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Reconsideration of the central nervous system pharmacology of amphetamine
TOXICOLOGY
AND
APPLIED
8, 550-5j7
PHARMACOLOGY
Reconsideration
of
the
Central
Pharmacology I. JOSEPH
F.
Toxicity
GARDOCKI,
(1966)
System
of Amphetamine
in Grouped MARGERY
Nervous
E.
and SCHULER,
Isolated AND
Mice LEONIDE
GOLDSTEIN
Biological Research Department, McNeil Laboratories, Inc., Fort Washington, Pennsylvania 19034, and Neuropharmacology Section, New Jersey Bureau of Research in Neurology and Psychiatry, Princeton, New Jersey 08540 Received
September
3, 1965
The variety of pharmacologic effects that have been reported for amphetamine would suggestthat it is a rather unusual drug. The following list of actions represents only a part of the spectrum reported for amphetamine: elevation of blood pressure (Alles, 1933), stimulation of the central nervous system (Alles, 1933), spasmolysis (Myerson and Ritvo, 1936), antidepressant activity (Wilbur et al., 1937), inhibition of appetite (Lessesand Myerson, 1938), mydriasis (Mayer, 1939), analgesia (Kiessig and Orzechowski, 1941) , antifatigue activity (Seashore and Ivy, 1953)) diuresis (Sapirstein et al., 1953), monoamineoxidase inhibition (Randall and Bagdon, 1959), hyperthermia (Askew, 1962), and catecholamine depletion (Moore, 1964). It is, therefore, no wonder that a great deal of research has been centered around this drug. However, it must be recognized that many of the features of its action are either poorly interpreted or not understood. It appeared, therefore, that a careful reexamination of certain aspectsof ,the effects of amphetamine would be in order. This report, the first of a series of papers on the toxicity and central nervous system effects of amphetamine, is devoted to one of the most unique properties of this drug, i.e., its acute toxicity in mice. The acute toxicity of amphetamine in mice has been studied during the last twenty years on a larger scale than perhaps any other compound in any other species.This came about following the discovery by Gunn and Gurd (1940) of the considerable enhancement of the toxicity of amphetamine in mice kept for a certain time in close proximity (so-called “grouped” conditions) ‘us.animals kept in separate cages. Subsequently, Chance (1946, 1947) reported on various factors playing a role in the group situation. Other investigators, Hiihn and Lasagna (1960), Swinyard et al. (1961), and Askew (1962), investigated extensively the effect of temperature on the toxicity of amphetamine. Numerous studies have been concerned with the types of drugs that block the lethality of amphetamine in the grouped situation (Lasagna and McCann, 1957; Burn and Hobbs, 1958; Moore, 1963). Generally, the LD5,, is used to express the lethality of amphetamine. The number of animals used by the forementioned investigators ranged from 3 to 10 per cage, 3 to 5 per cage being used most often. During studies in these laboratories with amphetamine, considerable variation was encountered. Doses which should not have been lethal were lethal. Multiples of a 550
AMPHETAMINE
TOXICITY
IN
MICE
531
theoretical LD50 were not found to be proportionally more lethal. A search of the literature revealed reports which contained data suggestive of a polyphasic response (Hiihn and Lasagna, 1960; Askew, 1962). Indeed, L’Huillier et al. (1963) made specific mention of a biphasic response for methamphetamine in grouped mice. In isolated mice, Cohen and La1 (1964) reported a dose of 25 mg/kg of d-amphetamine to be nonlethal, whereas Halpern et al. (1962) reported a dose of 14 mg/kg of &hetamine as killing 1 of 65 isolated mice. In the light of our own experiencesand the forementioned indications of variability in the mortality-response curve for amphetamine, we undertook the studies reported here. METHODS
