Phase I metabolites of mephedrone display biological activity as substrates at monoamine transporters

Article abstract of DOI:10.1111/bph.13547

Background and Purpose: 4-Methyl-N-methylcathinone (mephedrone) is a synthetic stimulant that acts as a substrate-type releaser at transporters for dopamine (DAT), noradrenaline (NET) and 5-HT (SERT). Upon systemic administration, mephedrone is metabolized to several phase I compounds: the N-demethylated metabolite, 4-methylcathinone (nor-mephedrone); the ring-hydroxylated metabolite, 4-hydroxytolylmephedrone (4-OH-mephedrone); and the reduced keto-metabolite, dihydromephedrone. Experimental Approach: We used in vitro assays to compare the effects of mephedrone and synthetically prepared metabolites on transporter-mediated uptake and release in HEK293 cells expressing human monoamine transporters and in rat brain synaptosomes. In vivo microdialysis was employed to examine the effects of i.v. metabolite injection (1 and 3?mg·kg^?1) on extracellular dopamine and 5-HT levels in rat nucleus accumbens. Key Results: In cells expressing transporters, mephedrone and its metabolites inhibited uptake, although dihydromephedrone was weak overall. In cells and synaptosomes, nor-mephedrone and 4-OH-mephedrone served as transportable substrates, inducing release via monoamine transporters. When administered to rats, mephedrone and nor-mephedrone produced elevations in extracellular dopamine and 5-HT, whereas 4-OH-mephedrone did not. Mephedrone and nor-mephedrone, but not 4-OH-mephedrone, induced locomotor activity. Conclusions and Implications: Our results demonstrate that phase I metabolites of mephedrone are transporter substrates (i.e. releasers) at DAT, NET and SERT, but dihydromephedrone is weak in this regard. When administered in vivo, nor-mephedrone increases extracellular dopamine and 5-HT in the brain whereas 4-OH-mephedrone does not, suggesting the latter metabolite does not penetrate the blood–brain barrier. Future studies should examine the pharmacokinetics of nor-mephedrone to determine its possible contribution to the in vivo effects produced by mephedrone.

Full text of DOI:10.1111/bph.13547

Phase I metabolites of mephedrone display biological activity as
substrates at monoamine transporters
Running short title: Bioactive metabolites of mephedrone
F P Mayer1, L Wimmer2, O Dillon-Carter3, J S Partilla3, N Burchardt1, M D Mihovilovic2, M H
Baumann3*, H H Sitte1, 4*
1 Medical University of Vienna, Center for Physiology and Pharmacology, Institute of Pharmacology,
Vienna, Austria
2 Institute of Applied Synthetic Chemistry, Vienna University of Technology, Vienna, Austria
3 Designer Drug Research Unit (DDRU), Intramural Research Program (IRP), NIDA, NIH,
Baltimore, MD, USA
4 Center for Addiction Research and Science - AddRess, Medical University Vienna,
Waehringerstrasse 13A, 1090 Vienna, Austria
*denotes equal contribution
Correspondence
Harald S Sitte, MD, Institute of Pharmacology, Center for Physiology and Pharmacology,
Medical University of Vienna, Währingerstrasse 13A, 1090 Vienna, Austria
E-mail: harald.sitte@meduniwien.ac.at
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/bph.13547
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BACKGROUND AND PURPOSE
4-Methyl-N-methylcathinone (mephedrone, MEPH) is a synthetic stimulant that acts as a
substrate-type releaser at transporters for dopamine (DAT), norepinephrine (NET) and 5-HT
(SERT). Upon systemic administration, MEPH is metabolized to several phase I compounds:
the N-demethylated metabolite, 4-methylcathinone (NOR-MEPH); the ring-hydroxylated
metabolite, 4-hydroxytolyl-mephedrone (4-OH-MEPH); and the reduced keto-metabolite,
dihydromephedrone (DIHYDRO-MEPH).
EXPERIMENTAL APPROACH
We used in vitro assays to compare the effects of MEPH and synthetically prepared
metabolites on transporter-mediated uptake and release in HEK293 cells expressing human
monoamine transporters and in rat brain synaptosomes. In vivo microdialysis was employed
to examine the effects of intravenous metabolite injection (1 and 3 mg kg-1) on extracellular
dopamine and 5-HT levels in rat nucleus accumbens.
KEY RESULTS
In cells expressing transporters, MEPH and its metabolites inhibited uptake, although
DIHYDRO-MEPH was weak overall. In cells and synaptosomes, NOR-MEPH and 4-OH-
MEPH served as transportable substrates, inducing release via monoamine transporters.
When administered to rats, MEPH and NOR-MEPH produced elevations in extracellular
dopamine and 5-HT, whereas 4-OH-MEPH did not. MEPH and NOR-MEPH, but not 4-OH-
MEPH, induced locomotor activity.
CONCLUSIONS AND IMPLICATIONS
Our results demonstrate that phase I metabolites of MEPH are transporter substrates (i.e.,
releasers) at DAT, NET and SERT, but DIHYDRO-MEPH is weak in this regard. When
administered in vivo, NOR-MEPH increases extracellular dopamine and 5-HT in the brain
whereas 4-OH-MEPH does not, suggesting the latter metabolite does not penetrate the blood-
brain-barrier. Future studies should examine the pharmacokinetics of NOR-MEPH to
determine its possible contribution to the in vivo effects produced by MEPH.
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TARGETS
LIGANDS
Transportersa
DAT, SLC6A3
NET , SLC6A2
SERT , SLC6A4
Enzymesb
Amphetamine
Citalopram
Cocaine
MDMA
MPP+
CYP2D6
Nomifensine
Norepinephrine
Desipramine
Dopamine
VMAT2,
SLC18A2
Serotonin,5-
hydroxytryptamine,
5-HT
GBR12935
These Tables of Links list key protein targets and ligands in this article that are hyperlinked to
corresponding entries in http://www.guidetopharmacology.org, the common portal for data from
the IUPHAR/BPS Guide to PHARMACOLOGY ((Southan et al., 2016)), and are permanently archived in
The Concise Guide to PHARMACOLOGY 2015/16 (a,b(Alexander et al., 2015a; Alexander et al.,
2015b)).
