D-578, an orally active triple monoamine reuptake inhibitor, displays antidepressant and anti-PTSD like effects in rats

Article abstract of DOI:10.1016/j.ejphar.2019.172632

Significant unmet needs exist for development of better pharmacotherapeutic agents for major depressive disorder (MDD) and post-traumatic stress disorder (PTSD) as the current drugs are inadequate. Our goal in this study is to investigate behavioral pharm

Full text of DOI:10.1016/j.ejphar.2019.172632

Journal Pre-proof
D-578, an orally active triple monoamine reuptake inhibitor, displays antidepressant
and anti-PTSD effects in rats
Aloke K. Dutta, Soumava Santra, Arman Harutyunyan, Banibrata Das, Michael
Lisieski, Liping Xu, Tamara Antonio, Maarten E.A. Reith, Shane A. Perrine
PII:
S0014-2999(19)30584-9
DOI:
https://doi.org/10.1016/j.ejphar.2019.172632
EJP 172632
Reference:
To appear in: European Journal of Pharmacology
Received Date: 23 March 2019
Revised Date: 5 August 2019
Accepted Date: 27 August 2019
Please cite this article as: Dutta, A.K., Santra, S., Harutyunyan, A., Das, B., Michael Lisieski, , Xu, L.,
Antonio, T., Reith, M.E.A., Perrine, S.A., D-578, an orally active triple monoamine reuptake inhibitor,
displays antidepressant and anti-PTSD effects in rats, European Journal of Pharmacology (2019), doi:
https://doi.org/10.1016/j.ejphar.2019.172632.
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition
of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of
record. This version will undergo additional copyediting, typesetting and review before it is published
in its final form, but we are providing this version to give early visibility of the article. Please note that,
during the production process, errors may be discovered which could affect the content, and all legal
disclaimers that apply to the journal pertain.
©
2019 Published by Elsevier B.V.

D-578, an orally active triple monoamine reuptake inhibitor, displays antidepressant
and anti-PTSD effects in rats.
2
Aloke K. Dutta,*1 Soumava Santra1, Arman Harutyunyan , Banibrata Das,1 Michael
2
Lisieski, Liping Xu1, Tamara Antonio3, Maarten E.A. Reith3, Shane A. Perrine2
1
. Wayne State University, Department of Pharmaceutical Sciences, Detroit, MI 48202
. Department of Psychiatry and Behavioral Neurosciences, School of Medicine, Wayne
2
State University
. New York University, Department of Psychiatry, New York, N.Y. 10016.
3
Corresponding author:
Aloke K. Dutta, Ph.D., Department of Pharmaceutical Sciences, Eugene Applebaum
College of Pharmacy & Health Sciences, Wayne State University, Detroit, MI, 48202.
Tel: 1-313-577-1064, Fax: 1-313-577-2033, e-mail: adutta@wayne.edu

Abstract
Significant unmet needs exist for development of better pharmacotherapeutic agents for
major depressive disorder (MDD) and post-traumatic stress disorder (PTSD) as the current
drugs are inadequate. Our goal in this study is to investigate behavioral pharmacological
characterization of a novel triple reuptake inhibitor (TRI) D-578 which exhibits nanomolar
potency at all three monoamine transporters (Ki; 16.2. 16.2, 3.23 nM, and 29.6, 20.6, 6.10
nM for the rat brain and cloned human dopamine, serotonin and norepinephrine
transporters, respectively) and exhibited little to no affinity for other off-target CNS
receptors. In a rat forced swim test, compound D-578 upon oral administration displayed
high efficacy and not stimulating in locomotor behavior. The effects of D-578 and
paroxetine were next evaluated in a rat model for traumatic stress exposure - the single
prolonged stress (SPS) model - which has been shown to have construct, predictive, and
behavioral validity in modeling aspects of PTSD. Our results show that SPS had no effect
on the acquisition of conditioned fear, but impaired extinction learning and extinction
retention of fear behavior compared to sham treatment. D-578, but not paroxetine,
attenuated the extinction and extinction-retention deficit induced by SPS. These findings
suggest that D-578 has greater efficacy in normalizing traumatic stress-induced extinction-
retention learning in a model for PTSD compared to paroxetine. Overall these results
suggest that D-578, in addition to producing a robust and efficacious antidepressant effect,
may attenuate maladaptive retention of fearful memories and support further testing of this
agent for the pharmacotherapy of depression and PTSD.
Key Words: Major depressive disorder; Post-traumatic stress disorder; Triple reuptake
inhibitors; Pharmacotherapy; selective serotonin reuptake inhibitor; Pharmacotherapy

1. Introduction
Both major depressive disorder (MDD) and post-traumatic stress disorder (PTSD) are
debilitating illnesses affecting greater than 15-20% of the population in the United States
(Kessler et al., 2005). It is believed that 20% of all individuals suffer from a MDD at least
once in their lifetime. Depression in some cases may lead to life threatening acts and
suicide (Pompili, 2019). PTSD is a chronic and debilitating illness which is caused by
exposure to traumatic events (Benjet et al., 2016). It has been estimated by
epidemiologic studies that PTSD exists in ~8% of the US population, and in up to 30%
of certain at-risk groups (Breslau et al., 1991; Kessler et al., 1995).
Over the years different classes of antidepressants have been developed. However,
a significant unmet need persists for improved therapy, as large numbers of people with
clinically-diagnosed depression, an estimated 15-30%, are still refractory to the current
existing therapies. Moreover, a number of people suffer from relapse after treatment with
current therapies (Rush et al., 2006). It is evident from the preclinical and clinical studies
that dopamine plays an important role in the pathophysiology and treatment of depression
(Dunlop and Nemeroff, 2007). However, one of the missing components in the current
pharmacotherapy of depression is a dopaminergic activity in spite of existing evidence
pointing to the presence of a strong dopaminergic component in MDD (Dunlop and
Nemeroff, 2007).
Similar to depression, there is a great unmet need in the treatment of PTSD. The
two FDA-approved pharmacotherapeutics currently used for PTSD are the selective
serotonin reuptake inhibitors (SSRIs) paroxetine and sertraline (Bandelow et al., 2012).
However, these agents are limited by their efficacy with low effect size (ES = 0.23, 95% CI,

