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The effects of cannabidiol or delta-9-tetrahydrocannabinol on phencyclidine-induced activity in mice
Toxicology Letters, 1(1978)331-335 o Elsevier/North-Holland Biomedical Press
331
THE EFFECTS OF CANNABIDIOL OR DELTA-9-TETRAHYDROCANNABINOL ON PHENCYCLIDINE-INDUCED ACTIVITY IN MICE
CONNIE J. STONE* and ROBERT B. FORNEY Department of Toxicology, 46202 (U.S.A.)
Indiana University School of Medicine, Indianapolis, Znd.
(Received January 6th, 1978) (Accepted January 9th, 1978)
SUMMARY
Phencyclidine (PCP) is a veterinary compound which acts as a psychotomimetic in man. This study examined the effects of A-9-tetrahydrocannabinol (THC) and cannabidiol (CBD) on PCP-induced activity in mice and followed the disappearance of PCP from whole mouse homogenates after PCP pretreatment. Male albino mice were injected with vehicle alone, 20 mg/ kg CBD or 20 mg/kg THC followed by 25 mg/kg PCP. Total activity was measured over a 2-h period. Both CBD and THC significantly attenuated the PCP-induced activity. In the disappearance study, mice were injected with 20 mg/kg CBD followed by 25 mg/kg PCP and their body tissues were homogenized at intervals ranging from O-160 min. The resulting disappearance curve was compared with that generated from PCP administered alone. Results showed a half-life for PCP of 46 min when administered alone and 66 min when administered after CBD pretreatment.
INTRODUCTION
PCP is a piperidine derivative initially developed as a general anesthetic and short-acting analgesic for use in man (Fig. 1). Postoperative side effects such as general agitation and hallucinations (Collins et al., 1960) resulted in its withdrawal from human usage. It is presently marketed as a veterinary anesthetic (Sernylan). PCP is often sold illicitly as more familiar psychoactive drugs such as LSD, THC, or mescaline or under its various street names, “angel dust”, “peace pill”, and “hog” [ 7,9]. Its usage is widespread in many areas of the country *Present address: Becton, Dickinson Research Center, Research Triangle Park, N.C. 27709 (U.S.A.). Abbreviations: CBD, cannabidiol; PCP, phencyclidine; THC, A-9-tetrahydrocannabinol.
332
PHENCYCLIDINE
Fig. 1. Structure
of phencyclidine.
[3,6]. Since PCP is sold and used with other abused drugs, overdose cases are difficult to both diagnose and treat. The response to PCP is species-specific. In general, mice and rats are stimulated by the drug while other species are depressed. This study was undertaken to examine the effects of two major marihuana constituents, THC and CBD, on PCP-induced activity in mice and the disappearance of PCP from the whole mouse after CBD pretreatment. MATERIALS
AND METHODS
Male albino Swiss-Webster mice (Cox Laboratory Supply) weighing between 20-25 g were used. The animals were maintained in an animal care facility on a 12-h light/l2-h dark schedule at 72°F. The experimental period was preceded by a 48-h animal equilibration period. PCP, as the hydrochloride salt, was administered i.p. in normal saline so that each animal received 0.5 ml/20 g. THC and CBD were administered in a 4% Tween 80 vehicle. Motor activity was measured using activity cages equipped with photoelectric cells. The cages were cylindrical in shape and each was equipped with 6 photoelectric cells which were evenly spaced around the cylinder. The photocells were connected to a digital counter. Each time a mouse broke the light beam, a count was recorded. These counts were then used as a measure of total activity in any given time period. The protocol for each test session called for first putting the mice in the cages (4/cage, representing an N of 1) and allowing a 30-min equilibration period. Each of the mice was then injected with either 4% Tween 80, 20 mg/ kg THC in 4% Tween 80, or 20 mg/kg CBD in 4% Tween 80 according to a randomized schedule. The electronic counters were activated and allowed to run for 30 min. At the end of this period, all counters were stopped for 5 min and the counts noted. These became the control counts. 12 mg/kg PCP was then given to all animals and the counters were restarted. Counts were taken every 30 min for the next 3.5 h. A cross-over design was used, i.e., the scheduling was arranged so that each of the 5 cages available saw each treatment once (N = 5) to control for any cage effect.