Abbreviations
4-OH-MEPH, 4-hydroxytolylmephedrone; 5-HT, 5-hydroxytryptamine or serotonin; DAT,
dopamine transporter; DIHYDRO-MEPH, dihydromephedrone; GBR12935, 1-(2-
diphenylmethoxyethyl)-4-(3-phenylpropyl)piperazine dihydrochloride; MEPH, 4-methyl-N-
methyl-cathinone or mephedrone; MPP+, 1-methyl-4-phenylpyridinium; NET,
norepinephrine transporter; NOR-MEPH, 4-methylcathinone; PDL, poly-D-lysine; SERT, 5-
HT transporter
Introduction
During the past decade, a variety of man-made “designer drugs” or “new psychoactive
substances” (NPS) have appeared in the recreational drug market as legal alternatives to more
traditional drugs of abuse (Baumann, Solis, Watterson, Marusich, Fantegrossi & Wiley, 2014;
Sitte & Freissmuth, 2015). Frequently, the chemical structures of NPS are based on known
illicit substances and mimic their psychoactive effects, but subtle structural modifications to
the drug molecules render them legal (Baumann & Volkow, 2016). In particular, a number of
NPS have been marketed as replacements for illicit stimulants like cocaine and 3,4-
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methylenedioxymethamphetamine (MDMA, “ecstasy”) (Green, King, Shortall & Fone,
2014). One of the most popular synthetic stimulants is the cathinone analog, 4-methyl-N-
methylcathinone (mephedrone, MEPH). MEPH first appeared in Israel as a “party drug”
during the early 2000s, and its recreational use spread to Europe, Australia and other parts of
the world (Kelly, 2011). In the United States, MEPH was a constituent of so-called “bath
salts” products which became popular during 2010-2011 (Spiller, Ryan, Weston & Jansen,
2011). Low doses of MEPH produce typical stimulant effects in humans, like increased
energy and mood elevation (Vardakou, Pistos & Spiliopoulou, 2011; Winstock, Mitcheson,
Ramsey, Davies, Puchnarewicz & Marsden, 2011), while high doses or chronic use can
produce life-threatening side-effects including tachycardia, hypertension, agitation and
seizures (James et al., 2011; Wood, Greene & Dargan, 2011). Deaths from MEPH are rare
but have been reported (Loi et al., 2015). In the interest of public health and safety,
legislation was passed in many countries to ban the sale, possession and use of MEPH (Drug
Enforcement Administration, 2011; Green, King, Shortall & Fone, 2014). Despite such bans,
MEPH continues to be abused in European countries (Archer, Dargan, Lee, Hudson & Wood,
2014; Hondebrink, Nugteren-van Lonkhuyzen, Van Der Gouwe & Brunt, 2015)
(http://www.emcdda.europa.eu/publications/edr/trends-developments/2014).
Similar to other stimulant drugs, MEPH exerts its effects by interacting with plasma
membrane monoamine transporter proteins of the solute carrier 6 family (SLC6) (Baumann et
al., 2012; Hadlock et al., 2011; Martinez-Clemente, Escubedo, Pubill & Camarasa, 2012),
namely the dopamine transporter (DAT, SLC6A3), norepinephrine transporter (NET,
SLC6A2) and serotonin (5-HT) transporter (SERT, SLC6A4). The normal role of
monoamine transporters is to capture previously released neurotransmitter molecules from
the extracellular space and move them back into the neuronal cytoplasm (i.e., uptake), thus
terminating monoamine signaling (Kristensen et al., 2011; Reith et al., 2015). Drugs that
interact with DAT, NET and SERT can be classified as either cocaine-like “blockers” or
amphetamine-like “substrates” (Rothman & Baumann, 2003; Sitte & Freissmuth, 2015). Both
types of compounds disrupt transporter function and produce elevations in extracellular
monoamine concentrations, but their precise modes of action are different. On a molecular
level, cocaine-like blockers act as non-transported inhibitors of monoamine transporters.
Consequently, blockers prevent the transporter-mediated uptake of released neurotransmitters
from the extracellular medium. In addition, cocaine is known to mobilize the intracellular
reserve pool of DA and stimulate its exocytotic release (Venton et al., 2006)(Venton et al.,
2006). In contrast, amphetamine-like compounds are transported substrates that not only act
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as competitive uptake inhibitors but also trigger neurotransmitter efflux by a complex process
involving reversal of transporter flux (Chen & Reith, 2004; Reith et al., 2015; Sitte &
Freissmuth, 2015). Consequently, drugs that act as transporter substrates are often referred to
as “releasers” as they induce a transporter-mediated efflux of neurotransmitters.
Studies using in vitro transporter assays in cells and rat brain synaptosomes have
shown that MEPH acts as a non-selective substrate at DAT, NET and SERT, thereby leading
to efflux of dopamine, norepinephrine and 5-HT (Baumann et al., 2012; Eshleman, Wolfrum,
Hatfield, Johnson, Murphy & Janowsky, 2013; Simmler et al., 2013). Systemic
administration of MEPH to rats increases the extracellular concentrations of dopamine and 5-
HT in the brain, with the effects on 5-HT being somewhat greater in magnitude (Baumann et
al., 2012; Kehr et al., 2011; Wright et al., 2012). Overall, the available preclinical data
indicate that MEPH displays neurochemical effects that mimic MDMA, but MEPH has a
number of physiological and toxicological properties that render it unique (Baumann et al.,
2012; Miller et al., 2013; Shortall, Green, Swift, Fone & King, 2013). For example, high-dose
administration of MEPH is less apt to produce robust hyperthermia and long-term depletions
of brain tissue 5-HT (Baumann et al., 2012; den Hollander, Rozov, Linden, Uusi-Oukari,
Ojanpera & Korpi, 2013; Motbey et al., 2012), effects that are well established for MDMA.
Importantly, MEPH has greatly reduced potency at the vesicular monoamine transporter 2
(VMAT2, SLC18A2) when compared to MDMA and other ring-substituted amphetamines
(Eshleman, Wolfrum, Hatfield, Johnson, Murphy & Janowsky, 2013; Pifl, Reither &
Hornykiewicz, 2015), suggesting MEPH is less likely to disrupt intracellular stores of
monoamine transmitters.
