The metabolism and excretion of Δ9-tetrahydrocannabinol in the rat

Life Sciences Vol . 10, Part I, pp. 49-59, 1971 . Printed in Great Britain

Pergamon Press

THE METABOLISM AND EXCRETION OF A ' -TETRAHYDROCANNABINOL IN THE RAT*+ Howard A . Klausner and James V . Dingell Department of Pharniacology, Vanderbilt University School of Medicine and Tennessee Neuropsychiatric Institute, Central State Hospital Nashville, Tennessee

(Received 20 October 1970; in final form 16 November 1970) THE AVAILABILITY of synthetic A 9 -tetrahydrocannabinol (THC), a psychotomimetic component of marihuana has stimulated the investigation of its behavioral effects and pharmacological actions .

Recent reports have described

the excretion of THC (1,2), the formation of the 11-hydroxy metabolite of THC by liver microsomes (3,4) and its excretion in urine (5) .

The present studies

were undertaken to further investigate the fundamental aspects of the fate of THC in rats .

These studies have confirmed the findings of Agurell et al, that

in rats THC is excreted almost completely in feces as more polar metabolites . Moreover, evidence is presented which shows that THC disappears from the tissues of rats in a biphasic fashion .

That is, immediately after its adminis-

tration the levels of THC in rats decline very rapidly ; after several hours, however, the compound disappears with a long half-life which resembles the rate of excretion of metabolites in urine and feces .

Moreover, it appears

that neither the hepatic metabolism nor excretion in bile are limiting factors in the excretion of metabolites of THC in feces .

The rate of excretion of

these compounds is probably determined by their rate of movement down the intestine possibly combined with an enterohepatic circulation . Methods '"C-e 9 -THC (5 .2 mCi/rtrral) labeled in positions 2 and 4, radlopurity greater than 95 percent, was obtained from the National upport y SPH Grants MH-11468 and GM-15431 + A preliminary report of this work has appeared :

49

Institute of Mental

Pharmacologist 12 :259, 1970 .

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Health, Center for Studies of Narcotic and Drug Abuse .

Male Sprague-Dawley

rats were used in these experiments . '"C THC (4 mg/kg, 0 .14 mCi/mnol) was administered intravenously in about 0 .5 ml of a mixture of 30 percent propylene glycol and 70 percent rat serum . Determination of Biolo~iç Half-Lives Rats were killed at various times after the administration of '"C THC and homogenized with 4 volumes of water in a Waring Blendor .

The homogenates

were filtered through several layers of gauze, and aliquots of the filtrates assayed as described below . Tissue Distribution Rats were killed by decapitation at various times after the administration of '"C THC .

Tissues were removed, chilled immediately on crushed ice

and homogenized with 4 volumes of water in a teflon-glass homogenizer . Aliquots of the homogenates were assayed for either total radioactivity or extractable radioactivity . Oxygen Combustion Total radioactivity was measured by liquid scintillation counting following combustion in a Peterson combustion apparatus (6,7) . Measurement of '"C THC and Metabolites The results presented in this paper and those of Agurell et al, (1,2) are in agreement that only THC is extracted from biological material into petroleum ether whereas metabolites as well are extracted into diethyl ether . Aliquots of homogenates, usually 5 ml, were pipetted into glass-stoppered shaking tubes containing 15 ml of organic solvent, the tubes shaken for 45 minutes and centrifuged .

When diethyl ether was used the tubes also con-

tained 1 .5 ml of a 2 percent solution of E .D .T .A . and ascorbic acid at pH 7 .4 to prevent oxidation of THC during extraction .

Moreover, to remove peroxides

diethyl ether was washed several times immediately before use by shaking with 1/5 volume of a saturated solution of ferrous sulfate .

Metabolism od A9 -THC

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51

After extraction, 10 ml aliquots of the organic phase were transferred to counting vials and evaporated to dryness under a stream of nitrogen .

The

residues were dissolved in 3 ml of ethanol and the radioactivity was measured by liquid scintillation counting after the addition of phosphor .

Using this

procedure the recoveries of 1 "C THC added to biological material were 90 - 95 percent with diethyl ether and 75 - 80 percent with petroleum ether .

Aliquots

of the injection solution of THC were carried through the procedure ; all data were corrected for counting efficiency by external standardization and for the appropriate recoveries . The Isolated Perfused Rat Liver Details of the perfusion procedure (8) and apparatus (9) have been published previously .