0.12–0.33) and do not work for a significant number (30%) of people and produce side
effects (Hoskins et al., 2015; Ipser and Stein, 2012; Marks et al., 2008b; Thomas and
Stein, 2017). Besides serotonergic neurotransmission, noradrenergic and dopaminergic
pathways have been implicated in PTSD symptomatology. Various studies indicated that
PTSD patients suffer from reward deficits with social anhedonia (Nawijn et al., 2015). It is
conceivable that augmentation of dopaminergic activity could address these untreated
symptoms related to anhedonia associated reward functioning in the PTSD population.
The concept behind the development of triple uptake inhibitors (TRIs) that target the
serotonin, norepinephrine, and dopamine transporters (SERT, NET, and DAT)
concurrently, has gained support based on the evidence from preclinical and clinical
studies for the treatment of depression. It has also been predicted that agents potentiating
all three monoaminergic systems might selectively benefit PTSD patients with the
dysphoric/ anhedonic phenotype (Friedman and Bernardy, 2017).
In our work towards the development of TRIs, we embarked on designing and
synthesizing novel pyran based asymmetric compounds (Dutta et al., 2014; Santra et al.,
2012; Zhang et al., 2006; Zhang et al., 2005). Our structure-activity relationship (SAR)
studies led to the identification of an earlier lead compound D-473 which showed high
efficacy in a rat model of depression (Dutta et al., 2014). In this manuscript, we report
development of an orally active TRI, D-578 and its pharmacological characterization in a
rat forced-swimming test, locomotor activity assay and conditioned fear behavior in a
rodent model of PTSD.

Fig. 1: Molecular structures of triple reuptake inhibitors.
2. Materials and Methods
2.1 Reagents and drugs
Synthesis of D-578 (2S,4R,5R)-5-(((2-aminobenzo[d]thiazol-6-yl)methyl)amino)-2-
(bis(4-fluorophenyl)methyl)tetrahydro-2H-pyran-4-ol are shown in Scheme 1 & 2 and is
described below. This compound was synthesized by asymmetric synthetic pathway as
3
developed by us (Santra et al., 2012; Zhang et al., 2005). [Ring 2,5,6- H]dopamine (45.0
3
Ci/mmol) and [1,2- H]serotonin (27.9 Ci/mmol) were obtained from Perkin-Elmer (Boston,
MA, U.S.A). Imipramine, desipramine, fluoxetine, and reboxetine, and GBR 12909
dihydrochloride (1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-[3-phenylpropyl]piperazine) were
purchased from SIGMA-ALDRICH (St. Louis, MO, U. S. A). All reagents used to perform

microdialysis (i.e. for aCSF) and saline solutions, as well as LC-MS reagents were
purchased from Sigma Chemical Co (St Louis, MO, U. S. A.).
2.2. Animals
Male Sprague-Dawley rats (200-225 g) were purchased from Harlan (Indianapolis,
IN, U. S. A.). Animals were group housed in a temperature and humidity controlled room
with 12 h light /dark cycle with lights on at 6 AM. Food and water were accessible to
animals freely throughout the duration of study except during behavioral testing. All testing
occurred during the light component of the light/dark cycle. All animal procedures were
reviewed and approved by Wayne State University Institutional Animal Care and Use
Committee consistent with AALAC guidelines.
2.3 Synthesis of D-578
Synthesis of 2-aminobenzo[d]thiazole-6-carbaldehyde
Scheme 1
Anhyd. THF
CH Cl , MeOH
(5:1)
2
2
EtO C
o
S
N
OHC
S
N
2
S
N
0 C, LiAlH
HO
4
NH2
NH2
NH
2
MnO , 72 h
24 h
2
2
3
1
Commercially available ester 1 (1.0 g, 4.5 mmol) was dissolved in anhydrous THF and the
o
RB flask was cooled to 0 C. Then LiAlH
4
was added slowly and the flask was stirred at 0
o
C for additional 10 min. The flask was then stirred at room temperature for 24 h. The
reaction was then cooled, quenched with methanol, NH Cl Rochelle’s salt and diluted with
4
ethyl acetate (10 ml). The organic layer was separated and the aqueous layer was
extracted with additional ethyl acetate (3 × 10 ml). The organic layers were combined,

