In vitro monoamine oxidase inhibition potential of alpha-methyltryptamine analog new psychoactive substances for assessing possible toxic risks

Article abstract of DOI:10.1016/j.toxlet.2017.03.007

Tryptamines have emerged as new psychoactive substances (NPS), which are distributed and consumed recreationally without preclinical studies or safety tests. Within the alpha-methylated tryptamines, some of the psychoactive effects of the prototypical alpha-methyltryptamine (AMT) have been described decades ago and a contributing factor of its acute toxicity appears to involve the inhibition of monoamine oxidase (MAO). However, detailed information about analogs is scarce. Therefore, thirteen AMT analogs were investigated for their potential to inhibit MAO. An in vitro assay analyzed using hydrophilic interaction liquid chromatography–high resolution-tandem mass spectrometry was developed and validated. The AMT analogs were incubated with recombinant human MAO-A or B and kynuramine, a non-selective MAO substrate to determine the IC₅₀values. The known MAO-A inhibitors 5-(2-aminopropyl)indole (5-IT), harmine, harmaline, yohimbine, and the MAO-B inhibitor selegiline were tested for comparison. AMT and all analogs showed MAO-A inhibition properties with IC₅₀values between 0.049 and 166?μM, whereas four analogs inhibited also MAO-B with IC₅₀values between 82 and 376?μM. 7-Me-AMT provided the lowest IC₅₀value against MAO-A comparable to harmine and harmaline and was identified as a competitive MAO-A inhibitor. Furthermore, AMT, 7-Me-AMT, and nine further analogs inhibited MAO activity in human hepatic S9 fraction used as model for the human liver which expresses both isoforms. The obtained results suggested that MAO inhibition induced by alpha-methylated tryptamines might be clinically relevant concerning possible serotonergic and adrenergic effects and interactions with drugs (of abuse) particularly acting as monoamine reuptake inhibitors. However, as in vitro assays have only limited conclusiveness, further studies are needed.

Full text of DOI:10.1016/j.toxlet.2017.03.007

Accepted Manuscript
Title: In vitro monoamine oxidase inhibition potential of
alpha-methyltryptamine analog new psychoactive substances
for assessing possible toxic risks
Authors: Lea Wagmann, Simon D. Brandt, Pierce V.
Kavanagh, Hans H. Maurer, Markus R. Meyer
PII:
S0378-4274(17)30100-5
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.toxlet.2017.03.007
TOXLET 9718
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Toxicology Letters
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27-1-2017
8-3-2017
10-3-2017
Please cite this article as: Wagmann, Lea, Brandt, Simon D., Kavanagh, Pierce V.,
Maurer, Hans H., Meyer, Markus R., In vitro monoamine oxidase inhibition potential
of alpha-methyltryptamine analog new psychoactive substances for assessing possible
toxic risks.Toxicology Letters http://dx.doi.org/10.1016/j.toxlet.2017.03.007
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In vitro monoamine oxidase inhibition potential of alpha-methyltryptamine
analog new psychoactive substances for assessing possible toxic risks
Lea Wagmanna, Simon D. Brandtb, Pierce V. Kavanaghc, Hans H. Maurera, and
Markus R. Meyera,*
a Department of Experimental and Clinical Toxicology, Institute of Experimental and
Clinical Pharmacology and Toxicology, Saarland University, Homburg (Saar),
Germany
b School of Pharmacy and Biomolecular Sciences, Liverpool John Moores University,
Liverpool, UK
c Department of Pharmacology and Therapeutics, Trinity Centre for Health and
Sciences, St. James’s Hospital, Dublin, Ireland
_________________________________________
*Corresponding author.
E-mail address: markus.meyer@uks.eu (M.R. Meyer).

Highlights
 Study provided comprehensive in vitro data on the inhibitory potential of
tryptamine-like new psychoactive substances (NPS) towards human MAO-A/B
 Studied compounds were shown to inhibit at least one MAO isoform
 7-Me-AMT was identified to have the lowest IC50 value against MAO-A and to
act as competitive inhibitor
 MAO inhibition by tested NPS was shown to be of possible clinical relevance
concerning serotonergic and adrenergic effects
 Interactions with drugs and drugs of abuse particularly acting as monoamine
reuptake inhibitors were shown to be likely
Abstract
Tryptamines have emerged as new psychoactive substances (NPS), which are
distributed and consumed recreationally without preclinical studies or safety tests.
Within the alpha-methylated tryptamines, some of the psychoactive effects of the
prototypical alpha-methyltryptamine (AMT) have been described decades ago and a
contributing factor of its acute toxicity appears to involve the inhibition of monoamine
oxidase (MAO). However, detailed information about analogs is scarce. Therefore,
thirteen AMT analogs were investigated for their potential to inhibit MAO. An in vitro
assay analyzed using hydrophilic interaction liquid chromatography-high resolution-
tandem mass spectrometry was developed and validated. The AMT analogs were
incubated with recombinant human MAO-A or B and kynuramine, a non-selective
MAO substrate to determine the IC50 values. The known MAO-A inhibitors 5-(2-
aminopropyl)indole (5-IT), harmine, harmaline, yohimbine, and the MAO-B inhibitor
selegiline were tested for comparison. AMT and all analogs showed MAO-A inhibition
properties with IC50 values between 0.049 and 166 µM, whereas four analogs
inhibited also MAO-B with IC50 values between 82 and 376 µM. 7-Me-AMT provided
the lowest IC50 value against MAO-A comparable to harmine and harmaline and was
identified as a competitive MAO-A inhibitor. Furthermore, AMT, 7-Me-AMT, and nine
further analogs inhibited MAO activity in human hepatic S9 fraction used as model for
the human liver which expresses both isoforms. The obtained results suggested that
MAO inhibition induced by alpha-methylated tryptamines might be clinically relevant
concerning possible serotonergic and adrenergic effects and interactions with drugs
(of abuse) particularly acting as monoamine reuptake inhibitors. However, as in vitro
assays have only limited conclusiveness, further studies are needed.
2

