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1521-0103/355/1/76–85$25.00 http://dx.doi.org/10.1124/jpet.115.225664
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS J Pharmacol Exp Ther 355:76–85, October 2015
Copyright ª2015 by The American Society for Pharmacology and Experimental Therapeutics
The Prodrug 4-Chlorokynurenine Causes Ketamine-Like
Antidepressant Effects, but Not Side Effects, by
NMDA/Glycine
B
-Site Inhibitions
Panos Zanos, Sean C. Piantadosi, Hui-Qiu Wu, Heather J. Pribut, Matthew J. Dell,
Adem Can, H. Ralph Snodgrass, Carlos A. Zarate, Jr., Robert Schwarcz,
and Todd D. Gould
Department of Psychiatry (P.Z., S.C.P., H.-Q.W., H.J.P., M.J.D., A.C., R.S., T.D.G.), Maryland Psychiatric Research Center
(H.-Q.W., R.S.), Department of Pharmacology (R.S., T.D.G.), Department of Anatomy and Neurobiology (T.D.G.), University of
Maryland School of Medicine, Baltimore, Maryland; VistaGen Therapeutics, Inc., San Francisco, California (H.R.S.); Experimental
Therapeutics and Pathophysiology Branch, Intramural Research Program, National Institute of Mental Health, National Institutes
of Health, Bethesda, Maryland (C.A.Z.)
Received May 3, 2015; accepted July 29, 2015
ABSTRACT
Currently approved antidepressant drug treatment typically
takes several weeks to be effective. The noncompetitive
N-methyl-D-aspartate (NMDA) receptor antagonist ketamine has
shown efficacy as a rapid-acting treatment of depression, but its
use is associated with significant side effects. We assessed
effects following blockade of the glycine
B
co-agonist site of the
NMDA receptor, located on the GluN1 subunit, by the selective
full antagonist 7-chloro-kynurenic acid (7-Cl-KYNA), delivered
by systemic administration of its brain-penetrant prodrug
4-chlorokynurenine (4-Cl-KYN) in mice. Following administration
of 4-Cl-KYN, 7-Cl-KYNA was promptly recovered extracellularly
in hippocampal microdialysate of freely moving animals. The
behavioral responses of the animals were assessed using
measures of ketamine-sensitive antidepressant efficacy (in-
cluding the 24-hour forced swim test, learned helplessness
test, and novelty-suppressed feeding test). In these tests,
distinct from fluoxetine, and similar to ketamine, 4-Cl-KYN
administration resulted in rapid, dose-dependent and persistent
antidepressant-like effects following a single treatment. The
antidepressant effects of 4-Cl-KYN were prevented by
pretreatment with glycine or the a-amino-3-hydroxy-5-methyl-
4-isoxazolepropionic acid (AMPA) receptor antagonist 2,3-dihydroxy-
6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione(NBQX).4-Cl-KYN
administration was not associated with the rewarding and
psychotomimetic effects of ketamine, and did not induce
locomotor sensitization or stereotypic behaviors. Our results
provide further support for antagonism of the glycine
B
site for the
rapid treatment of treatment-resistant depression without the
negative side effects seen with ketamine or other channel-
blocking NMDA receptor antagonists.
Introduction
Although interventions for major depressive disorder
(MDD) such as pharmacotherapies and cognitive behavioral
psychotherapies are available, more than 30% of patients
remain treatment-resistant. Even when effective, currently
used monoaminergic drugs often take up to several months to
exert their full therapeutic effects (Rush et al., 2006). More-
over, compelling evidence suggests a key role for the gluta-
matergic system in mood disorders (Popoli et al., 2012). Tissue
glutamate levels are higher in the brain of depressed patients
compared with healthy individuals (Sanacora et al., 2012),
and evidence indicates that the N-methyl-D-aspartate
(NMDA) subtype of glutamate receptors can be successfully
targeted for the treatment of MDD (Pittenger et al., 2007).
Placebo-controlled trials have demonstrated rapid-acting
antidepressant effects, within hours, of subanesthetic doses
of the noncompetitive NMDA-receptor antagonist ketamine in
treatment-resistant depressed patients [see Duman (2014)].
Moreover, antidepressant effects of ketamine have been
demonstrated in several relevant tests in experimental ani-
mals [see Browne and Lucki (2013)]. However, ketamine’s
potential as a long-term antidepressant medication is limited
by its addictive properties and its anesthetic, cognitive, and
psychotomimetic side effects (Krystal et al., 1994).
This study was supported by the National Institutes of Health National
Institute of Mental Health (Grant No. MH099345). This work was supported
by the Intramural Research Program of the National Institutes of Health.
dx.doi.org/10.1124/jpet.115.225664.
sThis article has supplemental material available at jpet.aspetjournals.org.
ABBREVIATIONS: 4-Cl-KYN, 4-chlorokynurenine; 7-Cl-KYNA, 7-chlorokynurenic acid; AMPA, a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic
acid; ANOVA, analysis of variance; FLX, fluoxetine; FST, forced-swim test; GLYX-13, (S)-N-[(2S,3R)-1-amino-3-hydroxy-1-oxobutan-2-yl]-1-[(S)-1-
((2S,3R)-2-amino-3-hydroxybutanoyl)pyrrolidine-2-carbonyl]pyrrolidine-2-carboxamide; MDD, major depressive disorder; MK-801, [5R,10S]-[1]-5-
methyl-10,11- dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine; NBQX, 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione; NMDA,
N-methyl-D-aspartate; NSF, novelty-suppressed feeding test; TST, tail-suspension test.
