In vitro metabolism and pharmacokinetic studies on...

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Title:

In vitro metabolism and pharmacokinetic studies on methylone

Anders Just Pedersen, MSc Pharm

Trine Hedebrink Petersen, MSc Pharm

Kristian Linnet, MD, PhD

Primary laboratory of origin: Section of Forensic Chemistry, Dept. of Forensic Medicine,

Faculty of Health Sciences, University of Copenhagen (A.J.P and K.L)

Department of Pharmaceutics and Analytical Chemistry, Faculty of Pharmaceutical Sciences,

University of Copenhagen (T.H.P)

DMD Fast Forward. Published on April 1, 2013 as doi:10.1124/dmd.112.050880

Copyright 2013 by the American Society for Pharmacology and Experimental Therapeutics.

This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on April 1, 2013 as DOI: 10.1124/dmd.112.050880

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Running title:

In vitro metabolism and kinetics studies on methylone

Corresponding author information: Anders Just Pedersen, Section of Forensic Chemistry,

Frederik Femte Vej 11, DK-2100 Copenhagen, Denmark.

E-mail: [email protected]

Phone: +45 35 32 61 09

Fax: +45 35 32 60 85

Number of text pages: 20

Number of tables: 2

Number of figures: 10

Number of references: 35

Number of words (Abstract): 190

Number of words (Introduction): 382

Number of words (Discussion): 1438

List of 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; MDMA, 3,4-

methylenedioyxmethamphetamine; LC-MS, liquid chromatography–mass spectrometry;


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LOD, limit of detection; LOQ, limit of quantification; MAO, monoamine oxidase; MRM,

multiple reaction monitoring; PPP, 2-phenyl-2-(1-piperidinyl)propane; α-PPP, α-

pyrrolidinopropiophenone; RAF, relative activity factor; RT, retention time; UHPLC-

TOF/MS, ultra-high performance liquid chromatography/time-of-flight mass spectrometry;

UHPLC-QTOF/MSE ultra-high performance liquid chromatography/quadrupole time-of-

flight mass spectrometry with fragmentation.


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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 min-1.


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Introduction

In the past decade, the group of cathinone (beta-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


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towards 4-HMMC and the enzymes responsible for the formation of DHMC have not yet

been identified.

Many amphetamine analogues are inhibitors of and substrates for CYP2D6 (Kreth et

al., 2000; Lin et al., 1997; Maurer et al., 2000; Wu et al., 1997). In addition, MDMA is a

mechanism-based inhibitor of CYP2D6 (Heydari et al., 2004; Wu et al., 1997), 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.


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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 (LC-MS) grade) and toluene (LC-MS 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

(Scotland, UK). Baculovirus-infected insect cell microsomes containing the cDNA-expressed

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, USA). From the same vendor, we also purchased

UltraPoolTM HLM 150, UltraPoolTM 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 U/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.


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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 CYP enzymes,

ICC, and the FMOs consisted of 1.3 mM NADP+, 3.3 mM glucose-6-phosphate, 3.3 mM

MgCl2, 0.4 U/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 min, 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 min at 4°C, and 7.5 µL

of the supernatant was analyzed directly by UHPLC-TOF/MS (ultra-high performance liquid

chromatography/time-of-flight mass spectrometry); a supplementary investigation of

fragmentation patterns was also carried out using UHPLC-QTOF/MSE (quadrupole time-of-

flight mass spectrometry with fragmentation).

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 min and then cooling with dry ice. This procedure is


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described to distinguish between CYP and FMO activity, as the FMO enzymes are highly

thermolabile (Cashman, 2005; Grothusen et al., 1996). The experimental conditions and

concentrations of co-factors were identical to those used with the recombinant enzymes. The

HLM incubations (1 mg protein/mL) were additionally performed with and without various

selective CYP enzyme inhibitors (Table 1 for inhibitors and concentrations) (Khojasteh et al.,

2011; Suzuki et al., 2002; Walsky and Obach, 2003; Zhang et al., 2007). HLM, co-factors,

and inhibitors were preincubated for 10 min 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 h at –78°C, the reaction was

quenched with 5 mL of MeOH added drop-wise, and the mixture was stirred for 2 h 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 U/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


