Cannabidiol, a major phytocannabinoid, as a potent atypical inhibitor for CYP2D6

Article abstract of DOI:10.1124/dmd.111.041384

Δ⁹-Tetrahydrocannabinol, cannabidiol (CBD), and cannabinol are the three major cannabinoids contained in marijuana, which are devoid of nitrogen atoms in their structures. In this study, we investigated the inhibitory effects of the major phytocannabinoids on the catalytic activity of human CYP2D6. These major cannabinoids inhibited the 3-[2-(N,N-diethyl-N- methylammonium)ethyl]-7-methoxy-4-methylcoumarin (AMMC) and dextromethorphan O-demethylase activities of recombinant CYP2D6 and pooled human liver microsomes in a concentration-dependent manner (IC₅₀ =4.01-24.9 μM), indicating the strongest inhibitory potency of CBD. However, these cannabinoids showed no or weak metabolism-dependent inhibition. CBD competitively inhibited the CYP2D6 activities with the apparent K_i values of 1.16 to 2.69 μM. To clarify the structural requirement for CBD-mediated CYP2D6 inhibition, effects of CBD-related compounds on the AMMC O-demethylase activity of recombinant CYP2D6 were examined. Olivetol (IC₅₀ =7.21 μM) inhibited CYP2D6 activity as potently as CBD did (IC₅₀ =6.52 μM), whereas d-limonene did not show any inhibitory effect. Pentylbenzene failed to inhibit CYP2D6 activity. Furthermore, neither monomethyl nor dimethyl ethers of CBD inhibited the activity. Cannabidivarin having a propyl side chain inhibited CYP2D6 activity; its inhibitory effect (IC₅₀ = 10.2 μM) was less potent than that of CBD. On the other hand, orcinol and resorcinol showed lack of inhibition. The inhibitory effect of CBD on CYP2D6 activity was more potent than those of 16 compounds without nitrogen atoms tested, such as progesterone. These results indicated that CBD caused potent direct CYP2D6 inhibition, in which two phenolic hydroxyl groups and the pentyl side chain of CBD may play important roles. Copyright

Full text of DOI:10.1124/dmd.111.041384

0
090-9556/11/3911-2049–2056$25.00
DRUG METABOLISM AND DISPOSITION
Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics
DMD 39:2049–2056, 2011
Vol. 39, No. 11
41384/3723377
Printed in U.S.A.
Cannabidiol, a Major Phytocannabinoid, As a Potent Atypical
Inhibitor for CYP2D6
Satoshi Yamaori, Yasuka Okamoto, Ikuo Yamamoto, and Kazuhito Watanabe
Department of Hygienic Chemistry, Faculty of Pharmaceutical Sciences, Hokuriku University, Kanazawa, Japan (S.Y., Y.O.,
K.W.); Department of Hygienic Chemistry, School of Pharmaceutical Sciences, Kyushu University of Health and Welfare,
Nobeoka, Japan (I.Y.); and Organization for Frontier Research in Preventive Pharmaceutical Sciences, Hokuriku University,
Kanazawa, Japan (K.W.)
Received June 26, 2011; accepted August 5, 2011
ABSTRACT:
⌬9
the three major cannabinoids contained in marijuana, which are 7.21 ␮M) inhibited CYP2D6 activity as potently as CBD did (IC50
-Tetrahydrocannabinol, cannabidiol (CBD), and cannabinol are activity of recombinant CYP2D6 were examined. Olivetol (IC50
؍
؍
devoid of nitrogen atoms in their structures. In this study, we 6.52 ␮M), whereas d-limonene did not show any inhibitory effect.
investigated the inhibitory effects of the major phytocannabinoids Pentylbenzene failed to inhibit CYP2D6 activity. Furthermore, nei-
on the catalytic activity of human CYP2D6. These major cannabi- ther monomethyl nor dimethyl ethers of CBD inhibited the activity.
noids inhibited the 3-[2-(N,N-diethyl-N-methylammonium)ethyl]-7- Cannabidivarin having a propyl side chain inhibited CYP2D6 activ-
methoxy-4-methylcoumarin (AMMC) and dextromethorphan O-de- ity; its inhibitory effect (IC50 ؍ 10.2 ␮M) was less potent than that of
methylase activities of recombinant CYP2D6 and pooled human CBD. On the other hand, orcinol and resorcinol showed lack of
liver microsomes in a concentration-dependent manner (IC50
؍
inhibition. The inhibitory effect of CBD on CYP2D6 activity was
4.01–24.9 ␮M), indicating the strongest inhibitory potency of CBD. more potent than those of 16 compounds without nitrogen atoms
However, these cannabinoids showed no or weak metabolism- tested, such as progesterone. These results indicated that CBD
dependent inhibition. CBD competitively inhibited the CYP2D6 ac- caused potent direct CYP2D6 inhibition, in which two phenolic
tivities with the apparent K values of 1.16 to 2.69 ␮M. To clarify the hydroxyl groups and the pentyl side chain of CBD may play impor-
i
structural requirement for CBD-mediated CYP2D6 inhibition, ef- tant roles.