The mice used in these experiments were male albinos of the Swiss-Webster strain. Body weight ranged from 18 to 24 g. The animals had accessto food and water up to the time of the experiments. A total of 100 mice were used per dosage. An additional 100 mice injected with saline served as controls. Koom temperature ranged from 22” to 24’C. The hydrochloride salt of &hetamine was used. The volume of solution injected ranged from 0.15 to 0.4 ml per 20-g mouse. Following an intraperitoneal injection of a given dose of amphetamine, the mice were placed either individually or in groups of 10 in an opaque plastic cage having a floor size of 14 X 25 cm. The mice were observed continuously during a 4.5hour period, and gross effects were recorded at 15-minute intervals during the first hour and at hourly intervals during the remainder of the observation period. Death time was recorded as that time at which respiration ceasedduring the 4.5hour observation; additional deaths were counted 18 hours after injection of the drug. RESULTS
The mortality-response curve observed with amphetamine in grouped mice appears to consist of five segments: (1) a rapidly rising portion corresponding to the 15-30 mg/kg range, (2) a plateau segmentin the range of 30-60 mg/kg, (3) a descending segment, 60-75 mg/kg, (4) a plateau segment, 75-90 mg/kg, and (5) an ascending segment, 90-150 mg/kg (Fig. 1). The mortality-response curve observed in isolated mice with amphetamine can be divided into the following segments: ( 1) an ascending segment, 1S-30 mg/kg range, (2) a plateau segment in the 30-45 mg/kg range, (3) a descending segment, 45-60 mgikg, (4) a plateau segment, 60-90 mg/kg, and (5) an ascending segment, 90-l 50 mg/ kg range. ,4 statistical analysis of these two curves is summarized in Table 1. As can be seen, the differences between the mean mortality for grouped and isolated animals have been found to be statistically significant (t test) at all dosagesexcept 150 mg/kg. Furthermore, statistical analysis of parallelism between the two curves (based on the coefficient of correlation of the successiveindices of change) has yielded the value 1’= 0.66. thus highly significant in view of the large number of degrees of freedom involved in these measurements.The significance of the decrease observed in both curves from 45 to 75 mg/kg was tested by chi-square analysis of actual values, and those that would be found if the decrease were replaced by a continuous p!ateau.
552
JOSEPH
F.
GARDOCKI
ET
AL.
The result of such calculations has shown that the probability for the decrease to be obtained by chance is less than 1 to 10,000. The cumulative lethality curves for each of the doses used in the curves in Fig. 1 are presented in Fig. 2. An examination of the shape of these curves for grouped mice reveals a slight decrease in survival time as the dose is increased from 15 to 30 mg/kg. With doses of 45, 60, and 75 mg/kg, this trend is reversed and replaced by an increase in overall survival. Finally, with the next three dosages employed there is once more a reversal with a decrease in survival time.
I 15
30
45
60 Dose
75 (mg/kg
90
105
I20
135
, 150
ip)
FIG. 1. Mean lethality (& SD) per 10 groups of 10 mice for 10 intraperitoneal amphetamine ranging from 15 to 150 mg/kg. Solid line = grouped animals. Broken animals. The vertical lines at each mean point represent one standard deviation.
doses of dlline = isolated
The trend in the cumulative mortality curves for the isolated mice appears also to follow similar trends. From 15 to 45 mg/kg there is a tendency for the slope of the curve to increase, especially because of the 18 hours delayed mortality. From 60 to 7.5 mg/kg this trend is reversed. Finally, from 90 to 150 mg/kg the third and last phase appears, which consists again in an accelerated time of death. A cross comparison of the cumulative mortality curves for the grouped and isolated mice indicates a decrease in survival time and number of animals surviving for doses of 15-75 mg/kg of amphetamine in grouped animals. In the range 90-150 mg/kg the shape of the curves for grouped and isolated mice appears to be similar. The gross effects observed following injection of amphetamine in grouped mice were qualitatively similar in the dosage range of 15-45 mg/kg. These were increased motor activity, Straub tail, sensitivity to touch. vocalization, head searching, saliva-
AMPHETAMINE
TOXICITY
IN
MICE
554
JOSEPH
F.
Amphetamine
GARDOCKI
ET
AL.
Cumulative
Lethality
Grouped
Isolated
Grouped
lsoloted
I5mg/hp
15mg/kg
75 mQ/kg
75mQlkQ
I I
l30",Q/kQ
L
30",Q/kQ
gO"TQ/kQ
go"lQ/kQ
45",Q/kQ
12O"lQ/kQ
120",Q/kQ
0') I
100
i 1
150mq/kq
60mQ/kq
Time
FIG. 2. Cumu!ative 15 to 150 mg/kg in time in minutes.
