Abstract
Abuse of the stimulant designer drug methylone (methylenedioxymethcathinone) has been documented in most parts of the world. As with many of the new designer drugs that continuously appear in the illicit drug market, little is known about the pharmacokinetics of methylone. Using in vitro studies, CYP2D6 was determined to be the primary enzyme that metabolizes methylone, with minor contributions from CYP1A2, CYP2B6, and CYP2C19. The major metabolite was identified as dihydroxymethcathinone, and the minor metabolites were N-hydroxy-methylone, nor-methylone, and dihydro-methylone. Measuring the formation of the major metabolite, biphasic Michaelis–Menten kinetic parameters were determined: Vmax,1 = 0.046 ± 0.005 (S.E.) nmol/min/mg protein, Km,1 = 19.0 ± 4.2 μM, Vmax,2 = 0.22 ± 0.04 nmol/min/mg protein, and Km,2 = 1953 ± 761 μM; the low-capacity and high-affinity contribution was assigned to the activity of CYP2D6. Additionally, a time-dependent loss of CYP2D6 activity was observed when the enzyme was preincubated with methylone, reaching a maximum rate of inactivation at high methylone concentrations, indicating that methylone is a mechanism-based inhibitor of CYP2D6. The inactivation parameters were determined to be KI = 15.1 ± 3.4 (S.E.) μM and kinact = 0.075 ± 0.005 minute−1.
Introduction
In the past decade, the group of cathinone (β-keto amphetamine) derivatives has become a major group of drugs on the illicit market. The beta-keto analog of 3,4-methylenedioyxmethamphetamine (MDMA), known as methylone, was patented in 1996 as an antidepressant and anti-Parkinsonian agent (Jacob and Shulgin, 1996). Methylone was never developed into a pharmaceutical product, but in the mid-2000s, it appeared in the illicit drug market in the Netherlands and Japan (Bossong et al., 2005; Kamata et al., 2006; Kelly, 2011). Abuse of methylone has been reported in most parts of the world, and it has been scheduled as illegal in many countries because of its associated health risks (Spiller et al., 2011; Zaitsu et al., 2011). Serious health risks linked to methylone abuse include long-term cognitive and neurochemical adverse effects demonstrated in rodents (den Hollander et al., 2013). Furthermore, acute methylone intoxication has led to at least eight confirmed human fatalities (Cawrse et al., 2012; Kovacs et al., 2012; Pearson et al., 2012). In three of these cases the cause of death was violent related with a confirmed methylone abuse, and methylone overdoses caused the remaining five fatalities. The increase in abuse together with these serious health effects demands a better understanding of the pharmacokinetics and dynamics. Thus, our main focus here is the metabolism and kinetics of methylone, for which studies have been limited until now.
Mueller and Rentsch (2012) showed that human liver microsomes (HLMs) metabolize methylone into nor-methylone, dihydro-methylone, and hydroxy-methylone. Figure 1 shows these in vitro–produced metabolites together with metabolites detected in vivo in rat and/or human urine, as determined by Meyer et al. (2010) and Kamata et al. (2006). The major metabolite in urine is a conjugated form of 4-hydroxy-3-methoxymethcathinone (4-HMMC) (Zaitsu et al., 2011). The intermediate (dihydroxymethcathinone; DHMC) in this pathway toward 4-HMMC and the enzymes responsible for the formation of DHMC have not previously been determined.
Many amphetamine analogs are inhibitors of and substrates for CYP2D6 (Lin et al., 1997; Wu et al., 1997; Kreth et al., 2000; Maurer et al., 2000). In addition, MDMA is a mechanism-based inhibitor of CYP2D6 (Wu et al., 1997; Heydari et al., 2004), which may also be expected for methylone because of their structural similarities. Here we elucidate enzyme phenotyping, metabolite identification, and the enzyme kinetics of methylone.
Materials and Methods
Reagents and Chemicals
Methylone used as standard and for the enzyme reactions was purchased from LGA (La Seyne-sur-Mer, France); methylone used for chemical synthesis of DHMC was taken from a seizure made by the Danish police (2008; purity >98%). α-Pyrrolidinopropiophenone (α-PPP) (SciGM, Shanghai, China) served as an internal standard. Other materials were obtained as follows: acetonitrile [liquid chromatography–mass spectrometry grade] and toluene (methylbenzene, liquid chromatography–mass spectrometry grade), Fisher Scientific (Leicestershire, UK); and formic acid (98–100%), Merck (Darmstadt, Germany). Purified water was obtained from a Millipore Synergy UV water purification system (Millipore A/S, Copenhagen, Denmark), and acidic water was prepared as 0.1% formic acid in water. All other chemicals were of analytical grade.
