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In vitro metabolism of a novel synthetic cannabinoid, EAM-2201, in human liver microsomes and human recombinant cytochrome P450s
Journal of Pharmaceutical and Biomedical Analysis 119 (2016) 50–58
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In vitro metabolism of a novel synthetic cannabinoid, EAM-2201, in human liver microsomes and human recombinant cytochrome P450s Ju Hyun Kim a,1 , Hee Seung Kim b,1 , Tae Yeon Kong a , Joo Young Lee a , Jin Young Kim b , Moon Kyo In b , Hye Suk Lee a,∗ a b
College of Pharmacy, The Catholic University of Korea, 43 Jibong-ro, Wonmi-gu, Bucheon-si, Gyeonggi-do 420-743, Republic of Korea Forensic Chemistry Laboratory, Forensic Science Division, Supreme Prosecutor’s Office, 157 Banpo-daero, Seocho-gu, Seoul 137-730, Republic of Korea
a r t i c l e
i n f o
Article history: Received 5 September 2015 Received in revised form 5 November 2015 Accepted 18 November 2015 Available online 22 November 2015 Keywords: EAM-2201 Synthetic cannabinoid In vitro metabolism LC–HRMS Human liver microsomes Cytochrome P450
a b s t r a c t 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. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Synthetic cannabinoids are referred to as a group of substances with similar chemical structures generally binding to cannabinoid receptor type 1 (CB1 ) or type 2 (CB2 ). Since the first identification of synthetic cannabinoids in ‘herbal mixtures’ in 2008 [1–3], their abuse has continuously increased worldwide and has become a major issue in the forensic community [4–9]. Possibly, the most prevalent synthetic cannabinoid is JWH-018, a naphthoylindole introduced in 2008, and its pharmacokinetic and pharmacological characteristics are well-known [10–16]. Owing to its great potential for abuse, JWH-018 is prohibited in most countries [13,17]. However, because of the well-established structure–activity relationship for synthetic cannabinoids [18–22], various modifications of JWH-018 have consistently appeared involving introduction of the fluorine atom (AM-2201) [23], and substitution of the naphthyl group to a cyclopropyl group (UR-144 and XLR-11) [9,24],
∗ Corresponding author at: Drug Metabolism and Bioanalysis Laboratory, College of Pharmacy, The Catholic University of Korea, 43 Jibong-ro, Wonmi-gu, Bucheon-si, Gyeonggi-do 420-743, Republic of Korea. Fax: +82 32 342 2013. E-mail address: [email protected] (H.S. Lee). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.jpba.2015.11.023 0731-7085/© 2015 Elsevier B.V. All rights reserved.
adamantyl group (APICA and 5F-APICA) [9] or quinolinyl group (QUPIC and QUCHIC) [25,26]. Synthetic cannabinoids are generally found in seized herbal materials, and the number of seizures has increased approximately 5-fold from 2009 to June 2013 in Korea [27]. Newly emerged synthetic cannabinoids are mostly used without the knowledge of their potential toxicity and pharmacokinetic properties, and several fatalities have been reported [28–30]. EAM-2201, (4-ethyl-1-naphthalenyl)[1-(5-fluoropentyl)-1Hindol-3-yl]-methanone, was first identified in 2012 as an ingredient of illegal synthetic cannabis smoking blends [9]. The pharmacological effects of EAM-2201 have not been investigated in detail, but psychotic effects can be easily presumed from its structural analogues such as JWH-210 and AM-2201 [10,23]. Therefore, the illegal use of EAM-2201 should be carefully monitored. The metabolism of naphthoylindole-type synthetic cannabinoids such as JWH-018, JWH-210, AM-2201, and MAM-2201 has been well-characterized in urine samples obtained from suspected users and/or in vitro studies using human liver microsomes and hepatocytes [12–16,31–37], but there are few reports on the characterization of drug-metabolizing enzymes responsible for the specific metabolic reactions of synthetic cannabinoids [15,34].
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Based on its structural similarity, EAM-2201 may be extensively metabolized. However, in vitro and in vivo EAM-2201 metabolism in animals and humans has not been reported. Therefore, identifying the metabolism of EAM-2201 and characterizing major metabolites as biomarkers of EAM-2201 are necessary. In this study, we identified EAM-2201 metabolites after incubation of EAM-2201 with human liver microsomes using liquid chromatography–high resolution mass spectrometry (LC–HRMS). Furthermore, the specific cytochrome P450 (CYP) isozymes responsible for the formation of each EAM-2201 metabolite were characterized using human cDNA-expressed CYPs. To the best of our knowledge, this is the first in vitro evaluation of the metabolic profile of EAM-2201. We believe the results will aid in the development of a screening method to determine EAM-2201 intake. 2. Materials and methods 2.1. Chemicals and reagents EAM-2201 was purchased from Cayman Chemical Company (Ann Arbor, MI, USA). Nicotinamide adenine dinucleotide phosphate (NADP+ ), glucose-6-phosphate and glucose-6-phosphate dehydrogenase were obtained from Sigma–Aldrich Co. (St. Louis, MO, USA). Pooled human liver microsomes and human cDNAoverexpressed CYPs 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 2J2, 3A4, and 3A5 were obtained from Corning Life Sciences (Woburn, MA, USA). Homoegonol was obtained from Toronto Research Chemicals (Toronto, Ontario, Canada). Methanol and water (LC–MS grade) were obtained from Fisher Scientific (Fair Lawn, NJ, USA). Other chemicals used were of the highest quality available. 2.2. Incubation of EAM-2201 with human liver microsomes Incubation mixtures containing 240 L of potassium phosphate buffer (pH 7.4; 50 mM), 12 L of 250 mM magnesium chloride, 30 L pooled human liver microsomes (3 mg/mL), 15 L of 1 mM NADPH, and 3 L of 2 mM EAM-2201 were prepared in a total incubation volume of 300 L. Control incubations were conducted under the same conditions with EAM-2201 in the absence of NADPH. The reaction mixtures were incubated at 37 ◦ C for 20 min in a shaking water bath and the reaction was terminated by the addition of 300 L ice-cold methanol. The reaction mixture was then centrifuged at 10,000 × g for 4 min at 4 ◦ C and 500 L supernatant was evaporated under a N2 gas stream. The residue was dissolved in 100 L 45% methanol, and a 5 mL aliquot was analyzed using LC–HRMS to identify the metabolites. 2.3. In vitro metabolism of EAM-2201 in human cDNA-expressed CYPs The major CYP enzymes responsible for the metabolism of EAM2201 were determined using reaction mixtures containing 10 L of 11 different human cDNA-expressed CYP enzymes (CYPs 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 2J2, 3A4, and 3A5; 40 pmol), 1 L of EAM-2201 stock solution (0.25 mM), 4 L of 250 mM magnesium chloride, and 80 L of potassium phosphate buffer (pH 7.4; 50 mM) in a total incubation volume of 95 L. The reaction was initiated by adding 5 L NADPH-generating system, and the mixtures were incubated in triplicate for 20 min at 37 ◦ C in a shaking water bath. The reaction was stopped by adding 100 L ice-cold methanol containing 50 ng/mL homoegonol (IS). The mixtures were centrifuged at 10,000 × g for 4 min at 4 ◦ C and 150 L supernatant was evaporated under a N2 gas stream. The residue was dissolved in 50 L 45% methanol and a 5 mL aliquot was analyzed using LC–MS/MS.
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2.4. LC–MS analysis To separate and identify the structures of EAM-2201 and its metabolites, Q-Exactive Orbitrap mass spectrometer equipped with an Accela UPLC system (Thermo Fisher Scientific Inc., Waltham, MA, USA) was used. For separation of EAM-2201 and its metabolites, various columns such as Halo C18 column (2.7 m, 2.1 mm i.d. × 100 mm, Advanced Materials Technology, Wilmington, DE, USA), Accucore MS C18 column (2.6 m, 2.1 mm i.d. × 50 mm, Thermo Scientific), Luna phenylhexyl column (5.0 m, 2.0 mm i.d. × 100 mm, Phenomenex, Torrance, CA, USA), Pinnacle DB biphenyl column (1.9 m, 2.1 mm i.d. × 50 mm, Restek Co., Bellefonte, PA, USA), and Kinetex PFP column (2.6 mm, 2.1 mm i.d. × 50 mm, Phenomenex) were tested using the gradient elution of methanol and 0.1% formic acid as the mobile phase. Optimum separation of the metabolites was obtained with a Halo C18 column using a gradient elution of 0.1% formic acid in 5% methanol in (mobile phase A) and 0.1% formic acid in 95% methanol (mobile phase B) at a flow rate of 0.4 mL/min. The gradient condition was as follows: 45% mobile phase B for 10 min, 45–65% mobile phase B for 15 min, 65–85% mobile phase B for 5 min, 85% mobile phase B for 5 min, 85–45% mobile phase B for 0.1 min, and 45% mobile phase B for 2.9 min. The column and autosampler were maintained at 40 ◦ C and 4 ◦ C, respectively. The mass spectra for EAM-2201 and its metabolites were obtained with the electrospray ionization source (ESI) in positive mode. The ESI source settings for EAM-2201 and its metabolites were optimized as follows: sheath gas flow rate, 35 (arbitrary units); auxiliary gas flow rate, 15 (arbitrary units); spray voltage, 4 kV; capillary voltage, 90 V; tube lens voltage, 125 V; skimmer voltage, 28 V; heater temperature, 350 ◦ C. Data were acquired using Xcalibur software (Thermo Fisher Scientific Inc.). Full MS scan data were obtained from m/z 100 to 500 at a resolution of 70,000, while data-dependent MS/MS spectra were acquired at a resolution of 35,000 using a normalized collision energy of 45 eV. The proposed compound structures were determined using Mass Frontier software (version 6.0; HighChem Ltd., Slovakia) with product ions of EAM-2201 and its metabolites. The relative amount of each metabolite was determined using an Agilent 6490 triple quadrupole MS coupled with Agilent 1290 Infinity LC (Agilent Technologies, Santa Clara, CA, USA). Chromatographic separation was performed as described above and the ESI source for EAM-2201 and its metabolites was operated in the positive mode setting as follows: gas temperature, 230 ◦ C; gas flow, 14 L/min; nebulizer gas pressure, 35 psi; sheath gas temperature, 350 ◦ C; sheath gas flow, 11 L/min; capillary voltage, 3500 V; nozzle voltage, 1500 V. Selected reaction monitoring (SRM) of the analytes was performed using N2 gas as a collision gas set to 32, and SRM mode was applied using mass transition of each protonated molecular ion to the most abundant product ion (the first diagnostic product ions in Table 1). The formation rates of each metabolite were estimated as the peak area ratio of metabolite to internal standard (IS). The metabolites showing a peak area less than 100 count were processed as not detected (N.D). Mass Hunter software (Agilent Technologies) was used for the LC–MS/MS system control and data processing.
