CBD is currently approved as adjunctive antiseizure therapy for DS and LGS in combination with clobazam, and as adjunctive therapy for TSC (12, 14, 19). However, many authors have investigated its potential role in other forms of DRE, where its use is off label. Indeed, literature data suggested that CBD antiseizure properties may be extended to a much broader spectrum of DREs (3, 20). CBD has demonstrated benefits in other epilepsy syndromes, both as long-term ASM and as treatment of acute manifestations (i.e., refractory status epilepticus) (23, 24). Over the years, clinical practice has provided clear evidence that CBD serves as an effective alternative for controlling seizures in patients with these pharmacoresistant epileptic conditions (18).

Our pediatric cohort of pharmacoresistant epilepsies included 7/15 (46.7%) DRE which were treated off label. Overall, 73.3% of cases (responders and partial responders) showed a reduction in seizure frequency with CBD treatment; in particular, 46.7% of patients achieved a significant reduction (>50%) in seizures (7/15 responders), with two cases displaying seizure freedom. Notably, none of the patients reported worsening of seizure frequency. These data were consistent with those reported in the literature. Particularly, real-world studies highlighted a significantly reduction in seizure frequency, with rates ranging from 36.9 to 68.8% of the patients investigated (23, 25), and were further confirmed by recent multicenter evidence supporting the efficacy of CBD in both approved and off-label genetic epilepsies (21). In line with the literature, drop seizures—defined as seizures characterized by an increase or decrease in muscle tone that leads or could lead to a fall, including tonic, atonic, and tonic–clonic seizures—were the types that showed the best response in terms of seizure reduction, with 5 out of 8 patients (62.5%) achieving complete remission from tonic seizures (9, 10, 26, 27).

In our sample, 8 patients were treated with a combination of clobazam and CBD, which demonstrated a good effectiveness in reducing seizure frequency. Specifically, 7/8 patients —comprising 3 with LGS, 1 with DS, and 3 with DRE — were identified as responders or partial responders. It has been proposed that the potential bidirectional drug interaction between CBD and clobazam may influence the overall effect and safety of CBD as an antiepileptic therapy, increasing the exposure to active metabolites of both drugs (28). Therefore, concomitant therapy with CBD and clobazam has been associated with a greater reduction in seizure frequency but also a higher incidence of side effects (18).

Although the mechanisms underlying CBD effects in humans are still not fully understood, evidence suggested that its action is multimodal, involving both cannabinoid and non-cannabinoid pathways, which may account for its strong anti-inflammatory and neuroprotective properties (15, 29). About 60 different neurological molecular targets of CBD have been identified, including enzymes, ion channels, and both ionotropic and metabotropic receptors (15, 29). Particularly, the endogenous cannabinoid system (including CB1 and CB2 receptors), the GPR55 receptor, and the TRPV1 calcium channel have been implicated in the CBD role as ASM (30).

Noteworthy, malformations of cortical development were observed in 6 of our patients: patient 1 had bilateral subcortical band heterotopia; patient 3 displayed a malformative pattern, including abnormalities in cortical gyration of the occipital-temporo-mesial regions; patient 4 showed lissencephaly; patient 8 had cortical tubers, consistent with the typical TSC neuroimaging pattern; patient 9 presented with frontal polymicrogyria in the context of Aicardi syndrome; and patient 10 exhibited bilateral parietal focal cortical dysplasia (FCD). It is crucial to point out that all these patients showed a good response to CBD, with 66.6% (4 out of 6 cases) experiencing a > 50% reduction in seizures, representing 57.1% of all the “best” responders (4 out of 7 cases – patients 1, 8, 9, and 10); the remaining 2 patients (33.3%) showed a seizure reduction between 30 and 50%, accounting for 40% of all partial responders (2 out of 5 patients – patients 3 and 4) (Figure 3). Malformations of cortical development are a heterogeneous group of brain malformations caused by disorders in neurogenesis, neuronal migration, post-migration neuronal development, and cortical organization. Over 75% of the patients with malformations of cortical development develop seizures over the course of their life, and in 40% of the cases present with pharmacoresistant epilepsies (31). Genetic factors play an important role in their etiology. Particularly, pathogenic variants of genes encoding regulators of the mammalian target of rapamycin (mTOR) cascade have been implicated in the occurrence of epilepsies, malformations of cortical development, and neurodevelopmental disorders (32). The involvement of mTOR pathway has been proved in FCD, in polymicrogyria, and in the TSC manifestations (31, 32). In fact, since they share histopathological features with FCD type II, cortical and subcortical tubers in TSC have been reclassified as FCD22 (33). FCD type II display an increase in CB1 receptor expression levels, especially in neurons with overactive mTOR Complex 1 (mTORC1) signaling (34). In addition, experiments conducted on Xenopus oocytes micro-transplanted with surgical and post-mortem tissue samples (including FCD type IIb) from DRE patients demonstrated that CBD has a modulatory effect on GABAergic currents. The authors suggested that this higher effect could be linked to specific cellular subtypes or different configurations of the GABA_A receptors (35). All this evidence may suggest that the receptor profile of patients with malformations of cortical development, characterized by increased CB1 receptor expression levels and a different configuration of the GABA_A receptors, favors the antiseizure effect of CBD, explaining the good response to treatment observed in this group within our cohort. To further explore this observation, we performed a Fisher’s exact test to evaluate whether patients with malformations of cortical development were more likely to respond to CBD. The analysis showed a trend toward significance, though it did not reach statistical thresholds, likely due to the limited sample size. These preliminary findings should be interpreted with caution, but they support the hypothesis of a differential treatment response in this subgroup and may serve as a foundation for future studies in larger, more homogeneous populations. Moreover, one of our patients, who did not have an evident malformation of cortical development, presented a de novo very large deletion including the YWHAG gene, which encodes for the tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein gamma. YWHAG is involved in the mitogen-activated protein kinases (MAPK) pathway through the RAF-1 bond and the mTORC1 signaling pathway, and its haploinsufficiency can lead to an altered expression of CB1 receptors in neurons (36). This patient showed a good response (>50% reduction of seizure) to CBD treatment, further enforcing the idea that genetic variants affecting the mTORC1 pathway may present a favorable profile of CB1 receptors that positively influence the clinical response to CBD treatment, similarly to malformations of cortical development such as FCD and TSC.