lethality grouped
UT-
150mQ/kq
in Minutes
folIowing 10 intraperitoneal doses of amphetamine ranging from and isolated animals. Ordinate = percentage lethality; abscissa =
AMPHETAMINE
TOXICITY
IN
MICE
55 5
tion, lacrimation, piloerection, exophthalmos, depression, and death. Quantitatively, there was an increase in the degree and duration of stimulation with an increase in dose in the range of 15-30 mg/kg. Following a dose of 45 mg/kg there was a decrease in the degree of stimulation and a concomitant decrease in the onset and depth of depression. In the dosage range of 60-75 mg/kg the increase in motor activity was not as marked, and pivoting behavior, tremors, and clonic convulsions were in evidence. With a further increase in dose there occurred a further reduction in augmented motor activity and an increase in the incidence and severity of tremors and convulsions. In general, the intensity, incidence, and nature of the effects paralleled the mortality curves. The effects observed in the isolated mice injected with amphetamine occurred in the same dosage ranges as did those observed in grouped mice. By contrast, isolated mice differed from grouped mice at doses of 15-45 mg/kg of amphetamine by: (1) a moderation of the increase in motor activity, (2) a concomitant increase in the occurrence of head searching, (3) absence of vocalization, (4) the incidence of tremors and convulsions, and (5) decrease in the incidence of depression. With doses of 60 and 75 mg/kg of amphetamine in isolated mice, the effects observed were similar to those seen in grouped animals except for a pronounced increase in head searching. The difference between the effects in grouped and isolated mice injected with 90-150 mg/kg was merely quantitative. DISCUSSION
It is generally accepted that grouping enhances the lethality of amphetamine in mice. The ratio of #the isolated to grouped LDS,-, as reported by Chance (1946) is approximately 10: 1. This large a difference would suggest that the LDr,,, in grouped mice would be nonlethal in isolated mice. The dose-response curves for d-amphetamine as reported by Moore (1963) support this contention. Furthermore, the mortality-response curve for grouped mice appears to be a lateral displacement of the one for isolated mice. The use of the LDr,,, in expressing the lethality of amphetamine would imply a linear dose-response effect. The data presented in this paper indicate that the mortality-response curve for amphetamine in grouped mice is vertically displaced from the one for isolated mice and not laterally: thus, a dose lethal in grouped mice is also lethal in isolated mice but to a lesser degree. Further, the dose-mortality curve for amphetamine in grouped and isolated mice is polyphasic in nature and not linear. Differences between successive doses of amphetamine were reflected not only by the total mortality counts: but also in the respective cumulative mortality-response curves and behavioral effects. The difference between the polyphasic mortality-response curves reported here a.nd the linear ones indicated by the LD;,(,‘s reported by other investigators (Lasagna and McCann, 1957; Swinyard et al., 1961; Chance, 1946; Moore, 1963) may be the result of a difference in methodology. The use of a larger number of mice per group, an increase in the floor area, and closely spaced doses are probably the major factors responsible for the character of the mortality-response curves obtained. Our findings are in agreement with the mortality-response curve for methamphetamine in grouped mice reported by L’Huillier et al. (1963). Another example of a polyphasic mortalityresponse curve is that reported for fentanyl citrate by Gardocki et al. (1964).
556
JOSEPH
F.
GARDOCKI
ET
AL.
The polyphasic nature of the mortalimty-response curve would suggest several different actions for amphetamine in the dosage range studied. Experiments are currently in progress that may hopefully uncover some of these possible differences. The mortality-response curves for amphetamine raise the question as to a suitable means of expressing ,the lethality of amphetamine in mice. Certainly, the classical LDr,O expression is not applicable in this case. How many compounds, particularly other stimulants, have polyphasic mortality-response curves remains to be determined. One good indication of their existence is the occurrence of considerable variability in repeating previously obtained mortality-response