Metabolic characterization of (1-(5-fluoropentyl)-1H-indol-3-yl)(4-methyl-1-naphthalenyl)-methanone (MAM-2201) using human liver microsomes and cDNA-overexpressed cytochrome P450 enzymes

Abstract

MAM-2201 is a synthetic cannabinoid that is increasingly found in recreational drug abusers and cases of severe intoxication. Thus, characterization of the metabolic pathways of MAM-2201 is necessary to predict individual pharmacokinetics and toxicity differences, and to avoid toxic drug-drug interactions. Collectively, 19 phase 1 metabolites of MAM-2201 were identified using liquid chromatography-Orbitrap mass spectrometry following human liver microsomal incubations in the presence of NADPH: 7 hydroxy-MAM-2201 (M1-M7), 4 dihydroxy-MAM-2201 (M8-M11), dihydrodiol-MAM-2201 (M12), N-(5-hydroxypentyl)-MAM-2201 (M13), hydroxy-M13 (M14), N-dealkyl-MAM-2201 (M15), 2 hydroxy-M15 (M16, M17), MAM-2201 N-pentanoic acid (M18), and hydroxy-M18 (M19). On the basis of intrinsic clearance values in human liver microsomes, hydroxy-MAM-2201 (M1), N-(5-hydroxypentyl)-MAM-2201 (M13), and hydroxy-M13 (M14) were the major metabolites. Based on an enzyme kinetics study using human cDNA-expressed cytochrome P450 (CYP) enzymes and an immunoinhibition study using selective CYP antibodies in human liver microsomes, CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 enzymes were responsible for MAM-2201 metabolism. The CYP3A4 enzyme played a prominent role in MAM-2201 metabolism, and CYP1A2, CYP2B6, CYP2C8, and CYP2C9 enzymes played major roles in the formation of some metabolites. MAM-2201 is extensively metabolized by multiple CYP enzymes, indicating that MAM-2201 and its metabolites should be used as markers of MAM-2201 abuse and toxicity. Graphical abstract In vitro metabolic pathways of MAM-2201 were characterized in human liver microsomes and recombinant CYPs using LC-HRMS analysis. Total 19 phase I metabolites were identified with predominant contribution of CYP3A4.

Targeted and non‐targeted metabolite identification of MAM‐2201 in human, mouse, and rat hepatocytes

“…Nineteen phase I metabolites previously identified in human liver microsomes 8 were also confirmed in human, mouse, and rat previously reported phase I metabolites, M1-M19, were identified in human, rat, or mouse hepatocytes by combining targeted and non-targeted metabolite profiling approaches. The metabolic fates of MAM-2201 in rat hepatocytes were quite different from those in human and mouse hepatocytes with eight rat-specific metabolites, i.e., M20, G1, G2, and GS1-GS5 (Table 1, Figure 4).…”

“…Nineteen phase I metabolites previously identified in human liver microsomes were also confirmed in human, mouse, and rat hepatocyte incubates: monohydroxy‐MAM‐2201 (M1–M7), dihydroxy‐MAM‐2201 (M8–M11), dihydrodiol‐MAM‐2201 (M12), oxidative defluorinated MAM‐2201 (M13), hydroxy‐M13 (M14), N ‐dealkyl‐MAM‐2201 (M15), hydroxy‐M15 (M16 and M17), MAM‐2201 N ‐pentanoic acid (M18), and hydroxy‐M18 (M19). The major phase I metabolic pathways of MAM‐2201 were similar to other structurally related synthetic cannabinoids, such as EAM‐2201, 5F‐PY‐PICA, and 5F‐CUMYL‐PICA …”

“…Detection and quantification of abused drug substances in biological samples are key steps in the diagnosis and treatment of emergency cases. However, most synthetic cannabinoids are extensively metabolized, and it is therefore difficult to identify such synthetic cannabinoids in biological samples . Therefore, in vitro metabolism studies of synthetic cannabinoids using human liver microsomes and hepatocytes are performed to identify their metabolites as relevant indicators of drug abuse.…”

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

“…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.…”

“…EAM-2201 showed potent time-dependent inhibition of CYP2C8-catalyzed amodiaquine N -deethylation, with a K i of 0.54 µM and k inact of 0.0633 min −1 ( Table 1 ) and its inactivation efficiency ( k inact / K i = 117.2 mL/µmol/min) was higher than those of its chemical derivatives, such as AM-2201 ( k inact / K i = 25.8 mL/µmol/min) [ 14 ] and MAM-2201 ( k inact / K i = 73.8 mL/µmol/min) [ 15 ] and of typical CYP2C8 inhibitors such as phenelzine ( k inact / K i = 3.13 mL/µmol/min) and amiodarone ( k inact / K i = 0.57 mL/µmol/min) [ 27 ]. These results indicate that EAM-2201 may be a potent time-dependent inhibitor of CYP2C8 and may inhibit the metabolism of CYP2C8 substrate drugs, including cerivastatin, MAM-2201, paclitaxel, repaglinide and sorafenib [ 26 , 28 ].…”