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SHORT RESEARCH ARTICLE

Cannabis in epilepsy: From clinical practice to basic
research focusing on the possible role of cannabidivarin
*,1Alessandra Morano, †,‡,1Pierangelo Cifelli, †Paolo Nencini, †Letizia Antonilli, *Jinane Fattouch,
†Gabriele Ruffolo, §Cristina Roseti, ¶,**Eleonora Aronica, †Cristina Limatola, *Carlo Di
Bonaventura, †,§,2Eleonora Palma, and *,2Anna Teresa Giallonardo

Epilepsia Open, 1(3-4):145–151, 2016
doi: 10.1002/epi4.12015

SUMMARY
Cannabidivarin (CBDV) and cannabidiol (CBD) have recently emerged among
cannabinoids for their potential antiepileptic properties, as shown in several animal
models. We report the case of a patient affected by symptomatic partial epilepsy who
used cannabis as self-medication after the failure of countless pharmacological/surgical
treatments. Clinical and video electroencephalogram (EEG) evaluations were periodi-
cally performed, and the serum levels of CBDV, CBD, and D9-tetrahydrocannabinol
were repeatedly measured. After cannabis administration, a dramatic clinical
improvement, in terms of both decrease in seizure frequency and recovery of cognitive
functions, was observed, which might parallel high CBDV plasma concentrations. To
Alessandra Morano, widen the spectrum of CBDV possible mechanisms of action, electrophysiological
MD, Department of methods were applied to investigate whether it could exert some effects on c-amino-
Neurology and Psy- butyric acid (GABA)A receptors. Our experiments showed that, in human hippocam-
chiatry, University of pal tissues of four patients affected by drug-resistant temporal lobe epilepsy (TLE)
Rome Sapienza. transplanted in Xenopus oocytes, there is decrease of current rundown (i.e., reduction
of use-dependent GABAA current) after prolonged exposure to CBDV. This result has
been confirmed using a single case of Rasmussen encephalitis (RE). Our patient’s elec-
troclinical improvement supports the hypothesis that cannabis could actually repre-
sent an effective, well-tolerated antiepileptic drug. Moreover, the experimental data
suggest that CBDV may greatly contribute to cannabis anticonvulsant effect through
its possible GABAergic action.
KEY WORDS: Epilepsy, Cannabidivarin, Antiepileptic, GABA.

Pierangelo Cifelli,
MD, PhD, Department
of Physiology and Phar-
macology, University of
Rome Sapienza.

Accepted August 8, 2016.
*Department of Neurology and Psychiatry; †Department of Physiology and Pharmacology, Pasteur Institute-Cenci Bolognetti Foundation, University
of Rome Sapienza, Rome, Italy; ‡Ri.MED Foundation, Palermo, Italy; §IRCCS San Raffaele Pisana, Rome, Italy; ¶Department of (Neuro) Pathology,
Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands; and **Stichting Epilepsie Instellingen Nederland (SEIN-Heemstede),
Amsterdam, the Netherlands
Address correspondence to Carlo Di Bonaventura, MD, Department of Neurology and Psychiatry, University di Roma Sapienza, P.le A. Moro 5, Rome
00185, Italy. E-mail: c_dibonaventura@yahoo.it
1, 2
Equal contribution.
© 2016 The Authors. Epilepsia Open published by Wiley Periodicals Inc. on behalf of International League Against Epilepsy.
This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution
in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

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1002/epi4. Epilepsia Open.146 A. 1(3-4):145–151. Morano et al.12015 . 2016 doi: 10.

