In Vitro Metabolism of 25B-NBF, 2-(4-Bromo-2,5-Dimethoxyphenyl)-N-(2-Fluorobenzyl)ethanamine, in Human Hepatocytes Using Liquid Chromatography–Mass Spectrometry

25B-NBF, 2-(4-bromo-2,5-dimethoxyphenyl)-N-(2-fluorobenzyl)ethanamine, is a new psychoactive substance classified as a phenethylamine. It is a potent agonist of the 5-hydroxytryptamine receptor, but little is known about its metabolism and elimination properties since it was discovered. To aid 25B-NBF abuse screening, the metabolic characteristics of 25B-NBF were investigated in human hepatocytes and human cDNA-expressed cytochrome P450 (CYP) and UDP-glucuronosyltransferase (UGT) enzymes using liquid chromatography–high resolution mass spectrometry. At a hepatic extraction ratio of 0.80, 25B-NBF was extensively metabolized into 33 metabolites via hydroxylation, O-demethylation, bis-O-demethylation, N-debenzylation, glucuronidation, sulfation, and acetylation after incubation with pooled human hepatocytes. The metabolism of 25B-NBF was catalyzed by CYP1A1, CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2J2, CYP3A4, and UGT2B7 enzymes. Based on these results, it is necessary to develop a bioanalytical method for the determination of not only 25B-NBF but also its metabolites in biological samples for the screening of 25B-NBF abuse.


Introduction
New psychoactive substances (NPSs) are abused compounds with effects similar to those of controlled drugs such as cannabis, morphine, cocaine, and amphetamine-type stimulants. Since the United Nations Office on Drugs and Crime (UNODC) launched the international NPS monitoring system in 2009, the amount of NPSs has increased by 3.7 times to 479 substances in 2017 [1]. Likewise, the European Monitoring Centre for Drugs and Drug Addiction (EMCDDA) was monitoring over 620 NPS at the end of 2016 and stated that more than 70% of these had been newly detected in the previous 5 years [2]. NPSs can be classified based on their chemical structure as synthetic cannabinoids, synthetic cathinones, tryptamines, phenethylamines, and others. Although synthetic cannabinoids remain the most commonly abused group, recently phenethylamine abuse has been on the rise, accounting for 28.4% of cases of NPS abuse at the end of 2017 according to the UNODC [1]. Phenethylamines are typical agonists of the 5-hydroxytryptamine 2 (5-HT2) receptor and their structure-activity relationships (SARs) are well-characterized [3]. Because N-benzyl substitution of phenethylamine results in a significant increase in binding affinity to 5-HT2A receptor and receptor activities, several N-benzyl-related analogs with methoxy, hydroxy, or fluorine moieties have been synthesized and their SARs have been evaluated [4,5]. 25B-NBF, 2-(4-bromo-2, 5-dimethoxyphenyl)-N-(2-fluorobenzyl)ethanamine, is an N-fluorobenzyl derivative of 2C-B [2-(4-bromo-2,5-dimethoxyphenyl)ethanamine] that binds to human 5-HT2A receptors and rat 5-HT2C receptors with pK i values of 8.57 and 7.77, respectively [4]. 25B-NBF is intended for research and forensic applications but is classified as a controlled substance in Sweden, the United Kingdom, and the Republic of Korea because of its potential for abuse [6,7]. Also, severe intoxication cases and the in vitro and in vivo metabolism of a structurally related substance, 25B-NBOMe [2-(4-bromo-2,5-dimethoxyphenyl)-N-(2-methoxybenzyl)-ethanamine], have been reported [8][9][10][11][12][13][14][15], but there are no published reports on the metabolism of 25B-NBF in humans and experimental animals.
For the control of illegal substances, it is necessary to develop bioanalytical methods for the sensitive and selective detection of illegal substances. Because many illegal substances are extensively metabolized, their metabolic profiles have been characterized to develop the bioanalytical methods for the simultaneous determination of illegal drugs and metabolites in forensic and emergency cases [13][14][15][16][17][18]. Therefore, it is necessary to understand the metabolic pathway of 25B-NBF in order to develop a bioanalytical method for monitoring 25B-NBF abuse. The purposes of this study were to identify in vitro metabolic profiles of 25B-NBF using human hepatocytes, the gold standard in vitro metabolism model, and liquid chromatography-high resolution mass spectrometry (LC-HRMS) [17][18][19] and to characterize the specific cytochrome P450 (CYP) and uridine 5 -diphospho-glucuronosyltransferase (UGT) enzymes responsible for 25B-NBF metabolism using human cDNA-expressed CYPs and UGTs in order to predict the pharmacokinetics and drug-interaction potential of 25B-NBF.
For the control of illegal substances, it is necessary to develop bioanalytical methods for the sensitive and selective detection of illegal substances. Because many illegal substances are extensively metabolized, their metabolic profiles have been characterized to develop the bioanalytical methods for the simultaneous determination of illegal drugs and metabolites in forensic and emergency cases [13][14][15][16][17][18]. Therefore, it is necessary to understand the metabolic pathway of 25B-NBF in order to develop a bioanalytical method for monitoring 25B-NBF abuse. The purposes of this study were to identify in vitro metabolic profiles of 25B-NBF using human hepatocytes, the gold standard in vitro metabolism model, and liquid chromatography-high resolution mass spectrometry (LC-HRMS) [17][18][19] and to characterize the specific cytochrome P450 (CYP) and uridine 5′-diphosphoglucuronosyltransferase (UGT) enzymes responsible for 25B-NBF metabolism using human cDNAexpressed CYPs and UGTs in order to predict the pharmacokinetics and drug-interaction potential of 25B-NBF.

