Oral and intravenous toxicity of Δ9-tetrahydrocannabinol in rhesus monkeys

TOXICOLOGYANTJAPPLIEDPHARMACOLCGY

Oral

and Intravenous

27,648-665(1974)

Toxicity of A9-Tetrahydrocannabinol in Rhesus Monkey+ 2

GEORGE R. THOMPSON,~ ROBERT W. FLEISCHMAN,~ HARRIS ROSENKRANTZ~ Mason

Research

Received

May

Institute, 29,1973;

AND MONIQUE C. BRAUDE~ Worcester, accepted

Massachusetts September

01608 &I973

Oral and Intravenous Toxicity of Ag-Tetrahydrocannabinol in Rhesus Monkeys. THOMPSON, G. R., FLEISCHMAN, R. W., ROSENKRANTZ, H. AND BRAUDE, M. C. (1974). Toxicol. Appl. Pharmacol. 27,648-665. The toxicity of Ag-THC was evaluated in 35 rhesus monkeys treated acutely po or iv; in 28 monkeys treated po for 28 days with 0, 50, 250 or 500 mg/kg/day; or in 16 monkeys treated iv for 28 days with 0, 5, 15 or 45 mg/kg/day. The high subacute doses selected from acute toxicity studies were chosen to establish toxicity, not to simulate human dosages. No deaths occurred in monkeys treated acutely po with up to 9000 mg/kg, but all monkeys treated acutely iv with 128 mg/kg or more died from respiratory arrest and cardiac failure. In the subacute oral study 2 of 8 monkeys treated with 500 mg/kg/day became moribund on days 10 and 14, and 1 of 6 monkeys treated with 50 mg/kg/day became moribund on day 16. Primary pathologic changes in these 3 moribund monkeys included atrophy of the pancreas, hemorrhagic ulcerative colitis, myeloid hyperplasia of the bone marrow, vacuolar nephrosis and severeserum electrolyte imbalance. All other monkeys were normal at necropsy. In the subacute iv trials, 2 of 4 monkeys treated with 45 mg/kg/day died on days 8 and 19 as a result of acute hemorrhagic pneumonia, but injection site edema, necrosis, ulceration and fibrosis also occurred. Behavioral and physiologic changes were similar in both studies and included lethargy, huddled posture, bradypnea, hypothermia, bradycardia, weight loss, anorexia and constipation. Tolerance and cumulative toxicity were also similar for the 2 studies, but monkeys treated iv showed dose-related anemia and increased BSP retention while no blood changes occurred in monkeys treated po. Pathologic changes in deceasedmonkeys were associated with the route of administration.

Recent proliferation of marihuana abuse coupled with relatively restrictive legal sanctions have created considerable social and medicolegal controversy about the hazards of marihuana consumption. Scientific contributions to this dispute have pertained primarily to defining behavioral effects. Most of these studies have contributed to understanding the mechanisms and sites of behavioral activity after short-term treatment with marihuana or its active constituent, d9-tetrahydrocannabinol (d9-THC) (Gaoni 1 Supported by NIMH Contracts HSM 42-70-95and HSM 42-71-79. ’ Presentedin part at the Fifth International Congresson Pharmacology,SanFrancisco,1972. 3 Abbot Laboratories, D-468, Abbott Park, North Chicago, Illinois 60064. 4 Mason ResearchInstitute, Worcester, Massachusetts01608. ’ National Institute of Mental Health, Rockville, Maryland 20015. Copyright 0 1974 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain

