Author Correction: Composition and Use of Cannabis Extracts for Childhood Epilepsy in the Australian Community.

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Composition and Use of Cannabis Extracts for Childhood Epilepsy in the Australian Community.

Recent surveys suggest that many parents are using illicit cannabis extracts in the hope of managing seizures in their children with epilepsy. In the current Australian study we conducted semi-structured interviews with families of children with diverse forms of epilepsy to explore their attitudes towards and experiences with using cannabis extracts. This included current or previous users of cannabis extracts to treat their child's seizures (n = 41 families), and families who had never used (n = 24 families). For those using cannabis, extracts were analysed for cannabinoid content, with specific comparison of samples rated by families as "effective" versus those rated "ineffective". Results showed that children given cannabis extracts tended to have more severe epilepsy historically and had trialled more anticonvulsants than those who had never received cannabis extracts. There was high variability in the cannabinoid content and profile of cannabis extracts rated as "effective", with no clear differences between extracts perceived as "effective" and "ineffective". Contrary to family's expectations, most samples contained low concentrations of cannabidiol, while Δ9-tetrahydrocannabinol was present in nearly every sample. These findings highlight profound variation in the illicit cannabis extracts being currently used in Australia and warrant further investigations into the therapeutic value of cannabinoids in epilepsy.

The direct actions of cannabidiol and 2-arachidonoyl glycerol at GABAA receptors.

Cannabidiol (CBD) is a major non-intoxicating component of cannabis and possesses anti-epileptic, anxiolytic and anti-hyperalgesic properties. The mechanism of action of CBD in producing such effects remains unclear. Despite evidence that some endogenous and synthetic cannabinoids interact with GABAA receptors, no-one has yet investigated the effects of CBD. Here we used two-electrode voltage clamp electrophysiology to compare the actions of CBD with those of the major central endocannabinoid, 2-arachidonoyl glycerol (2-AG) on human recombinant GABAA receptors (synaptic α1-6ßγ2 and extrasynaptic α4ß2δ) expressed on Xenopus oocytes. CBD and 2-AG were positive allosteric modulators at α1-6ßγ2 receptors, with low micromolar potencies. The maximal level of enhancement seen with either CBD or 2-AG were on α2-containing GABAA receptor subtypes, with approximately a 4-fold enhancement of the GABA EC5 evoked current, more than twice the potentiation seen with other α-subunit receptor combinations. Further we observed ß-subunit selectivity, whereby modulatory activity was higher at ß2/ß3 over ß1 subunits. The ß1-subunit homologous mutant ß2(V436T) substantially diminished the efficacy of both drugs to a third of that obtained with wild-type ß2 subunit combinations, but without changing potency. The potency of CBD increased and efficacy preserved in binary α1/α2ß2 receptors indicating that their effects do not involve the classic benzodiazepine site. Exploration of extrasynaptic α4ß2δ receptors revealed that both compounds enhanced GABA EC5 evoked currents at concentrations ranging from 0.01-1µM. Taken together these results reveal a mode of action of CBD on specifically configured GABAA receptors that may be relevant to the anticonvulsant and anxiolytic effects of the compound.

Neural correlates of interactions between cannabidiol and Δ(9) -tetrahydrocannabinol in mice: implications for medical cannabis.

BACKGROUND AND PURPOSE: It has been proposed that medicinal strains of cannabis and therapeutic preparations would be safer with a more balanced concentration ratio of Δ(9) -tetrahydrocannabinol (THC) to cannabidiol (CBD), as CBD reduces the adverse psychotropic effects of THC. However, our understanding of CBD and THC interactions is limited and the brain circuitry mediating interactions between CBD and THC are unknown. The aim of this study was to investigate whether CBD modulated the functional effects and c-Fos expression induced by THC, using a 1:1 dose ratio that approximates therapeutic strains of cannabis and nabiximols. EXPERIMENTAL APPROACH: Male C57BL/6 mice were treated with vehicle, CBD, THC or a combination of CBD and THC (10 mg·kg(-1) i.p. for both cannabinoids) to examine effects on locomotor activity, anxiety-related behaviour, body temperature and brain c-Fos expression (a marker of neuronal activation). KEY RESULTS: CBD potentiated THC-induced locomotor suppression but reduced the hypothermic and anxiogenic effects of THC. CBD alone had no effect on these measures. THC increased brain activation as measured by c-Fos expression in 11 of the 35 brain regions studied. CBD co-administration suppressed THC-induced c-Fos expression in six of these brain regions. This effect was most pronounced in the medial preoptic nucleus and lateral periaqueductal gray. Treatment with CBD alone diminished c-Fos expression only in the central nucleus of the amygdala compared with vehicle. CONCLUSIONS AND IMPLICATIONS: These data confirm that CBD modulated the pharmacological actions of THC and provide new information regarding brain regions involved in the interaction between CBD and THC.