Metabolic Stability of 25B-NBF and Prediction of Its Hepatic Clearance
The metabolic stability of 25B-NBF in human hepatocytes is illustrated in Figure 1. After 3 h incubation, 16.6% of 25B-NBF remained in the human hepatocyte suspension. The elimination slope (k) of degradation was estimated by linear regression and the elimination half-life (t1/2) of 25B-NBF was 29.7 min. Intrinsic clearance (Clint) and hepatic clearance (Clhep) of 25B-NBF were estimated using the well-stirred model as 83.4 and 16.6 mL/min/kg, respectively. The hepatic extraction ratio of 25B-NBF was 0.80, indicating that it is extensively metabolized in the liver. The Clint, Clhep, hepatic extraction ratio of honokiol, a positive control, using human hepatocytes were 168.4 mL/min/kg, 18.4 mL/min/kg, and 0.89, respectively, comparable to a previous report [19].

Metabolite Identification of 25B-NBF
The incubation of 25B-NBF with human hepatocytes for 2 h at 37 • C resulted in the formation of 33 metabolites from 25B-NBF. Representative extracted ion chromatograms of 25B-NBF and its metabolites are presented in Figure 2, and the retention times, molecular formulae, exact molecular ions ([M + H] + ), mass accuracies, fragment ions, and biotransformation are summarized in Table 1. 25B

Metabolite Identification of 25B-NBF
The incubation of 25B-NBF with human hepatocytes for 2 h at 37 °C resulted in the formation of 33 metabolites from 25B-NBF. Representative extracted ion chromatograms of 25B-NBF and its metabolites are presented in Figure 2, and the retention times, molecular formulae, exact molecular ions ([M + H] + ), mass accuracies, fragment ions, and biotransformation are summarized in Table 1.      Figure S4D).

Discussion
The in vitro metabolism of 25B-NBF, including metabolic stability and metabolite identification, was investigated using human hepatocytes. 25B-NBF was extensively metabolized in human hepatocytes, resulting in an elimination half-life and hepatic extraction ratio of 29.7 min and 0.80, respectively. The structure-related derivatives such as 25B-NBOMe, 25C-NBOMe, and 25I-NBOMe have been reported to undergo extensive metabolism in human, mouse, and rat [13][14][15]20].

Metabolic Stability of 25B-NBF in Human Hepatocytes
Pooled cryopreserved human hepatocytes were carefully thawed in thawing medium and resuspended in Krebs-Henseleit buffer to a final density of 1.0 × 10 6 cells/mL. Then a 60 µL aliquot of this hepatocyte suspension and equal volume of 25B-NBF (4 µM) in Krebs-Henseleit buffer were mixed into 96-well plates and incubated in triplicate for 0, 5, 15, 30, 60, 120, or 180 min in a CO 2 incubator at 37 • C. Incubation was quenched by addition of 120 µL ice-cold acetonitrile to each well, and the samples were centrifuged at 15,000× g for 10 min at 4 • C after 5 min sonication. Then 100 µL of supernatants were vortex-mixed with equal volume of water and 2 µL aliquots were analyzed by an LC-MS/MS system. Honokiol (2 µM) was separately incubated for the positive control of this system. The peak area ratios of substances versus IS at each sampling point were used in subsequent calculations of parameters. The following equations were used in the calculation of elimination parameters, including t 1/2 , Cl int , Cl hep , and hepatic extraction ratio of 25B-NBF.

