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Targeting the Endocannabinoid System for Gastrointestinal Diseases:

Expert Rev Clin Pharmacol. 2010;3(2):193-207.

Abstract and Introduction

Cannabinoids extracted from the marijuana plant (Cannabis sativa) and synthetic cannabinoids have numerous effects on
gastrointestinal (GI) functions. Recent experimental data support an important role for cannabinoids in GI diseases. Genetic
studies in humans have proven that defects in endocannabinoid metabolism underlie functional GI disorders. Mammalian cells
have machinery, the so-called endocannabinoid system (ECS), to produce and metabolize their own cannabinoids in order to
control homeostasis of the gut in a rapidly adapting manner. Pharmacological manipulation of the ECS by cannabinoids, or by
drugs that raise the levels of endogenous cannabinoids, have shown beneficial effects on GI pathophysiology. This review gives
an introduction into the functions of the ECS in the GI tract, highlights the role of the ECS in GI diseases and addresses its
potential pharmacological exploitation.

Endocannabinoid System in the Gastrointestinal System

The marijuana plant, Cannabis sativa, is one of the earliest known plants to have been used by humans for medicinal and
recreational purposes.[1] Owing to the analgesic, anti-inflammatory and antiemetic properties, it was used as a powerful remedy
in a variety of diseases, particularly those affecting the gastrointestinal (GI) system. Naturally, it also gained attention for its
psychotropic effects, which led to its criminalization in the USA in the late 1930s.[2] This event stopped the scientific exploitation
for decades until the identification of Δ9-tetrahydrocannabinol (Δ9-THC), the major bioactive constituent of Cannabis. It did not
take long until it was realized that the effects of Δ9-THC were not based on membrane disturbances, but on specific interactions
with a receptor that was later found to be widely expressed in the CNS, the cannabinoid receptor (CB)1. This discovery opened
the alley towards a new chapter in pharmacological research and led to the concept of the endocannabinoid system (ECS).[3]
This term encompasses the endogenous cannabinoids (endocannabinoids), their receptors (CBs) and the whole enzymatic set-
up for production, uptake and degradation of endocannabinoids (Figure 1).[4] It is getting clearer that the ECS has major
implications in the pathophysiology of GI diseases.[5–7] Finally, with the discoveries of new endocannabinoids and novel CBs,
this system is constantly expanding, offering new therapeutical targets for drugs from a plant already used in ancient medical
practice.
Figure 1.

Endocannabinoid system. Synthesis of endocannabinoids involves several steps using membrane phospholipids as
substrates. NAPE-PLD and lyso-PLD catalyze the final reaction in the production of anandamide. DAG lipase uses
diacylglycerols as substrates to produce 2-AG. After diffusion into the extracellular space, anandamide and 2-AG bind to CB1
and CB2, to TRPV1 and to a potentially novel CB, such as the GPR55 receptor. Their action is terminated by cellular reuptake
via the EMT or by passive diffusion. Anandamide is subsequently degraded by fatty acid amide hydrolase and 2-AG by
monoacylglycerol-lipase and FAAH, respectively. 2-AG: 2-arachidonoylglycerol; Δ9-THC: Δ9-tetrahydrocannabinol; AA-5-HT:
Arachidonoyl-serotonin; CB: Cannabinoid receptor; DAG: Diacylglycerol; EMT: Endocannabinoid membrane transporter; FAAH:
Fatty acid amide hydrolase; Lyso-PL: Lysophospholipid; MAGL: Monoacylglycerol lipase; NAPE-PLD: N-acyl
phospatidylethanolamine phospholipase D; OEA: Oleoylethanolamide; PEA: Palmitoylethanolamide.

Cannabinoid Receptors

The first CB was discovered almost two decades ago and was logically coined CB1.[8] The second was found by homology
cloning and named CB2. The interesting finding was that not only the amino sequence but also its distribution was quite
different to that of CB1.[9] CB1s are localized throughout the gut of various species and predominantly located in the enteric
nervous system (ENS).[10–16] They are expressed prejunctionally in cholinergic but not nitrergic neurons,[11,17] which is in
agreement with functional studies demonstrating that CB1 activation leads to depression of contractile transmitter release and
relaxation in muscle strips of the gut.[18] A new study also suggests that CB1 activation interferes with purinergic P2X receptor
signaling in cholinergic neurons of the ENS during relaxation of mouse ileum muscle strips.[19] CB1 is present in extrinsic fibers
of the ENS, originating from dorsal root ganglia,[20–22] nodose ganglia[23,24] and the dorsal motor nucleus[25] and also in
human tissue, particularly in parietal cells of the stomach mucosa,[26] epithelial and plasma cells of the colon and in blood
vessel smooth muscle cells of the colon wall.[12,27] CB2 is mainly expressed in immune cells[12,28] but can be found in human
epithelial cells during inflammatory bowel disease (IBD).[12] Some reports localize CB2 to myenteric neurons of rodents and
man[29,30] (a thorough review on the expression of CBs in the ENS is given by Duncan et al.[31]). CBs are coupled to Gi/o
proteins and their stimulation inhibits adenylate cyclase and voltage-gated Ca2+ channels of the P/Q-, L- and N-type, but
activate MAPK and inwardly rectifying K+ channels,[32,33] altogether leading to a inhibition of neuronal activity. A recent study
by Boesmans et al. describes CB1 signaling in the ENS for the first time.[34] The study shows that CB1 exerts tonic control over
ENS activity and provides evidence for the production of endocannabinoids in the ENS. CB1 in the ENS may therefore function
as a 'break' for neural over-reactivity and, consequently, for uncontrolled motility and secretion.