Female neuregulin 1 heterozygous mice require repeated exposure to Δ9-tetrahydrocannabinol to alter sensorimotor gating function.

INTRODUCTION: The schizophrenia susceptibility gene neuregulin 1 (NRG1) confers vulnerability to the neurobehavioural eff ects of cannabinoids differently across sexes. Male but not female Nrg1 heterozygous (HET) mice display facilitation of prepulse inhibition (PPI) to acute Δ9-tetrahydrocannabinol (THC) exposure compared to WT controls. We aim to observe whether repeated administration of THC may overcome the acute insensitivity of female Nrg1 HET mice to THC exposure. METHODS: Female Nrg1 HET mice and WT controls were administered THC daily for 21 days, with PPI and anxiety-related behaviour in the light-dark test (LD) examined on the fi rst and last day of treatment and 21 days after cessation of dosing. RESULTS: Following repeated, but not acute THC exposure, female Nrg1 HET mice displayed THC-induced facilitation of PPI which was not observed in WT mice treated with THC. There were no residual eff ects of THC on PPI in either genotype when assessed 21 days following the final THC dose. An anxiogenic response to THC was evident following repeated, but not acute, administration in the LD test in both genotypes. DISCUSSION: These findings show that the acute insensitivity of female Nrg1 HET mice to THC-induced PPI facilitation may be overcome following repeated THC exposure.

The yin and yang of cannabis-induced psychosis: the actions of Δ(9)-tetrahydrocannabinol and cannabidiol in rodent models of schizophrenia.

The link between cannabis and psychosis has often been debated with polarized views on the topic. There is substantial epidemiological evidence showing that cannabis increases the risk of psychosis, whereas other research suggests that schizophrenia patients self-medicate with the substance. These conflicting accounts may at least be partially explained by the two phytocannabinoids cannabidiol (CBD) and Δ(9)-tetrahydrocannabinol (THC) and their opposing actions on schizophrenia-related symptoms. In the present review we will first focus on how traditional rodent models of schizophrenia have been used to improve our understanding of the propsychotic actions of THC and the antipsychotic actions of CBD. We will also review novel rodent models used to address genetic vulnerability to cannabis-induced schizophrenia and show that specific genes are being uncovered that modulate cannabinoid action (e.g. the schizophrenia susceptibility gene neuregulin 1). We will also review rodent studies that have addressed interactions between THC and CBD. These animal studies underscore great complexity with some studies showing that CBD antagonises the neurobehavioural effects of THC, while others show the opposite, that CBD potentiates the actions of THC. Various mechanisms are put forth to explain these divergent effects such as CBD antagonism at central CB1 receptors or that CBD inhibits proteins that regulate THC disposition and metabolism (e.g. the ABC transporter, P-glycoprotein).

Life cycle assessment of the production and use of polypropylene tree shelters.

A detailed Life Cycle Assessment (LCA) has been conducted for the manufacture, use and disposal of polypropylene tree shelters, which are used to protect young seedlings in the first few years of growth. The LCA was conducted using Simapro software, the Ecoinvent database and ReCiPe assessment methodology. Detailed information on materials, manufacturing, packaging and distribution of shelters was obtained from Tubex Ltd. in South Wales, UK. Various scenarios based on different forest establishment methods, with or without tree shelters were derived and analysed using data from published literature and independent sources. The scenarios included commercial forestry in northern temperate conditions, amenity forest establishment in temperate conditions, and forest establishment in semi-arid conditions. For commercial forestry, a reduction in required seedling production and planting as well as additional time-averaged wood production led to significant benefits with tree shelters, both compared to unprotected and fenced cases. For the amenity forest scenarios, tree shelter use had a net environmental impact, while for semi-arid forestry, the benefits of reduction in water use outweighed shelter production impacts. The current practice of in-situ degradation was compared to collection and disposal and it was found that in-situ degradation was slightly preferable in terms of overall environmental impact. Use of biopolymer-based shelters would improve the environmental performance slightly.