One possible explanation for the distinct effects of MEPH is that metabolites of the
drug contribute to its in vivo profile of actions. Meyer et al. (2010) first reported that MEPH
is metabolized by three main hepatic mechanisms (see Figure 1): 1) N-demethylation to form
4-methylcathinone or nor-mephedrone (NOR-MEPH); 2) hydroxylation of the 4-methyl ring-
substitution to form 4-hydroxytolylmephedrone (4-OH-MEPH); and 3) reduction of the β-
keto-oxygen group, which forms dihydromephedrone (DIHYDRO-MEPH) (Meyer, Wilhelm,
Peters & Maurer, 2010). Pedersen and coworkers (2012) identified cytochrome P450 2D6
(CYP2D6) as the main enzyme responsible for the phase 1 metabolism of MEPH in humans,
and detected NOR-MEPH, 4-OH-MEPH and DIHYDRO-MEPH in human urine specimens
(Pedersen, Reitzel, Johansen & Linnet, 2013). As pointed out by Green et al. (Green, King,
Shortall & Fone, 2014), no studies have examined the pharmacology of MEPH metabolites.
Therefore in the present investigation, we used in vitro assays to compare the effects of
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MEPH and its metabolites on transporter-mediated uptake and release in cells expressing
human DAT, NET and SERT and in rat brain synaptosomes. Additionally, the in vivo
neurochemical effects of systemically administered MEPH, NOR-MEPH or 4-OH-MEPH
were examined using microdialysis in rat nucleus accumbens. Our data show that phase I
metabolites of MEPH are substrates at monoamine transporters when assessed in vitro, but
only NOR-MEPH displays substantial neurochemical actions in vivo which could contribute
to the behavioral effects of systemically administered MEPH.
Methods
Animals and housing
Male Sprague-Dawley rats from Harlan Laboratories (Frederick, MD, USA) weighing 250-300
g at arrival were housed three per cage for two weeks prior to being used in experiments. The
rats were housed under standard conditions (lights on from 0700-1900 h) with food and water
available ad libitum. Rats were maintained in facilities fully accredited by the Association for
Assessment and Accreditation of Laboratory Animal Care (AAALAC), and experiments were
performed in accordance with the Institutional Care and Use Committee of the NIDA IRP. Rats
used for brain tissue harvest to prepare synaptosomes were pair-housed, whereas those used in
microdialysis experiments were single-housed post-operatively (see below).
Rat studies described herein were carried out in accordance with ARRIVE guidelines for
reporting experiments involving animals. A total of 16 rats were used for in vitro synaptosome
assays, and an additional 28 rats were used for in vivo microdialysis experiments.
Cell culture
The generation of human embryonic kidney 293 cells (HEK293 cells) stably expressing the
human isoforms of DAT (hDAT) and NET (hNET) was carried out as described previously
(Scholze, Norregaard, Singer, Freissmuth, Gether & Sitte, 2002). For SERT, the human
isoform (hSERT) was cloned in frame with yellow fluorescent protein (YFP) (Schmid,
Scholze, Kudlacek, Freissmuth, Singer & Sitte, 2001). The generation of a stable cell line
was performed as described in Hilber and colleagues (Hilber et al., 2005). HEK293 cells
were maintained in humidified atmosphere (5% CO2, 37°C) in Dulbecco’s Modified Eagle
Medium (DMEM), supplemented with 10 % heat-inactivated fetal calf serum and penicillin
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(100 IU 100 mL-1) and streptomycin (100 µg 100 mL-1). Selection pressure was maintained
by adding geneticin (50 µg mL-1) to the cell culture media.
Transporter uptake assays in HEK293 cells
Uptake experiments were conducted as described previously (Sitte, Hiptmair, Zwach, Pifl,
Singer & Scholze, 2001) with minor modifications. In brief, HEK293 cells expressing
hDAT, hNET or hSERT were seeded into poly-D-lysine (PDL) coated 96-well plates at a
density of 40000 cells per well. The next day, DMEM was aspirated and replaced with Krebs
HEPES buffer (KHB, 25 mM HEPES, 120 mM NaCl, 5 mM KCl, 1.2 mM CaCl2, and 1.2
mM MgSO4, 5 mM D-glucose, pH adjusted to 7.3 with NaOH) (200 µL per well), and cells
were pre-incubated with various concentrations of MEPH or its metabolites for 5 min (50 µL
per well). Subsequently, 0.1 µM of [3H]-5HT or 0.02 µM of [3H]-MPP+ were added and
uptake was terminated after 1 (hSERT) or 3 min (hDAT, hNET) by washing the cells with
200 µL of ice-cold KHB. Cells were lysed with 1% sodium dodecyl sulphate (SDS) and
tritium uptake was determined by scintillation counting. Nonspecific uptake was determined
in presence of 10 µM paroxetine (hSERT) or 10 µM mazindol (hDAT and hNET).
Transporter release assays in HEK293 cells
Superfusion experiments were performed as described in Scholze and coworkers (Scholze,
Norregaard, Singer, Freissmuth, Gether & Sitte, 2002). Briefly, HEK293 cells expressing the
desired transporter were seeded at a density of 40000 cells per well onto poly-D-lysine-
coated 5mm glass cover slips in 96-well plates 24 h prior to the experiment. Cells were
preloaded with [3H]-MPP+ (0.1 µM, hDAT and hNET) or [3H]-5HT (0.4 µM, hSERT) for 20
min at 37°C in a final volume of 100 µL per well. Subsequently, glass coverslips were
transferred into small superfusion chambers (volume of 200 µL) and superfused with KHB at
25°C with a superfusion rate of 0.7 mL min-1 for 40 min to establish a stable basal efflux.
After washout, the collection of two-minute fractions was initiated. After the first three basal
fractions monensin (10 µM) or solvent were added for four fractions. Consequently, the cells
were challenged with test-drugs (10 µM) for five fractions in presence or absence of
monensin. Finally, the cells were lysed in 1% SDS to determine the total radioactivity.
Radioactivity per fraction was assessed by a liquid scintillation counter and expressed as
fractional release, i.e. the percentage of released 3H in relation to total 3H present at the
beginning of the fraction (Sitte, Scholze, Schloss, Pifl & Singer, 2000). For analysis, release
was expressed as area-under-the-curve (AUC). AUC was calculated for t=6 to 26 min and
normalized to basal efflux, i.e. t=0 to 4 min.
