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The lethal of Δ9-tetrahydrocannabinol in mice enhance pretreatment with SKF 525-A or chloramphenicol
EUROPEAN JOURNAL OF PHARMACOLOGY26 (1974) 383-385. NORTH-HOLLANDPUBLISHINGCOMPANY
Short communication THE LETHAL EFFECTS OF/x 9-TETRAHYDROCANNABINOL E N H A N C E D BY P R E T R E A T M E N T
IN M I C E
WITH SKF 525-A OR CHLORAMPHENICOL
R. Duane SOFIA Department of Pharmacology, WallaceLaboratories, Cranbury, New Jersey 08512, U.S.A.
Received 25 February 1974
Accepted 1 March 1974
R.D. SOFIA, The lethal effects of A 9-tetrahydrocannabinol in mice enhanced by pretreatment with SKF 525-A or chloramphenicol, European J. Pharmacol. 26 (1974) 383-385. The i.p. lethal activity of THC following single dose administration was significantly enhanced by pretreatment with SKF 525-A (30.0 mg/kg) or chloramphenicol (100.0 mg/kg) as evidenced by a significant reduction in the LDso value. Moreover, the onset to lethality was also sharply reduced. These results are discussed in light of the present knowledge concerning the metabolism of THC. Lethality
A 9-Tetrahydrocannabinol
1. Introduction Certain pharmacological effects of Ag-tetrahydro cannabinol (THC) have been reported to be enhanced following pretreatment with either SKF 525-A (Sofia and Barry, 1970; Kaymakcalan and Deneau, 1971; Sofia and Barry, 1972; Manno and Manno, 1973) or chloramphenicol (CHPC) (Adams and Sofia, 1973), both of which non-specifically inhibit liver microsomal enzymes responsible for drug metabolism (Dixon and Fouts, 1962). Likewise, Mantilla-Plata and Harbison (1971) reported that the incidence of death in mice following a single intraperitoneal (i.p.) dose of 200 mg/kg of THC increased from 20 to 90% in mice pretreatment 1 hr before with SKF 525-A (40.0 mg/kg, i.p.). The purpose of the following investigation was to determine whether the L D s o dose-response curve and onset to lethality following THC could be affected by pretreatment with SKF 525-A or CHPC.
2. Materials and methods These experiments were performed on Swiss male albino mice (Charles River) weighing 2 0 - 3 0 g at the time of dosing. The animals were housed in wire mesh
SKF 5 2 5 - A
Chloramphenicol
cages, 10 mice per cage, 5 days before use in a laboratory environment of 12 hr of light and 12 hr of dark, at a temperature and humidity of 22.0-24.0°C and 48-52% respectively, with food and water continuously available. Mice, in groups of 10 each, were administered SKF 525-A'HCI (30.0 mg/kg) or CHPC sodium succinate (100.0 mg/kg) i.p. 45 min prior to i.p. administration of several doses of THC or its vehicle (10% propylene glycol-l% Tween 80-0.9% saline). These doses of SKF 525-A and CHPC were chosen based on dose range and duration of action studies conducted for prior experiments (Sofia and Barry, 1972; Adams and Sofia, 1973). All doses of THC and the metabolic inhibitors SKF 525-A and CHPC (each prepared for injection as the base and solubilized in 0.9% saline) were prepared in such a concentration to permit a volume of 0.1 ml/10 g of body weight to be given each animal. Each group of mice were checked for deaths 1,2, 3; 4, 24, 48, 72 and 168 hr following THC administration. LDso and potency ratio (PR) values with 95% confidence limits were calculated by the method of Litchfield and Wilcoxon (1949).
