Blockade of endocannabinoid hydrolytic enzymes attenuates precipitated opioid withdrawal symptoms in mice

Abstract

Δ(9)-Tetrahydrocannbinol (THC), the primary active constituent of Cannabis sativa, has long been known to reduce opioid withdrawal symptoms. Although THC produces most of its pharmacological actions through the activation of CB(1) and CB(2) cannabinoid receptors, the role these receptors play in reducing the variety of opioid withdrawal symptoms remains unknown. The endogenous cannabinoids, N-arachidonoylethanolamine (anandamide; AEA) and 2-arachidonylglycerol (2-AG), activate both cannabinoid receptors but are rapidly metabolized by fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), respectively. The objective of this study was to test whether increasing AEA or 2-AG, via inhibition of their respective hydrolytic enzymes, reduces naloxone-precipitated morphine withdrawal symptoms in in vivo and in vitro models of opioid dependence. Morphine-dependent mice challenged with naloxone reliably displayed a profound withdrawal syndrome, consisting of jumping, paw tremors, diarrhea, and weight loss. THC and the MAGL inhibitor 4-nitrophenyl 4-(dibenzo[d][1,3]dioxol-5-yl(hydroxy)methyl)piperidine-1-carboxylate (JZL184) dose dependently reduced the intensity of most measures through the activation of CB(1) receptors. JZL184 also attenuated spontaneous withdrawal signs in morphine-dependent mice. The FAAH inhibitor N-(pyridin-3-yl)-4-(3-(5-(trifluoromethyl)pyridin-2-yloxy)benzyl)-piperdine-1-carboxamide (PF-3845) reduced the intensity of naloxone-precipitated jumps and paw flutters through the activation of CB(1) receptors but did not ameliorate incidence of diarrhea or weight loss. In the final series of experiments, we investigated whether JZL184 or PF-3845 would attenuate naloxone-precipitated contractions in morphine-dependent ilea. Both enzyme inhibitors attenuated the intensity of naloxone-induced contractions, although this model does not account mechanistically for the autonomic withdrawal responses (i.e., diarrhea) observed in vivo. These results indicate that endocannabinoid catabolic enzymes are promising targets to treat opioid dependence.

Fig. 1.

The primary psychoactive constituent of C. sativa THC and the irreversible MAGL inhibitor JZL184 attenuate the intensity of naloxone-precipitated withdrawal signs in a dose-dependent manner. THC (30-min pretreatment) or JZL184 (2-h pretreatment) was given before naloxone (1 mg/kg s.c.). Rimonabant (Rim; 3 mg/kg i.p.) given 30 min before naloxone antagonized the antiwithdrawal effects of THC (10 mg/kg i.p.). The withdrawal signs measured were number of jumps (A), number of paw flutters (B), weight loss (C), and occurrence of diarrhea (D). Data are expressed as means ± S.E.M. for A to C. ***, p < 0.001; **, p < 0.01; *, p < 0.05 versus vehicle; #, p < 0.05 versus 10 mg/kg THC; ##, p < 0.01 versus 10 mg/kg THC; †, p < 0.05 versus vehicle; ‡, p < 0.05 versus JZL184. n = 6 to 8 mice/group. Veh, vehicle.

Fig. 2.

The MAGL inhibitor JZL184 attenuated the intensity of naloxone-precipitated morphine withdrawal signs in a CB₁ receptor-dependent manner. Rimonabant (3 mg/kg i.p.) administered 90 min after JZL184 (40 mg/kg i.p.) blocked the antiwithdrawal effects of JZL184. The withdrawal signs measured were number of jumps (A), number of paw flutters (B), weight loss (C), and occurrence of diarrhea (D). Data are expressed as means ± S.E.M. for A to C. **, p < 0.01 versus vehicle; ##, p < 0.01 versus JZL184-vehicle; †, p < 0.05 versus vehicle; ‡, p < 0.05 versus JZL184-vehicle. n = 6 to 8 mice/group.

Fig. 3.

