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Modulation of peristalsis by cannabinoid CB1 ligands in the isolated guinea-pig ileum
Modulation of peristalsis by cannabinoid CB1 ligands in the isolated guinea-pig ileum
Angelo A Izzo, Nicola Mascolo,  Marcello Tonini, and Francesco Capasso
Department of Experimental Pharmacology, University of Naples ‘Federico II', via D. Montesano 49, 80131 Naples, Italy
Department of Pharmaceutical Sciences, University of Salerno, Via Ponte Don Melillo 84084 Fisciano (SA), Italy
Department of Internal Medicine and Therapeutics, Division of Pharmacology and Toxicology, University of Pavia, Piazza Botta 10, 27100 Pavia, Italy
Received June 2, 1999; Revised October 8, 1999; Accepted November 24, 1999.

Abstract
  • The effect of cannabinoid drugs on peristalsis in the guinea-pig ileum was studied. Peristalsis was induced by delivering fluid into the oral end of an isolated intestinal segment. Longitudinal muscle reflex contraction, threshold pressure and threshold volume to trigger peristalsis, compliance of the intestinal wall during the preparatory phase (a reflection of the resistance of the wall to distension) and maximal ejection pressure during the emptying phase of peristalsis were measured.
  • The cannabinoid agonists WIN 55,212-2 (0.3–300 nM) and CP55,940 (0.3–300 nM) significantly decreased longitudinal muscle reflex contraction, compliance and maximal ejection pressure, while increased threshold pressure and volume to elicit peristalsis. These effects were not modified by the opioid antagonist naloxone (1 μM) and by the α-adrenoceptor antagonist phentolamine (1 μM).
  • The inhibitory effect of both WIN 55,212-2 and CP55,940 on intestinal peristalsis was antagonized by the cannabinoid CB1 receptor antagonist SR141716A (0.1 μM), but not by the cannabinoid CB2 receptor antagonist SR144528 (0.1 μM).
  • In absence of other drugs, the CB1 receptor antagonists SR141716A (0.01–1 μM) and AM281 (0.01–1 μM) slightly (approximatively 20%) but significantly increased maximal ejection pressure during the empty phase of peristalsis without modifying longitudinal muscle reflex contraction, threshold pressure, threshold volume to trigger peristalsis and compliance.
  • It is concluded that activation of CB1 receptors reduces peristalsis efficiency in the isolated guinea-pig, and that the emptying phase of peristalsis could be tonically inhibited by the endogenous cannabinoid system.
Keywords: Cannabinoid, peristalsis, myenteric plexus, intestine, intestinal motility

Introduction
Cannabinoids are natural compounds found in the aerial parts of Cannabis sativa L. (Cannabinaceae). Medicinal properties of cannabis were recognized some 5000 years ago and potential therapeutic applications that are of either historical or contemporary interest include analgesia, attenuation of nausea and vomiting of cancer chemotherapy, antirheumatic and antipyretic actions, decreased bronchial constriction and decreased intestinal motility (Howlett, 1995). The synthetic cannabinoid drug nabilone and Δ9-tetrahydrocannabinol are already used clinically to suppress nausea and vomiting provoked by anticancer drugs or to boost the appetite of AIDS patients (Pertwee, 1998).
Many of the pharmacological effects previously reported for natural cannabinoids are mediated by specific receptors (Howlett, 1995). These are CB1, that are expressed mainly by central and peripheral neurons, and CB2 receptors, that occur mainly in immune cells (Pertwee, 1998). The discovery of these receptors has led to the demonstration that there are endogenous ligands (agonists) for these receptors in several mammalian tissues. The most important of the endogenous cannabinoids discovered to date are anandamide (Devane et al., 1992) and 2-arachidonylglycerol, the latter found in the intestine of the dog (Mechoullam et al., 1995).
The guinea-pig small intestine contains cannabinoid binding sites that closely resemble CB1 binding sites of guinea-pig brain (Ross et al., 1998). Prejunctionally located CB1 receptors regulate in a negative fashion the release of acetylcholine from myenteric neurons (Coutts & Pertwee, 1997). The activation of such receptors inhibits excitatory transmission in the guinea-pig (Coutts & Pertwee, 1997; Izzo et al., 1998) and human ileum (Croci et al., 1998), while SR141716A, a specific CB1 receptor antagonist, potentiates excitatory transmission in the guinea-pig (Coutts & Pertwee, 1997; Izzo et al., 1998) but not in the human ileum.
On the basis of these studies, which utilized the electrically-evoked contractions of the longitudinal and circular muscle or the release of tritiated acetylcholine from myenteric neurons, it is not clear whether cannabinoid receptors are really involved in the control of peristalsis as it occurs under physiological conditions. Indeed, there is evidence that some molecules (i.e. histamine H3 agonists) may suppress electrical twitch responses without affecting the distension-evoked peristaltic motility (Poli & Pozzoli, 1997).
In this work we have therefore studied the role of cannabinoid receptors on intestinal peristalsis in vitro. For this purpose, we have used the cannabinoid non-selective receptor agonists WIN 55,212-2 and CP55,940 (Compton et al., 1992; Gatley et al., 1997), the CB1 receptor antagonists SR141716A and AM840 (Rinaldi-Carmona et al., 1995; Gifford et al., 1997) and the CB2 receptor antagonist SR144528 (Rinaldi-Carmona et al., 1998).

Methods
Male guinea-pigs (300–450 g, Harlan Italy, Corezzana, Milano) were killed by asphyxiation with CO2. Portions of the ileum lying 5–15 cm proximal to the ileocaecal junction were excised, flushed of luminal contents and placed, for up to 4 h, in Krebs solution kept at room temperature and oxygenated with a mixture of 95% O2 and 5% CO2. The composition of the Krebs solution was (mM): NaCl 119, KCl 4.75, KH2PO4 1.2, NaHCO3 25, MgSO4 1.5, CaCl2 2.5 and glucose 11). After dissection, ileal segments were mounted in organ baths that contained oxygenated Krebs solution maintained at 37°C.
Peristalsis
Peristalsis was studied with a constant intraluminal perfusion system that has been described in detail previously (Waterman et al., 1992). Briefly, ileal segments (approximately 5–7 cm in length) were secured horizontally in an organ bath containing 80 ml of Krebs solution. Peristalsis was elicited by delivering Krebs solution into the oral end of the intestinal lumen; the infusion rate was 0.85 ml min−1. Longitudinal muscle contraction was recorded with an isotonic transducer (load 1 g). Two transducers measured aboral pressure; one recorded at high sensitivity and the other at low sensitivity. Each peristaltic wave caused the expulsion of fluid from the aboral end of the intestine via a one-way valve. The volume expelled at the end of each wave of contraction was measured with a measuring cylinder. Fluid expelled activated a photocell system that switched off the infusion pump. The arrest of the inflow, controlled by a timer, was maintained for 30 s in order not to force the contracted organ during propulsion and to allow a period of rest between two peristalses.
Physiological parameter of peristalsis
Two phases of peristalsis can be identified: the preparatory phase, when the intestine is being filled with fluid, causing the longitudinal muscle to contract, and the emptying phase, when the circular muscle contracts and forces fluid from the aboral end (Kosterlitz & Lees, 1964). The following parameters were measured: (i) longitudinal muscle contraction (calculated as the percentage shortening of the intestine at the threshold, relative to the resting length), (ii) threshold pressure value required for a peristaltic response, (iii) threshold volume to trigger peristalsis, (iv) compliance of the intestinal wall at the end of the preparatory phase, and (iv) maximal ejection pressure during the emptying phase. A schematic drawing of a peristaltic wave showing variables measured has been previously reported (Waterman et al., 1992; Holzer & Maggi, 1994). The compliance is defined as the change in intraluminal pressure in response to a given change in intraluminal volume and reflects the resistance of the intestinal wall to infused fluid (Waterman et al., 1992).
Experimental design
After stable control peristaltic activity had been recorded (at least 5 min), the response was observed in the presence of increasing cumulative concentration of WIN 55,212-2 (0.3–300 nM), CP55,940 (0.3–300 nM), SR141716A (0.01–1 μM), AM281 (0.01–1 μM) or single concentration of SR144528 (0.1 μM), atropine (1 μM), tetrodotoxin (0.3 μM), phentolamine (1 μM) and naloxone (1 μM). These concentrations were selected on the basis of previous work (Croci et al., 1998; Izzo et al., 1998).
The contact time for each concentration was 4 min for WIN 55,212-2, 6 min for CP55,940, 8 min for SR141716A and AM281, 10 min for tetrodotoxin and 20 min for the other drugs. Preliminary experiments showed that these contact times were sufficient to achieve maximal effect. In some experiments SR141716A (10, 30 and 100 nM), AM281 (0.1 μM) SR144528 (0.1 μM), phentolamine (1 μM) or naloxone (1 μM) were included in the Krebs.
Drugs
Drugs used were: WIN 55,212-2 mesylate, CP55,590, AM840 (Tocris Cookson, Bristol, U.K.), tetrodotoxin, atropine sulphate, naloxone hydrochloride, phentolamine hydrochloride (Sigma, Milan, Italy), SR141716A [(N-piperidin-1-yl)-5-(4-chlorophenyl)-1 - 2,4 - dichlorophenyl) - 4 - methyl -1H-pyrazole-3-carboxamide hydrochloride and SR144528 (N-[-1S-endo-1,3,3-trimethyl bicyclo [2.2.1] heptan-2-yl]-5- (4-chloro-3-methylphenyl) -1-(4-methylbenzyl) -pyrazole-3-carboxamide-3-carboxamide) were a gift from Dr Madaleine Mossè and Dr Francis Barth (SANOFI-Recherche, Montpellier, France). The drugs were dissolved in dimethyl sulphoxide (DMSO), with the exception of atropine, phentolamine, naloxone and tetrodotoxin which were dissolved in saline. All drugs were added in volumes less than 0.01% of the bath volume. DMSO (6 μl/80 ml) neither affected peristalsis efficiency nor modified the concentration-response to cannabinoid agonists.
Statistical analysis
Data are presented as the means±s.e.mean of experiment using n guinea-pigs. Each measurement was made with a separate segment of intestine. Comparisons between two sets of data were made by Student's t-test for paired data. When multiple comparisons against a single control were made, analysis of variance was used. Probability less than 0.05 was regarded as significant.

