The Berkeley Science Review: Articles

A Natural High
Harnessing the brain's own drugs (view PDF)
by Paul Hauser

The 1960s are remembered for many reasons—the civil rights movement, the Vietnam War, and the sexual revolution to name but a few. But there is a popular saying that if you remember the 1960s, you weren't there. Nowhere is this more pertinent than in the San Francisco Bay Area, where the marijuana- fueled counter-culture of the 1960s began. The movement sent tendrils of change through American academics, politics, and society, and Berkeley in the 1960s will forever be known for weaving the marijuana experience into the tapestry of our culture. Today, Berkeley is making marijuana-related discoveries that will again shape our culture. The latest interest in marijuana at Berkeley comes not from the drug, but rather the natural synthesis of compounds in the human brain and body that work in the same way. At this very moment, your body is producing compounds strikingly similar in action to components of marijuana. These natural "drugs" harbor the ability to control not only our mental and emotional health but also our most basic bodily functions-from the sensation of pain to the regulation of hunger and satiety.

At the leading edge of research into these compounds is a collaborative group with members in the Department of Environmental Science, Policy and Management (ESPM) and the Department of Nutritional Science and Toxicology (NST) at UC Berkeley. In a tale of scientific good fortune and interdisciplinary research, these investigators were able to use their knowledge of pesticiderelated compounds to elucidate the action of naturally synthesized compounds that mimic marijuana action. Researchers are now wagering that the inherent power of the marijuana high can be harnessed in novel drug therapies.

The history of the high Cannabis as a medicinal plant is reported to date back to prehistoric times, but its possession and use remains illegal in most of the world. The term cannabis (or marijuana) most commonly refers to the harvested flowers or buds from the Cannabis sativa species of the Cannabaceae, or hemp, family of plants. The diverse and agriculturally useful hemp family has been used throughout history not only to treat ailments, but also to make papers and textiles and to produce seeds and oils for cooking. Since its rise in consumption as a recreational and medical drug in the 20th century, scientists have become interested in understanding the plant's power, particularly amid continuing debates about the benefits and harms of the drug.

Marijuana research started simply enough in 1964 with the discovery that delta-9-tetrahydrocannabinol, or THC as it is better known, is the primary active ingredient in marijuana. Up to this point, it was generally assumed that a cocktail of compounds, categorically called "cannabinoids" because of their presence in cannabis, was collectively responsible for the central nervous effects of marijuana use. But work by Dr. Ralph Mechoulam and colleagues at the Weizmann Institute of Science in Israel demonstrated that THC alone is largely responsible for the changes in mood, perception, behavior, and consciousness known as the psychoactive effects of marijuana. With some 400 cannabinoids found in the marijuana plant (many of which are biologically innocuous or present in such low abundance as to have no effect), the cannabinoid compounds are now classified by their structural similarity to THC instead of their psychoactivity.

As public experimentation with marijuana increased dramatically throughout the 1960s and 70s, so too did scientific experimentation. In these two decades, studies flourished in an effort to understand the drug's cognitive and psychological effects. Early studies described the THC-induced feelings of euphoria, relaxation, fatigue, and noticeably altered perception. Other investigations focused on users' reports of increased appetite (the "munchies"), short-term memory impairment, and depressed motivation. Cannabinoid research was an interdisciplinary field from the start: chemists, biologists, and psychologists all got a piece of the descriptive action as the psychoactive profile of the "gateway" drug was born.

It would, however, be more than twenty years after the initial discovery of THC when the first biological mechanism for the cannabinoid response was proposed. In 1988, a natural brain receptor that seemed to be the target of THC was discovered. Researchers have since learned that the marijuana high is elicited when THC dissolves in the blood and passes into the brain. There it encounters the cannabinoid receptor in the outer membranes of cells that make up the tissue of the brain and nervous system. Its chemical composition and physical shape allow the THC molecule to bind and activate the receptor, thereby inducing its psychoactive effects. The discovery of THC and the cannabinoid receptor suggested a mechanism by which marijuana could produce its potent effects and raised an important question: why would the mammalian brain have evolved a receptor to bind this plant compound?

