Reducing neuronal excitability

How cannabinoids reduce neuronal excitability is not exactly known but the following options are possible:

• Neuronal activity induces a Cl^- influx through 2AG/Anandamide and CB2 (den Boon et al., 2014).
• Anandamide reduces burst-firing in neurons (Evans et al., 2008).
• cannabinoids reduce the number of neurotransmitter vesicles available for fusion (García-Morales et al., 2015).
• In human neuroblastoma cells (SH-SY5Y) and mouse cortical neurons CBD and CBG both blocked sodium channels Nav1.1, 1.2 and 1.5 (Hill et al., 2014). Interestingly, CBD but not CBG protected against pentyleneterzole (PTZ)-induced seizures in rat, suggesting that the anti-convulsant effect of CBD is not just through blocking sodium channels/blocking excitability.
• In HEK293 cells human CBD (and THC) were found to stabilize sodium channels in their inactive state. CBD was more effective at depolarized potentials thus particularly preventing over-excitation. Similar results were obtained in iPSC-neurons (Ghovanloo et al., 2018). IC₅₀ values were:
  
  • hNav1.1 – CBD: 2.0±0.1 μM
  • hNav1.2 – CBD: 2.9±0.1 μM
  • hNav1.3 – CBD: 3.3±0.1 μM
  • hNav1.4 – CBD: 1.9±0.1 μM
  • hNav1.5 – CBD: 3.8±0.2 μM
  • hNav1.6 – CBD: 3.0±0.1 μM
  • hNav1.7 – CBD: 2.9±0.1 μM
  • hNav1.2 – THC: 2.4±0.1 μM
  • mNav1.6 – CBD: 2.4±0.1 μM
• Both phytocannabinoids and endocannabinoids tend to reduce neuronal activity-dependent neurotransmitter release. This occurs both in excitatory synapses (Depolarisation-induced suppression of excitation/DSE) and inhibitory synapses (DSI). Although DSI tends to be more prominent in the brain, the combined effect of DSI and DSE often helps to suppress seizures (Alger, 2014).

Reducing neuronal synchronization

In rats, THC and other synthetic CB1 agonists, reduces synchronous firing of hippocampal principal neurons, suggesting a direct role for THC in seizure prevention (Goonawardena et al., 2011). Similarly, CB1 activation decreases synchrony in cortical neurons (Sales-Carbonell et al., 2013) suggesting THC (like substances) can be used to suppress seizures.

In healthy human volunteers, 10 mg oral THCV reduced functional network connectivity in the brain (measured by fMRI)(Rzepa et al., 2015). Although this does not prove anything in itself, it does support the idea that cannabinoids can reduce network synchronization.

In heterologous cells (HEK293), THC and CBD were found to inhibit T-type calcium channels with an IC50 of approximately 1μM (Ross et al., 2008). THC-mediated inhibition was frequency dependent where CBD-mediated inhibition was not. As T-type calcium channels function in thalamus-mediated synchronization of brain regions and are implicated in various types of epilepsy, THC and CBD are likely to suppress seizure generation.

Phytocannabinoids

Preclinical studies shows that, in addition to CBD, CBDV and THC also have anti-convulsant properties (Hill et al., 2013; Wallace et al., 2001).

CBD and CBG can both block Na_V 1.1, 1.2 and 1.5 at micromolar concentrations. However, neither CBD nog CBG had anti-convulsant effect in PTZ-induced seizures in mice at concentrations between 50 and 200 mg/kg, suggesting sodium channel inhibition is presumably not the main anti-convulsive action of CBD (or CBG) (Hill et al., 2014).

In the rat PTZ model of epilepsy 0.25 mg/kg THCV significantly reduced seizure incidence. Similarly, prior bath application of 10 μM THCV or acute application of > 20 μM THCV prevented complex burst firing and depolarizing shifts in slice experiments (Hill et al., 2010).

