About 20%–30% of patients with epilepsy are considered resistant to antiepileptic drugs (GBD-2016; Kitschen et al., 2023). Consequently, alternative therapies have been explored, including products derived from cannabis species, such as cannabidiol (CBD), which does not have the addictive properties of tetrahydrocannabinol (THC). Since 2018, the US Food and Drug Administration (FDA) has approved the use of a liquid formulation of CBD (100 mg/mL) in sesame oil (flavored). In combination with clobazam, it is indicated for the treatment of patients aged 2 years and older who have Lennox–Gastaut syndrome or Dravet syndrome. It is also prescribed to treat tuberous sclerosis complex with other epilepsy treatments in patients aged 2 years and older. These rare types of epilepsy begin in childhood and may last into adulthood (Golub and Reddy, 2021; Reddy, 2023).

Randomized clinical trials have shown the efficacy and safety of these drugs in children and adults who have seizures that are related to these conditions and are challenging to manage. Furthermore, non-randomized trials have also shown promising results for other types of epilepsy. However, the mechanisms of how these patients respond to the highly variable CBD are not entirely understood (Reddy, 2023). Research indicates that CBD has complicated pharmacokinetics and inconsistent bioavailability, and there is a lack of biomarkers for predicting its therapeutic effects.

The bioavailability of CBD varies greatly according to the route and mode of administration. In clinical trials and research studies, CBD has generally been administered orally in capsule form or dissolved in oily solutions. It can also be administered sublingually or intranasally. A wide range of oral doses has been reported in the literature, with most doses ranging from 10 mg/day to 800 mg/day (in high-dose cases) (Millar et al., 2020; Huestis, 2007; Fasinu et al., 2016). The absorption of CBD in the gastrointestinal tract is erratic, and the resulting pharmacokinetic profile is variable. These characteristics are probably due to the poor aqueous solubility of CBD, as well as the method of administration (food vs. beverage), co-administration with additional ingredients, and preparation (as a ready-made product, it is better than a powder that the consumer must mix with liquid before ingestion).

Once ingested, the circulating concentration of CBD is influenced by absorption rates in the gut, breakdown during first-pass metabolism, and potentially by the body size and composition of the consumer. The bioavailability from oral administration was estimated as 6% due to significant first-pass metabolism (Fasinu et al., 2016; Ujváry and Hanús, 2016). Further complications arise when patients combine CBD with other medications or nutrients that could potentially interact with it, which can limit its efficacy and possibly enhance its toxicity (Zendulka et al., 2016)

CBD is metabolized by cytochrome P450 isoenzymes (CYP450), particularly CYP2C19 and CYP3A4 and to a lesser extent CYP1A1, CYP1A2, CYP2C9, CYP2D6, and CYP3A5. The glucuronyltransferase activities of UDP-glucuronosyltransferase (UGT) enzymes UGT1A7, UGT1A9, and UGT2B7 also contribute (Harvey and Brown, 1991; reviewed by Chen et al., 2024). These reactions first oxidize the CBD molecule and form 7-OH-CBD, followed by other modifications that result in over 100 identifiable metabolites in several organisms (Jiang et al., 2011). CYP3A4 converts 7-OH-CBD into 7-COOH-CBD, a major inactive compound in human blood. Notably, CYP3A4 and CYP2C9 enzymes demonstrate higher affinity for CBD than other anti-seizure medication (ASM), which inhibit its metabolism by utilizing the same enzyme system (Figure 1; Harvey and Brown, 1991; Jiang et al., 2011; Ujvary and Hanus, 2016).

Previous reports have discussed how cannabinoids induce CYP450 enzymes to produce several metabolites. An in vitro study by Doohan et al. (2021) indicated that most cannabinoids did not affect CYP2D6 activity when dextromethorphan was used as a substrate. CBD partially inhibited CYP3A4 enzymatic activity when using triazolam, testosterone, or nifedipine as substrates. When observing CYP2C9-mediated tolbutamide metabolism, CBD caused more than 50% inhibition. CBD also impacted bupropion-mediated CYP2B6 activity, leading to an inhibition index (CI50) of 6.2 ± 1.7 μM. CBD also strongly inhibited CYPC19 activity, which is mediated by (S)-mephenytoin metabolism, with a CI50 of 2.1 ± 0.5 μM (Doohan et al., 2021).

