In vitro inhibition of carboxylesterase 1 by major cannabinoids and selected metabolites

Article abstract of DOI:10.1124/dmd.118.086074

The escalating use of medical cannabis and significant recreational use of cannabis in recent years has led to a higher potential for metabolic interactions between cannabis or one or more of its components and concurrently used medications. Although there have been a significant number of in vitro and in vivo assessments of the effects of cannabis on cytochrome P450 and UDP-glucuronosyltransferase enzyme systems, there is limited information regarding the effects of cannabis on the major hepatic esterase, carboxylesterase 1 (CES1). In this study, we investigated the in vitro inhibitory effects of the individual major cannabinoids and metabolites Δ9-tetrahydrocannabinol (THC), cannabidiol (CBD), cannabinol (CBN), 11-nor-THC-carboxylic acid, and 11-hydroxy-THC on CES1 activity. S9 fractions from human embryonic kidney 293 cells stably expressing CES1 were used in the assessment of cannabinoid inhibitory effects. THC, CBD, and CBN each exhibited substantial inhibitory potency, and were further studied to determine their mechanism of inhibition and kinetic parameters. The inhibition of CES1 by THC, CBD, and CBN was reversible and appears to proceed through a mixed competitive-noncompetitive mechanism. The inhibition constant (K_i) values for THC, CBD, and CBN inhibition were 0.541, 0.974, and 0.263 mM (0.170, 0.306, and 0.0817 mg/ml), respectively. Inhibition potency was increased when THC, CBD, and CBN were combined. Compared with the potential unbound plasma concentrations attainable clinically, the K_i values suggest a potential for clinically significant inhibition of CES1 by THC and CBD. CBN, however, is expected to have a limited impact on CES1. Carefully designed clinical studies are warranted to establish the clinical significance of these in vitro findings.

Full text of DOI:10.1124/dmd.118.086074

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In vitro inhibition of Carboxylesterase 1 by Major Cannabinoids and Selected
Metabolites
Yuli Qian, M.S.1 Xinwen Wang, Ph.D.2 and John S. Markowitz, Pharm.D.1ǂ
1 Department of Pharmacotherapy and Translational Research, University of Florida,
Gainesville, Florida, USA
2 Department of Clinical Pharmacy, University of Michigan, Ann Arbor, Michigan, USA
1

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Running title: Cannabinoid inhibition of carboxylesterase 1
ǂ Corresponding Author:
John S. Markowitz, Pharm.D.
Department of Pharmacotherapy and Translational Research
University of Florida College of Pharmacy
1600 SW Archer Road, RM PG-23
Gainesville, FL 32610-0486
Tel: 352-273-6262; Fax: 352-273-6121
E-mail: jmarkowitz@cop.ufl.edu
No. of text pages (including references): 26
No. of tables: 2
No. of figures: 6
No. of references: 61
No. of words in Abstract: 257
No. of words in Introduction: 750
No. of words in Discussion: 1556
Nonstandard abbreviations:
CES1, carboxylesterase 1; THC, ∆9-tetrahydrocannabinol; CBD, cannabidiol; CBN,
cannabinol; THC-COOH, 11-nor-∆9-tetrahydrocannabinol-carboxylic acid; 11-OH-THC,
11-Hydroxy-∆9-tetrahydrocannabinol; CYP, cytochrome P450; UGT, UDP-
glucuronosyltransferase; OST, Oseltamivir phosphate; OC, Oseltamivir carboxylate;
PBS, Phosphate buffered saline; HEPES, 4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid; Cmax, maximum plasma concentration; DDI, drug-drug
interaction.
