Introduction
AM1220, or {1-[(1-methylpiperidin-2-yl)methyl]-1
H-indol-3-yl}(naphthalen-1-yl)methanone, is a synthetic cannabinoid that was first synthesised in the 1990s to study the structure-activity relationship of cannabinoid receptors [
1]. The cannabinoid was shown to have a binding affinity (
Ki) of 3.88 and 73.4 nM to cannabinoid receptor type 1 (CB
1) and type 2 (CB
2) receptors, respectively [
2]. Because of the high affinity to cannabinoid receptors, AM1220 began to be sold and abused as “herbal products” and “research chemicals” on the recreational drug market among the continuous emergence of a myriad of new psychoactive substances [
3‐
8].
In these products, AM1220 is usually found together with its azepane isomer, [1-(1-methyl-3-azepanyl)-1
H-indol-3-yl](1-naphthyl)methanone [
4,
5], which is suggested to be present as a synthetic impurity [
4] or due to a rearrangement that occurs over time [
7]. The presence of the AM1220 azepane isomer may complicate interpretation of the pharmacological effects of AM1220, as the azepane isomer itself is shown to have binding affinities to CB
1 and CB
2 receptors [
9].
For detection of synthetic cannabinoids in humans, plasma samples are shown to be useful since the parent drugs can be found as they are without modifications [
10]. However, there are some issues with detection in plasma samples. Firstly, the window of detection of the parent drugs in blood is short [
10,
11]. Secondly, the concentrations of the parent drugs in plasma are reported to be lower than those of the major metabolites [
11]. In addition, plasma samples are not always obtainable due to invasiveness of collection method, and urine samples are often the preferred choice for drug testing. Therefore, suitable methods to analyse urine samples are desirable. Nevertheless, synthetic cannabinoids are highly lipophilic, and high distribution rate of parent drugs for tissue such as fat results in low excretion rate in urine. Furthermore, synthetic cannabinoids are extensively metabolised in humans and are generally not excreted in urine in the parent drug form. Consequently, metabolites need to be monitored for detecting synthetic cannabinoids in urine specimens.
Metabolism studies of synthetic cannabinoids have been performed using several approaches. Human liver microsome (HLM) incubation is the most common in vitro approach, and even though not reflective of the metabolism in a whole human body, it can generate a wide variety of human metabolites with advantages such as low cost and larger pools of donors [
11‐
13]. Human hepatocytes provide the metabolic profiles closest to the in vivo human data [
14‐
16], and animal models such as rats are valuable as a source of in vivo data, though not always consistent with human findings [
17‐
19]. Incubation with the fungus
Cunninghamella elegans (
C. elegans) has been shown to produce similar metabolic profiles to the human system with the advantage of low cost and production of large quantity of metabolites [
20‐
22].
Cunninghamella elegans is, however, not suitable for strict absorption-distribution-metabolism-elimination (ADME) studies, since it does not provide blood and urine as separate specimens as animal models do. The presence and abundance of the metabolites determined by these models may not be an accurate representation of in vivo metabolites. Thus, the in vitro metabolites should be confirmed in human urine, if available, by analysis of urine samples obtained from suspected users of synthetic cannabinoids, since analysis of human urine from controlled administration is difficult at this point without sufficient data to ensure safety [
11].
To date, there has been no in vitro metabolism study of AM1220. There is one in vivo study by Zaitsu et al. [
10] reporting two metabolites of AM1220 and two more potential metabolites in postmortem human plasma and urine specimens, respectively, from a fatal intoxication case. To complement the in vivo findings, which may have been affected by genotype, phenotype and/or inhibition of cytochrome P450 (CYP) enzymes by coadministration of drug, in vitro metabolism study will be useful [
23].
In this study, we report the metabolic stability of AM1220 based on HLM incubation and tentative structure elucidation of AM1220 metabolites obtained from HLM and
C. elegans incubation. Suitable markers for urinalysis are also suggested. Liquid chromatography–quadrupole time-of-flight mass spectrometry (LC–QTOF-MS) was used for analysis since high-resolution mass spectrometry has an advantage of providing accurate masses, enabling more confident characterisation of metabolites [
24].
