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Inhibition of Nicotine Metabolism by Cannabidiol (CBD) and
7‑Hydroxycannabidiol (7-OH-CBD)
Shamema Nasrin,
§
Shelby Coates,
§
Keti Bardhi,
§
Christy Watson, Joshua E. Muscat, and Philip Lazarus*
Cite This: https://doi.org/10.1021/acs.chemrestox.2c00259
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sı Supporting Information
ABSTRACT: Cannabis-based products have experienced notable increases in co-usage alongside tobacco products. Several
cannabinoids exhibit inhibition of a number of cytochrome P450 (CYP) and UDP glucuronosyltransferase (UGT) enzymes, but few
studies have examined their inhibition of enzymes involved in nicotine metabolism. The goal of the present study was to examine
potential drug−drug interactions occurring in the nicotine metabolism pathway perpetrated by cannabidiol (CBD) and its active
metabolite, 7-hydroxy-CBD (7-OH-CBD). The inhibitory eects of CBD and 7-OH-CBD were tested in microsomes from HEK293
cells overexpressing individual metabolizing enzymes and from human liver tissue. Assays with overexpressing microsomes
demonstrated that CBD and 7-OH-CBD inhibited CYP-mediated nicotine metabolism. Binding-corrected IC50,u values for CBD
inhibition of nicotine metabolism to cotinine and nornicotine, and cotinine metabolism to trans-3′-hydroxycotinine (3HC), were
0.27 ±0.060, 0.23 ±0.14, and 0.21 ±0.14 μM, respectively, for CYP2A6; and 0.26 ±0.17 and 0.029 ±0.0050 μM for cotinine and
nornicotine formation, respectively, for CYP2B6. 7-OH-CBD IC50,u values were 0.45 ±0.18, 0.16 ±0.08, and 0.78 ±0.23 μM for
cotinine, nornicotine, and 3HC formation, respectively, for CYP2A6, and 1.2 ±0.44 and 0.11 ±0.030 μM for cotinine and
nornicotine formation, respectively, for CYP2B6. Similar IC50,u values were observed in HLM. Inhibition (IC50,u = 0.37 ±0.06 μM)
of 3HC to 3HC-glucuronide formation by UGT1A9 was demonstrated by CBD. Significant inhibition of nicotine metabolism
pathways by CBD and 7-OH-CBD suggests that cannabinoids may inhibit nicotine metabolism, potentially impacting tobacco
addiction and cessation.
■INTRODUCTION
The co-use of cannabis and nicotine-based products has been
steadily increasing among adults in the United States,
coinciding with increased availability of cannabis and tobacco
vaping products.
1
In the National Survey on Drug Use and
Health, nearly all daily cannabis users are cigarette smokers.
2
It
is thought that this reflects shared psychosocial and environ-
mental risk factors for the use of these products.
3
While some
research has focused on adverse health outcomes of long-term
cannabis use and how co-use with tobacco may aect cannabis
outcomes, there is no data on the impact of cannabis use on
tobacco use behaviors. Specifically, the pharmacologic eect of
combined exposure to nicotine and cannabis compounds is
virtually unknown, with few studies focusing on how co-use
aects nicotine intake and metabolism. These patterns of co-
usage may result in unexpected drug−drug interactions (DDI)
as many of the compounds in tobacco and cannabis utilize the
same enzymatic systems for metabolism and detoxification.
Of the major cannabinoids in cannabis, cannabidiol (CBD)
interacts with the CB1 and CB2 receptors in the brain with a
much lower anity as compared to (−)-trans-Δ9-tetrahydro-
cannabinol (THC; the main psychoactive component of
cannabis), resulting in extremely low psychoactive eects.
4
However, CBD displays a broad range of potential therapeutic
Received: August 16, 2022
Articlepubs.acs.org/crt
© XXXX The Authors. Published by
American Chemical Society A
https://doi.org/10.1021/acs.chemrestox.2c00259
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applications, including anti-inflammatory properties, antipsy-
chotic and antiepileptic eects, as well as modulation of the
immune system and the central nervous system.
5
CBD usage is
rapidly expanding among many patient populations due in part
to its good safety profile, and recent industry statistics put
CBD usage higher than recreational marijuana usage among
the general population.
6
A 2019 Gallup poll found that 12% of
Americans consider themselves active users of recreational
marijuana, compared to 14% who reported regular CBD
usage.
7
CBD metabolism to its major active metabolite, 7-hydroxy-
CBD (7-OH-CBD), and its subsequent inactive metabolite, 7-
carboxy-CBD (CBD-COOH), is mediated by cytochromes
P450 (CYPs) 2C9, 2C19, and 3A4.
8
While the pharmacology
of 7-OH-CBD is not well studied, 7-OH-CBD exhibits a t1/2
that is longer than that of CBD and is found in the plasma of
CBD users. The plasma Cmax (maximum concentration) of
CBD following a single dose regimen of Epidiolex (6000 mg)
is 2.5 μM
9
with a calculated unbound plasma Cmax of 0.075
μM.
10
These values are similar for 7-OH-CBD, with the total
and calculated unbound plasma Cmax following this same
dosing regimen at 1.56
9
and 0.0936 μM,
10
respectively. 7-OH-
CBD exhibits anticonvulsant properties comparable to CBD
and is currently a patented medication for NAFLD, used to
lower blood triglyceride levels.
11,12
Nicotine is the major pharmacologically active substance in
tobacco products and e-cigarettes and is responsible for
mediating dependence. Nicotine is rapidly metabolized in vivo,
with a t1/2 of 1−2 h,
13
and smokers modulate their tobacco
consumption to achieve a brain nicotine concentration that
maintains the desired eects. The major metabolic pathway for
nicotine is oxidation by CYP2A6 to form the inactive
metabolite, cotinine (Figure 1), with some studies suggesting
that CYP2B6 plays a minor role in cotinine formation.