2 4
dried over Na SO and concentrated under vacuo on a rotary evaporator to obtain a yellow
solid. The crude product was purified via gradient silica gel column chromatography using
a mixture of CH Cl and methanol (100:1 to 5:1) to obtain the desired alcohol as yellow
2
2
solid 2 (420 mg, 52%).
1
H NMR (500 MHz, CDCl ): δ 7.57 (d, J = 0.9 Hz, 1 H), 7.35-7.46 (m, 1 H), 7.25 (dd, J =
3
8.2, 1.8 Hz, 1 H), 6.45 (br s, 1 H), 4.53 (s, 2 H), 4.0 (br s, 1 H).
13
C NMR (125 MHz, CDCl
The alcohol 2 (420 mg, 2.33 mmol) was dissolved in a mixture of CH
methanol (5:1) and MnO (81 mg, 9.32 mmol). The mixture was then stirred at room
3
): δ 167.6, 150.5, 134.9, 130.6, 125.1, 119.4, 117.7, 64.2,.
2
Cl and
2
2
temperature for 48 h following which additional amount of MnO (40 mg) was added after
2
TLC showed incompletion of the reaction. The mixture was stirred for additional 24 h and
then filtered through a whatman filter paper (grade 8) and the filtrate was concentrated
under vacuo on a rotary evaporator to obtain the desired aldehyde as a yellow solid 3 (400
mg, 95%).
1
3
H NMR (500 MHz, CDCl ): δ 9.81 (s,1 H), 8.01 (d, J = 1.5 Hz, 1 H), 7.70 (dd, J = 8.2, 1.5
Hz, 1 H), 7.42 (d, J = 8.5 Hz, 1 H), 3.70 (br s, 1 H).
13
C NMR (125 MHz, CDCl ): δ 191.3, 164.5, 157.1, 130.3, 128.7, 122.6, 118.0
3
Synthesis of (2S,4R,5R)-5-(((2-aminobenzo[d]thiazol-6-yl)methyl)amino)-2-(bis(4-
fluorophenyl)methyl)tetrahydro-2H-pyran-4-ol, D-578
Scheme 2

F
F
NH2
S
N
HN
OH
O
NH2
a)
O
OH
F
4
F
D-578
Reagents and conditions: a) RCHO, 1,2-dichloroethane,
MeOH, AcOH, Na (OAc) BH, rt.
3
Amine 4 (60 mg, 0.19 mmol), which was synthesized by following our published procedure
(Santra et al., 2012), was reacted with 2-aminobenzo[d]thiazole-6-carbaldehyde (37 mg,
0
.21 mmol), glacial acetic acid (16 µl, 0.22 mmol), and Na(OAc) BH (113 mg, 0.38 mmol)
3
in a mixture of 1,2-dichloroethane (4.5 ml) and methanol (1.5 ml). The residue was purified
by gradient silica gel column chromatography using a mixture of dichloromethane and
methanol (100:1 to 6:1) to afford corresponding compound (D-578) as light yellow solid
syrup (45 mg, 50%)
1
H NMR (500 MHz, CDCl ): δ 7.54 (s, 1 H), 7.30 (d, J = 8.2 Hz, 1 H), 7.20 (dd, J = 8.5, 6.1
3
Hz, 2 H), 7.16 (dd, J = 8.2, 1.1 Hz, 1 H), 7.09 (dd, J = 8.6, 5.5 Hz, 2 H), 6.79-6.96 (m, 4 H),
4
.38 (dt, J = 10.1, 2.4 Hz, 1 H), 4.06 (s, 1 H), 3.98 (d, J = 13.1 Hz, 1 H), 3.92 (dd, J = 12.8,
.5 Hz, 1 H), 3.82-3.92 (m, 1 H), 3.79 (d, J = 12.5 Hz, 1 H), 3.34 (br s, 2 H), 2.64 (br s, 1
1
H), 1.56-1.71 (m, 1 H), 1.35-1.47 (m, 1 H).
13
3
C NMR (125 MHz, CDCl ): δ 168.0, 162.4, 162.3, 160.5, 160.4, 151.4, 137.4, 137.2,
1
31.3, 129.7, 129.6, 126.9, 121.3, 118.2, 115.4, 115.3, 115.2, 115.0, 73.9, 64.5, 62.9,
5.8, 54.7, 50.2, 32.6.
5
25
[α]
= (-) 30.6°, c = 1 in MeOH. The product was converted into the corresponding
D
hydrochloride salt; mp: 210-215 °C. Anal. Calcd for [C H F N O S.3HCl.3H O] C, H, N.
26
25
2
3
2
2

Mesylate salt of D-578 was used for in vivo forced swimming study.
.4 Inhibition of uptake by monoamine transporters in brain synaptosomes and
heterologous cells
Inhibition of substrate uptake by monoamine transporters in synaptosome-enriched
2
fractions from rat brain was measured as we described previously (Santra et al., 2012;
Santra et al., 2015; Sharma et al., 2014). DAT in rat striatum and NET in rat cerebral
cortex was monitored with [3H]DA ([ring 2,5,6-3H]dopamine (45.0 Ci/mmol, Perkin-Elmer,
3
3
Boston, MA, USA), and SERT in rat cerebral cortex with [ H]5-HT ([1,2- H]serotonin (27.9
Ci/mmol, Perkin-Elmer). Inhibition of substrate uptake by cloned human transporters was
measured with stably transfected human embryonic kidney (HEK) 293 cells as described in
our previous work (Dutta et al., 2014; Reith et al., 2012). The cell lines were obtained and
used in uptake assays as described in the same papers (Dutta et al., 2014; Reith et al., 2012),
3
again with [ H]DA for DAT and NET and [3H]serotonin for SERT.
The use of [3H]DA instead of {3H]NE greatly reduced nonspecific uptake values for
rat synaptosomes; additionally, it is well established that DA is an excellent substrate for
NET, both in cells with cloned transporters (Buck and Amara, 1994; Gu et al., 1994;
Pacholczyk et al., 1991) and in rat tissue (Masserano et al., 1994; Snyder and Coyle,
1969; Williams and Steketee, 2004). Control experiments with various test compounds did
not show significant differences between potencies measured with [3H]DA and [3H]NE.
Drug stocks contained an additional 0.01% (w/v) bovine serum albumin in order to
reduce absorption of drug to the walls of the assay plates. At least five triplicate
concentrations of each test compound were studied, spaced evenly around the IC50 value
i
which was converted to K with the Cheng-Prusoff equation (Cheng and Prusoff, 1973)

