Differential roles of the two major endocannabinoid hydrolyzing enzymes in cannabinoid receptor tolerance and somatic withdrawal

While there is currently active debate over possible therapeutic applications of marijuana and cannabis-based compounds, consistently their primary drawbacks have been the psychoactive properties, dependence, and abuse potential. Prolonged administration of ∆9-tetrahydrocannabinol (THC), the primary psychoactive constituent in marijuana, demonstrates both tolerance and physical withdrawal in both preclinical and clinical studies. Repeated THC administration also produces CB1 receptor adaptations in the form of reduced activation of receptors, along with a downregulation of membrane surface receptors, in many brain regions involved in THC-associated behaviors. The increased need for drug to maintain therapeutic effects, and a withdrawal syndrome following discontinuation of use, are common risk factors in drugs of abuse. Recently, compounds have been developed that prolong the availability of the major naturally occurring endogenous cannabinoids, anandamide (AEA) and 2-arachidonoylglycerol (2-AG), through inhibition of their catabolic breakdown by fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), respectively. The overall objectives of this research are to elucidate the physiologic roles of these two endogenous ligands and to determine if either can produce beneficial therapeutic effects without negative cannabis-like CNS effects. Therefore, we tested the impact of acute and prolonged blockade of FAAH and MAGL on a variety of cannabinoid-mediated behaviors and on precipitated cannabinoid withdrawal. Despite that acute blockade of FAAH and MAGL produce similar efficacy in reducing nociceptive responses, and both can reduce THC-induced somatic withdrawal, sustained blockade of these enzymes leads to remarkably different adaptations in CB1 receptor functioning. Namely, prolonged elevations in brain 2-AG leads to marked antinociceptive tolerance, cross-tolerance to exogenous cannabinoid agonists, and physical dependence. In contrast, sustained elevations in brain anandamide continues to dampen pain responses without apparent signs of physical withdrawal, loss of CB1 receptor activation as measured by [35S]GTPγS, or receptor downregulation as measured by [3H]CP,55940. These results suggest that chronic 2-AG elicits greater compensatory changes in CB1 receptor functions than anandamide. With similar efficacy in most therapeutic endpoints tested, and evidence of reduced impact on long-term function of the endocannabinoid system, these results distinguish FAAH as a more promising therapeutic target to treat pain and other conditions than MAGL

Inactivation of anandamide signaling : A continuing Debate

Copyright 2011 Elsevier B.V., All rights reserved.Peer reviewedPublisher PD

Robust anti-nociceptive effects of MAG lipase inhibition in a model of osteoarthritis pain

BACKGROUND AND PURPOSE: Chronic pain is often a symptom of knee osteoarthritis (OA) for which current analgesics are either inadequate, or are associated with serious side effects. The endocannabinoid system may offer alternative targets for pain-relief. We evaluated the effects of a potent and selective MAG lipase inhibitor (MJN110) on OA pain behaviour, spinal mechanisms of action and joint histopathology in the rat. Experimental approach: Intra-articular injection of monosodium iodoacetate (MIA) models OA pain and mimics clinical joint pathology. Effects of MJN110 on MIA-induced weight bearing asymmetry and lowered paw withdrawal thresholds (PWTs), changes in spinal gene expression and brain levels of relevant lipids were determined. Key results: Acute MJN110 (5 mg·kg−1) significantly reversed MIA induced weight bearing asymmetry (MIA /vehicle: 68 ± 6g; MIA /MJN110: 35 ± 4g, p<0.05) and lowered ipsilateral PWTs (MIA /vehicle: 7 ± 0.8g; MIA /MJN110: 11 ± 0.6g, p<0.05), via both CB1 and CB2 receptors. Repeated treatment with MJN110 (5 mg·kg−1) resulted in anti-nociceptive tolerance. A lower dose of MJN110 (1 mg·kg−1) acutely inhibited pain behaviour, which was maintained for one week of repeated administration, but had no effect on joint histology. MJN110 significantly inhibited expression of MPGES1 (p<0.05) in the ipsilateral dorsal horn of the spinal cord of MIA rats, compared to vehicle treated MIA rats. Both doses of MJN110 significantly elevated brain levels of the endocannabinoid 2-AG. Conclusions and Implications: Our data support the further investigation of the therapeutic potential of MAG lipase inhibitors for the treatment of OA pain

Discovery of Prostamide F2α and Its Role in Inflammatory Pain and Dorsal Horn Nociceptive Neuron Hyperexcitability

It was suggested that endocannabinoids are metabolized by cyclooxygenase (COX)-2 in the spinal cord of rats with kaolin/λ-carrageenan-induced knee inflammation, and that this mechanism contributes to the analgesic effects of COX-2 inhibitors in this experimental model. We report the development of a specific method for the identification of endocannabinoid COX-2 metabolites, its application to measure the levels of these compounds in tissues, and the finding of prostamide F2α (PMF2α) in mice with knee inflammation. Whereas the levels of spinal endocannabinoids were not significantly altered by kaolin/λ-carrageenan-induced knee inflammation, those of the COX-2 metabolite of AEA, PMF2α, were strongly elevated. The formation of PMF2α was reduced by indomethacin (a non-selective COX inhibitor), NS-398 (a selective COX-2 inhibitor) and SC-560 (a selective COX-1 inhibitor). In healthy mice, spinal application of PMF2α increased the firing of nociceptive (NS) neurons, and correspondingly reduced the threshold of paw withdrawal latency (PWL). These effects were attenuated by the PMF2α receptor antagonist AGN211336, but not by the FP receptor antagonist AL8810. Also prostaglandin F2α increased NS neuron firing and reduced the threshold of PWL in healthy mice, and these effects were antagonized by AL8810, and not by AGN211336. In mice with kaolin/λ-carrageenan-induced knee inflammation, AGN211336, but not AL8810, reduced the inflammation-induced NS neuron firing and reduction of PWL. These findings suggest that inflammation-induced, and prostanoid-mediated, enhancement of dorsal horn NS neuron firing stimulates the production of spinal PMF2α, which in turn contributes to further NS neuron firing and pain transmission by activating specific receptors