fects of CBD-related compounds on the AMMC O-demethylase
9
9
Introduction
Major cannabinoids in marijuana are ⌬ -tetrahydrocannabinol (⌬ -
THC), cannabidiol (CBD), and cannabinol (CBN) (Fig. 1). The aver-
age contents of ⌬ -THC, CBD, and CBN in dried plant preparations
of marijuana confiscated from 1993 to 2008 in the United States are
Marijuana is the most widely abused drug in the world. Marijuana use
is a growing public health concern because of dependence, association
with polysubstance use, potential harmful effects on brain function and
several peripheral systems, and various health consequences (Hall and
Degenhardt, 2009). Epidemiological studies reveal that many abusers of
marijuana concurrently use other recreational drugs, such as metham-
phetamine and 3,4-methylenedioxymethamphetamine (MDMA) (Wu et
al., 2006; Grov et al., 2009). These facts prompted us to investigate how
marijuana and coadministered drugs interact one another.
9
4
.5, 0.4, and 0.3%, respectively, although these contents vary widely
(
Mehmedic et al., 2010). In marijuana resin, which is commonly
9
referred to as hashish, the mean contents of ⌬ -THC, CBD, and CBN
are 14.1, 2.5, and 1.9%, respectively (Mehmedic et al., 2010). ⌬ -
9
THC is a primary psychoactive component of marijuana, possessing
various pharmacological effects such as catalepsy, hypothermia, anti-
inflammation, and antinociception (Pertwee, 2008). CBD is not psy-
choactive but has several pharmacological effects including drug-
induced sleep prolongation and antiepileptic, anxiolytic, and
antiemetic actions (Mechoulam et al., 2002). CBN is thought to exert
Marijuana leaves contain at least 70 cannabinoids, which consist of
carbon, hydrogen, and oxygen atoms (ElSohly and Slade, 2005).
This work was supported in part by the Ministry of Education, Culture, Sports,
Science, and Technology of Japan [Grant-in-Aid for Scientific Research (C)
minimal pharmacological effects in the central nervous system.
9
⌬
-THC, CBD, and CBN have been reported to be extensively
20590127 and Grant-in-Aid for Young Scientists (B) 23790159]; and the Aca-
metabolized by human liver microsomes (HLMs) (Bornheim et al.,
992; Watanabe et al., 1995, 2006, 2007; Jiang et al., 2011). It has
demic Frontier Project for Private Universities from the Ministry of Education,
Culture, Sports, Science, and Technology of Japan (2005–2009).
Article, publication date, and citation information can be found at
http://dmd.aspetjournals.org.
1
been demonstrated that CYP2C and CYP3A enzymes are predomi-
nantly involved in the primary metabolism of the phytocannabinoids
9
doi:10.1124/dmd.111.041384.
in the liver microsomes. ⌬ -THC and CBN are mainly oxidized at the
ABBREVIATIONS: MDMA, 3,4-methylenedioxymethamphetamine; AMMC, 3-[2-(N,N-diethyl-N-methylammonium)ethyl]-7-methoxy-4-methyl-
coumarin; AHMC, 3-[2-(N,N-diethylamino)ethyl]-7-hydroxy-4-methylcoumarin; CBD, cannabidiol; P450, cytochrome P450; CBDM, CBD-2Ј-
monomethyl ether; CBDD, CBD-2Ј,6Ј-dimethyl ether; CBDV, cannabidivarin; CBN, cannabinol; DMSO, dimethyl sulfoxide; HLMs, human liver
9
9
microsomes; PAH, polycyclic aromatic hydrocarbon; ⌬ -THC, ⌬ -tetrahydrocannabinol; AUC, area under the curve.
2049

2
050
YAMAORI ET AL.
FIG. 1. Structures of three major phytocannabinoids.
1
1-position by CYP2C9 and at the 8-position by CYP3A4 (Bornheim naphthalene, and pyrene were purchased from Sigma-Aldrich (St. Louis, MO).
Orcinol and fluorene were obtained from Tokyo Chemical Industry Co., Ltd.
et al., 1992; Watanabe et al., 1995, 2007), whereas CBD is primarily
oxidized at the 7-position by CYP2C19 and at the 6-position by
CYP3A4 (Jiang et al., 2011). On the other hand, these major phyto-
cannabinoids have been shown to inhibit cytochrome P450 (P450)-
mediated drug oxidations (Jaeger et al., 1996; Yamaori et al., 2010,
(
Tokyo, Japan). NADP, glucose 6-phosphate, and glucose 6-phosphate dehy-
drogenase were obtained from Oriental Yeast Co. Ltd. (Tokyo, Japan). Other
chemicals and solvents used were of the highest quality that was commercially
available.