3. Results and discussion 3.1. Identification of EAM-2201 metabolites in human liver microsomes Incubation of EAM-2201 with human liver microsomes in the presence of NADPH resulted in 37 metabolites (M1-M37), as well as unchanged EAM-2201 according to LC–HRMS analysis (Fig. 1). The
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Table 1 Retention time, elemental composition, accurate mass, mass accuracy, and diagnostic product ions of EAM-2201 and its possible metabolites identified after incubation of EAM-2201 with human liver microsomes in the presence of NADPH. Biotransformation and metabolites
tR (min)
Formula
Exact mass [M + H]+ (m/z)
Error (ppm)
Diagnostic product ionsa (m/z)
Parent
31.4
C26 H26 FNO
388.2071
−1.2
183.0804, 232.1132, 155.0855, 144.0444
Monohydroxylation M1 M2 M3 M4 M5 M6 M7 M8 M9
23.0 24.1 26.0 26.0 27.3 27.6 28.0 28.5 30.1
C26 H26 FNO2 C26 H26 FNO2 C26 H26 FNO2 C26 H26 FNO2 C26 H26 FNO2 C26 H26 FNO2 C26 H26 FNO2 C26 H26 FNO2 C26 H26 FNO2
404.2020 404.2020 404.2020 404.2020 404.2020 404.2020 404.2020 404.2020 404.2020
−1.5 −1.1 −1.8 −1.8 −1.3 −1.7 −1.1 −1.8 −1.7
199.0754, 144.0444, 232.1132 199.0754, 144.0444, 232.1132 199.0754, 232.1132, 144.0444 183.0804, 144.0444, 248.1081 183.0804, 248.1081, 155.0855, 144.0444 199.0754, 232.1132, 144.0444 183.0804, 248.1081, 155.0855, 160.0393 199.0754, 232.1132, 144.0444 183.0804, 248.1081, 155.0855, 160.0393
Dihydroxylation M10 M11 M12 M13 M14
12.0 12.8 14.4 15.0 17.4
C26 H26 FNO3 C26 H26 FNO3 C26 H26 FNO3 C26 H26 FNO3 C26 H26 FNO3
420.1970 420.1970 420.1970 420.1970 420.1970
−1.4 −1.4 −1.3 −1.9 −1.4
199.0754, 248.1081, 144.0444 199.0754, 248.1081, 181.0648, 153.0699, 160.0393 199.0754, 248.1081, 144.0444 199.0754, 248.1081, 144.0444 215.0703, 232.1132, 144.0444
Dihydrodiol formation M15
17.3
C26 H28 FNO3
422.2126
−1.3
199.0754, 232.1132, 217.0859, 144.0444
Oxidative defluorination M16
27.8
C26 H27 NO2
386.2115
−1.3
183.0804, 155.0855, 230.1176
Oxidative defluorination + monohydroxylation 14.0 M17 M18 15.6
C26 H27 NO3 C26 H27 NO3
402.2064 402.2064
−1.3 −1.8
199.0754, 230.1176, 144.0444 199.0754, 144.0444, 230.1176
Oxidative defluorination + dihydroxylation 4.2 M19 4.5 M20 7.4 M21
C26 H27 NO4 C26 H27 NO4 C26 H27 NO4
418.2013 418.2013 418.2013
−1.1 −1.3 −1.1
199.0754, 246.1125, 160.0393 199.0754, 246.1125, 160.0393 215.0703, 230.1176, 144.0444
N-Dealkylation M22
C21 H17 NO
300.1383
−1.1
144.0444, 183.0804, 155.0855
C21 H17 NO2 C21 H17 NO2
316.1332 316.1332
−1.5 −1.4
144.0444, 199.0754 144.0444, 199.0754
C21 H17 NO3
332.1281
−1.3
144.0444, 215.0703
C26 H25 NO3
N-Dealkylation + monohydroxylation M23 M24 N-Dealkylation + dihydroxylation M25
22.6 5.6 6.8 3.0
Oxidative defluorination to carboxylic acid 27.2 M26
400.1907
−1.3
183.0804, 244.0968, 155.0855, 144.0444
Oxidative defluorination to carboxylic acid + monohydroxylation M27 14.4 C26 H25 NO4
416.1856
−1.4
199.0754, 144.0444, 244.0968
Dehydrogenation M28
C26 H24 FNO
386.1915
−1.0
181.0648, 153.0699, 144.0444, 232.1132
Dehydrogenation + monohydroxylation M29 25.6
C26 H24 FNO2
402.1864
−1.1
197.0597, 169.0648, 232.1132
Dehydrogenation + dihydroxylation M30 M31 M32 M33 M34 M35 M36
C26 H24 FNO3 C26 H24 FNO3 C26 H24 FNO3 C26 H24 FNO3 C26 H24 FNO3 C26 H24 FNO3 C26 H24 FNO3
418.1813 418.1813 418.1813 418.1813 418.1813 418.1813 418.1813
−1.2 −1.0 −1.4 −1.0 −1.7 −1.4 −1.2
197.0597, 169.0648, 248.1081, 144.0444 197.0597, 169.0648, 248.1081, 160.0393 197.0597, 169.0648, 248.1081, 144.0444 213.0546, 185.0597, 232.1132, 144.0444 213.0546, 185.0597, 232.1132, 144.0444 197.0597, 169.0648, 248.1081, 160.0393 213.0546, 232.1132, 185.0597, 144.0444
400.1907
−1.1
197.0597, 169.0648, 144.0444, 230.1176
30.9
14.4 15.3 16.7 17.6 20.0 20.6 22.5
Oxidative defluorination + monohydroxylation + dehydrogenation 17.4 C26 H25 NO3 M37 a
Product ions are arranged in the order of the intensity.
exact masses of molecular ion ([M + H]+ ) and diagnostic product ions, retention times, and biotransformation pathways of EAM2201 and its possible metabolites (M1–M37) are listed in Table 1. Diagnostic product ions are listed in the order of relative abundance. Chromatographic separation of the metabolites was needed to unambiguously identify the structure of the metabolites because many metabolites have the same [M + H]+ ions including M1–M9
at m/z 404.2020, M10–M14 at m/z 420.1970, M31–M36 at m/z 418.1813, and etc. (Fig. 1). After many trials with different columns and mobile phase combinations, EAM-2201 and its 37 metabolites were well separated on a Halo C18 column using a gradient elution of methanol and 0.1% formic acid (Fig. 1, Table 1). The MS/MS spectrum of EAM-2201 showed four characteristic product ions at m/z 183.0804 [(4-ethylnaphthalen-1-yl)(oxo) methylium ion], m/z 155.0855 (4-ethylnaphthalen-1-ylium ion),
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Fig. 1. Extracted ion chromatograms of EAM-2201 and its possible metabolites obtained following incubation of EAM-2201 with human liver microsomes in the presence of NADPH for 20 min at 37 ◦ C using 3 ppm accuracy.
m/z 232.1132 [(1-(5-fluoropentyl)-1H-indol-3-yl)(oxo) methylium ion], and m/z 144.0444 (loss of 5-fluoropentyl moiety from m/z 232.1132) (Fig. 2), serving as diagnostic product ions for the identification of EAM-2201 metabolites.
3.1.1. Monohydroxylation and dihydroxylation M1-M9 showed the [M + H]+ ion at m/z 404.2020, 16 amu more than the EAM-2201 [M + H]+ ion, indicating hydroxylation of EAM2201 (Fig. 1). Based on MS/MS spectra, the nine hydroxy-EAM-2201 (M1–M9) were classified into three categories due to hydroxylation positions at the ethylnaphthalene, aliphatic alkyl chain and indole moieties, although the exact hydroxylation positions were not identified. M1, M2, M3, M6, and M8 showed the characteristic product ion at m/z 199.0754 [hydroxy-(4-ethylnaphthalen-1-yl)(oxo) methylium ion], m/z 232.1132 [(1-(5-fluoropentyl)-1H-indol-3yl)(oxo) methylium ion] and m/z 144.0444 [(1H-indol-3-yl)(oxo) methylium ion], suggesting that hydroxylation in M1, M2, M3, M6, and M8 occurred at the ethylnaphthalene moiety (Fig. 2). M4 and M5 showed the product ions at m/z 248.1081, 16 amu higher than m/z 232.1132, m/z 144.0444, m/z 155.0855, and m/z 183.0804, indicating that hydroxylation occurred in the EAM-2201 fluoropentyl group (Fig. 2). M7 and M9 produced the characteristic product ions at m/z 183.0804, m/z 248.1081, m/z 155.0855, and m/z 160.0393 (loss of 5-fluoropentyl moiety from m/z 248.1081), indicating that hydroxylation of M7 and M9 occurred at the indole moiety, but the accurate hydroxylation positions were not identified (Fig. 2).
