Brain Microdialysis Coupled to LC-MS/MS Revealed That CVT-10216, a Selective Inhibitor of Aldehyde Dehydrogenase 2, Alters the Neurochemical and Behavioral Effects of Methamphetamine

Article abstract of DOI:10.1021/acschemneuro.1c00039

Methamphetamine (MA), a potent central nervous system stimulant, mainly affects the brain dopaminergic and serotoninergic systems. Monoamine oxidase, catechol-O-methyltransferase, and aldehyde dehydrogenase 2 (ALDH2) are important enzymes in the metabolis

Full text of DOI:10.1021/acschemneuro.1c00039

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Research Article
Brain Microdialysis Coupled to LC-MS/MS Revealed That CVT-10216,
a Selective Inhibitor of Aldehyde Dehydrogenase 2, Alters the
Neurochemical and Behavioral Effects of Methamphetamine
Seungju Kim,# Eun Young Jang,# Sang-Hoon Song, Ji Sun Kim, In Soo Ryu, Chul-Ho Jeong,*
and Sooyeun Lee*
Cite This: ACS Chem. Neurosci. 2021, 12, 1552−1562
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ABSTRACT: Methamphetamine (MA), a potent central nervous
system stimulant, mainly affects the brain dopaminergic and
serotoninergic systems. Monoamine oxidase, catechol-O-methyl-
transferase, and aldehyde dehydrogenase 2 (ALDH2) are
important enzymes in the metabolism of dopamine (DA) and
serotonin (5-HT); however, the role of ALDH2 in MA addiction
remains unclear. This study focused on the real-time changes in
DA, 5-HT, and their metabolites, including 3,4-dihydroxyphenyl-
acetic aldehyde and salsolinol, which are metabolites directly
related to ALDH2, to examine the effects of the inhibition of
ALDH2 on hyperlocomotion induced by MA. Locomotor activity
was evaluated in rats after administration of MA and/or CVT-
10216 (a selective ALDH2 inhibitor). Moreover, the simultaneous
quantification of DA, 5-HT, and their metabolites in brain microdialysates of the rats was performed using a derivatization-assisted
LC-MS/MS method after full validation. The validation results proved the method to be selective, sensitive, accurate, and precise,
with acceptable linearity within calibration ranges. Intraperitoneal (i.p.) administration of 10 or 20 mg/kg of CVT-10216
significantly decreased MA-induced hyperlocomotion (1 mg/kg, i.p.). The analytical results of rat brain microdialysates
demonstrated that the administration of CVT-10216 significantly downregulated DA levels, which were increased upon exposure to
MA. Moreover, the increase in 3-methoxytyramine levels following coadministration of CVT-10216 and MA could play a potential
role in antagonizing the hyperlocomotion induced by MA. All of these findings suggest that the inhibition of ALDH2 protects
against MA-induced hyperlocomotion and has therapeutic potential in MA addiction.
KEYWORDS: methamphetamine, drug addiction, aldehyde dehydrogenase 2, locomotor activity, dopamine, serotonin
The dopaminergic and serotoninergic systems in the brain
play an important role in the regulation of several aspects of
behaviors, such as motivation, reinforcement, mood, move-
ment, and various cognitive functions. Thus, the in vivo
measurement of these systems has received attention in the
field of brain pathology.6−9 The action of DA and serotonin
(5-HT), which are released from synaptic vesicles, is exerted
via the interaction with postsynaptic DA and 5-HT receptors,
respectively, and is lost by neuronal reuptake or degradation.
DA and 5-HT are degraded by MAO, catechol-O-methyl-
INTRODUCTION
■
Methamphetamine (MA), a psychostimulant with serious
abuse potential, has become an agent causing critical health
issues because of its increasing use and mortality in recent
years. The clinical diagnosis, without self-report, and
pharmacological treatment of MA use disorder remain
challenging despite the discovery of potential biomarkers and
therapeutic targets.1−3 MA provokes neurotoxic effects by the
release of monoamine neurotransmitters, mainly dopamine
(DA), and the inhibition of their reuptake. It also affects the
metabolizing or synthesizing enzymes of DA by inhibiting
monoamine oxidase (MAO) activity and increasing the activity
and expression of tyrosine hydroxylase (TH).1,3 The increase
in DA levels in the brain by exposure to psychostimulants,
including MA, contributes to their locomotor-activating and
rewarding properties, which has been proven in previous
animal studies.4,5
Received: January 20, 2021
Accepted: April 8, 2021
Published: April 19, 2021
© 2021 The Authors. Published by
American Chemical Society
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Figure 1. Pathways of dopamine (A) and serotonin (B) metabolism. *, target analytes.
transferase (COMT), and aldehyde dehydrogenase 2
(ALDH2). DA degrades in two ways: (1) transformation to
3-methoxytyramine (3-MT), followed by homovanillic acid
(HVA), and (2) transformation to 3,4-dihydroxyphenylacetic
aldehyde (DOPAL), followed by 3,4-dihydroxyphenylacetic
acid (DOPAC) and finally HVA. 5-HT is metabolized to 5-
hydroxyindoleacetic acid (5-HIAA) by MAO. Moreover, the
formation of DA derivatives is an alternative method of
transformation.10−12 In particular, salsolinol (SAL), the
endogenous condensation product of DA with acetaldehyde
or pyruvic acid, is regarded as an inhibitor of MAO and
COMT, as well as that of TH and L-tryptophan hydroxylase,
which are important enzymes for the synthesis of DA and 5-
HT, respectively.13
ALDH2 is well-known for its role in ethanol metabolism.
