Compliance with Ethical Standards
This trial was conducted in accordance with International Conference on Harmonisation Good Clinical Practice guidelines and ethical principles that have their origin in the Declaration of Helsinki. Protocols were approved by an Independent Ethics Committee (Foundation Evaluation of Ethics in Biomedical Research, Assen, The Netherlands) before eligibility screening. Written informed consent was obtained from each subject before any trial-related procedures were performed.
Healthy adult male or female subjects between 18 and 45 years of age with a body mass index between 18 and 28.0 kg/m2 were enrolled. Female subjects were of non-childbearing potential or were non-pregnant and non-lactating at each visit.
The trial was conducted between 27 May 2015 and 2 October 2015 at one site in The Netherlands. There were three arms to this trial: a single ascending dose (SAD) arm, a multiple dose (MD) arm, and an integrated food effect (FE) arm. Each arm investigated the safety, tolerability, and PK of a pharmaceutical formulation of highly purified CBD derived from Cannabis sativa L. plant in oral solution (100 mg/mL; Epidiolex® in the US; GW Research Ltd, Cambridge, UK), supplied as 100 mL in 105 mL amber glass bottles, and delivered orally using a syringe.
Single Ascending Dose Arm
The SAD arm was double-blind, randomized, and placebo-controlled. Four groups of eight healthy subjects were enrolled and received a single oral dose of 1500, 3000, 4500, or 6000 mg CBD (Groups 1–4, respectively; n = 6 per group) or matching placebo (Groups 1–4; n = 2 per group) after an overnight fast. Administration of each successive dose depended on the safety and tolerability of previous doses.
Multiple Dose Arm
The MD arm was double-blind, randomized, and placebo-controlled. A separate cohort of 24 subjects was enrolled, and two groups of 12 healthy subjects received multiple oral doses of 750 or 1500 mg CBD (n = 9 per group) or matching placebo (n = 3 per group) twice daily under fasted conditions. Morning doses were administered after an overnight fast of at least 10 h, and evening doses were administered after a fast of at least 2 h. Subjects received CBD or placebo twice daily for 6 days, with a single dose on the morning of day 7.
Food Effect Arm
The integrated FE arm was randomized (to a period; fed then fasted, or fasted then fed) and open-label. Twelve randomly selected subjects from the SAD arm, irrespective of group (excluding subjects randomized to the 6000 mg group) or investigational medicinal product (IMP) taken in the SAD arm (CBD or placebo), crossed over into the FE arm to receive a single oral dose of 1500 mg CBD in either the fed (high-fat breakfast) or fasted state before crossing over. The washout period between IMP administration in the SAD and integrated FE arms was ≥ 7 days.
In the fasted state, subjects were fasted overnight for ≥ 10 h prior to CBD administration. In the fed state, subjects were fasted overnight for ≥ 10 h then received a standardized high-fat breakfast of 918 kcal consisting of two fried eggs in 15 g butter (approximately 100 g), one portion of bacon (40 g), one portion of fried potatoes (115 g), two slices of toasted wheat bread with 15 g butter, and one glass (240 mL) of high-fat milk. The meal was consumed within 20 min, and subjects were dosed 30 min after starting breakfast, after which they fasted for 4 h. The washout period between CBD doses in the fed and fasted periods of the FE arm was 10 days.
All Trial Arms
Blood sampling for PK purposes was performed from pre-dose up to 72 h after IMP administration. Safety assessments were performed throughout the trial. Subjects were discharged after 48 (SAD and FE arms) or 72 h (MD arm) following the final IMP administration, and follow-up visits occurred 5–10 days after the last PK blood sample had been taken.
Determination of Sample Size
No prospective calculations of statistical power were made. The sample size was selected to provide information on safety, tolerability, and PK.
As THC is present as a trace impurity in the CBD formulation under development, plasma concentrations of THC and its metabolites, 11-hydroxy-Δ9-tetrahydrocannabinol (11-OH-THC) and 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (11-COOH-THC), were also determined. Reference and internal standards for 6-hydroxy-cannabidiol (6-OH-CBD), 7-hydroxy-cannabidiol (7-OH-CBD), 7-carboxy-cannabidiol (7-COOH-CBD), THC, 11-OH-THC, and 11-COOH-THC bioanalysis were supplied by GW Pharma Ltd (Cambridge, UK), Cerilliant (Round Rock, TX, USA) or BDG Synthesis (CA, USA).
Plasma Sample Preparations
CBD and metabolite samples were extracted from plasma by protein precipitation with isopropyl alcohol and acetonitrile. THC and metabolite samples were extracted by liquid–liquid extraction.
It was discovered that there may have been an overestimation of 7-OH-CBD (30–50% based on comparison with new standards) concentrations in SAD samples due to technical issues with reference standard material. As a result, data obtained for 7-OH-CBD in the SAD arm should be considered with a degree of caution.