Results
Continuous intraluminal infusion of Krebs solution resulted in peristaltic activity which was reproducible and stable during the experimental period of 20–30 min (n=5). In the absence of any drug the physiological parameters measured had the following values: threshold pressure: 118±5 Pa, threshold volume 381±31 μl, longitudinal muscle contraction: 16±2% inhibition, maximal ejection pressure 832±34 Pa, compliance 3.56±0.09 μl Pa−1. The application of tetrodotoxin (0.3 μM) or atropine (1 μM) completely prevented the appearance of peristaltic waves, while phentolamine (1 μM) or naloxone (1 μM) were ineffective (n=4 for each drug).
The cannabinoid agonists WIN 55,212-2 and CP55,940 affected all the measured parameter of peristalsis. WIN 55,212-2 (0.3–300 nM) and CP55,940 (0.3–300 nM) increased threshold pressure and volume to elicit peristalsis (Figure 1a,b), reduced in a concentration dependent fashion longitudinal muscle contraction (Figure 1c), and decreased maximal ejection pressure (Figure 1d). The compliance of the intestine during the preparatory phase was reduced by WIN 55,212-2 300 nM and CP55,940 300 nM by approximately 30% (Figure 2). WIN 55,212-2-induced changes in peristalsis were not modified by phentolamine (1 μM n=5) or naloxone (1 μM, n=5) (data not shown).
Figure 1
Figure 1
Effect of WIN 55,212-2 (0.3–300 nM) and CP55,940 (0.3–300 nM) alone or in combination with SR141716A (SR1 0.1 μM) or SR144528 (SR2 0.1 μM) on threshold pressure (Figure 1a), threshold volume (more ...)
Figure 2
Figure 2
Effect of WIN 55,212-2 (0.3–300 nM) and CP55,940 (0.3–300 nM) alone or in combination with SR141716A (SR1 0.1 μM) or SR144528 (SR2 0.1 μM) on intestinal compliance. The ordinates show the (more ...)
The emptying phase of peristalsis was abolished by 300 nM WIN 55,212-2 in two out of eight experiments. WIN 1 μM and CP55,940 1 μM also completely abolished peristalsis in five out of eight experiments and two out of five experiments, respectively. A significant rise in the triggering pressure, followed by a complete paralysis of the peristaltic motility was observed. The fluid moved straight through the intestine as if it were a passive tube. Figure 3 shows a recording of the effect of WIN 55,212-2 1 μM on peristalsis. In the experiments in which the emptying phase of peristalsis was not abolished, a strong inhibition of physiological parameter of peristalsis was observed (data not shown).
Figure 3
Figure 3
Trace showing the effect of WIN 55,212-2 (1 μM) on intestinal peristalsis (longitudinal muscle contraction, threshold pressure during the preparatory phase and maximal ejection pressure during the emptying phase) in the guinea-pig ileum. (more ...)
The effect of WIN 55,212-2 and CP55,940 on peristalsis was completely counteracted by SR141716A (0.1 μM) (Figures 1 and and2)2) and by AM281 (0.1 μM) (data not shown), two cannabinoid CB1 receptor antagonists, but not by the cannabinoid CB2 receptor antagonist SR144528 (0.1 μM) (Figures 1 and and2).2). Figure 4 shows the effect of SR141716A (10, 30 and 100 nM) on WIN 55,212-2 (300 nM)- and CP55,940 (300 nM)-induced changes threshold pressure (Figure 4a), threshold volume (Figure 4b), longitudinal muscle contraction (Figure 4c) and maximal ejection pressure (Figure 4d). SR141716A (10, 30 and 100 nM) also counteracted the effect of WIN (300 nM) and CP55,940 (300 nM) on compliance in a concentration-dependent manner (data not shown).
Figure 4
Figure 4
Effect of WIN 55,212-2 (300 nM) and CP55,940 (300 nM) alone or in the presence of various concentrations of SR141716A (10, 30 and 100 nM) on threshold pressure (Figure 4a), threshold volume (Figure 4b), longitudinal muscle contraction (more ...)
In absence of any drug, SR141716A and AM281 did not modify threshold pressure, threshold volume, longitudinal muscle contraction (Figure 5a–c) and compliance (data not shown), but significantly (P<0.05) increased maximal ejection pressure to elicit peristalsis. By contrast the CB2 receptor antagonist SR144528 (0.1 μM), when administered alone, did not modify the physiological parameter of peristalsis measured (per cent variation: threshold volume 1±4, threshold pressure −2±5, longitudinal muscle contraction 0±4%, maximal ejection pressure 2±5, compliance 5±4%, n=5).
Figure 5
Figure 5
Effect of SR141716A (0.01–1 μM and AM281 (0.01–1 μM) on threshold pressure (Figure 5a), threshold volume (Figure 5b), longitudinal muscle contraction (Figure 5c) and maximal ejection pressure (Figure 5d (more ...)
Discussion
Peristalsis is a coordinated pattern of motor behaviour which occurs in the gastrointestinal tract and allows the contents to be propelled in an anal direction. Two phases of peristalsis have been described in response to the slow infusion of liquid which radially stretches the intestinal wall: a preparatory phase, in which the intestine gradually distends until a threshold distension and an emptying phase, in which the circular muscle at the oral end of the intestine contracts, an effect followed by a wave of contraction that propagates aborally along the intestine. The pathways mediating peristalsis involve intrinsic primary enteric sensory neurons and interneurons, as well as excitatory and inhibitory motor neurons (Furness et al., 1998). Acetylcholine acting through both muscarinic and nicotinic receptors and tachykinins are excitatory neurotransmitters participating in the peristaltic activity (Tonini et al., 1981; Holzer & Maggi, 1994; Holzer et al., 1998), whereas vasoactive intestinal polypeptide, nitric oxide and an apamine-sensitive inhibitory transmitter act as inhibitory mediators (Grider, 1993; Waterman & Costa, 1994).
Prejunctional or presynaptic receptor systems, such as opioids, α2-adrenoceptors or adenosine A1 receptors are involved in the control of peristaltic movements (Kromer et al., 1980; Candura et al., 1992; Waterman et al., 1992; Poli & Pozzoli, 1997) while histamine H3 agonists are not able to modulate the reflex-evoked peristaltic response (Poli & Pozzoli, 1997). In the present study we have observed multiple actions of cannabinoid drugs on different parameters of peristalsis.
Longitudinal muscle contraction
The longitudinal shortening of the intestine during the preparatory phase is a reflex response elicited by radial distension; it is mediated largely by excitatory cholinergic motoneurons to the longitudinal muscle (Kosterlitz & Robinson, 1959). The present study indicates that this contraction is reduced in amplitude by activation of CB1 receptors. Consistent with this, a CB1-mediated inhibition of the longitudinal muscle in strips of the guinea-pig small intestine, associated with a reduction of acetylcholine release, has previously been reported (Coutts & Pertwee, 1997; Ross et al., 1998). In addition, WIN 55,212-2 and CP55,940 inhibit fast and slow excitatory synaptic transmission in myenteric S-neurons through activation of CB1 receptors (Lopez-Redondo et al., 1997). Therefore it is possible that cannabinoid agonists act at more than one site in the reflex pathways.
Compliance
The compliance of the intestinal wall reflects the resistance of the wall to dynamic distension. Compliance is an index of the activity of the circular muscle during the preparatory phase. The greater the tone of the circular muscle, the more resistant it is to distension and consequently the less compliance. Compliance is influenced both by neural and by muscular factors. Drugs which block excitatory nerves to the circular muscle increase compliance, while drugs which inhibit enteric inhibitory nerves decrease compliance (Waterman et al., 1994). On the basis of these data, it was expected that cannabinoid agonists, which inhibit excitatory transmission (Izzo et al., 1998) could increase compliance. However, we have shown that the cannabinoid agonists WIN 55,2122-2 and CP55,940 significantly reduced compliance, an effect counteracted by the CB1 antagonists SR141716A. The most likely explanation of these results is that cannabinoid agonists cause a decrease in the activity of enteric inhibitory nerves which mediates accommodation in the isolated guinea-pig small intestine (Waterman et al., 1994). There is no evidence in the literature about a modulation of enteric inhibitory nerves by cannabinoids drugs. However, it should be noted that central CB1 receptors mediate inhibition of nitric oxide production by rat microglial cells (Waksman et al., 1999) and nitric oxide inhibition is associated with decreased compliance in the guinea pig ileum (Waterman et al., 1994). It is unlikely that cannabinoid agonists decrease compliance by acting directly on smooth muscle since it has been recently shown that they are able to inhibit electrically-evoked contractions without modifying the contractions produced by exogenous acetylcholine or substance P (Izzo et al., 1998).
Threshold for triggering peristalsis
During the preparatory phase, activity of circular muscle motor neurones determines the tone of the circular muscle and the resistance of the muscle to further distension. We have recently shown that activation of CB1 receptors produces inhibition of cholinergic and tachykinergic transmission to the circular muscle of the guinea pig ileum (Izzo et al., 1998). In the present study we have demonstrated that the cannabinoid agonists WIN 55,212-2 and CP55,940 increased the threshold volume and pressure for triggering the emptying phase of peristalsis, an effect counteracted by the CB1 antagonists SR141716A and AM281 but not by the CB2 antagonist SR1445258. In some experiments, with high concentration of cannabinoid agonists (300 and 1 μM), a block of peristalsis was observed, in spite of an intraluminal pressure of 300 Pa.
Maximal ejection pressure
The extent of circular muscle contraction and its coordinated propagation determines the peak pressure generated by the intestine. This parameter was reduced, in a dose dependent fashion, by selective activation of CB1 but not CB2 receptors. These effects can be explained by the inhibitory action of the cannabinoid agonists on excitatory (both cholinergic and tackykinergic) motor neurones to the circular muscle recently reported (Izzo et al., 1998).
The role of the endogenous cannabinoid system
There is evidence in the literature that intestinal motility can be tonically inhibited by the endogenous cannabinoid system. Indeed the presence of the endocannabinoid agonist 2-arachidonylglicerol has been demonstrated in the canine gut (Mechoullam et al., 1995), while the rat small intestine contains high amounts of anandamide hydrolase, the enzyme responsible of anandamide inactivation (Katayama et al., 1997). Functional studies indicate that excitatory transmission to the circular and longitudinal muscle of the guinea-pig ileum is potentiated by SR141716A, a CB1 receptor antagonist (Izzo et al., 1998; Ross et al., 1998). Consistent with these in vitro results, SR141716A was found to increase upper gastrointestinal transit and defecation in mice (Colombo et al., 1998; Izzo et al., 1999a). The constitutive activity of CB1 receptors in these systems, however, could not be attributed unequivocally to displacement of endocannabinoids as SR141716A behaves as inverse agonist at the human CB1 receptors (MacLennan et al., 1998). In the present study we have observed a tendency of SR141716A and AM281 to decrease threshold pressure and volume to elicit peristalsis, but this effect was not significant. Therefore an activation of the endogenous cannabinoid system seems unlikely during the preparatory phase of the peristalsis. However, SR141716A and AM281, two selective CB1 receptor antagonists increased maximal ejection pressure during the empty phase of peristalsis, thus indicating the possible activation of the endogenous cannabinoid system during the emptying phase of peristalsis. In other studies, it has been shown that cholinergic transmission to the longitudinal muscle of the human ileum in vitro (Croci et al., 1998) and gastric emptying in rats in vivo were unaffected by SR141716A (Izzo et al., 1999b).
Conclusions
We have shown that activation of CB1 receptors, but not CB2 receptors, inhibits most of physiological parameters of peristalsis. This inhibitory effect does not involve the activation of α-adrenoceptors or opioid receptors and could be attributed, at least in part, to an action of cannabinoid agonists on excitatory motor neurones innervating the circular and longitudinal muscle as previously reported (Izzo et al., 1998; Ross et al., 1998). In addition, the emptying phase of peristalsis could be tonically inhibited by the endogenous cannabinoid system. This work emphasises the role of peripheral CB1 receptors in the control of intestinal motility under physiological conditions and opens the possibility to investigate in vivo selective, non-psychotropic cannabinoid agonists or antagonists that may decrease (e.g. spasm, diarrhoea) or increase gut motility (ileus), respectively.
Acknowledgments
This work was supported by Cofinanziamento Murst 1999 and Enrico and Enrica Sovena Foundation (Roma). We wish to thank Miss Emilia Nocerino and Marianna Amato for their help. SR141716A and SR144528 were a kind gift from SANOFI (Montpellier, France).
Abbreviations
 
DMSOdimethyl sulphoxide

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The synthetic cannabinoid WIN55,212-2 attenuates hyperalgesia and allodynia in a rat model of neuropathic pain

Daniel Bridges, Kamran Ahmad,  and Andrew S C Rice
Pain Research, Imperial College School of Medicine, Chelsea and Westminster Hospital Campus, London, SW10 9NH
Novartis Institute for Medical Sciences, 5 Gower Place, London, WC1
Received January 29, 2001; Revised April 9, 2001; Accepted April 9, 2001.
Abstract
  • The analgesic properties of the synthetic cannabinoid WIN55,212-2 were investigated in a model of neuropathic pain. In male Wistar rats, bilateral hind limb withdrawal thresholds to cold, mechanical and noxious thermal stimuli were measured. Following this, unilateral L5 spinal nerve ligation was performed. Seven days later, sensory thresholds were reassessed and the development of allodynia to cold and mechanical stimuli and hyperalgesia to a noxious thermal stimulus confirmed.
  • The effect of WIN55,212-2 (0.1  5.0 mg kg−1, i.p.) on the signs of neuropathy was then determined; there was a dose related reversal of all three signs of painful neuropathy at doses which did not generally alter sensory thresholds in the contralateral unligated limb. This effect was prevented by co-administration of the CB1 receptor antagonist SR141716a, but not by co-administration of the CB2 receptor antagonist SR144528, suggesting this action of WIN55,212-2 is mediated via the CB1 receptor. Administration of SR141716a alone had no affect on the observed allodynia and hyperalgesia, which does not support the concept of an endogenous analgesic tone.
  • These data indicate that cannabinoids may have therapeutic potential in neuropathic pain, and that this effect is mediated through the CB1 receptor.
Keywords: Cannabinoid, analgesia, neuropathic pain, hyperalgesia, allodynia, WIN55,212-2, CB1 receptor, SR141716a

Introduction
There have been suggestions that extracts of Cannabis sativa have medicinal properties for thousands of years, but until recently, little sound scientific data has been available to support this hypothesis. However, the structure of Δ9-tetrahydrocannabinol (THC), the major psychoactive component of the 66 known cannabinoids found in marijuana, was elucidated in the early 1960s and since then THC has been shown to be associated with a number of pharmacological effects, including analgesia (Gaoni & Mechoulam, 1964). A further breakthrough in the elucidation of cannabinoid pharmacology came in 1988 with the discovery of a cannabinoid receptor in rat brain (CB1, Devane et al., 1988). This receptor was subsequently cloned in 1990 (Matsuda et al., 1990) and was shown to have a seven transmembrane G-protein coupled structure (Howlett et al., 1986). A second cannabinoid receptor (CB2), with 44% sequence homology to CB1, was identified and cloned in 1993 (Munro et al., 1993) and was found to be predominantly expressed by cells of the immune system. Also in 1992, the endogenous cannabinoid anandamide (AEA) was extracted from the brain and spinal cord (Devane et al., 1992). More recently palmitoylethanolamide (PEA) and 2-arachidonyl glycerol (2-AG) have also been identified as potential endogenous cannabinoids (Mechoulam et al., 1995; Sugiura et al., 1995). The synthesis of the specific high affinity receptor antagonists, SR141716a (SR1) at CB1 (Rinaldi-Carmona et al., 1995; Welch et al., 1998) and SR144528 (SR2) at CB2 (Rinaldi-Carmona et al., 1998; Griffin et al., 1999), and the development of CB1 (Ledent et al., 1999) and CB2 ‘knockout mice' (Buckley et al., 2000) afforded new techniques for the elucidation of CB receptor mediated effects. Several studies have demonstrated an analgesic effect with synthetic and endogenous cannabinoids in inflammatory pain models (for example Jaggar et al., 1998a, 1998b; Calignano et al., 1998; Richardson et al., 1998.)
Neuropathic pain is defined as ‘pain initiated or caused by a primary lesion or dysfunction in the nervous system' (Merskey & Bogduk, 1994) and is an area of largely unmet therapeutic need. Tricyclic antidepressants and certain anticonvulsants are the mainstay of clinical therapy for neuropathic pain (McQuay et al., 1996; Sindrup & Jensen, 1999). However, systematic reviews reveal that only between 30  50% of patients suffering from neuropathic pain achieve clinically significant (>50%) pain relief with any available single therapy (McQuay et al., 1996; Sindrup & Jensen, 1999). Furthermore, side effects of these therapies often limit their usefulness (McQuay et al., 1996). Although controversial, it is generally accepted that opioids are less effective in neuropathic than inflammatory pain. (Rowbotham, 1999). One explanation for this observation is that spinal opioid receptor expression decreases after peripheral nerve injury (Besse et al., 1992). A recent study has demonstrated little decrease of spinal CB receptor binding when compared to μ-opioid receptor binding after neonatal capsaicin treatment (Hohmann & Herkenham, 1998). Furthermore, it has also been demonstrated using immunocytochemistry that, following dorsal rhizotomy, spinal CB1 receptor expression remains unaltered (Farquhar-Smith et al., 2000). This sparing of CB1 receptors following peripheral nerve injury could indicate a potential therapeutic advantage of cannabinoids over opioids in the treatment of neuropathic pain. This hypothesis is further supported by a recent study (Mao et al., 2000), which demonstrated an anti-hyperalgesic effect of THC in an animal model of neuropathic pain and suggested a therapeutic advantage of THC over opioids in painful neuropathy.
Various animal models of neuropathic pain have been developed (Kim et al., 1997). The three most commonly used models share partial injury of the sciatic nerve and a subsequent alteration in hind limb withdrawal thresholds to sensory stimuli as a common feature. In particular, hyperalgesia to a noxious thermal stimulus and allodynia to cold and mechanical stimuli are usually observed. The first model described was the chronic constriction injury (CCI) model in which loose chromic gut ligatures are placed around the sciatic nerve (Bennett & Xie, 1988). A more recent modification was the partial sciatic nerve ligation (PNL) model, in which a tight ligation is placed around 1/3 to 1/2 of the sciatic nerve trunk (Seltzer et al., 1990). A third model is the spinal nerve ligation (SNL) model, in which the L5 and L6 spinal nerves are tightly ligated (Kim & Chung, 1992). A direct comparison of various features of these three models of neuropathic pain has been reported (Kim et al., 1997). In this study, the latency and duration of signs of painful neuropathy were examined, and the authors also examined the effect of sympathectomy on these signs. This study showed that the partial nerve injury evoked signs with roughly the same onset in all three models, but that qualitatively the mechanical and cold allodynia were greatest in SNL. Ongoing pain, as determined by non-weight bearing behaviour, was greatest in CCI. Surgical lumbar sympathectomy caused a reduction in pain behaviour in all three models, with the largest decrease in SNL, suggesting a greater sympathetic nervous system involvement in this model.
There are two reports of the effectiveness of cannabinoids in an animal model of neuropathic pain. One study reported that the synthetic cannabinoid WIN55,212-2 alleviated the allodynia and hyperalgesia associated with the CCI model of neuropathic pain (Herzberg et al., 1997). It was confirmed that this effect was mediated through the CB1 receptor and occurred at a dose that was not associated with obvious side effects. This study also demonstrated an increase in thermal hyperalgesia and mechanical allodynia by administration of SR141716a alone, leading the authors to infer the presence of an endogenous tone of cannabinoid analgesia in neuropathy. However, the evidence for such tone during inflammation is controversial (Beaulieu et al., 2000). A separate study reported that THC, administered intrathecally, also alleviated the hyperalgesia of the CCI model (Mao et al., 2000). This effect was shown to be CB1 receptor mediated.
It has been suggested that the neuropathy in the CCI model is largely dependent upon an inflammatory reaction (Wagner et al., 1998) and therefore the anti-inflammatory effects of cannabinoids may have obfuscated the true effect on neuropathy in this model. Both the PNL and SNL are associated with a lesser inflammatory component. Therefore, in this study we examined whether the anti-allodynic and anti-hyperalgesic effects of cannabinoids were found in a model of neuropathic pain associated with less of an inflammatory component than the CCI model, namely the SNL model. We also investigated the concept of endogenous tone in this model.

