Jacob Bell
New Member
The effect of cannabinoids on intestinal motility and their antinociceptive effect in mice
G. B. CHESHER, C. J. DAHL,* M. EVERINGHAM, D. M. JACKSON,
H. MARCHANT-WILLIAMS AND G. A. STARMER
Department of Pharmacology, University of Sydney, Sydney,
New South Wales 2005, Australia
* Australian Government Analytical Laboratory, Department of Science, Melbourne, Victoria, Australia.
Summary
1. After oral administration to mice, pethidine, A8-tetrahydrocannabinol (THC), A9-THC, a cannabis extract and cannabinol had a dose-dependent antinociceptive effect when measured by the hot-plate method. Cannabidiol was inactive at 30 mg/kg. A8-THC, A9-THC and pethidine did not differ significantly in potency, but L 9-THC was 6.5 times more active than cannabinol.
2. After oral administration, three different cannabis extracts, L 8-THC, Q9-THC and morphine produced dose-dependent depressions of the passage of a charcoal meal in mice. p8-MC and A9-THC were equipotent and were about five times less potent than morphine. Cannabidiol was inactive up to 30 mg/kg. The effect of the three cannabis extracts on intestinal motility could be accounted for by their A9-THC content.
3. The antinociceptive effect of pethidine and the effect of morphine on intestinal motility were antagonized by nalorphine whilst the effects of the cannabis extracts and the pure cannabinoids were not.
4. From these results it is concluded that although cannabis and the narcotics share several common pharmacological properties, the mode of action of each is pharmacologically distinct.
Introduction
In pharmacological terms, cannabis and its derivatives are not considered to be narcotic analgesic drugs. There is evidence, however, that they do share with the narcotic analgesics the properties of analgesia and depression of intestinal motility. The analgesic effectiveness of cannabis derivatives has been reported in experimental animals (Bicher & Mechoulam, 1968 ; Buxbaum, Sanders-Bush & Efron, 1969 ; Buxbaum, 1972 ; Dewey, Harris & Kennedy, 1972) and in man (Walton, 1938). Several authors have reported that A9-tetrahydrocannabinol reduced defaecation in rats (Masur, Martz, Korte & Bieniek, 1971 ; Drew, Miller & Wikler, 1972) and Dewey et a!. (1972) reported that this substance delayed passage of a charcoal meal in mice.
In the present study, we describe an investigation of the effects of extracts of cannabis leaf and hashish, Q9-tetrahydrocannabinol (L 9-THC), A8-THC, cannabidiol, cannabinol acetate, morphine and pethidine on the threshold of the hot-plate test and intestinal motility in mice.
Preparation of cannabis extracts and materials
Extracts of cannabis leaf or hashish were prepared with light petroleum at room temperature. After concentration under reduced pressure at 40° C the soft extract was taken up in methanol and stored at -20° C for 24 hours. Filtra¬tion of this solution effected a satisfactory separation of solidified waxes. Other impurities were removed successively by adsorption chromatography on alumina (activity 1) from a chloroform solution and on Florisil (60-100 mesh) from a benzene solution. Removal of solvent produced a transparent ` red oil ', a sample of which was silylated and assayed for cannabinoids by gas-liquid chromatography. Three ` red oil ' extracts, each from a different sample of cannabis were prepared and the assay results are shown in Table 1.
TABLE I. The composition of three cannabis extracts.
Extract Source THC Content % of
CBD CBN
I Pakistan hashish 22 43 34
II Australian Cannabis leaf 52 39 5
III Australian Cannabis leaf 41 8 20
THC = A9-tetrahydrocannabinol; CBD = cannabidiol; CBN = cannabinol.
Cannabis extracts and the pure cannabinoids were dissolved or suspended in propylene glycol and kept at -20° C until required. Dilutions were made with a solution of Lissapol-Dispersol (ICI) (Whittle, 1964) to give a final concentration of 5% propylene glycol. Pethidine hydrochloride, morphine sulphate and nalorphine hydrochloride were dissolved in water or 0.9% w/v NaCI solution (saline) and doses given were calculated as the salt. All drugs were administered in a dose volume of 1 ml/ 100 g body weight.
Antinociceptive action
The mice (SW strain, males, 20-30 g) were allowed food and water ad libitum up to the time of the experiment. The method of Woolfe & MacDonald (1944) was used and the hot-plate was maintained at a constant temperature of 55 +1° C. Before dosing, all mice were tested individually and the time spent on the hot-plate before the animal elicited the end-point response (flicking of a hind paw) was recorded. Mice which failed to respond within 30 s were discarded. A mean reaction time and a critical reaction time (CRT, the mean pre-drug reaction time plus two standard deviations) were calculated for each group of mice used. The results were expressed as the number of mice in each group which remained after medication on the hot-plate beyond the CRT ; the EDso and its limits of error (P=0.05) were calculated by the method of Litchfield & Wilcoxon (1949). All drugs except pethidine were administered orally 60 min prior to testing. Pethidine was administered intraperitoneally 30 min before testing.
