Category Archives: CB65 Cannabinoid Receptor

Suppression of outward K+ currents by win55212-2 in rat retinal ganglion cells is independent of CB1/CB2 receptors.

Cannabinoid CB1 receptor (CB1R) signaling system is extensively distributed in the vertebrate retina. Activation of CB1Rs regulates a variety of functions of retinal neurons through modulating different ion channels. In the present work we studied effects of this receptor signaling on K+ channels in retinal ganglion cells by patch-clamp techniques. The CB1R agonist WIN 55212-2 (WIN) suppressed outward K+ currents in acutely isolated rat retinal ganglion cells in a dose-dependent manner, with an IC50 of 4.7 μM. We further showed that WIN mainly suppressed the tetraethylammonium (TEA)-sensitive K+ current component. Whilst CB1Rs were expressed in rat retinal ganglion cells, the WIN effect on K+ currents was not blocked by either AM251/SR141716, specific CB1R antagonists, or AM630, a selective CB2R antagonist. Consistently, cAMP-protein kinase A (PKA) and mitogen-activated protein kinase (MAPK)/ extracellular signal-regulated kinase (ERK) signaling pathways were unlikely involved in the WIN-induced suppression of the K+ currents because both PKA inhibitors H-89/Rp-cAMP and MAPK/ERK1/2 inhibitor U0126 failed to block the WIN effects. WIN-induced suppression of the K+ currents was not observed when WIN was intracellularly applied. Furthermore, an endogenous ligand of cannabinoid receptor anandamide (AEA), the specific CB1R agonist ACEA and the selective CB2R agonist CB65 also suppressed the K+ currents, and the effects were not blocked by AM251/SR141716 or AM630 respectively. All these results suggest that the WIN-induced suppression of the outward K+ currents in rat retinal ganglion cells, thereby regulating the cell excitability, were not through CB1R/CB2R signaling pathways.

Anti-Cancer Effects In Human Liver Cancer Cells Produced By Cannabis Agonists

According to preclinical data published online in the journal Toxicology Mechanisms and Methods, the administration of synthetic cannabinoid agonists minimizes cell viability in human hepacarcinoma cells and could be a potential option for treating liver cancer.

The anti-cancer properties of two synthetic cannabinoids, CB65 (CB2 receptor agonist) and ACEA (CB1 receptor agonist) in human hepacarcinoma cells were assessed by investigators from the Tehran University of Medical Sciences, Department of Toxicology and Pharmacology.

It was reported by the authors that the cannabinoid administration minimized malignant cell viability and cell invasion in a dose-dependent manner. “These data suggest ACEA and CB65 as an option for novel treatment of hepatocellular cancer,” they concluded.

Studies in the past have demonstrated that tumor cell growth is inhibited by cannabinoids that also inhibit selectively induced apoptosis by different cell signaling pathways in various types of malignant cells, including gliomas (brain cancers) and lymphomas, prostate, breast, lung, skin, and pancreatic cancer cells.

The study “Evaluation of Anti-invasion Effect of Cannabinoids on Human Hepatocarcinoma Cells,” is available in Toxicology Mechanisms and Methods.

The role of central CB2 cannabinoid receptors on food intake in neonatal chicks

Abstract
The endocannabinoids (ECBs) have diverse physiological functions including the regulation of food intake and metabolism. In mammals, ECBs regulate feeding primarily through the CB1 receptors within the brain whereas the CB2 receptors are primarily involved in the regulation of immune function by direct action on peripheral immune cells and central glia. The central effect of ECBs on feeding behavior has not been studied in non-mammalian species. Therefore, the present study investigated the effect of CB65, a selective CB2 receptors agonist, on food intake in the neonatal chicks. In addition, the effect of astressin, a CRF receptor antagonist, on CB65-induced food intake was also investigated. Intracerebroventricular injection of the CB65 (1.25 μg) increased the food intake at 30- and 60-min post-injection significantly as compared to the control group. Pretreatment with a selective CB2 receptor antagonist, AM630, but not astressin, significantly attenuated the CB65-induced food intake. These results suggested that CB2 receptor agonists act on the brain to induce food intake.

The role of central CB2 cannabinoid receptors on food intake in neonatal chicks.

