A high seizure burden increases several prostaglandin species in the hippocampus of a Scn1a+/- mouse model of Dravet syndrome.

Dravet syndrome is an intractable epilepsy with a high seizure burden that is resistant to current anti-seizure medications. There is evidence that neuroinflammation plays a role in epilepsy and seizures, however few studies have specifically examined neuroinflammation in Dravet syndrome under conditions of a higher seizure burden. Here we used an established genetic mouse model of Dravet syndrome (Scn1a+/- mice), to examine whether a higher seizure burden impacts the number and morphology of microglia in the hippocampus. Moreover, we examined whether a high seizure burden influences classical inflammatory mediators in this brain region. Scn1a+/- mice with a high seizure burden induced by thermal priming displayed a localised reduction in microglial cell density in the granule cell layer and subgranular zone of the dentate gyrus, regions important to postnatal neurogenesis. However, microglial cell number and morphology remained unchanged in other hippocampal subfields. The high seizure burden in Scn1a+/- mice did not affect hippocampal mRNA expression of classical inflammatory mediators such as interleukin 1ß and tumour necrosis factor α, but increased cyclooxygenase 2 (COX-2) expression. We then quantified hippocampal levels of prostanoids that arise from COX-2 mediated metabolism of fatty acids and found that Scn1a+/- mice with a high seizure burden displayed increased hippocampal concentrations of numerous prostaglandins, notably PGF2α, PGE2, PGD2, and 6-K-PGF1A, compared to Scn1a+/- mice with a low seizure burden. In conclusion, a high seizure burden increased hippocampal concentrations of various prostaglandin mediators in a mouse model of Dravet syndrome. Future studies could interrogate the prostaglandin pathways to further better understand their role in the pathophysiology of Dravet syndrome.

MEPIRAPIM-derived synthetic cannabinoids inhibit T-type calcium channels with divergent effects on seizures in rodent models of epilepsy.

Background: T-type Ca2+ channels (Cav3) represent emerging therapeutic targets for a range of neurological disorders, including epilepsy and pain. To aid the development and optimisation of new therapeutics, there is a need to identify novel chemical entities which act at these ion channels. A number of synthetic cannabinoid receptor agonists (SCRAs) have been found to exhibit activity at T-type channels, suggesting that cannabinoids may provide convenient chemical scaffolds on which to design novel Cav3 inhibitors. However, activity at cannabinoid type 1 (CB1) receptors can be problematic because of central and peripheral toxicities associated with potent SCRAs. The putative SCRA MEPIRAPIM and its analogues were recently identified as Cav3 inhibitors with only minimal activity at CB1 receptors, opening the possibility that this scaffold may be exploited to develop novel, selective Cav3 inhibitors. Here we present the pharmacological characterisation of SB2193 and SB2193F, two novel Cav3 inhibitors derived from MEPIRAPIM. Methods: The potency of SB2193 and SB2193F was evaluated in vitro using a fluorometric Ca2+ flux assay and confirmed using whole-cell patch-clamp electrophysiology. In silico docking to the cryo-EM structure of Cav3.1 was also performed to elucidate structural insights into T-type channel inhibition. Next, in vivo pharmacokinetic parameters in mouse brain and plasma were determined using liquid chromatography-mass spectroscopy. Finally, anticonvulsant activity was assayed in established genetic and electrically-induced rodent seizure models. Results: Both MEPIRAPIM derivatives produced potent inhibition of Cav3 channels and were brain penetrant, with SB2193 exhibiting a brain/plasma ratio of 2.7. SB2193 was further examined in mouse seizure models where it acutely protected against 6 Hz-induced seizures. However, SB2193 did not reduce spontaneous seizures in the Scn1a +/- mouse model of Dravet syndrome, nor absence seizures in the Genetic Absence Epilepsy Rat from Strasbourg (GAERS). Surprisingly, SB2193 appeared to increase the incidence and duration of spike-and-wave discharges in GAERS animals over a 4 h recording period. Conclusion: These results show that MEPIRAPIM analogues provide novel chemical scaffolds to advance Cav3 inhibitors against certain seizure types.