14
Nicotine undergoes additional metabolism through glucur-
onidation, catalyzed primarily by UGT2B10
15
to form nicotine
glucuronide (Nic-Gluc) and oxygenation by flavin monoox-
ygenases (FMOs) including FMO1 and FMO3
16
to form
nicotine-N′-oxide (NOX). Minor metabolites include norni-
cotine, formed primarily by CYPs 2A6 and 2B6, and 4-
hydroxy-4-(3-pyridyl)-butanoic acid (HPBA), possibly formed
by CYP2A6.
17−21
Cotinine is also metabolized by CYP2A6 to
form trans-3′-hydroxycotinine (3HC) and, like nicotine, can
undergo conversion by UGT2B10-mediated glucuronidation
to form cotinine glucuronide (Cot-Gluc)
22
or oxidation by
CYPs 2C19 and 2A6 to form cotinine N-oxide (COX).
16
3HC
is glucuronidated in vivo on the 3′-hydroxyl group, a reaction
mediated by UDP glucuronosyltransferases (UGTs) 1A9, 2B7,
and 2B17.
23
Functional variants of several of these metabolizing enzymes
were linked to altered nicotine metabolism in smokers,
15,16
and
individuals with reduced or nonfunctional CYP2A6 activity
were shown to be significantly less likely to become smokers,
and for those who do become smokers, they consumed fewer
cigarettes and scored lower for nicotine dependence.
24
Decreased expression or inhibition of CYP2A6 activity reduces
nicotine metabolism and has been suggested as a primary
target for tobacco cessation strategies, resulting in a reduction
Figure 1. Schematic of nicotine metabolic pathways.
Chemical Research in Toxicology pubs.acs.org/crt Article
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Chem. Res. Toxicol. XXXX, XXX, XXX−XXX
B
of the number of cigarettes necessary for a smoker to maintain
a given nicotine level leading to diminished smoking overall.
Previous studies suggest that CBD may be an eective
treatment for tobacco addiction.
25
In one study, the number of
cigarettes smoked was decreased by as much as 40% over the
course of treatment with a CBD inhaler.
26
Previous studies
have shown that cannabinoids and several major THC
metabolites can inhibit several important hepatic CYPs
27,28
as well as UGT enzymes.
29
The goal of the present study was
to examine the inhibitory potential of CBD and its major
metabolite, 7-OH-CBD, on nicotine metabolic pathways, with
the goal of better understanding the potential eect of these
cannabinoids on tobacco addiction.
■EXPERIMENTAL PROCEDURES
Chemicals and Reagents. CBD and 7-OH-CBD were purchased
from Cayman Chemicals (Ann Arbor, MI) or Sigma-Aldrich (St.
Louis, MO). Pooled human liver microsomes (HLM) [n= 50, mixed-
gender (21 female and 29 male), race (42 Caucasian, 4 Hispanic, 2
African American, and 2 Asian), and age (5−77 years old)] were
obtained from Sekisui Xenotech, LLC (Lenexa, Kansas). NADPH-
regenerating system (1.3 mM NADP, 3.3 mM glucose 6-phosphate,
and 0.4 U/mL glucose 6-phosphate dehydrogenase) was obtained
from Corning (Bedford, MA). Nicotine, nornicotine, HPBA, NOX,
Nic-Gluc, cotinine, COX, Cot-Gluc, 3HC, and 3HC glucuronide
(3HC-Gluc) standards were purchased from Sigma-Aldrich. Fluco-
nazole, benzydamine hydrochloride, clopidogrel, trifluoperazine,
diclofenac, amitriptyline, and tranylcypromine were also purchased
from Sigma-Aldrich. Optima grade methanol, acetonitrile, and formic
acid were obtained from Fisher Scientific (Waltham, MA). Ultra-low-
binding microcentrifuge tubes, Dulbecco’s modified Eagle’s medium,
Dulbecco’s phosphate-buered saline, UDP glucuronic acid
(UDPGA), alamethicin, MgCl2, and Geneticin (G418) were
purchased from VWR (Radnor, PA). BCA protein assays were
purchased from Pierce (Rockford, IL); premium-grade fetal bovine
serum (FBS) was purchased from Seradigm (Radnor, PA), and
ChromatoPur bovine serum albumin (BSA) was purchased from MB
Biomedicals (Santa Ana, CA).
Inhibition Assays. HEK293 cells individually overexpressing V5-
tagged CYP enzymes (CYP2A6, CYP2B6, and CYP2C19) and V5-
tagged FMO enzymes (FMO1 and FMO3) were developed and
described previously.
16
Microsomal membrane fractions of CYP- and
FMO-overexpressing cell lines were prepared by dierential
centrifugation as previously described, with protein concentrations
estimated using the BCA assay, as per the manufacturer’s
recommendations.
