Proline bis-amides as potent dual orexin receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2008.01.001

A series of OX₂R/OX₁R dual orexin antagonists was prepared based on a proline bis-amide identified as a screening lead. Through a combination of classical and library synthesis, potency enhancing replacements for both amide portions

Full text of DOI:10.1016/j.bmcl.2008.01.001

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 1425–1430
Proline bis-amides as potent dual orexin receptor antagonists
Jeffrey M. Bergman,a,* Anthony J. Roecker,a Swati P. Mercer,a Rodney A. Bednar,a
Duane R. Reiss,b Richard W. Ransom,b C. Meacham Harrell,b Douglas J. Pettibone,b
Wei Lemaire,b Kathy L. Murphy,b Chunze Li,c Thomayant Prueksaritanont,c
Christopher J. Winrow,b John J. Renger,b Kenneth S. Koblan,b
George D. Hartmana and Paul J. Colemana
aDepartment of Medicinal Chemistry, Merck Research Laboratories, West Point, PA 19486, USA
bDepartment of Depression and Circadian Disorders, Merck Research Laboratories, West Point, PA 19486, USA
cDepartment of Drug Metabolism, Merck Research Laboratories, West Point, PA 19486, USA
Received 30 October 2007; revised 27 December 2007; accepted 2 January 2008
Available online 8 January 2008
Abstract—A series of OX2R/OX1R dual orexin antagonists was prepared based on a proline bis-amide identified as a screening lead.
Through a combination of classical and library synthesis, potency enhancing replacements for both amide portions were discovered.
N-methylation of the benzimidazole moiety within the lead structure significantly reduced P-gp susceptibility while increasing
potency, giving rise to good brain penetration. A compound from this series has demonstrated in vivo central activity when dosed
peripherally in a pharmacodynamic model of orexin activity.
Published by Elsevier Ltd.
In 1998, two groups working independently discovered
two neuropeptides; orexin A and orexin B.1 Numerous
studies have implicated a wide range of functions for
orexin signaling in the CNS including regulating sleep–
wake and feeding behaviors.2 The two orexin peptides
bind to their respective G-coupled protein receptors,
OX1R and OX2R. While OX1R exhibits about a 10-
to 100-fold selectivity for orexin A over orexin B,
OX2R has an equal affinity for either peptide.1a
signaling with receptor antagonists may provide a new
mechanism for the treatment of insomnia. Actelion
has recently reported that a dual OX2R/OX1R antago-
nist (ACT-078573) induced somnolence and increased
electrophysiological markers for sleep in rats, dogs,
and humans.7 Herein, we describe a novel series of dual
orexin antagonists based on a proline bis-amide scaffold
and report their efficacy in a pharmacodynamic model
of orexin induced hyperlocomotion.
Orexin deficiencies have been implicated in narcolepsy.3
Patients with narcolepsy often show low or undetectable
levels of orexin A in the CSF and this decrease has been
traced to a reduction of the neurons responsible for
orexin production.4 Orexins promote arousal and wake-
fulness and it is thought that low orexin levels in the
brain are important in sleep initiation.5 Several groups
have developed orexin antagonist to explore the physio-
logical role of the orexin receptors.6 Inhibiting orexin
The primary screens that we utilized were an in vitro
binding assay measuring the ability of a compound to
displace a high affinity radioligand bound to human
orexin receptor (reported as Ki) and a cell based FLIPR
assay (fluorometric imaging plate reader) which mea-
sures Ca2+ flux as a functional determinant of orexin
binding.8,9
Recently, compound 1a (Fig. 1), a benzimidazole con-
taining proline bis-amide, was identified as a potent
inhibitor of orexins from high throughput screening ef-
forts. The original lead had unknown stereochemistry.
Resynthesis of this lead in enantiomerically pure form
identified the S stereochemistry as the more active enan-
tiomer. The S enantiomer, 1b (hOX2R Ki = 1 nM,
hOX1R Ki = 28 nM), was a potent dual antagonist in
Keywords: Orexin; Insomnia; Narcolepsy; Dual orexin antagonist;
Orexin inhibitor; Potent; Benzimidazole; Proline bis-amide; Brain
penetrant.
*
Corresponding author. Tel.: +1 215 652 5976; fax: +1 215 652
7310; e-mail: jeffrey_bergman@merck.com
0960-894X/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2008.01.001

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J. M. Bergman et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1425–1430
alytic hydrogenation to remove the benzyl group, giving
N
the primary alcohol in quantitative yield. Oxidation via
Dess–Martin periodinane afforded the aldehyde which
was condensed with a 1,2-phenylenediamine to give
the corresponding benzimidazole.11 N-methylation of
the benzimidazole was accomplished with iodomethane
and cesium carbonate in DMF to afford compound X.