ment was started.2 On the [VGKCR]. and the ratory findings suggesting a possible beneficial effect of dramatic evolution were highly suggestive of a focal variant CBDV. On the left: MRI scan before surgery. external clonazepam 4 mg/day) was unchanged. 147 Cannabidivarin in Treating Epilepsy Figure 1. Ma2. antiepileptic drugs (used in several associations) and the logue. sive care unit management. hippocampus. and were normal. age at onset. therefore. and partial consciousness impairment ( ). cal features (unilateral neurological deficits. and then by a pseudoperiodic slow spike-and-wave activity propagating over the midposterior temporal areas. oral and intravenous steroids were investigate whether CBDV may affect c-aminobutyric acid administered without improvement (for more details. mediated partial epilepsy begun at the age of 5. more specifically.000 mg/day. homemade oral infusions obtained by simmering 300– and speech and consciousness impairment. After the failure of all available in the scientific community and media.6 However. and basal ganglia involvement). and cerebrospinal fluid analyses THC) are thought to have a major anticonvulsant effect. During the following months. showing diffuse white matter alterations and left lateral ventricle dilatation.7 We report the case of a patient affected by anterior temporal lobe. and amygdala) were drug-resistant focal epilepsy treated with homemade canna. first interpreted as a perinatal Follow-up and outcome hypoxic/ischemic lesion (Fig. leve- trotemporal regions. [NMDAR].1. showing the presence of a lesion involving the left insula with concomitant slight hyperintensity and bulging of the mesial temporal area and atrophy of the head of the ipsilateral caudate nucleus. language functions was observed. (C) MRI scan (FLAIR and IR sequences).The neurological examination showed severe ideomotor slowing and an evident worsening of the preexistent aphasia. Cardiological evaluation.receptor [GABAB-R]. of Rasmussen encephalitis (RE) primarily involving the plantation from brain tissues to Xenopus laevis oocytes8 to temporal lobe. dystonic posturing of the right limbs (♦). The clinical correlate consisted of a seizure characterized by intense right perioral myoclonus (*). culty in word reaching. Although histological findings bis-containing infusions who underwent clinical.1002/epi4. vagus nerve stimulation and sur- data from well-designed clinical trials for both compounds gical treatments (specifically. EEG. Yo. clinical and radiologi- electroencephalogram (EEG) and laboratory monitoring.3–5 Its propyl ana. capsule. 1). see (GABA)-ergic transmission. characterized by right perioral myoclonia We report the case of a 19-year-old boy referred to our followed by stiffening of the right arm and increased diffi- Epilepsy Centre for a symptomatic. EEG recordings 400 mg of cannabis inflorescence in 250 ml of milk twice a showed abundant epileptiform activity on the left cen. carbamazepine 800 mg/day. we used the technique of membrane microtrans. phytocannabinoids hydroxy-5-methyl-4-isoxazolepropionic acid receptor other than the well-known D9-tetrahydrocannabinol (D9. and neuroimaging findings seizures a day. voltage-gated potassium channel receptor including epilepsy. different CV2. Epilepsia Open ILAE The potential role of cannabis in treating several diseases.12015 . his and motor aphasia. Seizures decreased in frequency soon after the treat- autoantibody screening (including anti-Hu. Ri. with increased difficulty in verbal comprehension and word reaching. removal of left insula. His medical Interictal video EEG showed a subcontinuous paroxysmal history was characterized by progressive unilateral neuro. [AMPAR] antibodies). the patient was experiencing dozens of Clinical. At that time. right motor signs. with prolonged postictal weakness. video were rather nonspecific. cannabidivarin (CBDV). On the right: MRI scan after left temporal lobectomy. 1). (A) EEG recording performed before the introduction of cannabis: the tracing shows a subcontinuous paroxysmal epileptiform activity characterized by discharges of spikes in the left hemisphere with rapid contralateral transmission. An improvement in cognitive and. activity involving the left hemisphere. 2016 doi: 10. day. GABAB. and insula along with atrophy of the head of the ipsilateral caudate nucleus. amphiphysin. followed by a brief voltage attenuation on the left frontocentral and anterior temporal regions. and a-amino-3- basis of in vitro and in vivo studies. unilateral sei- Considering the patient’s global improvement and the labo. right hemiparesis with dystonia tral and anterior temporal regions (Fig. probably immune. and recurrent partial seizures with parents decided to try cannabis and started giving him heterogeneous subjective manifestations. namely. Empirical use of cannabis Case Report In January 2014. clinically associated with isolated perioral myoclonus (*). has been recently proposed recurrence of convulsive status epilepticus requiring inten- to have an antiepileptic action in animal models. has been known for centuries. Magnetic resonance imaging (MRI) tiracetam 4. (B) EEG recording performed 1 year after the introduction of cannabis: the tracing shows diffuse discharges of spikes. unsuccessfully attempted. and focal theta-delta activity intermingled with spikes and spike-and-waves located on the frontal and anterior and middle temporal regions. followed by ipsilateral tonic deviation of the mouth (●). N-methyl-D-aspartate receptor strains of cannabis were used with variable clinical Epilepsia Open. especially frontocen- logical deficits. only homozygous prothrombin G20210A in particular cannabidiol (CBD) has received great attention mutation was detected. His previous therapy (topiramate 300 mg/day. the Supporting materials section). and scans documented a lesion involving left putamen. zure onset. 1(3-4):145–151. are still lacking.

