Structure-activity relationship, biological, and pharmacological characterization of the proline sulfonamide ACT-462206: A potent, brain-penetrant dual orexin 1/orexin 2 receptor antagonist

Article abstract of DOI:10.1002/cmdc.201402258

The orexin system consists of two G-protein-coupled receptors, the orexin 1 and orexin 2 receptors, widely expressed in diverse regions of the brain, and two peptide agonists, orexin A and orexin B, which are produced in a small assembly of neurons in the lateral hypothalamus. The orexin system plays an important role in the maintenance of wakefulness. Several compounds (almorexant, SB-649868, suvorexant) have been in advanced clinical trials for treating primary insomnia. ACT-462206 is a new, potent, and selective dual orexin receptor antagonist (DORA) that inhibits the stimulating effects of the orexin peptides at both the orexin 1 and 2 receptors. It decreases wakefulness and increases non-rapid eye movement (non-REM) and REM sleep while maintaining natural sleep architectures in rat and dog electroencephalography/electromyography (EEG/EMG) experiments. ACT-462206 shows anxiolytic-like properties in rats without affecting cognition and motor function. It is therefore a potential candidate for the treatment of insomnia.

Full text of DOI:10.1002/cmdc.201402258

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DOI: 10.1002/cmdc.201402258
Structure–Activity Relationship, Biological, and
Pharmacological Characterization of the Proline
Sulfonamide ACT-462206: a Potent, Brain-Penetrant Dual
Orexin 1/Orexin 2 Receptor Antagonist
Christoph Boss,* Catherine Roch-Brisbare, Michel A. Steiner, Alexander Treiber,
Hendrik Dietrich, Francois Jenck, Markus von Raumer, Thierry Sifferlen, Christine Brotschi,
Bibia Heidmann, Jodi T. Williams, Hamed Aissaoui, Romain Siegrist, and John Gatfield[a]
The orexin system consists of two G-protein-coupled receptors,
the orexin 1 and orexin 2 receptors, widely expressed in di-
verse regions of the brain, and two peptide agonists, orexin A
and orexin B, which are produced in a small assembly of neu-
rons in the lateral hypothalamus. The orexin system plays an
important role in the maintenance of wakefulness. Several
compounds (almorexant, SB-649868, suvorexant) have been in
advanced clinical trials for treating primary insomnia. ACT-
462206 is a new, potent, and selective dual orexin receptor an-
tagonist (DORA) that inhibits the stimulating effects of the
orexin peptides at both the orexin 1 and 2 receptors. It de-
creases wakefulness and increases non-rapid eye movement
(non-REM) and REM sleep while maintaining natural sleep ar-
chitectures in rat and dog electroencephalography/electro-
myography (EEG/EMG) experiments. ACT-462206 shows anxio-
lytic-like properties in rats without affecting cognition and
motor function. It is therefore a potential candidate for the
treatment of insomnia.
Introduction
The orexin (hypocretin) system is an evolutionarily conserved
neuropeptide–receptor system that acts as a central regulator
of wakefulness and modulates emotional states related to
stress or reward. The neuropeptides orexin A and orexin B are
biosynthesized by a discrete number of neurons in lateral hy-
pothalamic areas (LHA), regions historically implicated in arous-
al, emotional and metabolic regulation, and motivated behav-
iors such as feeding.[1–4] Orexins are released in a Ca2+-sensitive
manner at axonal terminals and can then bind to two closely
related G-protein-coupled receptors (GPCRs): orexin receptor
type 1 (OX1) and orexin receptor type 2 (OX2).[5–7] In neurons,
activation of OX1 and OX2 leads to the activation of the Gq/
phospholipase C/protein kinase C pathway which results in the
modulation of ion channel activities, cellular depolarization,
and increases in cytosolic Ca2+ concentrations.[8] Thus, orexin
receptor signaling enhances synaptic transmission.
tion of vigilance states and circadian activity. Nerve fibers from
orexin immunoreactive neurons of the LHA make wide and
dense projections to the basal forebrain, corticolimbic struc-
tures, and brainstem, particularly to those regions related to
waking/regulation of sleep (locus coeruleus, raphe nucleus, tu-
beromammillary nucleus), regions activated in anxiety/stress-
related conditions (paraventricular nucleus, amygdala) as well
as regions involved in reward processing and drug abuse (nu-
cleus accumbens, ventrotegmental area).[3,4,9–18] Accordingly, in-
fusing orexins intracerebrally in rats leads to enhanced behav-
ioral activity, arousal, delayed onset of REM sleep, and mainte-
nance of cortical activation. Furthermore, pharmacological in-
hibition of the orexin system in animal models of insomnia,
stress/anxiety as well as drug abuse has demonstrated a direct
role of an overactive orexin system in these pathologies and
suggests orexin receptors as therapeutic targets in insomnia,
stress/anxiety-related disorders and addiction.[19–30] Two dual
orexin receptor antagonists have been studied in advanced
clinical trials and demonstrated potential for the treatment of
sleep disorders. In insomnia patients, both almorexant (1) and
suvorexant (3) dose-dependently increased sleep efficiency
and total sleep time by decreasing latency to persistent sleep
and wake after sleep onset.[31–33] SB-649868 (2) has also been
studied in humans with primary insomnia. In initial studies it
exhibited a typical efficacy profile of dual orexin receptor an-
tagonists.[34] Other orexin receptor antagonists have recently
been tested by Merck as co-medication in depression, neuro-
pathic pain, and migraine (http://www.clinicaltrials.gov).
The orexin peptides and their receptors are highly conserved
across mammalian species, and the neuroanatomical distribu-
tion of OX1 and OX2 supports their essential role in the regula-
[a] Dr. C. Boss, Dr. C. Roch-Brisbare, Dr. M. A. Steiner, Dr. A. Treiber,
Dr. H. Dietrich, Dr. F. Jenck, Dr. M. von Raumer, Dr. T. Sifferlen,
Dr. C. Brotschi, Dr. B. Heidmann, Dr. J. T. Williams, Dr. H. Aissaoui,
Dr. R. Siegrist, Dr. J. Gatfield
Drug Discovery and Preclinical Research & Development
Actelion Pharmaceuticals Ltd.
Gewerbestrasse 16, 4123 Allschwil/BL (Switzerland)
E-mail: christoph.boss@actelion.com
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201402258.
