In vitro and in vivo efficacy of non-psychoactive cannabidiol in neuroblastoma.

BACKGROUND: Neuroblastoma (nbl) is one of the most common solid cancers in children. Prognosis in advanced nbl is still poor despite aggressive multimodality therapy. Furthermore, survivors experience severe long-term multi-organ sequelae. Hence, the identification of new therapeutic strategies is of utmost importance. Cannabinoids and their derivatives have been used for years in folk medicine and later in the field of palliative care. Recently, they were found to show pharmacologic activity in cancer, including cytostatic, apoptotic, and antiangiogenic effects. METHODS: We investigated, in vitro and in vivo, the anti-nbl effect of the most active compounds in Cannabis, Δ(9)-tetrahydrocannabinol (thc) and cannabidiol (cbd). We set out to experimentally determine the effects of those compounds on viability, invasiveness, cell cycle distribution, and programmed cell death in human nbl SK-N-SH cells. RESULTS: Both compounds have antitumourigenic activity in vitro and impeded the growth of tumour xenografts in vivo. Of the two cannabinoids tested, cbd was the more active. Treatment with cbd reduced the viability and invasiveness of treated tumour cells in vitro and induced apoptosis (as demonstrated by morphology changes, sub-G1 cell accumulation, and annexin V assay). Moreover, cbd elicited an increase in activated caspase 3 in treated cells and tumour xenografts. CONCLUSIONS: Our results demonstrate the antitumourigenic action of cbd on nbl cells. Because cbd is a nonpsychoactive cannabinoid that appears to be devoid of side effects, our results support its exploitation as an effective anticancer drug in the management of nbl.

Unraveling the molecular basis of cannabidiolic acid methyl Ester's anti-depressive effects in a rat model of treatment-resistant depression.

Major depressive disorder (MDD) stands as a significant cause of disability globally. Cannabidiolic Acid-Methyl Ester (CBDA-ME) (EPM-301, HU-580), a derivative of Cannabidiol, demonstrates immediate antidepressant-like effects, yet it has undergone only minimal evaluation in psychopharmacology. Our goal was to investigate the behavioral and potential molecular mechanisms associated with the chronic oral administration of this compound in the Wistar Kyoto (WKY) genetic model of treatment-resistant depression. Male WKY rats were subjected to behavioral assessments before and after receiving chronic (14-day) oral doses of CBDA-ME (0.5 mg/kg), 15 mg/kg of imipramine or vehicle. At the end of the study, plasma corticosterone levels and mRNA expression of various genes in the medial Prefrontal Cortex and Hippocampus were measured. Behavioral outcomes from CBDA-ME treatment indicated an antidepressant-like effect similar to imipramine, as oral ingestion reduced immobility and increased swimming duration in the Forced Swim Test. Neither treatment influenced locomotion in the Open Field Test nor preference in the Saccharin Preference Test. The behavioral impact in WKY rats coincided with reduced corticosterone serum levels, upregulated mRNA expression of Cannabinoid receptor 1, Fatty Acid Amide Hydrolase, and Corticotropin-Releasing Hormone Receptor 1, alongside downregulation of the Serotonin Transporter in the hippocampus. Additionally, there was an upregulation of CB1 mRNA expression and downregulation of Brain-Derived Neurotrophic Factor in the mPFC. These findings contribute to our limited understanding of the antidepressant effects of CBDA-ME and shed light on its potential psychopharmacological mechanisms. This discovery opens up possibilities for utilizing cannabinoids in the treatment of major depressive disorder and related conditions.

International Union of Basic and Clinical Pharmacology. LXXIX. Cannabinoid receptors and their ligands: beyond CB1 and CB2.