curves. SUMMARY Experiments were carried out in mice with intraperitoneal injections of &hetamine at 10 dosages between 15 and 150 mg/kg. In animals kept in isolated conditions, it was observed that lethality follows a triphasic pattern, increasing between 1S and 45 mg/kg, decreasing from 45 to 75 mg/kg, and increasing from ?5 to 150 mg/kg. In animals grouped by 10, the same evolution of dose-effect was found. However, lethality was greater in grouped animals at all levels except 150 mg/kg. These results are discussed, and it is pointed out that the concept of an LD,, cannot be validly used under such conditions. REFERENCES ALLES, G. A. (1933). The comparative physiological actions of dl-&phenylisopropylamines. I. Pressor effect and toxicity. J. Pharmacol. Erptl. Therap. 47, 339-354. ASKEW, B. M. (1962). Hyperpyrexia as a contributory factor in the toxicity of amphetamine in aggregated mice. Brit. J. Pharmacol. 19, 245-257. BURN, J. H., and HOBBS, R. (1958). A test for tranquilizing drugs. Arch. Intern. Pharmacodyn. 113, 290-295. CHANCE, M. R. A. (1946). Aggregation as a factor influencing the toxicity of sympathomimetic amines in mice. J. Pharmacol. Exptl. Therap. 87, 214-219. CHANCE. M. R. A. (1947). Factors influencing the toxicity of sympathomimetic amines to solitary mice. J. Phavmucol. Exptl. Therap. 89, 289-296. COHEN, M., and LAL, H. (1964). Effect of sensory stimuli on amphetamine toxicity in aggregated mice. Nature 241, 1037. GARDOCIU. J. F., YELNOSKY, J,, KUEHN, W. F., and GUNSTER, J. C. (1964). A study of the interaction of nalorphine with fentanyl and Innova@. Toxicol. fippl. Pharmacol. 6, 593-601. GUNN. J. A., and Guan, M. R. (1940). Action of some amines related to adrenalin. Cyclohexylalkylamines. J. Physiol. (London) 97, 453-470. HALPERN, B. N., DRUDI-BARACCO, C., and BESSIRARD, D. (1962). Toxicitt de groupe par l’amphetamine et action de la reserpine de la chlorpromazine et des inhibiteurs de la monoamineoxidase. Compt. Rend. SOL Biol. 156, 769-773. H~~HN, R., and LASAGNA, L. (1960). Effects of aggregation and temperature on amphetamine toxicity in mice. Psychophavmacologia 1, 210-220. KIESSIG: H. J., and ORZECHOWSKI, G. (1941). Untersuchungen iiber die Wirkungsweise der Sympathicomimetica; iiber die Beeinflussung der Schmerzempfindlichkeit durch Sympathicomimetica. drch. Expll. Pathol. Pkarmakol. 197, 391-404. LASAGNA, L., and MCCANN, W. P. (1957). Effect of “tranquilizing” drugs on amphetamine toxicity in aggregated mice. Science 125, 1241-1242. LESSES. M. F., and MYERSON, A. (1938). Human autonomic pharmacology. XVI. Benzedrine sulfate as aid in treatment of obesity. New, Engl. J. Med. 2l8, 119-124. L’HUILLIER, J,, GRANDJEAN, J. L., and THULLIER, J. (1963). Annulation par le procalmadiol des effets secondaires lethaux de la methamphetamine chez la souris: application a l’etude des psycholeptiques. Compt. Rend. Sot. Biol. 157, 2183-218s. MAYER. L. L. (1939). Dilation of the pupil for ophthalmologic examination. J. Am. Med. .4ssor. 113, 38-39.
AMPHETAMINE
TOXICITY
IN
MICE
557
K. E. (1963). Toxicity and catecholamine releasing actions of d- and Z-amphetamine in isolated and aggregated mice. J. Phavmacol. Exptl. Therap. 142, 6-12. MOORE, K. E. (1964). The role of endogenous norepinephrine in the toxicity of d-amphetamine in aggregated mice. J. Pharmacol. Ercptl. Therap. 144, 45-51. MYERSON, A., and RITVO, M. (1936). Benzedrine sulfate and its value in spasm of the gastrointestinal tract. J. Am. Med. Assoc. 107, 24-26. R4NDALL, L. O., and BAGDON, R. E. (1959). Pharmacology of iproniazid and other amine oxidase inhibitors. Ann. X.1’. Acad. Sci. 80, 626-642. SAPIRSTEIN, L. A., .%NDREWS, R., PULTZ, A., and RIDOLFO, A. (1953). Effects of amphetamine and its isomers on excretion of sodium and water in the rat. Proc. Sot. Exptl. Biol. Med. 82, 609-612. Effects of analeptic drugs in relieving fatigue. Psychol. SEASHORE, R. H., and IVY, .4. C. (1953). Monographs 67, 1-16. SWINYARD, E. A., CLARK, L. D., MIYAHAIW, J. T., and WOLF, H. H. (1961). Studies on the mechanism of amphetamine toxicity in aggregated mice. J. Pharmacol. Exptl. Therap. 132, 97102. WILBUR, D. L., MACLEAN, A. R., and ALLEN, E. V. (1937). Clinical observation on effect of Benzedrine sulfate; study of patients with states of chronic exhaustion, depression and psychoneurosis. J. Am. Med. Assoc. 109, 549-554. MOORE,
AND
APPLIED
8, 550-5j7
PHARMACOLOGY
Reconsideration
of
the
Central
Pharmacology I. JOSEPH
F.
