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.

Cannabidiol, a non-psychotropic component of cannabis, attenuates vomiting and nausea-like behaviour via indirect agonism of 5-HT(1A) somatodendritic autoreceptors in the dorsal raphe nucleus.

BACKGROUND AND PURPOSE: To evaluate the hypothesis that activation of somatodendritic 5-HT(1A) 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. EXPERIMENTAL APPROACH: The potential of systemic and intra-DRN administration of 5-HT(1A) receptor antagonists, WAY100135 or WAY100635, to prevent the anti-emetic effect of CBD in shrews (Suncus murinus) and the anti-nausea-like effects of CBD (conditioned gaping) in rats were evaluated. Also, the ability of intra-DRN administration of CBD to produce anti-nausea-like effects (and reversal by systemic WAY100635) was assessed. In vitro studies evaluated the potential of CBD to directly target 5-HT(1A) receptors and to modify the ability of the 5-HT(1A) agonist, 8-OH-DPAT, to stimulate [(35) S]GTPγS binding in rat brainstem membranes. KEY RESULTS: CBD suppressed nicotine-, lithium chloride (LiCl)- and cisplatin (20 mg·kg(-1) , but not 40 mg·kg(-1) )-induced vomiting in the S. murinus and LiCl-induced conditioned gaping in rats. Anti-emetic and anti-nausea-like effects of CBD were suppressed by WAY100135 and the latter by WAY100635. When administered to the DRN: (i) WAY100635 reversed anti-nausea-like effects of systemic CBD, and (ii) CBD suppressed nausea-like effects, an effect that was reversed by systemic WAY100635. CBD also displayed significant potency (in a bell-shaped dose-response curve) at enhancing the ability of 8-OH-DPAT to stimulate [(35) S]GTPγS binding to rat brainstem membranes in vitro. Systemically administered CBD and 8-OH-DPAT synergistically suppressed LiCl-induced conditioned gaping. CONCLUSIONS AND IMPLICATIONS: These results suggest that CBD produced its anti-emetic/anti-nausea effects by indirect activation of the somatodendritic 5-HT(1A) autoreceptors in the DRN. LINKED ARTICLES: This article is part of a themed section on Cannabinoids in Biology and Medicine. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.165.issue-8. To view Part I of Cannabinoids in Biology and Medicine visit http://dx.doi.org/10.1111/bph.2011.163.issue-7.

Omega-3 N-acylethanolamines are endogenously synthesised from omega-3 fatty acids in different human prostate and breast cancer cell lines.

Omega-3 (n-3) fatty acids inhibit breast and prostate cancer cell growth. We previously showed that N-acylethanolamine derivatives of n-3 (n-3-NAE) are endocannabinoids, which regulate cancer cell proliferation. These n-3-NAE are synthesised in certain cells/tissues, after supplementing with fatty acids, however, no one has assessed whether and to what extent this occurs in cancer cells. We determined levels of endogenous n-3-NAEs in hormone sensitive and insensitive prostate and breast cancer cells and subsequent effects on other endocannabinoids (anandamide and 2-arachidonoylglycerol), before and after supplementing with DHA and EPA fatty acids, using HPLC tandem mass spectrometry. This is the first study reporting that n-3-NAEs are synthesised from their parent n-3 fatty acids in cancer cells, regardless of tumour type, hormone status or the presence of fatty acid amide hydrolase. This could have important implications for the use of n-3 fatty acids as therapeutic agents in breast and prostate cancers expressing cannabinoid receptors.

Endocannabinoid signaling in neurotoxicity and neuroprotection.

The cannabis plant and products produced from it, such as marijuana and hashish, have been used for centuries for their psychoactive properties. The mechanism for how Delta(9)-tetrahydrocannabinol (THC), the active constituent of cannabis, elicits these neurological effects remained elusive until relatively recently, when specific G-protein coupled receptors were discovered that appeared to mediate cellular actions of THC. Shortly after discovery of these specific receptors, endogenous ligands (endocannabinoids) were identified. Since that time, an extensive number of papers have been published on the endocannabinoid signaling system, a widespread neuromodulatory mechanism that influences neurotransmission throughout the nervous system. This paper summarizes presentations given at the 12th International Neurotoxicology Association meeting that described the potential role of endocannabinoids in the expression of neurotoxicity. Dr. Raphael Mechoulam first gave an overview of the discovery of exogenous and endogenous cannabinoids and their potential for neuroprotection in a variety of conditions. Dr. Larry Parsons then described studies suggesting that endocannabinoid signaling may play a selective role in drug reinforcement. Dr. Carey Pope presented information on the role that endocannabinoid signaling may have in the expression of cholinergic toxicity following anticholinesterase exposures. Together, these presentations highlighted the diverse types of neurological insults that may be modulated by endocannabinoids and drugs/toxicants which might influence endocannabinoid signaling pathways.

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.

Suppressors of cancer cell proliferation from fig (Ficus carica) resin: isolation and structure elucidation.