Five EAM-2001 metabolites (M10-M14), which were 32 amu higher than EAM-2201, showed the [M + H]+ ion at m/z 420.1970, suggesting that M10-M14 may be dihydroxylated EAM-2201 metabolites (Fig. 1). M10, M12 and M13 showed the product ions at m/z 199.0754 [hydroxy-(4-ethyl1-naphthaldehyde)], m/z 248.1081 [hydroxy-(1-(5-fluoropentyl)1H-indole-3-carbaldehyde)] and m/z 144.0444 (1H-indole-3carbaldehyde), indicating that dihydroxylation in M10, M12 and M13 occurred at both the ethylnaphthalene moiety and 5fluoropentyl group (Supplement 1A). M11 showed product ions at m/z 199.0754, m/z 248.1081 and m/z 160.0393 [hydroxy-(1H-indole-3-carbaldehyde)], indicating the dihydroxylation in M11 occurred at the ethylnaphthalene and indole moieties (Supplement 1B). M11 showed two vinylnaphthalene product ions at m/z 153.0699 and m/z 181.0648 due to dehydrogenation of m/z 155.0855 and m/z 183.0804, respectively. M14 produced the product ions at m/z 215.0703 [dihydroxy(4-ethyl-1-naphthaldehyde)], m/z 232.1132 and m/z 144.0444, indicating dihydroxylation of EAM-2201 at the ethylnaphthalene moiety (Supplement 1C). 3.1.2. Dihydrodiol formation M15 showed the [M + H]+ ion at m/z 422.2126, which is 34 amu higher than EAM-2201 (Fig. 1), and generated product ions at m/z 217.0859, m/z 199.0754 (loss of water from m/z 217.0859), m/z 232.1132, and m/z 144.0444, suggesting that the dihydrodiol metabolite was formed (Supplement 1D). These results indicate that M15 is a dihydrodiolized EAM-2201 via epoxide formation at the naphthalene ring and subsequent hydrolysis of the epoxide
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Fig. 2. Product ion spectra of EAM-2201 and hydroxy-EAM-2201 (M1–M9) acquired following incubation of EAM-2201 with human liver microsomes in the presence of NADPH for 20 min at 37 ◦ C using a Q-Exactive mass spectrometer in NCE at 45 eV and proposed fragmentation pattern of principal ions.
moiety. The position of M15 hydroxyl groups was not accurately identified. 3.1.3. Oxidative defluorination and consecutive hydroxylation M16 showed the [M + H]+ ion at m/z 386.2115, which was 2 amu lower than the EAM-2201 [M + H]+ ion (Fig. 1). M16 produced product ions at m/z 230.1176 [(1-(5-hydroxypentyl)-1Hindol-3-yl)(oxo) methylium ion], m/z 183.0804 and m/z 155.0855, indicating that M16 was formed from EAM-2201 via oxidative defluorination (Supplement 2A). Interestingly, M16 was formed from EAM-2201 after incubation with human liver microsomes in both the absence and presence of NADPH, and the amount of M16 decreased in the presence of NADPH. This result suggests that M16 could be formed without NADPH, but further metabolic reactions such as hydroxylation of M16 to M17-M21 must be performed in the presence of NADPH. M17 and M18 showed the [M + H]+ ion at m/z 402.2064, which was 16 amu higher than the M16 [M + H]+ ion, suggesting additional hydroxylation of M16 (Fig. 1). M17 and M18 produced product ions at m/z 199.0754 [hydroxy-(4-ethyl-1-naphthaldehyde)], m/z 230.1176 and m/z 144.0444, indicating hydroxylation of M16 at the ethylnaphthalene moiety (Supplement 2B). M19-M21 showed the [M + H]+ ion at m/z 418.2013, which was 32 amu higher than the M16 [M + H]+ ion, indicating dihydroxylation of M16 (Fig. 1, Table 1). M19 and M20 showed the product ions of m/z 199.0754, m/z 246.1125 [hydroxy-(1-(5hydroxypentyl)-1H-indol-3-carbaldehyde)] and m/z 160.0393 (loss of 5-hydroxypentyl moiety from m/z 246.1125), suggesting that hydroxylation occurred at both the ethylnaphthalene and indole moieties (Supplement 2C). M21 showed the characteristic product ions at m/z 215.0703 [dihydroxy-(4-ethyl-1-naphthaldehyde)], m/z 230.1176 and m/z 144.0444, indicating that M21 (dihydroxy-M16) was formed from M16 via dihydroxylation of the ethylnaphthalene moiety (Supplement 2D).
3.1.4. N-Dealkylation and consecutive hydroxylation M22 showed the [M + H]+ ion at m/z 300.1383, which was 88 amu lower than the EAM-2201 [M + H]+ ion, suggesting the loss of the 5-fluoropentyl group (Fig. 1, Table 1). M22 showed the product ions at m/z 144.0444, m/z 183.0804 and m/z 155.0855, supporting that M22 is N-dealkyl-EAM-2201 due to the loss of the 5-fluoropentyl group (Supplement 3A). M23 and M24 showed the [M + H]+ ion at m/z 316.1332, which was 16 amu higher than the M22 [M + H]+ ion, suggesting hydroxylation of M22 (Fig. 1, Table 1). These metabolites showed the characteristic product ions at m/z 144.0444 and m/z 199.0754, suggesting hydroxylation of the ethylnaphthalene moiety (Supplement 3B). M25 showed the [M + H]+ ion at m/z 332.1281, which was 32 amu higher than the M22 [M + H]+ ion, suggesting dihydroxylation of M22 (Fig. 1, Table 1). M25 produced product ions at m/z 215.0703 and m/z 144.0444, indicating dihydroxylation of M22 at the ethylnaphthalene moiety to dihydroxy-M22 (M25) (Supplement 3C).
3.1.5. Oxidative defluorination to carboxylic acid and consecutive hydroxylation M26 showed the [M + H]+ ion at m/z 400.1907 and the product ions at m/z 144.0444, m/z 155.0855, m/z 183.0804, and m/z 244.0968 [(1-(4-carboxybutyl)-1H-indol-3-yl)(oxo) methylium ion] (Supplement 4A), indicating that M26 may be EAM-2201 pentanoic acid via the biotransformation of the fluoropentyl group to pentanoic acid. M27 showed the [M + H]+ ion at m/z 416.1856, which was 16 amu higher than the M26 [M + H]+ ion, indicating hydroxylation of M26 (Fig. 1, Table 1). M27 showed product ions at m/z 144.0444, m/z 199.0754 and m/z 244.0968, and therefore, M27 was tentatively identified as hydroxy-M26 via hydroxylation of the ethylnaphthalene moiety (Supplement 4B).
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Fig. 3. Product ion spectra of (A) dehydro-EAM-2201 (M28), (B) hydroxy-M28 (M29), (C)–(E) dihydroxy-M28 (M30-M36) acquired following incubation of EAM-2201 with human liver microsomes in the presence of NADPH for 20 min at 37 ◦ C using a Q-Exactive mass spectrometer in NCE at 45 eV.
3.1.6. Dehydrogenation and consecutive metabolism M28 showed the [M + H]+ ion at m/z 386.1915, which was 2 amu lower than the EAM-2201 [M + H]+ ion (Fig. 1), and produced product ions at m/z 232.1132 [(1-(5-fluoropentyl)-1H-indol-3-yl)(oxo) (methylium ion)], as well as m/z 153.0699 and m/z 181.0648, which were 2 amu lower than m/z 155.0855 and m/z 183.0804, respectively (Fig. 3A). These results suggest that M28 may be dehydro-EAM-2201 via dehydrogenation at the ethyl group. M29 showed the [M + H]+ ion at m/z 402.1864, which was 16 amu higher than the M28 [M + H]+ ion, suggesting it was hydroxy-M29 (Fig. 1). M29 showed product ions at m/z 232.1132, m/z 197.0597 (hydroxylation of m/z 181.0648) and m/z 169.0648 (hydroxylation of m/z 153.0699), indicating that hydroxylation occurred at the vinylnaphthalene moiety (Fig. 3B). Seven metabolites, M30-M36 showed the [M + H]+ ion at m/z 418.1813, which was 32 amu higher than the M28 [M + H]+ ion, suggesting that they were dihydroxy-M28 (Fig. 1, Table 1). M30 and M32 showed product ions at m/z 144.0444 (1H-indole-3carbaldehyde), m/z 169.0648 (hydroxylation of m/z 153.0699), m/z 197.0597 (hydroxylation of m/z 181.0648), and m/z 248.1081 (hydroxylation of m/z 232.1132), indicating that M30 and M32 were hydroxylated at the vinylnaphthalene and 5-fluoropentyl group, respectively (Fig. 3C). M31 and M35 showed the product ions at
m/z 160.0393 (hydroxylation of m/z 144.0444), m/z 169.0648, m/z 197.0597, and m/z 248.1081, indicating that M31 and M35 were hydroxylated at the vinylnaphthalene and indole moieties, respectively (Fig. 3D). However, M33, M34 and M36 produced product ions at m/z 144.0444, m/z 232.1132, m/z 185.0597 (dihydroxylation of m/z 153.0699), and m/z 213.0546 (dihydroxylation of m/z 181.0648), indicating that dihydroxylation of the vinylnaphthalene moiety resulted in M33, M34 and M36 (dihydroxy-M28; Fig. 3E). M37 showed the [M + H]+ ion at m/z 400.1907 (Fig. 1) and the product ions at m/z 144.0444, m/z 169.0648 (hydroxylation of m/z 153.0699), m/z 197.0597 (hydroxylation of m/z 181.0648), and m/z 230.1176 [(1-(5-hydroxypentyl)-1H-indol-3-yl)(oxo) methylium ion] (Table 1, Supplement 4C). Based on these results, M37 resulted from dehydrogenation of the ethyl group, the hydroxylation of the vinylnaphthalene moiety, and oxidative defluorination of the 5fluoropentyl group to 5-hydroxypentyl group. Based on these results, the proposed possible metabolic pathways of EAM-2201 in human liver microsomes are shown in Fig. 4. EAM-2201 was metabolized to 37 metabolites via mono- and dihydroxylation at the naphthalene moiety, indole moiety and pentyl chain, dehydrogenation at ethyl group, oxidative defluorination at the fluoropentyl chain, dihydrodiol formation, N-dealkylation, and carboxylation.