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Transporter release assays in rat brain synaptosomes
The ability of MEPH and its metabolites to evoke release via DAT, NET and SERT was
determined in rat brain synaptosomes as previously described (Baumann et al., 2012). Rats
were euthanized with CO2, decapitated, and brains were rapidly removed and dissected on
ice. Synaptosomes were prepared from striatum for DAT assays, whereas synaptosomes
were prepared from whole brain minus striatum and cerebellum for the NET and SERT
assays. [3H]-MPP+ (9 nM) was used as the radiolabeled substrate for DAT and NET,
whereas [3H]-5HT (5 nM) was used as the radiolabeled substrate for SERT. All buffers used
in the release assays contained 1 µM reserpine to block vesicular uptake of substrates. The
selectivity of assays was optimized for a single transporter by including unlabeled
compounds (nomifensine and 1-(2-diphenylmethoxyethyl)-4-(3-phenylpropyl)piperazine
dihydrochloride (GBR12935) for SERT; GBR12935 and citalopram for NET; citalopram and
desipramine for DAT) to prevent the uptake of [3H]-MPP+ or [3H]-5HT by competing
transporters. Synaptosomes were preloaded with radiolabeled substrate in Krebs-phosphate
buffer (KPB) which consisted of 126 mM NaCl, 2.4 mM KCl, 0.5 mM KH2PO4, 1.1 mM
CaCl2, 0.83 mM MgCl2, 0.5 mM Na2SO4, 11.1 mM glucose, 13.7 mM Na2HPO4, 1 mg mL-1
ascorbic acid, and 50 µM pargyline (pH=7.4) for 1 h (steady state). Assays were initiated by
adding 850 µL of preloaded synaptosomes to 150 µL of test drug. Dose-response curves were
generated using 8 different concentrations of MEPH, NOR-MEPH or 4-OH-MEPH. Assays
were terminated by vacuum filtration, and retained radioactivity was quantified by liquid
scintillation counting.
Microdialysis in Rat Nucleus Accumbens
In vivo microdialysis procedures were carried out as previously described with minor
modifications (Baumann et al., 2012). Briefly, male rats anesthetized with sodium
pentobarbital (60 mg kg-1, i.p.) received surgically-implanted jugular catheters and
intracerebral guide cannulae aimed at the nucleus accumbens (AP +1.6 mm, ML -1.7 mm
relative to bregma; -6.2 mm relative to dura)(Paxinos & Watson, 2007). After a 7-10 day
recovery, each rat was placed into a chamber equipped with photobeams for the detection of
motor parameters (TruScan, Harvard Apparatus, Holliston, MA, USA) and allowed to
acclimate overnight. Food and water were available ad libitum during the acclimation period.
On the following morning, catheters were attached to PE 50 extension tubes, and 0.5 x 2 mm
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microdialysis probes (CMA/12, Harvard Apparatus, Holliston, MA, USA) were inserted into
guide cannulae. Ringers' solution (150 mM NaCl , 2.8 mM KCl and 2.0 mM CaCl2) was
perfused through the probes at 0.6 µL min-1 for 3 h. To commence experiments, dialysate
samples (20 µL) were collected at 20 min intervals, and drug or saline treatments were given
after 3 baseline samples were obtained. Rats received two sequential intravenous (i.v.)
injections of MEPH or its metabolites, with 1 mg kg-1 administered at time zero, followed by
3 mg kg-1 60 min later. Saline was administered on the same schedule in a separate group of
rats. Dialysate concentrations of dopamine and 5-HT were quantified using high-pressure
liquid chromatography coupled to electrochemical detection (Baumann et al., 2012).
Chromatographic data were exported to an Empower software system (Waters, Inc., Milford,
MA, USA) for peak identification, integration and analysis.
Correct probe placements were assessed after the microdialysis experiments. Rats were
euthanized by CO2 narcosis then decapitated. Brains were quickly removed and immersion
fixed in 10% paraformaldehyde for one week. Subsequently, brains were sectioned on a
cryostat, and the location of each probe tip was verified by inspection of photographic images
of the brain taken with a digital camera using the macro lens setting.
Analysis
Calculations were performed using Microsoft Excel® 2010 (Microsoft Corporation,
Redmond, Washington, USA) and GraphPad Prism 5.0. (GraphPad Software Inc., La Jolla,
California, USA). IC50 values for uptake inhibition and EC50 values for release were
determined by nonlinear regression fits. Release data expressed as area-under-the-curve
(AUC) were analyzed by one-way analysis of variance (ANOVA) followed by Bonferroni’s
multiple comparison test. Microdialysis and locomotor data were analyzed by two-way
ANOVA (drug treatment x time) followed by Bonferroni’s test. The effect of monensin
treatment on basal efflux of tritiated substrate was analyzed with the Mann Whitney test. P
values less than 0.05 (i.e., p<0.05) were considered significant. The data and statistical
analysis comply with the recommendations on experimental design and analysis in
pharmacology(Curtis et al., 2015).
Materials
2-Methylamino-1-(p-tolyl)propan-1-one hydrochloride (MEPH, molecular weight (MW):
213.70), 2-amino-1-(p-tolyl)propan-1-one hydrochloride (NOR-MEPH, MW: 199.68) and 1-
(4-(hydroxymethyl)phenyl)-2-(methylamino)propan-1-one hydrochloride (4-OH-MEPH,
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MW: 229.70) were synthesized as racemic mixtures. In the case of 2-(methylamino)-1-(p-
tolyl)propan-1-ol hydrochloride (DIHYDRO-MEPH, MW: 215.72) all four stereoisomers
(syn-(1R,2R), syn-(1S,2S), anti-(1R,2S) and anti-(1S,2R)) were synthesized in their
enantiopure form (99%ee) and tested as 1:1:1:1 mixture. Synthetic procedures and chemical
characterization data are given in detail in the Supporting Information. Reagents used in the
experiments for uptake inhibition and release in HEK293 cells were used as mentioned in
previous work (Hofmaier et al., 2014). Plasmids encoding human SERT were a generous gift
of Dr. Randy D. Blakely. For uptake and release experiments in HEK293-cells and rat brain
synaptosomes, [3H]-1-methyl-4-phenylpyridinium ([3H]-MPP+; 80-85 µCi mmol-1) and [3H]-
5HT (28.3 µCi mmol-1) were purchased from American Radiolabeled Chemicals (St. Louis,
MO, USA) and Perkin Elmer (Boston, MA, USA), respectively. All other chemicals and cell
culture supplies were from Sigma-Aldrich (St. Louis, MO, USA) with the exception of cell-
culture dishes, which were obtained from Sarstedt (Nuembrecht, Germany).
