The study was approved by the King’s College London Research Ethics Committee (RESCMR-16/17-4163). All participants provided written informed consent and the study was conducted in compliance with the principles of Good Clinical Practice, the Declaration of Helsinki (1996). The study was registered on Open Science Framework (https://osf.io/kt3f7) and clinicaltrials.gov (NCT05170217).
Design
This randomised, double-blind, four-arm, within-subjects study was conducted at the NIHR Wellcome Trust Clinical Research Facility (CRF) at King’s College Hospital, London, UK (randomisation and masking described in Appendix pp2). Participants attended a baseline session, followed by four experimental visits, with a minimum one-week wash-out period between each experimental visit (average duration between experiments was 24 days).
Participants
Forty-six healthy volunteers (age 21–50 years), who had used cannabis at least once in the past, but had not used cannabis >1/week over the last 12 months, had never used synthetic cannabinoids, and did not have a substance use disorder were recruited. Additional inclusion/exclusion criteria are listed in Appendix pp2.
Procedure (Fig. 1)
At baseline, participants were assessed for study eligibility, and practiced the inhalation procedure. At baseline and all experimental visits, urine drug and pregnancy screen as well as alcohol and carbon monoxide breath tests (<10 ppm CO to verify 12 h tobacco abstinence) were completed. Participants were asked to avoid using cannabis and all other illicit drugs during the entire course of the study, including the periods between sessions.
Timeline of baseline and experimental sessions (baseline did not include bloods or return to sobriety).
Prior to each experimental visit participants had their usual breakfast and amount of caffeine – caffeine was not allowed again until completion of cognitive tests. An intravenous cannula was inserted before participants were administered vaporised cannabis (detailed below). Fifteen minutes after the completion of cannabis inhalation, participants completed a battery of cognitive tasks (30–35 min). This was followed by assessments of pleasurable responses to cannabis as well as a ‘hospital walk’ (15 min), a task previously been found to increase paranoia following THC [14]. In this task, participants were given £2 to purchase an item of their choice from a till operator in the hospital shop and to ask for a receipt before returning to the CRF. The research team observed from a distance for safety purposes. Participants were then given lunch and enough of a break to allow any intoxicating effects to wear off. When participants felt that at least 90% of the drug effect had subsided they completed the psychological questionnaires (CAPE, PSI and SSPS, detailed below) and a semi-structured clinical interview (PANSS-P, detailed below). This approach allows the scales to capture all the symptoms which have occurred throughout the experiment, as opposed to those that are evident at a particular time point. We have previously found that assessing participants after the maximal phase of acute intoxication has subsided increases the likelihood of them disclosing delusional thoughts or suspiciousness [10, 15]. Participants were discharged after a field sobriety test, having been informed of safety protocols, and provided with a 24 h emergency number.
Study drug and administration
The study drug was provided in the form of granulated cannabis inflorescence by Bedrocan BV (Netherlands) produced in accordance with Good Manufacturing Practice and confirms to the European Medicines Agency’s contaminant levels for products used in the respiratory tract. Each cannabis dose consisted of 10 mg of THC (two standard THC units [16]) and either 0 mg, 10 mg, 20 mg, or 30 mg of CBD. Participants were given preparations with CBD:THC ratios of 0:1, 1:1, 2:1, and 3:1, in a random order across visits. Bedrocan (22.6% THC, 0.1% CBD), Bedrolite (7.5% CBD, 0.3% THC) and Bedrocan placebo (<0.01% THC) were used to provide cannabis containing THC, CBD and placebo, respectively. The placebo cannabis was added to ensure that all preparations had the same weight (see Appendix pp4).
Cannabis preparations were administered using a Volcano® Medic Vaporiser (Storz-Bickel GmbH, Tüttlingen, Germany). Each preparation was vaporised at 210 °C into a transparent polythene bag. This temperature has been found to maximise cannabinoid delivery [17]. Once filled, the transparent bag was encased with an opaque bag to ensure blinding (a higher CBD:THC ratio produces a denser vapour). Inhalation was standardised by asking participants to hold their breath for 8 s before exhaling, with an 8 s break between inhalations (as described in [18]). Participants were asked to inhale a comfortable amount of vapour on each inhalation to minimise the risk of loss of study drug through coughing. The procedure continued until the contents of two bags had been emptied – all participants successfully inhaled the entire contents of both bags on all visits. The inhalation duration of each visit was recorded, and the severity of participant coughing was rated by the researchers using a visual analogue scale. A cup of warm lemon and honey water was provided to help with the abrasiveness of cannabis inhalation.
Blood collection and analysis
Venous blood samples were taken before drug administration, and at 0, 5, 15, and 90 min following the final exhalation, alongside blood pressure, heart rate and temperature. The concentration of Δ9-THC, 11-OH-Δ9-THC (OH-THC), 11-COOH-Δ9-THC (COOH-THC), CBD and 7-OH-CBD were determined using high performance LC/MS at the Mass Spectrometry Facility, KCL [19].
Cognitive tasks
Hopkins verbal learning task—Revised (HVLT-R) [20]
A researcher read out a list of 12 words to the participant, who then repeated the list back. This was repeated over three trials, with the total number of words recalled indexing immediate recall. 20–25 min later participants were asked to recall the words again, indexing delayed recall. The percentage of correctly recalled words indexed retention. Recalled words that were related to the words in the original list, but not part of it, were defined as intrusions. Repetitions referred to the number of times a correctly recalled word was repeated. A different word list was used on each study visit and the order was randomised.