648

TOXICITY

OF LI~-THCIN

MONKEYS

649

and Mechoulam, 1964), but they have not contributed significantly to defining toxicologic hazards associated with chronic administration. Haines and Green (1970) reported that 24.4 % of the moderate to heavy marihuana smokers in their survey used marihuana every day, and 94% smoked marihuana at least once a week. Despite this evidence, most scientific reports have pertained to acute or subacute treatment, including specific toxicologic investigations (Phillips et al., 1971a; Dewey et al., 1972; Thompson et al., 1973a). Manning et al. (1971) and Phillips et al. (1971b) described the effects of 30 daily treatments with A9-THC on behavior, food consumption and growth of rats. Thompson et al. (1973b) reported the chronic toxicologic and pathologic effects of large oral doses of A9-THC, d8-THC or a marihuana extract in rats treated for 119 consecutive days. Controlled studies of pathology or chronic toxicity in other species have not been reported, but Kew et al. (1969) reported a case of hepatotoxicity in a heavy marihuana smoker. Since most moderate or heavy marihuana smokers also occasionally eat marihuana as an ingredient in cookies, brownies or tea (Haines and Green, 1970) and since a small percentage of abusers also attempt iv injection of a marihuana broth (Henderson et al., 1968 ; King and Cowen, 1969) assessment of toxicity and pathology in primates treated po or iv would aid in evaluating the hazard of marihuana abuse. Consequently, the present study compares the toxicity of d9-THC administered po and iv to male and female rhesus monkeys for 28 consecutive days and evaluates various organs for systemic toxicity and pathologic changes. The high doses were chosen to establish toxicity and pathology, not to simulate human dosages. METHODS Animals. All rhesus monkeys (Macaca mulatta: 2.7-4.7 kg, 2.0-3.5 years old) were kept in individual stainless steel cages for 1 month prior to treatment and observed for abnormal behavioral or clinical signs. Three negative tuberculin tests were obtained during this period. The animals were fed Purina Monkey Chow supplemented with apples twice each day with water available ad libitum. The first daily feeding commenced approximately 1 hr after treatment. Ambient temperature was maintained between 67 and 73°C. Intraoenous formulations and treatments. For acute iv toxicity tests, A9-THC was suspended by sonication at 15 mg/ml in 10 % sesame oil-0.494 Tween 80 in saline or at 40 mg/ml in 13 % sesame oil-1.0% Tween 80 in saline (Rosenkrantz et al., 1972a).6 The acute studies were performed in 2 parts separated by 1 week with the first test utilizing the lower concentration of A9-THC for administration of 1, 4, 16 or 64 mg/kg. The higher concentration was used in the second acute iv study for administration of 64,92, 128, 192 or 256 mg/kg, and in the subacute iv toxicity test for administration of 5, 15 or 45 mg/kg!day. Additional groups in these acute and subacute studies were treated only with vehicle in volumes equivalent to the largest doses administered. In the acute studies, each group included 1 male and 1 female whereas the subacute study utilized 2 males and 2 females at each level. All dosage volumes were injected into the femoral vein of physically restrained monkeys at the rate of 2.0 ml/15 sec. In the subacute study, the 6 The d9-THC used in these experiments was obtained from the National Institute of Mental Health.

650

THOMPSONETAL.