There are at least two types of cannabinoid receptors (CB(1) and CB(2)). Ligands activating these G protein-coupled receptors (GPCRs) include the phytocannabinoid Δ(9)-tetrahydrocannabinol, numerous synthetic compounds, and endogenous compounds known as endocannabinoids. Cannabinoid receptor antagonists have also been developed. Some of these ligands activate or block one type of cannabinoid receptor more potently than the other type. This review summarizes current data indicating the extent to which cannabinoid receptor ligands undergo orthosteric or allosteric interactions with non-CB(1), non-CB(2) established GPCRs, deorphanized receptors such as GPR55, ligand-gated ion channels, transient receptor potential (TRP) channels, and other ion channels or peroxisome proliferator-activated nuclear receptors. From these data, it is clear that some ligands that interact similarly with CB(1) and/or CB(2) receptors are likely to display significantly different pharmacological profiles. The review also lists some criteria that any novel "CB(3)" cannabinoid receptor or channel should fulfil and concludes that these criteria are not currently met by any non-CB(1), non-CB(2) pharmacological receptor or channel. However, it does identify certain pharmacological targets that should be investigated further as potential CB(3) receptors or channels. These include TRP vanilloid 1, which possibly functions as an ionotropic cannabinoid receptor under physiological and/or pathological conditions, and some deorphanized GPCRs. Also discussed are 1) the ability of CB(1) receptors to form heteromeric complexes with certain other GPCRs, 2) phylogenetic relationships that exist between CB(1)/CB(2) receptors and other GPCRs, 3) evidence for the existence of several as-yet-uncharacterized non-CB(1), non-CB(2) cannabinoid receptors; and 4) current cannabinoid receptor nomenclature.

Cannabidiol and other cannabinoids reduce microglial activation in vitro and in vivo: relevance to Alzheimer's disease.

Microglial activation is an invariant feature of Alzheimer's disease (AD). It is noteworthy that cannabinoids are neuroprotective by preventing ß-amyloid (Aß)-induced microglial activation both in vitro and in vivo. On the other hand, the phytocannabinoid cannabidiol (CBD) has shown anti-inflammatory properties in different paradigms. In the present study, we compared the effects of CBD with those of other cannabinoids on microglial cell functions in vitro and on learning behavior and cytokine expression after Aß intraventricular administration to mice. CBD, (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl) pyrrolo-[1,2,3-d,e]-1,4-benzoxazin-6-yl]-1-naphthalenyl-methanone [WIN 55,212-2 (WIN)], a mixed CB(1)/CB(2) agonist, and 1,1-dimethylbutyl-1-deoxy-Δ(9)-tetrahydrocannabinol [JWH-133 (JWH)], a CB(2)-selective agonist, concentration-dependently decreased ATP-induced (400 µM) increase in intracellular calcium ([Ca(2+)](i)) in cultured N13 microglial cells and in rat primary microglia. In contrast, 4-[4-(1,1-dimethylheptyl)-2,6-dimethoxyphenyl]-6,6-dimethyl-bicyclo[3.1.1]hept-2-ene-2-methanol [HU-308 (HU)], another CB(2) agonist, was without effect. Cannabinoid and adenosine A(2A) receptors may be involved in the CBD action. CBD- and WIN-promoted primary microglia migration was blocked by CB(1) and/or CB(2) antagonists. JWH and HU-induced migration was blocked by a CB(2) antagonist only. All of the cannabinoids decreased lipopolysaccharide-induced nitrite generation, which was insensitive to cannabinoid antagonism. Finally, both CBD and WIN, after subchronic administration for 3 weeks, were able to prevent learning of a spatial navigation task and cytokine gene expression in ß-amyloid-injected mice. In summary, CBD is able to modulate microglial cell function in vitro and induce beneficial effects in an in vivo model of AD. Given that CBD lacks psychoactivity, it may represent a novel therapeutic approach for this neurological disease.

Identification of a C-glucuronide of delta6- tetrahydrocannabinol as a mouse liver conjugate in vivo.

Delta 6-Tetrahydrocannabinol-C-4-glucuronide was found in the livers of mice that had been administered delta 6-tetrahydrocannabinol. Thus, C-glucuronidation of a compound that contains a free hydroxyl group has been demonstrated in vivo.

Identification of a C-Glucuronide of Dgr6-Tetrahydrocannabinol as a Mouse Liver Conjugate in vivo.

Delta(6)-Tetrahydrocannabinol-C-4(')- glucuronide was found in the livers of mice that had been administered Delta(6)-tetrahydrocannabinol. Thus, C-glucuronidation of a compound that contains a free hydroxyl group has been demonstrated in vivo.

Chemical basis of hashish activity.

A sample of hashish was extracted consecutively with petroleum ether, benzene, and methanol. When tested intravenously in monkeys only the petroleum-ether fraction was active. This material was further fractionated. The only active compound isolated was Delta(1)-tetrahydrocannabinol. Cannabinol, cannabidiol, cannabichromene, cannabigerol, and cannabicyclol when administered together with Delta(1)-tetrahydrocannabinol do not cause a change in the activity of the latter, under the experimental conditions used. These results provide evidence that, except for Delta(1)-tetrahydrocannabinol, no other major, psychotomimetically active compounds are present in hashish.

