Drug Testing and Analysis
Short communication Received: 20 May 2014
Revised: 18 November 2014
Accepted: 20 November 2014
Published online in Wiley Online Library
(www.drugtestinganalysis.com) DOI 10.1002/dta.1765
Detection of main metabolites of XLR-11 and its thermal degradation product in human hepatoma HepaRG cells and human urine Tatsuyuki Kanamori,* Koji Kanda, Tadashi Yamamuro, Kenji Kuwayama, Kenji Tsujikawa, Yuko Togawa Iwata and Hiroyuki Inoue The metabolism of (1-(5-fluoropentyl)-1H-indol-3-yl)(2,2,3,3-tetramethylcyclopropyl)methanone (XLR-11), a novel synthetic cannabinoid, was studied using a HepaRG cell culture. The HepaRG cells were incubated with the drug for 48 hours and the metabolites were extracted from the culture medium by liquid-liquid extraction. The extract was analyzed by liquid chromatography/ mass spectrometry to detect the metabolites. N-(5-Hydroxypentyl) metabolite and N-pentanoic acid metabolite were identified in the culture medium of XLR-11, and several other metabolites, presumably formed by oxidation of the first two metabolites and XLR-11, were detected. The extract of an XLR-11 user’s urine was also analyzed; however, the metabolites detected in the urine were different from XLR-11 metabolites in the medium. A metabolic experiment with the thermal degradation product of XLR-11, XLR-11 degradant, using HepaRG cells revealed that the urinary metabolites were almost identical to the XLR-11 degradant metabolites. These findings suggest that most of the XLR-11 was degraded by heating when the user smoked the herbal product containing XLR-11. Copyright © 2015 John Wiley & Sons, Ltd. Keywords: synthetic cannabinoid; metabolism; hepatocyte; urine
Introduction Synthetic cannabinoids are chemically synthesized compounds that mimic the pharmacological effects of tetrahydrocannabinol (THC), the active ingredient of cannabis.[1–4] In the late 2000s, herbal products containing synthetic cannabinoids appeared on the black market,[5] and many new synthetic cannabinoids have subsequently been developed. The abuse of synthetic cannabinoids is, however, a serious problem worldwide. The synthetic cannabinoid (1-(5-fluoropentyl)-1H-indol-3-yl) (2,2,3,3-tetramethylcyclopropyl)methanone (XLR-11, Figure 1) was first reported in 2012 in the USA[6] and Japan.[7] XLR-11 shows a strong affinity for both cannabinoid receptors (CB1 and CB2) and activates these receptors as full agonists, producing THC-like effects.[8] In 2013, the US Drug Enforcement Administration placed XLR-11 into Schedule I of the Controlled Substances Act because of public safety concerns.[9] Since January 2014, XLR-11 has been controlled as a narcotic under the Narcotics and Psychotropics Control Law in Japan. The metabolic profile of XLR-11 was first reported by Wohlfarth et al. in 2013.[10] They treated XLR-11 with a suspension culture of human primary hepatocytes and analyzed the culture medium by liquid chromatography - time-of-flight mass spectrometry (LC-TOFMS), and they detected 30 metabolites, including 15 glucuronides. On the other hand, Adamowicz et al. and Grigoryev et al. reported detecting a thermal degradation product of UR-144 (Figure 1), a defluorinated analog of XLR-11, and its metabolites in blood and urine samples obtained from a UR-144 user.[11,12] As XLR-11 has a similar structure to UR-144 (both of these compounds have tetramethylcyclopropyl moiety), it is highly likely that XLR-11 undergo thermal degradation during smoking. In this study, a urine sample obtained from an XLR-11 user was analyzed to detect urinary metabolites. In addition, XLR-11 and its
Drug Test. Analysis (2015)
thermal degradation product (XLR-11 degradant, Figure 1) were treated with human hepatoma HepaRG cells[13,14] to investigate the metabolism of these compounds.
Materials and methods Chemicals Authentic standards of XLR-11, XLR-11 degradant, and N-(5hydroxypentyl) metabolite, N-pentanoic acid metabolite, and N-(4-hydroxypentyl) metabolite were purchased from Cayman Chemical Company (Ann Arbor, MI, USA). HepaRG cells, Medium 670, Medium 620, and Medium 650 were purchased from KAC Co., Ltd. (Kyoto, Japan). Medium 670 and Medium 620 contained 10% of fetal calf serum, and Medium 620 contained 1.7% of dimehylsulfoxide (DMSO). β-Glucuronidase/aryl sulfatase (from Helix pomatia; β-glucuronidase, 5.2 U/mL; aryl sulfatase, 2.1 U/mL) was purchased from Calbiochem-Novabiochem Co. Ltd (La Jolla, CA, USA). All other chemicals used were of analytical grade. Urine sample The urine sample was obtained from a man in his 20s who used XLR-11 for the purpose of forensic examination (suspicion of illicit drug use). After smoking an herbal product containing XLR-11, he
* Correspondence to: Tatsuyuki Kanamori, National Research Institute of Police Science, First Chemistry Section 6-3-1, Kashiwanoha, Kashiwa Chiba 277-0882 Japan. E-mail: [email protected] National Research Institute of Police Science, First Chemistry Section 6-3-1, Kashiwanoha Kashiwa Chiba 277-0882 Japan
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T. Kanamori et al. 20% to 90% B over 15 min, 90% B for 10 min, and 90% to 20% B over 1 min; flow rate, 0.3 mL/min; MS interface, positive electrospray ionization; analysis mode, scan (m/z 100–500) and product ion analysis (normalized collision energy, 35%; precursor ions, protonated molecule of drugs and putative metabolites); sheath gas flow rate, 40 arb; capillary temperature, 300 °C; capillary voltage, 9 V; tube lens, 65 V.
