Maria Grazia Cascio

Possible endocannabinoid control of colorectal cancer growth

BACKGROUND & AIMSnThe endocannabinoids anandamide and 2-arachidonoylglycerol (2-AG) inhibit cancer cell proliferation by acting at cannabinoid receptors (CBRs). We studied (1). the levels of endocannabinoids, cannabinoid CB(1) and CB(2) receptors, and fatty acid amide hydrolase (FAAH, which catalyzes endocannabinoid hydrolysis) in colorectal carcinomas (CRC), adenomatous polyps, and neighboring healthy mucosa; and (2). the effects of endocannabinoids, and of inhibitors of their inactivation, on human CRC cell proliferation.nnnMETHODSnTissues were obtained from 21 patients by biopsy during colonoscopy. Endocannabinoids were measured by liquid chromatography-mass spectrometry (LC-MS). CB(1), CB(2), and FAAH expression were analyzed by RT-PCR and Western immunoblotting. CRC cell lines (CaCo-2 and DLD-1) were used to test antiproliferative effects.nnnRESULTSnAll tissues and cells analyzed contain anandamide, 2-AG, CBRs, and FAAH. The levels of the endocannabinoids are 3- and 2-fold higher in adenomas and CRCs than normal mucosa. Anandamide, 2-AG, and the CBR agonist HU-210 potently inhibit CaCo-2 cell proliferation. This effect is blocked by the CB(1) antagonist SR141716A, but not by the CB(2) antagonist SR144528, and is mimicked by CB(1)-selective, but not CB(2)-selective, agonists. In DLD-1 cells, both CB(1) and CB(2) receptors mediate inhibition of proliferation. Inhibitors of endocannabinoid inactivation enhance CaCo-2 cell endocannabinoid levels and block cell proliferation, this effect being antagonized by SR141716A. CaCo-2 cell differentiation into noninvasive cells results in increased FAAH expression, lower endocannabinoid levels, and no responsiveness to cannabinoids.nnnCONCLUSIONSnEndocannabinoid levels are enhanced in transformed colon mucosa cells possibly to counteract proliferation via CBRs. Inhibitors of endocannabinoid inactivation may prove useful anticancer agents.

Cannabidiol, a non‐psychotropic component of cannabis, attenuates vomiting and nausea‐like behaviour via indirect agonism of 5‐HT1A somatodendritic autoreceptors in the dorsal raphe nucleus

BACKGROUND AND PURPOSE To evaluate the hypothesis that activation of somatodendritic 5‐HT1A autoreceptors in the dorsal raphe nucleus (DRN) produces the anti‐emetic/anti‐nausea effects of cannabidiol (CBD), a primary non‐psychoactive cannabinoid found in cannabis.

The endocannabinoid system and the molecular basis of paralytic ileus in mice

The endocannabinoid system (i.e., the cannabinoid receptors and their endogenous ligands) plays an important role in the physiological control of intestinal motility. However, its participation in intestinal pathological states is still poorly understood. In the present study, we investigated the possible role of the endocannabinoid system in the pathogenesis of paralytic ileus, a pathological state consisting of decreased intestinal motility following peritonitis, surgery, or other noxious situations. Ileus was induced by i.p. administration of acetic acid, and gastrointestinal propulsion was assessed by the charcoal method. Endocannabinoid levels were measured by isotope‐dilution gas chromatography‐mass spectrometry, whereas cannabinoid CB1 receptors were identified by immunohistochemistry. Acetic acid administration inhibited gastrointestinal transit (ileus), and this effect was accompanied by increased levels of the endocannabinoid anandamide compared with control mice and by overexpression of CB1 receptors in myenteric nerves. Furthermore, acetic acid‐induced ileus was alleviated by the CB1 receptor antagonist SR141716A and worsened by VDM11, a selective inhibitor of anandamide cellular uptake (and hence inactivation). From these findings, it can be concluded that the intestinal hypomotility typical of paralytic ileus is due, at least in part, to the enhancement of anandamide levels and CB1 expression during this condition, and that selective, nonpsychotropic CB1 receptor antagonists could represent new drugs to treat this disorder.

The plant cannabinoid Δ9-tetrahydrocannabivarin can decrease signs of inflammation and inflammatory pain in mice

Background and purpose:u2002 The phytocannabinoid, Δ9‐tetrahydrocannabivarin (THCV), can block cannabinoid CB1 receptors. This investigation explored its ability to activate CB2 receptors, there being evidence that combined CB2 activation/CB1 blockade would ameliorate certain disorders.

Evidence that the plant cannabinoid cannabigerol is a highly potent α2-adrenoceptor agonist and moderately potent 5HT1A receptor antagonist.

Background and purpose:u2002 Cannabis is the source of at least seventy phytocannabinoids. The pharmacology of most of these has been little investigated, three notable exceptions being Δ9‐tetrahydrocannabinol, cannabidiol and Δ9‐tetrahydrocannabivarin. This investigation addressed the question of whether the little‐studied phytocannabinoid, cannabigerol, can activate or block any G protein‐coupled receptor.

Chronic morphine modulates the contents of the endocannabinoid, 2-arachidonoyl glycerol, in rat brain.