Recombinant expressed human aldehyde oxidase (AO) was purchased from Cypex (Dundee, Scotland, UK). Baculovirus-infected insect cell microsomes containing the cDNA-expressed cytochrome P450 (P450) CYP isoenzymes (1A2, 2A6, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 3A4, 3A5, and Supermix), cDNA-expressed monoamine oxidases (MAO-A and MAO-B), and cDNA-expressed flavin-containing monooxygenases (FMO1, FMO3, and FMO5) were all purchased from BD Biosciences (Woburn, MA). From the same vendor, we also purchased UltraPool HLM 150, UltraPool Human Liver S9 150, insect cell control (ICC), and NADPH regeneration system solutions A and B. Solution A contained 31 mM NADP+, 66 mM glucose-6-phosphate, and 66 mM MgCl2 in H2O. Solution B consisted of 40 IU/ml glucose-6-phosphate dehydrogenase in 5 mM sodium citrate. The various enzymes and NAPDH regeneration system solutions were stored at −80°C and −25°C, respectively, until use.
In Vitro Metabolite Identification and Phenotyping
Methylone degradation and the formation of methylone metabolites were investigated by incubation with different liver fractions and various recombinant enzymes. All experiments were performed in duplicate. We included the following recombinant enzymes at the noted final assay concentrations: P450 CYP (1A2, 2A6, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 3A4, 3A5, and Supermix; each 50 pmol/ml, which is equivalent to the range 0.15–0.85 mg protein/ml, depending on the enzyme); ICC, FMO1, FMO3, and FMO5 (each 0.25 mg/ml); MAO-A and MAO-B (each 0.1 mg/ml); and AO (0.4 mg/ml). The incubation mixture with the P450 enzymes, ICC, and the FMOs consisted of 1.3 mM NADP+, 3.3 mM glucose-6-phosphate, 3.3 mM MgCl2, 0.4 IU/ml glucose-6-phosphate dehydrogenase, and 0.05 mM sodium citrate in 0.1 M phosphate buffer at pH 7.4. The incubations with the MAOs and AO were performed in 0.1 M phosphate buffer at pH 7.4. EDTA was added to the AO incubations to provide a final assay concentration of 0.1 mM. All enzyme assays were incubated with two concentrations of methylone (1.0 μM and 50 μM) in a final volume of 250 μl at 37°C. At the time points 0, 5, 10, 20, 35, 50, 70, 100, and 140 minutes, 20-μl aliquots of the incubations were quenched with 20 μl of ice-cold acidic acetonitrile containing 0.5% formic acid and α-PPP (internal standard) 300 μg/l. The quenched solutions were centrifuged for 10 minutes at 4°C, and 7.5 μl of the supernatant was analyzed directly by ultra-high performance liquid chromatography/time-of-flight mass spectrometry (UHPLC-TOF/MS); a supplementary investigation of fragmentation patterns was also carried out using quadrupole time-of-flight mass spectrometry with fragmentation (UHPLC-QTOF/MSE).
In addition, we investigated the metabolism of methylone at concentrations of 1 μM and 50 μM when incubated with pooled HLM, HLM with inactivated FMO, or S9 liver fraction, each at a final assay concentration of 1 mg protein/ml. FMO was inactivated by heating an aliquot of HLM to 50°C for 1 minutes and then cooling with dry ice. This procedure is described to distinguish between P450 and FMO activity, as the FMO enzymes are highly thermolabile (Grothusen et al., 1996; Cashman, 2005) The experimental conditions and concentrations of cofactors were identical to those used with the recombinant enzymes. The HLM incubations (1 mg protein/ml) were additionally performed with and without various selective P450 enzyme inhibitors (see Table 1 for inhibitors and concentrations) (Suzuki et al., 2002; Walsky and Obach, 2003; Zhang et al., 2007; Khojasteh et al., 2011). HLM, cofactors, and inhibitors were preincubated for 10 minutes at 37°C before the experiment was started with the addition of methylone. A positive-control incubation was performed with an HLM incubation without inhibitor, and the NADPH regeneration system was omitted for the negative control. All experiments were analyzed using UHPLC-TOF/MS and UHPLC-QTOF/MSE.
Chemical Synthesis of DHMC
The chemical synthesis was performed in accordance with the procedure for cleavage of the methylenedioxy group (Debernardis et al., 1987). A total of 120 mg (0.49 mmol) of methylone (HCl-salt) was dissolved in 10 ml dichloromethane and cooled to –78°C. A solution of BBr3 (2.0 mmol) in 2 ml dichloromethane was added drop-wise under nitrogen. After 4 hours at –78°C, the reaction was quenched with 5 ml of MeOH added drop-wise, and the mixture was stirred for 2 hours at room temperature. The mixture was evaporated and then purified with preparative LC, and the dried brown residue was analyzed using NMR, UHPLC-TOF/MS, and UHPLC-QTOF/MSE. The amount of DHMC was quantified using 1H-NMR, and used as the reference standard for the kinetics experiments.
