Pharmacological Research Communications, Vol. 10, No. 3, 1978
243
INTRAVENOUS COCAINE LETHALITY IN THE RATI, z
Marvin C. Wilson 3 and John M. Holbrook 4 Department of Pharmacology School of Pharmacy University of Mississippi University, MS 38677
Re~ived~fina/~rm 13 March 1978
Summary A series of experiments was conducted in order to azcertain several factors related to the convulsant {lethal) effect of cocaine following intravenous administration to freely moving rats.
The results of the initial study
demonstrated that as the concentration of a cocaine infusion is increased, the time to death and the mean lethal dosage are significantly reduced.
Pre-
treatment with non-depressing dosages of either alpha-methylparatyrosine, pimozide,or U-14,624 failed to alter either the convulsant action or the mean acute lethal dose of cocaine.
These results suggest that the convulsant and/
or lethal action of cocaine may not be mediated by catecholamines.
Subacute
intravenous treatment with cocaine for ~even consecutive days resulted in a significant elevatlon in the mean lethal dose 6f cocaine.
However)only half
ISupported in part by NIDA Grant No. DA 001S2-01 and by the Research Institute of Pharmaceutical Sciences and by a fellowship awarded to Dr. Holbrook by the American Foundation for Pharmaceutical Education. The authors express their sincere appreciati6nto Ms. Rita Ewing and Ms. Meredith Guinn for their assistance in the preparation Of this manuscript. 2parts of this manuscript were presented at the Fall Meeting of the American Society of Pharmacology and Experimental Therapeutihs, New Orleans, LA, 1976. 3Author to whom reprints should be requested at the above address. 4Presently located at the Southern School of Pharmacy, Mercer University, Atlanta, GA 30312.
0031-6989/78/0010-0243/$02.00/0
Pharmacological Research Communications, VoL 10, No. 3, 1978
244
of the subjects survived this treatment regimen.
In those subjects not sur-
viving, evidence of sensitization to the convulsant action of cocaine was demonstrated.
Introduction Although the illegal use of cocaine has increased dramatically during the last decade, knowledge of the toxic and lethal actions of this compound has not substantially increased in the last 55 years.
What data are available are
primarily based on studies using the intraperitoneal route of administration. Essentially no parametric lethality studies in which cocaine was administered intravenously,
have been reported.
is frequently abused by this route.
This lack of data is appalling for cocaine Furthermore2many of the current preclin-
ical and clinical cocaine studies involve the intravenous route of administration.
Since cocaine can induce local vasoconstriction and thereby retard
its own systemic absorption, generalizations between the results of studies in which cocaine was administered subcutaneously or intramuscularly, and intravenous results would appear inappropriate. Downs and Eddy (1932b) reported that the acute LDSO of intraperitoneal cocaine in the rat approximated 80 mg/kg,
In almost every case death was pre-
ceded by convulsions, however not every subject which convulsed died. Eidelberg et al. (1963) have determined that these convulsions originate in the limbic system, more precisely the amygdala.
Guerrero et al, (1965) admin-
istered a 1% cocaine solution continuously via the intraperitoneal route to rats.
These authors reported a mean convulsant dose of 102 mg/kg and a mean
lethal dose of 119 mg/kg.
Eidelberg e_~ta_~l. (1963) acutely treated rats intra-
peritoneally with cocaine and found that 50 mg/kg produced convulsions in half the subjects, whereas 65 mg/kg produced convulsions in all subjects. The ability of various drug treatments to antagonize the lethal action of cocaine in rats h a s b e e n reported by several investigators.
Pretreatment
Pharmacological Research Communications, Vol. 10, No. 3, 1978
245
with barbital (Downs and Eddy, 1932a), diazepam (Eidelberg et al., 1965~ and diphenylhydantoin, hydroxylamine, pyridoxine, reserpine, dibenamine, phenobarbital, and chlorpromazine (Eidelberg e_tta_~l., 1963~ have been shown to reduce cocaine lethality.
Pretreatment with the anticonvulsan~ e.g.
barbital,
phenobarbita% and diphenylhydantoin were less effective than the other agents in reducing the number of convulsions.