The major plant-derived cannabinoid Δ(9)-tetrahydrocannabinol promotes hypertrophy and macrophage infiltration in adipose tissue.

Synthetic cannabinoid receptor agonists activate lipoprotein lipase and the formation of lipid droplets in cultured adipocytes. Here we extend this work by examining whether Δ(9)-tetrahydrocannabinol (THC), a major plant-derived cannabinoid, increases adipocyte size in vivo. Further, possibly as a consequence of hypertrophy, we hypothesize that THC exposure promotes macrophage infiltration into adipose tissue, an inflammatory state observed in obese individuals. Rats repeatedly exposed to THC in vivo had reduced body weight, fat pad weight, and ingested less food over the drug injection period. However, THC promoted adipocyte hypertrophy that was accompanied by a significant increase in cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C) expression, an enzyme important in packaging triglycerides. We also showed that THC induced macrophage infiltration and increased expression of the inflammatory cytokine tumor necrosis factor alpha (TNF-α) in adipose tissue but did not induce apoptosis as measured by TUNEL staining. That THC increased adipocyte cell size in the absence of greater food intake, body weight and fat provides a unique model to explore mechanisms underlying changes in adipocyte size associated with a mild inflammatory state in fat tissue.

Resilience and reduced c-Fos expression in P2X7 receptor knockout mice exposed to repeated forced swim test.

There is considerable evidence suggesting genetic factors play an important role in the pathophysiology of depression, possibly by increasing susceptibility to repeated environmental stressors. Recent linkage studies have associated a polymorphism of the gene coding for the P2X7 receptor (P2X7R) with both major depressive disorder and bipolar disorder. Here we assessed whether P2X7 deletion affected the behavioural and neural response to repeated stress. P2X7R knockout (P2X7-/-) mice were subjected to the forced swim test for three consecutive days and neuronal activation in response to the third exposure was assessed using c-Fos immunohistochemistry. In addition, anxiety was evaluated in another group of P2X7-/- mice using the elevated plus maze (EPM) and light dark emergence (LDE) tests. Equivalent levels of immobility were observed in P2X7-/- mice and wild-type (WT) mice on the first exposure to forced swim, but much greater immobility was seen in WT mice on second and third exposures. This suggests that P2X7-/- mice exhibit an impaired adaptive coping response to repeated stress. Reinforcing this view, c-Fos expression in the dentate gyrus of the hippocampus and in the basolateral amygdala was seen in WT mice but not P2X7-/- mice following repeated forced swim. In addition, decreased locomotor activity was detected in P2X7-/- mice without any specific effects on anxiety in the LDE test. However, P2X7-/- mice showed greater anxiety-like behaviour in the EPM. These data suggest that the P2X7R may be involved in the adaptive mechanisms elicited by exposure to repeated environmental stressors that leads to the development of depression-like behaviours. This suggests that P2X7R antagonists may be useful therapeutics for the treatment of major depression, possibly by increasing resilience in the face of repeated stress.

Wafer-level self-aligned nano tubular structures and templates for device applications.

A method to fabricate self-aligned nano-scale tubular structures is introduced and investigated. These tubular structures, can be fabricated on wafer-level using common CMOS technologies, are robust and cannot be removed through standard etching or resist strip techniques. This method has shown the potential to be used in different nano device applications since the size of nano-scale tubular structures is adjustable. In addition, these structures can be fabricated as nano-scale templates in advanced device applications.

Reintoxication: the release of fat-stored delta(9)-tetrahydrocannabinol (THC) into blood is enhanced by food deprivation or ACTH exposure.