Results
MEPH metabolites inhibit transporter-mediated uptake in HEK293 cells
We first tested the effects of MEPH and its metabolites on transporter-mediated uptake.
Figure 2 shows that MEPH, NOR-MEPH, 4-OH-MEPH and DIHYDRO-MEPH were fully
efficacious inhibitors of uptake in HEK293 cells stably expressing hDAT, hNET and hSERT.
The potency of NOR-MEPH and 4-OH-MEPH to inhibit [3H]-MPP+ uptake via hDAT and
hNET was comparable to MEPH, with IC50 values in the low micromolar range, from 0.7 to 6
µM. The IC50 values for DIHYDRO-MEPH to inhibit uptake via hDAT and hNET were
much weaker (i.e., 24 µM). Uptake inhibition experiments carried out with hSERT-
expressing cells revealed that NOR-MEPH inhibited uptake in the low micromolar range with
an IC50 value of 10.6 µM, whereas 4-OH- and DIHYDRO-MEPH were much less active with
IC50 values exceeding 60 µM. The obtained IC50 values are shown in Table 1.
MEPH metabolites induce transporter-mediated release in HEK293 cells
Data from uptake inhibition assays cannot distinguish whether test drugs act as non-
transported inhibitors or transportable substrates which evoke release (Baumann et al., 2013;
Scholze, Zwach, Kattinger, Pifl, Singer & Sitte, 2000; Sitte, Scholze, Schloss, Pifl & Singer,
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2000). Therefore, MEPH and its metabolites were tested in release assays to further explore
their interaction with transporters. The release assays were performed with the same
transporter-expressing HEK293 cell lines described above, and used a superfusion system
(Sitte, Scholze, Schloss, Pifl & Singer, 2000). As described previously, efflux of preloaded
[3H]-MPP+ or [3H]-5HT was monitored in the presence or absence of monensin (10 µM)
(Scholze, Zwach, Kattinger, Pifl, Singer & Sitte, 2000). Monensin acts as a selective H+/Na+
ionophore and dissipates the Na+ gradient across cell membranes (Mollenhauer, Morre &
Rowe, 1990): This compound increases the intracellular Na+-concentration (Chen & Reith,
2004) and thus selectively enhances efflux triggered by transporter substrates. Importantly,
only substrate-induced release will be enhanced by the application of monensin, while the
effects of non-transported inhibitors will remain unchanged (Baumann et al., 2013; Sandtner
et al., 2016; Scholze, Zwach, Kattinger, Pifl, Singer & Sitte, 2000). The superfusion assays
performed here are a decisive tool to discriminate between inhibitors and substrates (Scholze,
Zwach, Kattinger, Pifl, Singer & Sitte, 2000).
Time-course experiments with MEPH and its metabolites (10 µM) demonstrated that
all of the agents evoked significant release of pre-loaded [3H]-MPP+ via hDAT and hNET,
and release of pre-loaded [3H]-5HT via hSERT. Figure 3A depicts a representative example
of time-course effects for DAT-mediated release of [3H]-MPP+ induced by NOR-MEPH in
the presence or absence of monensin. It is clear that monensin markedly enhanced the effects
of NOR-MEPH on [3H]-MPP+ efflux. Additionally, monensin alone elicited a significant
albeit modest increase in substrate release (p<0.05, Mann Whitney test), in agreement with
our previous publications (Scholze et al., 2000). As a means to summarize the overall effect
of test drugs on release, with and without monensin (10 µM), the data in Figure 3B-D are
expressed as AUC for the 9 fractions collected after drug treatment. One-way ANOVA
demonstrated that monensin significantly influenced the release of [3H]-MPP+ evoked by
MEPH and its metabolites at DAT (F7,91=24.61, p<0.001) and NET (F7,85=14.4, p<0.001).
Post-hoc analysis revealed that enhancement by monensin was significant for MEPH, NOR-
MEPH and 4-OH-MEPH at hDAT and hNET, but not for DIHYDRO-MEPH. One-way
ANOVA demonstrated that monensin significantly augmented the release of [3H]-5HT
(F7,71=31.68, p<0.001) via hSERT, and this effect was significant for MEPH and all of its
metabolites.
MEPH metabolites induce transporter-mediated release in synaptosomes
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Next, we examined the effects of MEPH and its metabolites in rat brain synaptosomes to: i)
analyze effects of test compounds in a native tissue preparation that contains plasma
membrane transporters in situ; and ii) compare data from the human and rat transporters.
MEPH, NOR- and 4-OH-MEPH were tested in release assays in rat brain synaptosomes,
under conditions which were optimized for each transporter as described in Baumann and
colleagues (Baumann et al., 2012). The dose-effect release data are depicted in Figure 4 and
the calculated EC50 values are shown in Table 2. In comparison to the parent compound
MEPH, NOR- and 4-OH-MEPH displayed only slightly reduced potencies as releasers of
preloaded [3H]-MPP+ at DAT and NET, with EC50s ranging from 0.05 µM to 0.22 µM
(Figure 4 and Table 2). At SERT, NOR-MEPH induced release of pre-loaded [3H]-5HT in a
manner comparable to MEPH (EC50 = 0.2 µM), whereas a 10-fold rightward shift was
detected for 4-OH-MEPH (EC50 = 2 µM).
NOR-MEPH, but not 4-OH-MEPH, affects neurochemistry and behavior in vivo
The findings from human and rat transporters agreed that MEPH, NOR-MEPH and 4-OH-
MEPH were potent substrates at monoamine transporters. Thus, we sought to examine the
neurochemical effects of these three compounds in vivo. Specifically, extracellular
concentrations of dopamine and 5HT were assessed by microdialysis in the nucleus
accumbens of freely-moving rats. As depicted in Figure 5, application of two-way ANOVA
(drug treatment x time) demonstrated that drug treatments significantly influenced dialysate
concentrations of dopamine (F3,24=63.22, p<0.001) and 5-HT (F3,24=83.83, p<0.001). Post-
hoc tests revealed that MEPH increased dopamine after 1 mg kg-1, whereas MEPH and NOR-
MEPH both elevated dopamine after 3 mg kg-1. 4-OH-MEPH had no significant impact on
dopamine at either dose tested. MEPH and NOR-MEPH elevated dialysate concentrations of
5-HT in a nearly identical manner, with increases of 15-fold and 25-fold above baseline for
the 1 and 3 mg kg-1 doses, respectively. Finally, drug treatments significantly affected motor
behavior (F3,24=36.05, p<0.001) such that MEPH and NOR-MEPH increased activity whereas
4-OH-MEPH did not. MEPH was more potent than NOR-MEPH as a locomotor stimulant,
but both compounds significantly stimulated motor activity at the 3 mg kg-1 dose.
