EFFECTS OF CANNABIDIOL IN ANIMAL MODELS PREDICTIVE OF ANTIPSYCHOTIC
ACTIVITY

Psychopharmacology (1991) 104: 260-264

A.W. Zuardi, J. Antunes Rodrigues, and J.M. Cunha

School of Medicine of Ribeirao Preto, USP, 14049 Ribeirao Preto, SP, Brazil
School of Medicine, UF, 38400 Uberlandia, MG, Brazil

Abstract. The effects of cannabidiol (CBD) were compared to those produced
by haloperidol in rats submitted to experimental models predictive of
antipsychotic activity. Several doses of CBD (15-480 mg/kg) and
haloperidol (0.062-1.0 mg/kg) were tested in each model. First, CBD
increased the effective doses 50% (or) ED-50 of apomorphine for induction
of the sniffing and biting stereotyped behaviors. In addition, both CBD
and haloperidol reduced the occurrence of stereotyped biting induced by
apomorphine (6.4 mg/k), increased plasma prolactin levels and produced
palpebral ptosis, as compared to control solutions. However, CBD did not
induce catalepsy even at the highest doses, in contrast to haloperidol.
Such a pharmacological profile is compatible with that of an "atypical"
antipsychotic agent, though the mechanism of action is uncertain and may
not be identical to that of the dopamine antagonists.

Key words: Cannabidiol---Cannabinoids---Antipsychotic
drugs---Antipsychotic screening---Stereotyped
behavior---Prolactin---Catalepsy---Palpebral ptosis

In a study of the interaction between a high delta-9-THC dose (0.5 mg / kg,
PO) and cannabidiol (CBD) carried out in normal volunteers, we observed
that the combination of the two cannabinoids significantly attenuated the
anxiety and psychotomimetic effects induced by delta-9-THC (Zuardi et al.
1982). Experimental evidence suggests that the antagonistic effect of CBD
did not result from pharmacokinetic interaction between the two
cannabinoids (Agurel et al. 1981; Zuardi et al. 1982). Therefore, CBD
may have anxiolytic and / or antipsychotic effects.
The hypothesis of a possible antipsychotic effect of CBD has also
been suggested by Rottanburg et al. (1982) to justify another type of
observation. These investigators, in a study carried out on patients at
the time of admission to apsychiatric hospital in South Africa, detected a
much higher frequency of acute psychotic episodes associated with the use
of Cannabis sativa than observed in other countries and attributed this
fact to the high potency of delta-9-THC and to the absence of CBD in the
plant variety occurring in that region. On this basis, lthey suggested
that the presence of CBD in Cannabis sativa samples consumed in other
countries may protect users against the occurrence of acute psychotic
episodes.
In the present study, we investigated the possible anti-psychotic
activity of CBD by studying the effect of this cannabinoid on animal models
frequently used in research on new compounds with potential antipsychotic
properties. We used apomorphine-induced stereotypy (Janssen et al. 1977),
prolactin secretion (Clemens et al. 1974), catalepsy (Janssen et al.
1965), and palpebral ptosis (Janssen and Van Bever 1975). The effects of
CBD on these four models were compared with those produced by haloperidol,
which is considered to be a representative drug of the group of "typical"
antipsychotics.

Material and methods

Animals

Male Wistar rats weighing about 350 g and not submitted to any previous
treatment or experimental manipulation were used. The animals were kept
under the following conditions for at least 1 week prior to the experiment:
three to a cage, free access to water and food, a 12-h light / 12-h dark
cycle, temperature between 22 and 25 degrees C. and daily handling for
weighing and cage cleaning.

Drugs

Powdered CBD, kindly supplied by Dr. R. Mecholam (Hebrew University,
Jerusalem, Israel), was dissolved in saline with the aid of Tween-80, at
concentrations proportional to the CBD dose, ranging from 1.5% (CBD < mg /
kg) to 12% (CBD = 480 mg /kg). The same amount of Tween-80 dissolved in
saline was used as control.
Haloperidol (5 mg / ml ampoules, Janssen Pharmaceutica) was
diluted with saline, and the vehicle in the ampoules was used as control.
Apomorphine hydrochloride (Merck) was dissolved in distilled
water.
All drugs were administered intraperitoneally in a volume of 2 ml /
kg body weight.

