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Spectral interactions of marihuana constituents (cannabinoids) with rat liver microsomal monooxygenase system
Chem.-Biol, Interactions Elsevier Publishing Company, Amsterdam Printed in The Netherlands
201
SPECTRAL INTERACTIONS OF M A R I H U A N A C O N S T I T U E N T S (CANNABINOIDS) WITH RAT LIVER M I C R O S O M A L M O N O O X Y G E N A S E SYSTEM
DAVID KUPFER, INGELA JANSSON AND STEN ORRENIUS
Worcester FoundationJbr Experimental Biology, Shrewsbury, Mass. 01545 (U.S.A.) and Department o f Forensic Medicine, Karolinska lnstitutet, Stoekhohn (Sweden) (Received March 13th, 1972) (Revision received April 27, 1972)
SUMMARY
Spectral interactions of various cannabinoids with rat liver microsomes were studied. A 1-Tetrahydrocannabinol (A ~-THC), A6-THC, and cannabinol (CBN) produced type I spectral changes indicating the formation of an enzyme-substrate complex with cytochrome P-450. The binding affinities of these compounds for cytochrome P-450, as determined by their spectral dissociation constants (K~), were found to be 17.7/~M, 13.0/~M and 11.9/~M, respectively. 7-Hydroxy-A6-THC, which is a metabolite of A6-THC, did not produce spectral changes with rat liver microsomes, suggesting that it is not a substrate for further oxidation by cytochrome P-450. Evidence was obtained that A 1-THC, A6-THC and CBN, as well as hexobarbital, bind to the same cytochrome P-450 species. Finally, it is suggested that the lack of inhibitory effect on A ~-THC hydroxylation by hexobarbital, previously reported, may be ascribed to substantial differences in binding affinity for cytochrome P-450 by the two substances.
INTRODUCTION
Evidence has been presented that various cannabinoids (constituents of cannabis) (Fig. 1) are metabolized by mammalian liver preparations supplemented with N A D P H 1-4. The prime metabolic pathway in vitro appears to be hydroxylation at C7. Furthermore, it has been observed that the 7-hydroxy metabolites of the biologically active cannabinoids, A 1-THC* and A6-THC * are also active and it has been postu• The accepted numbering systems for cannabinoids: . l l - T H C ~ .19-THC (trans-_ll-tetrahydrocannabinol) ;/I 6-THC ~ /I a-THC (trans-/I 6-tetrahydrocannabinol). Abbreviations: CBN, cannabinol; Ks, spectral dissociation constant(s); THC, tetrahydrocannabinol.
Chem.-Biol. Interactions, 5 (I 972) 20l 206
202
CH3
D. KUPFER, h JANSSON, S. ORRENIUS
6
~
AI- THC
A6- THe
CH3
7CHaOH
CBN
7-OH- A~THC
Fig. I. Structures of various cannabinoids.
lated that these metabolites, rather than the parent compounds, may be the active species 5,6. Recently, it has been demonstrated that the 7-hydroxylation of A I - T H C is catalyzed by liver microsomes and that this hydroxylation is carried out by a monooxygenase system most probably involving cytochrome P-450 (refs. 7, 8). A variety of substrates of the hepatic monooxygenase system bind to cytochrome P-450 as shown from spectral changes elicited by addition of these substrates to liver microsomes 9. The spectral changes produced by these substrates have been classified into two major types: type I (rain. at about 420 and max. at about 385 nm) and type 11 (rain. at about 390 and max. at about 430 nm). Recently, we presented preliminary evidence that A ~-THC elicits the Type 1 spectral change with rat liver microsomes ~°. The studies of COHEN et al. ~ confirmed this finding and in addition these investigators determined the K~ of A ' - T H C with liver microsomes from controls and phenobarbital-treated rats. The present investigation examines spectral interactions of rat liver microsomes with several cannabinoids: A~-THC, /J6-THC, CBN and 7-hydroxy-A6-THC (a metabolite of A%THC). MATERIALS AND METHODS
Male Sprague-Dawley rats (200 g) were used. The animals were killed by decapitation. Livers were immediately excised and microsomes were isolated as described by ERr~STERet a1.12. The microsomal pellets were washed with ice-cold 0.15 M KCI and resuspended in 0.25 M sucrose (10 mg protein per ml). Spectral changes. Each cuvette contained in 3 ml, microsomal suspension (5 mg protein, unless otherwise indicated) and 0.1 M Tris-HCl (pH 7.5) and 0.15 M KCI. The respective substance (for concentrations, see text) was added in ethanol to the sample cuvette and an equivalent amount of the ethanol was added to the reference cuvette. For determination of K~, 2-#1 aliquots of the ethanolic solution of the respective substance were added in succession to the sample cuvette, not to exceed 16kd. Measurements were made in an Aminco-Chance Duochrometer. Chem.-Biol. Interactions, 5 (1972) 201-206
203
INTERACTIONS OF CANNABINOIDS WITH RAT LIVER MICROSOMES
A6 - T E T R A H Y D R O C A N N A B I N O L
BINDING
AI -TETRAHYDROCANNABINOL
O.O2-
BINDING
0.02.