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methylone concentrations were 2, 5, 13, 32, 80, 200, 500, and 1250 µM. At 0, 3, 6, 9, 12, and

15 min, 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 min at 4°C, and 7.5 µL of each

supernatant was analyzed directly using UHPLC-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 (data not shown), 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

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:


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If CLint is determined for both a recombinant enzyme and for the same enzyme in

HLM, the relative activity factor (RAF) 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 U/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 min, 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 U/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 min. The quenched incubations were diluted with 40 µL of

purified water and centrifuged (rpm 3500) for 10 min at 4°C, and 5 µL of each supernatant

was analyzed using UHPLC-MS/MS to quantify the amount of dextrorphan metabolized

from dextromethorphan. The amount of dextrorphan was plotted against the 10-min and 20-


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min 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 (Heydari et al.,

2004; Silverman, 1998).

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 to 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 (Johansen and Hansen, 2012; Simonsen et al., 2010). 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 min

from 99.5% to 90% A; 4-5 min to 50% A; 5-5.3 min to 0% A; 5.3-5.5 min to 99.5% A; and

5.5-9 min isocratic at 99.5% A. Positive electrospray ionization operating in multiple reaction

monitoring (MRM) mode was used for detection. The determination was done with two

MRM transitions for the following compounds: methylone 208 > 160 (quantifier) and 208 >

132; DHMC 196 > 160 (quantifier) and 196 > 132; and dextrorphan 258 > 157 (quantifier)


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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 (LOQ)-1.5

µM and 0.02 (LOQ)-1.0 µM. LOD 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 (Manchester, UK). 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 min, to 50% of A from 4.0 to 6.0 min, to

5% of A from 6.0 to 7.0 min, and then increased back to 100% of A from 7.0 to 7.5 min. The

column was then reconditioned with 100% of A (7.5-9.5 min). 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 semi-quantitative LOD for new metabolites with no reference standard was

defined as three times the background noise, determined as absolute peak height.


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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 V to 40 V to create fragments. The rest of the detector

settings were as previously published (Reitzel et al., 2011). 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). The NMR data were recorded

using a Bruker 500.13 MHz instrument (Bruker, Rheinstetten, Germany). Methanol-d4 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 s 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.


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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 min. Figure 2 illustrates the remaining amount of methylone (1 µM) when

incubated with the same enzymes and liver fractions, and Figure 3 presents DHMC formation

from 50 µM of methylone when incubated with HLM and inhibited with various specific

inhibitors. With the same experimental setup, the degradation of 1 µM of methylone was

measured, and only quinidine was found to significantly reduce the degradation of methylone

(data not shown).

Four metabolites of methylone were detected in the in vitro experiments; Figure 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 min. Formula C11H13NO3; HRMS [M + H]+ calculated m/z

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., 2011). 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


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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 min), 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 min. Formula C11H13NO4; HRMS [M + H]+ calculated

m/z 224.0923, found 224.0927 (1.8 ppm); fragments observed with QTOF/MSE (listed in


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order of descending abundance) m/z 149.0243 (C3H9O loss), 121.0301 (C4H9O2 loss), and

176.0712 (CH4O2 loss).

Nor-methylone. RT 2.86 min. Formula 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 min. Formula 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 min for all experiments. The rate within this interval

was determined for all methylone concentrations, and the relationship is plotted in Figure 4

for the pooled HLM experiment. The same data are presented as the Eadie–Hofstee plot in

Figure 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

(data not presented). The respective monophasic kinetics parameters for each enzyme were


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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 RAF 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 Figure 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 Figure 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 min-1; for MDMA, KI = 5.1 ± 0.9 µM and

kinact = 0.146 ± 0.007 min-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


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use of kobs instead of V). Linearity was observed for both compounds, confirming the

assumed kinetics (data not shown). 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 min-1,

respectively, which is the same order of magnitude as our results for MDMA.


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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 (Kraemer and Maurer, 2002; Kreth et al., 2000).

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 to 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 (data not shown). However, when CYP2B6 is inhibited, the

incubation exhibits a significant reduction in the DHMC formation (Fig. 3); this finding is in


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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.


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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 demethylene 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 Figure 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 (Edlund and Baranczewski, 2004; Wu et

al., 2004; Yuan et al., 2002).