H
N
2
N 1
O
O
1a. screening lead (unknown stereochemistry)
1b. S enantiomer
S
N
1c. R enantiomer
HN
A large number of compounds were prepared in which
the 2-pyrrolylphenyl carboxamide was replaced with
various anilides (Table 1). Removal of the pyrrole group
resulted in a significant loss of potency toward both
orexin receptors (2). Replacement of the pyrrole with a
phenyl group yielded a compound similar in hOX2R
binding, but significantly enhanced hOX1R potency
afforded a more balanced dual inhibitor (3). Moving
the phenyl group to the meta (4) or para positions (5)
was not well tolerated. Changing the 2-biphenyl to a
2-phenyl ether (6) resulted in a small loss of activity to-
ward both receptors.
Figure 1. Proline bis-amide antagonists of orexin.
both the in vitro binding assay as well as the cell based
FLIPR assay. The corresponding R isomer, 1c (hOX2R
Ki = 131 nM, hOX1R Ki = 2500 nM), was significantly
less potent against both receptors. Fixing the L-proline
moiety (S stereochemistry) as the central part of our
molecule allowed us to explore the N-1 and C-2 amide
portions independently.
Synthesis of these proline bis-amides began with a cou-
pling of the appropriate aniline with Boc-L-proline
(Scheme 1). Classical peptide coupling methodology
(i.e. HOBt, mixed anhydride) was generally plagued
with poor yields. An improved procedure using phos-
phorus oxychloride in pyridine at 0 °C was developed
for the C-2 amide couplings.10 Removal of the BOC
group followed by standard peptide coupling conditions
put the N-1 amide in place. In some cases where the N-1
amide chain contained a thioether, acylation of the pro-
line with bromoacetyl bromide was followed by dis-
Compounds were prepared in which the sulfur was re-
placed with a carbon atom. These compounds were
somewhat less potent than their respective sulfur con-
taining counterparts but their SAR tracked similarly.
Compound 7 is about 10-fold less potent in hOX2R
binding and half as potent in the FLIPR assay than its
corresponding sulfur analogue (3). Additional C-1
amide replacements were evaluated with these carbon
chain benzimidazoles. The 2-biphenylamides were the
most promising compounds prepared in this series. Sub-
stitutions on the biphenyl such as o-methoxy (8) and o-
fluoro (9) were also tolerated.
placement of the a-bromide with
heterocyclic thiol.
a
suitable
For substituted benzimidazoles where the thioether was
replaced with a methylene unit, deviation from the first
route began with compound V (Scheme 2). Amide cou-
pling with 4-benzyloxybutyric acid was followed by cat-
The ability of a compound to cross the blood–brain bar-
rier and concentrate in the brain is an important consid-
eration in developing centrally active drugs.
A
significant concern for CNS activity is a compound’s
susceptibility to P-gp transport. A P-gp efflux assay
was used to determine if a compound is a substrate for
P-gp.12 It was determined that the benzimidazole N–H
was a P-gp liability. Compounds 1b (B to A/A to B ra-
tio = 6.8) and 10 (B to A/A to B ratio = 13) were sub-
strates for P-gp. Simply methylating 1b and 10 gave
compounds 11 (B to A/A to B ratio = 2) and 12 (B to
A/A to B ratio = 2), respectively. Both of these are only
moderate substrates for P-gp. An additional benefit of
this methylation was a boost in potency, often of the or-
der of 2- to 5-fold. Keeping the pyrrole fixed, a variety
of benzimidazole replacements were evaluated (Table
2). The benzothiazole (13) was similar in hOX2R po-
tency to the unmethylated benzimidazole while the benz-
oxazole (14) was less potent against both orexin
receptors.
N
OH
a, b
H
N
N
O
O
N
O
O
H
HCl
I
II
c
d
N
N
H
H
N
N
e
N
N
O
O
O
O
Modification of the benzimidazole by substitution at the
5 position afforded still greater enhancement in potency.
When a 5-methyl group is added to 1b (15), it becomes
equipotent to the N-methyl analogue 11. Methylation
of both positions (16) has an additive effect, yielding
one of the most potent dual orexin antagonists to date.