384
R.D. Sofia, THC lethality enhanced'by SKF 525-A or chloramphenicol
3. Results Table 1 gives a detailed account of the results o f this investigation. Close inspection o f these data reveal the onset to death for mice pretreated with saline then administered THC was delayed with the peak effect for all doses not observed until the 48 hr observation period. Only the 1000.0 mg/kg dose produced its peak effect, i.e., 100% mortality, at an earlier time (24 hr). On the other hand, the peak lethal activity o f THC in mice pretreated either with SKF 525-A (30.0 mg/kg) or CHPC (100.0 mg/kg) was observed Table 1 Effects of SKF 525-A and CHPC on the lethal effects of a single dose of THC in mice. Drug*
I.p. dose Percentage of animals dead at (mg/kg) various times (hr) after THC of THC administration 1
Saline + vehicle Saline + THC
62.5 125.0 250.0 500.0 1000.0
SKF 525-A + vehicle (30.0 mg/kg) SKF 525-A + THC (30.0
mg/l~)
CHPC + vehicle (100.0 mg/kg) CHPC + THC (100.0 mg/kg)
2
0 0 0 0 0 0
3
4
0 0 0 0 0 0 0 0 10 10 30 50
24
48
0 0 0 10 30 80
0 0 0 20 30 100
0 0 0 20 40 100
0
0
0
0
0
0
62.5
0
0
0
0
0
0
125.0 250.0 500.0 1000.0
0 0 0 20
10 20 20 40 20 70 50 90
20 60 80 100
20 60 80 100
20 60 80 100
62.5 125.0 250.0 500.0 1000.0
0 0 0 0 0 0 0 10 0 20 10 40
0 0 10 30 60 90
0 0 10 60 80 100
0 0 10 60 80 100
0 0 10 60 80 100
* Saline, SKF 525-A or CHPC were administered i.p. 45 min prior to THC or its vehicle.
Table 2 48 hr LDso values for THC given to mice pretreated i.p. with saline, SKF 525-A (30.0 mg/kg) or CHPC (100.0 mg/kg). Drug treatment
l.p. LDio (mg/kg) (95% confidence limits)
PR* (95% confidence limits)
Saline + THC
510.0 (532.0 - 740.0) -
SKF 525-A + THC
230.0 (118.0 - 418.0) 2.11 (1.18 - 4.17)t
CHPC + THC
250.0 (143.0 - 438.0) 2.04 (1.05 - 3.98)t
* Potency ratio (obtained by saline + THC/SKF 525-A + THC or CHPC + THC). t Statistically significant difference between LDso values (p < 0.05). by the 4 hr observation period. Neither o f these inhibitors o f drug metabolism interacted with the vehicle for THC to produce mortalities. Data obtained at the 72 and 168 hr time intervals for each treatment group were identical to those observed at 48 hr. Hence, these values were excluded from table I for the sake of clarity. Table 2 lists the i.p. LDs o values for THC in mice pretreated either with saline, SKF 525-A or CHPC. The lethal activity o f THC was markedly enhanced in mice pretreated with 30.0 mg/kg o f SKF 525-A as evidenced by the (saline + THC)/(SKF 525-A + THC) potency ratio (PR) o f 2.11. Likewise, prior administration of CHPC significantly increased THC lethality, i.e., lowered the LDs o from 510.0 mg/kg (in salinepretreated mice) to 250.0 mg/kg resulting in a PR of 2.04.
4. Discussion Results of the present investigation clearly indicate the lethal effect o f THC is markedly enhanced by pretreatment with the non-specific inhibitors o f liver microsomal drug metabolizing enzymes SKF 525-A or CHPC (Dixon and Fouts, 1962). This conclusion was drawn not only because the actual number o f mice which died was increased, hence a lower mg/kg LDs o value, but also the peak onset to lethal activity for all doses o f THC was reduced from 48 hr in saline-pretreated mice to 4 hr in mice receiving either
R.D. Sofia, THC lethality enhanced by SKF 525-,4 or chloramphenicol
SKF 525-A or CHPC. Saline pretreatment did not alter the LDs 0 o f THC since the 510.0 mg/kg value obtained in this study closely parallels the results obtained previously in these (Sofia, 1972) and other laboratories (Phillips et al., 1971). Augmentation o f the lethal activity of THC b y pretreatment with SKF 525-A as previously suggested by Mantilla-Plate and Harbison (1971)was confirmed and described in greater detail and extended to CHPC as a result of the present investigation. This effect on lethality was not totally unexpected since both SKF 525-A (Sofia and Barry, 1970) and CHPC (Adams and Sofia, 1973) have been shown to enhance the depressant effects o f THC, while the former has also increased the