333
In examining the whole-mouse disappearance of PCP after CBD pretreatment, 45 mice were injected i.p. with 20 mg/kg CBD in 4% Tween 30. 30 min later they were given 25 mgfkg PCP i.p. Following PCP adminis~ation, the mice were homogenized in a Waring blender at intervals from O-160 min. The blender contained a solution of 5% perchloric acid and benzphetamine as an internal standard. 5 mice were homogenized at each time period. An aliquot of the homogenate was extracted and analyzed using a gas chromatograph equipped with a flame ionization detector [ 81. The resulting disappearanee curve from PCP + CBD was then compared with that generated from PCP administration alone. RESULTS
Fig, 2 summarizes the data. The statistical test for significance used was the student-Newman-~uhls test, The control count referred to on the ordinate is the count immediately preceding PCP administration and 30 min post CBD or THC administration. 30 min after PCP administration, the CBD pretreated animals had counts significantly lower (P < 0.05) than either the THC or vehicle (4% Tween 80) pretreated animals, At 60 and 90 min post-PCP injection, both CBD- and 800
I
I
t
700
is g u
500
I
4%been
1o11 2
---
85 + PCP (lZmg/kg) THC (2Omg/k& + PCP (f2mg/kgf
-
CBD @Omg/kg)
f PCF ~~2mg~~
~Signfficantly different j from Tween 80 at p
405
*Significantly from THC
8 8 6”
I
600
;: c 2
----
different
at p
355
205
105
0 0 Control
30
85
90
120
150
180
210
TIME (MINUTES POST PCP INJECTION)
Fig. 2. The effects af eannabidiol or A-9-t~t~ydrocannabind induced activity in mice.
cm phencyclidine (PCPF
334
THC-pretreated animals showed significantly less activity than those animals given vehicle + PCP. At 2 h post-PCP administration, all counts had returned to control levels. Computer-generated regression lines were fitted to the data from the whole-mouse disappearance study (Fig. 3). The lines were generated from the terminal, or beta phase, of the disappearance curve only. The disappearance of PCP alone from whole mouse homogenates is shown as well as the disappearance of this drug after CBD pretreatment. The half-life for PCP when given alone is 46 min. After CBD pretreatment, the half-life is lengthened to 66 min. There was a significant difference between the slopes of the two regression lines (P < 0.05). 1.375 1.25
----
25 mg/kg
PCP
-
20 mg/kg
CBD
A
0
= Mean
of five
+ 25 mg/kg
O3
PCP
mice
0 1
SLOPES 0
’ i
DIFFER
SIGNIFICANTLY
@ < 0.05)
I
I
I
I
I
i0
i0
$0
80
100
TIME
POST
PCP
INJECTION
I
li0
I
I
140
160
’
(min)
Fig. 3. Disappearance of phencyclidine (PCP) from whole-mouse homogenates. DISCUSSION
This study demonstrates that both cannabinoids examined, CBD and THC, attenuate the excitation produced by PCP in mice. The lowered motor activity seen with THC is probably due to the central depressant effect of the drug. It has been shown that both THC and CBD are metabolized by liver microsomal enzymes but that CBD is a much more potent inhibitor of the cytochrome P-450 system than is THC [ 21. Therefore, CBD might be expected to enhance the activity of PCP by prolonging its metabolism. The study examining the disappearances of PCP from the whole mouse after CBD pretreatment indicated that PCP is metabolized more slowly because of CBD inhibition of microsomes. However, CBD does appear to attenuate the motor activity produced by PCP rather than enhancing it by prolonging its metabolism as would be
335
expected. This may indicate that CBD, while blocking PCP metabolism, is also exercising some central anticonvulsant effect since PCP does produce some clonic convulsant activity in mice at the dose administered in this study. CBD has been shown to be an effective anticonvulsant in mice and rats [4]. It is probable that, in its interaction with other drugs, PCP acts by a combination of metabolic and, central mechanisms to change either their pharmacologic actions or its own. In clinical overdose situations, such interactions would tend to confuse presenting symptoms leading to difficulties in diagnosis and treatment. ACKNOWLEDGEMENTS