BACKGROUND AND PURPOSE: Delta(9)-tetrahydrocannabinol (THC), the main psychoactive constituent of cannabis, accumulates in adipose tissue where it is stored for long periods of time. Here we investigated whether conditions that promote lipolysis can liberate THC from adipocytes to yield increased blood levels of THC. EXPERIMENTAL APPROACH: In vitro studies involved freshly isolated rat adipocytes that were incubated with THC before exposure to the lipolytic agent adrenocorticotrophic hormone (ACTH). A complementary in vivo approach examined the effects of both food deprivation and ACTH on blood levels of THC in rats that had been repeatedly injected with THC (10 mg.kg(-1)) for 10 consecutive days. Lipolysis promoted by ACTH or food deprivation was indexed by measurement of glycerol levels. KEY RESULTS: ACTH increased THC levels in the medium of THC-pretreated adipocytes in vitro. ACTH also enhanced THC release from adipocytes in vitro when taken from rats repeatedly pretreated with THC in vivo. Finally, in vivo ACTH exposure and 24 h food deprivation both enhanced the levels of THC and its metabolite, (-)-11-nor-9-carboxy-Delta(9)-tetrahydrocannabinol (THC-COOH) in the blood of rats that had been pre-exposed to repeated THC injections. CONCLUSIONS AND IMPLICATIONS: The present study shows that lipolysis enhances the release of THC from fat stores back into blood. This suggests the likelihood of 'reintoxication' whereby food deprivation or stress may raise blood THC levels in animals chronically exposed to the drug. Further research will need to confirm whether this can lead to functional effects, such as impaired cognitive function or 'flashbacks'.

The multidrug transporter ABCG2 (BCRP) is inhibited by plant-derived cannabinoids.

BACKGROUND AND PURPOSE: Cannabinoids are used therapeutically for the palliation of the adverse side effects associated with cancer chemotherapy. However, cannabinoids also inhibit both the activity and expression of the multidrug transporter, P-glycoprotein in vitro. Here we address the interaction of cannabinol (CBN), cannabidiol (CBD) and delta 9-tetrahydrocannabinol (THC) with the related multidrug transporter, ABCG2. EXPERIMENTAL APPROACH: Cannabinoid inhibition of Abcg2/ABCG2 was assessed using flow cytometric analysis of substrate accumulation and ATPase activity assays. The cytotoxicity and chemosensitization by cannabinoids was determined with cell viability assays. Expression of cannabinoid and vanilloid receptors was assessed using reverse transcriptase polymerase chain reaction, and cannabinoid modulation of ABCG2 expression was examined using immunoblotting. KEY RESULTS: CBN, CBD and THC increased the intracellular accumulation of the Abcg2/ABCG2 substrate, mitoxantrone, in an over-expressing cell line. The THC metabolite, (-)-11-nor-9-carboxy-delta 9-THC was much less potent. The plant cannabinoids inhibited both basal and substrate stimulated ATPase activity of human ABCG2. Cannabinoid cytotoxicity occurred in the absence of known cannabinoid cell surface receptors, and only at concentrations higher than those required for Abcg2/ABCG2 inhibition. Sub-toxic concentrations of the cannabinoids resensitized the overexpressing cell line to the cytotoxic effect of Abcg2/ABCG2 substrates, mitoxantrone and topotecan. This occurred in the absence of any effect on ABCG2 expression. CONCLUSIONS AND IMPLICATIONS: Cannabinoids are novel Abcg2/ABCG2 inhibitors, reversing the Abcg2-mediated multidrug-resistant phenotype in vitro. This finding may have implications for the co-administration of cannabinoids with pharmaceuticals that are ABCG2 substrates.

Heterozygous neuregulin 1 mice display greater baseline and Delta(9)-tetrahydrocannabinol-induced c-Fos expression.