Figure 1. Chemical structures of XLR-11, XLR-11 degradant, and UR-144.
became ill and was rushed to a police station, where urine sample was collected. The time from drug ingestion to urine sampling was estimated to be about 2 h. In this study, the urine sample was used with the consent of the XLR-11 user and all experiments were approved by the Research Ethical Review Board of National Research Institute of Police Science. Incubation of drug with HepaRG cells Cryopreserved HepaRG cells were thawed in warm Medium 670 and the cell viability was measured by trypan blue dye exclusion. The viability of HepaRG cells after thawing was higher than 95%. HepaRG cells were seeded (7.2 × 105 cells/mL, 0.3 mL/well) in BD BioCoat™ 48-well plate (Collagen Type I coated) and incubated for 7 days. The medium used to maintain HepaRG cells was Medium 620, which contained 10% of fetal calf serum and 1.7% of DMSO, and the medium was changed every 2 days. The medium was changed to Medium 650 with 0.7% of DMSO, and XLR-11 or XLR11 degradant (1 mM in DMSO) was added to each well of the plate at a final concentration of 10 μM and incubated for 48 h. The final concentration of DMSO was 1.7%. The medium was collected and stored at -30 °C until analysis. Extraction of the metabolites Water (300 μL) and 0.5 M acetate buffer (100 μL, pH 5.0) containing β-glucuronidase/aryl sulfatase (β-glucuronidase, 5.2 × 10 3 U) were added to a portion of medium (100 μL), and the mixture was incubated at 60 °C for 90 min to hydrolyze the conjugate. The hydrolyzed sample was extracted with chloroform/2-propanol (3:1 v/v, 3 × 1 mL) and the combined organic layer was evaporated to dryness under a stream of nitrogen at 40 °C. The residue was re-dissolved in 90% methanol (200 μL) and 10 μL of the sample was injected into the liquid chromatograph–mass spectrometer. 0.5 M acetate buffer (125 μL, pH 5.0) containing β-glucuronidase/ aryl sulfatase (β-glucuronidase, 2.6 × 10 2 U) was added to a portion of the urine sample (500 μL) and incubated at 60 °C for 90 min. The hydrolyzed sample was processed and analyzed in the same way as described above. Liquid chromatography-mass spectrometry (LC-MS) LC-MS analyses were carried out on an Accela high-performance liquid chromatograph system connected to an LCQ FLEET ion trap mass spectrometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). The conditions were as follows: column, XBridge BEH C18 (2.1 × 150 mm; particle diameter, 3.5 μm; Waters Corporation, Milford, MA, USA) maintained at 40 °C; mobile phase composition, 0.1% formic acid (A) and acetonitrile (B); linear gradient mode,
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Results and discussion According to a previous study,[10] XLR-11 is metabolized by hydroxylation, carboxylation, hemiketal, and hemiacetal formation, internal dehydration, and further glucuronidation of oxidative metabolites. Based on the finding, the extracts of culture medium and urine sample were analyzed by LC-MS using the scan mode, and extracted ion chromatograms (EICs) for protonated molecules ([M+H]+) of the putative metabolites were used to detect XLR-11 metabolites. For example, EIC of m/z 346 was drawn to search mono-oxidized metabolite of XLR-11 (molecular weight 345). In addition, product ion analysis was performed in order to obtain further structural information of each metabolite. The total ion current chromatogram (TIC) and EICs obtained from the XLR-11 culture medium extract are shown in Figure 2a. An unchanged XLR-11 peak was not detected in the EIC of m/z 330, indicating that XLR-11 was completely metabolized by HepaRG cells in 48 h. The EICs of m/z 328 and m/z 342 showed peaks corresponding to N-(5-hydroxypentyl) (M328) and N-pentanoic acid (M342) metabolites, respectively. The peaks detected in the EICs of m/z 344 and m/z 358 were assumed to correspond to the oxidized metabolites of M328 and M342, respectively. The metabolite peaks detected in the EIC of m/z 346 were thought to be the monooxidized metabolites of XLR-11. A di-oxidized metabolite (M362) was also detected in the medium (not shown in Figure 2). Other putative metabolites such as the hemiketal, hemiacetal, and dehydrated or carboxylated metabolites were not detected in the EICs of the protonated molecules of these compounds. The analytical data for XLR-11 and its metabolites are summarized in Table 1. Wohlfarth et al. detected 30 metabolites in the culture medium of human primary hepatocytes incubated with XLR-11.