individual total injected volumes were adjusted weekly to accommodate for changes in body weights. Oralformulationsand treatments. In acute po toxicity tests, A’-THC was dissolved in 100% sesame oil or in 9 % ethanol in sesame oil at concentrations ranging from 62.5 to 350 mg/ml for administration of dosages that ranged from 131 to 9000 mg/kg.6 Dosage volumes were generally 2.0 or 3.0 ml/kg; vehicle control animals were similarly treated. Sex-paired monkeys were treated at all doses in the acute toxicity study except at 7000, 8000 and 9000 mg/kg where only 1 monkey was treated with each dose. [The large volumes required for administration of these 3 massive doses (60-80 ml) were given as 3 divided doses at 15-min intervals. A control monkey was similarly treated with 80 ml of 100% sesame oil.] Monkeys treated po with Ag-THC for 28 consecutive days received 0,50,250 or 500 mg/kg/day of Ag-THC dissolved in 100% sesame oil at concentrations of 25, 125 or 250 mg/ml. Each animal received 2.0 ml/kg with dose adjustments made weekly to account for changes in body weights. Three males and 3 females were treated with the low and medium dosages while 4 sex-paired monkeys comprised the control and high dosage groups. All animals treated po (acute and subacute) were given the drug by intubation with a No. 20 French catheter. The gavage tube was always flushed after compound administration with 3.0 ml of sesame oil. Testparametersusedin theintravenous studies. In acute iv tests, clinical and behavioral observations were recorded at -1 hr, +5 or 15 min and at 1,2,6,24,48 and 168 hr post injection. Hematologic, biochemical and urinary parameters were determined once prior to treatment and on days 1 and 6 post-injection. Specific analyses performed by standard laboratory techniques included : hematocrit, hemoglobin, RBC count, WBC count, WBC differential, reticulocyte count, platelet count, blood urea nitrogen (BUN), blood sugar, bromosulfophthalein (BSP) retention, alkaline phosphatase, SGOT and SGPT activities, Na+, K+, Cl-, Ca’+, blood pH, prothrombin time, serum bilirubin, PCOZY COz, PO,, oxygen saturation, total protein and urinary specific gravity, glucose, bilirubin, ketone bodies, pH, protein and sediment. A necropsy was performed on all deceased monkeys and 1 vehicle-control monkey for histopathologic evaluation of lung, liver, brain and kidney. In the subacute iv experiment, clinical and behavioral observations were recorded twice prior to treatment and at 1 and 23 hr after each injection. Physical examination and hematologic, biochemical and urine analyses were performed twice prior to the treatment and weekly thereafter. Blood and urinary test parameters in this study were identical to those evaluated in the acute study. A necropsy was performed on the deceased monkeys as soon as possible after their discovery, and interim sacrifices were performed on days 20 and 23 for monkeys treated with vehicle, 5 or 15 mg/kg/day of A’-THC. In all other monkeys a necrospy was carried out shortly after termination of treatment. The following tissues were submitted for gross pathologic evaluation: adrenals, aorta, sternal bone marrow, brain (cerebral cortex, brain stem and cerebellum), colon, duodenum, esophagus, eye, gallbladder, gonads and accessories (ovaries, uterus, mammary glands or testes, epididymis, prostate gland), heart, ileum, jejunum, kidneys liver, lungs, mesenteric lymph nodes, optic nerve, pancreas, parathyroids, pituitary,

TOXICITY

OF A'-THY

651

IN MONKEYS

submaxillary and parotid salivary glands, skin, spleen, stomach, striated muscle, thymus, thyroids, trachea and urinary bladder. Tissues from high-dose, control and deceased monkeys were evaluated for histopathologic changes after fixation in 4% neutral buffered formaldehyde solution and preparation of paraffin sections which were stained with hematoxylin and eosin. Test parameters in the oral studies. After acute po treatment. behavioral and clinical observations were made at 15min intervals for the duration of the treatment day, and daily thereafter for 7-14 days. Histopathologic evaluations were performed on all monkeys that died; blood and urinary parameters were not evaluated in monkeys treated acutely po. In monkeys treated po for 28 consecutive days, physical examinations and behavioral observations were made prior to the start of the study and 5 hr post-treatment on days 1 and 7 and weekly thereafter. In addition, each animal was observed daily 24 hr posttreatment for clinical signs (e.g., anorexia, diarrhea, constipation, prostration). Hematologic, biochemical and urine analyses were performed twice prior to treatment and on day 28. The specific test parameters were identical to those monitored in monkeys treated iv. On day 29 a necropsy was performed on 1 male and 1 female monkey from each group; the remaining monkeys continued to receive treatment as part of a more extensive toxicity test to be reported later. Tissues evaluated for histopathology were also identical to those monitored in monkeys treated iv. RESULTS Mortality

Rate in the Acute and Subacute Study

Mortalities induced by iv or po administration of dg-THC are presented in Table 1. Six monkeys treated iv with a single dose of 128 mg/kg, or more, died within 15-180 min post-treatment as a result of respiratory arrest and subsequent cardiac failure. All monkeys survived acute iv treatment with 92 mg/kg or lessand recovered from all effects within 2-4 days. Deaths in the 2%day iv trial occurred only in animals treated with 45 mg/kg/day, and 2 of 4 animals (1 male and 1 female) in this group succumbed to treatment on days 8 and 19 as a result of acute hemorrhagic pneumonia. TABLE 1 MORTALITIESIN RHESUS MONKEYSTREATED INTRAVENOUSLY WITH A~-THC

ORALLY OR

Route of administration Number of treatments 1 28

Intravenous” 2of2at128mg/kg(15-180min) 2 of4 at 45 mg/kg/day (days 8 and 19)

(daily) OVehicle for iv administration: b Vehicle for po administration:

Oralb >9000 mg/kg 1 of 6 at 50 mg/kg/day (day 16) 2 of 8 at 500 mg/kg/day (days 10 and 14)

13 ‘A sesame oil, 1.O% Tween 80 in normal saline. lOO’/~ sesame oil.