Recently, the role of ALDH2 has been emphasized in various
diseases including cardiovascular diseases, diabetes, neuro-
degenerative diseases, stroke, and cancer,14 and ALDH2 has
been proposed as a promising therapeutic target for these
diseases.15 The pharmacological feasibility of both the
activators and the inhibitors of ALDH2 has been investigated.
In particular, DOPAL, a toxic aldehydic metabolite of DA in
the brain, has been suggested as a causative compound of
Parkinson’s disease; thus, an increased activity of ALDH2
provokes the conversion of DOPAL to DOPAC, which can
reduce toxic aldehyde load.14,16 The contribution of another
ALDH2-involved DA derivative, SAL, to Parkinson’s disease or
alcohol addiction as well as behavioral changes in animals has
also been reported despite of the fact that it cannot cross the
blood−brain barrier of exogenous SAL easily.13,17
The inhibition of ALDH2 is applied for the treatment of
alcohol addiction, for which disulfiram (an irreversible ALDH
inhibitor) is the approved medicine despite the fact that it
inhibits ALDH1A1 more potently than does ALDH2.18
A
previous study reported that 3-{[(3-{4-[(methylsulfonyl)-
amino]phenyl}-4-oxo-4H-chromen-7-yl)oxy]methyl}benzoic
acid (CVT-10216), a highly selective, reversible inhibitor of
ALDH2, prevented alcohol-seeking behavior and alcohol-
induced increases of DA levels in nucleus accumbens in
rats.19 The anxiolytic effects of CVT-10216 were also proved in
four unrelated rodent models: endogenous anxiety-like
̈
behavior in naive Fawn-Hooded rats, repeated alcohol-
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withdrawal-induced anxiety, restraint stress-induced anxiety,
and drug-induced anxiety Sprague−Dawley rats.20 Another
previous study demonstrated that ALDH2 inhibition by CVT-
10216 reduced cocaine-seeking behavior in rats and decreased
cocaine-induced DA production in vitro and in vivo.21
Brain microdialysis is a real-time in vivo sampling technique
for the extracellular brain fluid containing synaptically released
neurotransmitters and their metabolites as well as compounds
from nonsynaptic sources.12,22 The concentrations of neuro-
transmitters are regulated in the brain by reuptake and
metabolism. Although specific receptors or transporters to
regulate the actions of most of the neurotransmitters’
metabolites have not been identified, the extracellular levels
of DA, 5-HT, and their metabolites have often been measured
to investigate the effects of psychoactive drugs in the brain in
previous studies.23−26 Among the metabolites of DA, 3-MT
and SAL, which are found in the in the synaptic cleft, are
considered to be neuromodulators.27,28 Brain microdialysis
combined with mass spectrometry (MS) enables the accurate
and sensitive monitoring of changes in neurotransmitters
under the conditions of brain diseases or exposure to drugs
acting on the central nervous system.12 In previous studies,
quantitative analytical methods for DA, 5-HT, and their main
metabolites using liquid chromatography-tandem mass spec-
trometry (LC-MS/MS) in rat brain microdialysates have been
developed and validated;7,22,29,30 however, ALDH2-involved
metabolites, such as DOPAL and SAL, which are essential for
understanding brain pathology, were not investigated.
Figure 2. Total traveled distance over 60 min after administration of
MA and/or CVT-10216. Error bars represent SEM (n = 6, *<0.05,
#
**<0.01 vs the 1% CMC + saline group; < 0.05, ##< 0.01 vs the 1%
CMC + MA group; ns: not significant; one-way ANOVA followed by
a Tukey’s multiple comparisons post hoc test). CVT 10, CVT 20, and
MA represent intraperitoneal injections of 10 and 20 mg/kg of CVT-
10216 and 1 mg/kg of methamphetamine, respectively.
knowledge, this is the first study to evaluate the relationship
between behavioral changes caused by MA and ALDH2
inhibition.
LC-MS/MS Method Validation. The benzoylation of the
analytes produced retention times of 6−9 min, where no
interference was observed, as shown in Figure 3A. Figure 3B
shows the chromatograms obtained for the analytes in the
artificial cerebrospinal fluid (aCSF) spiked with limit of
quantification (LOQ) levels. Figure 3C displays those in a
rat brain microdialysate sample in which the endogenous
concentrations of DA, 5-HT, and their metabolites were clearly
detected. The results of other method validation parameters
are summarized in Table 1. The LOD values were 0.025 ng/
mL for all analytes except for 5-HIAA (0.5 ng/mL). The LOQ
values were 0.025 ng/mL for DA, DOPAL, DOPAC, 3-MT,
SAL, and 5-HT; 0.1 ng/mL for HVA; and 1 ng/mL for 5-
HIAA. Acceptable linearity was created within each calibration
range with the r value of each analyte ranging from 0.9906 to
0.9996. To improve the accuracy around the LOQ level,
weight factors of 1/x and 1/x2 were applied to 5-HIAA and
HVA, and the rest of the analytes, respectively. The mean
values of the matrix effect of each analyte at low, medium, and
high concentrations ranged from 89% (DOPAL) to 116%
(SAL). Minor ion suppression or enhancement was observed
with a coefficient of variation (CV) of less than 20% (range:
1.4−18.2%). The CVs of repeatability and intermediate
precision ranged from 1.0% (DOPAC) to 18.0% (DOPAL)
and 2.1% (DOPAC) to 18.8% (DOPAL), respectively, and
were within acceptable limits (i.e., < 20% CV at low
concentrations and < 15% CV at medium and high
concentrations, respectively). The accuracy of this method
ranged from −6.3 to 9.4% bias at the three levels and was
within acceptable limits (i.e., < 20% bias at low concen-
trations and 15% bias at medium and high concentrations).
