Interactions of Δ9-tetrahydrocannabinol with the hepatic microsomal drug metabolizing system

Life Sciences Vol. 10, Part I, pp . 1207-1215, 1971 . Priried in Great Britain .

Pergamon Press

INTERACTIONS OF A9 -TETRAHYDROCANNABINOL WITH THE HEPATIC MICROSOMAL DRIIG METÀHOLIZING SYSTEM G . M . Cohen, D. W. Peterson and G. J . Mannering Department of Pharmacologq, University of Minnesota, Medical School, Minneapolis, Minnesota 55455

(Received 2 August 1971; in final form 20 September 19?1) summary

~9 -Tetrahydrocannabinol (G9 -THC) was shown to combine with hepatic microsomes from both untreated and phenobarbital (Pb) treated male rats to give a typical ~ype I difference spectrum . The affinity of the microsomes for D -THC was very high, as reflected by the spectral dissociation constant (R s) values of 18 .5 and 9.1 uM for microeomes f mm untreated and Pb-treated animals, respectively . A 9 -THC inhibited competitively the microsomal metabolism of ethylmorphine, a typical type I substrate . The inhibitor constant (Ri) value obtained with untreated animals w~e vary low, 15 .4 uM . These studies support the view that -THC is metabolized by the hepatic mixed function oxidase 9 system im~olving cytochrome P-450. The high reactivity of ~ -THC with this system raises the possibility that D 9 -THC may interfere with the biotransformation of other drugs in vivo . Introduction A 9 -THC is believed by several investigators to be the major psychoactive principle of Cannabis sativa (1,2,3) .

Others have suggested that the activity 9

may reside in one or more of the metabolites, particularly 11-hydroxy-D -THC . Chrietensen et al . (4) showed that the 11-hydroxy metabolite was eighteen times more active than the parent compound on intracerebral administration and twice as active when given intravenously . Sofia et al .

This ie in conflict with the work of

(5) who demonstrated that when the metabolism of A 9-THC was

inhibited by 2-diethylaminoethyl 2,2-diphenylvalerate HC1 (SRF 525-A), a kmwn inhibitor of the microeomal enzyme system, the depressant effect of the drug was enhanced . Using the 10,000 z g supernatant fraction from Pb-treated female rabbits, 9

Nileson and coworkers (3) identified the major metabolite of D -THC as

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O9 -TfiC and Drug Metabolism

11-hydroxy-D 9 -THC .

Vol. 10, No. 21

More recently the hydroxylation of D 9 -THC by a hepatic

microsomal monooxygenase has been demonstrated and the involvmneat of cytochrome P-450 suggested (6) .

The current study presents further evidence

for an interaction of cytochrome P-450 aad D 9-THC . Methods Male Holtzman rats weighing 190-260 g were used .

Phenobarbital treated

animals received daily intraperitoneal injections of sodium phembarbital (40 mg/kg in 0 .9X saline) for 5 days . the last injection .

Animals were sacrificed 24 hours after

Microsomes were prepared ae described previously (7) and

used on the day of preparation .

Ethylmorphine N-demethylase activity was

determined as described previously (7) except that nicotinamide was excluded from the incubation medium .

p-Chloro-N-methylaniline N-demethylase activity

was determined by the same method .

When used as an inhibitor of ethylmorphiae

N-demethylase activity, 0 9-THC in alcohol was mixed with the mícrosomes before they were added to the incubation medium, thus facilitating its solution .

In

controls aliquots of alcohol were added to the enzyme and in all cases the incubation was started by adding the enzyme .

Difference spectra .produced by

binding of 09 -THC to hemoprotein were measured by the method of Remmer and coworkers (8) using a Shimadzu model MPS 50 spectrophotometer .

Protein

concentrations for the binding studies were 1 mg of microsomal protein/ml and the D 9-THC was added in 95X alcohol to the sample cuvette . (6-80 mM) of 95X alcohol was added to the reference cuvette .

An equal amount Cytochrome P-450

was determined by the method of Omura and Sato (9) .

Protein concentrations

were determined by the method of Lowry et al .

