Dual blockade of FAAH and MAGL identifies behavioral processes regulated by endocannabinoid crosstalk in vivo

Abstract

Delta(9)-tetrahydrocannabinol (THC), the psychoactive component of marijuana, and other direct cannabinoid receptor (CB1) agonists produce a number of neurobehavioral effects in mammals that range from the beneficial (analgesia) to the untoward (abuse potential). Why, however, this full spectrum of activities is not observed upon pharmacological inhibition or genetic deletion of either fatty acid amide hydrolase (FAAH) or monoacylglycerol lipase (MAGL), enzymes that regulate the two major endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG), respectively, has remained unclear. Here, we describe a selective and efficacious dual FAAH/MAGL inhibitor, JZL195, and show that this agent exhibits broad activity in the tetrad test for CB1 agonism, causing analgesia, hypomotilty, and catalepsy. Comparison of JZL195 to specific FAAH and MAGL inhibitors identified behavioral processes that were regulated by a single endocannabinoid pathway (e.g., hypomotility by the 2-AG/MAGL pathway) and, interestingly, those where disruption of both FAAH and MAGL produced additive effects that were reversed by a CB1 antagonist. Falling into this latter category was drug discrimination behavior, where dual FAAH/MAGL blockade, but not disruption of either FAAH or MAGL alone, produced THC-like responses that were reversed by a CB1 antagonist. These data indicate that AEA and 2-AG signaling pathways interact to regulate specific behavioral processes in vivo, including those relevant to drug abuse, thus providing a potential mechanistic basis for the distinct pharmacological profiles of direct CB1 agonists and inhibitors of individual endocannabinoid degradative enzymes.

Fig. 1.

Development of a dual FAAH and MAGL inhibitor, JZL195. (A) Structures of the FAAH-selective inhibitors PF-622 and PF-3845, the MAGL-selective inhibitor JZL184, and dual FAAH-MAGL inhibitors. (B) Serine hydrolase activity profiles of brain membranes after incubation with dual FAAH-MAGL inhibitors (1 μM) as determined by competitive ABPP using the serine hydrolase-directed probe fluorophosphonate-rhodamine (FP-Rh). (C) Concentration-dependent effects of JZL195 on mouse brain membrane serine hydrolases. (D and E) Blockade of AEA (D) and 2-AG (E) hydrolysis activity for MAGL and FAAH, respectively, recombinantly expressed in COS7 cells (black traces), or from mouse brain membranes (blue traces). For B–E, samples were treated with inhibitor for 30 min at 37 °C before addition of FP-Rh (1 μM) (B and C), 100 μM AEA (D), or 100 μM 2-AG (E). For D and E, data are presented as means ± SEM of three independent experiments.

Fig. 2.

JZL195 dose-responsively inhibits FAAH and MAGL in vivo and raises brain AEA and 2-AG levels. (A and B) Serine hydrolase activity profiles (A) and 2-AG and AEA hydrolytic activities (B) of brain membranes prepared from mice treated with JZL195 at the indicated doses (3–20 mg·kg⁻¹, i.p.) for 4 h. (C and D) Brain levels of AEA (C) and 2-AG (D) from mice treated with JZL195 at the indicated doses (3–20 mg·kg⁻¹, i.p.) for 4 h. For C and D, data from mice treated with selective MAGL (JZL184, 40 mg·kg⁻¹, i.p.) and FAAH (PF-3845, 10 mg·kg⁻¹, i.p.) inhibitors are also shown, respectively. For B–D, *, P < 0.05; **, P < 0.01; ***, P < 0.001 for inhibitor-treated versus vehicle-treated animals. Data are presented as means ± SEM. n = 3–5 mice per group.

Fig. 3.

Time course analysis of inhibitory activity of JZL195 in vivo. (A–C) Serine hydrolase activity profiles (A) and AEA (B) and 2-AG (C) hydrolytic activities of brain membranes prepared from mice treated with JZL195 (20 mg·kg⁻¹, i.p.) for the indicated times. (D–F) Brain levels of AEA (D), 2-AG (E), and arachidonic acid (F) from mice treated with JZL195 (20 mg·kg⁻¹, i.p.) for the indicated times. For B–F, *, P < 0.05; **, P < 0.01, ***, P < 0.001 for inhibitor-treated versus vehicle-treated control animals. Data are presented as means ± SEM. n = 3–5 mice per group.

Fig. 4.

Comparison of the effects of single versus dual inhibitors of FAAH and MAGL in the tetrad test for cannabinoid behavior. (A) Selective FAAH (PF-3845, 10 mg·kg⁻¹, i.p.) and MAGL (JZL184, 40 mg·kg⁻¹, i.p.) inhibitors produce antinociceptive effects in the tail immersion assay of thermal pain sensation. The dual FAAH/MAGL inhibitor JZL195 (20 mg·kg⁻¹, i.p) or dual treatment with individual inhibitors (JZL184 + PF-3845) produces a much greater antinociceptive effect in this assay. Average baseline latency was 0.56 ± 0.04 s and did not differ among treatment groups. (B) JZL195, or JZL184 + PF-3845, but not PF-3845 or JZL184 alone, produces robust catalepsy in the bar test. (C) JZL195 and JZL184, but not PF-3845, produce hypomotility an open-field test. (D) JZL195, JZL184, and PF-3845 do not cause hypothermia. For A–C, all FAAH/MAGL inhibitor effects were blocked by pretreatment with the CB1 antagonist rimonabant (RIM, 3 mg·kg⁻¹, i.p). **, P < 0.01, ***, P < 0.001 for vehicle–vehicle versus vehicle–JZL195, vehicle–JZL184, or vehicle–PF-3845 treated mice; †, P < 0.05; †††, P < 0.001 for vehicle–JZL195 versus vehicle–JZL184 or vehicle–PF-3845 treated mice; ##,P < 0.01, ###,P < 0.001; for vehicle–JZL195, vehicle–JZL184, or JZL184/PF-3845 versus rimonabant–JZL195, rimonabant–JZL184, or rimonabant–JZL184-PF 3845-treated respectively. Data are presented as means ± SEM. n = 6–14 mice per group.

Fig. 5.

Comparison of the behavioral effects of single versus dual inhibitors of FAAH and MAGL in a THC-appropriate drug discrimination assay. (A) JZL195 (40 mg·kg⁻¹, i.p.) produces THC-appropriate nose pokes at a magnitude similar to THC (5.6 mg·kg⁻¹, s.c.), which is reversed by rimonabant (3 mg·kg⁻¹, i.p.). (B) JZL184 (40 mg·kg⁻¹, i.p.) also shows full substitution for THC in FAAH(−/−) mice, but only incomplete (partial) substitution in FAAH(+/+) mice. For both A and B, an 80% cut-off was used to assign THC-appropriate responses. *, P < 0.05; ***, P < 0.001 for vehicle–vehicle versus vehicle–JZL195 or vehicle–JZL184 treated mice; #,P < 0.05; ###,P < 0.001; for vehicle–JZL195 or vehicle–JZL184 versus rimonabant–JZL195 or rimonabant–JZL184 treated mice, respectively. Data are presented as means ± SEM. n = 6–14 mice per group.