3
With respective K
m
values and total free [ H]ligand concentrations, the conversion factors
(
multipliers applied to IC for calculating K) were > 0.84.
50
i
2.5. Evaluation of broad receptors activity
Compound D-578 was characterized in several CNS receptor binding assays to assess
selective and specific interactions in the CNS. The assays were carried out generously by
the National Institute of Mental Health's Psychoactive Drug Screening Program (NIMH
PDSP). The NIMH PDSP is directed by Bryan L. Roth MD, PhD at the University of North
Carolina at Chapel Hill and Project Officer Jamie Driscol at NIMH, Bethesda MD, USA
(http://pdsp.med.unc.edu/).
Compound D-578 was first evaluated in primary binding assays targeting, among others,
cloned human dopamine receptor subtypes, serotonin receptor subtypes, α− and β-
adrenergic receptors, and opioid receptors. The description of all receptors targeted and
corresponding radioligand used, is provided in Table 3. The default concentration for
primary binding experiments was 10 µM. When observed inhibition under those conditions
was greater than 50%, secondary assays were conducted with full concentration curves of
the test compound in order to calculate the Ki value for inhibition. For experimental details
please refer to the PDSP web site <http://pdsp.med.unc.edu/> and click on "Binding
Assay" or "Functional Assay" on the menu bar. We have also added a brief description of
assay protocols in the supplementary material section.
2.6. hERG Binding Assay

hERG channel inhibition activity was carried out by following the Thallium Flux Assays by
using the FluxOR Potassium Ion Channel Assay kit (Invitrogen). The assay was carried out
by the National Institute of Mental Health's Psychoactive Drug Screening Program, The
protocol of the assay is posted on the PDSP website
(https://pdspdb.unc.edu/pdspWeb/content/PDSP%20Protocols%20II%202013-03-28.pdf
and was also published in detail (Huang et al., 2010).
2.7. Forced swimming test in rats after oral administration
The experiment was carried out in the same way as described in our previous publications
according to the Porsolt protocol (Dutta et al., 2011; Porsolt et al., 1977). The rats
weighing 200-225 g housed in cages for at least 1 week prior to testing. Animals were
maintained in a temperature-controlled environment under a 12 hr light-dark cycle. All
subjects were naive and were used only once.
Rats were transported to the testing room at least for one hour prior to testing for
acclimatization and adaptation purposes. Experimental sessions were conducted between
9
AM to 2 PM daily. Animals were assigned randomly and were placed individually in a
glass cylinder (24.5 cm X 35.5 cm) filled with water at room temperature to a depth of 22
cm. All the test sessions were recorded by video cameras. The water was changed in the
o
beginning of each session and the temperature was maintained constant at 24-25 C.
The test procedure consisted of a pretest and test session separated by 24 h
(
Porsolt et al., 1977). During the pretest period, rats were placed in the swim chamber for
5 min. Followed by the initial swim exposure; rats were patted dry and were transferred to
the individual cages. Drugs or vehicle were then orally administered (p.o) 15 min after the
1

initial swim exposure and were then transported to their home cages. On the following day
the rats were brought back to the testing room at least 1 h before the beginning of test
session. Rats were administered either drugs or vehicle 1 h before the swim test. Each rat
underwent a 5-min swim session, which was videotaped and scored later.
Drug solution was prepared freshly on the test days. Compound D-578 was
dissolved in 3% beta-hydroxy propyl cyclodextrin (BHCD) vehicle solution. The vehicle was
prepared by dissolving required amount of BHCD in saline. The drug and vehicle
preparations were administered orally. D-578 was administered at a dose of 10, 15 and 25
mg/kg. Depending upon the weight of the rats a slight variation of the administered drug
solution volume e.g. 1.2 ml/ rat, was maintained. Drug or vehicle was administered 1 h
prior to testing for forced swimming. Individuals, blinded to the treatment, scored the
videotapes for immobility. Rats exhibiting no activity other than that necessary to keep the
rat’s head above the water was considered as immobile. Immobility scores were analyzed
by one way ANOVA test.
2.8. Locomotor Activity
Sprague Dawley Rats were tested at 25 mg/kg oral dose of D-578 to monitor
changes in any locomotor activity in acrylic Auto-Track/Opto-Varimex-4 System (Columbus
Instrument; Columbus, Ohio). The purpose was to evaluate locomotor activity of the same
doses of drug that were used in the forced swimming test. Rats were acclimated in the test
chambers for 1 h prior to oral (gavage) administration of either D-578 or vehicle.
Locomotor activity of the drug was measured for a total of two hours post administration of

the drug which corresponded to the time of measurement in the forced swimming test
experiment.
2.9. Single Prolonged Stress and Fear Conditioning
In order to determine whether D-578 has effects on maladaptive alterations in fear
memory following exposure to traumatic stress, a cohort of rats were exposed to single
prolonged stress (SPS) or a control treatment, and then subjected to a cued fear
conditioning and extinction procedure with or without the administration of D-578. SPS is a
well-validated rodent model of changes in neuroendocrine function (Liberzon et al., 1997;
Liberzon et al., 1999) and fear behavior (Knox et al., 2012) commonly seen in
PTSD(Lisieski et al., 2018; Yamamoto et al., 2009).
After acclimating to the laboratory, male Sprague Dawley rats (Charles River
Laboratories, Kingston, NY) were given SPS as previously described (Eagle et al., 2015).
Rats were restrained for 2 h in cylindrical clear plastic restrainers. Immediately following
this restraint, they were put into 48 cm-diameter tub filled with 30 cm of room temperature
water for a 20-min group swim (6-8 rats at a time). Rats were then dried and allowed to
rest in a clean cage for 15 min. Finally, rats were exposed to diethyl ether vapor as a group
(6-8 rats) until loss of consciousness, which was confirmed by toe and tail pinch method.
Control animals were held in a separate room for an equal period of time, during which
they were weighed and handled for ~2 min. Following SPS or control exposure, rats were
returned to the vivarium and left undisturbed (except for normal care) for 7 days; this
undisturbed period is necessary for the development of SPS-related behavioral(Knox et
al., 2012) and neuroendocrine changes (Liberzon et al., 1997; Liberzon et al., 1999).