Moving the methyl to the 6 position (17) does not signif-
icantly change the orexin profile.13
IV
III
S
Br
het
Scheme 1. General synthesis of proline bis-amides (route A). Reagents
and conditions: (a) 1-(2-aminophenyl)pyrrole, POCl3, pyridine, 0 °C,
62%; (b) EtOAc, HCl, 100%; (c) carboxylic acid, EDC, HOBt, Et3N,
DMF, 135 °C in microwave, 9–85%; (d) bromoacetyl bromide, Et3N,
CH2Cl2, 0 °C, 91%; (e) HS-het, Cs2CO3, DMF, 60 °C in microwave,
30–60%.

J. M. Bergman et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1425–1430
1427
R'
R'
R'
HN
O
HN
O
a
b
HN
O
N
N
N
H
O
O
HCl
V
VI
VII
OH
OBn
c
R'
R'
R'
HN
HN
HN
O
e
d
N
N
N
O
O
O
O
O
X
VIII
IX
N
N
O
NH
N
CH3
R
R
Scheme 2. General synthesis of proline bis-amides (route B). Reagents and conditions: (a) 4-benzyloxybutyric acid, EDC, HOBt, Et3N, DMF 42%;
(b) Pearlman’s catalyst, MeOH, hydrogen, 89%; (c) Dess–Martin periodinane, NaHCO3, CH2Cl2 100%; (d) substituted 1,2-phenylenediamines,
Mn(OAc)3, HOAc, 15–30%; (e) MeI, Cs2CO3, DMF, 100%.
Table 1. SAR of C-2 amide
H
2
N
N 1
O
R
O
X
N
HN
Compound
R
X
hOX2R binding
(Ki, nM)a
hOX2R FLIPR
(IP, nM)a
hOX1R binding
(Ki, nM)a
hOX1R FLIPR
(IP, nM)a
1b
2
2-(1-Pyrrole)
S
1
560
0.6
280
11
3
6
4050
7
28
1400
10
240
20000
68
H
2-Ph
S
3
S
4
3-Ph
4-Ph
S
2640
143
16
500
2300
69
20000
1700
450
5
S
6
2-Phenoxy
2-Ph
S
7
C
C
C
6
20
5
40
86
240
210
8
2-(2-Methoxyphenyl)I
2-(2-Fluorophenyl)
2
9
3
4
27
68
a Values are means of two or more independent experiments.
Combining advantageous substitutions of the C-2 and
N-1 amides around the fixed proline constraint has an
additive effect on orexin activity. Combining the biphe-
nyl amide at C-2 with the N-methyl benzimidazole at N-
1 (18), increased hOX2R and hOX1R potency 5- and 9-
fold, respectively, compared to 1b. Replacement of the
sulfur in 18 with carbon (19) lowered the logP and im-
proved rat oral bioavailability with little consequence on
orexin binding. Both compounds are potent orexin
antagonists and have acceptably low P-gp susceptibility.
Compounds 18 and 19 represent optimized hOX2R/
hOX1R antagonists (Table 3).
are brain penetrant, affording 2.4 and 13 lM levels,
respectively, in the brain at 30 minutes after ip dosing
(100 mpk).14 Compounds 18 and 19 have water/octanol
partition coefficients of 3.4 and 2.6, respectively, and
neither compounds had any significant ancillary activity
as determined through Panlabs (MDS Pharma).
In order to determine if a dual orexin antagonist could
attenuate the actions of the ADL-orexin B peptide
in vivo, a rat locomotion study was developed.15 Move-
ment parameters were measured through use of a beam
box. After determining baseline activity for the rats, sig-
nificant increases in activity were observed when rats re-
ceived ICV injections of ADL-orexin B peptide.16 The
experiments were then performed with varying doses
Brain/plasma levels were determined following intra-
peritoneal dosing (ip) in rats. Compounds 18 and 19

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J. M. Bergman et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1425–1430
Table 2. SAR of N-1 amide
N
H
2
N
N 1
O
O
R
Compound
R
hOX2R binding
(Ki, nM)a
hOX2R FLIPR
(IP, nM)a
hOX1R binding
(Ki, nM)a
hOX1R FLIPR
(IP, nM)a
N
10
11
11
36
2
130
9
520b
22
N
H
N
S
S
0.3
N
N
12
13
2
2
3
49
39
480
545
N
N
11
S
N
14
15
16
17
8
28
2
120
3
1750
20
S
S
O
N
0.2
0.09
0.1
N
H
N
N
S
S
8
0.7
2
11
N
N
8
28
a Values are means of two or more independent experiments.
b Value is for rat OX1R FLIPR.