analgesic (Kaymakcalan and Deneau, 1971; Sofia and Barry, 1972) and cardiovascular activity (Manno and Manno, 1973) o f THC. Since the metabolism o f THC in vitro has been shown to be markedly inhibited by SKF 525-A (Burstein and Kupfer, 1971) it was suggested by Sofia and Barry (1970 and 1972) that the pharmacologic effects o f THC are primarily attributable to the parent compound rather than a metabolite(s), since the levels of unchanged THC in vivo presumably would increase. Likewise, Adams and Sofia (1973) by inference came to the same conclusion in their studies on the interaction o f CHPC and THC since CHPC, too, is a non-specific inhibitor o f liver microsomal drug metabolizing enzymes (Dixon and Fouts, 1962). However, Gill and Jones (1972) have shown that in mice pretreated with SKF 525-A brain levels of unchanged THC rose only 25%, while the level o f 11hydroxy-THC, the primary metabolite, was increased three-fold. Truitt (1972) gives evidence to explain this by indicating that further metabolism of l l hydroxy-THC to the inactive metabolite 7,11-dihydroxy-THC is more sensitive to SKF 525-A and other inhibitors. Hence, these authors suggest that an active metabolite theory may apply to the pharmacological properties o f THC. Although this controversy is not y e t completely resolved, the results o f the present investigation clearly illustrate that the lethal effect o f THC, like certain o f its pharmacological activities, is markedly increased in mice pretreated with SKF 525-A or CHPC.
385
Acknowledgements THC was generously supplied through the courtesy of Monique Braude, Ph.D., Executive Secretary, NIMH-FDA Psychotomimetic Agents Advisory Committee. The author extends his thanks to Smith Kline & French Laboratories, Philadelphia, Pennsylvania for their generous supply of SKF 525-A. The CHPC sodium succinate used in these studies is the brand of Chloromycetin ® used for i.v. administration made by Parke-Davis, Detroit, Michigan.
References Adams, H.R. and R.D. Sofia, 1973, Interaction of chloramphenicol and A l.tetrahydrocannabinol on barbital-anesthetized mice, Experientia 29, 181. Burstein, S.H. and D. Kupfer, 1971, Hydroxylation of trans-A t-tetrahydrocannabinol by a hepatic microsomal monoxygenase, Chem. Biol. Interact. 3,316. Dixon, R.L. and J.R. Fouts, 1962, Inhibition of microsomal drug metabolic pathways by chloramphenicol, Biochem. Pharmacol. 11,715. Gill, E.W. and G. Jones, 1972, Brain levels of A Ltetrahydrocannabinol and its metabolites in mice - correlation with behavior, and the effect of the metabolic inhibitors SKF 525-A and piperonyl butoxide, Biochem. Pharmacol. 21, 2237. Kaymakcalan, S. and G.A. Deneau, 1971, Some pharmacological effects of synthetic A9-tetrahydrocannabinol (THC), Pharmacologist 13,247. Litchfield, Jr., J.T. and F. Wilcoxon, 1949, A simplified method of evaluating dose-effect experiments, J. Pharmacol. Exptl. Therap. 96, 99. Manno, J.E. and B.R. Manno, 1973, The interaction of A 9tetrahydrocannabinol (THC), pentobarbital and SKF 525-A with the cardiovascular system of the rat, Federation Proc. 32,755. Mantilla-Plata, B. and R.D. Harbison, 1971, Phenobarbital and SKF 525-A effect on A 9-tetrahydrocannabinol (THC) toxicity and distribution in mice, Pharmacologist 13,297, Phillips, R.N., R.F. Turk and R.B. Forney, 1971, Acute toxicity of A9-tetrahydrocannabinol in rats and mice, Proc. Soc. Exptl. Biol. Med. 136,260. Sofia, R.D., 1972, The lethal effects of A 1-tetrahydrocannabinol in aggregated and isolated mice following single dose administration, European J. Pharmacol. 20, 139. Sofia, R.D. and H. Barry, III, 1970, Depressant effect of A Ltetrahydrocannabinol enhanced by inhibition of its metabolism, European J. Pharmacol. 13, 134. Sofia, R.D. and H. Barry, III, 1972, The influence of SKF 525-A on the analgesic actions of ALtetrahydrocannabinol, Federation Proc. 31,506. Truitt, Jr., E.B., 1972, Evaluation of an active metabolite hypothesis for ll-hydroxy-tetrahydrocannabinols, Abstr. Fifth Intern. Cong. Pharmacol., p. 236.
