Augmented tonic pain-related behavior in knockout mice lacking monoacylglycerol lipase, a major degrading enzyme for the endocannabinoid 2-arachidonoylglycerol

Highlights

• Selective inhibitors of MGL have antinociceptive effects upon acute administration.

• MGL knockout mice were tested in models of acute and chronic pain.

• KO mice had normal acute phasic pain and unaltered chronic pain.

• KO mice had augmented acute tonic pain caused by desensitization of CB₁ receptors.

• Long-term use of MGL inhibitors may lead to exacerbation of some types of pain.

Abstract

Monoacylglycerol lipase (MGL) is the main enzyme responsible for degradation of the endocannabinoid 2-arachidonoylglycerol (2-AG). Selective inhibitors of MGL have antinociceptive effects upon acute administration and, therefore, hold promise as analgesics. To gain insight into the possible consequences of their prolonged administration, genetically modified mice with the knocked-out MGL gene were tested in several models of acute (phasic, tonic) and chronic (inflammatory, neuropathic) pain. MGL knockout mice showed normal acute phasic pain perception (pain thresholds) and no alleviation of pain perception in models of inflammatory and neuropathic pain. However, compared with wild-type controls, they showed significantly augmented nociceptive behavior in models of acute somatic and visceral tonic pain (formalin and acetic acid tests). The observed proalgesic changes in perception of tonic pain in MGL knockouts could have resulted from desensitization of cannabinoid receptors (known to occur after genetic inactivation of MGL). Supporting this notion, chronic pretreatment with the selective CB₁ receptor antagonist AM 251 (employed to re-sensitize cannabinoid receptors in MGL knockouts) resulted in normalization of their tonic pain-related behaviors. Similar augmentation of tonic pain-related behaviors was replicated in C57BL/6N mice pretreated chronically with the selective MGL inhibitor JZL 184 (employed to pharmacologically desensitize CB₁ receptors). These findings imply that prolonged use of MGL inhibitors, at doses causing close to complete inhibition of MGL enzymatic activity, not only have no beneficial analgesic effects, they may lead to exacerbation of some types of pain (particularly those with a tonic component).

Introduction

The herb Cannabis sativa has been used in medicine since ancient times for its analgesic properties. The main psychoactive constituent of this herb, Δ⁹-tetrahydrocannabinol (Δ⁹-THC), acts on two G-protein-coupled cannabinoid (CB) receptors: CB₁ and CB₂. CB₁ receptors are found mainly in the central nervous system (CNS) and in moderate numbers in peripheral tissues. CB₂ receptors are found mainly in immune cells, but some reports suggest that they may also be present in the CNS [1], [2].

Endogenous ligands of cannabinoid receptors (“endocannabinoids”) are produced in living organisms and share some functional similarity with Δ⁹-THC and related exogenous cannabinoid agonists. Endocannabinoids such as 2-arachidonoylglycerol (2-AG) and anandamide are hydrophobic lipid molecules produced and released on demand that regulate synaptic transmission in the CNS [3], [4], [5]. They are also released constitutively in peripheral tissues where they act upon CB₁ receptors on nerve endings and serve to buffer arising pain signals at sites of tissue injury [6].

The effects of endocannabinoids are regulated tightly by the enzymes responsible for their biosynthesis and degradation. The main metabolizing enzyme for anandamide is fatty acid amide hydrolase (FAAH), whereas the main enzyme responsible for 2-AG hydrolysis is monoacylglycerol lipase (MGL). Pharmacological or genetic inactivation of FAAH has been shown to increase brain levels of anandamide and to produce CB₁ receptor-mediated analgesia in several pain assays [7]. FAAH inhibitors are being developed for the treatment of pain and CNS disorders [8].

MGL inhibitors are also regarded as promising analgesic drugs, but their pharmaceutical development has started only recently [9]. Similar to acute inhibition of FAAH, acute blockade of MGL is also accompanied by antinociception [10], [11], [12], [13]. Conversely, chronic pharmacological inactivation or genetic deletion of MGL leads to adaptation to increased levels of 2-AG by drastic alterations in the functioning of the endocannabinoid system as evidenced by the downregulation and desensitization of CB₁ receptors in the CNS (data on peripheral cannabinoid receptors is lacking) and development of cross-tolerance to CB₁ receptor agonists [14], [15]. Such a situation precludes simple characterization of the physiological roles of 2-AG using MGL knockout mice, and undermines efforts to increase our knowledge of the parts played by this endocannabinoid in the mechanisms of pain and other CNS disorders. However, despite the limitations of transgenic studies, the 100% selectivity obtained in the knockout approach remains its greatest advantage over conventional pharmacological tools [16]. Chronic pharmacologic blockade of MGL and genetic inactivation of its encoding gene cause similar alterations in endocannabinoid signaling, and no other secondary changes that could confound interpretation of the results in these animals have been reported. Hence, MGL knockout animals can serve as a behavioral model to help predict the effects of prolonged use of MGL inhibitors. The likelihood of such a situation arising in the near future is high given the reported efficacy of MGL inhibitors against pain and several other conditions (including cancer) [7], [9], [17]. Recently reported analgesic inefficiency at the clinical level of the potent and selective FAAH inhibitor PF-04457845 [18] has also contributed to increasing interest in the MGL system.

Here, to characterize the behavioral consequences that prolonged MGL inactivation may have for pain perception, several pain models representative of acute phasic, tonic, chronic inflammatory and neuropathic pain were applied to mice with knockout of the MGL gene. To ascertain if the phenotype observed in MGL knockout animals resulted from desensitization of CB₁ receptors, MGL knockout mice were pretreated chronically with the selective CB₁ receptor antagonist AM 251, which was employed to reverse desensitization of cannabinoid receptors by “protecting” them from elevated levels of 2-AG. Also, to provide additional proof for desensitization of CB₁ receptors being the primary cause underlying altered perception of pain after knockout of the MGL gene, the possibility of replication of a phenotype similar to that of the knockout was tested by chronic pretreatment of wild-type mice with the selective MGL inhibitor JZL 184. The potential implications of our findings for future research and clinical practice were then discussed.