Procedure

Apomorphine-induced stereotypy. We studied the effect of CBD on the
effective dose 50% (ED-50) of apomorphine for producing stereo-typed
behaviors, using the following treatments: control solution, CBD (15, 30
and 60 mg / kg) and apomorphine (0.1, 0.4, 1.6, 6.4 and 25.6 mg / kg) in
combination with either control solution or CBD at doses of 15, 30, and 60
mg / kg. Each treatment was administered to groups of six animals. The
experimental sessions were always held in the afternoon using 24 animals
(1 per treatment). Treatments were administered randomly at 1-mm intervals
to each animal. Immediately after being injected, the animals were placed
in individual cages (16 X 30 X 18 cm), and 30 min after the first animal
received the drug, a trained observer who was not aware of the treatments
started to observe animal behavior. The animals were observed in the same
sequence in which they had been injected for periods of 1 min at 30-min
intervals, for a total of six observation periods per animal over a period
of 3 h. The occurrence of the following behaviors was recorded during each
period: sniffing (vertical oscillation of the head accompanied by
movements of nostrils and vibrissae throughout the observation period) and
biting (open mouth approaching the cage bars with teeth exposed). The
number of animals exhibiting each stereotyped behavior during at least one
observation period was computed for each treatment.
For comparison with haloperidol we used the results obtained with
the 6.4 mg / kg dose of apomorphine (the dose that induced biting behavior
in 83% of the animals) in combination with CBD (0, 15, 30, and 60 mg /
kg). During an additional session we tested apomorphine (6.4 mg / kg) in
combination with control solution and increasing doses of haloperidol
(0.125, 0.23 and 0.5 mg / kg) on groups of six animals each.

Prolactin secretion. We tested the effects produced on prolactin secretion
by haloperidol (0.062, 0.125, 0.25, and 0.5 mg / kg) and CBD (15, 30, 60
120, and 240 mg / kg) and by their respective control solutions. The two
drugs were tested during independent experimental sessions. Each treatment
was administered to groups of ten animals each at random, though care was
taken to avoid that the three animals in the same cage would receive equal
treatments. The injections were made between 10:00 and 10:30 hours. One
hour after drug administration, the rats were taken individually to an
isolated room and decapitated with a guillotine. The time spent in
removing and sacrificing the three animals from the same cage was less than
1 min in order to avoid the stress of manipulation (Krulich 1974). Blood
was collected from the neck with a funnel into a heparinized tube and
maintained on ice until centrifugation, which was performed immediately
after all animals had been sacrificed. Plasma was stored at -20 degrees C
until the time for prolactin measurement. Plasma PRL levels were
determined by double-antibody radioimmunoassay (Niswender et al. 1969)
using material supplied by NIADDK. Absolute values are shown using RP3
(PRL) reference standards. The measurements were made in duplicate and
all samples from each experimental session were measured in the same assay.
Intrassay and interassay error were 4% and 12%, respectively. Assay
sensitivity was 0.8 ng / tube.

Catalepsy and palpebral ptosis. In this experiment we tested the following
treatments: CBD (60, 120, 240 and 480 mg / kg), haloperidol (0.125,
0.25, 0.5 and 1 mg / kg) and control solution (a mixture of the vehicles
for the two drugs). Each treatment was administered to groups of eight or
nine animals distributed at random among seven independent experimental
sessions.

After receiving the drugs, the animals were placed in individual
cages (30 X 16 X 16 cm) and were tested 1 h later. Testing time (2 h)
was divided into six periods of 20 min each and one palpebral ptosis
measurement plus one catalepsy measurement were made during each period.
At the beginning of each period, one observer unaware of the treatments
administered evaluated palpebral ptosis 1 min after a standardized sound
stimulus. Palpebral ptosis was classified into four grades by the
following criteria: 1 -fully open palpebral fissure, 2--partial occlusion
of <50% of the palpebral fissure, 3--partial occlusion of >50% of the
palpebral fissure, 4-- full occlusion of the palpebral fissure. After
observation of palpebral ptosis, the animals were positioned with the
forepaws resting on a horizontal bar (0.8 cm in diameter) 4.5 cm above
the floor surface. We then recorded the time the animals kept their paws
on the bar, with up to three attempts made to place the animals' paws on
the bar within each 20-min period.
For each animal we calculated one value for palpebral ptosis and
one for catalepsy. The highest score among six measurements was considered
to be the degree of palpebral ptosis. The catalepsy index was the total
time, in seconds, during which the animals stayed with their paws on the
bar during the observation period.