Beyond CBD: Inhibitory effects of lesser studied phytocannabinoids on human voltage-gated sodium channels.

Introduction: Cannabis contains cannabidiol (CBD), the main non-psychoactive phytocannabinoid, but also many other phytocannabinoids that have therapeutic potential in the treatment of epilepsy. Indeed, the phytocannabinoids cannabigerolic acid (CBGA), cannabidivarinic acid (CBDVA), cannabichromenic acid (CBCA) and cannabichromene (CBC) have recently been shown to have anti-convulsant effects in a mouse model of Dravet syndrome (DS), an intractable form of epilepsy. Recent studies demonstrate that CBD inhibits voltage-gated sodium channel function, however, whether these other anti-convulsant phytocannabinoids affect these classic epilepsy drug-targets is unknown. Voltage-gated sodium (NaV) channels play a pivotal role in initiation and propagation of the neuronal action potential and NaV1.1, NaV1.2, NaV1.6 and NaV1.7 are associated with the intractable epilepsies and pain conditions. Methods: In this study, using automated-planar patch-clamp technology, we assessed the profile of the phytocannabinoids CBGA, CBDVA, cannabigerol (CBG), CBCA and CBC against these human voltage-gated sodium channels subtypes expressed in mammalian cells and compared the effects to CBD. Results: CBD and CBGA inhibited peak current amplitude in the low micromolar range in a concentration-dependent manner, while CBG, CBCA and CBC revealed only modest inhibition for this subset of sodium channels. CBDVA inhibited NaV1.6 peak currents in the low micromolar range in a concentration-dependent fashion, while only exhibiting modest inhibitory effects on NaV1.1, NaV1.2, and NaV1.7 channels. CBD and CBGA non-selectively inhibited all channel subtypes examined, whereas CBDVA was selective for NaV1.6. In addition, to better understand the mechanism of this inhibition, we examined the biophysical properties of these channels in the presence of each cannabinoid. CBD reduced NaV1.1 and NaV1.7 channel availability by modulating the voltage-dependence of steady-state fast inactivation (SSFI, V0.5 inact), and for NaV1.7 channel conductance was reduced. CBGA also reduced NaV1.1 and NaV1.7 channel availability by shifting the voltage-dependence of activation (V0.5 act) to a more depolarized potential, and for NaV1.7 SSFI was shifted to a more hyperpolarized potential. CBDVA reduced channel availability by modifying conductance, SSFI and recovery from SSFI for all four channels, except for NaV1.2, where V0.5 inact was unaffected. Discussion: Collectively, these data advance our understanding of the molecular actions of lesser studied phytocannabinoids on voltage-gated sodium channel proteins.

Ictal vocalizations in the Scn1a+/- mouse model of Dravet syndrome.

OBJECTIVE: Ictal vocalizations have shown diagnostic utility in epilepsy patients. Audio recordings of seizures have also been used for seizure detection. The present study aimed to determine whether generalized tonic-clonic seizures in the Scn1a+/- mouse model of Dravet syndrome are associated with either audible mouse squeaks or ultrasonic vocalizations. METHODS: Acoustic recordings were captured from group-housed Scn1a+/- mice undergoing video-monitoring to quantify spontaneous seizure frequency. We generated audio clips (n = 129) during a generalized tonic-clonic seizure (GTCS) that included 30 seconds immediately prior to the GTCS (preictal) and 30 seconds following the conclusion of the seizure (postictal). Nonseizure clips (n = 129) were also exported from the acoustic recordings. A blinded reviewer manually reviewed the audio clips, and vocalizations were identified as either an audible (<20 kHz) mouse squeak or ultrasonic (>20 kHz). RESULTS: Spontaneous GTCS in Scn1a+/- mice were associated with a significantly higher number of total vocalizations. The number of audible mouse squeaks was significantly greater with GTCS activity. Nearly all (98%) the seizure clips contained ultrasonic vocalizations, whereas ultrasonic vocalizations were present in only 57% of nonseizure clips. The ultrasonic vocalizations emitted in the seizure clips were at a significantly higher frequency and were nearly twice as long in duration as those emitted in the nonseizure clips. Audible mouse squeaks were primarily emitted during the preictal phase. The greatest number of ultrasonic vocalizations was detected during the ictal phase. SIGNIFICANCE: Our study shows that ictal vocalizations are exhibited by Scn1a+/- mice. Quantitative audio analysis could be developed as a seizure detection tool for the Scn1a+/- mouse model of Dravet syndrome.

