AM-2201 Inhibits Multiple Cytochrome P450 and Uridine 5′-Diphospho-Glucuronosyltransferase Enzyme Activities in Human Liver Microsomes

AM-2201 is a synthetic cannabinoid that acts as a potent agonist at cannabinoid receptors and its abuse has increased. However, there are no reports of the inhibitory effect of AM-2201 on human cytochrome P450 (CYP) or uridine 5′-diphospho-glucuronosyltransferase (UGT) enzymes. We evaluated the inhibitory effect of AM-2201 on the activities of eight major human CYPs (1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4) and six major human UGTs (1A1, 1A3, 1A4, 1A6, 1A9, and 2B7) enzymes in pooled human liver microsomes using liquid chromatography–tandem mass spectrometry to investigate drug interaction potentials of AM-2201. AM-2201 potently inhibited CYP2C9-catalyzed diclofenac 4′-hydroxylation, CYP3A4-catalyzed midazolam 1′-hydroxylation, UGT1A3-catalyzed chenodeoxycholic acid 24-acyl-glucuronidation, and UGT2B7-catalyzed naloxone 3-glucuronidation with IC50 values of 3.9, 4.0, 4.3, and 10.0 µM, respectively, and showed mechanism-based inhibition of CYP2C8-catalyzed amodiaquine N-deethylation with a Ki value of 2.1 µM. It negligibly inhibited CYP1A2, CYP2A6, CYP2B6, CYP2C19, CYP2D6, UGT1A1, UGT1A4, UGT1A6, and UGT1A9 activities at 50 μM in human liver microsomes. These in vitro results indicate that AM-2201 needs to be examined for potential pharmacokinetic drug interactions in vivo due to its potent inhibition of CYP2C8, CYP2C9, CYP3A4, UGT1A3, and UGT2B7 enzyme activities.


Introduction
Synthetic cannabinoids are a group of substances with functionally similar effects to ∆9-tetrahydrocannabinol (THC), which is responsible for the major psychoactive effects of cannabis, and generally bind to cannabinoid receptor type 1 (CB 1 ) or 2 (CB 2 ) [1]. The synthetic cannabinoid JWH-018 was first detected in herbal smoking mixtures, called Spice, in 2008; 160 synthetic cannabinoids are now monitored by the European Monitoring Centre for Drugs and Drug Addiction (EMCDDA) through the EU Early Warning System [2]. The continued emergence of synthetic cannabinoids on the recreational and illicit drug markets has caused unexpected and serious events and has become a global public health issue [3][4][5][6][7][8][9]. AM-2201 ( Figure 1) is a third-generation synthetic cannabinoid, modified by introduction of a fluorine atom to JWH compounds, and exerts potent pharmacological actions on brain function, causing psychoactive and intoxicating effects [10]. compounds, and exerts potent pharmacological actions on brain function, causing psychoactive and intoxicating effects [10]. It has been increasingly found in recreational users and intoxication cases, as well as herbal products marketed for recreational use, such as incense blends [3,6,7,[11][12][13][14][15][16][17][18]. Cytochrome P450 (CYP) 1A2 and CYP2C9 enzymes play major roles in the metabolism of AM-2201 to 4-hydroxyfluoropentyl-AM-2201, AM-2201 pentanoic acid, and 5-hydroxypentyl-AM-2201 [19].
Variability of drug metabolism due to inhibition and induction of uridine 5'-diphospho-glucuronosyltransferase (UGT) enzymes, as well as CYP enzymes, is an important complicating factor in pharmacology and toxicology, drug therapy, environmental exposure, and risk assessment [20,21]. Phytocannabinoids, such as THC, cannabidiol, and cannabinol, inhibit CYPs 1A1, 1A2, 2A6, 2B6, 2C9, 2D6, 3A4, and 3A5 activities in human liver microsomes and recombinant CYP enzymes; cannabidiol is the most potent inhibitor of many CYPs [22][23][24][25][26][27][28][29]. Cannabidiol inhibited UGT1A9 and UGT2B7 activities, and cannabinol inhibited UGT1A9 activity in human liver and intestine microsomes and recombinant UGT enzymes [30]. Understanding the roles of synthetic cannabinoids in the regulation of CYP and UGT is necessary to predict individual differences in synthetic cannabinoid toxicity and to prevent toxic drug-drug interactions; however, the effects of synthetic cannabinoids, including AM-2201, on the regulation of CYP and UGT enzymes remain largely unknown.