652

THOMPSON ET AL.

No deaths occurred in 25 monkeys treated po with 13 1 to 9000 mg/kg. In the 28-day po study, 2 male monkeys treated with 500 mg/kg/day and 1 male monkey treated with 50 mg/kg/day became moribund and were sacrificed on days 10 and 14, and 16, respectively. All monkeys treated with 250 mg/kg/day survived. All vehicle control monkeys survived in all 4 studies. Pathologic Changes in Deceased Monkeys Gross and histopathologic lesions observed in the 3 monkeys treated with single iv injections of d9-THC included pulmonary hemorrhage and edema in 1 (Fig. 1 and 2), and severe emphysema and generalized organ congestion in 2. In all 3 monkeys oil-red-O (ORO) positive globules of sesame oil and golden yellow Garnet positive

FIG. 1. Focal and confluent petechial and ecchymotic hemorrhages in the lungs of a monkey treated iv with d9-THC.

globules of THC (Baker and Rosenkrantz, 1972) were observed in large pulmonary blood vessels and alveolar capillaries. In the first animal, OR0 and Garnet positive material were also observed in the alveoli. This suggested that capillary permeability had been altered by the combined vehicle and THC which in turn resulted in hemorrhage and edema. In vehicle controls, ORO-positive globules of sesame oil were also observed in the pulmonary capillaries, but no edema or hemorrhage occurred. The other 2 deceased monkeys that received the largest iv doses and died within minutes following injections, exhibited only gross and microscopic pulmonary emphysema. In both treated and vehicle control animals, ORO-positive material (presumably sesame oil) also OCcurred around lymphoid nodules in the spleen and in glomerular capillaries. However,

TOXICITY OF LI~-THC IN MONKEYS

653

no vehicle-related lesions other than small microemboli of sesame oil were observed in these acute studies. Gross and histopathologic lesions observed in 2 monkeys that received daily iv treatments and died on days 8 and 19 included acute hemorrhagic pneumonia and injection site edema, ulceration, necrosis, fibrosis and acute inflammation (Figs. 3 and 4). No injection site lesions occurred in vehicle control animals. Thymus atrophy and increased liver weights also occurred in monkeys treated with 45 mg/kg iv (Table 2). Sinusoidal congestion may partially explain the increase in liver weights, since the space of Disse was increased in size due to apparent separation of the sinusoidal lining cells from

FIG. 2. Pulmonary hemorrhages, edema and accumulation treated iv with &‘-THC; H and E. x540.

of formulation

(arrows) in a monkey

hepatocytes. The plasma like fluid in this extrasinusoidal space may have contributed to the increase in liver weight. In 1 animal, the liver showed degenerative changes characterized by vacuolization of hepatocytes and accumulation of pleomorphic eosinophilic oval and round inclusion-body-like material (Fig. 5). The empty vacuoles in hepatocytes contained neither fat nor PAS-positive material. Only 1 monkey treated po with a single dose of d9-THC (1050 mg/kg) was examined for histopathology, and no lesions were observed in this animal. Predominant pathologic changes in the 3 deceased monkeys treated po for IO-16 days included atrophy of the pancreas (Table 3) and ulcerative colitis (Fig. 6) with associated myeloid hyperplasia of the bone marrow, vacuolar nephrosis and severe serum electrolyte imbalance. Similar lesions have been seen in monkeys infected with Shigella, but colonic cultures were negative for this pathogen in the present study. Adrenal function appeared to be specifically

654

THOMPSON

ET AL.

affected in these 3 moribund monkeys as indicated by thymus atrophy, reduced body weights and enlarged adrenal glands (Table 3). Increased liver weight and testicular atrophy were also evident in several monkeys treated po. The apparent decrease in heart size resulted from general body weight losses; relative heart weights (g/kg FBW) were within the variability found in control animals. Prostate, ovary, pituitary, spleen, lung and thyroid weights were generally unaffected by oral treatment.