Statistical analysis. The ED-50 doses and the 95% confidence limits for
the stereotyped sniffing and biting behaviors induced by apomorphine plus
control solution and by apomorphine in combination with the three CBD doses
were calculated by the method of Weil (1952). We considered the ED-50
values for the groups that received CBD to differ significantly from those
obtained for the control group when there was no overlapping between their
95% confidence limits and those of the control group. The effects of
haloperidol (0.5 mg / kg) CBD (60 mg / kg) on the stereotyped biting
behavior induced by apomorphine (6.4 mg / kg) were compared with the
effects of the control solutions by the Fisher test (Siegel 1956). The
prolactin, catalepsy and palpebral ptosis data were submitted to
Kruskal-Wallis analysis of variance. Comparison between the results
obtained with each drug and their control groups were done by multiple
comparison based on the Kruskal-Wallis rank sum test (Hollander and Wolfe
1973). The significance level was P<0.05.

Results

Apomorphine-induced stereotypy

The control solution or CBD (15, 30 and 60 mg / kg) did not induce any
stereotyped behavior during the entire period of observation.
Figure 1 shows the ED-50 values and their 95% confidence limits for
apomorphine in combination with the control solution and with CBD (15, 30
and 60 mg / kg) in terms of induction of the sniffing and biting
stereotyped behaviors. For the sniffing behavior (Fig 1A), the ED-50
values of apomorphine plus CBD at the doses of 15, 30 and 60 mg / kg (1.3,
1.0 and 1.6, respectively) ranged around a higher value than the ED-50 for
apomorphine plus the control solution (0.7). For the biting behavior
(Fig. 1B), the addition of increasing CBD doses to apomorphine produced
progressively higher ED-50 values (4.8, 8.4 and 12.8) than those for the
control group (4.0). On the basis of the criterion used, the differences
obtained in relation to the control group were significant after the
highest CBD dose (60 mg / kg) both for sniffing and biting.

The results obtained for biting in the groups that received a
combination of apomorphine (6.4 mg / kg, the ED-83 for biting behavior
with: CBD (15, 30 and 60 mg / kg), haloperidol (o.125, 0.25 and 0.5 mg /
kg), and the respective control solutions are presented in Fig. 2A. With
increasing doses of both haloperidol and CBD, the number of animals showing
biting behavior was reduced, and this reduction was statistically
significant compared to controls at the highest doses of haloperidol
(P<0.01) and of CBD (P<0.05).


Prolactin secretion

Analysis of variance showed that plaxma prolactin levels changed
significantly with the various doses of CBD (H=25.1, df=5, P<0.001) and
of haloperidol (H=25.8, df=4, P<0.001).

Figure 2B shows the mean plasma prolactin levels detected in
animals injected with the various doses of haloperidol induced a
significant increase in plasma prolactin levels when compared with the
control group at the doses of 0.125, 0.25 and 0.5 mg / kg, whereas CBD
induced a significant increase in plasma prolactin levels only at the doses
of 120 and 240 mg / kg.

Catalepsy and palpebral ptosis

Analysis of variance showed significant differences between experimental
groups both in terms of catalepsy indices (H = 36.6; df = 8; P<0.001)
and the degrees of palpebral ptosis (H = 31.2; df =8; P<0.001).

Figure 2C shows the median times, in seconds, during which the
animals kept their paws on the bar (catalepsy index) after receiving the
control solution and the various doses of CBD and halopweidol. Catalepsy
time increased progressively with increasing haloperidol doses, with a
significant difference from the control group at the doses of 0.25, 0.5 and
1 mg / kg. CBD did not increase the catalepsy index in a significant
manner at any of the doses used.

The median values of degree of ptosis for the control group and for
the groups that received the various doses of CBD and haloperidol are shown
in Fig. 2D. There were significant increases in relation to the control
group at the doses of 120, 240 and 480 mg / kg CBD and 1.0 mg / kg
haloperido.