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Abstract
The escalating use of medical cannabis and significant recreational use of cannabis in
recent years has led to higher potential for metabolic interactions between cannabis or
one or more of its components and concurrently used medications. Although there have
been a significant number of in vitro and in vivo assessments of the effects of cannabis
on cytochrome P450 and UDP-glucuronosyltransferase enzyme systems, there is
limited information regarding the effects of cannabis on the major hepatic esterase,
carboxylesterase 1 (CES1). In this study, we investigated the in vitro inhibitory effects of
the individual major cannabinoids and metabolites ∆9-tetrahydrocannabinol (THC),
cannabidiol (CBD), cannabinol (CBN), 11-nor-∆9-tetrahydrocannabinol-carboxylic acid
(THC-COOH), and 11-Hydroxy-∆9-tetrahydrocannabinol (11-OH-THC) on CES1
activity. S9 fractions from human embryonic kidney (HEK 293) cells stably expressing
CES1 were utilized in the assessment of cannabinoid inhibitory effects. THC, CBD, and
CBN each exhibited substantial inhibitory potency, and were further studied to
determine their mechanism of inhibition and kinetic parameters. The inhibition of CES1
by THC, CBD, and CBN was reversible and appears to proceed through a mixed
competitive-noncompetitive mechanism. The Ki values for THC, CBD, and CBN
inhibition were 0.541, 0.974, and 0.263 µM (0.170, 0.306, and 0.0817 µg/ml),
respectively. Inhibition potency was increased when THC, CBD, and CBN were
combined. Compared to the potential unbound plasma concentrations attainable
clinically, the Ki values suggest a potential for clinically significant inhibition of CES1 by
THC and CBD. CBN however, is expected to have limited impact on CES1. Carefully
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designed clinical studies are warranted to establish the clinical significance of these in
vitro findings.
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Introduction
Cannabis (Cannabis sativa L.; marijuana) is the most commonly abused substance
globally and in the US (WHO, 2016; SAMHSA, 2017). In 2017, an estimated 26 million
Americans aged 12 or older reported current (past month) marijuana use, a significant
increase since 2002 (14.6 million) (SAMHSA, 2017). Beyond its widespread recreational
use, the use of cannabis or one or more of its components for therapeutic purposes, i.e.
“medical cannabis”, has attracted much interest in recent years from both the lay public
and medical community. Medical cannabis is most commonly utilized to alleviate the
symptoms (e.g. chronic pain, nausea, anorexia, seizure, etc.) associated with cancers,
epilepsy, glaucoma, human immunodeficiency virus/acquired immunodeficiency
syndrome (HIV/AIDS), and others (Bridgeman and Abazia, 2017). As of November
2018, the medical use of cannabis or its components in the US was legal in 33 US
States and the District of Columbia. Furthermore, in June 2018, the US Food and Drug
Administration (FDA) approved cannabidiol (CBD) oral solution for the treatment of
seizures associated with two rare and severe forms of epilepsy, Lennox-Gastaut
syndrome and Dravet syndrome, in patients two years of age and older (FDA News
Release, 2018).
With increasing cannabis use, the potential for cannabis or one or more of its physically
active constituents (cannabinoids) and their major metabolites interacting with other
therapeutic agents also increases. This risk of drug-drug interactions (DDI) could be
substantial given that the diseases and conditions most frequently targeted for
treatment with medical cannabis are chronic in nature and treated with conventional
medications concurrently. A number of studies have been conducted to evaluate the
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effect of cannabis/cannabinoids on major drug metabolizing enzymes (DMEs). In vitro,
the major cannabinoids ∆9-tetrahydrocannabinol (THC), CBD, and cannabinol (CBN)
were found to either inhibit or induce the cytochrome P450 (CYP) and UDP-
glucuronosyltransferase (UGT) through various mechanisms and extents. Specifically,
CYP3A4/5/7, 2D6, 2C9/19, 2A6, 2B6, 1A1/2, 1B1, and 2J2 were generally inhibited by
those cannabinoids (Yamaori et al., 2010; Yamaori et al., 2011a; Yamaori et al., 2011b;
Yamaori et al., 2011c; Yamaori et al., 2012; Jiang et al., 2013; Arnold et al., 2018), while
CYP2C9 and mRNA of CYP1A1 were induced by THC (Roth et al., 2001; Bland et al.,
2005). As for UGT, UGT1A9 was inhibited by both CBD and CBN, and UGT2B7 was
inhibited by CBD but induced by CBN (Al Saabi et al., 2013). In clinical assessments,
CBD oral solution was reported to significantly elevate blood/plasma/serum
concentrations of antiepileptic drugs including clobazam, N-desmethylclobazam (nCLB,
the primary active metabolite of clobazam), rufinamide, topiramate, zonisamide, and
eslicarbazepine (Geffrey et al., 2015; Gaston et al., 2017; Devinsky et al., 2018). In
addition, chronic exposure to smoked cannabis was associated with decreases in the
peak plasma concentration of indinavir and an increase in the clearance of theophylline
(Jusko et al., 1978; Jusko et al., 1979; Kosel et al., 2002), suggesting induction of the
expression of CYP3A4 and CYP1A2.