Enzyme Assays. AMMC O-demethylase activity was determined by using
6-well microtiter plates according to the instruction of BD Gentest. In brief,
an incubation mixture consisted of recombinant CYP2D6 (2 pmol), AMMC,
2
011a,b).
9
Some recreational drugs that are likely to be used concurrently with
marijuana are substrates for P450 enzymes. In particular, stimulants an NADPH-generating system (8.2 ␮M NADP, 0.41 mM glucose 6-phosphate,
e.g., methamphetamine and MDMA) and designer drugs including 0.41 mM magnesium chloride, and 1 unit/ml glucose 6-phosphate dehydroge-
-methoxy-N,N-diisopropyltryptamine are known to be mainly me- nase), and 100 mM potassium phosphate buffer (pH 7.4) in a final volume of
(
5
tabolized by CYP2D6 (Zhou et al., 2009). CYP2D6 is expressed 200 ␮l. After prewarming at 37°C for 5 min, reactions were initiated by the
predominantly in the liver, although the expression level is low (ϳ2%
of total P450 content) (Shimada et al., 1994). CYP2D6 is responsible
for the metabolism of a wide variety of drugs clinically used, such as
dextromethorphan, debrisoquine, bufuralol, promethazine, and co-
deine (Zhou et al., 2009). These findings indicate that CYP2D6 is one
of the most important P450 enzymes involved in hepatic drug metab-
olism. In addition, these findings imply the possibility of frequent
addition of the NADPH-generating system. Fluorescence derived from forma-
tion of an O-demethylated metabolite of AMMC was recorded every 3 min for
3
0 min using FLUOstar OPTIMA (BMG Labtech, Offenburg, Germany) with
excitation and emission filters at 390 and 460 nm, respectively. AHMC was
used as an external standard for quantification of the O-demethylated
metabolite of AMMC, i.e., 3-[2-(N,N-diethyl-N-methylammonium)ethyl]-
7
-hydroxy-4-methylcoumarin.
The activity of dextromethorphan O-demethylation was measured as re-
occurrence of CYP2D6-targeted drug interactions. Thus, it is toxico- ported previously (Yu and Haining, 2001) with minor modifications. Recom-
logically important to clarify possible interactions of marijuana or its binant CYP2D6 (0.25 pmol) and HLMs (12.5 ␮g of protein) were used as
components, including cannabinoids, with CYP2D6 substrates. How- enzyme sources. An incubation mixture consisted of an enzyme source, dex-
tromethorphan, an NADPH-generating system (500 ␮M NADP, 10 mM glu-
ever, the inhibitory effects of the cannabinoids on CYP2D6 activity
cose 6-phosphate, 10 mM magnesium chloride, and 1 unit/ml glucose 6-phos-
remain to be elucidated.
phate dehydrogenase), and 100 mM potassium phosphate buffer (pH 7.4) in a
In this study, we examined the potency and mechanism of CYP2D6
9
final volume of 200 ␮l. Incubations were performed at 37°C for 10 min and
were terminated by adding 10 ␮l of 70% (w/v) perchloric acid. After removal
of protein by centrifugation, 50 ␮l of the supernatant was subjected to a
high-performance liquid chromatography (D7500 integrator, L-7100 pump,
L-7200 autosampler, L-7300 column oven, and L-7485 fluorescence detector;
Hitachi, Tokyo, Japan) equipped with a Mightysil RP-18 GP column (4.6 ϫ
inhibition by the three major phytocannabinoids (⌬ -THC, CBD, and
CBN). We report herein that CBD is a potent competitive inhibitor for
CYP2D6. Furthermore, our study suggests that two phenolic hydroxyl
groups and the pentyl side chain of CBD may have structurally
important roles in CYP2D6 inhibition.
2
50 mm, 5 ␮m; Kanto Chemical, Tokyo, Japan). The mobile phase was the
Materials and Methods
mixture of acetonitrile/methanol: 10 mM potassium phosphate buffer adjusted
9
Materials. ⌬ -THC, CBD, and CBN were isolated from cannabis leaves to pH 3.5 with phosphoric acid (200:160:630). Elution was performed at a flow
using the method previously reported (Aramaki et al., 1968). CBD-2Ј-mono- rate of 1.0 ml/min. The formation of dextrorphan was monitored at an exci-
methyl ether (CBDM) and CBD-2Ј,6Ј-dimethyl ether (CBDD) were prepared tation of 280 nm and an emission of 310 nm.