w
0.01
0.0
z
== o
"~-0.0 I
-0.01
-0.0
-0.0~
3"f5
350
400 WAVELENGTH
425
450
350
375
(rim)
400 WAVELENGTH
425 (rim)
Fig. 2. Titration of the type I spectral change of~16-THC with rat liver microsomes. The procedure is described in MATERIALSAND METHODS. Concentrations of. 16-THC were: (1) 8.4/zM; (2) 16.8/tM; (3) 25.2 F M ; (4) 33.6 # M ; and (5) 50.4/~M. The concentration of cytochrome P-450 was 1.2 nmoles/ml. Fig. 3. Titration of the type 1 spectral change of, I ~-THC with rat liver microsomes. The procedure is described in MATERIALSAND METHODS. Concentrations of~I1-THC were (1) 6.7 tzM; (2) 13.3/~M; (3) 20.0 # M ; (4) 26.6/~M; (5) 40.0/~M; and (6) 46,6 tbM. The concentration of cytochrome P-450 was 1.2 nmoles/ml. TABLE 1 SPECTRAL INTERACTIONSOF CANNABINOIDSWITH RAT LIVER MICROSOMES
Substrate
Ks a (I~M )
~1 ~-TH C
17.7
16-THC CBN
13.0 11.9
a K~ was determined graphically (see Figs.) from the X intercept of a double inverse plot of.1 absorbance at 420 500 nm vs. concentration. RESULTS
The addition of ~I6-THC to rat liver microsomes elicited a type 1 spectrum (2 . . . . 390; )'rain. 421 rim) (Fig. 2). Similarly, AI-THC (Fig. 3) and CBN demonstrated identical type I spectra. The Ks for A6-THC, A1-THC and CBN were determined graphically as exemplified in Figs. 4 and 5, to be 13.0 #M, 17.7 # M and 11.9/~M, respectively (Table I). On the other hand, 7-hydroxy-A6-THC (7-OH-A6-THC)didnot demonstrate spectral interaction in several experiments (Table l l). Furthermore, the addition of 7-OH-A6-THC (25/~M) did not interfere with the spectral interaction of A6-THC (Table II). The addition of CBN at 25/~M or at saturating concentrations* interfered with * Saturating concentrations are defined as the concentrations above which there is no further increase in spectral change.
Chem.-Biol. Interactions, 5 (1972) 201-206
450
204
D. KUPFER, I. JANSSON, S. ORRENIUS I
L~ ABSORBANCE
80]
t
6O
5O
- 80
-60
- 40
- 20
0
20
I
40
60
s (m/q)
Fig. 4. Determination of the spectral dissociation constant (K~) of 16-THC with rat liver microsomes. The change in absorbance at 420 nm 500 nm occurring upon increase in ,16-THC concentration was recorded and its reciprocal was plotted against the reciprocal of substrate concentration. The concentration of cytochrome P-450 was 1.2 nmoles/ml. I
A ABSORBANCE 80 70 60 50
40
-60
-40
-20
0
20
40
60
80
I
S (m/q)
Fig. 5. Determination o f the spectral dissociation constant (Ks) o f I I - T H C with rat liver microsomes. The change in absorbance at 420 rim-500 nm occurring upon increase in 1 1 - T H C concentration was recorded and its reciprocal was plotted against the reciprocal of substrate concentration. The concentration of cytochrome P-450 was 1.2 nmoies/ml.
