A novel component of cannabis extract potentiates excitatory synaptic transmission in rat olfactory cortex in vitro

Cannabis is a potential treatment for epilepsy, although the few human studies supporting this use have proved inconclusive. Previously, we showed that a standardized cannabis extract (SCE), isolated Delta(9)-tetrahydrocannabinol (Delta(9)-THC), and even Delta(9)-THC-free SCE inhibited muscarinic agonist-induced epileptiform bursting in rat olfactory cortical brain slices, acting via CB1 receptors. The present work demonstrates that although Delta(9)-THC (1microM) significantly depressed evoked depolarizing postsynaptic potentials (PSPs) in rat olfactory cortex neurones, both SCE and Delta(9)-THC-free SCE significantly potentiated evoked PSPs (all results were fully reversed by the CB1 receptor antagonist SR141716A, 1microM); interestingly, the potentiation by Delta(9)-THC-free SCE was greater than that produced by SCE. On comparing the effects of Delta(9)-THC-free SCE upon evoked PSPs and artificial PSPs (aPSPs; evoked electrotonically following brief intracellular current injection), PSPs were enhanced, whereas aPSPs were unaffected, suggesting that the effect was not due to changes in background input resistance. Similar recordings made using CB1 receptor-deficient knockout mice (CB1(-/-)) and wild-type littermate controls revealed cannabinoid or extract-induced changes in membrane resistance, cell excitability and synaptic transmission in wild-type mice that were similar to those seen in rat neurones, but no effect on these properties were seen in CB1(-/-) cells. It appears that the unknown extract constituent(s) effects over-rode the suppressive effects of Delta(9)-THC on excitatory neurotransmitter release, which may explain some patients' preference for herbal cannabis rather than isolated Delta(9)-THC (due to attenuation of some of the central Delta(9)-THC side effects) and possibly account for the rare incidence of seizures in some individuals taking cannabis recreationally

Medicinal cannabis: is delta9-tetrahydrocannabinol necessary for all its effects?

Cannabis is under clinical investigation to assess its potential for medicinal use, but the question arises as to whether there is any advantage in using cannabis extracts compared with isolated Delta9-trans-tetrahydrocannabinol (Delta9THC), the major psychoactive component. We have compared the effect of a standardized cannabis extract (SCE) with pure Delta9THC, at matched concentrations of Delta9THC, and also with a Delta9THC-free extract (Delta9THC-free SCE), using two cannabinoid-sensitive models, a mouse model of multiple sclerosis (MS), and an in-vitro rat brain slice model of epilepsy. Whilst SCE inhibited spasticity in the mouse model of MS to a comparable level, it caused a more rapid onset of muscle relaxation, and a reduction in the time to maximum effect compared with Delta9THC alone. The Delta9THC-free extract or cannabidiol (CBD) caused no inhibition of spasticity. However, in the in-vitro epilepsy model, in which sustained epileptiform seizures were induced by the muscarinic receptor agonist oxotremorine-M in immature rat piriform cortical brain slices, SCE was a more potent and again more rapidly-acting anticonvulsant than isolated Delta9THC, but in this model, the Delta9THC-free extract also exhibited anticonvulsant activity. Cannabidiol did not inhibit seizures, nor did it modulate the activity of Delta9THC in this model. Therefore, as far as some actions of cannabis were concerned (e.g. antispasticity), Delta9THC was the active constituent, which might be modified by the presence of other components. However, for other effects (e.g. anticonvulsant properties) Delta9THC, although active, might not be necessary for the observed effect. Above all, these results demonstrated that not all of the therapeutic actions of cannabis herb might be due to the Delta9THC content

Potential of Brassic rapa, Cannabis sativa, Helianthus annuus and Zea mays for phytoextraction of heavy metals from calcareous dredged sediment derived soils

Remediation of soil pollution is one of the many current environmental challenges. Anthropogenic activity has resulted in the contamination of extended areas of land, the remediation of which is both invasive and expensive by conventional means. Phytoextraction of heavy metals from contaminated soils has the prospect of being a more economic in situ alternative. In addition, phytoextraction targets ecotoxicologically the most relevant soil fraction of these metals, i.e. the bioavailable fraction. Greenhouse experiments were carried out to evaluate the potential of four high biomass crop species in their potential for phytoextraction of heavy metals, with or without with the use of soil amendments (EDTA or EDDS). A calcareous dredged sediment derived surface soil, with high organic matter and clay content and moderate levels of heavy metal pollution, was used in the experiments. No growth depression was observed in EDTA or EDDS treated pots in comparison to untreated controls. Metal accumulation was considered to be low for phytoextraction purposes, despite the use of chelating agents. The low observed shoot concentrations of heavy metals were attributed to the low phytoavailability of heavy metals in this particular soil substrate. The mobilising effects induced by EDTA in the soil were found to be too long-lived for application as a soil amendment in phytoextraction. Although EDDS was found to be more biodegradable, higher effect half lives were observed than reported in literature or observed in previous experiments. These findings caution against the use of any amendment, biodegradable or otherwise, without proper investigation of its effects and the longevity thereof. (C) 2005 Elsevier Ltd. All rights reserved.