as described previously (Gohda et al., 1990). Purities of these cannabinoids
were determined to be greater than 97% by gas chromatography, except for
CBDD, or which the purity was 93% (Watanabe et al., 2005). Cannabidivarin
To determine the kinetic parameters for AMMC and dextromethorphan
O-demethylations by recombinant CYP2D6 and/or HLMs, AMMC at 0.24 to
15 ␮M or dextromethorphan at 0.15 to 24 ␮M were incubated with each
(
CBDV) was generously provided from Dr. Yukihiro Shoyama at Nagasaki enzyme source under the same conditions as mentioned above. In preliminary
International University (Sasebo, Japan). 3-[2-(N,N-Diethyl-N-methylammo- experiments, these reaction conditions were confirmed to ensure linear initial
nium)ethyl]-7-methoxy-4-methylcoumarin (AMMC), 3-[2-(N,N-diethylamino) rates for the formation of an AMMC metabolite or dextrorphan. Data points
ethyl]-7-hydroxy-4-methylcoumarin (AHMC), microsomes from baculovirus- were fitted to the Michaelis-Menten equation by nonlinear regression analysis
infected insect cells expressing CYP2D6 with NADPH-P450 reductase (Su- with GraphPad Prism 5.02 (GraphPad Software Inc., San Diego, CA).
persomes), and pooled HLMs were purchased from BD Gentest (Woburn,
Inhibition Studies. Recombinant CYP2D6 and HLMs were incubated with
MA). Dextromethorphan hydrobromide monohydrate, dextrorphan-D-tartrate, AMMC or dextromethorphan in the presence of test compounds including
resorcinol, acenaphthylene, benz[a]anthracene, benzo[a]pyrene, 1-methyl- cannabinoids (up to 100 ␮M) under the same manner as described under
naphthalene, 2-methylnaphthalene, phenanthrene, and quinidine sulfate dihy-
drate were obtained from Wako Pure Chemicals (Osaka, Japan). Olivetol,
Enzyme Assays. With the exception of quinidine that was dissolved in water,
all compounds used were dissolved in dimethyl sulfoxide (DMSO) and added
d-limonene, pentylbenzene, progesterone, allopregnanolone, 17␣-hydroxypro- to the incubation mixture at a final DMSO concentration of Յ1%. DMSO
gesterone, 5␣-dihydroprogesterone, acenaphthene, anthracene, fluoranthene, (Յ1%) in incubation mixtures caused less than 6% inhibition of AMMC and

INHIBITION OF CYP2D6 BY PHYTOCANNABINOIDS
2051
FIG. 2. Effects of the major phytocannabinoids on AMMC and the dextromethorphan O-demethylase activities of CYP2D6 and HLMs. Recombinant CYP2D6 was
9
incubated with 0.6 ␮M AMMC (A) or 0.6 ␮M dextromethorphan (B) in the presence of various amounts of ⌬ -THC, CBD, and CBN. C, HLMs were incubated with 4
9
␮
M dextromethorphan in the presence of various amounts of ⌬ -THC, CBD, and CBN. Each point is the mean of two determinations.
dextromethorphan O-demethylase activities under the current conditions. The
In eq. 3, AUCinhibited is the area under the curve for a given substrate probe
IC50 values were calculated by nonlinear regression analysis with GraphPad in the presence of an inhibitor, and AUCcontrol is the area under the curve for
Prism 5.02 (GraphPad Software Inc.) using the dose-response with variable the same probe substrate without inhibitor. The fraction of metabolism of the
slope function.
probe substrate by CYP2D6 and the magnitude of the potency of the inhibitor
The effects of three or four different inhibitor concentrations on AMMC and
are represented by fm(CYP2D6) and K , respectively. The value of unity was used
i
dextromethorphan O-demethylations were examined at five substrate concen- for the fm(CYP2D6) of dextromethorphan/dextrorphan urinary ratio (Obach et al.,
trations to characterize the enzyme kinetics for the inhibition of CYP2D6 by
2006). Thus, a ratio of AUCinhibited to AUCcontrol of dextromethorphan is equal
CBD and its structurally related compounds. The apparent K values and the
to 1 ϩ [I]in vivo/Ki.
i
mode of inhibition were determined by nonlinear regression analysis using eq.
1
for competitive inhibition with GraphPad Prism 5.02 (GraphPad Software
Results
Inc.):
Inhibition of CYP2D6 Activity by Major Phytocannabinoids.
To clarify the enzymatic characteristics of recombinant CYP2D6 and
pooled HLMs used in this study toward AMMC and dextrometho-
rphan O-demethylase activities, kinetic analysis was conducted with
v ϭ Vmax ⅐ [S]/͓K
m
⅐ (1 ϩ [I]/K
i
) ϩ [S]͔
(1)
where v, Vmax, [S], K , and [I] are the velocity, maximal velocity, substrate
m
concentration, Michaelis constant, and inhibitor concentration, respectively.