As part of the drug-resistant epilepsy (DRE) hypothesis, pharmacokinetic theory suggests that the concentration of ASM entering the brain changes due to the unique expression of specific allelic variants of CYP450 metabolizing enzymes, which metabolize around 90% of all drugs (Tang et al., 2017). Genetic changes are associated with variations in drug responses, ranging from complete ineffectiveness to susceptibility to adverse drug reactions (Berger and Iyengar, 2011). In a study on pediatric patients with epilepsy, the single-nucleotide polymorphism (SNP) rs2242480 in CYP3A4 (previously known as the CYP3A4 *1G allele) was associated with drug responsiveness. This SNP was identified in 75% of drug responders and 43.5% of non-responders (Feria-Romero et al., 2023).

Thus, the aim of the present study was to determine whether six CYP variants are associated with response to CBD combined with ASM among pediatric and adult patients with DRE, to describe the benefit in seizure control when CBD is added to ASM, and to identify how this treatment is used in these populations.

Pharmacogenetics is a factor that can influence the relative contribution of specific CYP450 enzymes to a drug-metabolism reaction. May result in significant interindividual and interethnic variability in the metabolism, disposition, and clinical response of various therapeutic agents (Tan et al., 2010). When a new drug is introduced, it is important to identify its response in the population. Therefore, the aim of this study has been to identify the genetic polymorphisms in CYP450 and assess their correlation with the anti-seizure drug metabolism. The results could help to explain the response to CBD, which was investigated among Mexican adults and pediatric patients with DRE from two hospitals. Our cohort was small due to difficulties in patient access to CBD and adherence to a treatment plan of at least 6 months, which were part of our inclusion criteria.

In addition to distinguishing drug-resistant patients from those responsive to CBD and other medications, we also investigated the most common genetic variations in Mexican pediatric patients with DRE, as previously identified in another study (Feria-Romero et al., 2023). We found no significant difference regarding seizure types between those who responded to the treatment and those who did not. This includes 2 cases of Lennox–Gastaut syndrome (4.25%), where there was no response, as well as 4 cases (8.5%) with juvenile myoclonic epilepsy (JME), which responded to the treatment. This suggests that the seizure type does not necessarily dictate the variability in the CBD treatment response.

Some syndromes appear to be more responsive to CBD treatment. This was shown in a randomized double-blind clinical trial involving 120 children and young adults with Dravet syndrome and drug-resistant seizures. That study demonstrated that oral administration of CBD (20 mg/kg/day) concomitantly with already established pharmacotherapy decreased seizure frequency compared to a placebo, but it increased the incidence of adverse effects (diarrhea, vomiting, fatigue, pyrexia, somnolence, and changes in liver function). The same study highlighted that the most frequently co-administered drugs were clobazam (65%), valproic acid/valproate (59%), stiripentol (42%), levetiracetam (28%), and topiramate (26%) (Lopera et al., 2022).

In the population included in this study, the most commonly used drugs in both groups were levetiracetam, valproic acid, and lamotrigine (Table 2). In combination with CBD, the adverse events reported were minimal, and the best responses were observed. It should be noted that CBD is also administered to patients who are treated with medications that have their own side-effect profiles. Co-administration increases the possibility of overlapping profiles through metabolic or transport pathways (Brown and Winterstein, 2019).