To investigate the possibility of cannabis-pethidine interactions, two experimental schemes were used. (a) Mice received cannabis extracts at various dose levels and some, in addition, received pethidine (6 mg/kg) ; the remainder, dosed with the vehicle only, served as controls. Cannabis extracts were administered 1 h and pethidine 0.5 h before testing. (b) The animals received pethidine at various dose
levels and some also received cannabis extract (60 mg/kg) ; the remainder, dosed with vehicle only, served as controls.
To determine if the antinociceptive effect produced by the cannabis extract could be antagonized by the narcotic antagonist, nalorphine, a group of mice was given a dose of cannabis extract (60 mg/kg) 1 h before testing on the hot-plate. These mice were then divided into two groups, one of which was given nalorphine (5 mg/kg) and the other saline, both by the intravenous route. A similar proce¬dure was used to study the interaction of pethidine (6 mg/kg, given 0.5 h before testing on the hot-plate) and nalorphine or saline.
Intestinal Motility
The effect of drugs on intestinal motility was determined by measuring the rate of passage of a charcoal meal (Macht & Barba-Gose, 1931). Mice received 0.2 ml of a meal, consisting of animal charcoal 12 g, tragacanth 2 g and water 130 ml by lavage and were killed 15 min later. The length of the small intestine from pylorus to the ilex-caecal junction was measured and the distance which the charcoal meal had travelled was expressed as a percentage of the total length of the small intestine. The ED50 and the limits of error (P=0.05) were calculated by the method of Litchfield & Wilcoxon (1949). The cannabis extracts, A8-THC and A9-THC were administered by lavage, 45 min before the charcoal meal.
To determine whether the activity of cannabis extracts on intestinal motility was of a morphine-like nature, a comparison was made of the effect of the narcotic antagonist, nalorphine, on the depression of intestinal motility produced by morphine and by cannabis extracts. Groups of mice were dosed with either cannabis extract, 1 h before they were killed, morphine 0.5 h before they were killed, or a vehicle control. Nalorphine (8 mg/kg) or a vehicle control was administered intraperitoneally to these animals 0.5 h before they were killed.
Results
Andinociceptive effects
Pethidine, A8-THC, A9-THC, cannabis extract, and cannabinol all exhibited dose-dependent antinociceptive activity (Table 2). Pethidine did not differ signifi-cantly in potency from A9-THC. A8-THC and A9-THC did not differ significantly in potency and A9-THC was estimated to be &5 times more active than cannabinol. Cannabis extract I had 70% of the potency of cannabinol and 11% of that of
TABLE 2. The effects of pethidine, pe-tetrahydrocannabinol (THC), p9-THC, cannabinol acetate and
cannabis extract I on antinociceptive activity measured by the hot plate method.
Drug
(Doses, mg/kg) Pethidine
(2, 4, 6, 8)
A9-THC
(4, 7.5, 15, 30, 60) p 5-THC
(4, 9.5, 15, 30)
Cannabinol acetate (10, 20, 40, 60) Cannabis extract I (7.5, 15, 30, 60, 100) ED5o (mg/kg)
(limits of
7.0 (4.8-10.3)
5.0
(2.9- 8.8) 5.0
(2.4-10.5) 32.5
(22.4-47.1) 47.0
(30.9-71.4) Slope Function error for P=0.05)
7.58
(1.46- 39.26) 7.07
(1.96- 25.45) 11.06
(0.64-190.20) 3.74
(1.68- 8.30) 13.74
(3.57- 52.90) No. of
observations
220
300 240 240 300
p9-MC. In terms of p9-THC present, the extract had approximately half the potency of A9-THC. Cannabidiol was inactive at a dose of 30 mg/kg. All the dose-response curves were parallel.
The antinociceptive effect of cannabis alone did not differ significantly from that produced by cannabis plus pethidine (Table 3). Another group of mice, in which the testing procedure was carried out 1.5 h after cannabis and 1 h after pethidine administration gave similar results. In the experiment where the pethidine
TABLE 3. The effect of interaction on the antinociceptive effects of cannabis and pethidine in mice.
Time
after
cannabis
before
testing
(min) ED60 (mg/kg)
(limits of error
(a) Cannabis Slope
function
for P=0.05) No. of
observations
Drug Administration
(mg/kg)
First Second
Cannabis Pethidine (6) 60 18.0 9.08 120
(5, 7.5, 15, 30) (10.3-31.5) (1.85- 44.49)
90 23.0 10.00 160
(13.9-38.2) (1.60- 62.50)
Cannabis Vehicle 60 21.2 15.12 118
(5, 7.5, 15, 30) Control (10.833.0) (0.66-346.2)
90 12.7 9.30 160
(7.9-20.3) (3.72- 23.25)
Cannabis (60) Pethidine 60 (b) Pethidine
4.6 9.21 230
(2, 4, 6, 8) (3.1- 6.9) (1.33- 63.73)
Vehicle Control Pethidine 60 7.0 7.58 230
(2, 4, 6, 8) (4.8-10.3) (4.76- 10.3)
dose was varied and the cannabis dose remained constant, the results were essentially similar to those reported above. Cannabis possibly potentiated the antinociceptive effect of pethidine, but the effect was statistically not significant.