[10] In the present study, we detected only 12 metabolites (Table 2). One reason why fewer metabolites were detected in our study was that we did not include tiny peaks that had a poor product ion spectrum. Additionally, we performed enzymatic hydrolysis prior to extracting the metabolites. The hydrolysis step was critical to the culture medium analysis because some metabolites (M328, M3441, 2, 3, 4, and M346-1, 2, 3, 4) were not detected if the hydrolysis step was omitted (data not shown). This indicates that most of these metabolites underwent glucuronidation or sulfoconjugation in HepaRG cells. Meanwhile, the metabolites that underwent carboxylation or hemiacetal formation were not detected in our study. This may be due to the difference in metabolic abilities between HepaRG cells and human primary hepatocytes. The TICs and EICs obtained from XLR-11 degradant culture medium extract are shown in Figure 2b. As was the case for XLR-11, no unchanged XLR-11 degradant peak was detected in the EIC of m/z 330, whereas several metabolites were detected in the EICs of m/z 328, 344, 342, 358, and 346. Based on the molecular weight and product ion spectra, M328d and M342d were assumed to be the XLR-11 degradant N-(5-hydroxypentyl) metabolite and the XLR-11 degradant N-pentanoic acid metabolite, respectively. The peaks detected in the EICs of m/z 344, m/z 358 and m/z 346 were
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Detection of metabolites of XLR-11 and its thermal degradation product
Figure 2. Total ion current chromatograms (TIC) and extracted ion chromatograms (EIC) obtained from the extracts of culture medium and urine sample. (a) culture medium (XLR-11), (b) culture medium (XLR-11 degradant), (c) urine sample. Table 1. Retention times, precursor ions and product ions of XLR-11, XLR-11 degradant and their metabolites in product ion analysis. Compound XLR-11 M328 M344-1 M344-2 M344-3 M344-4 M342 M358 M346-1 M346-2 M346-3 M346-4 M362 XLR-11 degradant M328d M344d M342d M358d-1 M358d-2 M346d-1 M346d-2 M362d-1 M362d-2
RTa (min)
Precursor ion (m/z)
14.6 11.9 5.1 7.8 8.1 9.8 11.6 7.7 7.2 10.7 11.3 12.3 7.8 13.8 11.1 5.1 10.8 5.2 7.7 7.9 10.7 5.6 6.4
330 328 344 344 344 344 342 358 346 346 346 346 362 330 328 344 342 358 358 346 346 362 362
Major product ions (m/z) 232, 312, 125, 274 230, 125 230, 314, 204, 186, 326 230, 326, 314, 270, 204 230, 326, 204 246, 125, 202, 190 244, 125, 324 244, 340, 328, 218, 200 232, 204 232, 328, 272, 316, 206 232, 328, 206 125, 248, 328 248, 344, 288 232 230 246, 186, 230, 326, 204 244 260, 200, 244, 218, 340 244, 340, 218, 200, 260 248, 232, 206, 328 248, 232, 206, 328 264, 248, 222, 344 264, 248, 222, 344
Sample
Description
b
ND Mc, Ud M M M M M, U M M M M M M ND M, U M, U M, U M, U U M, U M, U M, U M, U
N-(5-Hydroxypentyl) metabolitee N-(5-Hydroxypentyl) metabolite + (O) N-(5-Hydroxypentyl) metabolite + (O) N-(5-Hydroxypentyl) metabolite + (O) N-(5-Hydroxypentyl) metabolite + (O) N-Pentanoic acid metabolitee N-Pentanoic acid metabolite + (O) XLR-11 + (O) XLR-11 + (O) XLR-11 + (O) XLR-11 + (O) XLR-11 + (2O) N-(5-Hydroxypentyl) metabolite N-(5-Hydroxypentyl) metabolite + (O) N-Pentanoic acid metabolite N-Pentanoic acid metabolite + (O) N-Pentanoic acid metabolite + (O) XLR-11-degradant + (O) XLR-11-degradant + (O) XLR-11-degradant + (2O) XLR-11-degradant + (2O)
a
Retention time. Not detected in urine and culture medium. c Detected in culture medium. d Detected in urine. e Confirmed with authentic standards. b
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Drug Testing and Analysis
T. Kanamori et al.
Table 2. Comparison of the XLR-11 metabolites detected in cell culture medium. Metabolite
Protonated molecule (m/z)
Number of metabolites detected This study (HepaRG cell)
N-(5-Hydroxypentyl) metabolite (+GAa) N-(5-Hydroxypentyl) metabolite + (O) (+GA) N-(5-Hydroxypentyl) metabolite + carboxylation N-Pentanoic acid metabolite (+GA) N-Pentanoic acid metabolite + (O) (+GA) N-Pentanoic acid metabolite + carboxylation (+GA) N-Pentanoic acid metabolite + (2O) - > dehydration N-Pentanoic acid metabolite + (O) + hemiketal N-Pentanoic acid metabolite + (O) + aldehyde - > hemiacetal (+GA) XLR-11 + (O) (+GA) XLR-11 + (O) - > dehydration XLR-11 + (O) + aldehyde - > hemiacetal (+GA) XLR-11 + (2O) (+GA) XLR-11 + (3O) (+GA) Carboxylation (+GA) Carboxylation + (O)
328 (504)b 344 (520) 358 342 (518) 358 (534) 372 (548) 356 358 372 (548) 346 (522) 344 360 (536) 362 (538) 378 (554) 360 (536) 376
1c 4 0 1 1 0 0 0 0 4 0 0 1 0 0 0
Wohlfarth et al. (human primery hepatocyte) 1c (1)d 0 (3) 1 1 (1) 2 (1) 1 (1) 1 1 1 (1) 0 (3) 1 0 (1) 0 (1) 0 (1) 1 (2) 2
a
Glucuronic acid. Protonated molecule of glucuronide. c Number of free metabolite. d Number of glucuronide. b
assumed to correspond to the oxidized metabolites of M328, M342 and XLR-11 degradant, respectively. TICs and EICs obtained from the extract of the XLR-11 user’s urine are shown in Figure 2c. As was the case with hepatocyte cultures, the EIC of m/z 330 failed to show a parent XLR-11 or XLR-11 degradant peak, though several metabolite peaks were detected
in the EICs of m/z 328, 344, 342, 358, and 346. The peaks detected in the EICs of m/z 328 and 342 were identical to those of M328d and M342d detected in the XLR-11 degradant culture medium extract, respectively (Figures 2b and 2c). The other metabolites detected in the urine sample also were identical to the metabolites detected in the XLR-11 degradant culture medium (M344d, M358d-
Figure 3. Behavior of XLR-11 before and after ingestion.