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We carried out
a
high-
throughput screening program
using Ca2+ release assays in the
search for dual orexin receptor
antagonists. Herein we describe
the structural optimization of
our initial screening hit, leading
to the preclinical candidate ACT-
462206 (24), which is a new, se-
lective, and competitive antago-
nist at OX1 and OX2. It shows ex-
cellent brain penetration follow-
ing oral administration. It de-
creases wakefulness, decreases
sleep latency, and increases
Figure 2. Structure and data for the OX1/OX2 HTS hit.
sleep efficacy in rats and dogs.
Compound 24 also shows some
efficacy in paradigms of stress/anxiety and addiction.
All compounds shown in Figure 1 are or were in clinical
trials for primary insomnia. Compounds 1–4 are described as
potent dual OX1/OX2 antagonists, exhibiting an insurmounta-
ble inhibition profile at the orexin receptors.[34–38] Compounds
5 and 6 are reported to be selective OX2 antagonists. Based on
recent publications, selective OX2 antagonists might show simi-
lar clinical efficacy as dual OX1/OX2 antagonists.[39,40]
placement of the electron-rich thiophene by a bioisostere;
3) determining if replacement of the phenyl ring with nitro-
gen-containing heterocycles can avoid the anilide subunit; and
4) identification of replacements of the oxidatively labile S-
methyl substituent. After having scrutinized the available data,
we decided to keep the sulfonamide as well as the amide func-
tionality in the molecule.
The data collected for compound 7, with a molecular weight
of 461.42 Da and significant activity in the calcium release
assay, could be considered optimal and illustrate the high qual-
ity and attractiveness of this starting point for a medicinal
chemistry lead optimization program. Compound 7 exhibited
good brain penetration properties and no liabilities toward
being a P-glycoprotein (P-gp) substrate, reasonable in vitro
human liver microsomal metabolic stability, and no obvious cy-
tochrome inhibitory activity. From a synthetic chemistry point
of view, the preparation of compound 7 and its derivatives
was straightforward and is depicted in Scheme 1.
Results and Discussion
Our high-throughput screen for orexin receptor antagonist ac-
tivity on both OX1 and OX2 using calcium release assays result-
ed in the proline sulfonamide 7 as one of our preferred prom-
ising hit structures (Figure 2). Medicinal chemistry based opti-
mization focused on the following aspects (see arrows in
Figure 2): 1) effect of the bromine substituent exchange; 2) re-
The preparation of screening hit 7 for confirmation purpos-
es, and to obtain larger amounts for broader profiling, started
from commercially available l-proline methyl ester hydrochlo-
ride 8, which was treated with 5-bromothiophene-2-sulfonyl
Scheme 1. Synthesis of compound 7 and derivatives thereof (details given in
the Supporting Information and in Ref. [44]). Reagents and conditions:
a) DIPEA, CH2Cl2, RT, 12 h, 98%; b) MeOH/THF (1:1), 1m NaOH (2 equiv), RT,
12 h, 93%; c) POCl3, pyridine, 08C!RT, 30 min, 95%.
Figure 1. Compounds with clinical trial activity being reported.
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chloride (9) in dichloromethane in the presence of N,N-diiso-
propylethylamine at room temperature for 12 h to give the in-
termediate methyl ester 10 in 98% yield. Ester hydrolysis was
achieved under standard conditions[41] by dissolving 10 in a 1:1
mixture of THF/methanol and adding two equivalents of aque-
ous sodium hydroxide. The resulting mixture was stirred for
12 h at room temperature to give the acid precursor 11 in
93% yield. The final step, reaction with 3-(methylthio)aniline
(12), was achieved with phosphorous oxychloride in pyridine
starting at 08C and slowly warming the reaction mixture to
room temperature.[42,43] Screening hit 7 was obtained in 95%
yield. Selection of the reaction conditions to form the anilide
moiety was based on the potentially low nucleophilic reactivity
of the anilines used in this step.
Table 1. Structure–activity relationships: sulfonamide moiety part 1.
R1
Compd
IC50 [nm][a]
IC50 [mm][b]
OX1
OX2
3A4T
20
21
22
2.2
2.5
1.9
3.2
3.9
7.8
2.8
6.9
31
Scheme 2 represents the general possibilities to obtain final
proline sulfonamide dual orexin receptor antagonists 17. All
synthetic steps necessary by either way can be performed with
conditions mild enough to tolerate a diverse set of substitu-
ents at the aromatic or heteroaromatic rings contained in both
substituents introduced to the proline template. The route
chosen depended fully on the structure–activity relationship
(SAR) question to be investigated. Experimental details and an-
alytical data for the preparation of some of the final com-
pounds can be found in the Supporting Information and in
Ref. [44].
23
7
8.4
5.6
9.9
6.4
6.1
21
[a] Data are the geometric mean (ꢀ2-fold) of at least three independent
experiments and were determined by FLIPR assay (see the Supporting In-
formation for assay details). [b] Values are from single measurements with
testosterone used as the marker substrate.
Our optimization efforts started by looking for replacements
of the 5-bromothiophene unit present in the screening hit 7.
In an initial effort we kept the 3-methlythioanilide part fixed
and combined it with variously substituted phenyl moieties at
the sulfonamide end of the molecule, as depicted in Table 1.
These changes allowed maintenance of the promising inhibito-
ry activity at OX2 and clearly improved the potency at OX1.
With respect to orexin antagonistic activity, compounds 20
and 21 were superior to the other examples. Unfortunately,
the compounds containing a 3,4-disubstituted phenyl unit
showed rather prohibitive inhibitory potency in a cytochrome
P450 3A4 assay. Still very promising activities on both orexin
receptors, combined with lower inhibition of cytochrome P450
3A4 activity were found for compound 22, bearing a 4-me-
thoxyphenyl sulfonamide unit. Comparing the 3-bromophenyl
substituent present in 23 with the 2-bromothienyl substituent
from parent compound 7 shows that the phenyl unit is
a viable replacement for the thiophene moiety with respect to
orexin antagonist activity as well as cytochrome inhibition. In
the next step we investigated the SAR in the anilide area of
the orexin antagonists by fixing the sulfonamide to the previ-
ously identified 4-methoxyphenyl moiety 22. Table 2 summariz-
es the results obtained in this effort. Comparison of com-
pounds 24–28 with compound 22 shows that with respect to
orexin antagonist activity, exchanging the methylthio substitu-
ent generally results in decreased potency toward OX1. Poten-
cy toward OX2 suffered less from these changes or
could be maintained as shown in 24. In addition, in-
hibitory activity toward cytochrome P450 3A4 could
be significantly decreased in 24 relative to 22.