Novel CBs

Many examples exist where the pharmacology of cannabinoid compounds cannot be explained by the classical CBs, CB1 and
CB2. Additional targets for cannabinoids have been proposed and termed non-CB1/CB2s, which are probably responsible for
the many non-CB1/CB2 effects of cannabinoids seen in neuronal activity, vasorelaxation or immune responses.[35,36] Two of
these receptors, GPR119 and GPR55, have been characterized in more detail. Similar to the classical CBs, they belong to the
family of G-protein-coupled receptors and are activated by endocannabinoids, such as anandamide, oleoylethanolamide and
palmitoylethanolamide.[37] Both of them may be interesting in GI diseases because one of them, GPR119, is expressed in β-
cells of the pancreas and enteroendocrine cells of the GI tract of humans,[38,39] while the other one, GPR55, has been detected
in the GI tract of mice.[40] The role of these receptors in GI diseases, however, has not been determined yet.

CBs in Experimental & Human GI Disease

During pathophysiological conditions, CBs display a high degree of plasticity by changing their levels of expression in adaption
to the disease. Thus, increased expression of CB1 has been reported in several mouse models of intestinal inflammation, such
as in dextran sulfate sodium (DSS)-, oil-of-mustard- and dinitrobenzene sulfonic acid (DNBS)-induced colitis,[28,41] and in
croton oil-induced intestinal inflammation,[42] paralytic ileus[43] and cholera toxin-induced diarrhea.[44] The increase was strong
in myenteric ganglia, but was also noticed in colon epithelium.[28,44] Upregulation of CB2 transcripts has been reported in colon
full-thickness samples of trinitrobenzene sulfonic acid (TNBS)-induced colitis,[45] which is in agreement with the increased
expression of CB2 in inflammatory cell infiltrate during DSS and oil-of-mustard colitis.[28] In addition, CB2 upregulation was
demonstrated in biopsies of colonic mucosa from IBD patients.[12,27] Biopsies of duodenal mucosa from patients with active
celiac disease showed high levels of CB1,[46] whereas CB1 mRNA was unaltered in the colon tissue of patients with diverticular
disease[47] and colorectal carcinoma.[48] The increase of CB expression in GI diseases indicates that enhanced
endocannabinoid signaling occurs in some pathophysiological conditions to defend against the disease mechanisms.[4]

Endogenous Cannabinoids (Endocannabinoids)

Endocannabinoids are produced 'on demand' from fatty acid precursors and are released into the extracellular space to bind to
CBs (Figure 1). The first endocannabinoids investigated in the GI tract were anandamide and 2-arachidonoylglycerol (2-AG)
during croton oil-induced intestinal inflammation in mice.[42] Basal levels of 2-AG are higher than those of anandamide in
mouse and human gut[46,47,49] but despite this difference, anandamide may have more importance in CB signaling than 2-AG
owing to its stronger affinity to CB1 and higher activity of its synthetic and degradative enzymes.[43,44,49] Anandamide is also
known to activate transient receptor potential vanilloid receptor (TRPV)1, the capsaicin receptor,[50] which may be important in
the induction of neurogenic inflammation and hypersensitivity in the GI tract.[51]
Endocannabinoids in Experimental & Human GI Disease

As with CBs, levels of endocannabinoids increase during noxious and inflammatory events in the GI tract, and the increase is
assumed to have an ameliorating effect on the disease pathology.[52] An increase in anandamide levels was found in the colon
of DNBS-treated mice, TNBS-treated rats[52] and in the jejunum of methotrexate-treated rats.[46] Infection of rat ileum with
Clostridium difficile toxin A raised concentrations of anandamide and 2-AG, an effect that was enhanced by pretreatment with
an inhibitor of fatty acid amide hydrolase (FAAH), the main degrading enzyme of anandamide.[53] The latter report
demonstrated that, different from what has been observed in animal models of colitis and celiac disease, the increased level of
anandamide was not beneficial but caused enteritis via activation of TRPV1 and substance P release. Increased
endocannabinoid levels were also measured during inflammatory GI diseases and colon cancer in humans. In patients with
ulcerative colitis, diverticulitis and active celiac disease, anandamide was approximately two-fold higher than in control tissue
and, in the case of celiac disease, returned to normal levels after remission or after initiation of a gluten-free diet.[46,47,52] In
biopsies from adenomatous colon, levels of endocannabinoids were increased three- and two-fold compared with normal
mucosa,[48] pointing at a potential role of the ECS in colon carcinogenesis. Interestingly, a study by Izzo et al. recently
demonstrated that the carcinogen azoxymethane increased levels of 2-AG in mouse colon.[54] This increase was associated
with the formation of aberrant crypt foci, which are early indicators of colon carcinoma. High-fat diet in mice also affects
endocannabinoid levels in the GI tract by decreasing expression of anandamide in the stomach and small intestine and raising
2-AG in the small intestine.[55,56]

The site of the endocannabinoid production in the gut is less investigated. The colorectal carcinoma cell line CaCo-2 was
demonstrated to contain measurable levels of anandamide and 2-AG.[48] Endocannabinoids are, therefore, most probably
produced in colonic epithelium but may also originate from the vascular system and the inflammatory cells of the bowel wall,
since estrogen-stimulated human umbilical vein endothelial cells and lymphocytes are able to release anandamide.[57,58] In
addition, macrophages, basophiles and platelets are sources of anandamide and 2-AG.[59–62] Immunohistochemical detection
of enzymes that synthesize endocannabinoids has provided new information on the potential release sites of endocannabinoids
in the GI tract. A recent study in human colon localizes N-acyl phospatidylethanolamine phospholipase D (NAPE-PLD), the key
enzyme for the production of anandamide, and diacylglycerol lipase (DAGL), which is responsible for the production of 2-AG, to
many cell types of the colon, such as epithelial and lamina propria cells, muscle cells and myenteric plexus neurons.[27] In a
study of primary myenteric neuron culture, CB1 activation and treatment with FAAH antagonists led to a significant reduction in
spike activity from which the authors concluded that an endogenous ligand of CB1 must have been produced in myenteric
neurons.[34] Extrinsic sensory fibers represent a further source of anandamide, since NAPE-PLD was found in mouse dorsal
root ganglia.[63] The finding is supported by a study of KCl- and capsaicin-induced anandamide release in a primary neuronal
culture of capsaicin-sensitive primary afferents.[64] In summary, endocannabinoids derive from heterogenous sources of the GI
tract, which underlines that the ECS plays an important role in the functional regulation within, and probably between, various
cell systems of the gut.