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

Figure 1 is in agreement with the observed fragmentation and previous suggestions in the

literature (Kamata et al., 2006; Meyer et al., 2010; Mueller and Rentsch, 2012; Zaitsu et al.,

2011).


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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 non-

monophasic 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 to Figure 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 (Kraemer and Maurer, 2002; Kreth et al., 2000).

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


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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.


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Acknowledgments

Thanks to Christian Tortzen (Department of Chemistry, University of Copenhagen) for his

help with the chemical synthesis of DHMC.


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Authorship Contributions

Participated in research design and conducted experiments: Petersen and Pedersen.

Performed data analysis: Petersen and Pedersen.

Wrote or contributed to the writing of the manuscript: Linnet and Pedersen.


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Figure Legends

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 Meyer et al. (2010) and Kamata et al. (2006)

(4-HMC has only been detected in rat urine). Methylone and all of the metabolites detected in

urine were primarily observed as conjugated metabolites.

Fig. 2. The amount of methylone remaining after 140 min when 1 µM 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 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; SEM is indicated with error

bars.

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

µM methylone incubated in HLM with and without specific CYP inhibitors. All experiments

were performed in duplicate; SEM is indicated with error bars for the incubations with PPP

and quinidine 5 µM and 20 µM. (S)-(+)-N-3-Benzylnirvanol data are obscured under the data

from the positive control, ketoconazole, and furafylline.

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


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concentration of methylone. The curve is based on nonlinear regression assuming biphasic

kinetics.

Fig. 5. Data from Figure 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.

Fig. 6. Eadie–Hofstee plot of the kinetic data obtained from the incubation of 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 LOQ.

Fig. 7. The relationship between the preincubation time and the remaining CYP2D6 activity

at various methylone concentrations with respect to the control (0 µM). The experiment was

performed three times; the graph shows representative results from a single experiment.

Fig. 8. A plot of the inactivation constant (kobs) as a function of the methylone concentration.

The graph shows the mean ± SEM of the three replications of the experiment.

Fig. 9. Suggested structure of the most abundant fragment ion (149.0243) observed from the

QTOF/MSE fragmentation of N-hydroxy-methylone.

Fig. 10. A possible structure of hydroxy-methylone.


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Table 1

Specific inhibitors for cytochrome CYP enzymes and the concentrations (µM) used in the

assays.

Inhibitor µM Enzyme(s) Inhibited

Furafylline 5 CYP1A2

Benzylnirvanol a 1 CYP2C19

Quinidine 5 and 20 CYP2D6

Ketoconazole 2.5 CYP3A4

PPP b 20 CYP2B6

a (S)-(+)-N-3-Benzylnirvanol

b 2-phenyl-2-(1-piperidinyl)propane


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Table 2

The formation of methylone metabolites by incubation for 140 min with various recombinant

enzymes and liver fractions at a methylone concentration of 50 µM.

To calculate a semi-quantitative 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. [+] = Minor amount of metabolite detected, defined as RPA between LOD and

less than 0.02; [++] = RPA between 0.02 and 0.3; [+++] = Large amount of a metabolite

detected (RPA > 0.3); and ND (Not detected) was defined as less than LOD.

Enzyme or liver

fractions

Dihydro-

methylone

N-hydroxy-

methylone

Nor-

methylone

DHMC

CYP1A2 ND + + +

CYP 2A6 ND ND ND ND

CYP 2B6 ND + + +

CYP 2C8 ND ND ND ND

CYP 2C9 ND ND ND ND

CYP 2C18 ND ND ND ND

CYP 2C19 ND + + +

CYP 2D6 ND + ++ +++

CYP 2E1 ND + + ND

CYP 3A4 ND + + ND

CYP 3A5 ND ND + ND

Supermix ND + + ++

FMO 1 ND + + ND


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FMO 3 ND + + ND

FMO 5 ND ND ND ND

AO ND ND ND ND

MAO A ND ND ND ND

MAO B ND ND ND ND

ICC a ND ND ND ND

HLM + ++ + +++

HLM b + + + +++

HLM c ND ND ND ND

S9 + + + +

a Insect cell control

b FMOs inactivated

c no NADPH


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