Methods
Animal maintenance
All experiments were approved by the Home Office. Animals were housed, six per cage at constant temperature under a 14 : 10 h light/dark cycle, with free access to food and water at all times.
Surgery
A left L5 spinal nerve ligation, a modification of that described by Kim & Chung (1992), was performed on male Wistar rats of between 200  350 g in weight (n=126). The rats were anaesthetized (pentobarbitone sodium, 60 mg kg−1, i.p.) and the surgery performed using standard aseptic techniques. Using the transverse processes of L6 as a guide, the left paraspinal muscles were exposed and separated from the spinous processes of L4 to S2 by blunt dissection. The L6 transverse process was then removed by hemi-laminectomy and the L5 spinal nerve exposed and identified according to its size and position. This was then ligated tightly with a 3-0 silk suture and sectioned 1  2 mm distal to the suture before haemostasis was confirmed and the wound was sutured at both muscle and skin levels. Sham surgery (n=6) was performed by exposing the L5 spinal nerve as described above, but not damaging it.
Sensory testing
Three tests of hind limb withdrawal to thermal, cold and mechanical stimuli were employed in this study. Each test was repeated on both the operated hind paw and the contralateral hind paw with all sensory testing performed by a ‘blinded' investigator.
(i)
Cold allodynia was assessed using the acetone drop application technique modified from Carlton et al. (1994). Animals were placed in plexiglass boxes (23×18×14 cm) with 0.8 cm diameter mesh flooring and allowed to acclimatize for 15 min or until exploratory behaviour ceased. Sampling was performed by the application of a single bubble of acetone to the mid plantar surface of each hind paw from the tip of a 1 ml syringe. A positive response was recorded if the animal withdrew the paw following application. For each measurement, the paw was sampled five times and a mean calculated. At least 3 min elapsed between each test.
(ii)
Thermal hyperalgesia was assessed using an infrared noxious heat stimulus (Plantar test, Ugo Basile, Italy, Hargreaves et al., 1988). Briefly, animals were placed in a clear plexiglass box (23×18×14 cm) with a dry glass floor and allowed to acclimatize for 15 min or until exploratory behaviour ceased. A focused beam of radiant heat at a constant temperature of 46°C and wavelength of 50 nm was applied to the plantar surface of the paw. The hind paw withdrawal latency (s) to this stimulus was tested three times at intervals of not less than 3 min and a mean calculated. The device has an automatic cut-off at 21 s to avoid the risk of thermal injury to the skin.
(iii)
Mechanical allodynia was assessed using an electronic Von Frey device (Möller et al., 1998; Ahmad & Rice, 1999). Animals were placed into raised plexiglass boxes (23×18×14 cm) with 0.8 cm diameter mesh flooring and allowed to acclimatize for 15 min or until exploratory behaviour ceased. Sampling was conducted by a calibrated nylon electronic force transducer (1.0 mm diameter, type 739, Somedic Sales AB, Sweden) which was applied manually to the mid-plantar hind paw at a rate of 0.5  1.0 N s−1 with the withdrawal threshold amplified (Senselab 701, Somedic, Sweden) and displayed on a PC based chart recorder (AcqKnowledge v3.02, Biopac Systems Inc. U.S.A.). The mean withdrawal threshold was taken from a set of five applications, not less than 10 s apart.
Pharmacological interventions
Baseline sensory thresholds were measured for each group of animals (n=6) pre-operatively and 7 days post-operatively. Animals displaying thermal hyperalgesia or cold or mechanical allodynia were then administered the relevant drug according to a pre-determined randomization table and testing was re-performed at 20, 40, 60 and 90 min post drug administration. Each group of animals was used for only one drug administration protocol to ensure no ‘carry over' effects, hence they were used for only a single treatment. WIN55,212-2 (0.1  5.0 mg kg−1, i.p.) was administered at t=0, in 40% dimethylsulfoxide (DMSO) in saline solvent, SR141716a (0.5 mg kg−1, i.p.) and SR144528 (1 mg kg−1, i.p.) were administered at t=−5 min and dissolved in 40% DMSO in saline solvent. All animals were humanely culled at the end of the experiment. A summary of the treatment groups is displayed in Table 1.
Table 1
Table 1
Summary of treatment groups for experimental procedures
Statistical analysis
Statistical significance was determined for neuropathy by a paired t-test and for drug effects by one-way ANOVA (Dunnett, compared to post operative values), both taking P<0.05 as statistically significant (SigmaStat v2.0, Jandel Corporation, U.S.A.)
Drugs
WIN55,212-2 was supplied by Tocris Cookson Ltd., U.K., SR141716a was a gift from SRI  NIMH chemical synthesis program and SR144528 was a gift from Sanofi Recherche, France.
Results
All animals included in the analysis of study exhibited altered sensory thresholds 7 days following SNL (rejection rate=7%). In the sham surgery groups (20  22, Table 1) there was no significant difference from baseline sensory thresholds. In all animals in which solvent only was administered as a control, sensory thresholds were not subsequently altered in either the ipsilateral or contralateral paws.
WIN55,212-2 studies
(i)
Administration of WIN55,212-2 reversed the cold allodynia produced by SNL at a dose of 2.5 mg kg−1 over 90 min (Figure 1). This effect was observed to be both dose- and time-dependent with a maximum effect at 20  40 min. The dose dependency was observed with a non-significant trend towards an effect at 0.5 mg kg−1 WIN, but no effect at 0.1 mg kg−1.
Figure 1
Figure 1
Bilateral hind limb withdrawal responses to cold stimulation (acetone drop) in rats rendered neuropathic by L5 spinal nerve ligation administered WIN55,212-2 (0.1–2.5 mg kg−1, i.p., n=6 per dose). Attenuation of (more ...)
(ii)
WIN55,212-2 also reversed the thermal hyperalgesia produced by SNL. This effect was again observed to be both dose and time dependent with the results generally concurring with those from the cold allodynia studies (Figure 2). Thermal hyperalgesia was attenuated throughout the entire 90 min experiment at a dose of 2.5 mg kg−1 WIN55,212-2 and also between 20  40 min at a dose of 0.5 mg kg−1. No effect was seen at the 0.1 mg kg−1 dose.
Figure 2
Figure 2
Bilateral hind limb withdrawal responses to thermal stimulation (Hargreaves' device) in rats rendered neuropathic by L5 spinal nerve ligation administered WIN55,212-2 (0.1–2.5 mg kg−1, i.p., n=6 per dose). Attenuation (more ...)
(iii)
Mechanical allodynia produced by SNL was not as effectively attenuated by WIN55,212-2 as the cold allodynia and thermal hyperalgesia (Figure 3). A significant effect was only observed at the 5.0 mg kg−1 dose throughout the entire 90 min experiment. However, significant effects were also observed on the sensory thresholds of the contralateral limb at this dose. At 20 and 40 min post dosing the 2.5 and 0.5 mg kg−1 doses had no effect on mechanical allodynia.
Figure 3
Figure 3
Bilateral hind limb withdrawal responses to mechanical stimulation (electronic Von Frey) in rats rendered neuropathic by L5 spinal nerve ligation administered WIN55,212-2 (0.5–5.0 mg kg−1, i.p., n=6 per dose). Attenuation (more ...)
Receptor involvement
The CB1 receptor antagonist SR141716a (0.5 mg kg−1) prevented the anti-hyperalgesic or anti-allodynic effects seen at the highest doses of WIN55,212-2 (Figure 4). In the mechanical and thermal stimuli studies, SR141716a prevented the anti-allodynic and anti-hyperalgesic effects of WIN55,212-2 over the 90 min investigation period. In the cold stimuli studies, the anti-allodynic effects of WIN55,212-2 were prevented over 40 min, but persisted at 60 and 90 min at a much reduced level than the 2.5 mg kg−1 alone group. Administration of SR141716a alone had no significant effects on any of the sensory thresholds (Figure 5). Co-administration of WIN55,212-2 (2.5 mg kg−1) and the CB2 receptor antagonist SR144528 (1 mg kg−1) did not prevent the anti-allodynic effect of WIN55,212-2 in cold stimuli over 90 min (Figure 6).
Figure 4
Figure 4
Bilateral hind limb withdrawal responses to (a) cold (acetone drop), (b) thermal (Hargreaves' device) and (c) mechanical (electronic Von Frey) stimulation in rats rendered neuropathic by L5 spinal nerve ligation (n=6 per group) co-administered (more ...)
Figure 5
Figure 5
Bilateral hind limb withdrawal responses to (a) cold (acetone drop), (b) thermal (Hargreaves' device) and (c) mechanical (electronic Von Frey) stimulation in rats rendered neuropathic by L5 spinal nerve ligation (n=6 per group) administered SR141716a (more ...)
Figure 6
Figure 6
Bilateral hind limb withdrawal responses to cold (acetone drop) stimulation in rats rendered neuropathic by L5 spinal nerve ligation (n=6) co-administered SR144528 (1.0 mg kg−1, i.p.) and WIN55,212-2 (2.5 mg kg (more ...)
Discussion
This study has provided evidence for analgesic actions of the synthetic cannabinoid WIN55,212-2 in the spinal nerve ligation model of painful neuropathy. WIN55,212-2 attenuated cold allodynia and thermal hyperalgesia at a dose of 2.5 mg kg−1, i.p and mechanical allodynia at a dose of 5.0 mg kg−1, i.p. These effects are CB1 receptor mediated and have been observed at doses of WIN55,212-2 that cause no significant alteration in the sensory thresholds of the uninjured contralateral hind limb. Neither administration of SR141716a alone, administration of solvent nor sham surgery had an effect on the sensory thresholds. These results concur with a previous study which examined the analgesic actions of WIN55,212-2 in the CCI model of neuropathic pain (Herzberg et al., 1997) despite important differences between the models.
The blockade of the anti-hyperalgesic and anti-allodynic effects of WIN55,212-2 by the CB1 receptor antagonist, but not the CB2 receptor antagonist demonstrates that the analgesic effect of WIN55,212-2 is mediated through the CB1 receptor. This is despite the fact that WIN55,212-2, like most other cannabinoids, has significant affinity for the CB2 receptor, as well as CB1. The CB1 receptor is localized throughout the central nervous system, and has been mapped by immunocytochemistry in the brain (Tsou et al., 1998), spinal cord (Farquhar-Smith et al., 2000) and in cultured dorsal root ganglion cells (Ahluwalia et al., 2000). Immunocytochemical study of the spinal cord has shown that the expression of CB1 receptors is localized to areas associated with nociception, for example the superficial dorsal horn and lamina X (Farquhar-Smith et al., 2000). The localization of CB1 receptors in lamina I and lamina IIi/III coincides with the terminals of some of the neurons lost in peripheral nerve injury. It would therefore be expected that CB1 receptor expression would also decrease after peripheral nerve injury. However, Farquhar-Smith et al. (2000) observed no biologically relevant decrease in CB1 receptor after dorsal rhizotomy or rostral hemisection of the spinal cord. The authors postulate that this finding could be explained by the majority of spinal CB1 receptors being localized on spinal interneurons. This concept is supported by electrophysiological studies (Jennings et al., 2000) and changes in spinal cord receptor binding of [3H]-CP55,940 after neonatal capsaicin treatment (Hohmann & Herkenham, 1998), where the authors found only a 16% reduction in CB1 receptor binding after death of small diameter afferents was caused by capsaicin treatment. However, a multi-level dorsal rhizotomy (C3  T2) caused a 46% decrease in [3H]-CP55,940 binding, suggesting a presence of CB1 receptors on central termini of primary afferent neurons (Hohmann et al., 1999), although this finding could be explained by post-synaptic degeneration of neurons after such an extensive surgery.
A possible confounding factor in our experiments is a putative effect of WIN55,212-2 on the sensory thresholds per se. However, we have controlled for such an effect by measuring the sensory thresholds in the contralateral hind paw in all experiments. Only at the highest used dose of WIN55,212-2 was there a significant increase in the sensory threshold of the contralateral paw, suggesting that such an effect is not a confusing factor to the results at doses of WIN55,212-2 of 2.5 mg kg−1 or less.
The well-known psychotropic effects of cannabinoids are an obstacle to the development of cannabinoid based analgesics for the treatment of neuropathic pain (Perez-Reyes, 1999). Nevertheless, although not formally measured, we did not observe any obvious abnormal rodent behaviour indicative of psychotropic effects at the doses of WIN55,212-2 studies used in these experiments. However, there are a number of ways in which this issue may be circumvented: for example, structural engineering of cannabinoid molecules may permit certain cannabinoids to retain therapeutic activity whilst being devoid of psychotropic effects. One example of a cannabinoid which retains analgesic activity, in a model of inflammatory arthritis, in the absence of psychotropic effects is cannabidiol (Malfait et al., 2000). Furthermore, in a recent publication, Fox and colleagues have demonstrated that peripherally or spinal intrathecally administered WIN55,212-2, at doses that were not systemically active, attenuated the altered sensory thresholds associated with partial sciatic nerve ligation (Fox et al., 2001). This suggests that either selective delivery of cannabinoids by these routes, or systemic administration of cannabinoids that do not penetrate into the CSF, may be a method of divorcing analgesic from psychotropic effects. Finally, Baker et al. (2001) have demonstrated, using a model of multiple sclerosis, that concentrations of endocannabinoids are selectively elevated in areas of neuronal injury in spinal cord and brain. This finding indicates that it may be possible to selectively manipulate endocannabinoid concentrations in areas of neuronal injury, by inhibiting their degradation.
It has also been demonstrated that [3H]-DAMGO (a μ-opioid receptor agonist) binding was reduced by 60% following neonatal capsaicin treatment (Hohmann & Herkenham, 1998) and by 62% following multi-level dorsal rhizotomy (Hohmann et al., 1999). This consistent loss of opioid receptors following nerve injury is a factor for the relative ineffectiveness of morphine in neuropathic pain. Also, with several studies documenting a stable population of CB1 receptors in the superficial dorsal horn after peripheral nerve injury, a potential therapeutic advantage for cannabinoids over opioids has been found at the level of the spinal cord. This hypothesis is further supported by a study in which it was observed that THC alleviated hyperalgesia in the CCI model of neuropathic pain (Mao et al., 2000). This effect was shown to be on CB1 and not opioid receptor dependent, with no cross tolerance between opioids and cannabinoids, suggesting a different pathway of antinociception for cannabinoids and opioids.
In this study, we were unable to replicate the increase in allodynia or hyperalgesia associated with administration of the CB1 receptor antagonist SR141716a alone to animals with peripheral nerve injury that was observed by Herzberg et al. (1997). In our study, there was no significant alteration in sensory thresholds in SR141716a treated animals when compared to solvent treated controls. The increase of hyperalgesia or allodynia seen after administration of the receptor antagonist has been hypothesized to suggest the presence of an endogenous tone that is active during neuropathy  viz. locally synthesized endocannabinoids act at a low level on the receptors in response to peripheral nerve injury, causing a degree of attenuation of the hyperalgesia or allodynia. Thus, by blocking the action of these endogenous ligands by administration of the specific antagonist, the hyperalgesia or allodynia will be increased. The current data on an endogenous cannabinoid tone in pain models is controversial. Several studies have shown the presence of a SR141716a-associated enhancement of signs of pain or nociception in a variety of models, including the formalin test for inflammation (Calignano et al., 1998; Strangman et al., 1998). This could be interpreted as supporting the hypothesis that an endogenous tone exists in inflammatory pain, but not in neuropathic pain. If this is the case, then the inflammatory component of the CCI model could account for the difference seen between this and the findings of Herzberg et al. (1997). However, other studies of endogenous tone in inflammatory models have found no evidence supporting this concept, either in an inflammatory model (Beaulieu et al., 2000) or when administered to uninjured animals (Rinaldi-Carmona et al., 1995). The use of SR141716a as an experimental tool to reveal endogenous tone could be further confused by the findings of several groups that SR141716a is actually an inverse agonist at the CB1 receptor (Landsman et al., 1997; Pan et al., 1998). Further studies in CB1−/− knockout mice did not support the concept of an endogenous tone (Ledent et al., 1999). It also remains to be determined whether the levels of endogenous cannabinoids found in the spinal cord and dorsal root ganglia are sufficient to activate the receptors. Despite the controversial nature of the endogenous tone, our experiments seem to contradict its existence and agree with the study by Beaulieu et al. (2000) that there is a weak analgesic action at about 60 min post-administration, possibly due to SR141716a being able to show weak agonist activity under certain conditions.
This study has demonstrated the existence of CB1 receptor, but not CB2 receptor, mediated analgesia in an animal model of neuropathic pain. In light of the current therapeutic need for neuropathic pain treatments, this study provides evidence for the potential of cannabinoids, or inhibitors of degradation of endocannabinoids, as lead compounds for development of new therapies. The development of cannabinoid receptor specific agonists and antagonists will provide essential new tools for investigating the mechanism of this analgesic action.
Acknowledgments
D. Bridges is funded by an industrial collaborative grant from the MRC awarded to Novartis Institute of Medical Sciences. The authors are grateful to Dr J. Winter and Dr W.P. Farquhar-Smith for helpful discussions and to Dr J.W. Brooks for randomization. Part of this study has been previously presented at the 2000 Symposium on Cannabinoids, International Cannabinoid Research Society, 22/6/00, Baltimore, MD, U.S.A.
Abbreviations
 
2-AG2-arachidonyl glycerol
 
CB1cannabinoid 1 receptor
 
CB2cannabinoid 2 receptor
 
CCIchronic constriction injury
 
DMSOdimethylsulfoxide
 
PEApalmitoylethanolamide
 
PNLpartial nerve ligation
 
SNLspinal nerve ligation
 
SR1SR141716a
 
SR2SR144528
 
THCΔ9-tetrahydrocannabinol


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Cannabis sativa and dystonia secondary to Wilson's disease

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Uribe Roca MC; Micheli F; Viotti R
Parkinson's Disease and Movement Disorders Unit, Neurology Service, José de San Martín Clinicas Hospital, Buenos Aires, Argentina.