Synthesis, Degradation & Reuptake of Endocannabinoids

Specific phospholipases are responsible for the synthesis of endocannabinoids from fatty acid precursors. Key enzyme for the
production of anandamide is NAPE-PLD, which cleaves a membrane lipid precursor of anandamide, while the 2-AG
synthesizing enzyme, DAGL, cleaves diacylglycerols (reviewed along with an update on new enzymes in[65]). Within the GI
tract, NAPE-PLD has been observed in the stomach of mice,[66] in enterocytes and lamina propria cells of the rat small
intestine[67] and the human colon.[27] DAGL is present in the epithelium, lamina propria, smooth muscle and enteric neurons of
the human colon.[27] Breakdown of anandamide is catalyzed by FAAH,[68,69] while 2-AG is a substrate for both FAAH[70,71] and
monoacylglycerol lipase (MAGL) (Figure 1).[72] Whether the recently reported subtypes FAAH1 and FAAH2 display
physiological or pharmacological relevance remains to be established.[73] The endocannabinoid-degrading enzymes are
localized in enteric neurons and GI epithelial cells of rodents and man.[27,31,74] Endocannabinoids traverse the plasma
membrane unaided or with the help of an endocannabinoid membrane transporter (EMT) although its existence is still a matter
of controversy.[75] In a recent study, anandamide transporters have been characterized and identified as fatty acid binding
proteins, which transport anandamide from the plasma membrane to the endoplasmatic reticulum for inactivation by FAAH.[76]
These proteins may represent powerful future targets for pharmacological manipulation.

Enzymes & Transporters of Endocannabinoids in GI Disease

All the components of the ECS work in an orchestrated manner (i.e., not only endocannabinoids, but also the synthesizing and
degrading enzymes and their transporters display plastic behavior in GI pathophysiology). FAAH activity was demonstarted to
increase in croton oil-induced intestinal inflammation of the mouse,[42] and FAAH-positive immune cells were observed in the
colonic lamina propria of human ulcerative colitis.[27] MAGL has been investigated only under physiological conditions in mice
during whole-gut transit, which was attenuated by the (rather weak and unselective) MAGL inhibitor, URB602.[74] Other data
using a highly selective MAGL blocker, JZL184, suggest many overlapping functions of 2-AG and anandamide in vivo.[77] A
recent immunohistochemical study in human ulcerative colitis demonstrates increased expression of MAGL in colon epithelium
and immune cells but a decrease in NAPE-PLD immunoreactivity.[27] The EMT inhibitor, VDM11, prevented diarrhea by raising
anandamide levels and indirect activation of CB1,[44] and protected mice from DNBS-induced colitis.[52] By contrast, in a mouse
model of paralytic ileus, VDM11 worsened disease progress after raising endocannabinoid levels.[43] The role of the EMT in the
GI tract is, thus, still elusive and needs further investigation.

Regulation of ECS Components

Expression of the components of the ECS during physiological and pathophysiological conditions in the GI tract is subject to
regulation, which can occur at the levels of endocannabinoid production and release of the enzymes that catalyze
endocannabinoid synthesis (e.g., NAPE-PLD and DAGL). Expression can also be regulated at the level of CBs, transporters
and endocannabinoid-degrading enzymes (e.g., FAAH and MAGL). Although there is a considerable amount of data
demonstrating CB and endocannabinoid upregulation during GI pathophysiology, description on how this upregulation is
triggered is lacking. For instance, inflammatory mediators, such as IFN-γ, have been demonstrated to increase CB2 expression
in mouse immunocytes,[78] while cannabinoid agonists may upregulate CB1 in T cells.[79] In a model of neuropathic pain,
glucocorticoid receptors were demonstrated to regulate CB1 expression in the spinal cord.[80] Neuronal excitotoxicity induced
by intrastriatal injection of N-methyl-D-aspartic acid increased anandamide precursors,[81] indicating mechanisms aiming at
neuroprotection may have an impact on ECS components. Whether these mechanisms also apply for ECS regulation in the gut
remains to be explored.

Effects of Ligands on CBs in GI Physiology

Cannabinoids that act via central and peripheral CB1s influence fundamental features of gut physiology (i.e., food intake,
motility and mucosal homeostasis). Agonists of CB1 increase food intake and inhibit vomiting, while antagonists inhibit food
intake and induce vomiting.[25,82–87] Regulation of motility is a prominent task of CB1, and any pharmacological manipulation
has an impact on its effect. CB1 agonism inhibits gastric fundus contractions,[88] emptying of the stomach, lower esophageal
sphincter relaxation and contractions of the ileum and colon, and slows down intestinal and colonic transit (reviewed in[89]).
Conversely, CB1 antagonism increases gastric emptying and intestinal motility.[89] The effects on GI motility seen in rodents
also apply to human tissue. In vitro experiments in human ileum and colon confirm that CB1 ligands inhibit muscle contractility.
[47,90–92] Control of motility via CB can also be induced indirectly through inhibition of FAAH and an increase in
1
endocannabinoid levels.[93,94] CB2s are not involved in the physiological regulation of GI motility; however, they could come
into play in pathophysiological conditions.[30] CB1 not only inhibits motility but also gastric and intestinal secretion.[15,44,95] A
study by MacNaughton et al. in the guinea pig ileum has addressed how CB1 may be integrated in this inhibitory mechanism,
which involves activation of capsaicin-sensitive neurons.[16] The capsaicin receptor, TRPV1, which plays a significant
importance in visceral hypersensitivity,[96] is mainly expressed in extrinsic primary afferent fibers in the GI tract, where it
colocalizes with CB1.[16]

