Potent inhibition of human cytochrome P450 3A isoforms by cannabidiol: Role of phenolic hydroxyl groups in the resorcinol moiety

Article abstract of DOI:10.1016/j.lfs.2011.02.017

Aims: In this study, we examined the inhibitory effects of Δ⁹-tetrahydrocannabinol (Δ⁹-THC), cannabidiol (CBD), and cannabinol (CBN), the three major cannabinoids, on the activity of human cytochrome P450 (CYP) 3A enzymes. Furthermore, we investigated the kinetics and structural requirement for the inhibitory effect of CBD on the CYP3A activity. Main methods: Diltiazem N-demethylase activity of recombinant CYP3A4, CYP3A5, CYP3A7, and human liver microsomes (HLMs) in the presence of cannabinoids was determined. Key findings: Among the three major cannabinoids, CBD most potently inhibited CYP3A4 and CYP3A5 (IC₅₀ = 11.7 and 1.65 μM, respectively). The IC₅₀ values of Δ⁹-THC and CBN for CYP3A4 and CYP3A5 were higher than 35 μM. For CYP3A7, Δ⁹-THC, CBD, and CBN inhibited the activity to a similar extent (IC₅₀ = 23-31 μM). CBD competitively inhibited the activity of CYP3A4, CYP3A5, and HLMs (K_i = 1.00, 0.195, and 6.14 μM, respectively). On the other hand, CBD inhibited the CYP3A7 activity in a mixed manner (K_i = 12.3 μM). Olivetol partially inhibited all the CYP3A isoforms tested, whereas d-limonene showed lack of inhibition. The lesser inhibitory effects of monomethyl and dimethyl ethers of CBD indicated that the ability of CYP3A inhibition by the cannabinoid attenuated with the number of methylation on the phenolic hydroxyl groups in the resorcinol moiety. Significance: This study indicated that CBD most potently inhibited catalytic activity of human CYP3A enzymes, especially CYP3A4 and CYP3A5. These results suggest that two phenolic hydroxyl groups in the resorcinol moiety of CBD may play an important role in the CYP3A inhibition.

Full text of DOI:10.1016/j.lfs.2011.02.017

Life Sciences 88 (2011) 730–736
Contents lists available at ScienceDirect
Life Sciences
journal homepage: www.elsevier.com/locate/lifescie
Potent inhibition of human cytochrome P450 3A isoforms by cannabidiol: Role of
phenolic hydroxyl groups in the resorcinol moiety
Satoshi Yamaori a, Juri Ebisawa a, Yoshimi Okushima a, Ikuo Yamamoto b, Kazuhito Watanabe a,c,
⁎
a
Department of Hygienic Chemistry, Faculty of Pharmaceutical Sciences, Hokuriku University, Ho-3, Kanagawa-machi, Kanazawa 920-1181, Japan
b
Department of Hygienic Chemistry, School of Pharmaceutical Sciences, Kyushu University of Health and Welfare, 1714-1 Yoshino-machi, Nobeoka 882-8508, Japan
c
Organization for Frontier Research in Preventive Pharmaceutical Sciences, Hokuriku University, Ho-3, Kanagawa-machi, Kanazawa 920-1181, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 4 October 2010
Accepted 10 February 2011
Aims: In this study, we examined the inhibitory effects of Δ9-tetrahydrocannabinol (Δ9-THC), cannabidiol
(CBD), and cannabinol (CBN), the three major cannabinoids, on the activity of human cytochrome P450 (CYP)
3A enzymes. Furthermore, we investigated the kinetics and structural requirement for the inhibitory effect of
CBD on the CYP3A activity.
Main methods: Diltiazem N-demethylase activity of recombinant CYP3A4, CYP3A5, CYP3A7, and human liver
microsomes (HLMs) in the presence of cannabinoids was determined.
Key findings: Among the three major cannabinoids, CBD most potently inhibited CYP3A4 and CYP3A5
(IC50=11.7 and 1.65 μM, respectively). The IC50 values of Δ9-THC and CBN for CYP3A4 and CYP3A5 were higher
than 35 μM. For CYP3A7, Δ9-THC, CBD, and CBN inhibited the activity to a similar extent (IC50 =23–31 μM). CBD
competitively inhibited the activity of CYP3A4, CYP3A5, and HLMs (Ki=1.00, 0.195, and 6.14 μM, respectively).
On the other hand, CBD inhibited the CYP3A7 activity in a mixed manner (Ki=12.3 μM). Olivetol partially
inhibited all the CYP3A isoforms tested, whereas d-limonene showed lack of inhibition. The lesser inhibitory
effects of monomethyl and dimethyl ethers of CBD indicated that the ability of CYP3A inhibition by the
cannabinoid attenuated with the number of methylation on the phenolic hydroxyl groups in the resorcinol
moiety.
Keywords:
Cannabidiol
Δ9-Tetrahydrocannabinol
Cannabinol
CYP3A4
CYP3A5
CYP3A7
Inhibition
Significance: This study indicated that CBD most potently inhibited catalytic activity of human CYP3A enzymes,
especially CYP3A4 and CYP3A5. These results suggest that two phenolic hydroxyl groups in the resorcinol
moiety of CBD may play an important role in the CYP3A inhibition.
© 2011 Elsevier Inc. All rights reserved.
Introduction
1991; Bornheim et al., 1992; Watanabe et al., 1995, 2007). On the other
hand, these major cannabinoids have been reported to inhibit CYP-
Marijuana is one of the most widely abused drugs in the world.
