In 2009 in the United States (US), Neuropharmacology Post-doctoral Nick DiPatrizio was trying to identify exactly where and how endocannabinoids, endogenous molecules that bind to the same receptors as active ingredients in cannabis, were controlling food intake in rats. The young scientist persisted and eventually discovered hunger and the taste of fat led to increased endocannabinoid levels in the jejunum, a part of the small intestine. Endocannabinoid signalling in the gut, not the brain, was controlling food intake in the rodents in response to tasting fats. In 2011 he published his findings in the study, ‘Endocannabinoid signal in the gut controls dietary fat intake‘.
Ever since the first endocannabinoid receptor was identified in the late 1980’s, the field has been overwhelmingly focused on the central nervous system. The main endocannabinoid receptor, CB1, was first discovered in a rat brain and is now known to be among the most abundant G protein–coupled receptors in neurons there. However, the endocannabinoid system (ECS), a family of endogenous ligands, receptors and enzymes, isn’t exclusive to the brain. It is present everywhere scientists have looked, in the body: heart, liver, pancreas, skin, reproductive tract etc. Disrupted endocannabinoid signalling has been associated with many disorders, including diabetes, hypertension, infertility, liver disease and more. “There is so much that’s still unknown about this system. It looks to be regulating every physiological system in the body” said DiPatrizio.
Now an Assistant Professor at Riverside School of Medicine, University of California, DiPatrizio has trained his research on the gut, where the ECS appears to be a major player in human health and disease. His lab has suggested endocannabinoid signalling in the gut drives the overeating characteristic of Western diets. In a rodent model, chronic consumption of a high-fat, high-sugar diet led to elevated levels of endocannabinoids in the gut and blood, promoting further consumption of fatty foods. Blocking endocannabinoids from their receptors decreased over-eating in animals as reported in the 2017 study, ‘Peripheral endocannabinoid signaling controls hyperphagia in western diet-induced obesity‘.
Due to the link to appetite, pHARMaceutical companies have sought to target the ECS to create the ultimate diet pill, a drug to reduce appetite or treat metabolic disorders. Those efforts have been subdued by two tragic and highly visible failures. The ECS is a tantalising, elusive target for the pHARMaceutical industry, especially for conditions related to appetite and gut health. Sanofi-Aventis was the first to market an anti-obesity drug targeting endocannabinoid receptors. In 2006, the European Commission approved the CB1 antagonist rimonabant (Acomplia) as a treatment to curb hunger. But as a wider population of people began using it, dangerous side effects emerged. A small percentage of users suffered from serious psychiatric symptoms, including suicidal thoughts, evidenced in a meta-analysis published in 2007, ‘Efficacy and safety of the weight-loss drug rimonabant: a meta-analysis of randomised trials‘.
In 2008, the European Medicines Agency recommended suspension of the drug and the company withdrew it. That halted development of the whole class of CB1 antagonists, said George Kunos, M.D., Ph. D., Scientific Director of the National Institute on Alcohol Abuse and Alcoholism (NIAA), in the US. Yet the side effects should have been predictable, he argued, as CB1 receptors play an important role in brain reward pathways. Blocking them, therefore is likely to cause an inability to feel pleasure. Last January, the field was dealt a second blow. In France, six participants in a Phase 1 study of a compound known as BIA 10-2474 were hospitalised with neurological symptoms. Portuguese pharmaceutical company Bial was developing the drug as a candidate to treat a number of neurological disorders, including anxiety. But within days of receiving multiple daily doses of the drug, one participant was declared brain-dead, while others developed severe lesions on their brains.
BIA 10-2474 is an inhibitor of fatty acid amide hydrolase (FAAH), a key enzyme that breaks down endocannabinoids. Researchers had hoped by targeting a downstream part of the ECS, rather than the receptors themselves, they might avoid off-target effects in the brain and elsewhere. That was not the case. “That, again, scared regulators and the industry away from consideration of that system” said the University of Calgary’s Keith Sharkey. There is still potential for drug development in the field, but only under carefully controlled conditions with drugs that can be restricted to specific sites of action. But some scientists still hope that by understanding the true nature of this system, they might identify new treatments, especially for conditions related to gut health and metabolism.