Forward and reverse digit span
Digit span is a measure of verbal working memory and attention, involving the recall of sequences of numbers with increasing length (WAIS-III). Beginning with three digits on forward and two digits on reverse, the task ceased when the participant failed two consecutive attempts at a number sequence.
Spatial N-back [21]
Participants responded to a visual stimulus appearing in one of eight locations, with task demand varied across 0-back, 1-back, and 2-back conditions. Participants were required to indicate (by pressing a Yes or No button) whether the stimulus appeared at the 12 o’clock position (0-back), the same position as the previous visual stimulus (1-back), or the same position as the visual stimulus two previous (2-back).
Psychological measures
Positive and negative syndrome scale—positive subscale (PANSS-P) [22]
The PANSS-P is an investigator-rated semi-structured interview, which assesses positive psychotic symptoms (delusions, conceptual disorganisation, hallucinations, hyperactivity, grandiosity, suspiciousness, and hostility). Information from this assessment was supplemented by the researcher’s observations of, and interactions with the participant, while they were intoxicated.
State social paranoia scale (SSPS) [23]
The SSPS was used to assess persecutory thoughts.
Community assessment of Psychic Experiences—state (CAPE-state) [24]
The CAPE-state is a self-rated scale and was used to assess psychotic-like experiences.
Psychotomimetic states inventory (PSI) [25]
The PSI questionnaire was used to assess psychotic-like experiences following the use of cannabis use.
Visual analogue scales (VAS)
VAS were used to measure subjective effects along a continuum. Participants marked on a 100 mm horizontal line to indicate the level of a given feeling at that moment (0 mm ‘Not at all’ to 100 mm ‘Extremely’). The feeling states included: ‘feel drug effect’, ‘like drug effect’, ‘want more drug’, ‘mentally impaired’, ‘dry mouth’, ‘enhanced sound perception’, ‘enhanced colour perception’, ‘want food’, ‘want alcohol’, ‘high’, ‘calm and relaxed’, ‘tired’, ‘anxious’, ‘paranoid’, ‘stoned’, and ‘pleasure’. VAS were administered 5 times over the course of the experimental session: pre-drug, 10 min post-drug, after cognitive assessment, after the hospital walk, and finally before discharge. In order to explore drug effects over time, area under the curve (AUC) analyses we included as well as peak effects.
Pleasurable responses
Pleasurable effects of cannabis were assessed by the participant rating their enjoyment of a piece of either milk (Marabou) or dark (Lindt 70%) chocolate, and a self-selected piece of music, on a visual analogue scale (VAS), ranging from −5 to +5 on a 100 mm line. The centre of the line (indicated by 0) indicates that the chocolate and music is enjoyed as much as it was at baseline. A negative score indicates that they were enjoyed less compared to baseline, while a positive score indicates that they were more enjoyable.
Statistical analysis
According to our power calculation, at 80% power and Bonferroni adjusted alpha <0.008, a sample size of n = 45 will give a target ES of d = 0.5 as a minimum difference of interest for any of the 6 comparisons. The full power calculation for the study is presented in Appendix pp3.
The effect of THC was determined by comparing outcome scores from the baseline visit with those following administration with THC alone (0:1) using paired t-tests. For the primary analysis, we used linear mixed models to assess the effect of varying the CBD:THC ratio on delayed recall on the HVLT-R. The four CBD:THC ratios (0:1, 1:1, 2:1, 3:1) were included as a fixed effect, with participant as a random effect to account for the dependency between repeated measures. All 6 contrasts were of interest (0:1 vs 1:1, 0:1 vs 2:1, 0:1 vs 3:1, 1:1 vs 2:1, 1:1 vs 3:1, 2:1 vs 3:1) and alpha was set according to the results of our power calculation at p < 0.008 with the expectation that modulatory effects of CBD could emerge in any one of these comparisons. The same analysis was used for secondary pharmacokinetic, cognitive, psychological, pleasurable, and physiological outcomes. To account for any potential order effects, sensitivity analyses were conducted adding visit into the model as a fixed effect.
For pharmacokinetics, VAS scores and physiological outcomes, both peak effects (0 min for pharmacokinetic and physiological outcomes) and area under the curve (AUC) were investigated. For the AUC analyses, values were baseline corrected before using the spline method using the bayestestR package (version 0.7.5.1) [26]. Potential differences in VAS scores for ‘feel paranoid’ between the ‘post-cognition’ and ‘post-walk’ timepoints were assessed using paired t-tests to assess the effect of the walk on paranoia.
The relationships between both inhalation time and coughing with peak plasma THC and CBD, in addition with their respective AUCs, were assessed using Pearson’s correlation coefficients.
We additionally categorised clinically significant psychotic-like reactions as increases in PANSS scores from baseline of ≥3 points, as in previous studies due to floor effects [27, 28]. Similarly, we categorised any increase in SSPS score from baseline. The difference in the frequency of these reactions across CBD:THC ratios was analysed using Pearson’s Chi-square test.
Multiple imputation chain equations (MICE) were used to impute missing values in pharmacokinetic, cognitive, pleasurable, and physiological outcomes using the mice package (version 3.13.0) [29], following no detection of deviation from missing completely at random (MCAR) based on Little’s MCAR test.
All analyses were conducted using R version 3.5.3. lme4 (version 1.1-26) [30] was used to fit the linear mixed effects models and estimated marginal mean (EMM) contrasts were calculated using the emmeans package (version 1.5.2-1) [31].
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