Marijuana leaves contain at least 70 cannabinoids (ElSohly and Slade,
2005). Among them, Δ9-tetrahydrocannabinol (Δ9-THC), cannabidiol
(CBD), and cannabinol (CBN) are the three major constituents (Fig. 1).
Δ9-THC is a primary psychoactive component of marijuana (Pertwee,
2008). CBD is not psychoactive but has several pharmacological effects
such as drug-induced sleep prolongation, antiepileptic, anxiolytic, and
antiemetic actions (Mechoulam et al., 2002). CBN is thought to exert
minimal pharmacological effects in the central nervous system.
These major cannabinoids are known to be extensively metabo-
lized by hepatic microsomal cytochrome P450 (CYP) (Huestis, 2005;
Watanabe et al., 2007). Bornheim et al. and we previously demon-
strated that the hepatic metabolism of Δ9-THC, CBD, and CBN is
predominantly catalyzed by CYP2C and CYP3A (Bornheim and Correia,
mediated drug metabolism in livers from experimental animals
(Fernandes et al., 1973; Chan and Tse, 1978; Watanabe et al., 1981;
Hamajima et al., 1983). Furthermore, a previous clinical study has
shown that the administration of CBD decreases the systemic
clearance of hexobarbital, which is metabolized by CYP2C9, in
human subjects (Benowitz et al., 1980). Jaeger et al. (1996) have
reported that CBD inhibits cyclosporine and Δ9-THC oxidations
catalyzed by CYP3A in human liver microsomes (HLMs). More
recently, we have shown that Δ9-THC, CBD, and CBN inhibit catalytic
activities of human CYP1 enzymes (Yamaori et al., 2010). These
findings suggest that the administration of cannabinoids or marijuana
may lead to drug interactions with drugs or toxicants metabolized by
these CYP enzymes.
Human CYP3A subfamily is involved in the metabolism of more
than 50% of drugs clinically used. The human CYP3A subfamily
expressed in the liver consists of at least three members: CYP3A4
(Guengerich et al., 1986; Molowa et al., 1986), CYP3A5 (Aoyama et al.,
1989; Wrighton et al., 1989), and CYP3A7 (Kitada et al., 1985).
CYP3A4, which is the most abundant isoform expressed in adult
⁎
Corresponding author at: Department of Hygienic Chemistry, Faculty of Pharma-
ceutical Sciences, Hokuriku University, Ho-3, Kanagawa-machi, Kanazawa 920-1181,
Japan. Tel./fax: +81 76 229 6220.
E-mail address: k-watanabe@hokuriku-u.ac.jp (K. Watanabe).
0024-3205/$ – see front matter © 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.lfs.2011.02.017

S. Yamaori et al. / Life Sciences 88 (2011) 730–736
731
OH
OH
OH
O
HO
O
Δ9-THC
CBD
CBN
Fig. 1. Structures of three major cannabinoids.
human livers (Shimada et al., 1994), is capable of metabolizing
structurally diverse compounds, such as diltiazem, midazolam,
erythromycin, and cyclosporine (Rendic, 2002). CYP3A5, which is
polymorphically expressed in the liver (Kuehl et al., 2001), also can
catalyze oxidations of a variety of drugs (Aoyama et al., 1989;
Williams et al., 2002; Yamaori et al., 2003, 2004). The catalytic activity
of CYP3A5 is equal or lower as compared with that of CYP3A4,
although some drugs, such as diltiazem and midazolam, are more
efficiently metabolized by CYP3A5 than by CYP3A4 (Gorski et al.,
1994; Yamaori et al., 2004). CYP3A7, which is a major isoform
expressed in fetal human livers (Kitada et al., 1985; Komori et al.,
1990), is suggested to play a key role in the metabolism of
endogenous compounds and xenobiotics during the fetal period
(Kitada et al., 1985, 1987; Chen et al., 2000). These findings reveal that
human CYP3A subfamily is one of the most important enzyme groups
in the hepatic drug metabolism. Furthermore, these findings indicate
the possibility of frequent occurrence of drug interactions caused by
CYP3A enzymes. Thus, it is very important to elucidate possible
interactions of marijuana or its components including cannabinoids
with various compounds metabolized by CYP3A enzymes. However,
the potency and mechanism of cannabinoid inhibition against
individual isoforms of human CYP3A subfamily remain to be clarified.
In the present study, we investigated the inhibitory effects of the
three major cannabinoids (Δ9-THC, CBD, and CBN) on human CYP3A-
mediated oxidation. We report herein that CBD is a potent inhibitor
against human CYP3A enzymes. In addition, our study suggests that
two phenolic hydroxyl groups in the resorcinol moiety of CBD may
have structurally important role in the CYP3A inhibition.
Materials and methods
Materials
Δ9-THC, CBD, and CBN were isolated from cannabis leaves using the
method previously reported (Aramaki et al., 1968). CBD-2′-monomethyl
ether (CBDM) and CBD-2′,6′-dimethyl ether (CBDD) were prepared as
described previously (Gohda et al., 1990). N-Desmethyldiltiazem was a
gift from Tanabe Seiyaku (Osaka, Japan). Diltiazem was obtained from
Wako Pure Chemical Ind. (Osaka, Japan). Olivetol and d-limonene were
purchased from Sigma-Aldrich Corp. (St. Louis, MO). NADP, glucose 6-
phosphate, and glucose 6-phosphate dehydrogenase were obtained from
Oriental Yeast Co. Ltd. (Tokyo, Japan). Other chemicals and solvents used
were of the highest quality commercially available.