The antinociceptive effect of pethidine but not that of the cannabis extract was antagonized by nalorphine (Table 4).
TABLE 4. The effect of nalorphine (5 mg/kg) on the antinociceptive effects of pethidine and cannabis
extract I.
Cannabis Extract I
Pethidine (6 mg/kg) (60 mg/kg)
Mean Time on hot- Mean Time on hot
plate ± S.E.M. plate ± S.E.M.
Treatment (s) (s)
Before nalorphine 13.8±3.4 (41) 22.1±1.5 (37)
After nalorphine 8.1±0.9 (17) 22.8±2.0 (18)
After saline 16.0±1.4 (20) 25.3±1.7 (19)
Pethidine and cannabis extract were administered 35 and 65 min respectively before administration of nalorphine or saline. The numbers in brackets indicate number of observations.
Intestinal Motility
All cannabis extracts, Q9-THC and p9-THC had a dose-dependent effect on the passage of the charcoal meal (Table 5). Cannabidiol was inactive at all of five dose levels (6-30 mg/kg) tested. All the cannabis extracts and cannabinoids tested were significantly less potent than morphine but the regression lines were parallel. Q9-THC and p9-THC were not significantly different in potency. When the ED50 values for the three cannabis extracts were converted into their equivalent Q9-THC contents (Tables 1 and 5), the ED5o values calculated for Q9-THC were:
TABLE 5. The effects of morphine, A8-tetrahydrocannabinol (THC), A°-THC and cannabis extracts I,
II and Ill on the passage of a charcoal meal in mice.
Drug treatment & dose ED" (mg/kg) Slope function Potency
(mg/kg) (limits of error for P=0.05) (morphine =1)
Morphine (0.25, 0.5, 1.0, 3.4 0.186 1.0
2.0, 4.0, 8.0) (1.7- 6.7) (0.066-0.520)
A8-THC (2.0, 5.0, 10.0, 13.5 f k 0.072 0.25
20.0, 40.0) (10.9- 16.7) (0.007-0.756) (0.12-0.53)
A 9-THC (2.0, 5.0, 10.0, 20.0 0.370 0.17
20.0) (12.9- 31.0) (-250-0.55) (0.07-0.39)
Cannabis Extract I 86.0# 164 0.028
(4.5, 11.4, 22.7, 45.5,
91.0, 182.0) (46.0-160.8) (.059-0.459) (0.015-0.1)
Cannabis Extract II
(4.8, 9.7, 19.4, 38.8, 77.6) 21.5# -217 0.16
(13.6- 34.0) (0.095-0.494) (0.07-0.36)
Cannabis Extract III
(2.9, 7.25, 14.5, 29.0, 68.0#k 0.042 0.05
58.0) (29.8-155.3) (.003-0.671) (0.017-0.15)
A Significantly less potent than morphine at P<0.05.
The ED50 is that dose of compound required to slow the passage of a charcoal meal by 50% when compared with control animals which received the vehicle only.
I. 18.92 mg/kg ; II. 11.18 mg/kg ; III. 27.88 mg/kg. Thus, allowing for experi¬mental error, the effects of the extracts can probably be attributed to the content of A9-THC.
Whilst nalorphine effectively antagonized morphine-induced inhibition of gastro-intestinal motility (Table 6), it had no effect on the action of either cannabis extracts I or II.
TABLE 6. The effect of nalorphine (8 mg/kg) on the actions of morphine (8 mg/kg) and cannabis extracts
I and II (equivalent to 10 mg/kg tetrahydrocannabinol) on the passage of a charcoal meal in mice.
Passage of charcoal meal
expressed as % of total
length of small intestine
First treatment Second treatment +S.E. M.
Morphine Nil 9.0+0.6 (21)
Water Nalorphine 35.8+3.4 (20)
Morphine Nalorphine 24-7+1.9 (20)
Water Water 44.6+1.8 (20)
Extract I Water 33.2+2.2 (21)
Extract I Nalorphine 22.4+2.3 (19)
Vehicle control Water 48.6+2.5 (20)
Vehicle control Nalorphine 37.5+3.5 (20)
Extract II Water 29.4+2.0 (25)
Extract II Nalorphine 23.3+2.1 (25)
Vehicle control Nalorphine 43.6+3.3 (24)
Vehicle control Water 55.5+3.0 (10)
*The values are the means and their standard errors. The numbers in brackets are the number of
observations.