76
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Activity of the NMDA receptor requires binding of glycine or
D-serine to an obligatory co-agonist (“glycine
B
”) site, which is
located on the GluN1 subunit (Leeson and Iversen, 1994;
Danysz and Parsons, 1998). Although efficacy of glycine
B
receptor blockade by a full antagonist has been demonstrated
in tests in mice sensitive to selective serotonin reuptake
inhibition (Przegalinski et al., 1998; Poleszak et al., 2007),
limited work has assessed actions in ketamine-sensitive tests
predictive of rapid-acting antidepressant efficacy. 7-Chloro-
kynurenic acid (7-Cl-KYNA) is a potent and highly selective
competitive glycine
B
receptor antagonist that has been widely
used as a pharmacological probe to study the biology of the
NMDA receptor (Kemp et al., 1988). Although the therapeutic
use of 7-Cl-KYNA is limited by its poor penetration of the
blood-brain barrier, it can be enzymatically formed from its
prodrug 4-chlorokynurenine [4-Cl-KYN; Salituro et al. (1994)],
which readily enters the brain after systemic administration
(Hokari et al., 1996) and is then converted to 7-Cl-KYNA
within astrocytes [Wu et al. (1997); Fig. 1A].
In the present study, we evaluated the antidepressant
efficacy and side effect profile of 4-Cl-KYN in comparison with
ketamine and, in some experiments, with the selective
serotonin reuptake inhibitor fluoxetine (FLX). Our results
provide direct support for targeting the glycine
B
site in the
treatment of MDD and indicate that the prodrug approach
using 4-Cl-KYN holds promise for use in humans, without the
negative side effects seen with ketamine or related NMDA
receptor antagonists.
Materials and Methods
Animals
Male CD-1 mice (seven-weeks old on arrival; Charles River
Laboratories, MA) were housed in groups of four or five per cage with
a 12-hour light/dark cycle (lights on at 07:00). Food and water were
available ad libitum. Mice acclimatized to the new environment for
7 days prior to the start of the experiments. All experimental procedures
were approved by the University of Maryland, Baltimore Animal
Care and Use Committee and were conducted in full accordance with
the National Institutes of Health Guide for the Care and Use of
Laboratory Animals.
Drugs
Ketamine-HCl and glycine (Sigma-Aldrich, St. Louis, MO), NBQX,
and FLX (National Institute of Mental Health Chemical Synthesis
and Drug Supply Program) were dissolved in 0.9% saline. 4-Cl-KYN
(provided by VistaGen Therapeutics, Inc., South San Francisco, CA)
and 7-Cl-KYNA (Tocris Bioscience, Ellisville, MO) were suspended in
0.1 N NaOH until fully dissolved and neutralized with 0.1 N HCl
before injection. All drugs were administered intraperitoneally in
a volume of 7.5 ml/kg of body mass.
Measurement of 7-Cl-KYNA in Brain Microdialysate
Microdialysis in freely-moving mice was carried out as previously
described (Potter et al., 2010). Animals were anesthetized with chloral
hydrate (360 mg/kg) and mounted in a stereotaxic frame. A guide
cannula (outer diameter: 0.65 mm) was positioned over the dorsal
hippocampus (AP: 2.2 mm posterior to bregma, L: 2.0 mm from the
midline, V: 1.1 mm below the dura) and secured to the skull with an
anchor screw and acrylic dental cement. A concentric microdialysis
probe (membrane length: 1 mm; SciPro, Sanborn, NY) was then
inserted, extending 1 mm beyond the tip of the guide cannula. The
probe was subsequently connected to a microinfusion pump set to
a speed of 1 ml/min and perfused with Ringer solution containing
144 mM NaCl, 4.8 mM KCl, 1.2 mM MgSO
4
, and 1.7 mM CaCl
2
,
pH 6.7. Microdialysate samples were collected every 30 minutes for
eight hours. 4-Cl-KYN or 7-Cl-KYNA was administered 2 hours after
the initial collection of baseline fractions. 7-Cl-KYNA levels were
measured by high-performance liquid chromatography, using fluores-
cence detection (excitation wavelength: 344 nm; emission wavelength:
398 nm), as described (Lee and Schwarcz, 2001). Briefly, 20 mlof
the microdialysate were subjected to a 3-mm C18 reverse phase column
(80 4.6 mm; Thermo Scientific, Waltham, MA). 7-Cl-KYNA was
isocratically eluted at a flow rate of 1 ml/min using a mobile phase
containing 250 mM zinc acetate, 50 mM sodium acetate, and 8%
acetonitrile, pH 6.2. The retention time of 7-Cl-KYNA was ∼8 minutes.
Pharmacological Screening
Binding profiles and K
i
determination data of 7-Cl-KYNA and
4-Cl-KYN for a broad panel of receptors and channels were provided
by the National Institute of Mental Health Psychoactive Drug Screen-
ing Program (University of North Carolina, Chapel Hill, NC). For all
radioligand receptor assay methods, see the Psychoactive Drug
Screening Program web site (http://pdsp.med.unc.edu) as well as
previously published protocols (Besnard et al., 2012). Experimental
details for the binding assays are provided in Supplemental Table S2.
Fig. 1. Extracellular levels of 7-chorokynurenic acid studied by micro-
dialysis. (A) Schematic representation of 7-chlorok ynurenic acid (7-Cl-KYNA)
production from 4-chlorokynurenine (4-Cl-KYN) in the brain. 4-Cl-KYN
readily enters the brain from the circulation and is then converted to
7-Cl-KYNA by kynurenine aminotransferase (KAT) in astrocytes. (B)
Hippocampal microdialysis in freely-moving mice. The arrow indicates
intraperitoneal injection of 7-Cl-KYNA (25 mg/kg) or 4-Cl-KYN (25 mg/kg).
Data are the mean 6S.E.M. (n= 6/group). Inset: area under the curve
(AUC); unpaired Student’st-test. ***p,0.001.
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Behavioral Tests
All behavioral experiments were performed during the light phase.
Ketamine was used as a positive control in all of the behavioral
experiments. The dose used routinely (10 mg/kg) was previously
shown to exert antidepressant effects in mice (Maeng et al., 2008;
Ma et al., 2013; Antony et al., 2014).
Forced-Swim Test. Saline, FLX (20 mg/kg), ketamine (10 mg/kg),
or 4-Cl-KYN (0.2, 1, 5, 25, or 125 mg/kg) was injected, and mice were
tested in the forced-swim test (FST) 1 hour or 24 hours postinjection.