Although the subjects convulse~ the
convulsions less often resulted in death.
Pretreatment with chlorpromazine,
reserpin~ and dibenamine were equally effective in antagonizing both the convulsions and lethality.
These data suggest that catecholamines are involved
in mediating the convulsive action of cocaine.
Therefor~ pretreatment with
compounds capable of reducing catecholamine activity may be expected to increase the lethal dose of cocaine. Downs and Eddy (1932a) reported that during chronic treatment, rats becamo more sensitive to the convulsant action of cocaine.
Similar results
have been obtained in dogs {Tatum and Seevers, 1929; Downs and Eddy, 1932c) and monkeys (Tatum and Seevers, 1929; Post and Kopanda, 1975). Misra e_~ta_l. (1975) have reported preliminary results concerning the lethality of intravenous cocaine in rats.
The intravenous administration of
20 mg/kg resulted in convulsions and death in all subjects within five minutes. Lower dosages produced similar effects without mortality.
BecaUse of the lack
of reports concerning the intravenous lethality of cocaine in rodents, the present series of studies was conducted.
These studies were designed to
determine (I) the acute intravenous lethal dose of cocaine2and secondaril~ to ascertain the effects of infusate concentration on this dosage, (2) if pretreatment with agents which block central and peripheral norepinephrine and dopamine synthesis alter the acute lethal dose2and (33 if sensitization to the convulsant action of cocaine results during a subacute schedule of intravenous administration.
Pharmacological Research Communications, VoL 10, No. 3, 1978
246 Methods
Subjects were adult male albino rats of a Wistar-derived strain weighing 500-3S0 g (National Laboratory Animal Company; Creve Coeur, ~D).
Subjects
were individually housed in quarters in which the temperature was maintained at 22-24°C with a 12 hour light/dark cycle. were available ad libitum.
Food (Wayne Lab Chow) and water
All subjects were given at least two weeks to
adapt to this environment prior to experimental use.
Following this adapta-
tion period cannulae were surgically implanted in the external jugular vein. The preparation of the cannulae and surgical procedure were similar to methods described by Weeks (1971). pentobarbital
Cannulation of subjects was performed using sodium
(50 mg/kg, i.p.) as the anesthetic.
Following surgery I00,000
units of procaine penicillin G and 12S mg of dihydrostreptomycin
sulfate
(Franklin Laboratories Inc., Denver~ CO) was administered intramuscularly as a prophylactic anti-infective measure.
Subjects were then returned to the home
cages for 48-72 hours prior to use. Lethality studies were conducted in a 25.4 cm square cubicle constructed of sheet metal and plexiglas with a floor composed of 0.32 cm stainless steel rods, spaced 1.2S cm apart. through swivel
The top of the cubicle, which supported a feed-
(BRS-LVE #192-03, Beltsville, MD) for drug infusions, was con-
structed of mesh wire screen.
Drug delivery was accomplished with an infusion
pimp (Harvard Apparatus, Model 600-900, Millis, MA) which delivered 0.1 ml of solution per minute through a silicone rubber tubing (0.8 mm i.d. x 4.3 mm o.d.). From the swivel, solutions passed through polyethylene tubing (PE-20, C]ay Adams, Parsippany, NJ) encased in a flexible coiled-spring wire whic~ in turn s was attached to a restraining harness (BRS-LVE #191-10, Beltsville, MD) placed around the subject.
Preliminary studies had demonstrated that this
restraint system did not significantly affect,subject motility. Acute intravenous cocaine toxicity studies were conducted to determine
Pharmacological Research Communications, VoL 10, No. 3, 1978
247
the relationships between rate of infusion, lethal dose and latency to death. Three concentrations of cocaine hydrochloride [0.S, 1.0 and 2.0 mg/O.l ml) were used with a constant infusion rate of 0.i ml/minute. were tested with each concentration.
Eight subjects
Cocaine hydrochloride [Merck, Rahway,
NJ) solutions were prepared dail~ using an isotonic saline vehicle. Each subject was placed in the observation chamber, attached to the drug delivery system and allowed 5-10~minutes for exploratory behavior.
After ac-
tivity had reached a low level, the drug infusion pump and a timer were simultaneously activated.