Enzyme sources
Microsomes from baculovirus-infected insect cells expressing
CYP3A4, CYP3A5, and CYP3A7 each with NADPH-CYP reductase and
cytochrome b5 (Supersomes™) were purchased from BD Gentest
(Woburn, MA). A human liver sample was obtained from a 57-year-old
Japanese female who died in a traffic accident. The use of the human
liver for these studies was approved by the Ethics Committee of
Kanazawa University, Graduate School of Medical Science. HLMs were
prepared as reported previously (Watanabe et al., 1995). The protein
concentration was estimated by the method of Lowry et al. (1951),
using bovine serum albumin as a standard.
CYP3A4
CYP3A5
CYP3A7
HLMs
14
12
10
8
25
20
15
10
5
1.2
1.0
0.8
0.6
0.4
0.2
0
0.8
0.6
0.4
0.2
0
6
4
2
0
0
0
50
100
150
200
0
50 100 150 200 250 300
0
50 100 150 200 250 300
0
100 200 300 400 500
S (μM)
S (μM)
S (μM)
S (μM)
14
12
10
8
1.2
1.0
0.8
0.6
0.4
0.2
0
1.0
0.8
0.6
0.4
0.2
0
30
25
20
15
10
5
6
4
2
0
0
0
0.3
0.6
0.9
1.2
0
0.1 0.2 0.3 0.4 0.5 0.6
v/S (ml/min/nmol)
0
0.006 0.012 0.018 0.024
v/S (ml/min/nmol)
0
0.004 0.008 0.012 0.016
v/S (ml/min/mg protein)
v/S (ml/min/nmol)
Fig. 2. Kinetic analyses for diltiazem N-demethylation catalyzed by human CYP3A isoforms and liver microsomes. Recombinant CYP3A4, CYP3A5, CYP3A7, and HLMs were incubated
with diltiazem (5–500 μM). S and v indicate diltiazem and catalytic activity, respectively. Each point indicates an individual value.

732
S. Yamaori et al. / Life Sciences 88 (2011) 730–736
Table 1
was incubated with either of these enzyme sources under the same
conditions as mentioned above. In preliminary experiments, these
reaction conditions were confirmed to ensure linear initial rates for the
formation of N-desmethyldiltiazem. Data points were fitted to the
Michaelis–Menten equation by nonlinear least-squares regression
analysis with Origin 7.5J software (OriginLab, Northampton, MA).
Kinetic parameters for diltiazem N-demethylation catalyzed by recombinant human
CYP3A isoforms and HLMs.
Enzymes
Km
(μM)
Vmax
(nmol/min/nmol P450)
CYP3A4
CYP3A5
CYP3A7
12.5
48.4
50.8
13.6
29.3
1.27
Inhibition studies
(nmol/min/mg protein)
0.852
HLMs
50.2
Recombinant CYP3A isoforms and HLMs were incubated with
diltiazem (50 μM) in the presence of Δ9-THC, CBD, CBN, olivetol, d-
limonene, CBDM, or CBDD (0 to 50 μM) under the same manner as
described in the enzyme assays. All compounds were dissolved in
dimethylsulfoxide and added to the incubation mixture at a final
dimethylsulfoxide concentration of 0.5%. The IC50 value was calculat-
ed by nonlinear least-squares regression analysis with Origin 7.5J
software (OriginLab).
To characterize the enzyme kinetics for the inhibition of human
CYP3A enzymes by CBD, the effects of three different concentrations
of the cannabinoid on the formation of N-desmethyldiltiazem were
examined at five diltiazem concentrations. The apparent Ki value
(inhibition constant) was determined from the x-intercept of a plot of
apparent Km/Vmax (obtained from the slope of the Lineweaver–Burk
plots) versus inhibitor concentration. The x-intercept, which is equal
to −Ki, was calculated by linear regression using the Origin 7.5J
software (OriginLab). Lineweaver–Burk plots of the enzyme kinetic
data were generated to determine the mode of inhibition.
Data are derived from two replicates.
Enzyme assays
Diltiazem N-demethylase activity was determined as reported
previously (Yamaori et al., 2004) with minor modifications. Recom-
binant CYP3A4 (2 pmol), CYP3A5 (2 pmol), CYP3A7 (10 pmol), and
HLMs (50 μg protein) were used as enzyme sources. An incubation
mixture consisted of an enzyme source, diltiazem, an NADPH-
generating system (0.5 mM NADP, 10 mM glucose 6-phosphate,
10 mM magnesium chloride, and 1 unit/ml glucose 6-phosphate
dehydrogenase), 100 mM sodium potassium phosphate buffer (pH
7.4), and 50 μM EDTA in a final volume of 200 μl. Incubations were
carried out at 37 °C for 15 min (30 min for CYP3A7) and terminated
by adding 10 μl of 70% (v/v) perchloric acid. After the removal of
protein by centrifugation, 90 μl of the supernatant was subjected to a
high-performance liquid chromatography (Hitachi L-2130 pump, L-
2200 autosampler, and L-2400 UV detector, Hitachi, Tokyo, Japan)
equipped with a Mightysil RP-18 GP column (4.6×150 mm, 5 μm, Kanto
Chemical, Tokyo, Japan). The mobile phase was 50 mM potassium
phosphate buffer (pH 6.0) containing 28% (v/v) acetonitrile. Elution was
performed at a flow rate of 1.5 ml/min. The formation of N-desmethyl-
diltiazem was monitored at a wavelength of 238 nm.