During the FST, mice were subjected to a 6-minute swim session in
clear Plexiglas cylinders (30 cm height 20 cm diameter) filled with
15 cm of water (23 61°C). The FST was performed in normal light
conditions (800 lux). Sessions were recorded using a digital video-
camera. Immobility time, defined as passive floating with no addi-
tional activity other than that necessary to keep the head above water,
was scored for the last 4 minutes of the 6-minute test by a trained
observer blind to the treatment. In a separate cohort of animals,
glycine [1.6 g/kg, i.p.; Evoniuk et al. (1991); Javitt et al. (1999)] was
administered 10 minutes prior to injection of saline or 4-Cl-KYN. Since
a leading hypothesis of ketamine antidepressant action involves the
activation of AMPA receptors (Maeng et al., 2008; Li et al., 2010; Autry
et al., 2011), we also assessed the role of this receptor in the antidepres-
sant effects of 4-Cl-KYN by pretreatment with the AMPA receptor
antagonist NBQX (10 mg/kg) 5 minutes prior to injection of saline,
ketamine, or 4-Cl-KYN, and assessed behavior 1 hour later in the FST.
Tail-Suspension Test. The TST was performed as previously
described by Can et al. (2012). Animals were tested 1 hour post-
injection of saline, ketamine (10mg/kg), or 4-Cl-KYN (5, 25, or 125mg/kg).
Mice were individually suspended from the tail in suspension boxes
(55 cm height 60 cm width 11.5 cm depth; Four-Hour Day,
Baltimore, MD) for 6 minutes using a 15-cm long tape attached to the
end of their tails. TST was performed under normal light conditions
(800 lux). The test session was recorded using a digital videocamera.
All videos were scored by a trained observer blinded to the treatment.
Immobility time was measured for the entire 6-minute session.
A mouse was considered immobile when it hung passively.
Novelty-Suppressed Feeding. The novelty-suppressed feeding
test (NSF) was performed as described (Warner-Schmidt and Duman,
2007), with minor modifications. Briefly, mice were singly housed and
food-deprived for twenty-four hours in freshly made home-cages. Two
normal chow diet pellets were placed on a square food platform (10
10 cm) in the center of an open-field arena (40 40 cm). Thirty
minutes after saline, ketamine, or 4-Cl-KYN administration, mice
were introduced into a corner of the arena for 10 minutes. The time
needed for the mice to take a bite of food was recorded by a trained
observer blind to the treatment groups. After the test, the mice were
returned to their home-cages containing preweighed food pellets, and
latency to bite the food as well as consumption was recorded for
a period of 10 minutes.
Learned Helplessness. As in previously published procedures
(Maeng et al., 2008), the learned helplessness (LH) paradigm con-
sisted of three different phases, i.e., inescapable shock training, LH
screening, and the LH test. For the inescapable shock portion of the
test (Day 1), the animals were placed in one side of two-chambered
shuttle boxes (34 cm height 37 cm width 18 cm depth; Coulbourn
Instruments, Whitehall, PA), with the door between the chambers
closed. Following a 5-minute adaptation period, 120 inescapable foot-
shocks (0.45 mA, 15-second duration, randomized average intershock
interval of 45 seconds) were delivered through the grid floor. During the
screening session (Day 2), the mice were placed in one of the two
chambers of the apparatus for 5 minutes. A shock (0.45 mA) was then
delivered, and the door between the two chambers was raised simulta-
neously. Crossing over into the second chamber terminated the shock. If
the animal did not cross over, the shock terminated after 3 seconds. A
total of 30 screening trials of escapable shocks were presented to each
mouse with an average of 30-second delay between each trial. Mice that
developed helplessness behavior (.5 escape failures during the last 10
screening shocks) (Malberg and Duman, 2003) were treated with either
saline (7.5 ml/kg), FLX (20 mg/kg), ketamine (10 mg/kg), 4-Cl-KYN (5, 25
or 125 mg/kg), or 7-Cl-KYNA (1 or 25 mg/kg) 24 hours following screening
(Day 3). During the LH test phase (Day 4), the animals were placed in the
shuttle boxes and, after a 5-minute adaptation period, a 0.45-mA shock
was delivered concomitantly with door opening for the first five trials,
followed by a 2-second delay for the next 40 trials. Crossing over to the
second chamber terminated the shock. If the animal did not cross over to
the other chamber, the shock was terminated after 24 seconds. A total of
45 trials of escapable shock was presented to each mouse with 30-second
intertrial intervals. The number of escape failures was recorded for each
mouse. To determine the prolonged efficacy of the drugs, mice were
retested for helpless behavior after 7 days (i.e., on Day 11).
Open-Field Test Behaviors. Experiments were performed at 30
lux ambient light levels within the arena. Three days prior to the
locomotor sensitization experiments (Days 1–3), mice were placed into
individual open-field arenas (50 cm length 50 cm width 38 cm
height; San Diego Instruments, San Diego, CA) for a 30-minute daily
habituation period. Saline was then injected, and mice were assessed
for another 90 minutes. For the next 3 consecutive days (Days 4–6),
mice were randomly divided into treatment groups and received
injections after a 30-minute habituation period. Locomotor responses
were then recorded for an additional period of 90 minutes. During
Days 7–12 and 14–19, the animals received no injections. On Days 13
and 20, mice received a challenge treatment injection, and their
locomotor activity was recorded. On Day 21, all mice received a saline
injection to evaluate a possible contextual conditioning effect. Dis-
tance traveled was analyzed using TopScan v2.0 (CleverSys, Inc.,
Reston, VA) for a 30-minute period following the daily injection or 90
minutes for the acute effects of the drugs. Stereotypic rearing (vertical
activity) and circling behavior were scored for the 30 minutes following
the daily injections by a trained observer blind to the treatment
groups.
