The Peripheral Cannabinoid Receptor Type 1 (CB1) as a Molecular Target for Modulating Body Weight in Man

The cannabinoid 1 (CB1) receptor regulates appetite and body weight; however, unwanted central side effects of both agonists (in wasting disorders) or antagonists (in obesity and diabetes) have limited their therapeutic utility. At the peripheral level, CB1 receptor activation impacts the energy balance of mammals in a number of different ways: inhibiting satiety and emesis, increasing food intake, altering adipokine and satiety hormone levels, altering taste sensation, decreasing lipolysis (fat break down), and increasing lipogenesis (fat generation). The CB1 receptor also plays an important role in the gut–brain axis control of appetite and satiety. The combined effect of peripheral CB1 activation is to promote appetite, energy storage, and energy preservation (and the opposite is true for CB1 antagonists). Therefore, the next generation of CB1 receptor medicines (agonists and antagonists, and indirect modulators of the endocannabinoid system) have been peripherally restricted to mitigate these issues, and some of these are already in clinical stage development. These compounds also have demonstrated potential in other conditions such as alcoholic steatohepatitis and diabetic nephropathy (peripherally restricted CB1 antagonists) and pain conditions (peripherally restricted CB1 agonists and FAAH inhibitors). This review will discuss the mechanisms by which peripheral CB1 receptors regulate body weight, and the therapeutic utility of peripherally restricted drugs in the management of body weight and beyond.