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.

The anticonvulsant phytocannabinoids CBGVA and CBDVA inhibit recombinant T-type channels.

Introduction: Cannabidiol (CBD) has been clinically approved for intractable epilepsies, offering hope that novel anticonvulsants in the phytocannabinoid class might be developed. Looking beyond CBD, we have recently reported that a series of biosynthetic precursor molecules found in cannabis display anticonvulsant properties. However, information on the pharmacological activities of these compounds on CNS drug targets is limited. The current study aimed to fill this knowledge gap by investigating whether anticonvulsant phytocannabinoids affect T-type calcium channels, which are known to modulate neuronal excitability, and may be relevant to the anti-seizure effects of this class of compounds. Materials and methods: A fluorescence-based assay was used to screen the ability of the phytocannabinoids to inhibit human T-type calcium channels overexpressed in HEK-293 cells. A subset of compounds was further examined using patch-clamp electrophysiology. Alphascreen technology was used to characterise selected compounds against G-protein coupled-receptor 55 (GPR55) overexpressed in HEK-293 cells, as GPR55 is another target of the phytocannabinoids. Results: A single 10 µM concentration screen in the fluorescence-based assay showed that phytocannabinoids inhibited T-type channels with substantial effects on Cav3.1 and Cav3.2 channels compared to the Cav3.3 channel. The anticonvulsant phytocannabinoids cannabigerovarinic acid (CBGVA) and cannabidivarinic acid (CBDVA) had the greatest magnitudes of effect (≥80% inhibition against Cav3.1 and Cav3.2), so were fully characterized in concentration-response studies. CBGVA and CBDVA had IC50 values of 6 µM and 2 µM on Cav3.1 channels; 2 µM and 11 µM on Cav3.2 channels, respectively. Biophysical studies at Cav3.1 showed that CBGVA caused a hyperpolarisation shift of steady-state inhibition. Both CBGVA and CBDVA had a use-dependent effect and preferentially inhibited Cav3.1 current in a slow inactivated state. CBGVA and CBDVA were also shown to antagonise GPR55. Conclusion and implications: These findings show that CBGVA and CBDVA inhibit T-type calcium channels and GPR55. These compounds should be further investigated to develop novel therapeutics for treating diseases associated with dysfunctional T-type channel activity.

An Examination of the Anti-Cancer Properties of Plant Cannabinoids in Preclinical Models of Mesothelioma.

Mesothelioma is an aggressive cancer with limited treatment options and a poor prognosis. Phytocannabinoids possess anti-tumour and palliative properties in multiple cancers, however their effects in mesothelioma are unknown. We investigated the anti-cancer effects and potential mechanisms of action for several phytocannabinoids in mesothelioma cell lines. A panel of 13 phytocannabinoids inhibited growth of human (MSTO and H2452) and rat (II-45) mesothelioma cells in vitro, and cannabidiol (CBD) and cannabigerol (CBG) were the most potent compounds. Treatment with CBD or CBG resulted in G0/G1 arrest, delayed entry into S phase and induced apoptosis. CBD and CBG also significantly reduced mesothelioma cell migration and invasion. These effects were supported by changes in the expression of genes associated with the cell cycle, proliferation, and cell movement following CBD or CBG treatment. Gene expression levels of CNR1, GPR55, and 5HT1A also increased with CBD or CBG treatment. However, treatment with CBD or CBG in a syngeneic orthotopic rat mesothelioma model was unable to increase survival. Our data show that cannabinoids have anti-cancer effects on mesothelioma cells in vitro and alternatives of drug delivery may be needed to enhance their effects in vivo.