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Fig. 4. Possible metabolic pathways of EAM-2201 in human liver microsomes.
J.H. Kim et al. / Journal of Pharmaceutical and Biomedical Analysis 119 (2016) 50–58
57
Table 2 Relative formation rates of major EAM-2201 metabolites obtained from incubation of EAM-2201 with human cDNA-expressed CYPs in the presence of NADPH (n = 3). Relative formation rates of EAM-2201 metabolites (peak area ratios/min/fmol CYP, mean ± SD)
M1 M2 M3 M4 M5 M7 M8 M9 M10 M12 M14 M15 M16 M18 M22 M24 M26 M27 M28 M29 M30 M31 M32 M33 M34 M35 M36 M37
CYP1A2
CYP2B6
CYP2C8
CYP2C9
N.D 18.7 ± 3.3 N.D 1.5 ± 0.5 10.0 ± 1.2 297.0 ± 52.7 0.9 ± 0.3 24.1 ± 4.4 N.D N.D N.D 1.5 ± 0.4 2.3 ± 0.1 N.D 14.4 ± 2.3 0.9 ± 0.2 N.D N.D 4.2 ± 0.9 2.3 ± 0.3 N.D N.D N.D N.D N.D N.D N.D N.D
N.D N.D N.D 54.8 ± 3.7 1.1 ± 0.3 25.8 ± 3.4 N.D N.D N.D N.D N.D 1.3 ± 0.2 9.4 ± 1.3 N.D 3.4 ± 0.7 N.D N.D N.D 0.3 ± 0.0 N.D N.D N.D N.D N.D N.D N.D N.D N.D
N.D 3.0 ± 0.5 N.D 42.9 ± 6.9 17.7 ± 1.8 1.9 ± 0.1 N.D N.D N.D N.D N.D N.D 194.3 ± 34.8 N.D 0.5 ± 0.1 0.5 ± 0.1 6.3 ± 1.8 N.D 1.5 ± 0.3 N.D N.D N.D N.D N.D N.D N.D N.D N.D
37.7 ± 7.9 100.3 ± 16.4 N.D N.D N.D 5.2 ± 1.5 1.2 ± 0.3 N.D N.D N.D 6.1 ± 1.9 0.5 ± 0.1 N.D 0.6 ± 0.2 N.D N.D N.D N.D 120.9 ± 11.3 5.5 ± 2.3 N.D N.D N.D N.D 0.4 ± 0.2 N.D 0.7 ± 0.4 N.D
CYP2C19 1.2 ± 36.5 ± N.D 39.0 ± 39.8 ± 40.4 ± 0.7 ± 1.4 ± 1.0 ± 6.6 ± N.D 1.1 ± 14.2 ± 2.9 ± N.D N.D 1.2 ± 1.7 ± 33.8 ± 9.1 ± 10.0 ± 0.6 ± 1.9 ± N.D N.D N.D N.D 2.4 ±
0.2 3.1 4.9 2.6 2.9 0.2 0.1 0.7 0.8 0.1 1.5 0.4
0.2 0.4 5.1 1.2 2.1 0.5 0.1
0.3
CYP2D6
CYP2J2
CYP3A4
CYP3A5
0.4 ± 0.1 33.6 ± 5.6 N.D 0.7 ± 0.4 N.D 119.4 ± 15.2 2.0 ± 0.8 8.6 ± 2.2 N.D N.D 2.0 ± 0.3 1.9 ± 0.8 1.6 ± 0.2 N.D N.D N.D N.D N.D 83.6 ± 4.0 6.7 ± 0.8 N.D N.D N.D N.D N.D N.D N.D N.D
2.4 ± 0.2 5.4 ± 1.0 N.D 12.2 ± 2.4 0.4 ± 0.1 N.D N.D N.D N.D N.D N.D N.D 16.7 ± 2.4 N.D N.D N.D N.D N.D 2.8 ± 1.5 N.D N.D N.D N.D N.D N.D N.D N.D N.D
7.8 ± 2.7 3.9 ± 1.3 3.5 ± 0.8 N.D N.D 0.6 ± 0.0 N.D N.D N.D N.D 2.8 ± 0.7 N.D N.D N.D N.D 2.0 ± 0.3 N.D 3.8 ± 0.5 2.2 ± 1.1 252.4 ± 14.8 5.1 ± 1.2 7.2 ± 0.6 27.8 ± 2.4 5.7 ± 0.9 4.6 ± 0.4 2.1 ± 1.3 1.3 ± 0.2 1.9 ± 0.6
1.4 ± 0.1 67.0 ± 5.7 N.D 2.2 ± 0.4 4.7 ± 0.2 11.6 ± 0.9 14.4 ± 1.0 3.0 ± 0.8 N.D N.D N.D N.D 0.8 ± 0.3 N.D 2.6 ± 0.2 N.D N.D N.D 47.5 ± 16.3 14.5 ± 5.1 N.D N.D N.D N.D N.D N.D N.D N.D
N.D; <0.3 peak area ratios/min/fmol CYP.
3.2. Screening of CYPs responsible for the metabolism of EAM-2201 The calibration curve was linear over 0.5–500 pmol of EAM2201 in blank microsomal incubation mixture, and the coefficients of variation and relative errors (i.e., precision and accuracy) of the quality control samples at 0.5, 1.5, 100, and 400 pmol of EAM2201 in blank microsomal incubation mixture were ≤9.5% and −12.0% to 13.0%, respectively. The amounts of the metabolites were determined using peak area ratios since the authentic standards of EAM-2201 metabolites were not available. Therefore, there is a limitation in the accurate interpretation of the formation rates for the metabolites. To characterize CYP isozymes responsible for the metabolism of EAM-2201, EAM-2201 (2 M) was incubated with major human cDNA-expressed CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP2J2, CYP3A4, or CYP3A5 in the presence of NADPH. The metabolism of EAM-2201 was mediated by CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2J2, CYP3A4, and CYP3A5 (Table 2). CYP2A6 and CYP2E1 enzymes were not involved in the metabolism of EAM-2201. Hydroxylation of EAM-2201 was catalyzed by multiple CYP enzymes, but each isozyme appeared to have favorable hydroxylation sites. Hydroxylation of the ethylnaphthalene moiety to M1, M2, M3, and M8 was predominantly mediated by CYP2C9, CYP2C19, CYP2D6, and CYP3A4/5 enzymes, whereas the formation of M7 and M9 via hydroxylation of the indole ring was mediated by CYP1A2, CYP2C19, CYP2D6, and CYP3A5 enzymes. In addition, CYP1A2, CYP2B6, CYP2C8, CYP2C19, CYP2J2, and CYP3A5 were responsible for the hydroxylation of 5-fluoropentyl chain to M4 and M5. Although oxidative defluorination of EAM-2201 to M16 occurred in the absence of NADPH as described above, the formation of M16 from EAM-2201 was also catalyzed by CYP2C8 with minor contributions from CYP1A2, CYP2B6, CYP2C19, CYP2D6, CYP2J2, and CYP3A5. N-Dealkylation of EAM-2201 to M22 was mediated
by CYP1A2, CYP2B6, CYP2C8, and CYP3A5 enzymes. Dehydrogenation at the ethyl group of EAM-2201 to M28 was catalyzed by CYP2C9, CYP2C19, CYP2D6, and CYP3A5, with minor contributions from CYP1A2, CYP2B6, CYP2C8, and CYP2J2. Hydroxylation of M28 to M29 was mediated by CYP3A4 with minor contributions from CYP2C9, CYP2C19, CYP2D6, CYP3A5, and CYP1A2 enzymes. Hydroxylation of M29 to seven metabolites, M30-M36 was catalyzed by CYP3A4 enzyme. The formation of M6, M11, M13, M17, M19, M20, M21, M23, and M25 from EAM-2201 were very slight, and therefore, we could not characterize CYP enzymes responsible for the formation of these nine metabolites.
4. Conclusion Based on the exact mass of [M + H]+ and the diagnostic product ions, 37 metabolites of EAM-2201 were identified as nine hydroxyEAM-2201 (M1–M9), five dihydroxy-EAM-2201 (M10–M14), dihydrodiol-EAM-2201 (M15), defluorinated EAM-2201 (M16), two hydroxy-M16 (M17 and M18), three dihydroxy-M16 (M19M21), 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 defluorinated hydroxy-M28 (M37) (Table 1, Fig. 1). Multiple CYPs including CYP1A2, CYP2C8/9/19, CYP2B6, CYP2D6, and CYP3A4/5 were responsible for the metabolism of EAM-2201. The metabolic pathways of EAM-2201 may aid in the development of analytical methods for monitoring the abuse of EAM-2201 in biological samples such as urine and blood.