Introduction
A well characterized feature of cannabis use is the stimulation of appetite and suppression of nausea. This effect of cannabis was thought to be primarily mediated by the phytocannabinoid ∆ 9 -tetrahydrocannabinol (THC) binding to the CB 1 receptor in key areas of the brain that regulate feeding and nausea including the hypothalamus (feeding), dorsal vagal complex and insular cortex (nausea), and nucleus accumbens and limbic areas (reward and motivation aspects of feeding) [1,2]. For this reason, cannabis has been used to treat the loss of appetite and body weight in several disorders. Synthetic forms of THC (dronabinol and Nabilone ® ) are approved for chemotherapy-induced nausea and vomiting across many countries, supported by meta-analyses of trial data in cancer patients, showing cannabinoids are effective at treating nausea and vomiting [3] and increasing appetite [4]. Dronabinol also causes significant weight gain in patients who are HIV-positive [5,6] (and is approved for HIV/AIDS-induced anorexia in some regions), young anorexic women [7,8], and in patients with Alzheimer's disease [9].
Conversely, antagonising the CB 1 receptor suppresses appetite and causes weight loss, and this has also been exploited therapeutically. The CB 1 receptor blood-brain barrier (BBB) penetrable antagonist (and potentially inverse agonist [10]) Rimonabant (Acomplia ® ) was developed by Sanofi and licensed as an anti-obesity drug. Multiple randomized controlled trials (RCTs) showed that 20 mg rimonabant led to significant reductions in body At the peripheral level, extensive research has shown that CB 1 receptor activation impacts the overall energy balance of mammals in a number of different ways, inhibiting satiety and emesis, increasing food intake, altering adipokine and satiety hormone levels, altering taste sensation, decreasing lipolysis, and increasing lipogenesis. Table 1 summarizes some of the known effects of CB 1 activation in the various organs and body systems that play a role in body weight regulation, illustrated in Figure 1. The combined effect of peripheral CB 1 activation is to promote appetite and promote energy storage and preservation, ultimately leading to weight gain or weight maintenance. Important locations of peripheral CB 1 receptors include the oral cavity, gastrointestinal tract, afferent vagus nerves, adipose tissue, liver, and pancreas. Mendizabal-Zubiaga and colleagues demonstrated CB 1 to also be associated with mitochondria in skeletal, myocardial, and striated muscle, implicating CB 1 with direct involvement in peripheral energy metabolism [20]. Selective knockdown of CB 1 in adipose tissue [21], the liver [22], or skeletal muscle [23] all prevent diet-induced obesity or hyperphagia. Mice in whom CB 1 was selectively knocked down in the intestinal epithelium did not have the preference for a Western style diet (with reduced caloric intake and meal size) normally observed in wild-type mice [24]. In a preclinical model of cachexia, it was recently shown that the potent, selective CB 1 /CB 2 agonist WIN55,212-2 led to a significant reduction in the cachexia index and significantly prevented the cachexia-induced increase in gastric emptying [25].
There is strong correlative evidence from human studies that an active endocannabinoid system (ECS) is associated with visceral and subcutaneous fat accumulation [26], which is supported by many studies that CB 1 activation promotes fat cell differentiation and fat storage (see Table 1 and Figure 1 for details). For instance, in a human study by Côté and colleagues, plasma 2-arachidonoylglycerol levels correlate positively with body mass index (BMI), waist girth, intra-abdominal adiposity, fasting plasma triglyceride, and insulin levels but negatively with high-density lipoprotein cholesterol and adiponectin [27]. However, visceral fat accumulation is an important correlate with insulin resistance, and higher circulating endocannabinoids have been associated with insulin resistant obese patients [28]. The fact that there are abundant CB 1 receptors in visceral adipose tissue serves as means to target obesity and insulin resistance in human with peripheral CB 1 receptor antagonists or indeed promote weight gain with peripheral CB 1 receptor agonists. Important locations of peripheral CB1 receptors include the oral cavity, gastrointestinal tract, afferent vagus nerves, adipose tissue, liver, and pancreas. Mendizabal-Zubiaga and colleagues demonstrated CB1 to also be associated with mitochondria in skeletal, myocardial, and striated muscle, implicating CB1 with direct involvement in peripheral energy metabolism [20]. Selective knockdown of CB1 in adipose tissue [21], the liver [22], or skeletal muscle [23] all prevent diet-induced obesity or hyperphagia. Mice in whom CB1 was selectively knocked down in the intestinal epithelium did not have the preference for a Western style diet (with reduced caloric intake and meal size) normally observed in wild-type mice [24]. In a preclinical model of cachexia, it was recently shown that the potent, selective CB1/CB2 agonist WIN55,212-2 led to a significant reduction in the cachexia index and significantly prevented the cachexia-induced increase in gastric emptying [25].
There is strong correlative evidence from human studies that an active endocannabinoid system (ECS) is associated with visceral and subcutaneous fat accumulation [26], which is supported by many studies that CB1 activation promotes fat cell differentiation and fat storage (see Table 1 and Figure 1 for details). For instance, in a human study by Côté and colleagues, plasma 2-arachidonoylglycerol levels correlate positively with body mass index (BMI), waist girth, intra-abdominal adiposity, fasting plasma triglyceride, and insulin levels but negatively with high-density lipoprotein cholesterol and adiponectin [27]. However, visceral fat accumulation is an important correlate with insulin resistance, and higher circulating endocannabinoids have been associated with insulin resistant obese patients [28]. The fact that there are abundant CB1 receptors in visceral adipose tissue serves as means to target obesity and insulin resistance in human with peripheral CB1 receptor antagonists or indeed promote weight gain with peripheral CB1 receptor agonists.
Activation of hepatic CB1 has been shown to be associated with obesity and insulin Activation of hepatic CB 1 has been shown to be associated with obesity and insulin resistance (see Table 1). Measured observations include impaired metabolic function, impaired glucose and lipid metabolism, and augmentation of oxidative stress and inflammatory responses. Blocking peripheral CB 1 in liver not only has weight loss potential, but also the potential to increase insulin sensitivity and glucose metabolism in humans while reducing the potential for hepatic steatosis [29]. It is worth noting that medicines that activate the CB 1 receptor like nabilone may cause mild increase in serum liver enzymes but no cases of clinically apparent liver injury attributable to nabilone [30].
In human skeletal muscle studies, Eckardt and colleagues demonstrated that activation of the CB 1 receptor decreases insulin-mediated glucose uptake and AKT activation in cultured cells [31]. Cavuoto and colleagues also demonstrated an attenuating effect of cannabinoid signalling on cultured human muscle cell oxidative pathways in vitro, while CB 1 receptor antagonism increases whole body oxygen consumption [32]. In myotubes cultured from lean individuals, anandamide (AEA) treatment increases expression of pyruvate dehydrogenase kinase 4 (PDK4), an inhibitor of the pyruvate dehydrogenase complex, an enzyme which links glycolysis to the Krebs cycle, while CB 1 antagonism decreases PDK4 expression. PDK4 is a negative regulator of glucose oxidative metabolism in mitochondria, but is an enzyme that is also physiologically inhibited to facilitate fatty acid oxidation. A series of studies from Iannotti and colleagues show an important role of the CB 1 receptor in skeletal muscle cell differentiation and found that CB 1 receptor antagonism (using rimonabant, intra peritoneally) was beneficial at preventing the locomotor deficits in an animal model of Duchenne muscular dystrophy [33,34]. Genetic inhibition of skeletal muscle receptor was also found to improve mitochondrial performance, whole-body muscle energy expenditure, and physical endurance [23]. These studies indicate an important role for CB 1 in skeletal muscle function and metabolism.
Together, these data demonstrate that there are important direct effects of CB 1 receptor activation in adipose tissue, the GI tract, skeletal muscle, and the liver that drive the effects of CB 1 (agonism or antagonism) on body weight modulation. Table 1. An overview of some of the effects of CB 1 activation in various organs and body systems that play a role in metabolism. The combined effect of peripheral CB 1 activation is to promote appetite, and energy storage and preservation.