In a mouse model of epilepsy (Maximal Electro Shock), the following cannabinoids were found to be anti-convulsive (effective dose/ED50)(referenced within: Devinsky et al., 2014):

• CBD 120 mg/kg
• Δ9THC 100 mg/kg
• 11-OH-Δ9THC 14 mg/kg (This primary metabolite of THC is mostly produced in the liver and appears to be more effective in suppressing seizures than THC. Therefore, oral ingestion might prove to be a better route of application than sublingual application for instance, but this remains to be investigated).
• 8β-OH-Δ9THC 100 mg/kg
• Δ9THCA 200-400 mg/kg
• Δ8THC 80 mg/kg
• CBN 230 mg/kg
• Δ9α/β-OH-hexahydro-CBN 100 mg/kg

Apart from that the doses reported above are incredibly high, it does provide a proof of principle that many cannabinoids exert anti-convulsive effects.

In another Maximal Electro Shock experiment THC was anti-convulsive at an ED50 of 42 mg/kg. This was similar to the anti-convulsive effect of CB1 agonist WIN55,212-2 (ED50 47 mg/kg) and blocked by CB1 antagonist SR141716a (AD 2.5 mg/kg) suggesting a central role for CB1. CBD was also anti-convulsive (ED50 80 mg/kg) but in a CB1 independent way (Wallace et al., 2001).

In an epilepsy model comparison the anti-epileptic effect of i.p. CBD was tested (Klein et al., 2017):

• Acute mouse 6 Hz 44 mA: ED50 164 mg/kg
• Acute mouse MES: ED50 83.5 mg/kg
• Acute rat MES: ED50 88.8 mg/kg
• Chronic mouse corneal kindling: ED50 119 mg/kg
• Chronic rat amygdala kindling: no effect up to 300 mg/kg

Although THC/CB1 agonism is generally regarded as anti-convulsive it should be noted that in one study i.p. administration of 10 mg/kg THC or 2.5 mg/kg JWH-018 induced seizures in mice via activation of CB1 (Malyshevskaya et al., 2017).

In a comparative study, prolonged administration of THC-rich cannabis extracts caused spontaneous seizures in rats, but not dogs, suggesting inter-species differences (Whalley et al., 2018).

Terpenes

In the mouse PTZ model of epilepsy, 100 mg/kg β-caryophyllene increased seizure latency, suggesting an anti-epileptic effect (Oliveira et al., 2016).

In the rat PTZ model of epilepsy, 0.8 ml/kg of Cinnamosa madagascariensis essential oil completely blocked PTZ-induced convulsions (Rakotosaona et al., 2017). Linalool, limonene and myrcene are the main constituents of Cinnamosa madagascariensis essential oil and are therefore candidates for the treatment of epilepsy.

ECS involvement

As discussed above, 2AG and Anandamide can reduce neuronal excitability and drive DSI/DSE (Alger, 2014; den Boon et al., 2014; Evans et al., 2008).

In the adult PTZ model of epilepsy, extracellular accumulation of 2AG and Anandamide appeared anti-convulsive in a CB1-dependent manner. Intracellular Anandamide accumulation, however, appeared pro-convulsive in a TRPV1-dependent manner (Zareie et al., 2018).

In the rat PTZ model of epilepsy, PEA increased the latency to seizures and attenuated seizures. This effect was partially, but not entirely dependent on CB1 and CB2 receptors (Aghaei et al., 2015).

In the kainate mouse model of epilepsy, subchronic, but not acute, PEA administration reduced seizure intensity and neuronal damage (Post et al., 2018).

Audiogenic seizures in DBA/2 mice were reduced by i.p. PEA, mainly in a PPARα dependent way. CB1 agonists ACEA and WIN55,212-2 were also effective. PEA, ACEA and WIN55,212-2 also potentiated the efficacy of anti-epileptic drugs carbamazepine, diazepam, felbamate, gabapentin, phenobarbital, topiramate and valproate. In addition PEA also potentiated oxcabazepine and lamotrigine but not leviteracetam or phenytoin (Citraro et al., 2016).