A timeline of fear conditioning experiments is shown in Fig. 4A. Cued fear
th
th
th
conditioning, extinction, and extinction retention tests took place on the 8 , 9 , and 10
days, respectively, following SPS exposure. Rats were given i.p. injections of D-578 (10
mg/kg), paroxetine (5 mg/kg), or vehicle 90 minutes prior to each testing session. During
acquisition, rats were placed in a fear conditioning chamber (Coulbourn Instruments,
Whitehall, PA) and after a 180 s baseline period rats were exposed to 5 tones (10 s long, 2
kHz, 80 dB sine wave) each of which co-terminated with a 1 mA foot shock. Inter-trial
intervals were 50 s. Each rat was returned to its home cage at the conclusion of the
session. Digital videos were scored by two blinded raters for fear-related (i.e. freezing and
rearing) behaviors; data were averaged between raters (inter-rater reliability: Pearson
r>0.8 and quantified as percentage of time spent freezing and rearing during the 180s
baseline period and during each 10 s tone presentation. For extinction and extinction-
retention sessions, rats were placed in a different context, and after a 180 s baseline
period, 20 tones with properties identical to those played during acquisition were played
with inter-trial intervals of 50 s. FreezeFrame software (Coulbourn Instruments) was used
to detect freezing in the videos, which was quantified as percentage of time spent freezing
during the 180s baseline period and during each tone presentation. Data from the
extinction and extinction-retention, but not acquisition, sessions were collapsed into bins of
two-trials. Rater-observations were used for quantification of data for acquisition phase
because FreezeFrame does not include fear related rearing behavior which was present
during acquisition. Rearing behavior not related to fear observed during acquisition was
not included in the freezing data (see supplemental data). However, because fear related
rearing behavior was not observed during extinction or extinction retention (data not

shown), FreezeFrame was used to quantify data for these phases of conditioned fear
testing. Five animals that showed aberrantly high or low freezing behavior during multiple
sessions, defined as having values greater than +/- 2 standard deviations from the grand
mean for 5 or more time points in each of two or more sessions were removed from the
analysis; therefore, group sizes were control = 9, SPS+saline = 8, SPS+paroxetine = 10,
and SPS+D-578 = 9.
Shock-reactivity to the five 1 mA shocks (tone-shock pairings) presented during the
acquisition phase of fear learning was determined. The shock response ratings ranged
from 1 to 4, where: 1 - Flinch involving only head and/or forepaw, 2 - Whole body flinch, 3 -
Whole body flinch followed by ambulation, 4 - Whole body flinch and jump (all four feet in
air - usually followed by ambulation(Perrine et al., 2006) (Menard, Champagne,
Meaney,2004; Perrine, Hoshaw, Unterwald, 2006). The shock response for each animal
was averaged across the five shock presentations, and data from two blinded raters was
average (Pearson r>0.8).
Fear conditioning data from each session were analyzed using two-way repeated-
measures ANOVAs (between-subjects factor: treatment group, within-subjects factor: bin);
significant effects were followed up by post-hoc comparisons using Tukey’s HSD test. To
confirm that SPS caused changes in fear behavior, per-bin comparisons between the
control and SPS+saline groups were conducted for all sessions. To determine whether
administration of D-578 ameliorated any SPS-induced changes, per-bin comparisons
between the SPS+D-578 and the SPS+saline groups were conducted. Per-bin two-group
comparisons were conducted by t-test, using the Benjamini-Hochberg correction
(Benjamini and Hochberg, 1995; www.jstor.org/stable/2346101 ) to limit the family-wise

false discovery rate (FDR) to 0.1 for each session. All tests were two-tailed, except for
one-tailed posthoc tests that were used for per-bin comparisons between SPS and control
groups in the extinction-retention session, as we hypothesized a priori on the basis of
previous findings that SPS-exposed animals would show persistent freezing during
extinction-retention. The shock-reactivity data were analyzed by two-tailed, one-way
ANOVA (between-subjects factor: treatment group) on shock response ratings.
3. Results
3.1. Inhibition of monoamine uptake into rat brain synaptosomes and cells heterologously
expressing DAT, SERT and NET
Tables 1 and 2 show the potency of D-578 and other drugs in inhibiting uptake of
radiolabeled monoamines by their respective transporters, either measured in rat brain
synaptosomes or in cell lines expressing cloned human transporters. As shown in Table 1,
in rat brain synaptosomes, D-578 displays equipotency for DAT and SERT (Ki; 16.2 and
16.2 nM, respectively) whereas it exhibits an almost five-fold higher potency at NET (3.23
nM). The potency at SERT is comparable to that of fluoxetine, an SSRI. Compared to the
tricyclic antidepressant imipramine, D-578 is far more potent at DAT and NET. The well
known TRI DOV 21,947 under our assay conditions exhibited potencies at DAT and NET
comparable to previously reported values. However, we found DOV 21,947 to be
somewhat less potent at SERT in our assay condition than previously described (Skolnick
et al., 2003).
In the cell assays with cloned human transporters, the potency of D-578 is very
similar to the data found from rat tissue. Again D-578 is 3-4 times more potent at NET