Kinetic parameters, the

(10) .

Michaelie-Menten constant (Km ), the maximum velocity (V~), K s and ~ were determined by the method of Wilkinson (11), using an Olivetti computer . Results 9 As caa be seen in Fig . 1, D -THC caused a typical type I spectral change

mn, ~ 423 tam) i.n microsomea from both untreated and Pb-treated (amax ~ 390 arms

Vol . 10, No . 21 animals .

~9

-THC and Drug Metabolism

1209

The spectral chaages were concentration dependent in both cases.

w

U Z Q m

O N m Q G

WAVELENGTH (n m)

Mfference spectra produced by the addition of different concentrations of G9-THC to hepatic microsomes of untreated and Pb-treated rats . 1 mg of microsomal protein/ml was used is all cases. The concentrations of D9-THC used are shown in the figure . Ia drawing the spectra, deviations in the baseline were corrected mechanically . The magaitude of the spectral chaagee were measured and the Ks and A~ determiaed .

Ks values were 18 .5 and 9 .1 uM using microsomee from untreated

and Pb-treated animals, respectively (Table I) .

This high affinity of

e 9-THC

for the microsomal hemoprotein explains why we did not see binding in our earlier experiments .

9 In this earlier work the D -THC had been added is the

millimolar quantities normally used for binding studies, and these high concentrations caused aggregation of the micm comes . The low Ks value for A9-THC suggested that it might interfere with the metabolism of certain. of the multitude of compounds known to be metabolized by the hepatic microsomal mixed fuaction oxidase system .

9 The effects of D -THC

oa the metabolism of ethylmorphine, a typical type I compound, were therefore

1210 investigated .

O9 -THC a.~ Drug Metabolism

Vol. 10, No. 21

A 9 -THC proved to be a potent inhibitor of ethylanrphine

N-demethylase activity as ie seen by the low K1 value of 15 .4 uM (Table I) . When the data were plotted by the method of Lineweaver and Burk (13), the inhibition appeared to be competitive (Fig . 2) .

The

max

values obtained from

[Ethylmo~phine] x10-3 FIG . 2 Lineweaver-Burk plots showing competitive inhibition of ethylmorphine N-demethylase activity by G9 -THC . Microsomes from untreated animals ere employed (approaimately 1 mg of protein/ml) . The concentrations of ~ -THC are given with each curve . The final concentration of ethanol (used for the addition of D9 -THC) ìn all incubation mixtures, including those without D 9 -THC, was 17 mM . the control and A9 -THC inhibited reactions were not different statistically in microsomes obtained from untreated animals.

e9 -THC

was also a potent inhibitor

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A9

-THC and Drug Metabolism

1211

of ethylmorphiae N-demethylase activity is microsomes obtained from Pb-treated animals .

5 9 M), the inhibition At the lower concentration of D -THC (1 .27 % 10

was competitive but at the higher concentration the inhibition appeared to be mixed.

The %1 of 4 .2 uM reported in Tabla I for the higher concentration of

9 D -THC has been calculated fmm the kinetic parameters* obtained and is not a true%i as the

ms's

obtained were statistically different. TABLE I

9 Microsomel Binding and Inhibition %inetics of D -THC

Treatment

Untreated Phenobarbital

%sa (uM)

f 1 .9 (7) b

18 .5

9 .1 c t 0 .6 (9)

maze/nmole P-450

0 .041

± 0 .002 (7)

0 .033 c t 0 .001

Ge)

%i8 (uM)

15 .4

± 2.6

(11)

3 .6 c ' d t 1.3 (6) 4.2 c 'e ± 1.1 (6)

Concentrations of microsomal pm tein used is the binding and inhibition studies Binding studies employed concentrawere 1 and 0.9-1 .2 mg/ml, respectively . tione of A9-THC of 4 .3 to 64 uM . Inhibition studies employed concentratione of ethylmorphine of 0 .4 to 2 .0 mM . aMean t S .E . btiumber is parentheses represents the number of experiments . cSigaificantly different (P < 0.01) from the corresponding untreated group . d%i obtained using D 9-THC 1 .27 % 10-5 M. eRi obtained using D 9-THC 2.55 % 10-5 M; see text for explanation . A9-THC, is the concentrations used to inhibit ethylmorphine N-demethylase activity, had no effect on the demethylatioa of the type II compound p-chloroNrmethylaniline.

