Biomedical subjects
Publications and source records attributed to Raphael Mechoulam.
Most of the cannabinoids in Cannabis sativa L. have not been fully evaluated for their pharmacological activity. A publication in this issue presents evidence that a plant cannabinoid, Delta(9)-tetrahydrocannabivarin is a potent antagonist of anandamide, a major endogenous cannabinoid. It seems possible that many of the non-psychoactive constituents of this plant will be of biological interest.
We reported earlier that closed head injury (CHI) in mice causes a sharp elevation of brain 2-arachidonoylglycerol (2-AG) levels, and that exogenous 2-AG reduces brain edema, infarct volume and hippocampal death and improved clinical recovery after CHI. The beneficial effect of 2-AG was attenuated by SR141716A, a CB1 cannabinoid receptor antagonist, albeit at relatively high doses. In the present study, we further explored the role of CB1 receptors in mediating 2-AG neuroprotection. CB1 receptor knockout mice (CB1-/-) showed minor spontaneous recovery at 24 h after CHI, in contrast to the significant improvement in neurobehavioral function seen in wild-type (WT) mice. Moreover, administration of 2-AG did not improve neurological performance and edema formation in the CB1-/- mice. In addition, 2-AG abolished the three- to four-fold increase of nuclear factor kappaB (NF-kappa B) transactivation, at 24 h after CHI in the WT mice, while it had no effect on NF-kappaB in the CB1-/- mice, which was as high as in the WT vehicle-treated mice. We thus propose that 2-AG exerts its neuroprotection after CHI, at least in part, via CB1 receptor-mediated mechanisms that involve inhibition of intracellular inflammatory signaling pathways.
Cannabinoids, such as the delta9-tetrahydrocannabinol (THC), present in the cannabis plant, as well as anandamide and 2-arachidonoyl glycerol, produced by the mammalian body, have been shown to protect the brain from various insults and to improve several neurodegenerative diseases. The current review summarizes the evidence for cannabinoid neuroprotection in vivo, and refers to recent in vitro studies, which help elucidate possible molecular mechanisms underlying this protective effect. Some of these mechanisms involve the activation of CB1 and CB2 cannabinoid receptors, while others are not dependent on them. In some cases, protection is due to a direct effect of the cannabinoids on neuronal cells, while in others, it results from their effects on non-neuronal elements within the brain. In many experimental set-ups, cannabinoid neurotoxicity, particularly by THC, resides side by side with neuroprotection. The current review attempts to shed light on this dual activity, and to dissociate between the two contradictory effects.
Delta9-Tetrahydrocannabinol (Delta9-THC) and (-)-cannabidiol are major constituents of the Cannabis sativa plant with different pharmacological profiles: (-)-Delta9-tetrahydrocannabinol, but not (-)-cannabidiol, activates cannabinoid CB1 and CB2 receptors and induces psychoactive and peripheral effects. We have tested a series of (+)-cannabidiol derivatives, namely, (+)-cannabidiol-DMH (DMH-1,1-dimethylheptyl-), (+)-7-OH-cannabidiol-DMH, (+)-7-OH- cannabidiol, (+)-7-COOH- cannabidiol and (+)-7-COOH-cannabidiol-DMH, for central and peripheral (intestinal, antiinflammatory and peripheral pain) effects in mice. Although all (+)-cannabidiols bind to cannabinoid CB1 and CB2 receptors, only (+)-7-OH-cannabidiol-DMH was centrally active, while all (+)-cannabidiol analogues completely arrested defecation. The effects of (+)-cannabidiol-DMH and (+)-7-OH-cannabidiol-DMH were partially antagonized by the cannabinoid CB1 receptor antagonist N-(piperidiny-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (SR141716), but not by the cannabinoid CB2 receptor antagonist N-[-(1S)-endo-1,3,3-trimethil bicyclo [2.2.1] heptan-2-yl-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide (SR144528), and had no effect on CB1(-/-) receptor knockout mice. (+)-Cannabidiol-DMH inhibited the peripheral pain response and arachidonic-acid-induced inflammation of the ear. We conclude that centrally inactive (+)-cannabidiol analogues should be further developed as antidiarrheal, antiinflammatory and analgesic drugs for gastrointestinal and other peripheral conditions.
