Individuals aged 1–17 years who weighed between ≥ 9 and ≤ 90 kg and had treatment-resistant epilepsy were eligible for inclusion. Epilepsy was considered “treatment resistant” if patients had recurrent seizures despite adequate trials of three or more antiepileptic drugs and one or more prior adequate treatment courses with two or more antiepileptic drugs in combination. Patients were required to have been on a stable-dose antiepileptic drug regimen for ≥ 30 days prior to day 0 and to remain on that regimen for the entire duration of the study. Vagus nerve stimulation and adherence to a ketogenic diet were not counted as part of the antiepileptic drug regimen in this trial. However, the settings for vagus nerve stimulator treatment had to remain stable during the study. Similarly, for individuals on a ketogenic diet, the fat-to-carbohydrate ratio and daily protein intake needed to remain stable during the study. Additional inclusion criteria were use of contraception by both male and female individuals of childbearing age, lack of pregnancy in female individuals, willingness to comply with study procedures, and general good health, as determined by the investigator. Patients were excluded if they had any clinically significant abnormality, suicidal ideation or history of attempted suicide, history of allergic reaction to study drug, or known infection with hepatitis B or C or human immunodeficiency virus. The initiation of felbamate < 6 months before the screening visit and the use of any cannabinoids (e.g., cannabidiol, hemp oil, marijuana) in the 30 days before screening were not permitted. Patients with electrocardiogram results that were considered clinically significant by the investigator at time of enrollment, or a history of such results, were excluded.
Because cannabidiol inhibits or is a substrate for some members of the cytochrome P450 (CYP) protein family, particularly CYP2C19, and others such as CYP3A4, CYP3A5, and CYP3A7 [11, 23, 24], patients who received CYP inhibitors, inducers, or sensitive substrates with a narrow therapeutic index (Electronic Supplementary Material) were excluded from participation, as were patients with any disorder or history of a condition (e.g., malabsorption) that could have interfered with drug absorption, distribution, metabolism, or excretion. Patients were asked to refrain from consuming grapefruit, Seville oranges, and products made from these fruits (e.g., grapefruit juice, Seville orange marmalade).
This open-label, multiple-ascending dose, phase I/II study consisted of an up to 28-day screening phase, a 10-day treatment phase, and a 7-day follow-up period (Fig. 1). Patients who completed the study could be enrolled in an open-label extension study; data from that study will be published in a follow-on manuscript. Patients were randomly assigned to one of three cohorts: cannabidiol oral solution of 10 mg/kg/day (80 mg/mL; cohort 1), 20 mg/kg/day (300 mg/mL; cohort 2), or 40 mg/kg/day (300 mg/mL; cohort 3). These total daily doses were selected based on the tolerability and safety profiles reported for various cannabidiol product concentrations  and the anticipated body weight range of participants. The active ingredient in the cannabidiol oral solution is a synthetic pharmaceutical-grade cannabidiol produced by INSYS Manufacturing LLC (Chandler, AZ, USA), according to Current Good Manufacturing Practice. The three dosing cohorts were evaluated sequentially, starting with the 10-mg/kg cohort. Enrollment of approximately 20 patients per cohort was targeted, with each cohort consisting of five infants (aged 1 to < 2 years), nine children (aged 2 to < 12 years), and six adolescents (aged 12 to ≤ 17 years). Based on compatibility testing conducted by the sponsor, patients were allowed to receive cannabidiol oral solution via a gastronomy tube, if needed.
On day 1, patients received a single dose (morning; 5, 10, or 20 mg/kg) followed by a preset volume of water or clear liquid; no doses were received on days 2 and 3, and then multiple doses (morning and evening dose) were received on days 4 through 10 (10-, 20-, or 40-mg/kg total daily dose split into two daily doses; Fig. 1). The parent(s) or caregiver(s) of each eligible patient were offered the opportunity for all visits to be completed in an inpatient setting if they preferred. Otherwise, the following inpatient schedule was used: patients were admitted as inpatients on the final screening day (day 0) and remained in the medical facility at least until they received the morning dose on day 8. Patients were readmitted as inpatients on the morning of day 10 and released on the morning of day 11 (first day of the follow-up period) after completing the study assessments.