The %i was calculated from the slopes of the inhibited and uninhibited reactions using the formula : concentration and

max

and V'~ ar

and inhibited reactions,

KV'

_ 1

where I is the inhibitor

the maximum velo cities of the uninhibited

respectively, and

m

and %' m are the Michaelis-Menten

coaatants of the uninhibited and inhibited reactions, respectively .

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09 -THC and Drug Metabolism

Vol. 10, No. 21

Discussion The two findings reported in this study, namely the type I binding of 9 D -THC to cytochrome P-450 and the competitive inhibition of ethylmorphine N-demethylase by D9 -THC in microsomes from untreated animals, further implicate the microsomel mixed function oxidase system in the metabolism of D9 -THC . Several compounds which give type I binding spectra with cytochrome P-450 are known to be hydroxylated by this system, for example, cyclohexane is hydroxylated to cyclohexanol and hexobarbital to 3'-hydroxyhexobarbital . recent work of Dingell et al .

The

9

(13) showing that D -THC inhibited the microsomel

oxidation of aminopyrine and hexobarbital but enhanced the reduction of p-nitrobenzoic acid is consistent with our finding that 0 9 -THC is a type I compound . Rubin et al . (14) showed that a variety of structurally unrelated drugs known to be metabolized by hepatic microsomes competitively inhibited the N-demethylation of ethylmorphine .

They suggested that the inhibition could be

explained if the drugs were reacting with a common intermediate or with a single enzyme of low substrate specificity .

In the latter case, one drug

should inhibit competitively the metabolism of another drug by acting as an alternative substrate .

When a compound can act in this manner as both a

substrate and an inhibitor

of any enzyme, the Km of the metabolism of the

inhibitor should approximate the K 1 of the reaction being inhibited .

The

agreement between the K s and K i values (Table I) supports the hypothesis that 9 D -THC was acting as a substrate inhibitor .

As yet no suitable assay is

9 available for measuring the Km of D -THC, but if our interpretation of its inhibition kinetics is correct, it should be in the order of 15 NM when microsomes from untreated rats are employed .

A Km value of this magnitude

would explain why Buretein and Kupfer (6) observed no inhibition of D 9 -THC 4 3 M (14) . hydroxylation by hexobarbital (3 X lÒ M), which has a Km of 1 X 10 The change in K s and K 1 between control and Pb-treated animals suggests a qualitative change in the nature of the enzyme .

This supports the work of

voi. lo, xo. ai

n9

lals

-TIìC and Drug Metabolism

Guatino et al . (15) orho shared differences is the

â

and &s of aniline, a type

II compound, when the activities of microsomes from untreated sad Pb-treated animals vets compared .

The mixed inhibition observed at the higher

concentration of A9-THC, when microsomes from Pb-treated animals vets used, could have been due to the production of a metabolite with different inhibitory properties from the parent compound .

An alternative explanation vas

that the A9-THC or its metabolite in the higher concentrations used in this experimat were inactivating the enzyme . The results reported here shared that A9-THC is an effective in vitro inhibitor of microsomal drug metabolism .

Of greater practical interest,

however, is whether A9-THC can inhibit drug metabolism in vivo .

Should this

prove to be the case, the wide use of marihuana is our society could conceivably cause many drug interactions .

The known accumulation of A9-THC in rat

liver after inhalation (16) sad the low S credibility to this poaeibility .

s

and Ri values reported here lead

The observation of Garriott et al . (17) that

A9-THC potentiated hezobarbital sleeping time in rate may support the view 9 that 0 -THC can act ae an in viva inhibitor of drug metabolism .

The

potentiatioa could also be due to a central depressant action of the A9-THC . In preliminary is viva studies treated rate (190-220 g) received A9-THC (10 mg/kg; I .P .) dissolved is 1X pluronic F-68 (0 .4 ml ; üyandotte Chemical Corporation) .