Discussion
There is still some doubt as to the nature of the response of cannabis-treated animals when tested by standard pharmacological methods for analgesia. Evidence for antinociceptive effects has been reported in a number of species following administration of Q9-THC (Bicher & Mechoulam, 1968 ; Bukbaum, 1972 ; Buxbaum et al., 1969). Although Dewey et al. (1972) were unable to demonstrate a significant antinociceptive effect when using the tail flick method in mice (in
doses below 100 mg/kg), they reported a prolonged antinociceptive effect in the same species when using the hot-plate method.
In the present experiments we have found that A9-THC, A9-THC, a cannabis extract and cannabinol all produced a dose-dependent increase in reaction time when tested on the hot-plate, with A9-THC and A9-THC having equal potency and cannabinol approximately one sixth of the potency of A9-THC. The results for A9-THC and A9-THC are thus in broad agreement with those of Dewey et al. (1972).
The analgesic dose-response curve for pethidine was parallel to those for the cannabinoids ; both A9-THC and A9-THC being approximately equipotent with pethidine. These results agree more closely with those of Bicher & Mechoulam (1968) than those of Buxbaum (1972) who reported considerable deviation from parallelism between the dose-effect curves for cannabinoids and narcotic analgesics. These differences might have been due to the route of administration since Buxbaum (1972) injected mice subcutaneously and absorption of cannabinoids by this route is slower and less complete than by the oral route used in our studies (Ho, 1971).
The interaction between cannabinoids and pethidine in mice tested by the hot-plate method was only suggestive of an additive effect. This finding was quite unlike the interactions of cannabinoids with barbiturates or ether on the duration of anaesthesia in mice. Doses of cannabis extracts or A9-THC which themselves are not hypnotic, significantly potentiate the sleeping times of mice induced by barbiturate or ether (Paton & Pertwee, 1972 ; Chesher, Jackson & Starmer, 1974).
A possible effect of cannabis on intestinal motility had been noted in the observa-tion that A9-THC reduced the incidence of defaecation in rats including those considered to be ` high defaecators ' (Masur et al., 1971 ; Drew et al., 1972). A depressant effect of A9-THC and A8-THC administered subcutaneously on the passage of a charcoal meal in mice has been reported by Dewey et al. (1972), although a clear dose-response relationship was not apparent. In the present studies we have shown that oral administration of both A9-THC and A9-THC and three cannabis extracts produced parallel dose-dependent depressions of the passage of a charcoal meal. As with the results on the antinociceptive effects, A9-THC and A9-THC did not differ significantly in potency and the potency of the three cannabis extracts can reasonably be accounted for by their A9-THC contents. It appears therefore that other cannabinoids are exerting little effect on intestinal motility ; this concept is supported by our findings of the lack of activity of cannabidiol on intestinal motility and by the wide divergence in content of cannabidiol in the three extracts used. For the latter reason, cannabinol appears to be unimportant in the activity observed in these studies.
Although, when tested for the antinociceptive effects and the effects on intestinal motility, the dose-response curves of cannabinoids and narcotics were parallel, the response to nalorphine clearly suggests a different mode of action. For both effects, nalorphine antagonized the action of the narcotic analgesic but had no effect on the responses to the cannabinoids.
We wish to thank Professor R. H. Thorp for his constant encouragement and Miss Margaret Rutherford for her skilled technical assistance. This work was supported by a grant from the National Health and Medical Research Council. Samples of cannabis were obtained from seized material by courtesy of the N.S.W. Government. A9-THC and A9-THC were obtained from the WHO, Geneva, by courtesy of Dr. O. Braenden.
REFERENCES
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BUXBAUM, D. M. (1972). Analgesic activity of A'-tetrahydrocannabinol in the rat and mouse. Psychopharmacologia, 25, 275-280.
BUXBAUM, D., SANDERS-BUSH, E. & EFRON, D. H. (1969). Analgesic activity of tetrahydrocannabinol (THC) in the rat and mouse. Fedn. Proc., 28, 735.
CHESHER, G. B., JACKSON, D. M. & STARMER, G. A. (1974). Br. J. Pharmac. (in press).
DEWEY, W. L., HARRIS, L. S. & KENNEDY, J. S. (1972). Some pharmacological and toxicological
effects of 1-trans-A8 and 1-trans-A'-tetrahydrocannabinol in laboratory rodents. Archs. int.
Pharmacodyn. Thep. 196, 133-145.
DREW, W. G., MILLER, L. L. & WIKLER, A. (1972). Effects of A'-THC on the open-field activity of the rat. Psychopharmacologia, 23, 289-299.
Ho, B. T. (1971). Marihuana, importance of the route of administration. J. Pharm. Pharmac., 23, 309-310.
LITCHFIELD, J. T. & WILCOXON, F. (1949). A simplified method of evaluating dose-effect experiments. J. Pharmac. exp. Ther., 96, 99-113.