We followed up on these results by additional var-
iations in the sulfonamide part of the antagonists by
keeping the 3,5-dimethylanilide unit fixed. Results
are summarized in Table 3. Comparison of com-
pound 24 with the other mono-para-substituted resi-
dues, such as compound 30 containing a para-
methyl substituent or compound 35 containing
a para-bromo substituent, reveals that the para-me-
thoxy substituent remains the most favorable unit
with respect to orexin antagonistic activity. Moving
the para-methyl substituent of 30 into the ortho po-
sition as shown in 33, for example, results in a loss
of activity at OX2 and has no beneficial effect on
cytochrome P450 3A4 inhibition. Attaching two sub-
stituents to the phenyl ring of the sulfonamide
Scheme 2. Synthetic approaches toward proline sulfonamide dual orexin receptor antag-
onists: efficient strategies toward quick SAR development.
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Table 2. Structure–activity relationships: amide moiety part 1.
Table 3. Structure–activity relationships: sulfonamide moiety part 2.
R1
Compd
IC50 [nm][a]
IC50 [mm][b]
3A4T
R2
Compd
IC50 [nm][a]
IC50 [mm][b]
3A4T
OX1
OX2
OX1
OX2
24
29
60
17
11
15
22
24
25
31
60
61
6.9
7.8
15
16
ND
11
23
30
31
32
48
32
41
15
16
18
ND
3.7
ND
17
26
46
39
3.7
33
50
22
3.2
27
28
99
24
23
ND[c]
5.8
34
35
89
53
12
24
3.6
ND
157
[a] Data are the geometric mean (ꢀ2-fold) of at least three independent
experiments and were determined by FLIPR assay (see the Supporting In-
formation for assay details). [b] Values are from single measurements with
testosterone used as the marker substrate. [c] Not determined.
[a] Data are the geometric mean (ꢀ2-fold) of at least three independent
experiments and were determined by FLIPR assay (see the Supporting In-
formation for assay details). [b] Values are from single measurements with
testosterone used as the marker substrate. [c] Not determined.
moiety, as in 29, 31, 32, or 34, results in derivatives with prom-
ising antagonistic potency toward OX2. Activities at OX1 are
more variable, and for cases in which the inhibitory activity for
cytochrome P450 3A4 was determined, it was less advanta-
geous relative to compound 24. This effort finally confirmed
that derivative 24 was the best compound identified from this
series.
Compound 24 was further profiled in an in vivo blood–brain
barrier [BBB] penetration experiment in male Wistar rats (ex-
perimental details in the Supporting Information). We found
that 24 showed excellent brain penetration with high absolute
values for the brain concentration of [B]=1219 nggꢁ1 and the
plasma concentration of [P]=2667 ngmLꢁ1 when administered
orally at a dose of 100 mgkgꢁ1, resulting in a [B]/[P] ratio of
46%. In parallel, we checked the influence of the proline core
chirality and found that the non-natural R enantiomer 36
(Figure 3) exhibited almost no antagonistic activity for either of
the orexin receptors, but behaved very similarly in terms of
brain penetration properties, with [B]=1041 nggꢁ1 and [P]=
1309 ngmLꢁ1, and thus a [B]/[P] ratio of 79%. This result was
in agreement with theory and our expectations and excluded
active uptake phenomena of the natural proline-amino acid
based 24.
Figure 3. Influence of proline core chirality. BBB experiments were per-
formed in Wistar rats at a p.o. drug dosing of 100 mgkgꢁ1; sampling at 3 h;
drug formulation: PEG400.
derivatives for these experiments. Comparison of 24 with com-
pounds 22, 26, 27, 28, and 37 revealed that with respect to
absolute brain concentrations [B], compound 24 was by far
the best in this experimental setting. The same was true for
absolute plasma concentrations [P]. Analyzing the [B]/[P] ratios
revealed that the majority of the compounds exhibited good
to excellent values (with the exception of 28 and 22) pointing
toward the fact that, at least in the rat, the proline sulfonamide
derivatives did not seem to suffer from being efflux pump sub-
We then investigated several other derivatives from the pro-
line sulfonamide series for their brain penetration potential
(Figure 4). We selected mainly 4-methoxyphenylsulfonamide
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Figure 4. Structure–brain penetration analysis. BBB experiments were performed in Wistar rats at a p.o. drug dosing of 100 mgkgꢁ1; sampling at 3 h; drug for-
mulation: PEG400.
strates. Comparing 7 with 38 showed that replacement of the
2-bromothiophene moiety by the closest isostere, 4-bromo-
phenyl, resulted in clearly inferior absolute brain concentra-
tions without affecting the excellent [B]/[P] ratio. Finally, by
moving from 7 to 22 it became clear that replacement of the
2-bromothiophene unit with a 4-methoxyphenyl group only
resulted in increased plasma concentrations and only advanc-
ing one step, and replacing the 3-methylthiophenyl group by
the 3,5-dimethylphenyl moiety adjusted all values in the de-
sired range.
data (experiment done at 100 mgkgꢁ1 p.o.) for compound 43
([P]=130 ngmLꢁ1 and [B]=228 nggꢁ1) are exemplary for the
whole series based on the 3-methylene proline (more than 10
compounds investigated in vivo) with good [B]/[P] ratios, but
low to very low absolute concentrations. The bicyclic tem-
plates represented in 47 and 45 also did not exhibit advanta-
geous activity and added synthetic complexity for the scaffold
preparation. Therefore, it was decided not to continue investi-
gations with these templates. Similar trends were observed for
the compounds based on the tetrahydroisoquinoline template
as represented by 46, well in accordance with the results ob-
tained for 39 and 41. Based on these results it was decided to
characterize compound 24 in detail. Figure 6 summarizes the
data set.