FIG. 3. Inflammation, necrosis and accumulation of necrotic debris at an injection site in a monkey treated iv for 28 days; H and E. x140. This lesion occurred in monkeys treated with 5, 15, or 45 mg/kg/day. Clinical

Signs of Toxicity

The severity of changes in physiological parameters for monkeys treated iv or po with dg-THC for 28 days was either greater in monkeys treated iv or similar for both routes of administration (Table 4). The only exceptions were the more severe bradypnea in both groups treated po, which are presented in Table 4, and the persistent high incidence of anorexia in monkeys treated po with 50 mg/kg/day when compared to the incidence in monkeys treated iv with 5 mg/kg/day. Hypothermia, bradycardia and bradypnea generally occurred only during the first week of treatment in monkeys treated iv or po as

TOXICITY

OF L~~-THC

65.5

IN MONKEYS

tolerance to these effects developed rather rapidly. Maximal changes in these parameters occurred during the first 24 hr post-treatment and usually within the first 6 hr. The rapid development of tolerance in monkeys treated iv may explain the more severe bradypnea on day 1 in monkeys treated po since this parameter was not monitored prior to day 3 in monkeys treated iv. (In 2 monkeys treated iv with a single dose of 16 mg/kg, a 50 “/; reduction in respiration rate occurred within 6 hr.) Constipation was also more severe in monkeys treated iv and predominated during the first week of treatment, but this effect did occur throughout the 4-week treatment period. The incidences of anorexia and emesis were similar for both routes of administration, but anorexia was intermittent

FIG. 4. Perivascular fibrosis and proliferative treated iv for 28 days; H and E. x140.

obliterative

phlebitis at an injection site in a monkey

in monkeys treated iv and continuous in monkeys treated po. Emesis generally occurred only during the first week of treatment. A roughly dose-related incidence of anal passage of d9-THC occurred in monkeys treated po. The material appeared not to have been absorbed in that its physical appearance and sticky nature were identical to the formulation administered. Thinlayer chromatographic analyses revealed substantial amounts of dg-THC and minor amounts of 1 I-hydroxy-dg-THC.’ Efsects on Hematologic Biochemical and Urinary Parameters

Hematologic and biochemical changes in monkeys treated acutely iv with 64 mg/kg or less of dg-THC were limited to anemia, elevated BUN and increased BSP retention. ‘Analyses were performed Massachusetts. 22

by Dr. Francis J. Buliock

at Arthur

D. Little,

Inc., Cambridge,

3.6 + 0.1

3.6 + 0.1 3.3 & 0.1

5

15 4s

0.7

2.2

25.3 rt 2.3 66.0 + 18.5’

23.0 +

23.8 k

-~ Absolute’ -

IN MONKEYS

7.1 f 0.9 20.5 + 6.0’

6.4 -L 0.3

6.5 +- 0.5

-I__ Relatived -

WEIGHTS

Lung

ORGAN

7.9

Liver

WITH

27.9 _+ 0.8 38.0 _+ 4.0’

23.5 f 1.5

24.2 + 0.6

Relative

INTRAVENOUSLY

100.4 + 4.5 123.9 _t 14.2’

84.4 rt_ 5.0

81.5 +

Absolute

TREATED

1.43 f 0.33’ I .13 Ifr 0.25’

2.27 -I: 0.22

2.95 t 0.60

Absolute

dg-THC’

Relative

0.39 + 0.08” 0.35 + 0.07e

0.63 IL-0.07

0.80 2 0.13

Thymus

* Each group mean includes values for 2 male and 2 female monkeys. Values for the male and female monkeys that died after 19 and 8 days of treatment with 45 mg/kg/day, respectively, were included in the group means. ’ Average final body weight (mean + SE). c Absolute organ weight in g, mean + SE. d Relative organ weight in g/kg, mean + SE. c Significantly different from corresponding controls (p < 0.05, t test).