Results
Kinetic analysis for diltiazem N-demethylation by human CYP3A enzymes
To clarify enzymatic characteristics of recombinant CYP3A4,
CYP3A5, CYP3A7, and HLMs used in this study toward diltiazem N-
demethylase activity, kinetic analysis was conducted with these
enzyme sources. All the reactions examined followed the Michaelis–
To determine the kinetic parameters for the diltiazem N-demethyla-
tion in recombinant CYP3A isoforms and HLMs, 5 to 500 μM diltiazem
120
120
CYP3A4
CYP3A5
100
80
60
40
20
0
100
80
60
40
20
0
0
10
20
30
40
50
0
10
20
30
40
50
120
100
80
60
40
20
0
120
100
80
60
40
20
0
CYP3A7
HLMs
0
10
20
30
40
50
0
10
20
30
40
50
Cannabinoids (μM)
Cannabinoids (μM)
Fig. 3. Effects of major cannabinoids on diltiazem N-demethylase activity of human CYP3A isoforms and liver microsomes. Recombinants CYP3A4, CYP3A5, CYP3A7, and HLMs were
incubated with diltiazem (50 μM) in the presence or absence of Δ9-THC (triangles), CBD (circles), and CBN (squares). Each point indicates a mean value of duplicate determinations.

S. Yamaori et al. / Life Sciences 88 (2011) 730–736
733
Table 2
Table 3
IC50 values of major cannabinoids for inhibition of diltiazem N-demethylase activity of
Kinetic parameters for inhibition of diltiazem N-demethylase activity of human CYP3A
human CYP3A enzymes.
enzymes by CBD.
Enzymes
IC50 (μM)
Δ9-THC
Enzymes
Ki (μM)
Mode of inhibition
CBD
11.7
1.65
24.7
9.18
CBN
CYP3A4
CYP3A5
CYP3A7
HLMs
1.00
0.195
12.3
Competitive
Competitive
Mixed
CYP3A4
CYP3A5
CYP3A7
HLMs
N50
35.6
30.3
N50
N50
N50
23.8
N50
6.14
Competitive
Data are derived from two replicates.
Kinetic analysis for cannabidiol-mediated inhibition of human CYP3A
enzymes
Menten kinetics based on the Eadie–Hofstee plots (Fig. 2). Therefore,
kinetic parameters were determined from the Michaelis–Menten
equation. The Vmax values for CYP3A4, CYP3A5, and CYP3A7 were 13.6,
29.3, and 1.27 nmol/min/nmol P450, respectively, whereas the Vmax
value for HLMs was 0.852 nmol/min/mg protein (Table 1). The Km
value of diltiazem for CYP3A4 was 12.5 μM although those for the
other enzyme sources were approximately 50 μM (Table 1).
To determine the mode of inhibition of human CYP3A enzymes by
CBD, kinetic analysis for the inhibition was examined with recombi-
nant CYP3A isoforms and HLMs. The enzyme activities of CYP3A4,
CYP3A5, and HLMs were competitively inhibited by CBD, whereas the
CYP3A7 activity was inhibited by the cannabinoid in a mixed fashion
(Fig. 4). The lowest apparent Ki value of the cannabinoid was seen in
the inhibition of CYP3A5, followed by that of CYP3A4 (Table 3). The Ki
value for CYP3A7 was 12- and 63-fold higher than those for CYP3A4
and CYP3A5, respectively.
Effects of major cannabinoids on diltiazem N-demethylation by human
CYP3A enzymes
Effects of Δ9-THC, CBD, and CBN on diltiazem N-demethylase
activity of recombinant CYP3A isoforms and HLMs were investigat-
ed at a substrate concentration of 50 μM. All the cannabinoids tested
inhibited the activity of recombinant CYP3A4, CYP3A5, and CYP3A7
in a concentration-dependent manner (Fig. 3). CYP3A4 and CYP3A5
were most potently inhibited by CBD with the IC50 values of 11.7
and 1.65 μM, respectively, among the cannabinoids tested (Table 2).
The IC50 values of Δ9-THC and CBN for CYP3A4 and CYP3A5 were
higher than 35 μM. CYP3A7 was inhibited by Δ9-THC, CBD, and CBN
to a similar extent (Fig. 3). The IC50 values of these three
cannabinoids were between 23 and 31 μM (Table 2). When HLMs
were used as an enzyme source, the inhibitory effects of the
cannabinoids were similar to those on recombinants CYP3A4 and
CYP3A5 (Fig. 3).
Structural requirement for inhibitory effects of CBD and its derivatives on
CYP3A-mediated activity
CBD consists of the resorcinol and terpene moieties, as shown in
Fig. 1. To determine which moiety is important in the inhibition of
human CYP3A enzymes, inhibitory effects of olivetol and d-limonene
were examined. Olivetol, which corresponds to the resorcinol moiety
of CBD, inhibited the activity of CYP3A4, CYP3A5, and CYP3A7,
although its inhibitory effect was less potent than that of CBD (Fig. 5).
On the other hand, d-limonene, which corresponds to the terpene
moiety of CBD, did not exert appreciable inhibition of these three
CYP3A isoforms under the present conditions (Fig. 5).