Conflict of interest The authors have declared no conflict of interest.
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In vitro metabolism of a novel synthetic cannabinoid, EAM-2201, in human liver microsomes and human recombinant cytochrome P450s Ju Hyun Kim a,1 , Hee Seung Kim b,1 , Tae Yeon Kong a , Joo Young Lee a , Jin Young Kim b , Moon Kyo In b , Hye Suk Lee a,∗ a b
College of Pharmacy, The Catholic University of Korea, 43 Jibong-ro, Wonmi-gu, Bucheon-si, Gyeonggi-do 420-743, Republic of Korea Forensic Chemistry Laboratory, Forensic Science Division, Supreme Prosecutor’s Office, 157 Banpo-daero, Seocho-gu, Seoul 137-730, Republic of Korea
a r t i c l e
i n f o
Article history: Received 5 September 2015 Received in revised form 5 November 2015 Accepted 18 November 2015 Available online 22 November 2015 Keywords: EAM-2201 Synthetic cannabinoid In vitro metabolism LC–HRMS Human liver microsomes Cytochrome P450
a b s t r a c t 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. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Synthetic cannabinoids are referred to as a group of substances with similar chemical structures generally binding to cannabinoid receptor type 1 (CB1 ) or type 2 (CB2 ). Since the first identification of synthetic cannabinoids in ‘herbal mixtures’ in 2008 [1–3], their abuse has continuously increased worldwide and has become a major issue in the forensic community [4–9]. Possibly, the most prevalent synthetic cannabinoid is JWH-018, a naphthoylindole introduced in 2008, and its pharmacokinetic and pharmacological characteristics are well-known [10–16]. Owing to its great potential for abuse, JWH-018 is prohibited in most countries [13,17]. However, because of the well-established structure–activity relationship for synthetic cannabinoids [18–22], various modifications of JWH-018 have consistently appeared involving introduction of the fluorine atom (AM-2201) [23], and substitution of the naphthyl group to a cyclopropyl group (UR-144 and XLR-11) [9,24],
∗ Corresponding author at: Drug Metabolism and Bioanalysis Laboratory, College of Pharmacy, The Catholic University of Korea, 43 Jibong-ro, Wonmi-gu, Bucheon-si, Gyeonggi-do 420-743, Republic of Korea. Fax: +82 32 342 2013. E-mail address: [email protected] (H.S. Lee). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.jpba.2015.11.023 0731-7085/© 2015 Elsevier B.V. All rights reserved.
adamantyl group (APICA and 5F-APICA) [9] or quinolinyl group (QUPIC and QUCHIC) [25,26]. Synthetic cannabinoids are generally found in seized herbal materials, and the number of seizures has increased approximately 5-fold from 2009 to June 2013 in Korea [27]. Newly emerged synthetic cannabinoids are mostly used without the knowledge of their potential toxicity and pharmacokinetic properties, and several fatalities have been reported [28–30]. EAM-2201, (4-ethyl-1-naphthalenyl)[1-(5-fluoropentyl)-1Hindol-3-yl]-methanone, was first identified in 2012 as an ingredient of illegal synthetic cannabis smoking blends [9]. The pharmacological effects of EAM-2201 have not been investigated in detail, but psychotic effects can be easily presumed from its structural analogues such as JWH-210 and AM-2201 [10,23]. Therefore, the illegal use of EAM-2201 should be carefully monitored. The metabolism of naphthoylindole-type synthetic cannabinoids such as JWH-018, JWH-210, AM-2201, and MAM-2201 has been well-characterized in urine samples obtained from suspected users and/or in vitro studies using human liver microsomes and hepatocytes [12–16,31–37], but there are few reports on the characterization of drug-metabolizing enzymes responsible for the specific metabolic reactions of synthetic cannabinoids [15,34].
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Based on its structural similarity, EAM-2201 may be extensively metabolized. However, in vitro and in vivo EAM-2201 metabolism in animals and humans has not been reported. Therefore, identifying the metabolism of EAM-2201 and characterizing major metabolites as biomarkers of EAM-2201 are necessary. In this study, we identified EAM-2201 metabolites after incubation of EAM-2201 with human liver microsomes using liquid chromatography–high resolution mass spectrometry (LC–HRMS). Furthermore, the specific cytochrome P450 (CYP) isozymes responsible for the formation of each EAM-2201 metabolite were characterized using human cDNA-expressed CYPs. To the best of our knowledge, this is the first in vitro evaluation of the metabolic profile of EAM-2201. We believe the results will aid in the development of a screening method to determine EAM-2201 intake. 2. Materials and methods 2.1. Chemicals and reagents EAM-2201 was purchased from Cayman Chemical Company (Ann Arbor, MI, USA). Nicotinamide adenine dinucleotide phosphate (NADP+ ), glucose-6-phosphate and glucose-6-phosphate dehydrogenase were obtained from Sigma–Aldrich Co. (St. Louis, MO, USA). Pooled human liver microsomes and human cDNAoverexpressed CYPs 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 2J2, 3A4, and 3A5 were obtained from Corning Life Sciences (Woburn, MA, USA). Homoegonol was obtained from Toronto Research Chemicals (Toronto, Ontario, Canada). Methanol and water (LC–MS grade) were obtained from Fisher Scientific (Fair Lawn, NJ, USA). Other chemicals used were of the highest quality available. 2.2. Incubation of EAM-2201 with human liver microsomes Incubation mixtures containing 240 L of potassium phosphate buffer (pH 7.4; 50 mM), 12 L of 250 mM magnesium chloride, 30 L pooled human liver microsomes (3 mg/mL), 15 L of 1 mM NADPH, and 3 L of 2 mM EAM-2201 were prepared in a total incubation volume of 300 L. Control incubations were conducted under the same conditions with EAM-2201 in the absence of NADPH. The reaction mixtures were incubated at 37 ◦ C for 20 min in a shaking water bath and the reaction was terminated by the addition of 300 L ice-cold methanol. The reaction mixture was then centrifuged at 10,000 × g for 4 min at 4 ◦ C and 500 L supernatant was evaporated under a N2 gas stream. The residue was dissolved in 100 L 45% methanol, and a 5 mL aliquot was analyzed using LC–HRMS to identify the metabolites. 2.3. In vitro metabolism of EAM-2201 in human cDNA-expressed CYPs The major CYP enzymes responsible for the metabolism of EAM2201 were determined using reaction mixtures containing 10 L of 11 different human cDNA-expressed CYP enzymes (CYPs 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 2J2, 3A4, and 3A5; 40 pmol), 1 L of EAM-2201 stock solution (0.25 mM), 4 L of 250 mM magnesium chloride, and 80 L of potassium phosphate buffer (pH 7.4; 50 mM) in a total incubation volume of 95 L. The reaction was initiated by adding 5 L NADPH-generating system, and the mixtures were incubated in triplicate for 20 min at 37 ◦ C in a shaking water bath. The reaction was stopped by adding 100 L ice-cold methanol containing 50 ng/mL homoegonol (IS). The mixtures were centrifuged at 10,000 × g for 4 min at 4 ◦ C and 150 L supernatant was evaporated under a N2 gas stream. The residue was dissolved in 50 L 45% methanol and a 5 mL aliquot was analyzed using LC–MS/MS.
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2.4. LC–MS analysis To separate and identify the structures of EAM-2201 and its metabolites, Q-Exactive Orbitrap mass spectrometer equipped with an Accela UPLC system (Thermo Fisher Scientific Inc., Waltham, MA, USA) was used. For separation of EAM-2201 and its metabolites, various columns such as Halo C18 column (2.7 m, 2.1 mm i.d. × 100 mm, Advanced Materials Technology, Wilmington, DE, USA), Accucore MS C18 column (2.6 m, 2.1 mm i.d. × 50 mm, Thermo Scientific), Luna phenylhexyl column (5.0 m, 2.0 mm i.d. × 100 mm, Phenomenex, Torrance, CA, USA), Pinnacle DB biphenyl column (1.9 m, 2.1 mm i.d. × 50 mm, Restek Co., Bellefonte, PA, USA), and Kinetex PFP column (2.6 mm, 2.1 mm i.d. × 50 mm, Phenomenex) were tested using the gradient elution of methanol and 0.1% formic acid as the mobile phase. Optimum separation of the metabolites was obtained with a Halo C18 column using a gradient elution of 0.1% formic acid in 5% methanol in (mobile phase A) and 0.1% formic acid in 95% methanol (mobile phase B) at a flow rate of 0.4 mL/min. The gradient condition was as follows: 45% mobile phase B for 10 min, 45–65% mobile phase B for 15 min, 65–85% mobile phase B for 5 min, 85% mobile phase B for 5 min, 85–45% mobile phase B for 0.1 min, and 45% mobile phase B for 2.9 min. The column and autosampler were maintained at 40 ◦ C and 4 ◦ C, respectively. The mass spectra for EAM-2201 and its metabolites were obtained with the electrospray ionization source (ESI) in positive mode. The ESI source settings for EAM-2201 and its metabolites were optimized as follows: sheath gas flow rate, 35 (arbitrary units); auxiliary gas flow rate, 15 (arbitrary units); spray voltage, 4 kV; capillary voltage, 90 V; tube lens voltage, 125 V; skimmer voltage, 28 V; heater temperature, 350 ◦ C. Data were acquired using Xcalibur software (Thermo Fisher Scientific Inc.). Full MS scan data were obtained from m/z 100 to 500 at a resolution of 70,000, while data-dependent MS/MS spectra were acquired at a resolution of 35,000 using a normalized collision energy of 45 eV. The proposed compound structures were determined using Mass Frontier software (version 6.0; HighChem Ltd., Slovakia) with product ions of EAM-2201 and its metabolites. The relative amount of each metabolite was determined using an Agilent 6490 triple quadrupole MS coupled with Agilent 1290 Infinity LC (Agilent Technologies, Santa Clara, CA, USA). Chromatographic separation was performed as described above and the ESI source for EAM-2201 and its metabolites was operated in the positive mode setting as follows: gas temperature, 230 ◦ C; gas flow, 14 L/min; nebulizer gas pressure, 35 psi; sheath gas temperature, 350 ◦ C; sheath gas flow, 11 L/min; capillary voltage, 3500 V; nozzle voltage, 1500 V. Selected reaction monitoring (SRM) of the analytes was performed using N2 gas as a collision gas set to 32, and SRM mode was applied using mass transition of each protonated molecular ion to the most abundant product ion (the first diagnostic product ions in Table 1). The formation rates of each metabolite were estimated as the peak area ratio of metabolite to internal standard (IS). The metabolites showing a peak area less than 100 count were processed as not detected (N.D). Mass Hunter software (Agilent Technologies) was used for the LC–MS/MS system control and data processing.