According to the results of sample storage stability, quality
control (QC) samples stored at −80 °C were stable for up to
30 days with a stability range of 86−110%. Processed stability
was investigated stability for 12 h using QC samples spiked at
low and high concentrations. As a result, the stability after the
process showed no tendency of negative slope with a
significant difference from 0 (data not shown). Therefore,
In the present study, we focused on the real-time changes of
the DA and 5-HT metabolism to investigate the neurochemical
and behavioral effects of the inhibition of ALDH2 on
hyperlocomotion induced by MA. For this, a derivatization-
assisted LC-MS/MS method was developed and fully validated
for the simultaneous quantification of DA, 5-HT, and their
metabolites (DOPAL, DOPAC, 3-MT, HVA, 5-HT, 5-HIAA,
and SAL) (Figure 1) in nucleus accumbens (NAc) micro-
dialysates from rats.
RESULTS AND DISCUSSION
■
Changes of the MA-Induced Hyperlocomotion by
ALDH2 Inhibition. To investigate whether MA-induced
hyperlocomotion could be affected by ALDH2 inhibition, we
compared the locomotor activities of rats following the
administration of MA only with those following the
coadministration of CVT-10216 and MA. As shown in Figure
2, the administration of 1 mg/kg of MA significantly increased
locomotor activity as compared to the control group in which
1% carboxymethylcellulose (CMC) with saline vehicle was
administered. The administration of 20 mg/kg of CVT-10216
only did not induce any change in locomotion. As no changes
were observed in locomotion following the administration of
10 and 20 mg/kg of CVT-10216 in our preliminary test, the
higher dose (20 mg/kg) was chosen for comparison. The
coadministration of 10 or 20 mg/kg of CVT-10216 and 1 mg/
kg of MA significantly decreased MA-induced locomotor
activity. In a previous study, the administration of CVT-10216
only did not cause any behavioral changes in conditioned place
preference (CPP) in Long Evans rats.19 On the other hand, it
reduced intravenous cocaine self-administration, cocaine-
primed or cue-induced reinstatement, and MA-induced
reinstatement in Sprague−Dawley rats.21 This implies that
ALDH2 inhibition using CVT-10216 has potential in the
treatment and prevention of drug addiction. To the best of our
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Figure 3. Representative chromatograms of DA, 5HT, and their metabolites in a blank aCSF sample (A), a fortified aCSF sample (LOQ level, B),
and a microdialysate collected from a normal rat (C).
the entire analytical process was performed under optimum
conditions.
sample introduction or preparation techniques such as direct
injection, solid phase extraction, online enrichment, and
chemical derivatization using benzoyl chloride (BzCl) and
dansyl chloride were used to overcome low sensitivity.7,29−33
Most neurotransmitters retain weakly on reversed-phase
columns because of their high polarity. In this study, DA, 5-
HT, and their metabolites obtained in rat brain microdialysates
were analyzed by derivatization into relatively nonpolar
LC-MS/MS is an advanced analytical method that is widely
used to analyze biological samples owing to its advantages such
as high selectivity, sensitivity, and analytical throughput. DA, 5-
HT, and their metabolites are low-molecular-weight polar
analytes; thus, separation and detection using the LC-MS
system are not easy tasks. According to previous studies,
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a
Table 1. Summary of Validation Data
matrix effect
(%)
calibration regression concentrations of
range coefficient quality control
(ng/mL) (ng/mL) (ng/mL) (r, mean) samples (ng/mL) mean
intermediate
sample storage
b
c
compound
name
LOD
LOQ
repeatability
(CV, %)
precision
accuracy
stability (30
CV
8.3
(CV, %)
(bias, %) days, −80 °C)
DA
0.025
0.025
0.025
0.025
0.025
0.025
0.025
0.5
0.025
0.025
0.025
0.025
0.1
0.025−10
0.025−10
0.025−10
2.5−100
2.5−100
0.025−10
0.025−10
2.5−250
0.9943
0.9996
0.9906
0.9950
0.9970
0.9952
0.9939
0.9978
0.025
1.0
10
0.025
0.5
1.0
0.025
1.0
10
2.5
50
100
2.5
50
100
0.025
1.0
10
0.025
1.0
10
2.5
104
15.3
2.6
7.7
11.4
1.7
2.7
18.0
11.5
7.3
6.0
1.0
3.1
7.2
4.4
7.8
14.2
9.8
7.7
13.9
5.9
4.9
16.6
5.5
8.7
11.9
3.3
2.6
18.8
13.5
9.5
10.2
2.1
5.3
8.0
4.1
9.4
13.7
14.1
6.0
13.3
8.0
−5.8
−2.1
3.5
−6.2
−0.4
−0.4
6.2
−6.2
−2.4
3.9
97
115
90
11.4
11.9
92
97
3-MT
DOPAL
DOPAC
HVA
95
89
8.4
18.2
101
104
101
110
6.6
6.0
90
104
0.6
98
101
1.4
5.8
−3.9
−2.5
−1.2
5.3
7.0
−4.1
9.4
−1.0
−5.2
1.9
−6.3
−0.8
−1.0
105
103
101
105
4.2
10.1
101
104
SAL
0.025
0.025
1.0
116
93
12.1
12.4
104
110
5-HT
5-HIAA
110
103
9.7
4.2
8.0
8.7
3.7
5.7
97
86
6.5
3.9
3.3
50
250
101
1.7
94
a
Bz; Benzoylated, DA, dopamine; DOPAL, 3,4-dihydroxyphenylacetaldehyde; DOPAC, 3,4-dihydroxyphenylacetic acid; 3-MT, 3-methoxytyr-
amine; HVA, homovanillic acid; SAL, salsolinol; 5-HT, serotonin; 5-HIAA, 5-hydroxyindoleacetic acid; LOD, limit of detection; LOQ, limit of
b
c
quantification, CV; coefficient of variation. Within-day variation. Combination of within- and between-day variation.
