medRxiv preprint doi: https://doi.org/10.1101/2021.05.17.21257345; this version posted May 19, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license .

Title: Delta-9- and in psychosis: A balancing act of the principal phyto- on human brain and behavior? Short title: CBD-THC interaction in psychosis Suhas Ganesh1,2, Jose Cortes-Briones1,2, Ashley M. Schnakenberg Martin1,2, Patrick D Skosnik1,2, Deepak C D’Souza1,2, Mohini Ranganathan*1,2 1 Department of Psychiatry, Yale University School of Medicine 2 VA Connecticut Healthcare System, West Haven, CT 06516

*Corresponding Author Corresponding Author Contact Information: Mohini Ranganathan Associate Professor Department of Psychiatry Yale University School of Medicine VA Connecticut Healthcare System/116A 950 Campbell Ave West Haven, CT 06516 Email: [email protected] Tel: 203-932-5711 X 2546 Fax: 203-937-4860

Word Count: Abstract: 200 Manuscript without online methods: 4463 Figures: main – 3, supplement – 8 Tables: main – 2, supplement – 2

Keywords: delta-9-tetrahydrocannabinol, cannabidiol, psychosis, electrophysiology, neural noise, Lempel-Ziv complexity

1

NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice. medRxiv preprint doi: https://doi.org/10.1101/2021.05.17.21257345; this version posted May 19, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license .

Title: Delta-9-tetrahydrocannabinol and cannabidiol in psychosis: A balancing act of the principal phyto-cannabinoids on human brain and behavior? Abstract:

Delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD) are the principal phyto-

cannabinoids in the plant. The differential and possibly antagonistic effects of these

compounds on specific brain and behavioral responses, and the mechanisms underlying their

effects have generated extensive interest in pre-clinical and clinical neuroscience investigations.

In this double-blind randomized placebo-controlled counterbalanced human laboratory

experiment, we examined the effects of three different dose ratios of CBD: THC (1:1, 2:1 and

3:1) on ‘neural noise’, an electrophysiological biomarker of psychosis known to be sensitive to

cannabinoids as well as subjective and psychotomimetic effects. Interestingly, the lowest

CBD:THC ratio (1:1) resulted in maximal attenuation of both THC induced psychotomimetic

effects (PANSS positive - ATS = 7.83, df = 1, pcorr = 0.015) and neural noise (ATS = 8.83, df = 1,

pcorr = 0.009) with an inverse-linear dose response relationship. Further, in line with previous

studies, addition of CBD did not reduce the subjective experience of THC induced “high” (p >

0.05 for all CBD doses).

These novel results demonstrate that CBD attenuates THC induced subjective and objective

effects relevant to psychosis- but in a dose/ratio dependent manner. Given the increasing global

trend of cannabis liberalization and application for medical indications, these results assume

considerable significance given the potential dose related interactions of these key phyto-

cannabinoids.

2 medRxiv preprint doi: https://doi.org/10.1101/2021.05.17.21257345; this version posted May 19, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license .

Introduction

For millennia, , the cannabis plant, has been used for medicinal and

recreational purposes (1) and is one of the most widely used substances today (2, 3). Cannabis

contains over 500 distinct chemical compounds and ~120 phyto-cannabinoids (4) including Δ9-

tetrahydrocannabinol (THC), responsible for the psychoactive , and

cannabidiol (CBD), investigated for its therapeutic effects (5, 6).

The acute effects of cannabis include a wide array of subjective, behavioral, and physiological

effects including euphoria, relaxation, increased appetite, anxiolysis or increased anxiety,

tachycardia, altered spatial and time perception, paranoia, conceptual disorganization, and

hallucinations (7-9).

A large body of evidence supports an association between cannabis exposure and psychosis

outcomes ranging from acute mild psychosis-like symptoms to the development of a frank

psychotic disorder. While data supporting the association between cannabis and psychotic

disorders come from epidemiological studies and clinical observations, human laboratory

studies (HLS) have complemented this literature to provide temporally related evidence of an

association between cannabinoids and psychosis symptoms. Importantly, HLS also permit the

examination of underlying mechanisms and interactions between cannabinoids that are not

feasible in epidemiological studies. Over the past several decades, HLS have carefully

characterized the acute dose-related subjective, cognitive, physiological, neuroendocrine, and

psychophysiological effects of THC (10-16). These experimental studies demonstrate that THC

administration reliably induces transient psychotic-like symptoms (10, 11), cognitive deficits (11-

13), and psychomotor impairments (14, 15).

Unlike THC, CBD has no psychotomimetic or rewarding effects but appears to have sedative

and anxiolytic properties (17-21). Evidence suggests that CBD may have distinct

neurobiological effects compared to THC (22, 23). Interestingly, preclinical data suggest that

CBD appears to ameliorate psychotomimetic symptoms produced by THC (24), sensory gating

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deficits induced by NMDA receptor antagonists (25) and receptor agonists (26),

and hyperlocomotion induced by amphetamine, cocaine, and ketamine, and stereotyped

behavior induced by apomorphine in mice (27). Experimental studies in humans have

demonstrated mixed findings regarding its antipsychotic properties of CBD (28-33).

Cannabis that is high in THC and low in CBD is associated with a greater risk of psychosis (34-

36) and some studies suggest that CBD may attenuate the psychotomimetic and anxiogenic

effects of THC. Pretreatment with CBD has been observed to block the psychotomimetic effects

of THC (26, 37, 38), although with mixed, dose-dependent effects (39). The literature on the

behavioral, cognitive, and neurophysiological effects of THC, CBD, and their combination have

been previously reviewed by us and others (5, 6). We briefly present this literature relevant to

psychosis outcomes below.