Short communication THE LETHAL EFFECTS OF/x 9-TETRAHYDROCANNABINOL E N H A N C E D BY P R E T R E A T M E N T
IN M I C E
WITH SKF 525-A OR CHLORAMPHENICOL
R. Duane SOFIA Department of Pharmacology, WallaceLaboratories, Cranbury, New Jersey 08512, U.S.A.
Received 25 February 1974
Accepted 1 March 1974
R.D. SOFIA, The lethal effects of A 9-tetrahydrocannabinol in mice enhanced by pretreatment with SKF 525-A or chloramphenicol, European J. Pharmacol. 26 (1974) 383-385. The i.p. lethal activity of THC following single dose administration was significantly enhanced by pretreatment with SKF 525-A (30.0 mg/kg) or chloramphenicol (100.0 mg/kg) as evidenced by a significant reduction in the LDso value. Moreover, the onset to lethality was also sharply reduced. These results are discussed in light of the present knowledge concerning the metabolism of THC. Lethality
A 9-Tetrahydrocannabinol
1. Introduction Certain pharmacological effects of Ag-tetrahydro cannabinol (THC) have been reported to be enhanced following pretreatment with either SKF 525-A (Sofia and Barry, 1970; Kaymakcalan and Deneau, 1971; Sofia and Barry, 1972; Manno and Manno, 1973) or chloramphenicol (CHPC) (Adams and Sofia, 1973), both of which non-specifically inhibit liver microsomal enzymes responsible for drug metabolism (Dixon and Fouts, 1962). Likewise, Mantilla-Plata and Harbison (1971) reported that the incidence of death in mice following a single intraperitoneal (i.p.) dose of 200 mg/kg of THC increased from 20 to 90% in mice pretreatment 1 hr before with SKF 525-A (40.0 mg/kg, i.p.). The purpose of the following investigation was to determine whether the L D s o dose-response curve and onset to lethality following THC could be affected by pretreatment with SKF 525-A or CHPC.
2. Materials and methods These experiments were performed on Swiss male albino mice (Charles River) weighing 2 0 - 3 0 g at the time of dosing. The animals were housed in wire mesh
SKF 5 2 5 - A
Chloramphenicol
cages, 10 mice per cage, 5 days before use in a laboratory environment of 12 hr of light and 12 hr of dark, at a temperature and humidity of 22.0-24.0°C and 48-52% respectively, with food and water continuously available. Mice, in groups of 10 each, were administered SKF 525-A'HCI (30.0 mg/kg) or CHPC sodium succinate (100.0 mg/kg) i.p. 45 min prior to i.p. administration of several doses of THC or its vehicle (10% propylene glycol-l% Tween 80-0.9% saline). These doses of SKF 525-A and CHPC were chosen based on dose range and duration of action studies conducted for prior experiments (Sofia and Barry, 1972; Adams and Sofia, 1973). All doses of THC and the metabolic inhibitors SKF 525-A and CHPC (each prepared for injection as the base and solubilized in 0.9% saline) were prepared in such a concentration to permit a volume of 0.1 ml/10 g of body weight to be given each animal. Each group of mice were checked for deaths 1,2, 3; 4, 24, 48, 72 and 168 hr following THC administration. LDso and potency ratio (PR) values with 95% confidence limits were calculated by the method of Litchfield and Wilcoxon (1949).
