Heterozygous deletion of Gpr55 does not affect a hyperthermia-induced seizure, spontaneous seizures or survival in the Scn1a+/- mouse model of Dravet syndrome.

A purified preparation of cannabidiol (CBD), a cannabis constituent, has been approved for the treatment of intractable childhood epilepsies such as Dravet syndrome. Extensive pharmacological characterization of CBD shows activity at numerous molecular targets but its anticonvulsant mechanism(s) of action is yet to be delineated. Many suggest that the anticonvulsant action of CBD is the result of G protein-coupled receptor 55 (GPR55) inhibition. Here we assessed whether Gpr55 contributes to the strain-dependent seizure phenotypes of the Scn1a+/- mouse model of Dravet syndrome. The Scn1a+/- mice on a 129S6/SvEvTac (129) genetic background have no overt phenotype, while those on a [129 x C57BL/6J] F1 background exhibit a severe phenotype that includes hyperthermia-induced seizures, spontaneous seizures and reduced survival. We observed greater Gpr55 transcript expression in the cortex and hippocampus of mice on the seizure-susceptible F1 background compared to those on the seizure-resistant 129 genetic background, suggesting that Gpr55 might be a genetic modifier of Scn1a+/- mice. We examined the effect of heterozygous genetic deletion of Gpr55 and pharmacological inhibition of GPR55 on the seizure phenotypes of F1.Scn1a+/- mice. Heterozygous Gpr55 deletion and inhibition of GPR55 with CID2921524 did not affect the temperature threshold of a thermally-induced seizure in F1.Scn1a+/- mice. Neither was there an effect of heterozygous Gpr55 deletion observed on spontaneous seizure frequency or survival of F1.Scn1a+/- mice. Our results suggest that GPR55 antagonism may not be a suitable anticonvulsant target for Dravet syndrome drug development programs, although future research is needed to provide more definitive conclusions.

Hyperthermia-Induced Seizures Enhance Brain Concentrations of the Endocannabinoid-Related Linoleoyl Glycerols in a Scn1a+/- Mouse Model of Dravet Syndrome.

Introduction: The endocannabinoid system contributes to the homeostatic response to seizure activity in epilepsy, a disease of brain hyperexcitability. Indeed, studies using conventional epilepsy models have shown that seizures increase endocannabinoids in the brain. However, it is unknown whether endocannabinoids and structurally related fatty acid amides and monoacylglycerols are similarly released in response to acute seizures in animal models of drug-resistant epilepsy. Therefore, in this study, we investigated whether a hyperthermia-induced seizure increased concentrations of endocannabinoids and related signaling lipids in the Scn1a+/- mouse model of Dravet syndrome. Materials and Methods: We compared hippocampal concentrations of the major endocannabinoids and related monoglycerols and N-acylethanolamines in wild-type mice, naïve Scn1a+/- mice, and Scn1a+/- mice primed with a single, hyperthermia-induced, generalized tonic-clonic seizure. Samples were collected 5 and 60 min following the seizure and then analyzed with LC-MS/MS. Results: We found that a hyperthermia-induced seizure in Scn1a+/- mice did not affect hippocampal concentrations of the major endocannabinoids, 2-AG and anandamide, or the N-acylethanolamines studied, although the sampling of tissue 5 min postseizure may have been too late to capture any effect on these lipids. Heterozygous deletion of Scn1a alone did not affect these lipid signaling molecules. Notably, however, we found that a hyperthermia-induced seizure significantly increased hippocampal concentrations of the monoacylglycerols, 2-linoleoyl glycerol (2-LG) and 1-linoleoyl glycerol (1-LG), in Scn1a+/- mice. Conclusions: Our results show the unprecedented elevation of the lesser-studied endocannabinoid-related monoacylglycerols, 2-LG and 1-LG, following a hyperthermia-induced seizure in a mouse model of Dravet syndrome. Future research is needed to comprehensively explore the function of these lipid signaling molecules during seizure activity and whether their actions can be exploited to develop new therapeutics.

Cannabis constituents interact at the drug efflux pump BCRP to markedly increase plasma cannabidiolic acid concentrations.