Pharmacological Targets of the ECS in GI Disease

A prominent feature of eating disorders and cancer chemotherapy that can be influenced by cannabinoids is vomiting (emesis).
Animal models have demonstrated that cannabinoid ligands reduce vomiting.[85,86,97] The involved receptors, CB1 and TRPV1,
are located in the brainstem and have been suggested to play a key role in the regulation of emesis. In addition, CB2 receptors
are also present in the brainstem and inhibit vomiting in the ferret when stimulated together with CB1.[98] The maintenance of
an endogenous tone by anandamide-stimulated CB1s may be the most important mechanism in the control of emesis during
physiological states.[86] High levels of anandamide in the brainstem, therefore, may be useful in pathophysiological conditions,
such as nausea. They can be generated by FAAH and EMT blockers that prolong the action of anandamide by inhibiting its
degradation or its clearance from the extracellular space.[99] In fact, the FAAH blocker, URB597, was used successfully to
suppress lithium-induced nausea in rats[100] and decreased morphine 6-glucoronide-induced emesis in ferrets.[86] Blockade of
the EMT with VDM11 prevented emesis in the least shrew.[101]

It has been known for years that Cannabis inhibits vomiting induced by chemotherapy.[97] Cannabinoid compounds are now
being introduced into clinical practice for the prevention of emesis in patients receiving chemotherapy, especially when the
symptomatic control of emesis with 5-hydroxytryptamine (5-HT3) receptor antagonists, and dexamethasone has failed.[102] The
synthetic cannabinoid compound, nabilone, has been approved recently for the treatment of chemotherapy-induced nausea
(Cesamet®, Valeant Pharmaceuticals, CA, USA). A systematic review of the literature suggests nabilone as being superior to
the placebo, domperidone or prochlorperazine, but not to metoclopramide or chlorpromazine.[103] In a meta-analysis, nabilone
and dronabinol demonstrated clear superiority over neuroleptic antiemetics in cancer chemotherapy.[104] However, treating
emesis with cannabinoids may not be without concern, since administration of 2-AG – but not of anandamide – induces
vomiting in the least shrew,[101] maybe not directly via CB1 but via cyclooxygenase-dependent downstream metabolites.[105]
The main reason why the use of cannabinoids during chemotherapy remains unsatisfactory is because cannabinoids cause
severe psychotropic side effects, such as depression and hallucinations.[102] Nevertheless, patients show a clear preference to
cannabinoid medication and a 'high' sensation could be considered as a beneficial side effect.[106] To be comprehensive, it
needs to be mentioned that cases of paroxysmal vomiting with compulsive hot bathing have been reported in patients with
chronic Cannabis use.[107–109] The etiology of this symptom, however, is not yet known.

Gastroesophageal Reflux Disease & Gastric Ulcer

Gastroesophageal reflux is caused by transient lower esophageal sphincter relaxation (TLESR) triggered by postprandial
gastric distension. Derangement of the sphincter mechanism may also contribute to gastroesophageal reflux disease. CB1
agonists have shown to inhibit TLESR via brainstem mechanisms in dogs[110] and ferrets.[111] In a recent study in humans, Δ9-
THC significantly inhibited the increase in meal-induced TLESR and reduced basal lower esophageal sphincter pressure,
suggesting the involvement of CBs in TLESR.[112] Since CB1 agonists also inhibit gastric acid secretion,[15,95] they could
represent powerful drugs in the treatment of gastroesophageal reflux disease. In several experimental models of gastric ulcer
disease, CB1 activation has proved effective in the protection of the gastric mucosa.[113–115]

Irritable Bowel Syndrome

Irritable bowel syndrome (IBS) is a common disorder in the Western world and affects 10–20% of adults.[116] It belongs to the
group of so-called functional bowel disorders, which are characterized by the absence of organic abnormalities of the GI tract.
Typical symptoms of IBS include abdominal discomfort, pain, bloating and altered bowel habits.[117] The syndrome has been
defined recently in the Rome III criteria: recurrent abdominal pain or discomfort for at least 3 days per month over 3 months
associated with either an improvement with defecation or a change in stool frequency or stool form.[118] Despite the various
symptoms that constitute IBS, three clinical forms can be differentiated: diarrhea-predominant IBS (D-IBS), constipation-
predominant IBS (C-IBS) and mixed-form IBS.[117] The pathophysiology of IBS is complex and incorporates biological as well
as psychosocial factors (Figure 2).[118] Albeit the absence of organic failure, it is believed that altered gut motility, visceral
hypersensitivity and deranged gut–brain signaling underlies the etiology of IBS. Disturbances of motility and visceral
hypersensitivity would be suitable for a treatment with cannabinoids because of the important role the ECS plays herein.
Beneficial effects of cannabinoids should be mediated via CB1 located on enteric nerves, extrinsic sensory fibers and colon
epithelium, while CB2 could play a role in inflammatory conditions acting on cells involved in the immune response. Since IBS
consists of a multitude of symptoms, different ligands of CBs, also administered in combination, could fight individual symptoms
and, thus, lead to an improvement.
Figure 2.