Variability of drug metabolism due to inhibition and induction of uridine 5'-diphosphoglucuronosyltransferase (UGT) enzymes, as well as CYP enzymes, is an important complicating factor in pharmacology and toxicology, drug therapy, environmental exposure, and risk assessment [20,21]. Phytocannabinoids, such as THC, cannabidiol, and cannabinol, inhibit CYPs 1A1, 1A2, 2A6, 2B6, 2C9, 2D6, 3A4, and 3A5 activities in human liver microsomes and recombinant CYP enzymes; cannabidiol is the most potent inhibitor of many CYPs [22][23][24][25][26][27][28][29]. Cannabidiol inhibited UGT1A9 and UGT2B7 activities, and cannabinol inhibited UGT1A9 activity in human liver and intestine microsomes and recombinant UGT enzymes [30]. Understanding the roles of synthetic cannabinoids in the regulation of CYP and UGT is necessary to predict individual differences in synthetic cannabinoid toxicity and to prevent toxic drug-drug interactions; however, the effects of synthetic cannabinoids, including AM-2201, on the regulation of CYP and UGT enzymes remain largely unknown.
AM-2201 lowered the IC50 value of CYP2C8-catalyzed amodiaquine N-deethylation more than 2.5-fold after a 30-min pre-incubation with human liver microsomes and NADPH, compared with values obtained without pre-incubation (Table 1), indicating that AM-2201 acted as a potent mechanism-based inhibitor of CYP2C8. AM-2201 decreased CYP2C8-mediated amodiaquine N-deethylation with increasing pre-incubation time in a concentration-dependent manner ( Figure 3) with kinact and apparent Ki values of 0.052 min −1 and 2.1 µM, respectively ( Table 1).
The inhibitory effects of AM-2201 on six major human UGT enzymes were evaluated using human liver microsomes ( Figure 4). AM-2201 potently inhibited UGT1A3-catalyzed chenodeoxycholic acid 24-acyl-glucuronidation and UGT2B7-catalyzed naloxone 3-β-D-glucuronidation in human liver microsomes, with IC50 values of 6.4 and 14.5 µM, respectively ( Figure 4). AM-2201 lowered the IC 50 value of CYP2C8-catalyzed amodiaquine N-deethylation more than 2.5-fold after a 30-min pre-incubation with human liver microsomes and NADPH, compared with values obtained without pre-incubation (Table 1), indicating that AM-2201 acted as a potent mechanism-based inhibitor of CYP2C8. AM-2201 decreased CYP2C8-mediated amodiaquine N-deethylation with increasing pre-incubation time in a concentration-dependent manner ( Figure 3) with k inact and apparent K i values of 0.052 min −1 and 2.1 µM, respectively ( Table 1).
The inhibitory effects of AM-2201 on six major human UGT enzymes were evaluated using human liver microsomes ( Figure 4). AM-2201 potently inhibited UGT1A3-catalyzed chenodeoxycholic acid 24-acyl-glucuronidation and UGT2B7-catalyzed naloxone 3-β-D-glucuronidation in human liver microsomes, with IC 50 values of 6.4 and 14.5 µM, respectively ( Figure 4).       This in vitro study is the first to show the inhibitory potential of the popularly-used synthetic cannabinoid AM-2201 on CYP and UGT enzymes in human liver microsomes. AM-2201 was a potent competitive inhibitor of CYP2C9-catalyzed diclofenac hydroxylation with Ki value of 3.9 µM in human liver microsomes (Table 1, Figure 2). Ki values for the inhibition of THC, cannabinol, and cannabidiol on CYP2C9-catalyzed diclofenac hydroxylation in human liver microsomes were 1.31 µM, 1.29 µM, and 9.88 µM, respectively [27]. The Ki value (4.0 µM) for the competitive inhibition of AM-2201 on CYP3A4-catalyzed midazolam 1′-hydroxylation in human liver microsomes was comparable to the Ki (6.14 µM) of cannabidiol for CYP3A4-catalyzed diltiazem N-demethylation, but the IC50 values of THC and cannabinol for CYP3A4 activity were more than 50 µM [24].