The technical assistance of Betty Griffey was greatly appreciated. This work was supported in part by PHS GM 1089. REFERENCES 1 V.J. Collins, C.A. Gorospe and E.A. Rovenstine, Intravenous non-barbiturate, nonnarcotic analgesics: Preliminary studies, 1. Cyclohexylamines, Anesth. Analg., 39 (1960) 302-306. 2 M. Fernandes, N. Warning, W. Christ and R. Hill, Interaction of several cannabinoids with the hepatic drug metabolizing system, Biochem. Pharmacol., 22 (1973) 29812987. 3 J.P. Horwitz, E.B. Hells, D. Andrzejewski, W. Brukwinski, J. Penkala and S. Albert, Adjunct hospital emergency toxicology service, J. Am. Med. Ass., 235 (1976) 17081712. 4 R. Karler, W. Celg and S.A. Turkanis, The anticonvulsant activity of cannabidiol and cannabinol, Life Sci., 13 (1973) 1527-1531. 5 M. Perez-Reyes, M.C. Timmons, K.H. Davis and E.M. Wall, A comparison of the pharmacological activity in man of intravenously administered delta-g-tetrahydrocannabinol, cannabinol, and cannabidiol, Experientia, 29 (1973) 1368-1369. 6 J.M. Rainey and M.K. Crowder, Prevalence of phencyclidine in street drug preparations, N. Eng. J. Med., 290 (1974) 466-467. 7 A. Reed Jr. and A.W. Kane, Phencyclidine (PCP): Another illicit psychedelic drug, J. Psychedelic Drugs, 5 (1972) 8-12. 8 C.J. Stone and R.B. Forney, The effects of phencyclidine on ethanol and sodium hexobarbital in mice, Toxicol. Appl. Pharmacol., 40 (1977) 177-183. 9 H.J. Stuart and S. Bhatt, Analysis of street drugs, J. Drug Ed., 2 (1972) 197-210.
331
THE EFFECTS OF CANNABIDIOL OR DELTA-9-TETRAHYDROCANNABINOL ON PHENCYCLIDINE-INDUCED ACTIVITY IN MICE
CONNIE J. STONE* and ROBERT B. FORNEY Department of Toxicology, 46202 (U.S.A.)
Indiana University School of Medicine, Indianapolis, Znd.
(Received January 6th, 1978) (Accepted January 9th, 1978)
SUMMARY
Phencyclidine (PCP) is a veterinary compound which acts as a psychotomimetic in man. This study examined the effects of A-9-tetrahydrocannabinol (THC) and cannabidiol (CBD) on PCP-induced activity in mice and followed the disappearance of PCP from whole mouse homogenates after PCP pretreatment. Male albino mice were injected with vehicle alone, 20 mg/ kg CBD or 20 mg/kg THC followed by 25 mg/kg PCP. Total activity was measured over a 2-h period. Both CBD and THC significantly attenuated the PCP-induced activity. In the disappearance study, mice were injected with 20 mg/kg CBD followed by 25 mg/kg PCP and their body tissues were homogenized at intervals ranging from O-160 min. The resulting disappearance curve was compared with that generated from PCP administered alone. Results showed a half-life for PCP of 46 min when administered alone and 66 min when administered after CBD pretreatment.
INTRODUCTION
PCP is a piperidine derivative initially developed as a general anesthetic and short-acting analgesic for use in man (Fig. 1). Postoperative side effects such as general agitation and hallucinations (Collins et al., 1960) resulted in its withdrawal from human usage. It is presently marketed as a veterinary anesthetic (Sernylan). PCP is often sold illicitly as more familiar psychoactive drugs such as LSD, THC, or mescaline or under its various street names, “angel dust”, “peace pill”, and “hog” [ 7,9]. Its usage is widespread in many areas of the country *Present address: Becton, Dickinson Research Center, Research Triangle Park, N.C. 27709 (U.S.A.). Abbreviations: CBD, cannabidiol; PCP, phencyclidine; THC, A-9-tetrahydrocannabinol.