Conditioned-Place Preference. The conditioned-place prefer-
ence (CPP) apparatus consisted of a rectangular Plexiglas three-
chambered box (53 cm length 15 cm width 36 cm height) divided
into three compartments (two equal-sized end-chambers and a middle
“waiting”chamber) by two removable doors. One end-chamber (22 cm
length 15 cm width 36 cm height) was composed of a 1-cm-spaced
wired floor with four dark-gray walls (dark compartment), whereas
the other end-chamber (same dimensions) had a 0.3-cm-spaced wired
floor with white walls with black vertical stripes (1.5 cm; striped
compartment). The middle compartment (9 cm length 15 cm width
36 cm height) had white Plexiglas floor and walls. The CPP protocol
consisted of a habituation phase, a preconditioning test, six condi-
tioning sessions, and a postconditioning test. On Day 1, mice were
placed in the CPP apparatus for a 20-minute habituation period and
were allowed to freely explore all chambers. On Day 2 (preconditioning
phase), mice were placed in the CPP apparatus and were allowed to
explore all compartments for a period of 20 minutes. During the
conditioning phase, saline (7.5 ml/kg), ketamine (10 mg/kg), or 4-Cl-KYN
(25 or 125 mg/kg) was administered, and mice were placed in their
least-preferred compartment on alternating days (i.e., Days 3, 5, and
7). All groups of animals received saline (7.5 ml/kg) in their preferred
compartment on Days 4, 6, and 8. During the postconditioning test
session (i.e., Day 9), mice were placed in the CPP apparatus to freely
explore all three compartments for 20 minutes. Time spent in each
compartment was measured during the last 15 minutes of both pre- and
postconditioning sessions, as previously described (Zanos et al., 2014).
Prepulse Inhibition. The prepulse inhibition (PPI) paradigm
used was derived from previously published protocols (Chan et al.,
2008), with minor modifications. Mice were individually tested in
acoustic startle boxes (SR-LAB; San Diego Instruments, San Diego,
CA). The animals first received an injection of saline, ketamine, or
4-Cl-KYN and were placed in the startle chamber for a 30-minute
habituation period. The experiment started with a further 5-minute
adaptation period during which the mice were exposed to a constant
background noise (67 db), followed by five initial startle stimuli
(120 db, 40-millisecond duration each). Subsequently, animals were
78 Zanos et al.
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exposed to five different trial types: pulse alone trials (120 db,
40-millisecond duration), three prepulse trials of 76, 81, and 86 db of
white noise bursts (20-millisecond duration) preceding a 120-db pulse
by 100 milliseconds, and background (67 db) no-stimuli trials. Each of
these trials was randomly presented five times. The percentage
prepulse inhibition (% PPI) was calculated using the following
formula: [(magnitude on pulse alone trial –magnitude on prepulse 1
pulse trial)/magnitude on pulse alone trial] 100.
Statistics
All values are expressed as the mean 6S.E.M. For analyses of the
acute (i.e., 1-hour) and long-term (i.e., 24-hour) antidepressant-like
efficacy in the FST and TST, one-way ANOVA was performed. Glycine
pretreatment effects in the FST were assessed by a two-way ANOVA
with ‘pretreatment’(i.e., saline and glycine) and ‘treatment’(i.e., saline
and 4-Cl-KYN) as factors. Effects in the LH and NSF tests were analyzed
by one-way ANOVA. Acute and behavioral sensitization effects in the
open-field test were analyzed using two-way–repeated measures anal-
ysis of variance (ANOVA) with ‘treatment’and ‘time’as factors. CPP data
were assessed by two-way–repeated measures ANOVA for factors
‘treatment’and ‘CPP phase’(i.e., pre-Cond, post-Cond). The data of
startle amplitude and circling behavior were assessed by one-way
ANOVA. The % PPI data were analyzed by two-way ANOVA for factors
‘treatment’and ‘intensity’(i.e., 76 db, 81 db, and 86 db). A Student’st-test
was used to compare the area under the curve (AUC) following 4-Cl-KYN
or 7-Cl-KYNA administration, and to determine the effects of acute
(1 hour postinjection) FLX administration, in the FST. All ANOVAs were
followed by a Bonferroni post-hoc comparison when significance was
reached (i.e., p,0.05). All statistical analyses were performed using
STATISTICA 10 (StatSoft Inc., Tulsa, OK). ANOVA results are
presented in Table 1, while post-hoc comparisons are detailed in figures
and text.
Results
In Vivo Microdialysis. 7-Cl-KYNA levels were not mea-
surable in hippocampal microdialysates during the baseline
collection period (0–2 hours; Fig. 1B). Extracellular 7-Cl-KYNA
levels became detectable in mice that received 4-Cl-KYN or
7-Cl-KYNA (each 25 mg/kg), attaining peak concentrations of
100 616 nM 1.5 hours after the administration of 4-Cl-KYN
and 25 65 nM 1 hour after the administration of 7-Cl-KYNA.
Quantitative assessment of the AUC revealed approximately
10-times higher extracellular 7-Cl-KYNA levels for the
4-Cl-KYN-treated mice compared with 7-Cl-KYNA-treated
animals (inset, Fig. 1B). Notably, considering a typical
10–20% recovery and time lag from the microdialysis pro-
cedure (Stahle et al., 1991), the peak extracellular concen-
tration of 7-Cl-KYNA following administration of 4-Cl-KYN
approximated its IC
50
at the glycine
B
site [560 nM; Kemp et al.
(1988)].
Pharmacological Screening. In vitro screening revealed
only two receptors with K
i
values of ,10 mM for 7-Cl-KYNA
(Supplemental Table S1 and Supplemental Fig. S1): b-adrenergic
receptor subtype 1 (K
i
:3.95mM) and muscarinic receptor subtype
5(K
i
:2.81mM). This contrasts with the IC
50
of 7-Cl-KYNA at the
glycine
B
site [560 nM; Kemp et al. (1988)] and indicates a lack of
off-target effects of the compound. 4-Cl-KYN showed no affinity
(,10 mM)foranyofthepossibletargets assessed (Supplemental
Table S1 and Supplemental Fig. S2).