Discussion
The aim of the present study was to determine the pharmacological effects of phase I
metabolites of MEPH and decipher their precise mode of action at monoamine transporters.
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The synthetic cathinone MEPH has been shown to act as a non-selective, amphetamine-like
substrate at monoamine transporters, thereby triggering release of dopamine, norepinephrine
and 5-HT into the extracellular space (Baumann et al., 2012; Eshleman, Wolfrum, Hatfield,
Johnson, Murphy & Janowsky, 2013; Simmler et al., 2013). The neurochemical effects of
MEPH mimic those of MDMA (Baumann et al., 2012; Kehr et al., 2011; Wright et al., 2012)
but MEPH has a number of distinct pharmacological effects when compared to MDMA and
other ring-substituted amphetamines (reviewed by (Green, King, Shortall & Fone, 2014)).
Many therapeutic and abused stimulant drugs - including diethylpropion, phendimetrazine
and MDMA- are transformed by hepatic mechanisms into bioactive metabolites (Green,
Mechan, Elliott, O'Shea & Colado, 2003; Rothman et al., 2002; Yu, Rothman, Dersch,
Partilla & Rice, 2000). To examine whether metabolites of MEPH might be bioactive, we
tested the known metabolites NOR-MEPH, 4-OH-MEPH and DIHYDRO-MEPH for their
interactions with DAT, NET and SERT. It was found that all of the metabolites acted as
substrate-type releasers, but NOR-MEPH and 4-OH-MEPH were much more potent than
DIHYDRO-MEPH in this regard. Importantly, only NOR-MEPH influenced brain
neurochemistry and behavior upon systemic administration.
The present in vitro data from HEK293 cells show that MEPH metabolites inhibit
uptake in a concentration-dependent manner at all three plasma membrane monoamine
transporters. NOR-MEPH and 4-OH-MEPH inhibited uptake at hDAT and hNET with
potency comparable to MEPH, whereas DIHYDRO-MEPH was much weaker. Uptake
inhibition assays can identify compounds that interact with monoamine transporters, but
cannot discriminate whether such compounds act as inhibitors or substrates. Thus, we tested
the effects of MEPH metabolites using release assays in HEK293 cells and rat brain
synaptosomes. NOR-MEPH and 4-OH-MEPH evoked release of radiolabeled substrates
from HEK293 cells stably expressing hDAT, hNET or hSERT. The releasing action of the
drugs was augmented in the presence of monensin, an ionophore which dissipates Na+
gradients across plasma membranes. The enhancement of release by monensin provides
crucial mechanistic evidence that MEPH and its metabolites function mainly as transporter
substrates, not merely as inhibitors, and thus are capable of inducing release of monoamines
via their cognate transporters. Consistent with the data in HEK293 cells, NOR-MEPH and 4-
OH-MEPH induced release of [3H]-MPP+ via DAT and NET, and release of [3H]-5HT via
SERT, in rat brain synaptosomes. Our findings with NOR-MEPH in synaptosomes agree
with the recent findings of Hutsell et al. (Hutsell et al., 2015) who reported that stereoisomers
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of NOR-MEPH (i.e., stereoisomers of 4-methylcathinone) are non-selective transporter
substrates that evoke neurotransmitter release from synaptosomes in vitro.
Previous investigations have revealed that the corresponding IC50 and EC50 values for
a given drug to inhibit uptake or induce release may differ several-fold (Scholze, Zwach,
Kattinger, Pifl, Singer & Sitte, 2000; Sitte, Hiptmair, Zwach, Pifl, Singer & Scholze, 2001).
The apparent differences in potency that we observed here for inhibition of uptake (IC50
values in the µM range) versus stimulation of release (EC50 values in the nM range) might be
attributed to different assay systems and methods used in our studies. For example, uptake
assays in HEK293 cells use static incubation conditions, while release assays in HEK293
cells use dynamic perfusion conditions. Comparing results from release assays with HEK293
cells versus rat brain synaptosomes is even more problematic because the latter preparation
consists of homogenized tissue that maximizes the surface area for drug-protein interactions.
Additionally, HEK293 cells are non-neuronal in origin and do not possess all critical
components of the plasma membrane protein machinery that are present in neurons in vivo.
Despite the different assay systems and methods employed here, all of the findings agree that
MEPH and its metabolites are substrates at monoamine transporters.
Even though the tested MEPH metabolites acted as transporter substrates in vitro,
only NOR-MEPH significantly affected neurochemistry and behavior in vivo. The
neurochemical profile of NOR-MEPH closely resembled that of MEPH at the doses tested in
our study, but NOR-MEPH had weaker effects on extracellular dopamine and locomotion.
Thus, it seems NOR-MEPH displays a more serotonergic profile of activity than the parent
compound MEPH. The reduced locomotor response to NOR-MEPH as compared to MEPH
is probably linked to blunted dopaminergic effects of the metabolite, because previous studies
have shown that extracellular dopamine levels in the nucleus accumbens are tightly correlated
with the extent of motor activation produced by stimulant drugs (Baumann, Clark,
Woolverton, Wee, Blough & Rothman, 2011; Zolkowska et al., 2009). Surprisingly, the
systemic administration of 4-OH-MEPH had no significant effect on extracellular
neurotransmitters in the brain or on behavior. Taken together with the in vitro findings, our
in vivo data with 4-OH-MEPH suggest this metabolite may not penetrate through the blood-
brain-barrier. The likelihood of substances to enter the brain is correlated with their size and
lipid solubility (van Bree, de Boer, Danhof, Ginsel & Breimer, 1988; Waterhouse, 2003).