“We are now at a point where you have to understand how endocannabinoids can be so relevant in so many areas – literally everywhere in the body”, said Mauro Maccarrone, Head of Biochemistry and Molecular Biology at Campus Bio-Medico University of Rome, Italy, who has studied the molecules since 1995. “There must be a reason why these endocannabinoids are always there”. Researchers describe the ECS as the most complicated and most ubiquitous signalling system in our bodies, yet no one knew it was part of human physiology until the 1980’s. And that realisation came from an oft-derided effort to understand how cannabis gets us ‘high’, ‘Multiple Functions of Endocannabinoid Signaling in the Brain‘.
In 1964, researchers seeking to understand the ‘psychoactive’ component of the cannabis plant identified the compound Δ9-tetrahydrocannabinol, or THC. Over two decades later, in 1988, investigators found direct evidence of an endogenous signalling system for THC, a receptor in the rat brain that bound a synthetic version of THC with high affinity. Blocking the receptor with a chemical antagonist in humans effectively blocks the ‘high’ typically experienced after smoking cannabis. The receptor, called CB1, was subsequently identified in other mammalian brains, including those of humans and appeared to be present in similar density to receptors for other neurotransmitters, including glutamate, GABA and dopamine. A second cannabinoid receptor, CB2, was discovered in 1993. This receptor was first isolated in the rat spleen. That surprising finding was an omen of things to come; the ECS functions far afield from the brain, practically everywhere in the body.
The presence of these receptors sparked a quest to find natural ligands that bind to them. The first endocannabinoid identified, a fatty acid-based agonist for both receptors, was named anandamide, based on the Sanskrit word ananda meaning “inner bliss”. A second agonist, 2-arachidonoylglycerol (2-AG), appeared to be present at high levels in normal mammalian brains. By 1995, the so-called “grass route” was complete: over three decades, researchers had identified THC, its endogenous receptors and endogenous ligands for those receptors. Maccarrone suspects endocannabinoids are among the oldest signalling molecules to be used by eukaryotic cells. His team recently showed anandamide and its related enzymes are present in truffles, delectable fungi that first arrived on the evolutionary scene about 156 million years ago, suggesting endocannabinoids evolved even earlier than cannabis plants.
Putative mechanism of endocannabinoid-mediated retrograde signalling in the nervous system. Activation of metabotropic glutamate receptors (mGluR) by glutamate triggers the activation of the phospholipase C (PLC)-diacylglycerol lipase (DGL) pathway to generate the endocannabinoid 2-arachidonoylglycerol (2-AG). First, the 2-AG precursor diacylglycerol (DAG) is formed from PLC-mediated hydrolysis of membrane phospholipid precursors (PIPx). DAG is then hydrolysed by the enzyme DGL-α to generate 2-AG. 2-AG is released from the post-synaptic neuron and acts as a retrograde signalling molecule. Endocannabinoids activate pre-synaptic CB1 receptors which reside on terminals of glutamatergic and GABAergic neurons. Activation of CB1 by 2-AG, anandamide, or exogenous cannabinoids (e.g., tetrahydrocannabinol [THC]) inhibits calcium influx in the pre-synaptic terminal, thereby inhibiting release of the primary neurotransmitter (i.e., glutamate or GABA) from the synaptic vesicle. Endocannabinoids are then rapidly deactivated by transport into cells (via a putative endocannabinoid transporter) followed by intracellular hydrolysis. 2-AG is metabolized by the enzyme monoacylglycerol lipase (MGL), whereas anandamide is metabolised by a distinct enzyme, fatty-acid amide hydrolase (FAAH). MGL co-localises with CB1 in the presynaptic terminal, whereas FAAH is localised to post-synaptic sites. The existence of an endocannabinoid transporter remains controversial. Pharmacological inhibitors of either endocannabinoid deactivation (e.g., FAAH and MGL inhibitors) or transport (i.e., uptake inhibitors) have been developed to exploit the therapeutic potential of the endocannabinoid signalling system in the treatment of pain.
“They are kind of a master signalling system and other signals have learned to talk to these lipids” said Maccarrone. In the brain, endocannabinoids interact with other neurotransmitters; in the reproductive tract, with steroid hormones; in the muscles, with myokines; and so on. But even though researchers have documented the existence of the ECS throughout the body, they still don’t really know what role it plays outside the brain, where it is involved in synaptic signalling and plasticity. In healthy, non-obese animals, there is typically no consequence to knocking out endocannabinoid receptors in peripheral organs. “There is no detectable effect on any important biological function” said George Kunos.