384
R.D. Sofia, THC lethality enhanced'by SKF 525-A or chloramphenicol
3. Results Table 1 gives a detailed account of the results o f this investigation. Close inspection o f these data reveal the onset to death for mice pretreated with saline then administered THC was delayed with the peak effect for all doses not observed until the 48 hr observation period. Only the 1000.0 mg/kg dose produced its peak effect, i.e., 100% mortality, at an earlier time (24 hr). On the other hand, the peak lethal activity o f THC in mice pretreated either with SKF 525-A (30.0 mg/kg) or CHPC (100.0 mg/kg) was observed Table 1 Effects of SKF 525-A and CHPC on the lethal effects of a single dose of THC in mice. Drug*
I.p. dose Percentage of animals dead at (mg/kg) various times (hr) after THC of THC administration 1
Saline + vehicle Saline + THC
62.5 125.0 250.0 500.0 1000.0
SKF 525-A + vehicle (30.0 mg/kg) SKF 525-A + THC (30.0
mg/l~)
CHPC + vehicle (100.0 mg/kg) CHPC + THC (100.0 mg/kg)
2
0 0 0 0 0 0
3
4
0 0 0 0 0 0 0 0 10 10 30 50
24
48
0 0 0 10 30 80
0 0 0 20 30 100
0 0 0 20 40 100
0
0
0
0
0
0
62.5
0
0
0
0
0
0
125.0 250.0 500.0 1000.0
0 0 0 20
10 20 20 40 20 70 50 90
20 60 80 100
20 60 80 100
20 60 80 100
62.5 125.0 250.0 500.0 1000.0
0 0 0 0 0 0 0 10 0 20 10 40
0 0 10 30 60 90
0 0 10 60 80 100
0 0 10 60 80 100
0 0 10 60 80 100
* Saline, SKF 525-A or CHPC were administered i.p. 45 min prior to THC or its vehicle.
Table 2 48 hr LDso values for THC given to mice pretreated i.p. with saline, SKF 525-A (30.0 mg/kg) or CHPC (100.0 mg/kg). Drug treatment
l.p. LDio (mg/kg) (95% confidence limits)
PR* (95% confidence limits)
Saline + THC
510.0 (532.0 - 740.0) -
SKF 525-A + THC
230.0 (118.0 - 418.0) 2.11 (1.18 - 4.17)t
CHPC + THC
250.0 (143.0 - 438.0) 2.04 (1.05 - 3.98)t
* Potency ratio (obtained by saline + THC/SKF 525-A + THC or CHPC + THC). t Statistically significant difference between LDso values (p < 0.05). by the 4 hr observation period. Neither o f these inhibitors o f drug metabolism interacted with the vehicle for THC to produce mortalities. Data obtained at the 72 and 168 hr time intervals for each treatment group were identical to those observed at 48 hr. Hence, these values were excluded from table I for the sake of clarity. Table 2 lists the i.p. LDs o values for THC in mice pretreated either with saline, SKF 525-A or CHPC. The lethal activity o f THC was markedly enhanced in mice pretreated with 30.0 mg/kg o f SKF 525-A as evidenced by the (saline + THC)/(SKF 525-A + THC) potency ratio (PR) o f 2.11. Likewise, prior administration of CHPC significantly increased THC lethality, i.e., lowered the LDs o from 510.0 mg/kg (in salinepretreated mice) to 250.0 mg/kg resulting in a PR of 2.04.
4. Discussion Results of the present investigation clearly indicate the lethal effect o f THC is markedly enhanced by pretreatment with the non-specific inhibitors o f liver microsomal drug metabolizing enzymes SKF 525-A or CHPC (Dixon and Fouts, 1962). This conclusion was drawn not only because the actual number o f mice which died was increased, hence a lower mg/kg LDs o value, but also the peak onset to lethal activity for all doses o f THC was reduced from 48 hr in saline-pretreated mice to 4 hr in mice receiving either
R.D. Sofia, THC lethality enhanced by SKF 525-,4 or chloramphenicol
SKF 525-A or CHPC. Saline pretreatment did not alter the LDs 0 o f THC since the 510.0 mg/kg value obtained in this study closely parallels the results obtained previously in these (Sofia, 1972) and other laboratories (Phillips et al., 1971). Augmentation o f the lethal activity of THC b y pretreatment with SKF 525-A as previously suggested by Mantilla-Plate and Harbison (1971)was confirmed and described in greater detail and extended to CHPC as a result of the present investigation. This effect on lethality was not totally unexpected since both SKF 525-A (Sofia and Barry, 1970) and CHPC (Adams and Sofia, 1973) have been shown to enhance the depressant effects o f THC, while the former has also increased the analgesic (Kaymakcalan and Deneau, 1971; Sofia and Barry, 1972) and cardiovascular activity (Manno and Manno, 1973) o f THC. Since the metabolism o f THC in vitro has been shown to be markedly inhibited by SKF 525-A (Burstein and Kupfer, 1971) it was suggested by Sofia and Barry (1970 and 1972) that the pharmacologic effects o f THC are primarily attributable to the parent compound rather than a metabolite(s), since the levels of unchanged THC in vivo presumably would increase. Likewise, Adams and Sofia (1973) by inference came to the same conclusion in their studies on the interaction o f CHPC and THC since CHPC, too, is a non-specific inhibitor o f liver microsomal drug metabolizing enzymes (Dixon and Fouts, 1962). However, Gill and Jones (1972) have shown that in mice pretreated with SKF 525-A brain levels of unchanged THC rose only 25%, while the level o f 11hydroxy-THC, the primary metabolite, was increased three-fold. Truitt (1972) gives evidence to explain this by indicating that further metabolism of l l hydroxy-THC to the inactive metabolite 7,11-dihydroxy-THC is more sensitive to SKF 525-A and other inhibitors. Hence, these authors suggest that an active metabolite theory may apply to the pharmacological properties o f THC. Although this controversy is not y e t completely resolved, the results o f the present investigation clearly illustrate that the lethal effect o f THC, like certain o f its pharmacological activities, is markedly increased in mice pretreated with SKF 525-A or CHPC.