A patient with generalized dystonia due to Wilson's disease obtained marked improvement in response to smoking cannabis.

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Central and peripheral cannabinoid modulation of gastrointestinal transit in physiological states or during the diarrhoea induced by croton oil


Angelo A Izzo, Luisa Pinto,  Francesca Borrelli, Raffaele Capasso, Nicola Mascolo, and Francesco Capasso
Department of Experimental Pharmacology, University of Naples ‘Federico II', via D. Montesano 49, 80131 Naples, Italy
Department of Pharmaceutical Sciences, University of Salerno, Via Ponte Don Melillo 84084 Fisciano (SA), Italy
Received December 2, 1999; Revised January 25, 2000; Accepted January 28, 2000.

Abstract
  • We have evaluated the effect of cannabinoid drugs, administered intraperitoneally (i.p.) or intracerebroventricularly (i.c.v.) on upper gastrointestinal transit in control and in croton oil-treated mice.
  • The cannabinoid agonists, WIN 55,212-2 (2–239 nmol mouse−1) and cannabinol (24–4027 nmol mouse−1), decreased while the CB1 antagonist SR141716A (2–539 nmol mouse−1) increased transit in control mice. WIN 55,212-2, cannabinol and SR141716A had lower ED50 values when administered i.c.v., than when administered i.p. The CB2 antagonist SR144528 (52 nmol mouse−1, i.p.) was without effect.
  • During croton oil (0.01 ml mouse−1, p.o.)-induced diarrhoea, the ED50 values of i.p.-injected WIN 55,212-2 and cannabinol (but not SR141716A) were significantly decreased (compared to control mice). However, the ED50 values of WIN 55,212-2 were similar after i.p. or i.c.v. administration.
  • The inhibitory effects of WIN 55,212-2 and cannabinol were counteracted by SR141716A (16 nmol mouse−1, i.p.) but not by SR144528 (52 nmol mouse−1, i.p.) both in control and croton-oil treated mice.
  • Ganglionic blockade with hexamethonium (69 nmol mouse−1, i.p.) did not modify the inhibitory effect of i.p.-injected cannabinoid agonists either in control or in croton-oil treated mice.
  • The lower ED50 values of cannabinoid drugs after i.c.v. administration suggest a central (CB1) site of action. However, a peripheral site of action is suggested by the lack of effect of hexamethonium. In addition, croton oil-induced diarrhoea enhances the effect of cannabinoid agonists by a peripheral mechanism.
Keywords: Intestinal motility, cannabinoid receptors, inflammatory bowel disease, antidiarrhoeal drugs, small intestine

Introduction
Preparations of Cannabis sativa have been used medicinally for over 4000 years for the treatment of a variety of disorders, including migraine, muscle spasm, seizures, glaucoma, pain, nausea and diarrhoea (Felder & Glass, 1998). In 1964 Δ9-tetrahydrocannabinol (Δ9-THC) was isolated, which was later shown to be responsible for many of the pharmacological actions of Cannabis preparations (Mechoulam et al., 1998). With regard to the gastrointestinal tract, Dewey et al. (1972) were the first to report that Δ9-THC reduced the rate of passage of a charcoal meal in the mouse small intestine and these findings were confirmed by others (Chesher et al., 1973; Jackson et al., 1976; Shook & Burks, 1989).
Understanding of the mechanism by which Δ9-THC exerts its pharmacological actions has seen considerable progress in the last ten years following the discovery of two distinct cannabinoid receptors, named CB1, (expressed mainly by central and peripheral neurons) and CB2 (that occur mainly in immune cells) (Matsuda et al., 1990; Munro et al., 1993; Pertwee, 1998). The discovery of these receptors has led to the demonstration that there are endogenous agonists for these receptors, namely anandamide and 2-arachidonylglycerol (Devane et al., 1992; Stella et al., 1997), the latter found in the intestine of the dog (Mechoullam et al., 1995).
The myenteric plexus of the guinea-pig intestine contains CB1-, but not CB2-like cannabinoid receptor mRNA (Griffin et al., 1997). Activation of prejunctional CB1 receptors produces inhibition of excitatory transmission (Pertwee et al., 1996; Izzo et al., 1998) in the isolated guinea-pig ileum and these inhibitory effects are associated with a decrease in acetylcholine release from enteric nerves (Coutts & Pertwee, 1997). However, a preliminary report indicates that cannabinoid agonists potentiate electrically-induced contractions in the porcine ileum and this effect is mediated by CB2 receptors (Albasan et al., 1999).
The involvement of CB1 receptors in intestinal motility has been confirmed also in vivo. Indeed, the endogenous cannabinoid agonist anandamide (Calignano et al., 1997) and the synthetic cannabinoid agonist WIN 55,212-2 (Colombo et al., 1998; Izzo et al., 1999a) inhibited, whilst the CB1 receptor antagonist, SR141716A increased gastrointestinal transit in mice. However, in these studies, cannabinoid drugs were administered intraperitoneally or subcutaneously and therefore it was not clear if cannabinoids were acting at central or peripheral cannabinoid receptors. In addition, there are no data in the literature concerning the effects of cannabinoid drugs in the control of upper gastrointestinal motility during pathophysiological states.
The present study, therefore, has two objectives: (i) to compare the effect of cannabinoid drugs on intestinal motility after intracerebroventricular and intraperitoneal administration and (ii) to evaluate the effect of cannabinoid agonists on intestinal motility during experimental diarrhoea. In order to achieve this experimental condition, we have used croton oil, a well-known cathartic agent (Pol et al., 1996). The cannabinoid drugs used were: the natural agonist cannabinol (Petitet et al., 1998) and the synthetic agonist WIN 55,212-2 (Compton et al., 1992), the CB1 receptor antagonist SR141716A (Rinaldi-Carmona et al., 1995) and the CB2 receptor antagonist SR144528 (Rinaldi-Carmona et al., 1998).

Methods
Animals
Male ICR mice (Harlan Italy, Corezzana, MI) (24–26 g) were used after 1 week of acclimation (temperature 23±2°C; humidity 60%). Food was withheld 3 h before experiments but there was free access to drinking water.
Upper gastrointestinal transit
Gastrointestinal transit was measured in control mice or 3 h after treatment with croton oil (0.01 ml mouse−1). At this time, 0.1 ml of a black marker (10% charcoal suspension in 5% gum arabic) was administered orally to assess upper gastrointestinal transit as previously described (Pol et al., 1996; Izzo et al., 1999a). After 20 min the mice were killed by asphyxiation with CO2 and the gastrointestinal tract removed. The distance travelled by the marker was measured and expressed as a percentage of the total length of the small intestine from pylorus to caecum (Izzo et al., 1999a).
The cannabinoid agonists WIN 55,212-2 (2–239 nmol mouse−1), cannabinol (24–4027 nmol mouse−1), the CB1 receptor antagonist SR141716A (2–539 nmol mouse−1), the CB2 receptor antagonist SR144528 (52 nmol mouse−1) or vehicle (DMSO, 4–8 μl mouse−1) were given intraperitoneally (i.p.) or intracerebroventricularly (i.c.v.) 20 min before charcoal administration. In some experiments SR141716A (16 nmol mouse−1=0.3 mg kg−1), SR144528 (52 nmol mouse −1=1 mg kg−1) or hexamethonium (69 nmol mouse −1=1 mg kg−1) were given (i.p.) 10 min before the cannabinoid agonists. The doses of hexamethonium and SR144528 were selected on the basis of previous published work (Schirgi-Degen & Beubler, 1995; Rinaldi-Carmona et al., 1998)
Intracerebroventricular injections
Intracerebroventricular injections were performed as described by Haley & McCormick (1957)). Mice were briefly anaesthetized with enflurane and the drugs were delivered in a volume of 4 μl, using a Hamilton microlitre syringe fitted with 26-gauge needle.
Drugs
Drugs used were: WIN 55,212-2 mesylate (Tocris Cookson, Bristol, U.K.), hexamethonium bromide and cannabinol (SIGMA, Milan, Italy). SR141716A [(N-piperidin-l-yl)-5-(4-chlorophenyl)-1-2,4-dichlorophenyl) - 4 -methyl-lH-pyrazole-3-carboxamide hydrochloride and SR144528 (N-[-1S-endo-1,3,3-trimethyl bicyclo [2.2.1] heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl) - pyrazole - 3 - carboxamide-3-carboxamide) were a gift from Dr Madaleine Mossé and Dr Francis Barth (SANOFI-Recherche, Montpellier, France). Cannabinoid drugs were dissolved in DMSO, while hexamethonium was dissolved in saline.
Statistics
Data are mean±s.e.mean. To determine statistical significance, Student's t-test for unpaired data or one-way analysis of variance followed by Tukey–Kramer multiple comparisons test was used. A P-value less than 0.05 was considered significant. ED50 (dose which produced a 50% variation of gastrointestinal transit) and Emax (maximal effect) values were calculated using the computer program of Tallarida & Murray (1986).

Results
Effect of cannabinoid drugs on upper gastrointestinal transit in control mice
The effect of i.p.- or i.c.v.- injected WIN 55,212-2 (2–239 nmol mouse−1) and cannabinol (24–4027 nmol mouse−1) on percentage inhibition of upper gastrointestinal transit are presented in Figure 1. Both WIN 55,212-2 and cannabinol produce a dose-dependent inhibition of gastrointestinal transit. However, the ED50 values after i.p. or i.c.v. administration were statistically different. The ED50 and Emax values of cannabinoid drugs are shown in Table 1.
Figure 1
Figure 1
Dose related inhibition of upper gastrointestinal transit by WIN 55,212-2 and cannabinol after i.p. or i.c.v. administration in control mice. Each point represents the mean±s.e.mean of 10–13 animals for each experimental group. * (more ...)
Table 1
Table 1
ED50±s.e.mean and Emax±s.e.mean of cannabinoid drugs after i.p. or i.c.v. administration in control mice and in mice receiving croton oil (0.01 ml mouse−1, orally)
The CB1 receptor antagonist SR141716A (16 nmol mouse−1, i.p.), but not the CB2 receptor antagonist SR144528 (52 nmol mouse−1, i.p.) counteracted the inhibitory effect of WIN 55,212-2 (5 nmol mouse−1, i.c.v. or 50 nmol mouse−1, i.p.) and cannabinol (201 nmol mouse−1, i.c.v. or 2010 nmol mouse−1, i.p.) after both i.c.v. (Figure 2) and i.p. (Figure 3) routes of administration. Hexamethonium (69 nmol mouse−1, i.p.) abolished the effect of both WIN 55,212-2 and cannabinol after i.c.v. (Figure 2) but not after i.p. (Figure 3) administration.
Figure 2
Figure 2
Effect of WIN 55,212-2 (5 nmol mouse−1 i.c.v) and cannabinol (201 nmol mouse, i.c.v.) on upper gastrointestinal transit alone or in mice treated with SR141716A (16 nmol mouse−1, i.p.) or (more ...)
Figure 3
Figure 3
Effect of WIN 55,212-2 (50 nmol mouse−1, i.p.) and cannabinol (2010 nmol mouse−1, i.p.) on upper gastrointestinal transit alone or in mice treated with SR141716A (16 nmol mouse−1 (more ...)
SR 14176A (i.p. or i.c.v.), per se, dose-dependently increased upper gastrointestinal transit (Figure 4a). However, the ED50 value after i.c.v. administration was significantly (P<0.01) lower than the ED50 after i.p. administration (Table 1). At a dose of 16 nmol mouse−1, SR141716A (i.c.v.) significantly (P<0.05) increased intestinal motility (Figure 4a) and this effect was significantly (P<0.05) counteracted by hexamethonium (69 nmol mouse−1 i.p.) (per cent increase of SR141716A: 44±3; per cent increase of SR141716A in the presence of hexamethonium; 1±3, n=10).
Figure 4
Figure 4
Dose-related increase of upper gastrointestinal transit by SR141716A in control mice (a) or mice treated with croton oil (0.01 ml mouse−1, orally) (b). Results are mean±s.e.mean of 10–12 animals for each experimental (more ...)
The CB2 receptor antagonist SR144528 (52 nmol mouse−1, i.p.), given alone, did not significantly modify gastrointestinal transit (control 47±4%; SR144528 48±2%, n=10, P>0.2). Hexamethonium (69 nmol mouse−1 i.p.) did not significantly modify gastrointestinal transit (17±8% increase, n=12). DMSO (4 μl mouse−1 i.c.v. or 4–8 μl mouse−1 i.p.) had no effect on the response under study (data not shown).
Effect of cannabinoid drugs on upper gastrointestinal transit during croton oil-induced diarrhoea
Oral administration of croton oil produced diarrhoea which was associated with a significant increase in gastrointestinal transit (per cent transit: control 46±2; croton oil, 56±2, P<0.01, n=24). Both WIN 55,212-2 (2–239 nmol mouse−1, i.p.) and cannabinol (24–4027 nmol mouse−1, i.p.) produced a dose-related inhibition of transit (Figure 5) and both agonists had a lower ED50 value compared to the corresponding i.p. treatment in control mice (Table 1). In croton oil-treated animals, WIN 55,212-2 (i.p.) and cannabinol (i.p.) had a significant inhibitory effect with threshold doses of 5 nmol mouse−1 and 80 nmol mouse−1 doses respectively whilst in control mice, significant inhibitory effects were achieved at doses of 14 nmol mouse−1 (WIN 55,212-2) and 2010 nmol mouse−1 (cannabinol) respectively (Figure 5).
Figure 5
Figure 5
Dose-related inhibition of upper gastrointestinal transit by WIN 55,212-2 (i.p.) and cannabinol (i.p.) in control mice or in mice receiving croton oil (0.01 ml mouse−1, orally). Results are mean±s.e.mean of 10–12 (more ...)
Administered i.c.v. WIN 55,212-2 (2–239 nmol mouse−1) also decreased intestinal motility, but the ED50 value (74±10 nmol mouse−1) was not statistically different from the ED50 value (68±5 nmol mouse−1) after i.p. administration (Table 1).
The inhibitory effect of i.p.-injected WIN 55,212-2 (14 nmol mouse−1) or cannabinol (805 nmol mouse−1) was reduced by the CB1 receptor antagonist SR141716A (16 nmol mouse−1, i.p.) but not by the CB2 receptor antagonist SR144528 (52 nmol mouse−1, i.p.) or by the ganglion blocker hexamethonium (69 nmol mouse−1, i.p.) (Figure 6).
Figure 6
Figure 6
Upper gastrointestinal transit in mice with diarrhoea induced by croton oil (0.01 ml mouse−1, orally): effect of WIN 55,212-2 (14 nmol mouse−1, i.p.) and cannabinol (805 nmol mouse−1, i.p.) (more ...)
Figure 4b shows the potentiating effect of SR141716A (2–539 nmol mouse, i.p.) in mice treated with croton oil. The ED50 value (418±32 nmol mouse−1) was not statistically different from the corresponding ED50 value in control animals (375±31 nmol mouse−1). By contrast, SR144528 (52 nmol mouse−1, i.p.) or hexamethonium (69 nmol mouse−1, i.p.) did not modify gastrointestinal transit (per cent transit: croton oil: 58±6, croton oil+SR144528 61±5, croton oil+hexamethonium 68±4, n=6, P>0.2).