System/Organ
Tissue/Cell Effect of CB 1 Activation

GI system
Oral cavity CB 1 receptors are expressed in type II taste cells that also express the sweet-taste receptor, and their activation increases sweet sensitivity [35]. CB 1 receptors on the tongue increase gustatory nerve responses [35].
I cells of the small intestine CB 1 is expressed in enteroendocrine cells [41]. CB 1 inhibits the secretion of the satiation hormone cholecystokinin [41].

System/Organ
Tissue/Cell Effect of CB 1 Activation

Effects of Peripheral CB 1 Receptors on Appetite Hormones
In addition to the direct effects of CB 1 activation in peripheral tissues, there are humoral and neuronal links between peripheral CB 1 receptors and the central pathways controlling body weight through the modulation of key hormones that influence appetite.
Leptin is an adipose-derived hormone that acts on central receptors to reduce feeding and appetite, and leptin resistance is a feature of obesity. Cross-talk between central leptin and CB 1 receptors has been well documented, but leptin resistance in diet-induced obese mice can be reversed by the peripherally restricted CB 1 antagonist JD5037 [71], demonstrating that CB 1 receptors also modulate leptin sensitivity at a peripheral level, and this plays an important role in the ability of peripheral CB 1 blockade to mediate hypophagia and weight loss.
Ghrelin is a peptide hormone released in the gastrointestinal tract (mainly in the stomach and pancreas) and the brain that acts on receptors located on the vagus to stimulate appetite. The CB 1 receptor is expressed in the neuroendocrine cells of the stomach that secrete ghrelin, and CB 1 antagonism reduces ghrelin secretion, preventing appetite stimulation [40]. The peripheral-restricted CB 1 antagonist LH-21 was also found to block ghrelin-induced hyperphagia in free feeding animals [72]. Thus, the anorexigenic effect of CB 1 antagonists is at least partially a consequence of decreased gastric ghrelin secretion, and conversely CB 1 activation in the stomach will increase ghrelin, stimulating appetite and food intake through ghrelin's actions on the vagal nerve. This is supported by recent human studies that showed increased plasma levels of ghrelin after oral THC [73,74]. The ghrelin agonist anamorelin (Adlumiz ® ) has been approved in Japan for the treatment of cancer cachexia, demonstrating the utility of increasing ghrelin to improve anorexic and cachexic conditions [75].
Cholecystokinin (CCK) is a peptide hormone release from the duodenum during digestion, which acts as a hunger suppressant at receptors located on the vagus (mainly) and in the brain. The CB 1 receptor is expressed on endocrine cells of the intestinal epithelium that secrete CCK, and activation of CB 1 blocks the secretion of CCK (and the opposite true of CB 1 antagonists) [41]. The same study showed that the hypophagic effect of a peripherally restricted CB 1 antagonist in obese mice was reversed by co-administration with a CCK receptor antagonist, indicating the importance of CB 1 regulation over this appetite suppressant hormone.
Together, these studies show that peripheral activation of CB 1 modulates the activity of the key appetite-regulating hormones leptin, ghrelin, and CCK, whose receptors are located in the brain, or on the vagus nerve with direct influence on the brain via the gut-brain axis.