Cannabis is a complex mixture of hundreds of bioactive molecules. This provides the potential for pharmacological interactions between cannabis constituents, a phenomenon referred to as "the entourage effect" by the medicinal cannabis community. We hypothesize that pharmacokinetic interactions between cannabis constituents could substantially alter systemic cannabinoid concentrations. To address this hypothesis we compared pharmacokinetic parameters of cannabinoids administered orally in a cannabis extract to those administered as individual cannabinoids at equivalent doses in mice. Astonishingly, plasma cannabidiolic acid (CBDA) concentrations were 14-times higher following administration in the cannabis extract than when administered as a single molecule. In vitro transwell assays identified CBDA as a substrate of the drug efflux transporter breast cancer resistance protein (BCRP), and that cannabigerol and Δ9-tetrahydrocannabinol inhibited the BCRP-mediated transport of CBDA. Such a cannabinoid-cannabinoid interaction at BCRP transporters located in the intestine would inhibit efflux of CBDA, thus resulting in increased plasma concentrations. Our results suggest that cannabis extracts provide a natural vehicle to substantially enhance plasma CBDA concentrations. Moreover, CBDA might have a more significant contribution to the pharmacological effects of orally administered cannabis extracts than previously thought.

Citalopram and Cannabidiol: In Vitro and In Vivo Evidence of Pharmacokinetic Interactions Relevant to the Treatment of Anxiety Disorders in Young People.

BACKGROUND: Cannabidiol (CBD), a major nonintoxicating constituent of cannabis, exhibits anxiolytic properties in preclinical and human studies and is of interest as a novel intervention for treating anxiety disorders. Existing first-line pharmacotherapies for these disorders include selective serotonin reuptake inhibitor and other antidepressants. Cannabidiol has well-described inhibitory action on cytochrome P450 (CYP450) drug-metabolizing enzymes and significant drug-drug interactions (DDIs) between CBD and various anticonvulsant medications (eg, clobazam) have been described in the treatment of epilepsy. Here, we examined the likelihood of DDIs when CBD is added to medications prescribed in the treatment of anxiety. METHODS: The effect of CBD on CYP450-mediated metabolism of the commonly used antidepressants fluoxetine, sertraline, citalopram, and mirtazapine were examined in vitro. Cannabidiol-citalopram interactions were also examined in vivo in patients (n = 6) with anxiety disorders on stable treatment with citalopram or escitalopram who received ascending daily doses of adjunctive CBD (200-800 mg) over 12 weeks in a recent clinical trial. RESULTS: Cannabidiol minimally affected the metabolism of sertraline, fluoxetine, and mirtazapine in vitro. However, CBD significantly inhibited CYP3A4 and CYP2C19-mediated metabolism of citalopram and its stereoisomer escitalopram at physiologically relevant concentrations, suggesting a possible in vivo DDI. In patients on citalopram or escitalopram, the addition of CBD significantly increased citalopram plasma concentrations, although it was uncertain whether this also increased selective serotonin reuptake inhibitor-mediated adverse events. CONCLUSIONS: Further pharmacokinetic examination of the interaction between CBD and citalopram/escitalopram is clearly warranted, and clinicians should be vigilant around the possibility of treatment-emergent adverse effects when CBD is introduced to patients taking these antidepressants.

Medical cannabis use in the Australian community following introduction of legal access: the 2018-2019 Online Cross-Sectional Cannabis as Medicine Survey (CAMS-18).