ABSTRACT The endocannabinoids (ECBs) have diverse physiological functions including the regulation of food intake and metabolism. In mammals, ECBs regulate feeding primarily through the CB1 receptors within the brain whereas the CB2 receptors are primarily involved in the regulation of immune function by direct action on peripheral immune cells and central glia. The central effect of ECBs on feeding behavior has not been studied in non-mammalian species. Therefore, the present study investigated the effect of CB65, a selective CB2 receptors agonist, on food intake in the neonatal chicks. In addition, the effect of astressin, a CRF receptor antagonist, on CB65-induced food intake was also investigated. Intracerebroventricular injection of the CB65 (1.25 μg) increased the food intake at 30- and 60-min post-injection significantly as compared to the control group. Pretreatment with a selective CB2 receptor antagonist, AM630, but not astressin, significantly attenuated the CB65-induced food intake. These results suggested that CB2 receptor agonists act on the brain to induce food intake.

Study: Synthetic Cannabinoids Produce Anti-Cancer Effects In Human Liver Cancer Cells

As the debate continues in New Zealand about regulating the availability of products containing synthetic cannabinoids, a new study has shown two particular compounds exhibit anti-cancer effects in human liver cancer cells.

This report is from US NORML’s Paul Armentano.

Thursday, 11 October 2012

Study: Cannabis Agonists Produce Anti-Cancer Effects In Human Liver Cancer CellsTehran, Iran: The administration of synthetic cannabinoid agonists reduce cell viability in human hepacarcinoma cells and may be a potential option for the treatment of liver cancer, according to preclinical data published online in the journal Toxicology Mechanisms and Methods.

Investigators from the Tehran University of Medical Sciences, Department of Toxicology and Pharmacology assessed the anti-cancer properties of two synthetic cannabinoids, CB65 (CB2 receptor agonist) and ACEA (CB1 receptor agonist) in human hepacarcinoma cells.

Authors reported that the administration of cannabinoids reduced malignant cell viability and cell invasion in a dose-dependent manner. “These data suggest ACEA and CB65 as an option for novel treatment of hepatocellular cancer,” they concluded.

Previous studies have demonstrated that cannabinoids inhibit tumor cell growth and selectively induced apoptosis by different cell signaling pathways in various types of malignant cells, including gliomas (brain cancers) and lymphomas, prostate, breast, lung, skin, and pancreatic cancer cells.

For more information, please contact Paul Armentano, NORML Deputy Director, at: paul@norml.org. Full text of the study, “Evaluation of Anti-invasion Effect of Cannabinoids on Human Hepatocarcinoma Cells,” is available in Toxicology Mechanisms and Methods.

Neuroimmmune interactions of cannabinoids in neurogenesis: focus on interleukin

Neuroimmune networks and the brain endocannabinoid system contribute to the maintenance of neurogenesis. Activation of cannabinoid receptors suppresses chronic inflammatory responses through the attenuation of pro-inflammatory mediators. Moreover, the endocannabinoid system directs cell fate specification of NSCs (neural stem cells) in the CNS (central nervous sytem). The aim of our work is to understand better the relationship between the endocannabinoid and the IL-1β (interleukin-1β) associated signalling pathways and NSC biology, in order to develop therapeutical strategies on CNS diseases that may facilitate brain repair. NSCs express functional CB1 and CB2 cannabinoid receptors, DAGLα (diacylglycerol lipase α) and the NSC markers SOX-2 and nestin. We have investigated the role of CB1 and CB2 cannabinoid receptors in the control of NSC proliferation and in the release of immunomodulators [IL-1β and IL-1Ra (IL-1 receptor antagonist)] that control NSC fate decisions. Pharmacological blockade of CB1 and/or CB2 cannabinoid receptors abolish or decrease NSC proliferation, indicating a critical role for both CB1 and CB2 receptors in the proliferation of NSC via IL-1 signalling pathways. Thus the endocannabinoid system, which has neuroprotective and immunomodulatory actions mediated by IL-1 signalling cascades in the brain, could assist the process of proliferation and differentiation of embryonic or adult NSCs, and this may be of therapeutic interest in the emerging field of brain repair.

CB1 Cannabinoid Receptors Couple to Focal Adhesion Kinase to Control Insulin Release

Background: Endocannabinoids can affect pancreatic β cell physiology.

Results: Anandamide and 2-arachidonoylglycerol binding to CB1receptors induces focal adhesion kinase phosphorylation, which is a prerequisite of insulin release.

Conclusion: Focal adhesion kinase activation downstream from CB1receptors couples cytoskeletal reorganization to insulin release.