Materials and methods
Chemicals and reagents
AM1220 was obtained from Cayman Chemical (Ann Arbor, MI, USA). UR-144 was synthesised in-house following the methods previously reported [
25,
26] and characterised by mass spectrometry (MS) and 1D and 2D nuclear magnetic resonance spectroscopy techniques. Fifty-donor HLM pool, NADPH system solution A and NADPH system solution B were from Corning (Corning, NY, USA). Liquid chromatography–mass spectrometry (LC–MS) grade acetonitrile was obtained from Honeywell (Muskegon, MI, USA). Reagent grade dichloromethane and sodium chloride were purchased from Chemsupply (Gilman, SA, Australia). LC–MS grade formic acid was obtained from Sigma-Aldrich (St. Louis, MO, USA).
Cunninghamella elegans ATCC 10028b was from Cryosite Ltd. (South Granville, NSW, Australia). Glycerol and potassium dihydrogen phosphate and dipotassium hydrogen phosphate were from Ajax Chemicals (Auburn, NSW, Australia). Potato dextrose agar, glucose, peptone, and yeast extract were purchased from Oxoid Australia (Adelaide, SA, Australia).
AM1220 solution in acetonitrile/phosphate buffer (40 µM, pH 7.4, 25 µL, 0.1% acetonitrile), phosphate buffer (0.1 M, pH 7.4, 855 µL), NADPH-A (50 µL) and NADPH-B (20 µL) were mixed in an Eppendorf tube, to which HLM (50 µL = 1 mg protein) was added. The final concentration of AM1220 in the mixture was 1 µM with 0.003% acetonitrile. The mixture was incubated in triplicate at 37 °C in a shaking water bath. At time 0, 3, 8, 13, 20 and 30 min, a 100-µL aliquot was removed and placed into 100 µL ice-cold acetonitrile to quench the reaction. The mixture was centrifuged at 16,060 × g for 10 min and filtered with a 0.22 µm filter. Ten microliters of the filtrate was diluted in 990 µL water/acetonitrile (70:30, v/v) and 10 µL was injected into liquid chromatography–triple quadrupole mass spectrometer in triplicate.
Chromatographic separation was performed on an Agilent 1290 LC system with an Agilent Zorbax Eclipse XDBC18 analytical column (150 × 4.6 mm i.d., particle size 5 μm) (Agilent Technologies, Santa Clara, CA, USA). The mobile phase consisted of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). The gradient system was as follows: 30% B until 1 min, ramped to 40% B over 15 min, 95% B at 16.01 min and held until 19.1 min, ramped down to 30% B at 19.11 min and held until 23 min. The flow rate was 0.4 mL/min and the column temperature was kept at 30 °C.
Mass spectrometry was run in multiple reaction monitoring mode on an Agilent 6490 Triple Quadrupole mass spectrometer with electrospray ionisation (ESI) source in positive ion mode (Agilent Technologies). Two transitions (m/z 383 → 286 and m/z 383 → 98) were monitored with fragmentor voltage of 380 V and collision energy of 20 and 50 eV, respectively.
In vitro microsomal half-life (
t1/2) of AM1220 was calculated based on the plot of natural log of percentage of the drug remaining against time. Percentage of the drug remaining was calculated by dividing the peak area of the drug remaining at each time point by the peak area of the drug at time 0 min and multiplying by 100%. The slope of the line (−
k) was used to give
t1/2 = ln2/k. Intrinsic clearance (CL
int, in mL/min/kg) was calculated based on the following formula [
27]:
$${\text{CL}}_{\text{int}} = \frac{\ln 2}{{t_{1/2} }} \times \frac{\text{mL of incubation}}{\text{mg of microsomes}} \times \frac{{45{\text{ mg of microsomes}}}}{\text{g of liver}} \times \frac{{ 2 0 {\text{ g of liver}}}}{\text{kg of body weight}},$$
where
t1/2 (the only variable in the equation) was substituted.
Hepatic clearance (CL
H) and hepatic extraction ratio (
EH) were calculated based on the well-stirred model from the following formulae without considering blood protein and microsome binding [
27,
28]. The 21 mL/min/kg was used for human hepatic blood flow (
QH) [
27].
$${\text{CL}}_{\text{H}} = \frac{{Q_{\text{H}} \times {\text{CL}}_{\text{int}} }}{{Q_{\text{H}} + {\text{CL}}_{\text{int}} }},$$
$$E_{\text{H}} = \frac{{{\text{CL}}_{\text{H}} }}{{Q_{\text{H}} }}.$$
Tentative structure elucidation of metabolites
Human liver microsome incubation
The incubation mixture was prepared as described for the metabolic stability study using 1 mg/mL, i.e., 2.61 mM AM1220 solution (final concentration of acetonitrile was 0.2%). The mixture was incubated at 37 °C in a shaking water bath for 1 h. The reaction was quenched by adding ice-cold acetonitrile (1 mL) to the mixture and it was centrifuged at 16,060 × g for 10 min. The sample was filtered (0.22 µm) and injected to LC–QTOF-MS. A control sample without HLM, a control without AM1220 and a positive control using UR-144 were also incubated and analysed.