Antidepressant-Like Effects of 4-Cl-KYN in the
Forced-Swim Test, Tail-Suspension Test, and Novelty-
Suppressed Feeding Tests. To assess antidepressant-like
properties in classic tests of antidepressant efficacy, mice were
tested in the FST or TST 1 hour postinjection. Both ketamine
(10 mg/kg) and 4-Cl-KYN (25 and 125 mg/kg) administration
significantly decreased immobility time in the FST, compared
with the saline-treated controls (Fig. 2A). A similar decrease
in immobility was also observed after acute FLX administra-
tion (Fig. 2A). To examine whether systemic 7-Cl-KYNA
administration itself elicits antidepressant-like effects in the
FST, we administered the compound at three different doses
(25, 75, or 225 mg/kg). Tested 1 hour later, only the highest
dose of 7-Cl-KYNA resulted in significant antidepressant-like
effects (saline: 157.4 614.9 seconds; 25 mg/kg: 116.1 618.5
seconds; 75 mg/kg: 125.4 612.9 seconds; 225 mg/kg (P,0.05):
81.3 613.0 seconds; n57–8/group). The effective doses of
ketamine and 4-Cl-KYN in the FST also significantly decreased
immobility time in the TST (Fig. 2D). Notably, 4-Cl-KYN, in
apparent contrast to ketamine (Li et al., 2010), elicited antide-
pressant actions across a rather broad dose range; however, our
studies did not determine the maximally effective dose in either
FST or TST.
To determine whether these antidepressant-like effects
of 4-Cl-KYN are attributable to glycine
B
site inhibition, we
pretreated mice with glycine at a dose previously shown to
reverse the behavioral effects of NMDA channel blockers in
rodents [1.6 g/kg, i.p.; Evoniuk et al. (1991); Javitt et al. (1999)].
Whereas glycine administration alone did not change immo-
bility time of saline-treated animals in the FST, it prevented
the antidepressant-like effects of 4-Cl-KYN (25 mg/kg) in this
paradigm (Fig. 2B). Moreover, to mechanistically assess
downstream mechanisms mediating the antidepressant
effects of 4-Cl-KYN, we administered the AMPA receptor antag-
onist NBQX (10 mg/kg) 5 minutes prior to treatment with 4-
Cl-KYN and assessed immobility time in the FST 1 hour later.
Pretreatment with NBQX prevented the antidepressant ef-
fects of both ketamine and 4-Cl-KYN (Fig. 2C).
To determine the capacity for rapid antidepressant action of
4-Cl-KYN, we assessed its effects in the NSF test, which is
sensitive to monoamine-acting antidepressants only following
chronic administration (Santarelli et al., 2003). Administra-
tion of both ketamine and 4-Cl-KYN rapidly decreased the
latency to feed in the NSF arena compared with saline-treated
mice (Fig. 2E). There were no significant differences be-
tween the amounts of food consumed by saline-, ketamine-,
or 4-Cl-KYN-treated mice in their home cage assessed immediately
following the NSF test (Fig. 2F).
Sustained Antidepressant-Like Effects of 4-Cl-KYN
in the 24-Hour FST. It has previously been reported that, in
contrast to monoamine-acting antidepressants, ketamine
elicits antidepressant actions in the FST 24 hours after
administration, when ketamine is no longer present [e.g., Li
et al. (2010); Autry et al. (2011)]. We therefore compared the
antidepressant-like effects of ketamine and 4-Cl-KYN in the
FST 24 hours following a single injection. Similar to ketamine
(10 mg/kg), 4-Cl-KYN (25 or 125 mg/kg) significantly de-
creased immobility time compared with saline-treated ani-
mals (Fig. 3A). Consistent with its delayed time course of
action in humans [e.g., Barr et al. (1997)], FLX (20 mg/kg) had
no significant effect when it was administered a single time
24 hours prior to testing (Fig. 3A).
Antidepressant Effects of 4-Cl-KYN in the Learned
Helplessness Paradigm. Mice that developed helpless be-
havior (∼50% of the mice having experienced inescapable
foot shocks) were randomly assigned to treatment groups,
Fast-Onset Antidepressant Effects of 4-Chlorokynurenine 79
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receiving single injections of saline, ketamine (10 mg/kg),
4-Cl-KYN (25 mg/kg), FLX (20 mg/kg), or 7-Cl-KYNA (1 or
25 mg/kg). Both ketamine and 4-Cl-KYN significantly reduced
helpless behaviors as assessed by the number of escape
failures compared with saline-treated controls (Fig. 3, B and
D). Notably, the antidepressant-like effects of both ketamine
and 4-Cl-KYN persisted when the animals were retested 7 days
later (Fig. 3, C and E). In contrast, FLX- and 7-Cl-KYNA-treated
mice did not manifest antidepressant-like effects 24 hours or
7 days postinjection (Fig. 3, D and E).
4-Cl-KYN Does Not Affect Behaviors in the Open
Field. Acute administration of 4-Cl-KYN (25 or 125 mg/kg)
did not cause ketamine-like acute hyperlocomotor activity in
the open-field compared with saline-treated controls (Fig. 4B).
In the same mice, we assessed possible locomotor sensitization
effects following repeated saline, ketamine, or 4-Cl-KYN
injections. Administration of ketamine, but not of 4-Cl-KYN,
on Days 13 and 20 revealed a significant increase in locomotor
activity compared with the acute motor-enhancing effects
(Day 4), indicating locomotor sensitization (Fig. 4C). No effect
was seen when saline was administered on Day 21.
Similar to other compounds with stimulant properties [e.g.,
Morency et al. (1987)], administration of ketamine, but not of
4-Cl-KYN, caused significant circling behavior following acute
administration on Day 4, compared with saline controls (Fig.
4D). Animals did not manifest maintenance of this effect on
subsequent days (data not shown). Moreover, injection of
ketamine, but not of 4-Cl-KYN, induced a significant increase
in repetitive vertical activity (rearing) on the first day of drug
injection (Day 4; Fig. 4E). No sensitization to this effect was
observed (Fig. 4E).