Besides CYP450 and UGT enzymes, hydrolases are among the most essential
enzymes catalyzing the metabolism of numerous frequently prescribed therapeutic
agents (Williams et al., 2004; Laizure et al., 2013; Cerny, 2016). Human
carboxylesterase 1 (CES1) is the predominant hepatic esterase contributing to 80-95%
of total hydrolytic activity in human liver (Imai et al., 2006; Ross and Crow, 2007) and
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hydrolyzes therapeutic agents from nearly every drug class formulated as carboxylic
acid esters, amides, thioesters, and carbamates, among which specific agents may rely
on CES1 for both deactivation/detoxification (e.g. methylphenidate) or metabolic
activation (e.g. oseltamivir) (Takai et al., 1997; Satoh and Hosokawa, 2006; Laizure et
al., 2013). CES1 also catalyzes the hydrolysis of a large number of endogenous
compounds including short- and long-chain acyl-glycerols, long-chain acyl-carnitine, and
long-chain acyl-coenzyme A (CoA) esters (Lian et al., 2018). In vitro studies have
reported significant inhibition of CES1 by specific therapeutic agents suggesting
potential for clinically significant DDIs when used concurrently with other CES1
substrate medications (Fukami et al., 2010; Zhu et al., 2010; Rhoades et al., 2012;
Thomsen et al., 2014). Additionally, an expanding number of natural products and
traditional medicines have also been implicated as CES1 inhibitors (Wang et al., 2017).
Given the increasing use of medical and recreational cannabis, the potential for DDIs
between cannabis and CES1 substrates warranted investigation. In the present study,
we assessed the possible influence of three major cannabinoids (THC, CBD, and CBN)
and two major metabolites (11-nor-∆9-tetrahydrocannabinol-carboxylic acid (THC-
COOH) and 11-Hydroxy-∆9-tetrahydrocannabinol (11-OH-THC) (Fig. 1) on CES1
activity using an in vitro CES1 assay system. Also investigated were the mechanisms
and the in vitro potency of CES1 inhibition.
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Materials and Methods
Materials. Oseltamivir phosphate (OST) was purchased from Sequoia Research
Products Ltd. (Pangbourne, United Kingdom). Oseltamivir carboxylate (OC) was
purchased from Toronto Research Chemicals Inc. (Ontario, Canada). Ritalinic acid,
Montelukast, THC, CBD, CBN, THC-COOH, and 11-OH-THC were purchased from
Cayman Chemical (Ann Arbor, MI). Phosphate buffered saline (PBS) and 1M
HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) were the products from
Thermo Fisher Scientific Inc. (Waltham, MA). All other chemicals and reagents were of
the highest analytical grade and were commercially available.
Preparation of cell S9 fractions containing wild-type CES1. Human embryonic
kidney cells (Flp-In-293, Invitrogen, Carlsbad, CA) stably expressing wild-type CES1
have been established and described previously (Zhu et al., 2008). Cells from the
established cell line were cultured in Dulbecco’s modified Eagle’s medium with 10%
fetal bovine serum and 100 µg/ml hygromycin B until reaching approximately 90%
confluence. Cells were then harvested in PBS and sonicated. Afterwards, the cell
lysates were centrifuged at 9000xg for 30 min at 4 °C, from which the supernatant (S9
fraction) was collected and stored in -70°C freezer until use. The total protein
concentrations in S9 fractions were determined using a Pierce BCA assay kit (Thermo
Fisher Scientific Inc., Waltham, MA).
Metabolite formation. The active OST metabolite OC, a product of OST hydrolysis
(Fig. 2), was formed by incubating OST with S9 fractions in 2-ml tubes at a final volume
of 100 µl. Various concentrations of substrate and S9 fractions were prepared
separately in reaction buffers (PBS with 10 mM HEPES). The reaction was initiated by
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addition of substrate to S9 fractions mixture in a water bath at 37°C. Our preliminary
study indicated that the formation of OC from OST was linear over S9 protein
concentrations of 0 – 100 µg/ml and over incubation times of 0 – 30 min. Accordingly, a
final S9 protein concentration of 20 µg/ml and a 15-min incubation time were chosen as
the standard reaction condition for ensuing inhibition studies. After incubation, the
reaction was terminated by adding a 4-fold volume of methanol (400 µl) containing 100
nM ritalinic acid as the internal standard. The mixture was centrifuged at 16,100 × g for
5 min, and the supernatant was further diluted with water/methanol before being
transferred to HPLC vials for LC-MS/MS analysis.