Discussion
The role of cannabinoid receptors in control mice
It is now well known that cannabinoid agonists can reduce intestinal motility through activation of CB1 receptors. Indeed activation of CB1 receptors can mediate, (i) inhibition of electrically-evoked contractions in the isolated guinea-pig (Pertwee et al., 1996; Izzo et al., 1998) and human ileum (Croci et al., 1998), (ii) inhibition of fast and slow synaptic transmission in guinea-pig myenteric nerves (Lopez-Redondo et al., 1997), (iii) inhibition of electrically-evoked acetylcholine release from myenteric nerves (Coutts & Pertwee, 1997) and (iv) reduction of peristalsis efficiency in the isolated guinea-pig ileum (Heinemann et al., 1999; Izzo et al., 2000). These findings are in keeping with the presence of CB1, but not CB2-like receptor messenger RNA in the myenteric plexus of the guinea-pig small intestine (Griffin et al., 1997). Consistent with these in vitro findings, it has been shown that cannabinoid agonists reduced intestinal motility in mice (Calignano et al., 1997; Colombo et al., 1998; Izzo et al., 1999a) and rats (Izzo et al., 1999c) and this effect was counteracted by SR141716A, a specific CB1 antagonist. However, whether the effect of cannabinoid drugs in vivo is mediated via a central or a peripheral site of action was not demonstrated in these studies. Indeed the CB1 receptor is located within both the central nervous system (Matsuda et al., 1990) and within the enteric nervous system (Griffin et al., 1997).
In the present study we have shown that the synthetic cannabinoid agonist WIN 55,212-2 and the natural cannabinoid agonist cannabinol produced a dose-related inhibition of upper gastrointestinal transit when administered i.p. or i.c.v. The inhibitory effect of cannabinoid agonists was abolished by SR141716A, a specific CB1 antagonist, but not by SR144528, a CB2 receptor antagonist, indicating an involvement of CB1 but not CB2 receptors.
The ED50 values of WIN 55,212-2 and cannabinol after i.c.v. administration were significantly lower than the corresponding ED50 values after i.p. administration. The low doses that were needed to inhibit transit after i.c.v. injection implies that cannabinoid agonists may inhibit intestinal motility through activation of central CB1 receptors. However, the effect of i.p.-injected cannabinoid agonists was not modified by the ganglion blocker hexamethonium. These results probably indicate that the effect of i.p.-injected cannabinoid agonists is mediated by peripheral CB1 cannabinoid receptors.
Although some reports indicate that the CB1 receptor antagonist SR141716A does not affect intestinal motility in the isolated human ileum (Croci et al., 1998) and gastric emptying in the rat (Izzo et al., 1999b), other studies indicate that intestinal motility could be tonically inhibited by the endogenous cannabinoid system. Indeed SR141716A increased electrically-induced contractions in the isolated guinea-pig ileum (Pertwee et al., 1996; Izzo et al., 1998) and intestinal motility and defaecation in the mouse (Colombo et al., 1998; Izzo et al., 1999a). The observation that SR141716A, per se, increased intestinal motility does not necessary imply that endogenous cannabinoids are involved in the control of intestinal motility in view of the inverse agonist properties of SR141716A at human recombinant CB1 (Landsman et al., 1997) and both CB1 and CB2 receptors (MacLennan et al., 1998).
In the present study, we have shown that SR141716A (i.c.v. or i.p.) produced a dose-dependent increase in upper gastrointestinal transit. The ED50 value after i.c.v. administration was significantly lower than the ED50 value after i.p. administration, suggesting a central site of action of SR141716A. The most likely explanation of these results is that the endogenous cannabinoid system, within the central nervous system, can inhibit intestinal motility through activation of CB1 receptors. In a recent study, we have shown that SR141716A (i.p.)-induced changes in intestinal motility are not modified by the ganglionic blocker hexamethonium (Izzo et al., 1999a), suggesting a peripheral site of action of i.p.-injected SR141716A.
Effect of cannabinoid drugs during croton oil-induced diarrhoea
Croton oil is a well known irritant that has been widely used to produce experimental inflammation in different tissues, especially skin and mucosa, and induces diarrhoea associated with intestinal inflammation in the mouse small intestine (Pol et al., 1996). According to Pol et al., (1996), we have shown that croton oil increases upper gastrointestinal transit 3 h after oral administration. The cannabinoid agonists WIN 55,212-2 and cannabinol blocked the increase in intestinal motility induced by croton oil; in addition, the ED50 values of i.p.-injected WIN 55,212-2 and cannabinol were significantly decreased (compared to control mice). However, during croton oil-induced diarrhoea the ED50 value of WIN 55,212-2 was similar after i.p. or i.c.v. treatment and ganglionic blockade with hexamethonium did not alter the inhibitory effect of i.p.-injected cannabinoids.
Taken together, these results indicate that the enhanced effect of cannabinoid agonists are mediated by peripheral receptors. By contrast, using the castor oil test, we have recently shown that cannabinoid agonists possess either weak or no antidiarrhoeal activity in the rat (Izzo et al., 1999c). The use of a different cathartic (castor oil vs croton oil), different species (rat vs mouse) and different region of the gut (whole gut vs upper gastrointestinal tract) could explain this discrepancy. Consistent with this hypothesis, Shook & Burks (1989) showed that Δ9-THC produced a greater inhibition of small intestinal transit than large bowel transit.
In line with the result obtained in control mice and those reported in the isolated guinea-pig ileum (Pertwee et al., 1996; Izzo et al., 1998), the antitransit response of cannabinoid agonists involves CB1, but not CB2 receptors, as the inhibitory effect of both WIN 55,212-2 and cannabinol were reduced by SR141716A, but not SR144528. Administration of SR141716A (i.p.), per se, increased intestinal motility in control mice and those given croton oil with a similar ED50 value, thus indicating that during the experimental diarrhoea the endogenous cannabinoid system is activated as in control animals. By contrast, SR144524, a specific CB2 receptor antagonist, at doses previously shown to bind the CB2 receptor in the rat spleen (Rinaldi-Carmona et al., 1998), failed to modify the inhibitory effect of both WIN 55,212-2 and cannabinol and did not modify, per se, intestinal motility during the diarrhoea induced by croton oil. Thus, a role for CB2 receptors in modulating intestinal motility during experimental diarrhoea seems unlikely.
Conclusions
Our results suggest that both central and peripheral CB1 receptors can modulate upper gastrointestinal motility. However, the effect of systemic (i.p.) cannabinoid drugs is probably mediated by peripheral receptors. Diarrhoea induced by the irritant croton oil enhances the inhibitory effect of cannabinoid agonists by a peripheral mechanism, while CB2 receptors are not involved in the control of intestinal motility, either in physiological or in pathophysiological states. Thus, selective non-psychotropic CB1 agonists could represent novel drugs to treat motility disorders associated with inflammatory diarrhoea.
Acknowledgments
This work was supported by Cofinanziamento Murst 1999 and Enrico and Enrica Sovena Foundation (Roma). The Authors are grateful to Drs Antonio Calignano and Carla Cicala for their help. SR141716A and SR144528 were a kind gift from SANOFI (Montpellier, France).
Abbreviations
 
COcroton oil
 
Δ9-THCΔ9-tetrahydrocannabinol


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Anti-inflammatory property

Published in final edited form as:
PMCID: PMC1852513
NIHMSID: NIHMS18229
Anti-inflammatory property of the cannabinoid agonist WIN-55212-2 in a rodent model of chronic brain inflammation
Yannick Marchalant, Susanna Rosi, and Gary L Wenk
Arizona Research Laboratories, Division of Neural Systems Memory and Aging, University of Arizona, Tucson, AZ (Y.M., S.R.); Neurosurgery, Physical Therapy & Rehabilitation Science, University of California at San Francisco, San Francisco, CA (S.R.); Department of psychology, Ohio State University, Columbus, OH (Y.M., G.L.W.).

Abstract
Cannabinoid receptors (CBr) stimulation induces numerous central and peripheral effects. A growing interest in the beneficial properties of manipulating the endocannabinoid system has lead to the possible involvement of CBr in the control of brain inflammation. In the present study we examined the effect of the CBr agonist, (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)-pyrrolo[1,2,3-de]-1,4benzoxazin-6-yl]-1-naphthalenyl-methanone mesylate (WIN-55212-2), on microglial activation and spatial memory performance, using a well characterized animal model of chronic brain inflammation produced by the infusion of lipopolysaccharide (LPS, 250 ng/hr for 3 weeks) into the 4th ventricle of young rats. WIN-55212-2 (0.5 or 1.0 mg/kg/day, i.p.) was administered for three weeks. During the third week of treatment, spatial memory ability was examined using the Morris water-maze task. We found that 0.5 and 1 mg/kg WIN-55212-2 reduced the number of LPS-activated microglia, while 1 mg/kg WIN-55212-2 potentiated the LPS-induced impairment of performance in the watermaze task. CB1 receptors were not expressed by microglia and astrocytes, suggesting an indirect effect of WIN on microglia activation and memory impairment. Our results emphasize the potential use of CBr agonists in the regulation of inflammatory processes within the brain; this knowledge may lead to the use of CBr agonists in the treatment of neurodegenerative diseases associated with chronic neuroinflammation, such as Alzheimer disease.
Keywords: cannabinoid receptors, inflammation, activated microglia, Alzheimer’s disease, LPS, spatial memory

Microglial cells play a pivotal role as immune effectors in the central nervous system and may participate in the initiation and progression of neurological disorders, such as Alzheimer’s disease (AD), Parkinson’s disease and multiple sclerosis by releasing harmful molecules such as pro-inflammatory cytokines, reactive oxygen species or complement proteins (Akiyama et al., 2000; Kim and de Vellis, 2005). Many of the pathological, immunological, biochemical and behavioral changes seen in these and other neurodegenerative diseases can be reproduced in young rats by chronic infusion of lipopolysaccharide (LPS) into the 4th ventricle (Hauss-Wegrzyniak et al., 1998, Hauss-Wegrzyniak et al., 1999). Chronic infusion of LPS results in the activation of microglia within hippocampus and entorhinal cortex, brain regions involved in learning and memory formation (Hauss-Wegrzyniak et al., 1998; Rosi et al., 2004). Chronic brain inflammation is associated with impaired spatial memory, impaired induction of long-term potentiation, a loss of N-methyl-d-aspartate (NMDA) receptors, astrocytosis, elevated cytokines and related pro-inflammatory proteins and transcription factors (Hauss-Wegrzyniak et al., 1998, Hauss-Wegrzyniak et al., 1999; Rosi et al., 2004).
The endocannabinoid system may regulate many aspects of the brain’s inflammatory response, including the release of pro-inflammatory cytokines and modulation of microglial activation (Neumann, 2001; Klein, 2005). The endocannabinoid system is comprised of two G-protein-coupled receptors designated as CB1 and CB2, although not all endocannabinoid effects can be explained only by these two receptors (Begg et al., 2005). CB1 receptors are expressed in the brain and are responsible for most of the behavioral effects of the cannabinoids. CB2 receptors are expressed by immune and hematopoietic cells peripherally (Begg et al., 2005), and seem to be expressed on neurons in the brainstem and the brain (Van Sickle et al., 2005; Gong et al., 2006; Onaivi et al. 2006) although their presence in the brain is controversial (Munro et al., 1993). Two endogenous ligands for these receptors, arachydonylethanolamine and 2-arachidonoylglycerol (Stella, 2004), influence immune responses by inhibiting cytokine release and other anti-inflammatory actions (Klein et al., 2003; Klein, 2005). Microglia also express CBr and release cytokines in response to exposure to LPS or beta-amyloid protein; this effect can be inhibited by prior cannabinoid treatment (Facchinetti et al., 2003; Ramirez et al., 2005; Sheng et al., 2005). Astrocytes may also synthesize and release endocannabinoids (Walter et al., 2002). CB1 receptors have been widely studied because of their role in the psychoactive effects of the cannabis sativa plant (Δ9-etrahydrocannabinol or Δ9-THC). Δ9-THC can impair performance in rats, mice or monkeys under multiple experimental conditions (Castellano et al., 2003). Therefore, in the current study, we investigated the effect of a CBr agonist on microglial activation and spatial memory in a rodent model of chronic brain inflammation induced by LPS infusion into the 4th ventricle. We used the number of immunoreactive microglia (activated) as a biomarker of brain inflammation (Hauss-Wegrzyniak et al., 1998, Hauss-Wegrzyniak et al., 1999; Rosi et al., 2004, Rosi et al., 2005) to evaluate the potential anti-inflammatory properties of the WIN 55-212-2 compound.

EXPERIMENTAL PROCEDURES
Subjects and surgical procedures
Fifty-four young (3 months old) male F-344 rats (Harlan Sprague-Dawley, Indianapolis, IN) were singly housed in Plexiglas cages with free access to food and water. The rats were maintained on a 12/12-h light-dark cycle in a temperature-controlled room (22°C) with lights off at 0800. All rats were given health checks, handled upon arrival and allowed at least one week to adapt to their new environment prior to surgery. Artificial cerebrospinal fluid (aCSF n=26) or LPS (Sigma, St-Louis, MO, E. coli, serotype 055:B5, TCA extraction, 1.0 mg/ml dissolved in aCSF, n=28) were chronically infused for 21 days through a cannula implanted into the 4th ventricle that was attached (via Tygon tubing, 0.06 O.D., and an osmotic pump connect, Model 3280P, Plastics One, Roanoke, VA) to an osmotic minipump (Alzet, Cupertino, CA, model #2004, to deliver 0.25 μl/h, Hauss-Wegrzyniak et al., 1998). The aCSF vehicle contained (in mM) 140 NaCl; 3.0 KCl; 2.5 CaCl2; 1.0 MgCl2; 1.2 Na2HPO4, adjusted to pH 7.4. Rats infused with either aCSF or LPS were also administered daily the synthetic cannabinoid CB1/CB2 receptor agonist (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)-pyrrolo[1,2,3-de]-1,4benzoxazin-6-yl]-1-naphthalenyl-methanone mesylate (WIN-55212-2, Sigma, St-Louis, MO, 0.5 or 1.0 mg/kg, i.p.) or vehicle (Dimethylsulfoxide (100%), DMSO, Sigma, St-Louis, MO). Rats were assigned to one of the following six groups: aCSF + vehicle, LPS+ vehicle, aCSF + WIN (0.5 mg/kg), LPS + WIN (0.5mg/kg), aCSF + WIN (1 mg/kg), LPS + WIN (1 mg/kg). Drug or vehicle administration began the first day after surgery and continued throughout the behavioral testing, which began on day 14 of the LPS or aCSF infusion.
Behavioral Testing
Spatial learning ability was assessed using a 185 cm diameter water maze with white walls. The water was maintained at 26-28°C and made opaque by adding white, non-toxic, paint. The pool was in the center of a 2.3 x 2.73 x 2.5m room with multiple visual stimuli on the wall as distal cues, and a chair and a metal board against the wall of the pool as proximal cues. The circular escape platform was 11.5 cm in diameter. For the spatial (hidden-platform) version of the water task, a circular escape platform was present in a constant location, submerged about 2.5 cm below the water surface. The rats were tracked by an overhead video camera connected to a VP114 tracking unit (HVS Image, England). Custom software was used to store and analyze each rat’s latency to find the submerged platform during each trial.
Each rat performed three training blocks per day (two training trials per block) for 4 days (24 trials total), with a 60-min inter-block interval. On each trial, the rat was released into the water from one of seven locations spaced evenly at the side of the pool, which varied randomly from trial to trial. After the rat found the escape platform or swam for a maximum of 60 sec, it was allowed to remain on the platform for 30 sec. To control for possible drug-induced deficits in visual acuity and swimming ability, the same rats were also tested on a second version of this task. In this version, a visible platform raised 2 cm above the surface of the water was moved randomly to one of four locations in the tank after each trial. A total of 4 visible-platform trials were performed. Drug administration was performed 20 minutes prior to the behavioral testing. The results, i.e. latency (sec) to find the hidden platform, were analyzed by ANOVA followed by post hoc comparisons according to the method of Bonferonni/Dunn.
Histological procedures
After behavioral testing was completed, each rat was deeply anesthetized with isoflurane and prepared for a transcardiac perfusion of the brain with cold saline containing 1 U/ml heparin, followed by 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. Brains were then removed and the placement of the cannula in the 4th ventricle was confirmed. The brains were then post-fixed one hour in the same fixative and then stored (4°C) in phosphate buffer saline (PBS), pH 7.4. Free-obtained using a vibratome from perfused tissues for staining with standard avidin/biotin peroxydase or fluorescence labeling methods. The monoclonal antibody OX-6 (final dilution 1:400, Pharmigen, San Diego, CA) was used to visualize activated microglia cells. This antibody is directed against Class II major histocompatibility complex (MHC II) antigen. After quenching endogenous peroxydase/activity and blocking nonspecific binding, the sections were incubated (4°C) overnight with primary antibodies directed against the specific epitope (MCH II). Thereafter, the sections were incubated for 2h (22°C) with the secondary monoclonal antibody, rat adsorbed biotinylated horse anti-mouse immunoglobulin G (final dilution 1:200, Vector, Burlingame, CA). Sections were than incubated for 1h (22°C) with avidin-biotinylated horseradish peroxydase (Vectastain, Elite ABC kit, Vector, Burlingame, CA). After washing again in PBS, the sections were incubated with 0.05% 3, 3’-diaminobenzidine tetrahydrochloride (Vector, Burlingame, CA) as chromogen. The reaction was stopped by washing the section with buffer. No staining was detected in the absence of the primary or secondary antibodies. Sections were mounted on slides, air-dried and coverslipped with cytoseal (Allan Scientific, Kalamazoo, MI) mounting medium. The location of immunohistochemically-defined cells was examined by light microscopy. Quantification of cell density in the reconstructed hippocampal coronal sections was assessed with MetaMorph imaging software (Universal Image Corporation, West Chester, PA). Briefly, areas of interest were determined as previously reported in detail (Rosi et al., 2005), their surface measured, and the immunoreactive cells numerated, allowing use to determine a number of immunoreactive cells by millimeter square in the areas of interest.
A polyclonal antibody directed against the first ninety-nine amino-acid residue from the human CB1 (final dilution 1:500, Affinity Bioreagents, Golden, CO) was used to visualize CB1 receptors. After quenching endogenous peroxydase/activity and blocking nonspecific binding, the sections were rinsed in 0.1 M TRIS buffer (TB), pH 7.4, for 15min, in 0.1 M Tris buffered saline (TBS), pH 7.4, for 15 min, blocked using 2% avidin in TBS for 30 min, rinsed in TBS for 30 min, blocked using 2% biotin for 30min, and finally rinsed in TBS for 30 min. The tissue sections were then incubated in the anti-CB1 (diluted 1:500) overnight at 4°C. The antibody was diluted in a solution containing 0.1% Triton X-100 and 1% NGS in 0.1 M TBS. These sections were then rinsed in TBS for 60 min and incubated in biotinylated goat anti-rabbit IgG (diluted 1:200) for 90 min at room temperature. The sections were rinsed with TBS for 60 min and incubated for 1h (22°C) with avidin-biotinylated horseradish peroxydase (Vectastain, Elite ABC kit, Vector, Burlingame, CA). The sections were then rinsed with TBS for 30 min and then incubated with 0.05% 3,3’-diaminobenzidine tetrahydrochloride as chromogen. The reaction was stopped by washing the section with buffer. No staining was detected in the absence of the primary or secondary antibodies. Sections were mounted on slides, air-dried for 24 h.Counter staining (Cresyl Violet) was performed before slides were cover-slipped with cytoseal (Allan Scientific, Kalamazoo, MI) mounting medium.
Double immunofluorescence staining
Free floating sections were mounted on slides and air-dried under a hound. The tissues were than processed as described in details in Rosi et al., 2005. After washing in TBS solution the polyclonal rabbit anti-CB1 (Affinity Bioreagents, Golden, CO, dilution 1:500) was apply. After 48 h of incubation at 4°C, the sections were incubated for 2 h at room temperature with the secondary anti-rabbit biotinylated antibody (Vector, Burlingame, CA), followed by incubation with Avidin+Biotin amplification system (Vector, Burlingame, CA) for 45 minutes. The staining was visualized using the TSA fluorescence system CY3 (PerkinElmer Life Sciences, Emeryville, CA). After washing in TBS solution, the tissue was quenched and blocked again and incubated with the monoclonal antibody OX-6 (Pharmigen, San Diego, CA, final dilution 1:400) for 24 h or with the monoclonal antibody anti-GFAP (Chemicon, Temecula, CA, final dilution 1:500) for 24h. Before applying the biotinylated monoclonal secondary rat adsorbed antibody (Vector, Burlingame, CA) for 2 h, the tissue was incubated with Avidin Biotin Blocking Kit (Vector, Burlingame, CA) for 30 min to block cross reaction with the primary staining. Following treatment with an Avidin+Biotin amplification system (Vector, Burlingame, CA), the staining was visualized with TSA fluorescence system CY5 (PerkinElmer Life Sciences, Emeryville, CA) and the nuclei were counterstained with Sytox-Green (Molecular Probes, Eugene, OR). No staining was detected in the absence of the primary or secondary antibodies.