Determination of Michaelis–Menten Kinetics
The assay incubation conditions and concentrations were 1.0 mg protein/ml of pooled HLM, 1.3 mM NADP+, 3.3 mM glucose-6-phosphate, 3.3 mM MgCl2, 0.4 IU/ml glucose-6-phosphate dehydrogenase, and 0.05 mM sodium citrate in 0.1 M phosphate buffer at pH 7.4 in a final volume of 250 μl at 37°C. The methylone concentrations were 2, 5, 13, 32, 80, 200, 500, and 1250 μM. At 0, 3, 6, 9, 12, and 15 minutes, 20 μl-aliquots of each incubation were quenched with 30 μl of ice-cold 8% perchloric acid in purified water containing 4% acetonitrile and 300 μg/l α-PPP (internal standard). The quenched solutions were centrifuged for 10 minutes at 4°C, and 7.5 μl of each supernatant was analyzed directly using UHPLC-tandem mass spectrometry (MS/MS) to quantify the amount of DHMC. All incubations were performed in triplicate.
Prior to determination of the Michaelis–Menten kinetics, the DHMC formation from methylone (10 μM) was investigated with varying HLM concentrations (range, 0.25–2.0 mg protein/ml). DHMC formation was proportional to HLM concentrations in the range of 0.25–1.0 mg protein/ml (unpublished data), implying no binding of the substrate to liver microsomes, which would reduce the free fraction of substrate available to interact with the enzymes; therefore, 1.0 mg protein/ml was chosen as the assay concentration to determine the Michaelis–Menten kinetics. When Michaelis–Menten kinetics applies and only one enzyme is involved in the formation of the metabolite, the relationship between the substrate (methylone) concentration [S] and the rate of metabolite (DHMC) formation (V) can be described by monophasic kinetics:
Biphasic kinetics applies when two enzymes are forming the same metabolite, and the total rate is given by the sum of the rate from each enzyme:
Calculations of the Michaelis–Menten kinetics were performed using GraphPad Prism 5.04 (La Jolla, CA) with nonlinear regression. In vivo, [S] is usually much smaller than Km, and the intrinsic clearance (CLint) for each enzyme can then be calculated as:
If CLint is determined for both a recombinant enzyme and for the same enzyme in HLM, the relative activity factor can be determined as the ratio between CLint (HLM) and the CLint for the recombinant enzyme. Prior to these calculations, the absence of binding of the substrate to liver microsomes should be confirmed.
Determinations of the kinetics were also made using the recombinant P450 enzymes CYP2D6 and CYP2B6 (50 pmol/ml), as well as HLM inhibited with quinidine (5 μM) and 2-phenyl-2-(1-piperidinyl)propane (PPP) (20 μM) with the same incubation setup as described for the pooled HLM.
Investigation of Time-Dependent Mechanism-Based Inhibition of CYP2D6 by Methylone
The conversion of dextromethorphan into dextrorphan was used as a test substrate to investigate the remaining CYP2D6 activity after preincubation with methylone. HLM was preincubated with eight different concentrations of methylone (0, 3, 7.5, 15, 30, 50, 75, and 100 μM) in a mixture containing 1.3 mM NADP+, 3.3 mM glucose-6-phosphate, 3.3 mM MgCl2, 0.4 IU/ml glucose-6-phosphate dehydrogenase, and 0.05 mM sodium citrate in 0.1 M phosphate buffer at pH 7.4. The preincubations were started with the addition of HLM to give a concentration of 1.0 mg/ml. At 0, 2, 4, 7, and 10 minutes, 25 μl of these eight preincubations were transferred to a new incubation containing 80 μM dextromethorphan, 1.3 mM NADP+, 3.3 mM glucose-6-phosphate, 3.3 mM MgCl2, 0.4 IU/ml glucose-6-phosphate dehydrogenase, and 0.05 mM sodium citrate in 0.1 M phosphate buffer at pH 7.4, in a final volume of 250 μl. Aliquots of 20 μl of each dextromethorphan incubation were quenched with 20 μl ice-cold acidic acetonitrile containing 0.5% formic acid and 300 μg/l α-PPP (internal standard) at 10 and 20 minutes. The quenched incubations were diluted with 40 μl of purified water and centrifuged (rpm 3500) for 10 minutes at 4°C, and 5 μl of each supernatant was analyzed using ultra-high performance liquid chromatography/tandem mass spectrometry (UHPLC-MS/MS) to quantify the amount of dextrorphan metabolized from dextromethorphan. The amount of dextrorphan was plotted against the 10-minutes and 20-minutes time points, and CYP2D6 activity (slope) was calculated for each preincubation time in relation to the incubations without inhibitor. For all eight methylone concentrations, the remaining CYP2D6 activity was plotted against the five preincubation time points in a semi-log10 scale. The slope of these curves equals the rate of the inactivation constant (kobs) at each concentration. When mechanism-based inhibition kinetics applies, kobs can be described as:
where [I] is the inhibitor (methylone) concentration, kinact is the maximum rate constant when [I] approaches infinity, and KI is the [I] that gives one-half of the rate of kinact (Silverman, 1998; Heydari et al., 2004).
KI and kinact were calculated using GraphPad Prism 5.04 with nonlinear regression.
To verify the experimental setup, the assay was repeated with MDMA as the inhibitor in the preincubation instead of methylone. The calculated kinact and KI were compared with the MDMA results published by Heydari et al. (2004).