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Detection of metabolites of XLR-11 and its thermal degradation product 1, M346d-1, and M346d-2). In addition, two di-oxidized metabolites (M362d-1 and M362d-2) were detected in both the urine sample and the XLR-11 degradant culture medium (Table 1). These results suggest that the metabolites of XLR-11 degradant, but not of XLR-11, are mainly excreted in the urine of the XLR-11 user. Other putative metabolites (e.g. hemiketal, hemiacetal, and dehydrated or carboxylated metabolites) were not detected in the EICs of protonated molecules. The behaviour of XLR-11 before and after ingestion is summarized in Figure 3. The majority of XLR-11 was thermally degraded during smoking, and XLR-11 degradant was absorbed into the body. XLR-11 degradant was then metabolized in the body and excreted through urine. Small peaks for the XLR-11 metabolites M328 and M342 were also detected in the urine sample (Figure 2c), indicating that a small amount of XLR-11 escaped thermal degradation and was absorbed into the body. In the present case, the unchanged XLR-11 and XLR-11 degradant were not detected in urine. On the other hand, the XLR-11 degradant metabolites have the same molecular weight as the corresponding XLR-11 metabolites (e.g. M328d and M328, M342d and M342), which could lead to the misunderstanding that the XLR-11 degradant metabolites were the metabolites of XLR-11. However, the behaviour of XLR-11 was clarified because of the information obtained from our in vitro system regarding the metabolites of XLR-11 and XLR-11 degradant.
Conclusion The primary XLR-11 degradant metabolites formed by the in vitro system using HepaRG cells were also detected in the urine sample, indicating that XLR-11 thermally degraded during smoking and that XLR-11 degradant was metabolized and excreted through urine. This is the first report describing the metabolism of the XLR-11 thermal degradation product. In the field of forensic toxicology, it is important to clarify the metabolic fate of drugs in order to analyze drug metabolites in biological samples such as urine and blood. However, it is legally and ethically difficult to investigate the metabolism of new drugs of abuse in humans because of their potential harmful effects and toxicities. In the present study, the in vivo metabolism of XLR-11 degradant in a human subject was well replicated by HepaRG cell culture. HepaRG cells showed both phase I and phase II drugmetabolizing enzyme activities. Despite some limitations, the in vitro system using HepaRG cells appears to be an encouraging alternative to in vivo experiments with human subjects.
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Acknowledgement This work was supported by JSPS KAKENHI Grant Number 23590866.
References [1] D.G. Barceloux. MARIJUANA (Cannabis sativa L.) and Synthetic Cannabinoids, in Medical toxicology of drug abuse: synthesized chemicals and psychoactive plants, Wiley, Hoboken, 2012, pp. 915–919. [2] A. Weissman, G.M. Milne, L.S. Melvin Jr. Cannabimimetic activity from CP-47,497, a derivative of 3-phenylcyclohexanol. J. Pharmacol. Exp. Ther. 1982, 223, 516. [3] M.R. Johnson, L.S. Melvin, T.H. Althuis, J.S. Bindra, C.A. Harbert, G. M. Milne, A. Weissman. Selective and potent analgetics derived from cannabinoids. J. Clin. Pharmacol. 1981, 21(8-9 Suppl), 271S. [4] J.L. Wiley, D.R. Compton, D. Dai, J.A. Lainton, M. Phillips, J.W. Huffman, B. R. Martin. Structure-activity relationships of indole- and pyrrole-derived cannabinoids. J. Pharmacol. Exp. Ther. 1998, 285, 995. [5] K.A. Seely, J. Lapoint, J.H. Moran, L. Fattore. Spice drugs are more than harmless herbal blends: a review of the pharmacology and toxicology of synthetic cannabinoids. Prog. Neuropsychopharmacol. Biol. Psychiatry 2012, 39, 234. [6] NMS Labs. NMS Labs Trends Report: Changes in the Designer Drug Market Spring. 2012. Available at: http://www.nmslabs.com/uploads/PDF/ Designer%20Drug%20Spring%20Update_BKL%20Webinar_May% 202012.pdf [16 April 2014]. [7] N. Uchiyama, M. Kawamura, R. Kikura-Hanajiri, Y. Goda. URB-754: a new class of designer drug and 12 synthetic cannabinoids detected in illegal products. Forensic Sci. Int. 2013, 227, 21. [8] J.L. Wiley, J.A. Marusich, T.W. Lefever, M. Grabenauer, K.N. Moore, B. F. Thomas. Cannabinoids in disguise: Δ9-tetrahydrocannabinol-like effects of tetramethylcyclopropyl ketone indoles. Neuropharmacology 2013, 75, 145. [9] Drug Enforcement Administration, Department of justice. Schedules of controlled substances: temporary placement of three synthetic cannabinoids into Schedule I. Fed. Regist. 2013, 78, 21858. [10] A. Wohlfarth, S. Pang, M. Zhu, A.S. Gandhi, K.B. Scheidweiler, H.F. Liu, M. A. Huestis. First metabolic profile of XLR-11, a novel synthetic cannabinoid, obtained by using human hepatocytes and highresolution mass spectrometry. Clin. Chem. 2013, 59, 1638. [11] P. Adamowicz, D. Zuba, K. Sekuła. Analysis of UR-144 and its pyrolysis product in blood and their metabolites in urine. Forensic Sci. Int. 2013, 233, 320. [12] A. Grigoryev, P. Kavanagh, A. Melnik, S. Savchuk, A. Simonov. Gas and liquid chromatography-mass spectrometry detection of the urinary metabolites of UR-144 and its major pyrolysis product. J. Anal. Toxicol. 2013, 37, 265. [13] P. Gripon, S. Rumin, S. Urban, J. Le Seyec, D. Glaise, I. Cannie, C. Guyomard, J. Lucas, C. Trepo, C. Guguen-Guillouzo. Infection of a human hepatoma cell line by hepatitis B virus. Proc. Natl. Acad. Sci. U. S. A. 2002, 99, 15655. [14] K.P. Kanebratt, T.B. Andersson. Evaluation of HepaRG cells as an in vitro model for human drug metabolism studies. Drug Metab. Dispos. 2008, 36, 1444.