In addition to these molecular interactions, CBD has been shown to induce notable changes in brain connectivity. Specifically, studies have highlighted modifications in resting-state functional connectivity, particularly in inter-regional connections between the vermis, amygdala, hippocampus, and frontal cortex. Moreover, CBD appears to influence attentional processes by modulating the functional connectivity between the superior frontal gyrus and the insula/middle frontal gyrus (37, 38). Our study demonstrated significant benefits of CBD treatment, over seizure reduction, and improved social participation. Interestingly, the observed improvements in social functioning also contribute to better behavioral profiles. This is particularly interesting given that ASMs, especially in polytherapy, may show an adverse effect on the cognitive profile (39, 40). However, directly evaluating behavioral outcomes in our patient cohort proved challenging. Nonetheless, previous studies have already suggested a positive role of CBD in addressing neuropsychiatric symptoms (41, 42).

In our series, CBD treatment was generally well tolerated. The most common adverse event was transient irritability/hyperactivity (33%), which was dose-dependent or occurred during the titration phase; gastrointestinal disturbances were recorded in only one case (6.7%). Our results are in line with the literature, as side effects were reported in 10–47% of patients and encompassed somnolence, decreased appetite, diarrhea, transaminase elevations, fatigue, rash, sleep disorders, and infections (23, 29). In our cohort, only one patient has discontinued CBD treatment due to a lack of clinical benefit (6.7%). Cases of treatment discontinuation due to lack of effect have already been reported in other studies in literature; real-world studies have documented discontinuation in 37% of treated patients (43).

This study has several limitations that should be considered when interpreting the findings. The retrospective design may introduce biases related to data collection and the reliance on medical records and caregiver reports, potentially affecting the accuracy and consistency of the findings. The small sample size, which reduces statistical power, limits the further stratification by clinical variables (i.e., genetic etiology, epilepsy type) and affects the generalizability of our findings to the broader pediatric DRE population. Although improvements in seizure frequency and socio-environmental participation were documented, the assessment relied on subjective tools such as the CGI scale. In retrospective studies, this subjectivity—combined with potentially incomplete data—may increase the risk of bias. Another limitation lies in the heterogeneity of the cohort, including differences in genetic profiles and baseline characteristics, which complicates the interpretation of treatment response variability. Furthermore, the retrospective design also precluded monitoring of CBD metabolite serum levels (i.e., 7-OH-CBD, 7-COOH-CBD, 6-OH-CBD), limiting correlations between pharmacokinetics and clinical outcomes (44). Moreover, technical details of the genetic analyses, such as sequencing depth, were not available in the medical records, further limiting the ability to fully characterize the molecular findings. Given the role of several genes in epileptic encephalopathies (45), further research is also needed to investigate the correlation between genetic variants and treatment outcomes in order to implement more effective and personalized treatment approaches. Finally, the single-center nature of this study limits their applicability to broader populations with varying access to healthcare and treatment protocols. Future multicenter, prospective studies with larger sample sizes and standardized methodologies are necessary to validate these results.