Table 3. Optimized orexin inhibitors
H
N
H
N
N
O
N
O
O
O
S
N
N
N
N
Property
18
19
hOX2R Ki (nM)a
hOX1R Ki (nM)a
hOX2R FLIPR (nM)b
hOX1R FLIPR (nM)a
P-gp (B to A/A to B)
log P
0.2
3
0.8
15
4
17
5
98
1.4
3.4
3.7
0.3 h
11
2
2.6
Rat PK (Cl)c
35
0.3 h
Rat PK (t1/2)
Rat PK (%F)
23
13007/25705/860
2 hits < 10 lM
Brain/plasma/CSF (nM)d
2370/3500/43
3 hits < 10 lM
e
Panlabs (IC50
)
a See Ref. 8 for and assay description and relevant reference therein.
b See Ref. 9 for an assay description.
c Measured in mL/min/kg.
d 100 mpk ip 30 min. See Ref. 14 for an assay description and relevant reference therein.
e Panlabs (MDS Phama): compound 18: transporter, Monoamine: Protanoid, Thromboxane A2 (TP); Thyrotropin releasing Hormone (TRH) 19:
Sodium Channel, Site 2; Thyrotropin Releasing Hormone (TRH).

J. M. Bergman et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1425–1430
1429
Danielson, P. E.; Fukuhara, C.; Battenberg, E. L.;
Gautvik, V. T.; Bartlett, F. S., 2nd; Frankel, W. N.; van
den Pol, A. N.; Bloom, F. E.; Gautvik, K. M.; Sutcliffe, J.
G. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 322.
2. (a) Smart, D.; Jerman, J. C. Pharmacol. Ther. 2002, 94, 51;
(b) Baumann, C. R.; Bassetti, C. L. Lancet Neurol. 2005, 4,
673.
3. (a) Peyron, C.; Faraco, J.; Rogers, W.; Ripley, B.;
Overeem, S.; Charnay, Y.; Nevsimalova, S.; Aldich, M.;
Reynolds, D.; Albin, R.; Li, R.; Hungs, M.; Pedrazzoli,
M.; Padigaru, M.; Kucherlapati, M.; Fan, J.; Maki, R.;
Lammers, G. J.; Bouras, C.; Kucherlapati, R.; Nishino, S.;
Mignot, E. Nat. Med. 2000, 6, 991; (b) Nishino, S.; Ripley,
B.; Overeem, S.; Lammers, G. J.; Mignot, E. Lancet 2000,
355, 39.
4. (a) Thannickal, T. C.; Moore, R. Y.; Nienhuis, R.;
Ramanathan, L.; Gulyani, S.; Aldrich, M.; Cornford,
M.; Siegel, J. M. Neuron 2000, 27, 469; (b) Blouin, A. M.;
Thannickal, T. E.; Worley, P. F.; Baraban, J. M.; Reti, I.
M.; Siegel, J. M. Neurology 2005, 65, 1189; (c) Crocker,
A.; Espana, R. A.; Papadopoulon, M.; Saper, C. B.;
Faraco, J.; Sakurai, T.; Honda, M.; Mignot, E.; Scammell,
T. E. Neurology 2005, 65, 1184.
5. (a) Espana, R. A.; Baldo, B. A.; Kelley, A. E.; Berridge, C.
W. Neuroscience 2001, 106, 699; (b) Saper, C. B.; Choe, T.
C.; Scammet, T. E. Trends Neurosci. 2001, 24, 726; (c)
Sukurai, T. Nature Rev. 2007, 8, 171.
6. (a) Porter, R. A.; Chan, W. N.; Coulton, S.; Johns, A.;
Hadley, M. S.; Widdowson, K.; Jerman, J. C.; Brough, S.
J.; Coldwell, M.; Smart, D.; Jewitt, F.; Jeffrey, P.; Austin,
N. Bioorg. Med. Chem. Lett. 2001, 11, 1907; (b) Hirose,
M.; Egashira, S.; Goto, Y.; Hashihayata, T.; Ohtake, N.;
Iwaasa, H.; Hata, M.; Fukami, T.; Kanatani, A.; Yamada,
K. Bioorg. Med. Chem. Lett 2003, 13, 4497; (c) McAtee, L.
C.; Sutton, S. W.; Rudolph, D. A.; Li, X.; Aluisio, L. E.;
Phuong, V. K.; Dvorak, C. A.; Lovenberg, T. W.;
Carruthers, N. I.; Jones, T. K. Bioorg. Med. Chem. Lett.