Drug Analysis
UHPLC-MS/MS.
DHMC and dextrorphan were quantified with an Acquity UHPLC system interfaced to an Acquity TQD tandem mass spectrometer using an Acquity UHPLC BEH C18, 1.7 μm, 2.1 × 100 mm column, all from Waters (Manchester, UK). The method was developed from in-house validated methods that have previously been published (Simonsen et al., 2010; Johansen and Hansen, 2012). The flow rate was 0.6 ml/min, and the mobile phase consisted of acidic water (A) and acetonitrile (B), each containing 0.05% formic acid at 50°C. The gradient was programmed as follows: 0–4 minutes from 99.5 to 90% A; 4–5 minutes to 50% A; 5–5.3 minutes to 0% A; 5.3–5.5 minutes to 99.5% A; and 5.5–9 minutes isocratic at 99.5% A. Positive electrospray ionization operating in multiple-reaction-monitoring mode was used for detection. The determination was done with two multiple-reaction-monitoring transitions for the following compounds: methylone 208 > 160 (quantifier) and 208 > 132; DHMC 196 > 160 (quantifier) and 196 > 132; and dextrorphan 258 > 157 (quantifier) and 258 > 157. For the internal standard (α-PPP), only one transition was determined: 204 > 105. Argon was used as the collision gas at 0.45 Pa, and the desolvation gas flow was fixed at 1100 l/h. The source temperature was set at 120°C, and the desolvation temperature was set at 450°C. The respective linear ranges for DHMC and dextrorphan were 0.009 (limit of quantification)–1.5 μM and 0.02 (limit of quantification)–1.0 μM. Limit-of-detection values for DHMC and dextrorphan were determined to be 0.003 μM and 0.007, respectively. The relative intra- and interday standard deviations for DHMC were determined to be 6 and 7%, respectively, and were 3 and 10% for dextrorphan.
UHPLC-TOF/MS.
This instrumentation was primarily used in the qualitative search for metabolites, and all retention times (RTs) presented in this paper were determined with this system. UHPLC-TOF/MS analysis was performed using an Acquity UHPLC system coupled to an LCT premier XE time-of-flight mass spectrometer or a Synapt G2 QTOF/MSE, all from Waters. UHPLC separation and MS configuration were performed in accordance with our previously published protocols (Dalsgaard et al., 2012; Pedersen et al., 2012) with some changes in the gradient. The mobile phase consisted of 0.1% formic acid (solvent A) and 100% acetonitrile (solvent B). The gradient was decreased from 100 to 90% of A from 0.0 to 4.0 minutes, to 50% of A from 4.0 to 6.0 minutes, to 5% of A from 6.0 to 7.0 minutes, and then increased back to 100% of A from 7.0 to 7.5 minutes. The column was then reconditioned with 100% of A (7.5–9.5 minutes). Although TOF/MS was used in the positive mode, some samples were additionally injected in the negative mode to ensure that we did not fail to detect any acid or neutral metabolites. The search for metabolites was performed both with the use of Metabolynx XS (Waters V4.1 SCN803) software and manually by subtracting the mass of the expected and possible metabolites from the total ion chromatogram. A semiquantitative limit of detection for new metabolites with no reference standard was defined as three times the background noise, determined as absolute peak height. Additionally, new metabolites were identified as such only if they were produced during the incubation, as confirmed by following the reaction at the different time points.
Some supplementary qualitative studies were performed using UHPLC-QTOF/MSE to identify the fragmentation pattern for methylone and its metabolites. The collision cell of this instrument was switched between two functions: collision energy of 10 V for no fragmentation, and ramping from 20 to 40 V to create fragments. The rest of the detector settings were as previously published (Reitzel et al., 2012). Note that all exact and accurate masses presented in the present study have a deviation of 0.55 mDa from the true value, as the calculation performed by MassLynx software uses the mass of the hydrogen instead of the proton for the mass calculation of [M+H]+. Because this deviation is also applied during mass axis calibration, there is no negative impact on the presented mass errors.
NMR (Determination and Metabolite Quantification).
NMR data were recorded using a Bruker 500.13 MHz instrument (Bruker, Rheinstetten, Germany). Methanol-d4 (Sigma-Aldrich, Schnelldorf, Germany) was used as the solvent for all NMR experiments. NMR data were recorded for methylone and the synthesized DHMC [1H-NMR, 13C-NMR, correlation spectroscopy (COSY), and heteronuclear single quantum coherence spectroscopy (HSQC)]. A relaxation time of 1 second was adequate to fully relax all protons before the next impulse, which was especially important for the quantification experiment. An appropriate number of scans was set (64 or fewer in all experiments).
1H-NMR quantification of the chemically synthesized DHMC was performed as described by Dagnino and Schripsema (2005), with toluene as the internal standard. A solution of 2.9 mg/ml toluene in methanol-d4 was prepared, and the dried residue of DHMC was dissolved in 600 μl of this solution. 1H-NMR of the dissolved residue was recorded and DHMC quantified by comparing the intensities of the eight protons from toluene with the 12 aromatic and aliphatic protons from the chemically synthesized metabolite.