Effects of the endocannabinoid system (ECS) in irritable bowel syndrome. The ECS is involved in the regulation of many
factors during IBS. Arrows indicate each of these factors and show the components of the ECS that are involved. Each of these
components may represent targets for a potential treatment. AEA: Anandamide; CB: Cannabinoid receptor; EMT:
Endocannabinoid membrane transporter; FAAH: Fatty acid amide hydrolase.

IBS & Motility Gut motility and central pain processing has been investigated in IBS patients (reviewed in[119]). In D-IBS, for
instance, colonic transit time is accelerated and high-amplitude-propagated contractions are increased,[120–122] whereas in C-
IBS, transit is slower.[123,124] A consistent finding in IBS patients is that they display exaggerated phasic colonic contractions
after a meal,[121,122,125] although this alone cannot explain the entire syndrome. Two reasons would make it worthwhile to
investigate cannabinoids in IBS. First, cannabinoids have an important role in the motor control of the GI tract and, second,
altered gut motility is a potential factor in IBS etiology. Agonists of CB1s have all been demonstrated to inhibit gut motility in
vitro.[18,126] According to a new study, it also seems that the peristaltic reflex is under tonic control of CB1.[127] Therefore,
agonists of CB1 would be helpful in managing diarrhea, while antagonists of CB1 could be used in the treatment of constipation
and dyspepsia that frequently overlaps with IBS.[128] CB2 receptors may regulate gut motility during inflammatory conditions.[30]
Testing CB2 in IBS would be of interest owing to the changed bowel motility we observe in these patients.

IBS & Secretion Any disturbances in fluid secretion and water absorbance will lead to diarrhea. CB1 stimulation has been
demonstrated to inhibit electrical stimulation-induced ileal secretion in rats.[129] Castor oil- and cholera toxin-induced diarrhea in
rodents were also inhibited by CB1 agonists.[44,130] The role of endocannabinoids in secretion has not been yet evaluated in
IBS, although it would be worthwhile, considering the antidiarrheal abilities of CB1 agonists and the obvious fact that Cannabis
has been used for centuries in traditional medicine treating diarrhea. The antidiarrheal abilities of CB1 agonists are probably a
combination of their depressing effects on excitatory transmitter release and over-reactivity in the myenteric plexus[34] (resulting
in a slowdown of motility) and on transmitter release from extrinsic primary afferents that normally act on cholinergic secretory
pathways in submucosal ganglia[16] (resulting in reduced secretion).

IBS & Visceral Hypersensitivity Another common symptom of IBS is abdominal pain, and it is thought that both peripheral
and central mechanisms contribute to its development.[131–133] In particular, sensitization of visceral afferent fibers by acute or
chronic inflammation could be involved in the generation of abdominal pain.[132–135] Studies in animal models have shown that
cannabinoids produce analgesic effects and play an important role in visceral sensation.[136–138] Thus, activation of CBs by
CB1 and CB2 agonists reduced the visceromotor response in rodents to graded colorectal distension.[138] Hyperalgesia
induced by TNBS colitis lowered the amounts of CB agonists needed to reduce sensitivity to colorectal distension, suggesting
that, in hypersensitive states, patients might be more susceptible to cannabinoid treatment.[137,138] Where exactly CB1and CB2
agonists act to regulate visceral pain is still not clear. A peripheral site of action is possible, since bradykinin-induced activity in
mesenteric afferents were reduced by a CB2 agonist in vivo[139] and a peripherally restricted CB1 agonist was able to reduce
pain during colorectal distension.[140] It is not known whether these agonists would act the same way in humans but, in a
randomized placebo-controlled study, the CB1 and CB2 ligand dronabinol relaxed the colon, reduced postprandial colonic
motility and increased sensation, while gas sensation thresholds were unchanged.[141] To reduce visceral sensation, CBs could
interact with other analgesic receptors, such as k-opioid receptors (KORs), which colocalize with CBs in myenteric neuronal cell
cultures[13] and are able to reduce sensation to colorectal distension in IBS patients upon activation.[142] In this context, it is
interesting that Salivinorin A, the active ingredient of the plant Salvia divinorum, exerts both KOR- and CB1-/CB2-sensitive
effects on motility and secretion in the mouse colon in vitro,[143] and reduces ileitis-induced hypermotility via KOR and CB1in
vivo.[144] TRPV1, which can be activated by anandamide, also has been implicated in visceral hypersensitivity to colorectal
distension of rodents,[145] and is believed to act comparably in humans. An increased density of TRPV1 immunoreactive nerve
fibers has been demonstrated in biopsies of patients with IBS.[146] Blockade of TRPV1 in visceral hyperalgesia may be superior
to that of CB1 because application of the CB1 antagonist rimonabant causes hypoalgesia during repeated colorectal distension,
something that is not observed during treatment with TRPV1 antagonists.[147] Altogether, it would be worth targeting CB1, CB2
and TRPV1 simultaneously to see a synergistic or additive effect on pain sensation. FAAH blockers, such as N-arachidonoyl-
serotonin that also act as TRPV1 antagonists[148] may become useful drugs for the treatment of hypersensitivity associated
with IBS. In an animal model of visceral nocieception, pharmacological inhibition and genetic deletion of FAAH demonstrated
analgesic effects and reduced the severity of gastric irritations caused by diclofenac.[149]

IBS & Gut Microbiota An increasing number of studies support the concept that bacterial overgrowth plays a role in the
pathophysiology of IBS.[150] Certain probiotics have been observed to reduce visceral hypersensitivity in rats and mice[151,152]
and to alleviate symptoms in IBS patients.[153]Lactobacillus acidophilus increased CB2 mRNA expression in cultured epithelial
cells in vitro and in colonic epithelial cells in vivo after chronic treatment, while the CB2 antagonist, AM630, prevented the L.
acidophilus-induced reduction of rectal sensitivity.[154] It is however not clear how a change in epithelial expression of CB2
could influence visceral sensitivity. No data are available yet on CB1, FAAH or the roles they could play in the interaction with
microbiota. Further studies are needed to explore how microbiota influence the ECS and regulate CB expression.