and partial consciousness impairment) and severe ideomotor slowing with mixed aphasia.5 years.7  9. Figure 2. respectively. Data refer to 30 oocytes. THC and CBDV were monitored using positive mode electrospray ionization and multiple reaction monitoring (MRM).1 mm. In all experiments the holding poten- tial was 60 mV. The transitions were m/z 315 ? 193 and 287. mean age 32. dystonic posturing of the right limbs. PI: partial improvement. informed con- sent was obtained. Data refer to 4 TLE patients (4 males.01% formic acid in water and 0.2 ? 165.5 ng/ml for both analyses. TLE tissues with HS were provided by Amsterdam University (AMC) and used in accordance with the Declaration of Helsinki. cannabidiol (CBD). Plasma concentrations of D9-tetrahydrocannabinol (THC). (A) Cannabinoids serum levels and electroclinical correlations. Current amplitude normalized to the first response of rundown protocol. 2.2 for THC and CBDV. 1(3-4):145–151. For the clinical part of the study. with slight modifications. defined as the persistence of isolated perioral myoclonic jerks and the improvement of language fluency. subclinical ictal discharges.7  1 years).6 particle size) with a gradient of 0. B: baseline. I: improvement. 4 patients. Epilepsia Open ILAE Epilepsia Open. mean epilepsy onset 12. For the experiments. defined as the coexistence of perioral myoclo- nia. Morano et al. including CA1 and CA4 (HS. and motor aphasia. After solid phase extraction (SPE). (B) Normalized time-course of the averaged (mean  SEM) GABA current rundown in oocytes transplanted with hippocampal membranes in control condition (filled symbol) and after 50 nM CBDV application (empty symbol). All patients had mainly complex partial seizures resistant to maximal doses of different antiepileptic drugs and had hippocampal sclerosis (HS) with neu- ronal loss within all hippocampal subfields.12015 .148 A. ILAE type 1). and cannabidivarin (CBDV) were quantified by liquid chromatography tandem mass spectrometry (LC-MS/MS) according to Ferreir os et al. defined as the coexistence of subcontinuous motor seizures (right perioral myoclonus. chromatography was performed on KInetex C18 column (50 9 2.1002/epi4. ipsilateral tonic deviation of the mouth. Inset: Representative GABA current traces in one transplanted oocyte showing the first and sixth response to GABA 500 lM (black bars) before and after 50 nM CBDV application as indicated. The limit of quantification (LOQ) was 0.01% formic acid in methanol. 2016 doi: 10.