Results
In Vitro Metabolite Identification and Phenotyping
Table 2 presents the methylone metabolites that are formed when incubated with various enzymes and liver fractions for 140 minutes. Figure 2 illustrates the remaining amount of methylone (1 μM) when incubated with the same enzymes and liver fractions, and Fig. 3 presents DHMC formation from 50 μM methylone when incubated with HLM and inhibited with various specific inhibitors. With the same experimental setup, the degradation of 1 μM methylone was measured, and only quinidine was found to significantly reduce the degradation of methylone (unpublished data).
Four metabolites of methylone were detected in the in vitro experiments; Fig. 1 shows the suggested structures of these metabolites. NMR and fragmentation data are presented below for methylone and the four metabolites.
Methylone.
RT 3.28 minutes. Equation C11H13NO3; HRMS [M + H]+ calculated m/z (mass-to-charge ratio) 208.0974, found 208.0975 (0.5 ppm); fragments observed with QTOF/MSE (listed in order of descending abundance) m/z 160.0764 (CH4O2 loss), 132.0820 (C2H4O3 loss), 190.0869 (H2O loss), 117.0578 (C3H7O3• radical loss), and 91.0551 (C4H7NO3 loss). These fragments of methylone have previously been reported by our laboratory (Reitzel et al., 2012). 1H-NMR (500 MHz, CD3OD) δ 7.70 (dd, J = 8.3 Hz and 1.8 Hz, 1H, Ar-H); 7.50 (d, J = 1.8 Hz, 1H, Ar-H); 7.02 (d, J = 8.3 Hz, 1H, Ar-H); 6.13 (d, J = 1.1 Hz (geminal); 1H, -O-CHAHB-O-); 6.13 (d, J = 1.1 Hz (geminal); 1H, -O-CHAHB-O-); 5.01 (q, J = 7.1 Hz 1H, (C=O)-CH-NH); 2.75 (s, 3H, CH3-NH); and 1.57 ppm (d, J = 7.1 Hz, 3H, CH-CH3). Vicinal couplings between the aromatic protons and the coupling between the protons in CH-CH3 were also confirmed using H-H COSY (not shown). 13C-NMR (500 MHz, CD3OD) δ 195 (C=O), 155 and 150 (2 aromatic C adjacent to the methylenedioxy group), 129 (aromatic C adjacent to C=O), 127, 110, and 109 (3 aromatic C-H), 104 (-O-CH2-O-), 60 ((C=O)-CH-NHCH3), 32 (CH3-NH), and 17 ppm (CH3-CH-NHCH3). All outlined C-H bindings were confirmed using HSQC.
DHMC.
To our knowledge, this metabolite has not been reported before. The metabolite was detected in the in vitro enzyme experiments and was also synthesized chemically in preparative amounts. RT (1.45 minutes), accurate mass, and fragmentation pattern each verified that the enzymatic and chemically produced metabolites are identical, and only the accurate mass and fragmentation for the enzymatic product are presented: formula C10H13NO3; HRMS [M + H]+ calculated m/z 196.0974, found 196.0981 (3.6 ppm); fragments observed with QTOF/MSE (listed in order of descending abundance) m/z 160.0770 (2 × H2O loss), 178.0873 (H2O loss), 132.0823 (CH4O3 loss), 91.0548 (C3H7NO3 loss), and 117.0584 (C2H7O3• radical loss). The exact structure of the metabolite was determined by NMR data obtained from the chemically produced metabolite: 1H-NMR (500 MHz, CD3OD) δ 7.33 (m, 2H, Ar-H); 6.77 (d, J = 8.3 Hz, 1H, Ar-H); 4.82 (the peak is almost concealed in the solvent peak at 4.87, 1H, (C=O)-CH-NH); 2.58 (s, 3H, CH3-NH); and 1.42 ppm (d, J = 6.4 Hz, 3H, CH-CH3). Vicinal couplings between the aromatic protons and the coupling between the protons in CH-CH3 were further confirmed using H-H COSY (not shown). 13C-NMR (500 MHz, CD3OD) δ 195 (C=O), 154 and 147 (2 aromatic C-OH), 126 (aromatic C adjacent to C=O), 124, 116.3, and 116.2 (3 aromatic C-H), 60 ((C=O)-CH-NHCH3), 32 (CH3-NH), and 17 ppm (CH3-CH-NHCH3). All outlined C-H bindings were confirmed using HSQC (not shown). The chemical synthesis of DHMC produced 19 μg (95 μmol), determined by 1H-NMR, representing an efficacy of 19% in relation to the amount of methylone used for the synthesis.
N-Hydroxy-Methylone.
RT 4.56 minutes. Equation C11H13NO4; HRMS [M + H]+ calculated m/z 224.0923, found 224.0927 (1.8 ppm); fragments observed with QTOF/MSE (listed in order of descending abundance) m/z 149.0243 (C3H9O loss), 121.0301 (C4H9O2 loss), and 176.0712 (CH4O2 loss).
Nor-Methylone.