Next, to elucidate whether phenolic hydroxyl group(s) of CBD
contributes to the inhibition of human CYP3A enzymes, inhibitory
CYP3A4
CYP3A5
1.2
0.4
CBD
CBD
12 μM
7.5 μM
1.5 μM
1.0 μM
0.9
0.3
0 μM
0 μM
0.6
0.2
0.3
0.1
-0.08
0
0.08
1/S
0.16
0.24
-0.02
0
0.02
1/S
0.04
0.06
CYP3A7
HLMs
8
6
4
2
16
12
8
CBD
40 μM
25 μM
0 μM
CBD
25 μM
10 μM
0 μM
4
-0.012
0
0.012
1/S
0.024
0.036
-0.02
0
0.02
1/S
0.04
0.06
Fig. 4. Lineweaver–Burk plots for inhibition of human CYP3A isoforms and liver microsomes by CBD. Recombinant CYP3A4, CYP3A5, CYP3A7, and HLMs were incubated with
diltiazem in the presence of various amounts of CBD. S and v indicate diltiazem (μM) and catalytic activity (nmol/min/nmol P450 or mg protein), respectively. Each point indicates a
mean value of duplicate determinations.

734
S. Yamaori et al. / Life Sciences 88 (2011) 730–736
OH
HO
Olivetol
d-Limonene
120
100
80
60
40
20
0
120 CYP3A4
CYP3A5
120
100
80
60
40
20
0
CYP3A7
100
80
60
40
20
0
0
2
4
6
8
10
0
10
20
30
40
50
0
10
20
30
40
50
Compounds (μM)
Compounds (μM)
Compounds (μM)
Fig. 5. Effects of olivetol and d-limonene on diltiazem N-demethylase activity of human CYP3A isoforms. Recombinant CYP3A4, CYP3A5, and CYP3A7 were incubated with diltiazem
(50 μM) in the presence or absence of CBD (closed circles), olivetol (closed triangles), and d-limonene (open triangles). Each point indicates a mean value of duplicate
determinations.
effects of two methylated derivatives of CBD, CBDM and CBDD, were
investigated. The inhibitory effect of CBDM on the activity of CYP3A4,
CYP3A5, and CYP3A7 was smaller than that of CBD (Fig. 6). Furthermore,
CBDD exhibited no or less inhibitory effect on these CYP3A activities
(Fig. 6).
(Yamaori et al., 2010). This study indicates that the inhibition of
CYP3A5 by CBD is comparable to that of CYP1A1. In addition, CBD
caused a preferential inhibition against CYP3A5 among the human
CYP3A isoforms examined. Many CYP3A inhibitors preferentially
inhibit CYP3A4 activity (McConn et al., 2004; Granfors et al., 2006; Liu
et al., 2007), although the selectivity of ritonavir, nelfinavir, and
saquinavir to the inhibition of CYP3A4 and CYP3A5 is equivalent
(Granfors et al., 2006). Therefore, CBD may be a useful prototype as a
CYP3A5-selective inhibitor. The Ki value of CBD for CYP3A5 is
comparable to that of ketoconazole (Ki =0.219 μM) (McConn et al.,
2004).
Discussion
In this study, we demonstrated that Δ9-THC, CBD, and CBN
inhibited human CYP3A activity. Among these major cannabinoids,
CBD most potently inhibited the activity of human CYP3A enzymes,
especially CYP3A4 and CYP3A5. Recently, we have reported that the
CBD-mediated inhibition of 7-ethoxyresorufin O-deethylation by
recombinant human CYP1A1 showed the lowest Ki value (0.155 μM)
among the values of CBD for microsomal CYPs reported so far
The result that the kinetics for diltiazem N-demethylation by
recombinant CYP3A4, CYP3A5, and CYP3A7 followed the Michaelis–
Menten equation indicates that there is a site of diltiazem binding
within these CYP3A active sites. Since CBD competitively inhibited the
7
1
R1, R2 = H: CBD
R1 = CH3, R2 = H: CBDM
R1, R2 = CH3: CBDD
2
6
OR1
2’
1’
6’
5
9
4
3’
4’
3
8
10
R2O
5’
120
100
80
60
40
20
0
120
120
100
80
60
40
20
0
CYP3A4
CYP3A5
CYP3A7
100
80
60
40
20
0
0
10
20
30
40
50
0
10
20
30
40
50
0
2
4
6
8
10
Compounds (μM)
Compounds (μM)
Compounds (μM)
Fig. 6. Effects of CBDM and CBDD on diltiazem N-demethylase activity of human CYP3A isoforms. Recombinant CYP3A4, CYP3A5, and CYP3A7 were incubated with diltiazem (50 μM)
in the presence or absence of CBD (closed circles), CBDM (closed diamonds), and CBDD (open diamonds). Each point indicates a mean value of duplicate determinations.

S. Yamaori et al. / Life Sciences 88 (2011) 730–736
735
activity of CYP3A4 and CYP3A5, it is suggested that CBD may bind to
the catalytic sites of these CYP3A isoforms. On the other hand, CBD is
suggested to bind to the catalytic site and a site different from the
position of diltiazem binding within the CYP3A7 active site, because
the cannabinoid inhibited the CYP3A7 activity in a mixed manner.
Further investigations were conducted to characterize the struc-
tural requirement for the inhibitory effect of CBD on human CYP3A
activity. The partial inhibition of CYP3A4, CYP3A5, and CYP3A7 by
olivetol but not by d-limonene suggests that the resorcinol structure
in CBD is essential for the inhibition of these CYP3A isoforms, although
the whole structure of CBD is required for the overall inhibition of the
CYP3A activity. Inhibition studies with CBDM and CBDD indicate that
the degree of inhibitory effect of CBD on the CYP3A activity depends
on the number of free phenolic hydroxyl groups in the resorcinol
moiety of CBD. These results suggest that both phenolic hydroxyl
groups of CBD play an important role in the inhibition of CYP3A4,
CYP3A5, and CYP3A7.
might influence the activity of CYP3A7. Further studies are necessary
to clarify the effect of cannabinoid-mediated CYP3A7 inhibition on
fetal growth.