compared to DAT and SERT (Ki; 6,10 vs. 29.6 and 20.6, respectively). This cross
validation reaffirms a similar potency of D-578 across species.
Table 1. Uptake Inhibitory Potency at DAT, SERT, and NET in Rat Brain Synaptosomes.
Drugs
DAT uptake, Ki, nM, SERT uptake, Ki,
NET uptake, Ki, nM
3
a
3
a
3
a
[
H]DA
nM, [ H]5-HT
[ H]DA
D-578
16.2 ± 2.0
1,092 ± 98
2,908 + 136
16.2 ± 1.5
12.2 ± 2.4
503 ± 61
3.23 ± 0.99
b
Fluoxetine
Reboxetine
Desipramine
Imipramine
DOV 21,947
770 ± 100
0.79 ± 0.25
b
b
b
1,567 ± 260
1,696 ± 246
97.1 ± 19.6
106 ± 17
1.30 ± 0.22
b
b
b
20.0 ± 7.2
170 ± 48
53.1 ± 5.5
32.5 ± 6.2
c
c
c
DOV 21,947
96 ± 20
12 ± 2.8
23 ± 3
a
Uptake into rat brain synaptosomes was measured as described in Methods. Where
indicated (superscript b & c), values are from previous publications. Results are average +
S.E.M. for 3 to 6 independent determinations.
b
Data from (Dutta et al., 2008); c Data from(Skolnick et al., 2003)
Table 2. Uptake Inhibitory Potency In Cells Expressing Human Transporters.
Drugs
DAT uptake, Ki, nM, SERT uptake, Ki,
NET uptake, Ki, nM
3
a
3
a
3
a
[
H]DA
29.6 ± 6.95
nM, [ H]5-HT
[ H]DA
D-578
20.6 ± 3.8
6.10 ± 1.14
0.86 + .04
Fluoxetine
Desipramine
14.4 + 1.1

Citalopram
3.66 (6) ± 0.64
a
Uptake into cells was measured as described in Methods.
3.2 Broad screening CNS receptors data
Table 3: Binding affinity of D-578 for CNS receptors. The default concentration of drugs
for primary binding experiments was 10 µM, and % inhibition (mean of 4 determinations) is
shown. The study was carried out by the National Institute of Mental Health's Psychoactive
Drug Screening Program,
Target
Radioligand
% Inhibition of Ki for D-578
Receptor
Binding at 10
µM D-578
11
(nM)
3
D1
D2
D3
[ H]SCH 23390
3
[ H]N-methylspiperone
62.3
773
3
[ H]N-methylspiperone
56.4
2615
3
D
4
5
[ H]N-methylspiperone
20.9
3
D
[ H]SCH 23390
37.4
3
5HT1a
5HT1b
5HT1d
5HT2a
5HT2b
[ H]8-OH-DPAT
19.9
3
[ H]GR-125743
13.6
3
[ H]GR-125743
72
702
3
[ H]ketanserin
62.5
1,639.00
425
3
[ H]LSD
80.3

3
5
5
5
5
5
HT2c
HT3
HT5a
HT6
HT7
[ H]Mesulergine
96.3
8.5
73
3
[ H] LY 278,584
3
[ H]LSD
35.2
12.8
28.9
2.3
3
[ H]LSD
3
[ H]LSD
3
GABA
A
[ H]Muscimol
3
Alpha1A
Alpha1B
Alpha1D
Alpha2A
Alpha2B
Alpha2C
Beta1
[ H]Prazosin
18.6
38.3
24.7
22.6
6.3
3
[ H]Prazosin
3
[ H]Prazosin
3
[ H]Clonidine
3
[ H]Clonidine
3
[ H]Clonidine
72
2014
125
125
125
[
[
[
I]Iodopindolol
I]Iodopindolol
I]Iodopindolol
45.6
59.4
59.8
28.8
Beta
2
3
>10,000
2703
Beta
3
BZP Rat H-Flunitrazepam
Brain
Site
2+
3
Ca
[ H]Nitrendipine
Channel
δ-opioid
κ-opioid
3
[ H]DADLE
24.1
68.9
11
3
[ H]Bremazocine
1387
3
H
1
[ H]Pyrilamine

3
H2
H3
[ H]Tiotodine
94.1
19.6
42
3
[ H]Alpha-methyl
Histamine
3
H4
[ H]Histamine
2.4
3
µ-Opioid [ H]Diprenorphine
11.7
97.2
20.9
7.5
3
M
[ H]QNB
>10,000
3
M2
[ H]QNB
3
M3
[ H]QNB
3
M4
[ H]QNB
55
>10,000
>10,000
3
M5
[ H]QNB
84.4
NMDA
PCP Site
PBR
mGlur5
Oxytocin
V1A
MK801
10.6
52.8
53.1
44.6
7.8
600
>10,000
V1B
V2
21
D-578 was evaluated for its interaction with various CNS GPCR receptors and ion
channels with various radioligands as shown in Table 3. The compound did not exhibit any
appreciable affinity for dopamine receptor subtypes at 10 µM. However, for serotonin
receptor subtypes, full dose dependent binding inhibition experiment indicated moderate
affinity for 5HT2c (Ki = 73 nM) and very weak affinity for 5HT2b (Ki = 425 nM). For other