RT 2.86 minutes. Equation C10H11NO3; HRMS [M + H]+ calculated m/z 194.0817, found 194.0813 (–2.1 ppm); fragments observed with QTOF/MSE (listed in order of descending abundance) m/z 146.0603 (CH4O2 loss), 118.0652 (C2H4O3 loss), 176.0702 (H2O loss), 174.0547 (H4O loss), 117.0581 (C2H5O3• radical loss), and 91.0543 (C3H5NO3 loss).
Dihydro-Methylone.
RT 2.96 minutes. Equation C11H15NO3; HRMS [M + H]+ calculated m/z 210.1130, found 210.1131 (0.5 ppm). Fragments observed with QTOF/MSE (listed in order of descending abundance) m/z 192.1018 (H2O loss) and 177.0778 (CH5O• radical loss).
Determination of Michaelis–Menten Kinetics
The rate (V) of the formation of DHMC was linear in the range 0 to 9 minutes for all experiments. The rate within this interval was determined for all methylone concentrations, and the relationship is plotted in Fig. 4 for the pooled HLM experiment. The same data are presented as the Eadie–Hofstee plot in Fig. 5, showing that monophasic kinetics cannot explain the relationship between V and methylone concentration; therefore, biphasic kinetic parameters were calculated. The contribution from enzyme component E1 is Vmax,1 = 0.046 ± 0.005 (S.E.) nmol/min/mg protein and Km,1 = 19.0 ± 4.2 μM; for component E2, it is Vmax,2 = 0.22 ± 0.04 nmol/min/mg protein and Km,2 = 1953 ± 761 μM. CLint values for the two components E1 and E2 were calculated to be 2.4 μl/min/mg protein and 0.11 μl/min/mg protein, respectively, assuming [S] << Km. Consequently, 95% of the metabolism of methylone can be ascribed to E1, and E2 accounts for the remaining 5%.
Data obtained from the incubations performed with recombinant CYP2D6 and CYP2B6 (separately) were plotted as an Eadie–Hofstee plot, showing monophasic kinetics (unpublished data). The respective monophasic kinetics parameters for each enzyme were determined to be Vmax,2D6 = 3.49 ± 0.03 nmol/min/nmol P450 and Km,2D6 = 2.57 ± 0.17 μM, and Vmax,2B6 = 0.087 ± 0.007 nmol/min/nmol P450 and Km,2B6 = 25.2 ± 8.9 μM. CLint was calculated to be 1.4 ml/min/nmol CYP2D6 and 3.4 μl/min/nmol CYP2B6. Inhibition of CYP2D6 with quinidine in pooled HLM revealed approximately monophasic kinetics (Fig. 6), and the parameters were determined to be Vmax = 0.180 ± 0.014 nmol/min/mg protein and Km,1 = 1154 ± 154 μM. A t test failed to demonstrate a significant difference (P > 0.2) between these parameters and those obtained from the E2 component in pooled HLM without inhibitor. Therefore, the contribution observed from E1 could be ascribed to CYP2D6 because this contribution could be inhibited with quinidine. The relative activity factor for CYP2D6 was calculated to 1.8 pmol CYP2D6/mg protein. Inhibition of CYP2B6 with PPP in pooled HLM revealed biphasic kinetics, and the parameters were determined to be Vmax,1 = 0.034 ± 0.005 nmol/min/mg protein and Km,1 = 14.7 ± 4.7 μM, and Vmax,2 = 0.16 ± 0.01 nmol/min/mg protein and Km,2 = 840 ± 196 μM. These four parameters do not differ significantly (t test, P > 0.2) from the determinations made with pooled HLM without inhibitor; hence, the contribution from E2 cannot be ascribed to CYP2B6 activity alone, and the contribution must involve more enzymes.
Investigation of the Time-Dependent Mechanism-Based Inhibition of CYP2D6 by Methylone
CYP2D6 activity was determined from its ability to metabolize dextromethorphan into dextrorphan. In Fig. 7, the remaining activity is plotted as a function of the preincubation time for each methylone concentration. The slopes (kobs) of these curves are plotted against the methylone concentration in Fig. 8, and the inactivation parameters were calculated as described in Materials and Methods. For methylone, we found KI = 15.1 ± 3.4 (S.E) μM and kinact = 0.075 ± 0.005 minutes−1; for MDMA, KI = 5.1 ± 0.9 μM and kinact = 0.146 ± 0.007 minutes−1. To confirm the validity of the applied formula, kobs and the methylone concentrations were plotted in accordance with the Eadie–Hofstee plot (with the use of kobs instead of V). Linearity was observed for both compounds, confirming the assumed kinetics (unpublished data). Our determinations were made using an ultra-pool of HLM from 150 individuals. Heydari et al. (2004) previously determined the inactivation parameters for MDMA in HLM from three individuals, all phenotyped as extensive metabolizers. They found KI and kinact to be in the range of 8.8–45.3 μM and 0.12–0.26 minutes−1, respectively, which is the same order of magnitude as our results for MDMA.