3.7 + 0.2

Avg. FBWb (kg) -~

0

Dosage (m/Wday)

AVERAGE

TABLE 2

!2 b I‘

TOXICITY 0~ L~~-TEIC IN MONKEYS

657

These effects generally occurred only at 24 hr post-treatment and recovery was complete by day. 6. Monkeys treated iv for 28 days exhibited anemia with subsequent reticulocytosis, leukocytosis, hypocalcemia, hypochloremia, decreased P,, and occasionally increased BSP retention. The anemia was hypochromic and normocytic, and its severity and duration were dose-related. Anemia and BSP retention occurred only in monkeys treated iv (Table 4). The leukocytosis generally correlated with injection site

FIG. 5. Vacuolar change in the liver with accumulation of pleomorphic body-like material in hepatocytes (arrow); H and E. x540.

oval and round inclusion-

lesions. Urinary values were normal in monkeys treated acutely with doses administered iv, but treatment with 14 or 45 mg/kg/day for 28 days produced persistent proteinuria and decreased urine volume by day 7. Hematologic and biochemical changes in monkeys treated po for 28 days included leukocytosis, elevated BUN and increased prothrombin time. Moderate to marked leukocytosis occurred in monkeys treated at all dose levels, but the incidence of this effect predominated in monkeys treated with 500 mg/kg/day. Elevated BUN and increased prothrombin time occurred in nearly all vehicle-control and treated monkeys 22*

250

10

14

28

28

M

M

F

G

G

M

M

G

M

G

28

F

G

28

M28

G

16

28

M

G

G

M F

28

Stat&

M

28

F

Day

M

Sex

2.4

2.8

2.4

2.2

3.0

-0.4

-0.5

-0.6

-0.8

-0.2

-0.7

-0.2

3.0

2.8

-0.9 -0.2

-0.3

-0.2

AC

(kg)

2.9 2.8

3.2

3.2

Final

B.W.

All organ weights are expressed in grams. Status: G = sacriticed in good condition; A = change from initial body weight. ND = not determined.

500

’ * c d

0

50

(rn:rg)

Necropsy

0.8

0.3 1.1

1.1

1.0

1.1

1.4

1.0 3.0

1.6

1.6

Thymus

M = sacrificed

2.4

3.5

2.0

3.2

6.0

7.6

5.5

2.7 6.1

6.3

6.4

Pancreas

moribund.

0.4

0.5

1.1

1.2

0.7

0.6

0.7

0.6

0.8

0.5

0.5

Adrenal

59.5

83.5

81.1

93.7

83.2

81.3

0.7

0.5

0.7

1.0

2.3

1.2

ND

0.7

68.3

0.9

55.5

ND”

1.5

Testes, uterus

103.9

63.6

75.2

Liver

ORGANANDBODYWEIGHTCHANGESINMONKEYS

TABLE 3

0.1

0.2

0.3

ND

0.2

0.4

0.4

0.4

0.3

0.2

0.3

Prostate, ovaries

TREATED

10.4

11.1

16.7

15.4

11.2

14.4

13.3

11.6

16.9

14.2

13.4

Kidney

ORALLY

WITH

11.7

11.1

9.9

9.7

15.9

15.5

11.6

13.2

12.2

14.1

14.4

Heart

ND

0.05

0.05

ND

0.1

0.05

0.05

0.1

0.1

0.05

0.1

Pituitary

d9-THC”

1.8

3.4

2.0 1.9

2.6

2.8

2.0 4.3

2.1

4.0

2.0

Spleen

18.2

20.0 16.3

14.0

17.5

20.3

18.7

22.8

21.1

23.4

18.6

Lung

0.4

0.5 ND

0.3

0.3

0.7

1.0

0.5

0.4

0.3

0.3

Thyroid

TOXICITY OF L~~-THCINMONKEYS

659

and appeared, therefore, to be related to effects of the vehicles (sesame oil). However, the severity of elevated BUN values for several monkeys treated with 250 or 500 mg/kg/day was greater than observed in control animals. Urine values for all 28 monkeys treated po remained within normal limits. Behavioral Efects Changes in behavior were also similar in monkeys treated iv and po and were indicative of generalized depression (Table 5). The typical sequential development of depres-

FIG. 6. Focal ulcerative colitis in a monkey treated po for 14 days with 50 mg/kg/day d9-THC; H and E. x140.

sion commenced with drowsiness (ptosis), then lethargy characterized by slow movements and eventual retreat into a classical huddled posture (“thinker” position) which was generally maintained until the effects of the treatment subsided. Monkeys treated po occasionally would not masticate food in their cheek pouches for as much as 24 hr. Monkeys treated iv did not respond to pinching during the first 10 days of treatment, but this effect did not occur in monkeys treated po. The severity and duration of the other

THOMPSON

ET AL.