Membrane and from discontinuation.1 for CBDV. and he could GABA (Tocris. Fig. 2016 doi: 10. which con. 35 oocytes. 2B) and on and LEV 1 lM. In the attempt to find a con. down was affected by levetiracetam (LEV). we focused our attention monitoring with polygraphic study documented the dras.8. defined as the decrease. see Fig.5  4. C (PKC) pathway. rent amplitude remains. He was also administered a simplified ver. which had been intended only to reach were purchased from Sigma Italia. I% RE = 49%  7. Considering the proven effect of cannabinoids on eral times a day.6. on the modulation exerted by CBDV on GABAA-Rs. see Fig. of the cur- mances. 149 Cannabidivarin in Treating Epilepsy effects. In contrast. p < 0. focal motor seizures TLE tissues with hippocampal sclerosis (HS) and RE tis- involving the right hemisoma relapsed after about 4 days sues were transplanted in Xenopus oocytes.2 to 63. we observed a small. 25 years old). without remarkable changes oocytes.10 sistent correlation with the patient’s clinical evolution. 1(3-4):145–151. and Wisconsin All results are given as mean  standard error (SEM). Bristol.1002/epi4. when the patient’s tude range: 10 nA to 100 nA). p < 0.10 GABA current rundown (I%) was improved. cited bicuculline-sensitive inward currents (IGABA ampli- cally measured. in terms of both motor and cognitive perfor. prolonged exposure to CBDV (from 30 min to From Clinical Cues to 3 h)12 decreased IGABA rundown. 10 oocytes.11 induce a further improvement of rundown (data not shown).12015 . UK) and CBDV (THC Pharm. The patient’s participation in p < 0.0 to Epilepsia Open. Interestingly. for RE. rundown) of the GABAA receptor mediated spikes/spike-and-waves persisted.10 in the same conditions. However.10. no currents that was recovered by blocking the protein kinase side effects were reported. When oocytes were conditions improved. impairment was recorded as a strong use-dependent desen- although abundant slow abnormalities intermingled with sitization (i. 10 compared with the levels detected before cannabis oocytes. periodic neurological examinations rent peak amplitude after six 10-s applications of 500 lM showed an increase in verbal fluency/comprehension and GABA at 40-s intervals. 49 were always within range. from 49%  5. because of the unavailability of the patient’s own The described effect was not detected in brain tissues surgical specimens (temporal lobe tissue was already fixed obtained from 4 nonepileptic controls (data not shown).05. even graduate thanks to a simplified lesson plan and the Frankfurt. For an internal control. 4 patients. mediated action of cannabis. S1). Serum levels of the other RE tissues. CBD. see Fig. dramatically changed. When the same strain was used for oocyte preparation/injection procedures were performed as several months. previ- tic reduction of the usual subcontinuous paroxysmal ously reported to be impaired in these patients. but not statistically significant. other cells were incubated League Against Epilepsy (including Wechsler Adult with oocytes Ringer’s solution8.. Trail Making Test. Fig. Our results clearly indicate that the two specimens obtained from a single patient affected by RE compounds did not show a cumulative effect (from I% = (female. 4 patients. However.e. Oocytes were incubated with sion of the Neuropsychological Assessment Battery for CBDV (50 nM) for 120–150 min after the control rundown adolescents with epilepsy recommended by the Italian protocol. In particular. high concentrations of CBDV were injected with hippocampal membranes of TLE patients and detected in his blood (Fig. with the exception of socialization goals. 2B. Weekly/monthly prolonged video EEG GABA-evoked currents11 (IGABA). In these cells.9. 2A).12 In agreement with previous results. we performed mens resected from four drug-resistant temporal lobe experiments incubating oocytes with both CBDV 50 nM epilepsy (TLE) patients (for details. Two Card Sorting Test) and Aachen Aphasia Test. it was unmistakably observed that each Patients and methods time cannabis was withdrawn. According to the patient’s seizure diary. calculated in percentage. 2B.05 (analysis of variance [ANOVA] test). effect.10 This epileptiform activity over the left frontotemporal regions. During follow-up. ous application of CBDV (from 50 nM to 50 lM) and GABA did not alter the IGABA rundown (data not shown). we found a consistent IGABA rundown following antiepileptic drugs were repeatedly measured as well and repetitive application of GABA (I% TLE = 45%  12. help of a learning support teacher. 47%  5. at 10 nM some recent data emphasizing the possible role of GABA.7. application of GABA (500 lM) to transplanted oocytes eli- the serum levels of THC.9 Considering centrations lower than 5 nM did not exert effects. and embedded in paraffin) and the rarity of his pathology. Intelligence Scale. we decided to test CBDV while CBDV doses higher than 50 nM (up to 50 lM) did not effect on GABAergic transmission in human tissue. I% The potential mechanisms underlying CBDV clinical RE = 72%  6. Germany).9 the percentage of the original cur- praxic skills. and CBDV were periodi. All drugs school activities. Because it has been previously shown13 that GABAAR run- we performed our experiments on the hippocampal speci.05. S1). Electrophysiological results although isolated perioral myoclonic jerks occurred sev. with a maximal effect obtained 2 h after 50 nM CBDV treatment (I% TLE = Experimental Approach 65%  11. the patient’s general conditions steadily previously detailed. major motor seizures disappeared. Con- effects are several and only partially known. data sets were considered statistically different when firmed his improvement. the simultane- introduction.