Toxicity
GARDOCKI,
(1966)
System
of Amphetamine
in Grouped MARGERY
Nervous
E.
and SCHULER,
Isolated AND
Mice LEONIDE
GOLDSTEIN
Biological Research Department, McNeil Laboratories, Inc., Fort Washington, Pennsylvania 19034, and Neuropharmacology Section, New Jersey Bureau of Research in Neurology and Psychiatry, Princeton, New Jersey 08540 Received
September
3, 1965
The variety of pharmacologic effects that have been reported for amphetamine would suggestthat it is a rather unusual drug. The following list of actions represents only a part of the spectrum reported for amphetamine: elevation of blood pressure (Alles, 1933), stimulation of the central nervous system (Alles, 1933), spasmolysis (Myerson and Ritvo, 1936), antidepressant activity (Wilbur et al., 1937), inhibition of appetite (Lessesand Myerson, 1938), mydriasis (Mayer, 1939), analgesia (Kiessig and Orzechowski, 1941) , antifatigue activity (Seashore and Ivy, 1953)) diuresis (Sapirstein et al., 1953), monoamineoxidase inhibition (Randall and Bagdon, 1959), hyperthermia (Askew, 1962), and catecholamine depletion (Moore, 1964). It is, therefore, no wonder that a great deal of research has been centered around this drug. However, it must be recognized that many of the features of its action are either poorly interpreted or not understood. It appeared, therefore, that a careful reexamination of certain aspectsof ,the effects of amphetamine would be in order. This report, the first of a series of papers on the toxicity and central nervous system effects of amphetamine, is devoted to one of the most unique properties of this drug, i.e., its acute toxicity in mice. The acute toxicity of amphetamine in mice has been studied during the last twenty years on a larger scale than perhaps any other compound in any other species.This came about following the discovery by Gunn and Gurd (1940) of the considerable enhancement of the toxicity of amphetamine in mice kept for a certain time in close proximity (so-called “grouped” conditions) ‘us.animals kept in separate cages. Subsequently, Chance (1946, 1947) reported on various factors playing a role in the group situation. Other investigators, Hiihn and Lasagna (1960), Swinyard et al. (1961), and Askew (1962), investigated extensively the effect of temperature on the toxicity of amphetamine. Numerous studies have been concerned with the types of drugs that block the lethality of amphetamine in the grouped situation (Lasagna and McCann, 1957; Burn and Hobbs, 1958; Moore, 1963). Generally, the LD5,, is used to express the lethality of amphetamine. The number of animals used by the forementioned investigators ranged from 3 to 10 per cage, 3 to 5 per cage being used most often. During studies in these laboratories with amphetamine, considerable variation was encountered. Doses which should not have been lethal were lethal. Multiples of a 550
AMPHETAMINE
TOXICITY
IN
MICE
531
theoretical LD50 were not found to be proportionally more lethal. A search of the literature revealed reports which contained data suggestive of a polyphasic response (Hiihn and Lasagna, 1960; Askew, 1962). Indeed, L’Huillier et al. (1963) made specific mention of a biphasic response for methamphetamine in grouped mice. In isolated mice, Cohen and La1 (1964) reported a dose of 25 mg/kg of d-amphetamine to be nonlethal, whereas Halpern et al. (1962) reported a dose of 14 mg/kg of &hetamine as killing 1 of 65 isolated mice. In the light of our own experiencesand the forementioned indications of variability in the mortality-response curve for amphetamine, we undertook the studies reported here. METHODS
The mice used in these experiments were male albinos of the Swiss-Webster strain. Body weight ranged from 18 to 24 g. The animals had accessto food and water up to the time of the experiments. A total of 100 mice were used per dosage. An additional 100 mice injected with saline served as controls. Koom temperature ranged from 22” to 24’C. The hydrochloride salt of &hetamine was used. The volume of solution injected ranged from 0.15 to 0.4 ml per 20-g mouse. Following an intraperitoneal injection of a given dose of amphetamine, the mice were placed either individually or in groups of 10 in an opaque plastic cage having a floor size of 14 X 25 cm. The mice were observed continuously during a 4.5hour period, and gross effects were recorded at 15-minute intervals during the first hour and at hourly intervals during the remainder of the observation period. Death time was recorded as that time at which respiration ceasedduring the 4.5hour observation; additional deaths were counted 18 hours after injection of the drug. RESULTS