3. Results and discussion 3.1. Identification of EAM-2201 metabolites in human liver microsomes Incubation of EAM-2201 with human liver microsomes in the presence of NADPH resulted in 37 metabolites (M1-M37), as well as unchanged EAM-2201 according to LC–HRMS analysis (Fig. 1). The
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Table 1 Retention time, elemental composition, accurate mass, mass accuracy, and diagnostic product ions of EAM-2201 and its possible metabolites identified after incubation of EAM-2201 with human liver microsomes in the presence of NADPH. Biotransformation and metabolites
tR (min)
Formula
Exact mass [M + H]+ (m/z)
Error (ppm)
Diagnostic product ionsa (m/z)
Parent
31.4
C26 H26 FNO
388.2071
−1.2
183.0804, 232.1132, 155.0855, 144.0444
Monohydroxylation M1 M2 M3 M4 M5 M6 M7 M8 M9
23.0 24.1 26.0 26.0 27.3 27.6 28.0 28.5 30.1
C26 H26 FNO2 C26 H26 FNO2 C26 H26 FNO2 C26 H26 FNO2 C26 H26 FNO2 C26 H26 FNO2 C26 H26 FNO2 C26 H26 FNO2 C26 H26 FNO2
404.2020 404.2020 404.2020 404.2020 404.2020 404.2020 404.2020 404.2020 404.2020
−1.5 −1.1 −1.8 −1.8 −1.3 −1.7 −1.1 −1.8 −1.7
199.0754, 144.0444, 232.1132 199.0754, 144.0444, 232.1132 199.0754, 232.1132, 144.0444 183.0804, 144.0444, 248.1081 183.0804, 248.1081, 155.0855, 144.0444 199.0754, 232.1132, 144.0444 183.0804, 248.1081, 155.0855, 160.0393 199.0754, 232.1132, 144.0444 183.0804, 248.1081, 155.0855, 160.0393
Dihydroxylation M10 M11 M12 M13 M14
12.0 12.8 14.4 15.0 17.4
C26 H26 FNO3 C26 H26 FNO3 C26 H26 FNO3 C26 H26 FNO3 C26 H26 FNO3
420.1970 420.1970 420.1970 420.1970 420.1970
−1.4 −1.4 −1.3 −1.9 −1.4
199.0754, 248.1081, 144.0444 199.0754, 248.1081, 181.0648, 153.0699, 160.0393 199.0754, 248.1081, 144.0444 199.0754, 248.1081, 144.0444 215.0703, 232.1132, 144.0444
Dihydrodiol formation M15
17.3
C26 H28 FNO3
422.2126
−1.3
199.0754, 232.1132, 217.0859, 144.0444
Oxidative defluorination M16
27.8
C26 H27 NO2
386.2115
−1.3
183.0804, 155.0855, 230.1176
Oxidative defluorination + monohydroxylation 14.0 M17 M18 15.6
C26 H27 NO3 C26 H27 NO3
402.2064 402.2064
−1.3 −1.8
199.0754, 230.1176, 144.0444 199.0754, 144.0444, 230.1176
Oxidative defluorination + dihydroxylation 4.2 M19 4.5 M20 7.4 M21
C26 H27 NO4 C26 H27 NO4 C26 H27 NO4
418.2013 418.2013 418.2013
−1.1 −1.3 −1.1
199.0754, 246.1125, 160.0393 199.0754, 246.1125, 160.0393 215.0703, 230.1176, 144.0444
N-Dealkylation M22
C21 H17 NO
300.1383
−1.1
144.0444, 183.0804, 155.0855
C21 H17 NO2 C21 H17 NO2
316.1332 316.1332
−1.5 −1.4
144.0444, 199.0754 144.0444, 199.0754
C21 H17 NO3
332.1281
−1.3
144.0444, 215.0703
C26 H25 NO3
N-Dealkylation + monohydroxylation M23 M24 N-Dealkylation + dihydroxylation M25
22.6 5.6 6.8 3.0
Oxidative defluorination to carboxylic acid 27.2 M26
400.1907
−1.3
183.0804, 244.0968, 155.0855, 144.0444
Oxidative defluorination to carboxylic acid + monohydroxylation M27 14.4 C26 H25 NO4
416.1856
−1.4
199.0754, 144.0444, 244.0968
Dehydrogenation M28
C26 H24 FNO
386.1915
−1.0
181.0648, 153.0699, 144.0444, 232.1132
Dehydrogenation + monohydroxylation M29 25.6
C26 H24 FNO2
402.1864
−1.1
197.0597, 169.0648, 232.1132
Dehydrogenation + dihydroxylation M30 M31 M32 M33 M34 M35 M36
C26 H24 FNO3 C26 H24 FNO3 C26 H24 FNO3 C26 H24 FNO3 C26 H24 FNO3 C26 H24 FNO3 C26 H24 FNO3
418.1813 418.1813 418.1813 418.1813 418.1813 418.1813 418.1813
−1.2 −1.0 −1.4 −1.0 −1.7 −1.4 −1.2
197.0597, 169.0648, 248.1081, 144.0444 197.0597, 169.0648, 248.1081, 160.0393 197.0597, 169.0648, 248.1081, 144.0444 213.0546, 185.0597, 232.1132, 144.0444 213.0546, 185.0597, 232.1132, 144.0444 197.0597, 169.0648, 248.1081, 160.0393 213.0546, 232.1132, 185.0597, 144.0444
400.1907
−1.1
197.0597, 169.0648, 144.0444, 230.1176
30.9
14.4 15.3 16.7 17.6 20.0 20.6 22.5
Oxidative defluorination + monohydroxylation + dehydrogenation 17.4 C26 H25 NO3 M37 a
Product ions are arranged in the order of the intensity.
exact masses of molecular ion ([M + H]+ ) and diagnostic product ions, retention times, and biotransformation pathways of EAM2201 and its possible metabolites (M1–M37) are listed in Table 1. Diagnostic product ions are listed in the order of relative abundance. Chromatographic separation of the metabolites was needed to unambiguously identify the structure of the metabolites because many metabolites have the same [M + H]+ ions including M1–M9
at m/z 404.2020, M10–M14 at m/z 420.1970, M31–M36 at m/z 418.1813, and etc. (Fig. 1). After many trials with different columns and mobile phase combinations, EAM-2201 and its 37 metabolites were well separated on a Halo C18 column using a gradient elution of methanol and 0.1% formic acid (Fig. 1, Table 1). The MS/MS spectrum of EAM-2201 showed four characteristic product ions at m/z 183.0804 [(4-ethylnaphthalen-1-yl)(oxo) methylium ion], m/z 155.0855 (4-ethylnaphthalen-1-ylium ion),
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Fig. 1. Extracted ion chromatograms of EAM-2201 and its possible metabolites obtained following incubation of EAM-2201 with human liver microsomes in the presence of NADPH for 20 min at 37 ◦ C using 3 ppm accuracy.
m/z 232.1132 [(1-(5-fluoropentyl)-1H-indol-3-yl)(oxo) methylium ion], and m/z 144.0444 (loss of 5-fluoropentyl moiety from m/z 232.1132) (Fig. 2), serving as diagnostic product ions for the identification of EAM-2201 metabolites.
3.1.1. Monohydroxylation and dihydroxylation M1-M9 showed the [M + H]+ ion at m/z 404.2020, 16 amu more than the EAM-2201 [M + H]+ ion, indicating hydroxylation of EAM2201 (Fig. 1). Based on MS/MS spectra, the nine hydroxy-EAM-2201 (M1–M9) were classified into three categories due to hydroxylation positions at the ethylnaphthalene, aliphatic alkyl chain and indole moieties, although the exact hydroxylation positions were not identified. M1, M2, M3, M6, and M8 showed the characteristic product ion at m/z 199.0754 [hydroxy-(4-ethylnaphthalen-1-yl)(oxo) methylium ion], m/z 232.1132 [(1-(5-fluoropentyl)-1H-indol-3yl)(oxo) methylium ion] and m/z 144.0444 [(1H-indol-3-yl)(oxo) methylium ion], suggesting that hydroxylation in M1, M2, M3, M6, and M8 occurred at the ethylnaphthalene moiety (Fig. 2). M4 and M5 showed the product ions at m/z 248.1081, 16 amu higher than m/z 232.1132, m/z 144.0444, m/z 155.0855, and m/z 183.0804, indicating that hydroxylation occurred in the EAM-2201 fluoropentyl group (Fig. 2). M7 and M9 produced the characteristic product ions at m/z 183.0804, m/z 248.1081, m/z 155.0855, and m/z 160.0393 (loss of 5-fluoropentyl moiety from m/z 248.1081), indicating that hydroxylation of M7 and M9 occurred at the indole moiety, but the accurate hydroxylation positions were not identified (Fig. 2).