BACKGROUND: In 2016, the Australian federal government passed legislation enabling a range of cannabis-based products to be prescribed to patients by registered healthcare professionals. An online survey conducted immediately prior to these legislative changes found that the vast majority of respondents at the time were illicitly sourcing cannabis plant matter, smoking was the preferred route of administration and mental health, chronic pain, and sleep conditions were the most frequently cited reasons for medical cannabis use. This manuscript reports the results of a follow-up survey conducted in 2018-2019, the Cannabis As Medicine Survey (CAMS-18). The goal of this second questionnaire was to examine patterns of use and consumer perspectives regarding medical cannabis use in Australia, 2 years after the introduction of legal access pathways. METHODS: Anonymous online cross-sectional survey with convenience sample, recruited mainly through online media between September 2018 and March 2019. Participants were adults (18 years or over) residing in Australia who reported using a cannabis product for self-identified therapeutic reasons during the preceding 12 months. The survey measured consumer characteristics, indications and patterns of medical cannabis use, routes and frequency of administration, perceived benefits and harms, experiences and preferred models of access to medical cannabis. RESULTS: Data were available for 1388 respondents. The main categories of condition being treated with medical cannabis were pain (36.4%), mental health (32.8%), sleep (9.2%), neurological (5.2%) and cancer (3.8%). Respondents reported using medical cannabis on 15.8 (11.2) days in the past 28, by inhaled (71.4%) or oral (26.5%) routes and spending AUD$82.27 ($101.27) per week. There were high levels of self-reported effectiveness, but also high rates of side effects. There was uncertainty regarding the composition of illicit cannabinoid products and concerns regarding their possible contamination. Few respondents (2.7%) had accessed legally prescribed medical cannabis, with the main perceived barriers being cost, disinterest from the medical profession and stigma regarding cannabis use. CONCLUSIONS: Chronic pain, mental health and sleep remain the main clinical conditions for which consumers report using medical cannabis. Despite 2 years of legal availability, most consumers in Australia reported accessing illicit cannabis products, with uncertainty regarding the quality or composition of cannabis products.

Coadministered cannabidiol and clobazam: Preclinical evidence for both pharmacodynamic and pharmacokinetic interactions.

OBJECTIVE: Cannabidiol (CBD) has been approved by the US Food and Drug Administration (FDA) to treat intractable childhood epilepsies, such as Dravet syndrome and Lennox-Gastaut syndrome. However, the intrinsic anticonvulsant activity of CBD has been questioned due to a pharmacokinetic interaction between CBD and a first-line medication, clobazam. This recognized interaction has led to speculation that the anticonvulsant efficacy of CBD may simply reflect CBD augmenting clobazam exposure. The present study aimed to address the nature of the interaction between CBD and clobazam. METHODS: We examined whether CBD inhibits human CYP3A4 and CYP2C19 mediated metabolism of clobazam and N-desmethylclobazam (N-CLB), respectively, and performed studies assessing the effects of CBD on brain and plasma pharmacokinetics of clobazam in mice. We then used the Scn1a+/- mouse model of Dravet syndrome to examine how CBD and clobazam interact. We compared anticonvulsant effects of CBD-clobazam combination therapy to monotherapy against thermally-induced seizures, spontaneous seizures and mortality in Scn1a+/- mice. In addition, we used Xenopus oocytes expressing γ-aminobutyric acid (GABA)A receptors to investigate the activity of GABAA receptors when treated with CBD and clobazam together. RESULTS: CBD potently inhibited CYP3A4 mediated metabolism of clobazam and CYP2C19 mediated metabolism of N-CLB. Combination CBD-clobazam treatment resulted in greater anticonvulsant efficacy in Scn1a+/- mice, but only when an anticonvulsant dose of CBD was used. It is important to note that a sub-anticonvulsant dose of CBD did not promote greater anticonvulsant effects despite increasing plasma clobazam concentrations. In addition, we delineated a novel pharmacodynamic mechanism where CBD and clobazam together enhanced inhibitory GABAA receptor activation. SIGNIFICANCE: Our study highlights the involvement of both pharmacodynamic and pharmacokinetic interactions between CBD and clobazam that may contribute to its efficacy in Dravet syndrome.

Dark Classics in Chemical Neuroscience: Δ9-Tetrahydrocannabinol.

Cannabis ( Cannabis sativa) is the most widely used illicit drug in the world, with an estimated 192 million users globally. The main psychoactive component of cannabis is (-)- trans-Δ9-tetrahydrocannabinol (Δ9-THC), a compound with a diverse range of pharmacological actions. The unique and distinctive intoxication caused by Δ9-THC primarily reflects partial agonist action at central cannabinoid type 1 (CB1) receptors. Δ9-THC is an approved therapeutic treatment for a range of conditions, including chronic pain, chemotherapy-induced nausea and vomiting, and multiple sclerosis, and is being investigated in indications such as anorexia nervosa, agitation in dementia, and Tourette's syndrome. It is available as a regulated pharmaceutical in products such as Marinol, Sativex, and Namisol as well as in an ever-increasing range of unregistered medicinal and recreational cannabis products. While cannabis is an ancient medicament, contemporary use is embroiled in legal, scientific, and social controversy, much of which relates to the potential hazards and benefits of Δ9-THC itself. Robust contemporary debate surrounds the therapeutic value of Δ9-THC in different diseases, its capacity to produce psychosis and cognitive impairment, and the addictive and "gateway" potential of the drug. This review will provide a profile of the chemistry, pharmacology, and therapeutic uses of Δ9-THC as well as the historical and societal import of this unique, distinctive, and ubiquitous psychoactive substance.

