Shining a light on painkilling systems in the brain

New findings by Scripps Research and Virginia Commonwealth scientists could have implications for drug development and basic science

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LA JOLLA, Calif.—Research by scientists with the Scripps Research Institute and Virginia Commonwealth University has found that repeated boosting of brain levels of one natural painkiller results in shutting down the brain cell receptors that respond to it, thereby killing its painkilling effect.

The natural painkiller, 2-AG, is one of the two major "endocannabinoid" neurotransmitters. The other, anandamide, can be kept at high levels in the brain without losing its therapeutic effects, and researchers had hoped that the same would be true for 2-AG.

The study was led by Benjamin F. Cravatt III, professor and chairman of the Department of Chemical Physiology and member of the Skaggs Institute for Chemical Biology at Scripps Research in La Jolla, Calif. Co-author of the study was Aron Lichtman, a professor of pharmacology and toxicology at Virginia Commonwealth University in Richmond, Va.

The study, published in a recent issue of the journal Nature Neuroscience, has important implications for drug development, they say.

"Our study shows that acute inhibition of the endogenous cannabinoid catabolic enzymes fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) produces similar efficacy in short-term and neuropathic pain assays, but these effects are only sustained in chronically disrupted FAAH systems," Cravatt says. "Genetic deletion or chronic pharmacological blockade of MAGL results in tolerance as well as cross-tolerance to cannabinoid (i.e., marijuana-like drugs), that is the result of loss of function of the CB1 cannabinoid receptor."

Cravatt adds that the research team is interested in understanding complex physiology and behavior at the level of chemistry and molecules.

"At the center of cross-talk between different physiologic processes are endogenous compounds that serve as a molecular mode for intersystem communication," he explains. "However, many of these molecular messages remain unknown, and even in the instances in which the participating molecules have been defined, the mechanisms by which these compounds function and their modes of regulation are for the most part still a mystery."

Lichtman notes that the discovery can help lead to the development of drugs with greater efficacy.

"These findings indicate that the analgesic efficacy of FAAH inhibitors is unlikely to undergo tolerance following repeated drug administration," he says. "While our study finds that complete inhibition of MAGL results in massive loss of function of the CB1 receptor, our data do not preclude the possibility partial MAGL blockade could be used to achieve prolonged analgesic responses."

Cravatt points out that the levels and signaling function of endocannabinoids are tightly regulated by enzymes to maintain proper control over their influence on brain and body physiology.

Moreover, these findings have important clinical implications, as they point to specific therapeutic possibilities for each endocannabinoid pathway.

The researchers found that a key implication is that maximally elevating 2-AG levels in the brain might not provide a straightforward path to new pain drugs.

"But we remain optimistic that more modest elevations in 2-AG could produce sustained pain relief. Perhaps more importantly, on a basic science level, we've been able to tease apart a key difference between the two major endocannabinoid signaling pathways, since one can maximally elevate anandamide without observing tolerance," he notes.

Like the opioid system, the endocannabinoid system was discovered as a result of humans identifying a plant—in this case marijuana (cannabis sativa)—that artificially boosts its activity. Marijuana's main active ingredient, THC, typically reduces pain and anxiety.

Researchers have sought to develop drugs that reproduce such therapeutic effects while leaving out THC's unwanted side effects—which include memory impairment, locomotor dysfunction, and possibly addiction.

Cannabinoid research received a boost in 1990 with the description of the main cannabinoid receptor in the brain, CB1, and a few years later with the discoveries of the body's own (endo-) cannabinoids, anandamide and 2-AG, which exert most of their effects by binding to CB1.

Cannabinoid receptors are now known to be widely distributed in the brain, and when activated by anandamide or 2-AG, tend to calm the activity of the neurons where they reside. However, researchers so far have been unable to develop artificial cannabinoids that bind to CB1 without producing unwelcome THC-like side effects.

An alternative strategy has been to boost levels of the body's own cannabinoids by inhibiting the enzymes that normally break them down. And so far this has worked for anandamide. Inhibitors of its breakdown enzyme, fatty acid amide hydrolase (FAAH), have been shown to boost anandamide levels and reduce pain and inflammation without adverse side effects in animal tests and early clinical trials.

A similar strategy for boosting 2-AG may be promising, too, especially since 2-AG levels in the brain are naturally higher than anandamides. Two years ago, the Cravatt and Lichtman laboratories jointly reported the development of an inhibitor of 2-AG's breakdown enzyme, monoacylglycerol lipase (MAGL). When administered to mice, it boosted their brain levels of 2-AG on average by a factor of eight, and produced a pain-killing effect comparable to that of FAAH inhibitors.

Now the two labs report that 2-AG's pain-killing effect disappears after six days of treatment.

