Anu Mahadevan

Inhibition of monoacylglycerol lipase and fatty acid amide hydrolase by analogues of 2-arachidonoylglycerol.

The pharmacology of monoacylglycerol lipase (MAGL) is not well understood. In consequence, the abilities of a series of analogues of 2‐arachidonoylglycerol (2‐AG) to inhibit cytosolic 2‐oleoylglycerol and membrane‐bound anandamide hydolysis by MAGL and fatty acid amide hydrolase (FAAH), respectively, have been investigated. 2‐AG and its 1‐regioisomer (1‐AG) interacted with MAGL with similar affinities (IC50 values 13 and 17 μM, respectively). Shorter homologues of 2‐AG (2‐linoleoylglycerol and 2‐oleoylglycerol) had affinities for MAGL similar to 2‐AG. This pattern was also seen when the arachidonoyl side chain of arachidonoyl trifluoromethylketone was replaced by an oleoyl side chain. Arachidonoyl serinol (IC50 value 73 μM) was a weaker inhibitor of MAGL than 2‐AG. The IC50 values of noladin ether towards MAGL and FAAH were 36 and 3 μM, respectively. Arachidonoyl glycine interacted with FAAH (IC50 value 4.9 μM) but only weakly interacted with MAGL (IC50 value >100 μM). α‐Methyl‐1‐AG had similar potencies towards MAGL and FAAH (IC50 values of 11 and 33 μM, respectively). O‐2203 (1‐(20‐cyano‐16,16‐dimethyl‐eicosa‐5,8,11,14‐tetraenoyl) glycerol) and O‐2204 (2‐(20‐hydroxy‐16,16‐dimethyl‐eicosa‐5,8,11,14‐tetraenoyl) glycerol) were slightly less potent, but again affected both enzymes equally. α‐Methyl‐1‐AG, O‐2203 and O‐2204 interacted only weakly with cannabinoid CB1 receptors expressed in CHO cells (Ki values 1.8, 3.7 and 3.2 μM, respectively, compared with 0.24 μM for 1‐AG) and showed no evidence of central cannabinoid receptor activation in vivo at doses up to 30 mg kg−1 i.v. It is concluded that compounds like α‐Methyl‐1‐AG, O‐2203 and O‐2204 may be useful as leads for the discovery of selective MAGL inhibitors that lack direct effects upon cannabinoid receptors.

The monoacylglycerol lipase inhibitor JZL184 suppresses inflammatory pain in the mouse carrageenan model

AIMnThe present study tested whether the selective monoacylglycerol lipase (MAGL) inhibitor JZL184 would reduce allodynia and paw edema in the carrageenan test.nnnMAIN METHODSnThe anti-edematous and anti-allodynic effects of JZL184 were compared to those of PF-3845, an inhibitor of fatty acid amide hydrolase (FAAH), and diclofenac, a non-selective cyclooxygenase inhibitor. Cannabinoid receptor involvement in the anti-edematous and anti-allodynic effects of JZL184 was evaluated by administration of the respective CB1 and CB2 receptor antagonists rimonabant and SR144528 as well as with CB1(-/-) and CB2(-/-) mice. JZL184 (1.6, 4, 16, or 40mg/kg) was administered for six days to assess tolerance.nnnKEY FINDINGSnJZL184 administered before or after carrageenan significantly attenuated carrageenan-induced paw edema and mechanical allodynia. Complementary genetic and pharmacological approaches revealed that the anti-allodynic effects of JZL184 required both CB1 and CB2 receptors, but only CB2 receptors mediated its anti-edematous actions. Importantly, both the anti-edematous and anti-allodynic effects underwent tolerance following repeated injections of high dose JZL184 (16 or 40mg/kg), but repeated administration of low dose JZL184 (4mg/kg) retained efficacy.nnnSIGNIFICANCEnThese results suggest that the MAGL inhibitor JZL184 reduces inflammatory nociception through the activation of both CB1 and CB2 receptors, with no evidence of tolerance following repeated administration of low doses.

Inhibition of Monoacylglycerol Lipase Attenuates Nonsteroidal Anti-Inflammatory Drug-Induced Gastric Hemorrhages in Mice