The arachidonic acid derivative, 2-arachidonoyl-glycerol (2-AG), was initially isolated from gut and brain; it is also produced and released from blood and vascular cells. Many of the 2-AG-induced cellular responses (i.e., neuromodulation, cytoprotection and vasodilation) are mediated by cannabinoid receptors CB1 and CB2. The findings presented here demonstrate the expression of CB1, CB2 and TRPV1 receptors on cerebromicrovascular endothelial cells (HBEC). The expression of TRPV1, CB1 and CB2 receptor mRNA and proteins were demonstrated by RT-PCR and polyclonal antibodies, respectively. The endocannabinoid 2-AG, and other related compounds [anandamide (ANA), methanandamide (m-ANA), N-(4-hydroxyphenyl-arachidonyl-ethanolamide) (AM404) and capsaicin] dose-dependently stimulated Ca2+ influx in HBEC. The selective TRPV1 receptor antagonist (capsazepine), CB1 receptor antagonist (SR141716A) and CB2 receptor antagonist (SR144528) inhibited these responses. The effects of capsaicin, a specific agonist for TRPV1 receptors, were inhibited by capsazepine, but only weakly by CB1 or CB2 receptor antagonists. 2-AG also induced phosphorylation of vasodilator-stimulated phosphoprotein (VASP); this response was mediated by VR1 receptors. These studies clearly indicate that 2-AG and other related compounds may function as agonists on VR1 receptors, as well as CB1 and CB2 receptors, and implicated these factors in various HBEC functions.
Three cannabis constituents, cannabidiol (1), Delta(8)-tetrahydrocannabinol (3), and cannabinol (5), were oxidized to their respective para-quinones 2, 4, and 6. In the 1960s, the oxidized product 4 had been assigned a para-quinone structure, which was later modified to an ortho-quinone. To distinguish between the two possible quinone structures, a detailed NMR investigation was undertaken. The original para-quinone structure was confirmed. X-ray crystallography elucidated the structures of the crystalline 2 and 6. All three compounds displayed antiproliferative activity in several human cancer cell lines in vitro, and quinone 2 significantly reduced cancer growth of HT-29 cancer in nude mice.
OBJECTIVE: To explore the antiarthritic potential of a novel synthetic cannabinoid acid, Hebrew University-320 (HU-320), in the DBA/1 mouse model of arthritis, and to investigate in vitro antiinflammatory and immunosuppressive effects of HU-320 on macrophages and lymphocytes. METHODS: DBA/1 mice were immunized with bovine type II collagen (CII) to induce arthritis and then injected intraperitoneally daily with HU-320. The effects of treatment on arthritic changes in hind feet were assessed clinically and histologically, and draining lymph node responses to CII were assayed. Murine splenic and human blood lymphocytes were cultured to study the effect of HU-320 on polyclonal mitogenic stimulation. Macrophage cultures were set up to evaluate in vitro effects of HU-320 on production of tumor necrosis factor alpha (TNF alpha) and reactive oxygen intermediates (ROIs). The effect of HU-320 administration on lipopolysaccharide-induced serum TNF levels was assayed using C57BL/6 mice. Bioactive TNF production was measured using BALB/c clone 7 target cells. Evaluation of HU-320 psychoactivity was performed using established laboratory tests on Sabra mice. RESULTS: Systemic daily administration of 1 and 2 mg/kg HU-320 ameliorated established CII-induced arthritis. Hind foot joints of treated mice were protected from pathologic damage. CII-specific and polyclonal responses of murine and human lymphocytes were down-modulated. HU-320 inhibited production of TNF from mouse macrophages and of ROIs from RAW 264.7 cells and suppressed the rise in serum TNF level following endotoxin challenge. HU-320 administration yielded no adverse psychotropic effects in mice. CONCLUSION: Our studies show that the novel synthetic cannabinoid acid HU-320 has strong antiinflammatory and immunosuppressive properties while demonstrating no psychoactive effects. The profound suppressive effects on cellular immune responses and on the production of proinflammatory mediators all indicate its usefulness as a novel nonpsychoactive, synthetic antiinflammatory product.