A nutraceutical product, extracted from Cannabis sativa, modulates voltage-gated sodium channel function.

BACKGROUND: Purified cannabidiol (CBD), a non-psychoactive phytocannabinoid, has gained regulatory approval to treat intractable childhood epilepsies. Despite this, artisanal and commercial CBD-dominant hemp-based products continue to be used by epilepsy patients. Notably, the CBD doses used in these latter products are much lower than that found to be effective in reducing seizures in clinical trials with purified CBD. This might be because these CBD-dominant hemp products contain other bioactive compounds, including phytocannabinoids and terpenes, which may exert unique effects on epilepsy-relevant drug targets. Voltage-gated sodium (NaV) channels are vital for initiation of neuronal action potential propagation and genetic mutations in these channels result in epilepsy phenotypes. Recent studies suggest that NaV channels are inhibited by purified CBD. However, the effect of cannabis-based products on the function of NaV channels is unknown. METHODS: Using automated-planar patch-clamp technology, we profile a hemp-derived nutraceutical product (NP) against human NaV1.1-NaV1.8 expressed in mammalian cells to examine effects on the biophysical properties of channel conductance, steady-state fast inactivation and recovery from fast inactivation. RESULTS: NP modifies peak current amplitude of the NaV1.1-NaV1.7 subtypes and has variable effects on the biophysical properties for all channel subtypes tested. NP potently inhibits NaV channels revealing half-maximal inhibitory concentration (IC50) values of between 1.6 and 4.2 µg NP/mL. Purified CBD inhibits NaV1.1, NaV1.2, NaV1.6 and NaV1.7 to reveal IC50 values in the micromolar range. The CBD content of the product equates to IC50 values (93-245 nM), which are at least an order of magnitude lower than purified CBD. Unlike NP, hemp seed oil vehicle alone did not inhibit NaV channels, suggesting that the inhibitory effects of NP are independent of hemp seed oil. CONCLUSIONS: This CBD-dominant NP potently inhibits NaV channels. Future study of the individual elements of NP, including phytocannabinoids and terpenes, may reveal a potent individual component or that its components interact to modulate NaV channels.

In vitro evaluation of the interaction of the cannabis constituents cannabichromene and cannabichromenic acid with ABCG2 and ABCB1 transporters.

Cannabichromene (CBC) and cannabichromenic acid (CBCA) are cannabis constituents currently under evaluation for their therapeutic potential, but their pharmacological properties have not been thoroughly investigated. The most studied ATP-binding cassette (ABC) transporters, ABC subfamily G member 2 (ABCG2) and ABC subfamily B member 1 (ABCB1) limit absorption of substrate drugs in the gut and brain. Moreover, inhibitors of these proteins can lead to clinically significant drug-drug interactions (DDIs). The current study sought to examine whether CBC and CBCA affect ABCB1 and ABCG2 to advance their basic pharmacological characterisation. The plant cannabinoids CBC and CBCA were screened in vitro in a bidirectional transport assay to determine whether they were substrates and/or inhibitors of ABCB1 and ABCG2. Transwell assays with polarized epithelial Madin-Darby Canine Kidney II (MDCK) cells expressing ABCB1 or ABCG2 were used. Samples were measured using liquid chromatography tandem mass spectrometry (LC-MS/MS). CBCA was found to be an ABCB1 substrate, but not an ABCG2 substrate. CBC was not a substrate of either transporter. Neither CBCA nor CBC inhibited ABCB1 transport of prazosin or ABCG2 transport of digoxin. In silico molecular docking suggested CBCA binds ABCB1 in the access tunnel and the central binding pocket. CBC, an agent with anticonvulsant, anti-inflammatory and anti-depressant properties, is not a substrate or inhibitor of ABCB1 or ABCG2, which is favourable to its therapeutic development. CBCA is an ABCB1 substrate in vitro which might contribute to its poor absorption. These findings provide important basic pharmacological data to assist the therapeutic development of these cannabis constituents.

