Subthreshold Cannabidiol Potentiates Levetiracetam in the Kainic Acid Model of Temporal Lobe Epilepsy: A Pilot Study

Abstract

1. Introduction

Cannabidiol is an antiseizure medication (ASM) used as add-on drug for some types of pediatric epilepsies, including Dravet syndrome [1,2], Lennox–Gastaut syndrome [3,4], and tuberous sclerosis complex [5], disorders for which the use of cannabidiol has been approved by the U.S. Food and Drug Administration [6]. Although considered effective and safe, cannabidiol is currently not approved for other types of epilepsy in humans. Cannabidiol is also under investigation in veterinary medicine, and one recent trial suggested that this ASM is effective for dogs with idiopathic epilepsy. In these animals, cannabidiol was used as an add-on drug obtaining beneficial results, but at the cost of significant adverse events, mainly consisting of reduced food intake and vomiting [7]. In children with epilepsy, cannabidiol was also associated with adverse events such as diarrhea, increased occurrence of seizures, or sedation, just to mention the more frequent. Reduced appetite and vomiting were also present in the same cohort [8].

The effects of cannabidiol were not investigated in patients with temporal lobe epilepsy (TLE), in spite of the fact that this type of epilepsy is the most prevalent and also the less responsive to ASMs in the population of adults with epileptic disorders [9]. In contrast to the lack of clinical investigations on cannabidiol for TLE, this drug has been studied in various TLE animal models, including kindling in amygdala [10] and hippocampus [6], or the pilocarpine [11] and kainic acid [12] models of status epilepticus. In these previous experiments, and more recently in our rats treated with kainic acid [13], the antiseizure properties of cannabidiol have been consistently documented in animals reproducing human TLE.

The possible interest in clinical use of cannabidiol in TLE is related to the problem of drug refractoriness, which is frequently found in this type of epilepsy [9]. First-line drugs such as levetiracetam were shown to be poorly effective in controlling seizures in as much as 50% of patients with epilepsy [14]. Consistent with these clinical data, levetiracetam administered at 300 mg/kg/day was only partially effective in reducing seizures in epileptic rats that were previously treated with kainic acid [15]. In these rats, levetiracetam produced significant effects mainly on the seizure duration and not on seizure frequency, at least in the animals which were refractory to this ASM (i.e., with persistence of spontaneous recurrent seizures, SRSs). Instead, in the same model, cannabidiol reduced not only the seizure duration, but also the overall seizure occurrence when used at high doses (120 mg/kg bis in die, b.i.d.) [13].

In view of these partially positive findings obtained with levetiracetam in kainic acid-treated epileptic rats, we hypothesized that there could be a potentiation of levetiracetam by combining this ASM with cannabidiol. We also preferred to test a cannabidiol dose lower than that previously used in our experiment [13], so as to evaluate a condition in which the adverse effects of cannabidiol could be less probable. For this reason, and also to simplify the treatment protocol, we performed just one instead of two daily injections of cannabidiol. Here we illustrate an interesting interaction between levetiracetam (administered at 300 mg/kg/day) and cannabidiol, the latter at a dose (120 mg/kg/day) that resulted to be subthreshold in the absence of levetiracetam.

2. Results

We considered 26 rats for four groups of treatment (n = 6/7 rats per group). All of them survived to the kainic acid administration (15 mg/kg, intraperitoneally, i.p.), but four of them were non-responders (i.e., they did not develop SRSs). Thus, 22 rats were considered eligible for saline or drug treatment at the end of the monitoring period. After having induced the status epilepticus, epileptic activity was continuously recorded using video electrocorticography (vECoG) until the animals were implanted with osmotic minipumps releasing either saline or levetiracetam [15], with the exception of non-responders which were disregarded. The vECoG recording continued for one week following the treatment period. Subgroups of these rats were also treated with a single daily injection of cannabidiol (120 mg/kg, subcutaneously, s.c.; n = 4, since three additional rats belonging to this group did not develop SRSs) for 7 days, matching the full-time infusion interval by the minipumps. This protocol resulted in four sets of animals (saline released by minipumps, n = 6; levetiracetam (by minipumps), n = 7; saline (by minipumps) + cannabidiol (s.c.), n = 4; cannabidiol (s.c.) + levetiracetam (by minipumps), n = 5, this because one further rat was discarded in this group since it did not develop SRSs). Seizure occurrence and duration were analyzed starting one week before treatment (as previously detailed, four rats were disregarded for treatment, because they did not present SRSs in this week), during treatment for one week, and the following week after treatment. The four rats which did not present SRSs in the first week of monitoring were recorded, but not treated, also in the following two weeks, thus confirming the lack of epileptic activity.