The answer again came from Mechoulam's group in 1992, when they found a naturally occurring compound in the pig brain that also had the ability to bind the cannabinoid receptor. This compound, which they named anandamide, was the first example of a cannabinoid produced by animals. The compound was termed an "endocannabinoid" because of its natural, or endogenous, synthesis and its chemical resemblance to the canonical cannabinoid, THC.

This discovery was soon followed by the identification of a second endocannabinoid, 2-arachidonylglycerol (2-AG). In a surprising twist, this chemical, despite its similarity to other cannabinoids produced in the brain, was initially discovered in the canine intestine.

It was later shown that pure forms of either endocannabinoid elicit the standard response of increased appetite, depressed activity, reduced sensitivity to pain, and mild hypothermia. Knowing that the brain and intestine produce natural compounds capable of inducing these effects by triggering the cannabinoid receptor, researchers needed an explanation for what these compounds do in their natural settings. This was particularly intriguing given their presence in such seemingly disparate tissues as the brain and intestine.

How the brain makes its own bliss
Early work indicated that endocannabinoids are synthesized from common cellular molecules. Specialized fats found in the membranes of nerve cells provide the chemical starting material for the generation of the two naturally synthesized cannabinoids. Researchers became aware that this was a tightly regulated mechanism, able to provide controlled levels of these compounds to activate the cannabinoid receptor and regulate bodily functions.

Further developments came in the mid 1990s, when researchers learned how endocannabinoids are biologically degraded. Two enzymes, monacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH), which break down 2-AG and anandamide, respectively, were purified from rats and characterized. These proteins were later found in nearly all mammalian brains. Together these enzymes break down the endocannabinoids, reducing their biological availability and subsequently preventing them from binding their target receptor. This result explained how the body attenuates this natural chemical signal to prevent us from walking around high on our own cannabinoid tonic, but brought us only slightly closer to understanding the activity of endocannabinoids in the context of normal brain and body function.

In an unlikely twist, it was a UC Berkeley research group working on a class of chemically related compounds known as organophosphates that provided a key breakthrough toward understanding the natural role of endocannabinoid signaling. John Casida, professor in the ESPM and NST departments explains that his lab had "long been focused on the neurological and cytotoxic effects of organophosphatecontaining compounds, including common pesticides." It was the lab's investigation into this cellular toxicity, or cytotoxicity, that indirectly led them to the field of endocannabinoid signaling.

Although the consequences of longterm, low-level pesticide exposure are still being evaluated, it is clear that many organophosphates have the potential to harm animals. The side-effects of some of the compounds he was working with caused Casida to become "particularly suspicious that certain organophosphates were interfering with endocannabinoid signaling." From this hunch, says Casida, "we simply followed the chemical trail" from the poorly characterized side-effects of the suspicious pesticide-like compounds to understanding the relationship between organophosphate activity and cannabinoid signaling.

At the start of this connective trail was the synthesis of a diverse set of organophosphate compounds known as a chemical library. Casida's former graduate student Daniel Nomura assembled and tested the chemical library of compounds one by one for the ability to react with a collection of known brain enzymes. To test Casida's early suspicions, Nomura screened the library for compounds with the ability to act directly on the endocannabinoid system. Casida soon began to see that the endocannabinoid system disruption was "immediately obvious minute by minute and step by step," as he and Nomura observed that certain related compounds were able to inhibit a set of related brain enzymes.

Where it all breaks down for endocannabinoids
One of the compounds that produced a particularly marked cannabinoid-like effect was chosen for further investigation. This chemical, IDFP (or isopropyl dodecylfluorophosphonate as it is more formally known) elicits the classical cannabinoid intoxication profile. Curiously, however, the lab discovered that it was not working by the same mechanism as THC. "I was wondering how IDFP could induce stronger cannabinoid effects than THC without acting directly on the cannabinoid receptor," recalls Nomura. He soon realized that IDFP strongly inhibits both of the major endocannabinoid metabolizing enzymes. Nomura's later experiments showed that IDFP induces ten-fold increases in anandamide and 2-arachidonylglycerol levels, which meant that any IDFP traveling to the brain prevented endocannabinoid degradation. The resulting buildup of endocannabinoids in the nervous system causes the brain to get high on its own stupor-inducing tonic. IDFP produces cannabinoid effects not by directly targeting endocannabinoids or their receptor, but by inhibiting the breakdown enzymes that normally rid the body of these potent compounds.