Nonsteroidal anti-inflammatory drugs (NSAIDs) are commonly used analgesics, but can cause gastric and esophageal hemorrhages, erosion, and ulceration. The endogenous cannabinoid (endocannabinoid; eCB) system possesses several potential targets to reduce gastric inflammatory states, including cannabinoid receptor type 1 (CB1), cannabinoid receptor type 2 (CB2), and enzymes that regulate the eCB ligands 2-arachidonoylglycerol (2-AG) and N-arachidonoyl ethanolamine (anandamide; AEA). In the presented study, we tested whether 4-nitrophenyl 4-(dibenzo[d][1,3]dioxol-5-yl(hydroxy)methyl)piperidine-1-carboxylate (JZL184), a selective inhibitor of the primary catabolic enzyme of 2-AG, monoacylglycerol lipase (MAGL), would protect against NSAID-induced gastric damage. Food-deprived mice administered the nonselective cyclooxygenase inhibitor diclofenac sodium displayed gastric hemorrhages and increases in proinflammatory cytokines. JZL184, the proton pump inhibitor omeprazole (positive control), or the primary constituent of marijuana, Δ9-tetrahydrocannabinol (THC), significantly prevented diclofenac-induced gastric hemorrhages. JZL184 also increased stomach levels of 2-AG, but had no effect on AEA, arachidonic acid, or the prostaglandins E2 and D2. MAGL inhibition fully blocked diclofenac-induced increases in gastric levels of proinflammatory cytokines interleukin (IL)-1β, IL-6, tumor necrosis factor α, and granulocyte colony-stimulating factor, as well as IL-10. Pharmacological inhibition or genetic deletion of CB1 or CB2 revealed that the gastroprotective effects of JZL184 and THC were mediated via CB1. The antihemorrhagic effects of JZL184 persisted with repeated administration, indicating a lack of tolerance. These data indicate that increasing 2-AG protects against gastric damage induced by NSAIDs, and its primary catabolic enzyme MAGL offers a promising target for the development of analgesic therapeutics possessing gastroprotective properties.

New potent and selective inhibitors of anandamide reuptake with antispastic activity in a mouse model of multiple sclerosis

We previously reported that the compound O‐2093 is a selective inhibitor of the reuptake of the endocannabinoid anandamide (AEA). We have now re‐examined the activity of O‐2093 in vivo and synthesized four structural analogs (O‐2247, O‐2248, O‐3246, and O‐3262), whose activity was assessed in: (a) binding assays carried out with membranes from cells overexpressing the human CB1 and CB2 receptors; (b) assays of transient receptor potential of the vanilloid type‐1 (TRPV1) channel functional activity (measurement of [Ca2+]i); (c) [14C]AEA cellular uptake and hydrolysis assays in rat basophilic leukaemia (RBL‐2H3) cells; (d) the mouse ‘tetrad’ tests (analgesia on a hot plate, immobility on a ‘ring’, rectal hypothermia and hypolocomotion in an open field); and (e) the limb spasticity test in chronic relapsing experimental allergic encephalomyelitis (CREAE) mice, a model of multiple sclerosis (MS). O‐2093, either synthesized by us or commercially available, was inactive in the ‘tetrad’ up to a 20u2003mgu2003kg−1 dose (i.v.). Like O‐2093, the other four compounds exhibited low affinity in CB1 (Ki from 1.3 to >10u2003μM) and CB2 binding assays (1.310u2003μM), very low potency as fatty acid amide hydrolase (FAAH) inhibitors (IC50>25u2003μM) and were inactive in the ‘tetrad’ up to a 30u2003mgu2003kg−1 dose (i.v.). While O‐2247 and O‐2248 were poor inhibitors of [14C]AEA cellular uptake (IC50>40u2003μM), O‐3246 and O‐3262 were quite potent in this assay. O‐3246, which exhibits only a very subtle structural difference with O‐2093, is the most potent inhibitor of AEA uptake reported in vitro under our experimental conditions (IC50=1.4u2003μM) and is 12‐fold more potent than O‐2093. When injected intravenously O‐3246 and O‐3262, again like O‐2093 and unlike O‐2247 and O‐2248, significantly inhibited limb spasticity in mice with CREAE. These data confirm the potential utility of selective AEA uptake inhibitors as anti‐spasticity drugs in MS and, given the very subtle chemical differences between potent and weak inhibitors of uptake, support further the existence of a specific mechanism for this process.

SR-141716A-induced stimulation of locomotor activity: A structure–activity relationship study

The central cannabinoid receptor (CB(1)) antagonist, SR-141716A, has been used extensively to ascertain that cannabinoids interact with the CB(1) receptor. SR-141716A has been shown to produce effects opposite of cannabinoids when administered alone. It has been theorized that SR-141716A may act as an inverse agonist at the CB(1) receptor or by disinhibiting an endogenous cannabinoid tone. In an effort to ascertain the exact interaction between SR-141716A and the CB(1) receptor, we have conducted a structure-activity relationship study to compare CB(1) receptor affinity of SR-141716A analogs with their ability to produce an increase in locomotor activity. SR-141716A produced a significant increase in locomotor activity in mice within the first hour of administration. Twenty SR-141716A analogs from five different chemical series were also tested. Our data implicate particular regions of the SR-141716A molecule that may be involved in stimulation and depression of locomotor activity. When the K(I) of the analogs was plotted against the percent stimulation that each analog produced, it is evident that there is no correlation between the ability of the analogs to stimulate locomotor activity and their affinity for the CB(1) receptor. [35S]GTPgammaS binding data indicate that SR-141716A and five of the analogs are inverse agonists. However, none of the analogs demonstrating inverse agonism produce stimulation of locomotor activity. It is therefore concluded that the SR-141716A-induced stimulation in locomotor activity is not the result of inverse agonist activity at the CB(1) receptor or by disinhibition of an endogenous tone.