3. Discussion

In this study we found that: (i) cannabidiol is ineffective when administered once in a day in rats developing epilepsy after a status epilepticus induced by i.p. injecting kainic acid, even when a high dose is used. This finding contrasts sharply with our previous study, which showed a beneficial effect of cannabidiol administered b.i.d. at 120 mg/kg [13]; (ii) although cannabidiol was administered at a subthreshold dose, it potentiated the effects of levetiracetam, which was ineffective when administered alone in epileptic rats. This finding is of interest because it shows that a second-line ASM can be therapeutically useful even at a subthreshold dose, when combined with a first-line ASM (levetiracetam) toward which refractoriness is present. If confirmed by further experiments, this result might suggest that cannabidiol can be used at a dose able to reduce the occurrence of most serious adverse events of this drug.

This observation raises the question of the possible basis for the potentiation of levetiracetam by cannabidiol. While we do not have an answer, a suggestive hypothesis could be based on our previous results [15]. We previously noticed that levetiracetam significantly increases brain levels of allopregnanolone in epileptic rats, especially in the neocortex. Allopregnanolone is the major representative of a class of molecules known as neurosteroids that act as modulators of neuronal excitability [17]. Interestingly, allopregnanolone has been recognized to be an anticonvulsant in TLE [18] and led to development of an ASM, ganaxolone, presently used for seizures associated with the cyclin-dependent kinase-like 5 deficiency disorder [19,20,21].

Since cannabidiol can interact with a variety of targets in the brain [22], multiple mechanisms could be involved in the hypothesized synergism of cannabidiol and levetiracetam-induced allopregnanolone upregulation. Recently, cannabidiol was found to modulate γ-aminobutyric type A (GABA_A) receptor-mediated currents [23] generated in Xenopus laevis oocytes, transplanted with GABA_A receptors obtained from the neocortex of patients with TLE. GABA_A receptors are well-known targets for allopregnanolone, which is able to positively modulate the GABA_A-mediated inhibitory currents [24], thus reducing neuronal excitation in the brain. Interestingly, cannabidiol in the low micromolar range mainly modifies currents produced by α1-6βγ2 GABA_A receptors. The α2-containing GABA_A receptors appear to be more responsive to cannabidiol, which is able to produce a four-fold increase of their currents. A β-subunit selectivity was also reported by the same investigators, with a prevalence of β2/β3 over β1 subunits [22]. Several binding sites have been identified in the GABA_A receptor for allopregnanolone, both in α and β subunits [25], suggesting the possibility that the modulation of these GABA_A receptor components could underpin the reported synergism between cannabidiol and the allopregnanolone analog ganaxolone [26].

Other possible factors can be considered to explain the potentiation of levetiracetam effects by subthreshold cannabidiol. Cannabidiol was reported to alter kinetics and dynamics of various ASMs, including valproate, clobazam, and levetiracetam [27]. Specifically, in the case of levetiracetam a reduction in the antiseizure effects of this ASM was evidenced in presence of cannabidiol, when both these drugs were tested in mice with 6 Hz corneally induced seizures [28]. However, this finding was the opposite of that observed in our animals. Moreover, in a clinical study based on children and adult patients with epilepsy and treated with various ASMs, levetiracetam serum levels were increased by cannabidiol administered as an add-on drug, but only in the presence of pediatric epilepsy [29]. Although we could not exclude a similar phenomenon in our adult rats, we believe that it was improbable.