6 -hydroxy- 1 -tetrahydrocannabinol synthesis and biological activity.

6beta-Hydroxy-delta(1)-tetrahydrocannabinol, a metabolite of delta(1)-tetrahydrocannabinol has been synthesized from delta(6)-tetrahydrocannabinol. It shows high tetrahydrocannabinol-type activity in rhesus monkeys. The implications of this funding are discussed.

Isolation and structure of a brain constituent that binds to the cannabinoid receptor.

Arachidonylethanolamide, an arachidonic acid derivative in porcine brain, was identified in a screen for endogenous ligands for the cannabinoid receptor. The structure of this compound, which has been named "anandamide," was determined by mass spectrometry and nuclear magnetic resonance spectroscopy and was confirmed by synthesis. Anandamide inhibited the specific binding of a radiolabeled cannabinoid probe to synaptosomal membranes in a manner typical of competitive ligands and produced a concentration-dependent inhibition of the electrically evoked twitch response to the mouse vas deferens, a characteristic effect of psychotropic cannabinoids. These properties suggest that anandamide may function as a natural ligand for the cannabinoid receptor.

A study investigating the acute dose-response effects of 13 mg and 17 mg Delta 9- tetrahydrocannabinol on cognitive-motor skills, subjective and autonomic measures in regular users of marijuana.

Heavy use of marijuana is claimed to damage critical skills related to short-term memory, visual scanning and attention. Motor skills and driving safety may be compromised by the acute effects of marijuana. The aim of this study was to investigate the acute effects of 13 mg and 17 mg Delta 9-tetrahydrocannabinol (THC) on skills important for coordinated movement and driving and on subjective and autonomic measures in regular users of marijuana. Fourteen regular users of marijuana were enrolled. Each subject was tested on two separate days. On each test day, subjects smoked two low-nicotine cigarettes, one with and the other without THC. Seventeen mg THC was included in the cigarette on one test day and 13 mg on the other day. The sequence of cigarette types was unknown to the subject. During smoking, heart rate and blood pressure were monitored, and the subjects performed a virtual reality maze task requiring attention and motor coordination, followed by 3 other cognitive tasks (Wisconsin Card Sorting Test (WCST), a "gambling" task and estimation of time and distance from an approaching car). After smoking a cigarette with 17 mg THC, regular marijuana users hit the walls more often on the virtual maze task than after smoking cigarettes without THC; this effect was not seen in patients after they smoked cigarettes with 13 mg THC. Performance in the WCST was affected with 17 mg THC and to a lesser extent with the use of 13 mg THC. Decision making in the gambling task was affected after smoking cigarettes with 17 mg THC, but not with 13 m THC. Smoking cigarettes with 13 and 17 mg THC increased subjective ratings of pleasure and satisfaction, drug "effect" and drug "high". These findings imply that smoking of 17 mg THC results in impairment of cognitive-motor skills that could be important for coordinated movement and driving, whereas the lower dose of 13 mg THC appears to cause less impairment of such skills in regular users of marijuana.

Acute oral cannabidiolic acid methyl ester reduces depression-like behavior in two genetic animal models of depression.

BACKGROUND AND PURPOSE: Cannabidiolic acid methyl ester (HU-580) was recently shown to reduce stress-induced anxiety-like behavior in rats. The aim of this study was to examine the antidepressant effect of HU-580 in two different rat models of depression. EXPERIMENTAL APPROACH: Using the forced swim test (FST), we evaluated the effect of HU-580 in 43 Wistar-Kyoto (WKY) and 23 Flinders Sensitive Line (FSL) adult male rats. KEY RESULTS: 1 mg/kg HU-580 reduced immobility and increased swimming in WKY rats, compared to vehicle-treated controls (p < 0.05). This dose exerted similar effects in FSL rats (p < 0.05). CONCLUSION AND IMPLICATIONS: This is the first report of antidepressant efficacy of HU-580. These findings expand the very limited existent results, suggesting that HU-580 is a potent anxiolytic agent. Taken together with its chemical stability, HU-580 emerges as a candidate for a future antidepressant medication.

Endocannabinoids and traumatic brain injury.