Five EAM-2001 metabolites (M10-M14), which were 32 amu higher than EAM-2201, showed the [M + H]+ ion at m/z 420.1970, suggesting that M10-M14 may be dihydroxylated EAM-2201 metabolites (Fig. 1). M10, M12 and M13 showed the product ions at m/z 199.0754 [hydroxy-(4-ethyl1-naphthaldehyde)], m/z 248.1081 [hydroxy-(1-(5-fluoropentyl)1H-indole-3-carbaldehyde)] and m/z 144.0444 (1H-indole-3carbaldehyde), indicating that dihydroxylation in M10, M12 and M13 occurred at both the ethylnaphthalene moiety and 5fluoropentyl group (Supplement 1A). M11 showed product ions at m/z 199.0754, m/z 248.1081 and m/z 160.0393 [hydroxy-(1H-indole-3-carbaldehyde)], indicating the dihydroxylation in M11 occurred at the ethylnaphthalene and indole moieties (Supplement 1B). M11 showed two vinylnaphthalene product ions at m/z 153.0699 and m/z 181.0648 due to dehydrogenation of m/z 155.0855 and m/z 183.0804, respectively. M14 produced the product ions at m/z 215.0703 [dihydroxy(4-ethyl-1-naphthaldehyde)], m/z 232.1132 and m/z 144.0444, indicating dihydroxylation of EAM-2201 at the ethylnaphthalene moiety (Supplement 1C). 3.1.2. Dihydrodiol formation M15 showed the [M + H]+ ion at m/z 422.2126, which is 34 amu higher than EAM-2201 (Fig. 1), and generated product ions at m/z 217.0859, m/z 199.0754 (loss of water from m/z 217.0859), m/z 232.1132, and m/z 144.0444, suggesting that the dihydrodiol metabolite was formed (Supplement 1D). These results indicate that M15 is a dihydrodiolized EAM-2201 via epoxide formation at the naphthalene ring and subsequent hydrolysis of the epoxide
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Fig. 2. Product ion spectra of EAM-2201 and hydroxy-EAM-2201 (M1–M9) acquired following incubation of EAM-2201 with human liver microsomes in the presence of NADPH for 20 min at 37 ◦ C using a Q-Exactive mass spectrometer in NCE at 45 eV and proposed fragmentation pattern of principal ions.
moiety. The position of M15 hydroxyl groups was not accurately identified. 3.1.3. Oxidative defluorination and consecutive hydroxylation M16 showed the [M + H]+ ion at m/z 386.2115, which was 2 amu lower than the EAM-2201 [M + H]+ ion (Fig. 1). M16 produced product ions at m/z 230.1176 [(1-(5-hydroxypentyl)-1Hindol-3-yl)(oxo) methylium ion], m/z 183.0804 and m/z 155.0855, indicating that M16 was formed from EAM-2201 via oxidative defluorination (Supplement 2A). Interestingly, M16 was formed from EAM-2201 after incubation with human liver microsomes in both the absence and presence of NADPH, and the amount of M16 decreased in the presence of NADPH. This result suggests that M16 could be formed without NADPH, but further metabolic reactions such as hydroxylation of M16 to M17-M21 must be performed in the presence of NADPH. M17 and M18 showed the [M + H]+ ion at m/z 402.2064, which was 16 amu higher than the M16 [M + H]+ ion, suggesting additional hydroxylation of M16 (Fig. 1). M17 and M18 produced product ions at m/z 199.0754 [hydroxy-(4-ethyl-1-naphthaldehyde)], m/z 230.1176 and m/z 144.0444, indicating hydroxylation of M16 at the ethylnaphthalene moiety (Supplement 2B). M19-M21 showed the [M + H]+ ion at m/z 418.2013, which was 32 amu higher than the M16 [M + H]+ ion, indicating dihydroxylation of M16 (Fig. 1, Table 1). M19 and M20 showed the product ions of m/z 199.0754, m/z 246.1125 [hydroxy-(1-(5hydroxypentyl)-1H-indol-3-carbaldehyde)] and m/z 160.0393 (loss of 5-hydroxypentyl moiety from m/z 246.1125), suggesting that hydroxylation occurred at both the ethylnaphthalene and indole moieties (Supplement 2C). M21 showed the characteristic product ions at m/z 215.0703 [dihydroxy-(4-ethyl-1-naphthaldehyde)], m/z 230.1176 and m/z 144.0444, indicating that M21 (dihydroxy-M16) was formed from M16 via dihydroxylation of the ethylnaphthalene moiety (Supplement 2D).
3.1.4. N-Dealkylation and consecutive hydroxylation M22 showed the [M + H]+ ion at m/z 300.1383, which was 88 amu lower than the EAM-2201 [M + H]+ ion, suggesting the loss of the 5-fluoropentyl group (Fig. 1, Table 1). M22 showed the product ions at m/z 144.0444, m/z 183.0804 and m/z 155.0855, supporting that M22 is N-dealkyl-EAM-2201 due to the loss of the 5-fluoropentyl group (Supplement 3A). M23 and M24 showed the [M + H]+ ion at m/z 316.1332, which was 16 amu higher than the M22 [M + H]+ ion, suggesting hydroxylation of M22 (Fig. 1, Table 1). These metabolites showed the characteristic product ions at m/z 144.0444 and m/z 199.0754, suggesting hydroxylation of the ethylnaphthalene moiety (Supplement 3B). M25 showed the [M + H]+ ion at m/z 332.1281, which was 32 amu higher than the M22 [M + H]+ ion, suggesting dihydroxylation of M22 (Fig. 1, Table 1). M25 produced product ions at m/z 215.0703 and m/z 144.0444, indicating dihydroxylation of M22 at the ethylnaphthalene moiety to dihydroxy-M22 (M25) (Supplement 3C).
3.1.5. Oxidative defluorination to carboxylic acid and consecutive hydroxylation M26 showed the [M + H]+ ion at m/z 400.1907 and the product ions at m/z 144.0444, m/z 155.0855, m/z 183.0804, and m/z 244.0968 [(1-(4-carboxybutyl)-1H-indol-3-yl)(oxo) methylium ion] (Supplement 4A), indicating that M26 may be EAM-2201 pentanoic acid via the biotransformation of the fluoropentyl group to pentanoic acid. M27 showed the [M + H]+ ion at m/z 416.1856, which was 16 amu higher than the M26 [M + H]+ ion, indicating hydroxylation of M26 (Fig. 1, Table 1). M27 showed product ions at m/z 144.0444, m/z 199.0754 and m/z 244.0968, and therefore, M27 was tentatively identified as hydroxy-M26 via hydroxylation of the ethylnaphthalene moiety (Supplement 4B).
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Fig. 3. Product ion spectra of (A) dehydro-EAM-2201 (M28), (B) hydroxy-M28 (M29), (C)–(E) dihydroxy-M28 (M30-M36) acquired following incubation of EAM-2201 with human liver microsomes in the presence of NADPH for 20 min at 37 ◦ C using a Q-Exactive mass spectrometer in NCE at 45 eV.
3.1.6. Dehydrogenation and consecutive metabolism M28 showed the [M + H]+ ion at m/z 386.1915, which was 2 amu lower than the EAM-2201 [M + H]+ ion (Fig. 1), and produced product ions at m/z 232.1132 [(1-(5-fluoropentyl)-1H-indol-3-yl)(oxo) (methylium ion)], as well as m/z 153.0699 and m/z 181.0648, which were 2 amu lower than m/z 155.0855 and m/z 183.0804, respectively (Fig. 3A). These results suggest that M28 may be dehydro-EAM-2201 via dehydrogenation at the ethyl group. M29 showed the [M + H]+ ion at m/z 402.1864, which was 16 amu higher than the M28 [M + H]+ ion, suggesting it was hydroxy-M29 (Fig. 1). M29 showed product ions at m/z 232.1132, m/z 197.0597 (hydroxylation of m/z 181.0648) and m/z 169.0648 (hydroxylation of m/z 153.0699), indicating that hydroxylation occurred at the vinylnaphthalene moiety (Fig. 3B). Seven metabolites, M30-M36 showed the [M + H]+ ion at m/z 418.1813, which was 32 amu higher than the M28 [M + H]+ ion, suggesting that they were dihydroxy-M28 (Fig. 1, Table 1). M30 and M32 showed product ions at m/z 144.0444 (1H-indole-3carbaldehyde), m/z 169.0648 (hydroxylation of m/z 153.0699), m/z 197.0597 (hydroxylation of m/z 181.0648), and m/z 248.1081 (hydroxylation of m/z 232.1132), indicating that M30 and M32 were hydroxylated at the vinylnaphthalene and 5-fluoropentyl group, respectively (Fig. 3C). M31 and M35 showed the product ions at
m/z 160.0393 (hydroxylation of m/z 144.0444), m/z 169.0648, m/z 197.0597, and m/z 248.1081, indicating that M31 and M35 were hydroxylated at the vinylnaphthalene and indole moieties, respectively (Fig. 3D). However, M33, M34 and M36 produced product ions at m/z 144.0444, m/z 232.1132, m/z 185.0597 (dihydroxylation of m/z 153.0699), and m/z 213.0546 (dihydroxylation of m/z 181.0648), indicating that dihydroxylation of the vinylnaphthalene moiety resulted in M33, M34 and M36 (dihydroxy-M28; Fig. 3E). M37 showed the [M + H]+ ion at m/z 400.1907 (Fig. 1) and the product ions at m/z 144.0444, m/z 169.0648 (hydroxylation of m/z 153.0699), m/z 197.0597 (hydroxylation of m/z 181.0648), and m/z 230.1176 [(1-(5-hydroxypentyl)-1H-indol-3-yl)(oxo) methylium ion] (Table 1, Supplement 4C). Based on these results, M37 resulted from dehydrogenation of the ethyl group, the hydroxylation of the vinylnaphthalene moiety, and oxidative defluorination of the 5fluoropentyl group to 5-hydroxypentyl group. Based on these results, the proposed possible metabolic pathways of EAM-2201 in human liver microsomes are shown in Fig. 4. EAM-2201 was metabolized to 37 metabolites via mono- and dihydroxylation at the naphthalene moiety, indole moiety and pentyl chain, dehydrogenation at ethyl group, oxidative defluorination at the fluoropentyl chain, dihydrodiol formation, N-dealkylation, and carboxylation.