16
An initial screening of the inhibition potential
of CBD and 7-OH-CBD against individual CYP and FMO enzymes
was determined using microsomes (30−100 μg) from CYP- or FMO-
overexpressing HEK293 cell lines in reactions containing either 1 or
10 μM CBD or 7-OH-CBD as potential inhibitors, nicotine or
cotinine as substrates, 100 mM potassium phosphate buer (pH 7.4),
and 3 mM MgCl2in a final reaction volume of 30 μL. The nicotine
and cotinine concentrations used in these reactions are described in
Table 1 and were at or near their known Michaelis−Menten constant
values (KM) for a given enzyme (e.g., nicotine to cotinine, 100 μM;
cotinine to 3HC, 100 μM; nicotine to nornicotine, 50 μM; nicotine to
NOX, 1 mM; and cotinine to COX, 1 mM
14,16,19,30−33
). As CBD
exhibits extensive nonspecific binding (70−90%) to protein and
labware, low-binding 0.6 mL microcentrifuge tubes were used for all
reactions. Assays were preincubated for 5 min at 37 °C, initiated by
the addition of 1.8 μL of NADPH-regenerating system, and incubated
for 30 min at 37 °C. Reactions were terminated by the addition of 30
μL of ice-cold stop solution (acetonitrile/methanol; 1:1). Samples
were mixed on a vortex mixer and centrifuged at 17 000gfor 15 min at
4°C. The supernatant (∼50 μL) was transferred to an ultra-high-
performance liquid chromatography (UPLC) sample vial for
subsequent UPLC-MS/MS analysis. Reactions with HLM were
performed as described above for CYP- or FMO-overexpressing cell
microsomes except with a 15 min incubation time. As positive
controls for inhibition assays, tranylcypromine was added as an
inhibitor for CYP2A6,
34−36
fluconazole was added as an inhibitor for
CYP2C19,
37
and clopidogrel was added as an inhibitor of
CYP2B6.
38,39
As there are no known probe inhibitors of FMOs, a
probe substrate, the FMO3 substrate benzydamine, was added as a
potential inhibitor of FMOs 1 and 3.
40−43
Substrate (nicotine/
cotinine) without any inhibitor was used as the reference assay for
100% activity. All analyses were performed in triplicate.
Since purchased nicotine stocks had small amounts of nornicotine
as a contaminant, nornicotine peak areas were calculated after
subtracting out the “background” nornicotine peak observed within
the stock nicotine prep, as determined by UPLC.
HEK293 cells individually overexpressing UGT isoforms 1A4, 1A9,
2B7, 2B10, and 2B17 were developed and described previously.
44
Initial screenings of the inhibition potential of CBD and 7-OH-CBD
against UGTs 1A4, 1A9, 2B7, 2B10, and 2B17 were performed
similarly to that described above for CYP/FMO assays, using
microsomes from UGT-overexpressing HEK293 cell lines or HLM
(50−100 μg for both) in reactions containing either 10 or 100 μM
CBD or 7-OH-CBD, substrate at concentrations approaching their
known KM’s for a given enzyme (5 mM nicotine, 5 mM cotinine, or
30 mM 3HC
22,23,45
), 50 mM Tris−HCl buer (pH 7.4), and 5 mM
MgCl2in a final reaction volume of 25 μL. As higher concentrations
of cannabinoids were required for the UGT experiments, 2% BSA was
added to increase the solubility of cannabinoids as well as to sequester
inhibitory long-chain unsaturated fatty acids. Microsomes were
preincubated with alamethicin (50 μg/mg of microsomal protein)
on ice for 20 min prior to the addition of reaction components.
Reactions were then initiated by the addition of 4 mM UDPGA and
incubated for 60−120 min at 37 °C. Assays containing substrate
without any inhibitor were used as the reference assay for 100%
Table 1. IC50 Values
a
of CBD and 7-OH-CBD against in Microsomes from Recombinant CYP and UGT-Overexpressing Cells
and in HLM
CBD 7-OH-CBD
metabolite substrate substrate concentration
b
(μM) microsomes IC50 (μM) IC50,u
c
(μM) IC50 (μM) IC50,u
c
(μM)
cotinine nicotine 100 μM rec
d
CYP2A6 4.4 ±1.0 0.27 ±0.06 5.8 ±2.3 0.45 ±0.18
500 μM rec CYP2B6 4.2 ±2.7 0.26 ±0.17 15 ±5.7 1.2 ±0.44
500 μM HLM 10 ±2.9 0.98 ±0.28 21 ±5.2 2.0 ±0.49
3HC cotinine 100 μM rec CYP2A6 3.4 ±2.3 0.21 ±0.14 10 ±3.0 0.78 ±0.23
900 μM HLM 8.8 ±1.3 0.86 ±0.13 9.1 ±3.4 0.86 ±0.32
nornicotine nicotine 50 μM rec CYP2A6 3.7 ±2.2 0.23 ±0.14 2.1 ±0.97 0.16 ±0.08
100 μM rec CYP2B6 0.47 ±0.077 0.029 ±0.005 1.4 ±0.40 0.11 ±0.03
500 μM HLM 5.1 ±1.1 0.50 ±0.11 7.9 ±2.5 0.74 ±0.24
3HC-Gluc 3HC 30 mM rec UGT1A9 9.7 ±1.5 0.37 ±0.06 NA
e
NA
e
a
IC50 values are presented as mean ±SD of three independent experiments.
b
Shown are the concentrations of substrate used in the respective
assays for activity screenings and IC50 determinations.
c
IC50,u, binding-corrected IC50 values.
d
rec, recombinant.
e
NA, not analyzed.
Chemical Research in Toxicology pubs.acs.org/crt Article
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C
activity. All analyses were performed in triplicate. Reactions were
terminated by the addition of 25 μL of ice-cold stop solution
(acetonitrile/methanol; 1:1), mixed on a vortex mixer, and
centrifuged at 17 000gfor 15 min at 4 °C. The supernatant (∼40
μL) was transferred to a UPLC sample vial for subsequent UPLC-
MS/MS analysis. As positive controls for inhibition assays,
trifluoperazine was added as a potential inhibitor of UGT1A4,
46,47
diclofenac as a potential inhibitor of UGTs 1A9 and 2B7,
48,49
amitriptyline as a potential inhibitor of UGT2B10,
50
and imatinib as a
potential inhibitor of UGT2B17.
51
Incubation conditions were optimized for HLM and overexpressing
cell lines for amount of microsomal protein and reaction time, with
optimal conditions chosen based on the following criteria: (1)
Metabolite formation was linear with time and protein concentration.