Regarding the type of seizure, patients with focal seizures were the least responsive to CBD (60%). In the case of CBD^pos, patients with the 3 types of seizures (focal, generalized, and both origins) were the most responsive. In contrast, a separate report on adult patients with drug-resistant focal epilepsy found that 87% experienced a reduction in seizures by over 50% (Kochen et al., 2023). In comparison, 11% with focal seizures had no effect from CBD. In other research, an average seizure reduction of 67.8% was observed in a sample of 78 patients (median age 24 years) with various structural and genetic conditions who were receiving an average of three ASM treatments and 14 months of CBD. Furthermore, 68.8% of this group experienced a reduction in seizure frequency greater than 50%, while 11.5% became seizure-free (Espinosa-Jovel et al., 2023).

Another study on epileptic encephalopathies and cases of focal or multifocal epilepsy found that 29.4% of patients with encephalopathies and 22.7% of patients with focal or combined epilepsy responded positively to CBD (Perriguey et al., 2024). When comparing these studies to ours, it is clear that CBD can offer advantageous effects for several types of DRE, particularly in adult patients. Nevertheless, only a select few studies have reported patients becoming seizure-free after adding CBD treatment to their regimen (Espinosa-Jovel et al., 2023; Perriguey et al., 2024; Kochen et al., 2023).

The SNP rs2242480 is located in the intronic region C99763843T on chromosome 7 and was initially assigned the CYP3A4 *1G allele. The Pharmacogene Variation Consortium (www.pharmvar.org) reassigned it as allele CYP3A4 *36 to align with the official terminology before removing it from their list. Current studies regarding this variant refer solely to the SNP. This variant had the most notable difference between CBD^neg and CBD^pos, but the difference was not significant. However, its prevalence was higher in these patients than in drug-resistant (30%) and controlled (33%) patients in a previous study (Feria Romero et al., 2023).

Previous research has linked this SNP with varying levels of drug efficacy and toxicity, and it has been associated with poor response to carbamazepine (CBZ) in patients with epilepsy (Zhao et al., 2021). A meta-analysis of the rs2242480 polymorphism in CYP3A4 showed significant associations with the plasma CBZ concentration in the co-dominant heterozygous model (AG vs. GG, standardized mean difference (SMD) = −0.24, 95% confidence interval (CI) = −0.4 to −0.09, P = 0.002), dominant model (AA + AG vs. GG, SMD = −0.21, 95% CI = −0.36 to −0.06, P = 0.007), and overdominant model (AA + AG vs. CI = −0.36 to −0.06, P = 0.007). The results indicate that the G allele of this SNP could decrease the plasma CBZ concentration in cases of epilepsy (Zhang et al., 2021).

In contrast, the rs2242480 variant has been identified in patients who respond to valproic acid therapy (Feng et al., 2018). Most patients included in the study were treated with valproic acid in combination with other pharmacotherapies, including CBD. Valproic acid was associated with adverse events in 8.5% of patients when administered with CBD (Table 2). This polymorphism has also been associated with response to other drugs, such as nervous-system depressants. The pharmacogenetics of this SNP was evaluated in a sample of 200 gynecological patients requiring general fentanyl analgesia. Patients with the Ho mut (AA) genotype responded better to fentanyl than those with the He (GA) or Ho wild-type (GG) genotype (Yan et al., 2018).

In a subsequent study involving 59 patients with gastric or intestinal cancer, the efficacy of opioid anesthesia during laparoscopic surgery was examined (oxycodone, n = 30; sufentanil, n = 29). The results indicated a more significant response to sufentanil in patients with the AA genotype of SNP rs2242480 than those with the GA+AA genotypes (p < 0.05), but this was not observed for oxycodone (Pu et al., 2019). In the current study, the focus was on adjuvant therapy with CBD in anti-seizure treatment. The presence of this genetic variant could be considered a risk factor for poor response to CBD treatment, which contradicts previous observations in a sample of patients with controlled and drug-resistant epilepsy (Feria-Romero et al., 2023). However, this tendency needs to be corroborated with more patients.