IBS & Genetics A single-nucleotide polymorphism (C385A) has been discovered recently in the FAAH gene,[155] which
enhances sensitivity to proteolytic degradation.[156] In this study, the FAAH polymorphism was associated with accelerated
colonic transit in patients with D-IBS but not with C-IBS and mixed form-IBS. The functional alterations caused by the FAAH
polymorphism, however, are not yet fully characterized. It should be noted that IBD patients do not display such genetic
associations.[157]

Inflammatory Bowel Disease

There are approximately 2.2 million people in Europe and 1.4 million in North America who suffer from inflammatory bowel
attacks.[158] These attacks are often signs of an idiopathic, chronic and relapsing inflammation of the GI tract, termed IBD, and
manifest mainly in two clinical forms, ulcerative colitis and Crohn's disease. A growing number of studies demonstrated that the
ECS may play a significant role in GI inflammation and IBD. Levels of anandamide are elevated in rectum biopsies from
patients with ulcerative colitis.[52] In addition, CB expression is increased in response to GI inflammation in animal models. In
croton oil-induced intestinal inflammation and in DSS colitis of mice, CB1 is increased in myenteric ganglia of the colon,[28,41,42]
while mRNA for CB2 is increased in full-thickness samples of the colon during TNBS-induced colitis.[45] CB2 upregulation was
also noticed in infiltrated cells of the colon mucosa during DSS colitis[28] and in human colonic mucosa during IBD.[12]
Anecdotal reports exist that patients suffering from IBD experience relief when smoking marijuana,[5] indicating that stimulation
of CB receptors is protective in intestinal inflammation. This has been confirmed by studies in mice that demonstrated an
improvement of colitis parameters after CB1 or CB2 stimulation,[28,41,45] although some protective effects have been described
for the CB1 antagonist, rimonabant, in indomethacin-induced ileitis in rats.[159] Conversely, experimentally induced colitis is
worse in CB1- or CB2-knockout mice or mice treated with CB1 or CB2 antagonists.[41,45] How CB receptors contribute to the
improvement of colitis is not completely understood but an effect on the colon epithelium is quite conceivable. CB1s have been
observed to promote wound healing of colon epithelial cells[12] and activation of CB2 inhibited TNF-α-induced IL-8 release in
HT-29 cells.[160] CB1 and CB2 agonists may also reduce hypermotility and diarrhea during IBD, as previously shown in animal
models.[42,161] Blockade of FAAH activity and anandamide reuptake in order to increase endogenous anandamide levels are
also protective.[52,157] Owing to the changes in FAAH expression during experimental colitis[45,162] and the presence of a
FAAH gene polymorphism (C385A) in D-IBS, FAAH polymorphism was also investigated in patients with Crohn's disease.
However, no significant difference was detected between Crohn's patients and healthy people.[157] In summary, there is strong
indication that CBs and endocannabinoids play a key role in the protection against GI inflammation.[163] Pharmacological
intervention of the ECS for the treatment of IBD would, therefore, be extremely useful ().

Box 1. Potential therapeutic effects of cannabinoid compounds in gastrointestinal diseases.

CB1agonists

IBS: reduction of visceral pain,[138] secretion,[16,143] hypermotility and diarrhea[7,42,44]

Emesis: reduction of emesis[25,106]

IBD: reduction of intestinal inflammation, visceral pain, hypermotility and diarrhea[38,41,42]

CB2agonists

IBS: reduction of visceral hypersensitivity,[138,154] secretion[143] and hypermotility[30]

Emesis: inhibition of emesis[98]

IBD: reduction of inflammation and visceral hypersensitivity[12,28,45]

CB1antagonists

Diabetic gastroparesis: increase in gastric emptying[55]

Ileus: increase in gastrointestinal transit time in paralytic and septic ileus[43,182]

CB2antagonists

Ileus: increase in gastrointestinal transit in septic ileus[182]

FAAH blockers

Colon cancer: reduction of aberrant crypt foci[54]

Emesis: inhibition of toxin-induced vomiting[180]

IBD: reduction of gastrointestinal inflammation[41]

Endocannabinoid reuptake inhibitors

Emesis: inhibition of apomorphin-induced emesis[101]

IBD: reduction of gastrointestinal inflammation[52]

CB: Cannabinoid receptor; FAAH: Fatty acid amide hydrolase; IBD: Inflammatory bowel disease; IBS: Irritable bowel syndrome.
Colon Cancer