Cannabis constituents modulate ∆9-tetrahydrocannabinol-induced hyperphagia in rats

Rationale The hyperphagic effect of ∆9-tetrahydrocannabinol (∆9THC) in humans and rodents is well known. However, no studies have investigated the importance of ∆9THC composition and any influence other non-∆9THC cannabinoids present in Cannabis sativa may have. We therefore compared the effects of purified ∆9THC, synthetic ∆9THC (dronabinol), and ∆9THC botanical drug substance (∆9THC-BDS), a ∆9THC-rich standardized extract comparable in composition to recreationally used cannabis. Methods Adult male rats were orally dosed with purified ∆9THC, synthetic ∆9THC, or ∆9THC-BDS, matched for ∆9THC content (0.34–2.68 mg/kg). Prior to dosing, subjects were satiated, and food intake was recorded following ∆9THC administration. Data were then analyzed in terms of hourly intake and meal patterns. Results All three ∆9THC substances tested induced significant hyperphagic effects at doses ≥0.67 mg/kg. These effects included increased intake during hour one, a shorter latency to onset of feeding and a greater duration and consumption in the first meal. However, while some differences in vehicle control intakes were observed, there were significant, albeit subtle, differences in pattern of effects between the purified ∆9THC and ∆9THC-BDS. Conclusion All ∆9THC compounds displayed classical ∆9THC effects on feeding, significantly increasing short-term intake whilst decreasing latency to the first meal. We propose that the subtle adjustment to the meal patterns seen between the purified ∆9THC and ∆9THC-BDS are due to non-∆9THC cannabinoids present in ∆9THC-BDS. These compounds and other non-cannabinoids have an emerging and diverse pharmacology and can modulate ∆9THC-induced hyperphagia, making them worth further investigation for their therapeutic potential.

A low delta-9-tetrahydrocannabinol cannabis extract induces hyperphagia in rats

Appetite stimulation via partial agonism of cannabinoid type 1 receptors by Δ9tetrahydrocannabinol (Δ9THC) is well documented and can be modulated by non-Δ9THC phytocannabinoids. Δ9THC concentrations sufficient to elicit hyperphagia induce changes to both appetitive (reduced latency to feed) and consummatory (increased meal one size and duration) behaviours. Here, we show that a cannabis extract containing too little Δ9THC to stimulate appetite can induce hyperphagia solely by increasing appetitive behaviours. Twelve, male Lister hooded rats were presatiated before treatment with a low-Δ9THC cannabis extract (0.5, 1.0, 2.0 and 4.0 mg/kg). Hourly intake and meal pattern data were recorded and analyzed using one-way analyses of variance followed by Bonferroni post-hoc tests. The cannabis extract significantly increased food intake during the first hour of testing (at 4.0 mg/kg) and significantly reduced the latency to feed versus vehicle treatments (at doses ≥1.0 mg/kg). Meal size and duration were unaffected. These results show only the increase in appetitive behaviours, which could be attributed to non-Δ9THC phytocannabinoids in the extract rather than Δ9THC. Although further study is required to determine the constituents responsible for these effects, these results support the presence of non-Δ9THC cannabis constituent(s) that exert a stimulatory effect on appetite and likely lack the detrimental psychoactive effects of Δ9THC.

Cannabis sativa and the endogenous cannabinoid system: therapeutic potential for appetite regulation

The herb Cannabis sativa (C. sativa) has been used in China and on the Indian subcontinent for thousands of years as a medicine. However, since it was brought to the UK and then the rest of the western world in the late 19th century, its use has been a source of controversy. Indeed, its psychotropic side effects are well reported but only relatively recently has scientific endeavour begun to find valuable uses for either the whole plant or its individual components. Here, we discuss evidence describing the endocannabinoid system, its endogenous and exogenous ligands and their varied effects on feeding cycles and meal patterns. Furthermore we also critically consider the mounting evidence which suggests non‐tetrahydrocannabinol phytocannabinoids play a vital role in C. sativa‐induced feeding pattern changes. Indeed, given the wide range of phytocannabinoids present in C. sativa and their equally wide range of intra‐, inter‐ and extra‐cellular mechanisms of action, we demonstrate that non‐Δ9tetrahydrocannabinol phytocannabinoids retain an important and, as yet, untapped clinical potential.