4-Cl-KYN Does Not Elicit Conditioned Place Prefer-
ence. Ketamine-treated (10 mg/kg) mice exhibited a robust
CPP, as illustrated by an increase in the time spent in the
drug-paired compartment during the postconditioning phase
compared with the preconditioning phase (Fig. 5A). In con-
trast, administration of 4-Cl-KYN (25 or 125 mg/kg) did not
induce CPP (Fig. 5A). Additionally, analysis of the ratio of the
time mice spent in the drug-paired compartment during the
postconditioning phase and preconditioning session indicated
a significant increase in the preference of ketamine-treated
mice to spend their time in the drug-paired compartment
compared with saline-treated mice.
4-Cl-KYN Does Not Disrupt Prepulse Inhibition. Ket-
amine administration dose-dependently disrupted % PPI,
with 30 mg/kg found to be the lowest significantly effective
dose (Fig. 5B). In contrast, 4-Cl-KYN administration did not
induce significant alterations in % PPI at any dose tested (25,
TABLE 1
Statistical analyses
Factor Effect Interaction Effect
Overall effects for Fig. 2
Acute antidepressant effects Factor ‘treatment’Factor ‘pretreatment’Factor ‘pretreatment’‘treatment’
Forced-swim test F
[6,83]
= 4.10 P,0.01
Glycine, forced-swim test F
[1,53]
= 7.57 P,0.001 F
[1,53,]
= 7.50 P,0.001 F
[1,53]
= 6.55 P = 0.01
NBQX, forced-swim test F
[2,65]
= 4.51 P,0.05 F
[1,65]
= 16.80 P,0.001 F
[2,65]
= 6.16 P,0.01
Tail-suspension test F
[4,74]
= 3.57 P,0.05
NSF latency to feed F
[2,27]
= 10.51 P,0.001
Home-cage food consumption F
[2,27]
= 0.53 P = 0.60
Overall effects for Fig. 3
Sustained antidepressant effects,
24-hour FST Factor ‘treatment’
Immobility time F
[4,45]
= 11.84 P,0.001
Sustained antidepressant effects,
LH
Effects of 4-Cl-KYN (24 hours) F
[2,28]
= 71.68 P,0.001
Effects of 4-Cl-KYN (7 days) F
[2,28]
= 43.78 P,0.001
Effects of 7-Cl-KYN (24 hours) F
[4,39]
= 11.47 P,0.001
Effects of 7-Cl-KYN (7 days) F
[4,39]
= 4.23 P,0.01
Overall effects for Fig. 4
Effects of 4-Cl-KYN in the
open-field test Factor ‘treatment’Factor ‘time’Factor ‘treatment‘‘time‘
Acute locomotor effects F
[3,28]
= 0.35 P = 0.79 F
[23,644]
= 29.73 P,0.001 F
[69,644]
= 6.72 P,0.001
Locomotor sensitization F
[3,28]
= 16.08 P,0.001 F
[8,224]
= 8.87 P,0.001 F
[24,168]
= 11.34 P,0.001
Circling behavior F
[3,28]
= 8.04 P,0.001
Rearing activity F
[3,28]
= 3.39 P,0.05 F
[8,224]
= 7.86 P,0.001 F
[24,224]
= 2.87 P,0.001
Overall effects for Fig. 5
4-Cl-KYN effects in CPP Factor ‘treatment’Factor ‘CPP phase’Factor ‘treatment’‘CPP phase’
Time in drug-paired compartment F
[3,27]
= 1.71 P = 0.19 F
[1,27]
= 11.71 P,0.01 F
[3,27]
= 4.34 P,0.05
ratio (Post-Cond/Pre-Cond) F
[3,27]
= 4.40 P,0.05
PPI, Ketamine dose-response Factor ‘treatment’Factor ‘intensity’Factor ‘treatment’‘intensity’
% PPI F
[4,148]
= 15.83 P,0.001 F
[2,148]
= 25.40 P,0.001 F
[8,148]
= 0.33 P = 0.95
Startle amplitude F
[4,51]
= 0.51 P = 0.73
PPI, Effects of 4-Cl-KYN Factor ‘treatment’Factor ‘intensity’Factor ‘treatment’‘intensity’
% PPI F
[4,153]
= 23.33 P,0.001 F
[2,153]
= 16.57 P,0.001 F
[8,153]
= 0.74 P = 0.66
Startle amplitude F
[4,54]
= 0.99 P = 0.42
Effects of 7-Cl-KYNA in the FST
Antidepressant effects Factor ‘treatment’
Immobility time F
[3,27]
= 4.121 P,0.05
80 Zanos et al.
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125, or 375 mg/kg; Fig. 5D). Neither ketamine nor 4-Cl-KYN
affected startle amplitude at any dose (Fig. 5, C and E).
Discussion
The present study demonstrated that systemic administra-
tion of 4-Cl-KYN, the brain-penetrant prodrug of the glycine
B
-
site antagonist 7-Cl-KYNA, results in ketamine-like
antidepressant-like effects in mice. In particular, we observed
acute, dose-dependent effects of 4-Cl-KYN in the FST and the
TST, and showed that antidepressant-like efficacy was main-
tained for 24 hours in the FST. Moreover, 4-Cl-KYN showed
remarkable efficacy in the LH paradigm, where the effect was
evident up to 7 days after a single treatment, at a time point
past which 7-Cl-KYNA had become undetectable in brain
microdialysate (Fig. 1B). In addition, 4-Cl-KYN administra-
tion rapidly decreased the latency to feed in the NSF test. All
these antidepressant-like effects were qualitatively identical,
Fig. 2. Antidepressant effects in the forced-swim, tail-suspension, and novelty-suppressed feeding tests. Mice received intraperitoneal injections of
saline (SAL), fluoxetine (FLX), ketamine (KET), or 4-chlorokynurenine (4-Cl-KYN) and were tested in the FST 1-hour post-treatment. (A) Acute
administration of FLX (n= 9/group; unpaired Student’st-test) as well as KET and 4-Cl-KYN (n=12–16/group; one-way ANOVA followed by Bonferroni’s
multiple comparison) significantly reduced immobility in the FST. (B) Glycine pretreatment prevented the antidepressant -like effects of 4-Cl-KYNinthe
FST (n=13–15/group; two-way ANOVA followed by Bonferroni’s multiple comparison). (C) Administration of NBQX prevented the antidepressant
effects of both KET and 4-Cl-KYN in the FST (n=11–14/group, two-way ANOVA followed by Bonferroni’s multiple comparison). (D) In the TST,
administration of 4-Cl-KYN resulted in antidepressant-like effects 1 hour postinjection (n= 16/group; one-way ANOVA followed by Bonf erroni’s multiple
comparison). (E) Administration of KET and 4-Cl-KYN significantly reduced latency to feed in the NSF test, (F) without affecting home-cage food
consumption (n= 10/group; one-way ANOVA followed by Bonferroni’s multiple comparison). Data are the mean 6S.E.M. *p,0.05, **p,0.01, ***p,
0.001.
