Dual inhibition of endocannabinoid catabolic enzymes produces enhanced antiwithdrawal effects in morphine-dependent mice

Abstract

Inhibition of the endocannabinoid catabolic enzymes, monoacylglycerol lipase (MAGL) or fatty acid amide hydrolase (FAAH) attenuates naloxone-precipitated opioid withdrawal signs in mice via activation of CB1 receptors. Complete FAAH inhibition blocks only a subset of withdrawal signs, whereas complete MAGL inhibition elicits enhanced antiwithdrawal efficacy, but is accompanied with some cannabimimetic side effects. Thus, the primary objective of the present study was to determine whether combined, full FAAH inhibition and partial MAGL represents an optimal strategy to reduce opioid withdrawal. To test this hypothesis, we examined whether combined administration of high-dose of the FAAH inhibitor PF-3845 and low-dose of the MAGL inhibitor JZL184, as well as the novel dual FAAH-MAGL inhibitor SA-57, which is 100-fold more potent in inhibiting FAAH than MAGL, would prevent spontaneous withdrawal in morphine-dependent mice, a model with greater face validity than precipitating withdrawal with μ-opioid receptor antagonists. Strikingly, a combination of low-dose JZL184 and high-dose PF-3845 as well as the dual inhibitor SA-57 reduced all abrupt withdrawal signs (ie, platform jumping, paw flutters, head shakes, diarrhea, and total body weight loss), but did not elicit any cannabimimetic side effects. In addition, JZL184 or PF-3845 blocked naloxone-precipitated hypersecretion in morphine-dependent small intestinal tissue. Collectively, these results are the first to show that endocannabinoid catabolic enzyme inhibitors reduce abrupt withdrawal in morpine-dependent mice and are effective in a novel in vitro model of opioid withdrawal. More generally, these findings support the idea that joint MAGL and FAAH inhibition represents a promising approach for the treatment of opioid dependence.

Figure 1

The monoacylglycerol lipase (MAGL) inhibitor, JZL184 dose-dependently attenuated the occurrence of spontaneous withdrawal signs in morphine-dependent mice through the activation of CB₁ receptors. JZL184 dose-dependently increased whole-brain levels of 2-AG (a) and decreased arachidonic acid (AA; b), but did not relevantly affect anandamide (AEA) (c). JZL184 significantly decreased all measured withdrawal signs including: (d) jumps, (e) head shakes, (f) paw flutters, (g) diarrhea, and (h) weight loss. Rimonabant (3 mg/kg; Rim) significantly blocked each antiwithdrawal effect of JZL184 (40 mg/kg). Data expressed as mean±SEM for panels a–c, e, f, and h. *p<0.05, **p<0.01, ***p<0.001 vs vehicle control; ^#p<0.05, ^##p<0.01 vs JZL184(40)-treated group; n=7–9 mice/group.

Figure 2

The phytocannabinoid, tetrahydrocannabinol (THC) completely blocked all measured spontaneous withdrawal signs in morphine-dependent mice, while the FAAH inhibitor, PF-3845 attenuated only a subset of the withdrawal signs. The withdrawal signs measured include: (a) jumps, (b) head shakes, (c) paw flutters, (d) diarrhea, and (e) weight loss. Data are expressed as mean±SEM for panels b–d, and percentage scores for panels a and e. *p<0.05 vs morphine control; n=7–8 mice/group.

Figure 3

Combined administration of a threshold dose of JZL184 (4 mg/kg) and high dose of PF-3845 (10 mg/kg; JZL+PF) attenuated the occurrence of the spontaneous withdrawal signs in morphine-dependent mice to a greater extent than either inhibitor alone. The effects of vehicle or low-dose JZL184 (4 mg/kg) combined with vehicle or high-dose PF-3845 (10 mg/kg) on whole-brain levels of (a) 2-AG, (b) arachidonic acid (AA), and (c) AEA. The effects of high-dose JZL184 (40 mg/kg) are included for comparison. The withdrawal signs measured include: (d) jumps, (e) head shakes, (f) paw flutters, (g) diarrhea, and (h) weight loss. Data expressed as mean±SEM for panels a–c and e–f–h. *p<0.05, **p<0.01, ***p<0.001 vs morphine control; n=7–8 mice/group.

Figure 4

CB₁ receptors mediate the antiwithdrawal effects of combined administration of a threshold dose of JZL184 (4 mg/kg) and a high dose of PF-3845 (10 mg/kg; (JZL+PF)) in morphine-dependent mice. All subjects were implanted with morphine pellets for 72 h. One hour after pellet removal to elicit spontaneous withdrawal, subjects received the following two injections: (1) rimonabant (3 mg/kg; Rim) or vehicle, and (2) JZL+PF or vehicle. The withdrawal signs measured include: (a) Jumps, (b) head shakes, (c) paw flutters, (d) diarrhea, and (e) weight loss. Data are expressed as mean±SEM for panels b, c and e. *p<0.05, vs vehicle control, ^#p<0.05 vs JZL+PF condition; n=10–11 mice/group.

Figure 5

Combined administration of low dose JZL184 (4 mg/kg) and high-dose PF-3845 (10 mg/kg) attenuated the occurrence of the naloxone-precipitated withdrawal signs in morphine-dependent mice to a greater extent than either inhibitor alone. The withdrawal signs measured include: (a) jumps, (b) paw flutters, (c) diarrhea as well as (d) weight loss. Data expressed as mean±SEM for panels a, b and d. *p<0.05, **p<0.01 vs morphine control; n=6–8 mice/group.

Figure 6

The dual FAAH/MAGL inhibitor SA-57 reduced all measured signs of spontaneous morphine withdrawal. SA-57 dose-dependently increased whole-brain levels of 2-AG (a) and concomitantly increased arachidonic acid (AA; b), but was most potent in elevating brain AEA levels (c). The withdrawal signs measured include: (d) jumps, (e) head shakes, (f) paw flutters, (g) diarrhea, and (h) weight loss. Data are expressed as mean±SEM for panels a–c and e, f, and h, and percentage scores in panels d and h. *p<0.05, **p<0.01, ***p<0.001 vs morphine control; n=9 mice/group.

Figure 7

Effects of MAGL inhibition on naloxone-precipitated hypersecretion in CB₁ (+/+) and CB₁ (−/−) mice. (a) JZL184 blocked naloxone-precipitated hypersecretion in morphine-treated small intestine tissue from CB₁ (+/+) mice, did not alter naloxone-precipitated hypersecretion in tissue from CB₁ (−/−) mice (b). Finally, PF-3845 blocked naloxone-precipitated hypersecretion in morphine-treated small intestine tissue from C57/BL6 mice (c). Secretion was measured by change in I_sc and normalized to surface area. Data are expressed as mean±SEM. *p<0.05, **p<0.01, ***p<0.001 vs appropriate morphine control; ^#p<0.05 vs corresponding morphine-naloxone treated tissue; n=4–5 mice/group.