(2) Substrate consumption was no more than 20% of the initial
amount. (3) Metabolite formation was reliably and reproducibly
detected by the UPLC−MS/MS method utilized.
Metabolite Detection. For nicotine metabolite detection, we
used a method identical to that described and validated in previous
studies.
33
Briefly, nicotine, NOX, Nic-Gluc, HPBA, cotinine, COX,
Cot-Gluc, 3HC, and 3HC-Gluc were detected using a UPLC (Waters
Acquity; Waters Corp, Milford, MA) coupled to a triple-quadrupole
mass spectrometer equipped with a Zspray electrospray ionization
interface (Waters Xevo TQD; Waters Corp) by multiple reaction
monitoring (MRM). While a nornicotine standard was not available
for the present studies, nornicotine was identified using both parent
and daughter scans to develop and confirm an MRM method for
nornicotine detection. Except for the detection of NOX, all samples
were injected (2−5μL) onto an Acquity UPLC BEH C18 column (1.7
μM, 2.1 ×100 mm). A 9 min gradient elution was used with mobile
phases A (0.1% formic acid in water) and B (100% methanol) as
follows: 1 min at 95% A; 5% B followed by a linear gradient for 7 min
to 5% A; 95% B, 1 min at 5% A; 95% B and re-equilibration for 1 min
at 95% A; 5% B. The flow rate was 0.4 mL/min, and the column
temperature was 40 °C. A UPLC-BEH-HILIC column (2.1 ×100
mm, 1.7 μm) was used for the detection of NOX, with 5 mmol/L
NH4AC in 50% acetonitrile as buer A and 100% acetonitrile as
buer B, with elution as follows: 20% buer A for 1.5 min, a linear
gradient to 100% buer A from 1.5 to 2.5 min, maintenance of 100%
buer A for 3 min, and a re-equilibrium step to the initial 20% buer
A conditions from 5.5 to 7 min (flow rate = 0.4 mL/min). The
injection volume was 2 μL using a column temperature of 30 °C.
Analytes were detected with the mass spectrometer operated in the
positive ion mode for all metabolites tested in this study using dwell
times of 10−53 ms and collision energy and cone voltages of 15−28
and 20−40 V, respectively. Ultrapure argon was used for collision-
induced dissociation. The mass transitions (Q1/Q3), collision
energies, and cone voltages for each substrate and metabolite are
listed in Table S1. The desolvation temperature was 500 °C, with 600
L/h of nitrogen gas for desolvation and 30 L/h for the cone, while the
temperature of the source was 120 °C. Observed metabolite retention
times (see Table S1) were compared with the retention times of
corresponding substrate or metabolite standards.
Assay accuracy and precision accuracy were validated on LC-MS/
MS by repeated sample quantification by preparing five individual
samples of reaction matrix containing 1 ppm cotinine, COX, or 3HC
standard, processing them for LC-MS/MS, and immediately
measuring cotinine, COX, or 3HC concentrations for each standard
sample as compared to individually prepared standard curves all
measured by LC-MS/MS as previously described.
52
The mean
recovery for the five samples was >88% while the coecient of
variation (CV) was 3.1% for these samples, suggesting high accuracy
and precision for these studies.
Determination of IC50 Values. CBD or 7-OH-CBD concen-
trations that inhibited relative enzyme activity by ≥50% at 10 μM for
CYPs and FMOs and 100 μM for UGTs were further investigated by
the determination of IC50 values for each inhibitor−enzyme
combination. IC50 determinations were performed in HLM and
microsomes from HEK293-overexpressing cell lines using multiple
concentrations of CBD and 7-OH-CBD ranging between 0.5 and 120
μM, with all determinations performed in three independent
experiments. Nonspecific binding constants (fu,inc) for CBD in
HEK293 microsomes and HLM were determined previously.
29,53
For
the fu,inc for 7-OH-CBD, the previously determined fu,inc for the
structurally similar 11-OH-THC
53
was used as a surrogate. IC50
values were determined using the following equation:
= + +ybottom (top bottom)/(1 10 )
x( logIC )
50
where “bottom” is the maximum observed inhibition (lowest percent
activity), and “top” is the minimum observed inhibition (highest
percent activity) for a given cannabinoid.
Statistical Analysis. Data were exported and analyzed using an
Excel spreadsheet (Microsoft). The amount of metabolite formed at
each concentration of inhibitor relative to the control (% relative
activity) was calculated as
=
×
% relative activity peak area of metabolite with inhibitor
/peak area of metabolite without inhibitor
100%
IC50 values were calculated by plotting the relative activity of each
enzyme versus the log concentration of the test inhibitors using
GraphPad Prism 7.04 software (GraphPad Software Inc., San Diego,
CA). IC50 for unbound drug (IC50,u) values were calculated using the
following equation: IC50,u = IC50 ×fu,inc.