RATIONALE: The 5-HT3 antagonist, ondansetron (OND), and the cannabinoid, delta9-tetrahydrocannabinol (delta9-THC), have been shown to interfere with emesis; however, their relative and/or combined effectiveness in suppressing vomiting produced by the chemotherapeutic agent, cisplatin, is unknown. OBJECTIVE: To evaluate the potential of: 1) a broad range of doses of delta9-THC and OND to prevent cisplatin-induced vomiting and retching in the Suncus murinus (house musk shrew), 2) combined treatment with ineffective individual doses of delta9-THC and OND to prevent cisplatin-induced vomiting and retching, 3) the CB1 receptor antagonist, SR141716, to reverse the antiemetic effects of OND, and 4) cannabidiol (CBD), the principal non-psychoactive component of marijuana, to reverse cisplatin-induced vomiting in the shrew. METHODS: Shrews were injected with various doses of OND (0.02-6.0 mg/kg), delta9-THC (1.25-10 mg/kg) and a combination of ineffective doses of each (0.02 mg/kg OND+1.25 mg/kg delta9-THC) prior to being injected with cisplatin (20 mg/kg) which induces vomiting. Shrews were also injected with CBD (5 mg/kg and 40 mg/kg) prior to an injection of cisplatin. RESULTS: OND and delta9-THC both dose-dependently suppressed cisplatin-induced vomiting and retching. Furthermore, a combined pretreatment of doses of the two drugs that were ineffective alone completely suppressed vomiting and retching. CBD produced a biphasic effect, suppressing vomiting at 5 mg/kg and potentiating it at 40 mg/kg. CONCLUSIONS: A low dose of the non-intoxicating cannabinoid CBD may be an effective anti-emetic treatment and combined doses of OND and delta9-THC that are ineffective alone suppresses cisplatin-induced emetic reactions in shrews.
RATIONALE: Using the place-preference conditioning paradigm, we evaluated the potential of the two most prominent cannabinoids found in marijuana, the psychoactive component delta9-tetrahydrocannabinol (delta9-THC) and the nonpsychoactive component cannabidiol (CBD), to potentiate extinction of a cocaine-induced and an amphetamine-induced conditioned place preference in rats. METHODS: To determine the effects of pretreatment with delta9-THC or CBD on extinction, a cocaine-induced and amphetamine-induced place preference was first established. Rats were then given an extinction trial, during which they were confined to the treatment-paired floor for 15 min. Thirty minutes prior to the extinction trial, they were injected with a low dose of delta9-THC (0.5 mg/kg), CBD (5 mg/kg) or vehicle. The potential of the CB1 receptor antagonist, SR141716, to reverse the effects of delta9-THC or CBD was also evaluated. To determine the hedonic effects of CBD, one distinctive floor was paired with CBD (5 mg/kg) and another with saline. Finally, to determine the effect of delta9-THC.or CBD on the establishment and/or the expression of a place preference during four cycles of conditioning trials, rats were injected with delta9-THC (0.25-1 mg/kg), CBD (5 mg/kg) or vehicle 25 min prior to receiving an injection of amphetamine followed by placement on the treatment floor; on alternate days, they received injections of vehicle followed by saline and placement on the nontreatment floor. The rats then received two test trials; on one trial they were pretreated with the cannabinoid and on the other trial with vehicle. RESULTS: delta9-THC and CBD potentiated the extinction of both cocaine-induced and amphetamine-induced conditioned place preference learning, and this effect was not reversed by SR141716. The cannabinoids did not affect learning or retrieval, and CBD was not hedonic on its own. CONCLUSIONS: These results are the first to show that both delta9-THC, which acts on both CB 1 and CB2 receptors, and CBD, which does not bind to CB1 or CB2 receptors, potentiate the extinction of conditioned incentive learning.