Cannabinoids have recently received much interest as a potential therapy in colorectal cancer, which is one of the leading
causes of death after lung cancer in the Western hemisphere.[164] The interest is based on the cannabinoids' ability to suppress
proliferation and to induce apoptosis in tumor cells.[48,165,166] Studies in colorectal cancer cell lines indicate that the
antiproliferative and apoptotic effects are mediated via CB1 and, depending on the investigated cell line, maybe via CB2.[48,167]
Anandamide inhibits migration of colon carcinoma cells via CB1[168] and induces a nonapoptotic, non-necrotic cell death that
involves COX-2 expression.[169] Since colorectal tumor cells express high levels of COX-2,[170] which promotes tumorigenesis,
[171] anandamide would be very useful in the treatment of tumor cells that have become resistant to apoptosis.[169] CB -
1
induced apoptosis involves several pathways and mechanisms, such as inhibition of RAS–MAPK and PI3K–AKT pathways,[167]
downregulation of the antiapoptotic factor survivin[172] and activation of ceramide.[173] In particular, ceramide seems to be an
important downstream mediator of CB during apoptosis of human colon cancer cells. Both CB1 and CB2 activation have been
demonstrated to induce apoptosis in DLD-1 and HT29 cells via TNF-α-sensitive ceramide synthesis.[174] It should be also noted
that CB1 could play a role in the antiproliferative effects of estrogens because 17β-estradiol induces CB1 gene expression in a
variety of human colon cancer cells.[175] Adaptive changes of ECS components were also demonstrated during carcinogenesis.
Endocannabinoids, such as anandamide and 2-AG, were increased several-fold in adenomas and colorectal cancer compared
with normal mucosa,[48] while CB1 but not CB2 was downregulated in colon carcinoma, indicating that absence of CB1
promotes colon carcinogenesis.[172] The effects of cannabinoid drugs on colon carcinoma have been mostly studied in tumor
models using azoxymethane-treated and adenomatous polyposis coli (APC) mice. azoxymethane is a carcinogen that induces
early neoplastic lesions, so-called aberrant crypt foci, which occurs with a simultaneous rise in 2-AG levels.[54] The FAAH
inhibitor, N-arachidonoylserotonin, reduces aberrant crypt foci formation, indicating that high levels of endocannabinoids have
an impact on neoplastic lesion development.[54] APC mice are used as a model to study cancer progression. They have a
germ-line mutation in the APC (tumor-suppressor) gene that causes spontaneous development of intestinal polyps. Using this
model, Wang et al. could show that the antitumor effects of CB1 seen in vitro also apply to in vivo models of colon cancer.[172]
Knockout of the CB1 gene in APC mice or treatment with the CB1 antagonist, AM251, increased colonic cancer burden
compared with their littermates.[172] In this cancer model, the authors also found a high rate of DNA methylation of the CNR1
promotor, indicating epigenetic silencing of the CB1 gene.

In summary, cannabinoid drugs could be protective in colorectal cancer, either through activation of CB1 or through
enhancement of endocannabinoid levels by FAAH inhibition. Although the potential use of cannabinoids as antitumor drugs is
still in a preclinical phase, in the meantime, they may serve as valuable supportive drugs for cancer patients to alleviate pain
and nausea during chemotherapy.

Expert Commentary

The ECS represents a perfect pharmacological target for cannabinoid compounds. On the one hand, there is a pool of
cannabinoids that can be extracted from Cannabis plants and chemically modified; on the other hand, we have a pool of
endocannabinoids in our own organism. All molecules of a cell that participate in the orchestrated metabolism of
endocannabinoids belong to the ECS. This integrative system offers many hot targets for intervention and can be manipulated
at several stages (e.g., at the CBs, at their downstream pathways or at the levels of synthesis and degradation of their ligands –
the endocannabinoids). Despite the vast amount of cannabinoid literature, cannabinoid research in the GI tract is not greatly
represented. A PubMed search revealed more than 120 reviews related to cannabinoids for 2009, but only a handful were
related to the GI tract. The recent data on new safe and nonpsychotropic cannabinoids would justify entering clinical trials much
earlier. However, the reasons for the slow process from bench to bedside lies within the cannabinoid drug itself. There is still the
lingering problem that CB1 ligands cause psychotropic side effects, such as drowsiness, sedation, euphoria, dizziness and, in
their extreme forms, depression, hallucination and paranoia. Cannabis users are probably familiar with these signs. The
removal of the CB1 antagonist and antiobesity drug rimonabant (Acomplia®, Sanofi-Aventis, UK) from the European market
only exemplifies this unsolved issue.[176] The unwanted psychotropic effects may only be overcome by manipulating and
barring CB1 ligands from crossing the blood–brain barrier. A difficult task considering the lipophilicity of these molecules. In
addition, the manipulation may, at the same time, compromise the pharmacological response that now would entirely depend
on the stimulation of peripheral CB1. Given the large expression of CB1 in the brain, it is questionable whether antiobesity drugs
and drugs controlling emesis can work at their full potential if excluded from central binding sites. It would be necessary to
delineate how much of the effects of CB1 ligands are mediated via central and/or peripheral mechanisms. The ECS, however,
may hold more solutions to address this problem. One of these solutions is to focus on another class of cannabinoid
compounds with no or only very weak psychoactivity, such as cannabidiol and Δ9-tetrahydrocannabivarin.[177] Very little is
known regarding the effects of these compounds in GI pathophysiology. In mice, cannabidiol was protective in colitis[162] and
reduced hypermotility in ileitis,[178] while Δ9-tetrahydrocannabivarin caused reduced food intake and caused a decrease in
bodyweight.[179] Their mechanism is still unknown, but it is hypothesised that these drugs may act via new, unknown CBs, the
pharmacological potential of which we are just beginning to understand through research.[36] Finally, great hope lies in the use
of drugs, such as FAAH inhibitors, which are able to increase the level of endocannabinoids. They have proven effective in
animal models of emesis[100,180] and colon cancer ().[54] Their effectiveness in the protection against colitis[41,52,157] would
make them very useful for the treatment of severe inflammatory GI diseases in the future.

Box 1. Potential therapeutic effects of cannabinoid compounds in gastrointestinal diseases.