■RESULTS
Metabolite peaks were detected by UPLC-MS/MS (Figure 2)
in assays utilizing substrates from the nicotine metabolic
pathway, as described above. Using nicotine as the substrate,
preliminary screening studies demonstrated that, in assays
containing CYP2A6-overexpressing cell microsomes, CBD and
7-OH-CBD inhibited cotinine formation by 55% and 50% at 1
μM, and 70% and 75% at 10 μM, respectively, as compared to
100% activity control reactions without added cannabinoid
(Figure 3A). A similar pattern was observed when using
cotinine as the substrate in CYP2A6-overexpressing cell
microsomes, with CBD and 7-OH-CBD decreasing 3HC
formation by 55% and 45% at 1 μM, and 60% and 65% at 10
μM, respectively, as compared to 100% activity control
reactions (Figure 3B). A similar pattern was also observed
for microsomes from CYP2B6-overexpressing cells, with 1 and
10 μM CBD exhibiting 50% and 65% inhibition, respectively,
and 1 and 10 μM 7-OH-CBD exhibiting 35% and 50%
inhibition, respectively, against cotinine formation using
nicotine as the substrate (Figure 3A). No inhibitory eect on
NOX formation was observed with either cannabinoid in
FMO1- or FMO3-overexpressing cell microsomes in assays
where nicotine was the substrate (Figure 3C), while only
marginal inhibition was observed for 10 μM CBD and 7-OH-
CBD against COX formation in microsomes from cells
overexpressing either CYP2C19 (30% and 15% inhibition,
respectively) or CYP2A6 (25% and 40%, respectively) with
cotinine as the substrate (Figure 3D).
As controls for the inhibition assays, potential inhibitors
exhibited the following levels of inhibition of nicotine
metabolism using microsomes from recombinant enzymes:
70% and 95% inhibition of cotinine and nornicotine formation,
respectively, in CYP2A6 microsomes by 10 μM tranylcypro-
mine, 68% inhibition of Nic-Gluc formation in UGT1A4
microsomes by 100 μM trifluoperazine, and 50 μM clopidogrel
exhibited 60% inhibition of cotinine formation by CYP2B6.
Similar levels of inhibition were observed when using cotinine
as the substrate (results not shown), with 55% of COX
Chemical Research in Toxicology pubs.acs.org/crt Article
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Chem. Res. Toxicol. XXXX, XXX, XXX−XXX
D
formation inhibited by 10 μM fluconazole in CYP2C19
microsomes. In addition, up to 65% inhibition was observed
for 3HC-Gluc formation in UGT2B7, UGT1A9, or UGT2B17
microsomes containing 200 μM imatinib, and nicotine-Gluc or
cotinine-Gluc formation in UGT2B10 microsomes containing
100 μM amitriptyline. Known inhibitors of FMO activity have
not been previously described, and no significant inhibition
was observed when adding up to 50 μM benzydamine as a
potential inhibitor of FMO activities in the present studies.
Screening for inhibition by CBD and 7-OH-CBD was also
performed for two additional nicotine metabolites (nornicotine
and HPBA) in both CYP2A6- and CYP2B6-overexpressing
microsomes. Both CBD and 7-OH-CBD exhibited inhibition
of nornicotine formation in CYP2A6-overexpressing cell
microsomes, with 60% and 55% inhibition observed with 1
μM CBD and 7-OH-CBD, respectively, and 75% and 70%
inhibition observed with 10 μM CBD and 7-OH-CBD,
respectively (Figure 3E). Similarly strong inhibition was also
observed for nornicotine formation in CYP2B6-overexpressing
cell microsomes, with 1 and 10 μM CBD exhibiting 75% and
85% inhibition, respectively, and 1 and 10 μM 7-OH-CBD
exhibiting 64% and 72% inhibition, respectively (Figure 3E).
No HPBA formation was observed with either CYP2A6- or
CYP2B6-overexpressing microsomes when nicotine was used
as substrate in the present studies, a pattern consistent with
that observed in previous studies demonstrating a lack of
HPBA formation activity by CYP2A6 with nicotine as the
substrate.
Previous studies have shown that CBD does not inhibit
nicotine-Gluc formation by UGT2B10.
29
In the present study,
no significant inhibition was observed for either CBD or 7-
OH-CBD for nicotine-Gluc or cotinine-Gluc formation in
microsomes from UGT2B10- or UGT1A4-overexpressing cells
using nicotine or cotinine as the substrate (Figure 3F,G). No
eect on 3HC-Gluc formation was observed for either
cannabinoid using 3HC as the substrate in microsomes from
cells overexpressing UGTs 2B17, 1A9, or 2B7 for 7-OH-CBD.
In contrast, 1 and 10 μM CBD exhibited 55% and 65%
decreases, respectively, in 3HC-Gluc formation for UGT1A9
microsomes, and 35% and 55% decreases, respectively, in
3HC-Gluc formation for UGT2B7 microsomes (Figure 3H).
The inhibitory eects of CBD and 7-OH-CBD were
extended to establish IC50 and binding-corrected IC50 values
(IC50,u) for CBD and 7-OH-CBD against both CYP and UGT
enzymes shown to be inhibited in the screening assays
(described above). As described above, the known fu,inc for
CBD in HLM or microsomes containing CYP- or UGT-
overexpressed enzymes
29,53
and the fu,inc for 11-OH-THC
53
as
a surrogate for the unbound fraction of 7-OH-CBD in HLM or
microsomes containing CYP-overexpressing enzymes were
used to calculate IC50,u values. Since IC50 values were not
calculated for 7-OH-CBD in UGT microsomal incubations (it
exhibited no significant inhibition of UGT enzymes in the
screening studies described above), determinations of un-
bound fractions for UGT assays including 7-OH-CBD were
not performed.
Inhibition curves for enzymes shown to be inhibited by CBD
and 7-OH-CBD are shown in Figure 4. CBD showed strong
inhibition of CYP2A6 activity in CYP2A6-overexpressing
microsomes using either nicotine or cotinine as substrate,
with IC50,u values of 0.27 ±0.06 and 0.21 ±0.14 μM for
cotinine and 3HC formation, respectively (Table 1). Similarly,
CBD inhibited CYP2B6-mediated metabolism of nicotine to
cotinine with an IC50,u value of 0.26 ±0.17 μM. Moreover,
CBD inhibited CYP2A6- and CYP2B6-mediated nicotine to
nornicotine formation with an IC50,u value of 0.23 ±0.14 and
Figure 2. Chromatograms of nicotine and its metabolites formed in
assays containing microsomes from CYP-overexpressing HEK293
cells and standards. The metabolites examined are described with the
mass transition shown in brackets. (A) Nicotine standard; (B)
cotinine standard; (C) cotinine formation in CYP2A6 microsomes;
(D) 3HC standard; (E) 3HC formation in CYP2A6 microsomes; (F)
nicotine-N′-oxide standard; (G) cotinine-N′-oxide standard; (H)
nicotine-Gluc standard; (I) cotinine-Gluc standard; (J) 3HC-Gluc
standard; (K) HPBA standard; and (L) nornicotine formation in
CYP2A6 microsomes.