analytes by benzoylation of the primary and secondary amine
groups. As a result, the retention and resolution were improved
in the reverse phase column. Benzoylation using BzCl has the
advantages of simple sample preparation and short reaction
time compared to using dansyl chloride.34 This method that
can simultaneously analyze not only DOPAC and HVA, which
were the main metabolites monitored in DA metabolism in
previous studies,7,22,30 but also DOPAL and SAL, was
developed and proven to be a reliable method through
successful validation.
Changes of the MA-Induced DA and 5-HT Metabo-
lism by ALDH2 Inhibition. The developed method was
successfully applied to determine the concentrations of DA, 5-
HT, and their metabolites in rat brain microdialysates. The
kg) did not affect the levels of DOPAL, DOPAC, HVA, SAL,
5-HT, and 5-HIAA. MA induced significant increase in the
concentrations of DA (at 80 and 100 min), 3-MT (at 100
min), and HVA (at 20 min). Notable fluctuations were
observed in the concentrations of 5-HT and 5-HIAA, with 5-
HIAA showing a significant increase in levels at 80 min (p <
0.05).
Administration of CVT-10216 (10 or 20 mg/kg, i.p.) tended
to reduce DA concentrations, accentuated by exposure to MA,
compared to the basal level and showed a significant decrease
at 80 and 100 min (p < 0.05). SAL concentration was not
changed by CVT-10216 or MA exposure but was transiently
and significantly decreased by coadministration of CVT-10216
(10 mg/kg) and MA at 80 min compared to administration of
MA only. Notably, the concentration of 3-MT significantly
increased between 20 and 60 min following coadministration
of 10 mg/kg of CVT-10216 and MA, compared to
administration of vehicle, and reached a peak at 40 min (p <
0.05). The coadministration of 20 mg/kg of CVT-10216 and
MA also significantly increased the concentration of 3-MT at
40 min (p < 0.05). However, levels of DOPAL, DOPAC, and
HVA were not significantly affected by exposure to either
CVT-10216 or MA. These also showed high variations
between individuals despite employment of the analytical
method, which was fully validated in terms of matrix effects
and reproducibility. A higher number of animals might be
needed to clarify the effects of the treatments on the
concentrations of these metabolites. The coadministration of
10 mg/kg of CVT-10216 and MA induced significant increase
basal concentrations (mean
standard error of the mean
[SEM]) of the analytes in NAc microdialysates from rats (n =
4−6) in the 1% CMC + saline group were as follows: DA, 0.06
0.02 ng/mL; 3-MT, 0.02 0.01 ng/mL; DOPAL, 1.4 1.1
ng/mL; DOPAC, 18.6 5.1 ng/mL; HVA, 10.2 1.6 ng/mL;
SAL, 0.06 0.01 ng/mL; 5-HT, 0.02 0.01 ng/mL; and 5-
HIAA, 9.9
1.5 ng/mL. The real-time changes in
concentrations of DA, 5-HT, and their metabolites in rat
brain microdialysates after exposure to MA and/or CVT-
10216 are shown in Figure 4. CVT-10216 administration (20
mg/kg, i.p.) induced a significant decrease in DA concen-
trations at all times, except at the 100 min point, after injection
(p < 0.05). It also induced a significant temporal increase in
levels of 3-MT at 100 min compared to that in rats with vehicle
exposure (p < 0.05). Administration of CVT-10216 (20 mg/
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Figure 4. Changes in the concentrations of dopamine, serotonin, and their metabolites in rat microdialysates collected after administration of CVT-
10216 and/or methamphetamine. Error bars represent SEM (n = 4−6; *p < 0.05 vs 1% CMC + saline group; #< 0.05 vs 1% CMC + MA; two-
way ANOVA followed by a Fisher’s LSD post hoc test). Only the following interaction between drug and sampling time were significant: DA, p =
0.0016, F (DFn, DFd) = F (24, 115) = 2.322; 3-MT, p < 0.0001, F (DFn, DFd) = F (24, 111) = 3.612; others were not significant. CVT 10, CVT
20, and MA represent intraperitoneal injections of 10 and 20 mg/kg of CVT-10216 and 1 mg/kg of methamphetamine, respectively. The
significance of each group was expressed in the same color as the line. DA, dopamine; 3-MT, 3-methoxytyramine; DOPAL, 3,4-
dihydroxyphenylacetic aldehyde; DOAPC, 3,4-dihydroxyphenylacetic acid; DOPAL, 3,4-dihydroxyphenylacetic aldehyde; DOAPC, 3,4-
dihydroxyphenylacetic acid; HVA, homovanillic acid; SAL, salsolinol; 5-HT, serotonin; 5-HIAA, 5-hydroxyindoleacetic acid.