Cannabis use may increase the risk of developing schizophrenia by precipitating the disorder in genetically vulnerable individuals. Neuregulin 1 (NRG1) is a schizophrenia susceptibility gene and mutant mice heterozygous for the transmembrane domain of this gene (Nrg1 HET mice) exhibit a schizophrenia-related phenotype. We have recently shown that Nrg1 HET mice are more sensitive to the behavioral effects of the main psychoactive constituent of cannabis, Delta(9)-tetrahydrocannabinol (THC). In the present study, we examined the effects of THC (10 mg/kg i.p.) on neuronal activity in Nrg1 HET mice and wild type-like (WT) mice using c-Fos immunohistochemistry. In the lateral septum, THC selectively increased c-Fos expression in Nrg1 HET mice with no corresponding effect being observed in WT mice. In addition, THC promoted a greater increase in c-Fos expression in Nrg1 HET mice than WT mice in the central nucleus of the amygdala, the bed nucleus of the stria terminalis and the paraventricular nucleus of the hypothalamus. Consistent with Nrg1 HET mice exhibiting a schizophrenia-related phenotype, these mice expressed greater drug-free levels of c-Fos in two regions thought to be involved in schizophrenia, the shell of the nucleus accumbens and the lateral septum. Interestingly, the effects of genotype on c-Fos expression, drug-free or following THC exposure, were only observed when animals experienced behavioral testing prior to perfusion. This suggests an interaction with stress was necessary for the promotion of these effects. These data provide neurobiological correlates for the enhanced behavioral sensitivity of Nrg1 HET mice to THC and reinforce the existence of cannabinoid-neuregulin 1 interactions in the CNS. This research may enhance our understanding of how genetic factors increase individual vulnerability to schizophrenia and cannabis-induced psychosis.

Cannabis reward: biased towards the fairer sex?

In contrast to drugs such as alcohol, amphetamine and cocaine, cannabis use in humans has proven difficult to model in laboratory animals. Recent breakthrough discoveries of intravenous THC self-administration in rhesus monkeys and self-administration of the synthetic cannabinoid agonist WIN 55,212-2 in rats have allowed new studies of the genetic, neural and environmental determinants of cannabis use. In the present issue of BJP, Fattore and colleagues further demonstrate genetic (strain) differences in WIN 55,212-2 self-administration in rats, with Long Evans (LE) and Lister Hooded (LH), but not Sprague-Dawley, rats self-administering this drug. They then show that female LE and LH rats self-administer more WIN 55,212-2 than male rats. Ovariectomy abolished this sex difference, suggesting a permissive role for oestrogen in cannabis reward. This accompanying Commentary reviews recent progress in animal models of cannabis use and highlights the role of genetic, developmental and endocrine factors in driving cannabis use and dependence.

Prediction of the long-term creep behaviour of hydroxyapatite-filled polyethylmethacrylate bone cements.

The creep behaviour of bone cements based on polyethylmethacrylate, with and without addition of hydroxyapatite filler has been investigated, in order to determine the effect of hydroxyapatite filling and to investigate methods of predicting the long-term creep behaviour from short-term tests. The materials were produced under laboratory conditions and tested in tension in Ringer's solution, as the study was intended to investigate the inherent materials behaviour rather than to simulate realistic conditions. The effects of adding hydroxyapatite were to increase the short-term stiffness and more significantly to decrease the creep rate. Short-term creep tests of up to 10(6) s were conducted at various temperatures, stresses and ageing states. These were then used to investigate various methods of extrapolation to long-term behaviour. The use of time-temperature superposition was found to be useful, though it takes no account of ongoing physical ageing and so gives a significant overestimate of long-term creep strains. Stress-time superposition was less useful and also excludes ageing effects. The use of 'effective time' theory was more successful, but requires a large number of short-term tests. The most effective method was that of the 'integrated time' approach, which required fewer tests yet still gave good correlations with longer-term data.

Heterozygous neuregulin 1 mice are more sensitive to the behavioural effects of Delta9-tetrahydrocannabinol.