In a small within-subject study, Bhattacharya et al (23) demonstrated that pretreatment with

intravenous (IV) CBD (5 mg) attenuated the psychotomimetic effects of IV THC (1.25 mg). The

absence of pharmacokinetic interactions suggested that the observed attenuation was possibly

due to receptor-level interactions (23). In a between-subject, placebo-controlled study (n=48),

Englund et al (40) examined the effects of oral CBD pretreatment (600 mg) on the psychotic

symptoms induced by IV THC (1.5 mg) and found that a significantly lower proportion of

subjects experienced clinically significant THC induced positive symptoms and episodic memory

impairment with CBD pretreatment (40). Hindocha et al (41) examined the interaction of inhaled

THC (8 mg) and CBD (16 mg) on human facial affect recognition in 48 volunteers. The authors

noted that while the THC-CBD combination induced a subjective experience of feeling ‘stoned’

to the same extent as THC, the combination resulted in a significantly lower impairment in affect

processing (41). Haney et al (42) examined the effects of pretreatment with four doses of oral

CBD (0, 200, 400, 800 mg) on reinforcing, positive subjective, and physiological effects of

smoked THC (5.3% - 5.8%) in 31 cannabis smokers and noted that none of the CBD doses

altered any of the THC effects examined. More recently, Solowij et al (43) examined the effect

4 medRxiv preprint doi: https://doi.org/10.1101/2021.05.17.21257345; this version posted May 19, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license .

of low (4 mg) and high (400 mg) doses of vaporized CBD on the intoxicating effects of vaporized

THC (8 mg) in 36 frequent and infrequent cannabis users. Interestingly, the authors noted that

the low dose of CBD potentiated the intoxicating effects of THC while the high dose of CBD

attenuated the intoxicating effects of THC (43), suggesting that the proportion of THC and CBD

in cannabis may influence subjective and behavioral effects.

Thus far, most of the investigations of THC and CBD interaction in human studies have focused

on either behavioral or cognitive outcomes with only a few investigating neurophysiological

outcomes. In a previous study from our group, we found no differences between THC and

THC+CBD on P50 gating ratio (S2/S1) and evoked power (44). Preclinical studies on the

mechanisms of the interaction between THC and CBD also report mixed results (45-50). The

inherent challenges in modelling an acute behavioral response such as psychotomimetic effects

in preclinical models after exposure to clinically translatable doses of THC and CBD further

complicates the interpretability of these results.

In summary, the emerging data available thus far indicate that CBD may attenuate THC induced

psychotomimetic effects while sparing the other subjective experiences. However, how

CBD:THC ratio influences these interactions remains unstudied. Importantly, despite several

reports comparing THC and CBD effects on psychophysiological outcomes, there are limited

data examining the effects of CBD on THC induced psychosis relevant EEG outcomes. These

interactive effects may also be sensitive to the dose and route of administration of THC and

CBD. For instance, there is significantly greater variability in blood levels with the oral route

(42) compared to IV (23).

In this study, we aimed to examine the effect of IV CBD on a range of IV THC induced

subjective psychotomimetic and rewarding effects as well as a specific psychophysiological

index of psychosis in healthy human participants as described below.

5 medRxiv preprint doi: https://doi.org/10.1101/2021.05.17.21257345; this version posted May 19, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license .

We have previously shown that neural noise, i.e., the randomness of signals, as captured by

electroencephalography (EEG) signals with Lempel-Ziv complexity (LZC), is strongly related to

the acute psychosis-like effects of THC (51). LZC is a nonlinear measure from information

theory designed to assess the randomness (noise) of signals by counting the minimal number of

distinct signal patterns (words) that are required to fully reconstruct the signal without

information loss (52). LZC approaches zero in signals with regular and recurrent patterns (small

number of distinct patterns) and approaches one as the randomness (large number of distinct

patterns) of signals increases. In addition to predicting THC-induced psychotomimetic

symptoms, increased LZC has been demonstrated in schizophrenia (53, 54) and specifically in

patients in their early stages of illness with higher positive symptoms (55), potentially serving as

an EEG biomarker of psychosis.

We hypothesized that CBD would attenuate THC-induced psychotomimetic effects and neural

noise but not its rewarding effects. We further examined whether these interactions were dose

related by testing three different doses of CBD (CBD:THC ratio 1:1, 2:1, 3:1). Lastly, we

examined correlations between CBD’s effects on the subjective and electrophysiological

psychosis-relevant effects of THC i.e. a) THC-induced psychotomimetic effects and b) THC-

induced increase in neural noise.

Results

Sample Profile: The sample consisted of 28 (13 females) healthy volunteers with a median

(IQR) age of 25.5 (10.25) years. The demographic and clinical details including the race,

handedness, education, anthropometric measures, cannabis, and tobacco use patterns,

schizotypal personality and psychosis proneness scores are presented in table 1. None of the

subjects were cannabis naïve; 7 (25%) subjects reported cannabis use of two or more times per

week. Of the 28 subjects who participated in phase-1, 10 (35.71 %) participated in phase-2 and

received two additional doses of CBD:THC (1:1 and 3:1; supplementary figure 1). The

6 medRxiv preprint doi: https://doi.org/10.1101/2021.05.17.21257345; this version posted May 19, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license .

demographic and clinical details are presented in table 1 and supplementary table 2. There

were no statistically significant group differences between the 18 subjects who participated only

in the phase-1 and the 10 subjects who participated in both phases of the study (supplementary

table 2).

Behavioral, Subjective, and Physiological Measures

The outcomes of Positive and Negative Syndrome Scale (PANSS) positive, negative, general,

and total scores; Clinician Administered Dissociative Symptom Sale (CADSS) - patient rated

(PR) and clinician rated (CR) subscale scores; and subjective experiences measured with

Visual Analog Scale (VAS) – “VAS high” and “VAS anxious”, scores in phase-1 and phase-2 of

the study are listed in table 2.

THC-Induced Psychotomimetic Effects – PANSS Positive Symptom Scores (Table 2):

Phase -1: There was a significant main effect of treatment on PANSS positive change score

(Anova Type Statistic [ATS] = 16.01, df = 2.87, p < 0.00001). Compared to THC, CBD:THC-2:1

resulted in a decreased PANSS positive score, that was not statistically significant in a pairwise

post-hoc comparison (ATS = 0.58, df = 1, p = 0.44) (figure 1a). Thirteen of the 28 subjects had a

clinically meaningful psychotomimetic response to THC. In this ‘responder’ subgroup, the

CBD:THC-2:1 had a significantly lower PANSS positive change score compared to THC alone

(ATS = 9.73, df = 1, p = 0.0018) (figure 1b).

Phase -2: In the analysis of the phase-2 (figure 1c) comparing THC, CBD:THC-1:1, CBD:THC-

2:1, and CBD:THC-3:1, there was a significant main effect of the treatment on the PANSS

positive change score (ATS = 8.73, df = 3.37, p < 0.00001). In post-hoc analysis involving pair-

wise comparisons between THC and the three CBD:THC combinations, the lowest THC-

induced PANSS positive score was noted with CBD:THC-1:1 (ATS = 7.83, df = 1, pcorr = 0.015).