Cannabidivarin-rich cannabis extracts are anticonvulsant in mouse and rat via a CB 1 receptor-independent mechanism

BACKGROUND AND PURPOSE 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 CB 1 receptors. EXPERIMENTAL APPROACH The anticonvulsant profiles of two CBDV BDSs (50–422 mg·kg −1 ) were evaluated in three animal models of acute seizure. Purified CBDV and CBD were also evaluated in an isobolographic study to evaluate potential pharmacological interactions. CBDV BDS effects on motor function were also investigated using static beam and grip strength assays. Binding of CBDV BDSs to cannabinoid CB 1 receptors was evaluated using displacement binding assays. KEY RESULTS CBDV BDSs exerted significant anticonvulsant effects in the pentylenetetrazole (≥100 mg·kg −1 ) and audiogenic seizure models (≥87 mg·kg −1 ), and suppressed pilocarpine-induced convulsions (≥100 mg·kg −1 ). The isobolographic study revealed that the anticonvulsant effects of purified CBDV and CBD were linearly additive when co-administered. Some motor effects of CBDV BDSs were observed on static beam performance; no effects on grip strength were found. The Δ 9 -tetrahydrocannabinol and Δ 9 -tetrahydrocannabivarin content of CBDV BDS accounted for its greater affinity for CB 1 cannabinoid receptors than purified CBDV. CONCLUSIONS AND IMPLICATIONS CBDV BDSs exerted significant anticonvulsant effects in three models of seizure that were not mediated by the CB 1 cannabinoid receptor and were of comparable efficacy with purified CBDV. These findings strongly support the further clinical development of CBDV BDSs for the treatment of epilepsy.

The phytocannabinoid Delta(9)-tetrahydrocannabivarin modulates inhibitory neurotransmission in the cerebellum

Background and purposeThe phytocannabinoid Delta(9)-tetrahydrocannabivarin (Delta(9)-THCV) has been reported to exhibit a diverse pharmacology; here, we investigate functional effects of Delta(9)-THCV, extracted from Cannabis sativa, using electrophysiological techniques to define its mechanism of action in the CNS.Experimental approachEffects of Delta(9)-THCV and synthetic cannabinoid agents on inhibitory neurotransmission at interneurone-Purkinje cell (IN-PC) synapses were correlated with effects on spontaneous PC output using single-cell and multi-electrode array (MEA) electrophysiological recordings respectively, in mouse cerebellar brain slices in vitro.Key resultsThe cannabinoid receptor agonist WIN 55,212-2 (WIN55) decreased miniature inhibitory postsynaptic current (mIPSC) frequency at IN-PC synapses. WIN55-induced inhibition was reversed by Delta(9)-THCV, and also by the CB(1) receptor antagonist AM251; Delta(9)-THCV or AM251 acted to increase mIPSC frequency beyond basal values. When applied alone, Delta(9)-THCV, AM251 or rimonabant increased mIPSC frequency. Pre-incubation with Delta(9)-THCV blocked WIN55-induced inhibition. In MEA recordings, WIN55 increased PC spike firing rate; Delta(9)-THCV and AM251 acted in the opposite direction to decrease spike firing. The effects of Delta(9)-THCV and WIN55 were attenuated by the GABA(A) receptor antagonist bicuculline methiodide.Conclusions and implicationsWe show for the first time that Delta(9)-THCV acts as a functional CB(1) receptor antagonist in the CNS to modulate inhibitory neurotransmission at IN-PC synapses and spontaneous PC output. Delta(9)-THCV- and AM251-induced increases in mIPSC frequency beyond basal levels were consistent with basal CB(1) receptor activity. WIN55-induced increases in PC spike firing rate were consistent with synaptic disinhibition; whilst Delta(9)-THCV- and AM251-induced decreases in spike firing suggest a mechanism of PC inhibition.British Journal of Pharmacology advance online publication, 3 March 2008; doi:10.1038/bjp.2008.57.