Significance: Identifies the molecular blueprint of 2-arachidonoylglycerol signaling in the endocrine pancreas, and outlines a kinase activation cascade linking endocannabinoid signals to insulin release.

Drugs that Block Cannabinoid Receptors Seem Problematic

One of marijuana’s best known side effects is hunger, and the discovery of the brain-cell receptor that mediates this effect has led to the development of nearly a dozen drugs meant to block it and thereby treat obesity. However, it is now clear that CB1, the cannabinoid receptor targeted by these therapies, is responsible for much more than “the munchies.”

On Oct. 2, the drug company Merck announced that its CB1-blocking drug taranabant had caused unacceptable side effects in a Phase III trial in obese patients. Merck has stopped development of the drug. The chief side effects of taranabant are anxiety and depression. Merck’s action follows the FDA’s rejection in 2007, essentially for the same reasons, of Sanofi’s CB1-blocking drug rimonabant (brand name Acomplia).

“I was hoping that taranabant would do better than rimonabant, but I am not really surprised that it didn’t,” says Daniele Piomelli, a researcher at the University of California, Irvine, who has been in the forefront of research in this area. “It seems that the endogenous cannabinoid system is an important regulator of the stress-coping mechanism engaged in anxiety and depression.”

The CB1 receptor
Aside from being marijuana’s prime target, CB1 is one of the most widely expressed receptors on cells throughout the body, and it is particularly abundant in the brain. The receptor appears to have somewhat different functions in each organ system. In the brain, the binding of CB1 by its corresponding neurotransmitters, or “ligands,” also has multiple effects.

On the whole, however, CB1’s job seems to be to modulate the activity of other systems in the brain. Typically CB1 receptors stud the terminals of a neuronal output stalk, or axon, which delivers signals to another neuron across a synapse. Greater activity in the synapse tends to produce a buildup of the brain’s own cannabinoids, which bind to CB1, which in turn triggers a reduction in the flow of signals across the synapse.

Some evidence suggests, for example, that this braking or calming effect of the CB1 system is necessary to prevent the uncontrolled neuronal activity known as epilepsy. Lab tests with neuronal cultures made prone to seizure-like activity have shown that the blockade of CB1 leads to continuous firing, which stops when the blockade is lifted.

As evidence from marijuana users suggests, CB1 activation also tends to counteract the brain systems that produce anxiety, depression and pain. In one experiment in 2002, mice bred without the gene for CB1 and conditioned to associate a certain sound with pain were significantly less able to forget the fearful association than were normal mice. “Mutant mice lacking CB1 receptors are known to break down more easily when stressed, while drugs that boost endogenous cannabinoid activity strengthen the ability of animals to cope with stress,” Piomelli says.

The recent clinical trials of rimonabant and taranabant, both of which bind to CB1 in a way that reduces its activity, amount to large-scale tests of CB1’s function in humans. Both sets of trials indicate that blocking CB1 activity in humans produces harmful effects like those seen in animal experiments.

When an FDA panel unanimously rejected Sanofi’s application for rimonabant in 2007, it cited sharply increased rates of anxiety, depression, suicidal thoughts and mood disorders in patients taking the drug.

Although rimonabant was approved by the European Medicines Agency in 2006, a meta-analysis of clinical trial data, published in the Lancet in 2007, determined that people taking the drug, as opposed to a placebo, were two to three times more likely to stop taking it because of depression or anxiety. This was despite the fact that people reporting a depressed mood were not allowed into the trials.

“They have to exclude individuals with mood disorders in such studies because they don’t want the liability issues, and it enables a cleaner interpretation of the trial data,” explains Richard Deyo, a pharmacologist and cannabinoid expert at Winona State University in Minnesota. But to understand the full potential impact of side effects, he adds, “you’d have to look at the whole population.”

Better to boost CB1 than to block it?
Several other drug companies have CB1 blockers in early clinical trials, though the failure of rimonabant and taranabant casts a cloud over their further development.

Other academic and commercial researchers have sought not to block CB1 activity but to boost it, to relieve depression and anxiety. But this strategy, too, is fraught with complications.

The most obvious problem is that the CB1-binding molecule known as THC is potentially addictive, as are other cannabinoid molecules that directly bind and activate CB1. CB1 also appears to have different effects depending on which system it is modulating.

CB1 receptors are found on two broad classes of nerve terminals: excitatory, which increase the activity of target neurons; and inhibitory, which reduce activity, Piomelli says. CB1 therefore can either “put on the brakes” or “release the brakes” depending on which of these systems it modulates.