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Fig. 4. Possible metabolic pathways of EAM-2201 in human liver microsomes.
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Table 2 Relative formation rates of major EAM-2201 metabolites obtained from incubation of EAM-2201 with human cDNA-expressed CYPs in the presence of NADPH (n = 3). Relative formation rates of EAM-2201 metabolites (peak area ratios/min/fmol CYP, mean ± SD)
M1 M2 M3 M4 M5 M7 M8 M9 M10 M12 M14 M15 M16 M18 M22 M24 M26 M27 M28 M29 M30 M31 M32 M33 M34 M35 M36 M37
CYP1A2
CYP2B6
CYP2C8
CYP2C9
N.D 18.7 ± 3.3 N.D 1.5 ± 0.5 10.0 ± 1.2 297.0 ± 52.7 0.9 ± 0.3 24.1 ± 4.4 N.D N.D N.D 1.5 ± 0.4 2.3 ± 0.1 N.D 14.4 ± 2.3 0.9 ± 0.2 N.D N.D 4.2 ± 0.9 2.3 ± 0.3 N.D N.D N.D N.D N.D N.D N.D N.D
N.D N.D N.D 54.8 ± 3.7 1.1 ± 0.3 25.8 ± 3.4 N.D N.D N.D N.D N.D 1.3 ± 0.2 9.4 ± 1.3 N.D 3.4 ± 0.7 N.D N.D N.D 0.3 ± 0.0 N.D N.D N.D N.D N.D N.D N.D N.D N.D
N.D 3.0 ± 0.5 N.D 42.9 ± 6.9 17.7 ± 1.8 1.9 ± 0.1 N.D N.D N.D N.D N.D N.D 194.3 ± 34.8 N.D 0.5 ± 0.1 0.5 ± 0.1 6.3 ± 1.8 N.D 1.5 ± 0.3 N.D N.D N.D N.D N.D N.D N.D N.D N.D
37.7 ± 7.9 100.3 ± 16.4 N.D N.D N.D 5.2 ± 1.5 1.2 ± 0.3 N.D N.D N.D 6.1 ± 1.9 0.5 ± 0.1 N.D 0.6 ± 0.2 N.D N.D N.D N.D 120.9 ± 11.3 5.5 ± 2.3 N.D N.D N.D N.D 0.4 ± 0.2 N.D 0.7 ± 0.4 N.D
CYP2C19 1.2 ± 36.5 ± N.D 39.0 ± 39.8 ± 40.4 ± 0.7 ± 1.4 ± 1.0 ± 6.6 ± N.D 1.1 ± 14.2 ± 2.9 ± N.D N.D 1.2 ± 1.7 ± 33.8 ± 9.1 ± 10.0 ± 0.6 ± 1.9 ± N.D N.D N.D N.D 2.4 ±
0.2 3.1 4.9 2.6 2.9 0.2 0.1 0.7 0.8 0.1 1.5 0.4
0.2 0.4 5.1 1.2 2.1 0.5 0.1
0.3
CYP2D6
CYP2J2
CYP3A4
CYP3A5
0.4 ± 0.1 33.6 ± 5.6 N.D 0.7 ± 0.4 N.D 119.4 ± 15.2 2.0 ± 0.8 8.6 ± 2.2 N.D N.D 2.0 ± 0.3 1.9 ± 0.8 1.6 ± 0.2 N.D N.D N.D N.D N.D 83.6 ± 4.0 6.7 ± 0.8 N.D N.D N.D N.D N.D N.D N.D N.D
2.4 ± 0.2 5.4 ± 1.0 N.D 12.2 ± 2.4 0.4 ± 0.1 N.D N.D N.D N.D N.D N.D N.D 16.7 ± 2.4 N.D N.D N.D N.D N.D 2.8 ± 1.5 N.D N.D N.D N.D N.D N.D N.D N.D N.D
7.8 ± 2.7 3.9 ± 1.3 3.5 ± 0.8 N.D N.D 0.6 ± 0.0 N.D N.D N.D N.D 2.8 ± 0.7 N.D N.D N.D N.D 2.0 ± 0.3 N.D 3.8 ± 0.5 2.2 ± 1.1 252.4 ± 14.8 5.1 ± 1.2 7.2 ± 0.6 27.8 ± 2.4 5.7 ± 0.9 4.6 ± 0.4 2.1 ± 1.3 1.3 ± 0.2 1.9 ± 0.6
1.4 ± 0.1 67.0 ± 5.7 N.D 2.2 ± 0.4 4.7 ± 0.2 11.6 ± 0.9 14.4 ± 1.0 3.0 ± 0.8 N.D N.D N.D N.D 0.8 ± 0.3 N.D 2.6 ± 0.2 N.D N.D N.D 47.5 ± 16.3 14.5 ± 5.1 N.D N.D N.D N.D N.D N.D N.D N.D
N.D; <0.3 peak area ratios/min/fmol CYP.
3.2. Screening of CYPs responsible for the metabolism of EAM-2201 The calibration curve was linear over 0.5–500 pmol of EAM2201 in blank microsomal incubation mixture, and the coefficients of variation and relative errors (i.e., precision and accuracy) of the quality control samples at 0.5, 1.5, 100, and 400 pmol of EAM2201 in blank microsomal incubation mixture were ≤9.5% and −12.0% to 13.0%, respectively. The amounts of the metabolites were determined using peak area ratios since the authentic standards of EAM-2201 metabolites were not available. Therefore, there is a limitation in the accurate interpretation of the formation rates for the metabolites. To characterize CYP isozymes responsible for the metabolism of EAM-2201, EAM-2201 (2 M) was incubated with major human cDNA-expressed CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP2J2, CYP3A4, or CYP3A5 in the presence of NADPH. The metabolism of EAM-2201 was mediated by CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2J2, CYP3A4, and CYP3A5 (Table 2). CYP2A6 and CYP2E1 enzymes were not involved in the metabolism of EAM-2201. Hydroxylation of EAM-2201 was catalyzed by multiple CYP enzymes, but each isozyme appeared to have favorable hydroxylation sites. Hydroxylation of the ethylnaphthalene moiety to M1, M2, M3, and M8 was predominantly mediated by CYP2C9, CYP2C19, CYP2D6, and CYP3A4/5 enzymes, whereas the formation of M7 and M9 via hydroxylation of the indole ring was mediated by CYP1A2, CYP2C19, CYP2D6, and CYP3A5 enzymes. In addition, CYP1A2, CYP2B6, CYP2C8, CYP2C19, CYP2J2, and CYP3A5 were responsible for the hydroxylation of 5-fluoropentyl chain to M4 and M5. Although oxidative defluorination of EAM-2201 to M16 occurred in the absence of NADPH as described above, the formation of M16 from EAM-2201 was also catalyzed by CYP2C8 with minor contributions from CYP1A2, CYP2B6, CYP2C19, CYP2D6, CYP2J2, and CYP3A5. N-Dealkylation of EAM-2201 to M22 was mediated
by CYP1A2, CYP2B6, CYP2C8, and CYP3A5 enzymes. Dehydrogenation at the ethyl group of EAM-2201 to M28 was catalyzed by CYP2C9, CYP2C19, CYP2D6, and CYP3A5, with minor contributions from CYP1A2, CYP2B6, CYP2C8, and CYP2J2. Hydroxylation of M28 to M29 was mediated by CYP3A4 with minor contributions from CYP2C9, CYP2C19, CYP2D6, CYP3A5, and CYP1A2 enzymes. Hydroxylation of M29 to seven metabolites, M30-M36 was catalyzed by CYP3A4 enzyme. The formation of M6, M11, M13, M17, M19, M20, M21, M23, and M25 from EAM-2201 were very slight, and therefore, we could not characterize CYP enzymes responsible for the formation of these nine metabolites.
4. Conclusion Based on the exact mass of [M + H]+ and the diagnostic product ions, 37 metabolites of EAM-2201 were identified as nine hydroxyEAM-2201 (M1–M9), five dihydroxy-EAM-2201 (M10–M14), dihydrodiol-EAM-2201 (M15), defluorinated EAM-2201 (M16), two hydroxy-M16 (M17 and M18), three dihydroxy-M16 (M19M21), 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 defluorinated hydroxy-M28 (M37) (Table 1, Fig. 1). Multiple CYPs including CYP1A2, CYP2C8/9/19, CYP2B6, CYP2D6, and CYP3A4/5 were responsible for the metabolism of EAM-2201. The metabolic pathways of EAM-2201 may aid in the development of analytical methods for monitoring the abuse of EAM-2201 in biological samples such as urine and blood.
Conflict of interest The authors have declared no conflict of interest.
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