Opioids and cannabinoids are among the most widely consumed drugs of abuse in humans and the phenomena of cross-tolerance or mutual potentiation have been demonstrated between the two drugs. Several authors have suggested that both drugs share common links in their molecular mechanisms of action, although this has been a matter of controversy. Furthermore, no data exist on the possible adaptive changes in the contents of arachidonoylethanolamide (anandamide, AEA) and 2-arachidonoylglycerol (2-AG), the two major endogenous ligands for cannabinoid receptors, in morphine-tolerant rats. In the present work, we investigated the alterations in cannabinoid receptor functionality and endocannabinoid levels in rats chronically treated with morphine (5u2009mg/kg, s.c., twice a day for 5 days). Autoradiographic-binding studies using [3H]CP-55u2009940 revealed a slight but significant reduction in cannabinoid receptor level in the cerebellum and hippocampus of morphine-tolerant rats, while CP-55u2009940-stimulated [35S]GTPγS binding showed a strong decrease (40%) in receptor/G protein coupling in the limbic area of these animals. Moreover, in the same brain regions we measured, by isotope-dilution gas chromatography/mass spectrometry, the contents of AEA and 2-AG. Chronic morphine exposure produced a strong reduction in 2-AG contents without changes in AEA levels in several brain regions (ie striatum, cortex, hippocampus, limbic area, and hypothalamus). These findings clearly demonstrate that prolonged activation of opioid receptors could alter the cannabinoid system, in terms of both receptor functionality and endocannabinoid levels, and suggest the involvement of this system, alone or in combination with other mediators, in the phenomenon of morphine tolerance.

Inhibitory effect of salvinorin A, from Salvia divinorum, on ileitis‐induced hypermotility: cross‐talk between κ‐opioid and cannabinoid CB1 receptors

Salvinorin A, the active component of the hallucinogenic herb Salvia divinorum, inhibits intestinal motility through activation of κ‐opioid receptors (KORs). However, this compound may have target(s) other than the KORs in the inflamed gut. Because intestinal inflammation upregulates cannabinoid receptors and endogenous cannabinoids, in the present study we investigated the possible involvement of the endogenous cannabinoid system in salvinorin A‐induced delay in motility in the inflamed gut.

Cannabidivarin-rich cannabis extracts are anticonvulsant in mouse and rat via a CB1 receptor-independent mechanism

Epilepsy is the most prevalent neurological disease and is characterized by recurrent seizures. Here, we investigate (i) the anticonvulsant profiles of cannabis‐derived botanical drug substances (BDSs) rich in cannabidivarin (CBDV) and containing cannabidiol (CBD) in acute in vivo seizure models and (ii) the binding of CBDV BDSs and their components at cannabinoid CB1 receptors.

The cannabinoid TRPA1 agonist cannabichromene inhibits nitric oxide production in macrophages and ameliorates murine colitis

The non‐psychotropic cannabinoid cannabichromene is known to activate the transient receptor potential ankyrin‐type1 (TRPA1) and to inhibit endocannabinoid inactivation, both of which are involved in inflammatory processes. We examined here the effects of this phytocannabinoid on peritoneal macrophages and its efficacy in an experimental model of colitis.

New potent and selective inhibitors of anandamide reuptake with antispastic activity in a mouse model of multiple sclerosis

We previously reported that the compound O‐2093 is a selective inhibitor of the reuptake of the endocannabinoid anandamide (AEA). We have now re‐examined the activity of O‐2093 in vivo and synthesized four structural analogs (O‐2247, O‐2248, O‐3246, and O‐3262), whose activity was assessed in: (a) binding assays carried out with membranes from cells overexpressing the human CB1 and CB2 receptors; (b) assays of transient receptor potential of the vanilloid type‐1 (TRPV1) channel functional activity (measurement of [Ca2+]i); (c) [14C]AEA cellular uptake and hydrolysis assays in rat basophilic leukaemia (RBL‐2H3) cells; (d) the mouse ‘tetrad’ tests (analgesia on a hot plate, immobility on a ‘ring’, rectal hypothermia and hypolocomotion in an open field); and (e) the limb spasticity test in chronic relapsing experimental allergic encephalomyelitis (CREAE) mice, a model of multiple sclerosis (MS). O‐2093, either synthesized by us or commercially available, was inactive in the ‘tetrad’ up to a 20u2003mgu2003kg−1 dose (i.v.). Like O‐2093, the other four compounds exhibited low affinity in CB1 (Ki from 1.3 to >10u2003μM) and CB2 binding assays (1.310u2003μM), very low potency as fatty acid amide hydrolase (FAAH) inhibitors (IC50>25u2003μM) and were inactive in the ‘tetrad’ up to a 30u2003mgu2003kg−1 dose (i.v.). While O‐2247 and O‐2248 were poor inhibitors of [14C]AEA cellular uptake (IC50>40u2003μM), O‐3246 and O‐3262 were quite potent in this assay. O‐3246, which exhibits only a very subtle structural difference with O‐2093, is the most potent inhibitor of AEA uptake reported in vitro under our experimental conditions (IC50=1.4u2003μM) and is 12‐fold more potent than O‐2093. When injected intravenously O‐3246 and O‐3262, again like O‐2093 and unlike O‐2247 and O‐2248, significantly inhibited limb spasticity in mice with CREAE. These data confirm the potential utility of selective AEA uptake inhibitors as anti‐spasticity drugs in MS and, given the very subtle chemical differences between potent and weak inhibitors of uptake, support further the existence of a specific mechanism for this process.