Continuous observation of the subject permitted a
determination of the time at which convulsions and death occurred. In order to determine whether altering central catecholmnine function would affect acute cocaine lethality, a similar Study was performed using cocaine hydFochloride at a concentration of 0.5 mg/0.1 ml with the same infusion rate as indicated previously.
For this study, eight subjects each were
pretreated with either l-alpha-methylparatyrosine phenyl-3-[2-thiazolyl]-2-thiourea mg/kg, i.p.),
(AMPT) 50 mg/kg, i.p., l-
[U-14,624) 50 mg/kg, i.p~or pimozide [0.31
l-alpha-methy!paratyrosine [AMPT, Regis Chemical Co., Chicago,
IL) was suspended in saline [25 mg/ml) by sonification with a Polytron homogenizer.
U-14,624 [Aldrich Chemical Co., ~lwaukee,
WI) was suspended in
saline [25 mg/ml) using 1% Tween 80 as the suspending agent.
Pimozide [ORAP R,
McNeil Laboratories Inc., Fort Washington, PA) solutions were prepared by mixing 21.7 mg of pimozide base with 130.2 mg of tartaric acid in 20 ml of warm water.
T h e mixture was placed in a hot water bath and stirred until the
compounds were dissolved.
The solution was then diluted with hot water to
yield a final concentration of 0.31 mg/ml,
kMPT and U-14;624 were administered
4 hours prior to initiation of the cocaine infusion, whereas pimozide was administered thirty minutes prior to infusion. In subacute toxicity studies, eight subjects with intravenous cannulae were infused once daily for seven consecutive days With cocaine hydrochloride
Pharmacologicel Research Communications, VoL 10, No. 3, 1978
248
(0.5 mg/O.l ml/min) until a total of 15 mg/kg was administered or until convulsions occurred.
For each subject, infusion duration was altered daily
based on determinations of body weight.
~=
29'
~"
LETHAL
On the eighth day, cocaine was in-
DOSE
30
TIME TO DEATH
28
25
27 om..,
V
E
uJ co 0 D ..J .< -rl-uJ J
26
E
20
v
25 I
24
243
INTRAVENOUS COCAINE LETHALITY IN THE RATI, z
Marvin C. Wilson 3 and John M. Holbrook 4 Department of Pharmacology School of Pharmacy University of Mississippi University, MS 38677
Re~ived~fina/~rm 13 March 1978
Summary A series of experiments was conducted in order to azcertain several factors related to the convulsant {lethal) effect of cocaine following intravenous administration to freely moving rats.
The results of the initial study
demonstrated that as the concentration of a cocaine infusion is increased, the time to death and the mean lethal dosage are significantly reduced.
Pre-
treatment with non-depressing dosages of either alpha-methylparatyrosine, pimozide,or U-14,624 failed to alter either the convulsant action or the mean acute lethal dose of cocaine.
These results suggest that the convulsant and/
or lethal action of cocaine may not be mediated by catecholamines.
Subacute
intravenous treatment with cocaine for ~even consecutive days resulted in a significant elevatlon in the mean lethal dose 6f cocaine.
However)only half
ISupported in part by NIDA Grant No. DA 001S2-01 and by the Research Institute of Pharmaceutical Sciences and by a fellowship awarded to Dr. Holbrook by the American Foundation for Pharmaceutical Education. The authors express their sincere appreciati6nto Ms. Rita Ewing and Ms. Meredith Guinn for their assistance in the preparation Of this manuscript. 2parts of this manuscript were presented at the Fall Meeting of the American Society of Pharmacology and Experimental Therapeutihs, New Orleans, LA, 1976. 3Author to whom reprints should be requested at the above address. 4Presently located at the Southern School of Pharmacy, Mercer University, Atlanta, GA 30312.
0031-6989/78/0010-0243/$02.00/0
Pharmacological Research Communications, VoL 10, No. 3, 1978
244
of the subjects survived this treatment regimen.
In those subjects not sur-
viving, evidence of sensitization to the convulsant action of cocaine was demonstrated.
Introduction Although the illegal use of cocaine has increased dramatically during the last decade, knowledge of the toxic and lethal actions of this compound has not substantially increased in the last 55 years.