CYP2B6 is the only gene in the human CYP2B subfamily that encodes a functional enzyme (Nebert et al., 2013). The CYP2B6 consists of nine exons and is located on chromosome 19 at position 19q13.2. It is primarily expressed in the liver and catalyzes demethylation, hydroxylation, and oxidation to form active or inactive metabolites (Hidestrand et al., 2001; Ekins et al., 2008; Zhang et al., 2017). Substrates of CYP2B6 are found in ∼23 different therapeutic classes that are commonly used around the world.

When comparing the genetic variants with a Latin-American population of European descent (LA2) of rs8192709 and rs8192719, only the SNP rs8192709 had a significant difference (p < 0.0001) between the Alt allele (T) in the study population (0.4852) compared to LA2 (0.0256). Previous work found that the rate of the Alt allele (T) of this polymorphism was 7% in a Mexican pediatric population with solid embryonal tumors and poor response to ifosfamide treatment (Torres-Espindola et al., 2021), which is a lower rate than our results. Therefore, our results must be confirmed in the future with other molecular biology techniques. However, the clinical function of genetic variants in rs8192709 and rs8192719 has not been reported.

The CYP2D6 enzyme metabolizes about 20% of medications used in psychiatry, pain management, oncology, and cardiology (Taylor et al., 2020). Thus, pharmacogenetic consortia such as the Pharmacogenetic Guided Opioid Therapy advise against using these drugs for patients with specific genetic markers (Agullo et al., 2023). The SNP rs3892097 identifies the non-functional CYP2D6 *4 allele, which is linked to poor drug response, adverse effects, and comorbidities, although it was present in only 14% of patients in the CBD^pos group. Moreover, it has been described that CBD can inhibit various CYP enzymes, leading to potentially significant drug interactions, particularly for ASM (Brown and Winterstein, 2019; Landmark and Brandl, 2020).

CBD may notably inhibit CYP2C9, CYP2D6, and CYP2B6 (Bornheim et al., 1993; Jiang et al., 2013; Yamaori et al., 2010; Zendulka et al., 2016). Genetic alterations in these cytochromes directly affect the pharmacokinetics and metabolism of antiepileptic drugs (Kobayashi et al., 1999; Tan et al., 2010). Identifying altered cytochromes is particularly relevant for patients with DRE since these proteins are present in the blood–brain barrier and could affect drug metabolism directly at the target site (Ghosh et al., 2010; 2011).

The most important limitation of this study was the sample size. One of the reasons for the small sample size is the cost of commercial CBD (5,000 USD per year). Not all patients can afford it, and some use homemade preparations, in which the concentration of CBD is unknown. This limits the opportunity to meet the inclusion criteria of this work. In addition, although its medicinal use has been authorized, in public health programs, through which we recruited the patients, it is not included in the anti-seizure medications listed in the basic table for the treatment of epilepsy. Another limitation of this study was the lack of identification of the variant without CYP2C19*2 enzymatic activity in this population sample, which is discriminated by the detection of the SNP rs4244285. The reason was that in the analysis of polymorphisms by massive sequencing of a previous sample, we observed a lower frequency of the rs4244285 polymorphism in patients with drug-resistant epilepsy (0.16), compared to patients with controlled epilepsy (0.25). On the other hand, variants *3, *8, *9, and *10 without enzymatic activity and identified by polymorphisms rs4986893, rs41291556, rs17884712, and rs6413438, were not present in the previous population sample used as reference. Finally, the absence of the CYP2D6 enzyme known as the *5 variant or copies of CYP2D6 variants (xN) was also not evaluated in this work, which should be analyzed in a future study using other technologies.

In conclusion, treatment with CBD reduces seizures in most patients by 68%, regardless of the cause or type of seizure. The rs8192709 SNP may be a genetic contributor to drug resistance, but this finding needs to be confirmed through additional molecular biology techniques, given its high prevalence compared to other populations. The CYP3A4 *1/rs2242480 genotype was linked to a poor response to CBD in patients with DRE, which may be due to a stronger affinity between this enzyme and CBD, leading to its inactivation. This SNP is particularly relevant due to a higher occurrence in cases of DRE that do not respond to CBD in combination with ASM.