Akaike’s information criterion was used as a measure of goodness of fit. The these enzyme sources. All of the reactions examined followed the
mode of inhibition was verified by visual inspection of Lineweaver-Burk plots Michaelis-Menten kinetics based on the Eadie-Hofstee plots (data not
of the enzyme kinetic data.
shown). The apparent Km values were 0.555 ␮M for AMMC O-dem-
ethylation by recombinant CYP2D6, 0.551 ␮M for dextromethorphan
O-demethylation by recombinant CYP2D6, and 3.62 ␮M for dextro-
methorphan O-demethylation by HLMs.
Thus, the effects of ⌬ -THC, CBD, and CBN on CYP2D6-
mediated activity were investigated at substrate concentrations of 0.6
To identify potential metabolism-dependent inhibition of CYP2D6 by can-
nabinoids, the inhibition experiments were performed as described below. The
preincubation mixture contained recombinant CYP2D6 (2 pmol), each major
cannabinoid (2.5–50 ␮M), an NADPH-generating system (8.2 ␮M NADP,
9
0
.41 mM glucose 6-phosphate, 0.41 mM magnesium chloride, and 1 unit/ml
glucose 6-phosphate dehydrogenase), and 100 mM potassium phosphate buffer
pH 7.4) in a final volume of 180 ␮l. After prewarming at 37°C for 5 min,
␮M for recombinant CYP2D6 and 4 ␮M for pooled HLMs. All of the
(
reactions were initiated by the addition of the NADPH-generating system. cannabinoids tested inhibited AMMC and dextromethorphan O-dem-
After a 20-min preincubation, 20 ␮l of AMMC solution was added to the ethylase activities of recombinant CYP2D6 and HLMs in a concen-
preincubation mixture (final substrate concentration, 0.6 ␮M). Incubations tration-dependent manner (Fig. 2). Among the cannabinoids tested,
were conducted under the same manner as described under Enzyme Assays for
AMMC O-demethylase activity.
Prediction of In Vivo Drug Interactions for CYP2D6. An estimate of in
of quinidine, a CYP2D6-selective inhibitor, on CYP2D6 activities
vivo inhibition potency was determined by the methods of Obach et al. (2006).
The maximal unbound hepatic input concentration, Cmax, u, inlet, was deter-
CYP2D6 was strongly inhibited by quinidine with the IC50 value of
mined using the following equation:
CBD most potently inhibited the CYP2D6 activities, showing the IC50
values of 4.01 to 6.52 ␮M (Table 1). In addition, the inhibitory effect
was examined. The AMMC O-demethylase activity of recombinant
3
.33 nM, and the dextromethorphan O-demethylase activity of recom-
C
max,u,inlet ϭ f
u
⅐ (Cmax ϩ D ⅐ F
a
⅐ k
a
/Q
h
)
(2) binant CYP2D6 and HLMs was efficiently inhibited by quinidine with
the IC50 values of 1.86 and 20.3 nM, respectively (data not shown).
The inhibitory effects of quinidine on CYP2D6-mediated activity in
In eq. 2, Cmax is defined as the maximal systemic concentration, f is the
fraction unbound in the blood, D is the oral dose, F is the fraction of the oral
u
a
dose absorbed, ka is the first-order absorption rate constant, and Qh is the
hepatic blood flow. In the case of marijuana smoking, absorption from the
gastrointestinal tract is not taken into account. Thus, the Cmax, u, inlet for inhaled
cannabinoids is equal to f ⅐ C . The value of f for cannabinoids is at most
TABLE 1
IC50 values of the major phytocannabinoids for inhibition of AMMC and
dextromethorphan O-demethylase activities of recombinant CYP2D6 and HLMs
u
max
u
0
.05 because 95 to 99% of plasma THC is bound to plasma proteins, mainly
IC50 (␮M)
lipoproteins (Grotenhermen, 2003). A ratio of the area under the curve (AUC)
with inhibitor to control AUC could be estimated using the following equation:
Enzymes
Substrates
9
⌬ -THC
CBD
CBN
CYP2D6
CYP2D6
HLMs
AMMC
Dextromethorphan
Dextromethorphan
21.2
17.1
22.9
6.52
6.01
4.01
24.0
12.3
24.9
i
AUCinhibited/AUCcontrol ϭ 1/{͓fm(CYP2D6)/(1 ϩ [I]in vivo/K )͔ ϩ (1 Ϫ fm(CYP2D6))}
(
3)

2
052
YAMAORI ET AL.
FIG. 3. Lineweaver-Burk plots for the inhibition of CYP2D6 and HLMs by CBD. Recombinant CYP2D6 was incubated with AMMC (A) or dextromethorphan (B) in the
presence or absence of CBD. C, HLMs were incubated with dextromethorphan in the presence or absence of CBD. Each point is the mean of duplicate determinations.
this study were comparable to previous findings (Chauret et al., 2001; CYP2D6, 2.69 ␮M for dextromethorphan O-demethylation by recom-
Madeira et al., 2004).
binant CYP2D6, and 2.42 ␮M for dextromethorphan O-demethylation
Kinetic Analysis for Inhibition of CYP2D6 Activity by CBD. by HLMs.