Keywords Alpha-methyltryptamine analog new psychoactive substances, drugs of
abuse, MAO inhibition, LC-HR-MS/MS, IC50 value
1. Introduction
New psychoactive substances (NPS) are emerging drugs that are mainly
consumed as legal and easy available substitutes for traditional and controlled drugs
of abuse (Brandt et al. 2014; Meyer 2016). The detection of synthetic tryptamines
including alpha-methyltryptamine (AMT) has been frequently reported to the EU Early
Warning System coordinated by the European Monitoring Centre for Drugs and Drug
Addiction (EMCDDA) and continuously monitored as part of the EMCDDA’s
toxicovigilance system (EMCDDA 2015). AMT (-MT, 3-IT, IT-290) has been
investigated in the 1960s as a potential antidepressant and its popularity as a drug of
abuse came to light in the 1990s due to its hallucinogenic and stimulant properties
(Araujo et al. 2015). In late 2011, an isomer of AMT, 5-(2-aminopropyl)indole (5-IT, 5-
API), appeared on the European drug market (EMCDDA 2014). After intake of AMT
or 5-IT, symptoms related to monoaminergic toxicity were described, such as
restlessness, agitation, disorientation, shivering, sweating, mydriasis, vomiting,
tachycardia, or hyperthermia (EMCDDA 2015; Shulgin and Shulgin 1997). Both were
also involved in several fatalities (Boland et al. 2005; Elliott and Evans 2014). A key
factor involved in the occurrence of clinical features included the inhibition of
monoamine oxidase (MAO) enzymes, followed by increased monoamine levels
inducing a serotonin syndrome (Boyer and Shannon 2005; EMCDDA 2014). In 1968,
AMT, 5-IT, and four positional isomers were identified as inhibitors of guinea pig
MAO (Cerletti et al. 1968). More recently, 5-IT was confirmed as highly selective and
potent inhibitor of recombinant human MAO-A (Herraiz and Brandt 2014).
Furthermore, tryptamines were identified in NPS products combining different
NPS groups (UNODC 2016). Thus, the risk of encountering monoaminergic (side)
effects and drug-drug interactions is very likely although the extent to which this
might occur is difficult to predict, given that systematic data are not available. In
contrast to authorized medicines, NPS are marketed and consumed without
preclinical or clinical studies. Procedures employed for various substrates, mostly
therapeutic drugs, have been described for in vitro monitoring of MAO activity (Tipton
et al. 2006). Examples of drugs of abuse tested for human MAO inhibition activity
3

include MDMA and its metabolites, and 5-IT (Herraiz and Brandt 2014; Steuer et al.
2016).
Therefore, the aim of the present study was to develop a MAO inhibition assay
based on hydrophilic interaction liquid chromatography-high resolution-tandem mass
spectrometry (HILIC-HR-MS/MS), to investigate the in vitro inhibition potential of AMT
and 13 ring-substituted analogs (Fig. 1) on recombinant human MAO-A or B, and to
determine their IC50 values.
2. Materials and methods
2.1. Chemicals and enzymes
Harmine was obtained from THC Pharm (Frankfurt, Germany), amphetamine-d5
and AMT succinate from LGC Standards (Wesel, Germany), harmaline, yohimbine,
selegiline, kynuramine (KYN), 4-hydroxyquinoline (4-OHC), ammonium acetate,
potassium dihydrogenphosphate, and dipotassium hydrogenphosphate from Sigma-
Aldrich (Taufkirchen, Germany), formic acid (MS grade) from Fluka (Neu-Ulm,
Germany), acetonitrile, methanol (both LC-MS grade), and all other chemicals from
VWR (Darmstadt, Germany). 5-IT was synthesized (Scott et al. 2014) and kindly
provided by the Department of Pharmacology and Therapeutics, Trinity Centre for
Health Sciences, St James's Hospital, Dublin 8, Ireland, before it was scheduled. All
non-scheduled AMT analogs were prepared following procedures published
previously (Young 1958). Details can also be found in (Brandt et al. 2004).
The baculovirus-infected insect cell microsomes (Supersomes) containing
human complementary DNA-expressed MAO-A or MAO-B (5 mg protein/mL), wild-
type Supersomes as negative control without MAO activity (MAO control, 5 mg
protein/mL), and pooled human liver S9 (20 mg protein/mL) were obtained from
Corning (Amsterdam, The Netherlands). After delivery, enzyme preparations were
thawed at 37 °C, aliquoted, snap-frozen in liquid nitrogen, and stored at -80 °C until
use.
2.2. HILIC-HR-MS/MS apparatus
4