Any strategy that floods the brain with CB1-binding molecules will thus have a host of effects, depending on the systems affected, the dose that reaches each system, and the ongoing level of activity in those systems—which in turn can vary according to mood, personality and genetic differences in brain chemistry.

For example, at low to moderate levels, THC, which is the main active ingredient in marijuana, appears to have reliable pain- and anxiety-relieving effects, Deyo says. “As you increase the dose, though, you get memory blocking effects, you get a tendency towards depression and you also get the potential for hallucinations,” he adds. Furthermore, THC’s effects appear to vary according to gender.

The ‘make more of it where it’s needed’ strategy
To get past such obstacles, Piomelli and his colleagues at UC-Irvine recently devised an indirect CB1-activity boosting strategy, similar to that used for some other brain-cell receptors. The body’s main CB1-activating molecule, anandamide, is normally broken down quickly by enzymes near the synapses where it works. Piomelli reasoned that by preventing this continual degradation of anandamide, he could keep it more of it around synapses and thus maintain a higher level of CB1 activity. In this way he could also increase CB1 activity primarily where it was already occurring and needed, and to a lesser extent in other areas of the brain and body where the boosting of such activity could produce undesired side effects.

In a paper published in the journal Biological Psychiatry on Sept. 22, Piomelli and colleagues, together with a National Institutes of Health team led by Stephen Goldberg, reported on a set of experiments using this strategy in monkeys. Administering a drug that inhibits FAAH, an anandamide-eating enzyme, the researchers soon were able to measure higher levels of anandamide in the monkeys’ brains.

Using standard tests, they found that this approach did not reinforce other drug-taking behavior, as THC normally does. “Our results,” the researchers wrote, “suggest that FAAH inhibitors might be used therapeutically without risk of abuse or triggering of relapse to drug abuse.”

Piomelli explains that when the FAAH inhibitor, currently named URB597, is administered, anandamide levels rise and appear to reduce inhibitory controls on a midbrain region where neurons pump out the neurotransmitters serotonin and noradrenaline. The effect is to boost the levels of these neurotransmitters in the brain.

“This physiological effect is similar to that produced by certain antidepressant drugs,” says Piomelli, “and indeed URB597 displays profound antidepressant-like properties in rats and mice.”

Organon, a unit of pharmaceutical company Schering Plough, holds the rights to URB597 and is considering clinical trials of the drug for pain relief.

CB 65

Biological Activity

High affinity and selective CB2 receptor agonist; Ki values are 3.3 and > 1000 nM for CB2 and CB1receptors respectively.

Technical Data

M.Wt:

417.93

Formula:

C22H28ClN3O3

Solubility:

Soluble to 20 mM in DMSO with gentle warming and to 10 mM in ethanol with gentle warming

Purity:

>99 %

Storage:

Store at RT

CAS No:

913534-05-1

The technical data provided above is for guidance only.
For batch specific data refer to the Certificate of Analysis.

Role of cannabinoid and vanilloid receptors in invasion of human breast carcinoma cells.

It is known that the diversified effects of cannabinoid on the fate of carcinoma cells are mediated predominantly through receptors. However, little is known about the effects of the individual activities of cannabinoid and noncannabinoid receptors. Here we investigate the role of cannabinoid receptor (CB) 1, CB2, and transient receptor potential vanilloid type 1 in cell proliferation and invasion patterns in the MDA-MB-231 cell line. Our results showed that activation of CB1 and vanilloid receptors by methanandamide, a nonselective agonist, and arachidonyl-2′-choloroethylamide (ACEA) and N-oleoyldopamine, selective agonists, reduced invasion of MDA-MB-231 cells at pharmacological concentrations. Accordingly, CB1 activation resulted in decreased expression of matrix metalloproteinase (MMP) 2. On the other hand, administration of a CB2 agonist (CB65) increased cell invasion and expression of MMP2. The data obtained from MTT assay did not show any correlation between reduced invasion and cytotoxic effects of drugs. In addition, the level of vascular endothelial growth factor was significantly reduced in treatment with (R)-(+)-methanandamide, ACEA, CB65, and AM251 (a potent agonist for GPR55 and selective antagonist of CB1) compared with control. Elevated expression of cyclooxygenase-2 was observed in all of the MDA-MB-231 cells treated with agonists. These results underline the influence of cannabinoid and vanilloid receptors on the invasiveness of MDA-MB-231 human breast carcinoma cells.

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