In the 4-AP rat slice model of epilepsy Anandamide reuptake inhibitor AM404 and TRPV1 antagonist capsazepine suppressed seizure activity (Nazıroğlu et al., 2018).

In the rat PTZ model of epilepsy, acetaminophen/paracetamol/AM404 showed dose-dependent anti-convulsant activity which was suppressed by TRPV1 antagonists capsazepine and AMG9810 (Suemaru et al., 2018). The conflicting results of TRPV1 agonism and antagonism on seizure susceptibility suggest a complex role for TRPV1 in controlling neuronal excitability.

In mice, stimulating CB1 receptors (ACEA) or blocking TRPV1 receptors (capsazepine) protected against PTZ-induced seizures (Naderi et al., 2015). Interestingly, co-administration of both compounds attenuated the anti-convulsive effect, suggesting an interaction between CB1 and TRPV1 mediated signaling.

In the mouse pilocarpine model of epilepsy, CB1 agonist ACEA (10 mg/kg) increased the generation of new neurons where the classical anti-epileptic drug valproate did not (Andres-Mach et al., 2015, 2017). This may contribute to the anti-epileptic effect of ACEA.

In rats, the synthetic CB1 agonist WIN 55-212-2 was protective against the development of epilepsy when administered after an episode of pilocarpine-induced status epilepticus (Di Maio et al., 2014; Suleymanova et al., 2016). Sub-acute treatment with WIN 55-212-2 for 15 days dramatically reduced the frequency of spontaneous seizures, their duration and intensity and the incidence of neuronal oxidative damage.

In rats, WIN 55-212-2 delayed the onset of audiogenic epilepsy by two weeks suggesting a preventive effect of cannabinoids on the development of epilepsy as well as a curative effect (Vinogradova and van Rijn, 2015).

In P10 rat pups, CB1 agonism (ACEA) and CB1/2 agonism (WIN55,212-2) were anti-convulsant. CB1 and CB2 antagonism were pro-convulsant, while GPR55 agonism was ineffective. Unlike P10 pups and adults, CB1 agonism was ineffective in P20 pups suggesting variable efficacy of CB1 agonism in different stages of life (Huizenga et al., 2017).

In the rat PTZ and Maximal Electro Shock models of epilepsy CB1 agonist ACEA was anti-convulsant. Co-administration of BK channel antagonist paxilline attenuated the anti-convulsant effect of ACEA suggesting the involvement of BK channels in the action mechanism of ACEA (Asaadi et al., 2017).

In rats that were chronically treated with CP 55,940 (CB1/2 full agonist, GPR55 antagonist) during adolescence, PTZ-induced seizures in adulthood showed higher lethality suggesting a maladaptive effect of adolescent cannabinoid intake (Spring et al., 2015).

In the mouse maximal electroshock seizure threshold model of epilepsy various combinations of Anandamide reuptake inhibitors, FAAH inhibitors, CB1 and TRPV1 agonists showed anticonvulsant effects (Tutka et al., 2017). In descending order:

• Arvanil: Anandamide reuptake inhibitor and CB1 agonist
• Olvanil: CB1 and TRPV1 agonist
• AM1172: Anandamide reuptake inhibitor
• LY2183240: Anandamide reuptake inhibitor and FAAH inhibitor

In a mouse electroshock model of epilepsy 5 mg/kg WIN55,212-2 significantly potentiated the anti-convulsant effect of gabapentin and leviteracetam but not lacosamide, oxcabarzepine, pregabalin or tiagabine (Florek-Luszczki et al., 2014, 2015).

In a guinea pig kainate model of epilepsy AM404 (TRPV1 agonist and endocannabinoid reuptake inhibitor) and URB597 (FAAH inhibitor) were anti-convulsive whereas AM251 (CB1 antagonist) was not (Shubina et al., 2015). Inhibiting endocannabinoid reuptake or degradation also prevented hippocampal circuit remodeling normally seen during epileptogenesis (Shubina et al., 2017).