RESULTS
Chronic infusion of LPS and WIN-55212-2 injections were well tolerated by all rats: they gained weight normally for the duration of the study.
Behavior
No significant difference in locomotor activity (swim speed) was found across groups, p>0.1. An ANOVA performed on the latency results obtained in the water maze task revealed an overall main effect of testing day (F5, 218=16.057, p<0.0001) for all groups (See Figure 1) and an overall group effect (LPS versus aCSF) upon latency for each day of testing (F1, 328=11.367, p=0.0008 for day1; F1, 328=85.681, p<0.0001 for day2; F1,328=109.756, p<0.0001 for day3; F1, 328=96.25, p<0.0001 for day 4). There was no significant effect of treatment except for day 3 (F2, 328=5.788, p=0.0034). There was a significant interaction between group and drug on days 2 and 3 (F2, 328=3.982, p=0.0196 for day 2; F2, 328=12.641, p<0.0001 for day 3). Post-hoc analyses of each testing day revealed a significant treatment effect on day 1, where LPS+WIN 1 rats were significantly impaired compared to the aCSF+WIN 1 rats (p=0.0015). On days 2 and 3, all LPS-infused rats were significantly (p<0.0033) impaired, as compared to their respective control groups. On day 4, all LPS-infused rats were significantly (p<0.0033) impaired as compared to their respective aCSF controls. There was a significant interaction between treatment and LPS-infused animals (F2, 113=5.026, p=0.081). Post-hoc analyses of each testing day revealed a significant treatment effect on days 2 and 3; performance of LPS+WIN 1 rats was significantly (p<0.0033) worse as compared to the LPS+vehicle rats on those both days. Overall, the drug treatment (0.5 or 1 mg/kg) did not significantly impair performance of aCSF-infused rats and did not attenuate the impairment induced by the LPS infusion. The 1 mg/kg treatment, that did not cause any impairment in aCSF-infused rats, but did worsen the impairment observed in LPS-infused rats, demonstrating an interaction between inflammation and the highest dose of the drug used.
Figure 1
Figure 1
Water maze performance. On days 2 and 3, all LPS-infused animals (closed triangles, squares and circles) were significantly impaired (*p<0.0033, Bonferroni/Dunn post hoc test) compared to their control groups. On days 2 and 3, LPS+WIN 1 rats were (more ...)
Histology
Immunostaining for OX-6 (Figure 2A) revealed numerous highly activated microglia cells distributed throughout the hippocampus of LPS-infused rats (Figure 2A d). The activated microglia had a characteristic bushy morphology with increased cell body size and contracted and ramified processes (Figure 2A d). Rats infused with aCSF had very few mildly activated microglia evenly scattered throughout the brain, consistent with results from previous studies (Hauss-Wegrzyniak et al., 1998; Rosi et al., 2005). No difference in staining was evident between the aCSF group and the aCSF rats injected with either dose of WIN (Figure 2A a-c). In LPS-infused rats that were also treated with WIN (Figure 2A e-f) the OX-6 antibody stained fewer activated microglial cells.
Figure 2
Figure 2
(A.) Activated microglia in the dentate gyrus. Note the diminution of activated microglia cells in the dentate gyrus of animals treated with either doses of WIN 55212-2 (e,f) compare to the LPS+vehicle group (d) (B.) Density of activated microglial cells (more ...)
Activated microglia cell counts
The number of activated microglia per millimeter square was determined in 4 different brain regions: dentate gyrus (DG), CA3 and CA1 regions of the hippocampus and the entorhinal cortex (EC) (Figure 2B). These brain regions were examined for their known implication in spatial learning (Nadel and Land, 2000). An ANOVA of the data revealed an overall main region effect (F5,210=16.057, p<0.0001) for all groups and an overall main effect of the infusion of LPS in each region examined (F5,48=32.557, p<0.0001 for DG; F5,48=23.552, p<0.0001 for CA3; F5,48=3.828, p=0.0053 for CA1;F5,48=19.308, p<0.0001 for EC).
In the DG, CA3 and EC, all LPS infused rats had a significantly (p<0.0033) higher density of activated microglia compared to their respective control groups, consistent with our previous results (Rosi et al., 2005). In the DG and CA3 the number of activated microglia was significantly (p<0.0033) reduced in LPS-infused rats given either dose of WIN, as compared to rats in the LPS+vehicle group (DG, 41.4% reduction for 0.5mg/kg and 40.6% reduction for 1mg/kg; CA3, 43.7% reduction for WIN 0.5mg/kg and 49.4% reduction for WIN 1mg/kg). In the EC, despite a 24.2% reduction for WIN 0.5mg/kg and 32.4% reduction for WIN 1mg/kg, no significant difference was found between the LPS-infused groups. In the CA1 region of the hippocampus, there was a significant (p<0.0033) difference only between the aCSF and LPS+vehicle groups. Overall, the effects of WIN were not dose-dependent in the DG and CA3 and not significant within the CA1 or EC brain regions.
CB1 receptors
The distribution of the CB1 receptors observed following our staining is in accordance with previous studies (Tsou et al., 1998) (Figure 3a). Neuronal CB1 immunoreactivity was found in the hippocampus, striatum, amygdala as well as in the somatosensory, cingulate and entorhinal cortices. The apparent density of immunoreactive cells in the areas of interest (DG, CA3, CA1, or EC) did not vary across groups.
Figure 3
Figure 3
Immunoreactivity (IR) of CBr in the CA3 region of the hippocampus. (a) CB1-IR (DAB stain) is seen in the cytoplasm of all CA3 neuronal cell bodies (counterstaining with cresyl violet). (b) CB1-IR (red) and MCH-II-IR (green) do no co-localize, as indicated (more ...)
No co-localization between CB1 receptors and activated microglial cells
Double-immunofluorescence staining for CB1 and activated microglial cells performed on the brains of all LPS-infused groups did not show any co-localization (Figure 3b). These results indicate that CB1 receptors are not present on activated microglial cells in response to LPS infusion or treatment with a CBr agonist.
No co-localization between CB1 and astrocytes
Double-immunofluorescence staining for CB1 and astrocytes performed on the brains of all LPS-infused groups did not show any co-localization (Figure 3c). These results indicate that CB1 are not present on astrocytes in response to LPS infusion or treatment with a CBr agonist.

DISCUSSION
The results demonstrate that a CB1/CB2 agonist, WIN-55212-2, prevents microglial cell activation during LPS-induced chronic neuroinflammation in young rats. The effects of this agonist were not dependent on direct CB1 receptors stimulation of microglia or astrocytes, were region dependant and did not reverse the LPS-induced impairment in a spatial memory task. The neurodegeneration associated with AD may result from prolonged activation of microglia and a chronic elevation of cytokines and nitric oxide (Akiyama et al., 2000; Streit, 2004) leading to a cascade of self-propagating cellular events that alters the expression of cannabinoid receptors (Minghetti and Levi, 1998; Bernardino et al., 2005; Klein, 2005). Endocannabinoids are implicated in the modulation of the central inflammatory response by neurons and glia (Klein et al., 2003; Klein, 2005) and may have a neuroprotective role in several neuroinflammation related diseases (Klein, 2005). Cannabinoid agonists can prevent the activation of microglia by β-amyloid, reduce the subsequent release of TNF-α(Ramirez et al., 2005), and attenuate the induction and release of nitric oxide by cultured microglia (Waksman et al., 1999). An inhibition of glutamate release by cannabinoids and the subsequent reduction of the calcium influx via NMDA channels (Piomelli, 2003; Takahashi and Castillo, 2006) have also been demonstrated. Additionally, cannabinoids can attenuate oxidative stress and subsequent toxicity (Hampson and Grimaldi, 2001) and induce the expression of brain-derived neurotrophic factor following the infusion of kainic acid (Marsicano et al., 2003).
CBr are expressed in senile plaques (Benito et al., 2003; Ramirez et al., 2005) and the number of CB1 receptor-immunoreactive neurons is greatly reduced in areas of microglial activation in the AD brain (Ramirez et al., 2005). In contrast, we did not find evidence for changes in CB1 receptor expression on hippocampal neurons in response to brain inflammation or cannabinoid receptor stimulation. This absence of changes in CB1 receptors on neurons in our model may be related to the fact that the chronic infusion of LPS into young rats does not induce senile plaque formation (Hauss-Wegrzyniak et al., 1998) or neuronal loss in the hippocampus (Rosi et al., 2005), important factors that likely influence the expression of CBr-positive neuron in post-mortem studies on AD brains from aged humans. The LPS infusion into the 4th ventricle produces widespread activation of glia and impaired performance in the water maze task (Hauss-Wegrzyniak et al., 1998). In contrast, an infusion of amyloid into a lateral ventricle (Ramirez et al., 2005) produced a more localized and limited glial activation (frontal cortex) and performance impairment that responded positively to treatment with WIN 55-212-2 (Ramirez et al., 2005). This difference may explain why WIN 55-212-2 in our model did not reverse the impairment induced by LPS in the 4th ventricle.
In the current study, CB1 receptors were not co-localized with MCH II-positive microglia or GFAP-positive astrocytes within the hippocampus; the presence of these receptors appeared to be solely neuronal (Figure 3). This suggests an indirect role of CBr upon microglia that is linked to the modulation of neuronal activity by stimulation of the endocannabinoid system. Our results are consistent with previous in vivo findings (Tsou et al., 1998) that describe CBr receptors located on hippocampal neurons, and their modulator role upon glutamatergic and GABAergic function (Takahashi and Castillo, 2006; Katona et al., 1999). CBr are present on astrocytes and microglial cells taken from humans, monkeys, rats and mice when studied in vitro (Stella 2004). The contradictory findings we report may be related to many factors, such as the different species that have been studied, the microenvironment of the cells (in vivo vs in vitro), antibody sensitivity and selectivity, and the age or pathological condition under examination. Further experiments must be performed to determine clearly the characteristic and changes of the endocannabinoid system in our model and thus explain our present findings.
We have previously speculated that LPS induces a cascade of inflammatory processes that leads to an elevation in extracellular glutamate and activation of NMDA receptors (Wenk et al., 2006). The selective antagonism of NMDA receptors reduced microglia activation (Rosi et al., 2006) similar to that reported in the current study using WIN-55212-2. Taken together these findings are consistent with the hypothesis that the ability of endocannabinoids to reduce the release of glutamate within the hippocampus underlies the reduction of microglia activation by WIN-55212-2.
Interestingly, our results demonstrated an interaction between the presence of brain inflammation and the actions of the cannabinoid agonist at a dose (1 mg/kg/day i.p. of WIN 55-212-2) that did not impair performance in the control rats. Surprisingly, those doses of agonist (0.5 and 1 mg/kg/day i.p. of WIN 55-212-2) also did not improve performance, as might be expected given the results of our previous investigations using more typical anti-inflammatory drugs (Hauss-Wegrzyniak et al., 1999). We speculate that this was because the WIN 55-212-2 treatment was not able to reverse totally the activation of microglia that had been induced by the chronic LPS infusion; in the current study, the reduction in the number of activated microglia was only about 40-50%, in contrast to the ability of an non-steroidal anti-inflammatory drug’s ability to reduce the number of activated microglia almost completely. This partial reduction in microglia activation might thus not be sufficient to reverse the spatial memory impairment produced by the LPS infusion. This result is of particular importance with regard to patients suffering with a disease associated with brain inflammation, e.g. AD, Parkinson’s disease or multiple sclerosis, who are also using marijuana.
The current report is the first to our knowledge to demonstrate the modulatory role of cannabinoids in an animal model of chronic neuroinflammation, pointing out the effectiveness of a CBr agonist on the consequences of LPS mediated neuroinflammation at a dose (0.5 mg/kg/day i.p. of WIN-55212-2) that does not impair performance in a patial memory task. These results further advocate for the manipulation of the endocannabinoid system to diminish the consequences of neuroinflammation in progression of AD and others inflammation-related diseases (Klein, 2005).
Acknowledgments
We thank A. Sy, P. Raniero, F. Cerbai for technical assistance and Dr V. Ramirez-Amaya for critical experimental procedure assistance.
List of abbreviations
 
Δ9-THCΔ9-tetrahydrocannabinol
 
aCSFartificial cerebral spinal fluid
 
ADAlzheimer’s disease
 
CB1cannabinoid receptor 1
 
CB2cannabinoid receptor 2
 
CBrcannabinoid receptors
 
DGDentate gyrus
 
ECEntorhinal cortex
 
LPSLipopolysaccharide
 
NMDAN-methyl-d-aspartate
 
PBSphosphate buffer saline
 
TBSTris buffer saline
 
WIN-55212-2(R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)-pyrrolo[1,2,3-de]-1,4benzoxazin-6-yl]-1-naphthalenyl-methanone mesylate

 
Financial support:This study has been supported by AG10546 (GLW).
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Section editor: (j) Systems Neuroscience

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WIN 55,212-2- a synthetic cannabinoid

Anti-inflammatory property of the cannabinoid agonist WIN-55212-2 in a rodent model of chronic brain inflammation
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Central and peripheral cannabinoid modulation of gastrointestinal transit in physiological states or during the diarrhoea induced by croton oil
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Modulation of peristalsis by cannabinoid CB1 ligands in the isolated guinea-pig ileum
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The synthetic cannabinoid WIN55,212-2 attenuates hyperalgesia and allodynia in a rat model of neuropathic pain

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WITHDRAWAL SYNDROME

Excerpt from the Merck Manual
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Pot, Tobacco Withdrawal Equally Rough

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A Within-Subject Comparison of Withdrawal Symptoms During Abstinence From Cannabis, Tobacco, and Both Substances

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Cannabis withdrawal severity and short-term course among cannabis-dependent adolescent and young adult inpatients

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Excerpt from the Merck Manual

The following is an excerpt from the Merck Manual, the US military's field guide to medicine:

...no physical dependence [as a result of cannabis usage]; no abstinence syndrome when the drug is discontinued.