By going another step further in the analysis of the hit struc-
ture, further optimization possibilities consisted of scaffold
hopping, as summarized in Figure 5. Compounds 7, 22, 42,
and 24 are depicted as the proline-based parent reference
compounds for the respective substituents combined with
new scaffolds. Comparison of 7 with 39 showed that the pyrro-
lidine template was strongly preferred over the piperidine tem-
plate. Most of the activity lost in 39 was regained in 40, which
is based on a bridged bicyclic template containing a pyrrolidine
and a piperidine moiety. Ring enlargement to a morpholine
template was also not tolerated, as can be seen from compari-
son of 22 with 41.
Compounds 43 and 44 seemed to represent the group of
compounds with the most promising alternative scaffold. Fur-
ther investigations resulted in many potent dual OX1 and OX2
antagonists with slight selectivity toward OX2, but the series
was not superior to the initial proline series with respect to cy-
tochrome P450 inhibition profiles. In addition, in vivo rat BBB
The mode of antagonism of 24 at OX1 and OX2 was assessed
in greater detail using Ca2+ release assays and stably transfect-
ed Chinese hamster ovary (CHO) cells recombinantly express-
ing human, dog, or rat orexin 1 or orexin 2 receptors. Orexin A
concentration–response curves (CRC) were generated in the
presence of increasing concentrations of 24. The compound
induced rightward shifts of the orexin A CRCs, demonstrating
competitive antagonism, and Schild Kb values for 120 min an-
tagonist pre-incubation were calculated to be Kb =17 nm
(human OX1) and 2.4 nm (human OX2). Schild Kb values were
not different for 10 vs. 120 min antagonist pre-incubation time,
indicating rapid association and dissociation kinetics of 24
from the orexin receptors. No species differences in potency,
selectivity, or competitiveness were detected.
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Figure 5. Scaffold hopping SAR. Orexin IC50 data represent the geometric mean of at least three independent experiments and were determined by using
a fluorimetric imaging plate reader (FLIPR) assay. See the Supporting Information for assay details.
Compound 24 was tested on a panel of over 120 enzyme,
radioligand binding, and tissue assays for established central
and peripheral pharmacological targets. Compound 24
showed no significant activity against these targets at the
tested concentration of 10 mm, thus demonstrating high selec-
tivity for the orexin receptors. Detailed results are given in the
Supporting Information.
Beagle dogs. The intravenous dose was 1 mgkgꢁ1 in both spe-
cies. Oral doses were 3 mgkgꢁ1 in the dog (n=4) and
10 mgkgꢁ1 in the rat (n=5). Table 4 gives an overview of the
pharmacokinetic parameters in both species.
After intravenous dosing, compound 24 exhibited plasma
clearances of 29 and 11 mLminꢁ1 kg. Considering the blood-to-
plasma partitioning of ~0.6, blood clearances in both species
were between 50–70% of the respective liver blood flow. Nota-
bly, elevated clearance is required to achieve the appropriate
The pharmacokinetic characterization of compound 24 after
single dose administration was performed in Wistar rats and
Table 4. Pharmacokinetic parameters of 24 after single intravenous and oral dosing to male Wistar rats and Beagle dogs.[a]
Intravenous:
AUC0!1 [nghmLꢁ1
]
CL [mLminꢁ1 kgꢁ1
]
Vss [Lkgꢁ1
]
t1/2 [h]
rat
dog
586 (442–1020)
1490 (1160–1780)
29 (16–38)
11 (9.4–11)
1.8 (1.3–2.8)
1.4 (1.0–2.0)
1.9 (1.6–3.1)
1.7 (1.1–3.3)
Oral:
AUC0!1 [nghmLꢁ1
]
cmax [ngmLꢁ1
]
tmax [h]
F [%]
rat
dog
2310 (1600–3080)
2750 (1910–2960)
1600 (1290–1840)
426 (351–611)
0.5 (0.25–0.5)
0.5 (0.25–0.75)
39 (27–53)[b]
52 (43–57)[c]
[a] Data are the geometric mean (range in brackets); the median is given for tmax. [b] Bioavailability in rat was calculated with reference to the mean
AUC0!1 after an i.v. dose of 1 mgkgꢁ1. [c] Bioavailability in dog was calculated from individual animals (cross-over design).
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The sleep-promoting effects of 24 were evaluated in male
Wistar rats and in male Beagle dogs implanted with radiotele-
metry probes recording continuously EEG/EMG and locomotor
activity. Male Wistar rats were administered with single oral
doses of 0, 10, 30, 100, or 300 mgkgꢁ1 at the beginning of the
nocturnal active phase, when endogenous orexin levels in-
crease. Compound 24 significantly decreased the latency to
the first persistent episode of non-REM sleep (60 s) and the
first persistent episode of REM sleep (30 s) (one-way ANOVA;
p<0.001 and p<0.01 respectively). The first episode of persis-
tent non-REM sleep occurred within 10–15 min of treatment.
Over the 6 h following administration, 24 was found to dose-
dependently decrease total wake time and behavioral home
cage activity (one-way ANOVA; p<0.001), while increasing
REM and non-REM sleep times (one-way ANOVA; p=0.001 and
p<0.001) (Figure 7). The effect on sleep lasted between 2 and
12 h, depending on dose.
Non-REM and REM sleep increased in physiological propor-
tion. Under treatment with 24, sleep architecture was con-
served, as the relative proportion of non-REM and REM sleep
over the total sleep time were not significantly changed (one-
way ANOVA, p=0.12). Over the first 6 h night period post-ad-
ministration, vehicle-treated rats slept a total of 36.8% of the
time. Total sleep time was divided into 84.5% time spent in
non-REM sleep and 15.5% time spent in REM sleep. At the
highest tested dose, 300 mgkgꢁ1 p.o., rats slept in total 52.9%
of the first 6 h night period post administration. This total
sleep time was divided into 80.0% non-REM sleep and 20.0%
REM sleep.
Figure 6. Dataset generated with compound 24. Orexin activity data repre-
sent the geometric mean of at least three independent experiments and
were determined by using a fluorimetric imaging plate reader (FLIPR) assay.
See the Supporting Information for assay details. CYP3A4T: testosterone as
marker substrate; CYP3A4M: midazolam as marker substrate; HLM, RLM, and
DLM values represent a normalized rate constant.
Male Beagle dogs were treated with single oral doses of 0,
10, 30, 100, or 300 mg of 24 in a Cremophor RH40ꢁ formulation
during their active phase, when endogenous orexin levels are
naturally elevated. Compound 24 dose-dependently decreased
behavioral signs of activity and electrophysiological signs of
wakefulness (one-way ANOVA; p<0.001 and p<0.001). This
decrease in wakefulness was accompanied by increases in
pharmacokinetic/pharmacodynamic profile of a sleep-promot-
ing drug.