The CBD:THC 2:1 and 3:1 doses also resulted in lower PANSS positive scores compared to the

7 medRxiv preprint doi: https://doi.org/10.1101/2021.05.17.21257345; this version posted May 19, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license .

THC alone, but the differences were not statistically significant. Overall, there was an inverse

dose response relationship between the CBD dose in the CBD:THC combinations and the

magnitude of attenuation of THC-induced positive symptoms (figure 1c).

There were no differences between THC and CBD:THC conditions in the PANSS negative or

general symptom scores (supplementary figure 2 and 3). Although not statistically significant,

consistent with the above results, THC-induced PANSS general symptom score was also

maximally attenuated in the CBD:THC-1:1 condition (ATS = 5.15, df = 1, pcorr = 0.069). For

PANSS total score, phase -1 responder analyses revealed significantly lower scores with

CBD:THC-2:1 condition compared to THC (ATS = 6.78, df = 1, p = 0.009). In phase-2, PANSS

total score was significantly attenuated only in the CBD:THC-1:1 condition (ATS = 11.11, df = 1,

pcorr = 0.002) (table 2; supplementary figure 4).

Perceptual Alterations – CADSS - Patient Rated and Clinician Rated scales (Table 2):

Phase-1: There was no significant main effect on CBD treatment on THC induced perceptual

alterations. CBD:THC-2:1 resulted in a marginal reduction in the THC CADSS PR and CR

scales, but the differences were not statistically significant.

Phase-2: None of the three CBD:THC combinations were significantly different from the THC

condition on CADSS PR or CR. However, the largest relative reduction in THC-induced

perceptual alteration measured by CADSS CR was noted in CBD:THC-1:1 condition when

compared to the 2:1 and 3:1 ratios, consistent with the pattern observed on PANSS positive

scores (table 2, supplementary figures 6, 7).

Subjective Effects – VAS “High” and “Anxious” (Figures 2a and 2b):

“High”: In phase-1, there was a main effect of the treatment on subjective experience of high

(ATS = 43.03, df = 2.24, p <0.00001), but there was no significant difference between the THC

and the CBD:THC-2:1 in the post-hoc analysis (ATS = 0.13, df = 1, p = 0.72).

8 medRxiv preprint doi: https://doi.org/10.1101/2021.05.17.21257345; this version posted May 19, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license .

In phase-2, there was a main effect of treatment (ATS 17.57, df = 2.97, p < 0.00001) on “High”.

However, there was no difference between the THC and the three CBD:THC combinations –

CBD:THC-1:1 (ATS = 2.04, df = 1, pcorr = 0.45), 2:1 (ATS = 0.11, df = 1, pcorr > 0.99) and 3:1

ratios (ATS = 3.96, df = 1, pcorr = 0.14).

“Anxiety”: In phase-1, there was a main effect of treatment but no significant difference

between THC and CBD:THC-2:1 conditions (table 2). However, in phase-2, THC-induced

anxiety was maximally attenuated with the combination of CBD:THC-3:1 and this difference was

statistically significant (ATS = 10.41, df = 1, pcorr = 0.004) (supplementary figure 5).

Physiological Effects: In phase-1, there was a main effect of treatment (ATS = 37.9, df = 2.5,

p = <0.00001) and both THC and CBD:THC-2:1 resulted in a significant increase in heart rate at

+15 minutes. However, there was no difference between THC and CBD:THC-2:1 conditions in

the post hoc analysis (ATS = 2.44, df = 1, p = 0.12).

Similarly, in the phase-2, all treatment conditions resulted in a robust increase in heart rate

compare to placebo but there was no statistically significant difference between THC and

CBD:THC conditions (supplementary figure 8).

Neural Noise (Table 2 and Figures 3a and 3b):

Phase-1: There was a main effect of treatment on LZC with an increase in LZC noted in both

the THC and the CBD:THC-2:1 conditions compared to placebo (ATS = 5.66, df = 2.52, p =

0.0015). Post-hoc analysis revealed that the CBD:THC-2:1 condition had a lower median LZC

compared to THC alone, although the differences were not statistically significant (ATS = 0, df =

1, p = >.05) (figure 3A). A similar pattern was observed among THC PANSS responders, where

we noted a modest but statistically non-significant reduction in the reduction in LZC (ATS = 0.2,

df = 1, p > 0.05).

9 medRxiv preprint doi: https://doi.org/10.1101/2021.05.17.21257345; this version posted May 19, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license .

Phase-2: There was a main effect of treatment (ATS = 2.68, df = 3.3, p = 0.04) and all three

CBD:THC combinations resulted in a lower median LZC compared to THC alone (figure 3B).

Post-hoc analysis revealed that the maximal attenuation of THC-induced increase in LZC was in

the CBD:THC-1:1 condition and this difference was statistically significant (ATS = 8.83, df = 1,

pcorr = 0.009), consistent with the pattern observed with the THC induced PANSS positive

scores.

Relationship Between CBD Attenuation of Positive Symptoms and Attenuation of Neural

Noise:

Correlations between changes in PANSS positive and total scores and changes in LZC were

examined in the phase-1 subjects and phase-1 responders. Small statistically non-significant

positive correlations were noted between change in LZC and change in positive symptoms from

placebo to THC condition in phase-1 (r = 0.13, p > 0.05) and in the subsample of phase-1

responders (r = 0.28, p > 0.05). Among the responders a large, marginally significant correlation

was noted between change in LZC and change in the PANSS total symptoms (r = 0.64, p =

0.07). There were no correlations between change in positive or total symptom scores with

change in LZC from THC to CBD:THC conditions in Phase-1 or Phase-1 responders.

Discussion

We present novel results demonstrating dose dependent interactions between IV CBD and IV

THC on subjective and objective, psychosis and reward related measures, in this double-blind,

randomized, placebo-controlled two-phase human laboratory study in healthy individuals.