Kinetic analysis for the inhibition was performed to characterize the
Structural Requirements for Inhibition of CYP2D6 Activity by
mode of inhibition of CYP2D6 activity by CBD. The cannabinoid CBD. The inhibitory effects of CBD-related compounds on CYP2D6
competitively inhibited the CYP2D6 activities (Fig. 3). The apparent activity were investigated to clarify structural requirements for the
K values were 1.16 ␮M for AMMC O-demethylation by recombinant CYP2D6 inhibition by CBD (Fig. 4). In these inhibition studies, the
i
FIG. 4. Effects of CBD-related compounds
on the AMMC O-demethylase activity of
CYP2D6. A, structures of CBD-related com-
pounds. B to F, recombinant CYP2D6 was
incubated with 0.6 ␮M AMMC in the pres-
ence of various amounts of CBD-related com-
pounds: olivetol and D-limonene (B), CBDM and
CBDD (C), pentylbenzene (D), CBDV (E), and
orcinol and resorcinol (F). Each point is the mean
of two determinations.

INHIBITION OF CYP2D6 BY PHYTOCANNABINOIDS
2053
FIG. 5. Lineweaver-Burk plots for inhibition of
CYP2D6 by olivetol and CBDV. Recombinant
CYP2D6 was incubated with AMMC in the pres-
ence or absence of olivetol and CBDV. Each
point is the mean of duplicate determinations.
activity of AMMC O-demethylation by recombinant CYP2D6 was
CBD and olivetol have a pentyl side chain. To elucidate the
used as a marker of activity. CBD consists of the pentylresorcinol and importance of the pentyl group of CBD in the inhibition against
terpene moieties, as shown in Fig. 1. To determine which moiety of CYP2D6 activity, inhibition studies were conducted with CBDV,
CBD is important in the CYP2D6 inhibition, the effects of olivetol resorcinol, and orcinol. CBDV, possessing a shorter side chain (i.e.,
and d-limonene were examined. Olivetol, which corresponds to the propyl group; Fig. 4A), inhibited the CYP2D6 activity to some extent
pentylresorcinol moiety of CBD (Fig. 4A), inhibited the AMMC (Fig. 4E), although its inhibitory effect (IC50 ϭ 10.2 ␮M) was less
O-demethylase activity of recombinant CYP2D6 (Fig. 4B): its inhib- potent than that of CBD (Table 1). On the other hand, resorcinol and
itory effect (IC50 ϭ 7.21 ␮M) was comparable to that of CBD (Table its derivative orcinol, having a methyl group at the 5-position (Fig.
1
). On the other hand, d-limonene, which corresponds to the terpene 4A), did not exert any inhibitory effect (Fig. 4F).
moiety of CBD (Fig. 4A), showed no inhibitory effect (Fig. 4B). Kinetic analysis for the inhibition was conducted to characterize the
To determine whether the two free phenolic hydroxyl groups of mode of inhibition of CYP2D6 activity by olivetol and CBDV. These
CBD have a role in the inhibition of CYP2D6, the inhibitory effects compounds competitively inhibited the CYP2D6 activity (Fig. 5). The
of two methylated derivatives of CBD were investigated. The K values of olivetol and CBDV were 2.87 and 5.80 ␮M, respectively.
i
CYP2D6 activity was not inhibited by CBDM or CBDD (Fig. 4C),
Effects of Endogenous Steroids and Marijuana Smoke Contain-
which are monomethylated and dimethylated derivatives of CBD, ing Polycyclic Aromatic Hydrocarbons on CYP2D6 Activity. En-
respectively (Fig. 4A). In addition, pentylbenzene, which is free of dogenous steroids, polycyclic aromatic hydrocarbons (PAH), and
phenolic hydroxyl groups (Fig. 4A), did not inhibit the activity of cannabinoids do not have any basic nitrogen atom, the presence of
CYP2D6 at all (Fig. 4D).
which is a common characteristic of most CYP2D6 inhibitors. Nev-
FIG. 6. Effects of steroids and marijuana smoke containing PAH on CYP2D6 activities. A, recombinant CYP2D6 was incubated with 0.6 ␮M AMMC in the presence of
various amounts of progesterone (P), allopregnanolone, 17␣-hydroxy-P, and 5␣-dihydro-P. B, recombinant CYP2D6 was incubated with 0.6 ␮M AMMC in the presence
of various amounts of acenaphthene, acenaphthylene, fluorene, 1-methylnaphthalene, 2-methylnaphthalene, and naphthalene. C, recombinant CYP2D6 was incubated with
0
.6 ␮M dextromethorphan in the presence of various amounts of P. D, recombinant CYP2D6 was incubated with 0.6 ␮M dextromethorphan in the presence of various
amounts of anthracene, benz[a]anthracene, benzo[a]pyrene, fluoranthene, phenanthrene, and pyrene. Each point is the mean of two determinations.