What data are available are
primarily based on studies using the intraperitoneal route of administration. Essentially no parametric lethality studies in which cocaine was administered intravenously,
have been reported.
is frequently abused by this route.
This lack of data is appalling for cocaine Furthermore2many of the current preclin-
ical and clinical cocaine studies involve the intravenous route of administration.
Since cocaine can induce local vasoconstriction and thereby retard
its own systemic absorption, generalizations between the results of studies in which cocaine was administered subcutaneously or intramuscularly, and intravenous results would appear inappropriate. Downs and Eddy (1932b) reported that the acute LDSO of intraperitoneal cocaine in the rat approximated 80 mg/kg,
In almost every case death was pre-
ceded by convulsions, however not every subject which convulsed died. Eidelberg et al. (1963) have determined that these convulsions originate in the limbic system, more precisely the amygdala.
Guerrero et al, (1965) admin-
istered a 1% cocaine solution continuously via the intraperitoneal route to rats.
These authors reported a mean convulsant dose of 102 mg/kg and a mean
lethal dose of 119 mg/kg.
Eidelberg e_~ta_~l. (1963) acutely treated rats intra-
peritoneally with cocaine and found that 50 mg/kg produced convulsions in half the subjects, whereas 65 mg/kg produced convulsions in all subjects. The ability of various drug treatments to antagonize the lethal action of cocaine in rats h a s b e e n reported by several investigators.
Pretreatment
Pharmacological Research Communications, Vol. 10, No. 3, 1978
245
with barbital (Downs and Eddy, 1932a), diazepam (Eidelberg et al., 1965~ and diphenylhydantoin, hydroxylamine, pyridoxine, reserpine, dibenamine, phenobarbital, and chlorpromazine (Eidelberg e_tta_~l., 1963~ have been shown to reduce cocaine lethality.
Pretreatment with the anticonvulsan~ e.g.
barbital,
phenobarbita% and diphenylhydantoin were less effective than the other agents in reducing the number of convulsions.
Although the subjects convulse~ the
convulsions less often resulted in death.
Pretreatment with chlorpromazine,
reserpin~ and dibenamine were equally effective in antagonizing both the convulsions and lethality.
These data suggest that catecholamines are involved
in mediating the convulsive action of cocaine.
Therefor~ pretreatment with
compounds capable of reducing catecholamine activity may be expected to increase the lethal dose of cocaine. Downs and Eddy (1932a) reported that during chronic treatment, rats becamo more sensitive to the convulsant action of cocaine.
Similar results
have been obtained in dogs {Tatum and Seevers, 1929; Downs and Eddy, 1932c) and monkeys (Tatum and Seevers, 1929; Post and Kopanda, 1975). Misra e_~ta_l. (1975) have reported preliminary results concerning the lethality of intravenous cocaine in rats.
The intravenous administration of
20 mg/kg resulted in convulsions and death in all subjects within five minutes. Lower dosages produced similar effects without mortality.
BecaUse of the lack
of reports concerning the intravenous lethality of cocaine in rodents, the present series of studies was conducted.
These studies were designed to
determine (I) the acute intravenous lethal dose of cocaine2and secondaril~ to ascertain the effects of infusate concentration on this dosage, (2) if pretreatment with agents which block central and peripheral norepinephrine and dopamine synthesis alter the acute lethal dose2and (33 if sensitization to the convulsant action of cocaine results during a subacute schedule of intravenous administration.
Pharmacological Research Communications, VoL 10, No. 3, 1978
246 Methods
Subjects were adult male albino rats of a Wistar-derived strain weighing 500-3S0 g (National Laboratory Animal Company; Creve Coeur, ~D).
Subjects
were individually housed in quarters in which the temperature was maintained at 22-24°C with a 12 hour light/dark cycle. were available ad libitum.
Food (Wayne Lab Chow) and water
All subjects were given at least two weeks to
adapt to this environment prior to experimental use.
Following this adapta-
tion period cannulae were surgically implanted in the external jugular vein. The preparation of the cannulae and surgical procedure were similar to methods described by Weeks (1971). pentobarbital
Cannulation of subjects was performed using sodium
(50 mg/kg, i.p.) as the anesthetic.