serotonin receptor subtypes, the compound did not show any activity. When evaluated at
alpha adrenergic and beta adrenergic receptors, the compound was found to be inactive.
Similarly, when evaluated at muscarinic subtype receptors, the compound did not exhibit
any affinity for these receptors. The compound did not show any activity at three opioid
receptors and was also devoid of any activity at the GABA and benzazepine sites. The
only appreciable affinity uncovered was for the histamine receptor subtype 2 (H2): a Ki of
42 nM. In general, D-578, beyond interacting with the three monoamine transporters DAT,
SERT and NET, does not display appreciable affinity for other brain targets as assessed in
this screen.
3.3 hERG inhibition
The hERG potassium channel (human ether-a-go-go related gene) is expressed in the
human heart. The channel is a key effector of cardiac repolarization and contributes to the
QT interval measured by the electrocardiogram. Inhibition of hERG can lead to a
prolongation of the QT interval, widely considered a critical risk factor for torsades de
pointes (TdP) arrhythmia in non-cardiac drugs. Therefore, evaluation of hERG blocking
activity of test compounds early on in the drug discovery process is important to identify
any potential issue related to cardiotoxicity.
The hERG channel inhibition assay was carried out by following Thallium Flux FluxOR
Potassium Ion Channel Assay kit (Invitrogen). The assay was carried out by the National
Institute of Mental Health's Psychoactive Drug Screening Program. The results from the
assay show almost no inhibition of the hERG channel by D-578. On the other hand, the
reference compound cisapride exhibited potent inhibition of the channel.
Table 4: Assessment of Inhibition of hERG channel by Tl+ flux assay. Potency is

expressed in EC50 (µM). Results represent best fit values + SE taken directly from curve-
fittings in Prism. Multiple assays were normalized to percentage inhibition and pooled for
analysis
Tl+ Flux Assay
EC50 ( M)
Hill Slope
Compound
µ
Cisapride
D-578
0.205 ± 0.034
14.75 ± 3.310
1.01 ± 0.07
1.471 ± 0.23
3.4 In Vivo data from forced swimming test (oral administration)
Fig. 2: Effect of sub-chronic oral administration of vehicle (n = 18) and D-578 (n = 6-8 per
group) on the duration of immobility in the forced swimming test in rats. One way ANOVA
analysis demonstrates significant effect among treatments: F (3, 17) = 6.956 (P< 0.0029).
Dunnett’s analysis showed that the effect of D-578 at three doses (10, 15 and 25 mg/kg)

on immobility was statistically significant different compared to vehicle (P< 0.01 and
.001).
In order to evaluate dose dependent effect of D-578 on immobility, we decided to
0
use 10, 15 and 25 mg/kg doses of the drug. As shown in Fig. 2, D-578 dose-dependently
reduced immobility in the rat forced swimming test (FST) upon oral administration. The two
lowest doses (10 and 15 mg/kg) produced similar, but significantly reduced, immobility
compared to vehicle control. At the highest dose of 25 mg/kg, the compound produced the
greatest effect with marked reduction of immobility. Thus, the compound shows high
efficacy in the FST.
3.5 In Vivo data from locomotor activity test (oral administration)
Fig. 3: Effects of D-578 (PO) and vehicle on locomotor activity (Distance travelled, n = 4-7

rats/group). D-578 and vehicle were administered orally to the rats. The distance travelled
locomotor activities up to 120 min was measured and is represented by 5 min Bin block.
Unpaired two-tailed t-test analysis demonstrates non-significant effect between control and
D-578. Each treatment group contains four (vehicle) to seven (D-578 group) rats.
The dose which produced the highest efficacy in FST, was tested in locomotor
activity test to observe any effect in motor stimulation. Thus, distance travelled data from
oral administration of 25 mg/kg dose of D-578 is not significantly different from vehicle. It is
important to note that in the final hour of locomotor activity measurement which
corresponds to swim test measurement, neither D-578 or vehicle exhibited any locomotor
activity at all.
3.6 In Vivo data from Fear Conditioning studies in rodent PTSD model
The timeline of the conditioned fear experiments in the rat SPS model of PTSD is shown in
Fig. 4A. Fear-related behaviors from the acquisition, extinction, and extinction retention
sessions are shown in Fig. 4B-D. Shock-reactivity behavior during acquisition learning is
shown in Fig. 4E. The dose 10 mg/kg for D-578 was chosen as this dose produced
significant reduction of immobility compared to control in the FST test which reflects
sufficient behavioral alteration at that dose. For paroxetine, 5 mg/kg dose was chosen as
previous studies with another SPS model indicated efficacy of the drug at a lower dose
concentration administered chronically (Takahashi et al., 2006). Since D-578 at a 25 mg/kg
dose did not behave any differently than vehicle control in the locomotor activity study (Fig.
3), it was not included as a control in a non-SPS animals group.
Acquisition of conditioned fear