the spectral interaction of A6-THC; similarly, saturating concentrations of hexobarbital interfered with the spectral interaction of A 6-THC. Conversely, A 6-THC at saturating concentrations, entirely obliterated the spectral interaction of CBN (Table I1). DISCUSSION
The observation that cannabinoids are hydroxylated by liver preparations and that this oxidation is most probably catalyzed by the monooxygenase system involving cytochrome P-450 suggested that these compounds bind to cytochrome P-450 and thus may elicit spectral interactions. It is of interest that the three cannabinoids tested Chem.-Biol. Interactions, 5 (1972) 201 206
INTERACTIONS OF CANNABINOIDS WITH RAT LIVER MICROSOMES
205
T A B L E 11 T H E EFFECT OF V A R I O U S C A N N A B I N O I D S A N D O T H E R C O M P O U N D S ON T H E S P E C T R A L I N T E R A C T I O N S OF C A N N A B I N O I D S W I T H R A T LIVER MICROSOMES
Substrate
Mod([ier
Aamax. (. IA 420 500 ran)
16-THC • 16-THC 16-THC 16-THC CBN CBN 7-OH-16-THC
-CBN (saturation) h e x o b a r b i t a l (saturation) 7-OH- 16-THC (25 ~tM) -1 6 - T H C (saturation)
I1 • 10 - 3 0 3.5. 10- 3 I 1 • 10 - 3 8 ' 10 - 3 0 0
a Amax" is o b t a i n e d from the Y intercept of a doubl e inverse plot of. IA42o_5o onm VS. substrate concentration. Both sample and reference cuvettes c ont a i ne d mi c ros oma l suspension (3.0 mg c o n t a i n i n g 2.1 nmoles of c y t o c h r o m e P-450). The c o m p o u n d here called s ubs t ra t e was added successively in 2-/~1 aliquots to the sample cuvette and the Amax. obtained. In experiments where modifier was used, the modifier was added to both the reference and s a mpl e cuvette and the same procedure with substrate repeated. The c a n n a b i n o i d s were ad de d in e t h a n o l to a final c o n c e n t r a t i o n of 0.15 mM, whereas h e x o b a r b i t a l was added as an aqueous solution.
(A I-THC, A6-THC and CBN) all exhibited the type I spectral change, which is believed to be due to the formation of the enzyme-substrate complex 13, and that their K~ values were similar, being 17.7, 13.0 and l l .9/~Mrespectively. A similar K~ for A I-THC, 18.5 #M, was reported by COHEN e t a l . l l . The observation that the various cannabinoids interfere with the spectral interaction of each other and that hexobarbital interferes with the spectral interaction of A6-THC (cJ] Table II), suggests that they bind to the same cytochrome P-450. In our metabolic studies, we observed that whereas SKF-525A was a potent inhibitor of the 7-hydroxylation of A I-THC, hexobarbital at 3-fold the concentration of A ~-THC, caused little or no inhibition of the hydroxylation of 3 I-THCS. The present observation that the affinity with which A I-THC binds to cytochrome P-450 (K~ = 17.7/zM) is considerably higher than that reported for hexobarbital (K~ = 80/zM) 9, provides an explanation for the lack of inhibition of hydroxylation of A I-THC by hexobarbital. Similarly, the difference in affinity of the two substances for the enzyme is also supported by metabolic studies. Accordingly, a preliminary estimate of the K,, value of A I-THC with rat liver microsomes was found to be 28 ~ M (KUPFER AND BURSTEIN, unpublished observation) which is considerably lower than the two reported values for hexobarbital; 1.0 • 10 -4 M (ref. 9) and 1.2 • 10 -3 M (ref. 14). WALL e t al. i s observed that 6,7-dihydroxy-A6-THC is one of the metabolic products of incubation of A6-THC with rat liver preparations. Since 7-hydroxylation appears to be the major product of hydroxylation, we assumed that 6-hydroxylation occurs subsequent to the 7-hydroxylation and that 7-OH-A6-THC is a substrate for the 6-hydroxylation. The lack of spectral interaction of 7-OH-A6-THC with microsomes, however, does not support this hypothesis, but suggests that this compound does not interact with cytochrome P-450 and that other microsomal enzymes may be involved in its further metabolism. Whether the same holds true for 7-OH-A I-THC, which is a metabolite of the major constituent of marihuana, A 1-THC, remains to be Chem.-Biol. Interactions, 5 (1972)201-206