Distribution coefficients calculated for MEPH and its metabolites indicate a clear-cut
separation of lipohilic MEPH and NOR-MEPH on the one hand (logD7.4= 1.39 and 1.29,
respectively) and hydrophilic 4-OH-MEPH on the other hand (logD7.4=0.14). As a
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consequence, the increased hydrophilicity of 4-OH-MEPH, as compared to MEPH and NOR-
MEPH, likely precludes the hydroxylated metabolite from entering the brain. We have noted
a similar situation with the hydroxylated metabolites of MDMA, which are devoid of central
activity when administered systemically to rats (Schindler, Thorndike, Blough, Tella,
Goldberg & Baumann, 2014). Nevertheless, there is increasing evidence which points to the
presence of various CYPs in brain tissue. Even though the expression levels of CYPs in the
brain are low when compared to those in liver (Miksys & Tyndale, 2002), it is interesting to
speculate that in situ metabolism of MEPH and formation of phase 1 metabolites in brain
could impact on MEPH action in vivo. For instance, CYP2D6 has been detected in various
regions of human brain, including substantia nigra and hippocampus (Siegle, Fritz, Eckhardt,
Zanger & Eichelbaum, 2001). As a consequence, 4-OH-MEPH could be formed in close
proximity to monoamine transporters and thereby contribute to the effects of MEPH. At
present, there is no evidence that formation of metabolites in the central nervous system is of
any pharmacological relevance. Interestingly, the dihydroxy metabolite of MDMA, 3,4-
dihydroxymethamphetamine, displays potent stimulatory effects on heart rate and blood
pressure upon systemic administration (Schindler, Thorndike, Blough, Tella, Goldberg &
Baumann, 2014). Our results suggest that future investigations should examine the possible
cardiovascular effects 4-OH-MEPH.
Furthermore, the most abundant mephedrone metabolite detected in forensic traffic
cases was 4-OH-MEPH (Pedersen 2013). In two cases, however, the proportions of parental
drug:metabolite blood plasma concentrations was 28:2 and 29:9 (all in µg/kg). In a number of
cases, NOR-MEPH was detected up to five-fold higher as the internal standard amphetamine.
While NOR-MEPH and DIHYDRO-MEPH were both detected in blood and urine samples,
4-OH-MEPH was mostly limited to urine, highlighting its hydrophilicity.
On the contrary, incubating MEPH with Sprague-Dawley rat liver hepatocytes for 30
minutes (Khreit et al., 2013) displayed the relative amounts of the MEPH metabolites: MEPH
59%, NOR-MEPH 23.5% and 4-OH-MEPH 6.16%. After 120 minutes Khreit et al. report the
relative abundance as follows: MEPH 16%, NOR-MEPH 59% and 4-OH-MEPH 2.6%.
However, further studies are needed to precisely determine the quantitative abundance of
MEPH and its metabolites in vivo to estimate the contributions of the metabolites to the
effects of MEPH.
The present data alone cannot clarify whether NOR-MEPH contributes to the
psychoactive properties of systemically administered MEPH in animals or humans. Further
studies are needed to determine the blood and brain concentrations of NOR-MEPH after
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MEPH exposure. It is noteworthy that NOR-MEPH is the most abundant metabolite of
MEPH identified in rats (Khreit, Grant, Zhang, Henderson, Watson & Sutcliffe, 2013;
Martinez-Clemente, Lopez-Arnau, Carbo, Pubill, Camarasa & Escubedo, 2013) whereas 4-
OH-MEPH is the major metabolite in humans (Pedersen, Reitzel, Johansen & Linnet, 2013;
Pozo et al., 2015). Currently, no information is available on the pharmacokinetics and
bioavailability of the metabolites after MEPH administration in either species. The collective
results presented here demonstrate that phase I metabolites of MEPH are non-selective
transporter substrates at DAT, NET and SERT, similar to the parent compound. However,
only NOR-MEPH affects neurochemistry and behavior when administered peripherally,
suggesting this metabolite could contribute significantly to the unique profile of psychoactive
effects produced by MEPH. Further studies are warranted to examine this intriguing
hypothesis.
Author contributions
F.P.M., O.D-C., J.S.P., L.W. and N.B. performed all experimental work, F.P.M., L.W.,
M.D.M., M.H.B. and H.H.S. designed the experiments, F.P.M., M.H.B. and H.H.S. wrote the
manuscript and received significant input from all other authors.
Acknowledgements
We thank Dr. Oliver Kudlacek and Marion Holy for providing HEK293-cells stably
expressing the human serotonin transporter and Dr. Lars Richter for calculating the
distribution coefficients of mephedrone and its metabolites. The research was supported by
Austrian Research Fund/FWF grants F3506 and W1232 (H.H.S.), the Intramural Research
Program of the NIDA, NIH, grant DA000523-07 (M.H.B.), the FWF-DK MolTag, FWF
W1232 (M.D.M.) and F.P.M. is a recipient of a DOC-fellowship of the Austrian Academy of
Sciences.
Conflict of interest statement
HHS has received honoraria for lectures and consulting from AbbVie, Lundbeck, MSD,
Ratiopharm, Roche, Sanofi-Aventis and Serumwerk Bernburg (past 5 years). All other
authors declare no conflict of interest.
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Figures and Figure Legends:
Five Figures
Figure 1. Proposed pathways for metabolism of mephedrone (MEPH) to its phase I
metabolites. (1) N-demethylation forms 4-methylcathinone (NOR-MEPH); (2) para
hydroxylation forms 4-hydroxytolylmephedrone (4-OH-MEPH); (3) ß-keto reduction forms
dihydromephedrone (DIHYDRO-MEPH). Chemical synthesis started from non-chiral
precursors for the generation of racemic NOR-MEPH and 4-OH-MEPH and from chiral
precursors for DIHYDRO-MEPH (racemic diastereomers obtained by mixing of
enantiomers)
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NET
DAT
120
80
40
0
120
80
40
0
-3 -2 -1 0 1 2 3
-3 -2 -1 0 1 2 3
log [conc] µM
log [conc] µM
SERT
120
80
40
0
MEPH
NOR MEPH
4-OH-MEPH
DIHYDRO
-3 -2 -1 0 1 2 3
log [conc] µM
Figure 2. Effects of MEPH, NOR-MEPH, 4-OH-MEPH and DIHYDRO-MEPH on
transporter-mediated uptake in HEK293 cells expressing hDAT, hNET and hSERT. Uptake
of [3H]-MPP+ via hDAT and hNET, and uptake of [3H]-5HT by hSERT, was performed as
described in “Methods“; all symbols represent mean values ± SEM and the numbers in the
brackets indicate the number of individual experiments performed in triplicate: hDAT:
MEPH (3), NOR-MEPH (4), 4-OH-MEPH (4), DIHYDRO-MEPH (3); hNET: MEPH (4),
NOR-MEPH (4), 4-OH-MEPH (3), DIHYDRO-MEPH (4); hSERT: MEPH (3), NOR-MEPH
(3), 4-OH-MEPH (3), DIHYDRO-MEPH (3).