Fast-Onset Antidepressant Effects of 4-Chlorokynurenine 81
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and quantitatively very similar, to those of ketamine, which
was tested in parallel using a well-established antidepressant
dose for mice [10 mg/kg; Maeng et al. (2008); Ma et al. (2013)].
We confirmed that the antidepressant-like action of 4-Cl-KYN
in the acute FST involves the glycine
B
site, since pretreatment
with glycine prevented the effects at the same dose shown to
increase concentrations of glycine in the brain (albeit by less
than a 2-fold difference) and to reverse the behavioral effects of
NMDA channel blockers (Trullas and Skolnick, 1990; Evoniuk
et al., 1991; Javitt et al., 1999). In support, no off-site receptor
targets were identified by the extensive receptor screening
performed (Supplemental Figs. S1 and S2 and Supplemental
Table S1). Moreover, in line with the hypothesis that ket-
amine’s antidepressant mechanism involves enhanced synap-
tic glutamate release and activation of postsynaptic AMPA
receptors (Li et al., 2010; Duman and Aghajanian, 2012),
administration of an AMPA receptor antagonist completely
prevented the behavioral effects of 4-Cl-KYN in the FST.
Notably, effective doses of 4-Cl-KYN did not produce reward-
ing or aversive properties in the CPP paradigm, and were not
associated with locomotor stimulation, stereotypic behaviors,
or disruption of PPI, all of which are typically induced by
ketamine and other NMDA receptor channel blockers.
A major drawback of monoamine-based antidepressant med-
ications that are currently in clinical use is that they require
sustained treatment of several weeks or longer before thera-
peutic benefits are achieved (Rush et al., 2006). The ineffective-
ness of acute FLX administration in the 24-hour FST and LH
paradigms markedly contrasts with the actions of ketamine,
which have been documented in recent animal studies (Maeng
et al., 2008; Li et al., 2010; Autry et al., 2011; Browne and Lucki,
2013). On the basis of the theoretical construct introduced by
Skolnick and collaborators (1996), this pharmacological dispar-
ity in the effects of acute dosing can be explained at least partly
by the fact that clinical effects of monoaminergic drugs do not
become apparent until NMDA receptors are downregulated
following a delay period. Subsequently, the reduction of NMDA
receptor function initiates a cascade of molecular and cellular
events that may cumulatively account for the benefits seen in
MDD patients [see Skolnick et al. (1996)]. This conceptual
framework reconciles the monoamine and glutamate hypothe-
ses of MDD pathophysiology and accounts for the current
interest in glutamatergic modulators as rapid-acting medica-
tions for the treatment of the disease.
In the present study, 4-Cl-KYN was used as a prodrug of
7-Cl-KYNA, which is frequently employed as an experimental
tool to selectively probe the glycine
B
receptor in vitro, or
following intracerebral application in vivo (Kemp et al., 1988;
Wu et al., 1997). After peripheral administration, 4-Cl-KYN
gains rapid access to the brain via the large neutral amino acid
transporter (Hokari et al., 1996) and is then promptly
accumulated by astrocytes. Subsequently, 4-Cl-KYN is irre-
versibly converted to 7-Cl-KYNA by kynurenine aminotrans-
ferase, and the newly produced glycine
B
receptor antagonist is
then rapidly released into the extracellular milieu (Kiss et al.,
2003) (cf., Fig. 1A). Unlike 4-Cl-KYN (Hokari et al., 1996),
7-Cl-KYNA crosses the blood-brain barrier very poorly, prob-
ably because of its polar nature and the lack of an active
transport system (Leeson and Iversen, 1994). In the present
study, we confirmed the formation of 7-Cl-KYNA from sys-
temically administered 4-Cl-KYN by in vivo microdialysis in
the hippocampus of freely-moving mice.
A recent double-blind, placebo-controlled trial demon-
strated that long-term (6-week) administration of the glycine
B
partial agonist D-cycloserine has antidepressant effects in
humans (Heresco-Levy et al., 2013). Antidepressant-like
effects of glycine
B
-modulating compounds have been pre-
viously reported in behavioral tests that are also sensitive to
monoamine-acting drugs (e.g., the FST and TST). For exam-
ple, Poleszak et al. (2011) showed antidepressant-like efficacy
of the brain-penetrating, specific glycine
B
antagonist
L-701,324 and of D-cycloserine in the FST in mice. However,
few studies have addressed effects in “ketamine-sensitive”
paradigms (i.e., 24-hour FST, LH, and NSF). This is important
Fig. 3. Sustained antidepressant effects in the 24-hour forced-swim test
and learned helplessness paradigms. Mice received intraperitoneal injections
of saline (SAL), fluoxetine (FLX), ketamine (KET), 7-chlorokynurenic acid
(7-Cl-KYNA), or 4-chlorokynurenine (4-Cl-KYN) and were tested in the
FST 1 or 24 hours post-treatment, and in the LH paradigm 24 hours and
7 days following treatment. (A) While FLX administration did not induce
antidepressant effects, both KET and 4-Cl-KYN significantly reduced
immobility time in the FST and decreased the number of escape failures in
the LH paradigm at (B) 24 hours or (C) 7 days (n=10–11/group; one-way
ANOVA followed by Bonferroni’s multiple comparison). Injections of FLX
or 7-Cl-KYNA did not replicate the effects of 4-Cl-KYN in the LH paradigm
24 hours (D) or 7 days (E) post-treatment (n=8–9/group; one-way ANOVA
followed by Bonferroni’s multiple comparison). Data are the mean 6S.E.M.