Pretreatment with IV CBD 5mg (~ 2:1 CBD: THC ratio) attenuated THC induced

psychotomimetic symptoms without affecting any other subjective, behavioral or cognitive

effects. This attenuation was particularly notable in those ‘responders’ who developed a

clinically meaningful psychotomimetic response to THC alone (≥ 3 increase in PANSS positive

10 medRxiv preprint doi: https://doi.org/10.1101/2021.05.17.21257345; this version posted May 19, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license .

symptoms). Furthermore, we found that the maximal attenuation of THC induced

psychotomimetic symptoms resulted from the lowest tested dose of CBD (2.5 mg) (~ 1:1 CBD:

THC ratio) and an inverse linear dose-response relationship was observed between CBD dose

and attenuation of THC induced psychotomimetic response.

Consistent with the effects on THC induced psychotomimetic response, CBD treatment was

associated with a decrease in THC induced neural noise, a biomarker of drug induced

psychotomimetic effects (51). We observed that the largest decrease in neural noise was also

with the lowest dose of CBD (2.5 mg) (~ 1:1 CBD: THC ratio), similar to the psychotomimetic

response. However, there was no correlation between the decreases in the neural noise and

subjective psychotomimetic effects of THC. Finally, in contrast to psychotomimetic symptoms, it

is noteworthy that none of the doses of CBD decreased the rewarding effects of THC.

The results of this study provide novel insights into the intricate psychopharmacology of

cannabinoids important for future studies on cannabis and cannabinoid psychopharmacology as

discussed below.

CBD and THC Interactive Behavioral Effects: The attenuation of THC induced

psychotomimetic effects by CBD pretreatment, is in agreement with the findings of two earlier

studies (Bhattacharya et al (23), and Englund et al (40)). Both studies used PANSS positive

symptom subscale to measure THC-induced psychotomimetic effects similar to this study.

Bhattacharya et al examined the interactive effects of single doses of IV THC (1.25mg) and IV

CBD (5 mg), while Englund et al. tested 600 mg of oral CBD 210 minutes prior to IV THC (1.5

mg). Given the oral bioavailability of CBD is ~6% (56), the dose in Englund et al, would be

equivalent to ~3.6 mg of IV CBD although, additional variability of the peak plasma

concentration and half-life is recognized with the oral route (21, 56). Thus, both these studies

examined higher CBD:THC ratios ( ~2.4-4:1) compared to ~2:1 in phase-1 of our study and 1:1

and 3:1 in Phase -2. Further, it was the lowest CBD:THC ratio (1:1) examined in phase-2,

11 medRxiv preprint doi: https://doi.org/10.1101/2021.05.17.21257345; this version posted May 19, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license .

associated with the maximal attenuation of THC-induced psychotomimetic effects with an

inverse linear dose response relationship between CBD and THC induced psychotomimetic

effects. These data would suggest that equivalent ratio (1:1) of CBD:THC in herbal cannabis

may result in the least psychotomimetic effects. However, since we did not examine lower

CBD:THC ratio < 1 in this study, it is not possible to estimate the impact of lower CBD

concentrations in most current herbal cannabis.

In contrast to the psychotomimetic response, the experience of subjective effect of high was not

significantly different in any of the three CBD:THC dose ratios. This relative absence of effects

of CBD on ‘high’ and other related subjective effects has been demonstrated previously by

Hindocha et al (41) and Haney et al (42). Using inhalational administration of CBD 16 mg and

THC 8 mg (CBD: THC 2:1), Hindocha et al, noted that the subjective feeling of being ‘stoned’

was not altered by the combination of CBD and THC compared to THC alone. Similarly, Haney

et al, noted that oral CBD (200, 400 and 800 mg) administered 90 minutes prior to smoked

cannabis (5.3-5.8% THC), did not alter the subjective effects of ‘high’ and ‘good effect’

associated with THC. In contrast, Solowij et al (43), reported that low dose vaporized CBD (4

mg) augmented the intoxicating effects of vaporized THC (8 mg) while a higher dose of

vaporized CBD (400 mg) resulted in an attenuation of the intoxicating effects measured with

CADSS clinician-rated subscale. These effects were specifically prominent in the infrequent

cannabis users. In contrast, in our analyses none of the CBD:THC combinations were

significantly different on the CADSS subscale scores. It is of interest that Solowij et al observed

an augmentation of intoxication at a CBD:THC ratio of 1: 2 (THC dominant) and attenuation of

intoxication at a ratio of 50:1- both ratios that we did not test, but suggest that a wide dose

range of CBD:THC should be further studied. Lastly, we noted a statistically significant

difference between the THC and CBD:THC (3:1) condition on subjective effects of anxiety.

Although, both 2.5 mg and 7.5 mg produced greater anxiolysis compared to the 5 mg condition

12 medRxiv preprint doi: https://doi.org/10.1101/2021.05.17.21257345; this version posted May 19, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license .

in the phase-2 analysis (Supplementary figure 5)- this was statistically significant only with the

higher CBD dose. This is consistent with the inverted U-shaped dose-response of CBD on

anxiety that has been previously reported by Linares et al, using 150 mg, 300 mg and 600 mg of

oral cannabidiol in a simulated public speaking test (57).

CBD and THC and Neural Noise: To our knowledge, this is the first study to examine neural

noise (LZC), an electrophysiological correlate of psychosis, to assess the effects of THC, CBD,

and their interaction. Neural noise has been shown to be elevated in acute phases of

schizophrenia (54, 55) and we have previously demonstrated it to be strongly correlated with

THC-induced psychotomimetic effects, specifically with positive- and disorganization-like

symptoms (51). We now demonstrate that CBD-related attenuation of the psychotomimetic

effects of THC was mirrored by a similar attenuation of THC-induced noise. Further,

examination of dose-related effects demonstrates a trend towards an inverted U-shaped

response as CBD 2.5 mg and CBD 7.5 mg resulted in a larger attenuation of neural noise

compared to CBD 5 mg. Future studies in larger samples with a wider range of dose ratios of

CBD:THC will be needed to delineate this nonlinear dose-response relationship. CBD treatment

has been associated with improvement in psychotic symptoms in patients with psychotic

disorders in some clinical trials but not others (58-61). Whether CBD treatment has an effect on

the elevated neural noise observed in acute schizophrenia, has not been reported thus far.

Future studies that integrate psychophysiological measures of neural noise may shed further

light on the potential neural mechanisms by which CBD exerts its antipsychotic effects in

specific subpopulations of psychosis.