A Thermo Fisher Scientific (TF, Dreieich, Germany) Dionex UltiMate 3000
Rapid Separation (RS) UHPLC system with a quaternary UltiMate 3000 RS pump
and an UltiMate 3000 RS autosampler was used and controlled by the TF
Chromeleon software version 6.80. It was coupled to a TF Q-Exactive Plus equipped
with a heated electrospray ionization II source (HESI-II). The gradient elution was
performed on a Macherey-Nagel (Düren, Germany) HILIC Nucleodur column (125 ×
3 mm, 3 μm) using aqueous ammonium acetate (25 mM, eluent A) and acetonitrile
containing 0.1% (v/v) formic acid (eluent B). The flow rate was set to 500 µL/min and
the gradient was programmed as follows: 0-0.5 min hold 80% B, curve 5; 0.5-3.8 min
to 50% B, curve 5; 3.8-3.9 min to 40% B, curve 5; 3.9-5 min hold 40% B, curve 5; 5-
5.1 min to 80% B, curve 5; and 5.1-6 min hold 80% B, curve 5. Chromatography was
performed at 60 °C maintained by a Dionex UltiMate 3000 RS analytical column
heater. The injection volume for all samples was 1 µL. HESI-II conditions were
already described before (Richter et al. 2016): sheath gas, 53 arbitrary units (AU);
auxiliary gas, 14 AU; sweep gas, 3 AU; spray voltage, 3.50 kV; heater temperature,
438 °C; ion transfer capillary temperature, 269 °C; and S-lens RF level, 60.0. Mass
calibration was done prior to analysis according to the manufacturer’s
recommendations using external mass calibration. For evaluating the
chromatographic separation, a full scan experiment was used with the following scan
parameters: polarity, positive; micro scan, 1; resolution, 35,000; automatic gain
control (AGC) target, 3e6; maximum injection time (IT), 200 ms; and scan range, 50-
750. The final quantification was performed using a targeted single ion monitoring (t-
SIM) and a subsequent data-dependent MS2 (dd-MS2) mode with an inclusion list
containing the exact masses of positively charged KYN (m/z 165.1022), 4-OHC (m/z
146.0600), and the internal standard (IS) amphetamine-d5 (m/z 141.1434). The
settings for the t-SIM mode were as follows: micro scan, 1; resolution, 35,000; AGC
target, 5e4; maximum IT, 100 ms; and isolation window, 4 m/z. The settings for the
dd-MS2 mode were as follows: micro scan, 1; resolution, 35,000; AGC target, 2e5;
maximum IT, 100 ms; isolation window, 4 m/z; and dynamic exclusion, 4 s. TF
Xcalibur Qual Browser 2.2 software was used for data handling. The settings for
automated peak integration were as follows: peak detection algorithm, ICIS; area
noise factor, 5; and peak noise factor, 300. GraphPad QuickCalcs (GraphPad
Software, San Diego, USA) was used for outlier detection
5

(http://graphpad.com/quickcalcs/grubbs1), while GraphPad Prism 5.00 (GraphPad
Software) was used for statistical evaluation.
2.3. Preparation of stock solutions
Stock solutions of the MAO substrate KYN (6.25 mM) or its metabolite 4-OHC
(0.1 mM) were prepared in water. Stock solutions of harmine, harmaline, 5-IT,
selegiline, AMT (5 mM, respectively), or yohimbine (3 mM) were used in methanol.
Stock solutions of the AMT analogs (5 mM, respectively) were prepared in DMSO.
Afterwards, they were diluted 1:5 (v/v) with methanol, gently evaporated under
nitrogen, and resolved in water/methanol (9:1, v/v). To obtain the solutions for
incubation, the stock solutions were diluted in phosphate buffer. The organic solvent
content in the final incubation mixtures was always below 2.5% (Chauret et al. 1998).
Stock solutions were aliquoted and stored at -20 °C.
2.4. Method validation
The method for quantification of 4-OHC was validated in accordance to the
“Guideline on bioanalytical method validation” published by the European Medicines
Agency (EMA) (EMA 2011). For validation, 30 µL samples containing 1 µg/mL MAO
control in phosphate buffer were used. If needed, 10 µL of the phosphate buffer was
replaced by appropriate amounts of the calibration or quality control (QC) stock
solution. These samples were diluted with the same volume of acetonitrile with or
without IS. Briefly, the method was tested for selectivity (using ten blank samples
without IS), carry-over (using a blank sample without IS following the high QC), lower
limit of quantification (LLOQ) defined as lowest calibration standard, within-run
accuracy and precision (analyzed in a single run five samples per level at four
concentration levels: LLOQ QC, low QC, medium QC, and high QC), between-run
accuracy and precision (analyzed in three different runs on two different days six
samples per level at four concentration levels: LLOQ QC, low QC, medium QC, and
high QC), matrix effect (using six samples with matrix and six samples without matrix
at two concentration levels: low QC and high QC), and stability of processed samples
in the autosampler. The calibration consisted of six concentration points (50, 200,
6