Copyright © 2015 John Wiley & Sons, Ltd.
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Short communication Received: 20 May 2014
Revised: 18 November 2014
Accepted: 20 November 2014
Published online in Wiley Online Library
(www.drugtestinganalysis.com) DOI 10.1002/dta.1765
Detection of main metabolites of XLR-11 and its thermal degradation product in human hepatoma HepaRG cells and human urine Tatsuyuki Kanamori,* Koji Kanda, Tadashi Yamamuro, Kenji Kuwayama, Kenji Tsujikawa, Yuko Togawa Iwata and Hiroyuki Inoue The metabolism of (1-(5-fluoropentyl)-1H-indol-3-yl)(2,2,3,3-tetramethylcyclopropyl)methanone (XLR-11), a novel synthetic cannabinoid, was studied using a HepaRG cell culture. The HepaRG cells were incubated with the drug for 48 hours and the metabolites were extracted from the culture medium by liquid-liquid extraction. The extract was analyzed by liquid chromatography/ mass spectrometry to detect the metabolites. N-(5-Hydroxypentyl) metabolite and N-pentanoic acid metabolite were identified in the culture medium of XLR-11, and several other metabolites, presumably formed by oxidation of the first two metabolites and XLR-11, were detected. The extract of an XLR-11 user’s urine was also analyzed; however, the metabolites detected in the urine were different from XLR-11 metabolites in the medium. A metabolic experiment with the thermal degradation product of XLR-11, XLR-11 degradant, using HepaRG cells revealed that the urinary metabolites were almost identical to the XLR-11 degradant metabolites. These findings suggest that most of the XLR-11 was degraded by heating when the user smoked the herbal product containing XLR-11. Copyright © 2015 John Wiley & Sons, Ltd. Keywords: synthetic cannabinoid; metabolism; hepatocyte; urine
Introduction Synthetic cannabinoids are chemically synthesized compounds that mimic the pharmacological effects of tetrahydrocannabinol (THC), the active ingredient of cannabis.[1–4] In the late 2000s, herbal products containing synthetic cannabinoids appeared on the black market,[5] and many new synthetic cannabinoids have subsequently been developed. The abuse of synthetic cannabinoids is, however, a serious problem worldwide. The synthetic cannabinoid (1-(5-fluoropentyl)-1H-indol-3-yl) (2,2,3,3-tetramethylcyclopropyl)methanone (XLR-11, Figure 1) was first reported in 2012 in the USA[6] and Japan.[7] XLR-11 shows a strong affinity for both cannabinoid receptors (CB1 and CB2) and activates these receptors as full agonists, producing THC-like effects.[8] In 2013, the US Drug Enforcement Administration placed XLR-11 into Schedule I of the Controlled Substances Act because of public safety concerns.[9] Since January 2014, XLR-11 has been controlled as a narcotic under the Narcotics and Psychotropics Control Law in Japan. The metabolic profile of XLR-11 was first reported by Wohlfarth et al. in 2013.[10] They treated XLR-11 with a suspension culture of human primary hepatocytes and analyzed the culture medium by liquid chromatography - time-of-flight mass spectrometry (LC-TOFMS), and they detected 30 metabolites, including 15 glucuronides. On the other hand, Adamowicz et al. and Grigoryev et al. reported detecting a thermal degradation product of UR-144 (Figure 1), a defluorinated analog of XLR-11, and its metabolites in blood and urine samples obtained from a UR-144 user.[11,12] As XLR-11 has a similar structure to UR-144 (both of these compounds have tetramethylcyclopropyl moiety), it is highly likely that XLR-11 undergo thermal degradation during smoking. In this study, a urine sample obtained from an XLR-11 user was analyzed to detect urinary metabolites. In addition, XLR-11 and its
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thermal degradation product (XLR-11 degradant, Figure 1) were treated with human hepatoma HepaRG cells[13,14] to investigate the metabolism of these compounds.
Materials and methods Chemicals Authentic standards of XLR-11, XLR-11 degradant, and N-(5hydroxypentyl) metabolite, N-pentanoic acid metabolite, and N-(4-hydroxypentyl) metabolite were purchased from Cayman Chemical Company (Ann Arbor, MI, USA). HepaRG cells, Medium 670, Medium 620, and Medium 650 were purchased from KAC Co., Ltd. (Kyoto, Japan). Medium 670 and Medium 620 contained 10% of fetal calf serum, and Medium 620 contained 1.7% of dimehylsulfoxide (DMSO). β-Glucuronidase/aryl sulfatase (from Helix pomatia; β-glucuronidase, 5.2 U/mL; aryl sulfatase, 2.1 U/mL) was purchased from Calbiochem-Novabiochem Co. Ltd (La Jolla, CA, USA). All other chemicals used were of analytical grade. Urine sample The urine sample was obtained from a man in his 20s who used XLR-11 for the purpose of forensic examination (suspicion of illicit drug use). After smoking an herbal product containing XLR-11, he
* Correspondence to: Tatsuyuki Kanamori, National Research Institute of Police Science, First Chemistry Section 6-3-1, Kashiwanoha, Kashiwa Chiba 277-0882 Japan. E-mail: [email protected] National Research Institute of Police Science, First Chemistry Section 6-3-1, Kashiwanoha Kashiwa Chiba 277-0882 Japan
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T. Kanamori et al. 20% to 90% B over 15 min, 90% B for 10 min, and 90% to 20% B over 1 min; flow rate, 0.3 mL/min; MS interface, positive electrospray ionization; analysis mode, scan (m/z 100–500) and product ion analysis (normalized collision energy, 35%; precursor ions, protonated molecule of drugs and putative metabolites); sheath gas flow rate, 40 arb; capillary temperature, 300 °C; capillary voltage, 9 V; tube lens, 65 V.