In response to traumatic brain injury, there is local and transient accumulation of 2-AG at the site of injury, peaking at 4 h and sustained up to at least 24 h. Neuroprotection exerted by exogenous 2-AG suggests that the formation of 2-AG may serve as a molecular regulator of pathophysiological events, attenuating the brain damage. Inhibition of this protective effect by SR-141716A, a CB(1) cannabinoid receptor antagonist, and the lack of effect of 2-AG in CB(1) knockout mice suggest that 2-AG and the CB(1) receptor may be important in the pathophysiology of traumatic brain injury. 2-AG exerts its neuroprotective effect after traumatic brain injury, at least in part, by inhibition of NF-kappaB transactivation. 2-AG also inhibits, at an early stage (2-4 h), the expression of the main proinflammatory cytokines, TNF-alpha, IL-6, and IL-1beta, and is accompanied by reduction of BBB permeability. Moreover, the CB(1), CB(2), and TRVP1 receptors are expressed on microvascular endothelial cells, and their activation by 2-AG counteracts endothelin (ET-1)-induced cerebral microvascular responses (namely, Ca(2+) mobilization and cytoskeleton rearrangement). This suggests that the functional interaction between 2-AG and ET-1 may provide a potential alternative pathway for abrogating ET-1-inducible vasoconstriction after brain injury and play a role in the neuroprotective effects exerted by 2-AG, as a potent vasodilator.

Cannabidiol lowers incidence of diabetes in non-obese diabetic mice.

Cannabidinoids are components of the Cannabis sativa (marijuana) plant that have been shown capable of suppressing inflammation and various aspects of cell-mediated immunity. Cannabidiol (CBD), a non-psychoactive cannabidinoid has been previously shown by us to suppress cell-mediated autoimmune joint destruction in an animal model of rheumatoid arthritis. We now report that CBD treatment significantly reduces the incidence of diabetes in NOD mice from an incidence of 86% in non-treated control mice to an incidence of 30% in CBD-treated mice. CBD treatment also resulted in the significant reduction of plasma levels of the pro-inflammatory cytokines, IFN-gamma and TNF-alpha. Th1-associated cytokine production of in vitro activated T-cells and peritoneal macrophages was also significantly reduced in CBD-treated mice, whereas production of the Th2-associated cytokines, IL-4 and IL-10, was increased when compared to untreated control mice. Histological examination of the pancreatic islets of CBD-treated mice revealed significantly reduced insulitis. Our results indicate that CBD can inhibit and delay destructive insulitis and inflammatory Th1-associated cytokine production in NOD mice resulting in a decreased incidence of diabetes possibly through an immunomodulatory mechanism shifting the immune response from Th1 to Th2 dominance.

Endocannabinoids and neuroprotection.

Traumatic brain injury (TBI) releases harmful mediators that lead to secondary damage. On the other hand, neuroprotective mediators are also released, and the balance between these classes of mediators determines the final outcome after injury. Recently, it was shown that the endogenous brain cannabinoids anandamide and 2-Arachidonoyl glycerol (2-AG) are also formed after TBI in rat and mouse respectively, and when administered after TBI, they reduce brain damage. In the case of 2-AG, better results are seen when it is administered together with related fatty acid glycerol esters. Significant reduction of brain edema, better clinical recovery, and reduced infarct volume and hippocampal cell death are noted. This new neuroprotective mechanism may involve inhibition of transmitter release and of inflammatory response. 2-AG is also a potent modulator of vascular tone, and counteracts the endothelin (ET-1)-induced vasoconstriction that aggravates brain damage; it may thus help to restore blood supply to the injured brain.

The Jerusalem Balsam: from the Franciscan Monastery in the old city of Jerusalem to Martindale 33.

The Jerusalem Balsam, a remedy based on an ethanolic extract of a herbal mixture, was formulated in 1719 in the pharmacy of the Saint Savior monastery in the old city of Jerusalem. Having gained fame, the Jerusalem Balsam was replicated and prepared in Europe. One can still find variations of the formula in current pharmacopoeias (B.P., 1998. The Stationary Office, London, p. 1510; Sweetman, S.C., Blake, P.S., McGlashan, J.M., Parsons, A.V., 2002. Martindale: The Extra Pharmacopeia, 33rd ed. Pharmaceutical Press, London, p. 1101). We report here, five different formulas, all referred to as "The Jerusalem Balsam". Three of those formulas were translated and two of these translations are presented in the text. A third one is available as Supplementary data online. As the formulas originate from different historical periods, the Jerusalem Balsam may be a good case study of the development of pharmaceutical formulations over a 250 years period. One of the formulas, found in a manuscript form in the archive of the monastery, contains four plants: olibanum (Boswellia spp.), myrrh (Commiphora spp.), aloe (Aloe sp.) and mastic (Pistacia lentiscus L.). We conducted pharmacological assays on this four-plant formula. It showed anti-inflammatory, as well as anti-oxidative, and anti-septic properties.