A mixture of 6-O-acyl-beta-D-glucosyl-beta-sitosterols, the acyl moeity being primarily palmitoyl and linoleyl with minor amounts of stearyl and oleyl, has been isolated as a potent cytotoxic agent from fig (Ficuscarica) latex and soybeans. Identity was established by spectroscopic methods (NMR, MS) and confirmed by chemical synthesis. Both the natural and the synthetic compounds showed in vitro inhibitory effects on proliferation of various cancer cell lines.

The cannabinoids: an overview. Therapeutic implications in vomiting and nausea after cancer chemotherapy, in appetite promotion, in multiple sclerosis and in neuroprotection.

The present paper describes the historical use of cannabis, starting with its use in Assyria and China. Recent advances in the understanding of the molecular basis of cannabis action are explained, including the identification of the cannabinoid receptors CB(1) and CB(2), as well as the isolation of endogenous cannabinoids from the brain and periphery. The use of delta(9)-tetrahydrocannabinol as an anti-vomiting and anti-nausea drug for cancer chemotherapy, and as an appetite-enhancing agent is described. Clinical work in multiple sclerosis, which may lead to the approval of tetrahydrocannabinol as a drug for this condition, is presented. Preclinical and clinical investigations with cannabidiol, a non-psychotropic cannabis constituent, are also described. Recent work with cannabidiol in animal models of rheumatoid arthritis may lead to clinical investigations. A synthetic cannabinoid, HU-211 (Dexanabinol), is in advanced clinical stages of investigation as a neuroprotectant in head trauma. The above clinical approaches may ultimately lead to the realization that cannabinoids are valuable clinical drugs in numerous fields.

The nonpsychoactive cannabis constituent cannabidiol is an oral anti-arthritic therapeutic in murine collagen-induced arthritis.

The therapeutic potential of cannabidiol (CBD), the major nonpsychoactive component of cannabis, was explored in murine collagen-induced arthritis (CIA). CIA was elicited by immunizing DBA/1 mice with type II collagen (CII) in complete Freund's adjuvant. The CII used was either bovine or murine, resulting in classical acute CIA or in chronic relapsing CIA, respectively. CBD was administered after onset of clinical symptoms, and in both models of arthritis the treatment effectively blocked progression of arthritis. CBD was equally effective when administered i.p. or orally. The dose dependency showed a bell-shaped curve, with an optimal effect at 5 mg/kg per day i.p. or 25 mg/kg per day orally. Clinical improvement was associated with protection of the joints against severe damage. Ex vivo, draining lymph node cells from CBD-treated mice showed a diminished CII-specific proliferation and IFN-gamma production, as well as a decreased release of tumor necrosis factor by knee synovial cells. In vitro effects of CBD included a dose-dependent suppression of lymphocyte proliferation, both mitogen-stimulated and antigen-specific, and the blockade of the Zymosan-triggered reactive oxygen burst by peritoneal granulocytes. It also was found that CBD administration was capable of blocking the lipopolysaccharide-induced rise in serum tumor necrosis factor in C57/BL mice. Taken together, these data show that CBD, through its combined immunosuppressive and anti-inflammatory actions, has a potent anti-arthritic effect in CIA.

Low dose anandamide affects food intake, cognitive function, neurotransmitter and corticosterone levels in diet-restricted mice.

This investigation reports the possible role of the endocannabinoid anandamide on modulating the behavioral and neurochemical consequences of semi-starvation. We studied the effect of very low dose anandamide (0.001 mg/kg) administration on food intake, cognitive function and catecholaminergic and serotonergic pathways in two murine brain areas concerned with appetite (hypothalamus) and learning (hippocampus), and the peripheral corticosterone response to the stress of 40% diet restriction. Anandamide-treated mice consumed 44% more food (P<0.05) during 1 week of 2.5-h feeding each day. In the hypothalamus, there were significantly increased concentrations of norepinephrine (P<0.01), dopamine (P<0.05) and 5-hydroxytryptamine (5-HT) (P<0.001). In the hippocampus, anandamide increased significantly norepinephrine and dopamine, but decreased 5-HT (all at P<0.001). Diet restriction was accompanied in both areas by a significant decrease in all neurotransmitter concentrations that were partially restored by anandamide for dopamine and 5-HT, but not for norepinephrine. In animals on diet restriction, anandamide significantly improved impaired maze performance. Norepinephrine turnover and plasma corticosterone levels were also raised significantly by anandamide. The fact that low dose anandamide improved food intake, cognitive function and reversed some of the neurotransmitter changes caused by diet restriction, might have implications for the treatment of cachexia associated with acquired immunodeficiency syndrome (AIDS) and cancer, for mood changes sometimes associated with dieting, and in the extreme case, of patients with anorexia.

Recent advantages in cannabinoid research.

Although the active component of cannabis Delta9-THC was isolated by our group 35 years ago, until recently its mode of action remained obscure. In the last decade it was established that Delta9-THC acts through specific receptors - CB1 and CB2 - and mimics the physiological activity of endogenous cannabinoids of two types, the best known representatives being arachidonoylethanolamide (anandamide) and 2-arachidonoylglycerol (2-AG). THC is officially used against vomiting caused by cancer chemotherapy and for enhancing appetite, particularly in AIDS patients. Illegally, usually by smoking marijuana, it is used for ameliorating the symptoms of multiple sclerosis, against pain, and in a variety of other diseases. A synthetic cannabinoid, HU-211, is in advanced clinical tests against brain damage caused by closed head injury. It may prove to be valuable against stroke and other neurological diseases.