Discussion
It has been proposed that individuals who lack functional CYP2D6 might be prone to toxicities from drugs that are primarily metabolized by this enzyme, such as methylenedioxy-amphetamines. This lack of functionality is observed in poor metabolizers and when CYP2D6 is inhibited by a drug (e.g., mechanism-based inhibition). However, recent research and reviews disagree with this hypothesis, given that most of the drugs are also metabolized by enzymes other than CYP2D6 (Kreth et al., 2000; Kraemer and Maurer, 2002). Furthermore, many of the drugs are also excreted unmetabolized or as phase 2 conjugates, reducing the risk of drug accumulation to toxic levels. The literature offers no final conclusions; hence, research in this area is necessary for a more complete understanding of the mechanism underlying the toxicity of these compounds, and we present here our detailed studies concerning the in vitro metabolism of methylone.
The results of the experiments with recombinant enzymes show that methylone is primarily metabolized by CYP2D6, with some contribution from CYP1A2, CYP2B6, and CYP2C19, compared with the control (ICC; Fig. 2). Although a few other enzymes may also be able to form minor amounts of the metabolites (Table 2), these minor amounts do not contribute to the overall clearance of methylone when CYP2D6 is available. The same general conclusions can be made from the experiments with the inhibition of specific HLM enzymes, in which the inhibition of CYP2D6 with quinidine has the greatest effect in relation to both methylone degradation and DHMC formation. The inhibition of CYP2B6, CYP2C19, CYP1A2, and CYP3A4 (with PPP, S-benzylnirvanol, furafylline, and ketoconazole, respectively) reveals no significant difference in methylone degradation in relation to the positive control incubation (unpublished data). However, when CYP2B6 is inhibited, the incubation exhibits a significant reduction in the DHMC formation (Fig. 3); this finding is in agreement with the observations made with the recombinant CYP2B6 incubation, in which the contribution from CYP2B6 is also significant. No reduction is observed in the formation of DHMC in the experiments with S-benzylnirvanol and furafylline, which is somewhat unexpected in relation to the results from the recombinant experiments. This discrepancy might be due to the relatively large substrate concentration (50 μM methylone) that was used to form a sufficient amount of DHMC so that a reduction in the formation could be significantly detected. For inhibitory experiments like those presented here, substrate (methylone) concentrations are normally recommended to be at or below Km for the enzyme to be inhibited. Km for CYP2D6 in HLM was determined to be only 19 ± 4.2 μM, but apparently the methylone concentration of 50 μM was not a problem, as we could observe an effect when incubating with quinidine. The contribution with a Km of 1953 ± 761 μM was ascribed to different low-affinity enzymes (high Km), which means that a methylone concentration of 50 μM cannot explain why S-benzylnirvanol and furafylline do not significantly reduce DHMC formation. The most reasonable explanation is that 95% of the metabolism of methylone is ascribed to CYP2D6, implying that contributions from other enzymes are hard to identify.
The quantification experiments conducted with the UHPLC-MS/MS instrument reveal that DHMC is the primary in vitro metabolite of methylone, which agrees with the observations in Table 2, where DHMC also appears as the major metabolite. Kamata et al. (2006) previously identified 4-hydroxy-3-methoxymethcathinone (Fig. 1) as the major in vivo metabolite in human and rat urine. Its metabolic precursor is believed to be DHMC, which is then O-methylated by catechol-O-methyltransferase to form 4-HMMC (and 3-HMMC). To our knowledge, DHMC has not previously been identified; neither has the responsible enzyme, CYP2D6.
Concerning the identification of DHMC, we compared NMR data from the chemically produced metabolite with the data from methylone; this comparison reveals that both the 1H and 13C signals from the methylene group are missing from the metabolite data, confirming that the group has been cleaved off. This finding is further supported by the fact that the metabolite has lost the exact mass of a single carbon atom. Although the potential metabolite nor-dihydro-methylone (Fig. 1) also has the exact mass of m/z 196.0974 [M+H]+, no other peaks with this mass were identified, which thus excludes this metabolic pathway.
It was not possible to unambiguously determine the structure of the hydroxy-metabolite from our generated data; however, the most likely place for the hydroxylation is at the nitrogen side of the beta-keto group. This conclusion arises from our suggestion for the structure of the most abundant fragment (Fig. 9), which is a product of an alpha cleavage beside the beta-keto group, meaning that the hydroxy group is cleaved off together with the nitrogen part. Figure 10 shows another possible structure of the metabolite, which Mueller and Rentsch (2012) previously suggested. Although the differentiation between the N-hydroxy amine and the metabolite in Fig. 10 cannot be made unequivocally, the detected metabolite has a longer RT than methylone, (i.e., the metabolite is less polar), indicating that the N-hydroxy amine structure is the most likely one (Yuan et al., 2002; Edlund and Baranczewski, 2004; Wu et al., 2004).
