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

Abstract

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 K_i values of 0.54 µM (k_inact: 0.0633 min⁻¹), 3.0 µM (k_inact: 0.0462 min⁻¹), 3.8 µM (k_inact: 0.0264 min⁻¹) and 4.1 µM (k_inact: 0.0250 min⁻¹), respectively and competitively inhibited UGT1A3-catalyzed chenodeoxycholic acid 24-acyl-glucuronidation, with a K_i 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.

Keywords: EAM-2201; UDP-glucuronosyltransferase; cytochrome P450; drug–drug interaction; human liver microsomes.

Figure 1

Chemical structure of EAM-2201.

Figure 2

The inhibitory effects of EAM-2201 on CYP1A2-mediated phenacetin O-deethylase, CYP2A6-mediated coumarin 7-hydroxylase, CYP2B6-mediated bupropion hydroxylase, CYP2C8-catalyzed amodiaquine N-deethylase, CYP2C9-catalyzed diclofenac 4′-hydroxylase, CYP2C19-mediated [S]-mephenytoin 4′-hydroxylase, CYP2D6-mediated bufuralol 1′-hydroxylase and CYP3A4-mediated midazolam 1′-hydroxylase activities in ultrapooled human liver microsomes with (○) and without (●) 30-min preincubation in the presence of reduced β-nicotinamide adenine dinucleotide phosphate (NADPH) at 37 °C. The cocktail substrate concentrations used to assess IC₅₀ values were as follows: 50 µM phenacetin, 2.5 µM coumarin, 2.0 µM amodiaquine, 10 µM diclofenac, 100 µM [S]-mephenytoin, 5.0 µM bufuralol and 2.5 µM midazolam. Inhibition of CYP2B6 was evaluated separately using 50 µM bupropion. The data are presented as the mean ± standard deviation (SD; n = 3).

Figure 3

Inactivation kinetics of (a) N-desethylamodiaquine formation from amodiaquine; (b) 4′-hydroxydiclofenac formation from diclofenac; (c) 4′-hydroxy-[S]-mephenytoin from [S]-mephenytoin; and (d) 1′-hydroxymidazolam from midazolam in human microsomes by various concentrations of EAM-2201. Relationship between k_obs and EAM-2201 concentrations to estimate k_inact and K_i values for (e) CYP2C8-mediated amodiaquine N-deethylation; (f) CYP2C9-mediated diclofenac 4′-hydroxylation; (g) CYP2C19-mediated [S]-mephenytoin 4′-hydroxylation; and (h) CYP3A4-mediated midazolam 1′-hydroxylation.

Figure 4

The inhibitory effects of EAM-2201 on the activities of six uridine 5′-diphospho-glucuronosyltransferases (UGT) enzymes in ultrapooled human liver microsomes. The cocktail UGT substrate concentrations were as follows: 0.5 µM SN-38 for UGT1A1, 2 µM chenodeoxycholic acid for UGT1A3, 0.5 µM trifluoperazine for UGT1A4, 1 µM N-acetylserotonin for UGT1A6, 0.2 µM mycophenolic acid for UGT1A9 and 1 µM naloxone for UGT2B7. The data are presented as the mean ± SD (n = 3).

Figure 5

Representative Dixon plot showing the inhibitory effects of EAM-2201 on UGT1A3-catalyzed chenodeoxycholic acid 24-acylglucuronidation in human liver microsomes. Each symbol represents a particular concentration of chenodeoxycholic acid: ●, 1 µM; ○, 2 µM; ▼, 5 µM; and ▽, 10 µM. The data are presented as the mean ± SD (n = 3).