In vitro metabolism and pharmacokinetic studies on methylone

Article abstract of DOI:10.1124/dmd.112.050880

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 dihydromethylone. Measuring the formation of the major metabolite, biphasic Michaelis-Menten kinetic parameters were determined: V_max,1 = 0.046 ± 0.005 (S.E.) nmol/min/mg protein, K_m,1 = 19.0 ± 4.2 μM, V_max,2 = 0.22 ± 0.04 nmol/min/mg protein, and K_m,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 K_I = 15.1 ± 3.4 (S.E.) μM and k_inact = 0.075 ± 0.005 minute^-1. Copyright

Full text of DOI:10.1124/dmd.112.050880

1521-009X/41/6/1247–1255$25.00
http://dx.doi.org/10.1124/dmd.112.050880
DRUG METABOLISM AND DISPOSITION
Drug Metab Dispos 41:1247–1255, June 2013
Copyright ª 2013 by The American Society for Pharmacology and Experimental Therapeutics
In Vitro Metabolism and Pharmacokinetic Studies on Methylone
Anders Just Pedersen, Trine Hedebrink Petersen, and Kristian Linnet
Section of Forensic Chemistry, Department of Forensic Medicine, Faculty of Health Sciences (A.J.P., K.L.),
and Department of Pharmaceutics and Analytical Chemistry, Faculty of Pharmaceutical Sciences, University of Copenhagen,
Copenhagen, Denmark (T.H.P.)
Received January 2, 2013; accepted April 1, 2013
ABSTRACT
Abuse of the stimulant designer drug methylone (methylenediox-
ymethcathinone) has been documented in most parts of the world. 0.046 6 0.005 (S.E.) nmol/min/mg protein, Km,1 = 19.0 6 4.2 mM,
As with many of the new designer drugs that continuously appear in Vmax,2 = 0.22 6 0.04 nmol/min/mg protein, and Km,2 = 1953 6 761
the illicit drug market, little is known about the pharmacokinetics mM; the low-capacity and high-affinity contribution was assigned
of methylone. Using in vitro studies, CYP2D6 was determined to to the activity of CYP2D6. Additionally, a time-dependent loss of
be the primary enzyme that metabolizes methylone, with minor con- CYP2D6 activity was observed when the enzyme was preincubated with
Michaelis–Menten kinetic parameters were determined: Vmax,1 =
tributions from CYP1A2, CYP2B6, and CYP2C19. The major me-
tabolite was identified as dihydroxymethcathinone, and the minor
metabolites were N-hydroxy-methylone, nor-methylone, and dihydro-
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 =
methylone. Measuring the formation of the major metabolite, biphasic 15.1 6 3.4 (S.E.) mM and kinact = 0.075 6 0.005 minute21
.
Introduction
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 me-
tabolites 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 intermedi-
ate (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.
In the past decade, the group of cathinone (b-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 anti-
depressant 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.
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%). a-Pyrrolidinopropiophenone (a-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
dx.doi.org/10.1124/dmd.112.050880.
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; a-PPP, a-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.
1247