The perfusion medium consisted of 33 ml defibrinated

rat blood and sufficient Krebs-Henseleit bicarbonate buffer, pH 7 .4 (10) to make a final volume of 100 ml .

After equilibration

1

"C THC was added to the

medium in the recycling system in a mixture of 30 percent propylene glycol and 70 percent rat serum . Results Excretion of THC and its Metabolites About 60 percent of the radioactivity administered to rats is excreted in urine and feces 120 hours after the administration of 14 C THC (4 mg/kg, i .v .) . Only about 10 percent of the radioactivity which is excreted can be found in the urine .

The excretion of radioactivity in urine is essentially

complete 48 hours after the administration of

14

C THC and over 50 percent. of

this is excreted in the first 24 hours (Fig . lA) .

It is noteworthy that

only negligible amounts of radioactivity are extractable from urine into petroleum ether indicating that THC is excreted almost exclusively as metabolites by this route .

Moreover, the finding that about 60 percent of the

radioactivity in urine is extractable into diethyl ether is consistent with

s

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Metabolism ad D -THC

the view that this solvent extracts metabolites of THC . The major route of excretion of radioactivity after the administration of '"C THC is by way of the feces .

Fecal excretion of radioactivity is

essentially complete within 72 hours and similar to its urinary excretion, over 50 percent of the material excreted is found in feces within 24 hours (Fig . 1B) .

Moreover, it appears that THC is also excreted as metabolites

in feces, only a portion of which are extractable into diethyl ether . Disposition of THC in Rats After the administration of 1 "C THC (4 mg/kg, i .v .) to rats, the levels of both total radioactivity and that extractable into diethyl ether decline logarithnically with a biological half-life of about 16 hours (Fig . 2A) . Interestingly, THC itself disappears from rats in a biphasic fashion (Fig . 2B) . That is, immediately after the administration of 1 "C THC the petroleum ether extractable radioactivity declines very rapidly with a half-life of about 1/2 hour .

After about 2 hours, however, it declines far more slowly as is

evidenced by a biological half-life of about 21 hours .

These findings suggest

that initially a more available pool of THC is rapidly metabolized ; after several hours, however, the compound is less available for metabolism and undergoes transformation far more slowly .

The decreased availability of

THC could be the consequence of either a distribution into more slowly perfused tissues or intracellular binding . Fifteen minutes after the intravenous administration of 1 "C THC the concentration of diethyl ether extractable radioactivity in plasma is equivalent to less than 1 ug/ml of THC (Table 1) .

these levels remain re

latively constant for 1 hour and then decline to about one-half in the second hour .

The measurement of the diethyl ether extractable radioactivity

in tissues confirmed the rapid localization of the drug in tissues (Table 1) . For example, 15 minutes after the administration of THC, the tissue to plasma ratios of radioactivity for all tissues but brain were in excess of three . It is noteworthy that exceptionally high levels of diethyl ether extractable

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radioactivity were found in both lung and liver . In contrast to the relatively slow rate of decline of total radioactivity measured in the whole rat, the levels of total radioactivity in most tissues declines by about one-half in the second hour (Table 2) .

A

notable exception being the lung, the levels of radioactivity in this highly vascular tissue decrease strikingly in the second hour .

Since the data

presented for intestine does not include its contents, it is reasonable to speculate that the difference between the rate of disappearance of radioactivity from tissues and that measured in the whole rat is the consequence of the gradual excretion of radioactivity in feces perhaps combined with an enterohepatic circulation .

Moreover, it is noteworthy that the transient

appearance of radioactivity in brain is consistent with the short duration of the behavioral effects of THC observed in these rats .

FIG . 1 Urinary (lA) and fecal (1B) excretion of total and extractable radioactivity expressed as THC . Each point represents the average value obtained from at least three rats (average weight 119 g) .

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Total radioactivity, diethyl ether extractable radioactivity (2A) and petroleum ether extractable radioactivity (2B) expressed as THC . Each point represents the average value obtained by the assay of homo enates from at least three rats (average weight 119 g) . Vertical bars (2B~ indicate the range . Lines were drawn according to the method of least squares . Studies with the Isolated Perfused Rat Liver The extensive excretion of THC as metabolites in feces suggested the examination of its fate in the isolated perfused rat liver .

THC rapidly

disappears from the perfusate and accumulates in the liver .