Discussion

As expected, the animals predictive of antipsychotic activity used in the
present study proved to be sensitive to the effects of a "typical"
antipsychotic drug, i.e., haloperidol.
CBD attenuated the stereotypy induced by apomorphine, as
demonstrated by the increase in apomorphine ED-50 values for the induction
of sniffing (Fig 1A) and biting behaviors (Fig. 1B), and by the
significant decrease in the number of animals that exhibited biting
behavior after the 6.4 mg / kg dose of apomorphine (Fig. 2A). This effect
can be observed with most of the antipsychotic drugs available (Janssen et
al. 1977).

Another effect shared by antipsychotic drugs is the induction of
prolactin secretion (Clemens et al. 1974), which was also observed in the
present study with haloperidol and CBD (Fig. 2B). However, it can be seen
that the variability of the response obtained with CBD increased with
increasing doses, contrary to what occurred with the groups that received
haloperidol. The animals receiving the highest CBD doses (120 and 240 mg
/ kg) tended to show two types of response, i.e., either low or very high
prolactin levels, resulting in high standard errors of the means. The
responses to haloperidol were close to normal distribution. This
observation suggests that the effects of CBD and haloperidol on prolactin
secretion may involve different mechanisms.

The palpebral ptosis induced by both haloperidol and CBD is also
compatible with the effects produced by antipsychotic drugs (Janssen and
Van Bever 1975), though it reflects a sedative activity that is not
specific of these drugs. This result agrees with previous reports of the
sedative effects of CBD (Monti 1977; Pickens 1981; Colassanti et al.
1984).

The results obtained with CBD in catalepsy model differed from
those expected for a "typical" antipsychotic drug (Janssen et al. 1965).
Contrary to haloperidol, CBD did not induce a significant increase in
catalepsy index (Fig 2C). This absence of catalepsy induction by CBD in
rats confirms data reported by Fernandes et al. (1974). Reports of
catalepsy induction with CBD in mice are contradictory, with both negative
results (Jones and Pertwee 1972; Fairbairn and Pickens 1979) and one
positive result (Karniol and Carlini 1973).

Taken as a whole, the present results show that the CBD profile was
not exactly the same as observed with haloperidol, suggesting that CBD does
not belong to the group of "typical" antipsychotic drugs. The effects of
CBD may be compatible with those of an "atypical" antipsychotic drug such
as sulpiride, clozapine or thioridazine (Jenner and Marsden 1983). These
drugs stimulate prolactin secretion (Meltzer et al. 1980; Robertson and
MacDonald 1986), though they have also been reported to exert weak
antistereotypic abilities (Costall and Naylor 1975; Puech et al. 1976;
Jenner et al. 1978); but do not induce significant catalepsy (Costall and
Naylor 1975; Jenner et al. 1978; Zivkovic et al. 1980). In addition, the
potency of CBD in increasing prolactin levels was very low, as is also the
case for clozapine (Meltzer et al. 1979). Indeed, the minimum effective
dose of CBD for prolactin secretion was 2- to 24-fold higher than that
needed to produce other effects in rats such as decreased locomotor
(Karniol and Carlini 1973), anticonvulsive (Izquierdo et al. 1973) and
hypnotic (Monti 1977) activity, and increased corticosterone levels
(Zuardi et al. 1984).

The mechanism of action of CBD, which is responsible for the
effects observed in the present study, is not clear. Evidence about the
cellular effects of cannabinoids reviewed by Martin (1986) suggests that
CBD may not act as a typical dopaminergic antagonist. Other actions of CBD
may explain the present results, such as blockade of serotonin reuptake
(Johnson 1976) or increased GABA activity (Revuelta et al. 1979;
Benedito and Leite 1981). Drugs that increase serotoenergic activity may
attenuate the stereotypy induced by apomorphine (Carter and Pycock 1981;
Costall et al. 1981) and stimulate prolactin secretion (Weiner and Ganong
1978). Drugs that increase GABA-ergic activity have less consistent
effects on stereotypy (Scatton and Bartholini 1982; Dougherty and
Ellinwodd Jr. 1983) and on prolactin secretion (Casanueva et al. 1981).
The effects of CBD may also be due to its interaction with specific
cannabinoid receptors (Nye et al 1985; Herkenham et al. 1990).
In conclusion, the present results reveal a profile of CBD effects
compatible with that of "atypical" antipsychotic drugs, although its
mechannism of action is unknown and may involve neurotransmitter systems
other than the dopaminergic one.

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