MACHT, D. I. & BARBA-GOSE, J. (1931). Two new methods for pharmacological comparison in soluble purgatives. J. Am. pharm. Ass., 20, 558-564.
MASUR, J., MARTZ, R. M. W., KORTE, F. J. & BIENIEK, D. (1971). Influence of (-) A'-trans-tetra-hydrocannabinol and mescaline on the behaviour of rats submitted to food competition situations. Psychopharmacologia, 22, 187-194.
PATON, W. D. M. & PERTWEE, R. G. (1972). Effect of cannabis and certain of its constituents on
pentobarbitone sleeping time and phenazone metabolism. Br. J. Pharmac., 44, 250-261. WALTON, R. P. (1938). Marihuana, America's New Drug Problem. New York; Lipincott.
WHITTLE, B. A. (1964). The use of changes in capillary permeability in mice to distinguish between
narcotic and non-narcotic analgesics. Br. J. Pharmac. Chemother., 22, 246-253.
WOOLFE, G. & MACDONALD, A. D. (1944). The evaluation of the analgesic action of pethidine
hydrochloride (Demerol). J. Pharmac. exp. Then, 80, 300-307.
(Received March 20, 1973)
Source: The effect of cannabinoids on intestinal motility and their antinociceptive effect in mice
G. B. CHESHER, C. J. DAHL,* M. EVERINGHAM, D. M. JACKSON,
H. MARCHANT-WILLIAMS AND G. A. STARMER
Department of Pharmacology, University of Sydney, Sydney,
New South Wales 2005, Australia
* Australian Government Analytical Laboratory, Department of Science, Melbourne, Victoria, Australia.
Summary
1. After oral administration to mice, pethidine, A8-tetrahydrocannabinol (THC), A9-THC, a cannabis extract and cannabinol had a dose-dependent antinociceptive effect when measured by the hot-plate method. Cannabidiol was inactive at 30 mg/kg. A8-THC, A9-THC and pethidine did not differ significantly in potency, but L 9-THC was 6.5 times more active than cannabinol.
2. After oral administration, three different cannabis extracts, L 8-THC, Q9-THC and morphine produced dose-dependent depressions of the passage of a charcoal meal in mice. p8-MC and A9-THC were equipotent and were about five times less potent than morphine. Cannabidiol was inactive up to 30 mg/kg. The effect of the three cannabis extracts on intestinal motility could be accounted for by their A9-THC content.
3. The antinociceptive effect of pethidine and the effect of morphine on intestinal motility were antagonized by nalorphine whilst the effects of the cannabis extracts and the pure cannabinoids were not.
4. From these results it is concluded that although cannabis and the narcotics share several common pharmacological properties, the mode of action of each is pharmacologically distinct.
Introduction
In pharmacological terms, cannabis and its derivatives are not considered to be narcotic analgesic drugs. There is evidence, however, that they do share with the narcotic analgesics the properties of analgesia and depression of intestinal motility. The analgesic effectiveness of cannabis derivatives has been reported in experimental animals (Bicher & Mechoulam, 1968 ; Buxbaum, Sanders-Bush & Efron, 1969 ; Buxbaum, 1972 ; Dewey, Harris & Kennedy, 1972) and in man (Walton, 1938). Several authors have reported that A9-tetrahydrocannabinol reduced defaecation in rats (Masur, Martz, Korte & Bieniek, 1971 ; Drew, Miller & Wikler, 1972) and Dewey et a!. (1972) reported that this substance delayed passage of a charcoal meal in mice.
In the present study, we describe an investigation of the effects of extracts of cannabis leaf and hashish, Q9-tetrahydrocannabinol (L 9-THC), A8-THC, cannabidiol, cannabinol acetate, morphine and pethidine on the threshold of the hot-plate test and intestinal motility in mice.
Preparation of cannabis extracts and materials
Extracts of cannabis leaf or hashish were prepared with light petroleum at room temperature. After concentration under reduced pressure at 40° C the soft extract was taken up in methanol and stored at -20° C for 24 hours. Filtra¬tion of this solution effected a satisfactory separation of solidified waxes. Other impurities were removed successively by adsorption chromatography on alumina (activity 1) from a chloroform solution and on Florisil (60-100 mesh) from a benzene solution. Removal of solvent produced a transparent ` red oil ', a sample of which was silylated and assayed for cannabinoids by gas-liquid chromatography. Three ` red oil ' extracts, each from a different sample of cannabis were prepared and the assay results are shown in Table 1.
TABLE I. The composition of three cannabis extracts.
Extract Source THC Content % of
CBD CBN
I Pakistan hashish 22 43 34
II Australian Cannabis leaf 52 39 5
III Australian Cannabis leaf 41 8 20
THC = A9-tetrahydrocannabinol; CBD = cannabidiol; CBN = cannabinol.