385
Acknowledgements THC was generously supplied through the courtesy of Monique Braude, Ph.D., Executive Secretary, NIMH-FDA Psychotomimetic Agents Advisory Committee. The author extends his thanks to Smith Kline & French Laboratories, Philadelphia, Pennsylvania for their generous supply of SKF 525-A. The CHPC sodium succinate used in these studies is the brand of Chloromycetin ® used for i.v. administration made by Parke-Davis, Detroit, Michigan.
References Adams, H.R. and R.D. Sofia, 1973, Interaction of chloramphenicol and A l.tetrahydrocannabinol on barbital-anesthetized mice, Experientia 29, 181. Burstein, S.H. and D. Kupfer, 1971, Hydroxylation of trans-A t-tetrahydrocannabinol by a hepatic microsomal monoxygenase, Chem. Biol. Interact. 3,316. Dixon, R.L. and J.R. Fouts, 1962, Inhibition of microsomal drug metabolic pathways by chloramphenicol, Biochem. Pharmacol. 11,715. Gill, E.W. and G. Jones, 1972, Brain levels of A Ltetrahydrocannabinol and its metabolites in mice - correlation with behavior, and the effect of the metabolic inhibitors SKF 525-A and piperonyl butoxide, Biochem. Pharmacol. 21, 2237. Kaymakcalan, S. and G.A. Deneau, 1971, Some pharmacological effects of synthetic A9-tetrahydrocannabinol (THC), Pharmacologist 13,247. Litchfield, Jr., J.T. and F. Wilcoxon, 1949, A simplified method of evaluating dose-effect experiments, J. Pharmacol. Exptl. Therap. 96, 99. Manno, J.E. and B.R. Manno, 1973, The interaction of A 9tetrahydrocannabinol (THC), pentobarbital and SKF 525-A with the cardiovascular system of the rat, Federation Proc. 32,755. Mantilla-Plata, B. and R.D. Harbison, 1971, Phenobarbital and SKF 525-A effect on A 9-tetrahydrocannabinol (THC) toxicity and distribution in mice, Pharmacologist 13,297, Phillips, R.N., R.F. Turk and R.B. Forney, 1971, Acute toxicity of A9-tetrahydrocannabinol in rats and mice, Proc. Soc. Exptl. Biol. Med. 136,260. Sofia, R.D., 1972, The lethal effects of A 1-tetrahydrocannabinol in aggregated and isolated mice following single dose administration, European J. Pharmacol. 20, 139. Sofia, R.D. and H. Barry, III, 1970, Depressant effect of A Ltetrahydrocannabinol enhanced by inhibition of its metabolism, European J. Pharmacol. 13, 134. Sofia, R.D. and H. Barry, III, 1972, The influence of SKF 525-A on the analgesic actions of ALtetrahydrocannabinol, Federation Proc. 31,506. Truitt, Jr., E.B., 1972, Evaluation of an active metabolite hypothesis for ll-hydroxy-tetrahydrocannabinols, Abstr. Fifth Intern. Cong. Pharmacol., p. 236.