Gut-Brain Axis
In addition to the hormonal influence on the central integration of appetite, CB 1 receptors are expressed on vagal terminals throughout the GI tract, playing a direct role in the modulation of afferent information to the brain and the regulation of food intake (see [76] for an extensive review on this topic). GI vagal afferents play an important role in the peripheral regulation of food intake via signalling the degree of distension of the stomach, which leads to feelings of fullness and satiety. CB 1 activation inhibits the vagal afferent response to tension, thus preventing the feeling of fullness and allowing food consumption to continue [39,77].
Levels of the endogenous CB 1 agonists anandamide and 2-AG increase in the intestine in the starved state or by (lipid) feeding, and this stimulates feeding, which is abolished after sensory deafferentation or CB 1 receptor antagonism [48,78]. Argueta and DiPatrizio showed that the hyperphagia in mice given free access to a high-fat and sucrose diet was inhibited by a peripherally restricted CB 1 antagonist [45]. These researchers went on to show that mice in whom CB 1 was selectively knocked down in the intestinal epithelium did not have the preference for the high-fat and sucrose diet [24]. Thus, endogenous activation of CB 1 in the intestine increases the palatability of food through gut-brain communication.

Microbiome
A novel mechanism of action for CB 1 in the modulation of metabolism and body weight may be through modifications in the microbiome (see [79] for a recent review). Mehrpouya-Bahrami and colleagues found that a CB 1 antagonist caused changes in the gut microbial community with an increase in Akkermansia muciniphila (Verrucomicrobiaceae family) and a decrease in the Lanchnospiraceae and Erysipelotrichaceae families, although it is not clear if this was a direct effect or secondary to the improvements in metabolic dysfunction [49]. Chronic THC treatment prevented the diet-induced obesity changes in gut microbiota, particularly causing an increase in Akkermansia muciniphila [50]. Probiotic treatment has also been shown to increase CB 1 and CB 2 expression in colonic mucosa and adipose tissue [52], which was associated with improvements in disease activity in dogs with gut dysmotility disturbances [51]. Conversely, studies using germ-free mice have shown that there is an upregulation of CB 1 in the intestines that is reversed after faecal microbiota transfer [80]. These emerging studies suggest a link between the endocannabinoid system and gut bacteria that may play a role in the modulation of body weight by CB 1 at the peripheral level.