For example,

within 15 minutes after the addition of 1 mg 14 C THC to the perfusion system only about 10 percent of the petroleum ether extractable radioactivity remains in the perfusate . After perfusion for three hours, 95 percent of the radioactivity added to the system was recovered in the perfusate, bile and liver (Table 3) .

Since

only 5 percent or less of the radioactivity in these compartments was extrac table into petroleum ether it is evident that any THC reaching the liver is almost completely metabolized .

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TABLE 1 Diethyl Ether Extractable Radioactivity in Plasma as THC Minutes

ug/ml

15

30

60

0 .62

0 .78

0 .56

120 0 .29

Tissue to Plasma Ratios of Diethyl Ether Extractable Radioactivity after 15 Min . Lung

55 .2

Kidney

6 .5

Spleen

3 .5

Liver

12 .1

Fat

5 .5

Muscle

3 .0

Heart

6 .7

dntest .

3 .5

Brain

2 .7

Results are the mean values obtained with

Rats received 14 C THC 4 mg/kg, i .v . at least two rats .

TABLE 2 Total Radioactivity as THC u9/9 Time Minutes 15

30

60

120

Lung

70 .3

45 .5

37 .9

3 .3

Liver

12 .2

11 .0

7 .5

4 .3

Heart

5 .1

3 .6

2 .7

1 .1

Kidney

4 .7

4 .2

3 .1

1 .4

Spleen

2 .8

2 .6

2 .0

1 .3

Brain

1 .6

2 .6

1 .2

0 .6

Intest .

5 .9

7 .1

9 .2

1 .7

Muscle

2 .2

3 .0

2 .1

0 .7

Fat

2 .3

7 .9

4 .1

5 .4

Rats (average weight 184g) received 1 "C THC 4 mg/kg . i .v . the mean values obtained with at least two rats .

The levels are

vol . 10, No . 1

Metabolism ad A 9 -THC

5g

Almost 80 percent of the radioactivity added to the system is excreted in the bile within two hours with little additional excretion being observed over the next hour (Table 3) .

These findings are, thus, consistent with

the hypothesis that as far as the liver itself is concerned, neither the rate of metabolism of THC nor the excretion of metabolites in bile are responsible for the slow rate of disappearance of radioactivity observed in vivo . Enterohepatic Circulation of Metabolites of THC In preliminary studies, aliquots of bile from the liver perfusion experiments were injected into the duodenum of three rats under light ether anesthesia .

After two hours the diethyl ether extractable radioactivity in

the intestines and bodies of these rats was measured .

These studies revealed

that from 15 to 44 percent of the diethyl ether extractable radioactivity was present in the bodies of these rats .

Although these results are somewhat

variable, they are consistent with the view that metabolites of THC can be reabsorbed from the intestine .

TABLE 3 Isolated Perfused Rat Liver E xperiment 1 dpm x 10 -3 '"C THC Added

-

Percent

E~eriment 2

dpm x 10 -3

Percent

2432

100

2448

100

62

3

80

3

238

10

243

10

Bile 0 - 2 Hr .

1894

78

1863

76

2 - 3 Hr .

112

5

94

4

2306

95

2280

93

Radioactivity Recovered Perfusate Liver

Total Recovered

1 mg 1 "C THC added to recirculating system, livers were perfused 3 hr . a t 37°C .

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Discussion Although an understanding of the pharmacokinetic profile of a drug may often be essential to the interpretation of its pharmacological properties, it is only recently that the availability of radio labeled THC has made possible meaningful studies on its metabolic fate, distribution in tissues and excretion .

The major obstacle to the investigation of the fate of THC

has been the lack of suitable methodology for its measurement in biological material .

This is due in no small part to the highly lipid soluble nature of

the drug itself .

For example, we have found that the assay of THC in most

biological material by existing techniques of gas-liquid chromatography is made impractical by the large amount of interfering material which is also extracted from tissues .

The development of methodology for the separation

and measurement of THC and its metabolites in biological material will certainly continue to be both an important and challenging area of research . The results presented in this paper confirm the findings of Agurell et al

(1) that rats excrete THC almost exclusively as metabolites in feces .

There do, however, appear to be striking differences between the rates of excretion observed in the two laboratories .

For example, Agurell et al have

reported that THC is excreted very slowly by rats after intravenous administration ; less than 50 percent of the drug being eliminated in one week .

we

have found that the excretion of the drug is essentially complete in 72 hours, with about 60 percent excreted in five days .

