Ganesh A. Thakur

The Novel Cannabinoid CB1 Receptor Neutral Antagonist AM4113 Suppresses Food Intake and Food-Reinforced Behavior but Does not Induce Signs of Nausea in Rats

Drugs that interfere with cannabinoid CB1 transmission suppress various food-motivated behaviors, and it has been suggested that such drugs could be useful as appetite suppressants. Biochemical studies indicate that most of these drugs assessed thus far have been CB1 inverse agonists, and although they have been shown to suppress food intake, they also appear to induce nausea and malaise. The present studies were undertaken to characterize the behavioral effects of AM4113, which is a CB1 neutral antagonist, and to examine whether this drug can reduce food-reinforced behaviors and feeding on diets with varying macronutrient compositions. Biochemical data demonstrated that AM4113 binds to CB1 receptors, but does not show inverse agonist properties (ie no effects on cyclic-AMP production). In tests of spontaneous locomotion and analgesia, AM4113 reversed the effects of the CB1 agonist AM411. AM4113 suppressed food-reinforced operant responding with rats responding on fixed ratio (FR) 1 and 5 schedules of reinforcement in a dose-dependent manner, and also suppressed feeding on high-fat, high-carbohydrate, and lab chow diets. However, in the same dose range that suppressed feeding, AM4113 did not induce conditioned gaping, which is a sign of nausea and food-related malaise in rats. These results suggest that AM4113 may decrease appetite by blocking endogenous cannabinoid tone, and that this drug may be less associated with nausea than CB1 inverse agonists.

Latest advances in cannabinoid receptor agonists.

Background: Since the discovery of cannabinoid receptors and their endogenous ligands in early 1990s, the endocannabinoid system has been shown to play a vital role in several pathophysiological processes. It has been targeted for the treatment of several diseases including neurodegenerative diseases (Parkinsons disease, Alzheimers disease, Huntingtons disease and MS), cancer, obesity, inflammatory bowel disease, neuropathic and inflammatory pain. The last decade has witnessed remarkable advances in the development of cannabinergic ligands displaying high selectivity and potency towards two subtypes of cannabinoid receptors, namely CB1 and CB2. Objective: In this review, we highlight the latest advances made in the development of cannabinoid agonists and summarize recently disclosed, novel chemical scaffolds as CB-selective agonists in patents that appeared during January 2008 – June 2009. Methods: Data presented here are obtained through the search of PubMed for research articles and reviews, and the website of European patents (http://ep.espacenet.com), SciFinder Scholar™ and US patents (www.uspto.gov). Conclusions: Our analysis reveals prolific patenting activity mainly in the CB2 selective agonist area. Limiting the BBB penetrability, thereby, leading to peripherally restricted CB1/CB2 agonists and enhancing CB2-selectivity emerge as likely prerequisites for avoidance of adverse central CB1 mediated side effects.

Selective Activation of Cannabinoid CB2 Receptors Suppresses Neuropathic Nociception Induced by Treatment with the Chemotherapeutic Agent Paclitaxel in Rats

Activation of cannabinoid CB2 receptors suppresses neuropathic pain induced by traumatic nerve injury. The present studies were conducted to evaluate the efficacy of cannabinoid CB2 receptor activation in suppressing painful peripheral neuropathy evoked by chemotherapeutic treatment with the antitumor agent paclitaxel. Rats received paclitaxel (2 mg/kg i.p./day) on 4 alternate days to induce mechanical hypersensitivity (mechanical allodynia). Mechanical allodynia was defined as a lowering of the threshold for paw withdrawal to stimulation of the plantar hind paw surface with an electronic von Frey stimulator. Mechanical allodynia developed in paclitaxel-treated animals relative to groups receiving the Cremophor EL/ethanol/saline vehicle at the same times. Two structurally distinct cannabinoid CB2 agonists, the aminoalkylindole (R,S)-AM1241 [(R,S)-(2-iodo-5-nitrophenyl)-[1-((1-methyl-piperidin-2-yl)methyl)-1H-indol-3-yl]-methanone] and the cannabilactone AM1714 (1,9-dihydroxy-3-(1′,1′-dimethylheptyl)-6H-benzo[c]chromene-6-one), produced a dose-related suppression of established paclitaxel-evoked mechanical allodynia after systemic administration. Pretreatment with the CB2 antagonist SR144528 [5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-N-(1,3,3-trimethylbicyclo[2.2.1]heptan-2-yl)-1H-pyrazole-3-carboxamide], but not the CB1 antagonist SR141716 [5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-N-(piperidin-1-yl)-1H-pyrazole-3-carboxamide], blocked the antiallodynic effects of both (R,S)-AM1241 and AM1714. Moreover, (R)-AM1241, but not (S)-AM1241, suppressed paclitaxel-evoked mechanical allodynia relative to either vehicle treatment or preinjection thresholds, consistent with mediation by CB2. Administration of either the CB1 or CB2 antagonist alone failed to alter paclitaxel-evoked mechanical allodynia. Moreover, (R,S)-AM1241 did not alter paw withdrawal thresholds in rats that received the Cremophor EL vehicle in lieu of paclitaxel, whereas AM1714 induced a modest antinociceptive effect. Our data suggest that cannabinoid CB2 receptors may be important therapeutic targets for the treatment of chemotherapy-evoked neuropathy.