Possible Mechanisms of CBD Antagonism of THC-Induced Psychotomimetic Effects:

Several pre-clinical and clinical studies have demonstrated opposing effect of CBD on specific

THC-induced behavioral, cognitive, and biological outcomes. The opposing effects of THC and

CBD on human cognition has been extensively reviewed (5, 6, 62) and the opposing effects on

13 medRxiv preprint doi: https://doi.org/10.1101/2021.05.17.21257345; this version posted May 19, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license .

specific cognitive domains are possibly mediated via differential effects of these compounds on

functional connectivity between prefrontal cortex, hippocampus, and dorsal striatum (63). We

discuss findings from preclinical, molecular, human imaging studies relevant to the opposing

effects of CBD on THC-induced psychotomimetic effects. In rodents, co-administration of CBD

has been demonstrated to attenuate acute THC-induced c-Fos expression in multiple brain

regions (48). c-Fos is one of the immediate early genes with altered expression during learning

and memory and has also been proposed to be a biomarker of schizophrenia (64, 65). More

relevant to the timescale of the psychotomimetic response to THC, acute intra-PFC infusion of

CBD has been shown to reverse the cognitive impairments caused by glutamatergic

antagonism suggesting that the therapeutic effects of CBD may be relevant only during

pathologically aberrant states (47). In contrast, striatal glutamate increase has been shown in

human studies to be a marker of THC-induced acute psychotomimetic response (66). More

recently, a regional specificity in THC-CBD interaction relevant to alleviation of psychotomimetic

response has been demonstrated by Wall et al (67). Using resting state functional magnetic

resonance imaging, the authors noted that CBD:THC combination resulted in alleviation of THC

induced functional connectivity disruption, specifically in the limbic sub-division of the striatum

but not in the associative and sensorimotor sub-striatal networks (67). Taken together, the

findings suggest that restoring cortical-striatal balance of glutamatergic tone may be a potential

underlying mechanism of CBD action in attenuating THC-induced acute psychotomimetic

response.

CBD has been demonstrated to act via various receptor and enzymatic mechanisms in several

biological pathways. In addition to CB1 receptors, CBD has been shown to have effects on the

Adenosine receptor A2A and 5 hydroxy tryptamine receptor 1A (5HT-1A), GPR 55, and several

receptors of the TRPV family in the brain (68-70). However, the antagonism of THC induced

psychotomimetic effects by CBD is possibly mediated through negative allosteric modulation of

14 medRxiv preprint doi: https://doi.org/10.1101/2021.05.17.21257345; this version posted May 19, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license .

CB1 receptor (70). The precise mechanisms of THC and CBD interactions at the receptor level

in the brain still needs to be characterized in future studies that employ both human receptor

imaging studies as well as preclinical studies that permit in vivo assay of molecular and

neurochemical alterations under simultaneous drug exposure conditions.

Clinical and Public Health Relevance of the Findings: The results of our human laboratory

study align with and confirm some of the previous findings in epidemiological studies on the

effect of CBD content in cannabis on psychotic experience in the community. Morgan and

Curran analyzed hair samples in 140 individuals and noted higher schizophrenia-like positive

symptoms in persons with THC-alone detected in hair samples compared to persons with both

THC and CBD (71). In another study, Morgan et al, demonstrated that compared to low

CBD:THC (~ 1:100) cannabis users, high CBD:THC (~1:3) cannabis users had reduced

attentional bias towards food and drug stimuli. However, there were no differences between the

groups in the subjective feeling of being ‘stoned’ (72). The authors suggested that relatively

higher CBD:THC ratio may a have protective effect against developing cannabis dependence.

Schubart et al have also reported an inverse relationship between CBD content and the severity

of positive psychotic experiences in a large Dutch community study. Interestingly, highest CBD

concentration that the authors noted in their sample was CBD:THC of 1:2 and the lowest being

1: 81.5 (73). Taken together, the results of our investigation and these naturalistic studies

suggest that relatively higher CBD:THC ratio up to 1:1 may optimally protect against

psychotomimetic and dependence forming effects while minimally affecting the subjective

affective experiences of THC in cannabis. This hypothesis needs further investigation and has

the potential to inform harm reduction strategies and regulations for marketing of cannabis with

specific CBD:THC ratios especially given the increasing legalization of recreational cannabis

and medicinal cannabinoids in the US and the rest of the world. The attenuation of

psychotomimetic effects of THC at 1:1 ratio of CBD:THC is of particular interest as this is the

15 medRxiv preprint doi: https://doi.org/10.1101/2021.05.17.21257345; this version posted May 19, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license .

ratio of CBD:THC in the currently approved nabiximol preparations for the treatment of spasticity

in Multiple Sclerosis (74, 75).

A few limitations should be considered while interpreting the results of our study. The dose

related effects of CBD (phase 2) were tested in a small sub-sample of subjects. Although we

observed a large and significant effect for CBD attenuation of THC psychotomimetic symptoms

and neural noise at the lowest dose (1:1) tested, this needs to be replicated in a larger study

perhaps with a wider dose range CBD:THC ratios ( for instance ratios <1). Although consistent

with some population-based studies, the generalizability of these results to the effects of

cannabis exposure in the real-world setting remains complicated by the potential moderating

effect of other phyto-cannabinoids and chemical compounds in herbal cannabis. Further, IV

administration of THC and CBD, chosen for more precise and reliable delivery of these

compounds limits the generalizability to the typical smoked or oral routes of cannabis

consumption. This study focused on specific psychosis related behavioral and

psychophysiological interactions of CBD and THC. A recent study has demonstrated a

significant impairment in driving performance up to 2 hours after administration of CBD:THC

(1:1) cannabis (76). Thus, despite potential benefits of 1:1 CBD:THC ratio on psychosis

outcomes, a wider range of motor and real-life performance effects warrants further study.

Finally, future studies need to examine repeated or chronic dosing effects of CBD and THC

combinations to fully understand the range of interactions in humans.