A novel fluorophosphonate inhibitor of the biosynthesis of the endocannabinoid 2-arachidonoylglycerol with potential anti-obesity effects

The development of potent and selective inhibitors of the biosynthesis of the endocannabinoid 2‐arachidonoylglycerol (2‐AG) via DAG lipases (DAGL) α and β is just starting to be considered as a novel and promising source of pharmaceuticals for the treatment of disorders that might benefit from a reduction in endocannabinoid tone, such as hyperphagia in obese subjects.

Evaluation of Prevalent Phytocannabinoids in the Acetic Acid Model of Visceral Nociception

Considerable preclinical research has demonstrated the efficacy of Delta(9)-tetrahydrocannabinol (Delta(9)-THC), the primary psychoactive constituent of Cannabis sativa, in a wide variety of animal models of pain, but few studies have examined other phytocannabinoids. Indeed, other plant-derived cannabinoids, including cannabidiol (CBD), cannabinol (CBN), and cannabichromene (CBC) elicit antinociceptive effects in some assays. In contrast, tetrahydrocannabivarin (THCV), another component of cannabis, antagonizes the pharmacological effects of Delta(9)-THC. These results suggest that various constituents of this plant may interact in a complex manner to modulate pain. The primary purpose of the present study was to assess the antinociceptive effects of these other prevalent phytocannabinoids in the acetic acid stretching test, a rodent visceral pain model. Of the cannabinoid compounds tested, Delta(9)-THC and CBN bound to the CB(1) receptor and produced antinociceptive effects. The CB(1) receptor antagonist, rimonabant, but not the CB(2) receptor antagonist, SR144528, blocked the antinociceptive effects of both compounds. Although THCV bound to the CB(1) receptor with similar affinity as Delta(9)-THC, it had no effects when administered alone, but antagonized the antinociceptive effects of Delta(9)-THC when both drugs were given in combination. Importantly, the antinociceptive effects of Delta(9)-THC and CBN occurred at lower doses than those necessary to produce locomotor suppression, suggesting motor dysfunction did not account for the decreases in acetic acid-induced abdominal stretching. These data raise the intriguing possibility that other constituents of cannabis can be used to modify the pharmacological effects of Delta(9)-THC by either eliciting antinociceptive effects (i.e., CBN) or antagonizing (i.e., THCV) the actions of Delta(9)-THC.

The Synthesis of N-Vanillyl-arachidonoyl-amide (Arvanil) and its Analogs: An Improved Procedure for the Synthesis of the Key Synthon Methyl 14-Hydroxy-(all-cis)-5,8,11-tetradecatrienoate

Abstract Several arvanil analogs were synthesized where the end n -pentyl chain was branched and carried substituents at the terminal end of the chain. A high yielding total synthesis of these analogs was developed from methyl hex-5-ynoate, which was converted to the synthon 6 in a facile five step sequence (overall yield, 33%). The pharmacological profile of these novel analogs suggests that they may be acting through a novel site of action for anandamide (arachidonylethanolamide, AEA).

Combined inhibition of monoacylglycerol lipase and cyclooxygenases synergistically reduces neuropathic pain in mice

Neuropathic pain is commonly treated with GABA analogues, steroids or non‐steroidal anti‐inflammatory drugs (NSAIDs). NSAIDs inhibit one or more COX isozymes but chronic COX inhibition paradoxically increases gastrointestinal inflammation and risk of unwanted cardiovascular events. The cannabinoids also have analgesic and anti‐inflammatory properties and reduce neuropathic pain in animal models. The present study investigated the analgesic effects of inhibiting both monoacylglycerol lipase (MAGL) and COX enzymes, using low doses of both inhibitors.

Novel 3‐substituted rimonabant analogues lack Δ9‐tetrahydrocannabinol‐like abuse‐related behavioural effects in mice

Previous structure–activity relationship studies with analogues of the CB1 receptor antagonist rimonabant have demonstrated that a subset of these analogues with 3‐substituent replacements of rimonabants pyrazole core displayed cannabimimetic profiles seemingly independent of CB1 receptors. We sought to further evaluate these analogues in several behavioural models sensitive to detecting THC‐like abuse liability.