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In Vitro Inhibitory Effects of Synthetic Cannabinoid EAM-2201 on Cytochrome P450 and UDP-Glucuronosyltransferase Enzyme Activities in Human Liver Microsomes

EAM-2201, a synthetic cannabinoid, is a potent agonist of the cannabinoid receptors that is widely abused as an illicit recreational drug in combination with other drugs. To evaluate the potential of EAM-2201 as a perpetrator of drug–drug interactions, the inhibitory effects of EAM-2201 on major drug-metabolizing enzymes, cytochrome P450s (CYPs) and uridine 5′-diphospho-glucuronosyltransferases (UGTs) were evaluated in pooled human liver microsomes using liquid chromatography–tandem mass spectrometry (LC-MS/MS). EAM-2201 at doses up to 50 µM negligibly inhibited the activities of eight major human CYPs (1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6 and 3A4) and five UGTs (1A1, 1A4, 1A6, 1A9 and 2B7) in human liver microsomes. EAM-2201 exhibited time-dependent inhibition of CYP2C8-catalyzed amodiaquine N-deethylation, CYP2C9-catalyzed diclofenac 4′-hydroxylation, CYP2C19-catalyzed [S]-mephenytoin 4′-hydroxylation and CYP3A4-catalyzed midazolam 1′-hydroxylation with Ki values of 0.54 µM (kinact: 0.0633 min−1), 3.0 µM (kinact: 0.0462 min−1), 3.8 µM (kinact: 0.0264 min−1) and 4.1 µM (kinact: 0.0250 min−1), respectively and competitively inhibited UGT1A3-catalyzed chenodeoxycholic acid 24-acyl-glucuronidation, with a Ki value of 2.4 µM. Based on these in vitro results, we conclude that EAM-2201 has the potential to trigger in vivo pharmacokinetic drug interactions when co-administered with substrates of CYP2C8, CYP2C9, CYP2C19, CYP3A4 and UGT1A3.


Discussion
We evaluated the inhibitory effects of EAM-2201 on CYP and UGT activities for the first time, using human liver microsomes. EAM-2201 negligibly inhibited the activities of eight major human CYPs but showed time-dependent inhibition of CYP2C8, CYP2C9, CYP2C19 and CYP3A4 enzyme activities in human liver microsomes ( Figure 2). However, AM-2201 and MAM-2201, chemical derivatives of EAM-2201, potently inhibited CYP2C9 with K i values of 3.9 and 5.6 µM, respectively and CYP3A4 with K i values of 4.0 and 5.4 µM, respectively; and exhibited time-dependent inhibition of CYP2C8 activity in human liver microsomes [14,15]. The number of metabolites of EAM-2201 previously reported in human liver microsomes (37 metabolites) was greater than those of AM-2201 and MAM-2201 (19 metabolites) [10,26], suggesting that EAM-2201 is a better time-dependent inhibitor than AM-2201 and MAM-2201.
There have been no direct reports on human EAM-2201 pharmacokinetics, which would be necessary for predicting EAM-2201-induced drug interaction potential. Serum EAM-2201 concentrations in plasma samples from recreational users and a postmortem case ranged from 0.26 to 4.1 nM [8,9] but these plasma concentrations do not reflect tissue concentrations, particularly liver concentrations. Our in vitro results suggest that EAM-2201 should be examined in terms of potential in vivo pharmacokinetic drug-drug interactions caused by time-dependent inhibition of CYP2C8, CYP2C9, CYP2C19 and CYP3A4 activities and competitive inhibition of UGT1A3 activity.

Inhibitory Effects of EAM-2201 on Eight Major CYP Activities in Human Liver Microsomes
The inhibitory potential (IC 50 values) of EAM-2201 on CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 were evaluated using ultrapooled human liver microsomes and a cocktail of seven CYP substrates followed by LC-tandem mass spectrometry (LC-MS/MS) as previously described [41]. Each incubation mixture was prepared in a total volume of 100 µL as follows: pooled human liver microsomes (5.2 pmol CYP), 1.0 mM NADPH, 10 mM magnesium chloride, 50 mM potassium phosphate buffer (pH 7.4), various concentrations of EAM-2201 in DMSO (final concentrations of 0.1-50 µM, 0.5% DMSO) and a cocktail of seven CYP probe substrates in 50% acetonitrile (0.5% acetonitrile), including 50 µM phenacetin, 2.5 µM coumarin, 2.0 µM amodiaquine, 10 µM diclofenac, 100 µM [S]-mephenytoin, 5 µM bufuralol and 2.5 µM midazolam in 50% acetonitrile. After a 3-min preincubation at 37 • C, the reactions were initiated by adding NADPH and incubation proceeded for 15 min at 37 • C in a shaking water bath. The reactions were stopped by adding 100 µL of ice-cold methanol containing internal standards ( 13 C 2 , 15 N-acetaminophen for acetaminophen and N-desethylamodiaquine; d 9 -1 -hydroxybufuralol for 4 -hydroxydiclofenac, 7-hydroxycoumarin, 4 -hydroxymephenytoin, 1 -hydroxybufuralol and 1 -hydroxy-midazolam). The incubation mixtures were centrifuged at 13,000× g for 4 min at 4 • C and 50 µL of each supernatant was diluted in 50 µL of water. Aliquots (5 µL) of the diluted supernatants were analyzed using LC-MS/MS. All assays were performed in triplicate and the mean values were used in calculations. To measure the time-dependent inhibition of seven CYP activities, various concentrations of EAM-2201 (0.1-50 µM) were pre-incubated for 30 min with human liver microsomes in the presence of NADPH. Each reaction was initiated by adding the seven-CYP probe substrate cocktail described above.
The inhibitory effects (IC 50 values) of EAM-2201 on CYP2B6-catalyzed bupropion 4-hydroxylase activity were determined in ultrapooled human liver microsomes using LC-MS/MS [41]. Each incubation mixture was prepared in a total volume of 100 µL as follows: pooled human liver microsomes (5.