Following surgery I00,000
units of procaine penicillin G and 12S mg of dihydrostreptomycin
sulfate
(Franklin Laboratories Inc., Denver~ CO) was administered intramuscularly as a prophylactic anti-infective measure.
Subjects were then returned to the home
cages for 48-72 hours prior to use. Lethality studies were conducted in a 25.4 cm square cubicle constructed of sheet metal and plexiglas with a floor composed of 0.32 cm stainless steel rods, spaced 1.2S cm apart. through swivel
The top of the cubicle, which supported a feed-
(BRS-LVE #192-03, Beltsville, MD) for drug infusions, was con-
structed of mesh wire screen.
Drug delivery was accomplished with an infusion
pimp (Harvard Apparatus, Model 600-900, Millis, MA) which delivered 0.1 ml of solution per minute through a silicone rubber tubing (0.8 mm i.d. x 4.3 mm o.d.). From the swivel, solutions passed through polyethylene tubing (PE-20, C]ay Adams, Parsippany, NJ) encased in a flexible coiled-spring wire whic~ in turn s was attached to a restraining harness (BRS-LVE #191-10, Beltsville, MD) placed around the subject.
Preliminary studies had demonstrated that this
restraint system did not significantly affect,subject motility. Acute intravenous cocaine toxicity studies were conducted to determine
Pharmacological Research Communications, VoL 10, No. 3, 1978
247
the relationships between rate of infusion, lethal dose and latency to death. Three concentrations of cocaine hydrochloride [0.S, 1.0 and 2.0 mg/O.l ml) were used with a constant infusion rate of 0.i ml/minute. were tested with each concentration.
Eight subjects
Cocaine hydrochloride [Merck, Rahway,
NJ) solutions were prepared dail~ using an isotonic saline vehicle. Each subject was placed in the observation chamber, attached to the drug delivery system and allowed 5-10~minutes for exploratory behavior.
After ac-
tivity had reached a low level, the drug infusion pump and a timer were simultaneously activated.
Continuous observation of the subject permitted a
determination of the time at which convulsions and death occurred. In order to determine whether altering central catecholmnine function would affect acute cocaine lethality, a similar Study was performed using cocaine hydFochloride at a concentration of 0.5 mg/0.1 ml with the same infusion rate as indicated previously.
For this study, eight subjects each were
pretreated with either l-alpha-methylparatyrosine phenyl-3-[2-thiazolyl]-2-thiourea mg/kg, i.p.),
(AMPT) 50 mg/kg, i.p., l-
[U-14,624) 50 mg/kg, i.p~or pimozide [0.31
l-alpha-methy!paratyrosine [AMPT, Regis Chemical Co., Chicago,
IL) was suspended in saline [25 mg/ml) by sonification with a Polytron homogenizer.
U-14,624 [Aldrich Chemical Co., ~lwaukee,
WI) was suspended in
saline [25 mg/ml) using 1% Tween 80 as the suspending agent.
Pimozide [ORAP R,
McNeil Laboratories Inc., Fort Washington, PA) solutions were prepared by mixing 21.7 mg of pimozide base with 130.2 mg of tartaric acid in 20 ml of warm water.
T h e mixture was placed in a hot water bath and stirred until the
compounds were dissolved.
The solution was then diluted with hot water to
yield a final concentration of 0.31 mg/ml,
kMPT and U-14;624 were administered
4 hours prior to initiation of the cocaine infusion, whereas pimozide was administered thirty minutes prior to infusion. In subacute toxicity studies, eight subjects with intravenous cannulae were infused once daily for seven consecutive days With cocaine hydrochloride
Pharmacologicel Research Communications, VoL 10, No. 3, 1978
248
(0.5 mg/O.l ml/min) until a total of 15 mg/kg was administered or until convulsions occurred.
For each subject, infusion duration was altered daily
based on determinations of body weight.
~=
29'
~"
LETHAL
On the eighth day, cocaine was in-
DOSE
30
TIME TO DEATH
28
25
27 om..,
V
E
uJ co 0 D ..J .< -rl-uJ J
26
E
20
v
25 I
24