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in the concentrations of 5-HT at 20 and 40 min (p < 0.05),
compared to vehicle administration. However, exposure to 20
mg/kg of CVT-10216 did not induce any significant change in
the levels of 5-HT.
hyperactivity with a concomitant set of abnormal movements,
which are partially mediated by trace amine associated receptor
1.37 Paradoxically, it has also been proposed as an inhibitory
regulator of the behavioral activation produced by psychosti-
mulants such as cocaine.38 The authors of that study
emphasized findings on the decrease in the ratio of DA/3-
MT in an in vivo microdialysis study following the
administration of 1-methyl-1,2,3,4-tetrahydorisoquinoline (1-
MeTIQ) in cocaine-sensitized rats. The inhibitory effects of
1MeTIQ on morphine addiction and cocaine-induced
behavior stimulation have been previously reported.38 Our
results imply that a decrease in the DA levels as well as an
increase in the 3-MT levels following coadministration of
CVT-10216 and MA could play important roles in
antagonizing the hyperlocomotion induced by MA.
Exposure to psychomotor stimulants, including MA,
increases DA transmission in the mesocorticolimbic circuit,
which comprises dopaminergic cell bodies in the ventral
tegmental area; these bodies project to the limbic and cortical
regions, including the NAc, dorsal striatum, amygdala,
hippocampus, and prefrontal cortex. Functional interactions
between the medial prefrontal cortex, NAc, and ventral
tegmental area have an important role in locomotion and
reward.39−41 It has also been demonstrated that alterations in
DA transmission in the medial prefrontal cortex provoke
sensitization to psychomotorstimulants.42−44 Among the brain
regions in the mesocorticolimbic circuit, we focused on DA
metabolism in the NAc after administration of MA. It has been
previously reported that 3-MT injection into the medial
prefrontal cortex causes behavioral changes.45 3-MT is also
known as the major metabolite of released DA in not only the
frontal cortex but also the NAc and striatum.46 In a previous
study, MA and amphetamine increased 3-MT concentration in
both the striatum and NAc.47 Furthermore, Yoshikawa et al.
showed a relationship between changes in behavior and 3-MT
level in the NAc, which were induced by a challenge of MA.48
The coadministration of CVT-10216 (10 mg/kg) and MA
induced the release of 5-HT at 20 and 40 min and did not
affect the concentration of its metabolite, 5-HIAA. However,
the administration of MA alone did not affect the 5-HT and 5-
HIAA concentrations, except for a temporal increase in the
concentration of 5-HIAA at 80 min. 5-HT is metabolized by
MAO to 5-hydroxy-indole-3-acetaldehyde (5-HIAL) and then
by ALDH2 to 5-HIAA.49 It was assumed that simultaneous
inhibition of ALDH2 and MAO by CVT-10216 and MA,
respectively, might be responsible for the increased levels of 5-
HT observed. Further studies on the relationship between
ALDH2 suppression and 5-HT metabolism are also required
to better understand the role of ALDH2 in the neural system.
This study presents the analytical data of the analysis of rat
brain microdialysates at each collection time and provides a
snapshot of real-time changes in the targeted analytes.
Exposure to MA ultimately induced significant increase in
DA concentrations with temporal increase in the levels of 3-
MT, HVA, and 5-HIAA. It was previously reported that the
concentrations of DA in rat NAc microdialysates, collected at
20 min intervals for 2 h following i.p. injection of MA every
hour with a gradually increasing dose (0.3, 1, and 3 mg/kg),
significantly increased at most time points;23 this is similar to
results obtained in the present study. Unexpectedly, the
administration of CVT-10216 did not induce any alteration in
the concentrations of the ALDH2-related metabolites,
DOPAC, DOPAL, or SAL, but caused a significant decrease
in the concentrations of DA. The downregulation of DOPAL
by the inhibition of ALDH in PC12 cells was previously
reported,35 but few animal studies have been conducted. In
other previous studies, DA levels in PC12 cells (0.1, 0.3, and
1.0 μM)21 and Long−Evans rats (7.5 and 15 mg/kg, i.p.)
without induction of CPP19 were not changed by exposure to
CVT-10216. In the present study, a higher dose of CVT-10216
(20 mg/kg) decreased the release of DA without stimulation of
locomotion.