CB1agonists

IBS: reduction of visceral pain,[138] secretion,[16,143] hypermotility and diarrhea[7,42,44]

Emesis: reduction of emesis[25,106]

IBD: reduction of intestinal inflammation, visceral pain, hypermotility and diarrhea[38,41,42]

CB2agonists

IBS: reduction of visceral hypersensitivity,[138,154] secretion[143] and hypermotility[30]

Emesis: inhibition of emesis[98]

IBD: reduction of inflammation and visceral hypersensitivity[12,28,45]

CB1antagonists

Diabetic gastroparesis: increase in gastric emptying[55]

Ileus: increase in gastrointestinal transit time in paralytic and septic ileus[43,182]

CB2antagonists

Ileus: increase in gastrointestinal transit in septic ileus[182]

FAAH blockers

Colon cancer: reduction of aberrant crypt foci[54]

Emesis: inhibition of toxin-induced vomiting[180]

IBD: reduction of gastrointestinal inflammation[41]

Endocannabinoid reuptake inhibitors

Emesis: inhibition of apomorphin-induced emesis[101]

IBD: reduction of gastrointestinal inflammation[52]

CB: Cannabinoid receptor; FAAH: Fatty acid amide hydrolase; IBD: Inflammatory bowel disease; IBS: Irritable bowel syndrome.

Five-year View
Cannabinoid research is a rapidly expanding field and within the next few years, we can expect an increasing number of
cannabinoid drugs in trials addressing the GI diseases outlined in this review. The recent and important finding of a FAAH
polymorphism associated with accelerated colonic transit in patients with D-IBS will certainly lead to a higher interest in probing
cannabinoid drugs in IBS.[155] As a multifactorial disease related to hyperalgesia and pain perception, IBS offers a wide range
of targets for testing cannabinoids. A promising chapter in cannabinoid research that is set to be pursued in the coming years
will be the investigation of cannabinoid drugs that are effective in GI diseases, but operate independently of CB1 and CB2.
Since they are safe and centrally not active, two such compounds, cannabidiol and Δ9-tetrahydrocannabivarin, could already go
into clinical trials to test their efficacy in GI diseases, such as obesity, GI inflammation and colon cancer. A Phase III study is in
progress to test the ability of Sativex® () to alleviate pain in advanced cancer patients who are refractory to conventional pain
treatment.[165] The outcome of this study will give more insight into the usability of non-CB1/CB2-acting cannabinoids in GI
diseases.

Box 2. Currently available cannabinoid drugs.

Dronabinol (Marinol®, Solvay Pharmaceuticals, Brussels, Belgium): an agonist of both CB1and CB2and has been
marketed as an appetite stimulant and antiemetic. It is a synthetic Δ9-THC and taken orally as a capsule

Δ9-THC/cannabidiol: a combination of these two compounds is available in Canada under the brand name
Sativex®(GW Pharmaceuticals, Salisbury, UK). It is prescribed against pain and spasticity in multiple sclerosis and
provided as a sublingual oromucosal spray

Nabilone: a synthetic Δ9-THC analogue that is marketed under the brand name Cesamet®(Valeant Pharmaceuticals
International) and used for treating patients with chemotherapy-induced nausea and vomiting. It is taken orally as a
capsule

Δ9-THC: Δ9-tetrahydrocannabinol; CB: Cannabinoid receptor.

The main obstacle for the use of CB1 ligands lies in their psychotropic side effects, such as highs, hallucinations, depression or
anxiety. From targeting the CB1 receptor, we learned that both agonists and antagonists result in serious side effects and
strategies bypassing or minimizing central involvement of CB ligands are urgently needed. One strategy is to target the ECS at
sites distinct of the CB1 receptor and, thus, characterization of all structures in the ECS, including the novel cannabinoid
receptors – GPR55 and GPR119 – are wanted. Another strategy would be to modify cannabinoid drugs to stop them from
traversing the blood–brain barrier or to diminish their psychoactivity. This will dominate cannabinoid research in the
pharmaceutical industry in the near future. Furthermore, for most of the synthetic cannabinoid drugs, oral bioavailability will also
have to be addressed as well.

Certainly, there will be more basic research and clinical trials for drugs that enhance endocannabinoid levels, such as FAAH
inhibitors and EMT blockers, as these drugs seem to be devoid of psychotropic side effects, although they are still poorly
characterized. Both of them could be useful in any of the GI diseases discussed in this review. Owing to the low expression of
CB2 in the brain, CB2 agonists will become extremely interesting for the treatment of IBDs. Finally, the bottle of targeting
endocannabinoid synthesis and release has yet to be opened, and compounds, both activators and blockers that specifically
act on the involved enzymes, are warranted.[181] This would enable the investigation of the ECS from another site.

Sidebar
Key Issues

Phytocannabinoids from the plant Cannabis sativaor synthetic cannabinoid compounds have fundamental effects on
gastrointestinal (GI) physiology and pathophysiology.

These effects are mediated via two G-protein-coupled receptors, cannabinoid receptor (CB)1and CB2, which are widely
expressed in the GI tract.

The endocannabinoids anandamide and 2-arachidonoylglycerol are produced 'on demand' and act via (novel) CBs. Next
to endocannabinoids, many endocannabinoid-related lipid molecules are produced endogenously to act at CBs and
transient receptor potential vanilloid receptor 1, which may have an impact on GI function.
 Endocannabinoids and CBs display high plasticity of expression during GI diseases (e.g., they are upregulated in
inflammatory conditions). This boost in expression is probably aimed at protecting the homeostasis of the gut mucosa by
reducing the over-reactivity of the enteric nervous system network during hypermotility and diarrhea. The exact
underlying mechanisms are still elusive.

Two strategies can be followed in cannabinoid therapy: activation of CBs by exogenous ligands and/or inhibition of FAAH
activity or cellular reuptake of endocannabinoids with the aim of increasing endocannabinoid levels to drive CB activity.

Agonists of CB1may have therapeutical value in emesis, irritable bowel disease (IBD), colon cancer and functional
disorders associated with hypermotility and diarrhea. Agonists of CB2may be valuable in IBD.

Cannabinoids could be also valuable drugs in IBS; however, this still needs further investigation.

CB1stimulation as a therapeutical approach is still hampered by adverse psychotropic side effects.

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