Olivetolic acid, a cannabinoid precursor in Cannabis sativa, but not CBGA methyl ester exhibits a modest anticonvulsant effect in a mouse model of Dravet syndrome.

OBJECTIVE: Cannabigerolic acid (CBGA), a precursor cannabinoid in Cannabis sativa, has recently been found to have anticonvulsant properties in the Scn1a+/- mouse model of Dravet syndrome. Poor brain penetration and chemical instability of CBGA limits its potential as an anticonvulsant therapy. Here, we examined whether CBGA methyl ester, a more stable analogue of CBGA, might have superior pharmacokinetic and anticonvulsant properties. In addition, we examined whether olivetolic acid, the biosynthetic precursor to CBGA with a truncated (des-geranyl) form, might possess minimum structural requirements for anticonvulsant activity. We also examined whether olivetolic acid and CBGA methyl ester retain activity at the epilepsy-relevant drug targets of CBGA: G-protein-coupled receptor 55 (GPR55) and T-type calcium channels. METHODS: The brain and plasma pharmacokinetic profiles of CBGA methyl ester and olivetolic acid were examined following 10 mg/kg intraperitoneal (i.p.) administration in mice (n = 4). The anticonvulsant potential of each was examined in male and female Scn1a+/- mice (n = 17-19) against hyperthermia-induced seizures (10-100 mg/kg, i.p.). CBGA methyl ester and olivetolic acid were also screened in vitro against T-type calcium channels and GPR55 using intracellular calcium and ERK phosphorylation assays, respectively. RESULTS: CBGA methyl ester exhibited relatively limited brain penetration (13%), although somewhat superior to that of 2% for CBGA. No anticonvulsant effects were observed against thermally induced seizures in Scn1a+/- mice. Olivetolic acid also showed poor brain penetration (1%) but had a modest anticonvulsant effect in Scn1a+/- mice increasing the thermally induced seizure temperature threshold by approximately 0.4°C at a dose of 100 mg/kg. Neither CBGA methyl ester nor olivetolic acid displayed pharmacological activity at GPR55 or T-type calcium channels. CONCLUSIONS: Olivetolic acid displayed modest anticonvulsant activity against hyperthermia-induced seizures in the Scn1a+/- mouse model of Dravet syndrome despite poor brain penetration. The effect was, however, comparable to the known anticonvulsant cannabinoid cannabidiol in this model. Future studies could explore the anticonvulsant mechanism(s) of action of olivetolic acid and examine whether its anticonvulsant effect extends to other seizure types.

Evaluation of the Possible Anticonvulsant Effect of Δ9-Tetrahydrocannabinolic Acid in Murine Seizure Models.