*p ,0.05, **p,0.01, ***p,0.001. ns,notsignificant.
82 Zanos et al.
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as ketamine represents a first-in-class new generation rapid-
acting antidepressant drug. A notable exception is GLYX-13,
an amidated tetrapeptide, which can act as a partial agonist of
the glycine
B
receptor and has rapid-acting antidepressant
effects in the 24-hour FST, LH, and NSF tests (Burgdorf et al.,
2013). Since the endogenous ligands of the glycine
B
receptor—
glycine and D-serine—are present at high levels in brain, it is
hypothesized that at high concentrations glycine
B
receptor
partial agonists act as functional antagonists in vivo by
reducing the facilitating effect of glycine and/or D-serine (Clos
et al., 1996). However, the involvement of the glycine
B
site of
the NMDA receptor in the antidepressant effects of GLYX-13
has not been directly demonstrated so far.
Maj et al. (1994) described antidepressant-like effects
following repeated, but not single, administration of 7-Cl-KYNA
(20 mg/kg) in the FST, and acute antidepressant-like effects
were seen after a single injection of low doses of 7-Cl-KYNA
in rats (Zhu et al., 2013; Liu et al., 2015). However, the latter
findings were not replicated in our experiments. In contrast,
i.p. administration of lower doses of 4-Cl-KYN, the brain-
penetrant bioprecursor of 7-Cl-KYNA, exerted rapid and
sustained ketamine-like effects in tests that distinguish ket-
amine from monoamine-acting antidepressants. Reversal of
antidepressant effects with glycine supported the concept that
the antidepressant-like actions of 4-Cl-KYN involved inhibi-
tion of the glycine
B
site of the NMDA receptor.
Possible side effects of 4-Cl-KYN were examined by assess-
ing locomotor activity, stereotypic behaviors, PPI, CPP, and
behavioral sensitization. Unlike ketamine, 4-Cl-KYN produced
none of these effects at any of the doses tested. This indicates
that antagonism of the glycine
B
site with 4-Cl-KYN does not
appear to elicit the detrimental side effects (e.g., psychotomi-
metic and rewarding) that are associated with the use of
ketamine or other NMDA channel blockers (Mansbach, 1991;
Papp and Moryl, 1994). The advantages of targeting the
glycine
B
site may be linked to the different mechanisms by
which inhibition of the co-agonist site and channel blockade
reduce NMDA receptor function (Danysz and Parsons, 1998).
Alternatively or in addition, the detrimental effects of
ketamine, as well as other NMDA channel blockers, including
Fig. 4. Open-field behaviors. (A) Behavioral sensitization protocol. Acute intraperitoneal administration of 4-chlorokynurenine (4-Cl-KYN) did not have
effects similar to intraperitoneal ketamine (KET) on (B) acute hyperlocomotion (n=7–9/group; two-way ANOVA followed by Bonferroni’s multiple
comparison) or (C) locomotor sensitization (n=7–9/group; two-way ANOVA followed by Bonferroni’s multiple comparison). Unlike KET, 4-Cl-KYN
administration was not associated with (D) increased stereotypic circling(n=7–9/group; one-way ANOVA fo llowed by Bonferroni’s multiple comparison) or (E)
rearing behavior (n=7–9/group; two-way ANOVA followed by Bonferroni’s multiple comparison). Data are the mean 6S.E.M. **p,0.01, ***p,0.001.
Fast-Onset Antidepressant Effects of 4-Chlorokynurenine 83
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phencyclidine and MK-801, may be related to the fact that
these compounds have been reported to directly influence
dopaminergic transmission by reducing dopamine reuptake
(Nishimura and Sato, 1999) or facilitating dopamine release
(Uchihashi et al., 1992). Notably, we demonstrated here that
neither 4-Cl-KYN nor 7-Cl-KYNA have significant affinity for
a large number of monoaminergic and other possible targets.
In summary, the present study supports the hypothesis that
antagonism of the glycine
B
co-agonist site of the NMDA
receptor constitutes a promising approach for the treatment
of major depression. Specifically, our results highlight the
potential of 4-Cl-KYN as a next-generation, rapid-acting
antidepressant medication, with a safer side-effect profile
than ketamine. Since the prodrug uses astrocytes for delivery
of the active compound, the efficacy of our approach may be
further enhanced by the fact that it could specifically buttress
astrocyte function, which is impaired in individuals with MDD
(Etievant et al., 2013; Rajkowska and Stockmeier, 2013).
Importantly, in a Phase 1, randomized, dose-escalation study
evaluating safety and pharmacokinetics of single doses of
orally administered 4-Cl-KYN in healthy volunteers, the drug
did not cause any serious adverse events (ClinicalTrials.gov
Identifier: NCT01483846), indicating a potential for clinical
trials in MDD patients.
Acknowledgments
4-Cl-KYN was provided by VistaGen Therapeutics, Inc. Receptor
binding profiles and K
i
determinations were generously provided by
the NIMH Psychoactive Drug Screening Program, Contract No.
HHSN-271-2008-025C, directed by Dr. Bryan Roth (University of
North Carolina, Chapel Hill, NC) in conjunction with Ms. Jamie
Driscoll (NIMH, Bethesda, MD).
Authorship Contributions
Participated in research design: Zanos, Piantadosi, Zarate Jr.,
Schwarcz, Gould.
Conducted experiments: Zanos, Piantadosi, Wu, Pribut, Can.
Contributed new reagents or analytic tools: Snodgrass.
Performed data analysis: Zanos, Piantadosi, Dell.
Wrote or contributed to the writing of the manuscript: Zanos,
Piantadosi, Schwarcz, Gould.
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Address correspondence to: Dr. Todd D. Gould, Department of Psychiatry,
University of Maryland School of Medicine, Rm. 936 MSTF, 685 W. Baltimore
St., Baltimore, MD 21201. E-mail: gouldlab@me.com
Fast-Onset Antidepressant Effects of 4-Chlorokynurenine 85
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