206
D. KUPFER, 1. JANSSON, S. ORRENIUS
elucidated, since the difficulty in obtaining this compound hitherto precluded such studies. ACKNOWLEDGMENT
This study was supported by a grant from the Swedish Medical Research Council (Proj. No. 13K-247-1) and by U. S. Public Health Service Grant FR-05528. The authors wish to thank Dr. R. MECHOULAMfor supplying samples of the cannabinoids. Thanks are extended to Dr. S. BURSTHN for assaying the various cannabinoid samples and for helpful discussions. REFERENCES I 2
3
4 5 6
7 8 9 10 11 12 13 14 15
I . M . NILSSON, S. AGURELL, J. L. G. NILSSON, A. OHLSSON, F. SANDBERG AND M. WAHLQVIST, 1 l_Tetrahydrocannabinol : Structure of a major metabolite, Science, 168 (1970) 1228. R . L . FOLTZ, A. F. FENTIMAN, E. G. LEIGHTY, J. L. WALTER, H. R. DREWES, W. E. SCHWARTZ, T. F. PAGE AND E. B. TRUITT, Metabolism of trans- 18-tetrahydrocannabinol : Identification and synthesis, Science, 168 (1970) 844. M.E. WALL, D. R. BmNE, G. A. BRINE, C. G. PITT, R. 1. FREUDENTHALAND H. DIX CHRISTENSEN, Isolation, structure and biological activity of several metabolites of 19-tetrahydrocannabinol, J. Am. Chem. Soc., 92 (1970) 3466 3468. M. WIDMAN, 1. M. NILSSON, J. L. G. NILSSON, S. AGURELL AND K. LEANDEr, Metabolism of cannabis, Life Sci., 10 (1971) 157-162. Z. BEN-ZVl, R. MECHOUt.AM, S. BURSTEIN, Identification through synthesis of an active I ~-tG~tetrahydrocannabinol metabolite, J. Am. Chem. Sot., 92 (1970) 3468-3469. H . D . CHRISTENSEN, R. I. FREUDENTHAL, J. T. GIDLEY, R. ROSENFELD, G. BOEGLI, L. TESTINO, D. R. BRINE, C. G. PITT AND M. E. WALL, Activity of.1 s- and 1%tetrahydrocannabinol and related c o m p o u n d s in the mouse, Science, 172 (1971) 165 167. S . H . BURSTEIN AND D. KUPFER, Hydroxylation of trans-.l l-tetrahydrocannabinol by a hepatic microsomal monooxygenase, Chem.-Biol. Interactions, 3 (1971) 316. S. H. BURSTEIN AND D. KUPFER, Hydroxylation of trans-|l-tetrahydrocannabinol by hepatic microsomal oxygenase, Ann. N.Y. Acad. Sci., 191 (1971) 61 67. J. B. SCHENKMAN, H. REMMER AND R. W. ESTABROOK, Spectral studies of drug interaction with hepatic microsomal cytochrome, Mol. Pharmacol., 3 (1967) 113 123. S . H . BURSTEINAND O. KUPFER, Hydroxylation of trans-~| l_tetrahydrocannabinol by a hepatic microsomal monooxygenase, Chem.-Biol. Interactions, 3 (1971) 316. G . M . COHEN, O. W. PETERSON AND G. J. MANNERING, Interactions o f 19-tetrahydrocannabinol with the hepatic microsomal drug metabolizing system, 1., Life Sci., 10 ( 197 I) 1207 1215. L. ERNSTER, P. SIEKEVITZAND G. E. PALADE, Enzyme-structure relationships in the endoplasmic reticulum of rat liver, J. Cell. Biol., 15 (1962) 541-562. J . B . SCHENKMAN, Studies on the nature of Type 1 and Type II spectral changes in liver microsomes, Biochemistry, 9 (1970) 2081-209 I. A. RUBIN, T. R. TEPHLY AND G. J. MANNERING, Kinetics of drug metabolism by hepatic microsomes, Biochem. Pharmaeol., 13 (1964) 1007-1016. M . E . WALL, The in vitro and in vivo metabolism of tetrahydrocannabinol (THC), Anti. N. Y. Acad. Sei., 191 (1971) 23-39.
