In the present study, we have demonstrated the acute antiseizure efficacy of CBD in a battery of well‐defined and established acute seizure tests in both mice and rats following IP administration. Our findings are consistent with other reports of the acute antiseizure efficacy of CBD in numerous preclinical seizure models.10, 20 We have also demonstrated that acute IV administration of a subtherapeutic dose of CBD is able to significantly attenuate the MSS in the rat pilocarpine‐induced SE model to a similar degree as a supratherapeutic dose of the prototype ASD, PB. Furthermore, we have demonstrated for the first time that chronic oral administration of CBD improves seizure burden ratio, motor comorbidities, and cognitive function in the RISE‐SRS model of TLE in rats well after the onset of symptomatic seizures. Altogether, the present study further supports a growing body of evidence to demonstrate that CBD is a well‐tolerated and effective therapy for acute and chronic seizures,11 as well as now demonstrating the potential that chronic oral administration of CBD may confer disease‐modifying effects in an etiologically relevant preclinical model of TLE.

All of the FDA‐approved ASDs exhibit acute antiseizure efficacy in one or more of the acute seizure and epilepsy models presently used for the evaluation of CBD.16, 21 However, many of the FDA‐approved ASDs are associated with significant motor adverse effects that may limit clinical utility. In this regard, we presently provide pharmacological data that suggest differentiation between CBD and the FDA‐approved ASD, PB (Table 1). Of note, the activity profile of CBD in these acute seizure models is generally comparable to PB (Table 1). CBD is thus differentiated from numerous ASDs with broad‐spectrum efficacy, including another broad‐spectrum ASD, VPA (Table 1), because of this wide margin separating antiseizure efficacy and minimal motor impairment in numerous preclinical seizure models. The broad‐spectrum efficacy and margin of safety of CBD are more directly comparable to FBM (Table 1), which, like CBD, is also approved for patients with Lennox‐Gastaut syndrome.22 CBD presently demonstrated efficacy in the acute mouse MES, subcutaneous pentylenetetrazol, and 6‐Hz assays at both 32‐ and 44‐mA stimulus intensities. CBD also demonstrated a PI > 1.5 in the 6‐Hz model of pharmacoresistant epilepsy at a 44‐mA stimulus intensity, which substantially differentiates this compound from many other FDA‐approved ASDs, including VPA.23, 24 The activity of FBM is also preserved in the 44‐mA version of the 6‐Hz assay (Table 1); the PI of FBM is also much greater than CBD in this assay (4.6 for FBM vs 1.6 for CBD; Table 1). As the 6‐Hz stimulation intensity increases from 32 to 44 mA, most ASDs lose efficacy or are only effective at motor‐impairing doses16, 17, 23, 25; thus, the finding that CBD retained efficacy and potency at both stimulation intensities in this assay at doses well below the mouse TD50 (272 mg/kg IP) suggests that CBD is highly differentiated from other FDA‐approved ASDs (eg, PB and VPA; Table 1) and suggests potential for efficacy in pharmacoresistant patient populations akin to FBM. Unlike FBM, there have been no reports to date of hepatotoxicity or aplastic anemia with CBD use,26, 27 but whether other clinical adverse events will emerge with greater clinical use of this agent remains to be determined.

The presently reported ED50 values in these acute mouse assays align with work reported by Klein and colleagues using CBD provided by the US National Institute of Drug Abuse10 and by an independent laboratory using the same CF‐1 mouse strain.28 However, we presently report significantly greater potency of CBD in the rat MES test than that reported by Klein and colleagues (ED50 = 88.9 mg/kg [95% CI = 69‐124] IP10), likely due to differences in compound formulation, source, and purity. We also report that CBD exerted dose‐dependent reductions in seizure score in the corneal kindled mouse model of chronic focal seizures, further suggesting that CBD is effective as an antiseizure agent in an epileptic substrate (ie, kindled rodents). Altogether, the presently reported pharmacological profile of CBD supports further evaluation of its broad clinical utility for epilepsy.