Cannabidiol treatment reduces the motivation to self-administer methamphetamine and methamphetamine-primed relapse in rats.

BACKGROUND: Methamphetamine is an addictive stimulant that can cause many adverse physical, psychological and psychosocial effects. Preliminary evidence shows cannabidiol, a non-intoxicating constituent of the cannabis plant, may have efficacy in treating opioid and nicotine dependence. However, no study has yet examined whether cannabidiol treatment might impact on methamphetamine addiction. AIMS: The current study investigated whether cannabidiol administration reduces the motivation to self-administer methamphetamine and relapse to methamphetamine-seeking behavior following abstinence. METHODS: Thirty-two male Sprague Dawley rats with implanted jugular vein catheters were initially trained to self-administer methamphetamine via lever press during two-hour sessions on a fixed ratio 1 schedule of reinforcement. Rats in experiment 1 ( n=16) then advanced to a progressive ratio reinforcement schedule to examine the effects of cannabidiol (0, 20, 40, and 80 mg/kg intraperitoneal) on motivation to self-administer methamphetamine. Rats in experiment 2 ( n=16) were tested for cannabidiol effects on methamphetamine-primed reinstatement following extinction. RESULTS: Cannabidiol (80 mg/kg, but not 40 mg/kg, or 20 mg/kg) reduced the motivation to self-administer methamphetamine and attenuated methamphetamine-primed relapse to methamphetamine-seeking behavior after extinction. CONCLUSION: This is the first demonstration that cannabidiol can reduce the motivation to seek and consume methamphetamine, and suggests that cannabidiol might be worth trialing as a novel pharmacotherapy for methamphetamine dependence.

Medicinal cannabis in Australia, 2016: the Cannabis as Medicine Survey (CAMS-16).

OBJECTIVE: To explore patterns of cannabis use for medical purposes in Australia immediately prior to the 2016 legislation for frameworks for medical cannabis use. Design, setting: Anonymous online survey with convenience sample, April-October 2016. Participants were recruited through online media and at professional and consumer forums. PARTICIPANTS: Adults (at least 18 years of age) who reported using a cannabis product for self-identified medical or therapeutic reasons during the preceding 12 months. MAIN OUTCOME MEASURES: Consumer characteristics; indications and patterns of medical cannabis use; perceived benefits and harms; views on appropriate availability of medical cannabis. RESULTS: Most of the 1748 participants were men (68.1%) and employed (56.6%), with a mean age of 37.9 years (SD, 13.4 years) and mean reported period of medical cannabis use of 9.8 years (SD, 12.5 years). The most frequent reasons for medical cannabis use were anxiety (50.7%), back pain (50.0%), depression (49.3%), and sleep problems (43.5%). Respondents had used medical cannabis on a mean of 19.9 of the previous 28 days (SD, 10.0 days), spending a mean $68.60 (SD, $85.00) per week, and 83.4% had inhaled the substance. Participants reported high levels of clinical effectiveness and frequent side effects, including drowsiness, ocular irritation, lethargy and memory impairment; 17% met DSM-5 criteria for moderate or severe cannabis use disorder. Many reported harms or concerns related to the illicit status of cannabis. Participants believed that medical cannabis should be integrated into mainstream health care, and that products should be required to meet consistency and safety standards. CONCLUSION: Illicitly sourced cannabis is used to treat a broad range of medical conditions in Australia. Future models of prescribed medical cannabis take consumer patterns of use and demand into consideration.

Analysis of cannabis seizures in NSW, Australia: cannabis potency and cannabinoid profile.