Conclusions
We demonstrated that CBD is a potent inhibitor against human
CYP3A enzymes. This study indicates that two hydroxyl groups in the
resorcinol moiety of CBD play an important role in the CYP3A inhibition.
Our study will provide useful information to understand precise
mechanism(s) underlying the CBD-mediated CYP3A inhibition.
Conflict of interest statement
The authors have declared that no conflict of interest exists.
Acknowledgements
In contrast to CYP3A4 and CYP3A5, inhibitory effects of Δ9-THC,
CBD, and CBN on the CYP3A7 activity were almost equivalent. Δ9-THC
and CBN are structurally constrained because one of the two hydroxyl
groups in the resorcinol moiety is utilized for the formation of the
pyran ring, whereas CBD has free rotatable structure between the
resorcinol and terpene moieties. These findings suggest that CYP3A7
may recognize these three cannabinoids as nearly planar structures.
In general, marijuana is consumed by smoking. The average
contents of Δ9-THC, CBD, and CBN in dried plant preparations of
marijuana are 3.1, 0.3, and 0.3%, respectively although these contents
vary widely (ElSohly et al., 2000). In marijuana resin, which is
commonly referred to as hashish, the mean contents of Δ9-THC, CBD,
and CBN are 5.2, 4.2, and 1.7%, respectively (ElSohly et al., 2000). It has
been previously reported that smoking a single marijuana cigarette
containing 34 mg Δ9-THC (the content of 3.55%) shows a plasma peak
level of Δ9-THC at 162 ng/ml (0.516 μM) (Huestis et al., 1992). A peak
level of plasma concentration of CBD in human subjects has been
reported to be 114 ng/ml (0.363 μM) after smoking a placebo
marijuana cigarette spiked with 20 mg of the cannabinoid (Ohlsson
et al., 1984). The Ki value of CBD for CYP3A5 is lower than the plasma
concentration after marijuana smoking, although the values for
CYP3A4 and CYP3A7 are higher than the blood level. Since
cannabinoids are readily distributed in various tissues due to a high
lipophilicity (Leighty, 1973), tissue concentrations of CBD may be
even higher than the blood concentration. Thus, it is suggested that
the inhibition of human CYP3A isoforms by CBD might be caused
during and/or after marijuana smoking in some cases.
This work was supported in part by a Grant-in-Aid for Scientific
Research (C) (grant number 20590127) from the Ministry of
Education, Culture, Sports, Science, and Technology of Japan and by
the ‘Academic Frontier’ Project for Private Universities (grant number
05F016) from the Ministry of Education, Culture, Sports, Science, and
Technology of Japan (2005–2009). The authors thank Tanabe Seiyaku
for providing N-desmethyldiltiazem and Dr. Tohru Ohshima (Forensic
and Social Environmental Science, Graduate School of Medical
Science, Kanazawa University, Kanazawa, Japan) for generously
providing a human liver sample.
References
Anglicheau D, Le Corre D, Lechaton S, Laurent-Puig P, Kreis H, Beaune P, et al. Consequences of
genetic polymorphisms for sirolimus requirements after renal transplant in patients on
primary sirolimus therapy. Am J Transplant 2005;5:595–603.
Aoyama T, Yamano S, Waxman DJ, Lapenson DP, Meyer UA, Fischer V, et al. Cytochrome
P-450 hPCN3, a novel cytochrome P-450 IIIA gene product that is differentially
expressed in adult human liver. cDNA and deduced amino acid sequence and
distinct specificities of cDNA-expressed hPCN1 and hPCN3 for the metabolism of
steroid hormones and cyclosporine. J Biol Chem 1989;264:10388–95.
Aramaki H, Tomiyasu N, Yoshimura H, Tsukamoto H. Forensic chemical study on
marihuana. I. A detection method of the principal constituents by thin-layer and
gas chromatographies. Chem Pharm Bull (Tokyo) 1968;16:822–6.
Bailey JR, Cunny HC, Paule MG, Slikker Jr W. Fetal disposition of Δ9-tetrahydrocannab-
inol (THC) during late pregnancy in the rhesus monkey. Toxicol Appl Pharmacol
1987;90:315–21.
Benowitz NL, Nguyen TL, Jones RT, Herning RI, Bachman J. Metabolic and psychophys-
iologic studies of cannabidiol–hexobarbital interaction. Clin Pharmacol Ther
1980;28:115–20.
A lot of recent studies provide the evidence that the hepatic
expression of polymorphic CYP3A5 affects pharmacokinetics of
several CYP3A substrates (Thervet et al., 2003; Wong et al., 2004;
Anglicheau et al., 2005). Furthermore, CYP3A5 is a major isoform of
CYP3A expressed in extrahepatic tissues and is thought to play an
important role in the metabolism of endogenous and exogenous
compounds in these tissues (Koch et al., 2002). Thus, the inhibition of
CYP3A5 by CBD may not only cause its interactions with other drugs
but may also disturb normal metabolic pathways of endogenous
compounds.
CYP3A7 is involved in the metabolism of dehydroepiandrosterone
3-sulfate and all-trans-retinoic acid in fetal livers (Kitada et al., 1987;
Chen et al., 2000). These endogenous compounds are essential for
normal growth and development of the fetus (Schuetz et al., 1993;
Means and Gudas, 1995). It has been previously reported that
marijuana use during pregnancy affects fetal growth (Zuckerman
et al., 1989; Hurd et al., 2005). In addition, it has been demonstrated
that cannabinoids are detected in animal fetuses after maternal
exposure to cannabinoids (Harbison and Mantilla-Plata, 1972; Bailey
et al., 1987), although such an investigation has not been conducted
with humans. Taken together with these findings, it is possible that
cannabinoids transferred to the fetus by marijuana use in pregnancy
Bornheim LM, Correia MA. Purification and characterization of the major hepatic
cannabinoid hydroxylase in the mouse: a possible member of the cytochrome P-
450IIC subfamily. Mol Pharmacol 1991;40:228–34.