332
PHENCYCLIDINE
Fig. 1. Structure
of phencyclidine.
[3,6]. Since PCP is sold and used with other abused drugs, overdose cases are difficult to both diagnose and treat. The response to PCP is species-specific. In general, mice and rats are stimulated by the drug while other species are depressed. This study was undertaken to examine the effects of two major marihuana constituents, THC and CBD, on PCP-induced activity in mice and the disappearance of PCP from the whole mouse after CBD pretreatment. MATERIALS
AND METHODS
Male albino Swiss-Webster mice (Cox Laboratory Supply) weighing between 20-25 g were used. The animals were maintained in an animal care facility on a 12-h light/l2-h dark schedule at 72°F. The experimental period was preceded by a 48-h animal equilibration period. PCP, as the hydrochloride salt, was administered i.p. in normal saline so that each animal received 0.5 ml/20 g. THC and CBD were administered in a 4% Tween 80 vehicle. Motor activity was measured using activity cages equipped with photoelectric cells. The cages were cylindrical in shape and each was equipped with 6 photoelectric cells which were evenly spaced around the cylinder. The photocells were connected to a digital counter. Each time a mouse broke the light beam, a count was recorded. These counts were then used as a measure of total activity in any given time period. The protocol for each test session called for first putting the mice in the cages (4/cage, representing an N of 1) and allowing a 30-min equilibration period. Each of the mice was then injected with either 4% Tween 80, 20 mg/ kg THC in 4% Tween 80, or 20 mg/kg CBD in 4% Tween 80 according to a randomized schedule. The electronic counters were activated and allowed to run for 30 min. At the end of this period, all counters were stopped for 5 min and the counts noted. These became the control counts. 12 mg/kg PCP was then given to all animals and the counters were restarted. Counts were taken every 30 min for the next 3.5 h. A cross-over design was used, i.e., the scheduling was arranged so that each of the 5 cages available saw each treatment once (N = 5) to control for any cage effect.
333
In examining the whole-mouse disappearance of PCP after CBD pretreatment, 45 mice were injected i.p. with 20 mg/kg CBD in 4% Tween 30. 30 min later they were given 25 mgfkg PCP i.p. Following PCP adminis~ation, the mice were homogenized in a Waring blender at intervals from O-160 min. The blender contained a solution of 5% perchloric acid and benzphetamine as an internal standard. 5 mice were homogenized at each time period. An aliquot of the homogenate was extracted and analyzed using a gas chromatograph equipped with a flame ionization detector [ 81. The resulting disappearanee curve from PCP + CBD was then compared with that generated from PCP administration alone. RESULTS
Fig, 2 summarizes the data. The statistical test for significance used was the student-Newman-~uhls test, The control count referred to on the ordinate is the count immediately preceding PCP administration and 30 min post CBD or THC administration. 30 min after PCP administration, the CBD pretreated animals had counts significantly lower (P < 0.05) than either the THC or vehicle (4% Tween 80) pretreated animals, At 60 and 90 min post-PCP injection, both CBD- and 800
I
I
t
700
is g u
500
I
4%been
1o11 2
---
85 + PCP (lZmg/kg) THC (2Omg/k& + PCP (f2mg/kgf
-
CBD @Omg/kg)
f PCF ~~2mg~~
~Signfficantly different j from Tween 80 at p
405
*Significantly from THC
8 8 6”
I
600
;: c 2
----
different
at p
355
205
105
0 0 Control
30
85
90
120
150
180
210
TIME (MINUTES POST PCP INJECTION)
Fig. 2. The effects af eannabidiol or A-9-t~t~ydrocannabind induced activity in mice.