The molecular formulas of nor-methylone and dihydro-methylone were determined from the accurate mass data; the most likely metabolic pathways leading to these products are demethylation (CH2 loss) and reduction (2H gain), respectively. The proposed structure in Fig. 1 is in agreement with the observed fragmentation and previous suggestions in the literature (Kamata et al., 2006; Meyer et al., 2010; Zaitsu et al., 2011; Mueller and Rentsch, 2012).
Methylone does not follow simple monophasic kinetics in measures of the formation of the major metabolite DHMC. This deviation from monophasic kinetics may arise from the ability of more than one enzyme to form DHMC or from some atypical kinetics (e.g., homotropic cooperation). In the incubation with recombinant CYP2D6, this major metabolizing enzyme shows monophasic kinetics; therefore, the assumption is that the nonmonophasic kinetics is caused by the activity of additional enzymes apart from CYP2D6 rather than by atypical kinetics. Further supporting this conclusion is the fact that the incubation with HLM in the presence of quinidine (Fig. 6) yields approximately monophasic kinetics when compared with Fig. 5 with no inhibitor. The relatively small Vmax and Km determined for CYP2D6 in HLM (component E1) means that the enzyme has a low capacity and a high affinity for methylone, as previously described for MDMA (Kreth et al., 2000). Furthermore, at low methylone concentrations, 95% of the metabolism can be ascribed to CYP2D6, as calculated from the clearance. The contribution (5%) from the other component E2 is assumed to arise from a mixture of contributions from enzymes that all have high capacity and low affinity because it cannot be assigned to CYP2B6 alone. The assumption that more than one enzyme is contributing to component E2 is supported by the slight curvation of the Eadie–Hofstee plot observed with the incubation in HLM, in which CYP2D6 is inhibited (Fig. 6). In case of low CYP2D6 activity and/or at high methylone concentrations, metabolic switching will occur; i.e., these high-capacity and low-affinity enzymes will take over the metabolism of methylone. Whether this observation has any relevance in vivo requires further investigation; however, it emphasizes the recent hypothesis that the toxicity of compounds like methylone is unrelated to a reduction in CYP2D6 activity, because of metabolic switching (Kreth et al., 2000; Kraemer and Maurer, 2002).
Our experiments have shown that the inhibition of CYP2D6 with methylone is time-dependent and that the rate of inactivation reaches a maximum at high methylone concentrations. Both of these characteristics are fundamental criteria for mechanism-based inactivation; however, to unequivocally confirm that methylone is a mechanism-based inhibitor of CYP2D6, additional characteristics must be verified as described by Silverman (1998). Our experiments did not cover all of these characteristics, but our initial studies provide strong indications of mechanism-based inhibitor kinetics. We also determined the inactivation parameters for MDMA, which apparently features both a higher affinity (lower KI; P < 0.05, t test) and a greater capacity (higher kinact; P < 0.001, t test) for inactivation of CYP2D6 than methylone. This finding agrees with the observation that the introduction of a beta-keto group into amphetamine (forming cathinone) will reduce the compound’s affinity for CYP2D6 (Wu et al., 1997).
In conclusion, we have found that 95% of methylone is metabolized by CYP2D6, with some contribution from primarily CYP1A2, CYP2B6, and CYP2C19. The main metabolite is DHMC, which has not been identified before, and its formation shows biphasic Michaelis–Menten kinetics. Additionally, our experiments indicated that methylone is a mechanism-based inhibitor of CYP2D6, which makes the identification of the contribution from other enzymes important; because metabolic switching to these enzymes might prevent accumulation of methylone when CYP2D6 activity is reduced.
Acknowledgments
The authors thank Christian Tortzen (Department of Chemistry, University of Copenhagen) for help with the chemical synthesis of DHMC.
Authorship Contributions
Participated in research design: Petersen, Pedersen.
Conducted experiments: Petersen, Pedersen.
Performed data analysis: Petersen, Pedersen.
Wrote or contributed to the writing of the manuscript: Linnet, Pedersen.
Footnotes
- Received January 2, 2013.
- Accepted April 1, 2013.
Abbreviations
- AO
- human aldehyde oxidase
- COSY
- correlation spectroscopy
- DHMC
- dihydroxymethcathinone
- FMO
- flavin-containing monooxygenase
- HLM
- human liver microsomes
- 4-HMMC
- 4-hydroxy-3-methoxymethcathinone
- HSQC
- heteronuclear single quantum coherence spectroscopy
- ICC
- insect cell control
- MAO
- monoamine oxidase
- MDMA
- 3,4-methylenedioxymethamphetamine
- m/z
- mass-to-charge ratio
- P450
- cytochrome P450
- PPP
- 2-phenyl-2-(1-piperidinyl)propane
- α-PPP
- α-pyrrolidinopropiophenone
- RT
- retention time
- UPLC-MS/MS
- ultra-high performance liquid chromatography/tandem mass spectrometry
- UHPLC-QTOF/MSE
- ultra-high performance liquid chromatography/quadrupole time-of-flight mass spectrometry with fragmentation
- UHPLC-TOF/MS
- ultra-high performance liquid chromatography/time-of-flight mass spectrometry
- Copyright © 2013 by The American Society for Pharmacology and Experimental Therapeutics