1248
Pedersen et al.
Fig. 1. In vitro and in vivo metabolism of methylone.
The four metabolites marked with an asterisk (*) were
detected in our in vitro experiments. Detection of DHMC
has not been reported before. Mueller and Rentsch (2012)
previously detected nor-methylone, dihydro-methylone,
and the hydroxy metabolite in vitro. Metabolites marked
with the number sign (#) have been detected in rat and
human urine by Kamata et al. (2006) and Meyer et al.
(2010) (4-HMC has only been detected in rat urine).
Methylone and all of the metabolites detected in urine
were primarily observed as conjugated metabolites.
Scientific (Leicestershire, UK); and formic acid (98–100%), Merck (Darmstadt, The quenched solutions were centrifuged for 10 minutes at 4°C, and 7.5 ml
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-
of the supernatant was analyzed directly by ultra-high performance liquid
chromatography/time-of-flight mass spectrometry (UHPLC-TOF/MS); a supple-
mentary 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 mM and 50 mM 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
expressed flavin-containing monooxygenases (FMO1, FMO3, and FMO5) then cooling with dry ice. This procedure is described to distinguish be-
were all purchased from BD Biosciences (Woburn, MA). From the same tween P450 and FMO activity, as the FMO enzymes are highly thermolabile
vendor, we also purchased UltraPool HLM 150, UltraPool Human Liver S9 (Grothusen et al., 1996; Cashman, 2005) The experimental conditions and
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
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
dehydrogenase in 5 mM sodium citrate. The various enzymes and NAPDH inhibitors and concentrations) (Suzuki et al., 2002; Walsky and Obach, 2003;
regeneration system solutions were stored at 280°C and 225°C, respectively, Zhang et al., 2007; Khojasteh et al., 2011). HLM, cofactors, and inhibitors were
until use.
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.
In Vitro Metabolite Identification and Phenotyping
Methylone degradation and the formation of methylone metabolites were
investigated by incubation with different liver fractions and various recombi-
nant 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 mM and 50 mM) in a final volume of
250 ml at 37°C. At the time points 0, 5, 10, 20, 35, 50, 70, 100, and 140 minutes,
20-ml aliquots of the incubations were quenched with 20 ml of ice-cold acidic
acetonitrile containing 0.5% formic acid and a-PPP (internal standard) 300 mg/l.
TABLE 1
Specific inhibitors for P450 CYP enzymes and the concentrations (mM) used in
the assays.
Inhibitor
Enzyme(s) Inhibited
mM
Furafylline
Benzylnirvanol
Quinidine
5
1
CYP1A2
CYP2C19
CYP2D6
CYP3A4
CYP2B6
a
5 and 20
2.5
20
Ketoconazole
b
PPP
a
(S)-(+)-N-3-Benzylnirvanol.
2-Phenyl-2-(1-piperidinyl)propane.
b

In Vitro Metabolism and Kinetics Studies on Methylone
1249
Chemical Synthesis of DHMC
methylone. HLM was preincubated with eight different concentrations of
methylone (0, 3, 7.5, 15, 30, 50, 75, and 100 mM) 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 ml of these
eight preincubations were transferred to a new incubation containing 80 mM
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 ml.
Aliquots of 20 ml of each dextromethorphan incubation were quenched with 20
ml ice-cold acidic acetonitrile containing 0.5% formic acid and 300 mg/l a-PPP
(internal standard) at 10 and 20 minutes. The quenched incubations were
diluted with 40 ml of purified water and centrifuged (rpm 3500) for 10 minutes
at 4°C, and 5 ml of each supernatant was analyzed using ultra-high perfor-
mance 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:
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 ml at 37°C.
The methylone concentrations were 2, 5, 13, 32, 80, 200, 500, and 1250 mM.
At 0, 3, 6, 9, 12, and 15 minutes, 20 ml-aliquots of each incubation were
quenched with 30 ml of ice-cold 8% perchloric acid in purified water containing
4% acetonitrile and 300 mg/l a-PPP (internal standard). The quenched solutions
were centrifuged for 10 minutes at 4°C, and 7.5 ml 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 mM) 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:
k
inactꢀ ꢀ ꢀ ½Iꢁ
KIꢀ 1 ꢀ ½Iꢁ
kobs
¼
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).
V
maxꢀ ꢀ ꢀ ½Sꢁ
Drug Analysis
V ¼
Kmꢀ 1 ꢀ ½Sꢁ
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 mm, 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
(a-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 mM and 0.02 (limit of quantification)–1.0
mM. Limit-of-detection values for DHMC and dextrorphan were determined to
be 0.003 mM 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.
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:
V
max;1ꢀ ꢀ ½Sꢁ
V
max;2ꢀ ꢀ ½Sꢁ
V ¼
ꢀ þ ꢀ
K
K
m;1ꢀ þ ꢀ ½Sꢁ
m;2ꢀ þ ꢀ ½Sꢁ
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:
Vmax
CLintꢀ ¼ ꢀ
Km
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 mM) and 2-phenyl-2-(1-piperidinyl)propane (PPP) (20 mM) with
the same incubation setup as described for the pooled HLM.
UHPLC-TOF/MS. This instrumentation was primarily used in the quali-
tative search for metabolites, and all retention times (RTs) presented in this
paper were determined with this system. UHPLC-TOF/MS analysis was per-
formed 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.
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