Cannabis extracts and the pure cannabinoids were dissolved or suspended in propylene glycol and kept at -20° C until required. Dilutions were made with a solution of Lissapol-Dispersol (ICI) (Whittle, 1964) to give a final concentration of 5% propylene glycol. Pethidine hydrochloride, morphine sulphate and nalorphine hydrochloride were dissolved in water or 0.9% w/v NaCI solution (saline) and doses given were calculated as the salt. All drugs were administered in a dose volume of 1 ml/ 100 g body weight.
Antinociceptive action
The mice (SW strain, males, 20-30 g) were allowed food and water ad libitum up to the time of the experiment. The method of Woolfe & MacDonald (1944) was used and the hot-plate was maintained at a constant temperature of 55 +1° C. Before dosing, all mice were tested individually and the time spent on the hot-plate before the animal elicited the end-point response (flicking of a hind paw) was recorded. Mice which failed to respond within 30 s were discarded. A mean reaction time and a critical reaction time (CRT, the mean pre-drug reaction time plus two standard deviations) were calculated for each group of mice used. The results were expressed as the number of mice in each group which remained after medication on the hot-plate beyond the CRT ; the EDso and its limits of error (P=0.05) were calculated by the method of Litchfield & Wilcoxon (1949). All drugs except pethidine were administered orally 60 min prior to testing. Pethidine was administered intraperitoneally 30 min before testing.
To investigate the possibility of cannabis-pethidine interactions, two experimental schemes were used. (a) Mice received cannabis extracts at various dose levels and some, in addition, received pethidine (6 mg/kg) ; the remainder, dosed with the vehicle only, served as controls. Cannabis extracts were administered 1 h and pethidine 0.5 h before testing. (b) The animals received pethidine at various dose
levels and some also received cannabis extract (60 mg/kg) ; the remainder, dosed with vehicle only, served as controls.
To determine if the antinociceptive effect produced by the cannabis extract could be antagonized by the narcotic antagonist, nalorphine, a group of mice was given a dose of cannabis extract (60 mg/kg) 1 h before testing on the hot-plate. These mice were then divided into two groups, one of which was given nalorphine (5 mg/kg) and the other saline, both by the intravenous route. A similar proce¬dure was used to study the interaction of pethidine (6 mg/kg, given 0.5 h before testing on the hot-plate) and nalorphine or saline.
Intestinal Motility
The effect of drugs on intestinal motility was determined by measuring the rate of passage of a charcoal meal (Macht & Barba-Gose, 1931). Mice received 0.2 ml of a meal, consisting of animal charcoal 12 g, tragacanth 2 g and water 130 ml by lavage and were killed 15 min later. The length of the small intestine from pylorus to the ilex-caecal junction was measured and the distance which the charcoal meal had travelled was expressed as a percentage of the total length of the small intestine. The ED50 and the limits of error (P=0.05) were calculated by the method of Litchfield & Wilcoxon (1949). The cannabis extracts, A8-THC and A9-THC were administered by lavage, 45 min before the charcoal meal.
To determine whether the activity of cannabis extracts on intestinal motility was of a morphine-like nature, a comparison was made of the effect of the narcotic antagonist, nalorphine, on the depression of intestinal motility produced by morphine and by cannabis extracts. Groups of mice were dosed with either cannabis extract, 1 h before they were killed, morphine 0.5 h before they were killed, or a vehicle control. Nalorphine (8 mg/kg) or a vehicle control was administered intraperitoneally to these animals 0.5 h before they were killed.
Results
Andinociceptive effects
Pethidine, A8-THC, A9-THC, cannabis extract, and cannabinol all exhibited dose-dependent antinociceptive activity (Table 2). Pethidine did not differ signifi-cantly in potency from A9-THC. A8-THC and A9-THC did not differ significantly in potency and A9-THC was estimated to be &5 times more active than cannabinol. Cannabis extract I had 70% of the potency of cannabinol and 11% of that of
TABLE 2. The effects of pethidine, pe-tetrahydrocannabinol (THC), p9-THC, cannabinol acetate and
cannabis extract I on antinociceptive activity measured by the hot plate method.
Drug
(Doses, mg/kg) Pethidine
(2, 4, 6, 8)
A9-THC
(4, 7.5, 15, 30, 60) p 5-THC
(4, 9.5, 15, 30)
Cannabinol acetate (10, 20, 40, 60) Cannabis extract I (7.5, 15, 30, 60, 100) ED5o (mg/kg)
(limits of
7.0 (4.8-10.3)
5.0
(2.9- 8.8) 5.0
(2.4-10.5) 32.5
(22.4-47.1) 47.0
(30.9-71.4) Slope Function error for P=0.05)
7.58
(1.46- 39.26) 7.07
(1.96- 25.45) 11.06
(0.64-190.20) 3.74
(1.68- 8.30) 13.74
(3.57- 52.90) No. of
observations
220
300 240 240 300
p9-MC. In terms of p9-THC present, the extract had approximately half the potency of A9-THC. Cannabidiol was inactive at a dose of 30 mg/kg. All the dose-response curves were parallel.