Conclusions: CBD attenuates THC-induced psychotomimetic symptoms in healthy humans

and the effect is more notable in those who develop a robust psychotomimetic response with

THC. Further, amongst the CBD:THC dose ratios studied, a 1:1 ratio produces maximal

attenuation of psychotomimetic effects compared to 2:1 and 3:1 with an inverse linear dose

response. The attenuation of psychotomimetic symptoms is mirrored by a similar attenuation of

THC-induced increase in neural noise, an electrophysiological biomarker of psychosis. Lastly,

16 medRxiv preprint doi: https://doi.org/10.1101/2021.05.17.21257345; this version posted May 19, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license .

none of the CBD doses tested resulted in attenuation of the THC-induced subjective experience

of ‘high’. Future studies in larger samples with a wider range of dose ratios and longer dosing

paradigms are needed to systematically examine the spectrum of interactive effects between

CBD and THC.

Online methods

Study Design: This was a two-phase, double-blind, randomized, counterbalanced, placebo-

controlled cross-over trial. In phase-1, all subjects participated in the four test days where they

received one of 4 drug conditions (Placebo, THC [0.035mg/Kg; ~2.5mg in a 70kg individual],

CBD 5mg, THC ~2.5mg + CBD 5 mg) on each test day separated by at least 3 days. Subjective,

behavioral, physiological, and EEG data were collected before and at several time points after

drug administration on each test day. In phase-2, a subsample of subjects from phase-1

participated in two additional test-days to specifically examine the interactions of THC with two

additional doses of CBD (THC ~2.5mg + CBD 2.5 mg, THC ~2.5mg + CBD 7.5 mg). A

representation of the study design is presented in supplementary figure 1. The study protocol

was approved by the Human Investigations Committee at the Yale University and the Human

Subjects Study committee of the VA Connecticut Healthcare System (VACHS). The study was

carried out at the Neurobiological studies unit of the VACHS under an existing IND for the

administration of THC (#51671) and CBD (#101185).

Participants: The subjects consisted of 28 (13 females) healthy volunteers recruited using

methods consistent with our previous HLS (77, 78). After obtaining written informed consent,

subjects underwent a detailed medical and psychiatric evaluation, laboratory tests,

electrocardiogram, and urine toxicology. Structured Clinical Interview (SCID) for DSM-IV (non-

17 medRxiv preprint doi: https://doi.org/10.1101/2021.05.17.21257345; this version posted May 19, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license .

patient) for healthy controls was conducted to exclude psychiatric diagnoses. General

intelligence was assessed with National Adult Reading Test. Subjects were asked to explicitly

consent to identifying a collateral informant for verification of inclusion/exclusion criteria.

Cannabis naïve individuals were excluded from the study to avoid the theoretical risk of de novo

exposure in the laboratory. Similarly, subjects with major DSM IV axis 1 disorders were

excluded to avoid the potential risk of illness exacerbation.

Drugs - THC Dose, Rate, and Route of Administration: The route, dose, and rate of

administration of THC influence its behavioral and cognitive effects (78). The THC doses used

in our studies (78) produced blood levels comparable to those achieved by recreational

cannabis use and mimic the time course of plasma THC levels associated with the clinical “high”

from smoking 0.5-1.5 of a standard NIDA cannabis cigarette (79-81). Even with standardized-

pace smoking procedures, subjects are still able to titrate the dose of cannabinoids they receive

and in doing so, negate attempts to deliver a uniform dose (82). The IV route of administration

was chosen to reduce inter and intra-individual variability in plasma THC levels with the inhaled

route and to mimic the time course of plasma THC levels associated with the clinical “high” (78).

The dose chosen for this study was based on several previous studies conducted by our group

and has been demonstrated to be well tolerated by subjects while producing transient but

reliable psychotomimetic effects (83). THC [0.035 mg/kg (~ 2.5 mg in a 70 kg individual)], was

administered IV over 20 minutes into a rapidly flowing infusion of normal saline. Similar doses in

previous studies have produced clinically and statistically significant behavioral and cognitive

effects. Placebo administration involved an equal volume - 2ml of 190 proof ethanol.

CBD Dose, Rate, and Route of Administration: In experimental studies, CBD has been safely

administered to healthy humans in doses ranging from 15 mg to 600mg (oral) per day (20, 84-

97) alone and in combination with THC and is known to have been tolerated well with no

significant adverse effects. Previous studies have also demonstrated that IV CBD is well

18 medRxiv preprint doi: https://doi.org/10.1101/2021.05.17.21257345; this version posted May 19, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license .

tolerated at the dose ranges used in this study (2.5 – 7.5 mg), and have produced effects

similar to oral CBD (23). While most studies have administered CBD orally, the low and

variable oral bioavailability of CBD presents a limitation. For instance, the oral absorption of

CBD of 600 mg produces plasma levels of 4.7 (SD=7) and 17 (SD=29) ng/ml at 1 and 2 hours

post-administration, respectively (22). Thus, in this study 3 active doses of IV CBD were

administered, on separate test days. In an average weighing individual, the three doses of

CBD, 2.5mg, 5mg, and 7.5mg IV, resulted in CBD:THC ratios equivalent to 1:1, 2:1, and 3:1,

respectively. Additional details regarding the source, preparation, storage, stability, and

quantitative analysis of cannabidiol doses are presented in the supplementary methods.

Test-Day Procedures: An overview of the schedule of test-days is presented in supplementary

table 1. Subjects were instructed to fast overnight and to report to the test facility at 8 am.

Subjects were not permitted to drive to and from the test facility. Exclusions (like urine drug and

pregnancy test) were confirmed on each test morning. IV lines were inserted, and baseline

behavioral, subjective, physiological, and neurochemical assessments described below were

collected. CBD (active or placebo) was administered over 2 minutes immediately followed by

THC (active or placebo) through an IV line over 20 minutes. Outcome measures were repeated

periodically as described below and enumerated in supplementary table 1.

Behavioral and Subjective Measures

Psychosis and perceptual alterations: Positive, negative, and general symptoms were

assessed using the positive, negative, and general symptoms subscales of the Positive and

Negative Syndrome Scale (PANSS) (98). Perceptual alterations were measured using the

Clinician Administered Dissociative Symptoms Scale (CADSS) (99). The CADSS is a scale

consisting of 19 self-report items and 8 clinician-rated items (0 = not at all, 4 = extremely) that

we have previously shown to be sensitive to the psychotomimetic effects of THC (78) (fig. 1).

19 medRxiv preprint doi: https://doi.org/10.1101/2021.05.17.21257345; this version posted May 19, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license .

The scale captures alterations in environmental/time/body perception, feelings of unreality, and

memory impairment.