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E
0.030 ±0.0050 μM, respectively. To confirm that the eects
observed by CBD in CYP-overexpressing microsomes are also
observed in human liver, inhibition assays were performed in
HLM, with similar IC50,u values observed for CBD in HLM
(0.98 ±0.28 μM for cotinine formation and 0.50 ±0.11 μM
for nornicotine formation) as compared to that observed in
incubations with microsomes from CYP2A6- or CYP2B6-
overexpressing cells. CBD also exhibited strong inhibition of
glucuronidation activity in UGT1A9-overexpressing micro-
somes against 3HC, with an IC50,u value of 0.37 ±0.06 μM.
While 100 μM CBD exhibited >50% inhibition of UGT2B7
activity in the activity screenings described above, IC50 values
could not be calculated for UGT2B7 due to inconsistent
inhibition at higher CBD concentrations with this enzyme
(results not shown). IC50 values could also not be calculated
for 3HC-Gluc formation for CBD in HLM (inhibition reached
<45% with up to 100 μM CBD), likely due to the presence of
other UGTs (i.e., UGT2B17) which also glucuronidate 3HC
but which are not inhibited by CBD.
7-OH-CBD also exhibited significant inhibition against both
cotinine and 3HC formation in reactions containing nicotine
or cotinine, with IC50,u values of 0.45 ±0.18 and 0.78 ±0.23
μM, respectively, in incubations containing microsomes from
CYP2A6-overexpressing cells (Figure 4B and Table 1). 7-OH-
CBD also showed strong inhibition of cotinine formation
(IC50,u = 1.2 ±0.44 μM) in incubations containing
microsomes from CYP2B6-overexpressing cells and showed
strong inhibition of nornicotine formation in incubations
containing nicotine and microsomes overexpressing either
CYP2A6 (IC50,u = 0.16 ±0.08 μM) or CYP2B6 (IC50,u = 0.11
±0.03 μM). Comparable IC50,u values of 2.0 ±0.49, 0.86 ±
0.32, and 0.74 ±0.24 μM were also observed for 7-OH-CBD
inhibition of cotinine, 3HC, and nornicotine formation,
respectively, in incubations containing HLM.
■DISCUSSION
The present study is the first to conduct a comprehensive
examination of the inhibitory eects of CBD and its active
metabolite 7-OH-CBD on nicotine metabolism, with the
enzymatic activities of each of the major enzymes individually
assessed for potential drug−drug interactions with these
cannabinoids. The results indicate that CBD and 7-OH-CBD
strongly inhibit CYP2A6 activity, as both cannabinoids
inhibited the formation of cotinine and 3HC in incubations
Figure 3. Screening of CBD and 7-OH-CBD inhibition of major nicotine metabolic pathways in microsomes from CYP-, FMO-, and UGT-
overexpressing HEK293 cells. Incubations were performed using 1 or 10 μM cannabinoid, with nicotine, cotinine, and 3HC concentrations at or
close to their known KMfor their corresponding enzyme: 100 μM, 500 μM, 1 mM, 50 μM, 100 μM, and 5 mM nicotine for cotinine (CYP2A6),
cotinine (CYP2B6), NOX (FMOs 1 and 3), nornicotine (CYP2A6), nornicotine (CYP2B6), and nicotine-Gluc (UGT2B10 and UGT1A4)
formation reactions, respectively; 100 μM (CYP2A6), 1 mM (CYP2C19 and CYP2A6), and 5 mM (UGT2B17) cotinine for 3HC, COX, and
cotinine-Gluc formation reactions, respectively; and 30 mM 3HC for 3HC-Gluc formation reactions (UGT1A9 and UGT2B7). (A) Nicotine to
cotinine formation; (B) cotinine to 3HC formation; (C) nicotine to NOX formation; (D) cotinine to COX formation; (E) nicotine to nornicotine
formation; (F) nicotine to nicotine-Gluc formation; (G) cotinine to cotinine-Gluc formation; and (H) 3HC to 3HC-Gluc formation. Shown are
the mean inhibitions of three individual experiments performed for each cannabinoid against nicotine, cotinine, or 3HC as substrate. Data are
expressed as a percentage of metabolite formation formed in assays with cannabinoid compared to assays without cannabinoid.
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F
containing CYP2A6-overexpressing microsomes, with non-
specific binding-corrected IC50,u values <0.30 μM for CBD and
<0.80 μM for 7-OH-CBD. Interestingly, the IC50,u values for
the metabolism of nicotine and cotinine by CYP2A6 and
CYP2B6 are similar. In addition, both CBD and 7-OH-CBD
inhibited the formation of the secondary nicotine metabolite,
nornicotine, in CYP2A6- or CYP2B6-overexpressing micro-
somal reactions, suggesting that the inhibition observed for
these cannabinoids was not metabolite-specific for nicotine
metabolites. This pattern of inhibition of nicotine metabolism
by CYP2A6 and CYP2B6 by CBD and 7-OH-CBD was also
observed in HLM. While the strong inhibition observed for
CBD and 7-OH-CBD for both enzymes is similar to that
observed for CBD in previous studies of several major hepatic
CYP450 enzymes including CYP2A6 and CYP2B6 using probe
substrates,
28,53
dierences in IC50,u values were observed for
the substrates used in the present study for both enzymes as
compared to the probe substrates used in those studies. This
may be due to dierences in the substrates used between
studies as inhibition can be substrate specific.