IDFP's unique activity provided a new avenue through which to manipulate endocannabinoid degradation. Ben Cravatt of the Scripps Research Institute, a collaborating investigator in the study, showed that removing only one of the degradation enzymes, FAAH, had little noticeable effect. The only difference between Cravatt's and Nomura's experiments was the additional inhibition of MAGL. "We now strongly suspect that endocannabinoid degradation via the action of MAGL is critically important for proper brain and nerve function," says Nomura. As a result of this finding, 2-AG and its enzymatic degradation by MAGL are now front and center in the race to fully understand the endocannabinoid signaling pathway, a race for which the ultimate prize is the development of beneficial therapeutics.

Nomura and Casida have recently discovered that, in addition to increasing circulating endocannabinoid levels, suppression of endocannabinoid degradation in the nervous system has an unexpected secondary effect. When MAGL is inhibited, there is a corresponding reduction in arachidonic acid levels in the brain. Arachidonic acid is an essential fatty acid that is used by nearly all cells to produce signaling molecules as variable as blood clot promoting molecules, natural pain killers, and inflammatory compounds. The observed correlation between changes in arachidonic acid and endocannabinoid levels in this model suggests a strong but as yet not fully understood connection between MAGL activity and hormone signaling.

This discovery also defies conventional wisdom, which has long held that a different enzyme is responsible for maintenance of the brain arachidonic acid pool, and implies that MAGL is controlling not just endocannabinoid levels but a diverse set of brain activities associated with arachidonic acid metabolism. This challenge to the current dogma is ushering in a fresh wave of interest in endocannabinoids. If the evidence stands the test of time, it could have consequences that transcend the endocannabinoid field to enter the consciousness of researchers from fields as diverse as lipid biology, neurology, and pharmacology.

Not least of those interested in endocannabinoid research will be pharmaceutical companies, whose interest arises from the fact that arachidonic acid regulation has a proven track record in the development of therapeutics for pain, inflammation, and hormone signaling. Although Casida cautions that "in order to get [a pharmaceutical] that is highly selective, you have to walk a very thin line," the combined potential of the lab's findings reinforces the essential requirement of endocannabinoids for normal bodily function.

This formalized link between endocannabinoids and arachidonic acid provides strong support for the idea that the endocannabinoid system is not just a scientific hallucination, but rather it is a highly integrated network that facilitates vital cross talk between the brain and body. The endocannabinoid mind-body problem It's not just Casida and Nomura's findings that are fueling drug companies' interests in endocannabinoids. Mounting evidence suggests that in certain body systems there is potent endocannabinoid activity that is related to, but independent of, its brain effects. Particularly, researchers of the "brain-gut axis" are recognizing the regulatory power of endocannabinoids.

The brain-gut axis describes an exchange of chemical messengers between our digestive system and nervous system that communicates messages of hunger and satiety, and subsequently regulates body weight, metabolic rate, and energy management. The brain-gut axis is thought to have evolved to allow us to maintain unconscious communication between our internal metabolism, the external food supply, and our brain function. By extension, it has been suggested that disruptions in this communication con- tribute to metabolic diseases such as diabetes and obesity. Small, specialized proteins called neuropeptides, which have hormone-like activities, have been implicated in controlling the brain-gut axis, and it now seems that at least a few of these peptides are further regulated by endocannabinoid levels.

One of these peptides, ghrelin, induces a strong appetite signal when secreted from the gut. Recently, it was shown that this ghrelindependent hunger response is suppressed in animals that either lack the cannabinoid receptor or are treated with a compound that blocks cannabinoid receptor activity. This connection strongly suggests that the cannabinoid system is necessary for the proper function of the brain-gut axis.

In a parallel study, changes in a fat tissue secreted hormone, leptin, were implicated in the control of endocannabinoids in the body's fat stores. Interestingly, when leptin levels are increased, there is a parallel decrease in the endocannabinoid levels in fat tissue. This evidence connects other isolated studies showing that elevated leptin levels or lowered endocannabinoid levels can reduce fat stores and promote weight loss. While the connective mechanism remains uncertain, the suppression of appetite and weight gain signals associated with cannabinoids is now linked to the action of leptin, which already is known to have potent activities on insulin action, fatty acid and carbohydrate metabolism, and weight management.