Bornheim LM, Lasker JM, Raucy JL. Human hepatic microsomal metabolism of Δ1-
tetrahydrocannabinol. Drug Metab Dispos 1992;20:241–6.
Chan MY, Tse A. The effect of cannabinoids (Δ9-THC and Δ8-THC) on hepatic microsomal
metabolism of testosterone in vitro. Biochem Pharmacol 1978;27:1725–8.
Chen H, Fantel AG, Juchau MR. Catalysis of the 4-hydroxylation of retinoic acids by
CYP3A7 in human fetal hepatic tissues. Drug Metab Dispos 2000;28:1051–7.
ElSohly MA, Slade D. Chemical constituents of marijuana: the complex mixture of
natural cannabinoids. Life Sci 2005;78:539–48.
ElSohly MA, Ross SA, Mehmedic Z, Arafat R, Yi B, Banahan BF. Potency trends of delta9-
THC and other cannabinoids in confiscated marijuana from 1980–1997. J Forensic
Sci 2000;45:24–30.
Fernandes M, Warning N, Christ W, Hill R. Interactions of several cannabinoids with the
hepatic drug metabolizing system. Biochem Pharmacol 1973;22:2981–7.
Gohda H, Narimatsu S, Yamamoto I, Yoshimura H. In vivo and in vitro metabolism of
cannabidiol monomethyl ether and cannabidiol dimethyl ether in the guinea pig:
on the formation mechanism of cannabielsoin-type metabolite from cannabidiol.
Chem Pharm Bull (Tokyo) 1990;38:1697–701.
Gorski JC, Hall SD, Jones DR, VandenBranden M, Wrighton SA. Regioselective
biotransformation of midazolam by members of the human cytochrome P450 3A
(CYP3A) subfamily. Biochem Pharmacol 1994;47:1643–53.
Granfors MT, Wang JS, Kajosaari LI, Laitila J, Neuvonen PJ, Backman JT. Differential inhibition of
cytochrome P450 3A4, 3A5 and 3A7 by five human immunodeficiency virus (HIV)
protease inhibitors in vitro. Basic Clin Pharmacol Toxicol 2006;98:79–85.
Guengerich FP, Martin MV, Beaune PH, Kremers P, Wolff T, Waxman DJ. Characteriza-
tion of rat and human liver microsomal cytochrome P-450 forms involved in

736
S. Yamaori et al. / Life Sciences 88 (2011) 730–736
nifedipine oxidation, a prototype for genetic polymorphism in oxidative drug
metabolism. J Biol Chem 1986;261:5051–60.
Hamajima K, Watanabe K, Narimatsu S, Tateoka Y, Yamamoto I, Yoshimura H. Sex
difference in the effects of Δ9-tetrahydrocannabinol and cannabidiol on pentobar-
bital-induced sleeping time and hepatic microsomal drug metabolizing enzyme
systems in mice. Yakugaku Zasshi 1983;103:1289–97.
Harbison RD, Mantilla-Plata B. Prenatal toxicity, maternal distribution and placental
transfer of tetrahydrocannabinol. J Pharmacol Exp Ther 1972;180:446–53.
Huestis MA. Pharmacokinetics and metabolism of the plant cannabinoids, Δ9-tetrahydro-
cannabinol, cannabidiol and cannabinol. Handb Exp Pharmacol 2005;168:657–90.
Huestis MA, Henningfield JE, Cone EJ. Blood cannabinoids. I. absorption of THC and
formation of 11-OH-THC and THCCOOH during and after smoking marijuana. J Anal
Toxicol 1992;16:276–82.
Hurd YL, Wang X, Anderson V, Beck O, Minkoff H, Dow-Edwards D. Marijuana impairs
growth in mid-gestation fetuses. Neurotoxicol Teratol 2005;27:221–9.
Jaeger W, Benet LZ, Bornheim LM. Inhibition of cyclosporine and tetrahydrocannabinol
metabolism by cannabidiol in mouse and human microsomes. Xenobiotica
1996;26:275–84.
Kitada M, Kamataki T, Itahashi K, Rikihisa T, Kato R, Kanakubo Y. Purification and
properties of cytochrome P-450 from homogenates of human fetal livers. Arch
Biochem Biophys 1985;241:275–80.
Kitada M, Kamataki T, Itahashi K, Rikihisa T, Kanakubo Y. P-450 HFLa, a form of
cytochrome P-450 purified from human fetal livers, is the 16α-hydroxylase of
dehydroepiandrosterone 3-sulfate. J Biol Chem 1987;262:13534–7.
Koch I, Weil R, Wolbold R, Brockmöller J, Hustert E, Burk O, et al. Interindividual
variability and tissue-specificity in the expression of cytochrome P450 3A mRNA.
Drug Metab Dispos 2002;30:1108–14.
Komori M, Nishio K, Kitada M, Shiramatsu K, Muroya K, Soma M, et al. Fetus-specific
expression of a form of cytochrome P-450 in human livers. Biochemistry 1990;29:
4430–3.