400, 600, 800, and 1000 nM) equally distributed over the whole range. The
concentrations of LLOQ QC, low QC, medium QC, and high QC were as follows: 50,
100, 500, and 900 nM. Instead of MAO-A or B, MAO control was used for sample
preparation. For quantification, the ratios of 4-OHC versus IS were used. The
analytical runs consisted of a blank sample without IS, a blank sample with IS,
calibration standards, three levels of QC samples (low, medium, and high) in
duplicate, and the study samples. All calculations were done using GraphPad Prism
5.00.
2.5. Determination of Km values
KYN deamination is depicted in Fig. 2. The kinetic constants were derived from
incubations with MAO-A, MAO-B, or S9. Incubations were performed at 37 °C for 20
min using 1 µg/mL MAO-A or MAO-B, respectively, or 100 µg/mL S9, and the MAO
substrate KYN at concentrations of 0.1, 0.5, 1, 5, 10, 25, 50, 100, 150, 200, 250, and
500 µM. In addition, blank incubations with 1 µg/mL MAO control were prepared as
negative control. Besides enzyme preparations and substrate, the incubation
mixtures (final volume 30 µL) contained 100 mM phosphate buffer. Reactions were
initiated by addition of the ice-cold enzyme preparation and stopped with 30 μL of
ice-cold acetonitrile, containing 10 μM amphetamine-d5 as IS. The solution was
centrifuged for 2 min at 10,000×g, 50 μL of the supernatant were transferred to an
autosampler vial, and injected onto the HILIC-HR-MS/MS apparatus for analysis.
Enzyme kinetic constants were estimated by non-linear curve fitting using GraphPad
Prism 5.00 after validated quantification of the formed metabolite 4-OHC via six-point
calibration. The Michaelis-Menten equation (Eq. (1)) was used to calculate apparent
Km and Vmax values for MAO-A, MAO-B, or S9, where v is the initial reaction velocity,
S the substrate concentration, Vmax the maximal reaction velocity, and Km the
substrate concentration at half Vmax
.
푉
x 푆
max
(1) 푣 =
퐾 + 푆
m
7

2.6. Initial inhibition screening and determination of IC50 values and inhibition
constants
2.6.1. Initial inhibition screening
For inhibition studies, 10 µL of the phosphate buffer was replaced by
appropriate amounts of the potential inhibitor solution. KYN was used in
concentrations comparable to its Km value. All other incubation conditions were the
same as described above. To test for inhibition capability, MAO-A or B was incubated
with 10 µM of the potential inhibitor in triplicate (n = 3). In addition to these samples,
control samples without inhibitor, positive control samples with model inhibitors (10
µM 5-IT for MAO-A or 10 µM selegiline for MAO-B), blank samples without MAO
activity, and interfering samples were also prepared in triplicates. For preparation of
the interfering samples, control samples without inhibitor were incubated and the
reaction was terminated with ice-cold acetonitrile containing the IS and the potential
inhibitors. The 4-OHC formation in test samples was then compared to metabolite
formation in control samples. For statistical analysis of data, a one-way ANOVA
followed by Dunnett's multiple comparison test (significance level, P < 0.001, 99.9%
confidence intervals) by GraphPad Prism 5.00 was used.
2.6.2. Determination of IC50 values
Inhibitors were incubated at least at ten different concentrations (0.00004,
0.0002, 0.0006, 0.002, 0.01, 0.04, 0.2, 0.6, 2.5, 10, 40 160 640, 2560 µM),
depending on their expected inhibition strengths after initial inhibition screening. All
other incubation conditions were the same as described above. Control samples
were also prepared as described above. The IC50 values were calculated by plotting
the metabolite formation (relative to the control samples) over the logarithm of the
inhibitor concentration using GraphPad Prism 5.00.
2.6.3. Determination of inhibition constants (Ki values)
For experimental determination, four concentration levels of the test inhibitor
were incubated with KYN at five different concentrations (0.5, 5, 25, 100, and 500
8

µM). Inhibitor concentration levels depended on the previously determined IC50 value.
For selegiline and 7-Me-AMT, inhibitor concentration levels were: 0, 0.01, 0.05, and
0.1 µM, for 5-IT, 0, 0.1, 0.5, and 1 µM. All other incubation conditions were the same
as described above. 7-Me-AMT and 5-IT were incubated with MAO-A, whereas
MAO-B was used in the case of selegiline. The inhibition constants (Ki values) were
calculated using GraphPad Prism 5.00. Michaelis-Menten plots were transferred to
Lineweaver-Burk plots (1/V versus 1/S), and fit factors for different inhibition models
were compared to determine the most probable inhibition mode. If competitive
inhibition was identified as most probable inhibition model, the equation of Cheng
and Prusoff (Eq. (2)) (Cheng and Prusoff 1973) was additionally used to calculate Ki
from IC50 and Km values.
푆
(2) IC50 = 퐾 푥 (1 +
)
i
퐾
m
2.7. MAO inhibition in S9 fraction
Three concentration levels of the inhibitors were incubated with KYN at
concentrations at its Km value. Inhibitor concentration level one was the lowest
concentration and chosen based on plasma concentrations reported after intake
(Table 1). Concentration levels two and three were calculated to be ten and one
hundred times concentration level one, respectively. For selegiline, inhibitor
concentration levels were 0.005, 0.05, and 0.5 µM; for harmine, harmaline, and
yohimbine, 0.1, 1, and 10 µM inhibitor were chosen, and for all other compounds, 1,
10, and 100 µM. All other incubation conditions were the same as described above.
In addition, control samples without inhibitor and blank samples were prepared. All
incubations were performed in triplicate (n = 3). For evaluation, the metabolite
formation in test samples was compared to metabolite formation in control samples.
For statistical analysis of the data, a one-way ANOVA followed by Dunnett's multiple
comparison test (significance level, P < 0.05, 95% confidence intervals) by GraphPad
Prism 5.00 was used.
9