RATIONALE: Cannabis use may precipitate schizophrenia especially if the individual has a genetic vulnerability to this mental disorder. Human and animal research indicates that neuregulin 1 (Nrg1) is a susceptibility gene for schizophrenia. OBJECTIVES: The aim of this study was to investigate whether dysfunction in the Nrg1 gene modulates the behavioural effects of Delta(9)-tetrahydrocannabinol (THC), the major psychotropic component of cannabis. MATERIALS AND METHODS: Heterozygous Nrg1 transmembrane-domain knockout mice (Nrg1 HET) were treated with acute THC (0, 5 or 10 mg/kg i.p.) 30 min before being tested using open field (OF), hole board (HB), light-dark (LD), elevated plus maze (EPM), social interaction (SI) and prepulse inhibition (PPI) tests. RESULTS: Nrg1 HET mice showed differences in baseline behaviour with regard to locomotor activity, exploration and anxiety. More importantly, they were more sensitive to the locomotor suppressant actions of THC compared to wild type-like (WT) mice. In addition, Nrg1 HET mice expressed a greater THC-induced enhancement in % PPI than WT mice. The effects of THC on anxiety-related behaviour were task-dependent, with Nrg1 HET mice being more susceptible than WT mice to the anxiogenic effects of THC in LD, but not in the EPM, SI and OF tests. CONCLUSIONS: Nrg1 HET mice were more sensitive to the acute effects of THC in an array of different behaviours including those that model symptoms of schizophrenia. It appears that variation in the schizophrenia-related neuregulin 1 gene alters the sensitivity to the behavioural effects of cannabinoids.

The effects of cannabinoids on P-glycoprotein transport and expression in multidrug resistant cells.

Cannabis is the most widely used illicit drug in the world. Cannabinoids are used therapeutically by some patients as they have analgesic, anti-emetic and appetite stimulant properties which palliate adverse symptoms. Use of these agents in an oncology setting raises the question of whether they act to modulate the effectiveness of concurrently administered anti-cancer drugs. The transporter, P-glycoprotein (P-gp) confers multiple drug resistance (MDR) by effluxing a diverse array of anti-cancer agents. This study was undertaken to examine the effect of cannabinoids on P-gp. Unlike the known P-gp inhibitor, PSC833, short 1h exposure to three plant-derived cannabinoids, cannabinol (CBN), cannabidiol (CBD) and Delta(9)-tetrahydrocannabinol (THC) and the synthetic cannabinoid receptor agonist, WIN55, 212-2 (WIN) did not inhibit the efflux of the P-gp substrate Rhodamine 123 (Rh123) in either a drug-selected human T lymphoblastoid leukaemia cell line (CEM/VLB(100)) or in a mouse fibroblast MDR1 transfected cell line (77.1). However, in CEM/VLB(100) cells, prolonged 72 h exposure to the cannabinoids, THC and CBD, decreased P-gp expression to a similar extent as the flavonoid, curcumin (turmeric). This correlated with an increase in intracellular accumulation of Rh123 and enhanced sensitivity of the cells to the cytotoxic actions of the P-gp substrate, vinblastine. Taken together, these results provide preliminary evidence that cannabinoids do not exacerbate P-gp mediated MDR. Further, plant-derived cannabinoids are moderately effective in reversing MDR in CEM/VLB(100) cells by decreasing P-gp expression.

Staging of abdominal metastases in pancreatic carcinoma by diagnostic laparoscopy and magnetic resonance imaging.

BACKGROUND: Different noninvasive and invasive techniques are available for the detection of abdominal metastases in pancreatic carcinoma. Since small hepatic or peritoneal metastases are a crucial problem which precludes resection, accurate detection is mandatory. We compared laparoscopic staging of abdominal metastases in adenocarcinoma of the pancreas with MRI. METHODS: 55 patients with pancreatic adenocarcinoma had been screened and 49 entered the retrospective study. All patients had undergone MRI of the upper abdomen with concomitant MR-cholangiopancreatography (MRCP) and diagnostic laparoscopy under conscious sedation. Patients without histologic proof of abdominal metastases underwent laparotomy. RESULTS: In 6/10 patients, metastases were correctly detected by laparoscopy, yielding a sensitivity of 60% and a specificity of 92%.MRI predicted metastases in 6/10 patients with 60% sensitivity and 100% specificity. Laparoscopy showed metastases in 3 patients that were missed by MRI, but failed to identify metastases in 3 of 6 patients with metastases on MRI. Though only one patient showed previously undetected metastases during laparotomy, 3 patients with abdominal metastases underwent unnecessary laparotomy due to a lack of histologic proof of malignancy. CONCLUSIONS: Whereas diagnostic laparoscopy is sensitive for the detection of small metastases and offers histologic verification, intrahepatic alterations are not detectable by sole visual inspection.MRI may compensate for this deficiency, but histologic proof of malignancy may be problematic. To date, no definite decision in favor of one of the presented procedures for the staging of abdominal metastases can be given.