Mean volume of distribution at steady-state (Vss) as a general
measure of tissue distribution was 1.8 Lkgꢁ1 in rat and
1.4 Lkgꢁ1 in dog, i.e., exceeding
total body water volume and in-
dicating significant distribution
of 24 into tissues. Compound
24 was rapidly absorbed after
oral dosing as peak drug levels
were reached within the first
30 min after dosing. Absorption
from the gastrointestinal lumen
was considered complete. Bio-
availability was 39% in the rat
and 52% in the dog, which is
close to the maximally achieva-
ble bioavailability considering
the respective blood clearances.
Bioavailability of 24 therefore
appears to be limited by clear-
Figure 7. Effect of 24 on the relative time spent in sleep and wake stages (% of total time) during the first 6 h of
the active period post-administration in male Wistar rats. Rats were administered single oral doses of vehicle
(PEG400) or compound 24 at 10, 30, 100 or 300 mgkgꢁ1 at night, during their active phase. Data are the mean
ꢀSEM. Paired t-test with Sidak correction for multiple comparisons: *p<0.05, **p<0.01, ***p<0.001 relative to
matched vehicle-treated rats; n=7–15 per dose group. REM: rapid eye movement.
ance rather than by absorption
under the conditions employed
in this study.
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of 24, rats were trained to asso-
ciate an unpleasant electric foot
shock with a cue light (condi-
tioned stimulus) which was de-
livered through a metal grid
within an animal enclosure.
When these rats were later re-
exposed to the same enclosure
either under light (conditioned
stimulus; cued fear) or in the
dark (no conditioned stimulus;
contextual fear), compound 24
decreased the fear-potentiated
startle reflexes in response to
a sudden loud noise with effec-
tive doses of 100 and
300 mgkgꢁ1 (Figure 9a). Control
experiments revealed that star-
tle reflexes in naive rats re-
mained unaffected by 24
(300 mgkgꢁ1; Figure 9b), as well
Figure 8. Effect of 24 on the relative time spent in sleep and wake stages (% total time) during 9 h of the active
period post-administration in male Beagle dogs. Dogs were administered single oral doses of vehicle (Cremo-
phor RH40ꢁ-based formulation) or compound 24 at 10, 30, 100, or 300 mg in the daytime, during their active
phase. Data are the mean ꢀSEM. Bonferroni post-hoc analysis: *p<0.05, **p<0.01, ***p<0.001 relative to
matched vehicle-treated dogs; n=13/dose group. REM: rapid eye movement.
both REM and non-REM sleep times (one-way ANOVA; p<
0.001 and p<0.001) (Figure 8). Onset of daytime somnolence
occurred within 30 min of administration, and lasted between
2 and 9 h depending on the dose.
as forepaw grip strength, as an indicator of potential muscle
relaxation (Figure 9c).
Compound 24 (100 mgkgꢁ1) was also tested in a resident–
intruder rat model where it significantly decreased the social-
stress-induced increases of locomotion, body temperature, and
heart rate (Figure 10a–c). Mean arterial blood pressure re-
mained unaffected (Figure 10d).
In dogs, similar to rats, non-REM and REM sleep increased in
physiological proportions. Over the 9 h day period post-admin-
istration, vehicle-treated dogs slept in total 26.0% of the time.
This total sleep time was divided into 84.6% time spent in
non-REM sleep, and 15.4% time spent in REM sleep. At the
highest tested dose, 300 mg, dogs slept in total 41.1% of the
9 h day period post-administration. This total sleep time was
divided into 82.2% non-REM sleep and 17.8% REM sleep.
As mentioned in the introduction, the orexin system is impli-
cated in regulating stress and anxiety-like reactions in ro-
dents.[30] The dual orexin receptor antagonist 1 (almorexant)
shows anxiolytic-like behavioral effects in a cued fear model[28]
and decreases the autonomous nervous system response to
certain types of stressors.[25] To probe the anxiolytic potential
In addition to exerting anxiolytic-like effects, dual orexin re-
ceptor antagonists are also known to decrease certain types of
addiction-like behaviors in rodents.[27,29] Compound 24
(100 mgkgꢁ1) effectively decreased the expression of locomo-
tor sensitization to morphine (Figure 11a) without affecting
the conditioned place preference induced by conditioned mor-
phine reward (Figure 11b), mimicking previous findings with
1 (almorexant).[29]
Finally, as part of our pharmacological characterization of 24,
we also assessed potential effects on cognitive and motor
functions. In the Morris water maze, where rats are trained to
use spatial navigation along ex-
ternal cues to locate a hidden
platform in a large water tank,
repeated treatment with 24
(300 mgkgꢁ1) during training
was not different from vehicle
treatment. Both groups, but not
rats treated with the muscarinic
antagonist scopolamine, used
as positive control, established
spatial memory. This was indi-
cated by the larger amount of
Figure 9. a) Rats were exposed to the fear-potentiated startle test 2 h after oral treatment with vehicle (0 mgkgꢁ1
or increasing doses (30, 100, 300 mgkgꢁ1) of 24. The drug dose-dependently decreased the mean startle ampli-
tude both upon presentation of the cued fear-eliciting conditioned light stimulus (CS) and under dark conditions
(no CS) where only contextual fear traces remain. Data are the mean ꢀSEM; *p<0.05 vs. 0 mgkgꢁ1 under CS or
)
time spent searching for the
platform in the target quadrant
of the maze than in the other
quadrants, or chance level (Fig-
ure 12a). In comparison with
the positive GABAA receptor
no CS conditions, respectively; within subjects cross-over design, n=16. b) Compound 24 at 300 mgkgꢁ1 p.o. had
no effect on basal startle reactions in the dark in naive rats; within subjects cross-over design n=16; data are the
mean ꢀSEM. c) Compound 24 at 30, 100, 300 mgkgꢁ1 p.o. had no influence on forepaw grip strength in rats;
n=6–7; data are the mean ꢀSEM.