Cannabinoids inhibit human keratinocyte proliferation through a non-CB1/CB2 mechanism and have a potential therapeutic value in the treatment of psoriasis

Background: Cannabinoids from cannabis (Cannabis sativa) are anti-inflammatory and have inhibitory effects on the proliferation of a number of tumorigenic cell lines, some of which are mediated via cannabinoid receptors. Cannabinoid (CB) receptors are present in human skin and anandamide, an endogenous CB receptor ligand, inhibits epidermal keratinocyte differentiation. Psoriasis is an inflammatory disease also characterised in part by epidermal keratinocyte hyper-proliferation. Objective: We investigated the plant cannabinoids Delta-9 tetrahydrocannabinol, cannabidiol, cannabinol and cannabigerol for their ability to inhibit the proliferation of a hyper-proliferating human keratinocyte cell line and for any involvement of cannabinoid receptors. Methods: A keratinocyte proliferation assay was used to assess the effect of treatment with cannabinoids. Cell integrity and metabolic competence confirmed using lactate-dehydrogenase and adenosine tri-phosphate assays. To determine the involvement of the receptors, specific agonist and antagonist were used in conjunction with some phytocannabinoids. Western blot and RT-PCR analysis confirmed presence of CB1 and CB2 receptors. Results: The cannabinoids tested all inhibited keratinocyte proliferation in a concentration-dependent manner. The selective CB2 receptor agonists JWH015 and BML190 elicited only partial inhibition, the non-selective CB agonist HU210 produced a concentration-dependent response, the activity of theses agonists were not blocked by either C81 /C82 antagonists. Conclusion: The results indicate that while CB receptors may have a circumstantial role in keratinocyte proliferation, they do not contribute significantly to this process. Our results show that cannabinoids inhibit keratinocyte proliferation, and therefore support a potential role for cannabinoids in the treatment of psoriasis. (c) 2006 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

The phytocannabinoid Δ9-tetrahydrocannabivarin modulates inhibitory neurotransmission in the cerebellum

Background and purpose: The phytocannabinoid Delta(9)-tetrahydrocannabivarin (Delta(9)-THCV) has been reported to exhibit a diverse pharmacology; here, we investigate functional effects of Delta(9)-THCV, extracted from Cannabis sativa, using electrophysiological techniques to define its mechanism of action in the CNS. Experimental approach: Effects of Delta(9)-THCV and synthetic cannabinoid agents on inhibitory neurotransmission at interneurone-Purkinje cell (IN-PC) synapses were correlated with effects on spontaneous PC output using single-cell and multi-electrode array (MEA) electrophysiological recordings respectively, in mouse cerebellar brain slices in vitro. Key results: The cannabinoid receptor agonist WIN 55,212-2 (WIN55) decreased miniature inhibitory postsynaptic current (mIPSC) frequency at IN-PC synapses. WIN55-induced inhibition was reversed by Delta(9)-THCV, and also by the CB1 receptor antagonist AM251; Delta(9)-THCV or AM251 acted to increase mIPSC frequency beyond basal values. When applied alone, Delta(9)-THCV, AM251 or rimonabant increased mIPSC frequency. Pre-incubation with Delta(9)-THCV blocked WIN55-induced inhibition. In MEA recordings, WIN55 increased PC spike firing rate; Delta(9)-THCV and AM251 acted in the opposite direction to decrease spike firing. The effects of Delta(9)-THCV and WIN55 were attenuated by the GABA(A) receptor antagonist bicuculline methiodide. Conclusions and implications: We show for the first time that Delta(9)-THCV acts as a functional CB1 receptor antagonist in the CNS to modulate inhibitory neurotransmission at IN-PC synapses and spontaneous PC output. Delta(9)-THCV- and AM251-induced increases in mIPSC frequency beyond basal levels were consistent with basal CB1 receptor activity. WIN55-induced increases in PC spike firing rate were consistent with synaptic disinhibition; whilst Delta(9)-THCV-and AM251-induced decreases in spike firing suggest a mechanism of PC inhibition.

Phytocannabinoids as novel therapeutic agents in CNS disorders

The Cannabis sativa herb contains over 100 phytocannabinoid (pCB) compounds and has been used for thousands of years for both recreational and medicinal purposes. In the past two decades, characterisation of the body's endogenous cannabinoid (CB) (endocannabinoid, eCB) system (ECS) has highlighted activation of central CB1 receptors by the major pCB, Δ9-tetrahydrocannabinol (Δ9-THC) as the primary mediator of the psychoactive, hyperphagic and some of the potentially therapeutic properties of ingested cannabis. Whilst Δ9-THC is the most prevalent and widely studied pCB, it is also the predominant psychotropic component of cannabis, a property that likely limits its widespread therapeutic use as an isolated agent. In this regard, research focus has recently widened to include other pCBs including cannabidiol (CBD), cannabigerol (CBG), Δ9tetrahydrocannabivarin (Δ9-THCV) and cannabidivarin (CBDV), some of which show potential as therapeutic agents in preclinical models of CNS disease. Moreover, it is becoming evident that these non-Δ9-THC pCBs act at a wide range of pharmacological targets, not solely limited to CB receptors. Disorders that could be targeted include epilepsy, neurodegenerative diseases, affective disorders and the central modulation of feeding behaviour. Here, we review pCB effects in preclinical models of CNS disease and, where available, clinical trial data that support therapeutic effects. Such developments may soon yield the first non-Δ9-THC pCB-based medicines.

