Tae Yeon Kong

In vitro metabolism of a novel synthetic cannabinoid, EAM-2201, in human liver microsomes and human recombinant cytochrome P450s.

In vitro metabolism of a new synthetic cannabinoid, EAM-2201, has been investigated with human liver microsomes and major cDNA-expressed cytochrome P450 (CYP) isozymes using liquid chromatography-high resolution mass spectrometry (LC-HRMS). Incubation of EAM-2201 with human liver microsomes in the presence of NADPH resulted in the formation of 37 metabolites, including nine hydroxy-EAM-2201 (M1-M9), five dihydroxy-EAM-2201 (M10-M14), dihydrodiol-EAM-2201 (M15), oxidative defluorinated EAM-2201 (M16), two hydroxy-M16 (M17 and M18), three dihydroxy-M16 (M19-M21), N-dealkyl-EAM-2201 (M22), two hydroxy-M22 (M23 and M24), dihydroxy-M22 (M25), EAM-2201 N-pentanoic acid (M26), hydroxy-M26 (M27), dehydro-EAM-2201 (M28), hydroxy-M28 (M29), seven dihydroxy-M28 (M30-M36), and oxidative defluorinated hydroxy-M28 (M37). Multiple CYPs, including CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 2J2, 3A4, and 3A5, were involved in the metabolism of EAM-2201. In conclusion, EAM-2201 is extensively metabolized by CYPs and its metabolites can be used as an indicator of EAM-2201 abuse.

Inhibitory Effects of Cedrol, β-Cedrene, and Thujopsene on Cytochrome P450 Enzyme Activities in Human Liver Microsomes

Cedrol, β-cedrene, and thujopsene are bioactive sesquiterpenes found in cedar essential oil and exert antiseptic, anti-inflammatory, antispasmodic, tonic, astringent, diuretic, sedative, insecticidal, and antifungal activities. These compounds are used globally in traditional medicine and cosmetics. The aim of this study was to investigate the inhibitory effects of cedrol, β-cedrene, and thujopsene on the activities of eight major human cytochrome P-450 (CYP) enzymes using human liver microsomes to assess potential β-cedrene-, cedrol-, and thujopsene–drug interactions. Cedrol, β-cedrene, and thujopsene were found to be potent competitive inhibitors of CYP2B6-mediated bupropion hydroxylase with inhibition constant (Ki) values of 0.9, 1.6, and 0.8 μM, respectively, comparable with that of a selective CYP2B6 inhibitor, thioTEPA (Ki, 2.9 μM). Cedrol also markedly inhibited CYP3A4-mediated midazolam hydroxylation with a Ki value of 3.4 μM, whereas β-cedrene and thujopsene moderately blocked CYP3A4. Cedrol, β-cedrene, and thujopsene at 100 μM negligibly inhibited CYP1A2, CYP2A6, and CYP2D6 activities. Only thujopsene was found to be a mechanism-based inhibitor of CYP2C8, CYP2C9, and CYP2C19. Cedrol and thujopsene weakly inhibited CYP2C8, CYP2C9, and CYP2C19 activities, but β-cedrene did not. These in vitro results indicate that cedrol, β-cedrene, and thujopsene need to be examined for potential pharmacokinetic drug interactions in vivo due to their potent inhibition of CYP2B6 and CYP3A4.

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.

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.

AbstractMAM-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 abstractIn 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

Comparative metabolism of honokiol in mouse, rat, dog, monkey, and human hepatocytes.

Honokiol has antitumor, antioxidative, anti-inflammatory, and antithrombotic effects. Here we aimed to identify the metabolic profile of honokiol in mouse, rat, dog, monkey, and human hepatocytes and to characterize the enzymes responsible for the glucuronidation and sulfation of honokiol. Honokiol had a high hepatic extraction ratio in all five species, indicating that it was extensively metabolized. A total of 32 metabolites, including 17 common and 15 different metabolites, produced via glucuronidation, sulfation, and oxidation of honokiol allyl groups were tentatively identified using liquid chromatography–high resolution quadrupole Orbitrap mass spectrometry. Glucuronidation of honokiol to M8 (honokiol-4-glucuronide) and M9 (honokiol-2′-glucuronide) was the predominant metabolic pathway in hepatocytes of all five species; however, interspecies differences between 4- and 2′-glucuronidation of honokiol were observed. UGT1A1, 1A8, 1A9, 2B15, and 2B17 played major roles in M8 formation, whereas UGT1A7 and 1A9 played major roles in M9 formation. Human cDNA-expressed SULT1C4 played a major role in M10 formation (honokiol-2′-sulfate), whereas SULT1A1*1, 1A1*2, and 1A2 played major roles in M11 formation (honokiol-4-sulfate). In conclusion, honokiol metabolism showed interspecies differences.