2004, 14, 4225.
7. Brisbare-Roch, C.; Dingemanse, J.; Koberstein, R.;
Hoever, P.; Aissaoui, H.; Flores, S.; Mueller, C.; Nayler,
O.; van Gerven, J.; de Haas, S. L.; Hess, P.; Qiu, C.;
Buchmann, S.; Scherz, M.; Weller, T.; Fischli, W.; Clozel,
M.; Jenck, F. Nat. Med. 2007, 13, 150.
Figure 2. Induced Locomotion Inhibition.17
.
of compound 18 (15 mpk, 50 mpk, 100 mpk) adminis-
tered intraperitoneally, 30 minutes before ADL-orexin
B administration. Beam breaks were measured versus
time and the AUCs over a 180 minute window were
graphed (Fig. 2).18
A large increase in activity was observed in rats treated
with ADL-orexin B alone. Pretreatment with compound
18 showed a clear, dose dependent inhibition of this
excitation. At the 100 mpk dose locomotion was re-
turned to baseline.
We have described a novel series of dual orexin antago-
nists based on a proline bis-amide scaffold. Starting with
a screening lead, we were able to quickly identify antag-
onists with subnanomolar to low nanomolar potency for
hOX2R and hOX1R. The series demonstrated excellent
in vitro cell based activity, brain penetration, and low
to moderate bioavailability in rats. We further demon-
strated the ability of compound 18 to inhibit ADL-orex-
in B mediated locomotion, when dosed peripherally, in a
dose dependent manner. Further improvements will be
disclosed in due course.
8. (a) Radioligand binding assays for orexin receptors. [3H]-
compound 11 was used to determine the OX2R binding
and [3H]-compound 18 for OX1R binding. Membranes
were prepared from CHO/OX2R and CHO/OX1R cells. A
dose-inhibition radioligand binding assay was performed
by the Tecan robot by a standard protocol to test
compounds synthesized by medicinal chemistry pro-
grams.8b; (b) Mosser, S. D.; Gaul, S. L.; Bednar, B.;
Koblan, K. S.; Bednar, R. A. JALA 2003, 8, 54.
Acknowledgments
The authors are grateful to K.D. Anderson, T. Ander-
son, K.L. Hoffmann, J.S. Murphy, C.W. Ross III,
G.M. Smith, and M.M. Zrada for analytical support,
C. Lindsley, D. Winowski for library synthesis support,
and M.J. Bogusky for NMR assistance.
9. FLIPR assay. CHO-K1 cells expressing the orexin recep-
tor (hOX1R or hOX2R) were seeded into 96-well plates
and incubated with a cytoplasmic calcium indicator. After
washing the cells, intercellular Ca2+ mobilization was
monitored as a change in cell fluorescence intensity by
FLIPR (Molecular Devices). Differing concentrations of
orexin antagonists were added to the plates prior to
addition of orexin A and antagonistic activity calculated.
10. Rijkers, D.; Adam, H.; Hemker, H. C.; Tesser, G. I.
Tetrahedron 1995, 41, 11235.
References and notes
11. Ouyang, C.; Ouyang, Y.; Fujii, Y.; Nakano, Y.; Shoda, T.;
Nagano, T. J. Heterocycl. Chem. 2004, 41, 359.
1. (a) Sakurai, T.; Amemiya, A.; Ishii, M.; Matsuzaki, I.;
Chemelli, R. M.; Tanaka, H.; Williams, S. C.; Richardson,
J. A.; Kozlowski, G. P.; Wilson, S.; Arch, J.; Buckingham,
R. E.; Haynes, A. C.; Carr, S. A.; Annan, R. S.; McNulty,
D. E.; Liu, W.; Terrett, J. A.; Elshourbagy, N. A.;
Bergsma, D. J.; Yanagisawa, M. Cell 1998, 92, 573; (b) de
Lecea, L.; Kilduff, T. S.; Peyron, C.; Gao, X.; Foye, P. E.;
12. (a) P-gp is a N-glycosylated integral membrane protein
with two ATP binding sites, which function as an energy-
dependent drug efflux pump. P-gp efflux is determined by
a transcellular transport study using human MDR1 cells
and measuring the transport of a compound from basal to

1430
J. M. Bergman et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1425–1430
apical and apical to basal cells then determining the ratio
water. Experiments were conducted between 8 AM and
2 PM each day using Med Associates Open Field activity
chambers that employ infrared photobeam technology.