Kuehl P, Zhang J, Lin Y, Lamba J, Assem M, Schuetz J, et al. Sequence diversity in CYP3A
promoters and characterization of the genetic basis of polymorphic CYP3A5
expression. Nat Genet 2001;27:383–91.
Ohlsson A, Lindgren JE, Andersson S, Agurell S, Gillespie H, Hollister LE. Single dose
kinetics of cannabidiol in man. In: Agurell S, Dewey WL, Willette R, editors. The
cannabinoids: chemical, pharmacologic, and therapeutic aspects. New York:
Academic Press; 1984. p. 219–25.
Pertwee RG. The diverse CB1 and CB2 receptor pharmacology of three plant
cannabinoids: Δ9-tetrahydrocannabinol, cannabidiol and Δ9-tetrahydrocannabi-
varin. Br J Pharmacol 2008;153:199–215.
Rendic S. Summary of information on human CYP enzymes: human P450 metabolism
data. Drug Metab Rev 2002;34:83-448.
Schuetz JD, Kauma S, Guzelian PS. Identification of the fetal liver cytochrome CYP3A7 in
human endometrium and placenta. J Clin Invest 1993;92:1018–24.
Shimada T, Yamazaki H, Mimura M, Inui Y, Guengerich FP. Interindividual variations in
human liver cytochrome P-450 enzymes involved in the oxidation of drugs,
carcinogens and toxic chemicals: studies with liver microsomes of 30 Japanese and
30 Caucasians. J Pharmacol Exp Ther 1994;270:414–23.
Thervet E, Anglicheau D, King B, Schlageter MH, Cassinat B, Beaune P, et al. Impact of
cytochrome P450 3A5 genetic polymorphism on tacrolimus doses and concen-
tration-to-dose ratio in renal transplant recipients. Transplantation 2003;76:
1233–5.
Watanabe K, Yamamoto I, Oguri K, Yoshimura H. In vitro and in vivo interactions of Δ8-
tetrahydrocannabinol and its metabolites with hepatic microsomal drug metabolizing
enzyme systems of mice. J Pharmacobiodyn 1981;4:604–11.
Watanabe K, Matsunaga T, Yamamoto I, Funae Y, Yoshimura H. Involvement of CYP2C in
the metabolism of cannabinoids by human hepatic microsomes from an old
woman. Biol Pharm Bull 1995;18:1138–41.
Watanabe K, Yamaori S, Funahashi T, Kimura T, Yamamoto I. Cytochrome P450
enzymes involved in the metabolism of tetrahydrocannabinols and cannabinol by
human hepatic microsomes. Life Sci 2007;80:1415–9.
Williams JA, Ring BJ, Cantrell VE, Jones DR, Eckstein J, Ruterbories K, et al. Comparative
metabolic capabilities of CYP3A4, CYP3A5, and CYP3A7. Drug Metab Dispos 2002;30:
883–91.
Wong M, Balleine RL, Collins M, Liddle C, Clarke CL, Gurney H. CYP3A5 genotype and
midazolam clearance in Australian patients receiving chemotherapy. Clin Pharma-
col Ther 2004;75:529–38.
Leighty EG. Metabolism and distribution of cannabinoids in rats after different methods
of administration. Biochem Pharmacol 1973;22:1613–21.
Liu YT, Hao HP, Liu CX, Wang GJ, Xie HG. Drugs as CYP3A probes, inducers, and
inhibitors. Drug Metab Rev 2007;39:699–721.
Wrighton SA, Ring BJ, Watkins PB, VandenBranden M. Identification of a polymorphi-
cally expressed member of the human cytochrome P-450III family. Mol Pharmacol
1989;36:97-105.
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin
phenol reagent. J Biol Chem 1951;193:265–75.
McConn II DJ, Lin YS, Allen K, Kunze KL, Thummel KE. Differences in the inhibition of
cytochromes P450 3A4 and 3A5 by metabolite-inhibitor complex-forming drugs.
Drug Metab Dispos 2004;32:1083–91.
Means AL, Gudas LJ. The roles of retinoids in vertebrate development. Annu Rev
Biochem 1995;64:201–33.
Mechoulam R, Parker LA, Gallily R. Cannabidiol: an overview of some pharmacological
aspects. J Clin Pharmacol 2002;42:11S–9S.
Yamaori S, Yamazaki H, Suzuki A, Yamada A, Tani H, Kamidate T, et al. Effects of cytochrome b5
on drug oxidation activities of human cytochrome P450 (CYP) 3As: similarity of CYP3A5
with CYP3A4 but not CYP3A7. Biochem Pharmacol 2003;66:2333–40.
Yamaori S, Yamazaki H, Iwano S, Kiyotani K, Matsumura K, Honda G, et al. CYP3A5
contributes significantly to CYP3A-mediated drug oxidations in liver microsomes
from Japanese subjects. Drug Metab Pharmacokinet 2004;19:120–9.
Yamaori S, Kushihara M, Yamamoto I, Watanabe K. Characterization of major
phytocannabinoids, cannabidiol and cannabinol, as isoform-selective and potent
inhibitors of human CYP1 enzymes. Biochem Pharmacol 2010;79:1691–8.
Zuckerman B, Frank DA, Hingson R, Amaro H, Levenson SM, Kayne H, et al. Effects of
maternal marijuana and cocaine use on fetal growth. N Engl J Med 1989;320:
762–8.
Molowa DT, Schuetz EG, Wrighton SA, Watkins PB, Kremers P, Mendez-Picon G, et al.
Complete cDNA sequence of a cytochrome P-450 inducible by glucocorticoids in
human liver. Proc Natl Acad Sci USA 1986;83:5311–5.