Metabolite Identification in Human Hepatocytes
An amount of 60 µL 25B-NBF (20 µM) in Krebs-Henseleit buffer and human hepatocyte suspensions (60 µL; 1.0 × 10 6 cells/mL) were mixed in 96-well plates and incubated in triplicate for 2 h in a CO 2 incubator at 37 • C. Incubation was quenched by addition of 120 µL ice-cold acetonitrile to each well, and then the samples were centrifuged at 15,000× g for 10 min at 4 • C after 5 min sonication. Then the supernatants (200 µL) were dried using a speed-vac and residues were reconstituted with 10% methanol (100 µL). 5 µL Aliquots of each sample were analyzed using an LC-HRMS system.
By adding 100 µL of ice-cold methanol (containing 100 ng/mL of IS), the reaction was terminated. After centrifugation at 10,000× g for 4 min at 4 • C, 150 µL of supernatant was dried under N 2 gas. The residue was reconstituted in 50 µL of 45% methanol and a 5 µL of aliquot was injected into the LC-HRMS system.

LC-MS Analyses
To assess the metabolic stability of 25B-NBF, we used an Agilent 1290 Infinity UPLC coupled with Agilent 6495 triple quadrupole MS (Agilent Technologies, Santa Clara, CA, USA) in the quantification of 25B-NBF. For the chromatographic separation, 5% methanol containing 0.1% formic acid and 95% methanol containing 0.1% formic acid were used as mobile phase A and mobile phase B, respectively, and a Halo C 18 column (2.1 × 50 mm, 2.7 µm; Advanced Materials Technology, Wilmington, DE, USA) was used as a stationary phase. The flow rate used was 0.3 mL/min, and gradient elution was performed as follows: 10% B for 0-1 min, 10-90% B for 1-2.5 min, 90% B held for 2.5-4 min, 90-10% B for 4-4.1 min, and 10% B for 4.1-6 min. MS spectra were acquired in positive-ion mode using electrospray ionization (ESI). ESI conditions were optimized as follows: gas temperature 200 • C, gas flow 16 L/min, nebulizer 40 psi, sheath gas temp 380 • C, sheath gas flow 12 L/min, capillary voltage 4.5 kV, and nozzle voltage 500 V. Selective reaction monitoring transitions were 367.9→242.9 at a collision energy (CE) of 20 for 25B-NBF and 531.2→489.2 at a CE of 34 for ketoconazole.
A heated electrospray ionization (HESI) source was interfaced and the MS spectra were obtained in positive mode. For the metabolite identification, HESI source conditions were optimized as follows: 35 for sheath gas flow rate (arbitrary units), 10 for auxiliary gas flow rate (arbitrary units), 4 kV for spray voltage, and 350 • C for heater temperature. Xcalibur software (Thermo Fisher Scientific Inc.) was used in acquiring and processing MS spectra. Full MS spectra were acquired at a resolution of 70,000 (from m/z 100 to m/z 1500), and data-dependent MS/MS spectra were obtained at a resolution of 35,000 (normalized collision energies at 23 and 28). Prediction of fragment ions was aided by Mass Frontier (version 6.0; HighChem Ltd., Bratislava, Slovakia). Each metabolite was identified allowing 5 ppm mass error from the theoretical value. Owing to the absence of authentic standard of 25B-NBF metabolites, the formation rates of each metabolite were calculated using the standard curve of 25B-NBF.

Conclusions
25B-NBF was extensively metabolized into 33 metabolites by hydroxylation, O-demethylation, bis-O-demethylation, N-dearylation, cysteine conjugation, glucuronidation, sulfation, and acetylation alone or in combination in human hepatocytes. 2C-B, one of the most widespread NPSs, was identified as a major metabolite of 25B-NBF. Multiple CYPs including CYP1A1, CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2J2, and CYP3A4 were involved in hydroxylation, O-demethylation, bis-O-demethylation, and N-dearylation of 25B-NBF. UGT2B7 played a prominent role in glucuronidation of 25B-NBF to M9. The present study shows that it is necessary to determine the metabolites as well as 25B-NBF in biological samples to detect 25B-NBF abuse. These results may help to predict pharmacokinetic profiles aof 25B-NBF in humans and to address which metabolites should be synthesized for the analysis of 25B-NBF and metabolites in biological samples.

Conflicts of Interest:
The authors have no conflict of interest to declare.