Subjective Effects of Cannabis: Feeling states associated with cannabis intoxication were

measured using a self-reported visual analog scale of four feeling states (“high” and “anxious”)

associated with cannabis effects (99-101). Subjects were asked to score the perceived intensity

of their current feeling states by making a mark on a 100 mm line printed on a paper sheet (0

mm from the line’s left end = ‘not at all’; 100 mm from the left = ‘extremely’). These data were

captured to validate that the experiment is relevant to cannabis effects and to determine how

CBD pretreatment alters the subjective effects of THC.

Long-term follow-up of cannabis use: Subjects were evaluated 1 and 3 months after study

completion to document whether study participation altered future (78).

EEG Paradigm and Data Acquisition

Electroencephalography (EEG) data were recorded at the scalp using a 64-channel (extended

10-20 system) ActiveTwo Biosemi system (Biosemi B.V., Amsterdam, Netherlands) at a sampling

rate of 1024 Hz with an on-line low-pass filter of 256 Hz to prevent aliasing of high frequencies.

All electrodes were referenced during recording to a common-mode signal (CMS) electrode

between and then subsequently re-referenced to the nose offline.

During recordings, subjects were seated in a comfortable chair in front of a monitor in a

darkened, sound-attenuated, electromagnetically shielded booth while listening to auditory

stimuli were delivered through via insert earphones (Etymotic Research, Inc., Elk Grove Village,

IL, USA) as part of an auditory oddball task described elsewhere (102, 103). Briefly, the task

consisted of a series of frequent (83.33%) ‘standard’ tones, infrequent (8.33%) ‘target’ tones,

and infrequent (8.33%) task-irrelevant ‘novel’ distractor sounds presented with a 1250 ms

stimulus onset asynchrony in three separate blocks of 180 stimuli each. Standard tones were

500 ms in duration, target tones were 500 ms in duration, and novel distractor sounds ranged

20 medRxiv preprint doi: https://doi.org/10.1101/2021.05.17.21257345; this version posted May 19, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license .

between 175 and 250 ms in duration. All stimuli were presented binaurally using an intensity of

80 dB SPL (C weighting). The task was presented using Presentation software

(Neurobehavioral Systems, Berkeley, CA, USA). EEG collection was continuously monitored on

a screen outside the recording booth to detect drowsiness. Only EEG data preceding each

stimulus were used for neural noise (LZC) estimation.

EEG Preprocessing

In order to minimize the confounding effects that artifactual contamination could have on

measuring neural noise in EEG signals, raw data were cleaned and only Cz was used for

analyses, given that this electrode tends to be the least contaminated by artifactual noise

resulting from muscle activity (104). Raw EEG data were preprocessed following standard

procedures as described elsewhere (51, 103). Briefly, EEG data were band-pass filtered (0.5-

100Hz) with a windowed sinc FIR filter (-6dB/Hz), 60Hz line noise was removed using a multi-

tapering technique (105), muscle artifacts were cleaned using a canonical correlation analysis-

based blind source separation technique (106), and eye movement and blink artifacts were

removed with an adaptive filtering technique (107). Data were segmented in 1250ms epochs

time-locked to stimulation onset, with a 250ms pre-stimulus baseline. A ±95 μV criterion was

used to remove bad trials.

Neural Noise (LZC) Computation:

Following previous work from our group on the psychotic-like effects of THC (51), we calculated

neural noise (LZC) on the pre-stimulus (-250s to -1ms) preparatory baseline period of each trial

of an P300 oddball task. In that study, we showed that LZC during each trial’s the

expectation/focused attention period was strongly related to the psychotomimetic effects of THC

(51). The calculation of LZC of a real-valued signal requires to encode it into a symbolic

sequence. Following previous work (51), we used the median-crossing approach which is robust

21 medRxiv preprint doi: https://doi.org/10.1101/2021.05.17.21257345; this version posted May 19, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license .

against outliers to binarize the pre-stimulus segment (-250ms to -1ms) of each trial’s EEG signal

at Cz. LZC was calculated using Lempel and Ziv’s exhaustive parsing algorithm (52) and then

normalized between 0 and 1. Each subject’s LZC value was obtained by averaging LZC across

trials. EEG analyses were conducted using Matlab 2020b (MathWorks, Natick, MA,USA)

custom scripts and the EEGLab toolbox (108).

Statistical Analyses:

The distribution of each outcome variable was visually examined for normality with histogram,

box, and whisker plot and normality was tested using Kolmogorov-Smirnov statistics. The

behavioral and subjective data (PANSS, CADSS, VAS feeling states) were examined as the

change from baseline at post-infusion 15 minutes (P15) time-point, as both the physiological

and psychological effects of THC have been shown to peak around this time in IV infusion

studies (78). This time-point was also closest to the post-infusion EEG measure at +25 minutes.

The PANSS positive symptoms, VAS high, and EEG neural noise (LZC) were tested as the

primary outcomes and the remaining PANSS measures, CADSS patient and clinician rated

scores, VAS scores and physiological measures of outcome (pulse, blood pressure) were

examined as secondary outcomes. The effects of treatment condition on the outcome measures

were tested using linear mixed model with treatment as a within subject fixed factor and subject

ID included in the model as a random variable. For the analysis of phase-1 data, the drug-

condition factor involved four levels (Placebo, THC, CBD, CBD:THC-2:1) and in phase-2 it

involved testing two additional levels in a subsample of participants (CBD:THC-1:1 CBD:THC-

3:1). As previously described (40, 109), we examined the CBD attenuation of positive symptoms

in a subsample of “THC-responders”, i.e. those who had a clinically significant psychotomimetic

response to THC, defined as a change in PANSS positive score ≥ +3 with THC alone in phase-

1. In the event of non-normal distribution of outcome residuals or a singular model fit due to

22 medRxiv preprint doi: https://doi.org/10.1101/2021.05.17.21257345; this version posted May 19, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license .

invariant variance-covariance structure, non-parametric model for the analysis of factorial

experiments (Brunner et al, 2002) (110) was used to examine the drug-condition effects. Both

these models offer the advantage of greater flexibility in fitting correlated factor structure in

repeated measures and are optimal when data are missing at random. Further, the latter non-

parametric models are also robust for smaller sample sizes (111-113). Post-hoc comparisons

were defined a priori for the comparison between THC and CBD:THC-2:1 condition in the

analysis of phase-1 data and between THC and the three different CBD doses in the CBD:THC

combinations for the analysis of phase-2 data. Bonferroni corrections were applied for multiple

comparisons within outcome domains. The relationship between CBD attenuation of THC-

induced positive symptoms and neural noise was visually examined with scatter plots and the

relationship was measured using Pearson’s correlation coefficient. Data were plotted and

analyzed in R version 4.0.1 (R: A language and environment for statistical computing) using

packages Tidyverse (114), lme4 (115), lmerTest (116), and nparLD (113).

