Hee Seung Kim

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.

Rapid and sensitive determination of propofol glucuronide in hair by liquid chromatography and tandem mass spectrometry

A fast, sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the detection and quantitation of propofol glucuronide in human hair has been developed and validated. Propofol glucuronide was extracted from 10mg of hair using a simple methanol extraction method, with recovery greater than 91% at 3 quality control samples (15, 100, 4000 pg/mg). A reversed phase column (C8) was used to analyze and the mobile phase was composed of ammonium formate and acetonitrile gradient at a flow rate of 0.2 mL/min. The lower limit of quantitation (LLOQ) was 5 pg/mg and the assay was linear to 5000 pg/mg. The intra- and inter-day precision (% CV, coefficient of variation) ranged from 1.26 to 4.50% while the accuracy (% RE, relative error) were -4.24 to 4.4%. The matrix effects were monitored at 3 different concentrations and the %CV of the results for these concentrations was less than 10.6%. Propofol glucuronide was stable during processing and analysis in human hair. The procedure was validated and applied to the analysis of hair samples in human subjects previously administered in propofol.

Estimation of Measurement Uncertainty for the Quantification of 11-Nor-Delta 9-Tetrahydrocannabinol-9-Carboxylic Acid and Its Glucuronide in Urine Using Liquid Chromatography–Tandem Mass Spectrometry

Recently, the estimation of the measurement uncertainty has become a significant issue in the quality control of forensic drug testing. In the present study, the uncertainty of the measurement was calculated for the quantification of 11-nor-delta 9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) and its glucuronide conjugate (THC-COOH-glu) in urine using liquid chromatography-tandem mass spectrometry. The procedure was based on liquid-liquid extraction of a volume of urine (800 µL) with ethyl acetate. The sources of uncertainty were identified and classified into four major categories as follows: standard preparation, calibration curve, method precision and bias. The overall contribution of combined standard uncertainty on THC-COOH increased in the order of standard preparation (0.9%), method precision (10.4%), calibration curve (30.3%) and bias (58.4%) and, while calibration curve (53.0%) and bias (40.4%) gave the bigger contributions to the combined standard uncertainty for THC-COOH-glu than method precision and standard preparation, which accounted for 6.3 and 0.3%, respectively. The reliability of a measurement was expressed by stating the expanded uncertainty of the measurement result at 95% confidence level. The concentrations of THC-COOH and THC-COOH-glu in the urine sample with their expanded uncertainties were 10.20 ± 1.14 ng/mL and 25.42 ± 5.01 ng/mL, respectively.

Rapid analysis of drugs of abuse and their metabolites in human urine using dilute and shoot liquid chromatography–tandem mass spectrometry

Liquid chromatography-tandem mass spectrometric method for analysis of 113 abuse drugs and their metabolites in human urine was developed and validated. A simple sample clean-up procedure using the “dilute and shoot” approach, followed by reversed phase separation, provided a fast and reliable method for routine analysis. Drugs were separated in a Capcell Pak MG-III C18 column using a gradient elution of 1xa0mM ammonium formate with 0.1% formic acid in water and acetonitrile. The total time for analysis was 32xa0min. The multiple reaction monitoring mode using two transitions (e.g., quantifier and qualifier) was optimized for both identification and determination. The calibration curves for each analyte were linear over the concentration ranges of 1–100, 5–100, or 10–100xa0ng/mL using 400xa0μL of human urine sample with the coefficient of determination above 0.9921. The coefficient of variation and accuracy for the intra- and inter-assays of the tested drugs at three QC levels were 1.1–14.6 and 86.7–106.8%, respectively. The present method was successfully applied to the analysis of forensic urine samples obtained from 17 drug abusers. This method is useful for the rapid and accurate determination of multiple drug abuse with a small amount of urine in forensic and clinical toxicology.

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.n 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

Inhibition of cytochrome P450 and uridine 5′-diphospho-glucuronosyltransferases by MAM-2201 in human liver microsomes

MAM-2201, a synthetic cannabinoid, is a potent agonist of the cannabinoid receptors and is increasingly used as an illicit recreational drug. The inhibitory effects of MAM-2201 on major drug-metabolizing enzymes such as cytochrome P450s (CYPs) and uridine 5′-diphospho-glucuronosyltransferases (UGTs) have not yet been investigated although it is widely abused, sometimes in combination with other drugs. We evaluated the inhibitory effects of MAM-2201 on eight major human CYPs (CYPs 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4) and six UGTs (UGTs 1A1, 1A3, 1A4, 1A6, 1A9, and 2B7) of pooled human liver microsomes; we thus explored potential MAM-2201-induced drug interactions. MAM-2201 potently inhibited CYP2C9-catalyzed diclofenac 4′-hydroxylation, CYP3A4-catalyzed midazolam 1′-hydroxylation, and UGT1A3-catalyzed chenodeoxycholic acid 24-acyl-glucuronidation, with Ki values of 5.6, 5.4 and 5.0xa0µM, respectively. MAM-2201 exhibited mechanism-based inhibition of CYP2C8-catalyzed amodiaquine N-de-ethylation with Ki and kinact values of 1.0xa0µM and 0.0738xa0min−1, respectively. In human liver microsomes, MAM-2201 (50xa0µM) negligibly inhibited CYP1A2, CYP2A6, CYP2B6, CYP2C19, CYP2D6, UGT1A1, UGT1A4, UGT1A6, UGT1A9, and UGT2B7. Based on these in vitro results, we conclude that MAM-2201 has the potential to trigger in vivo pharmacokinetic drug interactions when co-administered with substrates of CYP2C8, CYP2C9, CYP3A4, and UGT1A3.

Simultaneous Measurement of Serum Chemical Castration Agents and Testosterone Levels Using Ultra-Performance Liquid Chromatography–Tandem Mass Spectrometry

Chemical castration involves administration of drugs to prevent pathological sexual behavior, reduce abnormal sexual drive and treat hormone-dependent cancers. Various drugs have been used for chemical castration; however, substantial interindividual variability and side effects are often observed. In this study, we proposed a useful monitoring method for the application of chemical castration agents using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS-MS). Testosterone, cyproterone acetate, medroxyprogesterone, goserelin acetate, leuprolide acetate and triptorelin acetate were analyzed by UPLC-MS-MS. The target drugs were extracted from serum samples by double protein precipitation using methanol. Testosterone-1,2-d2 and buserelin acetate were used as internal standards. Parameters of analytical performance were evaluated, including imprecision, linearity, ion suppression and detection capabilities. Testosterone measurements were compared with the results of immunoassays. Serum specimens from 51 subjects who underwent chemical castration were analyzed. All drugs and testosterone were well extracted and separated using our method. The method was essentially free from potential interferences and ion suppression. Within-run and between-run imprecision values were <15%. The lower limits of quantification were 0.125 and 0.5-1.0 ng/mL for testosterone and other drugs, respectively. Good correlations with pre-existing immunoassays for testosterone measurement were observed. Sera from subjects who underwent androgen deprivation therapy showed variable levels of drugs. We successfully developed a UPLC-MS-MS-based monitoring method for chemical castration. The performance of our method was generally acceptable. This method may provide a novel monitoring strategy for chemical castration to enhance expected effects while reducing unwanted side effects.