Cannabis can be used on an episodic but continuous basis without evidence of social or psychic dysfunction. In many users the term dependence with it's obvious connotations probably is misapplied.

Many of the claims regarding severe biological impact are still uncertain, but some others are not. Despite the acceptance of the 'new' dangers of marijuana, there is still little evidence of biologic damage even among relatively heavy users. This is true even in the areas intensively investigated, such aspulmonary, immunologic, and reproductive function.

Marijuana used in the USA has a higher THC content than in the past. Many critics have incorporated this fact into warnings, but the chief opposition to the drug rests on a moral and political, and not a toxicological, foundation.

(Merck Manual of Diagnosis and Therapy, 15th edition, 1987,Robert Berkow, MD, Editor-In-Chief. Published by Merck Sharp and Dohme Research Laboratories Division of Merck and Co, Inc)

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Comparison of Withdrawal Symptoms

A Within-Subject Comparison of Withdrawal Symptoms During Abstinence From Cannabis, Tobacco, and Both Substances

A cannabis withdrawal syndrome has been characterized, but its clinical significance remains uncertain. One method of assessing the significance of cannabis withdrawal is to compare it directly to an established withdrawal syndrome. The present study was a within-subject comparison of cannabis, tobacco, and combined cannabis and tobacco withdrawal among users of both substances. Participants (N=12) completed three 5-day periods of abstinence in a randomized order, separated by 9-day periods of usual substance use. Overall withdrawal severity associated with cannabis alone and tobacco alone was of a similar magnitude. Withdrawal during simultaneous cessation of both substances was more severe than for each substance alone, but these differences were of short duration and substantial individual differences were noted. These results are consistent with other evidence suggesting cannabis withdrawal is clinically important and warrants detailed description in the DSM-V and ICD-11. Additional research is needed to replicate these findings and to further investigate the effects of abstaining from multiple drugs simultaneously.
Keywords: cannabis, marijuana, tobacco, nicotine, withdrawal, dependence

1. Introduction
Cannabis is currently the most widely used illicit drug in the United States and treatment admissions in which cannabis was the primary problem substance have more than doubled since the early 1990’s and are now comparable in number with treatment admissions for cocaine and heroin (SAMHSA, 1998, 2001, 2003). A reliable cannabis withdrawal syndrome that involves increased anger and aggression, anxiety, depressed mood, irritability, restlessness, sleep difficulty and strange dreams, decreased appetite, and weight loss has been clearly demonstrated (Budney et al., 2004). Headaches, physical tension, sweating, stomach pain, and general physical discomfort have also been observed during cannabis withdrawal, but are less common (Budney et al., 2004). Most symptoms onset within the first 24 hours of cessation, peak within the first week, and last approximately 1–2 weeks (Budney et al., 2003; Haney et al., 1999; Kouri and Pope, 2000). Because these withdrawal symptoms are time-limited, occur shortly after cannabis cessation, and are reduced or eliminated following administration of the CB1 receptor agonist delta-9-tetrahydrocannabinol (THC), the primary psychoactive compound in cannabis, it appears that they represent a true withdrawal syndrome (Budney et al., 2007; Haney et al., 2004; Hart, 2005; Lichtman and Martin, 2002).
The clinical significance of cannabis withdrawal, however, has not been clearly established. Cannabis withdrawal is included, but not well defined, as a clinical diagnosis in the ICD-10 (World Health Organization, 1992), and is not included in the DSM-IV because the “clinical significance is uncertain” (American Psychiatric Association, 2000). However, a number of empirical observations are suggestive of its importance. The majority of adults and adolescents seeking outpatient treatment for cannabis dependence have great difficulty achieving initial periods of abstinence (Budney et al., 2000; Budney et al., in press; Copeland et al., 2001; Stephens et al., 2002). Many complain that withdrawal contributes to their inability to quit and report using cannabis or other substances to alleviate withdrawal symptoms (Budney and Hughes, 2006; Budney et al., 2004; Coffey et al., 2002; Copersino et al., 2006). Withdrawal symptoms are also observable to third-party observers, and comments made by these observers suggest that symptoms can be disruptive of daily living (Budney et al., 2001; Budney et al., 2003).
Another method of estimating the clinical significance of cannabis withdrawal is by comparing it with a syndrome of known clinical importance. In an initial archival study, we compared cannabis withdrawal with nicotine (tobacco) withdrawal effects observed in two separate outpatient studies that used similar methodologies (Vandrey et al., 2005). Close similarities in the type, magnitude, and time course of symptoms expressed during cannabis and tobacco withdrawal were observed on 6 participant-rated and 4 observer-rated withdrawal symptoms suggesting that the two syndromes showed comparable severity. Several important methodological limitations of this study (lack of inferential statistics, omission of some common symptoms, robust sample differences) limited confidence in the conclusions that could be drawn.
This paper describes a study designed to provide a more rigorous comparison of the cannabis and nicotine withdrawal syndromes. Comparisons were made for all symptoms commonly reported in prior studies of both cannabis and tobacco withdrawal (aggression, anger, depressed mood, irritability, anxiety/nervousness, restlessness, sleep difficulty, strange dreams, and tension). A within-subject design was used to eliminate inter-group variability, and because this required participation of regular users of both substances it also provided the opportunity to assess the effects of abstaining from both substances simultaneously. Examining withdrawal that occurs following cessation of both substances is important because approximately 9% of heavy smokers are daily or near daily cannabis users (Ford et al., 2002) and approximately 50% of heavy cannabis users also use tobacco regularly (Moore and Budney, 2001). Moreover, among cannabis-dependent individuals, those that smoke tobacco have increased psychosocial problems and poor cannabis cessation outcomes compared with non-smokers (Moore and Budney, 2001). Similarly, cannabis use among tobacco smokers is predictive of continued long-term tobacco use and is associated with poor tobacco cessation outcomes (Amos et al., 2004; Ford et al., 2002). Whether simultaneous drug abstinence results in significantly greater withdrawal or difficulty in achieving abstinence compared with abstinence from either drug alone has not been previously studied.

2. Methods
Two laboratories collaborated to conduct the experiment. Site 1 was the Treatment Research Center of the University of Vermont in Burlington, VT (UVM). Site 2 was the Department of Physiology and Pharmacology of the Wake Forest University School of Medicine in Winston-Salem, NC (WAKE).
2.1 Participants
Current users of cannabis and tobacco were recruited through newspaper advertisements and flyers posted on community bulletin boards. Criteria for participation included: age ≥18 years old; heavy use of cannabis (at least 25 days/month) and tobacco (at least 10 cigarettes/day) for the 6 months prior to participating; no intent to quit or change cannabis or tobacco use; not currently dependent on other substances; no use of illicit drugs (other than cannabis) in the prior month; not currently using any psychotropic medication; not meeting DSM-IV criteria for a current episode of an Axis I psychiatric disorder; and not pregnant. Treatment seekers were excluded because the protocol included periods during which participants were required to resume cannabis and tobacco use following brief periods of abstinence (see below). The cannabis and tobacco use criteria are consistent with that used in prior studies in which significant withdrawal effects were observed in a majority of users at these levels (e.g. Budney et al., 2001; Budney et al., 2003; Fernando et al., 2006; Pomerleau et al., 2000). The criteria requiring cannabis use on 25 of 30 days was used to enhance generality because a substantial number of heavy users are occasionally unable to obtain the drug for a day or two due to its illicit status.
At the UVM site, 23 participants were enrolled, and 9 completed the study. Of those who enrolled and did not complete, 8 failed to achieve the required periods of abstinence, 3 discontinued participation voluntarily prior to the first abstinence period, 2 were discontinued from the study due to use of restricted substances (e.g. cocaine), and 1 person was discontinued for testing negative for cannabis during the first baseline condition. At the WAKE site, 19 enrolled, and 3 completed the study. Of those who did not complete, 4 failed to achieve the required periods of abstinence, 6 discontinued participation voluntarily, 4 were discontinued from the study due to use of restricted substances, and 2 were discontinued from the study for using less tobacco during baseline than reported during screening. Voluntary discontinuation usually resulted from participant problems with time commitment or transportation to the laboratory. Detailed demographics of the 12 completers are provided in Table 1.
Table 1
 
2.2 Procedures
A brief telephone interview was conducted followed by an in depth laboratory screening to determine study eligibility. Informed consent was obtained at the beginning of the laboratory assessment, and the Committees on Human Research at the University of Vermont and Wake Forest University approved all study procedures.
An ABACAD within-subjects design was used to compare abstinence effects associated with cessation from cannabis only (cannabis), tobacco only (tobacco), and both cannabis and tobacco simultaneously (dual). In this design, A-conditions represent periods of cannabis and tobacco smoking-as-usual (SAU), and conditions B, C, and D correspond to randomly assigned 5-day periods of cannabis, tobacco, or dual abstinence. Participants attended 30-minute laboratory sessions each weekday to obtain self-reported affective and behavioral measures, physiological measures, and staff-observed urine and breath samples. The study always began on a Monday and each 5-day abstinence condition began following collection of a urine specimen on the prior Sunday and ended after the laboratory session the following Friday. Data collection did not occur on Saturdays or Sundays prior to SAU conditions. Participants were instructed to treat those days as SAU days and maintain use of cannabis and tobacco as they did prior to participation. Therefore, each 5-day abstinence condition was separated by a period of at least 9 days of usual substance use.
During the study, participants were asked to maintain their usual patterns of alcohol and caffeine use and abstain from using illegal drugs (other than cannabis) and psychoactive medications. To eliminate the influence of acute intoxication on subjective reporting, participants were instructed not to smoke cannabis or drink alcohol for at least 2 hours prior to each laboratory visit. Though there was no objective way to verify compliance with the requirement to not smoke cannabis during this time, compliance with the alcohol restriction was confirmed via negative breathalyzer readings obtained prior to all laboratory data collection. During periods of tobacco abstinence, participants were explicitly instructed not to use alternative tobacco or tobacco cessation products. Participants were also asked to not make significant changes to their diet or exercise patterns during the study.
For ethical reasons, on the last day of each abstinence period participants were asked if they intended to resume their usual use of cannabis and/or tobacco and would like to continue in the study, or whether they would instead like to remain abstinent and be referred for help with abstinence. During the study, all participants indicated they planned to resume use. At the end of the study, cannabis and tobacco treatment referral lists were offered and provided to those who would accept them.
2.3 Measures
Prior to enrollment, a drug history questionnaire assessed past and present patterns (e.g. age of first use and progression to daily use) and consequences (e.g. prior treatment episodes or experience of withdrawal) of substance use and abuse. The Time-Line Follow-Back method (Sobell and Sobell, 1992) was used to obtain the amount and frequency of substance use during the previous 6 months. The DSM Checklist (Hudziak et al., 1993) was used to diagnose current (past 6 months) Axis I psychiatric disorders.
During each weekday laboratory visit, participants completed a battery of self-report questionnaires. The Withdrawal Symptom Checklist (WSC), an adaptation of the Marijuana Withdrawal Checklist (Budney et al., 2001; Budney et al., 2003), was used to assess withdrawal symptoms prospectively over the course of the study. The WSC included common symptoms of both cannabis and tobacco withdrawal and several filler items rated on a 0–3 scale (0 = not at all, 1= mild, 2 = moderate, 3 = severe) based on their experience over the prior 24 hours. The primary outcome variable for this measure was a composite withdrawal discomfort score (WDS), a sum of 10 items previously reported as common symptoms of withdrawal for both cannabis and tobacco (aggression, anger, appetite change (increased for tobacco, decreased for cannabis), depressed mood, irritability, anxiety/nervousness, restlessness, sleep difficulty, and strange dreams).
The Profile of Mood States (POMS) (McNair et al., 1971) is a 65-item measure that provided composite scores of tension (range 0–36), depression (range 0–60), anger (range 0–48), and confusion (range 0–28) based on participants’ ratings of the previous 24 hours. Craving for cannabis and tobacco was measured by the short (12-item) versions of the Marijuana Craving Questionnaire (MCQ) (Heishman et al., 2001) and Tobacco Craving Questionnaire (TCQ) (Heishman et al., 2003) respectively. The MCQ and TCQ each yield 4 composite scores: purposefulness, expectancy, emotionality, and compulsivity. Participants kept a daily diary detailing their use of tobacco, cannabis, alcohol, and both prescription and OTC medications. Research staff obtained measures of resting heart rate, supine blood pressure, body weight, breath CO, and breath alcohol during each laboratory session. At the end of the final session, participants completed an End of Study Questionnaire. Participants rated the overall level of discomfort they experienced during each abstinence condition on a 0–3 scale, responded to an unstructured question asking what aspects of each abstinence period were most difficult for them, and then they rank-ordered the difficulty of completing each abstinence condition.
Observed urine samples were collected daily and qualitatively tested on-site using an enzyme immunoassay technique for evidence of cannabis, benzodiazepine, opiate, cocaine, methamphetamine, and amphetamine use. Specimens were then shipped overnight to Dominion Diagnostics (Kingstown, RI) for quantitative analysis (gas-chromatography-mass spectroscopy for cannabis, gas-chromatography for cotinine) of the primary metabolites of THC and nicotine (11-nor-9-carboxy-delta9-tetrahydrocannabinol (THCCOOH) and cotinine respectively) and creatinine. Results were typically received within 72 hours of specimen collection. During abstinence periods, participants were judged abstinent from cannabis using the previously validated criteria of a ratio of THCCOOH to creatinine that did not increase by 50% on consecutive days and decreased by at least 50% during the 5-day abstinence period (Huestis and Cone, 1998). Participants were judged abstinent from tobacco if cotinine levels decreased by at least 40% on consecutive days until specimens tested at or below a cutoff of 80ng/mL (Benowitz et al., 2002).
Participants were compensated up to $750 for study participation. During SAU periods compensation was $15 per laboratory visit, and during abstinence periods compensation was $25 per day, $12.50 of which was earned for completing study measures, and $12.50 of which was contingent on verified abstinence from the target substance(s). The payment for abstinence was delayed until verification was obtained from the off-site laboratory (usually 2–3 days). A $50 completion bonus was provided for completion of each of the three phases of the study.
2.4 Data Analysis
2.4.1 Preliminary Analyses
Quantitative urine results for each participant were evaluated based on the criteria described above to verify abstinence. A two-way repeated measures analysis of variance (ANOVA) was conducted to compare the mean variable scores obtained during each of the 3 SAU periods. Repeated measures ANOVA’s using mean scores collapsed across the SAU and abstinence study conditions were also conducted to assess effects due to abstinence condition order, and to assess the stability of self-reported cannabis, tobacco and alcohol use on days other than when abstinence was required.
2.4.2 Primary Analyses
Two-way repeated measures ANOVA’s (3 × 6; condition × day) were performed to examine effects of condition (cannabis, tobacco, and dual abstinence), day (Mean SAU collapsed across days and Abstinence Days 1–5), and condition by day interactions for each dependent variable. When significant main effects or interactions were detected, post-hoc analyses were conducted to test for abstinence manipulation effects and condition differences within each day using the corresponding ANOVA for testing simple effects. Bonferonni adjustments for multiple comparisons were used for post-hoc comparisons and statistical difference was determined based on α = .05 for all analyses.