Chem.-Biol. Interactions, 5 (1972) 201-206
201
SPECTRAL INTERACTIONS OF M A R I H U A N A C O N S T I T U E N T S (CANNABINOIDS) WITH RAT LIVER M I C R O S O M A L M O N O O X Y G E N A S E SYSTEM
DAVID KUPFER, INGELA JANSSON AND STEN ORRENIUS
Worcester FoundationJbr Experimental Biology, Shrewsbury, Mass. 01545 (U.S.A.) and Department o f Forensic Medicine, Karolinska lnstitutet, Stoekhohn (Sweden) (Received March 13th, 1972) (Revision received April 27, 1972)
SUMMARY
Spectral interactions of various cannabinoids with rat liver microsomes were studied. A 1-Tetrahydrocannabinol (A ~-THC), A6-THC, and cannabinol (CBN) produced type I spectral changes indicating the formation of an enzyme-substrate complex with cytochrome P-450. The binding affinities of these compounds for cytochrome P-450, as determined by their spectral dissociation constants (K~), were found to be 17.7/~M, 13.0/~M and 11.9/~M, respectively. 7-Hydroxy-A6-THC, which is a metabolite of A6-THC, did not produce spectral changes with rat liver microsomes, suggesting that it is not a substrate for further oxidation by cytochrome P-450. Evidence was obtained that A 1-THC, A6-THC and CBN, as well as hexobarbital, bind to the same cytochrome P-450 species. Finally, it is suggested that the lack of inhibitory effect on A ~-THC hydroxylation by hexobarbital, previously reported, may be ascribed to substantial differences in binding affinity for cytochrome P-450 by the two substances.
INTRODUCTION
Evidence has been presented that various cannabinoids (constituents of cannabis) (Fig. 1) are metabolized by mammalian liver preparations supplemented with N A D P H 1-4. The prime metabolic pathway in vitro appears to be hydroxylation at C7. Furthermore, it has been observed that the 7-hydroxy metabolites of the biologically active cannabinoids, A 1-THC* and A6-THC * are also active and it has been postu• The accepted numbering systems for cannabinoids: . l l - T H C ~ .19-THC (trans-_ll-tetrahydrocannabinol) ;/I 6-THC ~ /I a-THC (trans-/I 6-tetrahydrocannabinol). Abbreviations: CBN, cannabinol; Ks, spectral dissociation constant(s); THC, tetrahydrocannabinol.
Chem.-Biol. Interactions, 5 (I 972) 20l 206
202
CH3
D. KUPFER, h JANSSON, S. ORRENIUS
6
~
AI- THC
A6- THe
CH3
7CHaOH
CBN
7-OH- A~THC
Fig. I. Structures of various cannabinoids.
lated that these metabolites, rather than the parent compounds, may be the active species 5,6. Recently, it has been demonstrated that the 7-hydroxylation of A I - T H C is catalyzed by liver microsomes and that this hydroxylation is carried out by a monooxygenase system most probably involving cytochrome P-450 (refs. 7, 8). A variety of substrates of the hepatic monooxygenase system bind to cytochrome P-450 as shown from spectral changes elicited by addition of these substrates to liver microsomes 9. The spectral changes produced by these substrates have been classified into two major types: type I (rain. at about 420 and max. at about 385 nm) and type 11 (rain. at about 390 and max. at about 430 nm). Recently, we presented preliminary evidence that A ~-THC elicits the Type 1 spectral change with rat liver microsomes ~°. The studies of COHEN et al. ~ confirmed this finding and in addition these investigators determined the K~ of A ' - T H C with liver microsomes from controls and phenobarbital-treated rats. The present investigation examines spectral interactions of rat liver microsomes with several cannabinoids: A~-THC, /J6-THC, CBN and 7-hydroxy-A6-THC (a metabolite of A%THC). MATERIALS AND METHODS
Male Sprague-Dawley rats (200 g) were used. The animals were killed by decapitation. Livers were immediately excised and microsomes were isolated as described by ERr~STERet a1.12. The microsomal pellets were washed with ice-cold 0.15 M KCI and resuspended in 0.25 M sucrose (10 mg protein per ml). Spectral changes. Each cuvette contained in 3 ml, microsomal suspension (5 mg protein, unless otherwise indicated) and 0.1 M Tris-HCl (pH 7.5) and 0.15 M KCI. The respective substance (for concentrations, see text) was added in ethanol to the sample cuvette and an equivalent amount of the ethanol was added to the reference cuvette. For determination of K~, 2-#1 aliquots of the ethanolic solution of the respective substance were added in succession to the sample cuvette, not to exceed 16kd. Measurements were made in an Aminco-Chance Duochrometer. Chem.-Biol. Interactions, 5 (1972) 201-206
203
INTERACTIONS OF CANNABINOIDS WITH RAT LIVER MICROSOMES
A6 - T E T R A H Y D R O C A N N A B I N O L
BINDING
AI -TETRAHYDROCANNABINOL
O.O2-
BINDING
0.02.