Non-D9-tetrahydrocannabinol phytocannabinoids stimulate feeding in rats

Cannabinoid type 1 receptor-mediated appetite stimulation by D9tetrahydrocannabinol (D9THC) is well understood. Recently, it has become apparent that non-D9THC phytocannabinoids could also alter feeding patterns. Here, we show definitively that non-D9THC phytocannabinoids stimulate feeding. Twelve male, Lister-Hooded rats were prefed to satiety prior to administration of a standardized cannabis extract or to either of two mixtures of pure phytocannabinoids (extract analogues) comprising the phytocannabinoids present in the same proportions as the standardized extract (one with and one without D9THC). Hourly intake and meal pattern data were recorded and analysed using two-way analysis of variance followed by one-way analysis of variance and Bonferroni post-hoc tests. Administration of both extract analogues significantly increased feeding behaviours over the period of the test. All three agents increased hour-one intake and meal-one size and decreased the latency to feed, although the zero-D9THC extract analogue did so to a lesser degree than the high-D9THC analogue. Furthermore, only the analogue containing D9THC significantly increased meal duration. The data confirm that at least one non-D9THC phytocannabinoid induces feeding pattern changes in rats, although further trials using individual phytocannabinoids are required to fully understand the observed effects.

Cannabidiol exerts anti-convulsant effects in animal models of temporal lobe and partial seizures

Cannabis sativa has been associated with contradictory effects upon seizure states despite its medicinal use by numerous people with epilepsy. We have recently shown that the phytocannabinoid cannabidiol (CBD) reduces seizure severity and lethality in the well-established in vivo model of pentylenetetrazoleinduced generalised seizures, suggesting that earlier, small-scale clinical trials examining CBD effects in people with epilepsy warrant renewed attention. Here, we report the effects of pure CBD (1, 10 and 100 mg/kg) in two other established rodent seizure models, the acute pilocarpine model of temporal lobe seizure and the penicillin model of partial seizure. Seizure activity was video recorded and scored offline using model-specific seizure severity scales. In the pilocarpine model CBD (all doses) significantly reduced the percentage of animals experiencing the most severe seizures. In the penicillin model, CBD (�10 mg/kg) significantly decreased the percentage mortality as a result of seizures; CBD (all doses) also decreased the percentage of animals experiencing the most severe tonic–clonic seizures. These results extend the anticonvulsant profile of CBD; when combined with a reported absence of psychoactive effects, this evidence strongly supports CBD as a therapeutic candidate for a diverse range of human epilepsies.

Cannabidivarin is anticonvulsant in mouse and rat in vitro and in seizure models

Summary Background and purpose: Phytocannabinoids in Cannabis sativa have diverse pharmacological targets extending beyond cannabinoid receptors and several exert notable anticonvulsant effects. For the first time, we investigated the anticonvulsant profile of the phytocannabinoid cannabidivarin (CBDV) in vitro and in in vivo seizure models. Experimental approach: The effect of CBDV (1-100μM) on epileptiform local field potentials (LFPs) induced in rat hippocampal brain slices by 4-AP application or Mg2+-free conditions was assessed by in vitro multi-electrode array recordings. Additionally, the anticonvulsant profile of CBDV (50-200 mg kg-1) in vivo was investigated in four rodent seizure models: maximal electroshock (mES) and audiogenic seizures in mice, and pentylenetetrazole (PTZ) and pilocarpine-induced seizures in rat. CBDV effects in combination with commonly-used antiepileptic drugs were investigated in rat seizures. Finally, the motor side effect profile of CBDV was investigated using static beam and gripstrength assays. Key results: CDBV significantly attenuated status epilepticus-like epileptiform LFPs induced by 4-AP and Mg2+-free conditions. CBDV had significant anticonvulsant effects in mES (≥100 mg kg-1), audiogenic (≥50 mg kg-1) and PTZ-induced seizures (≥100 mg kg-1). CBDV alone had no effect against pilocarpine-induced seizures, but significantly attenuated these seizures when administered with valproate or phenobarbital at 200 mg kg-1 CBDV. CBDV had no effect on motor function. Conclusions and Implications: These results indicate that CBDV is an effective anticonvulsant across a broad range of seizure models, does not significantly affect normal motor function and therefore merits further investigation in chronic epilepsy models to justify human trials.