Screening of selected cannabinoids and their metabolites as CES1 inhibitors. A
preliminary screening assay was conducted to identify the selected cannabinoids and
metabolites for their potential to inhibit CES1 activity. THC, CBD, CBN, THC-COOH,
and 11-OH-THC were screened at a single concentration of 5 µg/ml. We have
previously shown the leukotriene receptor antagonist montelukast to significantly inhibit
CES1 activity and it was employed as a positive control (10 µM) (Rhoades et al., 2012).
The reaction was initiated by mixing S9 fractions (final concentration 20 µg/ml) with OST
(final concentration 500 µM) and tested compounds. The final incubation volume was
100 µl with 1% DMSO. Following the incubation at 37°C for 15 min, the reactions were
terminated and prepared for the LC-MS/MS analysis as described in the metabolite
formation section above.
Evaluation of time-dependent inhibition by selected cannabinoids. To determine
whether the inhibition on CES1 by any of the tested cannabinoids (THC, CBD, and
CBN) is irreversible (mechanism-based), the inhibitory potency of those cannabinoids
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was evaluated with and without a 30-min pre-incubation of them with S9 fractions before
addition of substrate (OST). The samples were divided into experimental and control
groups. In the experimental samples, cannabinoid (final concentration 0 – 50 µg/ml [0 –
161 µM], pre-dissolved in 0.5-µl DMSO due to its insolubility in aqueous buffer) was
mixed with S9 fractions (final concentration 20 µg/ml) for a 30-min pre-incubation at
37°C. After the pre-incubation, OST (final concentration1 mM) was added into the
mixture to initiate the reaction. In the control group, samples were prepared in the same
way as the experimental group except that cannabinoid was added together with OST
after the pre-incubation. To avoid the potential influence introduced by a varied solvent
(DMSO) effect among different samples, the final DMSO concentration (v/v) in all
samples were adjusted to 0.5% during the pre-incubation and 1% during the incubation.
After incubation for 15 min, the reactions were terminated and the analytical procedures
were as described above in the metabolite formation section.
In vitro inhibition study with tested cannabinoids. For determination of the inhibition
constant (Ki) and the type of inhibition, kinetic studies were performed with varying
concentrations of substrate (0, 100, 500, 1000, 2500, and 5000 µM) and tested
cannabinoids (THC, CBD, and CBN concentration, 0 – 10 µg/ml [0 – 32.2 µM]). The
pre-mixture contained S9 fractions (final concentration 20 µg/ml) and cannabinoid in
reaction buffer, and the reactions were initiated by adding substrate into the pre-mixture.
The final incubation volume was 100 µl with 1% DMSO. Following incubation at 37°C for
15 min, the samples were further prepared for the LC-MS/MS assay as described in the
metabolite formation section above.
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Assessment of CES1 inhibition by combined cannabinoids. Since multiple
cannabinoids are likely to present in the systemic circulation and liver after the more
common types of cannabis use and routes of administration (e.g. smoking), we further
assessed whether the inhibition on CES1 would be increased by the combination of the
major cannabinoid constituents THC, CBD, and CBN. The final concentration of THC,
CBD, and CBN utilized in experiments were 0.200 µg/ml (0.636 µM), 0.133 µg/ml (0.424
µM), and 0.133 µg/ml (0.430 µM), respectively. Those concentrations were
approximated with consideration to their calculated Ki and potentially achievable plasma
concentrations reported in different clinical studies (Ohlsson et al., 1986; Johansson et
al., 1987; Huestis et al., 1992). Cannabinoids were prepared in glass vials before mixing
with S9 fractions (final concentration 20 µg/ml) to minimize absorption into tube walls.
The reaction was initiated by adding OST (500 µM) to pre-mixture of S9 fractions and
cannabinoids. The final incubation volume was 100 µl with 1% DMSO. After a 15 min
incubation at 37°C, the samples were prepared for LC-MS/MS assay as described in
the metabolite formation section above. The degree of inhibition was compared
between samples containing individual cannabinoids versus the three combined
cannabinoids.