Peripherally Restricted CB 1 Antagonists
After the withdrawal of Rimonabant, researchers began developing peripherally restricted CB 1 antagonists in obesity and diabetes. Molecules such as URB447 (a mixed CB 1 /CB 2 neutral antagonist) [81], AM6545 (a CB 1 neutral antagonist) [82], TXX-522 (a CB 1 selective antagonist) [83], and LH-21 (a CB 1 neutral antagonist) [72,84] were shown to reduce feeding and body weight gain in rodents. In models of diabetes, peripherally restricted CB 1 antagonists improve glucose tolerance and insulin sensitivity [85]. This class of drugs also ameliorate other conditions associated with obesity and diabetes such as leptin resistance, fatty liver, and dyslipidemia [86,87] and reverse hyperphagia, body weight, and metabolic syndrome in a genetic model of Prader-Willi syndrome [88].
Another strategy to avoid the side effects of CB 1 antagonists is through allosteric modulation of the CB 1 receptor. The negative allosteric modulators ORG27569 [89], RVDhemopressin(α) [90], and PSNCBAM-1 [91] reduce food intake with or without a reduction in body weight in rats.
Several pharmaceutical companies are developing medicines to inhibit the peripheral CB 1 receptor (see Table 2).
Inversago Pharma has been granted rare paediatric disease designation by Food and Drug Administration (FDA) for the treatment of Prader-Willi syndrome with their periph-erally restricted CB 1 inverse agonist INV-101. The safety, tolerability, and pharmacokinetics of single ascending oral doses of INV-101 is being tested in healthy volunteers, although this trial is not recruiting at the time of writing (ClinicalTrials.gov Identifier: NCT04531150).
GFB-024 is a peripherally restricted CB 1 inverse agonist monoclonal antibody intended to treat patients with severe insulin-resistant diabetic nephropathy (DN) in development by Goldfinch Bio (https://www.goldfinchbio.com/pipeline/gfb-024/ (accessed on 10 September 2021)). Goldfinch Bio have just announced a phase 1 clinical trial to evaluate the safety and pharmacokinetics of single and repeated dosing of GFB-024 in overweight healthy volunteers (ClinicalTrials.gov Identifier: NCT04880291).
A phase 1 trial with the peripherally selective neutral CB 1 antagonist TM38837 from 7TM Pharma has been conducted in healthy subjects [104], although it is unclear whether this is an active drug development program.

Peripherally Restricted CB 1 Agonists
After the discovery of the CB 1 receptors and their important role in pain modulation, the first significant drug discovery program for peripherally restricted CB 1 agonists was analgesics. The concept was to utilize the analgesic effects of CB 1 activation without the CNS side effects, and extensive preclinical studies have demonstrated the analgesic effects of these compounds across various models of pain [126]. However, a lack of efficacy in clinical studies [124,125] meant the pharmaceutical development of these medicines was terminated. However, preclinical research with peripherally restricted CB 1 agonists continues in cancer-related pain [109,110] and migraine [111]. Other indications that have been investigated with a peripherally restricted CB 1 agonist included spasticity in multiple sclerosis [112], gastrointestinal motility issues [42,43], and anticipatory nausea [113], although none of these have been taken to clinic (see Table 2).
By contrast to the large number of peripherally restricted CB 1 antagonists in development for obesity and related metabolic disorders, far less work has been carried out to potential exploit CB 1 activation in the periphery to promote weight gain. Although appetite stimulants such as the progesterone megestrol acetate, and the steroid dexamethasone, have been used for treatment of anorexia associated with cancer, no drugs have been approved for this indication in the United States or Europe, with the exception of dronabinol, which is approved for HIV/AIDS-induced anorexia only. Thus, the development of novel pharmaceutical strategies to stimulate appetite in chronic states of anorexia (such as cancer, chronic kidney disease, and heart failure) is still a significant unmet need. ART27.13 is a CB 1 /CB 2 receptor agonist with reduced brain penetration originally developed by AstraZeneca for analgesia, now being developed by Artelo Biosciences. In a multiple-dose ascending study, a dose-dependent increase in body weight was observed (see Figure 2, ClinicalTrials.gov Identifier: NCT00689780, data on file) that was not explained by fluid retention; it was likely due to increased appetite and food intake. The clinical potential of ART27.13 to increase appetite leading to weight gain in patients with cancer anorexia is being trialed in a Phase 1b/2a study (EudraCT NUMBER:2020-000464-27).
of these compounds across various models of pain [126]. However, a lack of efficacy in clinical studies [124,125] meant the pharmaceutical development of these medicines was terminated. However, preclinical research with peripherally restricted CB1 agonists continues in cancer-related pain [109,110] and migraine [111]. Other indications that have been investigated with a peripherally restricted CB1 agonist included spasticity in multiple sclerosis [112], gastrointestinal motility issues [42,43], and anticipatory nausea [113], although none of these have been taken to clinic (see Table 2).
By contrast to the large number of peripherally restricted CB1 antagonists in development for obesity and related metabolic disorders, far less work has been carried out to potential exploit CB1 activation in the periphery to promote weight gain. Although appetite stimulants such as the progesterone megestrol acetate, and the steroid dexamethasone, have been used for treatment of anorexia associated with cancer, no drugs have been approved for this indication in the United States or Europe, with the exception of dronabinol, which is approved for HIV/AIDS-induced anorexia only. Thus, the development of novel pharmaceutical strategies to stimulate appetite in chronic states of anorexia (such as cancer, chronic kidney disease, and heart failure) is still a significant unmet need. ART27.13 is a CB1/CB2 receptor agonist with reduced brain penetration originally developed by AstraZeneca for analgesia, now being developed by Artelo Biosciences. In a multiple-dose ascending study, a dose-dependent increase in body weight was observed (see Figure 2, ClinicalTrials.gov Identifier: NCT00689780, data on file) that was not explained by fluid retention; it was likely due to increased appetite and food intake. The clinical potential of ART27.13 to increase appetite leading to weight gain in patients with cancer anorexia is being trialed in a Phase 1b/2a study (EudraCT NUMBER:2020-000464-27). The mean increase in body weight (kg) after 15 days daily treatment with AZD1940 (ART27.13) in healthy volunteers in a dose-ascending study (ClinicalTrials.gov Identifier: NCT00689780, data on file). Data are a presented as a scatterplot with mean and SD.