The antinociceptive effect of cannabis alone did not differ significantly from that produced by cannabis plus pethidine (Table 3). Another group of mice, in which the testing procedure was carried out 1.5 h after cannabis and 1 h after pethidine administration gave similar results. In the experiment where the pethidine
TABLE 3. The effect of interaction on the antinociceptive effects of cannabis and pethidine in mice.
Time
after
cannabis
before
testing
(min) ED60 (mg/kg)
(limits of error
(a) Cannabis Slope
function
for P=0.05) No. of
observations
Drug Administration
(mg/kg)
First Second
Cannabis Pethidine (6) 60 18.0 9.08 120
(5, 7.5, 15, 30) (10.3-31.5) (1.85- 44.49)
90 23.0 10.00 160
(13.9-38.2) (1.60- 62.50)
Cannabis Vehicle 60 21.2 15.12 118
(5, 7.5, 15, 30) Control (10.833.0) (0.66-346.2)
90 12.7 9.30 160
(7.9-20.3) (3.72- 23.25)
Cannabis (60) Pethidine 60 (b) Pethidine
4.6 9.21 230
(2, 4, 6, 8) (3.1- 6.9) (1.33- 63.73)
Vehicle Control Pethidine 60 7.0 7.58 230
(2, 4, 6, 8) (4.8-10.3) (4.76- 10.3)
dose was varied and the cannabis dose remained constant, the results were essentially similar to those reported above. Cannabis possibly potentiated the antinociceptive effect of pethidine, but the effect was statistically not significant.
The antinociceptive effect of pethidine but not that of the cannabis extract was antagonized by nalorphine (Table 4).
TABLE 4. The effect of nalorphine (5 mg/kg) on the antinociceptive effects of pethidine and cannabis
extract I.
Cannabis Extract I
Pethidine (6 mg/kg) (60 mg/kg)
Mean Time on hot- Mean Time on hot
plate ± S.E.M. plate ± S.E.M.
Treatment (s) (s)
Before nalorphine 13.8±3.4 (41) 22.1±1.5 (37)
After nalorphine 8.1±0.9 (17) 22.8±2.0 (18)
After saline 16.0±1.4 (20) 25.3±1.7 (19)
Pethidine and cannabis extract were administered 35 and 65 min respectively before administration of nalorphine or saline. The numbers in brackets indicate number of observations.
Intestinal Motility
All cannabis extracts, Q9-THC and p9-THC had a dose-dependent effect on the passage of the charcoal meal (Table 5). Cannabidiol was inactive at all of five dose levels (6-30 mg/kg) tested. All the cannabis extracts and cannabinoids tested were significantly less potent than morphine but the regression lines were parallel. Q9-THC and p9-THC were not significantly different in potency. When the ED50 values for the three cannabis extracts were converted into their equivalent Q9-THC contents (Tables 1 and 5), the ED5o values calculated for Q9-THC were:
TABLE 5. The effects of morphine, A8-tetrahydrocannabinol (THC), A°-THC and cannabis extracts I,
II and Ill on the passage of a charcoal meal in mice.
Drug treatment & dose ED" (mg/kg) Slope function Potency
(mg/kg) (limits of error for P=0.05) (morphine =1)
Morphine (0.25, 0.5, 1.0, 3.4 0.186 1.0
2.0, 4.0, 8.0) (1.7- 6.7) (0.066-0.520)
A8-THC (2.0, 5.0, 10.0, 13.5 f k 0.072 0.25
20.0, 40.0) (10.9- 16.7) (0.007-0.756) (0.12-0.53)
A 9-THC (2.0, 5.0, 10.0, 20.0 0.370 0.17
20.0) (12.9- 31.0) (-250-0.55) (0.07-0.39)
Cannabis Extract I 86.0# 164 0.028
(4.5, 11.4, 22.7, 45.5,
91.0, 182.0) (46.0-160.8) (.059-0.459) (0.015-0.1)
Cannabis Extract II
(4.8, 9.7, 19.4, 38.8, 77.6) 21.5# -217 0.16
(13.6- 34.0) (0.095-0.494) (0.07-0.36)
Cannabis Extract III
(2.9, 7.25, 14.5, 29.0, 68.0#k 0.042 0.05
58.0) (29.8-155.3) (.003-0.671) (0.017-0.15)
A Significantly less potent than morphine at P<0.05.
The ED50 is that dose of compound required to slow the passage of a charcoal meal by 50% when compared with control animals which received the vehicle only.
I. 18.92 mg/kg ; II. 11.18 mg/kg ; III. 27.88 mg/kg. Thus, allowing for experi¬mental error, the effects of the extracts can probably be attributed to the content of A9-THC.