IV administration of CBD also reduced MSS in the pilocarpine‐induced SE in rats (Figure 1A), consistent with earlier findings demonstrating efficacy of a preclinical CBD formulation administered via the IP route.9 We believe this is the first demonstration of the efficacy of IV CBD against the onset of SE, with this route of administration most commonly employed in the treatment of clinical SE.6, 9 Although further studies are needed to define whether IV CBD can effectively reduce MSS after the onset of SE (eg, against benzodiazepine‐resistant SE), the present results in the pilocarpine‐SE model further support the acute antiseizure efficacy of CBD in diverse preclinical models of seizure.

TLE is one of the most common forms of acquired epilepsy in humans.29 Therefore, we investigated the effect of a clinically relevant CBD administration protocol in the RISE‐SRS model of TLE.15 We presently demonstrate that CBD is disease‐modifying in this model, where all animals exhibit SRS before being assigned to treatment groups. Specifically, chronic oral administration of CBD significantly decreased the seizure burden ratio of epileptic animals and improved reference memory function. Although CBD did not significantly reduce seizure burden after 7 weeks of treatment, it did markedly modify the natural disease course following SE, as demonstrated by no overall increase in the seizure burden from the first to final seizure monitoring bin (Figure 2). These findings are in stark contrast to the vehicle‐treated post‐SE rats, which demonstrated a notable, time‐dependent increase in disease severity (ie, seizure burden ratio increased). Disease severity, as characterized by a seizure burden ratio < 1, was improved in 70% of the CBD‐treated animals, in contrast to only 10% of vehicle‐treated rats attaining such a ratio (Figure 2). Although several FDA‐approved ASDs have demonstrated disease‐modifying potential in preclinical models of chronic seizure,30, 31 and CBD has been found to be disease‐modifying in a mouse model of Dravet syndrome,32 to our knowledge, this is the first study to demonstrate such an effect with CBD in any preclinical TLE model. The third‐generation ASD topiramate has demonstrated some potential for cognitive sparing and modification of behavioral deficits when administered shortly after SE onset,33, 34 but no study has yet demonstrated such a disease‐modifying effect when treatment is initiated well after the SE insult (8 weeks in present study). Although our study did not include a CBD washout arm, the present results demonstrate that chronic oral administration of CBD is associated with long‐term improvements in disease trajectory in this rat model of epilepsy.

A number of ASDs (eg, PB, VPA, phenytoin) are reported to have an adverse effect on motor function characterized by dyskinesia in people with epilepsy.5, 35, 36, 37 Moreover, patients on phenytoin and VPA treatment sometimes exhibit parkinsonism.5 Although we presently demonstrate that acute administration of CBD is well tolerated at doses up to 500 mg/kg (IP) in naive rats (Table 1), in line with prior reports,9 no studies have yet been conducted to examine the long‐term effects of chronic CBD administration on tolerability and motor function in epileptic animals. Furthermore, no study has yet administered CBD for disease modification purposes after the onset of SRS. In this regard, the present study employed a clinically realistic treatment scenario in a preclinical model of TLE to demonstrate that chronic oral administration of CBD is associated with notable antiseizure efficacy, minimal adverse effects liability, and disease‐modifying potential well after the onset of symptomatic seizures.

It is well known that humans and animals with epilepsy are more sensitive to adverse effects of ASDs38; thus, our present findings that chronic oral administration of CBD in rats with epilepsy was not associated with any adverse effects further supports the potential of this agent for chronic clinical use in epilepsy patient populations. In addition to these drug‐induced adverse effects on motor function, motor deficits are one of the most common comorbidities exhibited by patients with epilepsy.39 Here, we used two well‐validated models of motor function to assess the fine motor control, balance, and gait of animals. Epileptic vehicle‐treated animals fell from the accelerating rotarod sooner than the naive vehicle‐treated ones, indicating that SRS produced significant motor dysfunction related to balance. Similar findings were reported previously in the rotarod40 test, where motor dysfunction was exhibited by epileptic rats. In contrast, CBD‐treated animals remained on the accelerating rotarod for a significantly longer time than the vehicle‐treated epileptic animals. Thus, we show for the first time that CBD is not only well‐tolerated by epileptic animals after prolonged oral administration, but that CBD reduces the severity of motor deficits induced by epilepsy.