cm phencyclidine (PCPF
334
THC-pretreated animals showed significantly less activity than those animals given vehicle + PCP. At 2 h post-PCP administration, all counts had returned to control levels. Computer-generated regression lines were fitted to the data from the whole-mouse disappearance study (Fig. 3). The lines were generated from the terminal, or beta phase, of the disappearance curve only. The disappearance of PCP alone from whole mouse homogenates is shown as well as the disappearance of this drug after CBD pretreatment. The half-life for PCP when given alone is 46 min. After CBD pretreatment, the half-life is lengthened to 66 min. There was a significant difference between the slopes of the two regression lines (P < 0.05). 1.375 1.25
----
25 mg/kg
PCP
-
20 mg/kg
CBD
A
0
= Mean
of five
+ 25 mg/kg
O3
PCP
mice
0 1
SLOPES 0
’ i
DIFFER
SIGNIFICANTLY
@ < 0.05)
I
I
I
I
I
i0
i0
$0
80
100
TIME
POST
PCP
INJECTION
I
li0
I
I
140
160
’
(min)
Fig. 3. Disappearance of phencyclidine (PCP) from whole-mouse homogenates. DISCUSSION
This study demonstrates that both cannabinoids examined, CBD and THC, attenuate the excitation produced by PCP in mice. The lowered motor activity seen with THC is probably due to the central depressant effect of the drug. It has been shown that both THC and CBD are metabolized by liver microsomal enzymes but that CBD is a much more potent inhibitor of the cytochrome P-450 system than is THC [ 21. Therefore, CBD might be expected to enhance the activity of PCP by prolonging its metabolism. The study examining the disappearances of PCP from the whole mouse after CBD pretreatment indicated that PCP is metabolized more slowly because of CBD inhibition of microsomes. However, CBD does appear to attenuate the motor activity produced by PCP rather than enhancing it by prolonging its metabolism as would be
335
expected. This may indicate that CBD, while blocking PCP metabolism, is also exercising some central anticonvulsant effect since PCP does produce some clonic convulsant activity in mice at the dose administered in this study. CBD has been shown to be an effective anticonvulsant in mice and rats [4]. It is probable that, in its interaction with other drugs, PCP acts by a combination of metabolic and, central mechanisms to change either their pharmacologic actions or its own. In clinical overdose situations, such interactions would tend to confuse presenting symptoms leading to difficulties in diagnosis and treatment. ACKNOWLEDGEMENTS
The technical assistance of Betty Griffey was greatly appreciated. This work was supported in part by PHS GM 1089. REFERENCES 1 V.J. Collins, C.A. Gorospe and E.A. Rovenstine, Intravenous non-barbiturate, nonnarcotic analgesics: Preliminary studies, 1. Cyclohexylamines, Anesth. Analg., 39 (1960) 302-306. 2 M. Fernandes, N. Warning, W. Christ and R. Hill, Interaction of several cannabinoids with the hepatic drug metabolizing system, Biochem. Pharmacol., 22 (1973) 29812987. 3 J.P. Horwitz, E.B. Hells, D. Andrzejewski, W. Brukwinski, J. Penkala and S. Albert, Adjunct hospital emergency toxicology service, J. Am. Med. Ass., 235 (1976) 17081712. 4 R. Karler, W. Celg and S.A. Turkanis, The anticonvulsant activity of cannabidiol and cannabinol, Life Sci., 13 (1973) 1527-1531. 5 M. Perez-Reyes, M.C. Timmons, K.H. Davis and E.M. Wall, A comparison of the pharmacological activity in man of intravenously administered delta-g-tetrahydrocannabinol, cannabinol, and cannabidiol, Experientia, 29 (1973) 1368-1369. 6 J.M. Rainey and M.K. Crowder, Prevalence of phencyclidine in street drug preparations, N. Eng. J. Med., 290 (1974) 466-467. 7 A. Reed Jr. and A.W. Kane, Phencyclidine (PCP): Another illicit psychedelic drug, J. Psychedelic Drugs, 5 (1972) 8-12. 8 C.J. Stone and R.B. Forney, The effects of phencyclidine on ethanol and sodium hexobarbital in mice, Toxicol. Appl. Pharmacol., 40 (1977) 177-183. 9 H.J. Stuart and S. Bhatt, Analysis of street drugs, J. Drug Ed., 2 (1972) 197-210.