Introduction: The cannabinoid Δ9-tetrahydrocannabinolic acid (Δ9-THCA) has long been suggested in review articles and anecdotal reports to be anticonvulsant; yet, there is scant evidence supporting this notion. The objective of this study was to interrogate the anticonvulsant potential of Δ9-THCA in various seizure models-the Scn1a+/- mouse model of Dravet syndrome, the 6-Hz model of psychomotor seizures and the maximal electroshock (MES) model of generalized tonic-clonic seizures. Materials and Methods: We examined the effect of acute Δ9-THCA treatment against hyperthermia-induced seizures, and subchronic treatment on spontaneous seizures and survival in the Scn1a+/- mice. We also studied the effect of acute Δ9-THCA treatment on the critical current thresholds in the 6-Hz and MES tests using outbred Swiss mice. Highly purified Δ9-THCA was used in the studies or a mixture of Δ9-THCA and Δ9-THC. Results: We observed mixed anticonvulsant and proconvulsant effects of Δ9-THCA across the seizure models. Highly pure Δ9-THCA did not affect hyperthermia-induced seizures in Scn1a+/- mice. A Δ9-THCA/Δ9-THC mixture was anticonvulsant in the 6-Hz threshold test, but purified Δ9-THCA and Δ9-THC had no effect. Conversely, both Δ9-THCA and Δ9-THC administered individually were proconvulsant in the MES threshold test but had no effect when administered as a Δ9-THCA/Δ9-THC mixture. The Δ9-THCA/Δ9-THC mixture, however, increased spontaneous seizure severity and increased mortality of Scn1a+/- mice. Discussion: The anticonvulsant profile of Δ9-THCA was variable depending on the seizure model used and presence of Δ9-THC. Because of the unstable nature of Δ9-THCA, further exploration of Δ9-THCA through formal anticonvulsant drug development is problematic without stabilization. Future studies may better focus on determining the mechanisms by which combined Δ9-THCA and Δ9-THC alters seizure thresholds, as this may uncover novel targets for the control of refractory partial seizures.

In Vitro Screening of Three Commercial Cannabis-Based Products on ATP-Binding Cassette and Solute-Carrier Transporter Function.

Introduction: Legalization of medicinal cannabis around the world has led to an increase in the use of commercial cannabis-based products in the community. These cannabis-based products are being used in combination with conventional drugs to treat a variety of health conditions. Moreover, recreational cannabis-based products may be used in combination with other drugs. In this setting, there is increased potential for drug-drug interactions (DDIs) involving commercial cannabis-based products. Since DDIs can lead to serious adverse events, drug regulatory bodies require that every investigational drug be evaluated for DDI potential at metabolic enzymes and transporters. However, this seldom occurs for cannabis-based products due to legislation in many jurisdictions allowing a direct pathway to market. This study aimed to examine the inhibitory potential of three commercially available cannabis-based products at human ATP-binding cassette (ABC) and solute-carrier (SLC) transporters. Materials and Methods: Three commercial cannabis-based products (Spectrum Yellow™, Tweed Argyle, and Spectrum Red™) that contain differing concentrations of cannabidiol (CBD) and Δ9-tetrahydrocannabinol (Δ9-THC) were evaluated for DDI potential at 12 drug transporters. HEK293 cells or vesicles expressing human ABC transporters (ABCB1, ABCC2, ABCG2, or ABCB11) and SLC transporters (SLC22A1, SLC22A2, SLC22A6, SLC22A8, SLCO1B1, SLCO1B3, SLC47A1, and SLC47A2) were used to measure transporter function. Results: Spectrum Yellow and Tweed Argyle inhibited ABCG2 transporter function. The IC50 value of Spectrum Yellow based on CBD and Δ9-THC content was 4.5 µM for CBD and 0.20 µM for Δ9-THC, and the IC50 value of Tweed Argyle was 9.3 µM for CBD and 6.0 µM for Δ9-THC. Tweed Argyle also inhibited ABCB11 transporter function with an IC50 value of 11.9 µM for CBD and 7.7 µM for Δ9-THC. SLC22A6, SLC22A1, SLC22A2, SLCO1B1, and SLCO1B3 transporter functions were modestly inhibited by high concentrations of the cannabis-based products. The three cannabis-based products did not inhibit ABCB1, ABCC2, SLC47A1, SLC47A2, or SLC22A8 transporters. Discussion: Novel findings were that the cannabis-based products inhibited ABCB11, SLC22A6, SLC22A1, SLC22A2, SLCO1B1, and SLCO1B3 (although modestly in most instances). Spectrum Yellow and Tweed Argyle potently inhibited ABCG2, and future in vivo DDI studies could be conducted to assess whether cannabis products affect the pharmacokinetics of medications that are ABCG2 substrates.