Peripherally Restricted Fatty Acid Amide Hydrolase (FAAH) Inhibitors
Indirect activation of peripheral cannabinoid receptors can also be achieved through peripherally restricted fatty acid amide hydrolase (FAAH) inhibitors, which increase endocannabinoid tone and promote activation of cannabinoid receptors. Such compounds have been shown in preclinical research models to be analgesic in many models, including neuropathic pain [115], diabetic neuropathy [119], chemotherapy (paclitaxel)-induced pain [116], inflammatory pain [115,117,119], visceral pain [115], and migraine and medication overuse headache [120,121] (see Table 2). Peripherally restricted FAAH inhibitors also reduce anticipatory nausea [113], protect against non-steroidal anti-inflammatory agent-induced gastric lesions [118], and reduce hyperactivity in the rat bladder induced by PGE prostaglandin E2 [123] and in an LPS model of cystitis [122].
The peripherally restricted FAAH inhibitor URB937 is in development by ExxelPharma for chronic neuropathic pain; although human clinical studies have not yet begun (https: //exxelpharma.com/pipeline/overview/ (accessed on 10 September 2021)), the use of this alternative strategy to activate peripheral cannabinoid receptors looks promising.

Conclusions
Drug discovery efforts to develop CB 1 agonists and antagonists were hampered by CNS-mediated side effects of these drugs. Second-and third-generation compounds in this area have tried to circumvent these adverse effects by selectively activating the CB 1 receptor expressed in the peripheral nervous system and major organ systems of the body. Preclinical investigation supports the importance of the CB 1 receptor throughout the gastrointestinal tract, adipose tissue, liver, pancreas, and skeletal muscle, as well as mediating humoral and afferent satiety signals to the brain. Preclinical efficacy data support the therapeutic utility of peripherally restricted CB 1 agonists in pain management, and antagonists in obesity, metabolic syndrome, and liver diseases. Preclinical data also support indirect activation of peripheral CB 1 receptors through peripherally-restricted FAAH inhibitors in pain management and bladder conditions. Translation of these findings into the clinical arena is emerging, with several pharmaceutical companies developing novel medicines in early phase 1 and 2 trials in weight gain in cancer anorexia (agonist: ART27.13), and in metabolic conditions (antagonists: INV-101, TM38837, and GFB-024), which, if successful, could result in novel, rationally designed synthetic cannabinoid medicines that demonstrate the appropriate benefit-risk profile to allow mainstream use in the modulation of weight by targeting CB 1 . Acknowledgments: In this section you can acknowledge any support given which is not covered by the author contribution or funding sections. This may include administrative and technical support, or donations in kind (e.g., materials used for experiments).

Conflicts of Interest:
S.E.O. is a paid scientific advisor to Artelo Biosciences and A.S.Y. is the Chief Scientific Officer for Artelo Biosciences.