CB1 cannabinoid receptor ligands.

The CB1 receptor is expressed in the central nervous system and numerous other tissues including heart, lung and uterus and has been recognized as an important therapeutic target for pain, appetite modulation, glaucoma, multiple sclerosis and other indications. An interesting feature of this GPCR is its ability to be activated by a number of structurally different classes of compounds, thus, raising the possibility of multiple activated forms of the receptor. Understanding of the structure-activity relationships of cannabinergic ligands has paved the road for the development of novel ligands exhibiting receptor subtype selectivity and efficacy. This review highlights the important CB1 cannabinergic ligands developed to date.

Mutation studies of Ser7.39 and Ser2.60 in the human CB1 cannabinoid receptor: evidence for a serine-induced bend in CB1 transmembrane helix 7.

Ligands of structurally diverse natures are able to bind at the CB1 cannabinoid receptor, suggesting the existence of multiple binding sites on the receptor. Modeling studies have implicated Ser2.60(173) and Ser7.39(383) as possible interaction site(s) for CB1 agonists. To test the importance of these residues for receptor recognition, recombinant human CB1 receptors, stably expressed in human embryonic kidney 293 cells, were used to investigate the consequences of mutating Ser2.60 (to S2.60A) or Ser7.39 (to S7.39A) in radioligand binding and guanosine 5′-3-O-(thio)triphosphate functional assays. The S7.39A mutant resulted in a total ablation of [3H](–)-3-[2-hydroxyl-4-(1,1-dimethylheptyl)phenyl]-4-[3-hydroxylpropyl] cyclohexan-1-ol (CP55,940) high-affinity binding. However, [3H](R)-(+)-[2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]-pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl](1-naphthalenyl)methanone (WIN55,212-2) binding properties at S7.39A were comparable with those of the wild-type (WT) receptor. The binding affinity of (–)-11β-hydroxy-3-(1′,1′-dimethylheptyl)hexahydrocannabinol (AM4056) and (–)-11-hydroxydimethylheptyl-Δ8-tetrahydrocannabinol (HU210) were drastically reduced (50- to 100-fold) at the S7.39A mutant. Likewise, the EC50 for HU210 and AM4056-mediated activation of the S7.39A receptor was increased by >200-fold. In contrast, the binding affinity and potency of WIN55,212-2, CP55,940, HU210, and AM4056 were unaltered at the S2.60A mutant compared with WT human CB1 receptors. These results clearly suggest that Ser7.39, but not Ser2.60, plays a crucial role in mediating ligand specific interactions for CP55,940, HU210, and AM4056 at the human CB1 receptor. Our modeling studies predict that Ser7.39 in a g–χ1 conformation may induce a helix bend in TMH7 that provides docking space for CP55,940 binding; the S7.39A mutation may alter this binding space, precluding CP55,940 binding.

Intrathecal cannabilactone CB2R agonist, AM1710, controls pathological pain and restores basal cytokine levels

Summary Spinal AM1710 suppresses allodynia with corresponding anti‐inflammatory and anti‐MAGL (monoacylglycerol lipase) effects in the spinal cord and dorsal root ganglia. ABSTRACT Spinal glial and proinflammatory cytokine actions are strongly implicated in pathological pain. Spinal administration of the anti‐inflammatory cytokine interleukin (IL)‐10 abolishes pathological pain and suppresses proinflammatory IL‐1β and tumor necrosis factor alpha (TNF‐α). Drugs that bind the cannabinoid type‐2 receptor (CB2R) expressed on spinal glia reduce mechanical hypersensitivity. To better understand the CB2R‐related anti‐inflammatory profile of key anatomical nociceptive regions, we assessed mechanical hypersensitivity and protein profiles following intrathecal application of the cannabilactone CB2R agonist, AM1710, in 2 animal models; unilateral sciatic nerve chronic constriction injury (CCI), and spinal application of human immunodeficiency virus‐1 glycoprotein 120 (gp120), a model of peri‐spinal immune activation. In CCI animals, lumbar dorsal spinal cord and corresponding dorsal root ganglia (DRG) were evaluated by immunohistochemistry for expression of IL‐10, IL‐1β, phosphorylated p38‐mitogen‐activated‐kinase (p‐p38MAPK), a pathway associated with proinflammatory cytokine production, glial cell markers, and degradative endocannabinoid enzymes, including monoacylglycerol lipase (MAGL). AM1710 reversed bilateral mechanical hypersensitivity. CCI revealed decreased IL‐10 expression in dorsal spinal cord and DRG, while AM1710 resulted in increased IL‐10, comparable to controls. Adjacent DRG and spinal sections revealed increased IL‐1β, p‐p38MAPK, glial markers, and/or MAGL expression, while AM1710 suppressed all but spinal p‐p38MAPK and microglial activation. In spinal gp120 animals, AM1710 prevented bilateral mechanical hypersensitivity. For comparison to immunohistochemistry, IL‐1β and TNF‐α protein quantification from lumbar spinal and DRG homogenates was determined, and revealed increased DRG IL‐1β protein levels from gp120, that was robustly prevented by AM1710 pretreatment. Cannabilactone CB2R agonists are emerging as anti‐inflammatory agents with pain therapeutic implications.