Coadministration of CVT-10216 and MA significantly
decreased DA levels compared to the administration of MA
only. Changes in the levels of ALDH2-related metabolites were
not observed with CVT-10216 and MA coadministration. The
decrease in MA-induced hyperlocomotion by CVT-10216 may
be linked to its ability to reduce MA-induced DA extracellular
levels. Yao et al.21 demonstrated that CVT-10216 produced
tetrahydropapaveroline (THP), a tetrahydroisoquinolinic
(TIQ) derivative formed through the condensation of DA
and DOPAL, which inhibits phosphorylated TH and reduces
DA levels via negative-feedback signaling. TIQs are products of
the condensation of biogenic amines (e.g., DA) and electro-
philic compounds (e.g., acetaldehyde); SAL is also a TIQ.28
Even though the changes in SAL concentrations induced by
the administration of CVT-10216 and/or MA (except for the
temporal inhibition at one time point compared to the
administration of MA) were investigated in the present study,
they fluctuated significantly in live animals, as shown on the y-
axis in Figure 4. Additional research is needed to investigate
the relationship between ALDH2 inhibition and DA
metabolism altered by MA (e.g., the effects of coadministration
of ethanol and an ALDH2 inhibitor on DA metabolism altered
by MA).
CONCLUSIONS
Notably, the coadministration of CVT-10216 and MA
significantly increased 3-MT levels compared to the admin-
istration of the vehicle, while the administration of CVT-10216
or MA alone did not affect them, except for one time point
(100 min) following the administration of MA. MAO could be
saturated by ALDH2 inhibition, which could upregulate the
alternative metabolism of DA to 3-MT by COMT. 3-MT is a
known neuromodulator that controls behavior. A previous
study demonstrated that it binds to rat noradrenergic cortical
α1 and striatal D1 and D2 receptors in the nanomolar
concentration range and to cortical α2 adrenoceptors at low
micromolar concentrations.36 Additionally, it has been
previously reported that 3-MT causes temporary mild
■
In the present study, we focused on the real-time changes in
DA and 5-HT metabolism to investigate the effects of the
inhibition of ALDH2 on MA-induced hyperlocomotion. To do
this, a derivatization-assisted LC-MS/MS method was
developed and successfully validated for the simultaneous
quantification of DA, 5-HT, and their metabolites (DOPAL,
DOPAC, 3-MT, HVA, 5-HT, 5-HIAA, and SAL) in rat brain
microdialysates. The analytical results of rat brain micro-
dialysates demonstrated that the administration of CVT-10216
significantly downregulated DA levels, which were increased
upon exposure to MA. Moreover, the increase in 3-MT levels
following coadministration of CVT-10216 and MA plays a
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potential role in antagonizing the hyperlocomotion induced by
MA. These findings suggest that the inhibition of ALDH2
protects against MA-induced hyperlocomotion and that the
measurement of extracellular levels of both DA and 3-MT in
the brain might provide a clue for the treatment of MA
addiction.
METHODS
■
Chemicals and Reagents. (+)-MA hydrochloride and CMC
were purchased from Sigma-Aldrich (St. Louis, MO, United States),
and CVT-10216 was purchased from Tocris Bioscience (Bristol, UK).
In the present study, 1% CMC was used as the suspending agent for
CVT-10216. Water and acetonitrile were of high-performance LC
grade. Sodium chloride, potassium chloride, calcium chloride
dihydrate, magnesium chloride hexahydrate, sodium phosphate
dibasic, and 1.0 M phosphoric acid solution used to prepare aCSF
were purchased from Sigma-Aldrich (St. Louis, MO, United States).
DA, DOPAC, 3-MT, HVA, 5-HT, 5-HIAA, DA-d4, DOPAC-d5, HVA-
d5, and 5-HIAA-d5 were also purchased from Sigma-Aldrich. 5-HT-d4
was purchased from TLC PharmaChem (Vaughan, Ontario, Canada).
DOPAL, 3-MT, and SAL were purchased from Cayman (Ann Arbor,
MI, United States), Cambridge Isotope Laboratories, Inc. (Tewks-
bury, MA, United States), and Santa Cruz Biotechnology (Dallas, TX,
United States), respectively. The aCSF (pH 7.4) prepared was a
mixture of 150 mM sodium chloride, 3.0 mM potassium chloride, 1.4
mM calcium chloride, and 0.8 mM magnesium chloride in 10 mM
phosphate buffer. The analytical stock solutions (1 mg/mL) for DA,
5-HT, and their metabolites were prepared in 1 mM ascorbic acid in a
1:1 mixture of water and methanol to prevent oxidation and stored at
−80 °C. A working standard solution consisting of DA, DOPAL,
DOPAC, 3-MT, HVA, 5-HT, and 5-HIAA (250 ng/mL for each) and
a working internal standard solution (DA-d4: 1 μg/mL; DOPAC-d5:
80 ng/mL; 3-MT-d4: 20 ng/mL; HVA-d5: 100 ng/mL; 5-HT-d4: 1
μg/mL and 5-HIAA-d5: 200 ng/mL for each) were prepared in aCSF
from the corresponding stock solutions and stored at −80 °C before
analysis. The BzCl and sodium carbonate used for derivatization were
purchased from Sigma-Aldrich. Fresh BzCl was prepared before
analysis.
Animals. Adult male Sprague−Dawley rats (Daehan Animal,
Seoul, Republic of Korea) of 310−350 g were housed individually in
cages in the laboratory animal facility under conditions of controlled
humidity (60 2%) and temperature (22 2 °C), with a 12 h light/
dark cycle (lights on at 7:00 AM). All rats were provided ad libitum
access to food and water. The experiments were performed in
accordance with the scientific research guidelines and regulations of
the Korea Institute of Toxicology. All procedures were approved by
the Institutional Animal Care and Use Committee of the Korea
Institute of Toxicology, Daegeon, Republic of Korea.