w
0.01
0.0
z
== o
"~-0.0 I
-0.01
-0.0
-0.0~
3"f5
350
400 WAVELENGTH
425
450
350
375
(rim)
400 WAVELENGTH
425 (rim)
Fig. 2. Titration of the type I spectral change of~16-THC with rat liver microsomes. The procedure is described in MATERIALSAND METHODS. Concentrations of. 16-THC were: (1) 8.4/zM; (2) 16.8/tM; (3) 25.2 F M ; (4) 33.6 # M ; and (5) 50.4/~M. The concentration of cytochrome P-450 was 1.2 nmoles/ml. Fig. 3. Titration of the type 1 spectral change of, I ~-THC with rat liver microsomes. The procedure is described in MATERIALSAND METHODS. Concentrations of~I1-THC were (1) 6.7 tzM; (2) 13.3/~M; (3) 20.0 # M ; (4) 26.6/~M; (5) 40.0/~M; and (6) 46,6 tbM. The concentration of cytochrome P-450 was 1.2 nmoles/ml. TABLE 1 SPECTRAL INTERACTIONSOF CANNABINOIDSWITH RAT LIVER MICROSOMES
Substrate
Ks a (I~M )
~1 ~-TH C
17.7
16-THC CBN
13.0 11.9
a K~ was determined graphically (see Figs.) from the X intercept of a double inverse plot of.1 absorbance at 420 500 nm vs. concentration. RESULTS
The addition of ~I6-THC to rat liver microsomes elicited a type 1 spectrum (2 . . . . 390; )'rain. 421 rim) (Fig. 2). Similarly, AI-THC (Fig. 3) and CBN demonstrated identical type I spectra. The Ks for A6-THC, A1-THC and CBN were determined graphically as exemplified in Figs. 4 and 5, to be 13.0 #M, 17.7 # M and 11.9/~M, respectively (Table I). On the other hand, 7-hydroxy-A6-THC (7-OH-A6-THC)didnot demonstrate spectral interaction in several experiments (Table l l). Furthermore, the addition of 7-OH-A6-THC (25/~M) did not interfere with the spectral interaction of A6-THC (Table II). The addition of CBN at 25/~M or at saturating concentrations* interfered with * Saturating concentrations are defined as the concentrations above which there is no further increase in spectral change.
Chem.-Biol. Interactions, 5 (1972) 201-206
450
204
D. KUPFER, I. JANSSON, S. ORRENIUS I
L~ ABSORBANCE
80]
t
6O
5O
- 80
-60
- 40
- 20
0
20
I
40
60
s (m/q)
Fig. 4. Determination of the spectral dissociation constant (K~) of 16-THC with rat liver microsomes. The change in absorbance at 420 nm 500 nm occurring upon increase in ,16-THC concentration was recorded and its reciprocal was plotted against the reciprocal of substrate concentration. The concentration of cytochrome P-450 was 1.2 nmoles/ml. I
A ABSORBANCE 80 70 60 50
40
-60
-40
-20
0
20
40
60
80
I
S (m/q)
Fig. 5. Determination o f the spectral dissociation constant (Ks) o f I I - T H C with rat liver microsomes. The change in absorbance at 420 rim-500 nm occurring upon increase in 1 1 - T H C concentration was recorded and its reciprocal was plotted against the reciprocal of substrate concentration. The concentration of cytochrome P-450 was 1.2 nmoies/ml.