The growth factors basic fibroblast growth factor (bFGF) and insulin-like growth factor 1 (IGF-I) have been implicated in the pathophysiology of atherosclerosis and restenosis. The Tibetan herbal preparation PADMA-28 (a mixture of 22 plants which is used as an anti-atherosclerosis agent) was tested for its ability to inhibit the mitogenic activity of bFGF and IGF-I, growth factors involved in restenosis, atherosclerosis and tumour progression. DNA synthesis and proliferation of vascular smooth muscle cells, in response to serum bFGF, thrombin, or combinations thereof, were abrogated in the presence of microgram amounts of both the aqueous and organic, partially purified, extracts of PADMA-28. These fractions also inhibited IGF-I-mediated proliferation, migration and invasion of tumour cells responsive to IGF-I. The inhibition by PADMA 28 was reversible upon removal of the PADMA extracts, indicating that the effects were not related to cell toxicity. These and other properties (i.e., anti-oxidant activity) of PADMA-28 may be responsible for its beneficial effect as an anti-atherosclerotic agent, suggesting that this herbal preparation may have potential applications in the prevention of intimal hyperplasia and arterial stenosis secondary to coronary angioplasty and bypass surgery, as well as in the prevention and treatment of other vascular diseases and tumour growth and metastasis.
RATIONALE: Marijuana has been reported to interfere with nausea and vomiting in chemotherapy patients. The principal cannabinoids found in marijuana include the psychoactive compound Delta-9-tetrahydrocannabinol (THC) and the non-psychoactive compound cannabidiol (CBD). The experiments reported here evaluated the potential of THC and CBD to interfere with vomiting in the Suncus murinus (house musk shrew) produced by lithium chloride (LiCl), which is the most commonly employed unconditioned stimulus for taste avoidance. OBJECTIVES: To evaluate the potential of the principal components of marijuana, THC and CBD, to suppress Li-induced vomiting in the house musk shrew. METHODS: Shrews were injected with vehicle or one of two cannabinoids [Delta-9-THC (1-20 mg/kg), or CBD (2.5-40 mg/kg)] 10 min prior to an injection of LiCl (390 mg/kg of 0.15 M) and were then observed for 45 min. The frequency of vomiting episodes and the latency to the first episode were measured. The role of the CB1 receptor in these effects was also evaluated by pretreatment with SR-141716. RESULTS: Delta-9-THC produced a dose-dependent suppression of Li-induced vomiting, with higher doses producing greater suppression than lower doses. CBD produced a biphasic effect with lower doses producing suppression and higher doses producing enhancement of Li-induced vomiting. The suppression of Li-induced vomiting by THC, but not by CBD, was reversed by SR-141716. CONCLUSIONS: These results indicate that two major cannabinoid compounds found in marijuana, THC and CBD, are effective treatments for Li-induced vomiting; however, only THC acts by the CB1 receptor. The effects of THC and CBD on vomiting were dose dependent; with THC the effect was linear, but with CBD the effect was biphasic.
2-Arachidonoyl glycerol (2-AG) levels in whole mouse brain and two of its regions-hippocampus and hypothalamus-were determined after diet restriction (between 60 and 40%) lasting 12 days. The diet restriction lowered the level of 2-AG, which in the hypothalamus depended on the severity of the diet restriction, while the level in the hippocampus was not dependent on the diet regimen. As these observations differ from previously published data showing elevation of 2-AG levels in rat brain after 24 h of severe food restriction, we measured 2-AG levels in whole mouse brain after a comparable period of full starvation (fasting). We confirmed the elevation of 2-AG levels. It seems possible that these time-dependent variations of 2-AG levels may be of importance as a general coping strategy by animals during periods of starvation.