AM-2201 was a potent mechanism-based inhibitor of CYP2C8, with a Ki value of 2.1 µM and its inhibitory potency was comparable to that of selective CYP2C8 inhibitor, quercetin (Ki, 2.0 µM) [33], but was less than those produced by phenelzine (Ki, 54.3 µM) [34] and gemfibrozil glucuronide (Ki, 20-52 µM) [35]. These results indicate that AM-2201 may inhibit the metabolism of drugs metabolized by CYP2C8, such as cerivastatin, paclitaxel, repaglinide, and sorafenib [36]. However, the inhibitory effects of THC, cannabinol, and cannabidiol on CYP2C8 activity were not evaluated, to our knowledge.
AM-2201 showed the potent competitive inhibition (Ki, 4.3 µM) of UGT1A3-catalyzed chenodeoxycholic acid 24-acyl glucuronidation similar to a selective UGT1A3 inhibitor, glycyrrhetinic acid (IC50, 4.3 µM) in human liver microsomes, indicating that this compound should be used carefully with UGT1A3 substrates, such as chenodeoxycholic acid, fimasartan, losartan, candesartan, zolarsartan, and JWH-018 [37][38][39][40], to avoid possible drug interactions. This in vitro study is the first to show the inhibitory potential of the popularly-used synthetic cannabinoid AM-2201 on CYP and UGT enzymes in human liver microsomes. AM-2201 was a potent competitive inhibitor of CYP2C9-catalyzed diclofenac hydroxylation with K i value of 3.9 µM in human liver microsomes (Table 1, Figure 2). K i values for the inhibition of THC, cannabinol, and cannabidiol on CYP2C9-catalyzed diclofenac hydroxylation in human liver microsomes were 1.31 µM, 1.29 µM, and 9.88 µM, respectively [27]. The K i value (4.0 µM) for the competitive inhibition of AM-2201 on CYP3A4-catalyzed midazolam 1 -hydroxylation in human liver microsomes was comparable to the K i (6.14 µM) of cannabidiol for CYP3A4-catalyzed diltiazem N-demethylation, but the IC 50 values of THC and cannabinol for CYP3A4 activity were more than 50 µM [24].
AM-2201 was a potent mechanism-based inhibitor of CYP2C8, with a K i value of 2.1 µM and its inhibitory potency was comparable to that of selective CYP2C8 inhibitor, quercetin (K i , 2.0 µM) [33], but was less than those produced by phenelzine (K i , 54.3 µM) [34] and gemfibrozil glucuronide (K i , 20-52 µM) [35]. These results indicate that AM-2201 may inhibit the metabolism of drugs metabolized by CYP2C8, such as cerivastatin, paclitaxel, repaglinide, and sorafenib [36]. However, the inhibitory effects of THC, cannabinol, and cannabidiol on CYP2C8 activity were not evaluated, to our knowledge.
There is no reported systemic information on human AM-2201 pharmacokinetics, essential for the prediction of AM-2201-induced drug interaction potential, but plasma and blood concentrations for AM-2201 were reported to be 0.14 nM to 26.4 nM in recreational users and intoxication cases [7,45]. However, plasma or blood concentrations do not reflect tissue concentrations, particularly liver concentrations. As AM-2201 is extensively metabolized [19], its metabolites may inhibit CYP and UGT activities. Consequently, these in vitro results suggest that AM-2201 should be examined in terms of potential in vivo pharmacokinetic drug interactions caused by inhibition of CYP2C8, CYP2C9, CYP3A4, UGT1A3, and UGT2B7 activities.
To measure the mechanism-based inhibition of CYP activities, various concentrations of AM-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.
The inhibitory effects (IC 50 values) of AM-2201 on CYP2B6-catalyzed bupropion 4-hydroxylase activity were determined in ultrapool human liver microsomes using LC-MS/MS [46]. The incubation mixtures were prepared in a total volume of 100 µL as follows: pooled human liver microsomes (0.2 mg/mL), 1.0 mM NADPH, 10 mM MgCl 2 , 50 mM potassium phosphate buffer (pH 7.4), various concentrations of AM-2201 (final concentrations of 0.1-50 µM, acetonitrile concentration <1% v/v), and the CYP2B6-selective substrate bupropion (50 µM). After a 3-min pre-incubation at 37 • C, the reactions were initiated by adding a NADPH generating system and incubated for 15 min at 37 • C in a shaking water bath. The reaction was stopped by placement of the tubes on ice and addition of 100 µL of ice-cold methanol containing internal standards (d 9 -1-hydroxybufuralol for 4-hydroxy-bupropion). The incubation mixtures were then centrifuged (13,000× g, 4 min, 4 • C). All incubations were performed in triplicate, and average values were used.