1250
Pedersen et al.
UHPLC separation and MS configuration were performed in accordance with A relaxation time of 1 second was adequate to fully relax all protons before the
our previously published protocols (Dalsgaard et al., 2012; Pedersen et al., next impulse, which was especially important for the quantification experiment.
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
An appropriate number of scans was set (64 or fewer in all experiments).
1H-NMR quantification of the chemically synthesized DHMC was per-
formed 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 ml of this
solution. 1H-NMR of the dissolved residue was recorded and DHMC quantified
mode, some samples were additionally injected in the negative mode to ensure by comparing the intensities of the eight protons from toluene with the 12
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 semiquanti-
tative 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)].
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 mM) when
incubated with the same enzymes and liver fractions, and Fig. 3
presents DHMC formation from 50 mM methylone when incubated
with HLM and inhibited with various specific inhibitors. With the
same experimental setup, the degradation of 1 mM 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 experi-
ments; 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
TABLE 2
The formation of methylone metabolites by incubation for 140 minutes with various recombinant enzymes and liver
fractions at a methylone 50 mM concentration
To calculate a semiquantitative measure of the amount of formed metabolite, the peak area for all detected metabolites was divided by
the internal standard area to give a relative peak area (RPA), without accounting for the differences in ionization efficiency of the different
metabolites.
Enzyme or Liver Fractions
Dihydro-Methylone
N-Hydroxy-Methylone
Nor-Methylone
DHMC
CYP1A2
CYP 2A6
CYP 2B6
CYP 2C8
CYP 2C9
CYP 2C18
CYP 2C19
CYP 2D6
CYP 2E1
CYP 3A4
CYP 3A5
Supermix
FMO 1
FMO 3
FMO 5
AO
MAO A
MAO B
ICCa
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
+
+
ND
+
ND
ND
ND
+
+
+
+
ND
+
+
ND
+
ND
ND
ND
+
++
+
+
+
+
+
+
ND
ND
ND
ND
ND
+
+
ND
+
+
ND
+
ND
ND
ND
+
+++
ND
ND
ND
++
ND
ND
ND
ND
ND
ND
ND
+++
+++
ND
+
+
+
ND
ND
ND
ND
ND
++
+
HLM
HLM (inactivated)b
+
ND
+
HLMc
ND
+
S9
+, Minor amount of metabolite detected, defined as RPA between LOD (limit of detection) and less than 0.02; ++, RPA between 0.02
and 0.3; +++, large amount of a metabolite detected (RPA . 0.3); ND (not detected) was defined as less than LOD.
a
Insect cell control.
FMOs inactivated.
No NADPH.
b
c