Since it appears that

biotransformation to more polar metabolites is required for the excretion of THC, it is reasonable that these differences are due in part to sex and strain differences in the rate of metabolism of THC .

They may, however, be

partially due to differences in the manner in which the drug was prepared for injection in the two laboratories .

Although we have not administered the

drug intravenously in emulsions of sesame oil, saline and lechithin, the results presented in this paper demonstrate the rapid absorption of THC after injection in mixtures of propylene glycol and serum .

Since THC has been ad-

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Metabolism od 0°-THC

ministered by various routes and prepared in a number of solutions and emulsions, the possibility that these factors may affect its rate of absorption suggests not only the need for further studies on the absorption of THC, but also the importance of relating pharmacological and behavioral effects to the levels of the drug in tissues . Immediately after its administration, THC is very rapidly metabolized in rats .

After several hours, however, a considerable fraction of the drug re-

mains and perhaps, because of intracellular binding or localization in poorly perfused tissues, such as fat, is metabolized at a very slow rate .

This

finding suggests the possibility that the repeated administration of the drug could result in its accumulation in certain tissues or cellular components . The localization of injected THC in lungs has also been reported by Agurell et al (2) and interestingly, Ho et al (11) have observed the accumulation and retention of radioactivity in lungs after the exposure of rats to the smoke of cigarettes which had been treated with 9 H-THC .

Although the

pharmacological or toxicological consequences of this localization remain to be established, it should be noted that this affinity for lung tissue is not an exclusive property of THC, but rather is shared with a large and heterogeneous group of lipid soluble drugs . The results obtained in the present study demonstrate the value of the isolated liver perfusion technique in the investigation of the fate of a drug . This preparation provided not only data which confirmed the extensive hepatic metabolism of THC and the .biliary excretion of its metabolites, but also a concentrated sample of these metabolites for the investigation enterohepatic circulation .

of their

The finding that metabolites of THC can be absorbed

from the intestine may be of pharmacological importance as it has been recently reported by Foltz et al (3) that the 11-hydroxy metabolite of THC evokes a behavioral profile in rats which resembles that seen after the administration of THC itself .

Studies are presently in progress to determine the identity

of the metabolites of THC which are excreted in bile from the isolated perfused

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Metabolism of 0° -THC

59

rat liver . Acknowledgements - The authors wish to express their appreciation to the following : Dr . Daniel Efron for his continued interest and assistance, Dr . John Scigliano for providing the 1 "C THC, Miss Joyce Ca11 and Miss Sharon Buff for valuable technical assistance . REFERENCES 1.

S . AGURELL, I .M . Nilsson, A . Ohlsson and F . Sandberg, Biochem . Pharniac . 18, 1195 (1969) .

2.

S . AGURELL, I .M . Nilsson, A . Ohlsson and F . Sandberg, Biochem . Pharmac . 19, 1333 (1970) .

3.

R .L . FOLTZ, A .F . Fentiman, E .G . Leighty, J .L . Walter, H .R . Drewes, W .F . Schwartz, T .F . Page and E .G . Truitt, Science 168, 844 (1970) .

4.

I .M . NILSSON, S . Agurell, J .L .G . Nilsson, A . Ohlsson, F . Sandberg and M . Wahlquist, Science 168, 1228 (1970) .

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S .H . BURSTEIN, F . Menezes, E . Willaimson and R . Mechoulam, Nature 225, 87 (1970) .

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J .I . PETERSON, F . Wagner, S . Siegel and W . Nixon, Anal . Biochem . 31, 189 (1969) .

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J .I . PETERSON, Anal . Biochem .

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M . HEIMBERG, I . Weinstein, H . Klausner and M .L . Watkins, Am . J . Physiol . 20~? , 353 (1962) .

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M . HEIMBERG, N .B . Fizette and H . Klausner, J . Am . 011 Chem . Soc . 41, 774 (1964) .

1 .0 .

H .A . KREBBS and K . Henseleit, Hoppe -Seyler's Z . Physiol . Chem . 21~, 33 (1932) .

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B .T . H0, G .E . Fritchie, P .M . Kralik, L .F . Englert, W .M . McIsaac and J . Idanpaan-Heikkila, J . Pharm . Pharmac . j 2? , 538 (1970) .

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204 (1969) .