3. Results
3.1. Method validation
The analytical procedure was based on HILIC-HR-MS/MS in t-SIM mode with a
subsequent dd-MS2 mode. The method was successfully validated in accordance to
the criteria of the EMA (EMA 2011). The method was selective at LLOQ levels, with a
response of interfering components less than 20% or 5% compared to the response
of the analyte at the LLOQ and the response of the IS, respectively. No carry-over
problems could be observed and the LLOQ for 4-OHC was defined as 50 nM. The
mean for within-run and between-run accuracies ranged from 3% to 19% but were
within 20% of the nominal values for the LLOQ QC and within 15% for the low,
medium, and high QC samples. The mean within-run and between-run precisions
ranged from 1% to 13%. Precisions were within 20% for the LLOQ QC and within
15% for the low, medium, and high QC samples. The coefficients of variation (CVs)
of matrix factors were 9% and 11% for the analytes and the IS at low QC level and
3% and 7% at high QC level. The CVs of IS-normalized matrix factor were 14% for
low and 9% at high QC level and thus not greater than 15%. Processed samples
showed sufficient stability in the autosampler for at least 10 h, corresponding to the
maximum duration of the analytical runs.
3.2. Determination of Km values
The kinetics for KYN deamination using MAO-A, MAO-B, or S9 followed classic
Michaelis-Menten behavior. Calculated Km values were 43 ± 5 µM, 23 ± 4 µM, and 34
± 2 µM for MAO-A, MAO-B, and S9, respectively. Vmax values were 75 ± 2
nmol/min/mg, 56 ± 2 nmol/min/mg, and 7 ± 0.1 nmol/min/mg for MAO-A, MAO-B, and
S9, respectively. R2 values were 0.9910, 0.9760, and 0.9980, respectively.
3.3. Initial inhibition screening results
The results are summarized in Fig. 3. All test tryptamines showed significant
inhibition of MAO-A resulting in residual activities below 50%. MAO-B was
additionally inhibited by 5-F-AMT, 6-F-AMT, 5-Cl-AMT, and 5-F-2-Me-AMT, resulting
10

in residual activities between 60 and 80%. Harmine, harmaline, and yohimbine also
showed inhibition of MAO-A in this assay. Whereas harmine and harmaline inhibited
MAO-A activity almost completely, yohimbine inhibition resulted in residual activities
of about 25%. No analytical interferences could be detected for all incubation sets
and the positive control samples showed almost complete inhibition of MAO-A or B
with residual activities of about 5%.
3.4. Determination of IC50 values
All results are given in Table 1. IC50 values of AMT and its analogs for MAO-A
inhibition were between 0.049 (7-Me-AMT) and 166 µM (1-Me-2-Ph-AMT), for MAO-
B inhibition, IC50 values of 5-Cl-AMT, 6-F-AMT, 5-F-2-Me-AMT, and 5-F-AMT were
82, 126, 223, and 376 µM, respectively. The known MAO-A inhibitors harmine,
harmaline, 5-IT, and yohimbine resulted in IC50 values of 0.006, 0.011, 0.20, and 6.0
µM, respectively, while the IC50 value of selegiline towards MAO-B was 0.017 µM.
3.5. Determination of Ki values
Michaelis-Menten and Lineweaver-Burk plots after incubation of 7-Me-AMT are
given in Fig. 4. The Ki value was 0.026 µM. A competitive inhibition model was
preferred, which was in line with the visual inspection of the intersection in the
Lineweaver-Burk plot. Ki value calculated from IC50 and Km values was 0.025 µM. For
5-IT and selegiline, experimentally determined Ki values were 0.18 and 0.011 µM,
respectively. Calculated Ki values were 0.10 and 0.009 µM, respectively. Visual
inspection and software based evaluation preferred competitive inhibition model for
both compounds.
3.6. MAO inhibition in S9 fraction
Results are summarized in Fig. 5. 7-Me-AMT exerted significant inhibition of
KYN deamination by MAO in S9 for all concentration levels. AMT, 5-Cl-AMT, and 5-
Br-AMT inhibited MAO activity when incubated at concentration levels two or three.
4-MeO-AMT, 2-Me-AMT, 5-Me-AMT, 5-F-AMT, 6-F-AMT, 5-F-2-Me-AMT, and 2-Ph-
11

AMT showed significant inhibition only when incubated in concentration level three.
5-MeO-AMT, 1-Me-AMT, and 1-Me-2-Ph-AMT showed no MAO inhibition in S9, even
at the highest concentration level. Harmine and harmaline showed significant
inhibition of MAO-A at all concentration levels, while in case of 5-IT and selegiline
concentration levels two or three led to significant inhibition, and for yohimbine only
concentration level three.
4. Discussion
MAO activity was assessed using KYN, a non-selective substrate for MAO-A
and B, which was transformed to the corresponding aldehyde, followed by non-
enzymatic condensation to 4-OHC (Fig. 2) (Weissbach et al. 1960). 4-OHC formation
was analyzed using HILIC-HR-MS/MS. Although literature described many MAO
activity assays (Tipton et al. 2006), Herraiz and Chaparro found that the needed
selectivity for analyzing 4-OHC could only be achieved by chromatographic
separation before detection (Herraiz and Chaparro 2006). Since HILIC was shown to
provide sufficient retention and separation of small and polar analytes (Steuer et al.
2016), it was anticipated that HILIC separation would be a suitable method in the
present study. Due to its high flexibility and sensitivity, HR-MS/MS required
comparatively low levels of recombinant MAO enzymes and thus reduction of the
total protein content, which can lead to non-specific protein binding of analytes
(Baranczewski et al. 2006). The t-SIM mode with a subsequent dd-MS2 acquisition
allowed simultaneous quantification and identification, respectively. The t-SIM
provided approximately 50 scans per peak by far sufficient for quantification, whereas
the dd-MS2 provided additional selectivity for further studies without the need for
revalidation. Experimental variability during sample preparation and analysis was
corrected by addition of the internal standard amphetamine-d5, which provided
comparable properties to 4-OHC. The whole analytical procedure was successfully
validated in accordance with international guidelines (EMA 2011).
For determination of Ki values, the substrate concentration should be used
below or at its Km value. Different Km values of KYN deamination by MAO-A or B
derived from diverse enzyme sources were published (Herraiz and Brandt 2014; Naoi
12