29,53
While IC50,u
values collected from in vitro experiments are not necessarily
good predictors of DDIs, the IC50,u values observed for both
CBD and 7-OH-CBD in both recombinant enzymes systems
and in HLM approach the physiological levels of CBD and 7-
OH-CBD in the plasma corrected for plasma protein binding
after a single clinical dose of CBD (0.06−0.15 μM CBD;
0.04−0.09 μM 7-OH-CBD
9,10
). With additional chronic or
frequent dosing of CBD, the physiologic unbound fractions of
plasma CBD and 7-OH-CBD are likely higher than the levels
necessary for an in vivo interaction. Together, this suggests that
these cannabinoids may be inhibiting overall nicotine
metabolism in smokers.
Significant inhibition of several other major nicotine
metabolism pathways was not observed by CBD or 7-OH-
CBD in the present studies. This includes the oxidation of
nicotine by FMOs 1 and 3 and the glucuronidation of nicotine
or cotinine by UGT2B10. This is consistent with previous
observations of FMOs not being shown to be subject to
induction or inhibition by most xenobiotics.
54
Additionally,
previous studies have also observed a lack of cannabinoid
inhibition of the UGT2B10-mediated glucuronidation of
nicotine.
29
The inhibition of UGT1A9-mediated 3HC-Gluc
formation by CBD observed in this study could potentially
cause increases in plasma 3HC in smokers. This could aect
the inherent ratio of plasma 3HC:cotinine, which is often used
as a biomarker of CYP2A6 activity.
55
A limitation of the present study was that the inhibition by
CBD or 7-OH-CBD of cotinine formation directly from the
nicotine Δ1′,5′-iminium ion was not tested in the present study.
While CYP2A6 has been previously shown to be active against
the iminium ion, aldehyde oxidase has also been shown to be
active.
56,57
However, it is likely that the inhibition of cotinine
formation from nicotine by CBD and 7-OH-CBD observed in
the present study was not due to an eect on aldehyde oxidase
since HLM and microsomal preparations of CYP2A6-over-
expressing HEK293 cells were used, which should have
minimal contamination by cytosolic proteins including
aldehyde oxidase. The results presented in this study are also
consistent with previous studies of both HLM and
recombinant CYP2A6 demonstrating cotinine formation
from nicotine.
19,58,59
Tobacco users are known to precisely titrate nicotine levels
throughout the day, finding the perfect balance between
reward and aversion.
60
While some nicotinic receptor agonists
Figure 4. Inhibitory eects of CBD and 7-OH-CBD in microsomes from CYP- and UGT-overexpressing HEK293 cells. (A) Incubations performed
using CBD as the inhibitor; (B) incubations performed using 7-OH-CBD as the inhibitor. Shown are averages of triplicate plots comparing CBD or
7OH-CBD concentration (X-axis) with the percent of control activity (Y-axis) against substrates in CYP- or UGT-overexpressing microsomes.
Incubations were performed for 30−120 min at 37 °C using 30−100 μg of microsomal protein, 0.5−120 μM of either CBD or 7-OH-CBD, and
100 μM (CYP2A6) and 500 μM (CYP2B6) nicotine for cotinine formation reactions, 50 μM (CYP2A6) and 100 μM (CYP2B6) nicotine for
nornicotine formation reactions, 100 μM cotinine for 3HC formation reactions, and 30 mM 3HC for 3HC-Gluc formation reactions. Individual
metabolites were analyzed by UPLC-MS/MS as described in the Experimental Procedures section.
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G
and partial agonists help tobacco users quit,
61
these therapies
have low success rates and significant side eects. Since greater
than 70% of nicotine is metabolized to cotinine by CYP2A6 in
the majority of tobacco users,
62
reducing CYP2A6-mediated
nicotine metabolism has been suggested as a primary target to
reduce the number of cigarettes necessary for a smoker to
maintain a given nicotine level, leading to diminished smoking
overall.
21,63
Functional variants of CYP2A6 (and consequent
variations in nicotine metabolism across individuals) are
known to aect human smoking behavior,
64
with individuals
carrying reduced-activity or nonfunctional CYP2A6 variants
(i.e., slow metabolizers) less likely to become smokers,
65−67
and those who do become smokers consuming significantly
fewer cigarettes
65,67,68
and scoring lower for nicotine depend-
ence.
65,67−69
The major endogenous function of CYP2A6 is
still unknown, and subjects homozygous for CYP2A6 low-
activity or deletion alleles (1:100 to 1:1000 people) have no
discernible biological or physical complications.
70
In addition,
there are few major drugs [selegiline (Ki= 4.6 μM) is one of
the few
71
] that are metabolized primarily by CYP2A6 and have
the potential for drug−drug interactions. While there are few
randomized trials of cessation stratified by CYP2A6 genotype,
the meta-relative risk of 6-month abstinence was 0.54 (95% CI
0.37−0.78) in normal vs slow metabolizers.
72
A systematic
review of the literature found that faster nicotine metabolizers
smoke more cigarettes per day (cpd) due to a decreased half-
life of nicotine.
73
Several studies focusing on CYP2A6 support
this, with CYP2A6 activity correlated with total cigarette
consumption in smokers of both Japanese and European
ancestry.
74
Faster CYP2A6-mediated nicotine metabolism was
shown to be associated with greater total daily pus and pu
volume.