3. Results
3.1 Results of Preliminary Analyses
Quantitative levels of urinary THCCOOH (adjusted for creatinine) and cotinine decreased as expected during each abstinence phase (Figure 1). No differences of SAU condition were found for any outcome variables indicating stable responding across the 3 baseline periods. Similarly, no effects of abstinence condition order were observed. No effects of study condition were detected in participants’ self-reported use of cannabis, tobacco, and alcohol (excluding abstinence condition days) were detected indicating that participants followed the instruction to not significantly alter their use of these drugs during the study except when instructed to abstain.
Figure 1
Figure 1
Mean creatinine-normalized tetrahydrocannabinol (THCCOOH) and cotinine levels. SAU values reflect the average of specimens collected on Days 1, 3, and 5 of the SAU condition preceding each abstinence condition.
3.2 Results of Primary Analyses
3.2.1 Withdrawal Symptom Checklist (WSC)
Main effects of study day were found for WSC items anxiety/nervousness (F = 4.99, p ≤ .05), decreased appetite (F = 3.03, p ≤ .05), depressed mood (F = 2.35, p ≤ .05), difficulty concentrating (F = 2.22, p ≤ .05), feverish (F = 2.62, p ≤ .05), increased anger (F = 2.55, p ≤ .05), irritability (F = 5.53, p ≤ .05), physical discomfort (F = 3.07, p ≤ .05), restlessness (F = 5.75, p ≤ .05), shakiness (F = 2.69, p ≤ .05), sleep difficulty (F = 5.50, p ≤ .05), stomach pain (F = 2.47, p ≤ .05), strange dreams (F = 2.49, p ≤ .05), sweating (F = 2.71, p ≤ .05), tension (F = 2.41, p ≤ .05), and the composite WDS (F = 8.76, p ≤ .05). Post-hoc tests comparing ratings by study day indicate that these differences reflect an abstinence manipulation or withdrawal effect (i.e. ratings during abstinence were significantly greater than SAU). Significant withdrawal effects were observed for anxiety/nervousness, decreased appetite, difficulty concentrating, irritability, sleep difficulty, strange dreams, and WDS in the cannabis abstinence condition. Significant withdrawal effects were observed for anxiety/nervousness, increased anger, irritability, physical discomfort, restlessness, shakiness, sleep difficulty, tension, and WDS in the tobacco abstinence condition.
Significant withdrawal effects were observed for each of these symptoms except decreased appetite in the dual abstinence condition. Significant condition by day interactions were observed for ratings of difficulty concentrating (F = 2.22, p ≤ .05), increased aggression (F = 2.37, p ≤ .05), increased anger (F = 2.80, p ≤ .05), irritability (F = 1.91, p ≤ .05), sleep difficulty (F = 2.33, p ≤ .05), and the composite WDS score (F = 2.72, p ≤ .05). Illustrations of these data are provided in Figure 2. Post-hoc analyses indicated that ratings of difficulty concentrating were greater on Days 4 and 5 in the tobacco and dual abstinence conditions compared with the cannabis abstinence condition. Increased aggression, increased anger, irritability, and WDS were greater in the dual abstinence condition compared with the cannabis and tobacco abstinence conditions on Day 2. Increased aggression was also greater in the dual abstinence condition compared with the cannabis abstinence condition on Day 4. Sleep difficulty was greater in the dual abstinence condition compared with the tobacco abstinence condition on Days 2 and 4.
Figure 2
Figure 2
Mean ratings for WSC items for which significant condition by day interactions were observed. Filled symbols indicate values significantly different from SAU. Subscripts designate differences by condition on a given study day (a = dual > cannabis (more ...)
3.2.2 POMS, MCQ, and TCQ
A main effect of study day was observed for the anger sub-scale of the POMS (F = 2.50, p ≤ .05). Post-hoc tests indicated a withdrawal effect during the tobacco and dual abstinence conditions. Significant condition by day interactions were observed for the anger (F = 2.28, p ≤ .05) and confusion (F = 2.05, p ≤ .05) sub-scales of the POMS. Post-hoc tests indicated that, on abstinence Day 2, scores on the anger sub-scale were greater in the dual abstinence condition compared to the cannabis and tobacco conditions, and that scores in the tobacco condition were greater compared with the cannabis condition. Scores on the confusion sub-scale were greater in the dual abstinence condition compared with the cannabis abstinence condition on Day 2, and in the tobacco condition compared with the cannabis condition on Days 4 and 5.
A main effect of study day was observed for the compulsivity (F = 5.03, p ≤ .05) and purposefulness (F = 3.64, p ≤ .05) sub-scales of the MCQ. Post-hoc tests indicated a withdrawal effect during the cannabis abstinence condition for both sub-scales.
Main effects of study day were observed for the compulsivity (F = 2.95, p ≤ .05), emotionality (F = 4.99, p ≤ .05), expectancy (F = 2.81, p ≤ .05), and purposefulness (F = 2.77, p ≤ .05) sub-scales of the TCQ. Post-hoc tests indicated a withdrawal effect during the tobacco and dual abstinence conditions for all 4 sub-scales. A significant interaction was also observed for the compulsivity sub-scale of the TCQ (F = 2.48, p ≤ .05), and post-hoc tests indicated greater ratings during the tobacco abstinence condition compared with the dual abstinence condition on Day 3.
3.2.3 Physiological Measures
Main effects for study day were observed for heart rate (F = 5.49, p ≤ .05) and systolic blood pressure (F = 2.43, p ≤ .05). Post-hoc tests indicated a withdrawal effect for heart rate during all three abstinence conditions and for systolic blood pressure during tobacco abstinence. Heart rate increased during cannabis abstinence and decreased during the tobacco and dual abstinence conditions. Systolic blood pressure decreased during tobacco abstinence.
Main effects of study condition were observed for systolic blood pressure (F = 9.26, p ≤ .05), diastolic blood pressure (F = 11.96, p ≤ .05), and body weight (F = 3.62, p ≤ .05). Post-hoc tests indicated that systolic and diastolic blood pressure was greater during cannabis abstinence compared with tobacco abstinence, while the opposite was true for body weight. Blood pressure and body weight during the dual abstinence condition was intermediate and did not differ from the cannabis or tobacco conditions.
A condition by day interaction was observed for heart rate (F = 3.27, p ≤ .05). Post-hoc tests indicated that heart rate was greater during the cannabis abstinence condition compared with the tobacco abstinence condition on all 5 abstinence days and compared with the dual abstinence condition on Days 1, 3, 4, and 5 of abstinence.
3.2.4 End of Study Questionnaire
No differences were detected for ratings of overall level of discomfort on the End of Study Questionnaire. Interestingly, notable individual variability was observed on the rank-order assessment of abstinence phase difficulty (Table 2). No single abstinence phase was consistently ranked as being more difficult than the others. Five participants rated the dual abstinence condition as the most difficult, 4 rated the cannabis condition as the most difficult, and 3 rated tobacco abstinence as the most difficult. Similarly, 5 participants rated the dual abstinence condition as least difficult, 4 rated the cannabis condition least difficult, and 3 rated tobacco abstinence as the least difficult.
Table 2
Table 2
Mean WDS, Rank-Order, and Substance Use by Participant

4. Discussion
Overall withdrawal discomfort (WDS) and individual symptom severity during cannabis abstinence was similar to that observed during tobacco abstinence in the present study. The differences observed between the cannabis and tobacco abstinence conditions were mostly for symptoms expected to differ based on previous studies (difficulty concentrating/confusion, heart rate, body weight). Exceptions to this were that ratings of anger and craving appeared to be higher during tobacco abstinence compared with cannabis abstinence. We believe the conclusion that the cannabis and tobacco withdrawal syndromes are of comparable severity is valid because the withdrawal effects observed in this study were similar in magnitude to that observed in prior studies using similar measures (Budney et al., 2001; Budney et al., 2003; Hughes, 1992; Hughes and Hatsukami, 1986). Moreover, these findings are consistent with our prior archival comparison (Vandrey et al., 2005).
This experiment provided the first examination of abstinence effects that occur following simultaneous cessation of cannabis and tobacco. The WDS and individual symptoms such as aggression, anger, and irritability appeared to be more severe during simultaneous abstinence compared with abstinence from either drug alone. However, this effect was not very robust (limited to Day 2 for most variables), and the participants’ general rating of discomfort across the three abstinence conditions indicated substantial individual differences. Although it is logical to expect greater withdrawal with dual abstinence, for a subset of participants, the dual abstinence condition was associated with less withdrawal and rated as less difficult. One possible reason for the lower withdrawal in the dual abstinence period for some participants may be that both substances are smoked and thus share smoking-related cues. Thus, the absence of smoking cues during dual abstinence might decrease withdrawal. Exposure to drug cues has been associated with increases in ratings of withdrawal and more rapid relapse in prior studies, but it is not known whether these cues generalize across substances (Childress et al., 1986; Droungas et al., 1995; Juliano et al., 2006).
Though significant methodological improvements over our previous cross-study comparison were made, important limitations remain with regards to the current experiment. First, the sample size was relatively small, which is potentially problematic with regard to the generality of the findings and when trying to demonstrate similarities (the null hypothesis). Replication with a larger sample size would be desirable, however, the feasibility of a larger scale study is uncertain given the high drop out rate and difficulty recruiting eligible participants encountered at both sites in the current study. The high drop out rate may also have resulted in the exclusion of people who experience more severe withdrawal. No clear pattern emerged with regard to which study condition participants were in when they quit the study early, but we cannot rule out the possibility that withdrawal effects observed in one of our abstinence conditions is a more conservative representation of withdrawal severity relative to the others.
Abstinence periods of 5 days duration might not have been sufficient to accurately assess the magnitude and importance of all abstinence effects. Prior research suggests that peak withdrawal effects for cannabis and tobacco occur within the first week of abstinence (Budney et al., 2004; Hughes, 1990; Kouri and Pope, 2000). However, these studies also suggest that some withdrawal symptoms (ex. anger and restlessness for cannabis; sleep difficulty and increased appetite/weight gain for tobacco) have a later onset of peak effects compared with other symptoms and may have been underestimated in the current study. It is also important to note that this study was not placebo controlled and was based on participants who are heavy daily users of cannabis and tobacco. Therefore, we cannot rule out the influence of expectancy effects across conditions, and these results may not generalize to less frequent cannabis and tobacco users.
Though we included measures of withdrawal based on prior cannabis and tobacco withdrawal research, the fact that mean WDS scores were not associated with greater rankings of abstinence difficulty in half the sample suggests our WDS did not capture some important facets of cannabis or tobacco withdrawal. Further research should examine what abstinence effects beyond the withdrawal symptoms included in the WDS may contribute to abstinence difficulty. Two such possibilities are negative consequences that may result from the absence of substance-related social activities or the disengagement of habitual substance use routines (e.g., no longer able to use substance after meals, after work, while driving, or before going to sleep) that accompany an abstinence attempt. Several participants described these types of consequences as being very difficult to endure on the End of Study Questionnaire.
These limitations acknowledged, the results do suggest that the severity of the cannabis withdrawal syndrome is of comparable magnitude to that of the tobacco withdrawal syndrome. Tobacco withdrawal has been extensively researched, and by its inclusion in DSM-IV, is judged to be clinically significant (substance withdrawal disorders must produce “clinically significant distress” to be included in DSM-IV). This observed similarity with tobacco withdrawal, combined with several lines of evidence that cannabis withdrawal is clinically significant as described in the introduction, suggest that cannabis withdrawal is clinically significant in a subset of heavy cannabis users and should be characterized in detail in the next iterations of the DSM and ICD manuals.
Several suggestions for future research can be made to extend this area of study. Prospective investigation of the relationships between cannabis withdrawal and relapse or failed quit attempts would provide additional information regarding its clinical significance. The implications of the dual abstinence effect findings are unclear, but also are of clinical importance. Interestingly, we could not locate another prospective study comparing withdrawal severity during cessation from one versus more than one drug simultaneously. Our data that drug withdrawal was not consistently greater across participants and that for some participants withdrawal was less severe with dual abstinence, clearly suggest further studies of single versus dual abstinence are warranted.
Acknowledgments
This research was supported by grants R01-DA12471, T32-DA07242 from the National Institute on Drug Abuse. The authors would also like to thank the research support staff at the University of Vermont and Wake Forest University for their invaluable contributions.
 
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Pot, Tobacco Withdrawal Equally Rough

Marijuana Withdrawal Symptoms Mirror Tobacco Withdrawal Symptoms, Study Shows
By Miranda Hitti
WebMD Health News
Reviewed by Louise Chang, MD

Jan. 30, 2008 -- Marijuana withdrawal symptoms are similar to tobacco withdrawal symptoms, new research shows.

Both types of withdrawal can prompt anxiety, irritability, restlessness, sleep problems, strange dreams, and other symptoms, according to Johns Hopkins University's Ryan Vandrey, PhD, and colleagues.

"These results indicate that some marijuana users experience withdrawal effects when they try to quit, and that these effects should be considered by clinicians treating people with problems related to heavy marijuana use," Vandrey says in a news release.

Vandrey's team studied 12 people who were heavy smokers of both tobacco and marijuana.

Participants reported smoking pot at least 25 days per month and smoking at least 10 tobacco cigarettes per day. And they had no plans to quit smoking either substance.

For the study's sake, participants stopped smoking marijuana -- but kept smoking tobacco -- for five days. Then they were free to smoke both substances for nine days.

Next, participants stopped smoking tobacco and kept smoking marijuana for five days. Then they smoked as they pleased for nine days, followed by five days without marijuana or tobacco.

Participants rated their withdrawal symptoms and took drug tests every day throughout the study. They noted similar symptoms when they stopped marijuana, tobacco, or both substances.

The three nonsmoking periods -- no marijuana, no tobacco, and no marijuana or tobacco -- were equally difficult, according to the study.

Some participants -- but not all -- found it easier to stop smoking marijuana and tobacco at the same time, instead of halting only one of those drugs. Smoking just one substance may have made them want the other one, too, Vandrey's team notes.

 

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Cannabis withdrawal severity

Preuss UW, Watzke AB, Zimmermann J, Wong JW, Schmidt CO 
Cannabis withdrawal severity and short-term course among cannabis-dependent adolescent and young adult inpatients. [Journal Article, Research Support, Non-U.S. Gov't]
Drug Alcohol Depend 2010 Jan 15; 106(2-3):133-41.


OBJECTIVE: While previous studies questioned the existence of a cannabis withdrawal syndrome (CWS), recent research provided increasing evidence of a number of clinical symptoms after cessation of frequent cannabis consumption. The aim of this study is to prospectively assess the course of CWS in a sample of cannabis-dependent inpatients and to provide an estimate of the proportion of subjects experiencing CWS.
METHODS: 118 subjects, aged 16-36 years, diagnosed with a cannabis dependence (DSM-IV, assessed by SCID I) were enrolled in the study. CWS was assessed prospectively over 10 days using a modified version of the Marijuana Withdrawal Checklist. Personality dimensions were assessed with the NEO-FFI.
RESULTS: 73 subjects (61.3%) completed all assessments over the observation period. Most symptoms peaked on day 1. Model-based analyses revealed a high and low intensity CWS group. Less than half of the patients belonged to the high intensity craving, psychological, or physical withdrawal symptoms group. Symptom intensity decreased almost linearly over time. Indicators of cannabis consumption intensity as well as personality dimensions, but not recalled withdrawal were related to membership in the high intensity CWS group.
DISCUSSION: A clinically relevant CWS may only be expected in a subgroup of cannabis-dependent patients. Most subjects with an elevated CWS experience physical and psychological symptoms. The small to negligible associations between recalled and prospectively assessed symptoms raise questions about the validity of the former approach.

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