Identifying candidate drivers of alcohol dependence-induced excessive drinking by assembly and interrogation of brain-specific regulatory networks

Background: A systems biology approach based on the assembly and interrogation of gene regulatory networks, or interactomes, was used to study neuroadaptation processes associated with the transition to alcohol dependence at the molecular level. Results: Using a rat model of dependent and non-dependent alcohol self-administration, we reverse engineered a global transcriptional regulatory network during protracted abstinence, a period when relapse rates are highest. We then interrogated the network to identify master regulator genes that mechanistically regulate brain region-specific signatures associated with dependent and non-dependent alcohol self-administration. Among these, the gene coding for the glucocorticoid receptor was independently identified as a master regulator in multiple brain regions, including the medial prefrontal cortex, nucleus accumbens, central nucleus of the amygdala, and ventral tegmental area, consistent with the view that brain reward and stress systems are dysregulated during protracted abstinence. Administration of the glucocorticoid antagonist mifepristone in either the nucleus accumbens or ventral tegmental area selectively decreased dependent, excessive, alcohol self-administration in rats but had no effect on non-dependent, moderate, alcohol self-administration. Conclusions: Our study suggests that assembly and analysis of regulatory networks is an effective strategy for the identification of key regulators of long-term neuroplastic changes within specific brain regions that play a functional role in alcohol dependence. More specifically, our results support a key role for regulatory networks downstream of the glucocorticoid receptor in excessive alcohol drinking during protracted alcohol abstinence

Paradoxical effects of JZL184, an inhibitor of monoacylglycerol lipase, on bone remodelling in healthy and cancer-bearing mice

Background Cancer-associated bone disease is a serious complication in bone sarcomas and metastatic carcinomas of breast and prostate origin. Monoacylglycerol lipase (MAGL) is an enzyme of the endocannabinoid system, and is responsible for the degradation of the most abundant endocannabinoid in bone, 2-arachidonoyl glycerol (2AG). Methods The effects of the verified MAGL inhibitor on bone remodelling were assessed in healthy mice and in mouse models of bone disease caused by prostate and breast cancers and osteosarcoma. Findings JZL184 reduced osteolytic bone metastasis in mouse models of breast and prostate cancers, and inhibited skeletal tumour growth, metastasis and the formation of ectopic bone in models of osteosarcoma. Additionally, JZL184 suppressed cachexia and prolonged survival in mice injected with metastatic osteosarcoma and osteotropic cancer cells. Functional and histological analysis revealed that the osteoprotective action of JZL184 in cancer models is predominately due to inhibition of tumour growth and metastasis. In the absence of cancer, however, exposure to JZL184 exerts a paradoxical reduction of bone volume via an effect that is mediated by both Cnr1 and Cnr2 cannabinoid receptors. Interpretation MAGL inhibitors such as JZL184, or its novel analogues, may be of value in the treatment of bone disease caused by primary bone cancer and bone metastasis, however, activation of the skeletal endocannabinoid system may limit their usefulness as osteoprotective agents

Assay of Monoacylglycerol Lipase Activity

Monoacylglycerol lipase (MGL) is a serine hydrolase involved in the biological deactivation of the endocannabinoid 2-arachidonoyl-sn-glycerol (2-AG). 2-AG is one of the main endogenous lipid agonists for cannabinoid receptors in the brain and elsewhere in the body. In the central nervous system (CNS), MGL is localized to presynaptic nerve terminals of both excitatory and inhibitory synapses, where it helps control the regulatory actions of 2-AG on synaptic transmission and plasticity. In this chapter, we describe an in vitro method to assess MGL activity by liquid chromatography/mass spectrometry (LC/MS)-based quantitation of the reaction product. This method may be used to determine the basal or altered MGL activity in various cells or animal tissues after pharmacological, genetic, or biological manipulations. In addition, this assay can be used for MGL inhibitor screening using purified recombinant enzyme or MGL-overexpressing cells

Keep off the grass?:Cannabis, cognition and addiction

This is the author accepted manuscript. The final version is available from the publisher via the DOI in this record.In an increasing number of states and countries, cannabis now stands poised to join alcohol and tobacco as a legal drug. Quantifying the relative adverse and beneficial effects of cannabis and its constituent cannabinoids should therefore be prioritized. Whereas newspaper headlines have focused on links between cannabis and psychosis, less attention has been paid to the much more common problem of cannabis addiction. Certain cognitive changes have also been attributed to cannabis use, although their causality and longevity are fiercely debated. Identifying why some individuals are more vulnerable than others to the adverse effects of cannabis is now of paramount importance to public health. Here, we review the current state of knowledge about such vulnerability factors, the variations in types of cannabis, and the relationship between these and cognition and addiction.This work was supported by grants from the US National Institutes of Health to L.H.P. (AA020404, AA006420, AA022249 and AA017447) and by grants from the UK Medical Research Council to H.V.C. and C.J.A.M. (G0800268; MR/K015524/1)