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modulator zolpidem, which is
frequently used as sleep medi-
cation in the clinic at present,
24 (300 mgkgꢁ1) did not impair
contextual memory acquisition
in the passive avoidance learn-
ing paradigm (Figure 12b), nor
forced motor performance in
the rotating rod test (Fig-
ure 12c). These data confirm the
particular mode of action of
DORAs, which induce sleep
without negative impact on
cognition or motor function in
rats.[45–49]
Figure 10. Rats equipped with telemetry transmitters were orally treated with vehicle (0 mgkgꢁ1) or 24
(100 mgkgꢁ1) 2 h before submission to social interaction stress with a male intruder for a duration of 1 h. During
stress, a) locomotion, b) body temperature, and c) heart rate were significantly decreased by 24, whereas d) mean
arterial blood pressure (MAP) remained unaffected. Data are the mean ꢀSEM of transmitter recordings during the
1 h period of social interaction. Dotted lines represent the respective baseline values before vehicle/drug treat-
ment and stress exposure. Within subjects cross-over design, n=12; *p<0.05 vs. 0 mgkgꢁ1
.
Importantly, DORAs primarily
promote sleep under little or
non-stimulating environmental
conditions (such as in the home
cage).[22] For instance, dogs
treated with DORAs are able to
wake up just fine when present-
ed with emotionally salient
acoustic stimuli,[50] and rats
treated with DORAs perform
normally on the rotarod after
being woken up during their
daytime sleeping phase (Fig-
ure 12c and Ref. [48]). It is there-
fore unlikely that the anxiolytic-
like effects of 24 observed in
the present study are merely
Figure 11. a) Locomotor sensitization was assessed as the increase in the distance moved in a box during a de-
fined period (45 min) upon repeated injections (four times) with morphine (Mor; 10 mgkgꢁ1 s.c.). Before the fifth
morphine injection rats received 24 (100 mgkgꢁ1 p.o., ꢁ2 h) which normalized the distance moved to those of
non-sensitized levels. After compound 24 washout and an additional (sixth) morphine exposure, the previous
level of locomotor sensitization was again observed; *p<0.05 vs. Mor (1st), n=16. b) Rats were tested for the ab-
sence of initial place preference for either compartment in a pretest before conditioning with saline or morphine
(10 mgkgꢁ1 s.c.) for eight days. After conditioning, at the conditioned place preference (CPP) test 1, treatment
with 24 (100 mgkgꢁ1 p.o., ꢁ2 h) failed to inhibit the induced CPP for the morphine (Mor)-paired compartment
over the saline (Sal)-paired compartment. Time spent in the neutral transition (Trans) compartment was, however,
increased. This was a specific effect that can be ascribed to dual OXR blockade, which was previously observed
with 1 (almorexant).[29] Indeed, after 24 washout, another control test (2) was conducted, which still revealed the
place preference for the morphine-paired compartment, but showed the expected low time spent in the third
neutral transition compartment. Data are the mean ꢀSEM; n=16.
a
consequence of increased
sleepiness.
Figure 12. a) Rats were trained for two consecutive weeks under drug treatment in the Morris water maze to find a hidden platform in a water tank through
orientation on external spatial cues. On the test day (probe trial) both vehicle- and 24 (300 mgkgꢁ1 p.o.)-treated rats spent significantly more time than
chance level (25%, indicated by the dotted line) in the target quadrant (black bars), where the platform was originally located, indicating successful spatial
memory formation. Scopolamine (0.7 mgkgꢁ1 s.c.)-treated rats failed to establish spatial memory; n=7–8 per group; *p<0.05 vs. chance level. b) In the pas-
sive avoidance paradigm rats that were treated during the training trial with vehicle or 24 (300 mgkgꢁ1 p.o.) showed significantly higher latency to step
through to the aversive side of the shuttle box on the test day than rats that had been treated with zolpidem (30 mgkgꢁ1 p.o.) during training, indicating
intact aversion learning under OXR blockade but not under GABAA receptor activation; n=9–12 per group; *p<0.05 vs. vehicle. c) Following oral treatment
the forced motor activity of rats was tested repeatedly on the rotarod up to 3 h. Whereas 24 (30 and 100 mgkgꢁ1)-treated rats behaved similarly to those
treated with vehicle, zolpidem (30 and 100 mgkgꢁ1)-treated rats showed impaired motor function in a dose-dependent manner; n=12 per group; *p<0.05
vs. vehicle at the respective time point. Data are the mean ꢀSEM.
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Smith, D. C. Piper, A. J. Hunter, R. A. Porter, N. Upton, Proc. Natl. Acad.
Sci. USA 1999, 96, 10911–10916.
Conclusions
[12] E. Eggermann, M. Serafin, L. Bayer, D. Machard, B. Saint-Mleux, B. E.
Jones, M. Muhlethaler, Neuroscience 2001, 108, 177–181.
[13] J. Fadel, A. Y. Deutch, Neuroscience 2002, 111, 379–387.
[14] A. Yamanaka, N. Tsujino, H. Funahashi, K. Honda, J. L. Guan, Q. P. Wang,
M. Tominaga, K. Goto, S. Shioda, T. Sakurai, Biochem. Biophys. Res.
Commun. 2002, 290, 1237–1245.
[15] J. C. Geerling, T. C. Mettenleiter, A. D. Loewy, Neuroscience 2003, 122,
541–550.
[16] R. Spinazzi, P. G. Andreis, G. P. Rossi, G. G. Nussdorfer, Pharmacol. Rev.
2006, 58, 46–57.
[17] M. G. Lee, O. K. Hassani, B. E. Jones, J. Neurosci. 2005, 25, 6716–6720.
[18] K. Yoshida, S. McCormack, R. A. Espana, A. Crocker, T. E. Scammell, J.
Comp. Neurol. 2006, 494, 845–861.
[19] G. C. Harris, M. Wimmer, G. Aston-Jones, Nature 2005, 437, 556–559.
[20] G. C. Harris, G. Aston-Jones, Trends Neurosci. 2006, 29, 571–577.
[21] M. Narita, Y. Nagumo, S. Hashimoto, J. Khotib, M. Miyatake, T. Sakurai,
M. Yanagisawa, T. Nakamachi, S. Shioda, T. Suzuki, J. Neurosci. 2006, 26,
398–405.
[22] C. Brisbare-Roch, J. Dingemanse, R. Koberstein, P. Hoever, H. Aissaoui, S.