Epileptic rats performed comparably to healthy animals in the gait test, which is consistent with gait disorders being rarely reported in adult epileptic patients with TLE. However, ASDs such as VPA and lacosamide have a detrimental effect on gait in human patients.35, 37 It should be emphasized that animals were in the active CBD administration period during this motor test, suggesting that CBD at the dose tested did not confer any adverse effects on motor coordination. Our results demonstrate that chronic oral administration of CBD did not have any adverse effect on gait in rats with epilepsy. Clinical trials conducted on Dravet syndrome and Lennox‐Gastaut syndrome patients also did not report any gait disturbances following CBD administration.11, 13 Whether these side effects may also be absent in the general TLE patient population remains to be further determined.

Cognitive decline is a common comorbidity associated with TLE.41 For example, patients with TLE often exhibit poor executive control and working memory deficits,42 and amnesia or accelerated long‐term loss of memory is also frequently reported in patients with TLE.43 Cognitive function in animals is typically assessed using spatial memory tasks measuring reference memory and working memory errors.32, 33 Reference memory can be defined as long‐term storage of acquired information that remains constant over successive training sessions, whereas working memory is a form of short‐term memory that refers to storage and manipulation of information acquired within a trial session.44 Several spatial memory tasks (eg, radial arm maze, Morris water maze, hole‐board task) have been used to evaluate these two types of memory in rodents.45, 46 Here, we employed the hole‐board task, where epileptic vehicle‐treated rats exhibited impairment of both the working and reference memory aspects of the hole‐board test. These findings are comparable with previous studies of spatial or hippocampus‐dependent memory in rats with TLE, for example, a delayed nonmatching to position task,47 eight arm radial maze,48 and Morris water maze.49 In contrast, CBD‐treated epileptic animals exhibited improved reference memory function compared to epileptic vehicle‐treated animals; however, reference memory function was not restored to levels seen in healthy animals. Interestingly, our study shows that chronic oral administration of CBD improved working memory function compared to both epileptic vehicle‐treated and naive vehicle‐treated animals. However, as we did not include a nonepileptic CBD treated group in our design, we are unable to ascertain whether CBD has memory enhancement properties in healthy animals. A memory‐enhancement effect would be unusual, as previous studies using a radial arm maze have shown that CBD does not improve working memory function in healthy mice.50, 51 Given that the integrity of the blood‐brain barrier is commonly disrupted in epilepsy,52 a limitation of the present study is that we did not determine whether higher CBD concentrations were present in the brains of these epileptic rats. Thus, the dose of CBD required to reach this concentration might not be therapeutically significant in healthy animals. Although SRS are the primary cause of cognitive decline in TLE,53 the improved cognitive function in the CBD‐treated epileptic group might be due to a seizure‐independent mechanism rather than solely by seizure reduction, for example, anti‐inflammatory/neuroprotective action.6 However, further detailed investigation is thus warranted to shed light on the mechanism by which CBD improved the presently tested behavioral comorbidities.

Epidiolex (CBD) was approved by the FDA in 2018 for the treatment of seizures in the catastrophic pediatric encephalopathies Dravet syndrome and Lennox‐Gastaut syndrome. The present study has demonstrated that CBD is also a good potential candidate drug for the treatment of spontaneous symptomatic seizures of TLE. Moreover, chronic oral administration of CBD also reduced severity of motor disorders and cognitive deficits typically associated with TLE, in line with prior demonstrations of disease‐modifying effects in a mouse model of Dravet syndrome.32 Taken altogether, this study further illustrates that CBD not only is a potent and broad‐spectrum ASD, but may also find use to attenuate cognitive deficits associated with TLE well after the onset of SRS.41 More detailed studies are thus required to investigate the potential mechanism of action for CBD as a disease‐modifying agent for the patient with epilepsy.