Selective blockade of the hydrolysis of the endocannabinoid 2-arachidonoylglycerol impairs learning and memory performance while producing antinociceptive activity in rodents

Abstract

Monoacylglycerol lipase (MAGL) represents a primary degradation enzyme of the endogenous cannabinoid (eCB), 2-arachidonoyglycerol (2-AG). This study reports a potent covalent MAGL inhibitor, SAR127303. The compound behaves as a selective and competitive inhibitor of mouse and human MAGL, which potently elevates hippocampal levels of 2-AG in mice. In vivo, SAR127303 produces antinociceptive effects in assays of inflammatory and visceral pain. In addition, the drug alters learning performance in several assays related to episodic, working and spatial memory. Moreover, long term potentiation (LTP) of CA1 synaptic transmission and acetylcholine release in the hippocampus, two hallmarks of memory function, are both decreased by SAR127303. Although inactive in acute seizure tests, repeated administration of SAR127303 delays the acquisition and decreases kindled seizures in mice, indicating that the drug slows down epileptogenesis, a finding deserving further investigation to evaluate the potential of MAGL inhibitors as antiepileptics. However, the observation that 2-AG hydrolysis blockade alters learning and memory performance, suggests that such drugs may have limited value as therapeutic agents.

Figure 1. Chemical structure of SAR127303.

Figure 2. Activity of SAR127303 in MAGL biochemical assay.

Data are presented as mean ± S.D.; N = 3. SAR127303 and JZL184 are MAGL inhibitors; SSR411298 is a FAAH inhibitor.

Figure 3. LC-MS evidence of covalent modification of MAGL Ser122 by SAR127303.

(A) MAGL protein sequence. Peptide containing Ser122 and control peptide sequences are underlined. (B) Dose-dependent decrease in unmodified Ser122-containing peptides in the presence of SAR127303.

Figure 4

A. Superimposition of SAR127303 active site in both molecules of the asymmetric unit (molecule A in cyan, molecule B in yellow). The flexible loops, interacting with the cholorophenyl and sulfonyl moieties of SAR127303 are pointed by arrows. B. MAGL-SAR127303 interactions in molecule A (cyan) and B (yellow). Hydrogen bonds are depicted as black dashes.

Figure 5. MAGL activity of brain homogenates from mice treated with SAR127303.

Data are presented as mean + S.E.M.; * P<0.05, *** P<0.001; N = 7 per group.

Figure 6. Brain lipid profiling.

Monoacylglycerol (MAG) abundance in SAR127303-treated samples was normalized to vehicle-treated samples harvested at the same time and expressed as fold induction. N = 4 mice per group. MAG 16:0, 1-hexadecanoyl-rac-glycerol; MAG 18:0, 1-monoacylglyceride; MAG 18:1, 1.1-(11Z-Octadecenoyl)-rac-glycerol; MAG 18:2, 1-(9Z,12Z-octadecadienoyl)-rac-glycerol; MAG 20:4, 2-arachidonylglycerol.

Figure 7. Levels of 2-AG, AEA, PEA and OEA in the hippocampus of mice treated with 8 mg/kg SAR127303.

Data are presented as mean ± S.E.M.; ***P<0.001 vs baseline level; N = 3 per group.

Figure 8. Effects of SAR127303 alone (A) and in combination with the CB1 receptor antagonist, rimonabant (B), or the CB2 receptor antagonist, SR144528 (C), against writhing induced by phenylbenzoquinone (PBQ) in mice.

Each bar represents the average (+ S.E.M) writhes. Post-hoc analyses following Kruskal-Wallis test: ** P<0.01, *** P<0.001 versus vehicle-treated controls. N = 10 mice per group.

Figure 9. Effects of acute and repeated (once-a-day for 5 consecutive days) dosing of SAR127303 on formalin-induced pain-like behavior in mice.

Each bar represents the average (+ S.E.M.) area under the curve (AUC) of flinching behavior. Two-sided Wilcoxon test for factor treatment: *** P<0.001 versus vehicle-treated controls. N = 8 mice per group.

Figure 10

Effects of 2-AG (A) and SAR127303 (B) on [³H]ACh release evoked by electrical depolarisation in rat hippocampal slices. (C) Blockade by the CB1 receptor antagonist rimonabant of the effects of 2-AG on [³H]ACh release evoked by electrical depolarisation in rat hippocampal slices. Data are expressed as a percentage of basal [³H]-ACh release. * P<0.05 vs control. N = 3 slices/group. (D) Effect of SAR127303 on hippocampal ACh release measured by microdialysis in the ventral hippocampus of freely-moving rats. Data are expressed as percent of basal period and are Mean ± S.E.M. of data.* P<0.05, *** P<0.001. Dunnett's test versus “Vehicle” treated group from 20 to 140 minutes for each level of “Time” with significant global effect of factor “Treatment”. N = 5 rats/group.

Figure 11. Effects of oral administration of SAR127303 on short-term visual episodic memory in mice using the novel object recognition test.

(A) Dose-response versus the NMDA receptor antagonist MK-801; (B) Antagonism of the effects of SAR127303 by the CB1 receptor antagonist, rimonabant; (C) Comparison of the effects of acute and repeated (5 days, twice-a-day) treatment of SAR127303. Each bar represents the average (+ S.E.M) novelty index ratio. Student's t-test: § P<0.05 versus chance level of 50; Post-hoc analyses following one-way ANOVA: * P<0.05, ** P<0.01 versus vehicle-treated controls. N = 9 to 13 mice per group.

Figure 12. Effects of oral administration of SAR127303 on spatial working memory in mice using the Y-maze test.

(A) Dose-response versus the non-competitive NMDA receptor antagonist, PCP; (B) Antagonism of the effects of SAR127303 by the CB1 receptor antagonist, rimonabant. Bars represent number of arm entries (mean + S.E.M.). Post-hoc analyses following one-way ANOVA: * P<0.05, ** P<0.01, *** P<0.001 versus vehicle-treated controls. N = 10 to 12 mice per group.

Figure 13. Effects of oral administration of SAR127303 on spatial reference memory in rats using the Morris water maze.

Each line represents the average (± S.E.M) latency to reach the platform across trial and learning sessions. Post-hoc analyses following a two-way ANOVA: * P<0.05 and ** P<0.01, vs Trial 1 on each day; $ P<0.05 and $$ P<0.01 vs control-treated rats on each Trial. N = 10 rats per group.

Figure 14

(A). Effect of different dosages of once daily treatment of SAR127303 on acquisition of kindling, i.e. development of seizure severity upon repeated transcorneal stimulation. Treatment and kindling were interrupted over the weekend on days 6 and 7. (B). Effect of SAR127303 at 30 mg/kg in fully-kindled vehicle-treated mice 11 days after the beginning of kindling. Animals were tested either 60 min or 24 h after drug administration. Data are mean values ± S.E.M shown for each stimulation session. # P<0.05, ## P<0.01, ### P<0.001, * P<0.05, ** P<0.01 compared to vehicle group. N = 26 to 58 mice per group.