Flores, C. Mueller, O. Nayler, J. van Gerven, S. L. de Haas, P. Hess, C. Qiu,
S. Buchmann, M. Scherz, T. Weller, W. Fischli, M. Clozel, F. Jenck, Nat.
Med. 2007, 13, 150–155.
[23] B. Boutrel, L. de Lecea, Physiol. Behav. 2008, 93, 947–951.
[24] S. G. Nair, S. A. Golden, Y. Shaham, Br. J. Pharmacol. 2008, 154, 406–416.
[25] T. M. Furlong, D. M. Vianna, L. Liu, P. Carrive, Eur. J. Neurosci. 2009, 30,
1603–1614.
[26] P. L. Johnson, W. Truitt, S. D. Fitz, P. E. Minick, A. Dietrich, S. Sanghani, L.
Traskman-Bendz, A. W. Goddard, L. Brundin, A. Shekhar, Nat. Med. 2010,
16, 111–115.
[27] C. J. Winrow, K. Q. Tanis, D. R. Reiss, A. M. Rigby, J. M. Uslaner, V. N.
Uebele, S. M. Doran, S. V. Fox, S. L. Garson, A. L. Gotter, D. M. Levine,
A. J. Roecker, P. J. Coleman, K. S. Koblan, J. J. Renger, Neuropharmacolo-
gy 2010, 58, 185–194.
[28] M. A. Steiner, H. Lecourt, F. Jenck, Psychopharmacology 2012, 223, 465–
475.
[29] M. A. Steiner, H. Lecourt, F. Jenck, Int. J. Neuropsychopharmacol. 2013,
16, 417–432.
In summary, we have identified and broadly characterized
compound 24 as a competitive small-molecule orexin receptor
antagonist displaying activity toward both the orexin 1 and
orexin 2 receptors. A broad receptor screen demonstrated that
24 was highly selective for the orexin receptors among more
than 100 other potential neuronal targets. The in vivo rat
blood–brain barrier penetration experiment confirmed the ex-
cellent brain penetration properties of 24, and in vitro tests
suggested that 24 is not a human P-gp efflux pump substrate.
Pharmacokinetic studies in rats and dogs revealed that 24 ex-
hibited a favorable pharmacokinetic profile for an insomnia
drug, and this translated into beneficial pharmacological ef-
fects in sleep studies in both rats and dogs. Compound 24
was further characterized in pharmacological rat experiments
assessing stress- and anxiety-related readouts exploring
a broader therapeutic potential for dual orexin receptor antag-
onists, besides insomnia.[49,51] In the meantime, 24 has been in-
vestigated in a phase I human clinical trial.[52]
Acknowledgements
The authors thank Pascal Rebmann, Marc Candreia, Daniela
Krꢀsi, Viktor Ribic, Eser Ilhan, Markus Gude, Stefan Abele, Simone
Tortoioli, Daniel Marchal, Celia Mꢀller-Grandjean, Katalin Meny-
hart, Thomas Sasse, Helene Massinet, Helene Roellinger, Cedric
Fischer, Hugues Lecourt, Rebekka Pietrek, Eileen Hubert, Monica
Gaiffi, Ruben de Kanter, Aude Weigel, Pꢁivi Aꢁnismaa, Stephan
Delahaye and Isabelle Weber for expert technical support, and
Martin Bolli and Thomas Weller for helpful and stimulating
discussions.
[30] E. M. Pich, S. Melotto, Front Neurosci. 2014, 8, 26.
[31] P. J. Coleman, C. D. Cox, A. J. Roecker, Curr. Top. Med. Chem. 2011, 11,
696–725.
ˇ
[32] P. Hoever, P. G. Dorffner, H. Benes, T. Penzel, H. Danker-Hopfe, M. J. Bar-
Keywords: insomnia · neurotransmitters · orexin receptor
antagonists · peptides · stress-related disorders
banoj, G. Pillar, B. Saletu, O. Polo, D. Kunz, J. Zeitlhofer, S. Berg, M. Parti-
nen, C. L. Bassetti, B. Hçgl, I. O. Ebrahim, E. Holsboer-Trachsler, H.
Bengtsson, Y. Peker, U. M. Hemmeter, E. Chiossi, G. Hajak, J. Dinge-
manse, Clin. Pharmacol. Ther. 2012, 91, 975–985.
[1] L. de Lecea, T. S. Kilduff, C. Peyron, X. Gao, P. E. Foye, P. E. Danielson, C.
Fukuhara, E. L. Battenberg, V. T. Gautvik, F. S. Bartlett II, W. N. Frankel,
A. N. van den Pol, F. E. Bloom, K. M. Gautvik, J. G. Sutcliffe, Proc. Natl.
Acad. Sci. USA 1998, 95, 322–327.
[33] D. Michelson, E. Snyder, E. Paradis, M. Chengan-Liu, D. B. Snavely, J. Hut-
zelmann, J. K. Walsh, A. D. Krystal, R. M. Benca, M. Cohn, C. Lines, T.
Roth, W. J. Herring, Lancet Neurol. 2014, 13, 461–471.
[34] P. Bettica, L. Squassante, S. Zamuner, G. Nucci, H. Danker-Hopfe, E. Ratti,
Sleep 2012, 8, 1097–1104.
[35] C. Boss, C. Brisbare-Roch, F. Jenck, H. Aissaoui, R. Koberstein, T. Sifferlen,
T. Weller, Chimia 2008, 62, 974–979.
[2] T. S. Kilduff, C. Peyron, Trends Neurosci. 2000, 23, 359–365.
[3] R. J. Liu, A. N. van den Pol, G. K. Aghajanian, J. Neurosci. 2002, 22, 9453–
9464.
[4] T. Sakurai, Nat. Rev. Neurosci. 2007, 8, 171–181.
[36] A. L. Gotter, C. J. Winrow, J. Brunner, S. L. Garson, S. V. Fox, J. Binns,
C. M. Harrell, D. Cui, K. L. Yee, M. Stiteler, J. Stevens, A. Savitz, P. L. Tan-
nenbaum, S. J. Tye, T. McDonald, L. Yao, S. D. Kuduk, J. Uslaner, P. J. Co-
leman, J. J. Renger, BMC Neurosci. 2013, 14, 90.
[37] P. J. Coleman, J. D. Schreier, C. D. Cox, M. J. Breslin, D. B. Whitman, M. J.