Time-Dependent Inhibition of CYP2C8, CYP2C9, CYP2C19 and CYP3A4 Activities by EAM-2201
The time-dependent inhibitory effects of EAM-2201 on CYP2C8, CYP2C9, CYP2C19 and CYP3A4 activities in ultrapooled human liver microsomes were evaluated using time-and concentration-dependent inhibition assays. Ultrapooled human liver microsomes (26.0 pmol CYP) were pre-incubated with various concentrations of EAM-2201 in DMSO (0.5% DMSO) in 50 mM potassium phosphate buffer (pH 7.4) in the presence of NADPH for predetermined time periods: 5-15 min for CYP2C8 activity, 5-20 min for CYP2C9 activity and 15-40 min for CYP2C19 and CYP3A4 activities. Aliquots (10 µL) of the preincubated mixtures were withdrawn at predetermined times after incubation and added to other tubes containing specific CYP substrates (2 µM amodiaquine for CYP2C8, 10 µM diclofenac for CYP2C9, 100 µM [S]-mephenytoin for CYP2C19, or 2 µM midazolam for CYP3A4 in 50% acetonitrile), 1 mM NADPH, 50 mM potassium phosphate buffer (pH 7.4) and 10 mM magnesium chloride in 90-µL reaction mixtures. The second reactions were terminated after 10 min by adding 100 µL of ice-cold methanol containing d 9 -1 -hydroxybufuralol (internal standard). The incubation mixtures were centrifuged at 13,000× g for 4 min at 4 • C and 50 µL of each supernatant was diluted in 50 µL of water. Aliquots (5 µL) of the diluted supernatants were analyzed using LC-MS/MS.

LC-MS/MS Analysis
The metabolites formed from CYP substrates were simultaneously quantified using our previously described LC-MS/MS method [41]. A tandem mass spectrometer (TSQ Quantum Access, Thermo Scientific, San Jose, CA, USA), equipped with an electrospray ionization (ESI) source coupled to a Nanospace SI-2 LC system (Tokyo, Japan) was used. The column and autosampler temperatures were 50 • C and 6 • C, respectively. Separations were performed on an Atlantis dC18 column (3 µm, 2.1 mm i.d. × 100 mm; Waters Corporation, Milford, MA, USA) using the gradient elution of a mixture of 5% methanol in 0.1% formic acid (v/v) (mobile phase A) and 95% methanol in 0.1% formic acid (v/v) (mobile phase B) at a flow rate of 0. 25  The metabolites formed from the six UGT cocktail substrates were simultaneously measured using LC-MS/MS [41]. Metabolites were separated on an Atlantis dC18 column (3 µm, 2.1 mm i.d. × 100 mm; Waters Corporation) via gradient elution using a mixture of 5% acetonitrile in 0.1% formic acid (mobile phase A) and 95% acetonitrile in 0.1% formic acid (mobile phase B) at a flow rate of 0.2 mL/min: 100% mobile phase A for 1.7 min, 0 to 98% mobile phase B for 0.1 min and 98% mobile phase B for 3.2 min. The ESI source settings in both the positive and negative ion modes were as follows: capillary voltage, 4200 V; vaporizer temperature, 350 • C; capillary temperature, 330 • C; sheath gas pressure, 35 psi; and auxiliary gas pressure, 15 psi. Each metabolite was quantified via SRM in the negative ion mode: chenodeoxycholic acid 24-acyl-β-glucuronide, 567.

Data Analysis
IC 50 values (the concentration of the inhibitor associated with 50% inhibition of the original enzyme activity) were calculated by nonlinear regression analysis with Sigma Plot 12.0 software (Systat Software Inc., San Jose, CA, USA).
To determine the reversible inhibition constant (K i ) and inhibition mode for UGT1A3, data obtained from enzyme kinetic inhibition were fitted to different built-in equations for competitive, noncompetitive, uncompetitive and mixed inhibition models using Enzyme Kinetics ver. 1.1 software (Systat Software Inc.), which automatically estimates the initial parameters for the selected models and uses the Levenberg-Marquardt algorithm to determine the parameter values. The best model was determined using Akaike's information criterion as a measure of goodness of fit. The inhibition mode was verified by visual inspection of Lineweaver-Burk plots of enzyme kinetic data provided by Enzyme Kinetics software.
For time-dependent inhibition, the observed rates of CYP2C8, CYP2C9, CYP2C19 and CYP3A4 inactivation (k obs ) at different EAM-2201 concentrations were calculated from the negative slopes of the lines using linear regression analysis of the natural logarithm of the remaining activity as a function of time. Then, the inhibitor concentration that supports half the maximal rate of inhibition (K i ) and maximal rate of enzyme inhibition (k inact ) values were calculated using the following equation with Enzyme Kinetics software: k obs = k inact × I/(K i + I), where I is the initial concentration of EAM-2201.