In Vitro Metabolism and Kinetics Studies on Methylone
1251
Fig. 2. The amount of methylone remaining after 140 minutes
when 1 mM is incubated with various enzymes and liver
fractions. HLM (inac) is HLM in which the FMO enzymes have
been heat-inactivated. Supermix is a balanced mixture of the
P450 CYP enzymes 1A2, 2C8, 2C9, 2C19, 2D6, and 2A4; the
activity is similar to that observed in pooled HLM. ICC denotes
insect cell control. All experiments were performed in duplicate;
S.E.M. is indicated with error bars. S9, UltraPool Human Liver
fraction.
91.0551 (C4H7NO3 loss). These fragments of methylone have pre- calculated m/z 196.0974, found 196.0981 (3.6 ppm); fragments observed
viously been reported by our laboratory (Reitzel et al., 2012). 1H-NMR with QTOF/MSE (listed in order of descending abundance) m/z 160.0770
(500 MHz, CD3OD) d 7.70 (dd, J = 8.3 Hz and 1.8 Hz, 1H, Ar-H); (2 ꢀ H2O loss), 178.0873 (H2O loss), 132.0823 (CH4O3 loss), 91.0548
•
7.50 (d, J = 1.8 Hz, 1H, Ar-H); 7.02 (d, J = 8.3 Hz, 1H, Ar-H); 6.13 (C3H7NO3 loss), and 117.0584 (C2H7O3 radical loss). The exact
(d, J = 1.1 Hz (geminal); 1H, -O-CHAHB-O-); 6.13 (d, J = 1.1 Hz structure of the metabolite was determined by NMR data obtained from
1
(geminal); 1H, -O-CHAHB-O-); 5.01 (q, J = 7.1 Hz 1H, (C=O)-CH- the chemically produced metabolite: H-NMR (500 MHz, CD3OD) d
NH); 2.75 (s, 3H, CH3-NH); and 1.57 ppm (d, J = 7.1 Hz, 3H, CH- 7.33 (m, 2H, Ar-H); 6.77 (d, J = 8.3 Hz, 1H, Ar-H); 4.82 (the peak is
CH3). Vicinal couplings between the aromatic protons and the coupling almost concealed in the solvent peak at 4.87, 1H, (C=O)-CH-NH); 2.58
between the protons in CH-CH3 were also confirmed using H-H COSY (s, 3H, CH3-NH); and 1.42 ppm (d, J = 6.4 Hz, 3H, CH-CH3). Vicinal
(not shown). 13C-NMR (500 MHz, CD3OD) d 195 (C=O), 155 and 150 couplings between the aromatic protons and the coupling between the
(2 aromatic C adjacent to the methylenedioxy group), 129 (aromatic C protons in CH-CH3 were further confirmed using H-H COSY (not
adjacent to C=O), 127, 110, and 109 (3 aromatic C-H), 104 (-O-CH2-O-), shown). 13C-NMR (500 MHz, CD3OD) d 195 (C=O), 154 and 147 (2
60 ((C=O)-CH-NHCH3), 32 (CH3-NH), and 17 ppm (CH3-CH-NHCH3). aromatic C-OH), 126 (aromatic C adjacent to C=O), 124, 116.3, and
All outlined C-H bindings were confirmed using HSQC.
116.2 (3 aromatic C-H), 60 ((C=O)-CH-NHCH3), 32 (CH3-NH), and 17
DHMC. To our knowledge, this metabolite has not been reported ppm (CH3-CH-NHCH3). All outlined C-H bindings were confirmed
before. The metabolite was detected in the in vitro enzyme experi- using HSQC (not shown). The chemical synthesis of DHMC pro-
ments and was also synthesized chemically in preparative amounts. duced 19 mg (95 mmol), determined by 1H-NMR, representing an
RT (1.45 minutes), accurate mass, and fragmentation pattern each efficacy of 19% in relation to the amount of methylone used for the
verified that the enzymatic and chemically produced metabolites are synthesis.
identical, and only the accurate mass and fragmentation for the
N-Hydroxy-Methylone. RT 4.56 minutes. Equation C11H13NO4;
enzymatic product are presented: formula C10H13NO3; HRMS [M + H]+ HRMS [M + H]+ calculated m/z 224.0923, found 224.0927 (1.8 ppm);
Fig. 3. The relative amount of the metabolite
DHMC, determined by UHPLC-TOF/MS, where
the area of DHMC is divided by the area of the
internal standard. The substrate is 50 mM methylone
incubated in HLM with and without specific P450
inhibitors. All experiments were performed in du-
plicate; S.E.M. is indicated with error bars for the
incubations with PPP and quinidine 5 and 20 mM.
(S)-(+)-N-3-Benzylnirvanol data are obscured under
the data from the positive control, ketoconazole, and
furafylline.