Whilst nalorphine effectively antagonized morphine-induced inhibition of gastro-intestinal motility (Table 6), it had no effect on the action of either cannabis extracts I or II.
TABLE 6. The effect of nalorphine (8 mg/kg) on the actions of morphine (8 mg/kg) and cannabis extracts
I and II (equivalent to 10 mg/kg tetrahydrocannabinol) on the passage of a charcoal meal in mice.
Passage of charcoal meal
expressed as % of total
length of small intestine
First treatment Second treatment +S.E. M.
Morphine Nil 9.0+0.6 (21)
Water Nalorphine 35.8+3.4 (20)
Morphine Nalorphine 24-7+1.9 (20)
Water Water 44.6+1.8 (20)
Extract I Water 33.2+2.2 (21)
Extract I Nalorphine 22.4+2.3 (19)
Vehicle control Water 48.6+2.5 (20)
Vehicle control Nalorphine 37.5+3.5 (20)
Extract II Water 29.4+2.0 (25)
Extract II Nalorphine 23.3+2.1 (25)
Vehicle control Nalorphine 43.6+3.3 (24)
Vehicle control Water 55.5+3.0 (10)
*The values are the means and their standard errors. The numbers in brackets are the number of
observations.
Discussion
There is still some doubt as to the nature of the response of cannabis-treated animals when tested by standard pharmacological methods for analgesia. Evidence for antinociceptive effects has been reported in a number of species following administration of Q9-THC (Bicher & Mechoulam, 1968 ; Bukbaum, 1972 ; Buxbaum et al., 1969). Although Dewey et al. (1972) were unable to demonstrate a significant antinociceptive effect when using the tail flick method in mice (in
doses below 100 mg/kg), they reported a prolonged antinociceptive effect in the same species when using the hot-plate method.
In the present experiments we have found that A9-THC, A9-THC, a cannabis extract and cannabinol all produced a dose-dependent increase in reaction time when tested on the hot-plate, with A9-THC and A9-THC having equal potency and cannabinol approximately one sixth of the potency of A9-THC. The results for A9-THC and A9-THC are thus in broad agreement with those of Dewey et al. (1972).
The analgesic dose-response curve for pethidine was parallel to those for the cannabinoids ; both A9-THC and A9-THC being approximately equipotent with pethidine. These results agree more closely with those of Bicher & Mechoulam (1968) than those of Buxbaum (1972) who reported considerable deviation from parallelism between the dose-effect curves for cannabinoids and narcotic analgesics. These differences might have been due to the route of administration since Buxbaum (1972) injected mice subcutaneously and absorption of cannabinoids by this route is slower and less complete than by the oral route used in our studies (Ho, 1971).
The interaction between cannabinoids and pethidine in mice tested by the hot-plate method was only suggestive of an additive effect. This finding was quite unlike the interactions of cannabinoids with barbiturates or ether on the duration of anaesthesia in mice. Doses of cannabis extracts or A9-THC which themselves are not hypnotic, significantly potentiate the sleeping times of mice induced by barbiturate or ether (Paton & Pertwee, 1972 ; Chesher, Jackson & Starmer, 1974).
A possible effect of cannabis on intestinal motility had been noted in the observa-tion that A9-THC reduced the incidence of defaecation in rats including those considered to be ` high defaecators ' (Masur et al., 1971 ; Drew et al., 1972). A depressant effect of A9-THC and A8-THC administered subcutaneously on the passage of a charcoal meal in mice has been reported by Dewey et al. (1972), although a clear dose-response relationship was not apparent. In the present studies we have shown that oral administration of both A9-THC and A9-THC and three cannabis extracts produced parallel dose-dependent depressions of the passage of a charcoal meal. As with the results on the antinociceptive effects, A9-THC and A9-THC did not differ significantly in potency and the potency of the three cannabis extracts can reasonably be accounted for by their A9-THC contents. It appears therefore that other cannabinoids are exerting little effect on intestinal motility ; this concept is supported by our findings of the lack of activity of cannabidiol on intestinal motility and by the wide divergence in content of cannabidiol in the three extracts used. For the latter reason, cannabinol appears to be unimportant in the activity observed in these studies.
Although, when tested for the antinociceptive effects and the effects on intestinal motility, the dose-response curves of cannabinoids and narcotics were parallel, the response to nalorphine clearly suggests a different mode of action. For both effects, nalorphine antagonized the action of the narcotic analgesic but had no effect on the responses to the cannabinoids.
We wish to thank Professor R. H. Thorp for his constant encouragement and Miss Margaret Rutherford for her skilled technical assistance. This work was supported by a grant from the National Health and Medical Research Council. Samples of cannabis were obtained from seized material by courtesy of the N.S.W. Government. A9-THC and A9-THC were obtained from the WHO, Geneva, by courtesy of Dr. O. Braenden.
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(Received March 20, 1973)
Source: The effect of cannabinoids on intestinal motility and their antinociceptive effect in mice