PADMA-28, a traditional tibetan herbal preparation inhibits the respiratory burst in human neutrophils, the killing of epithelial cells by mixtures of oxidants and pro-inflammatory agonists and peroxidation of lipids.

Both aqueous and methanolic fractions derived from the Tibetan preparation PADMA-28 (a mixture of 22 plants) used as an anti-atherosclerotic agent, and which is non-cytolytic to a variety of mammalian cells, were found to strongly inhibit (1) the killing of epithelial cells in culture induced by 'cocktails' comprising oxidants, membrane perforating agents and proteinases; (2) the generation of luminol-dependent chemiluminescence in human neutrophils stimulated by opsonized bacteria; (3) the peroxidation of intralipid (a preparation rich in phopholipids) induced in the presence of copper; and (4) the activity of neutrophil elastase. It is proposed that PADMA-28 might prove beneficial for the prevention of cell damage induced by synergism among pro-inflammatory agonists which is central in the initiation of tissue destruction in inflammatory and infectious conditions.

Developmental aspects of anandamide: ontogeny of response and prenatal exposure.

Recent breakthroughs in cannabinoid research, including the identification of two cannabinoid receptors (CB receptors) and a family of endogenous ligands, the anandamides, may shed new light on the sequelae of pre- and perinatal exposure to cannabinoid receptor ligands and enable the experimental manipulation of the endogenous ligand in the developing organism. In the present study we examined the behavioural response to anandamide (ANA) in developing mice from day 13 into adulthood. We observed that depression of ambulation in an open field and the analgetic response to ANA are not fully developed until adulthood. In a separate set of experiments, we administered five daily injections of ANA (SC, 20 mg/kg) during the last trimester of pregnancy. No effects on birth weight, litter size, sex ratio and eye opening were detected after maternal ANA treatment. Further, no effects on open field performance of the offspring were observed until 4 weeks of age. However, from 40 days of age, a number of differences between the prenatal ANA and control offspring were detected. Thus, the offspring from ANA-treated dams showed impaired responsiveness to a challenge with ANA or delta 0-THC expressed as a lack of immobility in the ring test for catalepsy, hypothermia and analgesia. On the other hand, without challenge, they exhibited a spontaneous decrease in open field activity, catalepsy, hypothermia and a hypoalgetic tendency. These data suggest that exposure to excessive amounts of ANA during gestation alters the functioning of the ANA-CB receptor system. Further experiments investigating responsivity of the immune system suggest an increased inflammatory response to arachidonic acid, and enhanced hypothermic response to lipopolysaccharide in prenatally treated offspring. The results are discussed in relation to other manipulations of the maternal milieu, especially prenatal stress. It is concluded that alterations induced by prenatal exposure to ANA, cannabinoids and other psychotropic drugs or prenatal stress, share common features, but the data also suggest specific effects on the ANA-CB receptor system.

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.

An efficient new cannabinoid antiemetic in pediatric oncology.

Delta-8-tetrahydrocannabinol (delta-8-THC), a cannabinoid with lower psychotropic potency than the main Cannabis constituent, delta-9-tetrahydrocannabinol (delta-9-THC), was administered (18 mg/m2 in edible oil, p.o.) to eight children, aged 3-13 years with various hematologic cancers, treated with different antineoplastic drugs for up to 8 months. The total number of treatments with delta-8-THC so far is 480. The THC treatment started two hours before each antineoplastic treatment and was continued every 6 hrs for 24 hours. Vomiting was completely prevented. The side effects observed were negligible.

Effect of the brain constituent anandamide, a cannabinoid receptor agonist, on the hypothalamo-pituitary-adrenal axis in the rat.

Anandamide (arachidonylethanolamide), an endogenous ligand of the cannabinoid receptor, was recently isolated from porcine brain. We report here for the first time on the effect of this ligand on the hypothalamo-pituitary adrenal (HPA) axis in comparison to that of the plant cannabinoid delta 9-tetrahydrocannabinol (THC). Intracerebroventricular injection of anandamide or THC (50 or 150 micrograms/rat) increased significantly the serum levels of ACTH and corticosterone in a dose-dependent manner and caused a pronounced depletion of CRF-41 in the median eminence. These data suggest that anandamide parallels THC in activating the HPA axis via mediation of a central mechanism which involves the secretion of CRF-41.

Toward drugs derived from cannabis.

Recent work aimed at the introduction of natural and synthetic cannabinoids as drugs is reviewed. Delta1-Tetrahydrocannabinol (delta1-THC) is mainly investigated as a potential drug against glaucoma and asthma, and as an antiemetic agent in cancer chemotherapy. Cannabidiol is being tried in the clinic against epilepsy and as a hypnotic. Numerous synthetic cannabinoids are currently being investigated as analgetics and as sedative-relaxants.