Fungus incubation
Cunninghamella elegans was cultured on potato dextrose agar plates at 27 °C for 5 days. The mycelia of the fungus were mixed in sterile physiological saline solution (1 plate of mycelia/5 mL). Growth medium was prepared [
29], and 1.5 mL of the fungus solution was added to 100 mL of medium in a conical flask. The culture was incubated for 48 h at 26 °C and 180 rpm on an Infors HT Multitron rotary shaker (In Vitro Technologies, Noble Park North, VIC, Australia). AM1220 (1 mg in 0.5 mL acetonitrile) was added to the flask and incubated for another 72 h. The solution was filtered, extracted with dichloromethane (3 × 50 mL) and evaporated using a rotary evaporator and a vacuum pump. The sample was reconstituted in 2 mL acetonitrile, which was further diluted in acetonitrile tenfold. A control without fungus and a control without AM1220 were also incubated.
LC–QTOF-MS
Chromatographic equipment and conditions were the same as described above for metabolic stability section, except for the following. The gradient started with 30% B, and was held until 1 min, ramped up to 40% B over 19 min, 90% B at 21 min, held until 24 min, ramped down to 30% B at 25 min and held until 30 min for re-equilibration. Injection volume was 2 µL for scan analysis and 10 µL for product ion scan analysis.
Mass spectra were acquired on an Agilent 6510 Accurate Mass Q-TOF mass spectrometer, equipped with a dual ESI source (Agilent Technologies). The parameters were as follows: scanning mass range, m/z 100–1000 (MS), m/z 80–1000 (MS/MS); capillary voltage, 3500 V; nebulizer pressure, 30 psig; gas temperature, 325 °C; gas flow, 5 L/min; fragmentor voltage, 160 V; collision energy for product ion scan analysis, 10, 20 and 40 eV; skimmer voltage, 65 V. Mass calibration was performed with the mixture provided by the manufacturer. Real-time mass calibration was enabled using the following reference masses: m/z 121.0509 and 922.0098.
Additional MS analyses were performed on an Agilent 6550A iFunnel Q-TOF with a dual AJS ESI source (Agilent Technologies) operated with the same parameters as above except for the following: gas temperature, 290 °C; gas flow, 11 L/min; sheath gas temperature, 350 °C; sheath gas flow, 11 L/min; injection volume for product ion scan analysis, 2 µL.
Extracted ion chromatograms and mass spectra were analysed using Agilent MassHunter Workstation Software Qualitative Analysis (version B.06.00). A personal compound database and library (PCDL) with known and potential metabolites of the drug was created with Agilent MassHunter PCDL Manager (version B.04.00) to search for the metabolites. Search parameters were as follows: mass tolerance, 20 ppm; maximum number of matches, 8; absolute peak area ≥ 5000. The criteria for metabolites were as follows: mass error of the protonated molecules ≤ 5 ppm; consistent fragmentation pattern with proposed structure; reasonable retention time relative to other biotransformations; absence of the metabolite in controls.
Conclusions
A potent synthetic cannabinoid AM1220 was incubated in HLM and C. elegans to elucidate the structures of the in vitro metabolites. Metabolic stability of AM1220 was estimated from HLM incubation and the estimated in vitro half-life and hepatic extraction ratio indicated AM1220 to be a high clearance drug. LC–QTOF-MS analysis of HLM and C. elegans samples resulted in detection of a total of 11 metabolites (nine and seven metabolites in respective samples) and they consisted of hydroxy, dihydroxy, desmethyl, dihydrodiol, and dihydrodiol-desmethyl metabolites. The results did not match the in vivo metabolism previously reported; however it should be noted that the results in the study were based on a single postmortem sample. Based on the in vitro data, hydroxy, desmethyl and dihydrodiol metabolites are deemed suitable urinary markers of AM1220 intake. These data should help toxicological and clinical laboratories to identify AM1220 consumption from human urine samples.