75
In addition, a predictive nicotine metabolism model
based on CYP2A6 haplotypes showed a similar association
between nicotine oxidation to cotinine and cpd in African
American smokers,
76
and recent clinical studies recommend
the targeting of CYP2A6 activity for tobacco use disorder
treatment.
77
Therefore, with the vast majority of the
population having active CYP2A6, pharmacological reduction
of CYP2A6 activity is expected to mimic the eects of naturally
occurring CYP2A6 variants in the human population, reducing
both smoking behavior and dependence.
In a previous randomized, double-blind placebo-controlled
study, the ad-hoc use of CBD by inhaler in cigarette smokers
resulted in a significant ∼40% reduction of the number of
cigarettes smoked during treatment.
26
In addition, Hindocha et
al. conducted a series of studies in which they discovered that
vaping cannabis was associated with decreased tobacco
consumption, and a single 800 mg oral dose of CBD reduced
the salience and pleasantness of cigarette cues, compared with
a placebo group.
78
These results are consistent with the
inhibitory eects by CBD and 7-OH-CBD on CYP2A6-
mediated nicotine metabolism described in the present study,
with decreases in nicotine metabolism potentially leading to
increased plasma nicotine levels per cigarette smoked and a
reduction in the number of cigarettes smoked, thus
diminishing the adverse health eects of smoking. Further
investigations will be required to determine the potential for
CBD and potentially other cannabinoids as agents for tobacco
cessation therapy.
■ASSOCIATED CONTENT
*
sı Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.chemrestox.2c00259.
Mass spectrometry conditions used to analyze individual
nicotine metabolites (PDF)
■AUTHOR INFORMATION
Corresponding Author
Philip Lazarus −Department of Pharmaceutical Sciences,
College of Pharmacy and Pharmaceutical Sciences,
Washington State University, Spokane, Washington 99223,
United States; orcid.org/0000-0002-8686-0874;
Email: phil.lazarus@wsu.edu
Authors
Shamema Nasrin −Department of Pharmaceutical Sciences,
College of Pharmacy and Pharmaceutical Sciences,
Washington State University, Spokane, Washington 99223,
United States
Shelby Coates −Department of Pharmaceutical Sciences,
College of Pharmacy and Pharmaceutical Sciences,
Washington State University, Spokane, Washington 99223,
United States
Keti Bardhi −Department of Pharmaceutical Sciences, College
of Pharmacy and Pharmaceutical Sciences, Washington State
University, Spokane, Washington 99223, United States
Christy Watson −Department of Pharmaceutical Sciences,
College of Pharmacy and Pharmaceutical Sciences,
Washington State University, Spokane, Washington 99223,
United States
Joshua E. Muscat −Penn State Cancer Institute, Department
of Public Health Sciences, Penn State University College of
Medicine, Hershey, Pennsylvania 17033, United States
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.chemrestox.2c00259
Author Contributions
§
S.N., S.C., and K.B. contributed equally to this work. CRediT:
Shamema Nasrin conceptualization, data curation, formal
analysis, investigation, methodology, validation, visualization,
writing-original draft, writing-review & editing; Shelby Coates
data curation, formal analysis, validation, visualization, writing-
review & editing; Keti Bardhi data curation, formal analysis,
validation, visualization, writing-review & editing; Christy
Watson methodology, writing-original draft; Joshua E. Muscat
writing-original draft, writing-review & editing; Philip Lazarus
conceptualization, data curation, formal analysis, funding
acquisition, investigation, methodology, project administration,
resources, supervision, validation, visualization, writing-original
draft, writing-review & editing.
Funding
This work was supported by the National Institutes of Health
National Institutes of Environmental Health Sciences [Grants
R01-ES025460] to P.L.
Notes
The authors declare no competing financial interest.
Significance Statement. The present study is the first to
comprehensively examine the eects of CBD and its major
active metabolite, 7-OH-CBD, on the metabolism of nicotine.
Results from this study demonstrate that both CBD and 7-OH-
CBD inhibit nicotine metabolism by inhibiting several enzymes
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Chem. Res. Toxicol. XXXX, XXX, XXX−XXX
H
important in nicotine metabolism, including the major nicotine
metabolizing enzyme, CYP2A6. This inhibition has implica-
tions for co-users of tobacco and CBD, with decreases in
overall nicotine metabolism potentially influencing tobacco
addiction and cessation strategies.
■ACKNOWLEDGMENTS
The authors would like to thank Gang Chen for his helpful
contributions to the study. Figure
1
was created with
BioRender.com.
■ABBREVIATIONS
AUC, area under the curve; BCA, bicinchoninic acid; BSA,
bovine serum albumin; CBD, cannabidiol; CBN, cannabinol;
CBD-COOH, 7-carboxy-cannabidiol; 7-OH-CBD, 7-hydroxy-
cannabidiol; CYP, cytochrome P450; COX, cotinine-N-oxide;
Cot-Gluc, cotinine glucuronide; DDI, drug−drug interaction;
FBS, fetal bovine serum; FMO, flavin monooxygenase; HEK,
human embryonic kidney; HLM, human liver microsomes;
IC50, half-maximal inhibitory concentration; LC-MS/MS,
liquid chromatography-tandem mass spectrometry; MRM,
multiple reaction monitoring; NADP, β-nicotinamide adenine
dinucleotide phosphate; NOX, nicotine-N′-oxide; Nic-Gluc,
nicotine glucuronide; THC, (−)-trans-Δ9-tetrahydrocannabi-
nol; 3HC, trans-3′-hydroxycotinine; UDPGA, UDP glucuronic
acid; UPLC, ultraperformance liquid chromatography; HPBA,
4-hydroxy-4-(3-pyridyl)-butanoic acid; 3HC-Gluc, 3HC glu-
curonide
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