Bogusky, G. B. McGaughey, R. A. Bednar, W. Lemaire, S. M. Doran, S. V.
Fox, S. L. Garson, A. L. Gotter, C. M. Harell, D. R. Reiss, T. D. Cabalu, D.
Cui, T. Prueksaritanont, J. Stevens, P. L. Tannenbaum, R. G. Ball, J. Stella-
bott, S. D. Young, G. D. Hartman, C. J. Winrow, J. J. Renger, ChemMed-
Chem 2012, 7, 415–424.
[5] T. Matsuki, T. Sakurai, Results Probl. Cell Differ. 2008, 46, 27–55.
[6] T. Sakurai, A. Amemiya, M. Ishii, I. Matsuzaki, R. M. Chemelli, H. Tanaka,
S. C. Williams, J. A. Richardson, G. P. Kozlowski, S. Wilson, J. R. Arch, R. E.
Buckingham, A. C. Haynes, S. A. Carr, R. S. Annan, D. E. McNulty, W. S.
Liu, J. A. Terrett, N. A. Elshourbagy, D. J. Bergsma, M. Yanagisawa, Cell
1998, 92, 573–585.
[7] K. N. Nilaweera, P. Barrett, J. G. Mercer, P. J. Morgan, Neuroscience 2003,
119, 713–720.
[8] J. Gatfield, C. Brisbare-Roch, F. Jenck, C. Boss, ChemMedChem 2010, 5,
1197–1214.
[9] A. S. Jansen, X. V. Nguyen, V. Karpitskiy, T. C. Mettenleiter, A. D. Loewy,
Science 1995, 270, 644–646.
[38] D. B. Whitman, C. D. Cox, M. J. Breslin, K. M. Brashear, J. D. Schreier, M. J.
Bogusky, R. A. Bednar, W. Lemaire, J. G. Bruno, G. D. Hartman, D. R.
Reiss, C. M. Harrell, R. L. Kraus, Y. Li, S. L. Garson, S. M. Doran, T. Prueksar-
itanont, C. Li, C. J. Winrow, K. S. Koblan, J. J. Renger, P. J. Coleman,
ChemMedChem 2009, 4, 1069–1074.
[10] C. Peyron, D. K. Tighe, A. N. van den Pol, L. de Lecea, H. C. Heller, J. G.
Sutcliffe, T. S. Kilduff, J. Neurosci. 1998, 18, 9996–10015.
[11] J. J. Hagan, R. A. Leslie, S. Patel, M. L. Evans, T. A. Wattam, S. Holmes,
C. D. Benham, S. G. Taylor, C. Routledge, P. Hemmati, R. P. Munton, T. E.
Ashmeade, A. S. Shah, J. P. Hatcher, P. D. Hatcher, D. N. Jones, M. I.
[39] A. J. Roecker, S. P. Mercer, J. D. Schreier, C. D. Cox, M. E. Fraley, J. T.
Stehen, W. Lemaire, J. G. Bruno, C. M. Harrell, S. L. Garson, A. J. Gotter,
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2014, 9, 2486 – 2496 2495

CHEMMEDCHEM
FULL PAPERS
www.chemmedchem.org
S. V. Fox, J. Stevens, P. L. Tannenbaum, T. Pruesksaritanont, T. D. Cabalu,
D. Cui, J. Stellabott, G. D. Hartman, S. D. Young, C. J. Winrow, J. J.
Renger, P. J. Coleman, ChemMedChem 2014, 9, 311–322.
[46] S. R. Morairty, A. J. Wilk, W. U. Lincoln, T. C. Neylan, T. S. Kilduff, Front.
Neurosci. 2014, 8, 3.
[47] H. Dietrich, F. Jenck, Psychopharmacology 2010, 212, 145–154.
[48] M. A. Steiner, H. Lecourt, D. S. Strasser, C. Brisbare-Roch, F. Jenck, Neuro-
psychopharmacology 2011, 36, 848–856.
[40] S. P. Mercer, A. J. Roecker, S. Garson, D. R. Reiss, C. M. Harrell, K. L.
Murphy, J. G. Bruno, R. A. Bednar, W. Lemaire, D. Cui, T. D. Cabalu, C.
Tang, T. Prueksaritanont, G. D. Hartman, S. D. Young, C. J. Winrow, J. J.
Renger, P. J. Coleman, Bioorg. Med. Chem. Lett. 2013, 23, 6620–6624.
[41] B. Jaun, M. Tanaka, P. Seiler, F. N. M. Kꢂhnle, C. Braun, D. Seebach, Liebigs
Ann./Recl. 1997, 1697–1710.
[49] S. D. Kuduk, C. J. Winrow, P. J. Coleman, Annu. Rev. Med. Chem. 2013, 48,
73–87.
[50] P. L. Tannenbaum, J. Stevens, J. Binns, A. T. Savitz, S. L. Garson, S. V. Fox,
P. Coleman, S. D. Kuduk, A. L. Gotter, M. Marino, S. J. Tye, J. M. Uslaner,
C. J. Winrow, J. J. Renger, Front. Behav. Neurosci. 2014, 8, 182.
[51] C. Boss, C. Brisbare-Roch, F. Jenck, J. Med. Chem. 2009, 52, 891–903.
[52] M. Hoch, H. van Gorsel, J van Gerven, J. Dingemanse, J. Clin. Pharmacol.
2014, 54, 979–986.
[42] J. Klosa, J. Prakt. Chem. 1963, 19, 45–55.
[43] C. A. G. N. Montalbetti, v. Falque, Tetrahedron 2005, 61, 10827–10852.
[44] C. Boss, C. Brotschi, J. Gatfield, M. Gude, B. Heidmann, T. Sifferlen, J. T.
Williams (Actelion Pharmaceuticals Ltd.), Int. PCT Pub. No. WO2012/
025877 A1, 2012.
[45] A. D. Ramirez, A. L. Gotter, S. V. Fox, P. L. Tannenbaum, L. Yao, S. J. Tye, T.
McDonald, J. Brunner, S. L. Garson, D. R. Reiss, S. D. Kuduk, P. J. Coleman,
J. M. Uslaner, R. Hodgson, S. E. Browne, J. J. Renger, C. J. Winrow, Front.
Neurosci. 2013, 7, 254.
Received: June 23, 2014
Published online on August 21, 2014
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