The one exception to this functional black box is the gastrointestinal tract. The idea cannabis, or endogenous cannabinoids, affects the gut is not surprising. Preparations derived from cannabis have long been used to treat digestive conditions such as inflammatory bowel disease and vomiting. Even before CB1 was discovered, scientists had suggested cannabinoids regulate the motility of the gastrointestinal tract, the orchestrated movements of muscles that churn and move food through the intestines. For example, in 1973, Australian researchers showed oral ingestion of THC slowed the passage of a meal through the intestines of mice. Conversely, knocking out parts of the system is associated with increased movement of food through the colon, a common symptom of irritable bowel syndrome (IBS). These pathways are conserved among many species.
Both CB1 and CB2 receptors are present and active in the gut, though they appear to be involved in different gut functions. At the University of Calgary, Keith Sharkey and colleagues found increased intestinal motility in the inflamed gut was reversed when CB2 receptors, but not CB1 receptors, were activated. To make things even more complicated, there is a group of non-classical receptors that interact with endocannabinoids in the gut, said Jakub Fichna, Head of the Department of Biochemistry at the Medical University of Lodz, Poland. His lab studies the role of these receptors in inflammatory bowel disease (IBD) and IBS. Depending on the conditions in the gut, some of these non-classical receptors don’t even need an agonist or antagonist to become active, Fichna says. “It can even be the change in pressure or pH of the neighbourhood. For example, if you have inflammation, most of the time you have decreasing pH and this is already enough for some of the endocannabinoid receptors to be activated”.
Endocannabinoids and their receptors also appear to be involved in gastric secretions, ion transport and cell proliferation in the gut. And then there is appetite. Cannabis users often experience the “munchies”, a sharp and sudden increase in appetite after inhaling or ingesting the herb. Kunos wondered whether endocannabinoids cause a similar increase in appetite. In 2001, with the help of collaborators, he confirmed the suspicion: endocannabinoids acting on CB1 receptors promoted appetite and mice with CB1 receptors knocked out ate less than their wild-type litter-mates. Additional research supported the idea endocannabinoids act as a general appetite-promoting signal and as DiPatrizio’s work showed, endocannabinoids control food intake not exclusively via the brain, but by way of signals generated in the gut. It’s a simple hypothesis with big implications for the management of obesity and other metabolic syndromes.
During his post-doctorate, DiPatrizio found when rodents tasted dietary fats (tasted, not swallowed), endocannabinoid levels increased in the rat small intestine, nowhere else. A CB1 receptor antagonist blocked that signal, leading the rodents to decrease their ingestion of fatty foods. “This suggests to us that this is a very important and critical mechanism that drives food intake” says DiPatrizio. From an evolutionary perspective, having a positive feedback mechanism for fat intake makes sense. When an animal in the wild detects high-energy foods, it is beneficial to stock up. However, that’s not true for people in today’s developed countries. “There’s no period of famine. It’s feast all the time, so now the system can drive us to over-consume” said DiPatrizio.
Sharkey sees the system as a regulator of homoeostasis within the body, especially considering its roles in maintenance of food intake, body weight and inflammation. “It seems to be very important in the conservation of energy. But in modern Western society in particular, those are the things that appear to have been dysregulated” said Sharkey. Although the job of the ECS remains mysterious in healthy tissues outside the brain and gut, diseases reveal clues. In obesity, both CB1 and CB2 receptors are up-regulated throughout the body, including in the liver and adipose tissue. And the activation of CB1 receptors increases food intake and affects energy metabolism in peripheral tissues. In type 2 diabetes, endocannabinoids and their receptors are up-regulated in circulating macrophages and contribute to the loss of pancreatic beta cells, which store and release insulin.