Pharmacokinetic sampling schemes were adjusted based on age to avoid excessive blood loss. All blood samples were collected in 2-mL potassium ethylene diamine tetra acetic acid (K2-EDTA) plasma tubes for patients aged < 2 years and in 4-mL K2-EDTA plasma tubes for patients aged ≥ 2 years. For the PK analysis of cannabidiol and its primary metabolite 7-hydroxy-cannabidiol (7-OH cannabidiol) concentrations, serial blood samples were collected on day 1 pre-dose (for patients aged 2 to < 6 years and patients aged 6 to ≤ 17 years) and at 2, 4, 8, and 12 h post-dose (for all patients; Table 1). Additional samples were obtained at 1, 3, 16, 24 (day 2), and 48 h (day 3) after initial dosing on day 1 in patients aged 2 to < 6 years and patients aged 6 to ≤ 17 years. Samples were also taken at 6, 36, and 72 h (before the scheduled morning cannabidiol dose on day 4) after initial dosing on day 1 in patients aged 6 to ≤ 17 years. On day 10 (multiple dose), plasma was collected pre-dose and at 2, 4, 8, and 12 h (before administration of the second daily dose) post-dose in all patients. On day 10, additional samples were collected at 6 h (age 6 to ≤ 17 years only), and 1, 3, and 24 h post-dose in patients aged ≥ 2 years (Table 1). In addition, samples for trough cannabidiol and 7-OH cannabidiol concentrations were evaluated on day 6 (for patients aged 12 to ≤ 17 years), day 8 (for patients aged 2 to ≤ 17 years), and day 9 (for patients aged 6 to ≤ 17 years) prior to the morning dose.
On plasma collection days, with the exception of preset volumes of water or clear liquid to be consumed immediately after drug administration, patients were not to have liquid or solid food for 1 h (age < 2 years) or 2 h (age ≥ 2 years) before and after drug administration.
Cannabidiol has been shown to impact the metabolism of clobazam in pediatric patients, resulting in elevated plasma concentrations of norclobazam, an active metabolite of clobazam [21, 26]. Therefore, plasma concentrations of clobazam and norclobazam were determined on days 1, 8, and 10 before dosing in patients aged ≥ 2 years who were receiving clobazam as part of their established antiepileptic drug regimen treatment.
Plasma concentrations of cannabidiol and 7-OH cannabidiol were determined using an API 5000™ liquid chromatography-mass spectrometry system (AB Sciex, Concord, ON, Canada) equipped with a high-performance liquid chromatography column. The procedure has been validated to detect cannabidiol and 7-OH cannabidiol concentrations ranging from 0.100 to 50.0 ng/mL, based on the analysis of 0.2 mL of plasma. Clobazam and norclobazam concentrations were analyzed with a validated assessment range from 1.00 to 500 ng/mL, based on the analysis of 0.1 mL of plasma. For both cannabidiol and clobazam, the quantitation was performed using a weighted 1/x2 linear least-squares regression analysis generated from calibration standards. Quality control samples from high, medium, and low pools were processed with each sample run; the sample run was validated when at least two-thirds of the qualifying quality control samples were within 15% of their theoretical values and ≥ 50% of quality control samples at each level met this criterion.
Safety assessments, including monitoring of treatment-emergent AEs and vital signs, were conducted throughout the study. Electrocardiogram measurements were obtained at screening and on days 1, 4, 8, and 11 before treatment administration. Clinical serum chemistry assessments included alanine aminotransferase, albumin, alkaline phosphatase, aspartate aminotransferase, bicarbonate, bilirubin (total and direct), blood urea nitrogen, calcium, chloride, creatinine, follicle-stimulating hormone, glucose, phosphorus, potassium, protein (total), sodium, and uric acid concentrations.
The PK population included all patients who received at least one dose of the study medication and had at least one post-dose cannabidiol and/or 7-OH cannabidiol plasma concentration measurement. The safety population included all patients who received at least one dose of the study medication. Pharmacokinetic analyses were performed using Phoenix® WinNonlin® Version 6.4 (Certara L.P., Princeton, NJ, USA) or SAS® Version 9.4 or higher (SAS Institute, Inc., Cary, NC, USA). A non-compartmental method was used to derive all PK parameters. Pharmacokinetic data were summarized by study day and scheduled time and stratified by dose using descriptive statistics (sample size [n], mean, standard deviation, coefficient of variation, median, minimum, maximum, and geometric mean and geometric coefficient of variation [as appropriate for PK parameters]). All observed data were analyzed; missing values were not imputed.