LC-MS/MS analysis. Oseltamivir carboxylate (OC), the active hydrolytic metabolite of
OST, was determined using a Shimadzu HPLC system (Shimadzu, Kyoto, Japan)
coupled to an AB Sciex API 3000 triple-quadrupole mass spectrometer (Applied
Biosystems, Foster City, CA). Chromatographic separation was achieved on a C18
reverse phase analytic column (Aqua, 50 × 2.0 mm, 5 µm, Phenomenex Inc., Torrance,
CA). A gradient mobile phase was employed, with the aqueous phase containing 0.1%
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formic acid in water, and the organic phase containing methanol. The mobile phase was
delivered at a total flow rate of 0.3 ml/min. Mass spectrometric analysis was performed
via electrospray ionization in positive mode, and the mass transitions of m/z 285.3 →
138.3 and 220.2 → 84.4 were monitored for OC and ritalinic acid, respectively. The
lower limit of quantification (LLOQ) of OC was estimated to be 25 nM. The inter- and
intra- day assay precision as well as accuracy were within 10%. A representative
chromatogram obtained from incubation of S9 fractions with 100 µM OST was shown in
Fig. 3.
Data analysis. In accordance with a previous report (Hoffmann et al., 2009), some
spontaneous hydrolysis of OST in aqueous buffer was observed in our study. Therefore,
in the kinetic analysis, the metabolite amount formed in control samples without S9
fractions was subtracted from that in samples with the same substrate concentrations.
In the time-dependent inhibition study, metabolite (OC) formation rate was expressed as
a percentage ratio (Rv) of the rate in the control sample without inhibitor. The
independent variable was inhibitor concentration ([I]). The half maximal inhibition
concentration (IC) was estimated by fitting the following equation (Eq.1) into the
experimental data using non-linear regression:
[ ]푏
+ 퐼퐶푏
퐼푚푎푥 ∙ 퐼
푅푣 = 100 × (1 −
)
(1)
[ ]푏
퐼
Additional iterated parameters were: Imax, the maximal degree of inhibition; and b, a
shape exponent. The IC50 (inhibitor concentration that achieves 50% Rv) was calculated
as follows (Eq. 2):
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1/푏
(
)
퐼퐶50 = 퐼퐶/ 2퐼푚푎푥 − 1
(2)
In the in vitro inhibition study, the metabolite formation rate (V) was expressed as the
dependent variable of the substrate ([S]) and inhibitor ([I]) concentration. A mixed
competitive-non-competitive inhibition model derived from Michaelis-Menten kinetics
(Eq.3) was utilized to evaluate the type of inhibition and to determine the inhibition
constant of tested cannabinoids. Non-linear regression analysis was performed to fit the
model into the experimental data:
푉
∙ [푆]
푚푎푥
푉 =
(3)
[ ]
[퐼]
훼 ∙ 퐾
퐼
퐾푚 (1 + ) + [푆](1 +
)
퐾
푖
푖
Iterated parameters were: Vmax, the maximum metabolite formation rate; Km, the
Michaelis-Menten constant; Ki, the inhibition constant for inhibitor; and α, an indicator of
the inhibition type (an α approaching positive infinity indicates competitive inhibition, an
α equal to 1 indicates non-competitive inhibition, and an α falling in between indicates
mixed inhibition). The majority of notations of kinetic parameters and symbols are
adapted from the recommendations of Segel (Segel, 1975).
The type of inhibition was also verified by visual inspection of Lineweaver-Burk plots of
the experimental data. The data points at lowest OST concentration (100 µM) were not
shown in the plots for easier visual assessment of the line cross point and thereby the
inhibition type. In addition, the data group with highest CBN concentration (10 µg/ml
[32.2 µM]) was excluded because too few metabolites were formed.
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The potential of clinical DDIs was assessed using the predicted ratio of the area under
plasma concentration-time curve (AUC) of substrate with the presence of inhibitor over
that without. The predicted ratio (RAUC) was calculated as follows (Eq. 4):
푅퐴푈퐶 = 1 + 퐼푚푎푥,푢/퐾
(4)
푖
Imax,u is the maximal unbound plasma concentration of the inhibitor observed in clinical,
and Ki is the estimated inhibition constant in vitro.