2
054
YAMAORI ET AL.
ertheless, some steroids have been reported to inhibit CYP2D6 activ-
ity (Hiroi et al., 2001). Thus, the inhibitory effects of endogenous
steroids and the major PAH contained in marijuana smoke (Moir et
al., 2008) on CYP2D6 activity were examined and compared with that
of CBD. Progesterone inhibited the AMMC and dextromethorphan
O-demethylase activities of recombinant CYP2D6 (Fig. 6), showing
the IC50 values of 68.8 and 94.3 ␮M, respectively (Table 2). Allo-
pregnanolone inhibited the AMMC O-demethylase activity of recombi-
nant CYP2D6 in a concentration-dependent manner (IC50 ϭ 44.6 ␮M)
TABLE 3
Effect of preincubation on inhibition of CYP2D6 activity by major
phytocannabinoids
All determinations were performed in duplicate.
IC50 (␮M)
Cannabinoids
Preincubation Time
B/A
0 min (A)
20 min (B)
9
⌬ -THC
19.6
6.65
16.0
22.7
4.64
21.9
1.16
0.698
1.37
and in a competitive type (K ϭ 35.2 ␮M). The other steroids tested,
CBD
CBN
i
17␣-hydroxyprogesterone and 5␣-dihydroprogesterone, exerted no inhib-
itory effect on the CYP2D6 activity under the current conditions. On the
other hand, benz[a]anthracene, benzo[a]pyrene, and fluoranthene inhib- potently inhibited CYP2D6 activity. However, the metabolism-depen-
ited CYP2D6 activity in a concentration-dependent manner, exhibiting dent inhibition of CYP2D6 by CBD was less effective. We have
3
7.9, 38.9, and 45.2%, respectively, inhibition at 100 ␮M. The other recently reported that CYP2D6 is capable of metabolizing CBD,
PAH examined—acenaphthene, acenaphthylene, anthracene, fluorene, although the enzyme is not a major one responsible for CBD metab-
-methylnaphthalene, 2-methylnaphthalene, naphthalene, phenanthrene, olism (Jiang et al., 2011). These results suggest that CBD itself may
and pyrene—did not show significant inhibition (Fig. 6). These results inhibit the activity of CYP2D6.
1
revealed that, among the compounds tested without nitrogen atoms, CBD
most potently inhibited CYP2D6 activity.
The structure of CBD contains the pentylresorcinol and terpene
moieties. The fact that the inhibitory effect of olivetol on CYP2D6
Metabolism-Dependent Inhibition of CYP2D6 Activity by Ma- activity was equal to that of CBD suggests that the pentylresorcinol
jor Phytocannabinoids. An effect of preincubation on inhibition by moiety may play a critical role in the CBD-mediated CYP2D6 inhi-
9
⌬
-THC, CBD, and CBN was investigated to determine whether these bition. The structure of the resorcinol moiety contains two phenolic
cannabinoids inhibit CYP2D6-mediated oxidation in a metabolism- hydroxyl groups and a phenyl ring, which are likely to interact with
9
dependent manner. A 20-min preincubation of ⌬ -THC and CBN in amino acids within the active site of CYP2D6. Then, we focused on
the presence of NADPH did not increase the inhibition of the the role of the phenolic hydroxyl groups of CBD in CYP2D6 inhibi-
CYP2D6 activity (Table 3). The preincubation of CBD slightly po- tion. The lack of both phenolic hydroxyl groups in olivetol completely
tentiated the CYP2D6 inhibition.
eliminated the inhibitory effect on CYP2D6 activity. Furthermore, the
methylation of either hydroxyl group in CBD failed to inhibit
CYP2D6 activity. A hydroxyl group at the 2Ј-position in CBD is
equivalent to the hydroxyl group at the 6Ј-position because there is
free rotation of the phenyl ring about the chemical bond at the
Discussion
9
In this study, we demonstrated that ⌬ -THC, CBD, and CBN, the
three major cannabinoids in marijuana, inhibited the catalytic activity
of human CYP2D6. Among the cannabinoids tested, CBD most
1
Ј-position (Fig. 1). These results suggest that both free phenolic
hydroxyl groups in the resorcinol moiety of CBD may be required for
CYP2D6 inhibition.
