensors; (C) Temperature: °C; (D) Ring immobility: time immobile in seconds. **P < 0.01, ***P<0.001. Data represent the mean ± SEM. N = 8 to 20.

Figure 7

Effects of SSR411298 on (A), spatial reference memory in the Morris water maze in rats. Each line represents the average (±SEM) latency to reach the platform across trial. N = 10 rats per group; (B) episodic memory in the object recognition test in mice. Each bar represents the average (+SEM) time spent exploring a familiar or a novel object. The interval between the acquisition and the recall session was 60 min. ***P < 0.001 novel vs familiar object at the considered treatment condition. N = 12 mice per group; (C) spatial working memory in the Y-maze test in mice. N = 12 mice per group.

Figure 8

Effects of SSR411298 and diazepam in several classical models of anxiety, including the (A), punished drinking test in rats (N = 11–12 rats per group); (B) the light/dark test in mice (N = 11–12 mice per group) and (C), the social interaction in gerbils (N = 5 gerbils per group). Data represent mean + SEM, *P < 0.05, **P < 0.01.

Figure 9

Effects of SSR411298 and diazepam in the mouse defense test battery on (A), locomotor activity prior to the exposure to the threat; (B) flight response in response to the approaching rat; (C) risk assessment when the rat was chasing the mouse, and (D), defensive attack reactions upon forced contact with the rat. Data represent mean+SEM, *P < 0.05, **P < 0.01 and ***P < 0.001. N = 7–11 mice per group.

Figure 10

Effects of SSR411298 in several models of anxiety involving prior stress exposure, including (A) social defeat induced anxiogenic-like behavior in mice in the elevated plus-maze test. *P < 0.05, **P < 0.01 (vs stressed vehicle) and +P<0.05, ++P < 0.01 (vs non-stressed vehicle). N = 8–13 mice per group; (B) ultrasonic distress vocalizations emitted during three minutes by 7-day-old rat pups separated from their mother and littermates. *P < 0.05, **P < 0.01 and ***P < 0.001. N = 7–10 rat pups/group; (C) short-term memory impairment induced by inescapable stress in an object recognition task mice. **P < 0.01, ***P < 0.001, new vs familiar object. N = 10 mice per group. Data represent mean+SEM.

Figure 11

Effect of SSR411298 and fluoxetine in two models of depression: (A) the chronic mild stress procedure in mice. Data are expressed as mean physical state score ± SEM. ^#P < 0.01 (Wilcoxon test versus non stressed control group), *P < 0.05, **P < 0.001 (Kruskal Wallis one sided upper multiple comparison tests with Bonferroni-Holm correction versus stressed control group). N = 14 to 20 mice per group; (B) the forced-swimming test in rats. SSR411298 was administered p.o. twice a day during 2 days, 60 minutes before pretest, immediately after, and 60 minutes before the second session of testing. Fluoxetine was administered p.o. twice (immediately after the first session, and 60 minutes before session 2). Data represent mean+SEM. *P < 0.05, **P < 0.01 and ***P < 0.001. N = 6–7 rats per group.