and Nagatsu 1988; Parikh et al. 2002; Ro et al. 2001) and none for S9. Therefore, Km
values were also determined in this study. The question as to whether the S9 fraction
would show MAO activity remained inconsistently answered. As enzymes of the
outer mitochondrial membrane, MAO was expected to be removed by centrifugation
at 9,000xg during production of S9 fraction (Lee and Zhu 2011). However, Salva et
al. reported NADPH-independent formation of metabolites even in pooled human
liver microsomes, which could be abolished by MAO inhibitors. They ascribed this
MAO activity to a contamination with mitochondrial enzymes (Salva et al. 2003).
Furthermore, the guidance for industry by the Food and Drug Administration
recommended the addition of the MAO inhibitor pargyline to the S9 fraction to identify
MAO contribution in the oxidative biotransformation of any given tested drug (FDA
2006). As 4-OHC formation in S9 was also observed in the present study, the Km
value of KYN transformation in S9 was determined. Resulting Km values for MAO-A
and MAO-B were similar, as expected for a non-selective substrate, and comparable
to published values (Herraiz and Brandt 2014; Naoi and Nagatsu 1988; Parikh et al.
2002; Ro et al. 2001). Initial experiments were conducted to choose incubation time
and enzyme concentration (data not shown) to be within the linear range of
metabolite formation. Substrate concentrations between 0.1 and 500 µM KYN
allowed modeling of enzyme kinetics. Less than 20% of substrate was metabolized in
all incubations, except of the lowest substrate concentrations. To avoid non-specific
protein binding, the protein concentrations were chosen as low as analytically
possible as recommended by (Baranczewski et al. 2006). Blank incubations should
be used to consider MAO-independent formation of 4-OHC.
Applicability of the initial inhibition screening procedure to assess the inhibition
by either MAO-A or MAO-B was tested using positive control samples containing 5-IT
or selegiline, known for inhibition of MAO-A or B, respectively. A one-way ANOVA
followed by Dunnett's multiple comparison test was used to decide whether 4-OHC
formation in test samples was statistically significantly different from 4-OHC formation
in control samples. Such an initial inhibition screening strategy was already described
by Dinger et al. for studying the cytochrome P450 (CYP) inhibition potential of 3,4-
methylenedioxy-derived designer drugs (Dinger et al. 2016a). In the present study, a
set of blank samples was also analyzed. A lower inhibitor concentration (10 µM) in
the positive control samples was chosen because plasma concentrations of known
MAO inhibitors were expected to be lower than those of CYP inhibitors. No significant
13

inhibition at 10 µM was thus considered as weak and probably not clinically relevant.
Interfering samples were used to exclude mass spectral ion suppression or
enhancement effects, caused by co-eluting analytes (Remane et al. 2010). These
samples were mandatory as only KYN, 4-OHC, and the IS were monitored by the
analytical method and co-eluting compounds could lead to false positive (in case of
ion suppression) or false negative (in case of ion enhancement) results.
In accordance to Cerletti et al. and Zirkle and Kaiser (Cerletti et al. 1968; Zirkle
and Kaiser 1964), AMT was identified as MAO-A inhibitor. All tested analogs also
revealed MAO-A inhibition properties, whereas 5-F-AMT, 6-F-AMT, 5-Cl-AMT, and 5-
F-2-Me-AMT inhibited MAO-B as well. Furthermore, harmine, harmaline, and
yohimbine showed MAO-A inhibition consistent with previous investigations e.g. (Dos
Santos et al. 2013). The initial inhibition screening was useful to realize that most
tested compounds inhibited only one MAO isoform. Hence, time and costs for further
IC50 determinations could be saved. Consequently, only for compounds showing a
statistically significant inhibition in the initial inhibition screening, IC50 values were
determined.
To predict a potential clinical relevance of the MAO inhibition based on IC50
values, the expected plasma levels of inhibitors (given in Table 1) of inhibitors should
be considered. In case of NPS, only scarce information is available, concerning
plasma concentrations after intake. The only information source was case reports of
fatal or non-fatal intoxications but particularly postmortem data is difficult to interpret,
due to postmortem redistribution and unclear cause of death. But also data of non-
fatal cases is problematic, as dosage and time of ingestion remain often unclear.
7-Me-AMT showed an IC50 value against MAO-A activity comparable to the
strong inhibitors harmine and harmaline. For 7-Me-AMT, plasma levels were not yet
published, but described plasma concentrations for AMT (Elliott and Evans 2014;
Ferec et al. 2015) were much higher than published harmine and harmaline plasma
concentrations (Baselt 2008; Oliveira et al. 2012). For AMT, Shulgin and Shulgin
(Shulgin and Shulgin 1997) described oral intake of 15 to 30 mg or smoking of 5 to
20 mg as common dosage. These amounts were comparable to oral dosages of 5-IT
(Shulgin and Shulgin 1997). In comparison, 5-MeO-AMT, however, was found to be
significantly more potent with an active oral dose in the 1.5-4.5 mg range (Kantor et
al. 1980; Shulgin 1979; Shulgin and Nichols 1978; Shulgin and Shulgin 1997).
Concerning other AMT analogs, no data about dosage or plasma concentrations are
14