TABLE 2
Next, we focused on a role of the pentyl side chain of CBD in
CYP2D6 inhibition. The substitution of the pentyl group of CBD to a
propyl group attenuated the inhibitory effect on CYP2D6 activity. In
addition, the side chain shortening to a methyl group resulted in the
loss of CYP2D6 inhibition. In our recent study, recombinant CYP2D6
has been shown to oxidize CBD at the 6-, 7-, 4Љ-, and 5Љ-positions
Summary of kinetic parameters for inhibition of CYP2D6 activity by steroids and
representative PAH contained in marijuana smoke
IC50a
K
i
Mode of
Compounds
Substrates
Inhibition
␮
M
␮M
Progesterone
AMMC
68.8
94.3
(
Jiang et al., 2011). This finding suggests two possible orientations in
Dextromethorphan
b
which the terpene moiety or pentyl side chain of CBD are positioned
near the heme in the CYP2D6 active site when the cannabinoid enters
into the active site. When the terpene moiety in CBD is located close
to the heme, the pentyl side chain in the cannabinoid is thought to be
fixed within the proposed hydrophobic region corresponding to a toe
area of the foot-shaped cavity in CYP2D6 (Rowland et al., 2006). On
the other hand, when the pentyl side chain of CBD is positioned near
the heme, the 4Љ- or 5Љ-position of the side chain is located above the
heme, and the proximal portion of the side chain is considered to
interact with the proposed hydrophobic region defined by hydropho-
bic residues, such as a Met residue at site 374 and Leu residues at sites
213 and 484 (de Graaf et al., 2007; Kotsuma et al., 2008). In both
cases, the side-chain shortening of CBD may lead to unstable binding
of the cannabinoid within the active site in CYP2D6 because of
attenuation of the hydrophobic interactions. These results suggest that
the pentyl side chain of CBD also plays an important role in CYP2D6
inhibition.
Bufuralol
33.2 Competitive
35.2 Competitive
62.6 Competitive
Allopregnanolone
Testosterone
Pregnanolone
Pregnenolone
AMMC
Bufuralol
Bufuralol
Bufuralol
44.6
b
b
134
Competitive
Competitive
Competitive
Competitive
b
163
307
391
b
1
1
7␤-Estradiol
7␣-Hydroxyprogesterone Bufuralol
Bufuralol
b
AMMC
Ͼ100
5
␣-Dihydroprogesterone
AMMC
AMMC
AMMC
Dextromethorphan Ͼ100
Dextromethorphan Ͼ100
Dextromethorphan Ͼ100
Dextromethorphan Ͼ100
AMMC
AMMC
AMMC
AMMC
Dextromethorphan Ͼ100
Dextromethorphan Ͼ100
Ͼ100
Ͼ100
Ͼ100
Acenaphthene
Acenaphthylene
Anthracene
Benz͓a͔anthracene
Benzo͓a͔pyrene
Fluoranthene
Fluorene
Ͼ100
Ͼ100
Ͼ100
Ͼ100
1
2
-Methylnaphthalene
-Methylnaphthalene
Naphthalene
Phenanthrene
Pyrene
a
Recombinant CYP2D6 was incubated with 0.6 ␮M AMMC or 0.6 ␮M dextromethorphan
in the presence of each compound at up to 100 ␮M. All determinations were performed in
duplicate.
Certain structural characteristics common to typical CYP2D6 in-
hibitors are known (Strobl et al., 1993). One of these is to contain at
least one basic nitrogen atom in structure. However, several CYP2D6
b
Hiroi et al. (2001).

INHIBITION OF CYP2D6 BY PHYTOCANNABINOIDS
2055
inhibitors do not have this characteristic. Some steroids such as Acknowledgments
progesterone and allopregnanolone are atypical inhibitors of CYP2D6
We thank Dr. Yukihiro Shoyama (Nagasaki International University,
Sasebo, Japan) for generously providing CBDV.
because they have no nitrogen atom in their structures (Hiroi et al.,
9
2
001). The three major phytocannabinoids (⌬ -THC, CBD, and CBN)
and some PAH (benz[a]anthracene, benzo[a]pyrene, and fluoran-
Authorship Contributions
thene), which inhibited CYP2D6 activity in this study, have no
Participated in research design: Yamaori, Okamoto, Yamamoto, and Wa-
nitrogen atom in their structures. Therefore, these compounds would tanabe.
be categorized as atypical CYP2D6 inhibitors. The most potent inhi-
bition reported so far of CYP2D6 by compounds without a nitrogen
Conducted experiments: Yamaori and Okamoto.
Performed data analysis: Yamaori and Okamoto.
Wrote or contributed to the writing of the manuscript: Yamaori, Yamamoto,
and Watanabe.
atom was observed in progesterone, with a K value of approximately
i
3
3 ␮M (Hiroi et al., 2001). In this study, CYP2D6-mediated activity
was most potently inhibited by CBD among the major phytocannabi-
noids, marijuana smoke containing PAH, and endogenous steroids
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