TOXICITY 0~

LI~-THc

661

IN MONKEYS

changes were similar for monkeys treated iv with 45 mg/kg/day or po with 500 mg/kg/ day except that drowsiness did not occur after day 7 in monkeys treated po, and the huddled posture was more exaggerated in monkeys treated iv. In addition, the onset of these effects and maximal changes occurred more rapidly in monkeys treated iv. The first signs of drowsiness appeared within 5-15 min in monkeys treated iv and within 30-90 min in monkeys treated po. Maximal changes occurred 2-4 hr after iv treatment and 6-8 hr after po treatment. The onset and duration of effects were dose-related in monkeys treated by either route. No behavioral changes occurred in vehicle control monkeys treated iv or po. TABLE 5 SIGNS

OF BEHAVIORAL DEPRESSION IN RHESUS MONKEYS TREATED INTRAVENOUSLY WITH dg-THC FOR 28 DAYS~

Maximum severityb and persistence (in days)

ORALLY

OR

Daily duration of effects (hr)

Signs

Intravenous

Oral

Days l-3

Days 10-14 Days 21-28

Drowsiness Lethargy Huddled posture Analgesia

++ (l-28) ++ (l-28) +++ (l-28) +-I-++ (l-10)

+t (l-7) ++ (l-28) ++ (l-28) -

12-20 24 24

0 4-6 4-6

0 1-2 l-2

24

0

0

a Valuesrepresentseverity,persistenceand duration in monkeystreated iv with 45 mg/kg/day or po with 500 mg/kg/day, but these toxic signs occurred at all dosage levels.

b (+) = Mild (borderline control), + = moderate, ++ = marked, +++ = severe,++++ = very severe

(maximal).

Development of Tolerance

After 2-5 days of iv or po treatment, the severity and/or duration of many behavioral and physiological effects decreased. Tolerance to physiological signs of depression was frequently more acute and continuous than for behavioral signs of depression since several physiological parameters were unaffected after the first week of treatment. Behavioral changes often occurred each treatment day, but the severity of maximal intoxication and the duration of effect after each treatment decreased (Table 5). For instance, lethargy and huddled posture occurred daily throughout the treatment period for both routes of administration, but the duration decreased from 24 hr during the first 3 days to l-2 hr during the last week of treatment. Tolerance was most evident in monkeys treated with the lowest iv or po dosage. Hyperactivity occurred after tolerance had developed to the initial depression in 3 out of 6 monkeys treated po with 250 mg/kg/day. This effect was manifested primarily as increased psychomotor activity and hyperaggressiveness. Hyperactivity was temporary and somewhat variable in 2 of the 3 monkeys, but the effect persisted for the duration of treatment in the third monkey. Hyperactivity did not occur in monkeys treated po with vehicle, 50 or 500 mg/kg/day, and did not occur in any monkeys treated iv. 22**

662

THOMPSON

ET AL.