Photobeam bars are mounted externally on the chamber
and locomotor activity was recorded in the xy-plane
(ambulations, fine movements, distance traveled) and z-
plane (rearing) as the number of beams interrupted. Each
rat was allowed to acclimate to the chamber for 90 min-
utes. The rat then received an intraperitoneal injection of
either vehicle or test compound. Thirty minutes later, each
rat received an ICV infusion of either vehicle (aCSF) or
ADL-OXB ([Ala11-D-Leu15]-orexin-B) using a microdialy-
sis pump delivering the peptide into the lateral ventricle.
The ICV cannula was capped and the rat was then
returned to the activity chamber where the locomotor
activity was recorded for an additional 180 minutes. The
data were tabulated in 10 minute bins. Mean responses
and SEM were calculated and graphed using Sigmaplot
graphing software. Area under the activity curve (AUC)
was calculated and graphed using GraphPad Prism.
16. Asahi, S.; Egashira, S.; Matsuda, M.; Iwaasa, H.; Kana-
tani, A.; Ohkubo, M.; Ihara, M.; Morishima, H. Bioorg.
Med. Chem. Lett. 2003, 13, 111.
17. This graph represents effects on fine movement activity in
ICV cannulated rats. Similar profiles were observed in the
other three locomotion parameters measured as well
(distance traveled, ambulatory movement, and vertical
movement). Although locomotor activity was recorded for
180 minutes (as described in Ref. 15), the control response
returns to baseline activity levels around 120 minutes. The
AUC is calculated during this time frame. This is
represented on the graph as what we considered to be
the measurable response.
(B to A/A to B); (b) Hochman, J. H.; Pudvah, Q.;
Yamazaki, M.; Tang, C.; Lin, J. H.; Prueksaritanont, T.
Pharm. Res. 2004, 21, 1686; (c) Yamazaki, M.; Neway, W.
E.; Ohe, T.; Chen, I.; Rowe, J. F.; Hochman, J. H.; Chiba,
M.; Lin, J. H. J. Pharmacol. Exp. Ther. 2001, 296, 723.
13. While the majority of the proline bis-amides described
are dual inhibitors of orexin, it is noteworthy that
considerable hOX2R selectivity can be achieved in this
series with certain modifications. When the upper amide
contains the 1-(4-phenyl)anilide (compound 5), a moder-
ately potent hOX2R antagonist with greater than 200-
fold selectivity for hOX2R versus hOX1R binding is
obtained.
14. (a) Brain/plasma/CSF methods. Male rats are dosed
intraperitoneally (ip) with the test article or vehicle
(control) and returned to their home cage for the test
article incubation period, either 30 or 120 minutes. At the
end of the incubation period, the rat is placed in an
anesthesia induction chamber and anesthetized with iso-
flurane. Once anesthetized, the rat is placed on a breathing
circuit (nose cone) and anesthesia is maintained. The head
is shaved and the shaved area wiped with an alcohol pad.
The rat is placed on an angled plexi-glass stand and the
head is flexed over the top of the stand. The CSF is
collected from the cisterna magna by needle puncture and
gentle aspiration. The CSF is dispensed and immediately
frozen on dry ice. The rat is then placed on its back and a
blood sample is obtained from the same anesthetized rat
via cardiac puncture. The blood is centrifuged and the
plasma is removed via pipette, dispensed into an appro-
priate vial, and frozen on dry ice. After the blood is
collected, the rat is euthanized and the brain is removed.
The olfactory bulbs and hind brain are then removed and
discarded. The CSF, plasma, and brain samples are stored
at À70 °C until assayed by Analytical Chemistry for
compound level; (b) Fujiki, N.; Yoshida, Y.; Ripley, B.;
Honda, K.; Mignot, Emmanuel; Nishino, S. NeuroReport
2001, 12, 993.
18. All animals used in these studies were cared for in
accordance with the NIH Guide for the Care and Use of
Laboratory Animals. The Merck Research Laboratories
Institutional Animal Care and Use Committee (IACUC)
approved all studies described in this manuscript and
experimental protocols were in accordance with all appli-
cable guidelines regarding the care and use of animals.
Animals were housed in an AALAC International
approved facility with free access to food and water.
15. Locomotion assay. Male ICV cannulated rats were indi-
vidually housed, maintained on a 12/12 light/dark cycle
with lights on at 6 AM, and had free access to food and