Interestingly, chronic cannabis users have no documented increased incidence of diabetes or obesity. Researchers speculate this is because chronic use results in down-regulation of CB1receptors, a form of pharmacological tolerance. Another possibility, explored by Sharkey and colleagues in 2015, is chronic THC exposure alters the gut microbiome, affecting food intake and preventing weight increase. In liver disease, up-regulation of CB1 appears to contribute to cell death and the accumulation of scar tissue (fibrosis). The two classical cannabinoid receptors, CB1 and CB2, are expressed by enteric neurons, immune cells and other cell types within the gastrointestinal tract. The gut and the liver also synthesise two key ligands, anandamide (AEA) and 2-arachidonoylglycerol (2-AG), for those receptors. Combined, this signalling system acts locally in the gut and liver, but also communicates with the brain to affect food intake, pain, inflammation and more.
In the liver, endocannabinoids are thought to act almost like hormones, stimulating cell division at some times, cell death at others. In the healthy liver, expression of CB receptors is very low, but in a diseased liver expression increases, and endocannabinoid ligands are released from all four cell types. Many ligands are produced and bind to CB1 receptors, causing lipid accumulation and insulin resistance in hepatocytes and increased proliferation of activated stellate cells, the major cell type involved in fibrosis (scarring) of the liver. Blocking CB1 receptors with drugs decreased the amount of fibrosis in mouse models.
Both CB1 and CB2 regulate the rhythmic contractions of the intestinal tract, called gut motility. In the healthy gut, CB1 predominates, but during intestinal inflammation, CB2 also contributes to motility. Conditions such as inflammatory bowel disease and coeliac disease often exhibit increased prevalence of these receptors, which results in decreased motility. Endocannabinoid signalling has also been shown to reduce inflammation, increase the permeability of gut epithelial cells and signal hunger to the brain.
Yet there remains debate as to whether endocannabinoid receptors are always the bad guys in disease. In some cases, endocannabinoid signalling appears to be therapeutic. Animal studies suggest endocannabinoids are effective pain relievers and the system has anti-inflammatory properties in certain contexts. In IBD, Sharkey’s team found activation of both CB1 and CB2 receptors resulted in reduced inflammation, suggesting the system may be activated as a protective force. Likewise, CB2 activation appears to be anti-inflammatory in cases of atherosclerosis, says O’Sullivan, who focuses on endocannabinoids in the cardiovascular system. “It’s a bit of a rescue receptor. In times of trouble, it gets upregulated”, she said. And several tantalising studies suggest cannabinoids from plants or synthetic compounds that mimic botanical molecules and the body’s own directly inhibit cancer growth by inducing cell death in tumour cells.
But the very thing that makes the ECS so interesting, its ubiquity and varied roles in the body, is also what makes it a difficult drug target. Within the last 10 years, two drugs targeting the ECS proved to have dire side effects in humans when the compounds crossed the blood-brain barrier. Off-target effects in other organ systems could also have long-term consequences. In a review of the pharmacology of 18 different CB2 ligands as potential drug candidates, Maccarrone and a large team of European researchers, in collaboration with Roche, concluded just three merited additional pre-clinical or clinical studies. Many of the other compounds engendered too many off-target effects.
Researchers are now working toward second-generation drugs that more specifically target peripheral systems. “If the scientific community faces the challenge of really understanding how to direct certain drugs to the right target, then we could have wonderful drugs for the future” says Maccarrone. Most of those compounds are in pre-clinical trials, though Kunos hopes to have an Investigational New Drug approval from the US Food and Drug Administration (FDA) soon for one agent his team has been working on as a possible treatment for non-alcoholic fatty liver disease. The compound does not penetrate the brain and is designed to accumulate in the liver, which may explain its efficacy in treating liver disease without causing psychiatric side effects in animal models, said Kunos.
If researchers can figure out how to avoid the devastating off-target effects, there is one more reason why endocannabinoids may effectively help treat disease: they provide an indirect link to the brain. “We’ve known, for some time, that the brain can modulate the gut” said Sharkey. With endocannabinoids, it appears the gut can also modify the brain. It is now clear, for example, there are very active communication pathways originating from peripheral nerves in the gut, able to modulate brain function. Numerous studies suggest the vagus nerve is a major information highway between the gut and brain. DiPatrizio is studying those communication pathways and hopes to identify ways to regulate feeding without ever getting near the brain with a drug. The research complements other evidence showing the gut is able to modulate pro-inflammatory cytokines in the blood and even influence central nervous system disorders. “We believe we can remotely control the brain from the gut, safely” says DiPatrizio. “That’s why, once again, endocannabinoid receptors are very attractive targets”.