BACKGROUND AND PURPOSE: Cannabinoids confer neuroprotection in several experimental paradigms, but the responsible mechanisms remain unknown. Therefore, we sought to examine whether the synthetic CB1 agonist HU-210 is capable of reducing ischemic damage and to determine the mechanisms responsible for such protection. METHODS: Sprague-Dawley rats underwent permanent middle cerebral artery occlusion (PMCAO). After dose-response and therapeutic time window-finding experiments, the rats were injected with HU-210 (45 microg/kg IV) or vehicle 1 hour after PMCAO. Physiological parameters and cerebral blood flow in the peri-infarct zone were monitored. The animals were examined with a motor disability scale, and the infarct volumes were measured 72 hours later. We also examined the effects of the selective CB1 antagonist SR-141716 and of controlled warming on the neuroprotection conferred by HU-210. RESULTS: HU-210 reduced blood pressure and heart rate but did not alter the cerebral blood flow in the infarct border zone. Motor disability and infarct volumes were significantly reduced (by up to 77%; P<0.05) in animals treated with HU-210. A single injection of HU-210 significantly lowered the body temperature compared with vehicle as measured both at 1 hour (32.3+/-1.3 degrees C versus 35+/-1.6 degrees C; P=0.0024) and at 24 hours (31.5+/-2.5 degrees C versus 37.25+/-0.3 degrees C; P=0.0031) after PMCAO. The protective effects of HU-210 were partially reversed by pretreatment with SR-141716 but were completely abolished by warming of the animals to the levels observed in controls. CONCLUSIONS: HU-210 confers robust protection against ischemic damage. This protection is mediated at least in part by binding to CB1 receptors and is also associated with the indirect protective effects of hypothermia.
Cannabinoids, whether plant-derived, synthetic or endogenous, have been shown to stimulate appetite in the adult organism. We have reported previously that cannabinoid receptors play a critical role during the early suckling period: The selective cannabinoid CB(1) receptor antagonist N-(piperidiny-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (SR141617A) permanently prevented milk ingestion in a dose-dependent manner, when administered to (Sabra, albino) mouse pups, within 1 day of birth. As a consequence, these pups died within the first week of life. We now generalize this finding to a different strain of mice (C57BL/6). Further, we show that cannabinoid CB(1) receptor blockade (20 mg/kg SR141716A) must occur within 24 h after birth as injection of SR141716A into 2- or 5-day-old pups had a much smaller effect or no effect at all, respectively. Cannabinoid CB(1) receptor knockout mice did not ingest milk on the first day of life, similarly to SR141716A-treated normal pups, as measured by the appearance of "milkbands". However, the knockout pups started to display milkbands from day 2 of life. Survival rates of cannabinoid CB(1) receptor knockout mice were affected significantly, but to a lesser extent than normal pups, by the administration of SR141716A. Daily administration of the endocannabinoid 2-arachidonoyl glycerol, or the synthetic agonists (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone (WIN55,212-2, 5 mg/kg) or (-)-cis-3-[2-Hydroxy4-(1,1-dimethylheptyl) phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol (CP55,940, 5 or 20 mg/kg) did not promote survival or weight gain in CB(1)(-/-) pups. Our data support previous evidence for a critical role of cannabinoid CB(1) receptors for the initiation of suckling. Further, the present observations support the existence of an unknown cannabinoid receptor, with partial control over milk ingestion in newborns. Our data also suggest that the CB(1)(-/-) neonates possess a compensatory mechanism which helps them overcome the lack of cannabinoid CB(1) receptors.