A repeated-measures ANOVA on rater-observed fear-behaviors during the acquisition
session revealed a main effect of bin, F(4,128) = 48.879, p < 0.001, but no main effect of
medication group, F(3,32) = 0.153, p=0.927 or bin x group interaction, F(12,128) = 0.795,
P =0.654. No posthoc tests were conducted given that no interaction was observed. As
expected, all animals showed good acquisition of conditioned fear behavior in a paradigm
intended to maximize acquisition learning to allow focus on extinction learning (Fig. 4B).
Extinction of conditioned fear
A repeated-measures ANOVA on freezing behavior during the extinction session
revealed a main effect of bin, F(10,320) = 20.055, P < 0.001; a main effect of medication
group, F(3,32) = 5.453, P = 0.004; and a bin x group interaction, F(30,320) = 2.0128, P =
0.001. Tukey’s HSD showed that rats given D-578 (i.p.) at a 10 mg/kg dose after SPS
froze less during the extinction session than rats given either saline (P = 0.007) or
paroxetine (i.p.) (P = 0.015) at a 5 mg/kg dose. Most importantly, false-discovery-rate-
corrected per-bin comparisons showed that in a majority of bins (all bins except for bins 1
and 7), the SPS+saline group showed significantly more freezing behavior than the control
group, and the SPS+D-578 group showed significantly less freezing behavior than the
SPS+saline group (Fig. 4C).
Extinction Retention of conditioned fear
A repeated-measures ANOVA on freezing behavior during the extinction retention
session revealed a main effect of bin, F(10,320) = 22.612, P <0.001; and a main effect of
medication group, F(3,32) = 3.229, P =0.035; and a significant bin x group interaction,
F(30,320) = 1.495, P =0.05. Tukey’s HSD showed that rats given D-578 after SPS froze
less during the retention session than rats given paroxetine (P = 0.034). One-tailed post-

hoc tests between SPS+saline and control groups showed differences between these
groups during bins 6 and 7, but only when not correcting for multiple comparisons. Most
importantly, false-discovery-rate-corrected per-bin comparisons showed that the SPS+D-
578 group froze significantly less than the SPS+saline group during the first two bins of the
session (Fig. 4D).
Shock-Reactivity during Acquisition
An ANOVA on rater-observed shock response behaviors during the acquisition
session revealed no main effects of treatment, F(3, 32) = 1.486, P = 0.237. As expected,
SPS-alone (i.e. SPS+saline) or either drug group (i.e. SPS+paroxetine or SPS+D-578) had
no effect on shock reactivity compared to the control group and therefor likely did not
influence acquisition or extinction of conditioned fear behaviors (Fig. 4D).

Fig. 4: A: Timeline of conditioning fear experiment in PTSD model. Rats were
exposed to single prolonged stress (SPS), allowed a 7-day incubation period, and then
subjected to a three-phase cued fear conditioning protocol with injections of D-578 (10
mg/kg), paroxetine (PRX, 5 mg/kg), or saline-vehicle (n = 8-10 per group). See Methods
for more details. B, C, and D: Effects of SPS and drugs on fear conditioning

behaviors. Rats given SPS+vehicle showed increased freezing relative to Control rats
throughout the extinction sessions (C) and during the later phase of extinction-retention
testing (D), but as expected did not affect fear-related behaviors during acquisition
sessions (B). Although paroxetine did not reverse the SPS-induced deficits during the
extinction and extinction-retention sessions, D-578 significantly reduced SPS-induced
freezing in response to the fear-conditioned cue in the extinction sessions, and reduced
freezing during the initial stage of the extinction retention session. E: Effects of SPS and
drugs on shock reactivity during acquisition of conditioned fear learning. Rats
significant difference between SPS+saline and control groups or SPS+saline and SPS+D-
78 groups, family-wise FDR = 0.1. : significant difference between SPS+saline and
control groups without multiple comparison correction.
*:
5
#
4
. Discussion
The general consensus is that current treatments for both MDD and PTSD are not
adequate, indicating significant unmet needs for development of effective therapies
Bandelow et al., 2012; Hoskins et al., 2015; Rush et al., 2006). The role of dopamine in
(
depression has been uncovered in an adjunct therapy approach: the combination of
bupropion and a SSRI turned out to be effective in patients refractory to SSRIs
(Mischoulon et al., 2000). Efficacy in such treatment strongly points towards the
involvement of a dopamine component since bupropion is a blocker of dopamine
transporter (Ascher et al., 1995). In another study, pramipexole, an antiparkinsonian drug
3
with D dopamine receptor preferring agonist activity, exhibited effectiveness in both
unipolar and bipolar depression (Sporn et al., 2000).

It is established that innervation of dopaminergic neurons in the cortex, limbic region, and
pituitary gland is linked with cognition, motivation and reward (D'Aquila et al., 2000; Dunlop
and Nemeroff, 2007; Nutt et al., 2007; Papakostas, 2006; Sharma et al., 2015). Mesolimbic
dopamine is associated with motivation and reward-related behavior and, therefore,
inclusion of dopamine activity in a TRI should improve anhedonia that is the central
component in depression (Nestler and Carlezon, 2006; Willner, 1983). As alluded above
such anhedonia is also implicated in PTSD. Thus, a rational strategy of inhibiting the
reuptake of all three monoamines with development of TRIs for the treatment of
depression as well as PTSD is warranted (Guiard et al., 2009; Marks et al., 2008a).
A number of TRIs have been developed in the past with varying potencies for
inhibition of monoamine transport (Sharma et al., 2015). Some of these compounds
including DOV 21,947, JNJ-7925476 and BMS-820836 (Fig. 1) have been well
characterized in animal models of depression (Risinger et al., 2014; Sharma et al., 2015;
Skolnick et al., 2003). DOV 21,947 exhibited promising results in initial clinical trial and has
undergone Phase IIb/IIIa studies (NCT01318434)(Tran et al., 2012). Another
norepinephrine preferring TRI, EB-1020, produced positive results in all subtypes of ADHD
population (NCT01939353)(Bymaster et al., 2012). In general TRIs have been shown to be
safe in human clinical studies when no off targets issues are involved (Sharma et al.,
2015). Thus, TRIs can potentially offer higher efficacy in these therapeutic areas compared
to the existing antidepressants and pharmacotherapies for PTSD. Although a number of
TRIs did not succeed in the clinical trials due to various issues including those related to
transporter occupancy and non-specific interaction, there are other TRIs which showed