DISCUSSION

Isbell et al. (1967) reported that dg-THC in man was 3 times more potent when smoked than when taken orally. As indicated by the severity of physiological and behavioral changes in the present study, dg-THC was approximately 10 times more potent when administered iv than when administered po. Part of this potency difference was probably related to incomplete absorption after po administration. Nevertheless, similar effects and severities of changes were induced by both routes of administration. Wi.irsch et al. (1972) reported that biotransformation of dg-THC in squirrel monkeys differed considerably from rhesus monkeys which exhibited a pattern similar to man. Metabolic differences between the 2 monkey species may have accounted for the increased mortality and CNS excitation observed in the squirrel monkey, as reported by Scheckel et al. (1968) and McIsaacs et al. (1971). Thompson et al. (1973a,b) reported greater incidences of mortalities in female than in male rats treated po with single or chronic doses of cannabinoids, and this sex-related difference probably resulted from sex-related differences in hydroxylation capabilities (Kato and Gillette, 1965; Schenkman et al., 1967). In addition, all rat mortalities occurred between 36 and 72 hr after the initial treatment in both the acute and chronic trials. In the present studies, no sex-related mortality differences occurred in acute iv or po trials or in the subacute iv study, but 3 male monkeys succumbed to po subacute treatments. The number of mortalities in the po monkey study was too small to support a firm conclusion about a sex-related effect, and the apparent trend in male monkeys was in opposition to the female susceptibility in rats. Furthermore, mortalities in the subacute iv and po monkey studies were more delayed (8-19 days) than in rats, and this temporal difference indicated a more protracted cumulative toxicity in monkeys, perhaps in different organs. However, species and sex differences in drug metabolizing capability are well known (Burns, 1970; Kato et al., 1970). Behavioral changes in monkeys treated po or iv for 28 days also differed substantially from changes reported in rats. Rats treated po for 119 days with cannabinoids exhibited a bimodal behavioral pattern characterized by depression to which tolerance developed, hyperactivity, fighting and convulsions (Thompson et al., 1973b). Rats also exhibited significantly decreased brain values for protein, RNA and acetyl-cholinesterase (Luthra et al., 1971). Monkeys treated iv or po exhibited depression to which tolerance developed, but only 3 monkeys treated po exhibited hyperactivity, and this was transient in 2 of the 3 monkeys. However, neurochemical changes in brains from monkeys treated iv were similar to the changes in rats and included decreased protein, RNA and total lipids (Rosenkrantz et al., 1972b). Hepatotoxicity observed in man by Kew et al. (1969), but not by Hochman and Brill (1971), was not a major effect in the present studies. However, liver enlargement occurred consistently in monkeys treated po and iv for 28 days, and hepatic glycogen was significantly decreased in monkeys treated iv, rabbits treated SC,and rats treated by inhalation (Sprague et al., 1973). Increased liver weights partially resulted from sinusoidal congestion and fluid entrapment in the extrasinusoidal space of Disse, but this change may have also resulted from a direct stimulatory effect of dg-THC on liver
TOXICITYOF Ag-~~c IN MONKEYS

663

tolerance. In addition, Ag-THC caused disruption and disorganization of rat liver mitochondria in vitro (Mahoney and Harris, 1972) and a similar effect in vivo may also have contributed to the subtle hepatic changes observed in the present studies with monkeys. Hormonal changes were evident in the 3 moribund monkeys treated po as indicated by testicular atrophy and enlarged adrenal glands. Thymus atrophy and decreased body weights correlated with the enlargement of adrenal glands. Thompson et al. (1973b) previously reported similar organ weight changes in rats treated chronically with AgTHC, A*-THC or a crude marihuana extract, and presented evidence that a compromised pituitary function could explain various hormonal effects in animals treated chronically with cannabinoids. However, hormonal assays in chronically treated animals have not been reported. The rapid development of behavioral and physiological tolerance in the present study superficially indicated a minimal hazard associated with subacute administration of A9-THC. However, the delayed mortalities in both the iv and po subacute trials indicated that Ag-THC was hazardous to monkeys, at least when large doses were administered. In addition, significant neurochemical changes in these monkeys (Rosenkrantz et al., 1972b) indicated functional rather than morphological changes. Neither light microscopy (present study) nor electron microscopy (unpublished data) revealed drug-related morphologic alterations. However, cerebral atrophy was also reported in chronic marihuana smokers (Campbell et al., 1971). Furthermore, at least one human fatality has been attributed to marihuana smoking (Heyndrickx et al., 1969) and recently a young student succumbed after chronic consumption of marihuana “tea” (DeGraffenreid, personal communication). Convulsions in rats (Thompson et al., 1973b) and psychoses in man (Talbott and Teague, 1969; Kaplan, 1971) also indicate some hazard associated with marihuana abuse. Although A9-THC potentiated the effects of barbiturates and amphetamine (Garriott et al., 1968), data are unavailable to enable evaluation of toxicity and pathology produced when marihuana or its constituents are administered in conjunction with other drugs of abuse. This may represent the greatest hazard to man since most users abuse several drugs simultaneously and the multiple abuse is frequently chronic (Carlin and Post, 1971). Obviously much work is needed before the hazard(s) associated with marihuana use can be properly evaluated.

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