The reduction in intensity of naloxone-precipitated morphine withdrawal signs is not CB₂ receptor-mediated. The CB₂ antagonist SR144528 (3 mg/kg i.p.) did not reverse the antiwithdrawal effects of JZL184 (40 mg/kg i.p.). The withdrawal signs measured were number of jumps (A), number of paw flutters (B), weight loss (C), and occurrence of diarrhea (D). Data are expressed as means ± S.E.M. for A to C. ***, p < 0.001; **, p < 0.01 versus vehicle; †, p < 0.05 versus vehicle. n = 7 to 8 mice/group.

Fig. 4.

JZL184 reduces spontaneous withdrawal signs in morphine-dependent mice. Removal of morphine pellets leads to increased spontaneous withdrawal signs compared with those in mice implanted with placebo pellets. The MAGL inhibitor JZL184 attenuated the intensity of the following spontaneous withdrawal signs in morphine-dependent mice: percentage of mice that displayed platform jumping (A); number of paw flutters (B); number of head shakes (C); and weight loss (D). Data are expressed as means ± S.E.M. for B to D. *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus placebo; #, p < 0.05; ##, p < 0.01; ###, p < 0.001 versus morphine control; †, p < 0.05 versus placebo; ‡, p < 0.05 versus morphine control. n = 6 to 9 mice/group.

Fig. 5.

FAAH(−/−) mice display attenuated naloxone-precipitated withdrawal signs. The withdrawal signs measured were number of jumps (A), number of paw flutters (B), weight loss (C), and occurrence of diarrhea (D). Data are expressed as means ± S.E.M. for A to C. **, p < 0.01; *p < 0.05 versus FAAH(+/+) group. n = 6 to 7 mice/group.

Fig. 6.

The FAAH inhibitor PF-3845 attenuated a subset of naloxone-precipitated morphine withdrawal signs in a CB₁ receptor-dependent manner. PF-3845 (10 mg/kg i.p.) reduced the intensity of jumps and paw flutters, which was reversed by rimonabant (3 mg/kg i.p.). The withdrawal signs measured were number of jumps (A), number of paw flutters (B), weight loss (C), and occurrence of diarrhea (D). Data are expressed as mean ± S.E.M. for A to C. **, p < 0.01; *, p < 0.05 versus vehicle; ###, p < 0.001; #, p < 0.05 versus PF-3845. n = 11 to 12 mice/group.

Fig. 7.

Inhibitors of endocannabinoid catabolic enzymes reduced naloxone-precipitated contractions of morphine-treated ilea, as measured by isometric tension recording. A, the MAGL inhibitor JZL184 (1 μM) attenuated the amplitude of naloxone-precipitated contraction in ileum treated with morphine. Rimonabant (Rim; 100 nM) did not block the antiwithdrawal effects of JZL184 (JZL). B, representative traces of contractions in each of the conditions before and after application of naloxone hydrochloride. C, the FAAH inhibitor PF-3845 (1 μM) attenuated the amplitude of naloxone-precipitated contraction. Rimonabant (100 nM) reversed the actions of PF-3845 (PF). D, representative traces of contractions in each of the conditions before and after application of naloxone hydrochloride. Data are expressed as means ± S.E.M. ***, p < 0.001; *, p < 0.05 versus morphine; ###, p < 0.001 versus PF-3845. n = 4 to 8 ilea/condition.

Fig. 8.

Evaluation of JZL184 and PF-3845 on EFS contractions on longitudinal smooth muscle preparations from naive ileum. A, representative trace of EFS contractions before and after addition of JZL184 (1 μM). B, representative trace of EFS contractions before and after addition of PF-3845 (1 μM). C, graphical representation of the amplitude of contractions represented as percentage of baseline values. JZL184 reduced the amplitude of EFS-stimulated contractions though the activation of CB₁ receptors, but PF-3845 had no effect. Data are expressed as mean ± S.E.M. *, p < 0.05; #, p < 0.05 versus JZL184. n = 4 to 5 ilea/group. Rim, rimonabant.