Epilepsia 2015.167:1629–1642. Phosphatase inhibi- than the PKC a. Neurol- ramate. which suggested that CBDV might play a istry of Health for Institutional Research (C. CBDV effect was not blocked by selective ments. but it was blocked by broad spectrum kinase into consideration. see Table S1). we showed 8. 4 patients) and by transient receptor potential matory properties of cannabis. for the first time we pro- Discussion pose GABAA-Rs as a possible new target for CBDV. Hill AJ. 8 oocytes each set of Although further investigations are needed. Prov Med J Retrosp Med Sci 1843. the concentrations of CBDV were always and UCB Pharma (E. desensitize transient receptor potential vanilloid 1 (TRPV1) channels in vitro: potential for the treatment of neuronal hyperexcitability. 1(3-4):145–151. 2 TLE patients. general action of CBDV. Di Angelantonio S.R.5:1131–1141.9 Chem Neurosci 2014. f. but it was modulated by a kinase or a PKC isoform other 10. zure frequency and epileptiform paroxysmal activity over time but. et al. ders. Eusebi F.R.1  5. Mathern GW.10. Ragozzino DA. PhD in Clinical/Experimental Neuroscience and Psychiatry at Sapienza University. paralleling electroclinical their contribution in data collection. Maa E. Morano et al.82:1556–1563.1002/epi4. References dent from THC but also that CBD and CBDV may have an additive effect. Hill CL. Nonpsychotropic plant cannabi- we can clearly state that CBDV and LEV did not act in a noids. be ruled out at present. clonazepam) could have also influenced ogy 2014. G. This have read the Journal’s position on issues involved in ethical publication hypothesis is in accordance with recent in vivo studies test. it was not possible to identify a “threshold efficacy level” for either CBD or CBDV. In addition. our data did not allow us to exclude that it was not a Disclosure single compound but the different molecules combined The authors have no conflict of interest to declare. The authors thank all the patients who made this study possible. et al. Devinsky O. Cannabinoids in the treatment of epilepsy. et al. Epilepsia 2014. 6. studies.C. potential efficacy of cannabis in the treatment of drug-resis- tant epilepsy and highlights the role of CBDV as an antiepileptic drug. Epilepsia 2014.373:11. The periodic electroclinical evaluations during randomized clinical trials (RCTs) and targeted experimental cannabis treatment showed a variable decrease in both sei.P. ings may be relevant for future pharmacological develop- Interestingly.). Iannotti FA. Hill TD. 66. Palma E. On the preparations of the Indian hemp. and metabolism cannot with other medical professionals and patients: result of Epilepsia’s sur- vey. b1.12 Notably. The reported clinical experience and the following exper- ant of RE who tried cannabis in addition to his medical ther. safety of medical marijuana in selected neurologic disorders. Accordingly. Interactions between cannabinoids and the other antiepilep. Devinsky O.MED Foundation (P. Fife T. As expected.). Rome. We report the case of a patient with a probable focal vari. the repeated analyses of cannabi. potential interferences with medical marijuana and CBD in treating epilepsy patients compared cannabinoid level. bioavailability. Amici M. and affirm that this report is consistent with those guidelines. Koppel BS. Albini and Dr. Moreover.150 A. ing enriched cannabis extracts. Ri. Palma E.5:363–369. erties. Interestingly. was supported by crucial role and greatly contribute to cannabis anticonvul. 4. Fanella for noid serum levels revealed that.14 1. Microtransplantation of ligand- a new possible mechanism of action elicited by CBDV on gated receptor-channels from fresh or frozen nervous tissue into Xeno- GABAA current rundown. et al.55:791–802. This work was supported by grants AICE-FIRE improvements. Friedman D. O’Shaughnessy WB. Cannabidivarin is anticonvulsant Based on the reported clinical experience and surprising in mouse and rat. most importantly. N Engl J Med 2015. nificant alterations in AED concentrations after the 5. which showed not only that cannabis anticonvulsant properties are most likely indepen. M.0. clobazam. unambiguous worsening of the patient’s conditions soon after cannabis Acknowledgments withdrawal. inhibitor staurosporine13 (from I% = 42  13 to I% = In conclusion. Cannabidiol: pharmacology and the clinical outcome. these find- experiments). sive effect.13 tors remove the run-down of gamma-aminobutyricacid type A Epilepsia Open. The case for medical marijuana in epilepsy.12015 . as proved by isobolographic methods. syndrome of our patient and the acknowledged anti-inflam- 19 oocytes. et al. Mercier MS.3 for CBDV plus LEV. and the Italian Min- particularly high. 2. Brust JCM. or Gun- jah.).55:783–786. considering the specific epileptic PKC antagonist GF 109203X 1 lM12 (I% = 60. Br J Pharmacol 2012. ACS this effect was not mediated by TRPV channels activity. 2016 doi: 10. 9. a well-known phenomenon pus oocytes: a potent tool for expanding functional information. LEV.56:1–6.d. 10 oocytes. Leo A.5%  9. beneficial effect of cannabis. Beninsig L. 3. Nehlig A. either through pharmacodynamic potential therapeutic role in epilepsy and other neuropsychiatric disor- mechanisms or reciprocal changes in pharmacokinetic prop. topi. M. et al. a consistent.88:32–40. described in drug-resistant epileptic patients. Indeed. Systematic review: efficacy and tic drugs (AEDs)15 used by the patient (namely. In addi- tion. imental approach confirm the need for both well-designed apy. we tried to better define the 7. Figi P. although periodic controls did not reveal sig. immune-modulating effects vanilloid (TRPV) channel antagonist capsazepine 10 lM9 contributing to the clinical improvement should be taken (see Table S1). We confirm that we together to have the most effective antiepileptic action. Cilio MR. The authors would like to thank Dr. cannabidivarin (CBDV) and cannabidiol (CBD). activate and synergistic fashion on rundown improvement. In a set of experiments. Fewer specialists support using introduction of cannabis. Cross H. Prog Neurobiol 2009. our case supports the tolerability and 44  11.

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