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DAT
*
A
B
+ MON
75
50
25
0
12
8
Control
*
*
4
+ NOR-MEPH
0
+ MON
0
5
10 15 20
Time [min]
NET
*
C
SERT
*
D
120
80
40
0
400
300
200
100
0
*
*
*
*
*
MEPH
MEPH + MON
NOR-MEPH
4-OH-MEPH
DIHYDRO-MEPH
DIHYDRO-MEPH + MON
NOR-MEPH + MON
4-OH-MEPH + MON
Figure 3. Effects of MEPH, NOR-MEPH, 4-OH-MEPH and DIHYDRO-MEPH on
transporter-mediated release of preloaded radiolabeled substrate in cells HEK293 expressing
hNET, hDAT and hSERT. [3H]-MPP+ was used as the radiolabeled substrate for hDAT and
hNET while release by hSERT-expressing cells was performed using [3H]-5HT as the
radiolabeled substrate. [A] Representative experiment showing the effect of NOR-MEPH (10
µM) in the presence or absence of monensin (10 µM) on DAT-mediated efflux of preloaded
[3H]-MPP+ (presence of substances indicated by black bar; n=5 independent experiments
performed in triplicate). [B to D] For each transporter, area-under-the-curve (AUC) was
calculated from 9 fractions collected after drug treatment (10 µM) in the absence or presence
of monensin (MON, 10 µM). Solid bars indicate vehicle + drug, whereas hatched bars
indicate MON + drug. Bars represent mean values ± SEM and the numbers in the brackets
indicate the number of individual experiments performed in triplicate: hDAT: MEPH (6),
NOR-MEPH (5), 4-OH-MEPH (5), DIHYDRO-MEPH (5); hNET: MEPH (5), NOR-MEPH
(5), 4-OH-MEPH (6), DIHYDRO-MEPH (5); hSERT: MEPH (5), NOR-MEPH (5), 4-OH-
MEPH (5), DIHYDRO-MEPH (5). * = p< 0.05 (Bonferroni’s) compared to corresponding
vehicle + drug group.
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NET
DAT
120
80
40
0
120
80
40
0
-3 -2 -1 0 1 2
-3 -2 -1 0 1 2
log [conc] µM
log [conc] µM
SERT
120
80
40
0
MEPH
NOR-MEPH
4-OH-MEPH
-3 -2 -1 0 1 2
log [conc] µM
Figure 4. Effects of MEPH, NOR-MEPH and 4-OH-MEPH on transporter-mediated release
of preloaded radiolabeled substrate in rat brain synaptosomes. [3H]-MPP+ was the
radiolabeled substrate for DAT and NET assays while [3H]-5HT was the radiolabeled
substrate for SERT assays. Symbols represent mean values ± SEM obtained from three
individual experiments performed in triplicate.
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600
400
200
0
3600
2400
1200
0
#
*
*
3
*
3
*
1
#
*
*
*
#
1
*
*
#
*
#
#
-40
0
40 80 120
-40
0
40 80 120
Time (min)
Time (min)
3600
2400
1200
0
*
MEPH
3
NOR-MEPH
#
*
*
1
4-OH-MEPH
SAL
-40
0
40 80 120
Time (min)
Figure 5. Effects of i.v. administration of MEPH, NOR-MEPH, 4-OH-MEPH or saline
(SAL) on neurochemistry and behavior in rats undergoing microdialysis in nucleus
accumbens. Drugs were administered intravenously at 1 mg kg-1 at time zero, followed by 3
mg kg-1 60 min later. Dopamine and 5-HT were detected by HPLC-EC as described in
Methods. Forward locomotion (Activity) was determined by photo-beam breaks. Data are
presented as mean ± SEM, n=6 rats in the control group (SAL) and n=7 rats for all other
groups (MEPH, NOR-MEPH and 4-OH-MEPH), arrows indicate time of drug administration.
Individual symbols represent significant differences from saline-treated control at
corresponding time points (p<0.05; Bonferroni’s): * denotes significance of MEPH over
saline, # denotes significance of NOR-MEPH over saline.
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Tables:
Two Tables
IC50 (µM)
NET
DAT
SERT
MEPH
0.77 (0.53-1.08)
6.35 (4.66 -8.64)
2.92 (2.35 – 3.6)
23.97 (8.65 – 66.46)
2.77 (1.92 -3.97)
5.46 (3.58 – 8.31)
4.85 (3.28 – 7.17)
23.53 (19.8 – 27.97)
7.83 (6.32 – 9.75)
10.61 (9.06 – 12.43)
73.53 (62.5 – 86.51)
64.98 (50.66 – 83.37)
NOR-MEPH
4-OH-MEPH
DIHYDRO-MEPH
Table 1: IC50 values of test drugs on uptake mediated by hDAT, hNET and hSERT, stably
expressed in HEK293 cells. Data are represented as the mean and 95 % confidence intervals
in brackets obtained from nonlinear regression fits as shown in Figure 2.
Table 2: EC50 values of test drugs on monoamine transporter mediated efflux
EC50 (µM)
DAT
NET
SERT
MEPH
0.052 (0.036-0.075)
0.22 (0.14 -0.32)
0.19 (0.13 – 0.267)
0.09 (0.08-0.11)
0.1 (0.08-0.13)
0.15 (0.11 – 0.19)
0.21 (0.17 – 0.26)
0.21 (0.13 – 0.32)
2.01 (1.390 – 2.91)
NOR-MEPH
4-OH-MEPH
Table 2: EC50 values of test drugs on transporter mediated efflux obtained in rat brain
synaptosomes. Data are presented as the mean and 95 % confidence intervals in brackets
obtained from nonlinear regression fits as shown in Figure 3.
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