The leptin results, taken together with findings that endocannabinoids are central to neuronal signaling, suggest possibilities for exciting therapeutic development. Casida has begun a collaboration with Ronald Krauss and graduate student Maxwell Ruby from the NST department to explore the peripheral effects (those outside the brain) of endocannabinoid signaling with the hopes of expanding therapeutic understanding of the system. "With all these pathways being impacted by the cannabinoid system, it gives a whole new dimension to exploiting this system therapeutically," says Krauss.

From cannabinoids to cures
Therapeutic exploitation is exactly what pharmaceutical companies are hoping for as they look for ways to harness the endocannabinoid system to develop a new class of commercial drugs. There has been significant interest in using direct cannabinoid receptor activators (such as THC-mimicking pharmaceuticals) to prevent pain and treat disease for some time, but only recently were the first commercial drugs brought to market as pharmacological proof of principle. Two recently developed endocannabinoid receptor blockers, Rimonabant from Sinofi-Aventis and Tiranabant from Roche Pharmaceuticals, have inspired hope for the future of endocannabinoid-based drugs and established major difficulties in separating the desirable and undesirable effects of cannabinoid- based therapies. According to their fact sheets, Rimonabant and Tiranabant are wonder drugs: the manufacturers claim everything from weight loss and suppressed appetite to improved blood cholesterol and fatty acid levels and increased short-term memory and energy levels. But if you've ever read the fine print on a drug label, you can be certain that life just isn't that simple.

While Rimonabant is approved for sale on the European market, neither cannabinoid receptor blocker fared well in recent US Food and Drug Administration trials. The US trials, independent scientific studies, and European consumer surveys all report that these drugs increase depression, anxiety, and even suicidal tendencies among a small but significant proportion of users. It appears that there is a critical psychological function for the natural, low levels of endocannabinoids, such that blocking their activity can create undesirable effects on brain function and mood.

Pharmacologists and drug companies, however, are not willing to concede that all is lost. The psychological side-effects cannot easily be overlooked and require careful investigation, but statistically significant weight loss, increases in beneficial cholesterol, and reduced arterial inflammatory signals among the Rimonabant trial participants are equally difficult to ignore. Pharmaceutical researchers, like their academic counterparts, are discovering that endocannabinoids have both complex central nervous system activities and beneficial whole-body effects. The endocannabinoid system is so diverse and far-reaching that it is challenging to isolate specific activities that render the system usable for beneficial treatments. Or as Krauss puts it, "there remains a lot of potential within the system for therapeutic intervention, but we don't yet have the best solutions that can take full advantage of that potential." Drug companies believe that selective activation of only the peripheral effects of endocannabinoid signaling could lead to effective treatments for obesity, metabolic disease, and disorders of hunger and satiety.

Pharmaceutical companies are now searching for ways to design drugs that would act in the peripheral body but would be prevented from passing into the brain. It is conceivable that targeted therapies could preferentially partition the whole-body benefits of endocannabinoid metabolism from the unwanted central nervous system complications.

With the recent growth in the endocannabinoid field, it's clear that researchers have made significant strides since the days of giving controlled doses of marijuana to willing volunteers. While endocannabinoid pharmacologists are still seeking the full path to their targets, there remains hope that understanding the endocannabinoid system could lead to more effective therapeutic tools for the treatment of diabetes, obesity, and cardiovascular disease. Undoubtedly, endocannabinoid research will remain an interdisciplinary affair, as pharmacologists, lipid biologists, and geneticists all examine the biological benefits and limitations of the natural high. With new discoveries in this emergent field, Berkeley is contributing to research on marijuana-type effects in a way that was never dreamed of in the 1960s. And although this tale is just beginning, it is certain that the latest research has taken the recreation out of cannabinoids. Getting to the bottom of the endocannabinoid story is now a serious business.

Paul Hauser is a student in molecular and biochemical nutrition.

Want to know more? Check out:

Dr. John Casida's research profile: ecnr.berkeley.edu/facPage/dispFP.php?I=466

Dr. Ronald Krauss's research profile: http://www.chori.org/Principal_Investigators/Krauss_Ronald/krauss_overview.html

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