Recent analysis of the cannabinoid content of cannabis plants suggests a shift towards use of high potency plant material with high levels of Δ(9)-tetrahydrocannabinol (THC) and low levels of other phytocannabinoids, particularly cannabidiol (CBD). Use of this type of cannabis is thought by some to predispose to greater adverse outcomes on mental health and fewer therapeutic benefits. Australia has one of the highest per capita rates of cannabis use in the world yet there has been no previous systematic analysis of the cannabis being used. In the present study we examined the cannabinoid content of 206 cannabis samples that had been confiscated by police from recreational users holding 15 g of cannabis or less, under the New South Wales "Cannabis Cautioning" scheme. A further 26 "Known Provenance" samples were analysed that had been seized by police from larger indoor or outdoor cultivation sites rather than from street level users. An HPLC method was used to determine the content of 9 cannabinoids: THC, CBD, cannabigerol (CBG), and their plant-based carboxylic acid precursors THC-A, CBD-A and CBG-A, as well as cannabichromene (CBC), cannabinol (CBN) and tetrahydrocannabivarin (THC-V). The "Cannabis Cautioning" samples showed high mean THC content (THC+THC-A = 14.88%) and low mean CBD content (CBD+CBD-A = 0.14%). A modest level of CBG was detected (CBG+CBG-A = 1.18%) and very low levels of CBC, CBN and THC-V (<0.1%). "Known Provenance" samples showed no significant differences in THC content between those seized from indoor versus outdoor cultivation sites. The present analysis echoes trends reported in other countries towards the use of high potency cannabis with very low CBD content. The implications for public health outcomes and harm reduction strategies are discussed.

Systemically administered oxytocin decreases methamphetamine activation of the subthalamic nucleus and accumbens core and stimulates oxytocinergic neurons in the hypothalamus.

Recent preclinical evidence indicates that the neuropeptide oxytocin may have potential in the treatment of drug dependence and drug withdrawal. Oxytocin reduces methamphetamine self-administration, conditioned place preference and hyperactivity in rodents. However, it is unclear how oxytocin acts in the brain to produce such effects. The present study examined how patterns of neural activation produced by methamphetamine were modified by co-administered oxytocin. Male Sprague-Dawley rats were pretreated with either 2 mg/kg oxytocin (IP) or saline and then injected with either 2 mg/kg methamphetamine (IP) or saline. After injection, locomotor activity was measured for 80 minutes prior to perfusion. As in previous studies, co-administered oxytocin significantly reduced methamphetamine-induced behaviors. Strikingly, oxytocin significantly reduced methamphetamine-induced Fos expression in two regions of the basal ganglia: the subthalamic nucleus and the nucleus accumbens core. The subthalamic nucleus is of particular interest given emerging evidence for this structure in compulsive, addiction-relevant behaviors. When administered alone, oxytocin increased Fos expression in several regions, most notably in the oxytocin-synthesizing neurons of the supraoptic nucleus and paraventricular nucleus of the hypothalamus. This provides new evidence for central actions of peripheral oxytocin and suggests a self-stimulation effect of exogenous oxytocin on its own hypothalamic circuitry. Overall, these results give further insight into the way in which oxytocin might moderate compulsive behaviors and demonstrate the capacity of peripherally administered oxytocin to induce widespread central effects.

Interaction of plant cannabinoids with the multidrug transporter ABCC1 (MRP1).

The ATP-binding cassette (ABC) transporter ABCC1, or multidrug resistance-related protein 1 (MRP1) is implicated in Phase II metabolism and multidrug resistance as it effluxes substrate anticancer drugs. As cannabinoids inhibit two related ABC transporters, P-glycoprotein and ABCG2, here we examined whether they also inhibit ABCC1. Indeed, the cannabinoids enhanced the intracellular accumulation of two ABCC1 substrates, Fluo3 and vincristine, in ovarian carcinoma cells over-expressing ABCC1 (2008/MRP1) with a rank order of potency: cannabidiol>cannabinol>Delta(9)-tetrahydrocannabinol. Cannabinoid inhibition of ABCC1 was confirmed using insect cell membrane MRP1 ATPase assays. These results demonstrate that cannabinoids inhibit ABCC1.