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

MAM-2201 is a fluorinated naphthoylindole synthetic cannabinoid with potent psychoactive properties that has been detected as an active ingredient in herbal incense blends. To gain a greater understanding of MAM-2201 metabolism and to compare its metabolic fate in humans with those in animals, the metabolism of MAM-2201 in human, mouse, and rat hepatocytes was investigated using liquid chromatography-high-resolution mass spectrometry combined with targeted and non-targeted metabolite profiling approaches. Nineteen phase I metabolites (M1-M19) reported previously in human liver microsomes and 13 novel metabolites were identified in human, mouse, and rat hepatocytes: 1 phase I metabolite (M20) and 12 phase II metabolites including 6 glucuronides (G1-G6), 1 sulfate (S1), and 5 glutathione (GSH) conjugates (GS1-GS5) of MAM-2201 metabolites. G3 was human-specific, but M20, G1, G2, and 5 GSH conjugates were rat-specific, indicating species-related differences in MAM-2201 metabolism. The findings in the present study can be useful for the experimental design and assessment of metabolism-mediated toxic risk of MAM-2201.

Simultaneous quantification of 18 saturated and unsaturated fatty acids and 7 sterols as their tert-butyldimethylsilyl derivatives in human saliva using gas chromatography-tandem mass spectrometry

The profiling of fatty acids (FAs) or sterols has been applied in clinical studies, but still needs to be improved to enable their simultaneous quantification. Moreover, little progress has been made in determining the levels of FAs and sterols in human saliva in a single run. In this study, gas chromatography-tandem mass spectrometry (GC-MS/MS) using one-step tert-butyldimethylsilyl (TBDMS) derivatization was developed for comprehensive profiling of 18 FAs (eight saturated, five monounsaturated, and five polyunsaturated FAs) and 7 sterols (cholesterol and its precursors). The TBDMS derivatization process was also optimized in terms of reaction solvent, catalyst, temperature, and reaction time. The optimized conditions resulted in better derivatization efficiency with good chromatographic separation through a high-temperature column within 23 min. The present method provided good linearity (r > 0.993), precision (coefficient of variation, 2.7% to 10.4%), and accuracy (91.5% to 103.4%). The overall recovery ranged from 73.8% to 114.3% for the 18 FAs, and from 68.9% to 79.8% for the 7 sterols. The validated method was applied to characterize FAs and sterols in human saliva samples. This is the first report of a GC-MS/MS method for the simultaneous determination of various FAs and sterols from a small volume (100 μL) of saliva. This approach can be used as a primary screening tool to examine the levels of both FAs and sterols in saliva, providing detailed information about their homeostasis for diagnostic and prognostic purposes.

Synthetic cannabinoids are substrates and inhibitors of multiple drug-metabolizing enzymes

Synthetic cannabinoids, a new class of psychoactive substances, are potent agonists of cannabinoid receptors, which mimic the psychoactive effects of the principal psychoactive component of cannabis, ∆9-tetrahydrocannabinol. Despite governmental scheduling as illicit drugs, new synthetic cannabinoids are being produced. The abuse of synthetic cannabinoids with several drugs containing different chemical groups has resulted in large numbers of poisonings. This has increased the urgency for forensic and public health laboratories to identify the metabolites of synthetic cannabinoids and apply this knowledge to the development of analytical methods and for toxicity prediction. It is necessary to determine whether synthetic cannabinoids are involved in drug-metabolizing enzyme-mediated drug–drug interactions. This review describes the metabolic pathways of 13 prevalent synthetic cannabinoids and various drug-metabolizing enzymes responsible for their metabolism, including cytochrome P450 (CYP), UDP-glucuronosyltransferases (UGTs), and carboxylesterases. The inhibitory effects of synthetic cannabinoids on CYP and UGT activities are also reviewed to predict the potential of synthetic cannabinoids for drug–drug interactions. The drug-metabolizing enzymes responsible for metabolism of synthetic cannabinoids should be characterized and the effects of synthetic cannabinoids on CYP and UGT activities should be determined to predict the pharmacokinetics of synthetic cannabinoids and synthetic cannabinoid-induced drug–drug interactions in the clinic.

Liquid Chromatography-tandem Mass Spectrometry for Quantification of Dioscin in Rat Plasma

Dioscin is a biologically active steroidal saponin with anticancer and hepatoprotective effects. A rapid, selective, and sensitive liquid chromatographic method with electrospray ionization tandem mass spectrometry was developed for the quantifi- cation of dioscin in rat plasma. Dioscin was extracted from rat plasma using ethyl acetate at acidic pH. The analytes were sepa- rated on a Halo C18 column using gradient elution of acetonitrile and 0.1% formic acid and detected by tandem mass spectrometry in selected reaction monitoring mode. The standard curve was linear (r 2 = 0.998) over the concentration range of 1�100 ng/mL. The lower limit of quantification was 1.0 ng/mL using 50 µL of plasma sample. The coefficient of variation and relative error for intra- and inter-assay at four QC levels were 1.3 to 8.0% and �5.4 to 10.0%, respectively. This method was applied successfully to the pharmacokinetic study of dioscin after oral administration of dioscin at a dose of 29.2 mg/kg in male Sprague-Dawley rats.