Cannabinoid Receptors as Therapeutic Targets

The cannabinoid receptors CB1 and CB2 are family A, G-protein Coupled Receptors that mediate the effects of cannabinoids, a class of compounds that are so named because the first members were isolates of the cannabis plant. In recent history, there has been much anecdotal evidence that the potent and diverse physiological responses produced by these compounds can be turned to therapeutic benefit for a wide variety of maladies. The remarkable abundance of cannabinoid receptors and the discovery of several endogenous ligands along with enzyme and transporter proteins for which they are substrates, suggests that an endogenous cannabinoid neuromodulatory system is an important mediator of biological function. For these reasons CB1 and CB2 receptors are attractive targets for the design of therapeutic ligands. The action of these receptors, however, may also be modulated by manipulating the enzymes and membrane transporters that regulate the endogenous ligands. Despite the range of physiological processes and activities that are mediated by cannabinoid receptors, it is clear that it is possible to produce ligands that result in differential responses. In this paper, we review the pharmacophoric elements that lead to these differential responses and in order to discuss them in context we present an overview of structural aspects governing cannabinoid receptor function, the cannabinergic system and its physiological functions.

Structural requirements for cannabinoid receptor probes.

The discovery and cloning of CB1 and CB2, the two known G(i/o) protein-coupled cannabinoid receptors, as well as the isolation and characterization of two families of endogenous cannabinergic ligands represented by arachidonoylethanolamide (anandamide) and 2-arachidonoylglycerol (2-AG), have opened new horizons in this newly discovered field of biology. Furthermore, a considerable number of cannabinoid analogs belonging to structurally diverse classes of compounds have been synthesized and tested, thus providing substantial information on the structural requirements for cannabinoid receptor recognition and activation. Experiments with site-directed mutated receptors and computer modeling studies have suggested that these diverse classes of ligands may interact with the receptors through different binding motifs. The information about the exact binding site may be obtained with the help of suitably designed molecular probes. These ligands either interact with the receptors in a reversible fashion (reversible probes) or alternatively attach at or near the receptor active site with the formation of covalent bonds (irreversible probes). This review focuses on structural requirements of cannabinoid receptor ligands and highlights their pharmacological and therapeutic potential.

In-vivo pharmacological evaluation of the CB1-receptor allosteric modulator Org-27569

Several allosteric modulators (AMs) of the CB1 receptor have been characterized in vitro, including Org27569, which enhances CB1-specific binding of [3H]CP55,940, but behaves as an insurmountable CB1-receptor antagonist in several biochemical assays. Although a growing body of research has investigated the molecular actions of this unusual AM, it is unknown whether these actions translate to the whole animal. The purpose of the present study was to determine whether Org27569 would produce effects in well-established mouse behavioral assays sensitive to CB1 orthosteric agonists and antagonists. Similar to the orthosteric CB1 antagonist/inverse agonist rimonabant, Org27569 reduced food intake; however, this anorectic effect occurred independently of the CB1 receptor. Org27569 did not elicit CB1-mediated effects alone and lacked efficacy in altering antinociceptive, cataleptic, and hypothermic actions of the orthosteric agonists anandamide, CP55,940, and &Dgr;9-tetrahydrocannabinol. Moreover, it did not alter the discriminative stimulus effects of anandamide in FAAH-deficient mice or &Dgr;9-tetrahydrocannabinol in wild-type mice in the drug discrimination paradigm. These findings question the utility of Org27569 as a ‘gold standard’ CB1 AM and underscore the need for the development of CB1 AMs with pharmacology that translates from the molecular level to the whole animal.

Behavioral effects of the novel cannabinoid full agonist AM 411

AM 411 ((-)-1-adamantyl-Delta8-tetrahydrocannabinol) is a novel full agonist at cannabinoid CB1 receptors. The present studies were conducted to provide behavioral characterization of this compound in rats. It was hypothesized that AM 411 should produce behavioral effects similar to known cannabinoid agonists, and that these effects should be inhibited by co-treatment with a CB1 antagonist. In Experiments 1 and 2, AM 411 dose-dependently produced behaviors consistent with CB1 agonism, including analgesia, hypothermia, catalepsy and reductions in locomotion, which were blocked by a CB1-selective antagonist. In Experiment 3, AM 411 produced a dose-dependent suppression of lever-pressing on a fixed-ratio 5 (FR5) schedule, a task known to be sensitive to administration of CB1 agonists. Detailed analysis of the temporal patterns of operant responding showed that AM 411 altered the distribution of interresponse times. Experiment 4 showed that AM 411 decreased relative interior activity in the open field, which is suggestive of an anxiogenic effect. It is concluded that AM 411 produces CB1 agonist-like behavior with potency between that of WIN 55,212-2 and AM 356. AM 411 could be a useful tool for understanding the behavioral and neural effects of CB1 receptor stimulation.