the spectral interaction of A6-THC; similarly, saturating concentrations of hexobarbital interfered with the spectral interaction of A 6-THC. Conversely, A 6-THC at saturating concentrations, entirely obliterated the spectral interaction of CBN (Table I1). DISCUSSION
The observation that cannabinoids are hydroxylated by liver preparations and that this oxidation is most probably catalyzed by the monooxygenase system involving cytochrome P-450 suggested that these compounds bind to cytochrome P-450 and thus may elicit spectral interactions. It is of interest that the three cannabinoids tested Chem.-Biol. Interactions, 5 (1972) 201 206
INTERACTIONS OF CANNABINOIDS WITH RAT LIVER MICROSOMES
205
T A B L E 11 T H E EFFECT OF V A R I O U S C A N N A B I N O I D S A N D O T H E R C O M P O U N D S ON T H E S P E C T R A L I N T E R A C T I O N S OF C A N N A B I N O I D S W I T H R A T LIVER MICROSOMES
Substrate
Mod([ier
Aamax. (. IA 420 500 ran)
16-THC • 16-THC 16-THC 16-THC CBN CBN 7-OH-16-THC
-CBN (saturation) h e x o b a r b i t a l (saturation) 7-OH- 16-THC (25 ~tM) -1 6 - T H C (saturation)
I1 • 10 - 3 0 3.5. 10- 3 I 1 • 10 - 3 8 ' 10 - 3 0 0
a Amax" is o b t a i n e d from the Y intercept of a doubl e inverse plot of. IA42o_5o onm VS. substrate concentration. Both sample and reference cuvettes c ont a i ne d mi c ros oma l suspension (3.0 mg c o n t a i n i n g 2.1 nmoles of c y t o c h r o m e P-450). The c o m p o u n d here called s ubs t ra t e was added successively in 2-/~1 aliquots to the sample cuvette and the Amax. obtained. In experiments where modifier was used, the modifier was added to both the reference and s a mpl e cuvette and the same procedure with substrate repeated. The c a n n a b i n o i d s were ad de d in e t h a n o l to a final c o n c e n t r a t i o n of 0.15 mM, whereas h e x o b a r b i t a l was added as an aqueous solution.
(A I-THC, A6-THC and CBN) all exhibited the type I spectral change, which is believed to be due to the formation of the enzyme-substrate complex 13, and that their K~ values were similar, being 17.7, 13.0 and l l .9/~Mrespectively. A similar K~ for A I-THC, 18.5 #M, was reported by COHEN e t a l . l l . The observation that the various cannabinoids interfere with the spectral interaction of each other and that hexobarbital interferes with the spectral interaction of A6-THC (cJ] Table II), suggests that they bind to the same cytochrome P-450. In our metabolic studies, we observed that whereas SKF-525A was a potent inhibitor of the 7-hydroxylation of A I-THC, hexobarbital at 3-fold the concentration of A ~-THC, caused little or no inhibition of the hydroxylation of 3 I-THCS. The present observation that the affinity with which A I-THC binds to cytochrome P-450 (K~ = 17.7/zM) is considerably higher than that reported for hexobarbital (K~ = 80/zM) 9, provides an explanation for the lack of inhibition of hydroxylation of A I-THC by hexobarbital. Similarly, the difference in affinity of the two substances for the enzyme is also supported by metabolic studies. Accordingly, a preliminary estimate of the K,, value of A I-THC with rat liver microsomes was found to be 28 ~ M (KUPFER AND BURSTEIN, unpublished observation) which is considerably lower than the two reported values for hexobarbital; 1.0 • 10 -4 M (ref. 9) and 1.2 • 10 -3 M (ref. 14). WALL e t al. i s observed that 6,7-dihydroxy-A6-THC is one of the metabolic products of incubation of A6-THC with rat liver preparations. Since 7-hydroxylation appears to be the major product of hydroxylation, we assumed that 6-hydroxylation occurs subsequent to the 7-hydroxylation and that 7-OH-A6-THC is a substrate for the 6-hydroxylation. The lack of spectral interaction of 7-OH-A6-THC with microsomes, however, does not support this hypothesis, but suggests that this compound does not interact with cytochrome P-450 and that other microsomal enzymes may be involved in its further metabolism. Whether the same holds true for 7-OH-A I-THC, which is a metabolite of the major constituent of marihuana, A 1-THC, remains to be Chem.-Biol. Interactions, 5 (1972)201-206
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D. KUPFER, 1. JANSSON, S. ORRENIUS
elucidated, since the difficulty in obtaining this compound hitherto precluded such studies. ACKNOWLEDGMENT
This study was supported by a grant from the Swedish Medical Research Council (Proj. No. 13K-247-1) and by U. S. Public Health Service Grant FR-05528. The authors wish to thank Dr. R. MECHOULAMfor supplying samples of the cannabinoids. Thanks are extended to Dr. S. BURSTHN for assaying the various cannabinoid samples and for helpful discussions. REFERENCES I 2
3
4 5 6
7 8 9 10 11 12 13 14 15
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