1252
Pedersen et al.
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); frag-
ments observed with QTOF/MSE (listed in order of descending abun-
dance) 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).
Fig. 5. Data from Fig. 4 presented as an Eadie–Hofstee plot. The means of the three
replicates are plotted together, with the contribution from each enzymatic component
presented as two separate linear curves determined from the nonlinear regression,
assuming biphasic kinetics.
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
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 param-
eters were determined to be Vmax,1 = 0.034 6 0.005 nmol/min/mg
protein and Km,1 = 14.7 6 4.7 mM, and Vmax,2 = 0.16 6 0.01
nmol/min/mg protein and Km,2 = 840 6 196 mM. 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.
calculated. The contribution from enzyme component E1 is Vmax,1
=
0.046 6 0.005 (S.E.) nmol/min/mg protein and Km,1 = 19.0 6 4.2
mM; for component E2, it is Vmax,2 = 0.22 6 0.04 nmol/min/mg
protein and Km,2 = 1953 6 761 mM. CLint values for the two
components E1 and E2 were calculated to be 2.4 ml/min/mg protein
and 0.11 ml/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 6 0.03 nmol/min/nmol P450 and Km,2D6 = 2.57 6
0.17 mM, and Vmax,2B6 = 0.087 6 0.007 nmol/min/nmol P450 and
Investigation of the Time-Dependent Mechanism-Based Inhibition
of CYP2D6 by Methylone
CYP2D6 activity was determined from its ability to metabolize
Km,2B6 = 25.2 6 8.9 mM. CLint was calculated to be 1.4 ml/min/nmol dextromethorphan into dextrorphan. In Fig. 7, the remaining activity is
CYP2D6 and 3.4 ml/min/nmol CYP2B6. Inhibition of CYP2D6 with plotted as a function of the preincubation time for each methylone
quinidine in pooled HLM revealed approximately monophasic concentration. The slopes (kobs) of these curves are plotted against
kinetics (Fig. 6), and the parameters were determined to be Vmax
=
the methylone concentration in Fig. 8, and the inactivation param-
0.180 6 0.014 nmol/min/mg protein and Km,1 = 1154 6 154 mM. eters were calculated as described in Materials and Methods. For
Fig. 6. Eadie–Hofstee plot of the kinetic data obtained from the incubation of
Fig. 4. The relationship between methylone concentration and the rate (V) of
DHMC formation when incubated with pooled HLM. Three replicates were
performed at each concentration of methylone. The curve is based on nonlinear
regression assuming biphasic kinetics.
methylone in pooled HLM inhibited with quinidine. Each data point represents the
mean of three replicates. Monophasic kinetics can approximately be assumed on the
basis of these plots when the uncertainty of the DHMC quantification is taken into
account near limit of quantification (LOQ).

In Vitro Metabolism and Kinetics Studies on Methylone
methylone, we found KI = 15.1 6 3.4 (S.E) mM and kinact = 0.075 6
1253
0.005 minutes21; for MDMA, KI = 5.1 6 0.9 mM and kinact = 0.146 6
0.007 minutes21. 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 mM and 0.12–0.26 minutes21
,
respectively, which is the same order of magnitude as our results for
MDMA.
Fig. 8. A plot of the inactivation constant (kobs) as a function of the methylone
concentration. The graph shows the mean 6 S.E.M. of the three replications of the
experiment.
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 con-
tribution 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 mM methylone)
that was used to form a sufficient amount of DHMC so that a reduc-
tion in the formation could be significantly detected. For inhibitory
experiments like those presented here, substrate (methylone) concen-
trations 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 6 4.2 mM, but apparently the methylone concentration of
50 mM was not a problem, as we could observe an effect when in-
cubating with quinidine. The contribution with a Km of 1953 6 761 mM
was ascribed to different low-affinity enzymes (high Km), which
means that a methylone concentration of 50 mM cannot explain why
S-benzylnirvanol and furafylline do not significantly reduce DHMC
formation. The most reasonable explanation is that 95% of the me-
tabolism of methylone is ascribed to CYP2D6, implying that con-
tributions 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;
1
this comparison reveals that both the H 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.
Fig. 7. The relationship between the preincubation time and the remaining CYP2D6
activity at various methylone concentrations with respect to the control (0 mM). The
experiment was performed three times; the graph shows representative results from
a single experiment.