The endocannabinoid system impacts seizures in a mouse model of Dravet syndrome.

Dravet syndrome is a catastrophic childhood epilepsy with multiple seizure types that are refractory to treatment. The endocannabinoid system regulates neuronal excitability so a deficit in endocannabinoid signaling could lead to hyperexcitability and seizures. Thus, we sought to determine whether a deficiency in the endocannabinoid system might contribute to seizure phenotypes in a mouse model of Dravet syndrome and whether enhancing endocannabinoid tone is anticonvulsant. Scn1a+/- mice model the clinical features of Dravet syndrome: hyperthermia-induced seizures, spontaneous seizures and reduced survival. We examined whether Scn1a+/- mice exhibit deficits in the endocannabinoid system by measuring brain cannabinoid receptor expression and endocannabinoid concentrations. Next, we determined whether pharmacologically enhanced endocannabinoid tone was anticonvulsant in Scn1a+/- mice. We used GAT229, a positive allosteric modulator of the cannabinoid (CB1) receptor, and ABX-1431, a compound that inhibits the degradation of the endocannabinoid 2-arachidonoylglycerol (2-AG). The Scn1a+/- phenotype is strain-dependent with mice on a 129S6/SvEvTac (129) genetic background having no overt phenotype and those on an F1 (129S6/SvEvTac x C57BL/6J) background exhibiting a severe epilepsy phenotype. We observed lower brain cannabinoid CB1 receptor expression in the seizure-susceptible F1 compared to seizure-resistant 129 strain, suggesting an endocannabinoid deficiency might contribute to seizure susceptibility. GAT229 and ABX-1431 were anticonvulsant against hyperthermia-induced seizures. However, subchronic ABX1431 treatment increased spontaneous seizure frequency despite reducing seizure severity. Cnr1 is a putative genetic modifier of epilepsy in the Scn1a+/- mouse model of Dravet syndrome. Compounds that increase endocannabinoid tone could be developed as novel treatments for Dravet syndrome.

Cannabigerolic acid, a major biosynthetic precursor molecule in cannabis, exhibits divergent effects on seizures in mouse models of epilepsy.

BACKGROUND AND PURPOSE: Cannabis has been used to treat epilepsy for millennia, with such use validated by regulatory approval of cannabidiol (CBD) for Dravet syndrome. Unregulated artisanal cannabis-based products used to treat children with intractable epilepsies often contain relatively low doses of CBD but are enriched in other phytocannabinoids. This raises the possibility that other cannabis constituents might have anticonvulsant properties. EXPERIMENTAL APPROACH: We used the Scn1a+/- mouse model of Dravet syndrome to investigate the cannabis plant for phytocannabinoids with anticonvulsant effects against hyperthermia-induced seizures. The most promising, cannabigerolic acid (CBGA), was further examined against spontaneous seizures and survival in Scn1a+/- mice and in electroshock seizure models. Pharmacological effects of CBGA were surveyed across multiple drug targets. KEY RESULTS: The initial screen identified three phytocannabinoids with novel anticonvulsant properties: CBGA, cannabidivarinic acid (CBDVA) and cannabigerovarinic acid (CBGVA). CBGA was most potent and potentiated the anticonvulsant effects of clobazam against hyperthermia-induced and spontaneous seizures, and was anticonvulsant in the MES threshold test. However, CBGA was proconvulsant in the 6-Hz threshold test and a high dose increased spontaneous seizure frequency in Scn1a+/- mice. CBGA was found to interact with numerous epilepsy-relevant targets including GPR55, TRPV1 channels and GABAA receptors. CONCLUSION AND IMPLICATIONS: These results suggest that CBGA, CBDVA and CBGVA may contribute to the effects of cannabis-based products in childhood epilepsy. Although these phytocannabinoids have anticonvulsant potential and could be lead compounds for drug development programmes, several liabilities would need to be overcome before CBD is superseded by another in this class.

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.