23 medRxiv preprint doi: https://doi.org/10.1101/2021.05.17.21257345; this version posted May 19, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license .

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Figure legends Figure 1: Box and jitter plots showing the change in PANSS positive symptoms from baseline the 15 minutes post infusion across the 4 treatment conditions. 1A. Phase-1 responders – a statistically significant group difference is noted between THC and CBD:THC(2:1) conditions; 1B. Phase-2 – a statistically significant group difference is noted between THC and CBD:THC(1:1) conditions but not the 2:1 and the 3:1 combinations. Figure 2: Box and jitter plots showing the change in subjective effects of high measured on Visual Analog Scale from baseline the 15 minutes post infusion across the 4 treatment conditions. 2A. Phase-1 – No significant group difference is noted between THC and CBD:THC(2:1) conditions; 2B. Phase-2 – No significant group differences are noted between THC and CBD:THC(1:1, 2:1 and 3:1) combinations. Figure 3: Box and jitter plots showing post infusion from neural noise measured as Limpelziv complexity across the 4 treatment conditions. 3A. Phase-1 – No significant group difference is noted between THC and CBD:THC(2:1) conditions. A similar pattern was noted in phase 1 responders (not shown); 1B. Phase-2 – a statistically significant group difference is noted between THC and CBD:THC(1:1) conditions but not the 2:1 and the 3:1 combinations.

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THCCBD main manuscript tables Table 1: Demographic variables Variable Summary – Phase 1 Summary – Phase 2 N = 28 N = 10 (subset of 28) Age – median (IQR) 25.5 (10.25) 25 (10) Gender – female - n (%) 12 (42.86%) 4 (40%) Race – Caucasian: black: other 16:8:4 6:3:1 (n) Education – mean (SD) 15.78 (2.41) 15 (2.24) Handedness – right – n (%) 27 (96.42%) 10 (100%) Cannabis – frequent user – n (%) 4 (14.28%) 0 (0) Smoker – n (%) 5 (17.86%) 2 (20%) BMI – mean (SD) 26.1 (4.8) 25.9 (4.84) NART – IQ – mean (SD) 113.3 (5.63) 111.8 (5.8) Psychosis proneness - median 19.5 (17.5) 20.5 (10.25) (IQR) SPQ score - median (IQR) 3 (5) 2.5 (6.75)

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Table 2: Behavioral, physiological and neural noise outcome measures

Phase 1 Phase 2 Variable Main effect of Post-hoc Main effect Post-hoc Post-hoc Post-hoc drug condition (THC vs of drug (THC vs (THC vs (THC vs - CBD 5 condition CBD 2.5 CBD 5 CBD 7.5 mg) mg) mg) mg)

PANSS 16.01 0.58 8.73 7.83 1.696 1.25 positive (<0.00001) (0.44) (<0.00001) (0.005) * (0.19) (0.26) ATS (p value) PANSS 21.2 9.73 NA NA NA NA positive (<0.00001) (0.0018) * responder # ATS (p value) PANSS 8.93 (0.00005) 0.84 4.91 (0.002) 1.35 3.38 0.36 negative (0.35) (0.24) (0.066) (0.55) ATS (p value) PANSS 26.74, 3.4 18.08 5.16 1.41 0.08 general (0.00001) (0.065) (<0.00001) (0.023) (0.24) (0.78) ATS (p value) PANSS total 25.82 1.6 (0.21) 17.58 11.11 1.48 0.002 ATS (p (<0.00001) (<0.00001) (0.0009) (0.22) (0.96) value) * PANSS total 18.64 6.78 NA NA NA NA responder # (<0.00001) (0.0092) * ATS (p value) CADSS – 17.25 0.07 4.48 (0.006) 0.67 1.12 0.21 PR (<0.00001) (0.79) (0.41) (0.29) (0.65) ATS (p value) CADSS – 29.98 1.93 11.11 3.03 0.91 3.02 CR (<0.00001) (0.16) (<0.00001) (0.08) (0.34) (0.08) ATS (p value) VAS – high 43.02 0.13 17.57 0.15 0.11 0.15 (<0.00001) (0.72) (<0.00001) (0.71) (0.73) (0.698)

33 medRxiv preprint doi: https://doi.org/10.1101/2021.05.17.21257345; this version posted May 19, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license .

ATS (p value) VAS – 7.24 (0.0002) 0.08 2.69 (0.04) 4.01 0.68 10.41 anxiety (0.78) (0.04) (0.41) (0.001) * ATS (p value) Pulse rate 37.9 2.44 8.85 0.09 2.53 0.08 ATS (p (<0.00001) (0.12) (<0.00001) (0.76) (0.11) (0.78) value) Neural noise 5.66 (0.0015) 0 (NA) 2.68 (0.04) 8.83 0.33 0.76 (LZC) (0.003) * (0.56) (0.38) ATS (p value)

# the primary outcome measures of PANSS positive and PANSS total scores were tested in a subgroup of responders in phase 1 with a PANSS positive response to THC with a score ≥ 3. * significant effects in post-hoc analysis. For phase-1, THC and CBD:THC-2:1 conditions were defined a priori for post hoc comparison. For phase-2, THC and 3 CBD:THC dose conditions were defined for post-hoc comparisons. Phase 2 alpha value was adjusted for three post-hoc comparisons and was considered significant at p <0.015. p values presented in the table are uncorrected.

34 medRxiv preprint doi: https://doi.org/10.1101/2021.05.17.21257345; this version posted May 19, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license .

* Figure 1A

Figure 1B * medRxiv preprint doi: https://doi.org/10.1101/2021.05.17.21257345; this version posted May 19, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license .

Figure 2A

Figure 2B medRxiv preprint doi: https://doi.org/10.1101/2021.05.17.21257345; this version posted May 19, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license .

Figure 3A

* Figure 3B