Figure 1. Chemical structures of XLR-11, XLR-11 degradant, and UR-144.
became ill and was rushed to a police station, where urine sample was collected. The time from drug ingestion to urine sampling was estimated to be about 2 h. In this study, the urine sample was used with the consent of the XLR-11 user and all experiments were approved by the Research Ethical Review Board of National Research Institute of Police Science. Incubation of drug with HepaRG cells Cryopreserved HepaRG cells were thawed in warm Medium 670 and the cell viability was measured by trypan blue dye exclusion. The viability of HepaRG cells after thawing was higher than 95%. HepaRG cells were seeded (7.2 × 105 cells/mL, 0.3 mL/well) in BD BioCoat™ 48-well plate (Collagen Type I coated) and incubated for 7 days. The medium used to maintain HepaRG cells was Medium 620, which contained 10% of fetal calf serum and 1.7% of DMSO, and the medium was changed every 2 days. The medium was changed to Medium 650 with 0.7% of DMSO, and XLR-11 or XLR11 degradant (1 mM in DMSO) was added to each well of the plate at a final concentration of 10 μM and incubated for 48 h. The final concentration of DMSO was 1.7%. The medium was collected and stored at -30 °C until analysis. Extraction of the metabolites Water (300 μL) and 0.5 M acetate buffer (100 μL, pH 5.0) containing β-glucuronidase/aryl sulfatase (β-glucuronidase, 5.2 × 10 3 U) were added to a portion of medium (100 μL), and the mixture was incubated at 60 °C for 90 min to hydrolyze the conjugate. The hydrolyzed sample was extracted with chloroform/2-propanol (3:1 v/v, 3 × 1 mL) and the combined organic layer was evaporated to dryness under a stream of nitrogen at 40 °C. The residue was re-dissolved in 90% methanol (200 μL) and 10 μL of the sample was injected into the liquid chromatograph–mass spectrometer. 0.5 M acetate buffer (125 μL, pH 5.0) containing β-glucuronidase/ aryl sulfatase (β-glucuronidase, 2.6 × 10 2 U) was added to a portion of the urine sample (500 μL) and incubated at 60 °C for 90 min. The hydrolyzed sample was processed and analyzed in the same way as described above. Liquid chromatography-mass spectrometry (LC-MS) LC-MS analyses were carried out on an Accela high-performance liquid chromatograph system connected to an LCQ FLEET ion trap mass spectrometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). The conditions were as follows: column, XBridge BEH C18 (2.1 × 150 mm; particle diameter, 3.5 μm; Waters Corporation, Milford, MA, USA) maintained at 40 °C; mobile phase composition, 0.1% formic acid (A) and acetonitrile (B); linear gradient mode,
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Results and discussion According to a previous study,[10] XLR-11 is metabolized by hydroxylation, carboxylation, hemiketal, and hemiacetal formation, internal dehydration, and further glucuronidation of oxidative metabolites. Based on the finding, the extracts of culture medium and urine sample were analyzed by LC-MS using the scan mode, and extracted ion chromatograms (EICs) for protonated molecules ([M+H]+) of the putative metabolites were used to detect XLR-11 metabolites. For example, EIC of m/z 346 was drawn to search mono-oxidized metabolite of XLR-11 (molecular weight 345). In addition, product ion analysis was performed in order to obtain further structural information of each metabolite. The total ion current chromatogram (TIC) and EICs obtained from the XLR-11 culture medium extract are shown in Figure 2a. An unchanged XLR-11 peak was not detected in the EIC of m/z 330, indicating that XLR-11 was completely metabolized by HepaRG cells in 48 h. The EICs of m/z 328 and m/z 342 showed peaks corresponding to N-(5-hydroxypentyl) (M328) and N-pentanoic acid (M342) metabolites, respectively. The peaks detected in the EICs of m/z 344 and m/z 358 were assumed to correspond to the oxidized metabolites of M328 and M342, respectively. The metabolite peaks detected in the EIC of m/z 346 were thought to be the monooxidized metabolites of XLR-11. A di-oxidized metabolite (M362) was also detected in the medium (not shown in Figure 2). Other putative metabolites such as the hemiketal, hemiacetal, and dehydrated or carboxylated metabolites were not detected in the EICs of the protonated molecules of these compounds. The analytical data for XLR-11 and its metabolites are summarized in Table 1. Wohlfarth et al. detected 30 metabolites in the culture medium of human primary hepatocytes incubated with XLR-11.