Measurement of Locomotor Activity. Locomotor activity was
measured with a video-tracking system (Ethovision, Nodus
Information Technology BV, Wageningen, The Netherlands) that
provided automatic measures of travel distance (cm). In summary,
each rat was placed in a square open-field box made of black acrylic
(40 × 40 × 45 cm) in a dimly lit room. On the test day, the animals
were habituated for 1 h in the open field. After measuring basal
activity for 30 min, the rats were administered vehicle (1% CMC, 1
mL/kg) or CVT-10216 (10 or 20 mg/kg, 1 mL/kg, i.p.), and 15 min
later, saline or MA (1 mg/kg, 1 mL/kg i.p.) was administered.
Thereafter, locomotor activity was measured for 1 h [Figure 5(A)].
Microdialysis. The microdialysis probe guide cannulae (CMA 11;
CMA Microdialysis AB, Kista, Sweden) were stereotaxically
implanted into the rat brains under sodium pentobarbital anesthesia
(50 mg/kg, i.p.) The rats had a recovery period of 6 days after
surgery. The microdialysis probes with a membrane length of 2 mm
(cutoff, 6 kDa; CMA Microdialysis AB) were inserted through the
guide cannulae into the NAc shell (anteroposterior +1.7 mm,
mediolateral +0.8 mm, from bregma; dorsoventral −6.0 mm, from
skull) of the unanesthetized rats and perfused with aCSF at a flow rate
of 1.5 μL/min (120 min), using a microinjection pump (CMA 100;
Figure 5. Schedule for locomotor activity measurement (A) and
microdialysate collection (B) after administration of CVT-10216 and/
or MA. ↓, time points at which rat microdialysate collection was
started; MA, methamphetamine.
CMA Microdialysis AB). After the stabilization period, six baseline
samples were collected at 20 min intervals into microcentrifuge tubes
before drug administration. After vehicle, CVT-10216 (10 or 20 mg/
kg, i.p.), and/or MA (1 mg/kg, i.p.) were administered, microdialysis
samples were collected every 20 min. The timetable for microdialysate
collection from rat brain is shown in Figure 5(B). The location of the
microdialysis probe was verified histologically at the end of the
microdialysis experiment.
LC-MS/MS Analysis of Rat Brain Microdialysates. Sample
Preparation. For the preparation of quality control samples, 25 μL of
aCSF containing each corresponding concentration of analytes was
derivatized by sequential addition of 5 μL of the working internal
standard solution, 12.5 μL of 100 mM sodium carbonate buffer, and
12.5 μL of BzCl (2% (v/v) in acetonitrile). For the rat brain
microdialysates, 25 μL were also derivatized as with the quality
control samples. Ten microliters of the sample was injected into the
LC-MS/MS system.
LC-MS/MS System. LC-MS/MS analysis was conducted using a
Nexera X2 LC-30AD and LCMS-8050 system (SHIMADZU
Corporation, Kyoto, Japan) coupled with an LC-30AD pump
degasser (DGU-205R) and a SIL-30AD autosampler (SHIMADZU
Corporation). Data were processed using LabSolutions (SHIMADZU
Corporation).
LC Conditions. The mobile phase consisted of 2 mM ammonium
formate/0.1% formic acid in water (A) and acetonitrile (B) at a flow
rate of 300 μL/min for the entire analysis. The temperature of the
autosampler was set to 4 °C. The separation of DA, 5-HT, and their
metabolites was performed using an Atlantis T3 (2.1 × 100 mm, 3
μm; Waters), and the temperature was maintained at 40 °C. Gradient
elution was performed using a linear gradient starting with 5% B,
holding at 5% B for 1 min, increasing linearly to 95% B for 8 min,
holding at 95% B until 10 min, returning to 5% B for 10.1 min, and
holding at 5% B for 15 min.
MS Conditions. The MS/MS system was operated using positive
electrospray ionization. The optimal conditions set were: nebulizing
gas flow, 3 L/min; drying gas flow, 10 L/min; heating gas flow, 10 L/
min; interface voltage, 4 kV; interface temperature, 300 °C;
desolvation line temperature, 250 °C; and heat block temperature,
400 °C. Two multiple reaction monitoring (MRM) transitions were
chosen for DA, 5-HT, their metabolites, SAL, and internal standards.
MRM transitions, retention times, and other conditions are shown in
Table 2.
Method Validation. The analytical method was validated using
aCSF as a blank matrix. The matrix effects of the analytes were
investigated by comparing the analytical responses of spiked aCSF
samples (n = 5, set 1) with those of neat standard samples in water (n
= 5, set 2) and calculated as a percentage of the responses of set 2
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(version 8.0.2, GraphPad Software, La Jolla, CA, United States). The
data were presented as mean SEM. The locomotor activity test was
statistically analyzed by Tukey’s post-test following a one-way analysis
of variance (ANOVA). Two-way ANOVA (mixed-effects analysis)
followed by Fisher’s LSD post-test was used to determine significant
differences in the data from the LC-MS/MS analysis of the
microdialysates. The baseline value was determined from three
consecutive microdialysates with less than 20% CV fluctuations in the
concentrations of DA, 5-HIAA, and HVA, and less than 30% CV
fluctuations in the concentrations of the other analytes. The
quantification of DA, 5-HT, and their metabolites was divided by
the selected baseline value and expressed as percentages.