"When you continually stimulate the endocannabinoid system by maximally raising 2-AG levels, you effectively desensitize the system," says Cravatt.

In one experiment, an injection of the MAGL inhibitor into mice showed evidence of pain relief on standard tests, but after six consecutive daily injections the drug could no longer achieve this effect. These chronically treated mice also lost much of their sensitivity to THC and to a synthetic CB1-binding compound, and showed a classic sign of drug dependency: when abruptly withdrawn from 2-AG's influence by having their CB1 receptors blocked, they developed paw flutters—a murine version of the shakes.

Jacqueline Blankman, a graduate student at the Scripps Research Kellogg School of Science and Technology who was co-first-author on the paper with Joel Schlosburg of the Lichtman lab, points out that when scientists investigated at the molecular level, they found that the number of CB1 receptors in the mouse brains had been reduced. This receptor "downregulation" occurred in some brain areas but not others.

To confirm this effect, the researchers utilized another experimental mouse model where the gene for MAGL was inactivated. This lifelong genetic disruption of MAGL also resulted in high 2-AG levels as well as a reduced and desensitized CB1 system.

"Because we're seeing downregulation of the whole cannabinoid system and tolerance to the anti-pain effects, it does raise some concern about whether MAGL would be a suitable pain target," says Blankman.

By contrast with the 2-AG experiments, chronically boosting anandamide had none of these effects on the CB1 system. Cravatt doesn't yet know why these two molecules have such different impacts when delivered chronically. He notes, however, that anandamide may be produced selectively under stress conditions, and perhaps for that reason is less likely to trigger a brain-wide CB1 downregulation.

"The question of why anandamide and 2-AG have such different effects when given chronically is certainly going to be motivating us from now on," says Cravatt. "But already with this finding and the development of these models we've taken a significant step forward in understanding and being able to manipulate this important neurotransmitter system."

The Cravatt and Lichtman laboratories have had a highly productive collaboration investigating catabolic enzymes regulating endogenous cannabinoids for over a decade.  

"The impetus for the research presented in this particular paper began in January 2009 from another research project between the Cravatt and Lichtman laboratories that demonstrated full blown THC-like effects when both FAAH and MAGL were simultaneously inhibited," explains Lichtman. "Dr. Qing-song Liu of the Department of Pharmacology and Toxicology, Medical College of Wisconsin, another key player in this collaborative effort, found that chronic that chronic MAGL inhibition, but not chronic FAAH inhibition, inhibition resulted in a loss of endocannabinoid-mediated short-term synaptic plasticity."

As the research progressed, the team was presented with several challenges along the way.

"Significant challenges throughout this project included technological issues related to the creation of the MAGL null mice and coordinating a tremendous array of multidisciplinary approaches (e.g., behavioral, pharmacological, biochemical, molecular, and electrophysiological) across three laboratories," notes Cravatt.

Going forward, Cravatt explains that further research is needed to investigate whether partial blockade of MAGL would produce sustained analgesia without tolerance or CB1 receptor loss of function.

"It will also be important to determine the impact of chronic inhibition of FAAH and MAGL on CB2 receptors, since agonists for this receptor have been shown to produce anti-nociception and anti-inflammatory effects," he says. "Moreover, the impact of chronic FAAH and MAGL inhibition needs to be examined in other pain models (e.g., rheumatoid arthritis, osteoarthritis, diabetic neuropathy, cancer pain, surgical pain, visceral pain)."  

Additionally, Cravatt notes that it will be vital to assess whether the same pattern of effects that we observed in mice also occurs in other mammalian species.

"In addition, it will be important to elucidate the impact of chronically blocking endocannabinoid catabolic enzymes in preclinical models of anxiety, depression, cognition, and neurodegenerative diseases," he says. "Finally, studies are needed to determine why the CB1 receptor undergoes profound adaptive changes in response to elevated brain levels of 2-AG, but not anandamide. Is it that these two endocannabinoids act differentially at the CB1 receptor to cause internalization, downregulation, desensitization etc.?  Is this differential effect simply a function of the relative mass quantity of these substances at the receptor, whereby 2-AG achieves much higher local concentrations than anandamide?  Is it that the enzymes responsible for anandamide and 2-AG biosynthesis are differentially distributed at synapses containing CB1 receptors synapses?"

As the research continues to move forward, Cravatt also noted that there is a very good chance that additional participants could come on board for the project.

"There is other ongoing work related to this project that is examining translational aspects to evaluate chronic inhibition of MAGL and FAAH on pain in nonhuman primates," he says. "This research will require the expertise of other scientists well versed with nonhuman primate models of pain."

(If you'd like to discuss this article or share your own insights, we have a copy of it at our blog, and you can visit it, and use the comments function, by clicking here.)

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