In mice FAAH inhibitor URB597 and CB1 agonist ACEA reduced cocaine-induced seizures whereas CB1 antagonist AM251 prevented this anti-epileptic effect (Vilela et al., 2015).

Interestingly, in a rat traumatic brain injury model CB1 antagonist SR141716a prevented long-term hyperexcitability, which is at least apparently at odds with a protective effect of CB1 agonism in the development of epilepsy (Wang et al., 2016).

In HEK293T cells, several epilepsy-associated sodium channel mutants were analysed (Nav1.1 arg1648his and asn1788lys and Nav1.6 asn1768asp and leu1331val). The Nav1.6, but not Nav1.1 mutants, showed increased resurgent sodium currents, which may increase neuronal excitability and thus cause the epileptic phenotype. CBD was found to specifically reduce resurgent sodium currents and action potential firing which may help explain the anti-epileptic effect of CBD (Patel et al., 2016).

In lymphocytes from Dravet syndrome patients, CBD targets within the ECS were analyzed. The voltage-dependent calcium channel alpha1h (Cav3.2) and CB2 were up-regulated (Rubio et al., 2016), suggesting their involvement in the disease or the bodies’ adaptive response to the disease.

In Xenopus oocytes expressing recombinant human GABA_A receptors the effect of CBD and 2AG was tested (Bakas et al., 2017). 2AG and CBD:

• were positive allosteric modulators at α1-6βγ2 receptors
• enhanced GABAergic current 4-fold at α2-containing receptors
• enhanced GABAergic current at concentrations ranging from 0.01 to 1 μM at α4β2δ receptors

In the mouse PTZ model of epilepsy 60 mg/kg CBD attenuated seizures. This attenuation was prevented by CB1, CB2 and TRPV1 antagonists suggesting the anti-epileptic effect has complex pharmacology (Vilela et al., 2017).

In patients with Dravet syndrome (SCN1A/Nav1.1 mutations) additional CACNA1A/Cav2.1 mutations produced more seizures, earlier onset of seizures and more prolonged seizures, suggesting a role for Cav2.1 in seizure development (Ohmori et al., 2013).

In a genetic mouse model of Dravet syndrome, CBD was found to decrease the duration and severity of spontaneous and thermally generated seizures and improve autistic-like social behavior. This effect was associated with restoration of interneuron excitability and was occluded by a GPR55 antagonist suggesting a beneficial CBD-GPR55 interaction (Kaplan et al., 2017).

In the rat kainate model of temporal lobe epilepsy 100 mg/kg (in vivo) or 10 μM (slices) CBD restored normal hippocampal interneuron excitability and prevented PV and CCK positive interneuron death (Khan et al., 2018).

In mice lacking DAGL/2AG production kainate-induced seizures were much more severe. The seizure suppressing effect appeared to depend on CB1 and presumably CB2 (Sugaya et al., 2016).

In the mouse pilocarpine model of epilepsy, 30 mg/kg CBD restored non-NMDA LTP. This effect was at least partially mediated by 5-HT1A receptors (Maggio et al., 2018).

In the rat pilocarpine model of epilepsy, status epilepticus strengthens GABAergic drive between interneurons. This strengthening can be inhibited via CB1 signalling (Yu et al., 2016).

Low frequency stimulation is known to have an anti-epileptic effect in experimental epilepsy models such as kindling. Kindling reduces CB1 expression in the brain wherease low frequency stimulation increases CB1 expression. Blocking CB1 receptors abolishes the effect of low frequency stimulation in kindling, suggesting an anti-epileptic effect of CB1 during low frequency stimulation (Mardani et al., 2018).

In mice with diazepam-resistent status epilepticus, inhibition of MAGL/2AG degradation reduced the duration of status epilepticus by 47%. In mice fed a ketogenic diet, MAGL inhibition immediately abolished seizures (Terrone et al., 2017).

In the rat PTZ model of epilepsy CB2 agonist AM1241 exacerbated seizures while CB2 antagonist AM630 was anti-convulsant (de Carvalho et al., 2016).

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