1254
Pedersen et al.
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
Fig. 10. A possible structure of hydroxy-methylone.
is less polar), indicating that the N-hydroxy amine structure is the most hypothesis that the toxicity of compounds like methylone is unrelated to
likely one (Yuan et al., 2002; Edlund and Baranczewski, 2004; Wu a reduction in CYP2D6 activity, because of metabolic switching (Kreth
et al., 2004).
et al., 2000; Kraemer and Maurer, 2002).
The molecular formulas of nor-methylone and dihydro-methylone
Our experiments have shown that the inhibition of CYP2D6 with
were determined from the accurate mass data; the most likely met- methylone is time-dependent and that the rate of inactivation reaches a
abolic pathways leading to these products are demethylation (CH2 maximum at high methylone concentrations. Both of these character-
loss) and reduction (2H gain), respectively. The proposed structure in istics are fundamental criteria for mechanism-based inactivation;
Fig. 1 is in agreement with the observed fragmentation and previous however, to unequivocally confirm that methylone is a mechanism-
suggestions in the literature (Kamata et al., 2006; Meyer et al., 2010; based inhibitor of CYP2D6, additional characteristics must be verified
Zaitsu et al., 2011; Mueller and Rentsch, 2012).
as described by Silverman (1998). Our experiments did not cover all
Methylone does not follow simple monophasic kinetics in measures of these characteristics, but our initial studies provide strong indi-
of the formation of the major metabolite DHMC. This deviation from cations of mechanism-based inhibitor kinetics. We also determined
monophasic kinetics may arise from the ability of more than one the inactivation parameters for MDMA, which apparently features
enzyme to form DHMC or from some atypical kinetics (e.g., homo- both a higher affinity (lower KI; P , 0.05, t test) and a greater
tropic cooperation). In the incubation with recombinant CYP2D6, capacity (higher kinact; P , 0.001, t test) for inactivation of CYP2D6
this major metabolizing enzyme shows monophasic kinetics; therefore, than methylone. This finding agrees with the observation that
the assumption is that the nonmonophasic kinetics is caused by the the introduction of a beta-keto group into amphetamine (forming
activity of additional enzymes apart from CYP2D6 rather than by cathinone) will reduce the compound’s affinity for CYP2D6 (Wu
atypical kinetics. Further supporting this conclusion is the fact that the et al., 1997).
incubation with HLM in the presence of quinidine (Fig. 6) yields
In conclusion, we have found that 95% of methylone is metabolized
approximately monophasic kinetics when compared with Fig. 5 with no by CYP2D6, with some contribution from primarily CYP1A2, CYP2B6,
inhibitor. The relatively small Vmax and Km determined for CYP2D6 in and CYP2C19. The main metabolite is DHMC, which has not been
HLM (component E1) means that the enzyme has a low capacity and identified before, and its formation shows biphasic Michaelis–Menten
a high affinity for methylone, as previously described for MDMA kinetics. Additionally, our experiments indicated that methylone is a
(Kreth et al., 2000). Furthermore, at low methylone concentrations, 95% mechanism-based inhibitor of CYP2D6, which makes the identification
of the metabolism can be ascribed to CYP2D6, as calculated from the of the contribution from other enzymes important; because metabolic
clearance. The contribution (5%) from the other component E2 is switching to these enzymes might prevent accumulation of methylone
assumed to arise from a mixture of contributions from enzymes that all when CYP2D6 activity is reduced.
have high capacity and low affinity because it cannot be assigned to
Acknowledgments
CYP2B6 alone. The assumption that more than one enzyme is con-
tributing 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 me-
tabolism of methylone. Whether this observation has any relevance in
vivo requires further investigation; however, it emphasizes the recent
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.
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Address correspondence to: Dr. Anders Just Pedersen, Section of Forensic
Chemistry, Frederik Femte Vej 11, DK-2100 Copenhagen, Denmark. E-mail:
anders.just@sund.ku.dk