Software and statistical methods. The parameter estimation was performed in R
3.4.2 (R Foundation for Statistical Computing, Vienna, Austria) using “nls” package with
least square approach. The estimation algorithm was Gauss-Newton. The model
performance was assessed by diagnostic residual plots, visual check, and certainty of
parameter estimates. Data visualization was performed in R 3.4.2 using “ggplot2”
package. Lineweaver-Burk plots were made using Microsoft Excel (Redmond, WA).
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Results
Screening of selected cannabinoids and their metabolites. The relative formation
velocities of OC in experimental samples were expressed as a percentage of the
results observed in the negative control containing only S9 fractions and substrate.
Decreased velocities were observed for all tested compounds (THC, CBD, CBN,
THC-COOH, 11-OH-THC) indicating their inhibitory effects on CES1 (Fig. 4). At a
concentration of 5 µg/ml, inhibition of greater than 90% was observed for THC, CBD,
and CBN, while only less than 50% CES1 inhibition was observed for THC-COOH
and 11-OH-THC. Considering the degree of systemic exposures to all of the tested
compounds in humans after cannabis/cannabinoid administration, only THC, CBD,
and CBN were further investigated.
THC, CBD, and CBN reversibly inhibited oseltamivir hydrolysis. The formation
rates of OC from OST were decreased by THC, CBD, and CBN in a concentration-
dependent manner (Fig. 5). The IC50 for each cannabinoid was computed and
compared between samples in which the S9 fractions were pre-incubated with
cannabinoids for 30 min before addition of substrate (Fig.5A), and those in which
cannabinoids were added after the pre-incubation (Fig.5B). A 30-min pre-incubation
with any of the tested cannabinoids did not increase their inhibition potency (Table 1),
indicating that their inhibition on CES1-mediated OST hydrolysis was not time-
dependent.
In vitro kinetic study on inhibition of CES1 activity by selected cannabinoids.
An in vitro study was conducted at varying concentrations of substrate (OST, 0 –
5000 µM) and tested cannabinoids (0 – 10 µg/ml [0 – 161 µM]). Nonlinear regression
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analysis was performed to characterize the type of inhibition and estimate kinetic
parameters (Fig. 6A1, B1, and C1), which were summarized in Table 2. The Ki values
for THC, CBD, and CBN inhibition of OST hydrolysis were 0.541, 0.974, and 0.263
µM (0.170, 0.306, and 0.0817 µg/ml), respectively. Based on the Ki values, the rank
order of in vitro inhibition potency of the tested cannabinoids was CBN > THC > CBD.
The values of α and Lineweaver-Burk plots (Fig. 6A2, B2, and C2) indicated that
inhibition of CES1 by all three cannabinoids followed a mixed competitive-
noncompetitive model, with characteristics more closely resembling a noncompetitive
model.
Increased inhibition by combined THC, CBD, and CBN. When THC, CBD, and
CBN were incubated individually and in combination with CES1, the combined
cannabinoids exhibited higher extent of inhibition than that observed in any individual
group (Fig. 7).
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Discussion
Increased medical and recreational use of cannabis/cannabinoids and concurrent use of
conventional medications poses a risk of DDI. We evaluated the potential for major
cannabinoids and selected metabolites to inhibit CES1. We identified the three major
cannabinoids, THC, CBD, and CBN, as potent inhibitors of CES1 in vitro. Pre-incubation
of S9 fractions with each of the compounds did not result in potentiation of inhibition
indicating reversible characteristics. Furthermore, the inhibition mechanisms were
revealed via the parameter α using the reversible inhibition model (Eq. 3) as well as
visual inspection of Lineweaver-Burk plots. The estimated α values for inhibition of
CES1 by THC, CBD, and CBN were 14.0, 5.8, and 12.0, respectively (Table 1),
demonstrating that the inhibition type was mixed competitive-noncompetitive with
characteristics more closely resembling noncompetitive inhibition for all three
cannabinoids. Lineweaver-Burk plots confirmed this result.
The potential for clinically relevant DDI between tested cannabinoids and CES1
substrates was evaluated comparing the in vitro inhibition constant (Ki) from our study
results to the potentially achievable inhibitor concentrations in humans. A predicted
change (RAUC) in the substrate drug AUC by tested cannabinoids was calculated by Eq.