The peripheral cannabinoid receptor: adenylate cyclase inhibition and G protein coupling.

Two cannabinoid receptors, designated neuronal (or CB1) and peripheral (or CB2), have recently been cloned. Activation of CB1 receptors leads to inhibition of adenylate cyclase and N-type voltage-dependent Ca2+ channels. Here we show, using a CB2 transfected Chinese hamster ovary cell line, that this receptor binds a variety of tricyclic cannabinoid ligands as well as the endogenous ligand anandamide. Activation of the CB2 receptor by various tricyclic cannabinoids inhibits adenylate cyclase activity and this inhibition is pertussis toxin sensitive indicating that this receptor is coupled to the Gi/G(o) GTP-binding proteins. Interestingly, contrary to results with CB1, anandamide did not inhibit the CB2 coupled adenylate cyclase activity and delta 9-tetrahydrocannabinol had only marginal effects. These results characterize the CB2 receptor as a functional and distinctive member of the cannabinoid receptor family.

Looking back at Cannabis research.

Research leading to the isolation of the plant cannabinoids during the 1960 s and to the endogenous cannabinoids, during the 1990 s is described. Investigations on two non-psychotropic, synthetic cannabinoids, HU-211, a neuroprotective agent and HU-308, a specific CB2 agonist are presented.

Suppression of neuropathic pain behavior in rats by a non-psychotropic synthetic cannabinoid with NMDA receptor-blocking properties.

HU211 is a novel synthetic derivative of tetrahydro-cannabinol (THC), the active marijuana ingredient. The stereochemistry of HU211 is enantiomeric to that of THC. In contrast to THC, HU211 is not psychotropic. This agent exhibits other types of biological activities; it is a non-competitive NMDA receptor blocker and has antinociceptive activity when injected with cupric chloride. This study examined its effects in autotomy, a behavioral model of neuropathic pain. Autotomy, a behavior of self-mutilation of denervated areas, was induced in Sabra rats by cutting the sciatic and saphenous nerves. We found that injections of HU211 (2.5 mg/kg) with cupric chloride (0.8 mg/kg) every 2nd day markedly suppressed autotomy during the injection period by delaying its average onset day and reducing the incidence of severe autotomy. Moreover, suppression of autotomy was retained in the postinjection period (for at least 30 days) but only when the drug was injected intraperitoneally. Lesser effects were achieved by subcutaneous injections. Cupric chloride or HU211 alone were ineffective. The general behavior and open field motor activity indicated that the effects of HU211 with Cu++ on autotomy were not due to sedation or ataxia but presumably due to antinociception mediated by NMDA receptor blockade.

Characterization of the hypothermic effect of the synthetic cannabinoid HU-210 in the rat. Relation to the adrenergic system and endogenous pyrogens.

In the present study we have characterized the hypothermic effect of the psychoactive cannabinoid HU-210, by investigating its interaction with the endogenous pyrogens, IL-1 and PGE2. We also studied the involvement of the adrenergic system in mediation of this hypothermic effect. Injection of HU-210 directly into the preoptic area caused a dose dependent reduction of rectal temperature from 37 to 32.1 degrees C. Injection of the non-psychoactive analog, HU-211 which does not bind to brain cannabinoid receptor, did not affect body temperature. Injection of the adrenergic agonists, CGP-12177 and clonidine (beta, and alpha adrenergic agonists, respectively) abrogated the hypothermia induced by HU-210. Injection of the adrenergic antagonists, prazosin (alpha 1) and propranolol (beta) enhanced the hypothermic effect of HU-210. Intracerebral administration of IL-1 or PGE2 to rats pretreated with HU-210 caused a transient inhibition of the hypothermia. The ex vivo rate of basal or bacterial endotoxin-induced synthesis of PGE2 by different brain regions, including the preoptic area was not affected by HU-210 administration. These results suggest that the synthetic cannabinoid HU-210 acts in the preoptic area, probably via the brain cannabinoid receptor to induce hypothermia. The hypothermic effect can be antagonized by adrenergic agonists and enhanced by adrenergic antagonists. HU-210 does not interfere with the pyrogenic effect of IL-1 or PGE2.