[10] In the present study, we detected only 12 metabolites (Table 2). One reason why fewer metabolites were detected in our study was that we did not include tiny peaks that had a poor product ion spectrum. Additionally, we performed enzymatic hydrolysis prior to extracting the metabolites. The hydrolysis step was critical to the culture medium analysis because some metabolites (M328, M3441, 2, 3, 4, and M346-1, 2, 3, 4) were not detected if the hydrolysis step was omitted (data not shown). This indicates that most of these metabolites underwent glucuronidation or sulfoconjugation in HepaRG cells. Meanwhile, the metabolites that underwent carboxylation or hemiacetal formation were not detected in our study. This may be due to the difference in metabolic abilities between HepaRG cells and human primary hepatocytes. The TICs and EICs obtained from XLR-11 degradant culture medium extract are shown in Figure 2b. As was the case for XLR-11, no unchanged XLR-11 degradant peak was detected in the EIC of m/z 330, whereas several metabolites were detected in the EICs of m/z 328, 344, 342, 358, and 346. Based on the molecular weight and product ion spectra, M328d and M342d were assumed to be the XLR-11 degradant N-(5-hydroxypentyl) metabolite and the XLR-11 degradant N-pentanoic acid metabolite, respectively. The peaks detected in the EICs of m/z 344, m/z 358 and m/z 346 were
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Detection of metabolites of XLR-11 and its thermal degradation product
Figure 2. Total ion current chromatograms (TIC) and extracted ion chromatograms (EIC) obtained from the extracts of culture medium and urine sample. (a) culture medium (XLR-11), (b) culture medium (XLR-11 degradant), (c) urine sample. Table 1. Retention times, precursor ions and product ions of XLR-11, XLR-11 degradant and their metabolites in product ion analysis. Compound XLR-11 M328 M344-1 M344-2 M344-3 M344-4 M342 M358 M346-1 M346-2 M346-3 M346-4 M362 XLR-11 degradant M328d M344d M342d M358d-1 M358d-2 M346d-1 M346d-2 M362d-1 M362d-2
RTa (min)
Precursor ion (m/z)
14.6 11.9 5.1 7.8 8.1 9.8 11.6 7.7 7.2 10.7 11.3 12.3 7.8 13.8 11.1 5.1 10.8 5.2 7.7 7.9 10.7 5.6 6.4
330 328 344 344 344 344 342 358 346 346 346 346 362 330 328 344 342 358 358 346 346 362 362
Major product ions (m/z) 232, 312, 125, 274 230, 125 230, 314, 204, 186, 326 230, 326, 314, 270, 204 230, 326, 204 246, 125, 202, 190 244, 125, 324 244, 340, 328, 218, 200 232, 204 232, 328, 272, 316, 206 232, 328, 206 125, 248, 328 248, 344, 288 232 230 246, 186, 230, 326, 204 244 260, 200, 244, 218, 340 244, 340, 218, 200, 260 248, 232, 206, 328 248, 232, 206, 328 264, 248, 222, 344 264, 248, 222, 344
Sample
Description
b
ND Mc, Ud M M M M M, U M M M M M M ND M, U M, U M, U M, U U M, U M, U M, U M, U
N-(5-Hydroxypentyl) metabolitee N-(5-Hydroxypentyl) metabolite + (O) N-(5-Hydroxypentyl) metabolite + (O) N-(5-Hydroxypentyl) metabolite + (O) N-(5-Hydroxypentyl) metabolite + (O) N-Pentanoic acid metabolitee N-Pentanoic acid metabolite + (O) XLR-11 + (O) XLR-11 + (O) XLR-11 + (O) XLR-11 + (O) XLR-11 + (2O) N-(5-Hydroxypentyl) metabolite N-(5-Hydroxypentyl) metabolite + (O) N-Pentanoic acid metabolite N-Pentanoic acid metabolite + (O) N-Pentanoic acid metabolite + (O) XLR-11-degradant + (O) XLR-11-degradant + (O) XLR-11-degradant + (2O) XLR-11-degradant + (2O)
a
Retention time. Not detected in urine and culture medium. c Detected in culture medium. d Detected in urine. e Confirmed with authentic standards. b
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T. Kanamori et al.
Table 2. Comparison of the XLR-11 metabolites detected in cell culture medium. Metabolite
Protonated molecule (m/z)
Number of metabolites detected This study (HepaRG cell)
N-(5-Hydroxypentyl) metabolite (+GAa) N-(5-Hydroxypentyl) metabolite + (O) (+GA) N-(5-Hydroxypentyl) metabolite + carboxylation N-Pentanoic acid metabolite (+GA) N-Pentanoic acid metabolite + (O) (+GA) N-Pentanoic acid metabolite + carboxylation (+GA) N-Pentanoic acid metabolite + (2O) - > dehydration N-Pentanoic acid metabolite + (O) + hemiketal N-Pentanoic acid metabolite + (O) + aldehyde - > hemiacetal (+GA) XLR-11 + (O) (+GA) XLR-11 + (O) - > dehydration XLR-11 + (O) + aldehyde - > hemiacetal (+GA) XLR-11 + (2O) (+GA) XLR-11 + (3O) (+GA) Carboxylation (+GA) Carboxylation + (O)
328 (504)b 344 (520) 358 342 (518) 358 (534) 372 (548) 356 358 372 (548) 346 (522) 344 360 (536) 362 (538) 378 (554) 360 (536) 376
1c 4 0 1 1 0 0 0 0 4 0 0 1 0 0 0
Wohlfarth et al. (human primery hepatocyte) 1c (1)d 0 (3) 1 1 (1) 2 (1) 1 (1) 1 1 1 (1) 0 (3) 1 0 (1) 0 (1) 0 (1) 1 (2) 2
a
Glucuronic acid. Protonated molecule of glucuronide. c Number of free metabolite. d Number of glucuronide. b
assumed to correspond to the oxidized metabolites of M328, M342 and XLR-11 degradant, respectively. TICs and EICs obtained from the extract of the XLR-11 user’s urine are shown in Figure 2c. As was the case with hepatocyte cultures, the EIC of m/z 330 failed to show a parent XLR-11 or XLR-11 degradant peak, though several metabolite peaks were detected
in the EICs of m/z 328, 344, 342, 358, and 346. The peaks detected in the EICs of m/z 328 and 342 were identical to those of M328d and M342d detected in the XLR-11 degradant culture medium extract, respectively (Figures 2b and 2c). The other metabolites detected in the urine sample also were identical to the metabolites detected in the XLR-11 degradant culture medium (M344d, M358d-
Figure 3. Behavior of XLR-11 before and after ingestion.