Bioanalysis and Pharmacokinetic (PK) Assessment
High-performance liquid chromatography (HPLC) with tandem mass spectrometry was performed using a Waters Acquity (Waters Corporation, Milford, MA, USA) system for HPLC, and Sciex API5000 (AB Sciex Pte. Ltd, Singapore [for analysis of CBD and metabolites]) or Waters Xevo TQS (Waters Corporation) and Sciex API5000 (AB Sciex Pte. Ltd [for analysis of THC and metabolites]) systems for mass spectrometry. These were used to quantify concentrations of CBD, THC and their metabolites 6-OH-CBD, 7-OH-CBD, 7-COOH-CBD, 11-OH-THC and 11-COOH-THC in human plasma using 200 µL sample volumes. Chromatographic separations were performed on an Acquity BEH Phenyl column (internal diameter 1.7 μm, 2.1 × 100 mm). The selectivity of the HPLC method was checked by comparing chromatograms from blank plasma samples with chromatograms from spiked samples to ensure that there were no potential interfering peaks with any analyte under investigation. Selectivity was ensured at the lower limit of quantification (LLOQ) for each analyte. Cross-interference between analytes was also assessed with samples spiked at the analytes’ upper level of quantification. The lower and upper limits of quantification for each analyte are given in Table 1.
The precision [coefficient of variation (%CV)] and accuracy [relative error (RE%)/mean % different (Bias%)] of the HPLC method was acceptable for all analytes [≤ 15% (20% at the LLOQ)]. Recovery was > 90% for CBD, 6-OH-CBD, 7-OH-CBD and 7-COOH-CBD, and was considered acceptable. The recovery of THC, 11-OH-THC and 11-COOH-THC was considered adequate (between 56.3 and 124%).
It should be noted that of the 1808 samples analyzed, 45 (2.5%) for 6-OH-CBD and 20 (1.1%) for 7-OH-CBD may have been affected by a hemolysis matrix effect, but this would not have any notable effect on the results shown.
Blood samples were taken from subjects via an indwelling intravenous catheter or direct venepuncture, and centrifuged for 10 min at 2600 g, at 18°C. The resultant plasma was stored in a freezer at − 80°C. Blood samples for PK analysis were taken at the following time points:
SAD arm: pre-dose and 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 8, 12, 24 and 48 h post-dose.
MD arm, as below:
Day 1: pre-morning dose, and 0.5, 1, 2, 2.5, 3, 4, 5, 6, 8 and 12 h post-morning dose, then at 0.5, 1, 2, 2.5, 3, 4, 5 and 12 h post-evening dose.
Days 3, 4, 5 and 6: pre-morning dose.
Day 7: pre-dose and 0.5, 1, 2, 2.5, 3, 4, 5, 6, 8, 12, 24, 48 and 72 h post-dose.
FE arm: pre-dose and 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 8, 12, 24, 48, and 72 h post-dose.
PK parameters were derived by non-compartmental analysis using WinNonlin® version 6.3. PK parameters evaluated included area under the plasma concentration-time curve from time zero to time t (AUCt), area under the concentration-time curve from time zero to infinity (AUC∞), area under the plasma concentration-time curve over a dosing interval (AUCτ; where τ is the dosing interval), maximum measured plasma concentration (Cmax), time to maximum plasma concentration (tmax), terminal elimination half-life (t½,z), oral clearance of drug from plasma (CL/F), apparent volume of distribution (Vz/F), elimination rate constant (from the central compartment; Kel), and accumulation ratio (Rac), based on AUCτ of day 7 versus day 1 (MD part only), measured using non-compartmental analysis.
The safety and tolerability of CBD were evaluated by recording the incidence and severity of AEs throughout the trial, review of clinical laboratory tests, vital signs, electrocardiogram (ECG), physical examination, sleep disruption 0–10 numerical rating scale (NRS) score, Epworth Sleepiness Scale (ESS) score, Cannabis Withdrawal Scale (CWS) scores, and Columbia-Suicide Severity Rating Scale (C-SSRS) questionnaire (MD part of the trial only). Safety data were recorded in case report forms by contract research organization staff at the clinical trial site.
The primary objective of this study was to evaluate the safety and tolerability of a SAD and MD of CBD. Descriptive statistics of subject demographics and outcomes were based on the safety analysis set (all subjects who received one or more doses of CBD).
Secondary objectives were to assess the PK of SAD and MD regimens of CBD, as well as the effect of food on CBD PK. Secondary safety assessments included sleep disruption 0–10 NRS, ESS, CWS, and C-SSRS, which were summarized descriptively or listed.
The PK parameters of CBD, THC and its metabolites were calculated for the PK analysis set (all subjects who received one or more doses of CBD and provided sufficient bioanalytical assessments to calculate reliable estimates of the PK parameters), using Phoenix® WinNonlin® version 6.3 or higher (Pharsight Corporation, Princeton, NJ, USA). Statistical testing was two-sided and used the 5% significance level. Non-compartmental methods estimated PK parameters for all analytes with sufficient data above LLOQ from the concentration-time profiles for the PK analysis set. A regression power model, relating log-transformed Cmax and area under the curve parameters to log-transformed dose, was used to investigate dose proportionality. Individual and geometric mean dose-normalized Cmax, AUCt and AUC∞ were plotted against dose level. To analyze the FE, an analysis of variance, with a model including fixed effects for treatment, period, and sequence, and a random effect for subject within sequence, was performed on natural log-transformed Cmax, AUCt and AUC∞ data. The back-transformed least-squares means for each treatment, and the ratio of least-squares geometric means between the test treatment and the reference, was calculated (fed over fasted). The FE arm was not powered to demonstrate statistical significance. Nevertheless, the potential for an FE would be suggested if the 90% confidence interval (CI) did not contain 1.