The mortality-response curve observed with amphetamine in grouped mice appears to consist of five segments: (1) a rapidly rising portion corresponding to the 15-30 mg/kg range, (2) a plateau segmentin the range of 30-60 mg/kg, (3) a descending segment, 60-75 mg/kg, (4) a plateau segment, 75-90 mg/kg, and (5) an ascending segment, 90-150 mg/kg (Fig. 1). The mortality-response curve observed in isolated mice with amphetamine can be divided into the following segments: ( 1) an ascending segment, 1S-30 mg/kg range, (2) a plateau segment in the 30-45 mg/kg range, (3) a descending segment, 45-60 mgikg, (4) a plateau segment, 60-90 mg/kg, and (5) an ascending segment, 90-l 50 mg/ kg range. ,4 statistical analysis of these two curves is summarized in Table 1. As can be seen, the differences between the mean mortality for grouped and isolated animals have been found to be statistically significant (t test) at all dosagesexcept 150 mg/kg. Furthermore, statistical analysis of parallelism between the two curves (based on the coefficient of correlation of the successiveindices of change) has yielded the value 1’= 0.66. thus highly significant in view of the large number of degrees of freedom involved in these measurements.The significance of the decrease observed in both curves from 45 to 75 mg/kg was tested by chi-square analysis of actual values, and those that would be found if the decrease were replaced by a continuous p!ateau.
552
JOSEPH
F.
GARDOCKI
ET
AL.
The result of such calculations has shown that the probability for the decrease to be obtained by chance is less than 1 to 10,000. The cumulative lethality curves for each of the doses used in the curves in Fig. 1 are presented in Fig. 2. An examination of the shape of these curves for grouped mice reveals a slight decrease in survival time as the dose is increased from 15 to 30 mg/kg. With doses of 45, 60, and 75 mg/kg, this trend is reversed and replaced by an increase in overall survival. Finally, with the next three dosages employed there is once more a reversal with a decrease in survival time.
I 15
30
45
60 Dose
75 (mg/kg
90
105
I20
135
, 150
ip)
FIG. 1. Mean lethality (& SD) per 10 groups of 10 mice for 10 intraperitoneal amphetamine ranging from 15 to 150 mg/kg. Solid line = grouped animals. Broken animals. The vertical lines at each mean point represent one standard deviation.
doses of dlline = isolated
The trend in the cumulative mortality curves for the isolated mice appears also to follow similar trends. From 15 to 45 mg/kg there is a tendency for the slope of the curve to increase, especially because of the 18 hours delayed mortality. From 60 to 7.5 mg/kg this trend is reversed. Finally, from 90 to 150 mg/kg the third and last phase appears, which consists again in an accelerated time of death. A cross comparison of the cumulative mortality curves for the grouped and isolated mice indicates a decrease in survival time and number of animals surviving for doses of 15-75 mg/kg of amphetamine in grouped animals. In the range 90-150 mg/kg the shape of the curves for grouped and isolated mice appears to be similar. The gross effects observed following injection of amphetamine in grouped mice were qualitatively similar in the dosage range of 15-45 mg/kg. These were increased motor activity, Straub tail, sensitivity to touch. vocalization, head searching, saliva-
AMPHETAMINE
TOXICITY
IN
MICE
554
JOSEPH
F.
Amphetamine
GARDOCKI
ET
AL.
Cumulative
Lethality
Grouped
Isolated
Grouped
lsoloted
I5mg/hp
15mg/kg
75 mQ/kg
75mQlkQ
I I
l30",Q/kQ
L
30",Q/kQ
gO"TQ/kQ
go"lQ/kQ
45",Q/kQ
12O"lQ/kQ
120",Q/kQ
0') I
100
i 1
150mq/kq
60mQ/kq
Time
FIG. 2. Cumu!ative 15 to 150 mg/kg in time in minutes.