available so far, but similar doses followed by similar plasma concentrations may be
expected. Considering assumed plasma levels, clinical relevance of the MAO-A
inhibition after intake of 7-Me-AMT could not be excluded. The same should be true
for 5-Cl-AMT, AMT, and 5-F-AMT, as their IC50 values against MAO-A were in the
same range as the IC50 value of 5-IT. Given that the other AMT analogs showed
higher IC50 values against MAO-A, clinical relevance should be unlikely. Comparing
the structural properties and IC50 values, a methylation in the 7-position of the indole
ring appeared to result in increased potency whereas compounds with a methylation
in position 1, 2, or 5 provided a higher IC50 value than AMT itself. The two
compounds carrying a methyl group in position 1 provided the highest IC50 values.
Therefore, an unchanged position 1 might be essential for the unhindered binding to
MAO-A and especially the combination of a methyl group in position 1 with a phenyl
group in position 2 reduced the inhibition potential. Furthermore, a methoxy group in
the 5-position led to an increased IC50 value, whereas the methoxy group located at
position 4, or insertion of a halogen atom in position 5, had nearly no effect on the
IC50 value, compared to AMT. Concerning MAO-B inhibition, determined IC50 values
of four AMT analogs were hundred to one thousand times higher than the IC50 value
of selegiline but also expected plasma concentrations of the AMT analogs were
hundred to thousand times higher than published selegiline plasma concentrations.
Additionally, these AMT analogs inhibited both MAO isoforms and thus, clinical
relevance could not be excluded.
Furthermore, the Ki values of 7-Me-AMT, 5-IT, and selegiline were
experimentally determined and additionally calculated from the IC50 values resulting
in comparable Ki values. Both Ki values for 5-IT were similar to those published by
Herraiz and Brandt (Herraiz and Brandt 2014). In the current study, all three
compounds were identified as competitive inhibitors and 7-Me-AMT confirmed as
potent MAO-A inhibitor, far stronger than 5-IT.
Moreover, MAO inhibition was tested in human hepatic S9 fraction given that
MAO-B is slightly overexpressed in human liver (Nishimura and Naito 2006; Wang et
al. 2013). In contrast to the artificial system of recombinant human MAO expressed in
baculovirus-infected insect cells, the S9 fraction represents an in vitro model of the
human liver, containing both isoforms in their natural environment and different
abundances. The test drugs were used at three different concentration levels.
Concentration level one represented expected plasma concentrations, as actual
15

concentrations in the liver were difficult to estimate. Being the main metabolizing
organ, a higher concentration than in plasma is more than likely (concentration levels
two and three). Obtained results correlated well with the determined IC50 values. 7-
Me-AMT, with the lowest IC50 value, showed significant inhibition of MAO activity for
all concentration levels, while 5-MeO-AMT, 1-Me-AMT, and 1-Me-2-Ph-AMT, with the
highest IC50 values towards MAO-A, showed no inhibition at all. Residual MAO
activity towards KYN in presence of the test inhibitor could be explained by inhibition
of only one isoform, which means that the effect might be more pronounced for a
MAO-A specific substrate such as serotonin or noradrenalin. As tryptamines were
often identified in NPS products containing different NPS groups (UNODC 2016), an
intake with substances that increase monoamine levels or inhibit MAO could lead to
severe intoxications as described by (Brush et al. 2004). They reported about an
adolescent who suffered from a severe intoxication exhibiting hyperthermia,
tachycardia, and massive agitation after consumption of the combination of a
hallucinogenic tryptamine and the monoamine oxidase inhibitor harmaline (Brush et
al. 2004). Therefore, clinical effects due to MAO inhibition by AMT or its analogs
could not be excluded for most of the tested compounds.
As controlled human trials for NPS are not feasible due to ethical reasons, in
vitro approaches for studying their toxicokinetics have to be used (Caspar et al. 2015;
Dinger et al. 2016b; Meyer 2016; Meyer et al. 2015; Michely et al. 2015; Wagmann et
al. 2016). Concerning MAO inhibition by drugs of abuse, only few studies were
published. Cerletti et al. identified AMT, 5-IT, and four positional isomers as inhibitors
of guinea pig MAO in an assay based on the manometric determination of the
oxygen uptake of guinea pig liver homogenates with serotonin as substrate (Cerletti
et al. 1968). Herraiz and Brandt confirmed 5-IT as highly selective and potent
inhibitor of recombinant human MAO-A by measuring the KYN deamination using
high-performance liquid chromatography coupled to diode array detection (Herraiz
and Brandt 2014). Steuer et al. investigated the MAO inhibition potential of MDMA
and its metabolites on the deamination of the neurotransmitters dopamine and
serotonin using HILIC-MS/MS. MDMA and MDA were identified as inhibitors of
recombinant human MAO-A (Steuer et al. 2016). However, MAO inhibition is not
always an effect of the abused drug itself. Users also intentionally consume MAO
inhibitors in order to enhance the activity of substances such as N,N-
dimethyltryptamine (DMT) that otherwise would be metabolically inactivated.
16