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Detection of metabolites of XLR-11 and its thermal degradation product 1, M346d-1, and M346d-2). In addition, two di-oxidized metabolites (M362d-1 and M362d-2) were detected in both the urine sample and the XLR-11 degradant culture medium (Table 1). These results suggest that the metabolites of XLR-11 degradant, but not of XLR-11, are mainly excreted in the urine of the XLR-11 user. Other putative metabolites (e.g. hemiketal, hemiacetal, and dehydrated or carboxylated metabolites) were not detected in the EICs of protonated molecules. The behaviour of XLR-11 before and after ingestion is summarized in Figure 3. The majority of XLR-11 was thermally degraded during smoking, and XLR-11 degradant was absorbed into the body. XLR-11 degradant was then metabolized in the body and excreted through urine. Small peaks for the XLR-11 metabolites M328 and M342 were also detected in the urine sample (Figure 2c), indicating that a small amount of XLR-11 escaped thermal degradation and was absorbed into the body. In the present case, the unchanged XLR-11 and XLR-11 degradant were not detected in urine. On the other hand, the XLR-11 degradant metabolites have the same molecular weight as the corresponding XLR-11 metabolites (e.g. M328d and M328, M342d and M342), which could lead to the misunderstanding that the XLR-11 degradant metabolites were the metabolites of XLR-11. However, the behaviour of XLR-11 was clarified because of the information obtained from our in vitro system regarding the metabolites of XLR-11 and XLR-11 degradant.
Conclusion The primary XLR-11 degradant metabolites formed by the in vitro system using HepaRG cells were also detected in the urine sample, indicating that XLR-11 thermally degraded during smoking and that XLR-11 degradant was metabolized and excreted through urine. This is the first report describing the metabolism of the XLR-11 thermal degradation product. In the field of forensic toxicology, it is important to clarify the metabolic fate of drugs in order to analyze drug metabolites in biological samples such as urine and blood. However, it is legally and ethically difficult to investigate the metabolism of new drugs of abuse in humans because of their potential harmful effects and toxicities. In the present study, the in vivo metabolism of XLR-11 degradant in a human subject was well replicated by HepaRG cell culture. HepaRG cells showed both phase I and phase II drugmetabolizing enzyme activities. Despite some limitations, the in vitro system using HepaRG cells appears to be an encouraging alternative to in vivo experiments with human subjects.
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Acknowledgement This work was supported by JSPS KAKENHI Grant Number 23590866.
References [1] D.G. Barceloux. MARIJUANA (Cannabis sativa L.) and Synthetic Cannabinoids, in Medical toxicology of drug abuse: synthesized chemicals and psychoactive plants, Wiley, Hoboken, 2012, pp. 915–919. [2] A. Weissman, G.M. Milne, L.S. Melvin Jr. Cannabimimetic activity from CP-47,497, a derivative of 3-phenylcyclohexanol. J. Pharmacol. Exp. Ther. 1982, 223, 516. [3] M.R. Johnson, L.S. Melvin, T.H. Althuis, J.S. Bindra, C.A. Harbert, G. M. Milne, A. Weissman. Selective and potent analgetics derived from cannabinoids. J. Clin. Pharmacol. 1981, 21(8-9 Suppl), 271S. [4] J.L. Wiley, D.R. Compton, D. Dai, J.A. Lainton, M. Phillips, J.W. Huffman, B. R. Martin. Structure-activity relationships of indole- and pyrrole-derived cannabinoids. J. Pharmacol. Exp. Ther. 1998, 285, 995. [5] K.A. Seely, J. Lapoint, J.H. Moran, L. Fattore. Spice drugs are more than harmless herbal blends: a review of the pharmacology and toxicology of synthetic cannabinoids. Prog. Neuropsychopharmacol. Biol. Psychiatry 2012, 39, 234. [6] NMS Labs. NMS Labs Trends Report: Changes in the Designer Drug Market Spring. 2012. Available at: http://www.nmslabs.com/uploads/PDF/ Designer%20Drug%20Spring%20Update_BKL%20Webinar_May% 202012.pdf [16 April 2014]. [7] N. Uchiyama, M. Kawamura, R. Kikura-Hanajiri, Y. Goda. URB-754: a new class of designer drug and 12 synthetic cannabinoids detected in illegal products. Forensic Sci. Int. 2013, 227, 21. [8] J.L. Wiley, J.A. Marusich, T.W. Lefever, M. Grabenauer, K.N. Moore, B. F. Thomas. Cannabinoids in disguise: Δ9-tetrahydrocannabinol-like effects of tetramethylcyclopropyl ketone indoles. Neuropharmacology 2013, 75, 145. [9] Drug Enforcement Administration, Department of justice. Schedules of controlled substances: temporary placement of three synthetic cannabinoids into Schedule I. Fed. Regist. 2013, 78, 21858. [10] A. Wohlfarth, S. Pang, M. Zhu, A.S. Gandhi, K.B. Scheidweiler, H.F. Liu, M. A. Huestis. First metabolic profile of XLR-11, a novel synthetic cannabinoid, obtained by using human hepatocytes and highresolution mass spectrometry. Clin. Chem. 2013, 59, 1638. [11] P. Adamowicz, D. Zuba, K. Sekuła. Analysis of UR-144 and its pyrolysis product in blood and their metabolites in urine. Forensic Sci. Int. 2013, 233, 320. [12] A. Grigoryev, P. Kavanagh, A. Melnik, S. Savchuk, A. Simonov. Gas and liquid chromatography-mass spectrometry detection of the urinary metabolites of UR-144 and its major pyrolysis product. J. Anal. Toxicol. 2013, 37, 265. [13] P. Gripon, S. Rumin, S. Urban, J. Le Seyec, D. Glaise, I. Cannie, C. Guyomard, J. Lucas, C. Trepo, C. Guguen-Guillouzo. Infection of a human hepatoma cell line by hepatitis B virus. Proc. Natl. Acad. Sci. U. S. A. 2002, 99, 15655. [14] K.P. Kanebratt, T.B. Andersson. Evaluation of HepaRG cells as an in vitro model for human drug metabolism studies. Drug Metab. Dispos. 2008, 36, 1444.
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