lethality grouped
UT-
150mQ/kq
in Minutes
folIowing 10 intraperitoneal doses of amphetamine ranging from and isolated animals. Ordinate = percentage lethality; abscissa =
AMPHETAMINE
TOXICITY
IN
MICE
55 5
tion, lacrimation, piloerection, exophthalmos, depression, and death. Quantitatively, there was an increase in the degree and duration of stimulation with an increase in dose in the range of 15-30 mg/kg. Following a dose of 45 mg/kg there was a decrease in the degree of stimulation and a concomitant decrease in the onset and depth of depression. In the dosage range of 60-75 mg/kg the increase in motor activity was not as marked, and pivoting behavior, tremors, and clonic convulsions were in evidence. With a further increase in dose there occurred a further reduction in augmented motor activity and an increase in the incidence and severity of tremors and convulsions. In general, the intensity, incidence, and nature of the effects paralleled the mortality curves. The effects observed in the isolated mice injected with amphetamine occurred in the same dosage ranges as did those observed in grouped mice. By contrast, isolated mice differed from grouped mice at doses of 15-45 mg/kg of amphetamine by: (1) a moderation of the increase in motor activity, (2) a concomitant increase in the occurrence of head searching, (3) absence of vocalization, (4) the incidence of tremors and convulsions, and (5) decrease in the incidence of depression. With doses of 60 and 75 mg/kg of amphetamine in isolated mice, the effects observed were similar to those seen in grouped animals except for a pronounced increase in head searching. The difference between the effects in grouped and isolated mice injected with 90-150 mg/kg was merely quantitative. DISCUSSION
It is generally accepted that grouping enhances the lethality of amphetamine in mice. The ratio of #the isolated to grouped LDS,-, as reported by Chance (1946) is approximately 10: 1. This large a difference would suggest that the LDr,,, in grouped mice would be nonlethal in isolated mice. The dose-response curves for d-amphetamine as reported by Moore (1963) support this contention. Furthermore, the mortality-response curve for grouped mice appears to be a lateral displacement of the one for isolated mice. The use of the LDr,,, in expressing the lethality of amphetamine would imply a linear dose-response effect. The data presented in this paper indicate that the mortality-response curve for amphetamine in grouped mice is vertically displaced from the one for isolated mice and not laterally: thus, a dose lethal in grouped mice is also lethal in isolated mice but to a lesser degree. Further, the dose-mortality curve for amphetamine in grouped and isolated mice is polyphasic in nature and not linear. Differences between successive doses of amphetamine were reflected not only by the total mortality counts: but also in the respective cumulative mortality-response curves and behavioral effects. The difference between the polyphasic mortality-response curves reported here a.nd the linear ones indicated by the LD;,(,‘s reported by other investigators (Lasagna and McCann, 1957; Swinyard et al., 1961; Chance, 1946; Moore, 1963) may be the result of a difference in methodology. The use of a larger number of mice per group, an increase in the floor area, and closely spaced doses are probably the major factors responsible for the character of the mortality-response curves obtained. Our findings are in agreement with the mortality-response curve for methamphetamine in grouped mice reported by L’Huillier et al. (1963). Another example of a polyphasic mortalityresponse curve is that reported for fentanyl citrate by Gardocki et al. (1964).
556
JOSEPH
F.
GARDOCKI
ET
AL.
The polyphasic nature of the mortalimty-response curve would suggest several different actions for amphetamine in the dosage range studied. Experiments are currently in progress that may hopefully uncover some of these possible differences. The mortality-response curves for amphetamine raise the question as to a suitable means of expressing ,the lethality of amphetamine in mice. Certainly, the classical LDr,O expression is not applicable in this case. How many compounds, particularly other stimulants, have polyphasic mortality-response curves remains to be determined. One good indication of their existence is the occurrence of considerable variability in