In our rats, we observed an incomplete response to the combined treatment, which means that levetiracetam and cannabidiol were able to significantly reduce the occurrence and severity of seizures, without abolishing them, a phenomenon well described in human patients [30]. Levetiracetam, instead, was not able to significantly modify symptoms of epilepsy when administered as single ASM, at least in our rats. This observation was consistent with other experiments in which both female [31] and male [32] mice, or male rats [33] developed levetiracetam refractory seizures after kainic acid administration, at a dose of this ASM comparable to that used in our rats. At variance, in a previous study we reported that approximately half of male rats completely responded to levetiracetam with seizure suppression, whereas the others did not respond, as in the present study [15]. Interestingly, in non-responders we observed a rebound effect due to levetiracetam withdrawal, consisting of increased seizure occurrence reaching a statistically significant level, as in the present experiment.

Other investigators found that much higher levetiracetam doses (up to 800 mg/kg) were effective in suppressing seizure in the intrahippocampal kainic acid model of TLE, in mice. The same investigators observed a large inter-individual variability in the response to levetiracetam [34], as also observed in our rats [15]. Unfortunately, variability in the response to levetiracetam in rodent models of epilepsy, and human patients [30], is a recognized problem that could not be ruled out even by increasing the number of investigated subjects, as we did in our previous study based on 15 levetiracetam-treated rats [15]. However, it is interesting to note that the combination of levetiracetam and subthreshold cannabidiol resulted in statistically significant beneficial results, in spite of the low number of considered epileptic rats. The therapeutic effects were on the number of seizures, whereas no significant changes concerned the mean seizure duration. Also in our previous experiment [15], the effects of levetiracetam on the seizure duration were inconsistent in the course of the treatment week, with only 3 days in which a reduction was statistically confirmed. Instead, cannabidiol was effective in reducing the seizure duration when administered b.i.d. at 120 mg/kg, but failed in the present experiment because administered at a single dose, even when combined with levetiracetam. This indicates that only certain features of epilepsy could be addressed by the tested levetiracetam and cannabidiol combination.

4. Materials and Methods

5. Conclusions

In epileptic rats, we unexpectedly found that cannabidiol can be used as a second-line ASM, at a dose not able to exert any effect without the concomitant administration of the first-line ASM levetiracetam. This discovery could be useful to make the cannabidiol administration more acceptable. However, not all the antiseizure properties of cannabidiol were preserved using this ASM at a subthreshold dose, thus making the decision to use a single administration of cannabidiol more questionable. In any case, our findings open the possibility to use cannabidiol in a more personalized way, so as to limit the sedative effect of this drug and all the other possible adverse events due to its administration at full doses.

Acknowledgments

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ph17091187/s1, Figure S1: Daily occurrence of spontaneous recurrent seizures (SRSs) in epileptic rats treated with saline subcutaneously (s.c., via osmotic minipumps [15]); Figure S2: SRSs after induction of status epilepticus in epileptic rats treated with saline s.c. (via osmotic minipumps [15]), saline (s.c.) and levetiracetam (LEV, s.c. via osmotic minipumps [15]), saline (s.c., via osmotic minipumps) and cannabidiol (CBD, 120 mg/kg s.c. [13]), and cannabidiol + levetiracetam (CBD+LEV; CBD s.c., LEV s.c. via osmotic minipumps).

Funding Statement

This research was funded by BPER Banca (project “Medicina Clinica e Sperimentale per il Trattamento delle Epilessie” to G.B.).

Author Contributions

Conceptualization, G.B. and G.C.; methodology, C.L. and A.-M.C.; software, C.L. and K.A.M.S.A.; validation, G.B. and C.L.; formal analysis, C.L., K.A.M.S.A. and M.M.; investigation, C.L., K.A.M.S.A., A.-M.C. and M.M.; resources, G.C. and G.B.; data curation, C.L.; writing—original draft preparation, C.L. and G.B.; writing—review and editing, C.L., G.B., G.C. and A.-M.C.; visualization, C.L., K.A.M.S.A., A.-M.C., G.B., G.C. and M.M.; supervision, C.L. and G.B.; project administration, G.B. and G.C.; funding acquisition, G.B. and G.C. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

The animal study protocol was approved by the university Animal Welfare Body and the Italian Ministry of Health (729/2021-PR).

Informed Consent Statement

Not applicable.

Data Availability Statement

The data is contained within article.

Conflicts of Interest

The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Footnotes

References