Table 2. Multiple Reaction Monitoring Transitions,
Retention Times, and Other Conditions of Benzoyl
Chloride-Derivatized Analytes Internal Standards
a
precursor ion
product ion
tR
frag
CE
b
compound name
Bz_DA
(m/z)
(m/z)
(min) (V)
(V)
466
378
394
376
304
492
385
313
470
399
380
309
389
318
105
77
105
77
105
77
105
77
105
287
105
77
105
264
105
296
105
77
105
77
105
77
105
292
105
268
105
301
8.0
7.9
7.3
7.4
6.6
8.4
7.3
6.3
8.0
7.3
7.4
6.6
7.3
6.3
20
16
14
14
20
14
20
20
11
21
13
18
20
20
11
21
18
18
14
14
20
20
17
15
20
20
12
16
25
55
21
55
25
55
25
53
21
8
22
55
35
20
27
9
22
55
22
55
25
54
21
9
35
20
28
10
Bz_DOPAL
Bz_DOPAC
Bz_3-MT
AUTHOR INFORMATION
Corresponding Authors
■
Bz_HVA
Chul-Ho Jeong − College of Pharmacy, Keimyung University,
Dalseo-gu, Daegu 704-701, Republic of Korea; Phone: +82
53 580 6638; Email: chjeong75@kmu.ac.kr; Fax: +82 53
580 5164
Bz_SAL
Bz_5-HT
Sooyeun Lee − College of Pharmacy, Keimyung University,
Dalseo-gu, Daegu 704-701, Republic of Korea; orcid.org/
0000-0002-7649-0910; Phone: +82 53 580 6651;
Email: sylee21@kmu.ac.kr; Fax: +82 53 580 5164
Bz_5-HIAA
Bz_DA-d4
Authors
Bz_DOPAC-d5
Bz_3-MT-d4
Bz_HVA-d5
Bz_5-HT-d4
Bz_5-HIAA-d5
Seungju Kim − College of Pharmacy, Keimyung University,
Dalseo-gu, Daegu 704-701, Republic of Korea
Eun Young Jang − Pharmacology and Drug Abuse Research
Group, Korea Institute of Toxicology, Yuseong-gu, Daegeon
34114, Republic of Korea
Sang-Hoon Song − College of Pharmacy, Keimyung
University, Dalseo-gu, Daegu 704-701, Republic of Korea
Ji Sun Kim − Pharmacology and Drug Abuse Research Group,
Korea Institute of Toxicology, Yuseong-gu, Daegeon 34114,
Republic of Korea
a
Bz; benzoylated, DA, dopamine; DOPAL, 3,4-dihydroxyphenylace-
In Soo Ryu − Pharmacology and Drug Abuse Research Group,
Korea Institute of Toxicology, Yuseong-gu, Daegeon 34114,
Republic of Korea
taldehyde; DOPAC, 3,4-dihydroxyphenylacetic acid; 3-MT, 3-
methoxytyramine; HVA, homovanillic acid; SAL, salsolinol; 5-HT,
serotonin; 5-HIAA, 5-hydroxyindoleacetic acid; tR, retention time;
b
frag, fragmentation voltage; CE, collision energy. Underlined
Complete contact information is available at:
https://pubs.acs.org/10.1021/acschemneuro.1c00039
transitions were used for quantification.
Author Contributions
samples in relation to those of set 1 samples.50 Other validation
parameters, such as selectivity, sensitivity, linearity, accuracy,
precision, and stability, were evaluated according to the guidelines.51
To evaluate the selectivity and sensitivity of the method, the blank
aCSF sample, an aCSF sample spiked at the expected LOQ level of
the analytes, and the rat brain microdialysates were compared.
Method sensitivity for the detection of basal concentrations of
analytes was also investigated. The analyte concentration at which the
signal-to-noise ratio was <3 was selected as the LOD, while the
analyte concentration at which the signal-to-noise ratio was <10, CV
(for precision) <20%, and bias < 20% were selected as the LOQ.
Linearity was demonstrated using five sets of calibration curves in the
ranges. The accuracy and precision were investigated in aCSF samples
spiked at low, medium, and high concentrations of analytes.
Repeatability and interday precision were estimated through one-
way variance analysis (ANOVA) using the grouping variable “day” at
each concentration level. Accuracy was calculated as the difference
between the average of all 25 measurements and the percentage of
concentrations spiked at each concentration. Stability was investigated
with aCSF samples spiked at the lowest and highest concentrations of
the calibration range of each analyte under storage conditions. Spiked
samples were stored at −80 °C for 30 days to evaluate sample storage
stability. Processed stability was investigated at 2 h intervals over 12 h.
Statistical Analysis. Statistical analysis of the locomotor activity
test and LC-MS/MS results was performed using GraphPad Prism8
#S.K. and E.J. contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the Basic Science Research
Program (NRF-2016R1A6A1A03011325 and NRF-
2018R1D1A1B07048159) through the National Research
Foundation funded by the Ministry of Education in Korea.
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NOTE ADDED AFTER ASAP PUBLICATION
■
This paper was published on April 19, 2021. Due to
production error, incorrect versions of Figures 1−5 were
used. The corrected version was posted on April 22, 2021.
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ACS Chem. Neurosci. 2021, 12, 1552−1562