repeating previously obtained mortality-response curves. SUMMARY Experiments were carried out in mice with intraperitoneal injections of &hetamine at 10 dosages between 15 and 150 mg/kg. In animals kept in isolated conditions, it was observed that lethality follows a triphasic pattern, increasing between 1S and 45 mg/kg, decreasing from 45 to 75 mg/kg, and increasing from ?5 to 150 mg/kg. In animals grouped by 10, the same evolution of dose-effect was found. However, lethality was greater in grouped animals at all levels except 150 mg/kg. These results are discussed, and it is pointed out that the concept of an LD,, cannot be validly used under such conditions. REFERENCES ALLES, G. A. (1933). The comparative physiological actions of dl-&phenylisopropylamines. I. Pressor effect and toxicity. J. Pharmacol. Erptl. Therap. 47, 339-354. ASKEW, B. M. (1962). Hyperpyrexia as a contributory factor in the toxicity of amphetamine in aggregated mice. Brit. J. Pharmacol. 19, 245-257. BURN, J. H., and HOBBS, R. (1958). A test for tranquilizing drugs. Arch. Intern. Pharmacodyn. 113, 290-295. CHANCE, M. R. A. (1946). Aggregation as a factor influencing the toxicity of sympathomimetic amines in mice. J. Pharmacol. Exptl. Therap. 87, 214-219. CHANCE. M. R. A. (1947). Factors influencing the toxicity of sympathomimetic amines to solitary mice. J. Phavmucol. Exptl. Therap. 89, 289-296. COHEN, M., and LAL, H. (1964). Effect of sensory stimuli on amphetamine toxicity in aggregated mice. Nature 241, 1037. GARDOCIU. J. F., YELNOSKY, J,, KUEHN, W. F., and GUNSTER, J. C. (1964). A study of the interaction of nalorphine with fentanyl and Innova@. Toxicol. fippl. Pharmacol. 6, 593-601. GUNN. J. A., and Guan, M. R. (1940). Action of some amines related to adrenalin. Cyclohexylalkylamines. J. Physiol. (London) 97, 453-470. HALPERN, B. N., DRUDI-BARACCO, C., and BESSIRARD, D. (1962). Toxicitt de groupe par l’amphetamine et action de la reserpine de la chlorpromazine et des inhibiteurs de la monoamineoxidase. Compt. Rend. SOL Biol. 156, 769-773. H~~HN, R., and LASAGNA, L. (1960). Effects of aggregation and temperature on amphetamine toxicity in mice. Psychophavmacologia 1, 210-220. KIESSIG: H. J., and ORZECHOWSKI, G. (1941). Untersuchungen iiber die Wirkungsweise der Sympathicomimetica; iiber die Beeinflussung der Schmerzempfindlichkeit durch Sympathicomimetica. drch. Expll. Pathol. Pkarmakol. 197, 391-404. LASAGNA, L., and MCCANN, W. P. (1957). Effect of “tranquilizing” drugs on amphetamine toxicity in aggregated mice. Science 125, 1241-1242. LESSES. M. F., and MYERSON, A. (1938). Human autonomic pharmacology. XVI. Benzedrine sulfate as aid in treatment of obesity. New, Engl. J. Med. 2l8, 119-124. L’HUILLIER, J,, GRANDJEAN, J. L., and THULLIER, J. (1963). Annulation par le procalmadiol des effets secondaires lethaux de la methamphetamine chez la souris: application a l’etude des psycholeptiques. Compt. Rend. Sot. Biol. 157, 2183-218s. MAYER. L. L. (1939). Dilation of the pupil for ophthalmologic examination. J. Am. Med. .4ssor. 113, 38-39.
AMPHETAMINE
TOXICITY
IN
MICE
557
K. E. (1963). Toxicity and catecholamine releasing actions of d- and Z-amphetamine in isolated and aggregated mice. J. Phavmacol. Exptl. Therap. 142, 6-12. MOORE, K. E. (1964). The role of endogenous norepinephrine in the toxicity of d-amphetamine in aggregated mice. J. Pharmacol. Ercptl. Therap. 144, 45-51. MYERSON, A., and RITVO, M. (1936). Benzedrine sulfate and its value in spasm of the gastrointestinal tract. J. Am. Med. Assoc. 107, 24-26. R4NDALL, L. O., and BAGDON, R. E. (1959). Pharmacology of iproniazid and other amine oxidase inhibitors. Ann. X.1’. Acad. Sci. 80, 626-642. SAPIRSTEIN, L. A., .%NDREWS, R., PULTZ, A., and RIDOLFO, A. (1953). Effects of amphetamine and its isomers on excretion of sodium and water in the rat. Proc. Sot. Exptl. Biol. Med. 82, 609-612. Effects of analeptic drugs in relieving fatigue. Psychol. SEASHORE, R. H., and IVY, .4. C. (1953). Monographs 67, 1-16. SWINYARD, E. A., CLARK, L. D., MIYAHAIW, J. T., and WOLF, H. H. (1961). Studies on the mechanism of amphetamine toxicity in aggregated mice. J. Pharmacol. Exptl. Therap. 132, 97102. WILBUR, D. L., MACLEAN, A. R., and ALLEN, E. V. (1937). Clinical observation on effect of Benzedrine sulfate; study of patients with states of chronic exhaustion, depression and psychoneurosis. J. Am. Med. Assoc. 109, 549-554. MOORE,