Cannabinoid Interactions with Cytochrome P450 Drug Metabolism: a Full-Spectrum Characterization.

Medicinal cannabis use has increased exponentially with widespread legalization around the world. Cannabis-based products are being used for numerous health conditions, often in conjunction with prescribed medications. The risk of clinically significant drug-drug interactions (DDIs) increases in this setting of polypharmacy, prompting concern among health care providers. Serious adverse events can result from DDIs, specifically those affecting CYP-mediated drug metabolism. Both cannabidiol (CBD) and Δ9-tetrahydrocannabinol (Δ9-THC), major constituents of cannabis, potently inhibit CYPs. Cannabis-based products contain an array of cannabinoids, many of which have limited data available regarding potential DDIs. This study assessed the inhibitory potential of 12 cannabinoids against CYP-mediated drug metabolism to predict the likelihood of clinically significant DDIs between cannabis-based therapies and conventional medications. Supersomes™ were used to screen the inhibitory potential of cannabinoids in vitro. Twelve cannabinoids were evaluated at the predominant drug-metabolizing isoforms: CYP3A4, CYP2D6, CYP2C9, CYP1A2, CYP2B6, and CYP2C19. The cannabinoids exhibited varied effects and potencies across the CYP isoforms. CYP2C9-mediated metabolism was inhibited by nearly all the cannabinoids with estimated Ki values of 0.2-3.2 µM. Most of the cannabinoids inhibited CYP2C19, whereas CYP2D6, CYP3A4, and CYP2B6 were either not affected or only partially inhibited by the cannabinoids. Effects of the cannabinoids on CYP2D6, CYP1A2, CYP2B6, and CYP3A4 metabolism were limited so in vivo DDIs mediated by these isoforms would not be predicted. CYP2C9-mediated metabolism was inhibited by cannabinoids at clinically relevant concentrations. In vivo DDI studies may be justified for CYP2C9 substrates with a narrow therapeutic index.

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.

The Heat Sensing Trpv1 Receptor Is Not a Viable Anticonvulsant Drug Target in the Scn1a +/- Mouse Model of Dravet Syndrome.

Cannabidiol has been approved for the treatment of drug-resistant childhood epilepsies including Dravet syndrome (DS). Although the mechanism of anticonvulsant action of cannabidiol is unknown, emerging data suggests involvement of the transient receptor potential cation channel subfamily V member 1 (Trpv1). Pharmacological and genetic studies in conventional seizure models suggest Trpv1 is a novel anticonvulsant target. However, whether targeting Trpv1 is anticonvulsant in animal models of drug-resistant epilepsies is not known. Thus, we examined whether Trpv1 affects the epilepsy phenotype of the F1.Scn1a +/- mouse model of DS. We found that cortical Trpv1 mRNA expression was increased in seizure susceptible F1.Scn1a +/- mice with a hybrid genetic background compared to seizure resistant 129.Scn1a +/- mice isogenic on 129S6/SvEvTac background, suggesting Trpv1 could be a genetic modifier. Previous studies show functional loss of Trpv1 is anticonvulsant. However, Trpv1 selective antagonist SB-705498 did not affect hyperthermia-induced seizure threshold, frequency of spontaneous seizures or survival of F1.Scn1a +/- mice. Surprisingly, Trpv1 deletion had both pro- and anti-seizure effects. Trpv1 deletion did not affect hyperthermia-induced seizure temperature thresholds of F1.Scn1a +/- ; Trpv1 +/- at P14-16 but was proconvulsant at P18 as it reduced seizure temperature thresholds. Conversely, Trpv1 deletion did not alter the frequency of spontaneous seizures but reduced their severity. These results suggest that Trpv1 is a modest genetic modifier of spontaneous seizure severity in the F1.Scn1a +/- model of DS. However, the opposing pro- and anti-seizure effects of Trpv1 deletion and the lack of effects of Trpv1 inhibition suggest that Trpv1 is unlikely a viable anticonvulsant drug target in DS.