Ayahuasca for example, a hallucinogenic beverage, is the combination of DMT and
the -carbolines MAO inhibitors harmine and harmaline (Araujo et al. 2015).
5. Conclusion
The presented study was the first to describe the MAO inhibition for a broad
range of NPS of the alpha-methylated tryptamine type. The workup and analysis
based on HILIC-HR-MS/MS were validated according to international guidelines. Due
to its high sensitivity, only minimal amounts of recombinant MAO enzymes were
needed, thus, reducing the risk of non-specific protein binding, as well as material
costs. All tested AMT analogs inhibited MAO-A activity, whereas four compounds
inhibited MAO-B as well. 7-Me-AMT was identified as potent and competitive inhibitor
of MAO-A. MAO inhibition by AMT and its analogs are expected to be clinically
relevant. Plasma levels of other MAO substrates, for example neurotransmitters or
drugs, could increase, especially if these substances were selective MAO-A
substrates. Further clinical studies are warranted to facilitate a more complete
assessment.
Conflict of interest
The authors declare that they have no conflict of interest.
Acknowledgements
The authors like to thank Achim T. Caspar, Julia Dinger, Andreas G. Helfer,
Sascha K. Manier, Julian A. Michely, Lilian H. J. Richter, and Armin A. Weber for
their support and fruitful discussion.
.
17

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20

Legends to the figures
Fig. 1 Chemical structures of tested alpha-methyltryptamines. Structural variations in
comparison to AMT were given in red (For interpretation of the reference to color in
this figure legend, the reader is referred to the web version of the article).
Fig. 2 Deamination of kynuramine catalyzed by MAO-A or MAO-B providing an
aldehyde, followed by non-enzymatic condensation to 4-hydroxyquinoline.
Fig. 3 Initial inhibition screening results using 10 µM each of the inhibitor. Percentage
of activity represented the percentage of metabolite formation in relation to control
incubations without inhibitor (100%). Values are expressed as mean and were tested
for significance (n = 3, ***, P < 0.001, **, P < 0.01, *. P < 0.1 for 4-hydroxyquinoline
formation in incubations with the test inhibitor versus 4-hydroxyquinoline formation in
control incubations).
Fig. 4 Michaelis-Menten plot (left part) of the deamination of kynuramine in absence
or presence of three given concentrations of 7-Me-AMT for determination of the
inhibition constant. Data points represent means and ranges (error bars) of duplicate
measurements. Transformation of data to a Lineweaver-Burk plot (right part) for
determination of the inhibition mode.
Fig. 5 Inhibition of MAO activity in S9 fraction by different inhibitor concentration
levels (level 3 represented highest inhibitor concentration). Percentage of activity
represented the percentage of metabolite formation in relation to control incubations
without inhibitor (100%). Values are expressed as mean and were tested for
significance (n = 3, ***, P < 0.001, **, P < 0.01, *. P < 0.1 for 4-hydroxyquinoline
formation in incubations with the test inhibitor versus 4-hydroxyquinoline formation in
control incubations).
21

Table 1 Test compounds, reference plasma concentrations, and IC50 values
(percentage errors in brackets) of AMT-type new psychoactive substances (NPS)
and known MAO inhibitors. Plasma concentrations in µM were calculated from the
published data (in mg/L or µg/L). Probable clinically relevant IC50 values for AMT-type
NPS are given in bold. PM: post-mortem cases
Test compounds
Reference plasma concentrations,
IC50 values, µM
MAO-A
µg/L
µM
MAO-B
AMT-type NPS
AMT
440
2.5
0.38 (8)
(Ferec et al. 2015)
160-1,300 (PM)
(Elliott and Evans 2014)
0.9-7.5
4-MeO-AMT
5-MeO-AMT
1-Me-AMT
2-Me-AMT
5-Me-AMT
7-Me-AMT
5-F-AMT
*
*
*
*
*
*
*
*
*
*
*
*
*
0.8-6.4
0.8-6.4
0.9-6.9
0.9-6.9
0.9-6.9
0.9-6.9
0.8-6.8
0.8-6.8
0.8-6.2
0.6-5.2
0.8-6.3
0.6-4.9
0.6-5.2
1.4 (11)
31 (4)
51 (3)
11 (7)
1.5 (13)
0.049 (2)
0.45 (23)
1.8 (19)
0.25 (7)
1.3 (7)
376 (1)
126 (2)
82 (2)
6-F-AMT
5-Cl-AMT
5-Br-AMT
5-F-2-Me-AMT
1-Me-2-Ph-AMT
2-Ph-AMT
Known inhibitors
5-IT
4.1 (7)
223 (3)
166 (2)
3.7 (18)
15-590
0.1-3.4
0.20 (1)
(Backberg et al. 2014)
700-5,100 (PM)
(EMCDDA 2014)
4.0-29
Selegiline
Harmine
0.3-1.5
(Barrett et al. 1996)
0.002-0.01
0.017 (2)
36-222
0.2-1.0
0.006 (2)
(Baselt 2008)
1.0-15.6
0.005-0.07
(Oliveira et al. 2012)
22

Harmaline
Yohimbine
2.7-15.7
(Oliveira et al. 2012)
0.01-0.07
0.01-0.5
0.011 (2)
6.0 (7)
3.7-171
(Baselt 2008)
* No plasma concentrations reported, thus concentrations estimated according to the
structure-related compound AMT
23