Control rate received 1X platonic only (0 .4 ml) .

later all rata received hezobarbital Na (80 mg/kg; L P .) .

Thirty minutes

No difference vas

observed is the rates of disappearance of hezobarbital from the blood of control or A9-THC pretreated animals .

9 The rapid metabolism of the D -THC sad

its bíliary ezcretion (18,19,20,21) could mesa that the drug does not remain is the liver a sufficient length of time to cause inhibition . A9-TSC is bound to many tissues sad it may be only when these sites are saturated after prolonged admiaietration that high enough levels are reached is the liver to cause inhibition . case .

Additional studies are planned to determine if this ie the

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09 -THC and Drug Metabolism

Vol. 10, No. 21

Açknowledgments The A 9-THC was kindly supplied by FDA-NIMH Psychotomimetic Agents Advisory Committee . This study was supported by U . S. Public Health Service Grant GM 15477 . References 1.

H. leben, C. W. Gorodetzaky, D . Jasinski, U . Claussen, F. von Spinale and F. Borte, Psychopharmacologie 11, 184 (1967) .

2.

R. Mechoulam, Science, N . Y. 168, 1159

3.

I . M. Nilsaon, S. Agurell, J . L . G. Nilsson, A. Ohleson, F . Sandberg and M. Wahlquiat, Science, N . Y. 168, 1228 (1970) .

4.

H. D . Chrietensen, R. I . Freudenthal, J . T . Gidley, R. Rosenfeld, G. Boegli, L . Testino, D. R. Brine, C . G . Pitt and M. E . Wall, Science N. Y . 172, 165 (1971) .

5.

R. D . Sofia and H . Barry III, Europ . J. Pharmac . 13, 134 (1970) .

6.

S . H . Burstein and D. Rupf er, Chem .-Biol . Interactions 3,

7.

M. W. Anders and G. J . Mannering, Molec . Pharmac . 2, 319 (1966)

8.

H. Remmer, J . Schenlanan, R. W . Eatabrook, H. Sesame, ~ . Gillete, S .Narasimhulu, D . Y. Cooper and 0 . Rosenthal, Molec. Pharmac. _2, 187 (1966) .

9.

T . Omura and R. Sato, J . biol . Chsm . 239, 2379 (1964) .

(1970) .

316 (1971) .

10 .

0 . H . Lowry, N . J . Rosebrough, A. L . Farr and R. J . Randall, J . biol . Chem . 193, 265 (1951) .

11 .

G. N . Wilkinson, Biochem. J. 80 , 324 (1961) .

12 .

H. Liaeweaver and D. Burk, J . Am . them . Soc. 56, 658 (1934) .

13 .

J . 0. Diagell, H. G. Wilcox and H. A . Klausner, Pharmacologiet 13 , 296 (1971) .

14 .A . Rubin, T . R . Tephly and G. J . Manneriag, Biochem. Pharmac . _13, 1007 (1964) . 15 .

A. M. Guatino, T. E . Gram, P . L . Gigon, F . E . Greene and J . R. Gillette, Molec. Pharmac . 5, 131 (1969) .

16 .

B . T . Ho, G . E . Fritchie, P . M. Kralik, L . F. Englert, W. M . Mclsaac and J . Idgnp~n-Heikkilg, J . Pharm . Pharmac . 22, 538 (1970) .

17 .

J . C.Garriott, L . J . ,King, R. B . Forney and F . W. Hughes, Life Sci . 6, 2119 (1967) .

18 .

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

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p9 -THC and Drug Metabolism

1215

19 .

S . Agurell, I . M. Nilsaon, A . Ohleson and F. Sandberg, Biochem. Pharmac . 18, 1195 (1969) .

20 .

L . Lemberger, S . D. Silberstein, J . Axelrod and I. Kopin, Science, N. Y . 170, 1320 (1970) .

21 .

H . A. Klausner and J . V . Diagell, Life Sci. lO c, 49 (1971) .