RATIONALE: Marijuana has been reported to suppress nausea produced by chemotherapy treatment in human cancer patients. Although there is abundant evidence that cannabinoid agonists attenuate vomiting in emetic species, there has been little experimental evidence of their anti-nausea potential. Considerable evidence suggests that conditioned rejection reactions in rats reflect nausea. The present experiments evaluated the potential of low doses of the cannabinoid agonists, delta-9-tetrahydrocannabinol (THC; 0.5 mg/kg, i.p.), and HU-210 (0.001 mg/kg and 0.01 mg/kg, i.p.), and the CB(1) antagonist SR-141716A in modulating the establishment and the expression of lithium-induced conditioned rejection reactions in rats. OBJECTIVES: To evaluate the effect of cannabinoids on conditioned rejection reactions, a rat model of nausea. METHODS: In experiments 1 and 2, respectively, rats were injected with cannabinoid agonists, THC (0.5 mg/kg, i.p.) and HU-210 (0.001, 0.005 or 0.01 mg/kg), 30 min prior to exposure to 0.1% saccharin solution by intraoral infusion. Immediately following saccharin exposure, they were injected with 20 ml/kg 0.15 M lithium chloride or saline. On each of two test trials, the rats were injected with the cannabinoid or vehicle 30 min prior to exposure to saccharin. In experiment 3, rats were injected with the CB(1) antagonist, SR-141716A (2.5 mg/kg) or a combination of SR-141716A and HU-210 (0.01 mg/kg) 30 min prior to an infusion of saccharin followed by injection of lithium or saline. They were given a single drug-free test trial. Experiment 4 replicated and extended the findings of experiment 3. RESULTS: delta-9-THC and HU-210 interfered with the establishment and the expression of lithium-induced conditioned rejection reactions. The suppressive effect of HU-210 on rejection reactions was reversed by pretreatment with SR-141716A. Administration of SR-141716A prior to conditioning potentiated lithium-induced conditioned rejection reactions. CONCLUSIONS: These results indicate that the establishment and the expression of lithium-induced conditioned rejection reactions are suppressed by pretreatment with cannabinoid agents. These effects appear to be mediated by their action on the CB(1) receptor, because they are reversed by pretreatment with SR-141716A. Finally, our results suggest that endogenous cannabinoids play a role in modulation of nausea, because the antagonist potentiated lithium-induced nausea.
Cannabinoids and alcohol activate the same reward pathways, and the cannabinoid CB(1) receptor system plays an important role in regulating the positive reinforcing properties of alcohol. Indeed, both cannabinoids and alcohol cause the release of dopamine in the nucleus accumbens. Recent research suggests that ethanol preference, which is dependent on CB(1) receptors, is higher in young mice than in old mice, and higher in female mice than in male mice.
Parkinson's disease is a chronic neurodegenerative disease of the extrapyramidal system associated with dopaminergic neuronal loss in the basal ganglia. However, several other neurotransmitters, such as serotonin, gamma-amino-butyric acid and glutamate, are also related to the symptoms of Parkinson's disease patients and their response to levodopa treatment. The co-expression of cannabinoid and dopamine receptors in the basal ganglia suggests a potential role for endocannabinoids in the control of voluntary movement in Parkinson's disease. In the present study we treated unilaterally 2,4,5-trihydroxyphenethylamine (6-hydroxydopamine)-lesioned rats with the enantiomers of the synthetic cannabinoid 7-hydroxy-delta6-tetrahydrocannabinol 1,1-dimethylheptyl. Treatment with its (-)- (3R, 4R) enantiomer (code-name HU-210), a potent cannabinoid receptor type 1 agonist, reduced the rotations induced by levodopa/carbidopa or apomorphine by 34% and 44%, respectively. In contrast, treatment with the (+)- (3S, 4S) enantiomer (code-name HU-211), an N-methyl-D-aspartate antagonist, as well as the psychotropically inactive cannabis constituent: cannabidiol and its primary metabolite, 7-hydroxy-cannabinol, did not show any reduction of rotational behavior. Our results indicate that activation of the CB1 stimulates the dopaminergic system ipsilaterally to the lesion, and may have implications in the treatment of Parkinson's disease.
Two non-psychotropic cannabinoids, cannabidiol (CBD) and cannabidiol-dimethylheptyl (CBD-DMH), induced apoptosis in a human acute myeloid leukemia (AML) HL-60 cell line. Apoptosis was determined by staining with bisBenzimide and propidium iodide. A dose dependent increase of apoptosis was noted, reaching 61 and 43% with 8 microg/ml CBD and 15 microg/ml CBD-DMH, respectively, after a 24 h treatment. Prior exposure of the cells to gamma-irradiation (800 cGy) markedly enhanced apoptosis, reaching values of 93 and 95%, respectively. Human monocytes from normal individuals were resistant to either cannabinoids or gamma-irradiation. Caspase-3 activation was observed after the cannabinoid treatment, and may represent a mechanism for the apoptosis. Our data suggest a possible new approach to treatment of AML.