To evaluate mechanism-based inhibition of CYP2B6 activity, various concentrations of AM-2201 (final concentrations of 0.1-50 µM, acetonitrile concentration <1% v/v) were pre-incubated for 30 min with human liver microsomes in the presence of NADPH. The reaction was started by addition of the CYP2B6 probe substrate, bupropion.

Mechanism-based Inhibition of CYP2C8 Activity by AM-2201
The mechanism-based inhibitory effect of AM-2201 on CYP2C8 activity was further evaluated using time-and concentration-dependent inhibition assays in human liver microsomes. The microsomes (1 mg/mL) were pre-incubated with various concentrations of AM-2201 in 50 mM potassium phosphate buffer (pH 7.4) in the presence of NADPH and aliquots (10 µL) of the pre-incubated mixtures were withdrawn at 15, 25, and 35 min after incubation commenced and added to other tubes containing 2 µM amodiaquine, 1 mM NADPH, 50 mM potassium phosphate buffer (pH 7.4), and 10 mM MgCl 2 in 90 µL reaction mixtures. The second reaction was terminated after incubation for 10 min by adding 100-µL amounts of ice-cold methanol containing d 9 -1 -hydroxybufuralol. The incubation mixtures were centrifuged (13,000× g, 4 min, 4 • C), and then 50 µL of each supernatant was diluted with 50 µL of water. Aliquots (5 µL) of the diluted supernatants were analyzed by LC-MS/MS.

Kinetic Analysis
To determine K i values of AM-2201 for CYP2C9 and CYP3A4, human liver microsomes (0.15 mg/mL) were incubated with various concentrations of substrates (2-20 µM diclofenac for CYP2C9 and 1-8 µM midazolam for CYP3A4), 1 mM NADPH, 10 mM MgCl 2 , and various concentrations of AM-2201 in 50 mM potassium phosphate buffer (pH 7.4) in a total incubation volume of 100 µL. The reactions were initiated by addition of NADPH at 37 • C and stopped after 10 min by placing the incubation tubes on ice and adding 100 µL of ice-cold methanol containing an internal standard (d 9 -1-hydroxybufuralol). The incubation mixtures were centrifuged (13,000× g, 4 min, 4 • C) and then 50 µL of the supernatant was diluted with 50 µL of water. Aliquots (5 µL) of the diluted supernatants were analyzed by LC-MS/MS.
To determine K i values of AM-2201 for UGT1A3 and UGT2B7 enzymes, human liver microsomes (0.15 mg/mL) were incubated with various concentrations of chenodeoxycholic acid (1-10 µM) for UGT1A3 or of naloxone (0.5-5 µM) for UGT2B7, 5 mM UDPGA, 10 mM MgCl 2 , and various concentrations of AM-2201 in 50 mM Tris buffer (pH 7.4) in a total incubation volume of 100 µL. The reactions were initiated by addition of UDPGA at 37 • C and stopped after 60 min by placing the incubation tubes on ice and adding 100 µL of ice-cold methanol containing propofol glucuronide (internal standard for UGT1A3) or meloxicam (internal standard for UGT2B7). The incubation mixtures were centrifuged (13,000× g, 4 min, 4 • C), and then, 50 µL of the supernatant was diluted with 50 µL of water. Aliquots (5 µL) of the diluted supernatants were analyzed by LC-MS/MS.

LC-MS/MS Analysis
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.
The metabolites formed from CYP substrates were quantified simultaneously by a LC-MS/MS method described previously [46]. The ESI source settings in positive-ion mode for metabolite ionization were: capillary voltage, 4200 V; vaporizer temperature, 350 • C; capillary temperature, The metabolites formed from the six UGT cocktail substrates were measured simultaneously using the LC-MS/MS method [47]. The ESI source settings in both positive-and negative-ion modes for metabolite ionization were: 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, 567.2 > 391.0, mycophenolic acid glucuronide, 495.2 > 318. 9

Data Analysis
IC 50 values (concentration of inhibitor causing 50% inhibition of the original enzyme activity) were calculated using the SigmaPlot program (ver. 11.0; Systat Software, Inc., San Jose, CA, USA). The apparent kinetic parameters for inhibitory potential (K i and k inact values) and inhibition mode were estimated from the fitted curves using the Enzyme Kinetics program (ver. 1.1; Systat Software Inc., San Jose, CA, USA).