4. According to both FDA and the European Medicines Agency, a RAUC value larger
than 1.02 warrants further investigations into the DDI potential using either mechanistic
models or formal clinical study (European Medicines Agency, 2012; Food and Drug
Administration, 2017). The exposure to the various cannabis constituents as determined
by blood sampling of the systemic circulation varies greatly and is influenced by the
formulation used, dosing routes (e.g. oral vs sublingual vs smoking, vs vaporizing) and
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dosing schedule (Huestis 2007). Maximal exposure scenarios were assumed in our
evaluation where the highest observed plasma concentrations of tested cannabinoids
were utilized. Smoking, the most common route of cannabis use, generally renders the
highest THC exposures relative to vaporized and orally ingested cannabis/cannabinoids
(Grotenhermen, 2003; Huestis, 2007; Lucas et al., 2018). In a clinical study the peak
plasma concentrations (Cmax) of THC in six healthy males averaged 162 ng/ml (0.515
µM) after smoking a cannabis cigarette containing 3.55% THC (Huestis et al., 1992).
Similar THC concentration ranges were observed in other studies where THC was
administered by smoking cannabis cigarettes (Lindgren et al., 1981; Perez-Reyes et al.,
1982; Lee et al., 2015). The contents of CBD and CBN in cannabis preparations were
much lower than that of THC (average 4.5% THC, 0.4% CBD, and 0.3% CBN in
cannabis during 1993 - 2008) (Mehmedic et al., 2010). After smoking a cigarette
containing 19 mg of deuterium-labeled CBD, a mean Cmax of 110 ng/ml (0.350 µM) was
observed in five healthy males (Ohlsson et al., 1986). In a more recently published
pharmacokinetic study on CBD oral solution (Epidiolex®), single dose of 1500 – 6000
mg was found to be well tolerated in six healthy subjects in each dose arm, and the
mean Cmax of CBD were measured 292 – 782 ng/ml (0.928 – 2.49 µM) (Taylor et al.,
2018). There is a dearth of information available on CBN pharmacokinetics in humans
with a few studies reporting low (< 12 ng/ml [0.0387 µM]) or unquantifiable (< LLOQ)
blood/plasma concentrations after smoked or vaporized doses (Schwope et al., 2011;
Desrosiers et al., 2014; Newmeyer et al., 2016). Johansson et al. reported mean CBN
Cmax of 126 ng/ml (0.406 µM) in six healthy males after smoking a cannabis cigarette
containing 19 mg of CBN (Johansson et al., 1987). However, since CBN is unlikely to
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be administered or used therapeutically in a relatively pure form, this concentration was
not considered clinically achievable for purposes of our assessment. In RAUC calculation,
the plasma protein binding of THC and CBD was assumed to be 97.2% and 94%,
respectively based on available data (Garrett and Hunt, 1974; Center for Drug
Evaluation and Research, 2018). We were unable to locate documentation of CBN
plasma protein binding so an empirical range of > 90% was chosen given its similarity in
structure and lipophilicity to THC and CBD. Taken altogether, the Imax,u (Eq. 4) was
calculated as (Cmax) × (1 – plasma protein binding), and the resulting RAUC for THC,
CBD, and CBN were 1.027, 1.057 – 1.153, and < 1.015, respectively. These results
suggest that THC and CBD may act as “perpetrator” constituents toward CES1
substrates, but this requires further clinical investigation and confirmation. Regarding
CBN, which also exhibited potent in vitro inhibitory effects on CES1, it appears less
likely to participate in clinically significant DDIs due to its low anticipated exposure in
individuals using cannabis for recreational or medical purposes.
Our study also demonstrated increased inhibition of CES1 activity when THC, CBD, and
CBN were combined versus individual constituents. Since individuals using cannabis or
multi-constituent extracts for medical reasons or on a recreational basis are more likely
to be exposed to a number of major cannabinoids rather than single constituents, this
approach may provide a better assessment of potential DDI liability.
Exposures to cannabis can vary depending on product content, dosing route and
schedule. Even within a single dosing route such as smoking, multiple factors influence
cannabinoid exposure including the cannabis potency, number, duration, and spacing of
puffs, hold time and inhalation volume (Huestis, 2007). Further, substantial intra- and
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inter-subject variability in cannabinoid exposures has been documented in
pharmacokinetic studies. In the present study we estimated the clinical DDI potential by
adopting the higher values we found reported in published literature which might be
achieved in actual use scenarios.
A further assumption made in our study was that hydrolysis rates of OST represented
only CES1 catalytic activity. This was a reasonable assumption since there is minimal
native expression of DMEs, including other esterases in the parent embryonic kidney
293 cells from which the CES1 overexpressing cells were made (Bouman et al., 2011).
In addition, OST was reported not to interact with either CYPs or UGTs (He et al.,
1999), which further validated our assumption.
Our study has several limitations. First, like most in vitro assessments, our evaluation
and interpretation were based only on reported systemic concentrations of cannabinoids
rather than actual tissue concentrations. CES1 is a ubiquitously expressed enzyme with
the liver being by far the predominant expression site (Satoh et al., 2002; Fagerberg et
al., 2014). Assessing DDIs based on concentrations reaching metabolic sites (i.e. liver)
could generally yield more accurate prediction. However, there are almost no data
regarding cannabinoid hepatic concentrations in humans beyond limited post-mortem
data suggesting certain cannabinoids (i.e. CBD, CBN) attain much higher
concentrations in the liver and bile relative to systemic concentrations. (Gronewold and
Skopp, 2011; Fabritius et al., 2012). Second, in the inhibition study conducted for Ki
determination, all inhibitor concentrations utilized were higher than the estimated Ki. We
believe that the relatively large uncertainty around the estimated α was partially due to
this limitation, and it could potentially introduce some error in the prediction of inhibition
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at lower inhibitor concentrations. However, the concentrations of THC, CBD, and CBN
in the combination assessment were in the range of their Ki and the inhibition results
were consistent with our model prediction, which increased the confidence in estimated
parameters.
Reversible inhibition was concluded for THC, CBD, and CBN since their IC50 did not
decrease after a 30-min pre-incubation with S9 fractions. On the contrary, an actual
increase of IC50 after the pre-incubation was observed. This phenomenon has also been
described in previous reports by another group (Yamaori et al., 2010; Yamaori et al.,
2011c; Yamaori et al., 2012). The exact cause(s) of this phenomenon are unclear. As a
technical note, we have observed that when prepared at lower concentrations (i.e.
around their Ki values), THC, CBD, and CBN prepared in plastic tubes exhibited lower
inhibition potency as compared to experiments using glass vials. Therefore, absorption
of the cannabinoids onto the plastic incubation tube walls was believed to be a likely
cause. This phenomenon has previously been noted by other investigators ( Garrett and
Hunt, 1974; Christophersen, 1986). Therefore, the incubation time was restricted to 15
min to minimize this potential influence.
Finally, an additional issue for consideration of our findings relates to the role of CES1
in the biotransformation of a large number of endogenous compounds. Indeed, CES1
mediated hydrolysis of esters (e.g. cholesteryl esters and triacylglycerols) is increasingly
viewed as an important role in lipid metabolism, cholesterol homeostasis, and possibly
fatty liver disease (Lian et al., 2018). Though speculative, a sustained inhibition of CES1
activity as a result of chronic exposure to cannabinoids could have implications for
these biological processes as well.
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In summary, our study identified THC, CBD, and CBN as potent inhibitors of CES1 in
vitro, with a reversible inhibition mechanism and mixed competitive-noncompetitive
characteristics. The calculated inhibition constant (Ki) values for THC, CBD, and CBN
were 0.541, 0.974, and 0.263 µM, respectively. An increase in the inhibition potency
was observed when THC, CBD, and CBN were combined for assessment. When
compared to the respective cannabinoid Cmax achievable physiologically, the Ki values
suggest that DDI may occur following the concomitant administration of CES1 substrate
medications and THC and CBD, but not by CBN.
The determination of whether the observed in vitro inhibitory effects of THC and CBD
translates into significant, or even detectable clinical DDIs requires further investigation
through formal clinical study. Such formal clinical studies would require the utilization of
a well-characterized cannabis product, a standardized dosing regimen, and incorporate
an appropriate CES1 substrate as a probe therapeutic agent.
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Authorship Contributions
Participated in research design: Qian, Wang, and Markowitz.
Conducted experiments: Qian
Performed data analysis: Qian, Wang, and Markowitz
Wrote and contributed to the writing of the manuscript: Qian and Markowitz
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